However, the pertinent mechanisms are unknown, especially at the single-cell level. Particularly, it is still a major challenge to get a comprehensive understanding of the effect of potential mechanosensitive molecular players on the cellular responses, including mechanosensitive ion channels, purinergic signaling and integrin ligation by extracellular matrix.Therapeutic ultrasound or shockwave has shown its great potential to stimulate neural and muscle tissue, where cavitation microbubble induced CaMethods: Here, laser-induced cavitation microbubble was used to stimulate individual HEK293T cells either genetically knocked out or expressing Piezo1 ion channels with different normalized bubble-cell distance. Ca2+ signaling and potential membrane poration were evaluated with a real-time fluorescence imaging system. Integrin-binding microbeads were attached to the apical surface of the cells at mild cavitation conditions, where the effect of Piezo1, P2X receptors and integrin ligation on single cell intracellular Ca2+ signaling was assessed.Results: Ca2+ responses were rare at normalized cell-bubble distances that avoided membrane poration, even with overexpression of Piezo1, but could be increased in frequency to 42% of cells by attaching integrin-binding beads. We identified key molecular players in the bead-enhanced Ca2+ response: increased integrin ligation by substrate ECM triggered ATP release and activation of P2X—but not Piezo1—ion channels. The resultant Ca2+ influx caused dynamic changes in cell spread area.Conclusion: This approach to safely eliciting a Ca2+ response with cavitation microbubbles and the uncovered mechanism by which increased integrin-ligation mediates ATP release and Ca2+ signaling will inform new strategies to stimulate tissues with ultrasound and shockwaves. Ultrasound-induced microbubble oscillation is attractive for its potential utility in neuromodulation and cancer immunotherapy Ultrasound can be focused deep in brain or other tissue to allow non-invasive imaging and therapy 2+ signaling plays a crucial role in mechanotransduction and regulates myriad cell processes including exocytosis, contraction, transcription, and proliferation 2+ signaling is one of the earliest events in mechanotransduction under quasi-static cell loading 2+ response 2+ signaling in high density cell culture from femtosecond laser exposure 2+ signaling in one cell can propagate to its neighbors via ATP release and purinergic receptors such as P2X 2+ signaling that results from the shear flow of cavitation bubble.Cells sense their physical environment through mechanotransduction, the process of converting mechanical forces into biochemical signals that control cell functions. Intracellular Ca2+ influx through ion channels; the third mechanism involves formation of focal adhesions (FAs) on the basal surface of cell and integrin-mediated signaling. Quasi-static loading of integrin-bound beads on the apical surfaces of cells can remotely alter basal focal adhesions via integrin-cytoskeleton interactions Three mechanisms are commonly observed in shear-induced mechanotransduction in single cells: 1) direct activation of mechanosensitive ion channels such as Piezo1 in vivo applications. In contrast, at regular ultrasound frequencies of 1-2 MHz, microbubbles are often required to elicit a Ca2+ response, as shown in C. elegans and in human mesenchymal stem cells and HEK293T cells High-frequency ultrasound (30-150 MHz) has been shown to directly activate mechanosensitive ion channels without microbubbles 2+ responses induced by impulsive shear flow from single cavitation microbubbles (SCBs). We used HEK293T cells with Piezo1 genetically knocked out (P1KO) 2+ response without membrane poration. We established that the cellular mechanical sensing induced by microbubbles is mediated by increased integrin ligation, which leads to release of extracellular ATP (eATP) and subsequent activation of P2X channels, resulting in Ca2+ entry and downstream dynamic cell spreading.In this work we aim to elucidate the determinants of intracellular Ca2. At 48 h before SCB treatment and recording, P1KO cells were transiently transfected in a 6-well plate (the cells were seeded 8 h before transfection) in the presence of 10 μM ruthenium red and mouse Piezo1 (3 μg) using Fugene6 . 20-30% of cells showed positive GFP expression indicating successful Piezo1 transfection.The HEK293T-P1KO cell line  and mouse Piezo1-pIRES-EGFP in pcDNA3.1 were obtained from Jorg Grandl's lab at Duke Neurobiology and have been described previously 5) washes with 1× PBS were used to remove unloaded fura-2 AM before adding media in various experiments. Propidium iodide (PI) was added at a final concentration of 100 μg/mL to monitor cell membrane poration.Two days after transfection, cells were seeded in Au/Ti-coated dishes pre-wetted with 1× PBS and coated with 50 μg/mL fibronectin to promote cell adhesion. To avoid intercellular calcium signaling, we seeded the cells with low density and only single cells were selected for SCB treatment. To assess the role of Piezo1, a mixture of P1KO and P1TF cells was seeded; these cells were distinguished by green fluorescence from GFP co-expressed with Piezo1 in P1TF cells. Cells were incubated in DMEM at 37 °C for 3 h to allow cell adhesion and spreading. The culture medium was replaced with 6 μM fura-2 AM  in Opti-MEM  and incubated at 37 °C in the dark for 30 min. Several . Unattached beads were removed by washing with 1× PBS.In experiments with beads, cells were incubated at 37 °C for 30 min with 6-μm carboxyl-functionalized polystyrene beads activated with water-soluble carbodiimide, precoated with BSA (2.5% in PBS) or RGD-containing peptide  in 1× PBS with Caλ = 532 nm, 5-ns pulse duration. Orion, New Wave Research) on the coated glass surface through a 20× objective . We treated single cells with distance > 150 μm from each other. Only the cells facing directly to cavitation bubble and located outside of the maximum bubble rim were chosen, see Fig. max and bubble-cell distance d, we adjusted our bubble size relative to the bubble-cell distance to match γ = d/ Rmax within desired range. The maximum bubble diameter ranges from 90 to 110 µm in this study with the laser energy around 9-11 µJ/pulse. The bubble production is associated with some fiducial on the coverslip due to laser ablation of the gold coating. Therefore, we can register and trace the sheared cells with this mark on the coverslip.Glass-bottom petri dishes  were coated with a 3 nm layer of Ti and a 7nm layer of gold by electron beam evaporation to promote SCB nucleation. SCBs were produced in medium near cells by focusing a pulsed Neodymium-doped yttrium aluminum garnet (Nd:YAG) laser beam (2+-free 1× PBS: (1) Treatment with anti-fibronectin antibodies followed protocols in the literature 2+ for 25 min at 37 °C before bubble treatment. (2) Cells were treated with Ca2+-free 1× PBS . (3) Gd3+ solution (100 μM GdCl3) in 1× PBS with Ca2+ was used. (4) For P2X channel inhibition, 30 μM iso-PPADS in 1× PBS with Ca2+ was used. (5) To deplete extracellular ATP, cells were incubated with 20 U/mL Apyrase in 1× PBS with Ca2+ for 10 min before bubble treatment in the same medium.The following treatments were performed for mechanistic studies after fura-2 loading, RGD bead attachment, and washing with Ca2+ for 25 min (37 °C). Cells were then treated with a non-hydrolyzable ATP analog, ATP-γ-S at 0 (control), 5, 10, and 40 μM for separate dishes, respectively, and Ca2+ response was recorded with fura-2 ratiometric imaging.After 3 h of cell spreading on a fibronectin -coated dish, P1KO cells were incubated with 20 μg/mL 13G12 or 16G3 in 1 × PBS with Ca2+ transients were captured with ratiometric imaging of Fura-2 and membrane poration was monitored with the uptake of propidium iodide (PI). Glass-bottom petri dishes with fura-2-loaded cells were fixed on the translation stage of an inverted microscope . Fluorescence excitation from a 75-W xenon lamp was controlled by a monochromator  using shutters at defined time intervals, and at excitation wavelengths of 340 and 380 nm for fura-2 imaging, 539 nm for PI imaging, and 465 nm for GFP imaging. Since PI could also be excited at 340 and 380 nm, a custom-made narrow bandwidth filter (510 ± 40 nm) was used for detection of fura-2 emission at 510 nm to avoid overlap with PI emission at 610 nm. The intracellular fura-2 and PI images were recorded by using an sCMOS camera  at a 5:1 frame ratio, with exposure times of 50 ms and 100 ms, respectively. The image recording sequence for fura-2 was similar to our previous study SCB-induced intracellular Ca2+ response and PI uptake in a cell  or a Kirana K1 —at a recording rate of 2 million frames per second with 200-ns exposure time. The triggers for laser bubble generation and high-speed image acquisition were synchronized by a delay generator . Fluorescence excitation, filter cubes, and image recording were automatically switched between fura-2 and PI with a switching interval ~0.7 s by μManager . The bas340/I380 was calculated with MATLAB at each pixel before obtaining the averaged ratio within each cell or ROI from a manually segmented region of the cell with ImageJ. The average Ca2+ response was quantified as (F-F0)/F0. Time traces of (F-F0)/F0 for each cell were processed with a one-dimensional median filter in MATLAB to reduce the noise. F0 is the fluorescence ratio calculated by averaging the data before SCB treatment. Error estimation for Ca2+ response amplitude (Fp-F0)/F0 is shown in Raw fura-2 images were corrected by background noise subtraction. The ratiometric value F = ICells were chosen from dishes on at least three independent days. Results were consistent across days. So data was pooled for further analysis.0) for normalization: Sn = S/S0. Time traces of normalized cell spread area Sn were processed with the one-dimensional median filter to reduce noise. The error analysis for cell spread area reduction is shown in Cell spread area (S) was calculated from fura-2 images with MATLAB by thresholding at each combined image frame from 340 and 380 nm excitation. The emissions from these two wavelengths have opposite trends of fluorescence change; therefore, adding them for thresholding can reduce artifacts from fluorescence intensity change. The spread areas from the image frames before bubble treatment were averaged (SPolystyrene beads (2 μm) in 1× PBS were used as tracers to map the flow field produced by SCBs. SCB dynamics and tracer bead movements were recorded by a Kirana camera with SL-LUX640 laser for illumination at 2 million frames per second and 100-ns exposure time. High-speed image sequences were analyzed offline using a commercial PIV software , then post-processed with MATLAB to obtain the time evolution of the flow velocity at different γ. For PIV software processing, the image field (370 × 370 μm) was divided into multiple interrogation windows of 20 × 20 μm each with 50% overlap, and multipass iterations and regional filters were applied to reduce the error in velocity field measurement Cells were fixed with 4% formaldehyde for 10 min, permeabilized with 0.1% Triton X100 for 5 min, and blocked with 2% BSA for 1 h. Cells were then incubated with primary antibodies to vinculin (Sigma V9131) or paxillin (Abcam 32084) for 2 h, labeled with secondary antibodies (30 min), and imaged via fluorescence microscopy .A-C). As both the bubble size  and bubble-cell distance (d) have effects on cell membrane deformation and the resultant bioeffects max). For more details on the experimental setup, see Materials and Methods and Supplementary Material, SCBs stimulated a mixture of isolated P1KO and P1TF cells simultaneously, increasing the throughput of the experiment. This experimental setup allowed us to assess the response of single cells located at various distances from a single microbubble . Particle image velocimetry (PIV) measurements revealed that the velocity of the SCB-generated impulsive radial flow decreases with increasing γ, ranging from a maximum of ~ 6 m/s at γ = 1.1 to ~ 1.6 m/s at γ = 1.9 . The radial flow imposes transient shear stress on adherent cells nearby (γ = 1-2)  is similar at the fixed γ for SCBs of different size . Cell injury was assessed by PI uptake and cell morphology change.Can Figure F. Cell i2+ response and PI uptake in individual P1KO and P1TF cells exposed to SCBs at γ = 1.0-1.5. Bright-field images of cell morphology before and after SCB treatment are also shown. For both P1KO and P1TF cells, distinct Ca2+ responses were observed depending on the cell injury. Fast Ca2+ responses with a short rise time and a large amplitude change were accompanied by PI uptake associated with membrane poration . Slower and milder Ca2+ responses were accompanied by negligible PI uptake (little or no membrane poration) . The Ca2+ response amplitude increased with PI uptake /F0, and on PI uptake. Most cells exhibiting a Ca2+ response were located within γ = 1.5 of the SCB, for both P1KO and P1TF cells . Ca2+ responses within the range of γ = 1.0-1.5 were further classified based as “injury response” (PI uptake), “non-injury response” (no PI uptake), and “no response”. The frequencies of these responses in P1KO and P1TF cells were similar: 53% and 41% for injury response, 5% and 6% for non-injury response, and 42% and 53% for no response for P1KO and P1TF cells, respectively . Within γ = 1-1.5, there were more injury than non-injury responses. Since there was no significant difference in Ca2+ response between Piezo1 knockout and Piezo1-expressing cells, Piezo1 does not contribute to Ca2+ responses elicited by SCB-induced impulsive shear flow.Since the shear flow strength decreased with γ, we examined the effect of γ on the peak Ca2+ response is to enhance the mechanical load applied to cells at normalized standoff distances at which no membrane poration occurs (γ = 1.5-1.8). Informed by our previous study, we explored the effects of treating the cells with 6-µm diameter RGD-coated polystyrene microbeads An alternative approach to elicit a CaA depicts a high-speed imaging sequence of SCB-cell interaction and the resultant displacement of RGD microbeads attached to the cell. Enlarged images  show that the bead closest to the bubble was displaced significantly from its original position. The bead moved away from its original position during bubble expansion, and back to its original position following bubble collapse, over a period of ~ 22 μs. The corresponding time-elapse images of Ca2+ response and PI uptake  indicate a non-injury Ca2+ response.Figure 32+ response at γ = 1.5-1.8 . Without RGD beads, none of the 16 P1KO cells and only 1 out of 38 P1TF cells showed a Ca2+ response at this distance . Notably, no PI uptake was observed in the RGD bead-treated cells—all Ca2+ responses were non-injury, for both P1KO and P1TF cells. The fraction of cells showing a Ca2+ response increased significantly, from 0% without beads to 42% with beads (p < 0.01) for P1KO, and from 2.6% to 19% (p < 0.05) for P1TF . No statistically significant difference was found between P1KO and P1TF cells (p > 0.05).Attachment of RGD beads enhanced the probability of eliciting a Ca8 Figure E. Withou2+ responses without cell injury. Again, no significant difference in Ca2+ response between Piezo1 knockout and Piezo-expressing cells was observed, indicating Piezo1 is not involved in the Ca2+ response.Attaching integrin-binding microbeads is thus an efficient approach to eliciting Ca2+ response to SCB with RGD beads, we examined spatiotemporal changes in Ca2+ signaling within P1KO cells at normalized distances causing a non-injury response (γ = 1.5-1.8). Figure 4A shows a contour map of Ca2+ signaling amplitude in a representative P1KO cell. Interestingly, the Ca2+ signaling appeared to propagate from the cell periphery toward the center, consistent with a previous study 2+ response, (F-F0)/F0, in each ROI versus time . The outermost ROI reached 50% of its maximum Ca2+ response earlier than the center ROI (~ 7.2 s versus ~ 12.5 s), with the Ca2+ wave propagating from the edge to the center of the cell at ~ 2.3 μm/s . This speed is faster than the diffusive rate of Ca2+ in cell 2+ wave propagated in the cytosol via a reaction-diffusion process from the endoplasmic reticulum To discern the processes that initiated the Ca2+ response is integrin-specific. Paxillin and vinculin are adaptor proteins in focal adhesions that connect transmembrane integrins with the actin cytoskeleton 4C). We examined whether integrin binding was required on the cell apical surface by comparing Ca2+ responses of P1KO cells exposed to beads coated with either RGD or BSA . BSA-coated beads non-specifically bind cell membranes but experience the same drag as RGD-coated beads. The Ca2+ response was significantly reduced in cells when using BSA beads, indicating that integrin-specific bead attachment was vital to the Ca2+ response. No Ca2+ response was observed when using Ca2+-free medium with RGD beads , indicating that the increased cytosolic Ca2+ was dependent upon extracellular Ca2+ entry.Since cells detect extracellular mechanical stimuli through integrins and focal adhesions 2+ response in our study. Therefore, we examined cell spreading as an indicator for integrin-ECM interactions following SCB treatment. We observed a transient increase in normalized cell spread area Sn immediately after SCB treatment, from 2.0 s to 2.4 s . A Ca2+ response was initiated concurrently with this increase in Sn . This increase in cell spread area indicates the effects are at the cell basal site, supporting our hypothesis of increased integrin ligation by substrate ECM.As the dynamics of our system is not compatible with significant changes in protein expression, we focus on the interaction of integrin at cell basal surface with the substrate ECM. Increased ligation of integrins by the substrate ECM has been associated with shear-induced mechanotransduction G). We examined this process in detail for three P1KO cells with RGD beads at γ = 1.5-1.8 before extending the analysis. The three cells showed three different Ca2+ responses . In the two cases with non-injury responses, the minimum spreading area (Sn)min was reached soon after or at nearly the same time as the Ca2+ peak . The reduction in cell spread area,1-(Sn)min, was greater for the cell showing the larger Ca2+ response , and was < 2% when there was no Ca2+ response . To reinforce this observation, we analyzed reduction in area for additional cells under the same conditions. Figure 5D and E summarize the dependence of area reduction on Ca2+ response and γ, respectively. The reduction in area was < 5% for no-response cases, but got larger with increasing Ca2+ response, reaching a maximum of 11% . No clear dependence on γ was observed for the no-response cases . In contrast, for non-injury response cases, the reduction in area decreased with increasing γ . Together, the results suggest that the reduction in cell spread area is a downstream effect of the Ca2+ response.After the transient increase in cell spreading immediately following SCB treatment, the spreading area decreased and then recovered to a plateau at ~ 150 s Figure G. We exa2+ response cases. In a P1KO cell without RGD beads that exhibited an injury Ca2+ response, the Ca2+ signal rose sharply upon SCB treatment at 0 s , a response that is distinct from the non-injury responses . However, there was little transient increase in cell spreading area. This result agreed with our previous finding 2+ influx occurred through pores in the plasma membrane in injury response cases. Despite the different route of Ca2+ entry, in both cases we observed a reduction and recovery of cell spread area following the Ca2+ response.We also examined whether the reduction of cell spread area occurs in other Cas Figure F, a resps Figure A-B. How2+ influx and integrin-binding RGD beads are necessary to induce the reduction in cell spreading, we extended the analysis to cases of P1KO cells with Ca2+-free medium or without RGD beads . There was no Ca2+ response in Ca2+-free medium, as observed previously. In all cases with no Ca2+ response, the reduction in cell area was < 6% and was often comparable to the error from measurement (1.8% - 4.3% standard deviation (SD), and 0.4%-0.9% standard error of the mean (SEM)), indicated no significant change. In contrast, for cells exhibiting a Ca2+ response, the reduction in cell area increased with the Ca2+ response amplitude and reached higher levels (up to > 15%).To assess whether Cas Figure G. There 2+ response; i.e. cell spreading is a result of 'inside-out' signaling from regulation of cell adhesion by increased intracellular Ca2+. Additional analysis of our previous data also showed transient reduction in cell spread area for tandem bubble-treated HeLa cells with or without RGD beads, which had a Ca2+ response Supplementary Material, 2+-dependent calpain activation that leads to focal adhesion disassembly This analysis suggested that the transient decrease in cell spread area is a downstream effect of the Ca2+ response. After P1KO cells had spread on fibronectin-coated dishes, the fibronectin-specific antibody 16G3 was added to block any new integrin αvβ3 and α5β1 binding sites. In this way, the cells could only maintain previously established integrin-ECM connections during SCB treatment. A non-blocking fibronectin-specific antibody, 13G12, was used as a control. These antibodies are well-validated and can be taken as direct evidence for the role of integrin ligation 2+ response, while 13G12 had no significant effect (p > 0.05) on the Ca2+ response . Moreover, a similar increase in Sn occurred but was not associated with a cytosolic increase in Ca2+ . However, there was no significant difference in the Ca2+ response between 16G3- and 13G12-treated cells stimulated with ATP-γ-S, suggesting that integrin ligation is upstream of eATP release, consistent with previous studies P2X is an ATP-gated channel. We depleted eATP with 20 U/mL apyrase (an ATP-diphosphatase) and found that the Ca2+ response : SCB-induced shear force is exerted on integrins on the cell apical surface by pulling on RGD-coated beads, causing new ligation of integrins by ECM proteins at the cell basal surface, which in turn triggers cellular release of eATP, which opens P2X ion channels, allowing Ca2+ influx that regulates a dynamic change in cell spreading.Altogether, our results suggest the following model for the SCB-elicited Cae Figure E: SCB-in2+ signaling have not been previously examined. In this study we developed a system in which laser-generated SCBs produce impulsive shear flow on multiple isolated cells nearby, allowing control of the normalized standoff distance between SCB and the cells, and used this system to dissect the key mechanisms underlying the elicited Ca2+ response.In therapeutic ultrasound and shockwave applications, the shear force experienced by cells likely involves a high strain rate 2+ signaling at normalized SCB-cell distances that did not cause membrane poration. Compared to the more gently varying shear stress induced by the flow alone, the displacement of the microbeads provides a localized source of mechanical stretch on the cell membrane. This enhanced mechanical loading led to increased integrin ligation to the substrate ECM, triggering eATP release that activated ATP-gated P2X channels, and resulting in Ca2+ influx and downstream signaling. This approach is also effective under impulsive jetting flow from tandem bubbles 2+ signaling initiated during the transient increase of the spread area. The involvement of integrin ligation by ECM and eATP release due to high strain rate loading has not been reported before. Moreover, under the same experimental conditions, BSA-coated beads did not induce Ca2+ signaling, suggesting that the signaling is not due to shear force transferred to the cell membrane or cell as a whole. Instead, mechanical transmission via integrins, likely linked to the cell basal surface via the cytoskeleton 2+ signaling. It is worth noting that new integrin ligation could happen at the RGD microbead surface or at the fibronectin-coated glass surface. We have focused on the glass surface as it is a larger surface area. Moreover, we have found that Ca2+ response was initiated during transient increase in cell spread area, indicating new ligation of integrin to substrate ECM leads to Ca2+ signaling.In general, shear-induced mechanotransduction can be mediated by several different intracellular or extracellular structures, including mechanosensitive ion channels, purinergic receptors, and integrins, ECM 2+ responses in cells stimulated by quasi-static shear flow 2+ response between Piezo1 knockout and Piezo1-expressing HEK293T cells, either with or without RGD beads under impulsive shear flow, indicating that Piezo1 is not involved under this shear loading regime. In general, Piezo1 enhances mechanotransduction at low strain rates and longer duration stimulations The mechanosensitive ion channel Piezo1 has been previously implicated in Ca2+ response in HEK293T cells transfected with Piezo1, but did evoke a response when integrin-binding microbubbles were attached to the cell membrane 2+ response, and were also shown to be important in Ca2+ signaling in human mesenchymal stem cells stimulated using low-intensity ultrasound Some elements of the mechanism identified in our study have been reported previously for ultrasound-induced mechanotransduction. For example, 2 MHz ultrasound alone did not evoke a Ca2+ response also can be evoked by ultrasound excitation of functionalized microbubbles, which were attached to cell membrane via RGD-integrin binding  2+ response. Future studies are warranted to determine which method is better for each specific application.It is worth mentioning that Ca2+ responses Compared to the current ultrasound/acoustic tweezers, the main difference is the robustness of our microbeads approach, where the beads can be stimulated multiple times without losing its mechanical integrity. In contrast, coated microbubbles used in acoustic tweezers may rupture or gradually leak out its gas content during/after insonification that lasts longer. Besides, the microstreaming produced by coated microbubble oscillation will be much weaker than that produced by inertial cavitation bubble, leading to a significantly diminished effective range of the approach. On the other hands, optical tweezers have also been used to elicit intracellular Ca2+ response in cavitation bubble-generated impulsive shear flow. We anticipate that the insights from this study will enable development of new strategies for better use of therapeutic ultrasound. First, this strategy does not require expression of exogenous mechanosensitive ion channels such as Piezo1 or MscL I92L in vivo. Second, since expression of integrins and P2X is widespread in mammalian brain, muscle, and other tissues Our results establish that applying forces to integrins and activating ATP-gated P2X ion channels is an efficient approach to elicit non-injury CaSupplementary figures and tables.Click here for additional data file.


Here, we use a multidisciplinary approach including protein biophysics, structural biology, confocal imaging, and patch-clamp electrophysiology to determine the effect of the disease-associated CaM mutation E140G on CaM structure and function. We present novel data showing that mutant-regulated CaMKIIδ kinase activity is impaired with a significant reduction in enzyme autophosphorylation rate. We report the first high-resolution crystal structure of a LQTS-associated CaM variant in complex with the CaMKIIδ peptide, which shows significant structural differences, compared to the WT complex. Furthermore, we demonstrate that the E140G mutation significantly disrupted Cav1.2 Ca2+/CaM-dependent inactivation, while cardiac ryanodine receptor (RyR2) activity remained unaffected. In addition, we show that the LQTS-associated mutation alters CaM’s Ca2+-binding characteristics, secondary structure content, and interaction with key partners involved in excitation-contraction coupling . In conclusion, LQTS-associated CaM mutation E140G severely impacts the structure-function relationship of CaM and its regulation of CaMKIIδ and Cav1.2. This provides a crucial insight into the molecular factors contributing to CaM-mediated arrhythmias with a central role for CaMKIIδ.Long QT syndrome (LQTS) is a human inherited heart condition that can cause life-threatening arrhythmia including sudden cardiac death. Mutations in the ubiquitous Ca More thchannels , 5, 6. H in LQTS , 18, 19.2+ sensing protein able to bind up to four Ca2+ ions through its Ca2+-binding EF-hand motifs: two located in its N-domain (EF-1 and EF-2) and two in its C-domain (EF-3 and EF-4). The associated structural transition to an open conformation upon Ca2+ binding mediates interaction with CaM’s targets and signal transduction (2+ channel (Cav1.2), voltage-gated Na+ channel (Nav1.5), voltage-gated K+ channel (Kv7.1), and ryanodine receptor (RyR2)  with the γ (CaMKIIγ) and δ (CaMKIIδ) isoforms present in heart  , 44, 45.in heart , 48, 49.v1.5 in cardiomyocytes results in cell membrane depolarization and activation of Cav1.2. Inward flux of Ca2+ into the cytoplasm via Cav1.2 triggers the opening of RyR2 embedded in the sarcoplasmic reticulum. This causes release of internal Ca2+ stores through a process called Ca2+-induced Ca2+ release (2+-induced Ca2+ release increases the cytosolic free Ca2+ concentration ([Ca2+]cyt) throughout the cardiomyocyte and binding of Ca2+ to myofilaments results in contraction. At high [Ca2+]cyt, Ca2+/CaM binding to RyR2 causes inhibition of RyR2 Ca2+ release  and terminates Ca2+ entry to avoid Ca2+ overload and arrhythmias  to offset partly reduced Ca2+ channel availability at high heart rates phosphorylation activity. We present novel electrophysiology and Ca2+ imaging data supporting that the E140G mutation significantly impairs Cav1.2 CDI, while RyR2 activity is unchanged. The data obtained help decipher the molecular mechanism of CaM-associated LQTS and highlights CaMKIIδ as a key player.Based on the extensive roles of CaM in the direct and indirect regulation of cardiac excitation contraction coupling, we hypothesize that CaM mutations can disrupt its regulatory functions resulting in cardiac arrhythmia. Here, we report the results of a comprehensive functional, biophysical, and structural analysis performed to determine the effect of LQTS-associated CaM mutation E140G on CaMKIIδ, Ca2+/CaM-E140G variant on CaMKIIδ kinase activity, we measured the amount of ADP produced using an end point fluorimetric assay. Using syntide2 as the enzyme substrate, commercial human CaMKIIδ (Abcam) as the enzyme, and CaM variants as activators, we find that phosphorylation levels decreased by ∼35% (A) for E140G when compared to CaM-WT. Further, the rate of CaMKIIδ autophosphorylation (Thr287) was determined using Western blotting and densitometry analysis , the relative CaMKIIδ autophosphorylation level at Thr287 was significantly decreased from 0.30 ± 0.05 (WT) to 0.01 ± 0.008 (E140G). We determined that even after a 60 min reaction, E140G showed ∼13-fold lower CaMKIIδ autophosphorylation levels than CaM-WT. These data suggest that the decrease in kinase activity could be attributed to impaired CaMKIIδ autophosphorylation in the presence of CaM-E140G variant.In order to evaluate the functional impact of the Ca by ∼35% A for E14analysis B and S1.2+/CaM and the E140G mutant were cocrystallized with CaMKIIδ294-315 to gain molecular insight into the interaction. The crystal structures were solved to a high resolution of 2.65 Å for Ca2+/CaM-WT–CaMKIIδ294-315 complex (PDB 7ZRP) and 1.68 Å for Ca2+/CaM-E140G–CaMKIIδ294-315 complex (PDB 7ZRQ) (A–C). Crystallographic data and refinement statistics are presented in 2+ ions at the N- and C-domains of CaM. The structural superimposition Ca2+/CaM-WT-CaMKIIδ294-315 with Ca2+/CaM-E140G-CaMKIIδ294-315 showed rather low RMSD of 0.949 Å with difference mainly at the N-terminal region of CaM. Almost one-half turn of the helix in the region Phe65 to Lys77 is missing from the E140G complex.CaDB 7ZRQ) , A–C. Cr2+/CaM-WT–CaMKIIδ294-315 and Ca2+/CaM-E140G–CaMKIIδ294-315 peptide complex structure (D–F). Glutamate at position 140 (E140) is one of the Ca2+ coordinating residues in EF-hand 4. Interestingly, in the LQTS variant structure (G140), this interaction is lost and replaced by a water molecule, HOH10, coordinated by the residues I130, N137, and Q135. The H-bonds and salt bridge interactions between the CaMKIIδ peptide and CaM were predicted using the QtPISA server  and the stoichiometry of binding (N) of interactions. In addition, from the thermodynamic parameters, the nature of the forces that drive the binding reaction can be determined . Using ITC, we showed that Ca2+-CaM can interact with CaMKIIδ294-315, with a stoichiometry of 1 (G). The dissociation constant of Ca2+/CaM for CaMKIIδ294-315  was significantly increased to 0.097 ± 0.008 μM for the E140G variant (n = 5), indicating a ∼2-fold reduction in binding affinity (H). The binding reaction was exothermic and enthalpy driven, with a significant increase of ΔH for CaM-E140G when compared to WT (I).Using isothermal titration calorimetry (ITC), we investigated the binding of CaM variants to CaMKIIδ-binding domain. ITC provides the dissociation constant (try of 1 G. The diaffinity H. The bied to WT I.v1.2 whole-cell currents (ICav1.2), patch-clamp electrophysiology was performed on HEK293-Cav1.2 cells transiently transfected to overexpress CaM-WT or E140G (A). In these experiments, CaM variants and the fluorescent marker (EGFP) were coexpressed under the control of the same promoter, as two distinct proteins and not as fusion proteins. We observed a bell-shaped current-voltage relationship, which is characteristic of ICav1.2 (B). The maximum peak current densities (measured at + 20 mV) were as follows: −5.34 ± 1.18 pA/pF ; −5.17 ± 0.60 pA/pF , and -3.50 ± 0.43 pA/pF . Using a two-way ANOVA with Tukey’s multiple comparisons test, we showed that the peak current densities at every voltage were not significantly affected by the overexpression of CaM-WT or the mutation. For both CaM-WT and E140G, activation curves showed a conductance increase from approximately −20 mV, with a maximum conductance achieved at the +40 mV test potential. V50 of activation, the voltage at which half-maximal conductance is reached, remained unchanged for E140G  when compared to CaM-WT  (C). V50 of voltage-dependent inactivation did not show any significant difference between CaM-WT  and E140G  (D). Together, this indicates that LQTS-associated CaM variant E140G does not affect the voltage-dependence of Cav1.2 activation and inactivation.In order to investigate the effect of the LQTS-associated variant CaM E140G on the voltage-dependent characteristics of Caor E140G A. In the ICav1.2 B. The ma, n = 6) C. V50 of n = 12) D. TogethA). In order to discriminate between Ca2+-dependent and Ca2+-independent inactivation, residual currents at the end of the test pulse were measured in the presence of either extracellular Ca2+ (r300Ca) or under conditions where all the extracellular Ca2+ was replaced by Ba2+ (r300Ba). Here, Ba2+ is able to enter the cell through the activated channel but is unable to induce CDI. Comparison of r300Ca and r300Ba across a range of different test potentials revealed a gradual decrease in r300Ba with increasing depolarization for both CaM-WT and E140G (B). Mean r300Ba at +10 mV for E140G remained unchanged, whereas mean r300Ca significantly increased for E140G from 0.21 ± 0.01 (n = 6) to 0.60 ± 0.03 (n = 12), when compared to CaM-WT (C). The difference between r300Ca and r300Ba, which indicates the proportion of total inactivation due to CDI, is denoted as f300 (D). A significant reduction in f300 for E140G was observed, from 0.76 ± 0.05 for WT (n = 4) to 0.18 ± 0.04 in E140G (n = 6). These data demonstrate a dramatic impairment in CDI in E140G, while Ca2+-independent inactivation is not affected (D).Whole-cell recordings showed that CDI during the 300 ms depolarizing test pulse was impaired in cells overexpressing CaM E140G A. In ordnd E140G B. Mean ro CaM-WT C. The di as f300 D. A signaffected D.Figure 2+/CaM can bind at multiple sites on the Cav1.2 channel. Through its N-lobe, CaM can interact with the N-terminal spatial Ca2+ transforming element (Cav1.2-NSCaTE51-67) and through the C-lobe with residues from the IQ domain (Cav1.2-IQ1665-1685) (v1.2-NSCaTE51-67 and N∼1 for Cav1.2-IQ1665-1685 (A and B). The disease-associated CaM variant E140G affected the interaction with both Cav1.2-binding domains. The Kd was significantly increased from 1.08 ± 0.01 μM  to 8.33 ± 0.47 μM  for Cav1.2-NSCaTE51-67. Interestingly, the interaction between the E140G variant and Cav1.2-IQ1665-1685 showed a significantly stronger binding affinity (∼2 fold) than WT (C). The thermodynamic parameters showed that the interaction of Ca2+/CaM-WT and E140G variant with Cav1.2-binding sites was exothermic and driven by enthalpic contributions (D). The E140G variant showed a significant increase in enthalpy upon binding to Cav1.2-NSCaTE51-67 domain, when compared to WT.Using ITC, we showed that Ca65-1685) , 60. The665-1685 , A and B than WT C. The thibutions D. The E1v1.2 channel complex, CaM is preassociated with higher affinity binding domains such as IQ. Therefore, we investigated the interaction between preassociated Ca2+/CaM-Cav1.2-IQ1665-1685 and Cav1.2-NSCaTE51-67 (E). In these conditions, the measured stoichiometry was N∼1 and the Kd was significantly increased from 2.23 ± 0.31 μM  to 25.2 ± 0.7 μM . The interaction of Ca2+/CaM-Cav1.2-IQ1665-1685 with Cav1.2-NSCaTE51-67 was exothermic and driven by enthalpic contributions, with the E140G variant showing a significant increase in enthalpy upon binding.Physiologically, in the full CaaTE51-67 E. In the3581-3608 peptide in the absence and in the presence of Ca2+ with a stoichiometry of 1 (A and B). The Kd of CaM for RyR2 is decreased from 2.59 ± 0.13 μM (n = 9) to 0.130 ± 0.001 μM (n = 6) upon Ca2+ binding, indicating a ∼20-fold stronger binding at saturating Ca2+ concentrations. The affinity of the E140G variant for RyR23581-3608 remained unchanged in the absence of Ca2+ , however the affinity was decreased when Ca2+ was present  (C). In apo condition, the binding of RyR23581-3608 to CaM variants was endothermic and entropy driven, whereas in the presence of Ca2+, the interaction was exothermic and enthalpy driven (D). In both Ca2+-free and Ca2+-bound conditions, the E140G mutant significantly increased the thermodynamic drive of the reaction, when compared to WT.Using ITC, we showed that CaM can bind to the RyR2try of 1 , A and B, n = 6) C. In apoy driven D. In bot2+ release from the endoplasmic reticulum, human RyR2 and CaM variants were transiently overexpressed in HEK293T cells. In these experiments, CaM variants and the fluorescent marker (dTomato) were coexpressed under the control of the same promoter, as two distinct proteins and not as fusion proteins. Spontaneous Ca2+ oscillations were measured using Calbryte 520-AM (AAT Bioquest) as a Ca2+ indicator and single-cell fluorescence confocal microscopy . Free Ca2+ concentrations ([Ca2+]) were calculated using the Maxchelator Web Maxc standard . Ca2+ titrations were normalized and fitted to Hill specific binding model to obtain the dissociation constant (Kd). Kd was significantly increased from 1.65 ± 0.08 μM  to 28.3 ± 0.9 μM , indicating a ∼17-fold decrease in Ca2+-binding affinity for the LQTS-associated variant. Cooperativity of binding (Hill coefficient) was reduced from 1.3 ± 0.1 (CaM-WT) to 0.9 ± 0.1 (E140G).Equilibrium Cae of CaM A. Free Cstandard  program via the DichroWeb online server (B and C). In the absence of Ca2+ (apo conditions), the secondary structure content of CaM-WT and the E140G variant were similar, both comprising of ∼40% of α-helices and 13% of β-sheets. When Ca2+ was present, we observed the characteristic increase in α-helical content for both variants, to 60 ± 2% for CaM-WT (n = 5) and 52 ± 1% for E140G (n = 5). Upon Ca2+ binding, CaM-E140G variant showed significantly lower α-helical content and more unordered structures, when compared to the WT protein (C).Secondary structure content was investigated by far-UV CD spectra analysis e server , B and C protein C.2+-free (apo-) CaM proteins. We found that the E140G mutation did not significantly affect the melting temperature (Tm) with a Tm of 42.3 ± 0.5 °C for CaM-WT (n = 8) and 42.0 ± 0.2 °C for CaM-E140G (n = 6) (A). In addition, we determined the stability of CaM proteins using trypsin and AspN-limited proteolysis (B and C). In the absence of Ca2+, 5 ng/μl trypsin and 0.5 ng/μl AspN were sufficient to achieve full protein degradation. We did not observe any significant difference in protease susceptibility for CaM-WT and E140G. When Ca2+ is present, CaM proteins are significantly more resistant to degradation with 4-fold more trypsin (20 ng/μl) required to achieve full degradation, while AspN did not completely digest the proteins at the concentrations tested. For Ca2+-bound proteins, we observed that E140G was significantly more sensitive to both trypsin and AspN cleavage (B and C).To determine whether the LQTS-associated mutation E140G induced significant 3D conformational changes to CaM, we investigated WT and mutant CaM’s susceptibility to temperature and protease digestion (trypsin or the endoproteinase AspN) . Using C (n = 6) A. In addteolysis , B and Ccleavage , B and CAltogether, these data suggest that the disease-associated mutation E140G may affect the 3D structure of the protein and the exposure of specific residues involved in protease digestion.2+ sensor that regulates several target proteins involved in cardiac muscle contraction, including RyR2, Cav1.2, and CaMKIIδ   and he is presenting speech and motor skill delay.CaM is a major Ca CaMKIIδ , 66, 67.iewed by ). In thi, CALM1) . CaM is  protein , 70. Thev1.2. Using a combination of electrophysiology, cell biology, biophysics, and structural biology techniques, we present novel data showing that the disease-associated mutation E140G has a significant effect on key functional properties of CaM, including the regulation of important targets such as CaMKIIδ and Ca2+ binding, Ca2+/CaM-E140G had a significantly lower α-helical content and was more susceptible to protease digestion than Ca2+/CaM-WT. Through its four EF-hand motifs, CaM can sense intracellular Ca2+ across a wide range of concentrations to trigger the appropriate functional outcome. Mutation in the residues at the EF-hands involved in direct binding with the Ca2+ ions can result in severe consequences. E140 is one of the residues that directly participate in Ca2+ binding at the C-terminal EF hand 4 of Ca2+/CaM. Our equilibrium Ca2+-binding titrations data showed a ∼17-fold decrease in Ca2+ affinity for the CaM-E140G variant compared to CaM-WT, which is in accordance with previous studies  was added to eliminate any effect arising due to impaired Ca2+ binding.Altogether, these data suggest a defect in Cavia phosphorylation. One of the major kinases involved in RyR2 and Cav1.2 channel regulation is CaMKIIδ   , a 2-fol2+ enters the cell through voltage-gated Ca2+ channels (Cav1.2) to trigger intracellular signaling cascades leading to cardiac muscle contraction. CaM can regulate the activity of Cav1.2 in a Ca2+-dependent manner  in the inactivation of the channel. We demonstrated a significant reduction in the CDI of Cav1.2 when CaM-E140G was present, which could be the result of a combination of defective Ca2+ sensing and altered interaction with the channel binding domains. This observation is in line with previously published data on LQTS-associated CaM mutants, implying a potential common mechanism of disease involving disrupted Cav1.2 inactivation , which would result in an increase of the QT interval, characteristic of LQTS. Interestingly, our cellular model still expressed endogenous CaM (as it would be in human patients), demonstrating that E140G detrimentally impacted Cav1.2 CDI even in the presence of CaM-WT. This is in accordance with a previous study which used a 1:3 ratio of CaM-E140G:CaM-WT protein introduced into murine ventricular cardiomyocytes where they observed a significant increase in ICa rate of inactivation  to Cav1.2-binding domains, suggesting subtle changes in the mechanism of interaction, when compared to CaM-WT. We observed a binding stoichiometry (N) of ∼2 for the Ca2+/CaM–Cav1.2-NSCaTE51-67 interaction which was in accordance with several studies ; therefore, CaM would present a stoichiometry of binding with Cav1.2–NSCaTE of only N∼1. For Ca2+/CaM–Cav1.2-IQ1665-1685 binding events, we measured a binding stoichiometry of ∼1, as shown in previous studies  , 84, 85  studies , 88, in of N = 1 . A stoic studies , 87. Usi studies .v1.2 peptides is not affected by the LQTS-associated mutation E140G. For Ca2+/CaM–Cav1.2-NSCaTE51-67, we measured a binding affinity of 1.08 ± 0.01 μM, which was in the same range as previously reported (Kd = 0.57–2.90 μM)  to 25.2 μM (CaM-E140G). These data suggest that the E140G mutation, while located in the C-lobe, induces global structural rearrangements which reduces CaM binding to NSCaTE and therefore could contribute to defects in Cav1.2 CDI. We measured a 2-fold increase in affinity for Ca2+/CaM-E140G–Cav1.2-IQ1665-1685 when compared to WT, which differs from the ∼3-fold reduction in affinity previously observed by GST-pull down assay  for the pre-IQ/IQ domain  , 88, 89.wn assay . HoweverQ domain .v1.2 IQ domain for regulating channel inactivation (CDI). It has been established that a single CaM prebound to the Cav1.2-IQ domain is necessary and sufficient to produce CDI of its associated channel  showed that inhibition of CaMKII (using AIP) has no effect on the AP in an ATX-II model of LQTS  CaM-E140G affinity for Ca2+ is reduced, therefore intracellular [Ca2+] changes are not sensed appropriately and 2) CaM-E140G has a reduced binding affinity for NSCaTE, the intracellular channel domain that Ca2+-bound CaM needs to dock onto to induce CDI. Additionally, at elevated intracellular [Ca2+], CaM-E140G has a higher affinity for Cav1.2-IQ, suggesting it may outcompete CaM-WT on the C-terminal domain of the channel. In combination, these alterations will severely impair CaM-dependent CDI. Importantly, we also show that CaMKIIδ activity is affected by CaM-E140G, adding another layer of complexity. CaMKIIδ bound to CaM-E140G shows significantly reduced kinase activity, which will reduce the phosphorylation of Cav1.2. As CaMKIIδ-mediated channel phosphorylation is required for CaM to bind to Cav1.2  into pHIV-IRES-dTomato or pHIV-IRES-EGFP  using Gibson Assembly as follows. The primers used to linearize the pHIV-IRES-dTomato or pHIV-IRES-EGFP backbone were forward 5′-AGGATCCGCCCCTCTCCC-3′ and reverse 5′-CGCTCACGACACCTGAAATG-3’; the primers to amplify the CaM genes were forward 5′-CATTTCAGGTGTCGTGAGCGATGGCTGACCAACTGACTG-3′ and reverse 5′-GAGGGAGAGGGGCGGATCCTTCACTTTGCTGTCATCATTTG-3’. PCR was performed using Phusion High-Fidelity DNA Polymerase (New England Biolabs). Fragments were assembled using the NEBuilder HiFi assembly kit according to the manufacturer's recommendations to generate pHIV-CaM-IRES-dTomato or pHIV-CaM-IRES-EGFP. In these constructs, CaM variants and the fluorescent marker (dTomato or EGFP) were coexpressed under the control of the same promoter, as two distinct proteins and not as fusion proteins. pHIV-CaM-IRES-dTomato was used in the CaThe molecular construct for expressing the human RyR2 (pcDNA-3/eGFP-hRyR2) was a kind gift from Dr Chris George .All resulting plasmids were confirmed by DNA sequencing .v1.2, and RyR2 as well as syntide2 (CaMKIIδ substrate) were chemically synthesized, HPLC-purified, and purity was estimated as >95% through mass spectrometry (GenicBio).The peptides encompassing CaM-binding domains in CaMKIIδ, Ca294-315: FNARRKLKGAILTTMLATRNFS; Cav1.2-NSCaTE51-67: SWQAAIDAARQAKLMGS; Cav1.2-IQ1665-1685: KFYATFLIQEYFRKFKKRKEQ; RyR23581-3608: RSKKAVWHKLLSKQRKRAVVACFRMAP; Syntide-2: PLARTLSVAGLPGKK  STAR and cultured in 2xYT media containing 100 μg/ml kanamycin. Expression was induced with 0.5 mM IPTG overnight at 18 °C. Cells were harvested by centrifugation and pellets were resuspended in 50 mM Hepes, 200 mM NaCl, pH 7.5 supplemented with protease inhibitor cocktail . Cells were lysed with lysozyme (1 mg/ml) for 30 min on ice followed by sonication. Lysates were further treated with BaseMuncher (Abcam) and clarified by ultracentrifugation.CaM-WT and E140G variant were expressed and purified as previously described . In shorClarified lysates were purified on a HisTrap HP column  using a linear gradient of 0 to 500 mM imidazole. Eluted proteins were dialyzed overnight at 4 °C (8 kDa) to remove the imidazole, and His-tag was removed by treatment with SUMO protease (ULP1). CaM proteins were then further purified by reverse HisTrap and size-exclusion chromatography . Fractions containing the purified proteins were concentrated using Amicon centrifugation units (3 kDa), flash-frozen in liquid nitrogen, and stored in -80 °C until used.0  = 2980 M−1 cm−1; ε0  = 5500 M−1 cm−1.Concentrations were obtained by measuring absorbance at 280 nm using a DS-11+ spectrophotometer (DeNovix) and molar extinction coefficients calculated from the amino acid composition (ExPASy/ProtParam program) . ε0  as per the manufacturer’s instruction.For CaMKIIδ2. The mixture was incubated at room temperature for 20 min prior to the addition of the ADP sensor. The reaction mix was further incubated at room temperature for 20 min before measuring the fluorescence intensity on a FLUOstar Omega (BMG Labtech) at λex 545 nm and λem 590 nm.CaMKIIδ phosphorylation activity was determined using the Amplite universal fluorimetric kinase assay kit (AAT Bioquest) following the manufacturer’s recommendations. The kinase reaction contained 4 μM CaM protein, 0.03 μM GST-CaMKIIδ (Abcam), 100 μM Syntide-2 peptide substrate (Genicbio), 200 μM ATP, and 2 mM CaCl+-Hepes, pH 7.5, 100 mM KCl, 2 mM MgCl2, 5 mM 1,4-DTT, 100 μM CaCl2 at room temperature. The reaction was started by adding 300 μM ATP and was terminated using SDS-containing sample buffer at predefined time points . Post separation of proteins by SDS-PAGE (NuPage 4–12% Bis-Tris), proteins were electro-transferred to a nitrocellulose membrane using iBlot2 gel transfer device . The membranes were blocked with 5% (w/v) fat-free powdered milk in 0.1% (v/v) Tween-20, 50 mM Tris–HCl (pH 7.6), 150 mM NaCl (T-TBS buffer). Membranes were then probed overnight at 4 °C with mouse anti-GST  and rabbit anti-phospho T287  monoclonal primary antibodies at 1/1000 and 1/500 dilutions, respectively. Next, membranes were washed in T-TBS and incubated for 1 h at room temperature with IRDye 680RD donkey anti-mouse  and IRDye 800CW donkey anti-rabbit  IgG secondary antibodies at 1/10,000 dilution. The bands were visualized using an Odyssey CLx infrared imaging system and the intensity of the bands were quantified by densitometry using Fiji (CaM protein (1 μM) and GST-CaMKIIδ (300 nM) were incubated in 50 mM King Fiji .2+/CaM-WT–CaMKIIδ294-315 and Ca2+/CaM-E140G–CaMKIIδ294-315 were grown at 20 °C . Ca2+/CaM–CaMKIIδ294-315 complexes crystallized using the following precipitant solutions: 0.2 M zinc acetate, 0.1 M imidazole, pH 8.0, and 18% w/v PEG 3000 for the WT; 0.1 M Na+-Hepes, 0.1 M MOPS (acid), pH 7.5, 0.03 M magnesium chloride hexahydrate, 0.03 M calcium chloride dihydrate, 12.5% v/v MPD; 12.5% PEG 1000; 12.5% w/v PEG 3350 for the E140G mutant.High-throughput screening was performed using a Screenmaker crystallization robot (Innovadyne) with a final drop volume of 400 nl  and the sitting drop vapor diffusion method. Five commercial crystallization screens were used: JCSG+ (Molecular Dimensions), Morpheus (Molecular Dimensions), PEGRx (Hampton Research), Structure (Molecular Dimensions), and Wizard Classic (Molecular Dimensions). Crystals for CaFor data collection, crystals were grown at 20 °C on a hanging drop format using 48-well plates with sealant (Hampton Research) and 12 mm siliconized glass circle coverslides (Hampton Research), with a final drop volume of 2 μl .2+/CaM-WT–CaMKIIδ294-315 was solved by molecular replacement with MOLREP  glycerol, and diffraction data were collected using the Diamond synchrotron beamline i04. Data were processed by xia2/DIALS or xia2/XDS/XSCALE , 103 andh MOLREP , using Pch model  and refich model . The str in COOT . The qua in COOT .7ZRP for Ca2+/CaM-WT–CaMKIIδ294-315 and 7ZRQ for Ca2+/CaM-E140G–CaMKIIδ294-315.A summary of diffraction data, refinement statistics, and the quality indicators for the structures are featured in 294-315; Cav1.2-NSCaTE51-67; Cav1.2-IQ1665-1685; RyR23581-3608) in 10- to 15-fold molar excess. For the preassociation experiments, CaM variants (100 μM) were mixed with an equimolar ratio of Cav1.2-IQ1665-1685 (100 μM) and titrated against 5- to 10-fold molar excess of Cav1.2-NSCaTE51-67.CaM proteins (WT or E140G) were used in the range of 10 to 50 μM and the peptides . The peptides were titrated against CaM proteins across 20 injections (2 μl each) lasting 4 seconds with a 180 s grace period in-between.Titrations were performed in 50 mM Hepes, 100 mM KCl, 2 mM MgClKd), stoichiometry of binding (N), and thermodynamic parameters .All titrations were performed using a MicroCal iTC200 and automated PEAQ-ITC systems  at 25 °C under continuous stirring at 800 rpm. Data were processed using the MicroCal PEAQ-ITC software and fitted to a one-site binding model to derive the dissociation constant  were purchased from B’SYS (Switzerland). Cells were cultured at 37 °C at 5% CO2, in Dulbecco’s Modified Eagle Medium/F12 GlutaMAX (Gibco) supplemented with 10% fetal bovine serum, 1× penicillin-streptomycin, 100 μg/ml Hygromycin B, 15 μg/ml Blasticidin, 0.4 μg/ml Puromycin, and 100 μg/ml Zeocin. Cells at approximately 40% confluency were transfected with 1 μg of pHIV- CaM-EGFP variants using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s guidelines. To induce expression of Cav1.2, 2.5 μg/ml tetracycline was applied to cells 24 h prior to experimental use.HEK293 cells stably expressing the Ca2+ current recordings. Currents were recorded using an Axopatch 200B amplifier (Molecular Devices), filtered at 2 kHz, and sampled at 10 kHz using a Digidata 1320A interface (Molecular Devices). All recordings were taken at room temperature. Internal pipette solution consisted of 140 mM CsMeSO4, 0.5 mM EGTA, 10 mM Hepes, 1 mM MgCl2, 1 mM Na-ATP, pH adjusted to 7.2 with CsOH. External solutions consisted of 140 mM NaCl, 5 mM CsCl, 0.33 mM NaH2PO4, 5 mM glucose, 10 mM Hepes, 1 mM MgCl2, and 2 mM of either CaCl2 or BaCl2 for the measurement of Ca2+ and Ba2+ currents, respectively. pH was adjusted to 7.4 with CsOH. Patch pipettes were pulled from borosilicate glass  and fire-polished to give a resistance of 3 to 5 MΩ once filled with pipette solution. HEK293-Cav1.2 cells transiently transfected with CaM were identified by EGFP fluorescence using a Nikon Eclipse TE200 inverted microscope with epifluorescence attachment. The Ca2+ current activation protocol consisted of 300 ms voltage steps ranging from −40 to +60 mV from a holding potential of −60 mV. Steady state inactivation was measured with a protocol comprising a 1 s conditioning prepulse to between −60 mV and +40 mV, from a holding potential of -60 mV, followed by a 300 ms test pulse to +10 mV. Half maximal activation and inactivation (V50) were calculated by fitting normalized peak conductance using the Boltzmann equation. For the determination of CDI, the residual current at the end of the 300 ms pulse from the activation protocol (r300) was calculated in extracellular solutions containing either Ca2+ (r300Ca) or Ba2+ (r300Ba). The proportion of current inactivation due to CDI (f300) was calculated using the following equation:The conventional whole-cell voltage-clamp configuration was used to obtain Ca2, in Dulbecco’s Modified Eagle Medium GlutaMAX (Gibco) supplemented with 10% fetal bovine serum, 1× penicillin-streptomycin (Gibco), and 1× nonessential amino acids (Gibco). For Ca2+ imaging experiments, cells were seeded onto 35 mm poly-lysine–treated glass-bottomed dishes (MatTek Corporation). Effectene reagent (Qiagen) was used to cotransfect eGFP-hRyR2 (pcDNA3) and CaM variant (dTomato) plasmids in a 1:2 M ratio, according to the manufacturer's instructions. Posttransfection (48 h), cells were loaded with 10 μM Calbryte 520-AM (AAT Bioquest) for ∼1 h. Then, cells were covered with Krebs Ringer Hepes buffer  and imaging was carried out at on a 3i Marianas spinning-disk. The confocal microscope was equipped with a Zeiss AxioObserver Z1, a 20 × /0.8 Plan-Apochromat air objective, and a 3i Laserstack as an excitation light source . Emitted light was collected through single bandpass filters  onto a digital complementary metal-oxide semiconductor camera .HEK293T cells (American Type Culture Collection) were cultured at 37 °C/5% CO2+ oscillations were recorded from cells coexpressing hRyR2 (eGFP) and CaM variants (dTomato). Calbryte 520 fluorescence signals were measured from regions of interest outlining individual cells using Fiji, and kinetic parameters of Ca2+ release were quantified using SignalFind .Data were acquired at 1024 × 1024 pixel resolution at a rate of ∼5 frames/sec, using SlideBook v.6 software. Spontaneous Ca2+ binding to the C-lobe of CaM. Proteins (6 μM) were titrated with increasing free [Ca2+] in 50 mM Hepes, 100 mM KCl, 1 mM MgCl2, 0.5 mM EGTA, 0.5 mM NTA, pH 7.4. Fluorescence emission spectra were recorded at λexc 277 nm and λem 300 to 320 nm (peak at 307 nm) on a JASCO FP-6300 spectrofluorometer. Free [Ca2+] were calculated using Maxchelator Web Maxc standard  was obtained using specific binding with Hill slope nonlinear fitting.Fluorescence emission at each Ca2 for apo- or Ca2+-bound CaM, respectively.Far-UV CD spectra (180–260 nm) were recorded at 20 °C in a 0.1 cm path length quartz cell using a JASCO J-1100 spectrometer equipped with a JASCO MCB-100 mini circulation bath for temperature control. Purified proteins (10 μM) were measured in 2 mM Hepes (pH 7.5) supplemented with either 1 mM EGTA or 1 mM CaCl-1) and buffer baseline subtracted prior to analysis. Data were normalized to mean residue ellipticity and the secondary structure content was estimated using the CDSSTR prediction algorithm   , 111.2+-free) to temperature was assessed by decrease in α-helical content measured by CD. Ellipticity at 222 nm for CaM variants was recorded in a 0.1 cm path length quartz cell using a JASCO J-1100 spectrometer equipped with a JASCO MCB-100 mini circulation bath for temperature control. Purified proteins (10 μM) were measured in 2 mM Hepes (pH 7.5) supplemented with 1 mM EGTA. Temperature ranged from 20 °C to 80 °C in 2 °C increments, with a ramp increase rate of 1 °C/min and a 180s equilibration period between recordings. Data were normalized and fitted to the Boltzmann sigmoid equation (GraphPad Prism) to derive the melting temperature of CaM (Tm).Sensitivity of apo-CaM (Ca2+-bound conditions (5 mM CaCl2). AspN digestions were performed in 50 mM Tris–HCl, 2.5 mM ZnSO4, pH 8 with protease concentration 0 to 5 ng/μl in apo conditions (10 mM EGTA) and 0 to 300 ng/μl in Ca2+-bound conditions (5 mM CaCl2).Sensitivity of CaM to enzymatic degradation by trypsin or AspN (New England Biolabs) was assessed by SDS-PAGE and densitometry analysis. Purified CaM (5 μM) were incubated with proteases for 30 min at 37 °C before rapid termination of the reaction by addition of SDS-containing sample buffer and heating to 95 °C for 10 min. Trypsin digestions were performed in 25 mM Hepes, 100 mM NaCl, pH 7.5 with protease concentration 0 to 10 ng/μl in apo conditions (10 mM EGTA) and 0 to 30 ng/μl in CaProteins were separated by NuPAGE 4 to 12% Bis-Tris (Life Technologies) and stained with InstantBlue (Thermo Fisher Scientific). Images were obtained on a ChemiDoc XRS+ transilluminator (Bio-Rad) and the amount of intact CaM was quantified by densitometry using Fiji software.t test, one-way ANOVA, or two-way ANOVA, as described in the appropriate figure legends. p-values are represented by stars with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. Structure representations were created using UCSF Chimera software and figures were generated using CorelDRAW 2021. Statistical parameters for all ANOVA tests are presented in Experiments were performed at least in triplicates and analyzed using GraphPad Prism. Statistical significance levels were obtained using a two-tailed unpaired Student's All data have been included within the article and supplementary information. Raw data files are to be shared upon request.This article contains The authors declare no competing or financial interests.


The maximum intensity of SHG in hybrid metasurface coupled MoS2 is more than ten thousand times larger than that of other structure-units coupled MoS2. The conversion efficiency is reported to be as high as 3.27 × 10−7. This work paves the way to optimize nonlinear light–matter interactions in low-dimensional structures coupled with semiconductors.Plasmonic nanostructures have been regarded as potential candidates for boosting the nonlinear up-conversion rate at the nanoscale level due to their strong near-field enhancement and inherent high design freedom. Here, we design a hybrid metasurface to realize the moderate interaction of Fano resonance and create the dual-resonant mode-matching condition to facilitate the nonlinear process of second harmonic generation (SHG). The hybrid metasurface presents dipolar and octupolar plasmonic modes near the fundamental and doubled-frequency wavelengths, respectively, further utilized to enhance the SHG of low-dimensional MoS Second-harmonic generation (SHG) is a kind of nonlinear optical process. Two photons of the same frequency coupled in nanostructures result in a single-photon emission with twice the fundamental frequency. System ground state information and pump light polarization are recorded via SHG signals, which have been used extensively in lasers , 2, holoA possible strategy to tackle this problem is to design hybrid nanostructures interacting with nonlinear materials , 8. Elec–10 W–1 for SHG [Plasmonic Fano resonance interferes with bright superradiant and dark subradiant modes, providing a platform for ultrasensitive signal detection applied in chemistry, physics, and biological sciences , 13. Gen for SHG . Compare for SHG .2 semiconductors. The hybrid metasurface is designed to behave with dual-resonance modes overlapping the fundamental and the SH wavelengths. Simulations of charge distribution at the doubled-frequency reveal that the hybridized Fano resonance mode has been established. The intensity of the SHG signal of hybrid metasurface coupled MoS2 is more than ten thousand times larger than that of metasurface units coupled MoS2 due to the combination of mode-matching conditions and moderate-interaction Fano resonance. Furthermore, the conversion efficiency of the proposed configuration is detected to be as high as 3.27 × 10–7. The hybrid metasurface proposed in this work may pave the way to explore the enhancement of nonlinear optical processes by inducing Fano resonance and mode matching conditions.Here, we propose a hybrid metasurface  having mode matching and plasmonic Fano resonance to enhance the SHG of low-dimensional MoS2 semiconductor located at the structure center. The generation and enhancement of SHG are realized by designing the plasmonic resonant modes matching the excitation wavelengths. Paired bull-eye rings are employed inside a wall ring to achieve stronger Fano resonance. Where eight parameters, namely, can characterize the structure geometry, the inner and outer radii of the wall ring are 2 semiconductor is in the gap between the paired bull-eye rings, and its size is chosen to ensure that they contact the bull-eye rings. The hybrid metasurface can be experimentally fabricated using the “Sketch and Peel” lithography to realize the high-resolution patterning [Figure tterning .Fig. 1Scx–z plane and the coaxial direction of bull-eye rings [2 [2 with refractive index of 1.5 is set be the substrate and support of the device.The Total-field scattered field (TFSF) as an incident light source is set to be linearly polarized parallel to the ye rings . In our ye rings . Perfectye rings  and the rings [2  was takeTo satisfy the mode-matching conditions, the resonance of the hybrid metasurface needs to be specifically optimized for both fundamental and SHG wavelengths. Scattering cross sections spectrum (SCSS) was simulated to discuss the plasmonic modes induced by structure units. Figure y-axis layout of Fig. The bi-coordinate of the ion mode . The resentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaTo comprehend the origin of this Fano resonance, the primary plasmon modes of metasurface units should be first understood. In Fig. 2 semiconductor, the SHG enhancement factor (τ) is introduced to describe the enhancement intensity, which is defined as the ratio of the SHG intensity converted from the metasurface to the intensity of the MoS2 nanoparticles. The volumetric effective second-order nonlinear polarizability 0.78 pm V−1 is used to simulate SHG emission from gold [2 chose to be 0.25 nm V−1 [8 V m−1, and the corresponding peak field strength is 3.2 GW cm−2, which is less than the damage threshold of gold [To discuss the nonlinear process of SHG from a hybrid metasurface coupled MoSrom gold , and the5 nm V−1 ; both co5 nm V−1 . In simu of gold . A narroτ) of the hybrid metasurface (red line), while the blue line corresponds to the ratio of pump light to SHG from MoS2 nanoparticles. The basic femtosecond excitation at 1600 nm produces an instantaneous SHG response centered at 800 nm. As shown in Fig. −7. Figure ħω) at the excitation wavelength assisted by a first plasmonic mode, results in a radiative frequency-doubled photon (2ħω) assisted by a second plasmonic mode, restoring the system ground state through the emission of a SH photon [Figure velength . The calH photon . The SHGH photon , 32. Fanx direction (the polarization angle θ = 0°), the largest SHG signal is obtained while the intensity of the SHG signal gradually decreases as the direction of the incident light moves away from x to the y-direction. Furthermore, when the polarization direction of the incident light is parallel to the y direction (the polarization angle θ = 90°), the SHG signal almost becomes zero. Thus, it clearly reveals that the SHG signal from the hybrid metasurface strongly depends on the polarization of the incident light. The polarization diagram of the SHG signal conversion efficiency generated by the hybrid metasurface is shown in Fig. θ = 0° reaches the maximum value, while for θ = 90° the conversion efficiency declines to a minimum value.In order to study the influence of incident light polarization on SHG intensity, the polarization distribution of the radiated SHG signals is plotted in Fig. 2 nanoparticles (red line), but the maximum intensity of SH enhancement appears to be 1.31 × 102 when the MoS2 nanoparticle is located at the center of the wall ring. The SH enhancements of paired bull-eye rings and designed hybrid structure without MoS2 are nearly zero and 60, respectively. In comparison, the maximum intensity of SH enhancement of bull-eye coupled MoS2 nanoparticle gets 2.7 times larger than that of wall ring coupled MoS2 nanoparticle. Remarkably, in hybrid metasurface-coupled MoS2 nanoparticles, the intensity is increased to 3.13 × 106, which is more than 104 times larger than other structure units coupled MoS2 nanoparticles. A previous report shows that the conversation efficiency of MoS2 monolayer enhanced by Au@SiOx nanoparticles is 1.24 × 10−7 at the 100 GW cm−2, and the SHG enhancement is 1.88 detected from experiments [2 on suspended metallic nanostructures are 1528 in experiments and 1898 in simulations, respectively [At the end of this discussion, here is a comparison Fig.  of SHG eeriments . Anotherectively . Our desAlthough hybrid plasmonic nanostructures with Fano resonant modes have been widely reported, the enhancement efficiency of SHG is still limited by finite electromagnetic field enhancement. In a fixed geometric dimension, the rational design of abundant plasmonic gaps can induce more “hot spots” creating stronger total-field-enhancement for SHG enhancement. Besides, the high-order plasmonic modes in our designed nanostructures can suppress the radiation loss and provide sharper line shape than dipole modes, which may help to increase the coupling sensitivity at resonant wavelengths. This rational design of plasmonic metasurface inducing matching conditions and Fano resonance can effectively enhance the SHG signals and conversion efficiency.–7. In this work, the SHG signal of hybrid metasurface coupled-MoS2 nanoparticle is more than ten thousand times larger than that of other metasurface unit coupled MoS2 nanoparticles. The combination of mode-matching conditions and moderate interaction of Fano resonance can enhance SHG conversion efficiency effectively. The related concept may also be applicable to other nonlinear processes, such as third harmonic generation, sum-frequency generation (SFG), and difference frequency. This paves the way to achieve infrared frequency up-conversion and overcome difficulties in infrared reception. Furthermore, combined with frequency doubling and frequency mixing technologies, it can achieve modulation in a wide range from infrared to ultraviolet regimes. Our designed hybrid metasurface opens a new avenue for optimizing nonlinear light–matter interactions in low-dimensional semiconducting materials and nanostructures.In summary, this contribution provides an understanding of a hybrid metasurface to realize the moderate interaction of Fano resonance and create the dual-resonant mode-matching condition to facilitate the nonlinear process of SH generation. Our hybrid metasurface presents the dipole and octupole plasmonic modes at the fundamental and doubled-frequency wavelengths. Simulations of charge distribution at the doubled-frequency reveal the hybridized Fano resonance mode formation. The designed metasurface produces a conversion efficiency of 3.27 × 10


FLT3-ITD mutation subtype, has a poorer prognosis and higher risk of recurrence, which seriously threatens human health. Ferroptosis, an iron-dependent regulated cell death, is involved in the development and progression of AML. The mechanism by which circRNAs regulate the pathogenesis and prognosis of FLT3-ITD mutant-type AML through ferroptosis-related genes (FerRGs) remains unclear. In the present study, we aimed to decipher the pathogenic role of circRNAs in AML with FLT3-ITD mutation. Encouragingly, we discovered a circRNA, hsa_circ_0015278, that regulates ferroptosis-related genes by sponging miRNAs to promote FLT3-ITD AML progression. Furthermore, ferroptosis-related genes with prognostic value contribute to FLT3-ITD AML progression by regulating the tumor microenvironment. In conclusion, we constructed a ceRNA regulatory network involving hsa_circ_0015278/miRNAs/FerRGs, and the hsa_circ_0015278 signaling axis contributes to the identification of potential diagnostic and prognostic biomarkers and provides new insight into the pathogenesis and therapeutic targets of AML with FLT3-ITD mutation.Acute myeloid leukemia (AML), especially the FLT3-ITD mutation seriously threatens human health. The mechanism by which circRNAs regulate the pathogenesis of FLT3-ITD mutant-type AML through ferroptosis-related genes (FerRGs) remains unclear. Differentially expressed circRNAs and mRNAs were identified from multiple integrated data sources. The target miRNAs and mRNAs of the circRNAs were predicted using various databases. The PPI network, ceRNA regulatory network, GO, and KEGG enrichment analyses were performed. The “survival” and the “pROC” R packages were used for K-M and ROC analysis, respectively. GSEA, immune infiltration analysis, and clinical subgroup analysis were performed. Finally, circRNAs were validated by Sanger sequencing and qRT-PCR. In our study, 77 DECircs-1 and 690 DECircs-2 were identified. Subsequently, 11 co-up-regulated DECircs were obtained by intersecting DECircs-1 and DECircs-2. The target miRNAs of the circRNAs were screened by CircInteractome, circbank, and circAtlas. Utilizing TargetScan, ENCORI, and miRWalk, the target mRNAs of the miRNAs were uncovered. Ultimately, 73 FerRGs were obtained, and the ceRNA regulatory network was constructed. Furthermore, MAPK3 and CD44 were significantly associated with prognosis. qRT-PCR results confirmed that has_circ_0015278 was significantly overexpressed in FLT3-ITD mutant-type AML. In summary, we constructed the hsa_circ_0015278/miRNAs/FerRGs signaling axis, which provides new insight into the pathogenesis and therapeutic targets of AML with FLT3-ITD mutation.AML with the  Acute myeloid leukemia (AML) is a hematological malignancy caused by the arrest of differentiation, uncontrolled proliferation, and delayed apoptosis of immature myeloid progenitor cells and myeloid blast cells, which is highly clinically heterogeneous and aggressive . It is aFLT3-ITD mutation), which promotes AML progression ) were cultured in RPMI 1640 (Sigma) and DMEM/F12 (gibco), respectively, with 10% fetal bovine serum (gibco). All cells were cultured in a humidified atmosphere at 37 °C containing 5% CO2. The circular structure of circRNAs was confirmed by Sanger sequencing and divergent primer PCR. Total RNA was extracted from AML and normal cell lines using the TRIzol reagent (Invitrogen), and complement DNA was generated using the ReverTra Ace qPCR RT Master Mix with the gDNA Remover kit (TOYOBO). qRT-PCR assays were performed using the SYBR Green Realtime PCR Master Mix (TOYOBO). The circRNA expression levels were normalized to GADPH and each sample was tested in triplicate. The relative expression was analyzed by the 2−ΔΔCt method. The primer sequences of circRNAs and GAPDH were synthesized by BGI . The primers used in this study are listed in Human FLT3-ITDhttps://www.R-project.org (accessed on 15 June 2021) and visualized by the “ggplot2” package v. 3.3.3 (https://www.rdocumentation.org/packages/ggplot2/ (accessed on 15 June 2021)). Data were presented as means ± standard error of the mean (SEM) in at least three independent experiments and analyzed with GraphPad Prism 9.3.1 . The Wilcoxon rank sum test and the Weltch t’ test were used to compare the expression differences of unpaired samples between the two groups. The Wilcoxon rank sum test was utilized to analyze the relations between the clinicopathological features and candidate FerRGs and miRNAs. Spearman correlation analysis was used to present the degree of immune cell infiltration. p < 0.05 was considered statistically significant.All analyses were performed using R v. 4.0.3 (The detailed flowchart is exhibited in FLT3-ITD mutant-type (FLT3+), 3 FLT3-ITD wild-type (FLT3−) AML samples, and 4 healthy controls (HCs). DECircs-1 between FLT3-ITD mutant AML samples and FLT3-ITD wild AML samples were screened with the cutoff threshold |LogFC| > 1 and adj. p value < 0.05. Consistently, DECircs-2 between FLT3-ITD mutant AML samples and HCs were screened with the same criteria. As shown in the Volcano plot and Heatmap, 77 DECircs-1 containing 60 up-regulated and 17 down-regulated DECircs (http://www.csbio.sjtu.edu.cn/bioinf/lncLocator/index.html (accessed on 5 July 2021). Surprisingly, except for hsa_circ_0060828 and hsa_circ_0049356, the remaining circRNAs were principally localized in the cytoplasm (The circRNA microarray datasets (GSE94591) contain 10 samples, namely, 3  DECircs A,C and 6 DECircs B,D, resp DECircs E. Ultima DECircs , and theytoplasm G. In addytoplasm .Many studies have reported that circRNA with abundant MREs (miRNA response elements) can regulate the expression of target genes by acting as a “sponge” for the adsorption of miRNA, thereby affecting the progression of the disease. For the sake of further elucidating the mechanism by which circRNAs affect the occurrence and progression of AML, we sought to establish a ceRNA regulatory network involving certain circRNAs. First, the bioinformatics software CircInteractome, CircBank, and circAtlas 2.0 were utilized to predict the target miRNAs of circRNAs. Then, the obtained miRNAs were used to predict the downstream target genes through TargetScan, ENCORI, and the miRWalk database. It is worth noting that only the miRNAs or target genes predicted by more than two prediction databases were considered credible and used for subsequent research. Simultaneously, 73 FerRGs were obtained by taking the intersection of DEGs, miRNA-predicted target genes, and ferroptosis genes downloaded from FerrDb . UltimatTo better understand the mechanism of 73 FerRGs in AML, GO, and KEGG enrichment analyses were performed (the detailed enrichment information was listed in FLT3-ITD mutated AML are listed in The “Cytohubba” plug-in in Cytoscape (version 3.9.0) was used to screen hub genes. As is well-known, there are 12 algorithms in the “Cytohubba” plug-in , and each algorithm can obtain the top 10 hub genes. Subsequently, we sorted the hub genes obtained by the 12 algorithms according to their frequencies and finally determined 15 hub genes (15 hub genes are shown in p = 0.022) and CD44 (Log-rank p = 0.001) were significantly associated with OS (TCGA (The Cancer Genome Atlas), which involves comprehensive and multidimensional data, including multi-omics and clinical data, is an essential database for Cancer researchers. The TCGA database was utilized for differential expression analysis of 15 hub genes A–O. The  with OS .+ AML cell line (MOLM-13 cell) compared with the FLT3− AML cell line (HL-60 cell). In addition, to confirm the circular structure of the candidate circRNAs, Sanger sequencing was performed. We then cross-checked the Sanger sequencing results with the base sequences of circRNAs provided by circBase, which confirmed the existence of back-splicing sites in the Sanger sequencing results  via FoxO3-mediated transactivation in FLT3-ITDsistance . Sykes esistance . These rMeanwhile, we screened two molecules with prognostic values, MAPK3 and CD44, using various algorithms of the “Cytohubba” plug-in and clinical prognostic analysis. MAPK3, alias ERK1, and Liu et al. found that the reduction of nuclear factor erythroid 2-related factor 2 (NRF2) elicited phosphorylation of ERK1/2 and then activated ferritinophagy, which led to the degradation of ferritin heavy chain 1 (FTH1). The proteasome releases free iron ions to trigger ferroptosis in macrophages . Gu et aFLT3-ITD mutation.Due to the upstream hsa_circ_0015278 of MAPK3 and CD44 exhibiting regulatory sophistication in the ceRNA network, we focused on hsa_circ_0015278 and constructed a sub-ceRNA regulatory network involving hsa_circ_0015278 in this study. The correlation analysis of clinicopathological parameters showed that the miRNAs and mRNAs in the sub-ceRNA regulatory network were closely related to clinicopathological parameters. In addition, accumulating evidence in the literature reveals that tumor-infiltrating immune cells affect the efficacy of chemotherapy and immunotherapy, thereby affecting patient outcomes ,54. It iFLT3-ITD mutation, and most importantly, its downstream MAPK3 and CD44 played a vital role in both ferroptosis and AML. These suggest that hsa_circ_0015278 can be used as a novel biomarker with diagnostic and prognostic values in AML with FLT3-ITD mutation.It is commonly believed that most of the circRNAs located in the cytoplasm can indirectly regulate mRNA expression through the ceRNA mechanism . It has FLT3-ITD mutations were focused on in our study. Furthermore, for the first time, we link the roles of circRNAs and ferroptosis-related genes in the progression of AML with FLT3-ITD mutation, providing a new perspective for targeted therapeutic strategies. However, we only detected the expression of hsa_circ_0015278 in AML cell lines, lacking validation in a large number of clinical samples. Moreover, in-depth mechanistic studies are lacking. Therefore, in the future, we will explore the underlying molecular mechanism of hsa_circ_0015278-mediated disease progression of AML with FLT3-ITD mutation through a series of functional experiments and animal models.To the best of our knowledge, some researchers have used the circRNA microarray GSE94591 to conduct AML-related research. However, the vast majority focused on the differentially expressed circRNAs between the AML and healthy control groups. For the first time, differentially expressed circRNAs in FLT3-ITD mutation. Encouragingly, MAPK3 and CD44, as hub genes, were not only enriched in “ferroptosis” and “acute myeloid leukemia” pathways but also significantly correlated with prognosis and immune cell infiltration in AML patients. Therefore, the hsa_circ_0015278 signaling axis contributes to the identification of potential diagnostic and prognostic biomarkers and provides new insights into the pathogenesis and therapeutic targets of AML with FLT3-ITD mutation and poor prognosis.In summary, based on GEO and TCGA database mining, bioinformatics analysis, and in vitro validation, we constructed a potential ceRNA regulatory network involving hsa_circ_0015278/miRNAs/FerRGs in AML with


With the slowdown of Moore’s law, many emerging electronic devices and computing architectures have been proposed to sustain the performance advancement of computing. Among them, the Ising machine is a non-von-Neumann solver that has received wide attention in recent years. It is capable of solving intractable combinatorial optimization (CO) problems, which are difficult to be solve using conventional digital computers. In fact, many CO problems can be mapped to finding the corresponding ground states of Ising model. At present, Ising machine prototypes based on different physical principles, such as emerging memristive oscillators, have been demonstrated, among which the Ising Hamiltonian solver based on the coupled oscillator network simultaneously holds the advantages of room-temperature operation, compact footprint, low power consumption, and fast speed to solution. This paper comprehensively surveys the recent developments in this important field, including the types of oscillators, the implementation principle of the Ising model, and the solver’s performance. Finally, methods to further improve the performance have also been suggested. Combinatorial optimization (CO) is an important branch of operations research and algorithm theory that has a wide spectrum of applications in the real world, including artificial intelligence (AI), circuit wiring, information network, route planning ,2,3, etcWith the slowdown of Moore’s law, emerging hardware and computing paradigms beyond traditional transistor-based von-Neumann architecture are now topical. Different from the optimization algorithms tailored for digital logic circuits, Ising machines have the ability to solve the Ising Hamiltonian equation through their internal physical evolution process, which may yield an optimal solution at a high speed . AccordiThanks to these advantages of oscillator-network-based Ising machines, there is a recent surge of interest in this field. This paper has reviewed the development of this field for the first time, including the merits of different kinds of oscillators and the theoretical proof on why oscillator networks are equivalent to the corresponding Ising model and their performances. Finally, methods to improve the performance of this kind of solver are also discussed.In general, a physical system that produces periodic outputs can be termed an oscillator. In electronics, oscillators are usually regarded as a circuit component that outputs a periodic analog electrical voltage or current signal under direct excitation, which has a wide spectrum of applications such as clocking. According to the circuits and device physics, they can be divided into resistor–capacitor (RC) oscillators, inductor–capacitor (LC) oscillators, ring oscillators, phase-transition oscillators, spin torque oscillators, spin Hall oscillators, etc. In addition, according to the output waveforms, they can be categorized into three types: sine wave, square wave, or triangle wave oscillators. The performance of these Ising machines is highly relevant to the type of oscillators, which largely determines the physical footprint, time to solution, energy consumption, accuracy, and other important attributes of the solvers.A RC oscillator, that is, an oscillator composed of an RC frequency selection network a, is genLike the RC oscillator, the LC oscillator is also very common, which employs a frequency selection network composed of LCs b and levt is the delay time of a single inverter, n is the number of inverters). This kind of oscillators hold the advantages of simple circuitry and robust oscillation. The ring oscillator is one of the most compact transistor-based oscillators, which is highly conducive to large-scale integration. In addition, it also has decent energy consumption, which is comparable to that of nanoscale oscillators [A ring oscillator is a closed-loop oscillation circuit composed of an odd number of inverters c. Its osillators . HoweverPhase-transition oscillators, as the name suggests, are oscillators made of phase-transition materials d, which Spin torque and spin Hall oscillators e are a nn represents the number of spins, i and j, if The Ising model is a mathematical model named after the German physicist Ernst Ising. As early as the 1920s, the Ising model was proposed to describe the formation of magnetic domains in ferromagnets . It contirection a. If Jijrections b. Jij=0 Hmin.In fact, many CO problems have been proven to root on this equation. With the appropriate coupling coefficients quations ,28. The V1, V2) [V1, the group connecting vertices in set V2, and the group linking vertices in V1 and those in V2. The sum of the weights in these three sets are represented by S1, S2, and Scut, respectively. By setting i and j, we can getThe max-cut problem, for example , refers V1, V2) . This isi∈V1, then i∈V2, then The vertices in two sets can be mapped to different spin values. For example, if itten as  follows:H is minimized, the sum of the cutting weights will be maximized. It should be noted that, for the max-cut problem, if the weight of the edge is positive, the mapped Ising model should be an antiferromagnetic system, that is Therefore, when This section introduces the theoretical formulation of a coupled oscillator network–based Ising machine and shows how the coupled oscillator networks automatically minimizes the Ising energy to yield the optimal solution. The implementation is mainly based on the fundamental injection locking (IL) and its variant second-harmonic injection locking (SHIL) ,32,33,34We will first mathematically derive the dynamics of a single oscillator. IL is a nonlinear phenomenon that exists in oscillator systems. The term stands for the fact that the phase of the oscillator will be pulled or locked by an external periodic perturbation signal. When the frequency of the external signal is close to the oscillation frequency, this phenomenon is called IL. If the frequency of the external signal is nearly twice the oscillation frequency, then it is called SHIL.A nonlinear oscillator is an autonomous dynamic system; when the oscillator is influenced by an external perturbation signal on (DAE) :(5)ddtq→ified as (6)x→pt=T0 is the steady-state solution without any perturbation, and In which, Here, terature ,37.Assuming that the oscillation frequency is When injecting an external periodic signal with a frequency The phase difference between the oscillator and the external signal is defined asThen, we haveCombining Equations (9) and (11), we obtainCombining Equation (10), we deriveIn Equation (13), we assume that Then,Equation (15) is the generalized Adler’s equation describing the IL phenomenon .Taking the LC oscillator as an example, when an LC oscillator (PPV: equation : (16)ddtQ is the quality factor of the oscillator.Here, When several oscillators are coupled together, the oscillator of interest is subject to the injection of output signals of other oscillators according to Equation (16): Then,N is the number of oscillators and Equation (18) is known as the Kuramoto model , where NIn addition, when the oscillator is locked by an external signal, From Equation (19), the range of IL and the phase difference between the oscillators can be easily determined.The total energy is determined by the Lyapunov equation :(20)dΔϕ(The derivative of energy with respect to time isFrom Equation (21), it can be seen that this system has a tendency to reduce the energy over time automatically.When Assuming that there are two LC oscillators with the same oscillation frequency,It can be seen from Equation (22) that, if the same a. Howeveopposite b. When meraction d. In thiWhen the oscillator is subjected to an external perturbation signal of about twice the base frequency (ation is (23)ddtΔIn which,In order to study its periodicity, the PPV and injection signal were expanded using Fourier series:Then,Assuming that It can be seen from When an oscillator network is perturbed by an injection signal with a frequency of The system’s energy isEquation (31) is very similar to the original Ising Hamiltonian equation. When In 2017, Wang et al. demonstrated for the first time that a self-sustaining LC oscillator network can be used as an Ising machine. They also showed how the global Lyapunov function of the oscillator phase macromodel can be mapped to the corresponding Ising equation . Moreove4–106 times faster, while the power consumption of the entire Ising machine was only 23 mW, corroborating its great potential for practical applications.Compared with LC oscillators, ring oscillators can easily achieve higher working frequencies and can be easily integrated. Ahmed et al. fabricated a programmable network consisting of 560 coupled ring oscillators and successfully realized a fully integrated chip-scale Ising machine ,43. EachRecently, Moy et al. reported a 1968-node coupled King’s graph ring oscillator–based Ising machine with five-level coupling strengths . Here, t2 memristors and investigated the impact of the coupling coefficients, the power of injection signals, and the annealing noise on solver’s performance [Dutta et al. developed a phase-transition nano-oscillator network using VOformance ,45,46. Iformance . TherefoIn terms of novel nanospintronic oscillators, McGoldrick et al. theoretically analyzed the performance of the Ising machine based on the electrically coupled spin Hall nano-oscillators and established a theoretical model to describe the process of IL phenomenon based on spin Hall oscillators . They alThanks to the unique advantages of these oscillator-network-based Ising machines in solving intractable CO problems, this research area is receiving increasing attention. In this paper, the research progress in this field was surveyed for the first time. Firstly, the pros and cons of different types of oscillators were discussed. Secondly, the theoretical formulation of the Ising model was derived, and the mapping between the coupled oscillator networks under SHIL and the Ising machine for solving max-cut problem was discussed. Last, the implementation and performance of the existing oscillator-network-based Ising machines were summarized. Compared with other types of Ising solvers, oscillator-network-based Ising machines have shown their advantages in fast solving speed and low power consumption. Although many breakthroughs have been made in this direction, the oscillator-network-based Ising machines are still relatively primitive compared to well-developed digital systems in terms of performance, hardware cost, and complexity. Performance-wise, there is large room for improvement. For example, the dynamic properties of oscillators have a profound influence on the performance of the Ising machine. Thus, there is a constant pursuit for faster, more compact, and less noisy oscillators. Hardware cost-wise, CMOS-based oscillators are relatively expensive in terms of fabrication. A possible solution is to employ emerging memristive nanoscale oscillators. However, such oscillators may suffer from intrinsic stochasticity, making it difficult to keep the oscillating frequency of memristive nanoscale oscillators in the network precisely the same using conventional fabrication techniques. Such frequency deviation may significantly impact the system performance by degrading the success rate of hitting the global optimum. Hardware complexity-wise, the current coupling weights are either not reconfigurable  or bulky . Implementing programmable weights using nonvolatile memristors is a promising solution to reduce the physical system footprint and idle power, while adapting to different tasks. Furthermore, small-scale prototypical hardware demonstrations based on different types of oscillators have proved this concept, while large-scale implementations are still rare and mostly simulation based.As CO problems are quickly growing in the era of Big Data and Internet of Things (IoT), novel hardware Ising solvers are of great interest to both academia and industry. In the near future, coupled oscillator network–based Ising machines with the advantages of high integration density, low power consumption, and fast solving speed are likely to have a wide spectrum of applications in mobile edge devices and data centers, which are expected to have a transformative impact on the computing technology.