Using confocal Ca2+ imaging, patch-clamp techniques, and immunocytochemistry, the relationship between t-tubule density and cholinergic regulation of intracellular Ca2+ in normal murine ventricular myocytes and myocytes with acute disruption of the t-tubule system caused by formamide treatment was studied. The inhibitory effect of muscarinic receptor agonist carbachol  on the amplitude of Ca2+ transients, evoked by field-stimulation in the presence of 100 nM isoproterenol (Iso), a β-adrenergic agonist, was directly proportional to the level of myocyte detubulation. The timing of the maximal rate of fluorescence increase of fluo-4, a Ca2+-sensitive dye, was used to classify image pixels into the regions functionally coupled or uncoupled to the sarcolemmal Ca2+ influx (ICa). CCh decreased the fraction of coupled regions and suppressed Ca2+ propagation from sarcolemma inside the cell. Formamide treatment reduced ICa density and decreased sarcoplasmic reticulum (SR) Ca2+ content. CCh did not change SR Ca2+ content in Iso-stimulated control and formamide-treated myocytes. CCh inhibited peak ICa recorded in the presence of Iso by ∼20% in both the control and detubulated myocytes. Reducing ICa amplitude up to 40% by changing the voltage step levels from 0 to –25 mV decreased Ca2+ transients in formamide-treated but not in control myocytes in the presence of Iso. CCh inhibited CaMKII activity, whereas CaMKII inhibition with KN93 mimicked the effect of CCh on Ca2+ transients in formamide-treated myocytes. It was concluded that the downregulation of t-tubules coupled with the diminished efficiency of excitation–contraction coupling, increases the sensitivity of Ca2+ release and propagation to muscarinic receptor-mediated inhibition of both ICa and CaMKII activity.Muscarinic receptors expressed in cardiac myocytes play a critical role in the regulation of heart function by the parasympathetic nervous system. How the structural organization of cardiac myocytes affects the regulation of Ca The juxignaling . Accordiignaling . Growingartments . However2+ channels, and decrease in CaMKII-dependent activity of RyR2.In the present study, the mechanisms underlying the regulation of ECC by muscarinic receptor stimulation in the setting of acute t-tubule disruption were addressed. Using formamide treatment, a well-characterized approach to detubulate ventricular myocytes, it was found that the downregulation of t-tubules amplifies the inhibitory effects of muscarinic receptor stimulation on ECC. Mechanistically, this effect was attributed to the increased sensitivity of ECC to muscarinic receptor-mediated effects, inhibition of the L-type Ca2, 10 HEPES, and 5.5 glucose with pH 7.4. Using the Langendorff apparatus, hearts were perfused with the a Ca2+-free solution at 37°C for 5 min that was followed by perfusion with a Ca2+-free solution containing Liberase TH . Following enzymatic digestion (10–20 min), hearts were minced and triturated in the perfusion solution containing 2% BSA. Following two rounds of gravity sedimentation, ventricular myocytes were plated on the laminin (40 μg/ml) and stored at room temperature (RT). Isolated cells were used for experiments within 6 h after isolation.All animal procedures were approved by the Ohio State University Institutional Animal Care and Use Committee and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health . Ventricular myocytes were isolated from 2 to 6-month-old C57BL/6J mice (Jackson Laboratory Stock No: 000664) of either sex. Mice were anesthetized with 5% isoflurane in 95% oxygen, hearts were rapidly excised and cannulated through the aorta for perfusion with an ice-cold calcium-free solution containing (in mM): 140 NaCl, 5.4 KCl, 0.5 MgCl2, 0.5 MgCl2, 5.6 glucose, and 10 HEPES (pH 7.4). Myocytes were incubated in a low Ca2+ external solution (0.5 mM CaCl2) containing 9 μM Fluo-4 AM  for 20 min at RT. Following 15–20 min of de-esterification in the dye-free external solution, intracellular Ca2+ transients were induced by electrical field stimulation  using a pair of platinum electrodes. The following extracellular solution was used for the field-stimulation experiments (mM): 140 NaCl, 5.4 KCl, 2.0 CaCl2, 0.5 MgCl2, 10 HEPES, and 5.6 glucose (pH 7.4).The cellular experiments were performed using an open bath imaging chamber  that was continuously perfused with an external solution containing the following (in mM): 140 NaCl, 5.4 KCl, 2.0 CaCl2+ channels by muscarinic receptor agonist, Ca2+ currents were recorded in the extracellular solution containing 1 mM CaCl2 and 5.4 mM CsCl replacing KCl. Patch pipettes were filled with a solution that contained in mM: 123 CsCl, 20 TEACl, 5 MgATP, 10 NaCl, 1 MgCl2, 0.1 Tris GTP, 5 EGTA, and 10 HEPES (pH 7.2). Whole-cell patch-clamp configuration was established using an Axopatch 200B amplifier coupled to Digidata1440 data acquisition system . In these experiments, the Ca2+ currents were evoked by 100 ms depolarization steps to 0 mV every 10 s. The depolarization steps were preceded by a 100 ms step from –80 to –50 mV to inactivate Na+ currents.In experiments directed to study the regulation of the L-type Ca2+ transients was performed using Olympus FluoView FV 1000  confocal microscope system equipped with x60 oil-immersion objective lens (NA 1.4). Fluo-4 was excited with a 488 nm line of argon laser, and the signal was collected at 500–600 nm wavelengths. Linescan images were acquired along the central axis of the myocytes at a speed of 2.1 ms per line. Fluorescence signals were normalized to the baseline cellular fluorescence (F0).Imaging of the intracellular Ca2+ currents simultaneously with the intracellular Ca2+ transients, the extracellular solution containing 1 mM CaCl2 was used and in the patch pipette solution, 5 mM EGTA was replaced with 0.1 mM fluo-3 K5 . In these experiments, the following voltage protocol was used: 100 ms voltage ramp was applied from the holding potential of –80 to –45 mV, at which level the voltage was kept for 500 ms followed by a 50 ms depolarization step to either –25, or –20, or 0 mV to induce the Ca2+ currents of different amplitude. The Ca2+ currents were evoked every 8 s. A train of 10 pulses from –80 to –10 mV was applied at 2 Hz to maintain steady SR Ca2+ loading before each Ca2+ current-inducing voltage protocol.To record Ca2+ content was assessed by the rapid application of an extracellular solution containing 5 mM caffeine and 20 mM 2,3-butanedione monoxime, as previously described  for 60 min at RT. Following overnight incubation at 4°C in the blocking solution containing primary antibody  antibody, ab32678, Abcam, Cambridge, United Kingdom) and several rounds of PBS washes, myocytes were incubated in the blocking solution containing secondary antibody  for 90 min at RT. After 3 × 5 min of PBS washes, the samples were mounted using ProLongTM Gold Antifade Mountant . Images were acquired using an Olympus FluoView FV 1000  confocal microscope system equipped with a ×60 oil-immersion objective lens (NA 1.4). Following subtraction of the non-cellular background signal, average fluorescence, excluding nuclear area, was calculated for each myocyte and normalized to the value of mean fluorescence value obtained from a group of myocytes incubated in 100 nM Iso.Plated on laminin cardiomyocytes were incubated for 10 min in the external solution containing either isoproterenol , or Iso (100 nM) plus carbachol (CCh) (10 μM), or Iso (100 nM) plus KN93 (2 μM). Myocytes were electrically stimulated for 1 min at 2 Hz and 1 min at 1 Hz. Following stimulation protocol, cells were immediately fixed with 4% paraformaldehyde (10 min at RT) and washed with phosphate-buffered saline . Fixed myocytes were permeabilized with 0.25% Triton X-100 in PBS  and incubated in blocking solution  during the rising phase of Ca2+ transients was calculated for each pixel. The obtained distribution of time to (dF/dt)max was used to classify pixels into “early” and “delayed,” as described in the “Results” section. T-tubule density was analyzed according to The activation time of Carcolemma . Time to1, 1997–2018) software. Aggregate data were analyzed using the R software environment  and OriginPro 2020b . Results are expressed as the mean ± SEM. Statistical significance was determined using either ANOVA with Tukey’s post hoc test or Student’s t-test with p-values of 0.05. Linear fits to the data were compared using the F test.Images were analyzed using MATLAB  and ImageJ  unless specified otherwise.2+ transients evoked by 1 Hz electrical field stimulation in ventricular myocytes isolated from WT mouse heart. In ventricular myocytes, the effect of muscarinic receptor stimulation is generally attributed to the modulation of cAMP-dependent responses coupled to β-adrenergic receptor stimulation  in control and formamide-treated myocytes and obtained time distribution of maximal rate of fluorescence increase (dF/dt)max for each line-scan pixel in each myocyte (2+ release was highly synchronous [(dF/dt)max for 98% of line-scan pixels occurred within 6.3 ms]. Formamide-treatment decreased the fraction of fast response regions (first 4.2 ms) by about 50% and significantly increased the fraction of regions displaying (dF/dt)max after 6.3 ms (2+ transients stem mostly from decreased t-tubule density (F/dt)max after 8.5 ms as “delayed,” or uncoupled from the t-tubules, whereas line-scan pixels with (dF/dt)max occurring within first 4.2 ms were classified as “early,” or coupled to t-tubule regions.The time delay between the electrical stimulus and activation of Carcolemma . We reco myocyte . As demor 6.3 ms . Assumin density , we clasp = 0.8). 2+ release regions. As illustrated in 2+ transients observed in the presence of 100 nM Iso. In contrast, peak Ca2+ transient recorded in the presence of Iso plus CCh inversely correlated with the proportion of the delayed regions . In the presence of 100 nM Iso plus 10 μM CCh, the relationship between the rate of Ca2+ transients decay and detubulation levels was fitted to the linear curve with the correlation coefficient –0.05 and the slope that was not different from 0 (p = 0.78).Following formamide treatment, the proportion of the delayed regions was on average 44% in myocytes treated with Iso alone and 42% in cells challenged with Iso plus CCh max recorded at the delayed regions to that recorded at the early regions, was significantly inhibited by the CCh , but significantly inhibited it in formamide-treated myocytes . Furthermore, CCh significantly reduced fractional release in myocytes with reduced t-tubules density ; Iso plus CCh group: 33.43 ± 0.79 ms in control vs. 53.84 ± 4.52 ms in formamide-treated myocytes (p < 0.001)  .2+ transients cannot be explained by changes in the SR Ca2+ load. Rather, these point to the potential role of decreased Ca2+ influx and/or reduced Ca2+ sensitivity of the SR Ca2+ release channels as potential mechanisms mediating the effect of a muscarinic receptor agonist.These results indicate that the inhibitory effect of CCh on the amplitude of Ca2+ channel current (ICa) was compared with Iso and CCh in control and formamide-treated ventricular myocytes. Formamide treatment reduced the cell capacitance by 33% and decreased the density of baseline Ca2+ current by 58%  reduction in the peak ICa. This experimental protocol produced similar decrease in ICa amplitude  in formamide-treated myocytes. However, in control ventricular myocytes, this decrease in ICa was not associated with the changes in Ca2+ transient . In contrast, in formamide-treated myocytes, Ca2+ transients were significantly reduced by such a decrease in ICa  (p = 0.05), whereas the gain recorded at –25 mV in formamide-treated myocytes was only 46 ± 6% of that measured in control myocytes  . Accordi2+ signal propagation is inhibited by CCh max, was indistinguishable from that recorded in the presence of CCh (F/dt)max at early regions and delayed regions fraction was similar in myocytes treated with Iso plus KN93 and Iso plus CCh max ratio was examined in myocytes treated with Iso plus CCh plus KN93. As shown in F/dt)max ratio was not significantly affected by CCh, suggesting that CCh had no effects on Ca2+ propagation under these conditions.It has been previously reported that in control and failing ventricular myocytes CCh reduced CaMKII-dependent phosphorylation of RyR2, a post-translational modification that is strongly associated with the increased sensitivity of RyR2 to Ca2+ . In this2+ . In cont2+ . For com2+ , reduced2+ . Next, te of CCh . Second,e of CCh . In addiplus CCh , 9B. To 2+ transients observed in myocytes with downregulated t-tubules can be attributed, at least partially, to the reduced levels of CaMKII-dependent phosphorylation of RyR2.Overall, these results suggest that the inhibitory effect of muscarinic receptor stimulation on Ca2+ release in cardiac disease  coupling in ventricular myocytes, whereas disorganization and downregulation of t-tubules have been consistently associated with the defective SR Ca disease . In this2+ transients and myocyte shortening (2+ transients occurred only during β-adrenergic receptor stimulation with Iso (2+ and Na+ signaling (2+ transients (2+ release sites and t-tubule sarcolemma (2+ transient and detubulation level for each myocyte. This analysis showed that the amplitude of Ca2+ transients recorded in the presence of 100 nM Iso alone was not significantly affected by the extent of detubulation (In ventricular myocytes, the negative ionotropic effect associated with the stimulation of muscarinic receptors is predominantly attributed to the inhibition of β-adrenergic receptor–mediated stimulatory effects on Caortening . Accordiwith Iso . In thiswith Iso . Growingignaling , but alsignaling . To testignaling . In our ignaling  and enhaansients . We usedrcolemma  to studybulation . In contbulation .Ca to Iso in control and detubulated rat ventricular myocytes. 2+ channels by signaling mediated via β2-adrenergic and muscarinic receptors in mouse model of heart failure. However, in our experiment we did not find a significant difference in the sensitivity of ICa to Iso between the control and detubulated myocytes (Ca to CCh in control and detubulated ventricular myocytes (2+ channel regulation.To explain this effect, we considered the possibility that β-adrenergic receptor and/or muscarinic receptor-mediated signaling is different at the surface sarcolemma vs. t-tubular domain. Indeed, myocytes , suggestmyocytes . Therefo2+ channels and RyR2, thus permitting the high fidelity coupling between the membrane depolarization and SR Ca2+ release (2+ influx, significantly increases the fraction of RyR2s uncoupled from the L-type Ca2+ channels, and thereby, increases the contribution of regenerative Ca2+ release to the global Ca2+ transient (Ca density in the presence of 100 nM Iso (2+ transients was not affected by detubulation (2+ influx and SR Ca2+ release observed at baseline becomes flattened during β-adrenergic stimulation (Ca amplitude beyond a certain level becomes redundant as it does not produce a further increase in the amplitude of Ca2+ transients. Accordingly, our experiments indicate that the presence of 100 nM Iso voltage-dependent reduction of ICa peak by 40% did not significantly change the amplitude of Ca2+ transients in control ventricular myocytes (2+ release becomes more sensitive to changes in ICa density as demonstrated in experiments illustrated in Ca in control and detubulated ventricular myocytes induced by CCh (2+ transients in formamide-treated than in control myocytes.In ventricular myocytes, t-tubules support the spatial alignment of the L-type Ca release . Downregransient . In the 0 nM Iso . Remarkabulation . These rmulation . In othemyocytes . In myocd by CCh  may have2+ release sites functionally coupled and uncoupled from the t-tubules. The initial experiments in formamide-treated myocytes indicated that muscarinic receptor stimulation did not affect the properties of coupled Ca2+ release sites (2+ transients in the presence of Iso and Iso plus CCh in the same myocytes revealed that CCh reduced the fraction of coupled Ca2+ release sites by only 6% (2+ transients (2+ release sites. In contrast, the CCh significantly inhibited Ca2+ release at the uncoupled Ca2+ release sites (2+ release sites, thus suggesting that CCh suppressed propagation of the Ca2+ signal from sarcolemma inside the cell. Propagation of Ca2+ under these conditions strongly relies on regenerative Ca2+ release at the uncoupled sites. This process is regulated by Ca2+ concentration inside the SR and by the sensitivity of RyR2 to cytosolic Ca2+ which is determined by both SR Ca2+ content and post-translational modifications of the protein (2+ content recorded in paced control and formamide-treated myocytes (2+ transients in formamide-treated ventricular myocytes (2+ transients in ventricular myocytes with disrupted t-tubules critically depends on inhibition of CaMKII-dependent phosphorylation of uncoupled RyR2 clusters.Analysis of time-dependent changes in fluorescence response to field stimulation  allowed se sites . Further only 6% . These ransients , 2, muscse sites . This ef protein . In our myocytes , suggestmyocytes  and oxidmyocytes . All of myocytes . This efmyocytes . Furthermyocytes , completmyocytes . Taken t2+ waves (2+ release site activity (2+ release sites was also found important for Ca-dependent arrhythmogenesis in heart failure (CaMKII-dependent phosphorylation of RyR2 has been associated mostly with pathological remodeling and manifested as the increased myocyte propensity to arrhythmogenic Ca2+ waves . The preactivity . CaMKII- failure . The resAcute disruption of t-tubule organization in ventricular myocytes by formamide has been an invaluable tool for the research of the functional role of t-tubule domain and domain-related proteins . Further2+ handling in ventricular myocytes that may be of importance in cardiac diseases, such as heart failure, which is associated with downregulated t-tubules (Heart failure has been associated with the diminished contribution of the parasympathetic nervous system in the regulation of cardiac function . Althoug-tubules . The pre-tubules  may be aThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by The Ohio State University Institutional Animal Care and Use Committee.AB, DT, and SG planned the research and wrote the manuscript. AB and VB designed and performed the experiments and analyzed the experimental data. All authors contributed to the article and approved the submitted 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.


In addition, rapid SHG imaging can be used to visualize laser-induced ablation of monolayer and bilayer MoS2 in situ under illumination by a strong femtosecond laser. Importantly, we observed a characteristic threshold behavior; the ablation process occurred for a very short time duration once the preheating condition was reached. We investigated the laser thinning of the bilayer MoS2 with different twist angles. When the twist angle was 0°, the SHG decreased by approximately one-fourth of the initial intensity when one layer was removed. Conversely, when the twist angle was approximately 60° (the SHG intensity was suppressed), the SHG increased abruptly close to that of the nearby monolayer when one layer was removed. Precise layer-by-layer control was possible because of the unique threshold behavior of the laser-induced ablation.We report an in situ characterization of transition-metal dichalcogenide (TMD) monolayers and twisted bilayers using a high-speed second-harmonic generation (SHG) imaging technique. High-frequency laser modulation and galvano scanning in the SHG imaging enabled a rapid identification of the crystallinity in the TMD, including the orientation and homogeneity with a speed of 1 frame/s. For a twisted bilayer MoS Conversely, in MoS2 with an even number of layers, the restoration of the crystal inversion symmetry largely suppresses the SHG signals [The noncentrosymmetric structures of transition-metal dichalcogenides (TMDs) enable an efficient second-harmonic generation (SHG). Hence, TMDs are advantageous in the development of next-generation optoelectronic devices with the increasing demand for smaller and thinner nonlinear media ,9,10,11. signals ,9,10,11. signals ,22,23,24 signals ,12,25. I signals ,25.In order to achieve SHG signals from the monolayer and few-layer TMDs, a femtosecond laser was focused through a microscope in which the SHG intensity was recorded as a function of the laser polarization angle. On the other hand, point-by-point imaging with SHG signals is a very powerful method for mapping of grain boundaries, homogeneity, structure-induced nonlinear enhancement, and interfaces between different layers ,14,15,162 as a function of the twist angle. In addition, we monitored the in situ ablation of monolayer and bilayer MoS2 induced by the femtosecond laser.In this study, we introduce a high-speed scanning SHG microscopy technique by incorporating galvano scanning combined with high-frequency laser modulation. Images were acquired with a typical scanning speed of 1 frame/s and a signal-to-noise ratio (SNR) of 30 dB. This enabled a rapid identification of the crystallographic direction in TMD materials as well as the grain boundaries and homogeneity of the crystallinity. In particular, we investigated the SHG intensity of an artificially stacked bilayer MoS2 flakes were grown on a SiO2/Si substrate by atmospheric-pressure CVD with MoO3  and S  powders as precursors. The substrate was placed facing down toward MoO3 (3 mg) on a ceramic boat, which was loaded into the center of the quartz tube in the CVD setup. A second ceramic boat containing 500 mg of sulfur powder was kept at the outside and upstream parts of the furnace, where the temperature could be controlled independently by another heating controller. The furnace and ceramic boat with the sulfur powder were heated to 650 and 200 °C with ramping rates of 32 and 10 °C/min, respectively. After 10 min of growth, the furnace was turned off and cooled to room temperature. Conversely, WSe2 flakes were grown on a SiO2/Si substrate by atmospheric-pressure CVD with WO3  and Se  powders as precursors. The furnace and ceramic boat with the selenide powder were heated to 850 and 250 °C, respectively. After 10 min of growth, the furnace was switched off and cooled to room temperature.MoS2 was prepared by wet transfer of the CVD-synthesized MoS2 flakes into another SiO2/Si substrate containing the MoS2 flakes. We used poly(methyl methacrylate) (PMMA) as a mechanical supporter, which was removed in acetone after the transfer. As there are hundreds of individual flakes on the substrate, each transfer creates dozens of bilayer heterostructures with various twist angles. Finally, the as-transferred MoS2 heterostructure was annealed in a vacuum environment at 400 °C for 1 h to remove PMMA residues.A twisted MoS2 and WSe2). We confirm that the SHG signals from WSe2 and MoS2 layers exhibited the six-fold symmetry as a function of polarization angle and a quadratic laser-power-dependence  images of the intensity and peak angle, as shown in 2 with a random orientation, fabricated artificially by stacking the CVD-grown monolayer MoS2, as shown in the microscopy image in 2 monolayer has been addressed and analyzed successfully in terms of polarization interference [2 layers possessing a strong interlayer coupling to investigate the possibility of the SHG enhancement effects deviating from the polarization interference model. We first show a photoluminescence (PL) image for comparison in We applied our technique to study the bilayer MoSrference . In thisθtwist = φ1 – φ2 using the angles φ1 and φ2 recorded for the adjacent layers in the image in θtwist = 0°, whereas it was suppressed virtually to zero for θtwist = 60°.As shown in 2 structures and studied the SHG peak intensity and angle as a function of θtwist (φs) in the twisted region exhibits a value that follows the relation φs = (φ1 – φ2)/2, as indicated by the dashed line in θtwist > 40° is likely due to errors originating from weak signals, which originates from the destructive interference.We tested more than 50 twisted bilayer MoSf θtwist a; the reneration . In addi2 bandgap (1.8 eV), a two-photon process is required for ablation. The ablation by the femtosecond laser is advantageous over those by continuous-wave or nanosecond lasers in general because it results in minimal collateral damage within the sample [2, as shown in 2 layer, while the SHG was monitored in situ using the galvano scanning method, as discussed above.The rapid SHG imaging can be used to visualize laser-induced ablation in situ under illumination by a strong femtosecond laser. As the energy of the laser (1.55 eV) is lower than the MoSe sample . We begaφ = 27°) before  before c the las in MoS2 ,40 folloperature .τ1 and τ2) once the ablation occurred. By fitting the curves, we summarize T and the time constants as functions of the laser fluence in τ1 (~10 s) did not vary with the laser fluence. In other words, the laser ablation of the MoS2 layer occurred for a very short period of time once the condition for chemical bond breakage was reached. This is characteristic threshold behavior, which has not been reported. It is likely that the lowering of the damage threshold due to the accumulated lattice heating caused the abrupt ablation processes, whereas the detailed mechanism during the ablation processes has to be addressed further [τ2) is likely due to the removal of the residues or the additional ablation around the edge of the spot  in 2. The SHG images acquired at t = 0, 280, and 880 s represent the times before ablation, when the top layer was removed, and when both layers were removed, respectively. This is more clearly shown in Finally, we monitored the thinning of the bilayer using the rapid SHG imaging technique, as shown in θtwist = 60°. Conversely, a strong signal appeared at the laser spot position at t = 180 s that is almost as strong as that of the nearby monolayer, which disappeared at t = 340 s  due to the unique threshold behavior shown in 2 bilayers and can help determine the optimal conditions for material processing for future electronic and optoelectronic device applications.In situ ablation images of the heterojunction with destructive interference are shown in 2, we studied the SHG peak intensity and peak angles as a function of the twist angle under a strong interlayer coupling confirmed by PL measurements. In addition, we studied the femtosecond ablation of the bilayer MoS2 in situ, which helped determine the optimal conditions for laser processing of the TMDs. We observed a characteristic threshold behavior: chemical bond breakage occurred for a very short time duration (~10 s) regardless of the presence of the ablation laser once the preheating condition was reached. We also investigated the thinning of the bilayer MoS2 for different twist angles. In the case of the homojunction, the SHG decreased by approximately one-fourth of the initial intensity when the upper layer was removed. Conversely, in the case of the heterojunction with the marginal SHG signal, the SHG increased abruptly and reached that of the monolayer when the upper layer was removed. Precise layer-by-layer control was possible due to the threshold behavior in the laser ablation. Therefore, our approach will be very useful for the in-line inspection of crystallinity in various TMDs and other functional nanomaterials and the determination of optimal conditions for their processing in the fabrication of optoelectronic devices.We reported the characterization of TMD bilayers through the rapid SHG imaging technique for the in situ identification of the crystallinity in the TMD, including the orientation and homogeneity. For the twisted bilayer MoS


Consequently, the mechanisms of initiating and regulating the normal spontaneous SA node beating rate are of vital importance. Spontaneous firing of the SA node is generated within sinoatrial nodal cells (SANC), which is regulated by the coupled-clock pacemaker system. Normal spontaneous beating of SANC is driven by a high level of cAMP-mediated PKA-dependent protein phosphorylation, which rely on the balance between high basal cAMP production by adenylyl cyclases and high basal cAMP degradation by cyclic nucleotide phosphodiesterases (PDEs). This diverse class of enzymes includes 11 families and PDE3 and PDE4 families dominate in both the SA node and cardiac myocardium, degrading cAMP and, consequently, regulating basal cardiac pacemaker function and excitation-contraction coupling. In this review, we will demonstrate similarities between expression, distribution, and colocalization of various PDE subtypes in SANC and cardiac myocytes of different species, including humans, focusing on PDE3 and PDE4. Here, we will describe specific targets of the coupled-clock pacemaker system modulated by dual PDE3 + PDE4 activation and provide evidence that concurrent activation of PDE3 + PDE4, operating in a synergistic manner, regulates the basal cardiac pacemaker function and provides control over normal spontaneous beating of SANCs through (PDE3 + PDE4)-dependent modulation of local subsarcolemmal Ca K1 as well as its stabilizing effect on the resting membrane potential. As a result, compared to atrial or ventricular myocytes, SANC have high membrane resistance, which allow small ion currents to produce a substantial effect on the membrane potential. Nowadays, it is well recognized that spontaneous beating of cardiac SANC is governed by a coupled-clock system (discussed below), which has overlapping and redundant systems to create robust and reliable automaticity i [2+]i. In contrast, AC1 and AC8 are stimulated by [Ca2+]i in a CaM-dependent manner, and AC 2/4/7/9 are Ca2+ insensitive. Different AC isoforms localize to distinct membrane compartments: Ca2+-sensitive ACs (AC1/3/5/6/8) were found in lipid rafts structures, while Ca2+ insensitive ACs (AC 2/4/7/9) were excluded [Though all ACs are inhibited by high intracellular Ca [Ca2+]i , AC5 andexcluded . Therefoexcluded .The idea that intracellular cAMP is required for cardiac pacemaker function was first proposed more than four decades ago and based on the observation that exposure to AC inhibitors suppressed spontaneous beating of isolated rabbit SA node , and thiDirect measurements of cAMP in isolated rabbit SANC were made three decades later and revealed that basal level of cAMP in intact isolated SANC is ~3-fold higher compared to ventricular myocytes , i.e., an increase in cytosolic [Ca2+]i was accompanied by an increase in the cAMP level, indicating activation of ACs and consequently an increase in the cAMP production i . Based oyR [2+]i  CaMKII cmyocytes . Close cmyocytes . The redmyocytes .2+ channels, PLB and RyR2  in guinea pig ventricular myocytes  (150–250 nmol/L) was ~2-fold larger compared to permeabilized ventricular myocytes despite similar SR Ca2+ content in both cell types i concentration is increased . T. TCa,L ihibition C. In conal state C [9].Ca,L amplitude by ~130%, ~140% and ~185% in different regions of the mouse myocardium, including ventricular, atrial myocytes, and SANC, respectively i was 2-fold larger in response to PDE3 inhibition and 4-fold larger when both PDE3 and PDE4 were concurrently inhibited nhibited , indicathibition . Anothernditions .Ca,L amplitude in response to PDE3 inhibitor in rabbit and especially human atrial myocytes, but not to PDE4 inhibitor in both species . NCX and leads to prolongation of the spontaneous SANC cycle length  pro pro2+-cA


DNMT3A mutations. In vitro studies implicated Grb2-associated binder 2 (GAB2) as FLT3-ITD effector. Utilizing a Flt3-ITD knock-in, Dnmt3a haploinsufficient mouse model, we demonstrate that Gab2 is essential for the development of Flt3-ITD driven AML in vivo, as Gab2 deficient mice displayed prolonged survival, presented with attenuated liver and spleen pathology and reduced blast counts. Furthermore, leukemic bone marrow from Gab2 deficient mice exhibited reduced colony-forming unit capacity and increased FLT3 inhibitor sensitivity. Using transcriptomics, we identify the genes encoding for Axl and the Ret co-receptor Gfra2 as targets of the Flt3-ITD/Gab2/Stat5 axis. We propose a pathomechanism in which Gab2 increases signaling of these receptors by inducing their expression and by serving as downstream effector. Thereby, Gab2 promotes AML aggressiveness and drug resistance as it incorporates these receptor tyrosine kinases into the Flt3-ITD signaling network. Consequently, our data identify GAB2 as a promising biomarker and therapeutic target in human AML.Internal tandem duplications (ITD) of the FMS-like tyrosine kinase 3 (FLT3) predict poor prognosis in acute myeloid leukemia (AML) and often co-exist with inactivating  Approximately 25% of all AML carry an internal tandem duplication (ITD) in the juxtamembrane domain of the receptor tyrosine kinase (RTK) FLT3, leading to constitutive PI3K/AKT, ERK and STAT5 signaling –3. GivenGAB2 in 70% of AML/myelodysplastic syndrome patients with MLL amplification provided the first hint for a role of GAB2 in AML [FLT3- and NPM1-mutant AML patients [The docking protein GAB2 amplifies signals downstream of cytokine and growth factor receptors, including FLT3-ITD . GAB2 is2 in AML . Further2 in AML , while i2 in AML . Moreovepatients .These findings suggest a crucial role of GAB2 in AML signaling pathways, especially as it amplifies signals from the FLT3-ITD oncoprotein and might thus contribute to the poor prognosis of this patient group and TKI resistance. However, all functional studies so far either used AML cell lines established decades ago or overexpressed FLT3-ITD in murine BM ectopically. Although the latter approach delivered novel insights, retroviral FLT3-ITD overexpression also induces transformed phenotypes distinct from AML . Therefoad libitum. Experiments were carried out in accordance with the German law for animal protection and approved by the government commission for animal protection and the local ethics committee . For genotyping primers see Supplementary methods.Mice (C57Bl/6 N background) were kept under specific pathogen-free conditions receiving standard diet and water 6 BM cells.Freshly isolated BM was transplanted into 8-week old male C57/Bl6N mice. Recipients underwent myeloablative body irradiation  followed by intravenous injection of 5 × 10PB was evaluated using a scil Vet ABC Plus+ hematology analyzer. Blood smears were examined with Pappenheim stain according to standard protocols. Results were assessed by an investigator blinded to the experimental groups.Paraffin-embedded sections were H&E stained according to standard protocols and imaged using a BZ9000 (Keyence) with ×20 and ×40 objectives. Images were processed and level corrected using BZ-II Analyzer software (Keyence).For immunophenotyping, freshly isolated cells from murine BM, spleens and PB were erythrocyte lysed, stained and analyzed using a BD LSR Fortessa. Propidium iodide (Invitrogen) was used for dead cell exclusion. For cell cycle staining, frozen BM was thawed and stained with surface antibodies. Next, cells were fixed, permeabilized and stained with an anti-Ki-67 antibody and DAPI (BioLegend). Analysis was performed using a BD LSR II Flow Cytometer. All antibodies are listed in Supplementary Methods.4 cells per duplicate assay were plated in MethoCult GF M3434 (Stemcell Technologies). Colonies were counted after 10 days. Quizartinib (Selleckchem) was dissolved in DMSO.Frozen BM was thawed one day prior to the experiment. Then, 2.5 × 10HL-60, KG-1a, MOLM-13, MV4–11 and THP-1 cells were a kind gift of Justus Duyster  and authenticated at Multiplexion . Kasumi-1 cells were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ). For culture conditions see Supplementary Methods.Western Blotting was performed as described previously . AntibodOaz1 expression. For primers see Supplementary Methods.Transcripts were amplified using SYBR™ Select Master Mix for CFX (Applied Biosystems) and quantified with the delta-delta Ct method normalizing to RNA was extracted from freshly isolated BM and sequenced on an Illumina HiSeq4000. Data curation was performed as described in Supplementary Methods. Results are available in the Gene Expression Omnibus (GEO) database under the accession number GSE182624 (token: mjutoqsqvhkjlsd).p values < 0.05 were considered statistically significant. Additional information, i.e., on tests used to correct for multiple comparisons, can be found in the Figure legends.Statistical analysis was performed using GraphPad Prism 9. If not stated otherwise, results were compared using an unpaired t test (two-tailed). In case of three or more groups to be analyzed, a one-way ANOVA was performed. Survival curves were compared by Mantel-Cox (log-rank) test. Data are presented as mean ± SD and Flt3-ITD knock-in allele only develop a mixture of myeloid and lymphoid neoplasms that do not progress into leukemia [DNA methyltransferase 3a (Dnmt3a) haploinsufficiency, however, transforms Flt3-ITD mutant murine myeloproliferative neoplasms (MPNs) into AML [Flt3-ITD knock-in mice [Dnmt3a (Dnmt3af/f) alleles [Dnmt3af/+/Flt3ITD/ITD as well as Dnmt3af/+/Flt3ITD/ITD/Mx-Cre mice  induced Cre expression generating Flt3ITD/ITD/Dnmt3a−/+ animals, which will from hereon be referred to as AML mice, while Dnmt3af/+/Flt3ITD/ITD mice that do not express Cre and therefore retain their floxed Dnmt3a allele will be designated as MPN mice. All animals were interbred with Gab2 knockout mice [Gab2-proficient (WT), -haploinsufficient (HET) and -deficient (KO) background, respectively. This allowed for a gene dosage-dependent analysis as Gab2 HET BM expresses ~50% of Gab2 protein compared to their WT littermates [Dnmt3a alleles in all AML mice regardless of their Gab2 genotype and without previous pIpC injections , MPN and AML mice in relation to their Gab2 genotype. White blood cell (WBC) counts  mice were reduced and comparable to those of controls with an insignificant differential between both groups. Commensurate with their haploinsufficient genotype, AML Gab2 HET (AML HET) mice showed an intermediate phenotype with significantly higher WBCs than AML KO, but significantly lower counts than AML WT mice. These results were confirmed upon AML BM transplantation  in control  and Lin−;Sca-1−;c-Kit+ (LK) cells were increased in AML WT mice compared to healthy controls, irrespective of their Gab2 genotype  was observed in the spleens and PB of AML WT mice compared to healthy controls Fig. . Here, tGab2 deficiency is maintained in older mice, we monitored several mice for at least 100 days  confirmed the immunophenotyping data, as the BM transcriptome reversed from an immature, HSC-enriched in AML WT back to a more mature, differentiated state in AML KO mice Fig. . These dAmong the differentially expressed genes with a large log2 fold change, we found the RTK Axl and Gfra2, a co-receptor of the RTK Ret, to be significantly decreased in AML KO compared to AML WT BM Fig. , which wGab2 genotype. Indeed, colony formation of AML WT BM was enhanced in the presence of the FLT3 inhibitor (FLT3i) quizartinib as compared to AML KO BM  STAT5 construct ).In this study, we show for the first time that rognosis . Our in ficiency , 36. Howignaling , a pathwignaling . Indeed,Gab2 deficient LSK cells to hematopoietic growth factors has been described [Interestingly, Gab2 KO mice possess the same absolute number of LK and LSK cells as Gab2 WT mice, indicating that Gab2 is dispensable for HSPC pool maintenance. However, an impaired response of escribed . This suTo unravel novel mechanisms by which Gab2 promotes AML aggressiveness, we compared the transcriptomes of AML WT and KO BM. An exciting finding, also from the angle of targeted therapy, is that Gab2 and its Stat5 effector arm act not only downstream but also upstream of RTK signaling Fig. . In the AXL emerges as promising biomarker and therapeutic target in oncology. In AML, AXL expression correlates with dismal prognosis and therapy resistance , 39. AXLGfra transcripts were only rarely found in leukemic blasts, while they were—in the absence of RET—detected in BM-derived stromal cells [q value 0.01 (gage)). Altogether, the mentioned studies and our present data suggest a second self-sustaining feed-forward loop in which Gab2—downstream of Flt3-ITD—increases Gfra2 expression, thereby promoting Ret activation and Flt3-ITD signaling clinical development blocking the SHP2, PI3K and STAT5 axes, could be used to indirectly counteract GAB2-mediated amplification of FLT3-ITD signaling. Indeed, FLT3-driven human AML cell lines and mouse models exhibited sensitivity against STAT5 and SHP2 inhibitors, respectively , 54. Furent mice , 56, Gabenotypes , 57. ThuBy inducing and interacting with RET and AXL , GAB2 could alleviate FLT3-ITD addiction and thereby contribute to FLT3 inhibitor resistance. Blocking GAB2 function or expression would be superior to targeting FLT3 directly, since the docking protein serves downstream of various leukemia-relevant RTKs. In that regard, GAB2 deficiency could mimic, at least in part, the effects of the AML relevant multi-kinase inhibitors sorafenib and midostaurin, while more selective drugs such as quizartinib rather imitate FLT3-ITD deficiency . MoreoveSupplementary Methods and FiguresSupplementary Table


MUT). Despite the fact that several FLT3 inhibitors have been developed, clinical responses are commonly partial or not durable, highlighting the need for new molecules targeting FLT3-ITDMUT. Here, we tested EC-70124, a hybrid indolocarbazole analog from the same chemical space as midostaurin (a well-known FLT3 inhibitor). Our in vitro and in vivo experiments showed that EC-70124 exerts a robust and specific antileukemia activity against FLT3-ITDMUT AML cells while sparing healthy hematopoietic cells. Collectively, EC-70124 is a promising and safe agent for the treatment of this aggressive type of AML.Patients with AML harboring constitutively active mutations in the FLT3 receptor generally have a poor prognosis (FLT3-ITDMUT) in the FMS-like kinase tyrosine kinase (FLT3) receptor generally have a poor prognosis. Several tyrosine kinase/FLT3 inhibitors have been developed and tested clinically, but very few (midostaurin and gilteritinib) have thus far been FDA/EMA-approved for patients with newly diagnosed or relapse/refractory FLT3-ITDMUT AML. Disappointingly, clinical responses are commonly partial or not durable, highlighting the need for new molecules targeting FLT3-ITDMUT AML. Here, we tested EC-70124, a hybrid indolocarbazole analog from the same chemical space as midostaurin with a potent and selective inhibitory effect on FLT3. In vitro, EC-70124 exerted a robust and specific antileukemia activity against FLT3-ITDMUT AML primary cells and cell lines with respect to cytotoxicity, CFU capacity, apoptosis and cell cycle while sparing healthy hematopoietic (stem/progenitor) cells. We also analyzed its efficacy in vivo as monotherapy using two different xenograft models: an aggressive and systemic model based on MOLM-13 cells and a patient-derived xenograft model. Orally disposable EC-70124 exerted a potent inhibitory effect on the growth of FLT3-ITDMUT AML cells, delaying disease progression and debulking the leukemia. Collectively, our findings show that EC-70124 is a promising and safe agent for the treatment of AML with FLT3-ITDMUT.Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Patients with AML harboring a constitutively active internal tandem duplication mutation (ITD MUT) represent approximately 80% of all FLT3 mutations and are associated with a high rate of relapse and poor clinical outcome [MUT AML [MUT AML.Acute myeloid leukemia (AML), the most common acute leukemia in adults, is a clinically and biologically heterogeneous disease characterized by the rapid proliferation and accumulation of immature myeloid blasts in bone marrow (BM) ,2,3. Int outcome ,7,8,9. A outcome . Despite[MUT AML ,12,13,14[MUT AML ,16. In aMUT AML primary cells in both in vitro and in vivo models. We show that EC-70124 has a potent and selective cytotoxic effect on FLT3-ITDMUT cells while sparing FLT3-ITDWT cells and healthy hematopoietic (stem/progenitor) cells (HSPC). Our results support EC-70124 as a novel and safe TKI in FLT3-ITDMUT.EC-70124 is a hybrid indolocarbazole analog from the same chemical space as midostaurin which has a potent multi-kinase inhibitory spectrum that impacts key signaling kinases implicated in pro-survival and proliferative cellular pathways . The antMUT AML cell line MV4;11 was obtained from the DSMZ . The FLT3-ITDWT cell line HL60 was kindly provided by Prof. Luciano Di Croce . The MOLM-13-Luc+ (MOML-13) and Baf3 cell lines were kindly provided by Prof. Michael Andreeff . All cell lines were cultured in RPMI medium supplemented with fetal bovine serum and antibiotics . A luciferase-expressing FLT3-ITDMUT AML patient-derived xenograft (PDX) (FLT3-ITDMUT-Luc+ AML-640 PDX) was generated and characterized as previously described by Jeremias’ Laboratory  [2 at 37 °C. The study was approved by the Institutional Review Board of the Clinic Hospital of Barcelona (HCB/2014/0687), and samples were accessed upon signed informed consent.The FLT3-ITDGermany) ,25. PrimGermany) ,27,28. TEC-70124 was synthesized by EntreChem S.L.  using a proprietary process . MidostaThe cytotoxic effect of the drugs on AML cell lines and Baf3 cells was assessed using the Cell Counting Kit-8    and MTT Cells were treated with 200 nM EC-70124 overnight (CB-CD34+ and primary AML cells) or for 48 h (AML cell lines). For the CFU formation assay, a total of 5 × 104 primary AML cells, 5 × 102 CB-CD34+ cells or 1 × 103 AML cells (cell lines) were plated onto methylcellulose H4434 medium . Colonies were counted and scored at day 14 (CB-CD34+ and primary AML cells) or day 7 (AML cell lines) using standard morphologic criteria, as described in ,29,30,317 MOLM-13 cells were treated with the different drugs at the indicated concentrations for 4 h. Cells were then lysed in 1% Triton X-100 lysis buffer supplemented with a complete protease inhibitor cocktail  and phosphatase inhibitors , as detailed in [A total of 10ailed in . ProteinMUT-AML PDX model, 105 cells were transplanted intravenously (i.v.) into sublethally (2 Gy) irradiated mice (n = 14 mice), as described in [All experimental procedures were approved by the Animal Care Committee of The Barcelona Biomedical Research Park (HRH-17-0007). We used 8–14-week-old NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice housed under pathogen-free conditions. EC-70124 was dosed daily by oral gavage at 20 mg/kg  and tumoribed in . Engraft®). Target regions were then captured and amplified by hybridizing the DNA library with the above-mentioned kit . The captured and amplified DNA was sequenced using NextSeq550 platform (Illumina®). The results were analyzed using DataGenomics platform as described elsewhere [A targeted NGSeq study was performed on ex vivo FACS-sorted AML cells retrieved from four mice (AML-640 PDX), treated (two mice) or untreated with EC-70124 (two mice) at the endpoint. The NGS panel included 76 genes related to myeloid disorders. Briefly, DNA was extracted from the AML cells and the libraries were prepared using the Haematology OncoKitDx . Differences between treatments along the days in PDX model were performed using multiple t test. Statistical analyses, normality test (Shapiro–Wilk) and IC50 were performed using GraphPad Prism v6.0 . Statistical significance was defined as a MUT AML cells. Cytotoxicity assays based on tetrazolium dye reduction (WST-8) revealed a comparable efficacy for both inhibitors, with similar levels of half-maximal inhibitory concentration (IC50) in FLT3-ITDMUT AML cells   a. EC-701ll lines b,c. Up tll lines c. Mechanll lines d–f. Althll lines f. Lysatell lines f. Taken WT AML cells using both FLT3-ITDWT AML primary cells and the cell lines HL60 and THP1. Cytotoxicity analysis revealed that the EC-70124 IC50 was considerably higher in FLT3-ITDWT cells than in FLT3-ITDMUT cells (72 µM (HL60) and 77 nM (THP1) vs. 17 nM (MOLM-13) and 14 nM (MV4:11)) (WT AML patient cells (MUT cells (WT AML patient cells (WT AML cell lines (WT cells (MUT AML cells.We next tested the specificity of EC-70124 by assaying its effects on FLT3-ITDMV4:11)) g. While nt cells h, the efUT cells b. Treatmnt cells h or FLT3ll lines i, AccordWT cells j,k. FinaWT cells l. The ICWe next evaluated the cytotoxicity of EC-70124 on healthy mature B cells, myeloid cells and T cells a and on MUT-Luc+ PDX model. For the MOLM-13 model, 1000 cells were IBM-transplanted into non-irradiated NSG mice, and due to the aggressiveness of this model, mice were randomized into groups and treatment was started the next day. Mice were dosed orally once daily with either vehicle or EC-70124 (20 mg/kg) for 15 days and were monitored by IVIS once weekly , a gene recently involved in AML pathogenesis, was specifically found in EC-7012-treated mice  or untreated with EC-70124 (n = 2) at the endpoint in order to genetically characterize the EC-70124 resistant outgrowing cells observed at relapse. The variant allelic frequency (VAF) of FLT3-ITD after EC-70124 or vehicle treatment in vivo remained almost identical to that at disease presentation (45.47 vs. 46.27), indicating that a FLT3-IDTted mice . These dMUT confers poor outcome in terms of overall and relapse-free survival in AML patients [MUT may function as an important oncogenic hit accelerating disease progression [MUT AML [MUT AML cells. We confirm in vitro the cytotoxic effect of EC-70124 on primary FLT3-ITDMUT AML patient cells and cell lines, revealing an even greater toxicity than midostaurin, which is in line with recent data by Puentes-Moncada et al. [MUT than midostaurin using kinase affinity assays, pointing to EC-70124 as a novel therapeutic for AML.AML is the most common hematological malignancy in adults, and the presence of FLT3-ITDpatients ,42. Indegression . Accordi[MUT AML . In linea et al. . These aMUT cell lines, and these effects were mediated through inhibition of FLT3 signaling. We found that EC-70124 inhibited p-AKT, p-ERK and pSTAT5, a master target in constitutively activated FLT3, and also inhibited the phosphorylation of S6 and BAD, which are downstream components of MAPK or PI3K pathways. This indicates a specific inhibitory function of EC-70124 on FLT3 signaling.EC-70124 is a potent multi-kinase inhibitor that can inhibit a broad spectrum of kinases including FLT3, JAK, SYK and PIM ,21,22,23MUT [WT cell lines, and barely (not significant) impacts FLT3-ITDWT AML patient cells, indicating a high specificity for FLT3-ITDMUT versus FLT3-ITDWT AML cells. This affinity may confer an advantage for treatment with FLT3 inhibitors, as it may uncouple specificity and potency from toxicity caused by off-target activity, the main limitation of first-generation inhibitors [Of note, it has been reported that EC-70124 has more affinity for FLT3 mutants in AML than both wild-type FLT3 and other kinases involved in the development of healthy hematopoietic cells, such as KIT, suggesting safety and specificity for FLT3-ITDMUT ,46. Indehibitors . Very imMUT PDX models. However, after treatment withdrawal, mice transplanted with FT3-ITDMUT PDX relapsed, with an action window of ~24 days, suggesting the eventual outgrowth of EC-70124-resistant AML cells. These results are in line with previous studies demonstrating that FT3-ITDMUT is subclonal and absent in the most immature AML-initiating/preleukemic cells, reinforcing that therapeutic targeting of subclonal mutations such as FLT3-ITDMUT might only have a transient clinical benefit in debulking the leukemia, but is unlikely to be curative, since it will not target the roots of the disease [MUT clone remains the major clone responsible for disease reoccurrence after EC-70124 discontinuation. Emergence of resistance poses a significant challenge in TKI-based treatments [EZH2 (VAFs: 6%), a gene recently involved in AML pathogenesis [Finally, in vivo analysis revealed that EC-70124 has antitumor activity and suppresses disease progression in both MOLM-13 and FT3-ITD disease . In facteatments . FLT3-TKeatments ; howevereatments ,49. Inteogenesis ,51, was MUT cells in vitro and in vivo, delaying disease progression and sparing FLT3-ITDWT cells and healthy mature hematopoietic cells and HSPCs. Collectively, our results support EC-70124 as a novel and safe TKI in FLT3-ITDMUT AML.Here, we show that EC-70124, a multi-kinase inhibitor, has a potent and selective cytotoxic effect on FLT3-ITD


Standard induction chemotherapy, consisting of an anthracycline and cytarabine, has been the first-line therapy for many years to treat acute myeloid leukemia (AML). Although this treatment induces complete remissions in the majority of patients, many face a relapse (adaptive resistance) or have refractory disease (primary resistance). Moreover, older patients are often unfit for cytotoxic-based treatment. AML relapse is due to the survival of therapy-resistant leukemia cells . Leukemia cells with stem cell features, named leukemic stem cells (LSCs), residing within MRD are thought to be at the origin of relapse initiation. It is increasingly recognized that leukemia “persisters” are caused by intra-leukemic heterogeneity and non-genetic factors leading to plasticity in therapy response. The BCL2 inhibitor venetoclax, combined with hypomethylating agents or low dose cytarabine, represents an important new therapy especially for older AML patients. However, often there is also a small population of AML cells refractory to venetoclax treatment. As AML MRD reflects the sum of therapy resistance mechanisms, the different faces of treatment “persisters” and LSCs might be exploited to reach an optimal therapy response and prevent the initiation of relapse. Here, we describe the different epigenetic, transcriptional, and metabolic states of therapy sensitive and resistant AML (stem) cell populations and LSCs, how these cell states are influenced by the microenvironment and affect treatment outcome of AML. Moreover, we discuss potential strategies to target dynamic treatment resistance and LSCs. The major problem with cancer treatment is that many patients obtain impressive remissions after a wide variety of treatments yet retain residual tumor cells after the initial therapy, which can develop into recurrence or metastasis. Intratumor heterogeneity in relation to therapy response is the key factor contributing to this treatment failure. For several decades, initial therapy for AML remained unchanged and typically consisted of repetitive courses of intensive combination chemotherapy with anthracyclines and cytarabine, the so-called “7 + 3” standard regimen, aiming at achieving complete remission . In general, and dependent on several risk factors, for patients under 60 years the 5-year overall survival (OS) rate after this treatment is 40–50%, while for patients older than 60 years the OS rate is only 15–20%. This poor OS rate in the elderly is partly explained by a higher proportion of patients with an unfavorable disease biology and an inability to tolerate intensive chemotherapy Figure 1Venetoclax-based therapy could induce responses in approximately 70% of untreated older AML patients , 14. HowThe biggest challenge in the treatment of AML is relapse and refractoriness caused by persistent AML cells that survive the initial treatment. The term “drug tolerant persisters” is frequently used to describe cancer cells with non-mutational mechanisms of drug resistance. Persistent leukemia cells might exist prior to drug treatment; however, they might also become resistant upon exposure to a treatment Figure 1AML has a hierarchical cellular organization, with a small fraction of self-renewing LSCs at the apex of the hierarchy. LSCs are defined as cells that can self-renew, which was experimentally shown by the capacity of re-initiation of leukemia when  transplanted into immunodeficient mice. Moreover, LSCs can differentiate into non-LSC blasts , 21. TheIn the past, the identification of cell surface markers differentially expressed between LSCs and HSCs has been intensively studied, but thus far no unique marker universally expressed on CD34+CD38− LSCs across AML patients but not on normal HSCs has been discovered. This is mainly due to the intra- and interpatient heterogeneity of AML. Many lymphoid and myeloid antigens are aberrantly expressed in AML, which give rise to complex leukemia-associated (immune)phenotypes (LAIPs) that are highly heterogeneous and differ between the individual AML patient . ExpressDuring the development of AML, genetic aberrancies induce epigenetic changes, leading to increased epigenetic plasticity in leukemic cells. While it was shown that genetic mutation-driven, unique epigenetic profiles in leukemic cells could be developed , LSCs deRecently, several studies challenged the idea that LSCs are less sensitive to chemotherapy than non-LSCs. These studies showed that LSCs are not selectively resistant to chemotherapy , 36, 37,). This reduced proliferative cell state might exist before therapy but can also be acquired as a result of treatment. For example, persistent cancer cells derived from human glioblastoma patients entered a slow-proliferation state following treatment with the tyrosine kinase inhibitor dasatinib -126 was identified as a critical regulator of LSC quiescence Table 1.nt state . Thus, mnt state . Multiplnt state , consistnt state , 45 , stably located on the LSC cell surface after chemotherapy treatment and present on cell cycle-quiescent AML-initiating cells residing within the endosteal niche .0 AML cells and proliferating cells   . Moreovehibition .Next to LSD1, expression of other HDMs, including the H3K27 demethylase KDM6B, the H3K36 demethylase KDM2B, and the H3K9/36me3 demethylase KDM4A correlated to treatment response. KDM6B is increased in AML as compared to normal BM, and positively correlated with poor survival. Treatment with the KDM6 inhibitor GSK-J4 enhanced the global levels of H3K27me3 and showed synergistic effects with cytarabine. GSK-J4 treatment decreased the expression of cell-cycle related pathways and HOX genes in AML cells . KDM2B ivia deregulation of HOX gene expression (PRC2 is one of the two classes of polycomb-group (PcG) protein complexes. PRC2 contains histone methyltransferase activity and primarily trimethylates H3K27, leading to silencing of target gene transcription, while PRC1 is able to condense nucleosomes, inducing stable gene silencing. The PcG proteins are required for long term epigenetic silencing and have an important role in maintaining “stemness” . The enhpression . Loss ofpression , 108. AMpression . Also prpression , which mpression . Interespression , 106. Hopression . Moreovepression . DZNEP rpression   proteins, which sustain Myc expression to promote aberrant self-renewal . BET famhigh AML cells are sensitized to LSD1 inhibition by venetoclax  responds to hypoxia by binding to hypoxia-response elements and by regulating expression of hypoxia-response genes, finally resulting in activation of enzymes involved in DNA repair, cell differentiation and apoptosis , CXC chemokine receptor-type 4 (CXCR4) and stem cell factor (SCF) . Since LThrough glycolysis, glucose is metabolized to pyruvate, and in the presence of oxygen pyruvate can be further metabolized to acetyl-CoA, that is oxidized in the tricarboxylic acid (TCA) cycle to drive OXPHOS and the generation of ATP. Although AML LSCs are mainly in a low oxidative and quiescent cell state, they often lack the ability to enhance glycolysis and therefore switch from anaerobic glycolysis to mitochondria-mediated OXPHOS as their major pathway to generate energy, while HSCs rely mainly on anaerobic glycolysis . Also, rInhibition of BCL2 by venetoclax efficiently affected LSC survival by inhibition of OXPHOS and impairing energy homeostasis, resulting in upregulation of myeloid differentiation genes, and downregulation of cell cycle and proliferation genes  Table , 64. To α . This resulted in increased ROS levels and activation of HIF-1α . LSCs re by 2-HG . However by 2-HG .α in the hypoxic BM microenvironment induces upregulation of CXCR4 on the membrane of LSCs, thereby enhancing their migration ability, their anchorage in the BM niche and their resistance to therapy  cells.Hypoxia-activating prodrugs (HAPs) are able to specifically target cells in hypoxic niches, as the active form of the drug is released under hypoxic conditions . The HAPA breaks . AdminisA breaks , suggestThere is emerging evidence that AML LSCs can remodel the BM niche into a leukemia-permissive microenvironment, thereby suppressing normal hematopoiesis. The complex interplay between LSCs and their microenvironment, including adhesion molecules, chemokines and cytokines, contribute to LSC survival, therapy resistance and disease relapse. Understanding these interactions is crucial for the development of effective drugs to overcome niche-mediated AML drug resistance.β-1 integrin receptor family members very late antigen-4 (VLA-4) and VLA-5, and the β-2 integrin LFA-1, which interact with the stromal ligands vascular cell adhesion molecule 1 (VCAM-1), fibronectin and intracellular adhesion molecule 1 (ICAM-1) to support attachment to the niche, thereby activating prosurvival and proliferative pathways in the leukemic blasts , interferon (IFN)-xpansion . Targetixpansion . In addixpansion , 144. Fuxpansion . Targetixpansion .β (TGFβ) family suppressed the growth of primary AML cells  is phosphorylated by the stress-responsive eIF2α kinases general control non-derepressible 2 (GCN2), heme-regulated inhibitor, protein kinase R, and protein kinase RNA-like ER kinase (PERK). Phosphorylated eIF2α reduces global protein synthesis while allowing translation of specific genes, including ATF4, ATF5 and C/EBP Homologous Protein  after initial therapy are caused by various mechanisms that co-exist, including epigenetic, transcriptional, and metabolic processes. Successful therapeutic strategies targeting AML MRD and LSCs will increase efficacy of treatment and finally the survival of patients. Increasing the knowledge on the mechanisms driving this persistence, and also on the changes in identity of MRD and LSCs during the course of the disease is crucial for the development of successful therapeutic strategies to overcome therapy resistance and to inhibit leukemia-initiating potential. Similarities and differences between normal HSCs and AML LSCs and studying LSC heterogeneity and intra- and interpatient plasticity are key for therapy development specifically eradicating LSCs at any time during the disease course. Persistent LSCs are characterized by their quiescent state and might exist prior to drug treatment; however, they might also become resistant upon exposure to therapy. At relapse, there are substantially more LSCs and phenotype diversities of LSCs than at diagnosis, indicating that the current therapeutic treatments induce dramatic changes in the LSC and relapse-initiating cell compartment. This indicates that there is a high degree of phenotypic plasticity to impose “stemness” on leukemia cells and shows the importance of studying leukemia re-initiating cells after the initial therapy. Future characterization of leukemia blasts and LSCs should therefore be performed after treatment to identify specific leukemia-relapse initiating markers that could be used not only to prognostically detect MRD, but also to early detect relapse and to be used as therapeutic targets delaying or even preventing relapse.There is a complex interplay between AML (stem) cells, their BM niche, and the outcome of treatment. This interplay is significantly influenced by signaling events from the BM niche affecting metabolism, epigenetic processes, stress responses and transcriptomes of the AML cells, and all subsequently affecting the level of leukemia (stem) cell death induced by therapy. Therefore, targeting the BM microenvironment by novel therapeutic strategies will also be crucial to overcome AML drug resistance. For example, targeting the energy consumption of LSCs in combination with inhibition of BCL2 can potentially eradicate residual chemotherapy-resistant LSC populations. Both FAO and BCL2 directly reduce ROS generation, promoting the survival of low ROS-producing quiescent LSCs. The combination of venetoclax and a FAO inhibitor might be a successful approach to eliminate LSCs with adapted energy homeostasis. Furthermore, the differentiation stage of the AML cells plays an important role in the response to therapy. AML cases originating from HSCs or MPPs, thus immature AML cases with high EVI-1 expression, may be successfully treated with venetoclax, while more differentiated AML cases may be successfully treated with alternative therapies that are more efficient in the eradication of differentiated AML cells.In conclusion, characterization of AML (stem) cells at the single cell level during the course of the disease and especially at MRD will provide valuable insights into the mechanisms of AML persistence and relapse-initiation and is key for development of successful treatment strategies that reduce or prevent relapse.NV, FD, and LS wrote the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the Dutch Cancer Society grant 12805.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.


C. elegans model. This models two cellular states that could contribute to development of PD. First the prolonged state with reduced cytosolic calcium that could deregulate multiple signaling pathways. Second the disease ER state with increased calcium concentration. We will discuss our hypothesis in the light of recent papers. First, a mechanistic study describing how variation in the Inositol-1,4,5-triphosphate (IP3) kinase B (ITPKB) may explain GWAS studies identifying the ITPKB gene as a protective factor toward PD. Here it was demonstrated that how increased ITPKB activity reduces influx of ER calcium to mitochondria via contact between IP3-receptors and the mitochondrial calcium uniporter complex in ER-mitochondria contact, known as mitochondria-associated membranes (MAMs). Secondly, it was demonstrated that astrocytes derived from PD patients contain α-synuclein accumulations. A recent study has demonstrated how human astrocytes derived from a few PD patients carrying the LRRK2-2019S mutation express more α-synuclein than control astrocytes, release more calcium from ER upon ryanodine receptor (RyR) stimulation, show changes in ER calcium channels and exhibit a decreased maximal and spare respiration indicating altered mitochondrial function in PD astrocytes. Here, we summarize the previous findings focusing the effect of α-synuclein to SERCA, RyR, IP3R, MCU subunits and other MAM-related channels. We also consider how the SOCE-related events could contribute to the development of PD.Neuronal calcium dyshomeostasis has been associated to Parkinson's disease (PD) development based on epidemiological studies on users of calcium channel antagonists and clinical trials are currently conducted exploring the hypothesis of increased calcium influx into neuronal cytosol as basic premise. We reported in 2018 an opposite hypothesis based on the demonstration that α-synuclein aggregates stimulate the endoplasmic reticulum (ER) calcium pump SERCA and demonstrated in cell models the existence of an α-synuclein-aggregate dependent neuronal state wherein cytosolic calcium is decreased due to an increased pumping of calcium into the ER. Inhibiting the SERCA pump protected both neurons and an α-synuclein transgenic  Parkinson's disease (PD) is the second most common neurodegenerative disorder with more than 6 million diagnosed cases, and more than 100 000 deaths/year . It beloSNCA (the gene encoding the α-syn protein) was identified as causing autosomal dominant PD  oscillation in this population of pacemaking dopaminergic (DA) neurons driven by Ca2+ influx through CaV1 Ca2+ channels, along with their low Ca2+ buffering capacity and increased oxidative stress formed part of the basis for an almost 30-year-old calcium hypothesis (2+ channel antagonists (The loss of the dopamine producing neurons of the substantia nigra pars compacta (SNpc) forms the basis for the motor symptoms that is diagnostic for PD. The demonstration of slow rhythmic calcium  Ca2+ pump SERCA thereby enhancing its pumping of Ca2+ from the cytosol into the ER. This activation causes an initial prolonged phase characterized by a decrease in cytosolic Ca2+ along with an increased Ca2+ loading of the ER that is followed by a second degenerative phase with increased cytosolic Ca2+ prior to cell death (C. elegans model with low doses of the SERCA inhibitor cyclopiazonic acid (CPA)  . The pro2+ regulatory molecules in the ER in relation to synucleinopathy-related pathology and discuss how our SERCA activation calcium hypothesis align with recent publications implicating mitochondria in this pathway.In the current review we will primarily focus on Ca2+ influx is a vital component in the process of neurotransmission from the regulation of dendritic responses to neurotransmitter binding, propagation of action potential along axons and transmitter release from nerve terminals; and also serves as a second messenger in signaling pathways regulating multiple processes, including metabolism, protein phosphorylation, exocytosis, gene transcription and programmed cell death  and membranes of different organelles such as ER, mitochondria, lysosomes, Golgi apparatus and others  carries the load. Together with the PM Ca2+ ATPase (PMCA), SERCA, and Na+/Ca2+ exchangers (NCX) contribute to maintain a low resting cytosolic Ca2+ concentrations in neurons  and Ryanodine receptors (RyRs) (3Rs show ubiquitous expression patterns.The release of Cas (RyRs) , 45. Con3R is a Ca2+ channel located in ER membrane that is activated by binding of Inositol-1,4,5-trisphosphate (IP3). The IP3 is generated by phospholipase C action on the membrane phospholipid Phosphatidylinositol 4,5-bisphosphate (PIP2) in a process often associated to G-protein coupled receptor activation. The IP3R is critically involved in the transport of Ca2+ from ER to mitochondria through its interaction with the major mitochondrial Ca2+ transport channel located in the outer mitochondrial membrane, called Voltage-dependent anion channel 1 (VDAC1) in a complex bridged by the chaperone GRP75 (75 kDa Glucose-regulated protein)  , 47. Thieostasis . There aectively , 50. Altectively .3R is not just regulated by phospholipase C dependent release of IP3 but can also be modulated by inactivating IP3 via its phosphorylation to IP4 by IP3 kinases. The IP3 kinase B (ITPKB) is such an inactivating kinase . In summary, this molecular study indicated that the genetic PD risk contributed by the ITPKB locus is caused by its modulation of the Ca2+ flux from ER to mitochondria . On the other hand, a detrimental decrease in cytosolic Ca2+, e.g., due increased activation of SERCA by α-syn aggregates  exists  exists . Pharmac) exists . Caffein release –65. RyR  release , named a neurons . Using s neurons .2+ level will alleviate PD-related pathophysiology, a caffeine-induced release of Ca2+ from the ER through RyR would be favorable. Interestingly, several epidemiological studies point toward a protective role of caffeine in relation to PD onset [for a review, see  that upon Ca2+ binding is able to modulate the function of SERCA and RyR (Another protein that may contribute to the interplay between molecules regulating Ca and RyR .2+ conducive membrane interphases with the PM (2+ entry (SOCE) into the ER and cytosol from the extracellular space with the tethering of the membranes mediated by the proteins ORAI and Stromal interaction molecules (STIMs)  are assembled by interaction between ER Ca2+-sensing STIM1/2 proteins and plasma membrane-associated ORAI1/2/3 proteins, which together form the PM Ca2+ release-activated Ca2+ (CRAC) channels, thereby allowing restoration of ER Ca2+ stores by influx from the extracellular space  . SOCE pl (STIMs) . Upon Caar space . In addiar space , 79. SER+ levels .2+ influx into the cytoplasm and ER as a consequence of ER Ca2+ depletion, SOCE plays an important role in maintaining neuronal Ca2+ homeostasis, and SOCE has been linked to a multitude of biological processes, including transcription, exocytosis, and metabolism , or idiopathic PD also show impaired SOCE relative to those of control patients , mRyR and the so-called rapid mode uptake channel (RaM) located in the inner mitochondrial membrane  and The EU Joint Programme-Neurodegenerative Disease Research (JPND) and The VELUX Foundations for the project OligoFIT. GK has been supported by the Novo Nordisk Foundation Distinguished Innovator 1-2020 award to Claus E. Olesen.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.


Nucleophosmin (NPM1) are the most common genetic abnormalities in adult acute myeloid leukaemia (AML), accounting for about 30% of cases. NPM1-mutated AML has been recognized as distinct entity in the 2017 World Health Organization (WHO) classification of lympho-haematopoietic neoplasms. WHO criteria allow recognition of this leukaemia entity and its distinction from AML with myelodysplasia-related changes, AML with BCR-ABL1 rearrangement and AML with RUNX1 mutations. Nevertheless, controversial issues include the percentage of blasts required for the diagnosis of NPM1-mutated AML and whether cases of NPM1-mutated myelodysplasia and chronic myelomonocytic leukaemia do exist. Evaluation of NPM1 and FLT3 status represents a major pillar of the European LeukemiaNet (ELN) genetic-based risk stratification model. Moreover, NPM1 mutations are particularly suitable for assessing measurable residual disease (MRD) since they are frequent, stable at relapse and do not drive clonal haematopoiesis. Ideally, combining monitoring of MRD with the ELN prognostication model can help to guide therapeutic decisions. Here, we provide examples of instructive cases of NPM1-mutated AML, in order to provide criteria for the appropriate diagnosis and therapy of this frequent leukaemia entity.Mutations of  He was NPM1 mutations have been reported in de novo AML with BCR-ABL1 but not in chronic myeloid leukaemia in blastic phase [NPM1-mutated AML and AML with BCR-ABL1 represent a ‘distinct’ and a ‘provisional’ entity, respectively [NPM1 mutations supersede BCR-ABL1 and our case should be diagnosed as NPM1-mutated AML, annotating the presence of BCR-ABL1 [NPM1-mutated AML [BCR-ABL1 AML [ic phase –76. How ic phase , NPM1-muectively . TherefoBCR-ABL1 . Notablyated AML  than witABL1 AML . The relBCR-ABL1 is regarded as high-risk disease [NPM1-mutated AML without FLT3-ITD has a relatively good outcome [NPM1 mutations and BCR-ABL1. Interestingly, two patients carrying NPM1 mutation and BCR-ABL1 were alive 36 and 71 months after diagnosis [NPM1-mutated AML than as de novo AML with BCR-ABL1. However, further studies are required.What is the prognostic impact of these genetic alterations? In the 2017 ELN model, AML with  disease  whilst N outcome . ELN rec outcome  do not ciagnosis , suggestNPM1-mutated AML should be suspected in a middle-age or older patient who presents with M4-M5 morphology [FLT3 or RAS. A low WBC count does not exclude NPM1-mutated AML since cases with FLT3 wild-type may have this presentation, despite the BM tends to remain not hypocellular [NPM1 or/and FLT3-ITD mutations may associate with disseminated intravascular coagulation [rphology , cup-likrphology  or multirphology  (case 1)cellular . Indepengulation . Extramegulation , especiaNPM1 mutations occurring at exons other than 12 and even NPM1-containg fusion transcripts [In haematological centers performing BM biopsy at presentation, demonstration by IHC of cytoplasmic NPM1 may serve as surrogate to molecular techniques in case of dry tap or myeloid sarcoma  (case 2)nscripts .NPM1 mutation by Sanger or the more sensitive NGS technique . NGS can also identify concomitant driver mutations, even within subclones. Identifying the exact type of NPM1 mutation [Confirmation of diagnosis requires the identification of mutation , 82 is cNPM1 mutation has been recognized, the definitive diagnosis of NPM1-mutated AML requires that other distinct and provisional AML entities of the 2017 WHO classification, such as AML-MRC (case 2), AML with BCR-ABL1  cases [NPM1 mutation allelic burden [NPM1-mutated AML presenting with marked monocytosis.Once a BL1 case , Fig. 6 M blasts  n to AML  and respn to AML , suggestrcentage . SimilarL) cases , 87 ofteL) cases , especiaNPM1-mutated AML has been established, the patient should be assigned to one of the ELN categories, either favorable (if FLT3 is wild-type or low ratio) or intermediate (FLT3-ITDhigh). Cases co-mutated for NPM1 and FLT3 should be possibly analyzed for DNMT3A to exclude the triple mutated genotype, which shows a particularly adverse outcome. As discussed in cases 1, 2, 3 and 4, risk stratification is important in guiding frontline (whether to use or not a FLT3 inhibitor) and post-remission therapeutic decisions . Patient’s age and fitness are other important parameters to be considered for choosing between CHT and venetoclax-based regimens or for deciding whether to perform or not allo-HSCT.Once the diagnosis of NPM1-mutated AML [NPM1 mutation points to a second AML [As discussed in cases 1 and 2, MRD monitoring Fig.  during aated AML . In factated AML , 24, 88.ated AML –33, althcond AML –91 , available data suggest that it probably supersedes the value of blast count. Indeed, the rare cases diagnosed as NPM1-mutated MDS or CMML often show features overlapping with AML [NPM1 mutations as genetic abnormalities sufficient to diagnose acute myeloid leukaemia regardless of the blast count, joining t, inv(16), t, and t.Although the presence of with AML –87. TherNPM1-mutated AML remain a medical need. MRD monitoring is usually done by RT-qPCR [NPM1 MRD have become available [NPM1 mutations [Despite great progresses, improving standardization of MRD monitoring and therapy of  RT-qPCR . Howevervailable –94. Digiutations .NPM1-mutated AML [NPM1-mutated AML. The good outcome of NPM1-mutated AML with FLT3-ITDlow was questioned in recent CHT-based studies [Incorporation of gemtuzumab ozogamicin has been advocated in the frontline treatment of ated AML –98. Poteated AML –101 and ated AML  should b studies –105, whi studies . Thus, t studies  and MLL- studies , 108, al studies  is warra studies .


Mentoring continues to be a salient conversation in academia among junior and senior faculty and administrators. Mentors provide guidance and structure to junior faculty so that they can meet their academic and professional goals. Mentors also convey skills in balancing life and academic pursuits. Therefore, the purpose of this descriptive study was to provide additional insight from a training program called Leading Emerging and Diverse Scientists to Success (LEADS) regarding successful strategies and challenges of mentoring relating to lessons learned from the scholars and mentees’ perspective. The LEADS program provided multiple training platforms to increase skills and knowledge regarding research to promote expertise in grant writing and submission for funding opportunities among diverse scientists. These findings reinforce the knowledge about the value of a mentor in helping define the research pathway of their mentee and underscoring the importance of mentoring. While career development and mentoring programs are not new concepts in higher education, innovative approaches that increase the success of the next generation of underrepresented (UR) academicians are needed. African Americans/Blacks, Hispanics, Native Americans, Alaska Natives, Native Hawaiians, and Native Pacific Islanders make up almost 30 percent of the US population, but only account for 9% of Science, Technology, Engineering, and Mathematics (STEM) faculty at US colleges and universities . FurtherIt is well known that strong networks of support within and outside the institution , formal To this end, the Leading Emerging and Diverse Scientists to success (LEADS) program was established at the University of Pittsburgh in 2015 through a grant from the National Institutes of Health (R25 GM116740). We forged a partnership with nine MSIs , working with a senior leader at each site and focusing on junior faculty and postdoctoral fellows wanting to launch their research careers. LEADS focused on developing the skill set of early career investigators while simultaneously providing mentoring and coaching from trained senior faculty, as well as resources that allowed them to partake in research dissemination, networking opportunities, and additional training. In another paper, we describe LEADS in detail .Since the establishment of the program, the LEADS administration collected data that explore the benefits and barriers related to mentoring. Through an iterative process, the program was modified as we applied lessons learned over the years. In this manuscript, we describe lessons learned, successful strategies, and provide insight from the participants’ perspective. We also explore the impact of the COVID-19 pandemic on mentoring. We are using descriptive research methods as required by the nature of the data we have.Collectively, the authors have noticed that mentoring programs at MSI face numerous challenges, such as limited time from senior faculty, finding mentors whose area of research aligns with the mentee, and competing demands on mentees’ time to establish their research. COVID-19 compounded the problem as research labs were closed and access to patients was impossible. These challenges are even more relevant for mentoring programs focused on UR faculty at MSIs. For instance, UR junior faculty at MSIs have reduced access to funded investigators for mentoring as there are not as many at well-resourced institutions. They also have increased teaching demands with heavier course loads. The LEADS program has been described in detail elsewhere . One parThe LEADS program provided a forum to share best practices across institutions, particularly around mentoring. The program also augments the success of MSIs senior leaders and mentors who are often burdened with leadership, research, and instruction responsibilities. Equally important is the networking that occurs among scholars in the purposefully planned LEADS environment, resulting in cross-institutional collaboration. Collaboration among MSIs promotes institutional excellence to enrich their respective communities. LEADS does just this by not trying to transform the MSIs into a micro-University of Pittsburgh, but rather to have each institution live up to its vision and embrace their strengths. The purpose of this descriptive study was to provide additional insight, from the LEADS scholars’ perspective, regarding the challenges to and strategies of successful mentoring learned through the LEADS program. In short, the LEADS program was a one-year fellowship where scholars participated in online modules focused on developing their research. We also had an annual Summit for all of the LEADS scholars and site leaders for more intensive training and networking. In addition, scholars were expected to meet with the site leader at their MSI who provided mentoring and career coaching. Site leaders were also responsible for recruiting scholars for the program. Each year, the LEADS program solicits applications from the participating MSIs. Applications were also advertised across other MSIs, particularly those institutions that had a Research Center in Minority Institutions (RCMI). Over the five years, the range of applications we received was 14–27. Once we received the applications, the Institute for Clinical Research Education (ICRE) Diversity Committee at the University of Pittsburgh reviewed the applications for appropriateness for the program. Applicants were reviewed based on their readiness to embark on a research career and time commitment to fully engage in LEADS. The program required that participants have 20% protected time for the program. Each applicant was asked to upload a letter from their department chair documenting their protected time.Each year we accepted 13–17 scholars. We tried to keep enrollment limited to 15 as that was the maximum number that the instructors could accommodate without diluting the impact of the training. Participant characteristics are provided under the results.To understand the impact of this training program, the LEADS Administration Team surveyed the scholars at baseline (the start of the program) and then annually thereafter. Each scholar received an email with a unique link to the online survey. They were sent reminder emails every week for five weeks. We created a survey to collect data on scholars’ characteristics related to research skills, research experience, mentoring structure, number of times meeting with the mentor, methods of communication with the mentor, and effectiveness of the mentor. Other data were collected on other factors such as burnout, motivation, and leadership. However, for the purposes of this study, we are only focusing on mentoring.For this descriptive study, we have self-reported data from the LEADS scholars. In addition to the site leaders, many of the scholars had mentors in their area of research. Scholars responded to the mentoring questions with a focus on their primary mentor or mentoring team. We analyzed baseline data for 61 scholars from five cohorts that participated in the LEADS program over five years and the exit survey data from 26 scholars . The medOverall, most of the scholars (70%) rated their mentor as effective/very effective at baseline, which increased to 76% at the exit survey. Scholars rated their mentors’ advice about balance between professional and personal life as effective/very effective (baseline = 47% and exit = 64%) and career development and balancing professional responsibility (baseline = 61% and exit = 85%), and receiving help in developing research skills (baseline = 60% and exit = 77%). The scholars agreed or strongly agreed that their mentors were accessible (baseline = 83% and exit = 84%), provided useful critique of their work (baseline = 90% and exit = 95%), and motivated them to improve their work product (baseline = 80% and exit = 81%). For the mentoring team, scholars agreed or strongly agreed that the team contributed and effectively communicated more than one mentor to their professional development  and provided research knowledge and skills (baseline = 69% and exit = 77%). Through the engagement in online modules and annual Summit, scholars learned how to become effective academic researchers. They participated in grant writing workshops, team science training, and cohort development activities to foster networking. In addition, the site leaders met with their respective scholars to provide career coaching and mentoring. Additionally, each scholar had their own mentor in their research content area.Unfortunately, LEADS scholars and mentors at MSIs were often burdened with heavy teaching loads or disproportionate administrative duties, resulting in inadequate time for research, professional development, and mentoring. Demands placed on junior faculty often preclude them from spending significant time on research endeavors, hampering efforts in remaining relevant in their field. COVID-19 has disrupted the mentoring relationship . As a reDespite these challenges, the majority of LEADS scholars rated the effectiveness of their mentor as high, which improved at the exit survey. In fact, many of the questions regarding mentoring improved at the exit survey. One reason that might explain this is the focus of the online modules in helping them drive their own careers and navigate the mentoring relationship. Though the LEADS program, scholars were able to connect with the teaching faculty of the modules, LEADS leadership, and site leaders. This expanded their mentoring network and could explain the more positive responses at the exit survey. The frequency of meeting with the mentor also increased at the exit survey as compared to baseline, as did the accessibility of the mentors. One explanation could be that the scholars learned how to make better use of their mentors through the LEADS program. The online modules are focused on the different components needed to launch a successful career. Therefore, as scholars become more knowledgeable, they become empowered to interact on a different level with their mentors. One area where this is seen is in their rating of the mentors’ effectiveness in helping them develop research skills. Only 19% rated them as very effective at baseline, but 46% rated them very effective at the exit survey. This dramatic increase could be a result of the scholars being able to engage with the mentors in a more focused way with the new knowledge they gained through the program. There are countless articles on mentoring and how it impacts mentees careers. It is common knowledge that good mentoring can promote the careers of mentees. In a study on mentoring indigenous mentees, Murry et al. found that mentorship differs across cultures . This isThese findings can be used by other academic institutions, particularly MSIs, if they are looking to improve their mentoring programs. This study suggests that by training postdoctoral fellows and junior faculty and providing career coaching, these scholars learn how to drive their own careers and more effectively work with their mentors. On 30 January 2020, the World Health Organization declared the coronavirus (COVID-19) a global health emergency and a global pandemic on 11 March 2020 . The panThe most common barrier to mentoring is also possibly the most underappreciated. It is difficult to be a mentor or a mentee during an existential crisis. Faced with a generational health and financial crisis, it not surprising that nearly 50% of the general population report anxiety or depressive symptoms, with 32% reporting concern about finances and 80% being at least slightly worried about their employment . The psyAnother set of barriers are related to the technological advances that were rapidly adopted as alternatives to face-to-face meetings. Virtual meetings, virtual private networks (VPNs), voice over Internet protocols (VoIPs) and cloud technology have made remote work and distance learning feasible, even as it increases the risk of social isolation, lower work performance, demotivation, disengagement, and conflicts between work and family as we work from home ,17. EarlThe third set of COVID-19-related barriers to mentoring are more system-related. COVID-19 has forced many universities and health care organizations to re-prioritize and institute work guidelines for health and safety. For some investigators, long-term experiments have been halted or terminated, with reduced or frozen start-up funding and suspended hiring of research staff to meet budgetary restrictions. Scientists and laboratory staff may be restricted from performing their work on-site or re-deployed to assist in other high-need areas [This study has several limitations. First, it did not use any theoretical framework to inform the descriptive study. Since this is a descriptive study, we are not positing causal relationships, which a theoretical framework could inform. Second, LEADS is a career development program for postdoctoral fellows and junior faculty at MSIs. People opt to apply to the program. The program is somewhat competitive, so we do not know if this would help all postdoctoral fellows and junior faculty at MSIs. However, we have had five cohorts of scholars over five years and the results are consistent. Finally, we did not survey the mentors, so we do not know what their perspective is of mentoring or how their mentoring has changed since COVID. However, many of the authors of this manuscript are mentors and have provided their perspective. The LEADS program partnered with nine MSIs to train their postdoctoral fellows and junior faculty so that they could launch a successful research career. The LEADS scholars engaged in online modules that were focused on topics such as team science, grant writing, and critical and creative thinking. In addition, the scholars participated in an annual summit where special speakers gave a variety of presentations all focused on advancing their research careers. In addition, the LEADS scholars received career coaching from licensed career coaches who worked one on one with the scholars to plan their careers. Site leaders and mentors dedicated time to working with the LEADS scholars to guide them through their careers. Through this descriptive study, we have shown that mentoring can improve as junior faculty are trained to better navigate the mentoring relationship and have learned how to receive better mentoring from their mentors. This indicates that professional development is a critical component that positively impacts mentoring, particularly at MSIs where scholars have limited access to mentors. With COVID-19, many additional challenges surfaced as faculty moved to sheltering at home. This limited access to mentors, as scholars would not see their mentors in the hall nor could they walk to their office. Anxiety and depression also significantly increased, which added additional challenges for scholars to navigate. However, these results suggest that a program, such as LEADS, can improve mentoring by training the scholars how to navigate the mentoring relationship better. Additionally, with career coaching, they learned how to drive their own careers.