Delamination is one of the most common types of defects for carbon fiber reinforced plastic (CFRP) composites. The application of laser techniques to detect delamination faces difficulties with ultrasonic wave excitation because of its low thermal conductivity. Much of the research that can be found in the literature has only focused on the detection of a single delamination. In this study, aluminum foil was pasted onto the surface of the composite so that it was vulnerable to ablation and could acquire a usable signal. Using a fully noncontact system with laser excitation at a fixed point and a scanning laser sensor, the effects of different aluminum foil sizes and shapes on the wavefield were studied for the composites; we decided to use a rectangle with 3 mm length and 5 mm width for laser excitation experiments. Wavefield characteristics of the composite plates were analyzed with single- and multi-layered Teflon inserts. Taking the time window for standard ultrasonic testing as a reference, the algorithms for localized wave energy with appropriate time windows are presented for the detection of single and multiple defects. The appropriate time window is meaningful for identifying each delamination defect. The algorithm performs well in delamination detection of the composites with one or multiple Teflon inserts. In the aircraft and wind turbine industries, carbon fiber reinforced plastic (CFRP) composites are widely used as structural components thanks to their light weight and high strength. Since CFRP structures are fabricated by bonding multiple layers of laminates with resin, one of the most common types of defects is delamination. Such defects can grow and affect the mechanical properties and structural integrity. Delamination often occurs between the composed laminates, so it is invisible from the outside; therefore, non-destructive testing (NDT) of composites is essential and challenging.Ultrasonic testing is the most commonly used method for detecting defects in materials. Ultrasonic structural health monitoring (SHM) techniques ,2,3 applFor the detection of defects in CFRP composites, the use of laser techniques for ultrasonic wave excitation faces difficulties because of it low thermal conductivity. Relevant studies have shown that a laser beam with a wavelength near 3.2 μm is the best for generation of ultrasonic waves in CFRPs ,13, but There are many methods for delamination detection using the wavefield of a guided wave ,29,30,31In this study, aluminum foil was attached to the surface of the composite, and the effects of different aluminum foil sizes and shapes on the wavefield were studied for the composites. Taking the time window for standard ultrasonic testing as a reference , the algThis article is organized as follows. The scheme of fixed-point laser excitation and scanning laser sensor is one style of four excitation and sensing systems . Such a −6 nm(W/Hz)1/2. The system is manufactured by Intelligent Optical System Inc. . The measurement head was installed on the X-Y scanning guide rail. The X and Y direction and origin of the scanning region are shown in A Nd:YAG pulse laser is used to excite ultrasonic waves. The wavelength is 1064 nm, the pulse duration is 8 ns, and the maximum pulse energy is 55 mJ. Pulse laser was fixed on the optical platform and the energy (about 20 mJ) was set for excitation. The beam size needs to be adjusted by placing optical lens in front of the pulse laser. As the size of the laser beam becomes large, the amplitude of the direct waves and reflect waves increases when the peak energy density of the laser beam is constant . In thisIn the experiment, aluminum foil was attached on the excitation point to protect the surface from burning. The glue layer was made of acrylic acid with strong adhesion. The thickness of the bonded aluminum foil was 0.1 mm. Before the aluminum foil was pasted onto the composite, mark the excitation position. The aluminum foils with the 6 mm-diameter circle or the rectangle of 3 mm length and 5 mm width were studied for laser excitation. To get a high SNR, an average method of ten measurements was used at each sensing point. After the laser hits the aluminum foils several times in succession, it was also easy to cause ablation or breakdown. Therefore, the aluminum foil should be replaced regularly. In the experiment, the aluminum foil was replaced every two lines of laser scanning.The specimens were the 1.5 mm crossply CFRP made of T300 prepreg materials with a stacking sequence of [0/±45/90] s, as shown in x = 40 mm in the intact composite plate and three specimens. The total signal includes guided wave and acoustic signal. The acoustic wave here refers to the signal propagated through the air. The measurement time of the acoustic signal coincides with the time taken for the wave to travel a distance of a certain distance at a speed of 340 m/s in the air. In addition to the acoustical signal, the prominent component of guided wave is the A0 mode because it is the main component of the out-of-plane displacement. The guided wave velocities in the intact composite plate are different from one in three damaged composite plates. Additionally, the velocity of the guided wave becomes slower for specimen I, II and III, because the slope of the line is smaller compared to one in the intact composite.When the surface of a solid is illuminated by the laser beam, the thermal energy of the electromagnetic radiation is absorbed by the region and local temperature is produced. Then, the physical process of thermal expansion occurs and it produces stresses and strains in the solid. The sudden stresses in the illuminated region excite a transient displacement field. For the composite, the thermal conductivity is low and it is vulnerable to ablation. The aluminum foil with thickness 0.1 mm was pasted onto the surface of the composite to obtain the wavefield. In order to eliminate the influence of aluminum foil size and shape on the wavefield, the circular aluminum foil with 6 mm diameter and the rectangular one with 3 mm length and 5 mm width were prepared in the experiment, as shown in The aluminum foil was replaced every two lines of laser scanning and the acquired wavefield snapshots at 40, 60 and 80 μs by laser shot on two aluminum foils are shown in The guided wave excited by pulse laser on the composite plate is dispersive and the frequency or thickness affect the propagation velocity. The wavefield characteristics of the composite with delamination can be useful for the analysis of damage factor and it can be used for quantitative assessment of damage. In this section, wavefield snapshots of the composites with single and multiple delamination defects are analyzed in space–time and frequency–wavenumber domain.0 mode. A0 mode with higher amplitude has reached the delamination region at t = 50 μs, and the wavelength is reduced. This is because the delamination causes the incident waves to split and propagate independently through the upper and lower laminates [For specimen I, Teflon insert is between the fifth and sixth layer. aminates , and thet = 70 μs.For specimen II, Teflon insert is between the second and third layer. t = 50 μs, the guided wave mainly propagates in the composite with first delamination. The propagation distance for the second crest of the guided wave is farther than specimen I and it is almost the same as specimen II. The first Teflon insert is added between the second and third layer. At t = 70 μs, the guided wave has passed through two delamination defects. Additionally, the wave pattern anomalies are similar to specimen I. The second Teflon insert is added between the second and third layer.For specimen III, two Teflon inserts are added, one between the fifth and sixth layer, and the other second and third layer. xk and yk at a specific frequency. x-kyk spectrum at 98 kHz of the intact plate and three specimens. In the damaged plates, as shown in Frequency–wavenumber analysis methods can enable further analysis of wave modes and wavenumber distribution. By using the three-dimensional (3D) Fourier transform (FT), the acquired time–space wavefield is converted to a frequency–wavenumber field. The change in wavenumber can be extracted from the diagram of xk-yk spectrums at 98 kHz. The wavenumber 85.94 m−1 is in all four specimens. The wavenumber 93.75 m−1 is in specimen II and III. The wavenumber 117.2 m−1 is in specimen I and III. It has been known in advance that the delamination defect in specimen I is preset between the fifth and sixth layer and specimen II between the second and third. From the value of increased wavenumber 93.75 m−1 and 117.2 m−1, two delamination defects exist in specimen III, one between the fifth and sixth layer, and the other between the second and third.The main wavenumber that exits in the intact plate, specimen I, II and III, is shown in Taking the time window for standard ultrasonic testing as a reference, the algorithms of localized wave energy with appropriate time windows are present. The algorithm gathers wave energy anomalies caused by delamination defects at a certain frequency. First, experimental equipment is used to acquire the 3D measurement matrix. Then, the signal processing steps below are followed.(1) The 3D full-wavefield matrix Threshold is decided by the wavenumber of A0 mode at frequency (2) Wavenumber filter (3) An inverse Fourier transform is then applied, so that the wavefield with increased wavenumber components can be obtained by Equation (5). By observing the energy diagrams at different times, the appropriate time windows t = 40 μs to t = 70 μs, the phenomenon of energy anomalies is obvious, indicating that these are the appropriate time windows. The test result of specimen I using localized wave energy algorithm is shown in −1) indicates that the Teflon insert is between the fifth and sixth layer.In specimen I, the energy caused by increased wavenumber component is becoming larger when passing through the delamination, as shown in t = 40 μs to t = 70 μs can also be used for specimen II. The test result of specimen II using the localized wave energy algorithm is shown in −1) indicates that the Teflon insert is between the second and third layer. The result can show a successful identification of delamination location, size and depth.Similar to the phenomenon of specimen I, the energy caused by increased wavenumber component in specimen II is also becoming larger when passing through the delamination and it is less obvious, as shown in t = 40~90 μs with 10 μs intervals is shown in t = 40 μs to t = 60 μs. The energy discontinuity of the defect area at 70 μs is obviously different from previous time and the contour of the energy accumulation has changed, indicating that the guided wave has spread to the second defect. When the wavefield caused by the increased wavenumber has passed through the second defect, shown at 90 μs, some energy still remains inside the delamination. The second time windows are from t = 60 μs to t = 90 μs. t = 40 μs to 90 μs, and the result is not affected by the choice of time windows. The defect overlap region shows high energy anomalies. The imaging results of two defects are shown in For specimen III, there are two artificial delamination defects, one located between the fifth and sixth layer, and the other between the second and third. The energy distribution caused by the increased wavenumber at −1 at Combined with the wavefield characteristics in In this paper, aluminum foil was pasted onto the surface of the composite because of its low thermal conductivity. Using the fully noncontact system of fixed-point laser excitation and scanning laser sensor, the circular aluminum foil with a 6 mm diameter and the rectangular one with 3 mm length and 5 mm width were tested in the experiment to study the effects of their sizes and shapes on the wavefield. By comparing the wavefield snapshots obtained by two aluminum foils, the rectangle with 3 mm length and 5 mm width is a better choice because the replacement position has a small deviation from the preset.Taking the time window for standard ultrasonic testing as a reference, the algorithms of localized wave energy with appropriate time windows are present for the detection of single and multiple delamination defects. The composites with multiple delamination defects were first researched using the method of localized wave energy. Since the material properties of CFRP are not known in advance, two specimens with single delamination defects were used to study the wavenumber change related to material properties. For two specimens with different layered Teflon inserts, the propagation characteristics of the guided wave is different. For specimen I where Teflon is inserted between the fifth and sixth layer, the wavefield distortion is more obvious and the value of increased wavenumber is higher than in specimen II where Teflon is inserted between the second and third layer. Energy distribution caused by the increased wavenumber is becoming large when passing through the delamination. For specimen III, where two Teflon inserts are between the fifth/second and sixth/third layer, the frequency–wavenumber curve shows two increased wavenumber components appearing in two composites with a single delamination defect. The algorithms of localized wave energy with appropriate time windows are not only suitable for the detection of single delamination defect, but also for multiple delamination defects. For the detection of multiple delamination defects, the total localized wave energy distribution is not affected by the choice of time windows and the defect overlap region shows high energy anomalies. The appropriate time windows are meaningful for understanding the distribution of each delamination defect. They show a successful identification of the delamination location and size. Combined with the analysis of the wavefield and the value of the increased wavenumber, the position in the thickness direction can be determined.A fully noncontact inspection method for the composites was present through non-ablative way. The limitation of the method of pasting aluminum foil onto composite material is that aluminum foil would be ablated after a period of time and it needs to be replaced by another material. The algorithms of localized wave energy with appropriate time windows have only been verified in composites with two regular-shaped delamination defects, and it should be verified in irregular-shaped delamination defects. In the future, we will focus on the influence of different materials on the ultrasonic wavefield of CFRP as well as the detection of multiple and irregular delamination defects.


Modern cancer therapies increased the survival rates of acute myeloid leukemia (AML) patients tremendously. However, the complexity of the disease and the identification of new targets require the adaptation of treatment protocols to reduce side effects and increase benefit for the patients. One key regulator of leukemogenesis and chemotherapy resistance in AML is the Hedgehog (HH) signaling pathway. It is deregulated in numerous cancer entities and inhibition of its downstream transcription factors GLI translates into anti-leukemic effects. One major regulator of GLI is BRD4, a BET family member with epigenetic functions. We investigated the effect of ZEN-3365, a novel BRD4 inhibitor, on AML cells in regard to the HH pathway. We show that ZEN-3365 alone or in combination with GANT-61 reduced GLI promoter activity, cell proliferation and colony formation in AML cell lines and primary cells. Our findings strongly support the evaluation of the BRD4 inhibitor ZEN-3365 as a new therapeutic option in AML. Modern chemotherapeutics and newly developed personalized and targeted therapies enhanced the overall survival of cancer patients tremendously within the last decades. However, acute myeloid leukemia (AML) remains as a highly complex and heterogeneous disorder with still insufficient survival rates. This is especially true for elderly patients . Thus, nDue to the frequently occurring dysregulations of the Hedgehog (HH) signaling cascade in cancer, it has become an interesting target for therapeutic interventions. HH signaling plays a key role in embryogenesis and stem cell maintenance. It is activated by ligand binding to the cell surface receptor Patched (PTCH) that normally inhibits Smoothened (SMO), which mediates downstream signal transduction towards the GLI trancription factors GLI1-3) . Several . SeveraHowever, the Hedgehog cascade can also be activated non-canonically through a variety of pathways including the TGF-β, PI3K/AKT/mTOR, and RAS/RAF/MEK/MAPK cascades . Most ofIn the recent years, targeting epigenetic regulators had come into focus as therapeutic strategy in oncology. Bromodomain and extraterminal domain (BET) family members represent one group of epigenetic modulators regulating gene transcription through recruitment of regulatory complexes to acetylated chromatin regions and interaction with RNA polymerase II . The BETHere we examined the impact of BRD4 inhibition on GLI signaling in AML. Utilizing the new BRD4 inhibitor ZEN-3365, we could show a significant role of epigenetic regulation for AML cell survival. Furthermore, we have found that GLI inhibition via GANT-61 and the combinatorial blocking of BRD4 have a strong effect on AML cell proliferation and colony forming capacities. Thus, our data provide the evidence that the BET inhibitor ZEN-3365 should be evaluated as therapeutic option in AML.2 at 37 °C.The following human AML cell lines were used: MV4-11 , MOLM13 , HL60 , KG-1 , Kasumi-1 , OCI-AML3 . Verifications were performed with multiplex human cell line authentification test (MCA) from multiplexion. MV4-11, MOLM-13, HL60, and KG-1 were cultured in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum . The AML cell line Kasumi-1 was cultured in RPMI-1640 medium supplemented with 20% FBS. The AML cell line OCI-AML3 was cultured in α-MEM medium (Gibco) supplemented with 20% FBS. Primary AML cells were obtained after patient’s informed consent and approval of the study by the ethics committee . Cells were isolated from bone marrow using density gradient centrifugation and cultured as described elsewhere . All celProteins of MV4-11, MOLM-13, and OCI-AML3 cells were extracted using the trichloroacetic acid method. Protein extracts were applied to a 4–20% SDS-PAGE  followed by electrotransfer to nitrocellulose membranes . Blots were incubated with either rabbit anti-human GLI1  or mouse anti-human β-Actin  at 4 °C overnight. The subsequent incubation with the peroxidase-conjugated secondary antibodies  was followed by detection using ECL Western blotting detection reagents  and the FusionSL 4 3500 WL detection system .Stable GLI reporter AML cell lines were produced using lentiviral particles containing the firefly luciferase gene under the control of GLI1 and GLI 2 transcriptional response elements and as internal control the renilla luciferase gene under CMV promoter elements  followed by puromycin and hygromycin selection. Stable GLI reporter cells were treated with ZEN-3365 and the GLI promoter activity was measured after 24 h or at different time-points up to 84 h using the Dual-GLO Luciferase Assay Kit (Promega) and the Infinite F200 PRO reader (Tecan). The firefly luciferase-mediated GLI promoter activity was normalized to the renilla luciferase-mediated CMV promoter activity.AML cell lines or primary AML blasts were cultured with different concentrations of the GLI inhibitor GANT-61 -1,3-pyrimidinediyl]bis(methylene)]bis; Tocris Bioscience) or the BET inhibitor ZEN-3365 (Zenith Epigenetics) either alone or in combination or dimethyl sulfoxide  as solvent control. Cell numbers were determined after 3 and 7 days using the cell viability analyzer Vi-Cell TM XR (Beckman Coulter).AML cell lines were seeded with GANT-61, ZEN-3365, or their combination in Methocult . DMSO was used as solvent control. The number of colonies was counted after 7 days using an inverted microscope  and normalized to DMSO control.zed by Welch’s t-test (SPSS Inc.). A P value less than 0.05 was considered to be statistically significant.Figures were created with GraphPad PRISM version 6.2. Data was statistically analyFirst, we investigated the impact of the BET bromodomain inhibitor ZEN-3365 on the expression on the HH transcription factors GLI using immunoblotting and GLI reporter assays. For western blot analysis, AML cell lines MV4-11, MOLM13, and OCI-AML3 were incubated with 100 or 200 nM ZEN-3365 for 3 days. ZEN-3365 treatment led to a dose-dependent decrease of the GLI1 protein expression in all three cell lines Fig. . Next, wSo far, direct GLI antagonists such as GANT-61 have not entered clinical stages yet. Nevertheless, combination therapies might represent a promising clinical strategy. Therefore, we investigated whether the combination of GANT-61 with ZEN-3365 augmented the inhibitory effect on the HH signaling using the GLI reporter cell lines. The AML cell lines MV4-11, MOLM13, Kasumi-1, and HL60 were treated with 2500 or 5000 nM of GANT-61 and 50 or 100 nM of ZEN-3365, the combinations of both substances or the solvent control. The GLI reporter activity was measured after 24 h. The combination of GANT-61 with ZEN-3365 resulted in a significantly decreased GLI reporter activity in all analyzed cell lines. While this effect was only observed for the low GANT-61 concentration in MV4-11 Fig. , the comNext, the influence of the ZEN-3365 and GANT-61 combination on the growth and survival of AML cells was investigated in proliferation assays. The combination of 5 or 10 µM GANT-61 with 100 or 200 nM ZEN-3365 led to a significantly reduced growth of the AML cell lines MV4-11, HL60, KG-1, MOLM13, Kasumi-1, and OCI-AML3 in comparison to GANT-61 treatment alone Fig. . FurtherSubfractions of AML cell lines have the potential to develop colonies in semisolid methylcellulose medium, comparable to hematopoietic stem and progenitor cells. The number of colonies is proportional to the size of the subfraction with progenitor capacities. Since HH signaling is regulating the development, maintenance, and expansion of leukemic stem cells , we analIn the last decade, numerous new treatment strategies for AML were developed, representing a significant advantage in the therapeutic options for leukemic patients. However, emergence of resistant clones enables the clinical recurrence of the disease, leading frequently to fatal outcome for the individual patient . A key dHere, we show for the first time that the BRD4 inhibitor ZEN-3365 can reduce the activity of the Hedgehog signaling cascade through inhibition of the GLI transcription factors in AML cells. Furthermore, the combined treatment of AML cells with ZEN-3365 and GANT-61 resulted in significantly reduced cell growth and colony formation capacity compared to the single-agent treatment, leading to the conclusion that BRD4 inhibition with ZEN-3365 represents a promising strategy for rational combinatorial AML therapy.BRD4, a part of the BET protein family, is known as an epigenetic reader for acetylated histones and regulates global transcription . In AML,Previous studies have shown that BET inhibitors such as OTX015 attenuate the mRNA and protein expression of several oncogenes including c-MYC, BCL-2, or CDK6 on the one hand and induce cell differentiation via induction of p21 or HEXIM1 –31. At pTherefore, several combinations of BET inhibitors with other targeted therapies have been investigated. Saenz et al. described synergistic effects when combining BET inhibitors with the Jak2 inhibitor ruxolitinib in post-MPN sAML cells , 31. FurIncreased anti-leukemic effects have also been observed upon combination of BET inhibitors with FLT3 kinase inhibitors in FLT3-mutated AML cells . In our In summary, AML is a heterogenous disease with aberant regulations of numerous pathways referable to genetic or epigenetic abnormalities. Among them, the HH signaling pathway represents a promising target for cancer therapy, especially the downstream transcription factors of the GLI family. Our results emphasize that the specific inhibition of BRD4 by ZEN-3365 leads to a decrease of GLI expression and a downregulation of HH signaling, resulting in a decrease of proliferation and colony formation potential. Future in vivo studies have to confirm our findings, proving the promising anti-leukemic potential of ZEN-3365 as a single drug but also in combination with other chemotherapeutic applications.


Growing evidence show that the Ca2+ entry mediated by mechanosensitive ion channels is also precisely regulated by a variety of key proteins which are distributed in the cell membrane or endoplasmic reticulum. Recent studies have revealed that mechanosensitive ion channels can even physically interact with Ca2+ regulatory proteins and these interactions have wide implications for physiology and pathophysiology. Therefore, this paper reviews the cross-talk between mechanosensitive ion channels and some key Ca2+ regulatory proteins in the maintenance of calcium homeostasis and its relevance to cardiovascular health and disease.Mechanosensitive ion channels are widely expressed in the cardiovascular system. They translate mechanical forces including shear stress and stretch into biological signals. The most prominent biological signal through which the cardiovascular physiological activity is initiated or maintained are intracellular calcium ions (Ca For example, dietary calcium supplement and its retention reduce cardiovascular response to sodium stress in Black people [2+ intake in the diet, and whether the relationship between Ca2+ intake and cardiovascular disease risk is J- or U-shape [2+ is pivotal in maintaining the functions of endothelial cells, smooth muscle cells (SMCs) and cardiomyocytes. It controls the contraction or relaxation of arteries and heart and regulates blood pressure and cardiac functions [Despite decades of efforts, cardiovascular disease is still the number one killer in the world. The latest data show that one in six elderly people dies of cardiovascular disease. In 2019, ischemic heart disease and stroke were reported to be the leading causes for disability in the age groups of 50–74 and 75 years or above [ U-shape . Ca2+ isunctions ,7. 2+ via various proteins, and thus maintains Ca2+ homeostasis. Plasma membrane Ca2+ ATPase (PMCA), voltage-gated calcium channel (VGCC), Na+/Ca2+ exchanger (NCX), and Orai have been identified as the main calcium regulatory proteins on the cell membrane [2+ reservoir in the cell also contains some calcium regulatory proteins. Such proteins in the ER include stromal interaction molecules (STIM), inositol 1,4,5-trisphosphate receptor (IP3R), and sarco/endoplasmic reticulum calcium-ATPase (SERCA). All these proteins are critical in controlling cell functions, such as growth, migration, apoptosis, and metabolism [2+ regulatory proteins are also regulated by intracellular Ca2+ level.The cell membrane controls the balance between intracellular and extracellular Camembrane ,9,10,11.tabolism ,13,14. A2+, in response to the stimulus from shear stress of blood flow [Mechanical forces are crucial for cardiovascular functions and therefore the discoveries of mechanosensitive ion channels represent a major breakthrough in understanding cardiovascular mechanobiology. In particular, the endothelium in the cardiovascular system is subjected to regular mechanical stimulus evoked by blood flow. The discovered mechanosensitive ion channels including Piezo channels and transient receptor potential (TRP) channels can conduct the entry of cationic ions, particularly Caood flow ,16. Bothood flow . 2+ regulatory proteins sensitize any subtle change of intracellular Ca2+. It has emerged that these proteins can cross-talk to mechanosensitive ion channels -binding domains 1 and 2 [2+ oscillation [2+ extrusion usually leads to smooth muscle relaxation, so that the vascular tension is reduced [+ or high positive membrane potential, NCX works in a reverse mode to evoke Ca2+ influx instead of the typical extrusion [+ and membrane potential [NCX mainly works in the forward mode, which uses the electrochemical gradient driven by Naa+:1Ca2+ . There aa+:1Ca2+ . Structu 1 and 2 ,22, whicillation ,23,24. T reduced . Howeverxtrusion ,27. Inteotential , suggestotential .2+ homeostasis and initiate molecular and cellular remodeling in various tissues, which is related to hypertension and heart failure. Inhibitors of NCX can improve myocardial function in patients with heart failure and bring the Ca2+ hyper-responsiveness back to normal in vascular SMCs from hypertensive patients [2+ in cultured endothelial cells, indicating that the activation of NCX reverse mode can play an important role in mediating the acetylcholine-induced vasodilation in resistance arterial endothelial cells [NCX regulates many essential physiological events, such as muscle excitation-contraction or blood pressure regulation ,30. The patients ,31. NCX1patients . The incpatients . Furtherpatients , which spatients . In the al cells .2+)-selective ion channel in the plasma membrane formed by four transmembrane domains. Orai family includes Orai1, Orai2, and Orai3 [2+ entry (SOCE) [2+ channels fine tune Ca2+ entry in both cardiomyocytes and SMCs, and they are activated once the Ca2+ store in ER or sarcoplasmic reticulum (SR) is depleted or the level of cytosolic Ca2+ is lowered, thereby facilitating agonist-induced Ca2+ influx. It has also been suggested that STIM1, Orai and TRPC channels could form the molecular basis of SOCE in some types of cells and their intricate interactions control the entry of Ca2+ into cells to regulate numerous physiological processes [2+ store is depleted, STIM1 protein can move to the plasma membrane and activate Orai and TRPC channels, allowing extracellular Ca2+ to enter the cytoplasm [2+ oscillations induced by physiological receptor activation, while Orai1 is not necessarily involved in this process. However, the binding of Orai2 and Orai3 to Orai1 could expand the sensitivity range of receptor-activated Ca2+ signals [Orai is a highly  ,34. Storrocesses . Orai1 irocesses ,36. Onceytoplasm ,36. Orai signals . 2+ entry and drives the fibroproliferative gene program [2+ entry and is a potential therapeutic target for the treatment of hypertension [2+ signaling pathways have emerged as important regulators of smooth muscle, endothelial, epithelial, platelet, and immune cell remodeling [2+-permeable Orai channel is also important for endothelial cell proliferation and angiogenesis [Orai plays a critical role in regulating cardiovascular function in both health and disease ,38,39. O program . Orai fartension . Most camodeling . Ca2+-peogenesis ,47. In togenesis . ApoE knogenesis . The migogenesis . All theogenesis ,39,50. 2+ in ER and makes appropriate response through conformational change to regulate Ca2+ homeostasis [2+ and transitions to an open state when Ca2+ in the ER lumen is decreased [2+-sensitive domain in the STIM N-terminal senses Ca2+ level of ER ranging from 100 to 400 µM [2+ influx [STIM is a single pass transmembrane protein residing in the ER. It contains two homologous proteins, STIM1 and STIM2 ,52. The eostasis ,53. STIMecreased . The Ca2o 400 µM , and the+ influx . As ment+ influx ,39,50.2+ signaling generates the Ca2+ microdomain that regulates myofilament remodeling and activates pro-hypertrophic factors locally, and as a consequence, pathological cardiac hypertrophy is induced [2+ signaling is also involved in thrombosis. The aggregation of platelets at the site of thrombosis requires the increase of intracellular Ca2+ concentration. STIM1 is involved in this process through maintaining a high Ca2+ concentration. In addition, STIM1 stabilizes the thrombus by promoting the expression of phosphatidylserine in plasma membrane [STIM is involved in both the cardiac physiological functions and the cardiac disease development. STIM is essential for the maintenance of myocardial contractility, and its knockout leads to a reduction in left ventricular contractility. STIM1 is expressed more abundantly in early cardiomyocytes than in somatic cells. Cardiomyocyte-STIM1-specific knockout mice exhibit dilated cardiomyopathy and cardiac fibrosis with increased stress biomarkers and altered organelle morphology in the heart, suggesting that STIM1 can regulate myocardial development and heart function . However induced . The STImembrane ,58. The membrane . The celmembrane ,60. Oxidmembrane ,60. The membrane .STIM2 hamembrane . 2+ [2+ release. The overexpression of IP3R enhances ER Ca2+ depletion, which reduces ER intraluminal Ca2+ concentration in the vicinity of STIM1 and then activates Orai ion channels [2+ in ER, IP3Rs empty Ca2+ stored in the ER and activate Ca2+ inward flow [2+ from ER to mitochondria. Each isoform of IP3R can mediate this contact and Ca2+ transport, but IP3R2 is the most efficient one in delivering Ca2+ to mitochondria from ER [2+ accumulation through physical interaction with IP3R [IP3R is a tetrameric channel consisting of four glycoproteins in the ER or SR. So far, three types of IP3R channels have been identified: IP3R1, IP3R2, and IP3R3 . IP3R ha2+ . All the2+ . IP3R ischannels . In respard flow . IP3R al from ER ,69. The ith IP3R , which i2+ depletion in ER. The association of IP3R-STIM1 increases Ca2+ inward flow [2+ released from the ER. The deletion of IP3Rs reduces the contractile response to vasoconstrictors and even reverses the pathological states [2+ release ensures the integrity of cardiac excitation-contraction coupling, which forms the basis of the heartbeat [2+ homeostasis, which is closely related to congenital diseases, increased risk of arrhythmia, decreased contractility, or heart failure related arrhythmias [Under physiological conditions, IP3R signal controls the contraction, migration, and proliferation of vascular SMCs. However, under the pathological conditions, IP3R is involved in the development of atherosclerosis and hypertension . IP3R isard flow . When IPl states . In the eartbeat . The dyshythmias . The exphythmias . 2+ transporter located on SR/ER and is mainly responsible for the transport of cytoplasmic Ca2+ back to SR/ER. SERCA isoform is encoded by SERCA1, SERCA2, or SERCA3 genes. Each isoform may have differential roles in different tissues or cells [2+ binding sites, while N, P and A located in the sarcoplasm are responsible for ATP hydrolysis [2+ to the ER lumen in exchange for 1 H+ [SERCA is a Caor cells . There adrolysis . Each ATfor 1 H+ . SERCA2a2+ into SR/ER mediated by SERCA2 is necessary for the relaxation of cardiomyocytes and blood vessels. The disruption of SERCA2 activity leads to ER stress and cardiovascular disease [2+ at low cytoplasmic Ca2+ concentration [2+ by SR, which would improve the diastolic dysfunction of myocardium, and result in strong antiarrhythmic effect [S-glutathiolation of the amino acid residue Cys674 (C674) is key to the increase of the activity in SERCA2 under physiological conditions [The influx of Ca disease . Hormonentration . The downtration . The decntration . Activatc effect ,82. Our nditions ,84, but nditions ,85,86,87nditions ,89, incrnditions , aggravanditions . All the2+ regulatory proteins in the cardiovascular system, as shown in Currently, there are some ongoing clinical trials for the drugs specifically targeting these Ca2+ [Piezo channels are cation channels activated by mechanical force. They have two subtypes: Piezo1 and Piezo2. Piezo1 is the major one distributed in cardiovascular system. Upon mechanical stimulus, Piezo channels convert the mechanical signals into biological signals to participate in cellular physiological events. When Piezo1 channels sense shear stress in cardiovascular system, they conduct the inward flow of cationic ions, especially Ca2+ . Piezo1 2+ ,100. Sin2+ ,101,102.2+ ,105,106.2+ and Na+. The latter can depolarize endothelial cells. In this case, depolarization in the endothelium can be transduced to the vascular SMCs in which voltage-gated Ca2+ channels could be activated to drive vasoconstriction [The systemic knockout or specific endothelial disruption of Piezo1 in mice severely disrupts angiogenesis and results in embryonic death within days of cardiac pulsation . Endothetriction . Such vatriction . The imp2+-permeable, but their Ca2+ permeability varies [2+ is much greater than that of TRPC6 [2+ [2+ stores [2+ channels with a contractile phenotype independently of Orai1 in vascular SMCs [Based on amino acid sequence, TRP channels can be classified into seven subfamilies: TRPC, TRPV, TRPM, TRPA, TRPP, TRPML, and TRPN. They share some common structural features including six transmembrane domains and a pore lining between the fifth and sixth transmembrane domains ,112. Mosy varies . For exaof TRPC6 , while TRPC6 [2+ . As mechRPC6 [2+ . Meanwhi+ stores ,117. Reclar SMCs . 2+ signaling, which is relevant to physiological and pathological functions in the heart [2+ entry following stimulation of shear stress mediates the vasodilatory response in endothelial cells [2+ entry [2+ inward flow and activates Ca2+-dependent pathways, which would lead to diastolic-mediated heart failure in cardiomyocytes [2+ concentration under physiological conditions, which then induces nitric oxide release and vasodilation [TRP channels control acute hemodynamics and regulate cardiac remodeling through the modulation of Cahe heart . Shear sal cells ,121. TRP2+ entry . TRPA1 cmyocytes ,124. TRPmyocytes . TRPA1 cdilation . TRPA1 hdilation ,128. FroClinical trials for drugs specifically targeting these mechanosensitive ion channels are being run in the cardiovascular system, as shown in 2+ regulatory proteins in the regulation of intracellular Ca2+. To date, many scientific reports have shown that they both can cross-talk to each other so that intracellular Ca2+ signaling can be precisely coordinated in cardiovascular health and diseases (as listed in 2+ concentration. There are many similarities between mechanosensitive ion channels and Ca2+ stores by SERCA RNAi results in severe reproductive defects [2+ measurement experiments showed that nearly 72% of the increase in Ca2+ is from the mechanically activated Piezo1 while 28% comes from the Ca2+ store depletion due to SERCA inactivation [2+ signaling, albeit rather differently, in these diseases.Pizeo and SERCA have been found to cross-talk to each other through physical interaction in endothelial cells. SERCA2 inhibits Piezo1-dependent endothelial cell migration through a 14-residue linker region . Mutatio defects , reiterativation , suggest2+ by triggering the Ca2+ entry mediated by TRP-formed SOC channels. Pulmonary hypertension, as an example, is closely related to both NCX and TRP proteins. Dysfunction of either protein disrupts the Ca2+ homeostasis, which changes contraction, proliferation, and migration in SMCs of pulmonary artery. Gradually the hypertension is developed [NCX is involved in the regulation of intracellular Caeveloped ,137. 2+ homeostasis. Co-immunoprecipitation experiments show the co-localization of TRPC3 and NCX1, suggesting that TRPC3 cross-talks to NCX1 through physical interaction in the Ca2+ regulation [2+ signaling is significantly reduced in cells with negative TRPC3 expression [2+ entry is induced. The increased intracellular Ca2+ concentration in turn activates NCX to extrude the cytoplasmic Ca2+ [2+ entry by cooperatively mediating the hypoxia-induced increase in intracellular Ca2+ and then causing vasoconstriction [The cross-talk between TRP channels and NCX can regulate contractile dysfunction and spontaneous ectopic . Growinggulation . Glutathpression . In addimic Ca2+ . NCX andtriction . Howevertriction . Howevertriction . TRP and Orai are both located in the cell membrane . The int2+ change. Upon depletion of Ca2+ stores in the ER, STIM1 can form a dimer and then activate TRPC channels. However, the TRPC channel function is not always dependent on STIM1 and therefore the interaction between STIM1 and TRPC may be absent in some cells [2+ signal to regulate another protein. It has been observed that TRPC1, TRPC2, or TRPC4 can interact directly with the ezrin/radixin/moesin domain of STIM1 [2+ stores. In addition, the Orai1-mediated Ca2+ entry into the cytosol can also trigger TRPC1 interaction with STIM1. In this case, each TRPC1 tetramer binds to two STIM1s and the 639DD640 of TRPC1 C-terminus is then directly interacted by the 684KK689 region of STIM1 C-terminus [2+ store depletion. When the EF-hand of STIM1 is mutated, it spontaneously aggregates with TRPC1 and then activates TRPC1 [TRP can cross-talk to STIM1 through physical interaction between them . The intme cells . When thof STIM1 . Interacof STIM1 . Fluoresterminus . Caveoliterminus . Though terminus . The intes TRPC1 . The intes TRPC1 ,145,146.2+ depletion is followed by activation of TRP channels to promote Ca2+ influx, which supports the notion that IP3R cross-talks to TRP channels to regulate intracellular Ca2+ [2+-dependent manner. Also, IP3R has differential affinity to various TRP member. This affinity ranges from 10 nm for TRP2 to 290 nm for TRP6 [The IP3R-initiated Calar Ca2+ . This islar Ca2+ . The intfor TRP6 . Prior sfor TRP6 . Co-immufor TRP6 . The coufor TRP6 ,148. Thefor TRP6 ,164.2+ signaling compensatory mechanism in which the transcription of TRPC4 and TRPC5 are increased after SERCA silencing has been proposed to be responsible for cardiac development and hypertrophy [2+ influx through TRPC6 [2+ can be compensated by TRP channels when the Ca2+ in the store is insufficient [2+ concentration during cardiac pulsation is a consequential result of temporal cross-talk between SERCA and TRP. Thus these proteins could be promising targets in treating cardiac disease [Though the direct physical interaction between TRP channels and SERCA has not yet reported, numerous studies have shown that they both can cross-talk to each other. SERCA pump inhibitor thapsigargin blocks the activation of TRPC7 channel by diacylglycerol or the activation of TRPC3 channel . The SERertrophy . A similgh TRPC6 . The expgh TRPC6 . SERCA afficient . However disease .2+ signaling pathway in both physiology and pathology require the dynamic cross-talk between mechanosensitive ion channels and Ca2+ regulatory proteins. Ca2+ regulatory proteins are diverse and widely distributed in the cells, mostly on the cell membrane or in ER. Notably, certain pathogenic factors are largely activated by changes in intracellular Ca2+ concentrations, and pharmacological modulation of these proteins has emerged as an attractive strategy to prevent cellular dysfunction and tissue remodeling, and eventually to treat cardiovascular disease. Mechanosensitive ion channels play a dominant role in endothelium-dependent vascular development and blood pressure regulation, vascular tension control, angiogenesis and the atherosclerotic process. When either mechanosensitive ion channel or Ca2+ regulatory protein is activated, a Ca2+-related pathway is often triggered and then the cross-talk between both proteins is frequently initiated. In addition, it is interesting to observe that in some cardiovascular diseases, such as hypertension, atherosclerosis or aneurysms, mechanical stress or shear stress is altered, and inherent activation of the mechanosensitive ion channels are reported. Accordingly, Ca2+ regulatory proteins are communicated by the channels on a temporal and spatial basis. In some cells, when mechanosensitive ion channel expression is defective or disrupted, the inability to sense the mechanical stimuli directly affects the regulation of downstream Ca2+-related pathways, which leads to defects in some physiological functions. The cross-talk between these mechanosensitive ion channels and Ca2+ regulatory proteins in the cardiovascular system is therefore not only relevant to the maintenance of health but is also involved in the development of diseases.The multifaceted effects of the Ca2+ regulatory proteins in cardiovascular health and disease through the regulation of Ca2+ related pathways reveals an apparent cross-talk between both types of proteins. The cross-talk occurs with either physical interaction or functional coupling. Especially, the physical interaction in such cross-talk has emerged to be important for the cardiovascular physiology or pathology. We have summarized some interactions between mechanosensitive ion channels and Ca2+ regulatory proteins. The study of protein–protein interaction depends on FRET, TIRFM, and immunoprecipitation techniques. With the comprehensive application of protein-protein interaction methods and the development of electron microscopy technology, we expect that more interactions between mechanosensitive ion channels and Ca2+ regulatory proteins will be discovered. In addition, the functional coupling in the cross-talk between the channels and Ca2+ regulatory proteins will also be unraveled with functional assays combined with appropriate animal models. These studies will certainly be useful in identifying novel therapeutic approaches for treatment of cardiovascular diseases.The summary of recent articles on the role of mechanosensitive ion channels and Ca


Herein, we propose that 2D Ca2+ exchange diffusion on the negatively charged phospholipid surface lining nano-junctions participates in guiding Ca2+ from its source (channel or carrier) to its target (transport protein or enzyme). Evidence provided by in vitro Ca2+ flux experiments using an artificial phospholipid membrane is presented in support of the above proposed concept, and results from stochastic simulations of Ca2+ trajectories within nano-junctions are discussed in order to substantiate its possible requirements. Finally, we analyze recent literature on Ca2+ lipid interactions, which suggests that 2D interfacial Ca2+ diffusion may represent an important mechanism of signal transduction in biological systems characterized by high phospholipid surface to aqueous volume ratios.Nano-junctions between the endoplasmic reticulum and cytoplasmic surfaces of the plasma membrane and other organelles shape the spatiotemporal features of biological Ca The ultrastructure of the NJ optimizes SERCA Ca2+ uptake by reducing leakage into the bulk cytoplasm [2+ entry via rNCX/SERCA was indeed sufficient for replacing the Ca2+ released by periodic opening and closing of inositol 1,4,5-trisphosphate receptors (IP3Rs) during the activation of asynchronous cytoplasmic Ca2+ waves that stimulate contraction [2+ target size on SERCA of approximately 20 nm2. This is 2500 times larger than the area occupied by the dehydrated Ca2+ with a diameter of 1Å. Assuming that short-range local electrostatic forces of attraction between fixed negatively charged binding sites on the SERCA macromolecule and the positively charged Ca2+ would increase the effective target size to an area several times larger than the size of the non-hydrated Ca2+, it would still be orders of magnitude smaller than 20 nm2. Therefore, in order to achieve effective functional transfer of Ca2+ from NCX on the PM to SERCA on the SR, it appears that an additional, yet ignored, mechanism is operative for supporting the linkage between Ca2+ signaling elements (sources and sinks) within NJs. To this end, all available computational-modeling data prompt us to conclude that, by itself, a 3D random walk of Ca2+ between its sources and sinks on the two closely apposing membrane surfaces may be insufficient for efficient NJ Ca2+ signaling. Thus, we propose that, in addition to the three NJ-related mechanisms mentioned above, a fourth requirement consists of 2D exchange diffusion of Ca2+ on the targeted phospholipid membrane surface.NJs have been defined as cytoplasmic sub-compartments where membranes of different organelles appose each other within the nanometer scale. Typically, limiting membranes are separated by 20 nm or less and the specialized signaling function has been shown to fail at separation distances greater than 50 nm ,7,8. Theytoplasm . By empl2+ can move rapidly through negatively charged solid lattices, such as fluorapatite, by the process of exchange diffusion [2+ movements through narrow aqueous passages lined by PLs. Its salient feature is that it supports net transfer of Ca2+ through relatively long PL-lined pores at a much faster rate than would be possible for free diffusion within the limited adjoining aqueous phase. The mechanism that was presented to explain Ca2+ transport through this solid ion-exchange membrane involves the association of Ca2+ with a negatively charged phosphate or carboxyl group of the PL surface on the cis-side of the membrane, followed by transfer of Ca2+ within a 2D matrix of similar sites, constituted by pore-lining PL layers, and a final step of dissociation of Ca2+ from the negatively charged PL head groups on the trans-side of the membrane. The rate of this PL-mediated transport of radioactive-labeled Ca2+ through the membrane decreased by removal of Ca2+ from the buffered solution on the trans-side of the membrane. Paradoxically, further removal of the remaining Ca2+ from the trans-solution by the addition of the soluble but non-permeant chelator EDTA increased the rate of PL-mediated net Ca2+ transport across the PL-lined Millipore filter by more than one order of magnitude. The mechanism proposed to explain the latter observation is that dissociation of Ca2+ from PL head groups on the trans-side of the membrane is rate-limiting, and the addition of a freely diffusible, impermeant Ca2+ binding site on EDTA to the trans-side of the flux chamber facilitates the dissociation of PL-bound Ca2+. Once Ca2+ transported from the cis-side of the flux chamber has been chelated by EDTA, which is dissolved in a large volume of buffered solution on the trans-side, it cannot rebind to the PL membrane, but is replaced by the next Ca2+arriving from the cis-side. In this model system, electroneutrality is preserved by the movement of Mg2+ and monovalent cations in the opposite direction. The mechanism envisioned for the transfer of Ca2+ from the PL membrane to EDTA involves an intermediary step of partial dissociation from the negative PL site and simultaneous association with a carboxyl group of EDTA. We propose, herein, that a similar mechanism is involved in the transfer of Ca2+ bound to PL head groups of the membranes lining the NJ to its biological target site on the Ca2+-receptor protein.It is well established that Caiffusion . An analiffusion ,12,13. T2+ transport in the model described above with biological PL-lined nano-spaces, some striking parallels become obvious. For example, rod outer segments of the bovine eye feature a 15 nanometer-wide cytoplasmic phase between the intercalated discs, which stretch for several microns [2+ released during stimulation, 90% to 99% was bound to the PL head groups of membranes lining the nano-spaces between the intercalated discs, which is similar to the high ratio of bound/free Ca2+ in the above model membrane. Although historically it has been accepted that such binding slows diffusion due to a drastic decrease in freely diffusible Ca2+ in the aqueous phase, it is also possible that PL-bound Ca2+ within the water–PL membrane interface of the nano-space continues its trajectory by the mechanism of 2D Ca2+ exchange diffusion. We, therefore, propose that the mechanism of 2D Ca2+/Mg2+, K+ exchange diffusion at the aqueous–phospholipid interfaces of NJs facilitates targeting Ca2+ receptors located on organellar membranes and the inner PM. When we compare the artificial PL-mediated Ca microns . The lip microns . In this2+ enters the NJ via the reverse mode of NCX located in the junctional domain of the PM. It will then perform a 3D random walk and when it hits the negatively charged PL-membrane boundary of the junctional nano-space then proceeds along the lipid¬–water interface by a series of steps of reversible binding to negatively charged oxygen molecules for some variable time before being released back into the aqueous phase to resume its 3D random walk. After a number of such cycles, Ca2+ is envisioned to hit the ER-membrane surface in the proximity of its target protein, in this case SERCA, which can then be reached more effectively by 2D surface exchange diffusion than would be expected if it depended solely on 3D diffusion in the aqueous phase.Returning to the example of NCX-mediated SR refilling in vascular smooth muscle, we propose that Ca2+ surface exchange diffusion, described for the artificial membrane [2+ refilling [2+ in reaching its target by two means: (i) by augmenting the rate of Ca2+ movement towards its target and thus enhancing the speed and intensity of the signal and (ii) by decreasing the loss of Ca2+ at NJ edges, further increasing the speed as well as the selectivity of Ca2+ signaling.A simplified model for this mechanism, which incorporates the PL Camembrane , with thefilling , is illu2+ exchange diffusion, specifically for its role in junctional Ca2+ signaling. On the other hand, non-Brownian interfacial diffusion has been intensively studied in physical chemistry, using state-of-the-art techniques of single-molecule tracking and dynamic modeling [2+ signaling, it is particularly relevant that the ultrastructure of the endoplasmic reticulum as well as its appositions to other organelles exhibit features such as tortuosity, binding affinity and high surface to aqueous volume ratios, a combination of which is a prerequisite for elementary processes of intermittent surface diffusion.Due to the novelty of this concept, there are currently no experimental or computational tests reported with respect to the biological implications of 2D Camodeling . A complmodeling . With re2+ interactions with lipid bilayers has also been extensively studied and shows that Ca2+ binding can lower negative surface charge [2+ binding to PLs require millimolar concentrations of Ca2+, such as those observed in extracellular space and the ER lumen [2+ concentrations, which are three orders of magnitude lower than the cytoplasmic concentration of Mg2+ and, at least under resting conditions, will contain minimal bound Ca2+. Nonetheless, phosphate, carboxyl and carbonyl groups are effective Ca2+ binders at low concentrations and will buffer Ca2+ during transient elevations typically associated with Ca2+-signaling events. Recent studies in model membranes suggest that Ca2+-induced PIP2 clustering and Ca2+ coordination within a hydrogen-bond network formed by PL head groups occur at membrane surfaces at micromolar divalent cation concentrations [2+ rises. The physical chemistry of Cae charge ,16, cause charge  and prome charge . The strtrations . Hence, 2+ exchange diffusion, which could promote efficient coupling between Ca2+ sources and Ca2+ targets during activation. A parallel mechanism of lateral diffusion of Ca2+ tightly bound to PLs in a monolayer cannot be excluded at this time, although the lateral mobility of free PLs in cell membranes is limited to a diffusivity of approximately 10 µm2/s, which is further slowed down in complexes with divalent cations [2+ signaling complexes in the context of PL signaling [2+ sources to downstream targets via 2D surface Ca2+ exchange diffusion. Support in favor of such a membrane-delimited transfer of Ca2+ signals is derived from current concepts of tight interactions between membrane phospholipids and Ca2+ transport proteins and the well-documented functional relevance of these interactions. In this context, it is intriguing that PLs are a prominent component of the “annular lipid” shells, which typically surround transmembrane proteins involved in Ca2+ handling [2+-signaling complexes by crystallography and single-particle cryo-EM studies identified “non-annular” lipids, including PLs, which protrude into fenestrations and crevices of integral membrane proteins to govern Ca2+ transport and Ca2+ regulatory or sensory functions [2+ within the transporter [2+ from the membrane surface through hydrophilic clefts towards the central binding pocket [2+ to the transporter via 2D exchange diffusion. We hypothesize that the unique architecture of NJs, along with the organization of membrane proteins within a specialized lipid environment, allow for exceptionally efficient and specific transfer of Ca2+ between sources and target sites. This proposed concept of NJ Ca2+ transfer combines 3D random walk of Ca2+ within the junctional nano-space with a process of 2D interfacial surface diffusion that feeds Ca2+ into the acceptor and guidance machinery of target molecules.In essence, the negatively charged cytoplasmic surface of NJs provides all the features required for 2D surface Ca cations  and pres cations . On the ignaling , may prohandling ,23,24. Thandling . Recent unctions ,27,28,29nsporter . Interesg pocket . It is t2+ ions at the surface of cell membranes by bridging the head groups of negatively charged lipids and (ii) the displacement of monovalent cations and Mg2+ at lipid headgroups by Ca2+. These molecular principles have been demonstrated by experiments in mammalian cells as well as by simulation [2+ by exchange diffusion within a phospholipid matrix, has been demonstrated in cell-free experimental systems [2+ exchange diffusion hypothesis by computational as well as experimental approaches and, thereby, promote a conceptional advance towards understanding NJ Ca2+ signaling.Hence, we propose a novel signaling concept, for which individual mechanistic steps appear plausible in view of current knowledge on the interaction of cations with biological membranes. Key steps include (i) the accumulation of Camulation ,17,31. A systems . We expe2+ exchange diffusion on PL membranes containing functional Ca2+ receptors is expected to enhance the speed and efficacy of Ca2+ signaling, it holds the promise of improved therapy. The specific example described in this communication focuses on refilling the ER/SR during rapid oscillatory Ca2+ release stimulating vasoconstriction. This mechanism has also been demonstrated in human blood vessels [This Special Issue on “Calcium Signaling in Human Health and Disease” presents burgeoning research in channelopathies and aberrant ionic signaling caused by genetic mutations, chronic disease and aging. Since the proposed process of Ca vessels  and decl vessels .2+ homeostasis resulting in a drop of luminal Ca2+ is a hallmark of ER stress in chronic disease. Upregulation of rNCX to promote ER refilling, by a mechanism similar to that described in 2+ signaling units, or nano-junctions, interact in health and disease. Ultimately, such research can be expected to result in improvements in the management and therapy of both physical and mental diseases.Dysregulation of ER Ca2+ exchange diffusion on negatively charged surfaces of biological PLs, as documented earlier in an inanimate model membrane [2+ to its receptor sites embedded on biological membranes. This constitutes a new basic component of cellular Ca2+ signaling; however, direct experimental or computational evidence for an involvement of this process of NJ Ca2+ transfer is still awaited. Nonetheless, by combining evidence from stochastic modeling and various computational as well as experimental studies with membrane models, it appears that 2D surface diffusion of Ca2+ at the interfaces of PL membranes and narrow aqueous phases of NJs is both feasible and essential for specific targeting of calcium-sensitive functional proteins embedded in PL bilayers. Stochastic modeling of the targeting of Ca2+ receptors in NJs predicts that Ca2+ transfer from source to target cannot be accomplished on the sole basis of 3D random walk, but becomes plausible when involving 2D exchange diffusion at the membrane’s surface, which accommodates the target. In addition, 2D interfacial Ca2+ diffusion may play an important role in signaling functions of other membrane systems characterized by high PL surface to aqueous volume ratios, such as diads and triads in cardiac and skeletal muscle [2+ tunnelling [2+ waves [We propose that 2D Camembrane , functiol muscle ; the narnnelling ; the narnnelling ; the lum2+ waves .


Dear Editor,Acute lymphoblastic leukemia (ALL) in infants differs from ALL in older children  leading Genomic DNA was isolated from leukemic cells for molecular characterization. A MLL-MLLT3/AF9 rearrangement was detected and used as a marker for minimal residual disease (MRD). For further molecular characterization, targeted deep next-generation sequencing was performed for a panel of 54 leukemia-associated genes [2), being well investigated in children with AML [2) being used in adult ALL, but not yet in infants [Since the CD33 antigen was strongly expressed in the first immunophenotyping, we administered the anti-CD33 monoclonal antibody gemtuzumab ozogamicin (GO) twice, two weeks apart (6 mg/mwith AML . As CD38with AML  and show infants . AfterwaA combination of three different monoclonal antibodies according to the immunophenotype of the leukemic cells can effectively eliminate leukemic cells in chemotherapy-refractory leukemia and serve as bridging to transplant to induce a complete molecular remission afterwards even in infants.


NPM1 is involved in tumorigenesis via chromosomal translocations, deletions, or mutation. Acute myeloid leukemia (AML) with mutated NPM1, a distinct diagnostic entity by the current WHO Classification of myeloid neoplasm, represents the most common diagnostic subtype in AML and is associated with a favorable prognosis. The persistence of NPM1 mutation in AML at relapse makes this mutation an ideal target for minimal measurable disease (MRD) detection. The clinical implication of this is far-reaching because NPM1-mutated AML is currently classified as being of standard risk, with the best treatment strategy (transplantation versus chemotherapy) yet undefined. Myeloid neoplasms with NPM1 mutations and <20% blasts are characterized by an aggressive clinical course and a rapid progression to AML. The pathological classification of these cases remains controversial. Future studies will determine whether NPM1 gene mutation may be sufficient for diagnosing NPM1-mutated AML independent of the blast count. This review aims to summarize the role of NPM1 in normal cells and in human cancer and discusses its current role in clinical management of AML and related myeloid neoplasms.Nucleophosmin (NPM1) is a multifunctional protein with both proliferative and growth-suppressive roles in the cell. In humans,  NPM1 cDNA encoding a 294-amino acid protein was cloned in 1989 [NPM1 results in gene developmental defects and embryonic lethality at mid-gestation [NPM1 is involved in tumorigenesis. The NPM1 gene can undergo a variety of genetic alterations, explaining its contributions to molecular pathogenesis of tumors of diverse histological origins. Genetic changes of the NPM1 gene include chromosomal rearrangements and deletions in various hematological and solid tumors. The mutation of NPM1 plays a unique role in the pathogenesis of acute myeloid leukemia (AML) and is seen in about 35% of AML patients [NPM1-mutated AML the single largest unique group of AML. NPM1 has a greatly heterogeneous role in the cell and interacts with both oncogenic and tumor-suppressing cellular functions.Nucleophosmin NPM1), also called B23 or numatrin, is a phosphoprotein expressed at high levels in the granular region of the nucleolus [, also cap53 and ARF.In the normal cell, NPM1, an abundant phosphoprotein of 37 kDa that is highly concentrated in the nucleolus, shuttles rapidly between the nucleus and the cytoplasm ,7. The sWithin the structure of NPM1 protein, several functional domains account for various biochemical functions .The N-terminal region carries a conserved non-polar sequence, with an important role as chaperone for proteins and nucleic acids ,9,10. InNPM1 in human cancer is remarkable for its involvement in tumors of diverse histological origins. At least three well-characterized genetic mechanisms can contribute: overexpression of the NPM1 protein, chromosomal translocations resulting in fusion products with NPM1, or mutation of the NPM1 gene. In addition, NPM1 maps to the 5q35 locus of chromosome 5, a region that is deleted in a subset of patients with de novo myelodysplastic syndromes (MDS). The partial or complete loss of chromosome 5 is also commonly observed in therapy-related MDS. The specific role of NPM1 is yet to be explored in these subsets of MDS.The role of NPM protein is known to be overexpressed in various tumors, including carcinomas of gastric , colonicNPM1 gene are common genomic alterations in both lymphoid and myeloid hematologic neoplasms [NPM1 mutation being essentially unique to AML [NPM-ALK in anaplastic large cell lymphoma, NPM-RARα  in acute promyelocytic leukemia (APL), and NPM-MLF1 (myelodysplasia/myeloid leukemia factor 1) in MDS and AML. The mutant NPM1 in AML leads to abnormal subcellular localization of the protein in the cytoplasm. A common feature in both mechanisms is the reduction in the total amount of NPM1 protein due to decrease in NPM1 dosage, secondary to heterozygosity. The decreased dosage of NPM1 could be potentially augmented if a negative-dominant effect of the mutated protein exists over the wild-type protein.On the other hand, chromosomal translocation and mutation of the eoplasms ,27,28,29e to AML . ChromosNPM1 will function as an oncogene, a tumor suppressor, or both in any specific scenario. The role of NPM1 as a proto-oncogene is supported by the observation that cancer cells in the phase of growth and proliferation overexpress NPM1, and at the same time, apoptosis is inhibited. The level of NPM1 protein is higher in proliferating and cancerous cells than in quiescent cells [MYC oncogene, since MYC is one of the regulators of NPM1 transcription [NPM1 [NPM1 in supporting cell growth relates to its role in the synthesis of ribosomes. The ability of NPM1 protein to bind nucleic acids [NPM1 has various options for promoting tumor genesis and function as a proto-oncogene.The different mechanisms described above do not necessarily identify whether nt cells . This mecription ,32, or ton [NPM1 . Althougic acids ,34, to aic acids ,7, and tic acids ,36 desigic acids . These mic acids . In summNPM1 function as a tumor-suppressor gene? NPM1 protein stabilizes ARF by binding to it inside the nucleolus and thus inhibiting its degradation [NPM1 may be an important contributor to the ARF-mediated regulation of oncogenic stress, and as such, it could be considered as a tumor suppressor. Furthermore, increasing evidence suggests that during cellular stress, such as UV irradiation, NPM1 increases the stability of p53 by binding to MDM2 [NPM1 haploinsufficient mice (Npm+/−) show increased genomic instability and increased susceptibility to oncogenic transformation [NPM1 has a capability to act either as a tumor suppressor or a proto-oncogene, and its actual role may be determined by the balance between its various molecular partners and the level of NPM1 expression  and lack of expression of CD34 and CD13 by flow cytometry [While studying subcellular localization of NPM1 protein, Falini et al. observed a correlation between the presence of cytoplasmic NPM1 and certain clinical and biological features in AML . In theiytometry .NPM1 gene results in cytoplasmic localization of NPM1. Several different types of mutations exist. The most common type , representing about 70–80% of all mutations, features a four base pair nucleotide (TCTG) insertion at the position encoding the 288th amino acid residue, resulting in a frame shift of the downstream sequence [It turns out that a novel mutation in exon 12 of the sequence . As a cosequence  , all mutant proteins feature a critically important motif, notably the loss of at least one of W288 and W290, and they all share the last five amino acids [NPM1-mutated AML, it was the cytoplasmic staining of NPM1 that directed attention to a subgroup of AML that turned out to become the single largest subtype of AML defined by a mutation [NPM1 mutations were identified in 52.9% of AML cases [NPM1 mutations with FLT3 mutations and additionally reported that MLL tandem duplication, NRAS, KIT, and CEBPA mutations were not common in NPM1-mutated AML [NPM1 mutations were less frequent than in those who were older than 35 years. Subsequent studies continued to add further layers of observations. Verhaak et al. reported that NPM1-mutated AML, while associated with normal karyotype and FLT3 mutation is also associated with higher white blood cell counts [NPM1 and FLT3 mutations, NPM1-mutated AML patients without FLT3-ITD mutation represent a distinct subgroup of young adult patients with AML [Though numerous different sequence mutations exist in the no acids . Thus, dno acids . Falini no acids . The cormutation . The resML cases . Their rated AML . There sated AML . In AML l counts . In contwith AML .NPM1, raising a differential diagnosis of AML with multilineage dysplasia [NPM1-mutated AML this type of dysplasia does not signify a worse prognosis and that the observation of dysplasia should not overwrite the diagnosis of AML with mutated NPM1 [NPM1 mutations over time, reliably present at disease relapse and typically expressed across the entire leukemic population [Of particular interest to diagnostic hematopathologists, multilineage dysplasia is observed quite frequently in bone marrow cells of AML with mutated ysplasia . It has ted NPM1 ,56. Anotpulation ,57,58,59NPM1 typically shows a good response to induction chemotherapy [FLT3-ITD mutation have a favorable prognosis [FLT3-ITD mutation, the prognosis is comparable with that of patients with AML with t or AML with inv(16) [NPM1 mutations [FLT3, IDH1, IDH2, and DNMT3A genes, may significantly alter the prognosis [FLT3-ITD mutations negatively influence the prognosis, it is to be pointed out that in younger adult patients, the prognosis is still better than in those patients who have AML with FLT3-ITD and wild-type NPM1, especially when the FLT3-ITD mutation is present at a low allelic ratio [FLT3-ITD mutation in NPM1-mutated AML is less clear [NPM1, FLT3-ITD, and DNMT3A [AML with mutated otherapy . Cases wrognosis ,60,61,623.1;q22) . The preutations ,63,64. Arognosis . Though ic ratio ,66,67. Iss clear . The lead DNMT3A .NPM1 mutations are stable over time, usually documented at disease relapse and commonly expressed in the whole leukemic population [NPM1 mutation may be considered an ideal leukemia-specific target for minimal residual disease/minimal measurable disease (MRD) detection [NPM1-mutated gene copies [NPM1 mutations type A, B, and D because these three mutations together represent the vast majority of AML cases  [NPM1 mutations undergo molecular MRD evaluation at the following clinical timepoints: at diagnosis, after 2 cycles of induction/consolidation chemotherapy, and at the end of treatment [NPM1-mutated MRD may sometimes be undetectable in PB but that a BM sample shows detectable MRD at the end of treatment. Balsat et al. reported discordant results (a positive MRD in BM and negative result in PB) in 24.6% of cases [NPM1 MRD monitoring is recommended every 4 weeks for at least 3 months to evaluate for MRD increase [Unlike many other myeloid associated mutations, pulation ,57,58,59etection . Since tetection , RQ-PCR e copies ,75,76,77 15–20%) ,74,77. Treatment . The selof cases . If suchincrease . OverallNPM1 mutation levels at diagnosis as measured by RQ-PCR did not have an impact on complete remission (CR) rate after induction chemotherapy, and other survival measures such as event-free survival (EFS) and overall survival (OS) were not influenced either [NPM1 variant allelic frequency (VAF) (>0.44) detected by next-generation sequencing (NGS) correlated with shortened median OS and shortened EFS. High NPM1-mutated allele burden at diagnosis by NGS was associated with unfavorable clinical outcomes [NPM1 mutations in the BM.d either ,65,81,82outcomes . This suNPM1-mutated AML patients who previously reached an MRD negative status, serial molecular MRD evaluation may offer the benefit of predicting an impending relapse [NPM1 mutation may be documented as early as 2–3 months before a morphologic relapse becomes evident [NPM1-mutated transcripts to the overt morphologic relapse was highly variable  [In  relapse ,84,85. I evident ,92,93,94 months) .NPM1-mutated AML is still controversial. The prognostic value of pretransplant NPM1-mutated MRD has been evaluated in several studies [FLT3-ITD mutational status did not influence this difference [NPM1-mutated MRD for transplant selection. Notably, patients who achieved a >4-log NPM1-mutated MRD clearance did not benefit from allo-HSCT, but a survival benefit existed for those who failed to achieve clearance [NPM1-mutated AML represents the largest uniform subgroup within AML but also because these patients are classified as being of standard risk, for whom the best treatment strategy (transplantation versus chemotherapy) in unclear. It is now possible that the detection of persistent residual NPM1-mutated transcripts may provide additional guidelines for selecting the best treatment approach.The role for allogeneic hematopoietic cell transplantation  in  studies ,95,96,97 studies . In a stlearance ,80. The NPM1-mutated MRD monitoring in predicting relapse after allo-HSCT [NPM1 mutation MRD detection in predicting relapse after allo-HCT. Based on the study of Zhou et al., highly sensitive NGS for NPM1-mutated transcript performed around day 28 after an allo-HSCT predicted a morphologic relapse in 15 of 18 cases (83%) [NPM1 MRD at 3 months after allo-HSCT and observed that MRD positive result predicted relapse and was predictive of OS [NPM1-mutated AML who received an allo-HSCT could benefit from NPM1 MRD monitoring after transplant. Unfortunately, this benefit is decreased by the fact that preventive therapies, such as tapering if immune suppression, donor lymphocyte infusion, and HMA are not yet standardized [Finally, there may be a role for llo-HSCT ,94,97,98dardized ,93,95,99NPM1-based MRD monitoring in AML, various challenges exist. NPM1 mutation is stable over the course of disease [NPM1 mutation may be lost at relapse [NPM1 mutations has been extremely variable, ranging from 1% [NPM1 mutations were believed to represent a new, therapy-related AML rather than a true relapse [NPM1 mutation at relapse [DNMT3A mutations were present in both the primary and the relapse samples in every case with absent NPM1 mutation at relapse [DNMT3A mutation before acquiring an NPM1 mutation, and the NPM1 wild-type/DNMT3A-mutated clone gave rise to the relapsed AML [NPM1 MRD monitoring will result in negative results [NPM1 MRD monitoring is a potential switch of NPM1 mutation subtype from D to A, a rare scenario that has been recently reported [Despite the many advantages of  disease ,100,101, disease ,100,101. relapse . The fre from 1%  to 25% i from 1% ,104. Cas relapse ,72,91. L relapse . This stpsed AML . In such results ,106. Anoreported .NPM1 in de novo AML by Falini et al., a strong and specific association between NPM1 mutations and AML has been observed [NPM1 mutations were not observed in any non-myeloid neoplasm. Though less frequent compared with de novo AML, NPM1 mutations have also been observed in secondary AML (defined as progressing from either MDS or myelodysplastic/myeloproliferative neoplasm (MDS/MPN)) with a variable incidence ranging from 4.5% to 27% of cases [NPM1 mutations were commonly detectable during the antecedent MDS phase, while in other cases, the MDS stage lacked the NPM1 mutation [NPM1 mutation has been detected in MDS (2%) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) (3%), with MDS/MPN represented by mainly chronic myelomonocytic leukemia (CMML). Of note, NPM1-mutated MDS most commonly represented MDS with excess blasts (MDS-EB) at diagnosis [Since the original discovery of cytoplasmic observed . NPM1 muof cases ,112,113.mutation . In myeliagnosis .NPM1 mutation and <20% blasts has been characterized by a rapid progression to AML, usually within 12 months of diagnosis [NPM1-mutated MN requires additional mutations, especially FLT3 mutations [NPM1 mutation and <20% blasts rapidly progress into AML even without additional FLT3 mutations [Several small retrospective studies reported that the rare cases of MN with iagnosis ,119,120.utations . Later, utations ,122,123.NPM1 mutation (VAT 45%) and U2AF1 mutation (VAT 45%). Treatment was started with decitabine and venetoclax. NPM1-mutated CMML cases were characterized by more severe anemia, higher BM monocyte percentage, a more rapid transformation to AML, and decreased overall survival (OS) then non-NPM1-mutated cases, though this study did not reach statistical significance [NPM1-mutated CMML patients—and additionally described that DNMT3A and FLT3 mutations were more common, while TET2 and ASXL1 mutations were less common than in CMML cases with no NPM1 mutation [NPM1-mutated CMML (63%) in comparison with NPM1 wild-type CMML (18%) at a median of 5 months after initial diagnosis [A study of 144 CMML patients by Peng et al. described that ificance . Anothermutation . This stiagnosis .NPM1 and less than 20% blasts was reported by Patel et al. [NPM1 mutations occurred in younger patients and were more commonly associated with a normal karyotype when compared with NPM1 wild-type cases. The mutation landscape was also different, as NPM1-mutated MN more commonly showed DNMT3A and PTPN11 mutations, while mutations of ASXL1, RUNX1, TP53, IDH1, IDH2, FLT3, NRAS, and KRAS mutations were less common than in the NPM1 wild-type neoplasms [NPM1-mutated MN (73%) were treated with hypomethylating agents (HMA) upfront, and 39% progressed to AML at a median time of 5.2 months. As a contrast, none of the patients treated with intensive induction chemotherapy progressed to AML. These data suggest that a more intensive chemotherapy upfront may benefit patients with NPM1-mutated MN [NPM1-mutated MN were younger, had lower hemoglobin levels, and more commonly had a normal karyotype compared with NPM1 wild-type cases. Interestingly, the clearance of mutation after therapy paralleled the disappearance of the dysplastic morphologic features. Complete remission (CR) rates were significantly higher after intensive chemotherapy than after HMA treatment [NPM1 mutation who achieved a favorable outcome after decitabine [NPM1-mutated MN with less than 20% blasts cannot be drawn. The limited number of data available were derived from small retrospective studies, and they lacked a controlled clinical trial design [NPM1-mutated MN patients, HMA-based moderate intensity therapy could be considered inadequate. More intensive therapy could be considered for those NPM1-mutated MN patients who are fit, potentially including induction chemotherapy rather than MDS-directed treatment modalities [The largest multicenter cohort of MN with mutated l et al. . Myeloideoplasms . When lotated MN . Montalbtated MN . In theireatment . Not evecitabine . At presl design ,121,123.dalities ,121,123.NPM1 gene mutation and less than 20% PB and BM blasts. The pathological diagnosis and subclassification has a direct impact on subsequent therapy. It has been long accepted that the presence of certain recurrent cytogenetic abnormalities including t, t, or inv(16)(p13.1q22)/t is enough to establish a diagnosis of AML even in the absence of 20% blasts [NPM1 gene mutation: myeloid neoplasms with less than 20% blasts do not meet current criteria for a diagnosis of NPM1-mutated AML. To further complicate the issue, multilineage dysplasia is a common finding in NPM1-mutated AML, yet the presence of NPM1 mutation should have a priority over multilineage dysplasia as a disease-defining criterion [NPM1 mutation or biallelic mutation of CEBPA superseded multilineage dysplasia in the diagnostic classification. Based on presence of multilineage dysplasia, a case of NPM1-mutated MN with <20% blasts could be easily classified as MDS-EB or MDS/MPN. This diagnosis might sidetrack the patient for a subsequent diagnosis of AML with myelodysplasia-related changes (rather than AML with mutated NPM1) at the time when a transformation into AML occurred. Overall, AML with mutated NPM1 may remain underdiagnosed. Forghieri et al. recommended an integrated molecular and immunohistochemical approach by demonstrating NPM1 mutation in the bone marrow and extensive NPMc+ staining in more than 20% blasts in a bone marrow core biopsy to increase a successful diagnosis of AML with mutated NPM1 [NPM1 gene mutation may be sufficient to diagnose NPM1-mutated AML independent of the blast count [An additional challenge relates to the pathological classification of these uncommon cases of MN showing % blasts . In contriterion . The 201ted NPM1 . Future st count ,118. TheNPM1 is remarkable for its specificity to a subtype of AML that is recognized as a specific diagnostic entity by the current WHO classification of myeloid neoplasms. In addition to its role in diagnostic subclassification, NPM1 mutation determines a subgroup of AML with favorable prognosis, though this is influenced by the presence or absence of certain concomitant mutations, in particular FLT3-ITD and DNMT3. The NPM1 mutation exhibits stability during persistent disease and relapse and has provided an opportunity for MRD testing in this subgroup in AML. Considering that NPM1-mutated AML represents the largest subgroup of AML and is classified as being of standard risk, the detection of persistent residual NPM1-mutated transcripts may provide additional guidelines for decision making between chemotherapy and hematopoietic cell transplantation. The best approach to the pathologic diagnosis and clinical management of myeloid neoplasms with NPM1 mutation and less than 20% blasts is still unclear. These cases have been characterized with an aggressive clinical course and a rapid-progression AML, and they may benefit from aggressive treatment upfront. Future studies will determine whether NPM1 gene mutation may be sufficient for diagnosing NPM1-mutated AML independent of the blast count.The availability of new molecular techniques, especially next-generation sequencing, introduced mutation testing into the routine diagnostic arena of myeloid neoplasms. Unfortunately, most of myeloid-associated mutations are not specific to any diagnosis, as they can be seen in individuals without disease and can be present in many different subgroups of disease . Unlike most myeloid mutations,


Oxytropis falcata Bunge is a plant used in traditional Tibetan medicine, with reported anti-inflammatory and antioxidants effects and alleviation of myocardial ischemia reperfusion injury (MIRI). However, the underlying mechanism against MIRI and the phytochemical composition of O. falcata are vague. One fraction named OFF1 with anti-MIRI activity was obtained from O. falcata, and the chemical constituents were identified by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS). The potential targets and signaling pathways involved in the action of O. falcata against MIRI were predicted by network pharmacology analysis, and its molecular mechanism on MIRI was determined by in vitro assays. The results revealed that flavonoids are the dominant constituents of OFF1. A total of 92 flavonoids reported in O. falcata targeted 213 potential MIRI-associated factors, including tumor necrosis factor (TNF), prostaglandin-endoperoxide synthase 2 (PTGS2), and the NF-κB signaling pathway. The in vitro assay on H9c2 cardiomyocytes subjected to hypoxia/reoxygenation injury confirmed that the flavonoids in OFF1 reduced myocardial marker levels, apoptotic rate, and the inflammatory response triggered by oxidative stress. Moreover, OFF1 attenuated MIRI by downregulating the ROS-mediated JNK/p38MAPK/NF-κB pathway. Collectively, these findings provide novel insights into the molecular mechanism of O. falcata in alleviating MIRI, being a potential therapeutic candidate. Ischemic heart disease is a highly prevalent threat worldwide, with a mortality rate of 16% ,3. MultiOxytropis falcata Bunge, belonging to the Leguminosae family of perennial herbs, is widely distributed in the northwestern and southwestern regions of China, including Qinghai, Gansu, Tibet, Sichuan, and Xinjiang provinces −) with a mass error of 5 ppm, and qualitative analyses were mainly completed with MS/MS fragments. The results showed that OFF1 was mainly composed of flavonoids, and 31 of them including daidzein, 7-hydroxyflavone, maackiain, isoliquiritigenin, naringenin, and luteolin were tentatively identified, as shown in O. falcata.The chemical components of OFF1 were analyzed by UHPLC in the ESI negative and positive ion modes . Their aO. falcata was found through the search of the relevant literature [A total of 115 flavonoids from terature ,27,43,47terature . Myricetterature ,50  in MIRI (yellow node) corresponding to 92 active compounds (purple nodes) of O. falcata (blue node) was found, as shown in O. falcata , cellular response to nitrogen compound , positive regulation of protein phosphorylation , response to hypoxia , regulation of inflammatory response , ROS metabolic process , and apoptotic signaling pathway  (p < 0.001), perinuclear region of cytoplasm , side of membrane , distal axon , apical part of cell , and nuclear envelope . The significantly enriched MF terms included kinase binding , oxidoreductase activity , nitric-oxide synthase regulator activity , ubiquitin-like protein ligase binding , protein phosphatase 2A binding , growth factor receptor binding , and G protein-coupled peptide receptor activity .The GO enrichment analysis of the overlapping targets between flavonoids and MIRI was mainly categorized into the biological process , cellular component , and molecular function  . As rega< 0.001) . In addip < 0.001), cAMP signaling pathway , platelet activation , NF-κB signaling pathway , adrenergic signaling in cardiomyocytes , renin–angiotensin system , and apelin signaling pathway . Since the NF-κB pathway plays a pivotal role in preventing MIRI [KEGG pathway analysis B furthering MIRI , our hypp < 0.01; p < 0.01; p < 0.01; p < 0.01; p < 0.01; The mechanism of action of OFF1 in the protection against MIRI was investigated in vitro according to the insights provided by the network pharmacology analysis. H9c2 cells subjected to H/R injury resulted in a significant increase in the cytokine levels of the pro-inflammatory interleukins (IL)-1β and IL-6, monocyte chemoattractant protein (MCP)-1, and tumor necrosis factor (TNF)-α in the supernatant of the cultured cardiomyocytes by 4.30-fold, 2.59-fold, 2.20-fold, and 1.96-fold, respectively, compared to the Con group , as shown in H9c2 cells subjected to H/R showed significantly reduced superoxide dismutase (SOD) levels by approximately 64.13% and increased levels of cyclooxygenase (COX-2), inducible nitric oxide synthase (iNOS), and malondialdehyde (MDA) by 2.20-fold, 4.03-fold, and 2.57-fold, respectively, as shown in p < 0.01; Previous studies showed that Nrf2 activation protects cardiomyocytes from oxidative stress-induced damage, and the level of intracellular ROS depends on the expression of the endogenous antioxidant Nrf2 ,53. Therp < 0.01; p < 0.01) and decreased in cells pretreated with Dil and OFF1, as seen in p < 0.01 or p < 0.01), as shown in The NF-κB pathway was analyzed in the H9c2 cells subjected to H/R injury to further clarify the cardioprotective mechanism of OFF1. The levels of NF-κBp65 in the Con, H/R, Dil, and OFF1 (25 and 50 μg/mL) groups were 0.93 ± 0.07, 1.84 ± 0.06, 1.30 ± 0.04, 1.86 ± 0.08, 1.47 ± 0.07, and 1.25 ± 0.05 (U/L), respectively. The NF-κBp65 levels were decreased by approximately 29.18%, 19.71%, and 31.79% in the Dil and OFF1 (25 and 50 μg/mL) groups, respectively, when compared with the H/R group (p < 0.01), and it was reduced by OFF1 (p < 0.05 or p < 0.01), as shown in p < 0.05 or p < 0.01). Taken together, OFF1 mitigated the effects of H/R injury on cardiomyocytes by inhibiting the JNK/p38 MAPK signaling.Previous studies showed that JNK and p38MAPK are involved in the inhibition of NF-κB induced by different compounds ,55. The O. falcata is used in Tibetan Medicine to treat several diseases, including leprosy, rheumatic pain, back pain, influenza, and tonsillitis [O. falcata have a protective effect on MIRI, although the specific mechanism was not clear [MIRI is a complex physiological and pathological process, mainly due to arterial bypass, thrombolytic therapy, and percutaneous coronary angioplasty in the recovery of cardiac tissue and organ blood flow . In recesillitis . Severalsillitis ,61,62. Iot clear ,31,33. Sot clear , it coulO. falcata called OFF1, which could protect H9c2 cell subjected to H/R from cell death, inhibit the release of CK and LDH, reduce apoptotic proteins such as Caspase9, Caspase3, and Bcl-2, as well as increase the levels of Bax and the ratio of Bax/Bcl-2. These findings demonstrated that OFF1 could protect H9c2 cell injury by suppressing cell apoptosis. The predominant component were flavonoids, including daidzein, 7-hydroxyflavone, maackiain, isoliquiritigenin, naringenin, and luteolin, which were identified by UHPLC–MS/MS analysis. Therefore, flavonoids probably exerted a crucial role in alleviating MIRI by O. falcata, accordingly providing a basis for the prediction of targets by network pharmacology analysis. Thus, the role of flavonoids of O. falcata were investigated. The analysis of network pharmacology revealed the intricate interactions among drug components, disease targets, signaling pathways, and molecular mechanisms of TCMs [The present study evaluated a bioactive fraction of  of TCMs ,63.O. falcata was evaluated using Lipinsky’s RO5, and myricetin and robinin were additionally included due to their established pharmacological effects. For example, myricetin alleviates MIRI by up-regulating COX-2 and cytochrome P450 and p38, and downregulating fatty acid synthase antibody and glucose 6-phosphatedehydrogenase signaling pathway, thus reducing myocardial infarction, apoptosis, inflammation response, and oxidative stress [O. falcata were identified by the compound–target–disease network , oxidative stress-related cytokines , and the relative expression and phosphorylation of NF-κBp65, JNK, and p38 were analyzed in the cardiomyocytes subjected to H/R injury. Our findings revealed that OFF1 protects cardiomyocytes from H/R-induced injury by inhibiting cell apoptosis, inflammation response, and oxidative stress by regulating the ROS-mediated JNK/p38MAPK/NF-κB pathway . These eO. falcata was collected in 2015 from Gonghe County, Qinghai Province, China, and identified by professor Yongchang Yang, Northwest Plateau Institute of Biology. A voucher specimen of O. falcata was stored at College of Eco-Environmental Engineering, Qinghai University, as ZHANG2015-O18. One kilogram of the plant matter was ground into powder and extracted with 95% ethanol (10 L each time) at 75 °C under reflux for 3 h three times. The extracts were filtered, combined, and evaporated under reduced pressure to obtain a concentrated O. falcata extract [ extract . The ethWe obtained an OFF1 of 4.6 g. The fraction OFF1 was selected and identified by ultra-high-performance liquid chromatography (thermoultimate 3000) coupled to mass spectrometry  according to our previous report . The chrO. falcata were obtained from the relevant literature. The 2D structure of the flavonoids was drawn using Chemdraw 18.0, and the Smile structure was derived. Lipinski’s Rule of five (RO5), including molecular weight (MW) < 500, the number of hydrogen bond donors (nOHNH) < 5, number of hydrogen bond acceptors (nOH) < 10, lipid–water partition coefficient (miLogP) < 5, and number of rotatable keys (nRotb) ≤ 10 was applied to screen for flavonoids relevant to drug development . In addition, a few active compounds that did not meet the RO5 were included due to their high bioactivity.The flavonoids in http://www.swisstargetprediction.ch/, accessed on 15 April 2021), SEA , and TargetNet  databases. The criterion for the selection was the probability value , Max Tc (SEA) or prob score (TargetNet) ≥ 0.4. MIRI-related targets were collected from GeneCards , Disgnet , GeneCLip 3 , and OMIM  databases. UniprotKB (https://www.uniprot.org/) was used to standardize all targets to UniProt ID and gene name. The active compounds overlapping with MIRI-related targets were identified through Venn diagrams using Omicshare  to further evaluate the cardioprotective bioactive compounds.The compound-related targets in Homo sapiens were downloaded from the SwissTarget Prediction , and those with connection score 0.4 were used to construct the PPI network. Cytoscape 3.7.2 was used to visualize the PPI network. The hub targets were calculated by the Degree method of the Cytohubba plugin. Higher node degree was indicative of a stronger association among the target genes.The shared protein targets were incorporated into the STRING database , MIRI, active compounds, and potential targets. The network was analyzed to predict the hub active compounds of O. falcata against MIRI.Cytoscape 3.7.2. was used to construct the “compound–gene–disease” network to determine the relationship between the drug  to clarify the biological function and pathways associated with the potential targets. The GO terms and KEGG pathways with a p-value < 0.05 were considered significantly enriched.GO and KEGG pathway analyses were performed using the metascape network tool (v/v) fetal bovine serum  and 0.1% penicillin/streptomycin  and cultured at 37 °C under 5% CO2. The cells were used for different experiments once they reached 80% confluency. The cells were cultured in serum-free medium under hypoxic conditions consisting of 2% O2, 5% CO2, and 94% N2 for 12 h, and thereafter under normoxic conditions (95% air and 5% CO2) for 6 h for reoxygenation to simulate MIRI in vitro.H9c2 cells  were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10%  for 12 h, except for the control. A CCK8 assay kit  was used to evaluate cell viability. Briefly, 10 μL CCK-8 and 100 μL DMEM were added to each well after H/R induction, and the cells were incubated for 1 h at 37 °C. The absorbance of each well was measured at 450 nm using a microplate reader . After that, cells were divided into six groups: (a) control, (b) H/R, (c) H/R + Dil (20 μmol/L), (d) H/R + 10 μg/mL OFF1, (e) H/R + 25 μg/mL OFF1, and (f) H/R + 50 μg/mL OFF1.CK and LDH levels in the supernatants of H9c2 cells were detected using the corresponding kits , and the absorbance was measured at 450 nm. Commercial kits were also used to measure SOD, COX-2, and iNOS levels, as well as MDA activity  according to the manufacturer’s protocols. IL-1β, IL-6, MCP-1, TNF-α, and NF-κBp65 levels were measured by ELISA using the corresponding kits  according to the manufacturer’s instructions.Intracellular ROS levels were measured by the ROS Assay Kit  using the fluorescence indicator 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe . BrieflyTotal protein was extracted from H9c2 cells using RIPA lysis buffer and quantified with the BCA method . The protein samples were resolved by SDS-PAGE and then transferred onto a PVDF membrane . After blocking in 5% skim milk at room temperature for 1 h, the membrane was incubated overnight with antibodies targeting p-NFκBp65, NFκBp65, p-JNK, JNK, p38 , p-p38, Caspase-9, Caspase-3 , Bax , and Bcl-2  at 4 °C. The following day, the membrane was washed thrice with TBST for 10 min each and incubated with the secondary antibody  for 1 h at room temperature. After washing thrice with TBST, the membrane was imaged using the ChemiDoc MP system . The grey scale values of the protein bands were quantified using ImagePro Plus 7.0 software, and β-actin was used as the internal reference.An immunofluorescence assay was performed as previously described. Briefly, the cell climbing slices were rinsed twice with PBS for 3 min each and fixed in 4% paraformaldehyde for 15 min. After washing again with PBS, the slices were blocked in PBS containing 5% goat serum for 1 h and incubated overnight with anti-Nrf2  and anti-NF-kBp65  antibodies in a humidified box at 4 °C. After the incubation with the secondary antibodies for 1 h, the cells were washed with PBS and counterstained with DAPI  for 5 min at room temperature. The slides were washed thrice with PBS for 5 min each and observed under a fluorescence microscope at 400x magnification. The average fluorescence intensity was measured using the Image-Pro Plus 6.0 software.p < 005 was considered statistically significant.Statistical analysis was performed using SPSS software 22.0 . The results were obtained from at least three independent experiments and indicated as mean ± standard deviation (SD). The comparison of multiple groups was performed by one-way analysis of variance. A value of O. falcata, and the results were confirmed by in vitro experiments. O. falcata protected against MIRI by regulating inflammatory response, oxidative stress, and apoptosis by targeting the ROS mediated JNK/p38MAPK/NF-κB signaling pathway. These findings lay the foundation for the clinical application of O. falcata against MIRI.In this study, UHPLC–MS and network pharmacology were used to evaluate the multiple chemical components, multiple biological processes, and multiple signaling pathways involved in MIRI and in the effect of