The problem of prognostic stratification in acute myeloid leukemia (AML) patients still has limitations. R software.  The expression profile data and clinical features of AML patients were obtained from multiple publicly available sources, including GSE71014, TCGA-LAML, and TARGET-AML. Single-cell analysis was performed using the TISCH project. All the analysis was conducted in the  In our study, three public AML cohorts, GSE71014, TARGET-AML, and TCGA-AML, were selected. Then, we identified the prognosis-related molecules through bioinformatic analysis. Finally, the DUSP7 was noticed as a risk factor for AML patients, which has not been reported previously. Biological enrichment analysis and immune-related analysis were performed to illustrate the role of DUSP7 in AML. Single-cell analysis indicated that the DUSP7 was widely distributed in various cells, especially in monocyte/macrophages and malignant. Following this, a prognosis model based on DUSP7-derived genes was constructed, which showed a good prognosis prediction ability in all cohorts.  Our results preliminarily reveal the role and potential mechanism of DUSP7 in AML, providing direction for future research. Acute myeloid leukemia (AML) is a highly heterogeneous malignant clonal disease that seriously affects human health . The patα, CBF, NPM1, WNT, RUNX1, WT1, RB, PU.1, p53, MYC, MPL, JUNB, GATA-1, FOS, FES, N-RAS, CEBPA, KIT, and FLT3 [α, BCR-ABL1, ASXL1, TP53, FLT3-ITD, and MLL [β1, VEGF, CSRP2, and others are all associated with the prognosis of AML [At present, studies have found that the factors affecting the prognosis of AML patients not only include general clinical characteristics such as age and FAB typing, but also include molecular genetics, cytogenetics, and mutations of specific molecules . For insand FLT3 –10. Some and MLL , 12. How and MLL . Numerou and MLL . As earl and MLL . Paschka and MLL . Recent s of AML –19. As is of AML –22.In our study, three public AML cohorts, GSE71014, TARGET-AML, and TCGA-AML, were selected. Then, we identified the prognosis-related molecules through bioinformatic analysis. Finally, the DUSP7 was noticed as a risk factor for AML patients, which has not been reported previously. Biological enrichment analysis, single-cell analysis, and immune-related analysis were performed to illustrate the role of DUSP7 in AML. Following this, a prognosis model based on DUSP7-derived genes was constructed, which showed a good prognosis prediction ability in all cohorts.R code. The human genomic reference file obtained from the Ensembl project (https://www.ensembl.org/) was utilized for probe annotation. Clinical information was downloaded in the “bcr-xml” form and organized using Perl code. For GSE71014, the expression profile of patients was downloaded from the direct link “Series Matrix File(s)” of the website https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE71014. Probe annotation was conducted by the GPL10558 platform. For all downloaded data, data quality control and standardization processing are carried out before data analysis. The interaction data of multiple proteins were downloaded from the STRING database and then visualized through Cytoscape software [The expression profile data and clinical features of AML patients were obtained from multiple publicly available sources. For TCGA-LAML (access time 22/3/2023) and TARGET-AML (access time 22/3/2023), the expression profile of each AML patient was downloaded in a “STAR-Count” form and then extracted into a merge matrix through P value <0.05) [DEGs analysis was performed using the limma package with the specific threshold (|logFC| > 1 and e <0.05) .P < 0.05). Furthermore, LASSO regression analysis was performed for variable optimization. Ultimately, multivariate Cox regression analysis was used to screen independent prognosis markers and model construction. The TCGA-AML cohort was set as the training cohort. The GSE71014 and TARGET-LAML cohorts were set as the validation cohort. Kaplan–Meier (KM) survival curves were used to compare the prognosis difference in different groups. The prediction ability of specific variables on patients' survival was performed using the receiver operating characteristic (ROC) curves.For a specific gene list, the expression profile of these genes was firstly combined with the corresponding survival information. Next, the molecules remarkably correlated with AML patients' survival were identified using the univariate Cox regression analysis (R packages. Clusterprofiler package was used for the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis [Evaluation of biological function was performed using specific analysis . Gene seanalysis .The single-cell data of GSE116256, GSE135851, GSE154109, PBMC_8K, PBMC_30K, and PBMC_60K were analyzed in the TISCH project .Estimate package was used to quantify the stromal score, immune score, and estimate score of the tissue microenvironment, respectively, representing the corresponding cell components . The micR software. Analysis with a statistic P value <0.05 was regarded as statistically significant. For data with different distributions, different analysis methods are used according to statistical requirements.All the statistical analyses were performed in the The flowchart of the whole study is shown in Firstly, after a comprehensive search, we have finally identified three AML datasets with complete transcriptional profiles and clinical information, including GSE71014, TCGA-LAML, and TARGET-AML . Then, wP < 0.001) .Then, based on the identified risk genes, we performed LASSO regression analysis to optimize variables Figures . Followi< 0.001) . ConsideThen, we tried to explore the potential biological function of DUSP7 in AML. Limma package was used to screen the DEGs in patients with high and low DUSP7 expression . The heaP < 0.001; P=0.012; P < 0.001). Next, the ssGSEA algorithm was used to quantify the tissue microenvironment of AML . ROC curves indicated that the 1-year AUC = 0.809, 3-year AUC = 0.851, and 5-year AUC = 0.935 .We then identified the top 100 molecules positively and negatively correlated with DUSP7 in AML patients Figures . Univaripatients . KM surv Figures . In the AML is a highly heterogeneous hematological malignancy caused by dysgenesis of myeloid hematopoietic stem/progenitor cells, characterized by abnormal clonal proliferation and differentiation arrest of primitive and immature myeloid cells in the bone marrow and peripheral blood –32. AML In our study, three public AML cohorts, GSE71014, TARGET-AML, and TCGA-AML, were selected. Then, we identified the prognosis-related molecules through bioinformatic analysis. Finally, the DUSP7 was noticed as a risk factor for AML patients, which has not been reported previously. Biological enrichment analysis, single-cell analysis, and immune-related analysis were performed to illustrate the role of DUSP7 in AML. Following this, a prognosis model based on DUSP7-derived genes was constructed, which showed a good prognosis prediction ability in all cohorts.DUSP7, whose full name is dual specificity phosphatase 7, has been reported involved in various pathophysiological processes, especially in cancers –38. For α can effectively reduce the recurrence rate of AML patients [α can prevent recurrence and improve the survival rate [α response can partially explain the impact of DUSP7 on the prognosis of AML patients. However, further research is needed to confirm the relationship between DUSP7 and IFN-α response.Biological enrichment analysis showed that the IL6_JAK_STAT3 signaling, interferon alpha response, bile acid metabolism, coagulation, and UV response DN were the top five pathways DUSP7 was involved in. This suggests the importance of metabolism and immunity in AML. AML cells have an atypical metabolic phenotype, characterized by increased mitochondrial mass, and more dependence on oxidative phosphorylation and fatty acid oxidation for survival. Tcheng et al. indicated that AML has a special fatty acid metabolism manner and level . Moreovepatients . Also, bval rate . TherefoImmune-related analysis showed that the DUSP7 was positively correlated with B cells, NK CD56 dim cells, Tem, Th17 cells, macrophages, and Treg. Some studies have begun to focus on the role of immune cells in AML. Goswami et al. found that after chemotherapy, AML patients experienced irreversible immune damage to their B cells . MeanwhiThe continuous development of bioinformatics and the massive data in the era of big data have improved the convenience of data secondary mining. Although our results are based on reliable data and algorithms, some limitations cannot be ignored. Firstly, most patients are of Western ethnicity. There are specific differences in genomics among populations of different ethnicities; therefore, racial bias will reduce the universality of our conclusions. Secondly, it cannot be denied that bioinformatics analysis cannot fully reflect the true situation in the body. Therefore, most conclusions only have indicative significance and still require subsequent experimental verification.


They are ubiquitously expressed and play an essential role in maintaining cytosolic Ca2+ homeostasis. Although extensively studied, little is known about the global structural arrangement of eukaryotic NCXs and the structural mechanisms underlying their regulation by various cellular cues including cytosolic Na+ and Ca2+. Here we present the cryo-EM structures of human cardiac NCX1 in both inactivated and activated states, elucidating key structural elements important for NCX ion exchange function and its modulation by cytosolic Ca2+ and Na+. We demonstrate that the interactions between the ion-transporting transmembrane (TM) domain and the cytosolic regulatory domain define the activity of NCX. In the inward-facing state with low cytosolic [Ca2+], a TM-associated four-stranded β-hub mediates a tight packing between the TM and cytosolic domains, resulting in the formation of a stable inactivation assembly that blocks the TM movement required for ion exchange function. Ca2+ binding to the cytosolic second Ca2+-binding domain (CBD2) disrupts this inactivation assembly which releases its constraint on the TM domain, yielding an active exchanger. Thus, the current NCX1 structures provide an essential framework for the mechanistic understanding of the ion transport and cellular regulation of NCX family proteins.Na + and Na+.Here authors present the cryo-EM structures of human cardiac NCX1, elucidating key structural elements for ion exchange and modulation by cytosolic Ca2 They are ubiquitously expressed and play a central role in maintaining cellular calcium homeostasis for cell signaling6. Dysfunctions of NCXs are associated with many human pathologies, including cardiac hypertrophy, arrhythmia, and postischemic brain damage9. NCX-catalyzed ion exchange reaction is electrogenic with a stoichiometry of 3 Na+ for 1 Ca2+. NCX normally functions to extrude Ca2+, but it can be reversed depending on the chemical gradient of Na+ and Ca2+ and the membrane potential16. Three NCX isoforms (NCX1–3) are found in mammals, and each isoform carries various splice variants that are expressed in specific tissues and exhibit distinct regulatory phenotypes22. The cardiac exchanger NCX1.1 has been the most extensively studied variant whose function is central to the cardiac contractile activity26.Sodium-calcium exchangers (NCX) are membrane antiporters that control the extrusion or entry of Ca30. The TM domain is responsible for the ion exchange function in NCX31 and has been modeled by the crystal structures of the archaebacterial exchanger NCX_Mj32. The intracellular domain is involved in the allosteric regulation of the exchanger by cytosolic Ca2+ and Na+31. In cardiac NCX1, cytosolic Ca2+ binds to two calcium-binding domains (CBDs) within the intracellular domain to increase transport activity35, while cytosolic Na+ inhibits transport activity by driving the exchanger into an inactivated state known as Na+-dependent inactivation37. The Na+-dependent inactivation occurs when three Na+ ions are bound to the inward-facing exchanger39. An amphipathic region, known as eXchanger Inhibitory Peptide (XIP) at the N-terminus of the intracellular domain, plays a central role in the inactivation process41. NCX1 activity can also be modulated by PIP244 as well as posttranslational modification such as palmitoylation48.The eukaryotic NCX consists of a TM domain with 10 TM helices and a large intracellular regulatory domain which, in primary sequence, separates the TM domain into two homologous halves (TMs 1–5 and TMs 6–10)2+-activated states, revealing the molecular basis underlying the complex regulation of NCX1.Despite the extensive investigations, little is known about the architecture of the eukaryotic NCX1 and the coupling between its TM and intracellular regulatory domains. Here we present the structures of human cardiac NCX1 in both the inactivated and Ca+ and nominal Ca2+-free condition, the structure of NCX1 in complex with Fab was determined to an overall resolution of 3.1 Å . Using the protein sample prepared in 200 mM Nager Fig. .Fig. 1OvAs expected, the human NCX1 contains two major structural components, the TM domain for ion exchange and the cytosolic regulatory domain Fig. . The TM 30. Eight of the TM helices (TMs 2–5 and 7–10) form a tightly packed core with two highly conserved α-repeats (TMs 2–3 as α1 and TMs 7–8 as α2) bundled at the center; TMs 1 and 6 are much longer helices at the periphery of the TM domain and loosely pack against the core at a 45° angle  superimposes well with that of outward-facing NCX_Mj Fig. , suggest54; it forms two arch-shaped helices (TMH1 and TMH2) partially embedded in the inner leaflet of the membrane  constitute the major part of the cytosolic domain 57 suggests the potential Ca2+-induced conformational changes activate NCX1 by destabilizing the inactivation assembly  between βE and βF as well as the C-terminus of βG, both of which undergo a concurrent swing motion from apo to Ca2+-bound state. This swing motion is not a simple rigid body movement but involves main-chain and side-chain rearrangement at defined residues, which can be summarized into three key movements. First, the side chains and peptide bonds of both E582 and K583 flip between apo and Ca2+-bound states. K583 sidechain, initially pointing towards Ca2+ site 2 and making direct interaction with D552 in the apo state, subsequently flips away from D552 towards the bulk solution upon Ca2+ binding. Conversely, the E582 sidechain, which is initially exposed to the bulk solution in the apo state, subsequently flips inwardly to form a salt bridge with K585 upon Ca2+ binding. The side-chain rearrangement of both residues liberates D552 from salt-bridging with K583 and K585, allowing it to directly chelate site 2 Ca2+. Second, the swing motion of E580 and its two neighbors (E579 and Y581) from apo to Ca2+-bound state removes its occlusion of site 1 Ca2+. Third, the swing motion of the Ca2+ binding loop from apo to Ca2+-bound conformation also yields space for the concurrent movement at the C-terminus of βG (a.a. 683-685), allowing the E683 side chain to be re-orientated for direct chelation with site 1 Ca2+. Although not present in the CBD2 crystal structure, the CH2 helix immediately following βG is expected to undergo a swing motion similar to the tail of βG  with low cytosolic [Ca2+] and the exchanger is in a Na+-loaded, inward-facing state. Only when in this state the linker β-hairpin between TMs 1 and 2ab would be in a position to interact with XIP and form the β-hub. Once formed, the β-hub can readily interact with the cytosolic domain mainly through the CH2 helix of CBD2 and generate a stable inactivation assembly that locks the TMs 1 and 6 in the inward-facing conformation, preventing the TM module from transporting ions. At higher cytosolic [Ca2+], the conformational change induced by Ca2+ binding at CBD2 causes a direct collision between its CH2 helix and the β-hub, resulting in the disassembly of the β-hub and the detachment of the entire cytosolic domain from the TM module. This detachment likely also pulls the XIP region away from the TM and releases TMs 1 and 6 from any constraint, yielding a constitutively active TM module for ion transport.In summary, we determined the structures of the human cardiac NCX1, which revealed the structural basis underlying Naion Fig. . NCX1 inNP_066920.1) containing a C-terminal Strep-tag was cloned into a pEZT-BM vector65 and expressed in HEK293S GnTI- cells using the BacMam system (ATCC). Bacmids were synthesized using E. coli DH10Bac cells (Thermo Fisher Scientific), and baculoviruses were produced in Sf9 cells using Cellfectin II reagent (Thermo Fisher Scientific). For protein expression, cultured GnTI- cells were infected with the baculoviruses at a ratio of 1:20  for 10 h. 10 mM sodium butyrate was then introduced to boost protein expression level, and cells were cultured in suspension at 30 °C for another 60 h and harvested by centrifugation at 4000g for 15 min. All purification procedures were carried out at 4 °C. The cell pellet was resuspended in lysis buffer  and homogenized by sonication. NCX1 was extracted with 2% (w/v) N-dodecyl-β-d-maltopyranoside  supplemented with 0.2% (w/v) cholesteryl hemisuccinate  by gentle agitation for 2 h. After extraction, the supernatant was collected by centrifugation at 40,000g for 30 min and incubated with Strep-Tactin affinity resin (IBA) for 1 h. The resin was then collected on a disposable gravity column (Bio-Rad) and washed with 30 column volumes of buffer A  supplemented with 0.06% (w/v) digitonin (Fisher Scientific) or 0.03% (w/v) lauryl maltose neopentyl glycol . NCX1 was eluted in a wash buffer supplemented with 50 mM biotin. The protein eluate was concentrated and further purified by size-exclusion chromatography on a Superdex200 10/300 GL column  in buffer A with 0.06% digitonin or 0.03% LMNG.Full-length or truncated human NCX1  for 2 h at 37 °C. The reaction was quenched by adding 30 mM iodoacetamide to the digestion solution and placing it at RT for 30 min in the dark. After verifying the complete papain cleavage by SDS-PAGE, the Fc fragment was separated from Fab using anion exchange chromatography column HiTrap Q, and the flow through containing Fab was collected and concentrated for complex formation and structural analysis.Monoclonal antibodies against full-length human NCX1 were raised by the Vector and Gene Therapy Institute (VGTI) at Oregon Health & Science University (OHSU) using standard methods. The purified NCX1 in LMNG was used for immunization. Antibodies were screened by ELISA using both the native and denatured exchanger proteins as substrate and western blot to select clones that recognized natively folded NCX1. 2E4 IgG was identified as an NCX1-specific monoclonal antibody from the screening, and subsequent fluorescence-based size-exclusion chromatography (FSEC) analysis confirmed the complex formation between NCX1 and 2E4 IgG. To generate the Fab fragment, the purified 2E4 IgG (provided by VGTI) was subject to papain digestion in PBS buffer containing 10 mM EDTA, 10 mM 66. Sequences of the resulting PCR products with the size of ~500 bp were analyzed using the IMGT database (http://www.imgt.org/) to determine possible variable regions of the light chain and heavy chain. The sequence of the 2E4 clone was verified by mapping the Fv to the EM density of the Fab.To obtain the sequence of the 2E4 antibody, total RNA was isolated from the hybridoma using PureLink RNA Mini Kit (Invitrogen) following the manufacturer’s protocol. Total RNA was subjected to reverse transcription reactions using Superscript III reverse transcription kit (Invitrogen), and the resulting cDNA was used as a template in PCR reactions with degenerated primers to amplify the antibody variable regions2+-activated exchanger proteins, 0.5 mM CaCl2 was added to protein samples 1 h before grid preparation.For the generation of NCX1-Fab 2E4 complex, NCX1 in digitonin was incubated with purified Fab in a molar ratio of 1:1.2 (NCX1:2E4 Fab) for 3 h. The samples were further purified by size-exclusion chromatography in buffer A supplemented with 0.06% digitonin. The peak fractions were collected and concentrated to 5–6 mg/ml for cryo-EM analysis. To obtain the CaHuman NCX1-Fab 2E4 samples (5–6 mg/ml) in various conditions were applied to a glow-discharged Quantifoil R1.2/1.3 300-mesh gold holey carbon grid , blotted for 4.0 s under 100% humidity at 4 °C and plunged into liquid ethane using a Mark IV Vitrobot (FEI).−/Å2. All data were collected using the CDS mode of the K3 camera with electron dose rates between 7 and 9 e-/pixel/s.For the data of NCX1-Fab (2E4) samples in various states, movies were acquired on a Titan Krios microscope (FEI) operated at 300 kV with a K3 Summit direct electron detector (Gatan), using a slit width of 20 eV on a GIF-Quantum energy filter. Images were recorded with Serial EM in super-resolution counting mode with a super-resolution pixel size of 0.415 Å. The defocus range was set from −0.9 to −2.2 μm. Each movie was recorded for about 5 s in 60 subframes with a total dose of 60 e67. The CTF parameters of the micrographs were estimated using the GCTF program68. After CTF estimation, micrographs were manually inspected to remove images with bad defocus values, ice contamination, or carbon. Particles were selected using Gautomatch  and extracted using a binning factor of 3. Next, particles were subjected to 2D classification, ab initio modeling, and 3D classification. Classes that showed clear features of the NCX1-Fab complex were subjected to 3D auto-refinement and another round of 3D classification without performing particle alignment using a soft mask around the protein portion of the density.Cryo-EM data were processed following the general scheme described below with some modifications to different datasets  using the 0.143 criterion72. Local resolution was estimated using cryoSPARC.The best-resolving class was then re-extracted with the original pixel size and further refined. Beam tilt, anisotropic magnification, and per-particle CTF estimations were performed. After the initial processing in Relion 3.173, facilitated by the predicted TM structure from Alphafold (based on NCX_Mj) and previous X-ray structure of the CBD domain (PDB: 3US9)58. Models were manually adjusted in Coot and refined against maps using the phenix.real_space_refine with secondary structure restraints applied74. The final NCX1structural model contains residues 17–248, 370–467, 482–644, 652–698, 707–718, and 738–935. For the bound Fab fragment, only the variable region (Fv) was modeled.The EM map of human NCX1 in the apo inactivated state shows high-quality density for de novo model building in Coot2+ were classified into two groups. The apo-inactivated NCX1 structure could be directly docked into the EM map from group I particles without adjustment. The EM maps of NCX1 in the Ca2+-activated state from group II particles are relatively poor, particularly at the TM region To model the Ca2+-activated structure, the TM structure from apo NCX1 and the X-ray structures of the Ca2+-bound CBD1 (PDB: 2DPK) and CBD2 (PDB: 2QVM)57 were fitted into the focused maps of the TM region and cytosolic region, respectively, as rigid-bodies followed by manual adjustment in Coot and refinement in Phenix against the overall map. The statistics of the geometries of the models were generated using MolProbity75. All the figures were prepared in PyMol , Chimera76, and ChimeraX77. The solvent-accessible cavities were calculated using the program Caver78.The particles from the NCX1 sample prepared in the presence of 0.5 mM Ca53. Oocytes were kept at 18 °C for 4–7 days. Outward human Na+–Ca2+ exchanger (NCX1) currents were recorded using the giant patch technique in the inside-out configuration. The external solution (pipette solution) contained the following (mM): 100 NMG (N-methylglucamine), 10 HEPES, 20 TEAOH (tetraethyl-ammonium hydroxide), 0.2 niflumic acid, 0.2 ouabain, 8 Ca(OH)2, pH = 7 (using MES); bath solution (mM): 100 CsOH or 100 NaOH, 20 TEAOH, 10 HEPES, 10 EGTA or HEDTA (N-(2-Hydroxyethyl) ethylenediamine-N,N’,N’-triacetic acid) and different Ca(OH)2 concentrations to obtain the desired final free Ca2+ concentrations, pH = 7 (using MES). Free Ca2+ concentrations were calculated according to the MAXc program and confirmed with a Ca2+ electrode.The wild-type human NCX1 and its mutants were cloned into a pGEMHE vector and expressed in oocytes for electrophysiological recordings. RNA was synthesized using mMessage mMachine (Ambion) and injected into Xenopus laevis oocytes as described in+ with 100 mM Na+ using a computer-controlled 20-channel solution switcher. As NCX1 does not transport Cs+, there is no current, and only upon application of Na+ does the exchange cycle initiate. Data were acquired online at 4 ms/point and filtered at 50 Hz using an 8-pole Bessel filter. Experiments were performed at 35 °C and at a holding potential of 0 mV. The effects of the Na+-dependent inactivation were analyzed by measuring fractional currents calculated as the ratio of the steady-state current to the peak current  for the recordings at 100 mM Na+ and 12 µM Ca2+. All P values were calculated using an unpaired, two-sided Welch’s t-test.NCX1 currents were evoked by the rapid replacement of 100 mM CsFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Reporting SummarySource Data


In addition, calcium signals participate in the accumulation process of GABA under stress. Moreover, GABA also transmits calcium signals through receptors to trigger downstream signaling cascades. In conclusion, understanding the role of GABA in this defense response provides a theoretical basis for applying GABA in agriculture and forestry and feasible coping strategies for plants in complex and changeable environments.Gamma-aminobutyric acid (GABA) is a four-carbon non-protein amino acid that acts as a defense substance and a signaling molecule in various physiological processes, and which helps plants respond to biotic and abiotic stresses. This review focuses on the role of GABA’s synthetic and metabolic pathways in regulating primary plant metabolism, redistributing carbon and nitrogen resources, reducing the accumulation of reactive oxygen species, and improving plants’ tolerance of oxidative stress. This review also highlights the way in which GABA maintains intracellular pH homeostasis by acting as a buffer and activating H Intracellular pH homeostasis is the basic cell activity required to maintain normal plant growth, regulate plant growth and development, and respond to environmental stress. ROS enhances resistance by inducing cell damage through oxidative properties to promote plant morphology and structural change. It can also interact with transcription factors to regulate the expression of defense-related genes cyt is pumped out of the cell by a “pump out” mechanism, and [Ca2+]cyt returns to its resting state [Under different environmental stimuli, intracellular Caome time . In normessenger ; this pressenger . Calciumng state .2+/CaM. When plants are subjected to temperature stress, drought, salinity, or mechanical treatment, the intracellular Ca2+ concentration increases, promoting the stimulation of Ca2+/CaM to GAD and causing the accumulation of GABA. For example, the accumulation of GABA in apples stored under controlled atmosphere conditions is due to the highly conserved CaMBD of MdGAD1 and MdGAD2, whose activity and spectral properties are regulated by Ca2+/CaM and acidic pH [2+/CaM complex in rice bran; when the concentrations of Ca2+ and CaM are 200 μmol L−1 and 150 nmol L−1, respectively, the GAD of rice bran is significantly increased, by three times [2+ and BoCaM2 levels in Broccoli sprouts have been raised to form Ca2+/CaM complexes that bind to GAD. Similarly, exogenous calcium-activated GAD promoted GABA accumulation in shredded carrots and fresh-cut pears [The accumulation of GABA under biotic and abiotic stress is related to the regulation of Cacidic pH . GAD is ee times . In the ut pears ,112,113.A receptors, which leads to calcium increases in these cells [+ dependent. ATP induces an increase in intracellular Ca2+ concentration which in turn increases the activity of the Na+/Ca2+ exchanger (NCX), and the accumulation of Na+ in astrocytes then leads to the decrease of GAT activity [GABA transmits calcium signals through receptors. The process of transporting GABA into the cell by sodium-dependent GABA receptors in horizontal cells from the skate retina causes depolarization of membrane potential and activates L-type voltage-gated calcium channels to transport extracellular calcium ions into the cell, thus triggering the release of calcium ions from the intracellular calcium pool . Similarse cells . In planse cells . ALMTs ase cells ,117. Forse cells . In addiactivity . In planactivity .+-ATPase and can affect Ca2+ transport through ALMTs, thus triggering downstream signaling cascades. Although progress has been made in the study of GABA in plants, there are still many questions to be explored, and the polyamine pathway synthesis of GABA has not been thoroughly studied. There are many Ca2+ channels in the plasma membrane of cells, but which calcium channel is activated by GABA remains unclear. Few studies have examined GABA’s signaling function through ROS.In recent years, an increasing number of studies have been conducted on the role of GABA in plant defense response. The synthesis of GABA uses Glu as a precursor and participates in the TCA cycle. Therefore, GABA can be used as a bridge to regulate carbon and nitrogen balance and participate in plant carbon and nitrogen metabolism resistance to various stresses. The accumulation of GABA reduces excess ROS in cells and improves the plant’s tolerance to oxidative stress. GABA can also act as a signaling molecule in response to various stresses. For example, GABA can maintain pH balance in plants by activating HOverall, the positive role of GABA in plant resistance was analyzed in detail in this paper, providing a theoretical basis for the application of GABA in agriculture and forestry. Exogenous GABA can be applied as an effective and sustainable strategy to improve plant tolerance under field conditions to combat complex and changeable environmental problems.


However, excessive mitochondrial Ca2+ uptake causes toxic mitochondrial Ca2+ overloading that triggers numerous cell death pathways which destroy the NVU. Mitochondrial damage is one of the earliest pathological events in stroke. Drugs that preserve mitochondrial integrity and function should therefore confer profound NVU protection by blocking the initiation of numerous injury events. We have shown that mitochondrial Ca2+ uptake and efflux in the brain are mediated by the mitochondrial Ca2+ uniporter complex (MCUcx) and sodium/Ca2+/lithium exchanger (NCLX), respectively. Moreover, our recent pharmacological studies have demonstrated that MCUcx inhibition and NCLX activation suppress ischemic and excitotoxic neuronal cell death by blocking mitochondrial Ca2+ overloading. These findings suggest that combining MCUcx inhibition with NCLX activation should markedly protect the NVU. In terms of promoting NVU repair, nuclear hormone receptor activation is a promising approach. Retinoid X receptor (RXR) and thyroid hormone receptor (TR) agonists activate complementary transcriptional programs that stimulate mitochondrial biogenesis, suppress inflammation, and enhance the production of new vascular cells, glia, and neurons. RXR and TR agonism should thus further improve the clinical benefits of MCUcx inhibition and NCLX activation by increasing NVU repair. However, drugs that either inhibit the MCUcx, or stimulate the NCLX, or activate the RXR or TR, suffer from adverse effects caused by undesired actions on healthy tissues. To overcome this problem, we describe the use of nanoparticle drug formulations that preferentially target metabolically compromised and damaged NVUs after an ischemic or hemorrhagic stroke. These nanoparticle-based approaches have the potential to improve clinical safety and efficacy by maximizing drug delivery to diseased NVUs and minimizing drug exposure in healthy brain and peripheral tissues.The neurovascular unit (NVU) is composed of vascular cells, glia, and neurons that form the basic component of the blood brain barrier. This intricate structure rapidly adjusts cerebral blood flow to match the metabolic needs of brain activity. However, the NVU is exquisitely sensitive to damage and displays limited repair after a stroke. To effectively treat stroke, it is therefore considered crucial to both protect and repair the NVU. Mitochondrial calcium (Ca Stroke is the third leading cause of death and the tenth major contributor to disabilities requiring long-term care . Most stThe NVU is the basic component of the blood brain barrier . This in2+ uptake to rapidly buffer cytosolic Ca2+ and stimulate energy production in multiple cellular components of the NVU  and sodium/Ca2+/lithium exchanger (NCLX), respectively  mobilize diverse vascular and glial cell subtypes that suppress inflammation and rebuild the NVU ; essential MCUcx regulator (EMRE), and MCU regulator 1 (MCUR1)  . Three-d (MCUR1) . Each MC (MCUR1) . The carhe MCUcx . MCUb acnel pore . EMRE, fnel pore . In brainel pore . Importaactivity . MCUR1 avity . In these studies, CRISPR-Cas9 was employed to knockout MICU1 in mouse embryonic fibroblasts  is composed of two compartments known as cristae and the inner boundary membrane (IBM) . Cristae2+ influx into the cytosol  via the voltage-dependent anion-selective channel (VDAC)  on the cell surface triggers Ca cytosol . Ca2+ thl (VDAC) . Howevernd MICOS . Under re matrix , 2022. Wf the CJ . MICU1 tactivity , 2022.Drosophila, a loss-of-function mutation in the MICU1 caused lethality that was not mitigated by a loss-of-function mutation in the MCU which blocks mitochondrial Ca2+ uptake  in peaks along the cristae was lost. This suggests that disruption of the CJ in MICU1 knockout cells allows protons to escape resulting in depolarization of the Ψm  toxicity  concentrations. The subsequent induction of Na+/hydrogen exchange by elevated Na+ concentrations in matrix would thus increase the ΔΨm by enhancing proton pumping into the IMS  proteases cause neurodegeneration in spinocerebellar ataxia type 28, hereditary spastic paraplegia 7, and spastic ataxia 5 . Heterozrloading , 2015. Lhe MICU1 . This oche MICU1 . These f2+/H+ uniporter that utilizes the high proton gradient generated by the Ψm to power Ca2+/H+ exchange  MCU ablation, inducible MCU deletion in cardiac myocytes at maturity protected the heart from ischemic/reperfusion injury . These d2+ from mitochondria  superfamily , glia , and neurons that communicate with each other to regulate CBF . As cerex topics .Endothelial cells form the wall of cerebral blood vessels . Tight jPericytes are located within the endothelial basement membrane that surrounds blood vessels . This poAstrocytes are situated between endothelial cells and neurons . This st2+ concentrations are thought to activate Ca2+-sensitive phospholipase A2 resulting in the increased production and release of prostaglandins that dilate the cerebrovasculature by acting on vascular smooth muscle cells and pericytes  capillaries . Norepinericytes . Neurovaericytes .Oligodendrocyte progenitor cells (OPCs) differentiate into myelin-producing oligodendrocytes essential for functional recovery after a demyelinating injury . HoweverMicroglia are resident macrophages in the brain . Approxi2+, producing ATP, and synthesizing lipids in vast amounts  are preferentially located in the astrocyte endfoot  to enter them by way of Ca2+-permeable ionotropic glutamate receptors  from hemoglobin (2+ uptake by the MCUcx (Box 5) that induces further ROS over-production and mitochondrial injury (2+ buffering and ATP synthesis (Box 7) exacerbate the death of vascular cells, glia, and neurons that comprise the NVU  resulting in profound neural cell protection , improved functional recovery 21 days later compared to the control group (n = 25).Demyelination of large white matters tracts in the brain is also major pathological feature of ischemic and hemorrhagic stroke . Human sHowever, T3 has not always been found to promote remyelination. Deletion of myelin regulatory factor results in pervasive CNS demyelination over a 10- to 12-week period followed by gradual but incomplete myelin repair . In thisThyroid hormone produces a variety of adverse side effects such as changes in body weight, sweating, diarrhea, cold intolerance, tachycardia, irregular heart beats, menstrual changes, joint pain, skin rash, and bone loss . As a reMost putative neuroprotective and restorative drugs fail in the clinic because of lack of efficacy . AdverseDelivery of drugs to the brain after intravascular administration is difficult due to the BBB, which poses both a structural and a metabolic restriction on drug transport and uptake into the brain. In the normal BBB, permeation is highly controlled through transcellular transport mechanisms such as passive diffusion, carrier-mediated transport and various efflux transporters, or transcytosis  are typically constructed using polymers of different molecular weights  such as poly (lactic acid) (PLA), poly (glycolic acid) (PGA), poly (lactic-co-glycolic acid) (PLGA), and polyethylene glycol (PEG) and lipid-PEGs, or PEG alternatives with reduced immunogenicity . These pDespite the strong therapeutic potential of NP drug delivery for stroke, a viable clinical approach has yet to be developed. One strategy that has received considerable attention is the use of cell-penetrating peptides (CPPs) to promote NP uptake and cellular targeting in the brain . PerhapsTransferrin receptors are attractive targets because these receptors are present on NVU endothelial cells but not endothelial cells in other parts of the body . The GYRThese CPPs can be attached to the end of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-maleimide PEG (DSPE-PEG2-5K) using maleimide chemistry to produce DSPE-PEG2-5K-TAT, DSPE-PEG2-5K-GYR, and DSPE-PEG2-5K-CLE . CombiniCompared to the IV route, the IN route is more convenient and reduces systemic drug exposure . More imFor example, to increase the retention time at the nasal mucosal surface and enhance absorption by nasal epithelia, NPs have been coated with low MW polysaccharides (PSACC), chitosan, carboxymethylcellulose, and dextrin . This caA variety of obstacles for NP-based drug delivery have been elegantly described in several recent reviews . Among tSystemic NPs are often designed with a stealth component using PEG or PEG-conjugated lipids to enhance the circulation time and access to the target site. However, the use of PEG is limited by the immunogenic potential of this polymer resulting in adverse allergic reactions . AntibodAfter exposure to biological fluids, a protein corona rapidly forms on the surface of NPs . Over 301–45 (Amyloid β25–35) that specifically interacts with the lipid-binding domain of apolipoproteins  and investigational (IRX4204) drugs for the treatment of ischemic and hemorrhagic stroke, we have described the use of NP-based drug delivery strategies. For example, NPs can be designed to preferentially deliver their payload to metabolically compromised (vulnerable) or damaged NVUs. Furthermore, we have discussed how combining the rapid IV administration of NPs containing Ru265 and Bay-607550 with the repeated IN delivery of NPs loaded with IRX4204 and T3, should achieve positive clinical outcomes in the treatment of ischemic and hemorrhagic stroke by protecting vulnerable and repairing damaged NVUs.First, there is a demand for therapeutic agents with novel mechanisms of action. More specifically, there is an urgent need for promising drug candidates to treat ischemic and hemorrhagic stroke. To tackle this problem, we have outlined the various neural protective benefits of preventing mitochondria CaSecond, the development of NP-based drug products has its own challenges and complexity. However, the remarkable success of NP-based vaccines in combating the recent COVID-19 pandemic has demonstrated that the large-scale production of NPs is an achievable goal. During the development of neuroprotective and restorative treatments for stroke, it will be crucial to employ rigorous preclinical testing guidelines for effective translational research in stroke . Further2+ handling and nuclear hormone receptors with small molecules and preferentially delivering them to where they are required in the brain may very well tip the risk-benefit balance toward renewed investment in therapeutic development for ischemic and hemorrhagic stroke.Third, drug development for neurodegenerative disorders still suffers from an exceptional high failure rate. Hence, major biotechnology companies and pharmaceutical firms are reluctant to commit the considerable resources required for such risky drug discovery efforts. Nevertheless, we have described how recent innovations in targeting mitochondrial CaAll authors contributed to the article, agreed to be accountable for the content of this work, and approved the submitted version.


BCR::ABL1 has recently been recognized as a distinct subtype in international classifications. Distinguishing it from myeloid blast crisis chronic myeloid leukemia (BC-CML) without evidence of a chronic phase (CP), remains challenging. We aimed to better characterize this entity by integrating clonal architecture analysis, mutational landscape assessment, and gene expression profiling. We analyzed a large retrospective cohort study including CML and AML patients. Two AML patients harboring a BCR::ABL1 fusion were included in the study. We identified BCR::ABL1 fusion as a primary event in one patient and a secondary one in the other. AML-specific variants were identified in both. Real-time RT-PCR experiments demonstrated that CD25 mRNA is overexpressed in advanced-phase CML compared to AML. Unsupervised principal component analysis showed that AML harboring a BCR::ABL1 fusion was clustered within AML. An AML vs. myeloid BC-CML differential expression signature was highlighted, and while ID4 (inhibitor of DNA binding 4) mRNA appears undetectable in most myeloid BC-CML samples, low levels are detected in AML samples. Therefore, CD25 and ID4 mRNA expression might differentiate AML with BCR::ABL1 from BC-CML and assign it to the AML group. A method for identifying this new WHO entity is then proposed. Finally, the hypothesis of AML with BCR::ABL1 arising from driver mutations on a BCR::ABL1 background behaving as a clonal hematopoiesis mutation is discussed. Validation of our data in larger cohorts and basic research are needed to better understand the molecular and cellular aspects of AML with a BCR::ABL1 entity.Acute myeloid leukemia (AML) with  BCR::ABL1 rearrangement, encoded by the Philadelphia (Ph1) chromosome and resulting from t translocation, is the molecular hallmark of chronic myeloid leukemia (CML), a well-known myeloproliferative neoplasm [The  from t9;2 translocation in recent international classifications [BCR::ABL1 rearrangement can, therefore, be detected in AML arising from BC-CML, in de novo AML, and throughout the course of AML, as a secondary event. In this context, we believe that improved molecular analysis of AMLs harboring a BCR::ABL1 rearrangement holds excellent promise for refining their classification and selecting the best therapeutic strategies.The  with t9;2 has paved the way for accurate characterization of the mutational landscape of AMLs ,7,8. MorCD25 mRNA expression was higher in BC-CML patients than in AML patients.CML stem cells (LSCs) are characterized by several surface markers, including CD25 , CD26 (dipeptidyl peptidase-4 or DPP4), and IL1-RAP (interleukin-1 receptor accessory protein) ,12. CD25BCR::ABL1 could be molecularly distinguished from acute transformations of CML and definitively assigned as typical AML. To this end, we used targeted NGS to determine the molecular landscape of AML harboring a BCR::ABL1 fusion. We performed colony-forming cell (CFC) assays to understand the clonal architecture of these leukemias. We also examined CD25, CD26, and IL1-RAP mRNA expression to highlight a potential marker for BC-CML or AML. Finally, we analyzed the mRNA expression data obtained using RNA-Seq in an attempt to stratify patients according to their disease.In this paper, we tried to determine whether AML with BCR::ABL1 rearrangement have often been described as de novo leukemias and, more rarely, as a secondary event during disease relapse. Two AML patients harboring a BCR::ABL1 fusion were identified in our institution and extensively analyzed. The first patient (AML/BA-1) is representative of a de novo AML with BCR::ABL1, without CML history. As such, it can be distinguished from BC-CML. He was a 47 year old man with hyperleukocytosis (292 × 109/L) and 75% peripheral blasts at diagnosis  but died rapidly of multi-visceral failure associated with cerebral hematoma.AMLs carrying the iagnosis . Bone maBCR::ABL1 acquisition upon first AML relapse. She was a 61 year old woman with pancytopenia and 63.5% blasts in the peripheral blood at diagnosis. Bone marrow aspiration revealed massive infiltration by granular myeloblasts (92%), and flow cytometry confirmed the myeloid phenotype (The second patient (AML/BA-2) represents an example of henotype .TET2 (two variants) and NPM1 genes. The patient achieved complete cytologic remission after receiving the standard induction regimen (daunorubicin + cytarabine) and consolidation therapy (cytarabine). A hematologic relapse was observed 9 months after the disease onset, and a Philadelphia chromosome was detected. Molecular analyses characterized an atypical BCR::ABL1 e6a2 rearrangement in addition to previously identified variants. Before allogeneic stem cell transplantation, the patient was treated with salvage chemotherapy . Five years later, the patient remains in molecular remission (NPM1 and BCR::ABL1 mRNA transcript follow-up).The patient was subsequently diagnosed with AML-FAB-M1. A normal karyotype was found, and NGS identified three pathogenic variants in the BCR::ABL1 fusion and specific mutations were researched in each CFC using qRT-PCR. The clonal evolution of the disease was then described using CFC assays and NGS/RNA-Seq data.To determine the sequence of mutation acquisition at the hematopoietic stem cell (HSC)/progenitor level, CFC assays were performed using a bone marrow sample at diagnosis (patient AML/BA-1) or at relapse (patient AML/BA-2). At day +14, clonogenic cells (CFU-GM and CFU-G) were harvested from the methylcellulose. BCR::ABL1 fusion and the IDH2/NPM1 variants was evaluated from 50 individual CFCs. These experiments revealed a major BCR::ABL1-NPM1-IDH2 sequence and a minor BCR::ABL1-IDH2 one  as secondary cooperative events.For the AML/BA-1 patient, the presence of IDH2 one . Most HSClonogenic assays were also used to understand the clonal architecture from the bone marrow NGS or RNA-Seq results. In contrast to the CFCs, the bone marrow bulk cells presented all three genetic abnormalities with equal frequency .TET2/NPM1 variants and BCR::ABL1 fusion was estimated from 44 individual CFCs representative of the relapse. Clonogenic assays highlighted a TET2 (K148fs)-TET2 (G1275R)-NPM1-BCR::ABL1 sequence (BCR::ABL1 fusion operated as a secondary event (proliferating mutation), as expected. NGS and RNA-Seq analyses should also be interpreted based on the clonal architecture determined by the CFC assay.For patient AML/BA-2, the presence of the sequence . It shouBCR::ABL1-expressing cells.As shown in BCR::ABL1.The major clonal characteristics of the AML/BA-1 and AML/BA-2 patients are summarized in CD25 mRNA expression was higher in advanced-phase CML than in CP-CML and AML [CD25, CD26, and IL1-RAP mRNA expressions as potential biomarkers to distinguish advanced-phase CML from AML. Three sub-cohorts, including 26 AML patients, 33 CP-CML patients, and 15 AP/BC-CML patients, were analyzed. It should be noted that the advanced phases of CML include AP-CML and BC-CML, as detailed in BCR-ABL1-positive AML were not included in the analysis.We previously showed that  and AML . Here, wCD25 mRNA was significantly overexpressed in advanced-phase CML compared to CP-CML and AML  or a lack of expression in all samples (ROS1).The RNA-Seq expression data from AML and CML patients already analyzed for fusions and variant detection were extracted and subsequently analyzed . These dBCR::ABL1-positive AML samples were found to be clustered within the AML samples . Regarding the AML/BA-2 patient, the progression from de novo AML  to relapse  occurred without any significant changes in the gene expression profile. The same investigations based on the sample subtypes allowed for good stratification of the CML samples   B. Converp-values, nine genes appear to be differentially expressed between AML and M-BC-CML. In particular, MYD88, PICALM, and RARA are overexpressed in M-BC-CML with log2 fold changes of 1.22, 1.16, and 1.37, respectively  is a very attractive marker for distinguishing BC-CML from AML.The transcriptome-normalized matrix was used for supervised analysis, highlighting the genes differentially expressed between the myeloid blast crisis CML and AML samples A. After ectively . On the istances B. These ID4 using RNA-Seq and CD25 (or CD26) using qRT-PCR. To verify their efficacy in distinguishing BCR::ABL1 AMLs, we performed CD25 mRNA quantification on the entire RNAseq sub-cohort. The results for the CD25 and ID4 expression in the AML and M-BC-CML samples are presented in This study identified two molecular markers: BCR::ABL1-positive AML and other AML samples  is observed (ABL1 expression) in the BCR::ABL1-positive AML subtype. We also show a significant down-regulation of PDCD1LG2 in BCR::ABL1-expressing AML . PDCD1LG2 (programmed cell death 1 ligand 2) encodes an immune checkpoint receptor ligand. It should be noted that this gene was not differentially expressed between the BC-CML and AML samples.Lastly, differential expression analyses were performed between the  samples B. After observed . This reBCR::ABL1”, also called “AML with t”, as a distinct entity [BCR::ABL1 could be established without evidence of CML history. Compared to myeloid BC-CML, AML with BCR::ABL1 seemed to be characterized by the absence of basophilia and a karyotype with less than 100% Ph1 chromosomes [BCR::ABL1 from M-BC-CML.New AML classifications have validated “AML with  with t9;2 to verify that BCR::ABL1 is the first event of the multi-step process , (2) to highlight the presence of typical AML abnormalities in addition to BCR::ABL1 (using NGS and RNA-Seq), but also (3) to eliminate “specific” BC-CML mutations, such as ABL1 TKD mutations.Although other mutations may play a role in disease prognosis, the  for CML ,17. AMLs for CML . Acquisi is rare . In thisiagnosis ,25,26,27nhibitor ,29. Not BCR::ABL1 share a characteristic mutational landscape. Various approaches are available today to answer these questions, from older methods such as fragment analysis or Sanger sequencing to more global methods such as NGS or RNA-Seq. Overall, the AML driver genes include (according to frequency) FLT3, NPM1, DNMT3A, NRAS, IDH1/2, RUNX1, TET2, WT1, ASXL1, PTPN11, SRSF2, TP53, CEBPA, BCOR, KMT2A, and KRAS [One of the major issues is whether or not AMLs with and KRAS . Howeverand KRAS ,31. It mand KRAS .BCR::ABL1) is detected during the progression to the blast phase. Pathogenic variants of RUNX1, ASXL1, and BCR::ABL1 TKD mutations (involved in TKI resistance) are frequently found. BCR::ABL1 mutations are a well-known characteristic feature of TKI-resistant CMLs. RUNX1 mutations may not be a specific marker for M-BC-CML, as they are also found in AMLs . Interestingly, NPM1 mutations (common in AML) appear to be absent or extremely rare in BC-CML [BCR::ABL1 by their mutational landscape? Recent studies based on NGS experiments have shown that the genetic profiles of AMLs harboring a BCR::ABL1 fusion are not significantly different from those of M-BC-CML [NPM1 variant and the absence of a BCR::ABL1-TKD mutation can distinguish Ph1-positive AMLs from M-BC-CMLs and link them to Ph1-negative AMLs [Several studies focusing on M-BC-CML, particularly those based on high-throughput sequencing approaches, have been published in the last decade ,35,36,37n BC-CML ,39,40,41M-BC-CML . Therefoive AMLs .+CD38−) using multicolor flow cytometry [CD25. These findings were confirmed in patients with CP-CML, BC-CML, AML, B-ALL (B-acute lymphoblastic leukemia), and healthy controls. CD25 mRNA transcripts were found to be over-expressed in BC-CML in comparison to all other categories. Based on independent patient cohorts, these data were corroborated in the present study, highlighting their relevance. Notably, CD25 mRNA was shown to be expressed in all samples and significantly overexpressed in BC-CML. All in all, CD25 mRNA expression may be an attractive marker to differentiate BC-CML from AML.Some cell surface markers have been shown to distinguish CML LSCs from AML LSCs  entity  and the absence of ABL1 TKD point mutations. CD25 and ID4 mRNA expression may also be helpful, but this needs to be confirmed in larger patient cohorts.Our work shows that a comprehensive molecular analysis might be necessary to characterize leukemias more likely to correspond to AMLs with the ) entity . IdentifBCR::ABL1 within the AML category. However, the identification of the BCR::ABL1 fusion  as the primary oncogenic event through the clonal ontogeny of these cases of acute leukemia raises several concerns. BCR::ABL1 rearrangement has been sporadically detected at low levels in the blood of healthy individuals [BCR::ABL1 oncogene. Some studies have suggested that BCR::ABL1 expression alone (or low-level BCR::ABL1 expression) may not be sufficient to trigger CML leukemogenesis [BCR::ABL1 fusion could occur within a committed progenitor that has lost its stem cell properties [BCR::ABL1 could be considered a CHOP mutation likely to progress to overt leukemia subsequent to a variable latency period.Overall, our results support the inclusion of AML with ividuals ,50,51,52ogenesis ,54,55,56operties ,58,59. Soperties . ConcernBCR::ABL1, we can speculate that the BCR::ABL1 fusion (primitive event) would act as a clonal hematopoiesis mutation. The subsequent acquisition of leukemogenesis-driving abnormalities such as NPM1 or IDH1/2 mutations could lead to the sudden development of de novo AML without passing through CP-CML. While this hypothesis appears attractive, it nevertheless requires further validation.Based on these data, we can formulate a hypothesis worthy of discussion. In AMLs with BCR::ABL1. Distinguishing these AMLs from myeloid BC-CMLs remains challenging, and fundamental research is required to better understand the molecular and cellular specificity of AMLs with BCR::ABL1. Given the rarity of these AMLs, a multicenter study is needed to analyze a significant number of patients. It would also be helpful to consider a single-cell analysis approach to assess the clonal architecture. In addition, this work highlights the importance of gene expression signatures. In this context, a more global transcriptomic approach could be an attractive perspective for better characterization of AMLs with BCR::ABL1.In conclusion, our study provides some new molecular clues helping to characterize AML with CD25, CD26 or IL1-RAP). Two patients with AML and BCR::ABL1 rearrangements (AML/BA-1 and AML/BA-2) had their bone marrow and peripheral blood extensively analyzed. The details about the patients  are provided in In this study, we analyzed bone marrow or peripheral blood samples from 63 CML patients  and 54 AML patients (37 de novo AML and 17 secondary AML or sAML). Molecular and cellular experiments  were performed on residual sample material . Among all patients, sub-cohort studies provided expression data using RNA-Seq or RT-qPCR . RNA-Seq was performed using the Archer® FusionPlex® Myeloid kit with a target set of 84 genes . After sequencing, the raw data were analyzed using the Archer Analysis bioinformatics system. All libraries (DNA/RNA) were sequenced on the Illumina MiSeq platform .Myeloid-targeted NGS was used to characterize pathogenic variants with their VAFs . The NGS analyses were performed using the Agilent SureSelect Target Enrichment System and a personalized myeloid panel of 85 genes . An in-house bioinformatics pipeline was used to analyze the raw data after sequencing. RNA-Seq was used to characterize the fusion transcripts, to identify some pathogenic variants with their TAFs , and to estimate the mRNA expression of the targeted genes (expressed as log2 expressions using BCR::ABL1 rearrangement. The CFC assays were performed on bone marrow samples at diagnosis (patient AML/BA-1) or relapse (patient AML/BA-2) as previously reported [ABL1 was used as an internal control to discard the samples where RNA extraction had failed. The experiment aimed to classify the clones according to their distinct genetic expression profiles.CFC assays were used to characterize the sequence of genetic events in AML patients carrying a reported . At day CD25, CD26, IL1-RAP, and ABL1 are described in CD25/ABL1, CD26/ABL1, and IL1-RAP/ABL1 were determined using the ΔΔCt method using ABL1 as a housekeeping gene, and a calibrator sample was analyzed in each experiment to ensure good reproducibility of the assays [The total RNA from the whole blood or bone marrow samples was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit , and the qRT-PCR experiments were performed using the StepOnePlus Real-Time PCR System . The primers and TaqMan probes for e assays .www.graphpad.com). Sample logistic regression, total area under the curve (AUC), and statistical analyses were performed using GraphPad Prism. The statistical parameters, including the statistical significance , are reported in the figures, and a p-value  <  0.05 was used as the cutoff.The scatter plots, box-and-whisker charts, and receiver operating characteristic (ROC) curves were generated using GraphPad Prism 9 for macOS  in the R software 4.2.1 environment. Principal component analysis (PCA) was carried out using the “pcatrans” function from the R package transpipe and depended on the “prcomp” R-based function and on the “autoplot” function from the ggfortify R package. Differentially expressed gene analyses were carried out with the “deg” function from the transpipe R package, which depends on the limma algorithm [The bioinformatic analyses performed on the normalized complete transcriptome matrix were carried out with “transpipe” R package version 1.4.0 (available at lgorithm . The vol


IKZF1 are frequent events and well-established markers of adverse risk in acute lymphoblastic leukemia. However, their function in the pathophysiology and impact on patient outcome in acute myeloid leukemia (AML) remains elusive. In a multicenter cohort of 1606 newly diagnosed and intensively treated adult AML patients, we found IKZF1 alterations in 45 cases with a mutational hotspot at N159S. AML with mutated IKZF1 was associated with alterations in RUNX1, GATA2, KRAS, KIT, SF3B1, and ETV6, while alterations of NPM1, TET2, FLT3-ITD, and normal karyotypes were less frequent. The clinical phenotype of IKZF1-mutated AML was dominated by anemia and thrombocytopenia. In both univariable and multivariable analyses adjusting for age, de novo and secondary AML, and ELN2022 risk categories, we found mutated IKZF1 to be an independent marker of adverse risk regarding complete remission rate, event-free, relapse-free, and overall survival. The deleterious effects of mutated IKZF1 also prevailed in patients who underwent allogeneic hematopoietic stem cell transplantation (n = 519) in both univariable and multivariable models. These dismal outcomes are only partially explained by the hotspot mutation N159S. Our findings suggest a role for IKZF1 mutation status in AML risk modeling.Genetic lesions of  Acute myeloid leukemia (AML) is driven and maintained by a heterogenous set of genetic lesions that affect clinical phenotypes and patient outcomes. The recently revised European Leukemia Net recommendations  broaden IKZF1 gene is located on chromosome 7 at 7p12.2 [GATA1 and RUNX1 as well as lineage determination and cell survival [The Ikaros zinc finger (IKZF) family comprises a set of zinc-finger proteins including five members: IKAROS (IKZF1), HELIOS (IKZF2), AIOLOS IKZF3), EOS IKZF4), and PEGASUS IKZF5) [, EOS  conferring poor prognosis [IKZF1 are reported in ~15% of cases while this frequency rises to 30% in high risk pediatric populations [IKZF1 deletions reach 30–50% [IKZF1 to be an independent marker of adverse risk in ALL adjusting for age and cytogenetic ALL subtype, resulting in higher risk of relapse and substantially shortened survival [Genetic lesions of rognosis , 13. In ulations –24. In ah 30–50% , 25, 26,h 30–50% –29. Numesurvival , 30–35.IKZF1 alterations is less clear in AML. Previous studies have reported the frequency of altered IKZF1 in AML to range between 1.2% in a pediatric cohort of 258 patients [IKZF1 in AML in a large scale study seems warranted.While its frequency and impact on patient outcome are well established in ALL, the clinical significance of patients  and 2.6%patients –39. GiveWe retrospectively investigated a cohort of 1606 newly diagnosed and intensively treated AML patients from previously reported multicenter trials (AML96  NCT0018 NCT0018 NCT 001 NCT0089. When noIKZF1 according to the manufacturer’s recommendations as previously reported [IKZF1-mutated patients) confirmed the myeloid phenotype  covering 54 genes Table  that arereported , 46. DNApe Table . An extet-test. If the assumption of normality was violated, continuous variables between two groups were analyzed using the Wilcoxon rank sum test. Univariate analysis was carried out using logistic regression to obtain odds ratios (OR). Time-to-event analysis was performed using Cox-proportional hazard models to obtain hazard ratios (HR). Additionally, the Kaplan–Meier-method and the log-rank-test were used. For survival times, OR and HR, 95%-confidence-intervals (95%-CI) are reported. Median follow-up time was calculated using the reverse Kaplan–Meier method. Statistical analysis was performed using STATA BE 17.0 .Statistical significance was determined using a significance level α of 0.05. All tests were carried out as two-sided tests. Fisher’s exact test was used to compare categorical variables. Normality was assessed using the Shapiro–Wilk test. If the assumption of normality was met, continuous variables between two groups were analyzed using the two-sided unpaired IKZF1 to be altered in 45 cases (2.8%). Alterations were almost entirely heterozygous . Single nucleotide variants (SNV) were the predominant mode of alteration  while insertions  were rare. Indels or deletions were only found in one instance each. Only four alterations lead to a frameshift (8.9%), all of which were predicted to resulted in premature truncation. Alterations of IKZF1 represented more often missense  rather than nonsense  mutations , and exon 8 , while alterations in exon 4 , exon 6 , as well as exon 7  were rare and no alterations were found in exons 1–3. IKZF1 harbors four N-terminal zinc finger domains which enable DNA binding and two C-terminal zinc finger domains for homo- and heterodimerization with other Ikaros proteins [n = 19, 42.2%, Fig. n = 4, 8.9%, Fig. n = 3, 6.7%, respectively) while the second C-terminal zinc finger domain was only altered in one patient  mutations, while the majority  of mutations were detected in dominant clonal constellations , GATA2 , KRAS , KIT , SF3B1 , and ETV6 . In contrast, co-occurring mutations of NPM1 , FLT3-ITD , and TET2  were significantly less prevalent  and were more frequently categorized within the ELN2022 adverse risk group . Table IKZF1-mutated AML.In our cohort of 1606 AML patients, we found ons Fig. . The mosons Fig. . The medons Fig. . IKZF1-ment Fig. . PatientIKZF1 to less frequently present with de novo AML , while there was no significant difference with regard to sAML or tAML. Patients harboring mutated IKZF1 had significantly lower median Hb  and platelet count  at initial diagnosis while white blood cell count, peripheral and bone marrow blast count did not differ. There was no significant difference in age, sex or presence of extramedullary disease manifestations. Table IKZF1 mutation status.Regarding clinical parameters, we found patients with mutated IKZF1 had significantly lower odds to achieve complete remission after intensive induction therapy compared to IKZF1-wildtype patients  compared to non-N159S  and wild-type patients  harbored alterations in IKZF1. The rates between patients with altered and wildtype IKZF1 that underwent allo-HSCT either in first CR or as a salvage therapy did not differ significantly , 3.5% of them , OS was significantly reduced [IKZF1. In RNA sequencing, they delineated three clusters of IKFZ-mutated patients: N159S (40%), co-occurring CEBPA mutations (43%), and others (17%) [The role and implications of d in ALL , 13, whiient age –29, howeient age , 37, whifunction , 33, 47,function . In chrot crisis . Among 2t crisis  found elg et al.  investigted NPM1 . While t reduced . Wang et reduced  found 23rs (17%) . They rers (17%) .IKZF1. In accordance with previous results [IKZF1 to be altered in dominant clonal constellations suggesting these mutations to be earlier events in leukemogenesis. With regard to the co-mutational landscape, we found alterations of IKZF1 to be associated with alterations in RUNX1, GATA2, KRAS, KIT, SF3B1, and ETV6 while concomitant alterations in NPM1, TET2 as well as FLT3-ITD and normal karyotypes were less frequent. The high co-occurrence of alterations in RUNX1 and GATA2 hints at a synergistic pathway in leukemogenesis, arguably converging on NOTCH signaling, with possible dysregulation of lineage determination and perturbance of erythropoiesis and megakaryopoiesis as well as survival regulation in myeloid progenitors [SF3B1 have also been described by Zhang et al. [CEBPA. Although we also observed a substantial number of CEBPA-mutant patients (n = 10), this association did not reach statistical significance. Interestingly, most patients with IKZF1 mutations in the CEBPA-cohort had mutations outside exon 5. IKZF1-mutated AML patients less frequently had de novo AML, however, the rates of sAML or tAML were not significantly increased in our cohort. Jäger et al. [IKZF1 to occur in ~20% of AML cases that arose secondary to myeloproliferative neoplasms suggesting a differential role of deletions and mutations in myeloid leukemogenesis.In our large cohort of 1606 adult AML patients, we found heterozygous SNVs to be the most common mode of alteration while we observed only four frame-shift mutations and only one small deletion of  results , 39, we genitors –21. Co-og et al. , 38, howr et al.  found deIKZF1-mutated AML to show a significantly lower Hb and platelet count upon initial diagnosis, possibly corresponding to the suggested dysregulation of erythro- and megakaryopoiesis. In our cohort, patients with IKZF1-mutated AML were more frequently categorized within the ELN2022 adverse risk group. While deletions of IKZF1 are a well-established marker of adverse outcomes in ALL portraying substantially higher relapse rates and shortened survival [IKZF1 alterations in AML is sparse. In pediatric AML, de Rooij et al. [IKZF1 or monosomy 7 compared to non-affected patients. In the studies by Zhang et al. [IKZF1 was limited to high VAF and only demonstrated for overall survival. In a comprehensive study of multiple genetic lesions, Milosevic et al. [IKZF1 on overall survival in 203 AML cases.With regard to clinical phenotypes, we found patients with survival –35, evidj et al.  found nog et al. , 38, thec et al.  did not IKZF1 mutations to be an independent marker of adverse outcomes in AML. Univariable analyses revealed patients with IKZF1-mutated AML to have significantly lower odds of achieving CR upon intensive induction therapy in line with recent findings by Zhang et al. [IKZF1-wildtype patients. These dismal effects of IKZF1-mutated AML on CR rate, EFS, RFS, and OS persisted in multivariable analyses adjusting for age, de novo or sAML, and ELN2022 categories (which include monosomy 7 in the adverse risk group) for all outcome variables. Interestingly, for the hotspot mutation N159S, we only found significant effects on EFS and OS in multivariable models, while the effect on CR rate and RFS was only present in univariable analysis. This hints at considerable heterogeneity within IKZF1-altered AML. Since the N-terminal zinc finger domains are critical for IKZF1’s DNA-binding function, an alteration in these domains could arguably reduce IKZF1’s ability to bind to DNA and thus impair its role as a tumor suppressor by disrupted regulation of target genes [IKZF1 alterations or IKZF1-wildtype. Even considering our large sample size, the differential effects of other IKZF1 alterations than the N159S hotspot mutation still remain elusive. The heterogeneity of the functional aspects of different IKZF1-mutants has been previously documented for several germline variants, with mutations affecting the highly conserved region in zinc finger 2 appearing to affect most physiological roles of IKZF1, including DNA-binding, transcriptional repression, adhesion, and protein localization [IKZF1 alterations is warranted both in an in vitro and clinical setting in an even larger cohort to elucidate the potential effect of the IKZF1 mutation type. Our findings are, however, limited by the fact that we investigated a Caucasian adult patient sample and thus our results may not necessarily be generalizable to pediatric or non-Caucasian populations. Further, all patients in our analysis received intensive induction regimens while hypomethylating agents or targeted therapy was not applied except for a minority of patients from the SORAML study who received sorafenib in addition to intensive chemotherapy. However, sorafenib did not impact CR rate or OS in the original report [IKZF1 mutations and deletions in such populations as well as external validation in comparable cohorts. Furthermore, preclinical evidence suggests a therapeutic implication of immunomodulatory imide drugs (IMiDs) and targeted therapy in the context of altered IKZF1 in a variety of hematological neoplasms. For instance, lenalidomide causes selective ubiquitination and degradation of IKZF1 and IKZF3 conferring cytotoxicity in multiple myeloma cells [IKZF1 mutation status. Further, IKZF1 cooperates with MLL1/MENIN and combined degradation of IKZF1 via IMiDs as well as MENIN inhibition, i.e., via ziftometinib (KO539) or VTP-50469, has been demonstrated to effectively kill leukemic cells in preclinical studies [IKZF1 mutation status. Moreover, BTX-1188, a myc inhibitor and specific degrader of GSPT1 and IKZF1/3, is currently under investigation in a phase 1 dose-escalation trial (NCT05144334) enrolling patients with advanced solid tumors, non-Hodgkin-lymphomas and AML [IKZF1 mutation status.In our cohort, we found g et al. . Furtherlization . Intereslization . Thus, al report . This wama cells , 55. Thema cells  as well ma cells . The so  studies , 59. Thi and AML , howeverIKZF1 mutations to be recurrent events in a large multicenter cohort of adult AML patients with a hotspot lesion at N159S. AML with mutated IKZF1 displayed a distinct co-mutational pattern hinting at synergistic and convergent pathways contributing to leukemogenesis and resulting in clinical phenotypes associated with cytopenia. Further, we identified mutated IKZF1 to be an independent marker of adverse outcomes in multivariable analyses demonstrating a substantially decreased CR rate and shortened EFS, RFS, and OS, which can only partially be attributed to the hotspot lesion N159S. These findings warrant the further evaluation of IKZF1 mutation status for clinical decision making as well as the development of therapeutic strategies to alleviate the dismal outcomes of IKZF1-mutated AML, for example, using combinatorial strategies including IMiDs.In summary, we found Supplements


We found that PA induced accumulation of mitochondrial ROS and that mitochondrial ROS induced release of lysosomal Ca2+ due to lysosomal Ca2+ exit channel activation. Lysosomal Ca2+ release led to increased cytosolic Ca2+ which induced mitochondrial permeability transition (mPT). Chelation of cytoplasmic Ca2+ or blockade of mPT with olesoxime or decylubiquinone (DUB) suppressed lipotoxicity. Lysosomal Ca2+ release led to reduced lysosomal Ca2+ content which was replenished by ER Ca2+, the largest intracellular Ca2+ reservoir (ER → lysosome Ca2+ refilling), which in turn activated store-operated Ca2+ entry (SOCE). Inhibition of ER → lysosome Ca2+ refilling by blockade of ER Ca2+ exit channel using dantrolene or inhibition of SOCE using BTP2 inhibited lipotoxicity in vitro. Dantrolene or DUB also inhibited lipotoxic death of hepatocytes in vivo induced by administration of ethyl palmitate together with LPS. These results suggest a novel pathway of lipotoxicity characterized by mPT due to lysosomal Ca2+ release which was supplemented by ER → lysosome Ca2+ refilling and subsequent SOCE, and also suggest the potential role of modulation of ER → lysosome Ca2+ refilling by dantrolene or other blockers of ER Ca2+ exit channels in disease conditions characterized by lipotoxicity such as metabolic syndrome, diabetes, cardiomyopathy or nonalcoholic steatohepatitis.While the mechanism of lipotoxicity by palmitic acid (PA), an effector of metabolic stress in vitro and in vivo, has been extensively investigated, molecular details of lipotoxicity are still not fully characterized. Since recent studies reported that PA can exert lysosomal stress in addition to well-known ER and mitochondrial stress, we studied the role of lysosomal events in lipotoxicity by PA, focusing on lysosomal Ca Lysresponse , 12 or dresponse –20. Henc2+ after PA treatment, concentration of lysosomal Ca2+ ([Ca2+]Lys) stained with Oregon Green-488 BAPTA-1 dextran (OGBD) was significantly decreased in HepG2 cells treated with PA  of cytosolic Ca2+ concentration ([Ca2+]i) tracing using Fluo-3 AM staining after treatment with Gly-Phe β-naphthylamide (GPN), a lysosomotropic agent i estimated by Fluo-3 AM staining was significantly increased by PA , the largest intracellular Ca2+ reservoir [2+ emptying occurs  [2+]ER was determined after PA treatment of HepG2 cells using GEM-CEPIA1er [2+-free Krebs-Ringer bicarbonate (KRB) buffer to abolish possible store-operated Ca2+ entry (SOCE) [2+ concentration in the ER ([Ca2+]ER) was significantly reduced  after ER Ca2+emptying  , 31. WheCEPIA1er  in a Ca2y (SOCE) , Ca2+ coced Fig. , suggesting Fig. , which slar Ca2+  and elim2+ depletion by ER Ca2+, we next studied whether ER to lysosomal Ca2+ movement indeed occurs after PA treatment inducing lysosomal Ca2+ release. When PA was removed after treatment for 6 h, a decrease of [Ca2+]Lys by PA treatment was recovered  ethylene diamine (TPEN) that has a low Ca2+ affinity and can chelate ER Ca2+ but not cytosolic Ca2+ [2+]Lys after removal of PA was significantly inhibited /aspartate aminotransferase (AST)  Fig. , similarene Fig. . Elevateene Fig. . In addiene Fig. , which iene Fig. . We alsoene Fig. , indicatene Fig. . These r2+ content due to release of Ca2+ from lysosome. Increased cytosolic Ca2+ can mediate diverse beneficial or harmful effects on cells. Since Ca2+ is a well-known inducer of mPT i was increased, which imposed mPT and subsequent death. Cell death due to mPT caused by PA-induced lysosomal Ca2+ release was inhibited by mPT inhibitors such as olesoxime or DUB, showing the role of lysosomal Ca2+ release and consequent mPT in lipotoxicity, which is consistent with previous papers reporting mPT induction by PA Lys is comparable to [Ca2+]ER, lysosomal Ca2+ content might not be sufficient for progression of events requiring Ca2+ flux. Besides ER Ca2+ pool, another source of lysosomal Ca2+ could be endocytic pathway. However, most of endocytic Ca2+ has been reported to be dissipated before reaching lysosome ER and [Ca2+]Lys during incubation in a Ca2+-free medium ensuring recovery of [Ca2+]Lys after removal of PA. Furthermore, blockade of ER → lysosome Ca2+ refilling with dantrolene, an inhibitor of RyR Ca2+ exit channel on ER or chelation of ER Ca2+ by TPEN could inhibit lipotoxicity, which indicates that intracellular Ca2+ flux from ER occurs to sustain lysosomal Ca2+ release, resulting in cell death. Ca2+ flux from ER to lysosome, in turn, induced ER Ca2+ emptying which activated SOCE. Occurrence of SOCE after PA treatment of HepG2 cells was evidenced by no decrease of [Ca2+]ER in cells treated with PA without removal of extracellular Ca2+, reappearance of the decrease of [Ca2+]ER by BTP2 even in the presence of extracellular Ca2+ and STIM1 aggregation co-localized with ORAI1. Functional role of SOCE after treatment with PA was demonstrated by inhibition of lipotoxicity by BTP2 or extracellular Ca2+ chelation with EGTA. Furthermore, occurrence of SOCE and no decrease of [Ca2+]ER in cells treated with PA without removal of extracellular Ca2+ supports that increase of [Ca2+]i after PA treatment is not due to direct release of Ca2+ from ER through SERCA inhibition, since increase [Ca2+]i due to SERCA inhibition would not be affected by extracellular Ca2+ i increase and cell death in vitro by PA could inhibit lipotoxic death of hepatocytes in vivo. Reduction of mitochondrial ROS by dantrolene or DUB on mitochondrial ROS could be due to a feed-forward response leading to further ROS release following Ca2+-mediated mPT Lys, HepG2 cells grown on a chambered coverglass  were loaded with 100 μg/ml OGBD, an indicator of lysosomal luminal Ca2+ for 16 h. After incubation in a fresh media for 4 h, cells were treated with BSA or 500 μM PA for 6 h. After washing with Ca2+-free HEPES-buffered saline (HBS), fluorescence was measured using an LSM780 confocal microscope  and quantified using ImageJ software.To measure [Ca2+]i by confocal microscopy, HepG2 cells grown on chambered coverglass were loaded with 5 μM Fluo-3 AM for 30 min, an indicator of cytosolic Ca2+. After treatment with 500 μM PA for 6 h, cells were washed with Ca2+-free PBS twice, and fluorescence was recorded using an LSM780 confocal microscope. Fluorescence intensity was quantified using ImageJ software. For ratiometric [Ca2+]i measurement, cells were loaded with 2 μM of the acetoxymethyl ester form of Fura-2 in DMEM at 37 °C for 30 min and then washed with a basal Ca2+ solution . Measurements were conducted using MetaFluor on an Axio Observer A1 (Zeiss) equipped with a 150 W xenon lamp Polychrome V , a CoolSNAP-Hq2 digital camera , and a Fura-2 filter set. Fluorescence at 340/380 nm was measured in phenol red-free medium, and converted to [Ca2+]i using the following equation ER), HepG2 cells were grown on chambered coverglass and transfected with a GEM-CEPIA1er plasmid, a ratiometric fluorescent indicator of ER Ca2+. After 24 h, cells were treated with BSA or 500 μM PA for 6 h, and then washed with Ca2+-free KRBB . GEM-CEPIA1er fluorescence was measured using an LSM780 confocal microscope at an excitation wavelength of 405 nm and an emission wavelength of 466 or 520 nm. Fluorescence ratio F466/F520 was calculated as an index of [Ca2+]ER [To determine ER Ca[Ca2+]ER .2+ release, HepG2 cells were transfected with a GCaMP3-ML1 Ca2+ probe, a lysosome-targeted genetically-encoded Ca2+ sensor. Twenty-four h after transfection, cells were treated with BSA or 500 μM PA for 6 h. Perilysosomal Ca2+ release was recorded by monitoring GCaMP3 fluorescence intensity at 470 nm in a basal Ca2+ solution  using a Zeiss LSM780 confocal microscope. GPN, a lysosomotropic agent, of 200 μM was added to evoke lysosomal Ca2+ release. GCaMP3-ML1 fluorescence was calculated as a change GCaMP3 fluorescence (ΔF) over baseline fluorescence (F0).To detect perilysosomal CaYFP-STIM1 and 3xFLAG-mCherry Red-Orai1/P3XFLAG7.1 (provided by Yuan J through Cha S-G) plasmids for 48 h, HepG2 cells were treated with BSA or 500 μM PA for 6 h. Cells were then fixed with 4% paraformaldehyde (PFA) at room temperature for 10 min. Fluorescence images were acquired with an LSM780 confocal microscope, and the co-localization between STIM1 and ORAI1 was examined by calculating Pearson’s correlation coefficient using ZEN software .After transfection with TM Red. Briefly, HepG2 cells were pretreated with 10 μM MitoTEMPO for 1 h, and then treated with 500 μM PA for 24 h. After incubation with 5 μM MitoSOXTM Red at 37 °C for 15 min, fluorescence was measured by flow cytometry using BD FACSVerse or FACSCanto II. Data were analyzed using FlowJo software.Mitochondrial ROS was measured using MitoSOXTM Red at 37 °C for 15 min, and then washed with PBS. Fluorescence images were observed with an LSM710 confocal microscope and fluorescence intensity was quantified using ImageJ software .To determine mitochondrial ROS accumulation in vivo, frozen sections of the liver tissue were incubated with MitoSOX2+ quenching of calcein-AM fluorescence. Cells were treated with PA (500 μM) with or without DUB (200 μM) or olesoxime (100 μM) for 24 h. Cells were then loaded with calcein-AM (1 μM) and CoCl2 (2 mM) at 37 °C for 15 min. Calcein fluorescence was measured by flow cytometry employing BD FACSVerse and FACSCanto II and analyzed using FlowJo software.mPTP opening was assessed by CoMitochondrial membrane potential was determined using tetramethylrhodamine ethyl ester perchlorate (TMRE). After treatment of HepG2 cells with PA for 24 h, cells were incubated with 100 nM TMRE at 37 °C for 30 min, and fluorescence was measured using LSM710 confocal microscope. Fluorescence intensity was quantified using ImageJ software.Eight-week-old C57BL/6N male mice were purchased from Orient Bio . All experiments using mice were performed in accordance with the guidelines of the Public Health Service Policy in Humane Care and Use of Laboratory Animals. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the Department of Laboratory Animal Resources of Soonchunhyang Institute of Medi-bio Science. Ethyl palmitate  was dissolved in water with 4.8% lecithin  and 10% glycerol  to make a mixture containing ethyl palmitate at a concentration of 300 mM. LPS  was dissolved in PBS, and 0.025 mg/kg LPS was injected intraperitoneally into mice. Dantrolene and DUB were dissolved in DMSO and diluted with PBS. For in vivo administration, dantrolene (10 mg/kg), DUB (5 mg/kg) or DMSO solution diluted in PBS was injected into mice that were fasted for 24 h. After 1 h, 300 mM ethyl palmitate or vehicle was administered, followed by LPS injection 1 h later. Blood and liver tissue samples were obtained 24 h later.Serum ALT and AST levels were measured using a Fuji Dri-Chem NX500i chemistry Analyzer .Paraffin-embedded liver tissue blocks were prepared by fixing in 10% neutral buffered formalin . TUNEL staining was conducted using In Situ Cell Death Detection kit  according to the manufacturer’s instructions. Cell death index was expressed as the number of TUNEL-positive cells per field counted from more than 20 fields randomly selected (×200).t-test was used to compare values between two groups. One-way ANOVA with Tukey’s test was used to compare values between multiple groups. Data were considered significant when P < 0.05. Data analysis was performed using GraphPad Prism 8 software .Data are presented as means ± SD of three independent experiments. Two-tailed Student’s