Alternatively, NO could increase the sensitivity of the contractile response to [Ca2+]cyt (Ca2+ sensitivity). Arginase regulates NO production by competing with NO synthase (NOS), and thus, arginase inhibition should increase cardiac contractility by increasing NO production. We hypothesized that arginase inhibition increases cardiac contractility by increasing both [Ca2+]cyt and Ca2+ sensitivity. [Ca2+]cyt and contractile (sarcomere length [SL] shortening) responses to electrical stimulation were measured simultaneously in isolated rat cardiomyocytes using an IonOptix system. In the same cardiomyocytes, measurements were obtained at baseline, following 3‐min exposure to an arginase inhibitor  and following 3‐min exposure to BEC plus a NOS inhibitor . These responses were compared to time‐matched control cardiomyocytes that were untreated. Compared to baseline, BEC increased the amplitude and the total amount of evoked [Ca2+]cyt, and the extent and velocity of SL shortening in cardiomyocytes, whereas addition of l‐NAME mitigated these effects. The [Ca2+]cyt at 50% contraction and relaxation were not different across treatment groups indicating no effect of BEC on Ca2+ sensitivity. The [Ca2+]cyt and SL shortening responses in time‐matched controls did not vary with time. Arginase inhibition by BEC significantly increased the amplitude and the total amount of evoked [Ca2+]cyt, and the extent and velocity of SL shortening in cardiomyocytes, but did not affect Ca2+ sensitivity. These effects of BEC were mitigated by l‐NAME. Together, these results indicate an effect of NO on [Ca2+]cyt responses that then increase the contractile response of cardiomyocytes.Nitric oxide (NO) is thought to increase cardiac contractility by increasing cytosolic Ca 2+]cyt to electrical stimulation. It enhanced myocardial contractility by increasing the extent and velocity of SL shortening in cardiomyocytes, but did not affect Ca2+ sensitivity. These effects were mitigated by NOS inhibition, supporting a role of NO on [Ca2+]cyt and contractile responses.Arginase inhibition significantly increased the amplitude and total amount of evoked [Ca Enhanced generation of both NO and superoxide, and as a consequence, peroxynitrite, has been demonstrated to play critical roles in the etiology of cardiovascular diseases  is one of the key molecular players in cardiac contractility via modulation of Cal‐arginine to mutually modulate NOS activity, thus regulating NO and reactive oxygen species production. Among NOS isoforms, NOS1 positively modulates cardiac contractility, whereas NOS3 inhibits β‐adrenergic receptor‐mediated increases in cardiac contractility  in the sarcoplasmic reticulum (SR)  and Ca2+ sequestration also plays an important role in regulation of [Ca2+]cyt  male Sprague–Dawley rats weighing 250–350 g (Envigo). The animals were anesthetized with intraperitoneal injection of a mixture of ketamine  and xylazine  and provided 100% O2 through a nasal cannula. After confirming the anesthetic status by verifying the lack of a toe pinch reflex withdrawal, a thoracotomy was performed, the heart excised and the animal was euthanized by exsanguination.The experimental protocol was approved by the Mayo Clinic Institutional Animal Care and Use Committee (ref #. AA2521‐17‐R19). Studies were performed using 12  responses to electrical field stimulation were measured simultaneously in cardiomyocytes using an IonOptix system (Ionoptix LCC), as previously described , and perfused with Ca2+ Tyrode's solution kept at 37°C and aerated with a 95% O2–5% CO2 gas mixture. Evoked [Ca2+]cyt responses in cardiomyocytes were measured using a dual‐excitation spectrofluorometer (IonOpitx). Fura‐2 fluorescence was excited using alternating 340 and 380 nm wavelengths, and emission fluorescence at 510 nm was detected using a photomultiplier tube. The [Ca2+]cyt was determined based on calibration of the ratio of Fura‐2 fluorescence (R = 340/380 nm) using the equation described by Grynkiewicz et al. (Kd) was determined at 37°C based on an in vitro (cell‐free) calibration performed using Fura‐2 pentapotassium (Fura‐2 PP) salt in which Kd is reflected by the x‐intercept of the double‐log plot of free Ca2+ vs. Fura‐2 fluorescence. The isolated cardiomyocytes were stimulated using platinum wires positioned on the side of the chamber with 15 V 5 ms duration pulses delivered in 2 s duration trains at a frequency of 0.5 Hz using the MyoPacer stimulator (IonOptix). The cardiomyocytes were initially stimulated for 15 min to allow sufficient time for stabilization of [Ca2+]cyt and contractile responses (baseline). The amplitude of evoked [Ca2+]cyt responses was measured as well as the total the area under the curve of the transient response. In addition, the time to peak [Ca2+] and tau were determined. Contractile responses were assessed by measuring the extent and velocity of SL shortening in a region of interest within the cardiomyocyte encompassing at least seven sarcomeres and calculated based on the fast Fourier transform algorithm. Continuous measurements of SL were determined from rest (diastolic) to peak contractile (systolic) back to resting state during each stimulation cycle. In addition, the times to SL shortening peak 10%, 50%, and 90%, and times to SL relaxation 10%, 50%, and 90% were measured. Ten evoked [Ca2+]cyt and SL responses were obtained at each time point in the experimental protocol  to the perfusion solution. After determining the effect of BEC, the same cardiomyocytes were perfused with a NOS inhibitor, NG‐nitro‐l‐arginine‐methyl ester  together with BEC (10−5 M). The concentrations of BEC and l‐NAME were determined based on previous reports demonstrating increasing and offsetting effects on contractility in isolated cardiomyocytes, respectively  consisted of 137 NaCl, 5.4 KCl, 0.5 MgCl2, 1.8 CaCl2, 0.33 NaH2PO4, 10.0 HEPES, and 10.0 glucose and was adjusted to pH 7.4 with NaOH. Enzyme solution consisted of zero‐Ca2+ Tyrode solution supplemented with 0.2 mM CaCl2, 0.6 mg/ml type II collagenase (Worthington), and 0.1 mg/ml protease with 1% bovine serum albumin. Kraft‐Brühe solution (in mM) contained 70 KOH, 50 l‐glutamic acid, 40 KCl, 0.5 MgCl2, 1 KH2PO4, 0.5 EGTA, 10 HEPES, 5 pyruvic acid, 5 Na2ATP, 5.5 glucose, 20 taurine, and 5 creatinine, and was adjusted to pH 7.38 with KOH. Cell medium  consisted of 500 ml of basal medium, 25 ml of fetal bovine serum (Cat. No. 0025), 5 ml of cardiac myocyte growth supplement  and 5 ml of penicillin/streptomycin solution .BEC and 2.6n = 6 per group) were assigned to treatment and time‐matched control groups. Because there were differences in the number of cardiomyocytes studied per animal, inter‐animal variability was considered in the statistical/experimental design. In addition, at each time point, 10 evoked responses were obtained for each cardiomyocyte. Thus, variance across the responses in each cardiomyocyte was also considered in the statistical/experimental design. In a preliminary study, the mean and standard deviation (SD) of 10 evoked [Ca2+]cyt and SL shortening responses were measured in untreated cardiomyocytes and used in a power analysis to determine the number of cardiomyocytes and animals required to measure an effect size of 20% . The results of this study were based on an n = 6 animals per group, an n = 10 of cardiomyocytes per group and an n = 10 evoked response per cardiomyocyte. A mixed linear model analysis of variance was used to compare the effects of BEC−, BEC + l‐NAME (treatment group) and time (time‐matched controls) to baseline with animal as a random effect and treatment group, time and group‐by‐time as fixed effects. When variables showed significant differences between groups, post hoc analyses with Bonferroni correction were performed for the adjustment of multiple comparisons. Statistical analysis was performed using Prism 9.0.2 software (GraphPad). Results were presented as mean ± SD. A p‐value <0.05 was considered significant.One or two cardiomyocytes were studied per animal and animals (33.12+]cyt (no stimulation) remained constant at 180.6 ± 23.2 nM across the experimental period (14 min) and was comparable between experimental and time‐matched control groups  was 606.2 ± 28.5 nM and increased following BEC treatment, but the change across time did not reach statistical significance (p = 0.087)  increased following BEC treatment , and this effect was mitigated following BEC + l‐NAME treatment. The amplitude of [Ca2+]cyt did not change across time in the time‐matched controls  Figure . Compares Figure .2+]cyt was 60.7 ± 15.7 nM. Compared to baseline, the total amount of evoked [Ca2+]cyt expressed in percent change from baseline increased following BEC treatment , and this effect was mitigated following BEC + l‐NAME treatment (p = 0.016). The total amount of evoked [Ca2+]cyt remained constant across time in time‐matched control cardiomyocytes. Time to peak [Ca2+]cyt and tau were not affected by treatment or time , whereas this enhancement of contractile response was mitigated following BEC + l‐NAME treatment (p = 0.001). Compared to baseline, the extent of SL shortening evoked by stimulation remained constant across time in the time‐matched control cardiomyocytes  mice exhibited reduced SR Ca2+ release and SR Ca2+ stores in response to increasing frequency of stimulation .


However, the role of P2-type Ca2+ ATPases in stress response and stomatal regulation is still elusive in soybean. In this study, a total of 12 P2-type Ca2+ ATPases genes (GmACAs and GmECAs) were identified from the genome of Glycine max. We analyzed the evolutionary relationship, conserved motif, functional domain, gene structure and location, and promoter elements of the family. Chlorophyll fluorescence imaging analysis showed that vegetable soybean leaves are damaged to different extents under salt, drought, cold, and shade stresses. Real-time quantitative PCR (RT-qPCR) analysis demonstrated that most of the GmACAs and GmECAs are up-regulated after drought, cold, and NaCl treatment, but are down-regulated after shading stress. Microscopic observation showed that different stresses caused significant stomatal closure. Spatial location and temporal expression analysis suggested that GmACA8, GmACA9, GmACA10, GmACA12, GmACA13, and GmACA11 might promote stomatal closure under drought, cold, and salt stress. GmECA1 might regulate stomatal closure in shading stress. GmACA1 and GmECA3 might have a negative function on cold stress. The results laid an important foundation for further study on the function of P2-type Ca2+ ATPase genes GmACAs and GmECAs for breeding abiotic stress-tolerant vegetable soybean.P Glycine max L., also named “Maodou” in China and “edamame” in Japan) serves as a fresh vegetable and has higher nutritional content than grain- and oil-type soybean (Vegetable soybean ( soybean . In rece soybean . However2+]cyt) is a key cellular second messenger that plays a crucial role in plant growth, development, and stress response -type ATPases located at the plasma membrane (PM), tonoplast, endoplasmic reticulum (ER), and Golgi  and autoinhibited Ca2+ ATPases  in plants, which have a higher affinity for Ca2+ transportation than Ca2+ channels and Ca2+ antiporters  that have the main role in signal transduction -dependent programmed cell death regulated by AtACA4 and AtACA11 in Arabidopsis  was highly and rapidly induced by salt stress and a fungal elicitor  positively regulated the activities of AtACA8 and AtACA10. The bon1, aca10, and aca8 knock-out mutants have defects in stomatal closure in response to environmental stimuli  on double-layer filter papers (20 × 30 cm) with 3 cm away from the upper and lower edges. Then a layer of wet filter paper was covered on the seeds. The three-layer filter paper with seeds was rolled up from left to right and placed in a covered bucket (20 × 20 cm). Sterile water was added to the bucket every day. The bucket was placed in a light incubator under the conditions of 25°C/25°C (day/night), 12 h/12 h photoperiod (day/night) and 70% relative humidity for germination. Seedings with consistent germination were then transplanted into a pot filled with peat/vermiculite (2:1). Three seedlings in each pot were cultured in a light incubator in the same condition for germination and irrigated with water.For expression pattern analysis of PGmACAs and GmECAs after salt, drought, cold and shading stresses. Soybean seedlings under normal growth conditions were used as a control. In salt treatment, soybean seedlings were transferred to 250 mM NaCl solution for root soaking treatment  were used to study the expression patterns of reatment . For droreatment . Potted reatment . Moreovereatment . The firG. max, Ostreococcus tauri, Chlamydomonas reinhardtii, P. patens, Selaginella moellendorffii, Oryza sativa, Zea mays, Capsicum annuum, and Arabidopsis thaliana were downloaded from the National Center for Biotechnology Information (NCBI) database.2-type Ca2+ ATPase genes. The gene family sequence feature file E1-E2 ATPase (PF00122.20) was obtained from Pfam.−10. The obtained protein sequences were searched for protein domains through the alignment Pfam-A database of the Hmmscan program. Sequences that did not contain family characteristic domains were deleted. BLASTp programA. thaliana and O. sativa for orthologs. Computation of the theoretical isoelectric point (pI) and molecular weight (Mw) of soybean P2-type Ca2+ ATPase proteins was performed using the compute pI/Mw online tool.The genome sequence and gene annotation of 2-type Ca2+ ATPase genes were aligned using ClustalX2.1.0.0 software  were utilized the neighbor-joining method and a graphical representation was produced with MEGA-X software and bootstrap analysis. The robustness of each node in the tree was determined using 1,000 bootstrap replicates with the pairwise deletion option. The phylogenetic trees comparing soybean and A. thaliana were also constructed with Mega-X by the parameters above. The MEME 5.0.5 online program , 7.5 μl of Hieff® qPCR SYBR Green Master Mix, and 0.4 μl of each of the two gene specific primers (10 μM), 4.7 μl ddH2O. The reactions were performed as follows: 95°C for 30 s, 40 cycles at 95°C for 10 s, 60°C for 15 s, and 72°C for 20 s. A melting curve analysis was conducted following each assay to confirm the specificity of the amplicon for each primer pair. Gene-specific primers were designed using Primer 3  following the manufacturer’s instructions. RNA integrity was verified with 1% agar gel electrophoresis and the RNA concentration was measured using BioDrop uLite . The first cDNA strand was synthesized from 1 μg of total RNA using Hifair® III 1st Strand cDNA Synthesis Kit with dsDNase  according to the manufacturer’s instructions. Real-time quantitative PCR (qPCR) was conducted in Bio-rad CFX96™  using Hieff® qPCR SYBR Green Master Mix . The 15 μl reaction mixture contained 2 μl of a diluted template . Significance levels: 0.01 < *p ≤ 0.05, 0.001 < **p ≤ 0.01, ***p ≤ 0.001. The relative transcript expression levels of each vegetable soybean P2-type Ca2+ ATPase genes were transformed to log2. The data clustering analysis and the quantitative color scheme were performed by Amazing Heatmap.14Student’s 2-type Ca2+ ATPase gene sequences from the G. max genome by HmmerWeb version 2.39.0. Twelve P2-type calcium ATPase genes from G. max were identified by further BLASTp methods of NCBI. We renamed the Glycine max P2B-type Ca2+ ATPase genes  and P2A-type Ca2+ ATPase genes  based on close homology to the corresponding Arabidopsis genes  to 1,105 (GmACA10) residues with an average of 1,053 amino acids. Molecular weights of GmACAs and GmECAs ranged from 109.9 to 120.8 kDa and their theoretical isoelectric points (pIs) ranged from 5.22 (GmECA4) to 8.06 (GmACA8).In this study, we first obtained the Pis genes . The resomosomes . The len2-type Ca2+ ATPase genes, a phylogenetic analysis was performed for nine green plant species  19 had the most genes, including GmACA11, GmACA12, GmACA13, and GmECA4. There was no tandem duplication in the GmACAs and GmECAs, but there were nine pairs of segmental duplicates. For instance, GmACA2 has three homologous genes on chromosomes 5, 11, and 17, respectively  from leaves indicated a significant difference in damage to soybean plants under drought, cold, salt, and shading stresses , as well as GmACA8, GmACA9, and GmACA11  in algae is less than the number of species distributed by GmACA8 (129 species). The results suggested that there are differences between P2-type Ca2+ ATPase genes in algae and vascular plants, which might be a sign of functional differentiation in calcium signal regulation and stress tolerance.Pd influx . In the he Golgi . Ostreocructures , indicatd GmECA3 , it was 2+, and atmospheric CO2 all induce stomatal closure. [Ca2+]cyt signals could be generated by influx and efflux of the ion from the extracellular space, or by storage and release from intracellular compartments, such as the endoplasmic reticulum (ER), Golgi, plastid, and vacuole. By recruiting different stores, distinct spatial patterns of [Ca2+]cyt could be generated (2+ pumps (CAs and ECAs) are responsible for the release of [Ca2+]cyt to the extracellular space and the influx to the intracellular Ca2+ stores. In rice, OsCAs family genes were regulated by four different osmotic-related abiotic stresses , hydrogen peroxide, cold, the elevation of external Caenerated . Ca2+ puc stress . Correla closure . Further closure , which i closure . We specmovement , 7.2+ pump in vegetable soybean cells , endoplasmic reticulum , tonoplast (GmACA11), and Golgi (GmECA3) intracellular membrane systems, respectively. According to the temporal and spatial expression of GmACAs and GmECAs under different stresses, all the genes located on the plasma membrane  and the tonoplast (GmACA11) were up-regulated by drought, cold, and salt stress, and down-regulated by shading stress. Among the genes located at the endoplasmic reticulum, only GmECA2 and GmECA4 showed similar expression profiles. The results revealed that P2-type Ca2+ ATPase genes located at the endoplasmic reticulum  might produce functional differentiation in response to different abiotic stresses. It has also shown that when plants are severely stressed, Ca2+ pumps located on the plasma membrane and tonoplast are preferentially mobilized in a unified manner, which can rapidly generate Ca2+ oscillation to regulate plant responses to external stresses, such as stomata closure. Moreover, GmACA1 might be involved in the negative regulation of cold and shading stress. GmECA1 was up-regulated under four abiotic stresses, especially under shading stress, which might positively regulate stomatal closure under shading stress. SCA1, the homologous gene of GmACA2 in this study, was up-regulated after salt stress . The effect of the Ca2+ pump on stomatal opening under different stress was speculated through the spatial location and temporal expression of the P2-type Ca2+ ATPase family genes in soybean. GmACA8, GmACA9, GmACA10, GmACA12, GmACA13, and GmACA11 might promote stomatal closure under drought, cold and salt stress; GmECA1 might regulate stomatal closure in shading stress. GmACA1 and GmECA3 might have a negative function on cold stress. The roles of GmACAs in regulating tolerance to different stress should be further evaluated through genetic transformation and functional analysis in soybean and other model organisms such as Arabidopsis, yeast, and Xenopus oocytes.This study was the first genome-wide analysis of the P2+ pump belongs to the P2-type calcium ATPases gene family, which is responsible for cellular Ca2+ transport and plays an important role in plant development and response to biotic and abiotic stresses. The results laid an important foundation for further study on the function of P2-type calcium ATPases genes GmACAs and GmECAs for soybean abiotic-resistant breeding.The CaThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/SX, JW, XF, and GC conceived and designed the experiments. JW, XF, SZ, SL, TS, and YZ performed the experiments. JW, GC, Z-HC, and SX analyzed the data and wrote the paper. JW, YZ, and FX contributed to reagents/materials/analysis tools. All authors contributed to the article and approved the submitted version.The research was supported by grants from the Key R&D Program of Zhejiang Province (2021C02009) and National Key R&D Program of China (2018YFD0100901).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.


Dvornikov et al. test a new high-throughput fluorescence lifetime-based assay using a human cardiac troponin C probe in cardiac myofibrils. The assay sensitivity and precision allow for the detection of key structural changes in cTnC that reports changes in thick and filament proteins in health and disease, which promises to have utility in screening therapeutic compounds that target these proteins. 2+-induced structural changes of the thin filaments to permit myosin cross-bridge cycling driven by ATP hydrolysis in the sarcomere. In congestive heart failure, contraction is weakened, and thus targeting the contractile proteins of the sarcomere is a promising approach to therapy. However, development of novel therapeutic interventions has been challenging due to a lack of precise discovery tools. We have developed a fluorescence lifetime-based assay using an existing site-directed probe, N,N′-dimethyl-N-(iodoacetyl)-N′-ethylenediamine (IANBD) attached to human cardiac troponin C (cTnC) mutant cTnCT53C, exchanged into porcine cardiac myofibrils. We hypothesized that IANBD-cTnCT53C fluorescence lifetime measurements provide insight into the activation state of the thin filament. The sensitivity and precision of detecting structural changes in cTnC due to physiological and therapeutic modulators of thick and thin filament functions were determined. The effects of Ca2+ binding to cTnC and myosin binding to the thin filament were readily detected by this assay in mock high-throughput screen tests using a fluorescence lifetime plate reader. We then evaluated known effectors of altered cTnC-Ca2+ binding, W7 and pimobendan, and myosin-binding drugs, mavacamten and omecamtiv mecarbil, used to treat cardiac diseases. Screening assays were determined to be of high quality as indicated by the Z′ factor. We conclude that cTnC lifetime-based probes allow for precise evaluation of the thin filament activation in functioning myofibrils that can be used in future high-throughput screens of small-molecule modulators of function of the thin and thick filaments.Cardiac muscle contraction is regulated by Ca As a result, tropomyosin (Tm) moves from the “blocked” to “closed” position, according to the three-state model of thin filament activation , myosin inhibitor mavacamten (Mava), and myosin activator omecamtiv mecarbil (OM) to illustrate the usefulness of this new TR-F assay in functioning myocardium.In the present study, we hypothesized that changes in human IANBD-cTnCAll reagents were purchased from Fisher Scientific unless noted otherwise.T53C was previously developed for use in fluorescence measurements of myofilaments (T53C were expressed in Escherichia coli BL21(DE3)-competent cells. Wild-type cTnT was expressed in Rosetta (DE3)-competent cells (New England Biolabs). Purification of cTnC was performed as described in Plasmids used to express wild-type human cardiac troponin protein subunits cTnC, cTnI, and cTnT (TnT3) were provided by Dr. Jil Tardiff (University of Arizona). cTnCilaments  and myofilaments . RecombiT53C was labeled with IANBD (Thermo Fisher Scientific) in Tris-Urea-KCl-EDTA (TUKE) buffer . Prior to labeling, 200 μM of Tris(2-carboxyethyl)phosphine hydrochloride  was added and incubated for 30 min at room temperature (23°C) to reduce cysteines. Then, IANBD was added to a final concentration of 150 μM (from a 20 mM stock in dimethylformamide). Labeling was performed for ∼1 h at room temperature. The reaction was terminated by the addition of 750 μM DL-dithiothreitol . Excess dye was removed by exhaustive dialyses against TUKE buffer and IANBD-labeled cTnC was clarified at 14,000 × g for 15 min at 4°C. 74 ± 3% of the cTnC cysteines were labeled with IANBD under these conditions as determined on a UV-Vis spectrophotometer.50 μM of cTnCT53C was combined with wild-type cTnI and cTnT to form the recombinant troponin complex (Tn) as described in 2, pH 7.0 with KOH; Sigma-Aldrich), and then mixed at a molar ratio of 1:1.2:1.2, respectively. Refolding of the Tn subunits and complex formation was carried out by dialysis against buffers with decreasing urea concentration (6 to 4 to 0 M urea). Next, Tn was dialyzed against Tn reconstition buffer 2 with KCl reduced from 0.5 to 0.4 M . Finally, the Tn complex was dialyzed against hypotonic rigor buffer  used for the Tn exchange in cardiac myofibrils. Tn was then centrifuged at 14,000 rpm  in an Eppendorf centrifuge (5424R) for 20 min at 4°C to remove uncomplexed cTnI and cTnT.IANBD-cTnC2, 6.2 mM ATP [Cat. No. BP413-25], and 10 mM Na2CrP [Sigma-Aldrich], pH 7) containing Sigma Protease Inhibitor Cocktail (Sigma-Aldrich) at 1/1,000 dilution, 0.2 mM PMSF, 1 mM benzamidine HCl, 1 mM DTT (Thermo Fisher Scientific), 1 mM NaN3, and 1% Triton X-100 to resuspend the tissue. The membrane permeabilization (skinning) was carried out on a rocker overnight at 4°C. The next morning, detergents were washed out by three changes of 15 ml of fresh, ice-cold relaxing buffer after the tissue was allowed to settle for 2 min. Tissue in relaxing buffer was transferred using a pipette with a wide bore tip into several glass tubes  for homogenization by a 5-mm homogenizer probe  with three pulses of 10 s each at 20,000 rpm (position 4). Homogenization in small volumes (batches) increased homogeneity of the myofibril preparations. Myofibrils from each tube were then passed through 200-μm nylon mesh placed on a 15-ml Dounce homogenizer tube to remove remaining chunks of tissue. The combined filtrate was homogenized in a Dounce tube by five pumps using the B pestle. The resulting suspension was diluted with relaxing buffer to a final myofibrillar concentration of ∼1 mg/ml. Finally, myofibrils were centrifuged at 1,000 × g for 5 min to remove the mitochondrial supernatant. Typically, ∼500 mg of wet-frozen ventricle tissue resulted in 16 pellets of myofibrils, each ∼12 mg of wet mass  or ∼1 mg dry protein mass.Frozen cryoground pig ventricle heart tissue was obtained from Pel-Freez Biologicals and stored at −80°C until use. The day before the experiment, tissue was thawed in ice-cold relaxing buffer . The pellets were centrifuged/resuspended two more times before final resuspension in 0-EGTA rigor buffer (to ∼0.75 mg/ml myofibrillar protein). Lastly, the appropriate 10X Ca-EGTA buffer and drug (or 1% DMSO as a vehicle) was added to the myofibril suspension. Percent of exchanged Tn and total myofilament protein were determined by 18% SDS-PAGE of myofibrils and labeled-Tn standards dissolved in 7 M Tris-urea sample buffer . After electrophoresis, fluorescence of labeled Tn in the gel was detected in a UV-box and followed by Coomassie staining of myofilament proteins.Human recombinant IANBD-Tn was exchanged for endogenous porcine Tn in permeabilized myofibrils by a 4-h incubation on ice. Briefly, ∼48 mg of wet myofibrils  were resuspended and combined in a total of 200 μl of 23 ± 3 μM IANBD-labeled Tn in hypotonic rigor buffer containing of 6 mM ATP and 1 mg/ml BSA (to prevent clumping of myofibrils). The myofibril protein concentration during the exchange was ∼20 mg/ml. Following exchange, excess/non-exchanged Tn was removed by centrifuging myofibrils for 1,000 × 2+ conditions by adding 10× Ca2+-EGTA. Pre-warmed ATP was added to a final concentration of 0.1 mM to start the myosin ATPase reaction (t = 0 min). Every 1 or 2 min for 10–20 min, 10 μl of the reaction suspension was removed (out of 100 μl) and placed in the reaction stop solution (90 μl of ice-cold 0.2 M perchloric acid). 100 μl of malachite green solution was added to the 100 μl reaction, gently vortexed, and allowed to incubate for 10 min at room temperature (23°C) on a shaker. The malachite green solution was prepared by dissolving 2 g of ammonium molybdate (Sigma-Aldrich) in 700 ml of 1 M HCl, followed by the addition of 0.3 g of malachite green oxalate (Sigma-Aldrich), 5 ml of 10% Triton X-100 (Thermo Fisher Scientific), and double distilled H2O to a final volume of 1 liter. To determine the concentration of Pi, absorbance was measured on a BioTek plate reader at 655 nm. Total protein concentration of myofibril samples was obtained by the BCA Protein Assay  or a Coomassie-stained SDS-PAGE. Results were expressed as nmol Pi per mg of protein per min (Pi·mg−1·min−1), calculated using a Pi standard curve using NaH2PO4 standards. Three technical replicates were performed for high and low Ca2+ and measured in the presence of 1% DMSO or 100 μM Mava in DMSO.To ensure that the Tn exchange does not perturb the normal function of myofibrils, ATPase activity was evaluated by a modified malachite green assay . BrieflyFor TR-F experiments, 50 μl myofibrils  were loaded into wells of 384-well flat, black-bottom polypropylene plates (Greiner Bio-One) using a P200 pipette and regular tips. The plate was sealed with a sheet of plate tape sealant and spun down at 1,000 rpm for ∼10 s to remove bubbles. In cases of high variability of fluorescence intensity, due to non-homogeneity of the myofibrillar suspension, several scans with different laser intensities were carried out to make sure that lifetimes were measured and compared under similar intensity levels. No photobleaching was observed as a result of repeated scans. Fluorescence lifetime measurements were acquired at room temperature (∼23°C) using a high-precision FLTPR . pCa (−log [Ca2+]) solutions tested were high calcium (pCa 4.5) and low calcium (pCa 9). Intermediate [Ca2+] of 7.4, 7, 6.6, and 6 were also tested. Concentrations of free Ca2+ were calculated by the WEBMAXC STANDARD resource (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/webmaxc/webmaxcS.htm).For cTnCAfter the first scan (“rigor” condition), we added 3 μl of ATP (Cat. No. BP413-25) to each well to a final concentration of 0.1 mM ATP using a multi-channel pipette. Plates were read at 20 min after addition of the nucleotide.W7 hydrochloride (Bio-Techne), Pimo (Sigma-Aldrich), Mava (MedChemExpress), or OM (MedChemExpress) were added to the myofibrils in rigor buffer to a final concentration of 100 μM (from 10 mM stocks in DMSO). All compounds were tested for fluorescence, at the concentrations used in each experiment. Background fluorescence of all compounds did not exceed 5% threshold of the signal intensity. The final concentration of DMSO was 1% and no drug controls also contained 1% DMSO. Drugs were added to a tube of myofibrils and then pipetted into the microplate wells. Plates were read before the addition of ATP and at 3–4 and 20 min following the addition of ATP .Inhomogeneities in myofibrils can result in high-fluorescence intensity in some wells due to chunks of tissue or clusters of labeled proteins not specifically bound to cardiac muscle tissues. These bright clusters/chunks can be seen by epifluorescence microscopy (data not shown). Wells with intensities above 200% of average or below 50%, due to pipetting flaws/air bubbles, were excluded. The percentage of samples in wells typically rejected based on aggregates was ∼3%.SD(t), consisting of single-exponential decay model MD(t), characterized by pre-exponential factor A and lifetime τ, convolved with the IRF(t), acquired by recording scatter from the same volume double distilled H2O in a well of the 384-well plate.Following data acquisition, TR-F waveforms observed for each well were analyzed using least squares minimization global analysis software  and fittThe decay of the IANBD fluorescence was calculated as:T53C-IANBD TR-F assay quality, for use in HTS, was determined by calculating the Z′ factor. This compares the average lifetimes and SD values of two states measured multiple times. Here, the two states include low versus high Ca2+; rigor versus ATP (or ADP) conditions; 1% DMSO versus drug  plus 1% DMSO. The Z′ factor was calculated as1 and σ2 are the SDs and μ1 and μ2 are the averages of the lifetimes (τ1 and τ2) from multiple samples of the two states. A value of 0–0.5 indicates feasible assay quality and 0.5–1 indicates excellent assay quality (Z′ is positive (0 < Z′ < 1) when the difference between the average signals  of two conditions  is separated by more than three times SDs for each condition and Z′ is negative (Z′ < 0) when there is no separation  between three times SDs for each condition.The cTnC quality . In othet test was used to make comparisons between two independent groups. A one-way ANOVA using Dunnett’s post-hoc test was used for comparisons of three groups . P < 0.05 was considered significant. For each experiment, n = technical replicates or the number of wells containing myofibrils from a single mixture containing either high or low Ca2+ or DMSO versus drugs. Each n corresponds to one reading per well. Nucleotide was added to each well so that each well represents an independent response to the nucleotide. N = biological repeats or the number of independent purifications of recombinant proteins and/or preparations of skinned myofibrils.Average data are provided as mean ± SD. We performed four sets of myofibril experiments (separately exchanged preparations) from three separate batches of Tn. A two-tailed 2+) of mock screens using the four drugs. 2+ and ATP on fluorescence intensity and lifetime in IANBD-cTnCT53C exchanged myofibrils. 2+ and ATP on fluorescence intensity and lifetime in IANBD-cTnCT53C exchanged myofibrils. T53C exchanged myofibrils in a mock screen illustrates Z′ quality of high-throughput assays. T53C fluorescence lifetime changes due to W7 and Pimo in low Ca2+. T53C fluorescence lifetime changes due to W7 and Pimo in high Ca2+. T53C fluorescence lifetime changes due to Mava and OM in low Ca2+. T53C fluorescence lifetime changes due to Mava and OM in high Ca2+. T53C fluorescence lifetime changes due to Mava and OM by well-to-well analysis at high Ca2+.The supplemental material contains results of representative fluorescence waveforms, changes in intensities and lifetimes zoomed-out and plotted from zero for reference, and additional conditions  suggesting the incorporation of IANBD-Tn onto the thin filaments between Z-discs  and maximal ATPase activity in high Ca2+ (pCa 4.5: ∼11–14 nmol Pi·mg−1·min−1) were not different at each respective Ca2+ level between non-exchanged and exchanged myofibrils with 0.1 mM ATP . ADP was present at 20 min in high Ca2+ conditions . Following exchange, addition of 100 μM Mava reduced myofibril ATPase to ∼2 nmol Pi·mg−1·min−1 in low Ca2+ and to ∼4 nmol Pi·mg−1·min−1 in high Ca2+ , the relatively large well-to-well variations result in a negative Z-factor  that precludes its use in HTS designed to identify compounds that modify troponin’s response to Ca2+  compared to intensity, and with low well-to-well variability (reflected in a greatly reduced SD of ∼0.01 ns and C.V. of ∼0.4%), thereby offering a suitable assay for use in HTS. This was demonstrated by determining a Z-factor (Z′) and is illustrated in 2+ and the Z′ of 0.7 indicates excellent conditions for high throughput screening. Repeating this experiment of comparing intensity and lifetime changes of IANBD-cTnCT53C four times with three independent preparations of IANBD-Tn and myofibrils gave similar results  complicates examination of Ca2+ effects due to variable myosin binding (see next section). Despite this complication we observed that Ca2+ caused a reduction in the lifetime of IANBD on cTnCT53C in the presence of nucleotide , the IANBD lifetime was reduced by 6.7%  of the ATP remains unhydrolyzed and myosin only weakly binds actin. At high Ca2+, by 20 min, myosin has hydrolyzed the ATP to ADP  and high Ca2+ (pCa 4.5) first in rigor conditions and then again 20 min after the addition of 0.1 mM ATP. In low Ca2+, the rigor bound-myosin showed a cTnCT53C-IANBD lifetime that was 3.3% greater than that seen when ATP was present (−27) and large enough to be used in an HTS as shown by a positive Z′ (0.16) in this experiment (Z′ of 0.11 for four experiments (Myosin binding to the thin filament in rigor conditions increased the lifetime of IANBD on cTnCcleotide  at eithe present . This diperiment  and an aeriments .2+ (pCa 9), W7 increased lifetime by 11.4% as compared to DMSO control , W7 increased the lifetime by 6.1% relative to DMSO (P = 1.5 × 10−33). Moreover, the change in lifetime from low to high Ca2+ was 64% larger with W7 as compared to DMSO . In a representative mock screen of 24 wells, the Z′ factor was 0.76  and 2.5% in high Ca2+ . In one mock screen, Z′ factor was 0.22 at low Ca2+ (−6) with a Z′ factor of −2.7  and again the average Z′ factor was negative (−0.58) indicating that the conditions are not useful for an HTS assay that would reliably detect Pimo.In contrast to W7, Pimo decreased the lifetime as compared to DMSO by 3.2% in low Calow Ca2+ . Repeati of −2.7 . Combinifeasible . At highible , with 0.1 mM ATP, the lifetime of IANBD-cTnC decreased from 2.61 ± 0.01 ns in the absence of Mava to 2.49 ± 0.02 ns in the presence of Mava. This 4.4% decrease in lifetime is highly significant (P = 1.6 × 10−27). Z′ for this experiment (2+ ± Mava) was 0.19 and the average of three tests at low Ca2+ was 0.39 . The average Z′ for four tests was −0.48, indicating that this test condition is not useful for an HTS assay at high Ca2+  to be useful in HTS  drug candidates that modulate cTnC binding to Ca2+ and (2) drug candidates that modulate myosin, actin, Tm, cTnT, cTnI, and cTnC—the myofilament proteins responsible for transmitting the structural changes taking place following myosin binding to actin that result in changes in cTnC structure. Finally, our results suggest that compounds like W7, that desensitize Ca2+ binding to cTnC, and Mava that regulates the disordered relaxed (DRX) and ordered super-relaxed (SRX) states of myosin, can be identified by this platform. As cardiac myosin-binding protein C (cMyBP-C) is a known modulator of the SRX state, drugs modulating its function might also be detected by this platform.In the present study, we demonstrate that lifetime measurements of fluorescent IANBD-cTnC incorporated into cardiac myofibrils can sense Ca2+ or myosin binding. The earlier work by Davis and colleagues  but human tissue studies are also feasible  decreased the IANBD intensity, while myosin binding increased the intensity  than it is in the absence of Ca2+ or when the thin filament is impacted by myosin binding. More broadly, this result suggests that the response of cTnC to myosin binding to the thin filament is fundamentally different from its response to binding Ca2+.In striated muscle, Caen state . In the tate  and similar magnitudes of changes in IANBD lifetime. Additionally, changes in conformations of TnC may or may not result in different lifetimes.2+ where developed force would also be intermediate  but also between the proposed SRX and DRX myosin states, the latter being promoted by the drug Mava  and this will be seen as an increase in IANBD-cTnCormation . OM showlow Ca2+ , consistOur assay adds to a very small number of cTnC fluorescence-based assays conducive to HTS for therapeutic compounds to treat heart failure . At thisTable S1T53C fluorescence lifetime changes due to W7 and Pimo in low Ca2+shows IANBD-cTnCClick here for additional data file.Table S2T53C fluorescence lifetime changes due to W7 and Pimo in high Ca2+shows IANBD-cTnCClick here for additional data file.Table S3T53C fluorescence lifetime changes due to Mava and OM in low Ca2+shows IANBD-cTnCClick here for additional data file.Table S4T53C fluorescence lifetime changes due to Mava and OM in high Ca2+shows IANBD-cTnCClick here for additional data file.Table S5T53C fluorescence lifetime changes due to Mava and OM by well-to-well analysis at high Ca2+shows IANBD-cTnCClick here for additional data file.SourceData F1is the source file for Fig. 1.Click here for additional data file.


Calcium imaging experiments showed that intracellular Ca2+ increased to a peak due to calcium influx in the extracellular fluid through L-type and N-type voltage-gated calcium channels when HT22 cells were treated with 100 nM T for 20 min. Subsequently, we investigated whether the Ca2+/CaMKII signaling pathway mediates the rapid effect of T, promoting the expression of the synaptic protein PSD95. Immunofluorescence cytochemical staining and western blotting results showed that T promoted CaMKII phosphorylation by rapidly increasing extracellular Ca2+ influx, thus increasing PSD95 expression. This study demonstrated that CaMKII acts as a mediator assisting androgen which regulates the synaptic protein PSD95Also, it provides evidence for the neuroprotective mechanisms of androgens in synaptic plasticity and reveals the gated and pharmacological mechanisms of the voltage-gated Ca2+ channel family for androgen replacement therapy.Androgens rapidly regulate synaptic plasticity in hippocampal neurones, but the underlying mechanisms remain unclear. In this study, we carried out a comprehensive bioinformatics analysis of functional similarities between androgen receptor (AR) and the synaptic protein postsynaptic density 95 (PSD95) to evaluate the effect. Using different measurements and thresholds, we obtained consistent results illustrating that the two proteins were significantly involved in similar pathways. We further identified CaMKII plays a critical role in mediating the rapid effect of androgen and promoting the expression of PSD95. We used mouse hippocampal neurone HT22 cells as a cell model to investigate the effect of testosterone (T) on intracellular Ca The complex then acts on specific DNA androgen reaction elements, initiating and regulating the transcription of related genes and affecting mRNA expression and protein synthesis . This hoAndrogens play an important role in the regulation of synaptic plasticity in the hippocampus. In male mice, the density of dendritic spines in the hippocampal CA1 region decreased significantly after castration. Androgen supplementation increased dendritic spine density to the level observed in mice with intact gonads . The eff2+ is an important secondary messenger in neurones. Many extracellular signals, such as neurotransmitters and hormones, regulate Ca2+ content after interacting with receptors on cell membranes. Ca2+ activates protein phosphatase and participates in synaptic plasticity regulation  is a complex composed of signal transduction proteins, neurotransmitter receptors, and coupled scaffold proteins. Postsynaptic density 95 (PSD95) is an important scaffold protein, which is usually used as a marker protein of postsynaptic plasticity and is involved in synaptic plasticity regulation . In thisgulation . As a reThe protein–protein interaction data were collected from the STRING database (v11.5) . To obtaThe functional similarity of genes or proteins can be calculated by Gene Ontology (GO) . Currentp-value of semantic similarity between two sets of proteins by randomly picking up protein set(s) of the same size and calculating the random similarities. The p-value is defined as the ratio of random values that greater than the real value over the random times. Two random sampling categories were used, i.e., creating random protein sets 1,000 times for both real sets or only one set.Monte Carlo simulations are typically used to model the probability of different outcomes in a process that cannot easily be predicted due to the intervention of random variables . In this2. When the cells reached 80–90% confluence, they were harvested through treatment with a solution containing 0.25% trypsin and resuspended in appropriate culture plates.Mouse hippocampal neurone cell line HT22 cells were cultured in DMEM containing 10% fetal bovine serum  and 1% penicillin/streptomycin at 37°C and 5% CO2+ was measured via fluorescence imaging using the Ca2+ indicator dye, Fluo-4 AM. A 5 mM stock solution of Fluo-4 AM  and 20% Pluronic F-127  was prepared in DMSO , diluted 1:1000 with HBSS and used within a week. When HT22 cells were 60–70% confluent, they were treated with 5 μM working solution Fluo-4 AM containing 0.02% Pluronic F-127 for 30 min at 37°C in the dark. Afterward, the Fluo-4 AM working solution was removed, and the cells were incubated for an additional 30 min in HBSS at 37 °C in the dark. Intracellular free calcium imaging was performed using confocal microscopy  at an excitation wavelength of 488 nm and an emission wavelength of 530 nm. Images of 800 × 800 pixels were acquired in XYZ scan mode using a ×60 objective . The cells were perfused with HBSS for 5 min to obtain a basal fluorescence intensity level of intracellular Ca2+ (F0). This was followed by a further 55 min of treatment, including varying concentrations of testosterone , and EGTA , a chelating agent selective for Ca2+  and 1 μM CgTx  for 1 min before the start of T treatment, and those in the control group were treated with an equal volume of DMSO. The cells were incubated at 37°C for 20 min, and immunofluorescence cytochemistry and western blotting were performed.HT22 cells were cultured on coverslips until 60–70% confluence. The cells were then cultured in the presence or absence of 5 μM KN-93  for 2 h before treatment with T, and those in the control group were treated with equal volumes of DMSO. The cells were incubated at 37°C for 20 min, and immunofluorescence cytochemistry and western blotting were performed.HT22 cells plated on coverslips were fixed with 4% paraformaldehyde at room temperature for 15 min and sealed with 10% donkey serum at room temperature for 1 h. Afterward, the cells were incubated overnight at 4°C with the following primary antibodies: anti-PSD95  or anti-p-CaMKII , incubated with donkey anti-rabbit fluorescent secondary antibody  for 2 h at room temperature in the dark, and counterstained with DAPI  for 10 min. Fluorescence images were obtained by using an inverted Olympus FV1200 confocal microscope system. The relative mean fluorescence intensity was measured using Image-Pro Plus software (Media Cybernetics).RIPA lysis buffer containing phenylmethylsulfonyl fluoride was added to lyse HT22 cells, and proteins were extracted for quantification. Proteins were separated using SDS-PAGE and transferred onto a PVDF membrane. Then, the membranes were blocked with 5% milk for 2 h and incubated overnight at 4°C with the following primary antibodies: anti-PSD95 , anti-p-CaMKII , anti-CaMKII , and anti-GAPDH . Subsequently, they were incubated with DylightTM 800 goat anti-rabbit fluorescent secondary antibodies  in the dark for 2 h. Finally, imaging analysis was performed using an Odyssey imaging system . The relative expression of the target protein was calculated using the grey value of GADPH as a reference.p > 0.1) and homogeneity of variance (p > 0.1), and post-hoc multiple comparisons were performed using the SNK-q test. Differences were considered statistically significant at p < 0.05.All statistical analyses were performed using SPSS 21.0 statistical software. Results are expressed as the means ± standard deviation (SD). Data sets were subjected to normality testing using the Shapiro-Wilk normality test. Data from multiple groups were tested for homogeneity of variance using Levene’s test. One-way ANOVA was performed for data with a normal distribution  between AR interactors and PSD95 interactors is 0.818 when using the Interaction Confidence Score (ICS) greater than 0.95 . The SSSFor the above result, the SSS was computed using the Wang measurement. On top of that, four other methods were also used for calculating SSS, including Resnik, Rel, Lin, and Jiang. As shown in 2+/calmodulin-dependent protein kinase subfamily. Ca2+/calmodulin-dependent protein kinase that functions autonomously after Ca2+/calmodulin-binding and autophosphorylation, and is involved in the dendritic spine and synapse formation, neuronal plasticity, and regulation of sarcoplasmic reticulum Ca2+ transport in skeletal muscle. In neurones, it plays an essential structural role in the reorganization of the actin cytoskeleton during plasticity by binding and bundling actin filaments in a kinase-independent manner. In consequence, we performed the differential analysis of CaMK2B in two Alzheimer’s Disease RNA-sequencing (RNA-seq) datasets GSE173955 and GSE159699, and observed that CaMK2B was significantly differentially expressed in the two datasets   . Therefo2+ concentration in HT22 cells, Fluo-4AM, a calcium indicator, was used to monitor intracellular calcium signals. The calcium signal of HT22 cells in the T treatment group increased significantly and reached its peak at 20 min, whereas DMSO did not cause fluorescence changes in the cells  or N-type calcium channel blocker CgTx (1 μM), compared with T alone. This suggested that voltage-gated Ca2+ channels, especially L-type calcium channels, are involved in T-induced increase in intracellular Ca2+ levels , activate CaMKII, and affect synaptic plasticity through a series of cascade reactions (Androgens cause rapid external calcium influx and increase intracellular Caeactions . CaMKII eactions . CaMKII eactions . Changeseactions , decreaseactions , and deceactions . PSD95, eactions . PSD95 ieactions . PSD95 reactions  and play2+/CaMKII pathway in HT22 cells for the first time. To begin with, we inhibited L- and N-type voltage-gated Ca2+ channels and observed that the T-induced upregulation of PSD95 was significantly inhibited, suggesting that the T-induced increase in PSD95 protein partially depended on calcium influx caused by voltage-gated Ca2+ channel opening. This study confirmed that T could rapidly activate CaMKII phosphorylation in HT22 cells, while inhibition of L- or N-type voltage-gated calcium channels weakened the activation of CaMKII. In the central nervous system, activation of NMDA receptors on the postsynaptic membrane leads to an increase in the local concentration of postsynaptic Ca2+, which binds to and activates CaM. Activated CaM activates Ca2+/CaM-dependent CaMKII, which plays an important role in synaptic plasticity, learning, and memory (305 (In this study, immunofluorescence cytochemistry and western blotting were performed to determine whether T rapidly affects the expression of the synaptic protein PSD95 through the Cad memory . KN-93 iory (305 . In this2+ and upregulating the expression of PSD95. This study reveals the pharmacological mechanism of androgen replacement therapy mediated by the voltage-gated Ca2+ channel family, which contributes to a full understanding of the physiological role of androgens and provides evidence for further research on the neuroprotective mechanism of androgens.The results of this study showed that T promoted CaMKII phosphorylation by rapidly increasing the influx of external Ca


Recently, using an in vitro model of hypoosmotic hyponatremia, we have found that a decrease in sodium ion concentration in the perfusate to 121 mmol L−1 relaxes the isolated rat middle cerebral artery (MCA). The aim of the present study was to explore the mechanism responsible for this relaxation. Isolated, pressurized, and perfused MCAs placed in a vessel chamber were subjected to a decrease in sodium ion concentration to 121 mmol L−1. Changes in the diameter of the vessels were monitored with a video camera. The removal of the endothelium and inhibition of nitric oxide-dependent signaling or the reverse mode sodium-calcium exchanger (NCX) were used to study the mechanism of the dilation of the vessel during hyponatremia. The dilation of the MCA  in a low-sodium buffer was absent after removal of the endothelium or administration of the inhibitor of the reverse mode of sodium-calcium exchange and was reversed to constriction after the inhibition of nitric oxide (NO)/cGMP signaling. The dilation of the middle cerebral artery of the rat in a 121 mmol L−1 Na+ buffer depends on NO signaling and reverse mode of sodium-calcium exchange. These results suggest that constriction of large cerebral arteries with impaired NO-dependent signaling may be observed in response to hypoosmotic hyponatremia.A decrease in serum sodium ion concentration below 135 mmol L The difference in Na+ concentration across the cell membrane is essential for the maintenance of cell volume, resting membrane potential, and sodium-dependent transport systems oxadiozoloquinoxalin-1-one  to the extravascular perfusate 30 min before the lowering of Na+ concentration. Series VI was designed to study the participation of the reverse mode of the Na+/Ca2+ exchanger in the response of the MCA to low sodium environment. In this series, (2-[2-[4-(4-nitrobenzyloxy)phenyl] ethyl isothiourea mesylate  was administered to the normal sodium MOPS-buffered saline solution prior to the lowering of sodium ion concentration and maintained throughout the observation time. The lower dose, 1 µmol L−1 KB-R7943, has been used to inhibit nonselective mechanosensitive cation channels (TRPC) in order to find out if these channels can provide a route for Ca2+ influx during hyponatremia in our experiments. It has been shown that KB-R7943 blocks TRPC at submicromolar concentrations, whereas a half-maximal block of the Ca2+ entry mode of the NCX is between 1 and 5 µmol L−1 [−1, has been used to inhibit the Ca2+ entry mode of the Na+/Ca2+ exchanger [Induction of hyponatremia by lowering the concentration of NaCl by 24 mmol Lµmol L−1 . The hig−1), a selective membrane permeable chelator of intracellular Ca2+, was administered intraluminally to the normal sodium MOPS-buffered saline solution prior to the lowering of sodium ion concentration and maintained throughout the observation time. At the dose 10 µmol L−1, BAPTA-AM fully buffers the intracellular Ca2+ in endothelial cells and minimally chelates Ca2+ in smooth muscle cells [2+ to the response of the MCA to hypoosmotic hyponatremia.In series VII, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester),  144.0 NaCl, 3.0 KCl, 2.5 CaCl2, 1.5 MgSO4, 1.21 NaH2PO4, 0.02 EDTA, 2.0 pyruvate, 5.0 glucose, and 2.0 MOPS. The concentration of solutes in the hyponatremic MOPS buffer was the same as in the normonatremic one except for NaCl, which was lowered to 120 mmol L−1 to obtain a Na+ concentration in the buffer equal to 121 mmol L−1. The hyponatremic MOPS buffer was hypoosmotic in all but one series (series II) of the experiments in which the osmotic pressure was maintained at 300 ± 3 mOsm kg−1 as mentioned above. Acetylcholine chloride, NMDG, L-NAME, BAPTA-AM, Tris, and nimodipine were purchased from Sigma-Aldrich. ODQ and KB-R7943 mesylate were obtained from Tocris Cookson Ltd. The drugs were dissolved in a MOPS buffered saline solution except for ODQ, BAPTA-AM, and KB-R7943 mesylate, which were prepared in DMSO and added to the buffer. The final concentration of DMSO in the MOPS buffered saline did not exceed 0.005%. According to the results of a pilot study, this concentration of DMSO does not affect the diameter of the MCA.The normonatremic MOPS buffered saline solution contained  followed by post hoc Tukey’s comparison (Statistica 10). Single comparisons were made using Student’s t-test for paired or unpaired data when appropriate. The differences were considered statistically significant at p < 0.05.All the data is presented as mean ± standard error (S.E.), and The results of time control series 0 demonstrate that during the observation time of 180 min the diameter of pressurized and perfused MCA maintained at 37 °C and pH = 7.4  of the normonatremic control  or the Tris–HCl buffer did not affect the response  observed in endothelium-intact vessels. The diameter of the MCA without endothelium did not change when a 121-mmol L−1 Na+ MOPS-buffered saline solution was applied intra- and extraluminally . This dose of L-NAME induced a 25 ± 6% constriction  during normonatremia. During the subsequent lowering of Na+ concentration to 121 mmol L−1, the MCA constricted by an additional 10 ± 2% (p < 0.05). When ODQ (5 µmol L−1), an inhibitor of the synthesis of cGMP, was administered during normonatremia, the MCA constricted by 35 ± 7% . The low-sodium buffer administered on the background of ODQ induced a further constriction of the vessel by 12 ± 5% (p < 0.05).The successful removal of the endothelium was confirmed with 100 µmol L+ concentration resulted in the stable and comparable dilation of the MCA by, on average, 23% (p < 0.02) in response to 1 µmol L−1 and 19% (p < 0.02) in response to 10 µmol L−1 KB-R7943. These diameters were considered new baselines.The addition of KB-R7943 to the normal sodium buffer prior to the lowering of Na−1 Na+ MOPS buffer containing 1 µmol L−1 KB-R7943, the MCA dilated by 15 ± 5%, which was not different from the response in the control group  to the normonatremic buffer resulted in the constriction of the MCA by 15 ± 2% (p < 0.01). In the 121-mmol L−1 Na+ MOPS buffer with BAPTA-AM, the MCA dilated by 26 ± 6% (p < 0.05), which was not statistically different from the response observed in the control series I, i.e., BAPTA-AM did not affect the response of the MCA to decreased Na+ concentration.To assess the possible role of the intracellular Ca+ concentration in the perfusate i) depends on the lowering of extracellular sodium ion concentration and not on the decrease in osmolality and ii) requires the presence of an intact endothelium and nitric oxide/cGMP signaling. Our results suggest that the concentration of intracellular calcium ions required for the stimulation of the synthesis of nitric oxide by endothelial cells increases in a low sodium environment due to the activation of the reverse mode of the sodium/calcium exchanger. To the best of our knowledge, this is the first study that demonstrates normal cerebral blood vessels relaxing response to the lowering of extracellular sodium ion concentration through an endothelium- and NO/cGMP-dependent mechanism.We have demonstrated that the dilatation of the isolated, pressurized, and perfused rat middle cerebral artery in response to the lowering of NaIt is well established that extravascular osmolarity affects the tone of blood vessels. In hypoosmotic conditions, they constrict, whereas in a hyperosmotic environment, they relax . Local i2+ concentration. The increase in the intracellular concentration of Ca2+ in the low sodium/hypoosmotic buffer may be related to cell swelling (RVD does not completely restore the volume) and opening of stretch-activated Ca2+ channels such as, e.g., nonselective cation transient receptor potential (TRPC) membrane channels [2+ influx” mode. The present study shows for the first time that the “Ca2+ influx” mode of NCX participates in maintenance of the myogenic tone of isolated middle cerebral artery, since NCX inhibitor KB-R7943 administered at a dose of 10 µmol L−1, known to block the Ca2+ entry mode of the exchanger [−1 KB-R7943 in normonatremia. This lower dose of KB-R7943 was chosen by us to block TRPC channels based on the publication by Kraft who showed, as already mentioned, that half maximal inhibitory concentration (IC50) to block TRPC is below micromolar concentrations, whereas the IC50 of the Ca2+ entry mode of NCX is between 1 and 5 µmol L−1 [NO is the most prominent endothelium-dependent vasodilator, produced with the participation of a specific endothelial synthase, eNOS, and released from the endothelium to activate soluble guanylate cyclase in the vascular smooth muscle . The esschannels . Our prechannels , 24. Thechannels , 45, 52.channels  and to tchannels . Accordichannels  who revixchanger , 50, 52,µmol L−1 .+/Ca+2 exchanger caused by decrease in the Na+ concentration gradient across the cell membrane also participates in the transport of Ca2+ into the endothelium. They also suggest that the reverse mode of the neuronal isoform of NCX is activated during lowering of Na+ concentration in the perfusate and participates in NO/cGMP-dependent dilation of the middle cerebral artery in response to hyponatremia. This suggestion is legitimate on the basis of the observation that removal of the endothelium eliminates dilation of the MCA in response to hyponatremia, whereas inhibition of NOS-dependent signaling with a nonselective NOS inhibitor  or blockade of cGMP synthesis leads to MCA constriction in hyponatremia. This discrepancy may be best explained by the stimulation of both eNOS and nNOS in response to activation of the reverse-mode NCX. The more so, presence of functional nitrergic nerve fibers in the isolated rat MCA has been well documented [It has been suggested that the reverse mode of NCX is involved in activation of endothelial NOS and release of NO by rat aortic endothelial cells , porcinecumented , 31.2+ entry to the endothelium or nitrergic nerve endings during hyponatremia is the abovementioned TRP channels. These channels are commonly found in cellular membranes, participate in maintenance of basal myogenic tone, and as is also confirmed by our current data, are sensitive to stretch and participate in the dilation of cerebral blood vessels [2+ to cells in response to hyponatremia. KB-R7943, administered at a dose which blocks both the reverse mode of NCX and TPRC, eliminated the dilation of the MCA in response to hyponatremia, whereas inhibition of TPRC using a lower dose of KB-R7943 did not affect this response. However, further studies using well established TRPC inhibitors are needed to confirm this result. On the other hand, the experimental data published by some groups strongly suggest that the reverse mode of NCX is ideally situated in the cell membrane to deliver Ca2+ for the activation NOS. It has been shown that the Ca2+ influx associated with activation of the reverse mode of NCX does not lead to global increase in intracellular concentration of Ca2+ but occurs locally in the subplasmalemmal domain [Another possible route of Ca vessels , 38, 51.l domain . The impl domain , 45.2+ in the endothelium on the response of the MCA to hyponatremia strongly support the extracellular origin of the calcium signal required to activate NOS. The administration of BAPTA-AM at a dose which fully buffers the intracellular Ca2+ in endothelial cells and minimally chelates the Ca2+ in smooth muscle cells [+ concentration. Thus, intracellular Ca2+ does not participate in the response of the MCA to hyponatremia.Our results showing no effect of the buffering of intracellular Cale cells  did not 2+ concentration does not increase limits the possibility of participation of endothelial Ca2+-activated hyperpolarizing K+ currents such as IKCa and SKCa in the response of the MCA to hyponatremia [The results of our experiment with BAPTA-AM and the observations by others that during activation of the reverse mode of NCX in the endothelium intracellular Canatremia . Howevernatremia . The red2+ channel of the cell membrane [2+ channel does not seem to be present in the endothelium of the rat MCA [2+-activated K+ channel (BKCa) [Although there are data in the literature questioning KB-R7943 selectivity, they do not apply to the endothelial aspect of our study. It has been demonstrated that, in addition to the inhibition of the reverse mode of NCX, KB-R7943 may block the voltage-dependent, L-type Camembrane . However rat MCA . Anotherl (BKCa) . This ar− concentration. The result of this experiment confirmed that decrease in osmolality is irrelevant to the response of the MCA to hyponatremia.One may also question the use of NMDG to compensate for the osmolality in our study, as it was shown to activate outward currents , which, −1 decrease in plasma sodium concentration. This, in turn, suggests that endothelial NCX is much more sensitive to decrease in Na+ gradient across the plasma membrane than the one operating in smooth muscle cells. The elucidation of this issue requires a further study.Our results strongly suggest that reverse mode of NCX can be stimulated in endothelial cells of intact, isolated large cerebral arteries by a 24 mmol L2+ entry” mode of the Na+/Ca2+ exchanger in the endothelium.In summary, these results demonstrate for the first time that during hypoosmotic hyponatremia, normal cerebral blood vessels dilate through an endothelium- and NO/cGMP-dependent mechanism. They also suggest that stimulation of the NO/cGMP signaling pathway in the middle cerebral artery of the rat during short-lasting lowering of the extracellular sodium concentration is associated with activation of the “Ca+ concentration in the extracellular fluid.Last but not least, these results demonstrate that when NO-dependent regulation of the middle cerebral artery is impaired, the vessel constricts in response to the lowering of NaHowever, it should be stressed that the experiments reported here were performed on the middle cerebral artery of male rats. Taking into consideration the reported sexual differences in the response to hyponatremia and protective effects of estrogens on the regulation of cerebral blood vessels , 15, 34,


Complex karyotypes in acute myeloid leukemia (AML) are characterized by an overall low response rate with frequent relapses after clinical treatment. Here, we describe the case of a 61-year-old obese female with clinically diagnosed AML who presented a complex karyotype involving an uncommon abnormality: ring chromosome 11. Immunophenotypic analysis confirmed the diagnosis. Classical and molecular cytogenetic analyses, using GTG banding and FISH (fluorescence in situ hybridization), revealed the presence of complex structural rearrangement involving r(11), add(12)(p13), der(5) and der(13).  Molecular cytogenetic analysis is suitable for better identification and characterization of chromosomal rearrangements in AML. Case reports like this, as well as population-based studies, are necessary for understanding the karyotypic changes that occur in humans. In some cases, patients have satisfactory survival, whereas in others, the course has a dismal prognosis. The risk factors include obesity and chromosomal aberrations. Recent studies have suggested that obesity is a risk factor associated with AML.,Classically, karyotyping has formed a powerful independent prognostic indicator in this group of diseases.AML with normal karyotype;AML with primary balanced chromosomal aberrations; andAML with unbalanced karyotype abnormalities characterized by gains and/or losses of usually larger regions of the genome and no known primary balanced abnormality.Complex karyotypes are defined as three or more independent chromosomal abnormalities in one genome. AML patients with such abnormalities are characterized by a low overall response rate, and often present relapses after clinical treatment.Here, we describe a case with a complex karyotype involving uncommon chromosomal abnormalities in an obese patient. We also present a review of the literature focusing on studies in which ring chromosome 11 was found in AML cases.3/µl and leukocyte count of 19.58 x 103/µl. Serological tests were negative for anti-HIV1/2, anti-HTLV1/2, anti-HBc and anti-HBS. Bone marrow immunophenotypic analyses revealed 23% blasts; positivity for CD34, HLADR and CD33; and negativity for CD7, CD19, CD10, CD117, CD36 and CD15, characterizing acute myeloid leukemia (AML), FAB classification M4.A 61-year-old woman was registered at Pedro Ernesto University Hospital (HUPE) in December 2010, with a three-month history of dizziness, precordial pain and adynamia, a weight loss of 5.5 kg and fever. Her medical history was remarkable for obesity and hypertension. Physical examination showed pallor, rare petechiae and no palpable lymph nodes. Laboratory analysis revealed a hemoglobin concentration of 5.5 g/dl, platelet count of 25 x 10The patient was treated with one cycle of cytarabine and daunorubicin to induce remission and four consolidation cycles of cytarabine. Hematological remission was achieved after the first cycle of cytarabine/daunorubicin (one month afterwards).Nevertheless, she relapsed in August 2011. At that time, a myelogram showed the presence of blasts  and an iEthics committee approval: CAAE #56621716.5.0000.5259.The literature was reviewed through the MEDLINE (PubMed) and LILACS databases . Obesity is a chronic inflammatory condition, characterized by increased production of pro-inflammatory cytokines and adipokines, presence of hyperinsulinemia and insulin resistance and elevated levels of insulin-like growth factors.,,20,,,At the time of relapse, our patient also presented a complex chromosomal karyotype involving at least four chromosomes. She was positive at the molecular level for AML-ETO [t] rearrangement, e.g. der(12), and the marker had the capacity to carry parts of chromosomes 8 or 21.For the chromosomal abnormalities found here, i.e. ring chromosome and translocations, it needs to be noted that ring chromosomes are considered to be rare in hematopoietic cancer (less than 10%).Ring chromosome 11 may carry the important leukemia-related gene MLL (mixed lineage leukemia)/KMT2A, which encodes a DNA-binding protein that methylates histone H3. MLL is a frequent target for recurrent translocations in acute leukemia cases, which can be classified as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), or mixed lineage (biphenotypic) leukemia (MLL). Interestingly, leukemia with translocations involving MLL shows poor prognosis. More than 50 different MLL fusion partners have been identified, and it has been observed that MLL fusion proteins lose H3K4 methyltransferase activity, thus generating transformation capacity.Molecular cytogenetic analysis is suitable for better identification and characterization of chromosomal rearrangements in acute leukemia. Single case reports, as well as population-based studies, are necessary for providing further insights into karyotypic changes that take place in human leukemogenesis.


Calcium (Ca2+) is a ubiquitous intracellular mediator involved in various cellular functions such as releasing neurotransmitters from neurons, contraction of muscle cells, fertilization, and cell differentiation and morphogenesis. The multiple roles played by this ion in mediating signals can be primarily explained by its spatiotemporal dynamics that are kept tightly checked by mechanisms that control its entry through plasma membrane and its storage on intracellular stores. Given the large electrochemical gradient of the ion across the plasma membrane and intracellular stores, signals that can modulate Ca2+ entry channels or Ca2+ receptors in the stores will cause Ca2+ to be elevated in the cytosol and consequently activating downstream Ca2+-responsive proteins resulting in specific cellular responses. This review aims to provide an overview of the reported neurotransmitters and neuropeptides that participate in early stages of development and their association with Ca2+ signaling.A primary reason behind the high level of complexity we embody as multicellular organisms is a highly complex intracellular and intercellular communication system. As a result, the activities of multiple cell types and tissues can be modulated resulting in a specific physiological function. One of the key players in this communication process is extracellular signaling molecules that can act in autocrine, paracrine, and endocrine fashion to regulate distinct physiological responses. Neurotransmitters and neuropeptides are signaling molecules that renders long-range communication possible. In normal conditions, neurotransmitters are involved in normal responses such as development and normal physiological aspects; however, the dysregulation of neurotransmitters mediated signaling has been associated with several pathologies such as neurodegenerative, neurological, psychiatric disorders, and other pathologies. One of the interesting topics that is not yet fully explored is the connection between neuronal signaling and physiological changes during oocyte maturation and fertilization. Knowing the importance of Ca Based on this, we focus this literature review on the association of neuronal signals with Ca2+ in oocyte mutation and early development.Oocyte maturation and embryogenesis has always been an area of interest for many researchers. Despite the abundant research on the topic, many aspects of the development process are yet to be studied. One specific area of interest is investigating the interplay between neurotransmitters and neuropeptides and oocyte maturation and early development . SimilarOocyte maturation is a process by which an immature oocyte arrested at prophase of first meiotic division resumes meiosis to reach competence for normal fertilization after ovulation . In almo2+ signaling in the oocyte physiology is described in many animal species .2+ signaling during oocyte maturation and fertilization . On the other hand, neuropeptides are a specific type of large-molecule neurotransmitters identified as dense-core vesicles for their appearance under electron microscopy .There are two different classes of neurotransmitter receptors, ionotropic and metabotropic. Ionotropic receptors are neurotransmitter-gated ion channels. This class of channels will open after neurotransmitter binding, and messages between neurons using ionotropic receptors will be delivered in less than a millisecond . On the 2+ signaling during these important processes.Different signaling pathways have been recognized as vital regulators of early organism development, such as fibroblast growth factor (FGF), Hedgehog, Wnt, transforming growth factor-β (TGF), and Notch . These sIn mammals, the gonads of both males and females and other reproductive organs are innervated by the parasympathetic and sympathetic efferents . A growiInnervation of the ovaries is mainly through the nerves from the ovarian plexus. Most of these nerves are noradrenergic nerves . Some stApostichopus japonicus, injected two neuropeptides, NGIWYamide and NGLWYamide into the abdominal cavities of sexually mature males and females, which resulted in induction of gamete spawning. Despite these valuable findings, the exact sites of action and synthesis of NGIWYamide are still unknown. The study was only able to illustrate the importance of the ovarian wall for the function of the peptide  and octopamine (OA)  .Different hormonal signals have been recognized for their vital role in the various reproductive processes, such as ovulation. Oocyte maturation in vertebrates is triggered by luteinizing hormone (LH) surge secreted by the anterior pituitary in response to the pulsatile release of gonadotropin releasing hormone (GnRH) by the hypothalamus . Howeverαs and Gαolf  is another important neurotransmitter that plays a significant role in regulating the immune system as it controls inflammation by preventing specific inflammatory cells’ effects in non-neuronal cells . Moreoveq or Gi . Ach is A, or the metabotropic receptor, GABAB. GABAA receptor is a chloride ion channel that when activated, results in cell membrane hyperpolarization. In contrast, GABAB is a G-protein coupled receptor that decreases presynaptic Ca2+ conductance and increases postsynaptic K+ conductance , was reported to play a role in promoting synapse formation, cell survival, and plasticity, specifically in the olfactory bulb. The study by Garcia et al. revealed that manipulating the CRH signaling levels can affect the formation of synaptic proteins and the maintenance of active synapses. .2+ concentrations. Eshra et al. studied the effects of different Ca2+ concentration levels on vesicle priming, fusion, and replenishment at mossy fiber synapses in the rodent cerebellum. This study showed Ca2+ dependent vesicle fusion, as demonstrated in previous studies. In terms of vesicle priming, the results indicated the presence of a high-affinity Ca2+ sensor at the studied synapse that initiated the process of priming at intracellular Ca2+ concentrations in the range of 30 and 180 nM. Finally, there is a low association between intracellular Ca2+ concentration and vesicle recycling. Overall, the study illustrated the high dependence of the neuronal signaling process, specifically the phases of priming and fusion, on the intracellular Ca2+ levels  mainly regulated intracellular Ca2+ release, which led to the activation of IP3 receptors and, subsequently, the ryanodine receptors attached to the endoplasmic reticulum, increasing Ca2+ concentrations in the cytosol. Moreover, to exclude the presence of other pathways triggered by NPSR1 activation, the researchers incorporated into the culture receptor-specific and PLC blockers and noted that the Ca2+ concentration levels did not increase, thereby concluding that NPSR1 mediates intracellular Ca2+ release only through Gq signaling (IP3-DAG Pathway). Overall, several studies link the NPSR1 gene to the development of panic and anxiety disorders. Thus, the authors indicated that their study design and findings will benefit future research in developing a better understanding of the clinical implications of the gene in anxiety and panic disorders (The role of the neuropeptide S (NPS) and NPS receptor 1 (NPSR1) complex in activating the intracellular pathways of Caisorders .2+ signaling in serotonin (5-HT) mediated neurite outgrowth  neurons  promoted a synchronic, transient increase in Ca2+ in the nucleus of hippocampal neurons. This increase translated into elevated levels of phosphorylated CREB, Nps4, and RyR2 mRNA. This study proved the imperative role of RyR2 and Ca2+ induced- Ca2+ release in the generation of nuclear Ca2+ transients following neuronal stimulation either through GBZ or glutamate uncaging  or Norepinephrine (NE). In Caenorhabditis, the pivotal role of OA in maintaining oocytes’ quiescence was proven through mutant nematodes lacking endogenous OA. The study identified SER-3, a G-protein-coupled receptor (Gq), as the receptor targeted by OA and allowing the transduction of downstream inhibitory signals. In Drosophila melanogaster, the balance between this noradrenergic and the nutrient signals determines the onset of oogenesis. Mutant forms of Drosophila melanogaster revealed the importance of OA in inhibiting the progression of egg chambers during nutrient insufficiency. This action, in turn, is mediated via either alpha or beta-adrenergic receptors. Like Drosophila melanogaster, the maintenance of oocyte quiescence in Danio rerio females is governed by the balance between the nutrient and noradrenergic signals. The only difference is that the ovaries in this species are innervated by NE-producing cells  release   acts on the pituitary gonadotrophs to induce biphasic Ca release . The Ca2channels . Other schannels , and indchannels . In addi C (PKC) .2+ dynamic in oocytes expressing rat TRH receptors  generation, they induce Ca2+ mobilization from distinct cellular stores in the Xenopus oocyte, suggesting that these different compartments are associated with different receptors  is another peptide that can modulate pituitary secretion and was shown to trigger a biphasic Caeceptors . TRH receceptors . TRH-medectively . Shapiraeceptors . Unfortu2+ signaling and the neurotransmitter serotonin (5-HT), in triggering oocyte maturation, spawning, and early development in bivalve mollusks , that can suppress 5-HT-induced oocyte maturation. The factor was detected with serotonin-containing neurons in the cerebral and pedal ganglia in the CNS of these bivalve mollusks  concentrations  and Neuronal Per Arnt Sim domain protein 4 (Npas4)  . On the ogenesis . Sato ettal rats . Additioelopment .2+ changes in modulating processes during oocyte maturation and early development. Further studies are needed to understand the molecular machinery modulated by Ca2+-dependent neuropeptides signaling. Equally important is to identify new gonadal neuropeptides and their receptors. Understanding the role of neuropeptide signaling in abnormalities associated with fertility and early development could pave the way for targeting these pathways during therapeutic interventions.Several neurotransmitters including neuropeptides are known to play a role in oocyte maturation and early development in vertebrates and invertebrates. Examples are noradrenergic molecules, serotonin, neuropeptides coupled to G-protein coupled receptor, GABA, and dopamine. However, few of these neurotransmitters were demonstrated to be dependent on Ca


RUNX1 mutations, and high leukemic stem cell frequency as well as poor outcome, whereas granulocyte–monocyte progenitor-like AMLs have CEBPA mutations, RUNX1-RUNX1T1 or CBFB-MYH11 translocations, lower leukemic stem cell frequency, higher chemosensitivity, and better outcome. NPM1 mutations correlate with most mature stages of leukemia arrest together with TET2 or IDH mutations in granulocyte progenitors-like AML or with DNMT3A mutations in monocyte progenitors-like AML. Overall, we demonstrate that AML is arrested at specific stages of myeloid differentiation (SLA classification) that significantly correlate with AML genetic lesions, clinical presentation, stem cell properties, chemosensitivity, response to therapy, and outcome.Classifications of acute myeloid leukemia (AML) patients rely on morphologic, cytogenetic, and molecular features. Here we have established a novel flow cytometry-based immunophenotypic stratification showing that AML blasts are blocked at specific stages of differentiation where features of normal myelopoiesis are preserved. Six stages of leukemia differentiation-arrest categories based on CD34, CD117, CD13, CD33, MPO, and HLA-DR expression were identified in two independent cohorts of 2087 and 1209 AML patients. Hematopoietic stem cell/multipotent progenitor-like AMLs display low proliferation rate, inv(3) or  Immunophenotypically, human HSCs are enriched in a population of LinA− cells –4. Upon A− cells  postulatDNMT3A, TET2, or ASXL1, which then accumulate a series of secondary mutations some of which will block differentiation while others induce uncontrolled proliferation [Acute myeloid leukemia (AML), the neoplastic counterpart of early hematopoiesis, is caused by the excessive proliferation of transformed hematopoietic progenitors which show great heterogeneity at the morphological, immunophenotypic, cytogenetic, and molecular levels . Recent feration , 9. Impoferation –12.Despite a better characterization of AML and higher efficacy of new AML therapies, biomarkers for response prediction are currently lacking, in part because genomic aberrations have shown only limited predictive value . More acThe proteome and the surfaceome have been considered a promising and complementary fields to the genome for elucidating cancer biology and identifying diagnostic and predictive biomarkers , 15. HowHere, we did a phenotypic, cytogenetic, and molecular correlative analysis of a very large cohort of AML patients to capture the AML surfaceome that appears to be caused by the stage of leukemia arrest (SLA) and by specific genomic features. We hypothesized that our approach may add to current AML classifications by identifying relevant phenotypic AML subtypes with specific clinical and molecular features as well as information on outcomes after standard treatment. Overall, we anticipated that phenotypic classification associated with the morphological analysis, available within 24 h on the day of diagnosis, could be very useful to begin planning therapeutic strategy in daily practice.Between January 1, 2000 and December 31, 2019, 2448 AML patients (>15 years) were included in the AML database of Toulouse University Hospital (TUH) and 1700 AML patients in the AML database of Bordeaux University Hospital (BUH). Immunophenotyping at diagnosis was available for 2087 TUH patients (median age: 63 years) of which 1266 received intensive chemotherapy , 22.Tabl5 cells were stained per analysis tube, and data were acquired on at least 1 × 104 blasts when specimen quality permitted. Data on standardized 8- to 10-color staining combinations were acquired on FACSCanto II cytometers using FACSDiva software (BD Biosciences) or Navios instruments analyzed using Kaluza (Beckman-Coulter). Several different tube configurations were used through the course of the study, all with staining for CD13, CD33, CD34, CD45, CD117, HLA-DR, and cytoplasmic MPO. A blast gate including CD45 dim mononuclear cells was analyzed according to cytomorphologic data.Multi-parameter flow cytometry (MFC) was performed on whole bone marrow (BM) or blood specimens using a standard stain-lyse-wash procedure with ammonium chloride lysis. 1 × 10FLT3-ITD was tested as described [CEBPA screening was performed by classical Sanger sequencing [ASXL1 (NM_015338.6), ASXL2 (NM_018263.6), BCOR (NM_001123383.1), BCORL1 (NM_021946.5), CBL (NM_005188.4), CCND2 (NM_001759.4), CEBPA (NM_004364.5), CSF3R (NM_156039.3), DHX15 (NM_001358.3), DNMT3A (NM_022552.5), EP300 (NM_001429.4), ETV6 (NM_001987.5), EZH2 (NM_004456.5), FLT3 (NM_004119.3), GATA1 (NM_002049.4), GATA2 (NM_032638.5), IDH1 (NM_005896.4), IDH2 (NM_002168.4), JAK2 (NM_004972.4), KDM5A (NM_001042603.3), KDM6A (NM_021140.4), KIT (NM_000222.3), KMT2D (NM_003482.4), KRAS (NM_004985.5), MGA (NM_001164273.2), MYC (NM_002467.6), NF1 (NM_000267.3), NPM1 (NM_002520.7), NRAS (NM_002524.5), PHF6 (NM_032458.3), PIGA (NM_002641.4), PTPN11 (NM_002834.5), RAD21 (NM_006265.3), RUNX1 (NM_001754.5), SETBP1 (NM_015559.3), SF3B1 (NM_012433.4), SMC1A (NM_006306.4), SMC3 (NM_005445.4), SRSF2 (NM_003016.4), STAG2 (NM_001042749.2), TET2 (NM_001127208.3), TP53 (NM_000546.6), U2AF1 (NM_006758.3), WT1 (NM_024426.6), ZBTB7A (NM_015898.4), ZRSR2 (NM_005089.4). Data were processed through two algorithms from GATK (https://software.broadinstitute.org/gatk), HaplotypeCaller, and Mutect2, and also through Agilent Surecall software, with a sensitivity of 1% [DNA samples from 409 AML patients have been obtained after informed consent and stored at the HIMIP collection (BB-0033-00060). Briefly, genomic DNA was extracted from the baseline bone marrow sample using a Qiagen DNA extraction kit (Qiagen). The presence of escribed . Electroquencing . An exteMYC NM_0067.6, NF1T3A NM_0252.5, EP3Freely available gene expression datasets for normal hematopoietic stem and progenitor cells GEO:GSE42414 , Array Ep < 0.05, **p < 0.01, ***p < 0.001.Complete response and relapse rates were defined according to the Cheson criteria . CompariDisease-free survival was measured from the date of complete remission until the date of relapse or death. The cumulative incidence of relapse was measured from the date of complete remission until the date of relapse, with death regarded as a competitive event. Overall survival was measured from the date of diagnosis until death. Patients in complete remission were censored at the time of the last contact. The risk groups for prognosis were evaluated for overall and disease-free survival by univariate analysis (log-rank test) using a multivariate model of Cox regression and for the cumulative incidence of relapse by the Fine and Gray test. All calculations were performed using STATA version 13 software , all graphs were done using Graph Pad Prism.More details are provided in Supplemental Information.To define a surrogate phenotype for each stage of normal myeloid maturation, we need to assess by flow cytometry, the expression of markers used to characterize AML routinely on HSPCs. HSC, MPP, CMP, GMP, and GP/MP can be characterized by gating in the lineage negative cell population according to their expression of CD34, CD38, CD90, and CD45RA  represented 0.9% Fig. . TUH cohFollowing induction of the differentiation process, HSPCs lose their capacity to self-renew, in favor of proliferation and migration. We, therefore, investigated the functional characteristics of the six SLA using clinical data and clonogenic properties as surrogate markers of migration (extramedullary involvement) and proliferation (leucocytosis) capacities. Cell migration capacities and emigration from the bone marrow are known to be features acquired during differentiation . As a reBAALC, ERG, and MN1 [BAALC, ERG, and MN1 were overexpressed in HSC-L and MPP-L and repressed in GP-L and MP-L .We have previously shown that the level of CD34otherapy , 34. To p = 0.0001, Fig. p = 0.0027, Fig. p = 0.0002, Fig. To evaluate the stem properties of SLA subsets, we injected leukemic cells from 70 AML in 446 NGS mice . Early SLA  had higher number of engrafted mice .To identify oncogenic events linked to specific SLA, we studied point mutations and cytogenetic anomalies. We screened 46 genes commonly mutated in myeloid malignancies from 409 patients of the TUH cohort and identified 1363 mutations or cytogenetic anomalies Fig. , with ovn = 1967, Fig. n = 409, Fig. Although co-mutation or mutual exclusivity profiles have been previously described in AML , 36, our+ AML, positive for myeloid markers (CD13+CD33+CD117+); and differ by their expression of cytoplasmic MPO (<10% for MPP-L and within the range of 10–70% for CMP-L). MPP-L and CMP-L show more often cytogenetic abnormalities of AML MRC , del(17p)  and del(12p) . MPP-L and CMP-L are also enriched in secondary AML (s-AML) mutations [n = 200, Supplemental Fig. ASXL1 , SRSF2 , EZH2 , ZRSR2 , STAG2 , and SF3B1 , BCOR . In order to investigate the relationship between secondary AML and SLA, we rigorously classified 409 AML patients as clinical s-AML (post-MDS or MPN), molecular s-AML . In addition, inv(3) , t , CSF3R  and RUNX1 mutations  were enriched in MPP-L.MPP-L and CMP-L are phenotypically defined as CD34MRC Fig.  such as utations  in normad in MDS ) or karyp = 0.001), spliceosome  and myeloid transcription factors .Gene mutations can be further functionally classified into eight categories  Fig. 4DC. MPP-L +CD13+CD33+CD117+ and high expression of cytoplasmic MPO (>70%). Astonishingly, it was very specific of three abnormalities , t  and CEBPA mutations . We further studied CEBPA mutations in 871 AML from the TUH cohort and found the mutation in 35.7% of GMP-L − AML, positive for myeloid markers (CD13+CD33+CD117+/−); and differ by their expression of HLA-DR (≥20% for MP-L and <20% for GP-L). Both groups frequently expressed NPM1 mutation  and FLT3  in MP-L, and with TET2 mutations in GP-L, . Mutations in TET2, IDH1, and IDH2 are largely mutually exclusive and lead to similar epigenetic changes [TET2 mutations were enriched in GP-L, we looked at the distribution of IDH1 and IDH2 mutations in the TUH cohort and found that these mutations were also enriched in GP-L . Of note, besides the NPM1-mutated MP-L subset which accounts for 64% of all MP-L and 82% of normal karyotype MP-L, MLL fusions were enriched in this SLA . As a result, overall survival was significantly worse in patients with immature compared to mature SLA  was identical or more immature to diagnostic, in most of the cases  showed that MPP-L and CMP-L had a significantly higher ICIn multivariate models, SLA classification retained independent prognostic values for overall survival, event-free survival, and cumulative incidence of relapse Tables S and S6. Altogether, these data indicate that the chemoresistance of AML cells is, at least in part, a consequence of innate (SLA imprint) and acquired (oncogenic events) mechanisms   are associated with MPP-L, abnormalities of secondary AML with MPP-L and CMP-L, CEBPA mutations, RUNX1-RUNX1T1 or CBFB-MYH11 translocations with GMP-L, NPM1, and TET2 or IDH mutations with GP-L and NPM1 and DNMT3A mutations and t(11q23) with MP-L. As noted, future studies will need to further clarify the genotype–phenotype correlations of AML as improved myeloid maturation markers are developed.Our findings also suggest that few oncogenic events may be responsible for the SLA. The subclonal architecture of AML has been already described . HypotheRUNX1-RUNX1T1, CBFB-MYH11, CEBPA, and NPM1 mutations) do benefit from intensive chemotherapy as compared with HSC-L/MPP-L/CMP-L. Obviously, it will be very interesting to describe the impact of new therapeutic combinations such as azacitidine and venetoclax in this context [In vitro studies of chemosensitivity and clinical data also demonstrated that the SLA classification could predict response to the main therapeutic strategy used in AML. Indeed, GMP-L/GP-L/MP-L encompass the more chemosensitive genetic subgroups  in CR according to SLA.Comparison of SLA at diagnosis and at relapseCox regression model for overall survival of TUH cohort .Competing risk regression model for cumulative incidence of relapse of TUH cohort .Summary of AML patient characteristics according to the SLA classification.Cox regression model for overall survival of BUH cohort .