To use this approach, the cell has to be loaded with a Ca2+ sensitive dye  via the patch pipette, which is usually done in the whole-cell patch clamp configuration. However, determining Ca2+ handling properties can be complex and frequently unsuccessful due to the wash-out of intracellular components  during whole-cell patch clamp recordings. We present two Ca2+ imaging datasets from adult substantia nigra dopamine neurons where the 'added buffer approach' was either combined with the 'conventional' whole-cell configuration or with a β-escin based perforated patch clamp configuration. These data can be used to compare the two methods or to draw comparisons with the Ca2+ handling properties of other neuron types. Further details and an in-depth analysis of the new combination of the ‘added buffer approach’ with the β-escin based perforated patch clamp configuration can be found in our companion manuscripts “Analysis of neuronal Ca2+ handling properties by combining perforated patch clamp recordings and the added buffer approach” Multiple processes shape calcium signals in neurons. The spatial and temporal dynamics of these signals are determined by various cellular parameters, including the calcium influx, calcium buffering, and calcium extrusion. The different Ca Specifications TableValue of the Data•The data demonstrate the gain resulting from the proposed β-escin based perforated patch method when investigating calcium dynamics. They constitute a reference against which calcium dynamics studied in other cell types can be compared.•The data are interesting for scientists working on neuronal  calcium dynamics and scientists developing analysis methods for fluorescence indicator-based calcium concentration studies.•The data can be used for investigating the pros and cons of several acquisition protocols in fura-2 calcium measurement, e.g., the excitation wavelengths to use (three wavelengths were used here) or the sampling frequency. They can also be used for experiment planning: how long will a complete set of measurements require with the β-escin perforated patch method, how many transients can or should be used?•2+ handling properties by combining perforated patch clamp recordings and the added buffer approach” in combination with the publicly available analysis software .The data allow anyone to reproduce the analysis presented in the companion paper “Analysis of neuronal Ca1www.hdf5.com) is used throughout. There is one file per experiment. Our file names DA_YMD_EN.h5 have the following meaning: ‘DA’ stands for dopaminergic neuron; ‘Y’ is the year (after 2000); ‘M’ is the month; ‘D’ is the day; ‘E’ stands for experiment and ‘N’ is the experiment number at that date.Our data are stored in two directories containing β-escin perforated patch recordings (‘data_beta_escin’) and whole-cell recordings (‘data_whole_cell’) of substantia nigra dopamine neurons. The HDF5 file format .-S_RO: the read-out standard deviation of the CCD chip .-P: the number of pixels in the `Region Of Interest' (ROI); the read-out variance should be added as many times as there are pixels when one computes the measurement's variance.-P_B: the number of pixels in the `Background Measurement Region' (BMR).The `CCD' group relates to acquisition features with the CCD chip and contains the following 1.2.2scalar datasets resulting from independent calibration experiments .-K_eff_se: the estimated K_eff standard error in (µM).-K_d_hat: the estimated K_d parameter in (µM).-K_d_se: the estimated K_d standard error in (µM).-pipette_concentration: the dye concentration in the pipette in (µM).The DYE group contains parameters relating to the fluorescent dye, namely the following 1.2.3scalar datasets:-T_340: illumination duration at 340 nm in seconds.-T_360: illumination duration at 360 nm in seconds.-T_380: illumination duration at 380 nm in seconds.The ILLUMINATION group contains parameters describing the illumination used, that is the following 1.2.4-AREA: the brain area where the experiment was performed.-DATE: the experiment date.-EXPNAME: the experiment name or reference in the lab book.-LAB: the lab where the experiment was performed.-OBJECT: the type of neuron recorded.-OBSERVER: the person who did the experiment.-ORIGIN: the lab location.-PROTOCOL: a brief description of the experiment type.-WEB: URL of the lab where the experiment was performed.The EXPERIMENT group contains the following datasets:1.2.5X = 1,2,…) as stimulations were applied.The DATA group consists of several subgroups which contain the actual calcium imaging data. There is always a ‘load’ group containing the measurements used for monitoring the loading of Fura-2. There are then as many ‘stimX’ groups , then comes the ADU340B measurements (measurements at 340 nm in the BMR), the ADU360 measurements (at 360 nm in the ROI), the ADU360B measurements (at 360 nm in the BMR), the ADU380 measurements (at 380 nm in the ROI), the ADU380B measurements (at 380 nm in the BMR).-TIME_DELTA: a scalar (see below).-TIME_OFFSET: a scalar (see below).Each of these groups contains 3 data sets:To get the real time of each measurement, multiply the first column of ADU by TIME_DELTA and add TIME_OFFSET. The other columns of the ADU matrix contain the ‘raw’ readings of the P of P_B pixels from the CCD chip.22.12+ imaging, see the companion paper: “2+ handling properties by combining perforated patch clamp recordings and the added buffer approachAnalysis of neuronal Ca” [2]. In brief: Electrophysiological data were recorded with an EPC10 patch-clamp amplifier , which was controlled by the PatchMaster software . The sampling frequency was 10 kHz. The data were low-pass filtered at 2 kHz with a four-pole Bessel filter. The patch pipettes were fabricated using a vertical pipette puller  from borosilicate glass  and had tip resistances between 3 and 5 MΩ. The pipette solution for the whole-cell recordings contained (in mM): 141 K-gluconate, 10 KCl, 10 HEPES, 0.1 EGTA, 2 MgCl2, 3 K-ATP, 0.3 Na-ATP and adjusted to pH 7.2 with KOH. Since ATP and GTP can impair the measurement of Ca2+ handling properties 2+ imaging. In Ca2+ imaging experiments, EGTA was substituted by 100 or 200 µM fura-2 . The protocols for the perforated patch recordings were adapted from Fan and Palade −1; E1378; Sigma). The liquid junction potential, which was calculated with the Patcher's Power Tools plug-in from http://www.mpibpc.mpg.de/groups/neher/index.php?page=software for IGOR Pro 6 , equaled 14.6 mV for normal aCSF and was compensated.For methodical details of the brain slices preparation, patch-clamp recordings, and Ca2.1.1The fluorometric measurements were performed using an Imago/SensiCam CCD camera with a 640 × 480 chip  and a Polychromator IV (Till Photonics), both controlled by the program Vision . The filter and mirror settings for the experiments are provided below. Details of the upright microscope are provided in the companion paper 2.1.22+ indicator fura-2 was used to determine the intracellular Ca2+ concentrations.Fura-2 is a ratiometric indicator suitable to determine absolute Ca2+ concentration when calibrated [via the patch pipette (100 or 200 µM). It was excited at 340 nm, 360 nm, or 380 nm . Emission was detected through a 440 nm long-pass filter (LP440).The Calibrated ,9. Fura-2.1.32+ signals strongly rely on the cell's endogenous and exogenous (added) Ca2+ buffers. The amplitude and decay rate of the free intracellular Ca2+ change with increasing exogenous buffer concentration: The amplitude of free Ca2+ decreases while the time constant increases. With a known added buffer capacity, the time constant of decay can be used to extrapolate the Ca2+ signal to conditions, with only endogenous buffers present. The procedure is fully described in the companion manuscript The kinetics of cytosolic CaAll animal procedures were conducted in compliance with protocols approved by local government authorities  under §4.17.020.Simon Hess: Conceptualization, Investigation, Methodology, Visualization, Writing – review & editing. Christophe Pouzat: Conceptualization, Methodology, Software, Formal analysis, Data curation, Writing – review & editing. Peter Kloppenburg: Conceptualization, Resources, Funding acquisition, Writing – original draft.The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article.


CXC chemokine ligand-12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) and acute myeloid leukemia (AML). However, large-scale data analysis of potential relationship between CXCL12 and AML remains insufficient.Accumulating studies have been made to understand the association between CXCL12 expression data and AML samples from several publicly available datasets. The CIBERSORT algorithm was used to quantify immune cell fractions and the online website of STRING was utilized for gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The statistical analysis and graphical work were mainly performed via the R software.We collected abundant CXCL12 expression was extremely down-regulated in AML. Clinically, low CXCL12 expression was correlated with higher white blood cells (WBCs) (P < 0.0001), more blasts in bone marrow (BM) (P < 0.001) and peripheral blood (PB) (P < 0.0001), FLT3-internal tandem duplications (FLT3-ITD) (P = 0.010) and NPM1 mutations (P = 0.015). More importantly, reduced CXCL12 expression predicted worse overall survival (OS) and event-free survival (EFS) in all AML, non-M3-AML, and cytogenetically normal (CN)-AML patients in three independent cohorts. As for immune cell infiltration, high CXCL12 expressed groups tended to harbor more memory B cells and plasma cells infiltration while low CXCL12 expressed groups exhibited more eosinophils infiltration. GO enrichment and KEGG pathways analysis revealed the potential biological progress the gene participating in.CXCL12 is significantly down-regulated in AML and low CXCL12 expression is an independent and poor predictor of AML prognosis. CXCL12 expression level correlates with clinical and immune characteristics of AML, which could provide potential assistance for treatment. Prospective studies are needed to further validate the impact of CXCL12 expression before routine clinical application in AML. FLT3, NPM1 and CEBPA (Acute myeloid leukemia (AML) represents a heterogeneous disease characterizing by complex molecular signatures, complicated pathogeny and aggressive progression, as well as a poor clinical outcome . Bone mand CEBPA . Unfortund CEBPA . ConsideCXC chemokine ligand-12 (CXCL12), also known as stromal cell-derived factor-1 (SDF-1), is a CXC chemokine that belongs to the large family of chemotactic cytokines. The protein, functioning through its receptor CXC chemokine receptor 4 (CXCR4), plays an important role in hematopoietic cell development, immune system foundation, and retention of hematopoietic stem and progenitor cells in the BM microenvironment (CXCL12 expression has been found to be related to at least 23 different types of tumors (CXCL12 is reported to be overexpressed in chronic lymphocytic leukemia (CLL)  , as wellia (CLL) . Also, Spatients . All theCXCR4 and AML, studies concentrating on CXCL12 and AML remain insufficient, and large-scale analysis of the potential association between the gene and AML hasn’t been performed before. Herein, we adopted similar methods of our previous research  and The Cancer Genome Atlas  in this study. Of these datasets, three of them consist of expression data for bulk primary AML samples , two involve expression data for healthy and AML bone marrow samples (GSE30029 and GSE63270) and one includes expression data for major hematopoietic lineages during the differentiation (GSE42519), respectively. Expression data for FAB AML subtypes and patient characteristics were acquired from TCGA sequencing data. Normal mRNA expression data from Genotype-Tissue Expression (GTEx) database and pan-cancer cell line sequencing data from Broad Institute Cancer Cell Line Encyclopedia (CCLE) database were extracted through their portal websites for analysis.We used six publicly available datasets from Gene Expression Omnibus  was used to quantify immune cell fractions according to the expression pattern of CXCL12. More detailed information for this method is available in Newman AM et al.’s introduction  enrichment and Kyoto encyclopedia of genes and genomes (KEGG) analysis were conducted using the online website of STRING (http://string-db.org). One-way ANOVA was used to test for differences among at least three groups and the Wilcoxon test was used to determine differences in each two-group comparison. All statistical analyses and figure plotting were performed using the R software version 3.5.3 (https://www.r-project.org/). All differences were considered statistically significant at the level of P < 0.05.Low and high eviously . DiffereGSE30029, and all genes were screened in p-value < 0.05 and |logFC| > 0. As obviously displayed in CXCL12 gene was one of the most significantly different genes between AML patients and normal controls. Then, we further verified the differences in GSE63270 . Interestingly, cervix cancer cells showed exclusively lower CXCL12 expression levels than AML and CML, as shown in To explore how database . Apparenll lines , we founCXCL12 expression difference between healthy people and AML patients. A significant decrease of CXCL12 expression was identified in AML cells  in comparison to normal cells  , as indicated in CXCL12 expression difference in eight FAB AML subtypes from M0 to M7 , multipotent progenitor cells (MPPs), granulo-monocyte progenitor cells (GMPs), common myeloid progenitor cells (CMPs), megakaryocyte-erythroid progenitor cells (MEPs), monocytes, and erythrocytes using the single-cell data sequenced by CXCL12. Also, we studied CXCL12 and CXCR4 expression status in the single-cell sequenced data and found CXCR4 expression was abundant in single cells from all patients while CXCL12 was rarely expressed  counts (P < 0.0001) and more percentages of blasts in BM (P < 0.001) and peripheral blood (PB) (P < 0.0001). With regard to frequently mutated genes in AML, low CXCL12 expressers were more likely to have FLT3-internal tandem duplications (FLT3-ITD) (P = 0.010) and NPM1 mutations (P = 0.015) and less likely to have mutated TP53 (P = 0.046) than those with high CXCL12 expression.The patient characteristics with respect to mmarized . Low CXCGSE6891, and GSE10358), and all cohorts were divided base on optional cut-off values determined by the x-tile method. In the TCGA whole-AML cohort, CXCL12low patients showed significantly decreased overall survival (OS)  and NPM1 mutated  patients, which could provide more subdivision proofs for AML based on the dichotomous stratification of CXCL12 expression.The potential relationship between low CXCL12 expression and known clinical factors, we first conducted a univariate analysis in the TCGA dataset and then covered variables with a univariable P ≤ 0.20 in the multivariate Cox proportional hazard model. The multivariate analysis result of CXCL12 expression for OS was displayed in CXCL12 expression maintained a high hazard rate for whole-AML patients , after adjusting age (P < 0.0001), WBC count (P < 0.0001), cytogenetic risk (P = 0.002) and mutation status of TP53 (P = 0.004), RUNX1 (P = 0.023) and DNMT3A (P = 0.012). The predictive significance also applied to CN-AML patients , as indicated.To evaluate whether there were potential interrelationships between CXCL12 expression and immune cell infiltration was systematically evaluated by the CIBERSORT algorithm , T cells gamma delta, and NK cells were rarely infiltrated in both high and low CXCL12 expressed groups.The landscape of the relationship between lgorithm . We founCXCL12 expression, and totally 16 significantly correlated results were displayed in P < 0.001) and B cells  had a positive correlation with CXCL12 expression.To verify the signatures of immune infiltration, we adopted multiple deconvolution methods including TIMER, quanTIseq, xCell, MCP-counter, and EPIC to cross-check the analytic results of CIBERSORT. Then, we made a Pearson correlation analysis between all infiltration results and CXCL12 for biological process (BP), cellular component (CC) and molecular function (MF) were performed on the online website of STRING (http://string-db.org) ; extracellular region, extracellular space, extracellular matrix, intrinsic component of plasma membrane, integral component of plasma membrane, cell periphery, plasma membrane, collagen-containing extracellular matrix, basement membrane, and collagen trimer (CC); binding, growth factor binding, signaling receptor binding, calcium ion binding, cell adhesion molecule binding, glycosaminoglycan binding, chemokine activity, heparin binding, receptor ligand activity, and receptor regulator activity (MF)  , using gity (MF) . GO analanalysis .CXCL12 expression was extremely low in blood and bone marrow compared with other normal tissues and also downgraded in hematological neoplasms among pan-carcinomas. For our purpose, we then validated the expression differences between AML and their normal contrasts in two independent cohorts and found a consistent result. Herein, we concluded that CXCL12 was notably down-regulated in AML. Hitherto, there hasn’t been enough researches to investigate CXCL12 expression in AML, although its receptor CXCR4 has been demonstrated to be overexpressed in AML  and low-risk groups (with CXCL12 up-regulation) respectively based on the expression level of CXCL12.Concerning prognostic relevance, we identified and validated low s of AML  and CXCRn of AML . However signals , and ManML cells , which mCXCL12 and the result showed that the gene dynamically participated in anatomical structure morphogenesis for biological process, extracellular region for cellular component, and binding for molecular function. It was also reasonable to extrapolate protein digestion and absorption pathway was curbed molecularly in some way during the development of AML. It has been widely evidenced that CXCL12 plays an essential role in stem cell anchorage to the BM microenvironment and reduction of CXCL12 concentrations within the BM may interfere with retention and facilitate the egress of cells , multipotent progenitor cells (MPPs), granulo-monocyte progenitor cells (GMPs), common myeloid progenitor cells (CMPs), megakaryocyte-erythroid progenitor cells (MEPs), Monocytes, and Erythrocytes. The dot diameter represents the expression percentage, and the color represents the average expression level. (B) Uniform manifold approximation and projection (UMAP) dimension 1 and 2 plots display CXCL12 (left) and CXCR4 (right) expression overlay onto UMAP space in single cells including HSCs, MPPs, GMPs, CMPs, MEPs, Monocytes, and Erythrocytes.(A) Click here for additional data file.10.7717/peerj.11820/supp-4Supplemental Information 4 Kaplan-Meier estimate for OS (A) and EFS (D) of whole-AML.  Kaplan-Meier estimate for OS (B) and EFS (E) of non-M3-AML.  Kaplan-Meier estimate for OS (C) and EFS (F) of CN-AML.Click here for additional data file.10.7717/peerj.11820/supp-5Supplemental Information 5 Kaplan-Meier estimate for OS (A) and EFS (D) of whole-AML.  Kaplan-Meier estimate for OS (B) and EFS (E) of non-M3-AML.  Kaplan-Meier estimate for OS (C) and EFS (F) of CN-AML.Click here for additional data file.10.7717/peerj.11820/supp-6Supplemental Information 6Multiple deconvolution methods including TIMER, quanTIseq, xCell, MCP-counter, and EPIC are adopted. Each dot represents a single AML patient, and a regression line was delineated to fit the dot plot.Click here for additional data file.10.7717/peerj.11820/supp-7Supplemental Information 7A total of 261 cytokine and cytokine receptor genes are listed.Click here for additional data file.10.7717/peerj.11820/supp-8Supplemental Information 8p-value < 0.05 and |logFC| ＞ 0.All genes were screened with the difference considered significant if adjusted Click here for additional data file.10.7717/peerj.11820/supp-9Supplemental Information 9p-value < 0.05 and |logFC| ＞ 0.All genes were screened with the difference considered significant if adjusted Click here for additional data file.10.7717/peerj.11820/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.11820/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.11820/supp-12Supplemental Information 12Click here for additional data file.10.7717/peerj.11820/supp-13Supplemental Information 13Click here for additional data file.


Using genetically encoded Förster resonance energy transfer calcium sensors for the peroxisome, this study dissects the previously contentious peroxisome involvement in calcium handling in HeLa cells and in cardiomyocytes derived from human induced pluripotent stem cells. Peroxisomes communicate with other cellular compartments by transfer of various metabolites. However, whether peroxisomes are sites for calcium handling and exchange has remained contentious. Here we generated sensors for assessment of peroxisomal calcium and applied them for single cell-based calcium imaging in HeLa cells and cardiomyocytes. We found that peroxisomes in HeLa cells take up calcium upon depletion of intracellular calcium stores and upon calcium influx across the plasma membrane. Furthermore, we show that peroxisomes of neonatal rat cardiomyocytes and human induced pluripotent stem cell–derived cardiomyocytes can take up calcium. Our results indicate that peroxisomal and cytosolic calcium signals are tightly interconnected both in HeLa cells and in cardiomyocytes. Cardiac peroxisomes take up calcium on beat-to-beat basis. Hence, peroxisomes may play an important role in shaping cellular calcium dynamics of cardiomyocytes. In CMs,d in CMs , highligf the ER .2+ can be detrimental for health. Elevated mitochondrial uptake increases mitochondrial reactive oxygen species (ROS) production and is associated with heart failure and ischemic brain injury   as fluorophore  and acceptor  enabling FRET (Measurement of Ca (GECIs) . Ca2+ dyng GECIs . GECIs horophore . GECIs ping FRET  Fig 1A)2+ dynami2+ homeostasis, they may have also antiarrhythmic effects independent of their metabolic roles.Patients with adult Refsum disease due to peroxisome biogenesis disorder develop cardiac arrhythmias and heart failure at advanced disease stages . As the 2+ sensors, and we measure intraperoxisomal Ca2+ after pharmacological stimulation in non-excitable and excitable cells. We show that peroxisomes take up Ca2+ upon cytosolic Ca2+ increase after both ER Ca2+-store depletion and Ca2+ entry to the cells through PM. We also demonstrate that peroxisomes take up Ca2+- in rat CMs and hiPSC-CMs.This work combines the advantages of organelle-targeted GECIs and human induced pluripotent stem cells (hiPSCs). We develop several peroxisomal Ca2+, we used three GECIs with different affinities to Ca2+: D3cpV, D1cpV, and ratiometric pericam  (To test if D3cpV-px senses CaET ratio . To illule (LUT) .2+ addition, showing that the D1cpV-px construct is Ca2+ sensitive  as a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) inhibitor , the product of the cascade, binds to the IP3 receptor on the ER membrane, triggering Ca2+ store release. The cells are then exposed to 1 mM extracellular Ca2+, which leads to store-operated Ca2+ entry and a second Ca2+ elevation in the cytosol.Based on the Caganelles , we deveituation . The sti2+-binding site. Neither histamine addition nor addition of 1 mM Ca2+ showed signal change. We detected, however, mild photobleaching over time (To confirm that this stimulation protocol does not result in changes of the acceptor fluorophore (YFP or Venus) signal of the GECI , we applied the treatment protocol from ver time .2+ based on depletion of the ER. Addition of extracellular Ca2+ resulted in more gradual increase and gradual return to basal levels 2+ concenntration  applying µM Ca2+ . The Ca2d 2.4 µM . For betd 2.4 µM , we deci2+ concentration, and to be able to directly compare peroxisomal Ca2+ handling with that of the cytosol, cells were co-transfected with D3cpV-px and the mApple-based cytosolic Ca2+ sensor R-GECO1, which increases in intensity when binding Ca2+  the hypothesis that Cas on CMs .2+ in these cells is the ER. We used thapsigargin (Tg) to chemically stimulate the CMs  in T-tubules  pump Ca2+ back to the intracellular Ca2+ stores and out of the cells  into the peroxisomes resulting in relatively high peroxisomal Ca2+. This second mechanism is known from the mitochondrial Ca2+ handling, where ER–mitochondria contact sites with tethering proteins generate microdomain with locally high Ca2+ concentration  was generated from (pcDNA-)D3cpV , oligonucleotides OST801 (CACCCCTGTC ATAGGAAGTC TGAAATCGAA GCTCTAG) and OST802 (CTAGAGCTTC GATTTCAGAC TTCCTATGAC AGGGGTG) encoding the C-terminal decapeptide ACOX3 were annealed and cloned into the pENTR/D-TOPO cloning vector, yielding pENTR-ACOX3dp (PST1209). The resulting insert was transferred to pDEST-Venus using the Gateway cloning system.2 at 37°C. For immunofluorescent analysis, cells were fixed with 4% paraformaldehyde for 30 min, and permeabilized using 1% Triton X-100 in PBS for 10 min. After blocking for 30 min with 10% BSA in PBS (blocking buffer) at 37°C, antigens were labelled with primary antibodies at room temperature for 1 h. Rabbit anti-PEX14 (ProteinTech) and mouse anti-PMP70 (Sigma-Aldrich) primary antibody dilution in blocking buffer was 1:500, and 1:200 for mouse anti-RyR (Invitrogen) and goat anti-LTCC (Santa Cruz). Labelling with the secondary antibodies conjugated to Cy3 (Life Technologies), Alexa Fluor 488 (Life Technologies), Alexa Fluor 633 (Invitrogen), or Alexa Fluor 647 (Jackson ImmunoResearch) was done for 1 h (1:500). Cover slips were mounted with ProLong Gold mounting medium with or without DAPI (Thermo Fisher Scientific). Images were taken with Axio Observer Z1 (equipped Zeiss Colibri 7 and with 63× oil Fluar) and deconvoluted. Colocalisation analysis was performed with Fiji (http://fiji.sc/) according to ImageJ User Guide. Mandor’s colocalisation coefficient was measured after applying MaxEntropy thresholding on images.HeLa cells were cultured in low glucose DMEM medium (Biochrom) supplemented with 1% Pen/Strep (100 U/ml penicillin and 100 µg/ml streptomycin), 1% (wt/vol) glutamine, and 10% (vol/vol) FCS in 5% CO2). The fibroblasts adhered and NRCMs remained in the supernatant. NRCMs were then seeded on glass cover slips covered by Geltrex (Thermo Fisher Scientific).NRCMs were isolated from newborn rats. Briefly, after the rats were euthanized, hearts were removed from the thoracic cavity, homogenized mechanically and digested in 1 mg/ml collagenase type II containing calcium- and magnesium-free PBS at 37°C with magnetic stirring. Supernatant was taken every 20 min and transferred to DMEM medium supplemented with Glutamax (Thermo Fisher Scientific), 10% FCS and 1% Pen/Strep. Cells were then centrifuged, the cell pellet resuspended in fresh medium and transferred to a Petri dish for 45 min (37°C and 5% CO+) or by morphology . 3 million cells were transfected by nucleofection  using the SE Cell Line Kit (#V4XC-1012) according to manufacturer’s instructions with 4 μl of a 20 μM siRNA stock solution (for MCU using 2 μl siMCU_1 and 2 μl siMCU_2). All measurements were performed 72 h after transfection and the knockdown efficiency was confirmed using the same cells for qPCR.−ΔCT method. Primers used: TBP_forw: 5′-CGGAGAGTTCTGGGATTGT-3′, TBP_rev: 5′-GGTTCGTGGCTCTCTTATC-3′, MCU_A_forw: 5′-CACACAGTTTGGCATTTTGG-3′, MCU_A_rev: 5′-TGTCTGTCTCTGGCTTCTGG-3′.Total isolated RNA (800 ng) was reverse transcribed to cDNA using SuperscriptIV  and 1 μl was used for RT-qPCR using the GoTaq qPCR Master Mix  and Realtime PCR System . TBP (TATA box–binding protein) was used as a housekeeping gene. Data were analysed using the 22+-free imaging buffer  24 h (HeLa and NRCMs) or 48 h (hiPSC-CMs) after transfection. Where indicated, NRCMs were field-stimulated at 1 Hz with MyoPacer ES (IonOptix). Data were analysed with AxioVision (Zeiss) and ZEN (Zeiss) software. Background and bleed-through (BT) were corrected in the FRET/donor ratio:Cells  were seeded on glass cover slips and transfected with sensor plasmids using Effectene (QIAGEN) (HeLa) or Lipofectamine LTX Reagent (Thermo Fisher Scientific) (hiPSC-CMs and NRCMs) according to the manufacturers’ instructions. Cells were imaged using a Zeiss Observer D1  or Axio Observer Z1  at 37°C in a Ca2+ in the imaging buffer was increased to 1 mM by doubling the buffer volume to the cells  by the addition of Ca2+-containing buffer .Excitation 420 ± 20 and 505 ± 15 nm with emission filters 483 ± 16 and 542 ± 14 nm, or excitation 438 ± 12 and 508 ± 11 nm with emission filters 479 ± 20 and 544 ± 14 nm were used. For R-GECO measurements excitation was 550 ± 16 nm and emission 630 ± 46 nm. Where indicated, the concentration of Cad value in the experimental setup was determined based on the titration protocol described for cytosolic GECI  and incubated in Ca2+- and Mg2+-free buffer (pH 7.2) containing 3 mM EGTA and 5 µM ionomycin until the FRET signal reached its minimum Rfree (for both D3cpV and D3cpV-px 5–6 min). D3cpV-px expressing cells were additionally incubated in 0.01% digitonin, 3 mM EGTA, and 5 µM ionomycin for 50 s then washed with Ca2+-, Mg2+-, and EGTA-free buffer (pH 7.2). Cells were immediately exposed to different Ca2+ concentrations (once per experiment) and the Rfinal was acquired. Buffers with different Ca2+ concentrations were made as described previously  before the measurements. FRET ratios  were calculated by subtracting the background intensity and correcting for crosstalk. ER-store depletion in the cells was induced by 100 μM histamine (HeLa) in Ca2+ in the buffers here was substituted with 1 mM EGTA. Before the experiments, cells were incubated in the Ca2+-free imaging buffer, which was then removed. After washing the cells once with the according pH buffer, fresh buffer containing 10 µM nigericin was added to the cells. Images were taken with the same settings as for Ca2+ measurements but every 15 s. After the stabilisation of the FRET ratio values the imaging was continued for another two-three minutes from which the mean value of five to seven time points was taken.The preparation of buffers for the experiments is described elsewhere , with tht test when comparing two groups, or one-way ANOVA followed by Tukey’s post hoc test when three groups were compared. Data were presented as Tukey’s box plots: the box is limited by 25th and 75th percentiles. Data points larger than 75th percentile plus 1.5 IQR (interquartile range) or smaller than 25th percentile minus 1.5 IQR are presented as outliers. The extreme outliers were excluded from the graphical presentation of data. The whiskers cover all other data.Statistical significance was assessed using two-sided unpaired


Eye reflexes that stabilize gaze are essential in navigating daily life. One such reflex is the cervico-ocular reflex (COR). An important neural structure involved in the COR is the cerebellum, which facilitates proper gaze stability through sensorimotor integration to adjust eye movements accordingly. This reflex is tested by fixating the head in space and rotating the body around the neck. Thus, a rotating chair is needed to elicit proper cervical rotation while keeping the head fixed. The chair that was developed for this project was able to rotate to the specified amplitude (±0.5º of accuracy) and frequency. The parameters of the rotation amount, frequency, and amplitude can be adjusted as desired by the project guidelines. Our project aimed to improve upon existing chair models and develop a chair that can be used to assess the COR in neck pain populations. The cervico-ocular reflex (COR) is a reflexive eye movement that stabilizes gaze during trunk rotation . SensoryResearch investigating the COR is growing, thus it is imperative to develop a chair that is appropriate for the research population being studied. Prior studies have utilized various methods to stabilize the head during trunk rotation. For example, one study had the researcher hold the participant’s head still during trunk rotation . FurtherConditions, inputs, process, and outcomeThe chair was designed to measure the COR in those with subclinical neck pain (SCNP) in order to determine how cerebellar deficits previously observed in this population impact ocular movements such as the COR . ExperimParticipantsOperation of the chair requires trained experimenters that have ethical approval to conduct research involving humans. Participants can be anyone that are appropriate for the parameters of the study . Participants are informed about the nature of the research and sign a written consent prior to the study.Setting and equipment The COR chair is a custom-designed chair made by the research team at Ontario Tech University, Oshawa, Canada. The materials used to build the chair were available off-the-shelf components, which also allowed us to easily make any modifications or adjustments.SpecificationsThe chair structure was built using T-slotted framing aluminum extrusion Figure . The oveComponents and FunctionsThe chair mechanical system consists of aluminum structural frame, pulley system, belt and tensioner, chair, safety harness, and headrest Figure . The majThe electrical system consists of stepper motor, motor driver, power supply, and Arduino Uno Figure . The defThe software required are Arduino IDE version 1.8.16  and COR Chair Controller UI. Arduino IDE is used to program the Arduino Uno to receive commands from COR chair controller UI through USB serial port, and then translate the commands into signals to the stepper driver. The UI application allows the operator to quickly define a rotational parameter and initiate it Figure . The UI Oscillation rotation is defined using cycle frequency, amplitude, and number of cycles. Oscillation starts in the center, rotates to negative amplitude (left), then to positive amplitude (right). Each cycle is defined as completing one negative amplitude, one positive amplitude, and then returning to the center at 0º. Figure Scenario templateA rigid body containing three infrared light-emitting diodes (IRED) markers is attached to the right shoulder area of the chair such that the participant’s shoulder will not impede the visibility of the rigid body. Motion capture camera setup is completed prior to the arrival of the participant. The cameras collect at a frequency of 100 Hz for 120 seconds .The participant is secured with safety belts around each shoulder and the waist. The eye tracker is placed on the participant’s head to measure eye movements and head is fixed to the headrest using a headband to avoid rotation. The target apparatus appears on a screen roughly 160 cm from the chair using Experiment Builder software. Once the eye cameras are adjusted, the researcher sets the parameters of chair rotation using the UI.The protocol involves 25 rotations split evenly between five trials  at an amplitude of 5º with a frequency of 0.04 Hz . Prior tSupplemental materialsThe Optotrak 3D Investigator™ motion capture system  was used to validate the accuracy of the seat rotation. A rigid body consisting of three IRED markers was placed on the seat and tracked using the Optotrak 3D cameras. After positional data has been collected, MATLAB software  was used to calculate the rotation and velocity of the chair. The Eyelink II eye-tracking device is used to track the movement of the eyes during rotation. Eye data is analyzed using custom MATLAB code.The purpose of this technical report was to provide a comprehensive outline for the creation, implementation, and validation of the rotating chair necessary to test the COR. Preliminary test data show that the chair is able to rotate to the specified amplitude within ±0.5º accuracy. Accurate rotation of the chair is necessary as prior studies have illustrated that the optimal parameters to induce COR require low rotation amplitude and frequency . In addiFurthermore, the addition of the UI allows for the parameters of the rotation amplitude and frequency to be changed to match the parameters set at the beginning of the research study. This provides additional benefit to seamlessly test the impact of varying frequencies on the COR on a trial-to-trial basis. For example, this can be incorporated into future studies testing a participant’s ability for COR adaption to changing stimuli  and how this is impacted in populations with altered sensorimotor integration. In addition, this can be incorporated into future studies that can test passive VOR or trunk proprioception. For example, prior studies have tested VOR using both passive and active head rotations ,11. In tSome limitations include a slight jerk in the movement during chair deceleration. As mentioned previously, some unanticipated outcomes could be a large shift in amplitude due to the movement of the participant during collection. These shifts in amplitude present in a noticeable atypical shift in the amplitude graph. To avoid this, emphasize to the participant that they must remain still throughout the rotation of the chair to avoid large shifts in rotational amplitude.The designed chair was able to function adequately to allow collection of COR data which is important for investigating cerebellar deficits in special populations and in response to experimental perturbations. This design represented a low-cost and open-sourced solution for COR data collection and research, an alternative to the limited and costly options currently available in the medical equipment market. Furthermore, this design accommodates for those with neck pain and mitigates the risk for neck fatigue while performing the study. Future redesign and development can be based on this first COR chair iteration, where rotational accuracy and stability can be improved further.


Nearly 1 in every 100 children born have a congenital heart defect. Many of these defects primarily affect the right heart causing pressure overload of the right ventricle (RV). The RV maintains function by adapting to the increased pressure; however, many of these adaptations eventually lead to RV hypertrophy and failure. In this study, we aim to identify the cellular and molecular mechanisms of these adaptions. We utilized a surgical animal model of pulmonary artery banding (PAB) in juvenile rats that has been shown to accurately recapitulate the physiology of right ventricular pressure overload in young hearts. Using this model, we examined changes in cardiac myocyte protein expression as a result of pressure overload with mass spectrometry 4 weeks post‐banding. We found pressure overload of the RV induced significant downregulation of cardiac myosin light chain kinase (cMLCK). Single myocyte calcium and contractility recordings showed impaired contraction and relaxation in PAB RV myocytes, consistent with the loss of cMLCK. In the PAB myocytes, calcium transients were of smaller amplitude and decayed at a slower rate compared to controls. We also identified miR‐200c, which has been shown to regulate cMLCK expression, as upregulated in the RV in response to pressure overload. These results indicate the loss of cMLCK is a critical maladaptation of the RV to pressure overload and represents a novel target for therapeutic approaches to treat RV hypertrophy and failure associated with congenital heart defects. Many congenital heart defects primarily affect the right heart causing pressure overload of the right ventricle (RV) and eventually lead to RV hypertrophy and failure. We found pressure overload of the RV induced significant downregulation of cardiac myosin light chain kinase (cMLCK), which lead to impaired contraction and relaxation in PAB RV myocytes consistent with cMLCK loss, and also found upregulation of miR‐200c, which has been shown to regulate cMLCK expression. These results indicate the loss of cMLCK is a critical maladaptation of the RV to pressure overload and represents a novel target for therapeutic approaches to treat RV hypertrophy and failure associated with congenital heart defects. However, much of this basic and clinical research has focused on the LV, and our understanding of the adaptions of the RV to pressure overload and the cellular and molecular mechanisms of these adaptions are critically lacking.Nearly 1% of babies born in the US will be diagnosed with a congenital heart defect (CHD). CHDs are the most common birth defect and are the number one cause of death from birth defects during the first year of life , we examined changes in cardiac myocyte protein expression as a result of pressure overload with mass spectrometry 4 weeks post‐banding. We found pressure overload of the RV induced significant downregulation of cardiac myosin light chain kinase (cMLCK) in the PAB myocytes through RV proteomic analysis. cMLCK is an abundantly expressed protein that is critical for normal contractile function of cardiac myocytes and loss of cMLCK has been shown to play a role in the development of left ventricular hypertrophy  138 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES; pH 7.4 with NaOH. A cMLCK inhibitor, ML‐7 (Tocris), was used at a concentration of 10 µM in normal Tyrode's solution.All chemicals and reagents were purchased from Sigma‐Aldrich unless otherwise stated. Fluo‐4/AM Ca2.2n = 18). Sham operated control (CTL) animals underwent the same procedure without banding the PA (n = 16). Transthoracic echocardiography was performed prior to surgery and at 4 weeks post‐banding using a Vevo 2100 digital high‐frequency ultrasound system  equipped with a probe (MS250) suited for rat imaging. Tricuspid annular plane systolic excursion (TAPSE) was measured in the apical four‐chamber view in M‐mode. RV end diastolic diameter (RVEDD) was measured from the interventricular septum to the RV free wall in the apical four‐chamber view in B‐mode. RV wall thickness during diastole  was measured in the two‐dimensional short‐axis view in M‐mode. As previously shown, this surgery produces severe right ventricular dysfunction within two weeks post‐banding  Sprague‐Dawley rats (~120–150 g) were obtained from Envigo (RRID:RGD_737903). All rats exhibited normal RV function on echocardiography at the time of pulmonary artery banding (PAB) surgery (4–5 weeks old). Rats were anesthetized with 2% isoflurane until no response from toe pinch reflex, USP , and a limited left thoracotomy was performed to expose the pulmonary artery (PA). The PA was dissected from the aorta and partially ligated over an 18‐gauge angiocatheter. The sizer was then promptly removed to allow for antegrade flow through the banded area, and thoracotomy performed was closed under positive pressure ventilation to evacuate pleural air  solution containing 20 µM Ca2+ and 22.5 µg/ml Liberase Blendzyme TH (Roche Applied Science) for 25–45 min at 37°C. The right ventricular free wall was removed from the heart, minced, filtered and washed in MEM solution containing 50 µM Ca2+ and 10 mg/ml bovine serum albumin. Isolated cells were kept in MEM solution with 50 µM Ca2+ at room temperature (22–24°C) until indicator dye loading and subsequent experimentation. For some experiments, the cMLCK inhibitor ML‐7 (Tocris) or the PKA inhibitor Rp‐cAMPS (Santa Cruz) was used. Myocytes were treated for 5 min with 10 µM ML‐7 or 100 µM Rp‐cAMPS in normal Tyrode's solution before data acquisition.Single right ventricular myocytes were isolated from CTL and PAB rats. Rats were anesthetized with ketamine (0.1 mg/g) and xylazine (0.01 mg/g) until loss of toe pinch reflex, and hearts were excised and mounted on a Langendorff apparatus. Hearts were retrogradely perfused with nominally Ca2.42PO4, pH 8.5), including 3 µL (100x stock) HALT(‐EDTA) protease and phosphatase inhibitor cocktail (Pierce) was added to the cell pellets. Samples were sonicated  three times for 5 sec each with 5 sec intervals of rest at 30% amplitude to disrupt nucleic acids and were subsequently centrifuged at 4°C. Protein concentration was determined by the bicinchoninic acid (BCA) method, and samples were frozen in aliquots at −80°C. Protein homogenates (100 µg) were treated with 1 mM dithiothreitol (DTT) at room temperature for 30 min, followed by 5 mM iodoacetimide at room temperature for 30 min in the dark. Protein samples were digested with 1:100 (w/w) lysyl endopeptidase (Wako) at room temperature for overnight. Next day, samples were diluted with 50 mM NH4HCO3 to a final concentration of less than 2 M urea and were further digested overnight with 1:50 (w/w) trypsin (Promega) at room temperature. Resulting peptides were desalted with HLB column (Waters) and were dried under vacuum.300 µL of urea lysis buffer  were collected at a resolution of 70,000 at m/z 200 in profile mode. The HCD MS/MS spectra  were acquired at a resolution of 17,500 at m/z 200. Dynamic exclusion was set to exclude previously sequenced precursor ions for 20 s within a 10 ppm window. Precursor ions with +1, and +7, or higher charge states were excluded from sequencing.The data acquisition by LC‐MS/MS protocol was adapted from a published procedure . The protein concentration of the lysate was determined with the BCA Protein Assay (Thermo Fisher Scientific). Approximately 30 μg of lysate was mixed with 4× sample buffer, boiled for 5 min, and resolved on 7.5% Tris‐Glycine stain‐free SDS‐PAGE (Bio‐Rad). Stain‐free gels were activated with UV light for 5 min after SDS‐PAGE to allow for visualization of total protein and eventual signal normalization. After transfer to nitrocellulose membranes, the membranes were incubated with primary antibody followed by incubation with HRP‐conjugated secondary antibodies. Primary antibodies used were anti‐cardiac myosin light chain kinase , anti‐myosin light chain 2v , and anti‐phospho‐myosin light chain 2v (Ser15) . The secondary antibody used was goat‐anti‐rabbit HRP‐conjugated . Visualization was accomplished using ECL reagents (Bio‐Rad) and Bio‐Rad ChemiDoc XRS+. Stain‐free gel technology provided total protein for each lane and allowed for normalization of the antibody signal to the total protein loaded for each sample.2.6Total RNA was extracted from myocyte cell homogenates using TRIzol reagent (Invitrogen) per manufacturer's protocol, followed by DNase I treatment and first‐strand cDNA synthesis with M‐MLV reverse transcriptase (Invitrogen) primed by random hexamers and oligo(dT)18. Real‐time PCR was then performed on the StepOne Plus System (Applied Biosystems) based on SYBR Green fluorescence detection of PCR products. The following primer sequences were used (rat species sequences):cMLCK: forward 5′‐CCAACGTGTCCCTAATCTACAA‐3’ and reverse 5’‐CCAAGCTCCTGACTCCATAAA‐3′GAPDH: forward 5′‐ACTCCCATTCTTCCACCTTTG‐3’ and reverse 5’‐CCCTGTTGCTGTAGCCATATT‐3′rno‐miR‐200c‐3p Qiagen Catalog #YP00205505 and U6 snRNA Qiagen Catalog #YP00203907.2.7Cell shortening was measured with an IonOptix Myocyte Calcium Photometry and Contractility System connected to an Olympus IX81 microscope. Sarcomere shortening was measured by Fourier analysis of the myocyte image in real time and analyzed with IonWizard software.2.82+ sparks, with excitation at 488 nm and emission collected at >500 nm. Cardiac myocytes were loaded with 20 µm fluo‐4/AM for 20 min at room temperature, followed by a 20 min wash in 0 Ca2+ Tyrode's at room temperature. Ca2+ spark measurements were acquired from intact myocytes perfused with Tyrode's solution during rest after 1 Hz stimulation in line scan mode at 2 ms/line with a pixel size of 0.155 µm. All fluorescent signals were background subtracted. Changes in [Ca2+]i are expressed as ΔF/F0, where ΔF is the change in fluorescence (measured fluorescence [F] – F0) and F0 is resting baseline fluo‐4 fluorescence. Calcium release synchrony analysis was performed as previously described  along the scan line were normalized to the fluorescence intensity at diastole , and each line of the time‐averaged line scan image was then divided by the averaged F0 line. F50 was defined as the half‐maximum of the normalized overall peak Ca2+‐dependent fluorescence. Sections of the line where F did not reach F50 within 200 ms were excluded from analysis. For all Ca2+ imaging and contractility experiments, cells were placed on laminin‐coated coverslips. Action potentials and global Ca2+ transients were elicited by electrical field stimulation using a pair of platinum electrodes. Experiments were conducted at room temperature (22–24°C). Sparks were detected and analyzed by Sparkmaster  was used to image Ca2.9Sarcolemma and t‐tubular membranes were visualized with the membrane‐bound fluorescent probe di‐8‐ANEPPS (Molecular Probes/Life Technologies) by two‐dimensional confocal microscopy. Cells were loaded for 15 min with di‐8‐ANEPPS (5 μm) in Tyrode solution and the indicator was excited at 488 nm, and emission was measured at >600 nm. Transverse tubule analysis was performed using the ImageJ/Fiji plugin TTorg  as noted. Differences were considered statistically significant at p < 0.05.Data are presented as mean ± SD unless otherwise noted in the legend. Statistical analysis was performed using unpaired 3Myh7 and Acta1 were observed in the RV myocytes of PAB animals, validating our surgical model  was also downregulated in PAB animals compared to CTL  pressure overload remains in some patients and eventually impairs RV function and influences long‐term mortality and morbidity. Although compensated hypertrophy develops initially, ultimately RV failure will occur. The mechanisms underlying the progression from compensated to decompensated RV hypertrophy have not been well defined. To recapitulate the human disease, we utilized a juvenile animal model of right ventricular pressure overload created by partial pulmonary artery banding in juvenile rats. We first sought to identify the changes in right ventricular cardiac myocyte protein expression as a result of pressure overload on the juvenile right ventricle. To enrich for myocyte‐specific proteins, cardiac myocytes were isolated and purified from sham operated control (CTL) and pulmonary artery banded (PAB) right ventricles and subjected to mass spectrometry analysis 4 weeks post‐surgery. Tandem MS/MS analysis revealed several key changes in protein expression between CTL and PAB RV cardiac myocytes Figure . UpregulL Figure . PreviouL Figure . In agreL Figure  and mylkA Figure  in the RA Figure , consistLoss of cMLCK expression has been reported in left ventricular hypertrophy and heart failure as a result of pressure overload  handling could also be playing a role. To test this, we next characterized cardiac myocyte Ca2+ handling in CTL and PAB RV myocytes using confocal microscopy and intracellular Ca2+ recordings. Representative line scans of fluo‐4/AM fluorescence are shown in Figure 2+ transient amplitude  structures in CTL  in LV pressure‐overloaded rats and cultured cells  in the RV in response to pressure overload.Previous studies have implicated both microRNAs (miRs) and the ubiquitin‐proteasome machinery in the regulation of cMLCK expression in LV pressure‐overload  pressure overload remains in some patients after surgical correction of the defects and eventually impairs RV function and influences long‐term mortality and morbidity due to the limited treatment options are available.In this study, we aimed to identify the changes in RV cardiac myocyte protein expression as a result of pressure overload. To enrich for myocyte‐specific proteins, cardiac myocytes were isolated and purified from sham operated control (CTL) and pulmonary artery banded (PAB) right ventricles and subjected to mass spectrometry analysis 4‐weeks post‐surgery. Our results presented here show for the first time that cMLCK protein is downregulated in RV myocytes in response to pressure overload and may represent a part of the early hypertrophic response in the RV. cMLCK is an abundantly expressed protein that is critical for normal contractile function of cardiac myocytes and loss of cMLCK has been shown to play a role in the development of left ventricular hypertrophy  at Ser15  and the slower Ca2+ transient decay times. Overall, our data indicate that the Ca2+ release mechanism is dysregulated during diastole and the loss of cMLCK in the pressure‐overloaded RV contributes to the impaired contraction and relaxation of cardiac myocytes. While our Ca2+ handling data do not indicate any dysfunction in Ca2+ release kinetics, the increased diastolic Ca2+ leak in the form of Ca2+ sparks could also contribute to slowing both the Ca2+ transient decay kinetics and the myofilament relaxation kinetics. However, as we do not see any impairment in Ca2+ release kinetics, we hypothesize that the loss of cMLCK is responsible for the slowed contraction kinetics in PAB RV myocytes. While impaired SERCA function, expression, and regulation by phospholamban could also contribute to decreased SR Ca2+ content and slower Ca2+ transient decay times, we did not see significant changes in SERCA expression in PAB RV myocytes via mass spectrometry. Finally, the relative contributions of decreased cMLCK levels and reduced Ca2+ transient amplitude to the decreased sarcomere shortening in PAB RV myocytes are still unclear; however, we observed decreased sarcomere shortening in CTL cells when cMLCK was inhibited and did not see changes in Ca2+ transient amplitudes in these cells (data not shown). Overall, these findings warrant further examination of the mechanisms of dysregulated Ca2+ handling in PAB RV myocytes.In addition to impaired contractility, we also observed dysregulated Camylk3 mRNA was significantly downregulated and miR‐200c was significantly upregulated in response to pressure‐overload in the RV, however, whether miR‐200c is directly responsible for the decreased levels of mylk3 is yet to be determined. Further studies are needed to fully understand the relationship of miR‐200c and mylk3/cMLCK expression in RV pressure overload. Overall, these data provide a potential link between the metabolic dysregulation and impaired cardiac myocyte contractility seen in RV failure and provide sights to the underlying cellular mechanisms leading to RV failure due to congenital heart defects (CHDs). Of interesting note, the mylk3 gene is regulated by the cardiac homeobox protein Nkx2‐5 which is involved in the pathology of many CHDs (Chan et al., In previous studies, RV hypertrophy and failure as a result of pressure‐overload were characterized by decreased transcription and activity of electron transport chain complexes, altered mitochondrial function, and increased oxidative stress (Hwang et al., In conclusion, our results indicate the loss of cMLCK is a critical maladaptation and early hypertrophic response of the RV to pressure overload and represents a novel target for therapeutic approaches to treat RV hypertrophy and failure associated with congenital heart defects. Improved understanding of the mechanisms of cardiac myocyte dysfunction in RV pressure overload will aid in the development of novel therapeutic targets to improve contractility and preserve long‐term RV function.None declared.V.P., N.M., and J.T.M conceived and planned the experiments. P.B. performed the mass spec analysis. V.P., N.M., R.R., A.P., B.M., R.K., J.R., J.D., and J.T.M. performed the experiments and analyzed the data. V.P., N.M., and J.T.M. wrote the manuscript and created the figures. The final version of the manuscript was approved by all authors.Fig S1Click here for additional data file.Fig S2Click here for additional data file.


TP53 mutation was associated with a poor prognosis, and its mutation displayed exclusiveness with other common mutated genes in both datasets. Moreover, TP53 mutation correlated with TMB and the immune microenvironment. Gene Set Enrichment Analysis (GSEA) showed that TP53 mutation upregulated signaling pathways involved in the immune system. In summary, TP53 mutation is frequently mutated in AML, and its mutation is associated with dismal outcome, TMB, and immunological features, which may serve as a biomarker to predict immune response in AML.Acute myeloid leukemia (AML) is a heterogeneous disease related to a broad spectrum of molecular alterations. The successes of immunotherapies treating solid tumors and a deeper understanding of the immune systems of patients with hematologic malignancies have promoted the development of immunotherapies for the treatment of AML. And high tumor mutational burden (TMB) is an emerging predictive biomarker for response to immunotherapy. However, the association of gene mutation in AML with TMB and immunological features still has not been clearly elucidated. In our study, based on The Cancer Genome Atlas (TCGA) and BeatAML cohorts, 20 frequently mutated genes were found to be covered by both datasets in AML. And  Immunotherapy of cancers has opened a new era in cancer treatment. A great quantity of immunomodulatory strategies has gradually been explored in the past decade such as engineered viruses and cells with enhanced functionalities, immune checkpoint inhibitors (ICIs), and so on . And ICIAt present, several clinical trials using anti-PD-1 or PD-L1 antibodies treat patients with acute myeloid leukemia (AML) , 7. It iTP53 aberrations have been addressed to modulate both the immune and inflammatory responses in malignant tumors, involving the regulatory T cell (Treg) recruitment and T-cell differentiation  dataset and BeatAML dataset and then identified the common mutant genes in the two cohorts. Furthermore, the association of these gene mutations with TMB and prognosis was investigated. Finally, we explored whether https://cancergenome.nih.gov/) (n = 164). Meanwhile, BeatAML mutation, clinical, and sample annotation data were downloaded from source data . And somatic interactions, oncoplot, and Lollipop plot were performed by the “maftools” package. Somatic interaction function that performed pairwise Fisher’s exact test was applied to survey mutually exclusive or co-occurring sets of genes. Lollipop plots were drawn for TP53 mutation. Then, Mutation Annotation Format data files based on TCGA and BeatAML cohorts were functioned by “maftools” package to extract detailed mutational information. TP53 mutation in AML was also evaluated by the cbioportal dataset . Gene Set Enrichment Analysis (GSEA) (http://www.gsea-msigdb.org/gsea/index.jsp) was used based on analysis of Hallmark gene sets and curated gene sets.Circos plot was acquired by the use of Circos  cells, macrophages, dendritic cells, and myeloid subsets. Difference in immune cell abundance between wild group and mutation group was analyzed using a violin map. TIDE, a novel computational method that can evaluate the potential of tumor immune escape, was used to calculate immune measures (http://tide.dfcl.harvard.edu/).For TCGA and BeatAML datasets, the edgeR voom algorithm was used to convert the RNA sequencing data and the count data to values that were closer to the microarray results . CIBERSOhttps://www.r-project.org/). The data were shown as either boxplots or violin plots using the ggplot2 package concerning comparison of two continuous variables, and the association between two categorical variables was evaluated by chi-square test and Fisher’s exact test. Univariate Cox regression was used to test whether gene mutations had prognostic value in AML. The prognostic effects of gene mutations were verified through Kaplan–Meier survival analysis using a log-rank test. We employed the survival package to generate Kaplan–Meier survival curves. The correlation between mutant genes and TMB was examined by Wilcoxon rank sum test. The TMB value of each patient was calculated with the TMB function of the maftools package. The two-sided with a significance level of 0.05 in all comparisons was defined as statistically significant.Statistical analyses in this study were performed with R software version 4.0.4 (FLT3 (27%), DNMT3A (25%), NPM1 (17%), IDH2 (10%), IDH1 (9%), TET2 (9%), RUNX1 (8%), TP53 (8%), NRAS (8%), and CEBPA (7%) (FLT3 (29%), DNMT3A (23%), NPM1 (22%), NRAS (13%), TET2 (12%), RUNX1 (12%), IDH2 (12%), SRSF2 (12%), WT1 (9%), and TP53 (9%). Interestingly, there were 20 genes mutated in both TCGA and BeatAML cohorts of AML  in both TCGA and BeatAML cohorts  (TP53 mutation was associated with a poor prognosis (p < 0.001 and p = 0). Meanwhile, DNMT3A and RUNX1 gene mutations predicted poor survival probability  in TCGA cohort, and patients with TET2 and U2AF1 gene mutations had shorter survival time  in the BeatAML cohort using Kaplan–Meier methods   (We compared TMB data from public databases (TCGA and BeatAML). Among commonly mutated genes, patients with mutation in TP53 mutant subtypes and the TP53 expression level, the results demonstrated that TP53 mRNA expression was related to TP53 mutant subtypes in TCGA (p = 0.050) (TP53 group and the TP53 expression level in BeatAML (p = 0.201) , while activated mast cells showed a tendency to be enriched in wild-type group (p = 0.051). Nevertheless, the TP53 wild-type group had more naive CD4 T cells, activated mast cells, and eosinophils from BeatAML dataset  (TP53 wild-type group than TP53 mutant group (p = 0.05), which would hamper the immune antitumor effect. Also, the FLT3 mutant group had more M2 macrophages and eosinophils in both datasets . TET2, RUNX1, ASXL1, U2AF1, and PHF6 mutations presented increased TMB in both TCGA and BeatAML cohorts. We also evaluated the differences of CD8+ T cells of these gene mutated and wild–type groups; interestingly enough, no statistical difference was found in AML and other cancers  data from TCGA, which showed a prominent enrichment in TP53 mutant group of signatures related to wnt beta catenin signaling, IL2 signal transducer and activator of transcription (stat)5 signaling, notch signaling, and inflammatory response  TP53 mutant group showed significant enrichment of genes downregulated in C57MG cells (mammary epithelium) by overexpression of WNT1 gene. 3) TP53 mutant group revealed enrichment of genes upregulated in HCT116 cells (colon carcinoma) upon knockdown of PTEN by RNAi. Together, these results further demonstrated that TP53 mutation was associated with distinct molecular features in AML. For the C7 immunologic collection, the TP53 mutated group had strong enrichment in genes downregulated in monocytes compared to macrophages treated with interleukin (IL)-4  rates of 20%–40% (TP53 is much higher in therapy-related AML (t-AML) than in de novo AML. The P53 protein contains three key regions: the N-terminal region, the central DNA-binding domain (DBD), and the C-terminal region  were components of the microenvironment of hematologic human tumors -β1 activates STAT5 binding to the promoter of Foxp3 to induce the differentiation of Tregs via IL-2  by overexpression of WNT1 gene, and a prominent enrichment of signatures related to wnt beta catenin signaling in the present study. Deregulation of Wnt/β-catenin signaling is well-connected with AML initiation and progression and important to leukemia stem cell (LSC) self-renewal and survival , and other experiments to detect cytokines, cell proliferation and invasion, and so on in the following research to further validate these findings.We analyzed the somatic mutation landscape of 164 AML samples from TCGA dataset and 558 AML patients from BeatAML dataset. We discovered that The original contributions presented in the study are included in the article/JQ conceived and designed the research. Z-JX and J-CM collected and analyzed data. X-MW, YJ, and P-HX prepared the figures and performed data analysis. X-MW and Z-JX drafted the article. JL and WQ participated in study supervision. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China , Medical Innovation Team of Jiangsu Province (CXTDB2017002), Zhenjiang Clinical Research Center of Hematology (SS2018009), Social Development Foundation of Zhenjiang , and Scientific Research Project of The Fifth 169 Project of Zhenjiang .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In skeletal and cardiac muscle this triggers contraction via ryanodine-receptor (RyR)-mediated sarcoplasmic reticular (SR) Ca2+ release. We here review potential feedback actions of intracellular [Ca2+] ([Ca2+]i) on Na+ channel activity, surveying their structural, genetic and cellular and functional implications, translating these to their possible clinical importance. In addition to phosphorylation sites, both Nav1.4 and Nav1.5 possess potentially regulatory binding sites for Ca2+ and/or the Ca2+-sensor calmodulin in their inactivating III–IV linker and C-terminal domains (CTD), where mutations are associated with a range of skeletal and cardiac muscle diseases. We summarize in vitro cell-attached patch clamp studies reporting correspondingly diverse, direct and indirect, Ca2+ effects upon maximal Nav1.4 and Nav1.5 currents (Imax) and their half-maximal voltages (V1/2) characterizing channel gating, in cellular expression systems and isolated myocytes. Interventions increasing cytoplasmic [Ca2+]i down-regulated Imax leaving V1/2 constant in native loose patch clamped, wild-type murine skeletal and cardiac myocytes. They correspondingly reduced action potential upstroke rates and conduction velocities, causing pro-arrhythmic effects in intact perfused hearts. Genetically modified murine RyR2-P2328S hearts modelling catecholaminergic polymorphic ventricular tachycardia (CPVT), recapitulated clinical ventricular and atrial pro-arrhythmic phenotypes following catecholaminergic challenge. These accompanied reductions in action potential conduction velocities. The latter were reversed by flecainide at RyR-blocking concentrations specifically in RyR2-P2328S as opposed to wild-type hearts, suggesting a basis for its recent therapeutic application in CPVT. We finally explore the relevance of these mechanisms in further genetic paradigms for commoner metabolic and structural cardiac disease.Voltage-dependent Na The activation process feeds forward into a ryanodine receptor (RyR) mediated release of sarcoplasmic reticular (SR) store Ca2+. The consequent elevation of cytosolic Ca2+ concentration [Ca2+]i is central to initiation of myocyte contraction. Ca2+ is additionally a strategic second messenger with signalling actions regulating protein activity through widespread cell types. This article addresses recent interest in possible Ca2+ feedback signalling on the Na+ channel itself, its possible physiological significance, and implications for human disease in skeletal and cardiac muscle. We relate the voltage sensing, and channel opening and inactivation processes in skeletal, Nav1.4 and cardiac Nav1.5 to their potential regulation at direct and indirect Ca2+ binding and phosphorylation sites. This includes its III–IV linker region and its interactions with its C-terminal domain, whose different regions are associated with widespread mutations related to skeletal and cardiac muscle disease. We examine in vitro studies in expression systems exploring for direct and indirect effects of Ca2+ on channel properties, then extend these to physiological studies in both skeletal and cardiac myocytes in situ, from experimental platforms using normal hearts, and those modelling genetic Ca2+ homeostatic disease, broadening these to genetic exemplars for more common human disease types.Transmembrane action potential initiation and propagation, mediated by surface membrane Naclosed to an open, activated, state. The latter permits the inward, depolarizing, transmembrane Na+ fluxes driving cell excitation.Voltage-gated sodium channels (Navs), expressed in excitable cells including neurons and skeletal and cardiac myocytes, initiate action potentials underlying electrical excitation and its propagation. Their principal α-subunits (Mwt ∼220–250 kDa) each include four homologous domains, DI-IV, each containing six transmembrane α-helices, S1–S6, following a S0 helix just preceding the S1 segment . High-reinactivated state, and restoring the resting membrane potential i from ∼100 nM to 1–10 µM causing contractile activation. In addition, recent reports implicated cytosolic Ca2+ in a feedback Nav modulation whether through Ca2+ by itself or following its binding to the modulator protein calmodulin . Th. Th2+-caing site . This coing site . In Nav1ing site . In the ing site .2+-calmodulin C-lobe  residues within the DI-DII intracellular linker region increases late INa delaying action potential repolarization, characteristic of LQT3 [INa recovery from inactivation [INa and shifted (by −15 mV) steady state inactivation V1/2 [INa [2+] may also up-regulate Nedd4-2 in turn targeting Nav1.5 for degradation via a CTD PY motif i and calmodulin often used buffered, Ca2+-containing (0–10 µM), pipette solutions, to test for Ca2+, Ca2+-calmodulin or apo-calmodulin-mediated actions, on Nav1.4 and/or Nav1.5 C-terminal EF-hand or IQ domains, with some differences between reports  often significantly exceeded the Ca2+ dissociation constant, Kd of either the EGTA (67 nM) or 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA) (192 nM) pipette buffer, even as determined in the absence of Mg2+ i by Nitr-photo-uncaging, or activating co-expressed Cav1.2, contrastingly all reduced Imax in Nav1.4, or Nav1.5 chimeras expressing the Nav1.4 CTD  diminished Ca2+-calmodulin-regulation of Nav1.4 expressed in HEK293 cells, possibly involving allosteric sites within upstream CTD regions distinct from the calmodulin-binding interface i elevations produced by Ca2+-BAPTA (0–500 nM)-buffered patch-clamp electrode solutions or caffeine challenge caused parallel reductions in INa density, unit channel amplitudes and maximum action potential upstroke rates (dV/dt)max, without altering steady state voltage dependences of INa activation or inactivation [2+ homeostasis. Nav1.5 mRNA levels then altered in parallel with decreases or increases in whole cell patch-clamp INa with 24 h sustained elevations (10 mM) or BAPTA-AM-mediated reductions of culture media [Ca2+]. These also occurred without alterations in single conductance, or activation and inactivation properties i-mediated increases in electrogenic Na+/Ca2+ exchanger (NCX) activity in cardiac muscle under pro-arrhythmic conditions [+]i thereby influencing transmembrane Na+ electrochemical gradients. However, this would involve µM-levels corresponding to the altered [Ca2+]i as opposed to normal background nM-[Na+]i levels. Furthermore, NCX activity is not a prominent normal skeletal as opposed to cardiac muscle feature. Nevertheless, in either event, over the long term, reduced or increased background [Ca2+]i resulting from sustained low or high firing rates could furnish a form of Ca2+ memory modifying Nav expression or gating and therefore its availability for driving action potential upstroke and propagation. In skeletal muscle, this could reduce cell excitability permitting recovery from fatiguing stimulation. However, the accompanying conduction velocity (CV) reductions could contribute to pathological cardiac arrhythmic or epileptiform nerve cell phenotypes.These varied observations could arise from a range of possible Nav Caike hand ,15,35, o the CTD ,36. The  the CTD ,43. Fina the CTD . At all nditions . The latIn vivo Ca2+-dependent Nav modulation was observed in native cardiac or skeletal myocytes in intact physiological systems and clinical disease models. Use of loose, as opposed to conventional cell-attached, patch-clamp methods, avoided Ca2+ perturbations produced by the measurement method itself. INa families recorded from voltage steps from resting to sequentially depolarized activating test potentials, followed by further pulses to a fixed depolarized level to evaluate the resulting channel inactivation  by the activator 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate  i i 47; . These ca values  also abr on INa  induced Ca2+ homeostatic changes manifesting as spectrofluometrically measured spontaneous Ca2+ waves in murine atrial myocytes (INa (INa at pre-treatment levels. Inactivation V1/2 and k (+ current recovery from inactivation remained unchanged . Th. Th2+]i tes max . Action V/dt)max .SCN4A, mutation associated with human disease is cold-aggravated myotonia, which causes transient myotonic stiffness or renders fibres transiently inexcitable resulting in a periodic paralysis , calsequestrin (CASQ2), triadin (TRDN) or calmodulin  [RyR2-P2328S ventricles showed abnormal RyR2-mediated diastolic [Ca2+]i elevations . Fl. Fl2+ ho periods  while mu with WT . Intrinsas in WT . Gain-ofenotypes .RyR2-P2328S/P2328S murine atria. Flecainide blocks both Nav1.5 and RyR2 with IC50s of 2–7 µM and 5–11 µM, respectively [2+]i. On the one hand, flecainide's Class Ic Nav1.5 blocking action causes a pro-arrhythmic CV slowing; however, action of a consequently reduced [Na+]i on NCX could reduce pro-arrhythmic [Ca2+]i elevations . Th. ThRyR2-f Nav1.5 ,68–71.2+-mediated regulation of Nav1.5 may also contribute to commoner pro-arrhythmic cardiac conditions associated with spontaneous SR Ca2+ leak. The latter was reported in peroxisome proliferator activated receptor-γ coactivator-1 (PGC-1) transcriptional coactivator deficient (Pgc1-β−/−) murine models for pro-arrhythmic metabolic changes related to ageing, obesity and diabetes mellitus [2+ leak. Classically, SR Ca2+ leak is implicated in a pro-arrhythmic activation of inward depolarizing, NCX current [Pgc1-β−/− atria and ventricles were also associated with reduced INa [V/dt)max and CVs [INa in these experimental conditions as well as in clinical heart failure or atrial fibrillation slowing action potential CV could contribute pro-arrhythmic substrate.Camellitus . Atrial  current . Howeveruced INa ,74,  activation and the resulting release of intracellular Ca2+ stores underly skeletal and cardiac myocyte excitation-contraction coupling abnormalities which underly a wide range of human genetic diseases.Action potential generation by Na2+ potentially of regulatory importance in their reciprocal Ca2+-mediated feedback regulation. Evidence from cell expression systems, native myocytes and normal and disease models demonstrate such Ca2+-mediated Nav regulation effects.Nav channels possess sites directly or indirectly binding Ca+ channel function with associated clinical conditions.Future studies may correlate this molecular evidence bearing particularly on the Nav C-terminal and III–IV linker domains and biophysical studies of Na


DUBR in AML pathogenic mechanism. As a result, lncRNA DUBR showed high expression level within AML, resulting in poor prognosis, especially in M4 AML. In vitro studies elucidated that knockdown of DUBR with small interfering RNA (siRNA) resulted in the suppression of survival and colony formation ability, as well as induction of apoptosis, in AML cells. RNA pull-down assay and computational revealed that DUBR could sponge with miRNA-142-3P and interact with FUS protein. MiRNA-142-3P have a negative correlation with DUBR and overexpression of miRNA-142-3P inhibited cell growth in AML. Meanwhile, DUBR promoted the expression of FUS protein, targeting inhibition of FUS significantly promoted cell apoptosis in AML cell lines. In conclusion, these results revealed new mechanism of lncRNA DUBR in AML malignant behavior, and suggested that the manipulation of DUBR expression could serve as a potential strategy in AML therapy.Acute myeloid leukemia (AML) represents a frequently occurring adulthood acute leukemia (AL). Great progresses have been achieved in the treatment of AML, but its pathogenic mechanism remains unclear. This study reported the biological functions of lncRNA  Acute myeloid leukemia (AML) represents the heterogeneous myeloid cancers with high aggressiveness. It has the features of fast cell proliferation, aggressiveness, and great mortality . Its incHOTAIRM1, UCA1 (MEG3 (Non-coding RNAs (ncRNAs), the short RNAs, can be classified into circular RNAs (circRNAs), long ncRNAs (lncRNAs), and microRNAs (miRNAs). These ncRNAs can not encode proteins and may be used for diagnosis, prognosis, and therapy . LncRNAsM1, UCA1 , and MEGA1  protein, which could potentially be an essential biomarker and therapeutic target for AML.The present work examined https://portal.gdc. cancer.gov/). This study utilized the online bioinformatics analysis tool Gene Expression Profiling Interactive Analysis 2 (GEPIA2) to analyze differentially expressed genes (DEGs) as well as patient overall survival (OS) and to identify correlations. To analyze DEGs, we utilized normal TCGA and Genotype-Tissue Expression  data for reference. To analyze OS, we compared distributions by log-rank test, where median level was adopted to be the threshold. Finally, we use the Xiantao academic website to make figures.Public RNA-seq data and clinical manifestations of LAML (which corresponds to AML) were downloaded from The Cancer Genome Atlas (TCGA) project  containing 1% penicillin–streptomycin and 10% fetal bovine serum  under 37°C and 5% CODUBR, FUS siRNA, and miRNA-142-3P mimic, purchased from RiboBio  by the use of Lipofectamine 3000  in line with specific protocols. At 48 h later, we harvested cells in later analysis.AML cells were transfected with scramble, 2 conditions, colony formation was observed. Then, colonies (>50 cells) were observed and counted under the light microscope  in the 24-well plates, followed by complete mixing with 0.6% Agar solution within IMDM that contained 20% FBS. Later, we randomized single cells, followed by even distribution in all wells. When cells were incubated for 1–2 weeks under 37°C and 5% COcroscope .DUBR siRNA, FUS siRNA, for 48 h, over 105 of cells were harvested by EDTA-free trypsin, washed with PBS and re-suspended in 55 μl of binding buffer containing 5 μl of Annexin V-FITC. 450 μlof binding buffer and 10 μl of PI were then mixed with cells and stained in the dark for 5 min. The apoptotic cells were immediately analyzed and quantified with flow cytometry within 1 h   .−ΔΔCT approach, with U6 and GAPDH being references for lncRNA, gene, or miRNA. Besides, data were normalized relative to GAPDH. Data were expressed in a form of normalized mean ± SD.Total RNA was isolated from cells and tissues using the RNeasy kit  in line with specific protocols. In qRT-PCR assay, MCE qRT-PCR Kits were used to prepare cDNA using 1 μg of the extracted total RNA from reverse transcription as per manufacturer recommendations. For miR-142-3P detection, Qiagen miRNA extraction kits  were employed. The levels of miRNAs were determined by applying Qiagen miRNA detection kits  according to the kits’ protocols. The expressing levels of lncRNA, gene and miRNA were defined based on the threshold cycle (Ct), and determiend by 2The radioimmunoprecipitation assay (RIPA) buffer that contained the protease inhibitor cocktail  was utilized for cell lysis. Total protein was quantified using the bicinchoninic acid (BCA) protein detection kit . Later, SDS-PAGE was conducted to separate proteins, then proteins were transferred on PVDF membranes, and membranes were probed with the corresponding primary antibodies at 4°C overnight. After washing, secondary antibodies were utilized to incubate membranes for another 1 h under ambient temperature. Then, enhanced chemiluminescence reagent  was utilized to visualize protein bands.in vitro. After purification, the protein lysates were adopted for cultivating biotinylated cells. Thereafter, streptavidin agarose beads  were utilized for treating cells for another 1 h under ambient temperature, followed by bead elution within Biotin Elution Buffer three times and boiling within SDS buffer. Later, we conducted qRT-PCR and WB assays for RNA and protein analyses, with IgG being the reference.The Biotin RNA Labeling Mix  was used for biotin labeling of RNA, and later T7 RNA polymerase (Roche) was utilized for transcription p < 0.05 stood for statistical significance.Data were displayed in a form of mean ± SD from at least three biological duplicates. This study adopted GraphPad Prism software  for statistical analysis. Significant differences were determined by unpaired student’s t-test (two-tailed). DUBR expression and cancer, we analyzed GEPIA data and found that DUBR was significantly upregulated in LAML, cholangiocarcinoma (CHOL), brain lower grade glioma (LGG), sarcoma (SARC), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), pancreatic cancer (PAAD), gastric cancer (STAD), and thymoma (THYM). In contrast, DUBR was markedly down-regulated in bladder urothelial carcinoma (BLCA), kidney chromophobe (KICH), endocervical adenocarcinoma and cervical squamous cell carcinoma (CESC), lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), prostate adenocarcinoma (PRAD), ovarian serous cystadenocarcinoma (OV), skin cutaneous melanoma (SKCM), rectum adenocarcinoma (READ), thyroid carcinoma (THCA), testicular germ cell tumors (TGCT), uterine carcinosarcoma (UCS) and uterine corpus endometrial carcinoma (UCEC)  . These rrognosis . TherefoDUBR’s effect on the genesis and progression of AML, this study examined the DUBR levels within 173 AML and 70 normal tissue samples derived from GEPIA, the extensively utilized online bioinformatic approach. As a result, DUBR level significantly increased within AML tissues  and different AML cell lines  via qRT-PCR. The results demonstrated that DUBR level significantly higher within AML cells compared with healthy PBMC cell lines, especially in Molm-13 and KG-1 cells, so we choose these two cell lines for the further experiments , M1 (0.331), M2 (0.626), M3 (0.943), M4 (0.003), and M5 (0.07), it means DUBR can accurately predict the malignancy of M4 patients  on the colony-forming, proliferation and apoptosis assays of KG-1 and Molm-13 cells   . Taken tTo explore the roles of miR-142-3P in AML cell lines, miR-142-3P mimic was transfected into Molm-13 and KG-1 cells, then CCK8 assay was performed to measure the cell viability. As a result, miR-142-3P treatment markedly suppressed cell proliferation in both two cells, compared to the control .DUBR significantly affected AML growth, yet the underlying mechanism of DUBR in affecting cancer growth remains unknown. While exploring this, we first predicted the RNA-binding protein (RBP) FUS as the candidate target for lncRNA DUBR, based on StarBase (http://starbase.sysu.edu.cn/index.php), RBPsuit (http://www.csbio.sjtu.edu.cn/bioinf/RBPsuite/), and RBPDB (http://rbpdb.ccbr.utoronto.ca/) databases. Two possible binding proteins of DUBR were found, including quaking (QKI) and FUS   , middle. and FUS , right. DUBR with FUS protein, this study examined FUS protein level following DUBR overexpression in AML cells. We found that the FUS protein was upregulated by DUBR overexpression in KG-1 and Molm-13 cells, which implies that DUBR promoted the FUS expression  were transfected into KG-1 and Molm-13 cell lines to reduce FUS expression  than miR-142-3p mimic or siFUS alone (*p < 0.05)  .DUBR is first suggested to participate in regenerating and differentiating muscle cells  cell growth and metastasis by inhibiting oxidative phosphorylation regulated by ZBTB11  in vitro  cell proliferation  is suggested to relate to several RNA metabolic processes, such as splicing, transcription, local translation, miRNA processing, and mRNA transport (TLS/FUS-ERG fusion gene, owing to which patients are resistant to conventional chemotherapy (LINC00319 regulates post-transcriptional SIRT6 expression by the FUS-mediated pathway (siFUS; as a result, FUS exerted an important effect on enhancing AML growth.Furthermore, we found that feration . Anotherferation . As discithin GC . Herein,ransport . Accordiransport . Brooke ransport . FUS hasransport . FUS serransport . The reaotherapy , and FUSotherapy . A numbe pathway . Our resDUBR, miR-142-3P and FUS, it is not a DUBR/miR-142-3P/FUS axis, it is meaningful because we will get a better inhibition rate by regulation of FUS and miR142-3P genes simultaneously.In order to observe the candidate target genes of miR142-3P, we applied target prediction based on miWalk, starbase, mitarbase, and miRDB database , FUS is DUBR expression increased within AML cells, which was related to AML prognosis via DUBR-miRNA-142-3P and DUBR-FUS interaction. We suggest that the DUBR-miRNA-142-3P and DUBR-FUS interaction promotes AML cell proliferation, which can serve as the candidate anti-AML therapeutic target.Based on the work presented here, we confirmed that