The adsorption of MGITC to DOPC liposome nanoparticlesin water is driven by electrostatic and dipole–dipole interactionsbetween the positively charged MGITC molecules and the zwitterionicphospholipid membranes. The SHG intensity increases as the added MGITCdye concentration is increased, reaching a maximum as the MGITC adsorbateat the DOPC bilayer interface approaches a saturation value. The experimentaladsorption isotherms are fit using the modified Langmuir model toobtain the adsorption free energies, adsorption equilibrium constants,and the adsorbate site densities to the DOPC liposomes both with andwithout APAP. The addition of APAP is shown to increase MGITC adsorptionto the liposome interface, resulting in a larger adsorption equilibriumconstant and a higher adsorption site density. The MGITC transporttimes are also measured, showing that APAP decreases the transportrate across the DOPC liposome bilayer, especially at higher MGITCconcentrations. Studying molecular interactions at the colloidal liposomeinterface using SHG spectroscopy provides a detailed foundation fordeveloping potential liposome-based drug-delivery systems.Time-resolved secondharmonic generation (SHG) spectroscopy isused to investigate acetaminophen (APAP)-induced changes in the adsorptionand transport properties of malachite green isothiocyanate (MGITC)dye to the surface of unilamellar 1,2-dioleoyl- Understanding potential drug–druginteractions at lipid bilayer interfaces is crucial in the furtherdevelopment of liposome-based and lipid nanoparticle-based drug-deliveryapplications, especially for ensuring the overall safety and effectivenessof these powerful emerging nanomedicine technologies.Several examples highlight the growing role of liposome systemsfor drug-delivery applications as a subset of nanomedicine.27 In our firststudy, comparisons of adsorption and transport of malachite green(MG) and methyl green (MetG) dye molecules in 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)(DOPG), dioleoyl-sn-glycero-3-phospho-l-serine (DOPS), trimethylquinone-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (QPADOPE), andDOPC liposomes under different buffer and salt conditions highlightedthe influence of several interrelated factors on molecular translocationsuch as electrostatic interactions, the molecular structures of thelipid headgroups, effects from buffer concentrations, adsorbate–adsorbaterepulsions, and ion-pair formation.25 Inour second study, time-dependent SHG measurements were combined withmolecular dynamics (MD) simulations to elucidate fundamental eventsassociated with adsorption and transport of the small molecular cation,malachite green isothiocyanate (MGITC), in comparison to MG in DOPG,DOPC, DOPS, and QPADOPE colloidal liposomes, focusing on changes dueto added chemical functional group isothiocyanate.26 In our group’s most recent work, SHG spectroscopyand MD simulations were used to study molecular adsorption and transportof MG at the surface of DOPG liposomes in water at different temperaturesto determine the thermodynamic properties of adsorption enthalpy andadsorption entropy at the bilayer surface.27 In the current study, presented here, we build on our previous workto investigate changes to molecular translocation caused by the presenceof a drug molecule acetaminophen (APAP).In ourprevious work, time-resolved second harmonic generation(SHG) spectroscopy was used to investigate molecular adsorption andtransport kinetics of drug-like organic dye molecules in differenttypes of liposomes prepared in aqueous solution.N-acetyl-p-aminophenol andparacetamol, is one of the most commonly used over-the-counter drugsfor pain and fever relief. APAP is very safe and effective when usedas directed.30 However, despite its wide and common analgesic andantipyretic uses, APAP also shows a variety of side effects and toxicitieswith potential for overdose when used incorrectly. APAP overdose isthe most common cause of acute liver failure and the leading causeof chronic liver damage requiring liver transplantation in developedcountries.31 In animal studies, APAP overdose induced a dramaticchange of many phosphatidylcholine and phosphatidylethanolamine speciesin the plasma membrane of liver cells, resulting in damaged hepatocytesand interference with phospholipid metabolism.32 Studies have shown that APAP can interact with zwitterionicphospholipid bilayers, leading to altered membrane fluidity, rigidity,permeability, and morphology.35 Understanding the unique effectsof APAP on mammalian cells, particularly related to molecular-leveldetails of the drug’s influences on physicochemical propertiesof the cell-membrane integrity and fluidity, is important for mitigatingpotential toxicity and for developing safer therapeutic treatments.33 Additionally, studying changes in molecularadsorption and transport properties of small, drug-like molecularprobes with liposome bilayers both with and without added APAP canform the basis for fundamental research on potential drug–druginteractions at cell membranes and in liposome-based drug-deliveryapplications.APAP, also known as 37 SHG is a frequency-doubling process where two photons of frequency ω are added coherently to generate a third photonof frequency 2ω.38 In the dipole approximation,SHG is forbidden in bulk media with centrosymmetric symmetry, suchas in isotropic solutions, while SHG is allowed at surfaces wherethe symmetry is broken.40 SHG measurements have been used to investigate freely adsorbingmolecules at the surface of colloidal nanoparticles as well as theadsorption and transport kinetics at phospholipid bilayer membranesin liposomes and living cells,45 where most of this research focuses on cationic dyes such MG,43 MGITC,26 and hemicyanine46 because of their strongSHG signals when adsorbed to the outer membrane surface.47 The triphenylmethane dyes of MG, MGITC, and brilliant green alsohave very low two-photon fluorescence signals because of their ultrafastexcited-state relaxation dynamics,50 making them excellentSHG-active probes for liposome studies. After adsorption, these moleculescan transport through the bilayer membrane and adsorb onto the innersurface of the liposome with an opposite orientation compared to dyemolecules on the outer surface.51 Because the lipid bilayer thickness is approximately 5 nm, whichis much smaller than the SHG coherence length, the second harmonicpolarizations of the oppositely oriented molecules on the inner andouter surfaces of the membrane effectively cancel resulting in a decreasein SHG intensity.47 Therefore, the time-dependentincrease and subsequent decrease in the SHG signal provide surface-specificinformation on the molecular adsorption and transport properties ofthese SHG-active probe molecules at the liposome bilayer interface.52 The SHG electric field ESHG generated at frequency 2ω is linearlyproportional to the difference in the population of dye moleculeson the outer surface N0 and the innersurface iN and given by the equation,53Eω is the incident opticalelectric field at frequencyω. The measured intensity of the SHG signal ISHG at frequency 2ω is given by ISHG = ESHG2.SHG is a powerful, nondestructive, nonlinear spectroscopictechniquewhich can be applied to characterize surfaces and interfaces of colloidalnanoparticles, microparticles, and liposomes.56 The adsorption of MGITC to DOPCliposomes is driven by electrostatic and dipole–dipole interactionsbetween the positively charged MGITC molecules and the zwitterionicphospholipid membranes.26 Time-resolvedSHG signals are used to determine the adsorption equilibrium constants,adsorbate site densities, and molecular transport times, characterizingthe detailed molecular interactions with the liposome surface. Additionally,these measurements are repeated after adding APAP to determine correspondingchanges to these molecular interactions caused by the presence ofthis added drug molecule. These surface-sensitive nonlinear opticalmeasurements of molecular adsorption and transport at colloidal liposomeinterfaces provide a detailed foundation for understanding molecularinteractions with phospholipid bilayers and drug–drug interactionsfor designing safe and effective drug-delivery applications.In this study, SHG spectroscopyis used to monitor the adsorptionand transport kinetics of the drug-like dye molecule MGITC in DOPCliposomes in water both with and without added APAP. DOPC is a commonphospholipid in mammalian cell membranes and has been used in severaldrug-delivery applications.33 DOPC lyophilized powder, purchasedfrom NOF America Corporation, and APAP powder, purchased from SpectrumChemical MFG Corp, are dissolved in HPLC-grade chloroform (0.4 mg/mL),purchased from Sigma-Aldrich. After dissolving, the DOPC and APAPare mixed to obtain specific molar ratios of 1:0, using 75 μMDOPC and 0 μM APAP, and 3:1, using 75 μM DOPC and 25 μMAPAP. The samples are placed under a nitrogen stream until most ofthe solvent is evaporated and then placed in a vacuum oven overnightto remove the remaining organic solvent traces. The obtained dry lipidcakes are hydrated with 5 mL of ultrapure water for each sample. Thevesicle suspensions then undergo eight freeze–thaw cycles at−20 and 50 °C in 10 min intervals. Finally, the vesiclesare extruded using an Avanti Mini-Extruder with 100 nm polycarbonatemembranes passing the vesicle suspension 33 times through the membraneto obtain large unilamellar vesicles with diameters of approximately100 nm. After extrusion, 95 mL of ultrapure water is added to eachsample, making a total of 100 mL per sample. The DOPC liposomes arein the fluid phase at ambient temperature, with a transition meltingtemperature Tm of −16.5 °Cwhere the ordered gel phase changes to the more disordered fluid phase.57 Additional characterization of DOPC liposomesis discussed in the Supporting Information. The molecular structures of DOPC, MGITC, and APAP are also shownin Figure S1.The synthesisof large unilamellar vesicles of DOPC liposomes has been previouslyreported.58 Briefly, a titanium:sapphire oscillator laser output centered at800 nm with 75 fs pulses at a repetition rate of 80 MHz is attenuatedto 1 W using a neutral density filter and is focused into a 1 cm quartzcuvette containing the colloidal DOPC liposomes in aqueous solution.The SHG signal is collected in the forward direction and is detectedas a function of time using a high-sensitivity spectroscopy charge-coupleddevice connected to a monochromator spectrograph. Additional detailsof the SHG setup are provided in the Supporting Information.TheSHG spectroscopy setup has been describedpreviously.26 The extinction spectrum of MGITC in water is displayed in the Supporting Information. As shown in 60 The SHG signal of MGITCin DOPC liposomes is approximately 3.5 times greater than the HRSfrom MGITC alone because of adsorption of MGITC at the liposome surface,in general agreement with our previous observations.2653 According to our previous work, MG, whichhas a similar molecular structure to MGITC, demonstrates no adsorptionor transport in DOPC liposomes.25 In thiscase, MGITC adsorbs and transports through the DOPC liposome membranemuch more efficiently than MG because of the isothiocyanate groupin MGITC molecules and the added dipole–dipole interactionswith the zwitterionic DOPC bilayer.26As shown in (2) second-order nonlinear susceptibility at theliposome surface. The decrease of the SHG signal is the result ofMGITC molecules migrating across the DOPC membrane and adsorbing atthe inner bilayer surface with an opposite orientation compared tothe outer surface, causing a cancelation and an overall decrease ofthe SHG signal.62The SHG time traces of MGITC added to DOPC liposomes with and withoutAPAP in water at various dye concentrations are shown in ESHG(t) is the SHG electric field at experimental time t after MGITC addition, and A0 and A1 are proportionality constants. Each SHG timetrace uses a fresh liposome sample. The obtained transport times areplotted as a function of the MGITC concentration for each liposomesample, as shown in Table S1 in the Supporting Information.The experimental SHG time traces are plotted in 64 to obtain the corresponding adsorption equilibrium constants andthe adsorbate site densities. The modified Langmuir model is an extendedform of the Langmuir model, which accounts for bulk depletion of theadsorbate because of a large cumulative liposome surface area. Thismodel assumes that freely adsorbing molecules form a single monolayerwith a corresponding maximum adsorbate site density at the liposomesurface.65 The modified Langmuir modelis given byN is the concentrationof MGITC dye molecules adsorbed on the DOPC liposomesurface, Nmax is the maximum adsorptionsite concentration, A is the SHG intensity at saturation, B is the baseline offset due to the SHG signal from liposomesin water without the addition of the dye, M is theconcentration of free dye molecules in solution, α is the slopeobtained from the plot of SHG intensity of dye alone as a functionof concentration C, 55.5 is the molar concentrationof water, and K is the adsorption equilibrium constant.The adsorption isotherms are fit using three fit parameters, A, Nmax, and K. The experimental data are corrected to account for the contributionfrom HRS from free MGITC molecules in water, which is displayed inthe Supporting Information, to give theα values. The free energy of adsorption is obtained using ΔG = – RT ln K. The modified Langmuir fits are shown as dotted black lines in Nmax values, thelipids per adsorption site are obtained for each sample, and theseresults are also listed in The sudden rise in the SHG signalupon MGITC addition to the DOPCliposome samples at time zero, as shown in 7 and (9 ± 1) × 107 for pure DOPC and for DOPC with APAP, respectively. Thisshows that APAP increases the electrostatic and dipole–dipoleattractions of MGITC adsorption to the liposome surface. The correspondingfree energy of adsorption is −10.3 ± 0.1 and −10.6± 0.1 kcal/mole for DOPC liposomes with and without added APAP,respectively. The maximum adsorption site concentration Nmax of MGITC adsorbing to pure DOPC liposomes is 2.9 ±0.5 μM compared to the corresponding Nmax value of 3.6 ± 0.3 μM for DOPC liposomes withadded APAP. These values are consistent with the interpretation ofincreased adsorption of MGITC when APAP is present. The correspondingnumber of lipid molecules per adsorption site (lipid/site) is 25.9± 4.5 and 20.8 ± 1.7 for pure DOPC liposomes and DOPC liposomeswith added APAP, respectively. A positive attraction of MGITC to APAPcan also have synergistic effects, where increased MGITC concentrationsat the liposome surface can draw more APAP from the aqueous bulk tothe lipid bilayer. The SHG intensity A at adsorbatesaturation is 174 ± 2 and 241 ± 3 for liposomes of pureDOPC and DOPC with APAP, respectively. The ratio of –2 for liposomes of pureDOPC and DOPC with APAP, respectively. Although the liposomes withAPAP have greater SHG signals at saturation, the SHG signal is shownto scale quadratically according to the Nmax adsorbate surface coverage, to within experimental uncertainty,indicating a similar MGITC angular distribution at the surface forboth liposome samples. Overall, these SHG measurements show that APAPincreases MGITC adsorption to the DOPC liposome surface resultingin larger magnitudes of adsorption free energy and higher adsorptionsite densities.As shown in Ka value of 9.5 and a relatively low lipophilicity.66 The octanol/water partition coefficient for APAP has alogP value of 0.38,66 indicatingthat APAP is more soluble in water than in octanol. MGITC has a higherlogP value of 1.67, showing a higher lipophilicity.67 Previous SHG studies of liposomes observed thatthe transport times of the similar dye molecule MG increase becauseof effects such as ion pairing with anions such as citrate and chloride43 and higher membrane rigidity by the additionof cholesterol68 or by decreased temperatures.27 However, in our results presented here, thetransport times of MGITC at concentrations of 1 to 2.5 μM areapproximately the same for the liposome samples both with and withoutadded APAP, to within experimental uncertainty, while the transporttimes are significantly longer when APAP is added at higher MGITCconcentrations of 3.5 μM and above, closer to the adsorptionsaturation level. This indicates a thresholding effect, where largerinteractions between APAP and MGITC are activated at higher MGITCconcentrations. Our recent investigation of DOPC liposomes with APAPusing small-angle X-ray and neutron scattering showed a slight decreaseof the diameter and that the number of lamellae did not change afterthe addition of APAP, while cryo-transmission electron microscopyrevealed increased heterogeneity after the addition of APAP, mirroredby the occurrence of irregular shape morphologies.33 Neutron spin echo spectroscopy revealed a strong decreaseof the bending modulus and the space explored by the lipid tails withincreasing APAP concentration.33 An increasein DOPC membrane fluidity is expected to decrease the MGITC transporttime. However, the APAP in the lipid bilayer can also hinder transportbecause of attractive electrostatic and dipole–dipole interactionsbetween APAP and MGITC, or through more complicated interactions involvingAPAP with the DOPC membrane, creating counter-balancing effects tothe increased membrane fluidity. These results highlight the complicatedchemical and physical interactions that can occur both at the surfaceand within phospholipid bilayers, which depend on many factors includingelectrostatics, hydrophobic–hydrophilic interactions, dipole–dipoleinteractions, and membrane fluidity. In addition, this research demonstratesa new experimental framework for investigating chemical interactionswith drug molecules at phospholipid bilayers using SHG spectroscopyto study molecular translocation processes for advancing potentialdrug-delivery applications.A comparison of the adsorption isotherm measurementswith the moleculartransport times highlights the complicated interactions between MGITC,DOPC, and APAP. APAP is a neutral, weak acid with a pThe effect of APAP onthe adsorption and transport properties ofMGITC in DOPC liposomes is investigated using time-dependent, surface-sensitiveSHG spectroscopy. The SHG results are used to determine the adsorptionequilibrium constants, adsorption site densities, and transport kineticsof MGITC at the DOPC liposome surface, both with and without APAP.The molecular transport of MGITC is found to be more rapid in pureDOPC liposome samples compared to DOPC liposomes with added APAP,but this effect occurs predominantly at higher MGITC concentrations.The SHG adsorption isotherms are fit using the modified Langmuir model,showing that the free energy of adsorption increases slightly in magnitudealong with a corresponding increase in adsorption site density whenAPAP is added to the DOPC liposomes. These results highlight the complicatedmolecular interactions between MGITC, APAP, and the DOPC liposomesurface, with factors that depend on electrostatics, dipole–dipoleinteractions, hydrophobic/hydrophilic interactions, and membrane fluidity.This research also demonstrates a pathway for investigating fundamentalproperties of molecular translocation at biological membranes, includingdrug–drug interactions, that are important in the developmentof drug-delivery technologies.


However, the delineation of the role of Ca2+ signaling within disease and disease progression is less defined.The importance of intracellular calcium , Parkinson’s disease (PD) and Huntington’s disease (HD) [2+ signaling dysregulation also associated with autoimmunity, asthma and even pulmonary cancer. In particular, the pulmonary consequences of deregulated Ca2+ homeostasis can include the development of pathology: airway inflammation, chronic obstructive pulmonary disease (COPD), acute lung injury (ALI), acute respiratory disease syndrome (ARDS) and lung cancer [2+ concentration fluxes play in these pulmonary diseases. Herein, we will briefly highlight our current understanding of the part that Ca2+-signaling abnormalities play within the context of pulmonary disease, with specific emphasis on the role of endoplasmic reticulum (ER)-resident Ca2+ entry and its potential as a therapeutic target for treating pulmonary disease.Recent evidence demonstrates that Caase (HD) , with Ca2+ is important for normal pulmonary physiology as well as regulating inflammatory responses against both environmental and pathogenic exposures [2+ levels are tightly regulated and maintained. However, when appropriately stimulated, intracellular Ca2+ release occurs. In particular, when Ca2+ is released inappropriately from the ER, significant depletion of ER-resident Ca2+ is associated with cellular apoptosis, pro-inflammatory responses and dysregulation of ciliary beat frequencies [2+ signaling derangement has been shown to be associated with disease across the body. Of note, a recent emphasis has been placed upon electronic cigarette  use or “vaping”, which presents but one vignette of Ca2+ signaling and disease.As previously mentioned, cellular Caxposures . Under bquencies . As such2+ signaling and its dysregulation, which ultimately results in the cytotoxicity of pulmonary epithelia. In brief, we (and others) have utilized both in vitro and in vivo models to demonstrate that exposure to e-cigarette liquids  can elevate cytosolic Ca2+ levels and result in significant cytotoxicity and/or pathology [2+ influx is diminished within the bronchial epithelia of traditional smokers, which is due to decreased ORAI3-dependent Ca2+ mobilization [2+ levels within both the human bronchial epithelial cell line CALU-3 and primary-derived human bronchial epithelial cells. These latter observations correlate with increased cytotoxicity [2+ levels, increased levels of the inflammatory cytokine IL-6 as well as apoptosis and cell death were observed in both CALU-3 cells and a 2nd human epithelial cell line, A549 [2+ release and the clear exacerbation of pulmonary disease when using either a bacterial or viral challenge, which was related to a prior acute vape exposure. These data strongly suggest that vaping may dysregulate intracellular Ca2+ stores, thus contributing to increased pathogenesis and morbidity after a pulmonary pathogen challenge. We have further utilized pharmacology, that is, small molecule treatments, to strengthen the association between a dual exposure, i.e., bacterial or viral challenge with prior vaping exposure, and Ca2+-mediated pathology. Finally, vaping has been theorized to induce the polarization of M0 macrophages to pro-inflammatory M1 macrophages, which is a process documented to be mediated by increased intracellular Ca2+ levels [The recent literature has presented a convincing association between the use of either the traditional combustible cigarette or e-cigarette use/vaping, and Caathology ,5,6,7,8.2+ signaling is intrinsically important to general biological homeostasis, therapeutics cannot be designed to generally blunt signaling in the presence of a stimulating factor. This former description would be more akin to using a proverbial “hammer” to treat an ailment. Indeed, specific and targeted approaches must be pursued to modulate Ca2+ signaling and to diminish inflammation when warranted. However, such an approach does provide a level of complexity to studies involving Ca2+-dependent pathologies, thus requiring new approaches and innovative and, undoubtedly, intensive studies to develop the appropriate “rheostat” for therapeutic design. Because Ca2+ signaling is dysregulated and the resultant pathology that can result. The contained works will bring to light the new cutting edge and experimental results that will drive future study in this area, which will elucidate the framework of Ca2+ signaling and how its dysregulation can result in disease. Further, this issue is meant to highlight potential novel therapeutic applications and/or directions to ameliorate Ca2+ dysregulation, and thus disease, which will require interdisciplinary studies in the areas of genetics, toxicology, physiology and beyond.Thus, this special issue on “Calcium Signaling Derangement and Disease Development and Progression” addresses the complex set of circumstances that result when such an intrinsically important signaling cascade such as Ca


CEBPG), a gene implicated in myeloid differentiation and AML progression.Acute myeloid leukemia (AML) is a myeloid neoplasm accounts for 7.6% of hematopoietic malignancies. AML is a complex disease, and understanding its pathophysiology is contributing to the improvement in the treatment and prognosis of AML. In this study, we assessed the expression profile and molecular functions of CCAAT enhancer binding protein gamma , a known repressor of translation. Knockdown of EIF4EBP1 was used to assess its potential role in AML progression downstream of CEBPG.shRNA mediated gene interference was used to down-regulate the expression of CEBPG was activated through its distal enhancer in AML cell lines. Using the public transcriptomic dataset, the Cancer Cell Line Encyclopedia (CCLE) and western blotting, we also found CEBPG was overexpressed in AML. Moreover, we observed that CEBPG promotes AML cell proliferation by activating EIF4EBP1, thus contributing to the progression of AML. These findings indicate that CEBPG could act as a potential therapeutic target for AML patients.We explored the ChIP-Seq data of AML cell lines and non-AML hematopoietic cells, and found CEBPG in AML and identified CEBPG as a potential therapeutic target for AML patients. Our findings provide novel insights into the pathophysiology of AML and indicate a key role for CEBPG in promoting AML progression.In summary, we systematically explored the molecular characteristics of The online version contains supplementary material available at 10.1186/s12935-021-02305-z. Acute myeloid leukemia (AML) is a myeloid neoplasm that accounts for 7.6% of hematopoietic malignancies. It is caused by the oncogenic transformation of hematopoietic progenitors in the bone marrow (BM), which results in the destruction of blood tissue. AML is reported to have a long-term survival of less than 20% –3. EveryCCAAT enhancer binding proteins (CEBPs) including CEBPA, CEBPB, CEBPD, CEBPE, CEBPG and CEBPZ, are suggested as potential biomarkers for cancer prognosis –14. CEBPCEBPG suppressed tumor growth [CEBPG is suggested as a biomarker for lung cancer risk [CEBPG in several types of cancer have been revealed, its expression profile and molecular functions in AML remain unresolved. Therefore, in this study we assess the role of CEBPG in AML progression.Among CEBPs, CEBPA, CEBPE and CEBPZ have been reported to function in AML development , 19, 20,r growth . CEBPG icer risk . It is acer risk , 28. AltCEBPG in AML cell lines, and the knockdown efficiency was detected by RT-qPCR and western blotting. The effect of CEBPG knockdown on the growth of AML cell lines was evaluated by Cell Counting Kit-8 (CCK-8) assays. Western blotting was used to detect poly(ADP-ribose) polymerase (PARP) cleavage, and flow cytometry was used to determine the effect of CEBPG knockdown on apoptosis of AML cells. Genes and pathways affected by knockdown of CEBPG were identified by gene expression analysis using RNA-seq.In the present study, shRNA mediated gene interference was used to down-regulate the expression of CEBPG was Eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1). EIF4EBP1 is a translation repressor protein [EIF4EBP1 was used to assess its potential role in AML progression downstream of CEBPG.One of the genes affected by knockdown of  protein  that pla protein –33. For CEBPG was activated through its distal enhancer in AML cell lines. Using the public transcriptomic dataset, the Cancer Cell Line Encyclopedia (CCLE) and western blotting, we also found that CEBPG was overexpressed in AML. Moreover, CEBPG promotes AML cell proliferation by activating EIF4EBP1, thus contributing to the progression of AML. These findings indicate that CEBPG could act as a potential therapeutic target for AML patients.In the present study, we explored the ChIP-Seq data of AML cell lines and non-AML hematopoietic cells and found Human AML cell lines, including NB4,THP-1, MV4-11, and K562 which was from blastic crisis of chronic myelogenous leukemia were obtained from the cell bank of the American type culture collection and cultured in RPMI medium (Termo Fisher Scientifc) containing 10% fetal bovine serum , and 1% penicillin–streptomycin  at 37 °C in a humidified incubator with an atmosphere of 5% CO2 and tested routinely for mycoplasma.CEBPG and EIF4EBP1 (Table Short hairpin RNA (shRNA) targeting 3 cells per well. The cell viability was determined by Cell Counting kit-8 (CCK8) assay  according to the manufacturer’s instructions. Cell proliferation was calculated as a percentage of that in cells in control medium. Each concentration was tested in triplicate and repeated in at least three independent experiments. The calculation was performed by Graph Prism software 7.0 .Leukemia cells were seeded in 96-well plates at a density of 1 × 10Total RNA was extracted from cell pellets using TRIzol®reagent , according to the manufacturer’s protocol. For cDNA synthesis, 1 µg of total RNA was converted to cDNA using a High-Capacity cDNA Reverse Transcription Kit . Quantitative real-time PCR analysis was carried out using LightCycler® 480 SYBR Green I Master mix  with a LightCycler 480 Real Time System (Roche), according to the manufacturer’s protocol. mRNA expression levels were calculated using the Ct method with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression as an internal reference. Primer sequences are listed in Table Western blotting analysis was conducted using the following primary antibodies: CEBPG , EIF4EBP1 , and PARP , with glyceraldehyde 3-phosphate dehydrogenase (GAPDH)  as a reference protein. Peroxidase-conjugated Afniure goat anti-rabbit IgG (H + L)  and goat anti-mouse IgG (H + L)  secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. . ImageJ software was used for band quantifcation. Then, protein levels were determined using a GAPDH antibody for normalization.Leukemia cells  were infected with lentivirus in the presence of 10 μg/mL Polybrene (Sigma-Aldrich) for 24 h. Stable cell lines were selected with puromycin (Sigma-Aldrich). Following 4 days incubation, leukemia cells were harvested and washed with cold PBS, suspended in 1 × binding bufer, and stained with fuorescein isothiocyanate (FITC)-Annexin V antibody and PI solution using an FITC-Annexin V apoptosis kit , according to the manufacturer’s instructions. Cell apoptosis was analyzed by flow cytometry .https://david.ncifcrf.gov).RNA-seq was carried out according to the protocols suggested by Novogene, Beijing, China. First, total RNA was reverse transcribed to cDNA for library construction, and the cDNA library was then sequenced. The raw reads were filtered and clean reads were mapped according to HISAT. The gene expression level (as fragments per kilobase of exon model per million reads mapped) was then calculated. Differentially expressed genes (P < 0.05 and fold-change > 2 or fold-change < 0.5) were identified using DESeq2 analysis. For enrichment analysis, differentially expressed genes were analyzed using the DAVID Bioinformatics Resources v6.8 online server . Sonicated chromatin was incubated with anti-histone H3 (acetyl K27) antibody  overnight at 4 °C. DNA was eluted and purified using a QIAquick PCR purification kit . Samples were sequenced on a novaseq 6000 platform . Raw data of ChIP-Seq H3K27ac analysis for NB4 cell line was aligned to the reference genome (UCSC hg38) using Bowtie2 (v 2.3.5) [3–5 × 10v 2.3.5) , with alv 2.3.5) , with pav 2.3.5) .http://www.cistrome.org/). The ChIP-Seq datasets of H3K27ac and CEBPG in K562 cell line were also obtained in the Cistrome database. The bigwig files of those datasets obtained  were further visualized by Integrative Genomics Viewer (IGV) [In this study, we searched public ChIP-Seq H3K27ac datasets of AML cell lines and non-AML hematopoietic cells in the Cistrome database (er (IGV) .EIF4EBP1 expression and overall survival of AML patients were assessed using the Kaplan–Meier analysis. Comparison between two groups was carried out using the Student’s t-test or the Mann–Whitney u test. Statistical analysis was carried out by GraphPad Prism 7.0 . Statistically significant P values are indicated as *P < 0.05, ** P < 0.01, ***P < 0.001, and ****P < 0.0001.The association between CEBPG in AML cell lines showed coincident H3K27ac signals that were not present in non-AML hematopoietic cells, suggesting a potential role in transcription regulation. Then, we assessed the expression pattern of CEBPG between AML patients and healthy controls in a public transcriptomic dataset (GSE114868) [CEBPG was more highly expressed in AML samples  which includes CEBPG mRNA expression profiles for multiple cancer cell lines, showed that CEBPG was highly expressed in hematologic malignancies including AML  and non-AML hematopoietic cells  , and fouCEBPG, we selected three AML cell lines with high CEBPG protein levels shown in Fig. CEBPG using three independent shRNAs  were identified upon CEBPG knockdown, in NB4 and MV4-11 cell lines respectively , which involves EIF4EBP1  from the EGFR tyrosine kinase inhibitor resistance signaling pathway was selected for qRT-PCR validation. In accordance with the RNA-Seq results, the mRNA levels of 6 of these genes, including EIF4EBP1, were downregulated while 2 genes were upregulated in NB4 cells in response to CEBPG silencing  and knocked down EIF4EBP1 in both cell lines using three independent shRNAs  [EIF4EBP1 was significantly overexpressed in AML samples in both datasets  . The resets Fig. a and b. ion Fig. c. These AML is an aggressive malignancy with poor prognosis . It is aCCAAT enhancer binding proteins (CEBPs) including CEBPA, CEBPB, CEBPD, CEBPE, CEBPG and CEBPZ, are suggested as potential biomarkers for cancer prognosis –14. AmonEIF4EBP1 in AML cell lines.Although a role for CEBPG in myeloid differentiation has been demonstrated , 28, if EIF4EBP1) gene encodes a translation repressor protein [EIF4EBP1 significantly inhibited proliferation and increases apoptosis in NB4 and K562 cells. Furthermore, in two public transcriptomic datasets (GSE114868 and GSE142700) [EIF4EBP1 was observed to be significantly overexpressed in AML samples. EIF4EBP1 was also identified as an unfavorable prognostic factor for AML patients using the online tool http://gepia.cancer-pku.cn/. Taken together, these results suggested that EIF4EBP1 is involved in the pathogenesis of AML and represents a negative prognostic factor for AML patients.Eukaryotic translation initiation factor 4E binding protein 1 ( protein . This pr protein –33. For E142700) , EIF4EBPIn summary, we explored the function of CEBPG in AML and identified CEBPG as a potential therapeutic target for AML patients. Our findings provide novel insights into the pathophysiology of AML and elucidated a crucial role of CEBPG in promoting AML progression.Additional file 1: Table S1. Differentially expressed genes between AML and control samples in dataset GSE114868.


Sensorless and sensor-based upper limb exoskeletons that enhance or support daily motor function are limited for children. This review presents the different needs in pediatrics and the latest trends when developing an upper limb exoskeleton and discusses future prospects to improve accessibility. First, the principal diagnoses in pediatrics and their respective challenge are presented. A total of 14 upper limb exoskeletons aimed for pediatric use were identified in the literature. The exoskeletons were then classified as sensorless or sensor-based, and categorized with respect to the application domain, the motorization solution, the targeted population(s), and the supported movement(s). The relative absence of upper limb exoskeleton in pediatrics is mainly due to the additional complexity required in order to adapt to children’s growth and answer their specific needs and usage. This review highlights that research should focus on sensor-based exoskeletons, which would benefit the majority of children by allowing easier adjustment to the children’s needs. Sensor-based exoskeletons are often the best solution for children to improve their participation in activities of daily living and limit cognitive, social, and motor impairments during their development. There is a wide variety of diagnoses that impair the arm movements of children, such as muscular dystrophy , spinal For children, the difficulty of moving the upper limb and, hence, interacting with their environment can have great consequences for the learning process. Indeed, it is known that children with limited exploration ability are at higher risk of developing cognitive, social, and motor impairments . MoreoveTraditional interventions to improve upper extremity function in children consist of strength training and aquatic therapy ,10,11. TIn the last few years, numerous sensorless and sensor-based exoskeletons have been developed to improve the quality of life of people with impairments at the upper limb, by acting both for rehabilitation, i.e., enhancement of the motor function, and assistance, i.e., support of the motor function. Furthermore, exoskeletons showed potential to increase intensive therapy  and reduDespite the increasing number of rehabilitation and assistance exoskeletons developed for adults, the options available for children are limited ,23. ThisIn the last few years, numerous reviews on upper limb exoskeletons have been published. However, these reviews mostly addressed the trends and challenges of exoskeletons and robotic rehabilitation devices for adults ,29,30,31The aim of this study is to (1) identify the different needs for children with an upper limb impairment, (2) present the latest development trends in sensorless and sensor-based upper limb exoskeletons, and (3) discuss future prospects to meet the specific needs in pediatrics.An exoskeleton is a device that works as an external skeleton to support the user . These dThe upper limb can be divided into four separate structures: shoulder complex, elbow complex, wrist joint, and fingers/hand . The shoThe elbow complex  is compoThe wrist  allows fThere are several diagnoses in pediatrics that could benefit from sensorless or sensor-based exoskeletons. Although each one has certain specific characteristics, the common symptom is weakness in the upper limb that makes it difficult to move freely one or more DOF, as presented in With a prevalence of 2.5 per 1000 children , cerebraThere are multiple ways to classify CP, but in regard to upper limb function, the Manual Ability Classification System (MACS) is the most widely used by experts . A MACS There exist a variety of types of muscular dystrophy, each affecting specific muscle groups, and with signs and symptoms appearing at different ages. The most common type of muscular dystrophy is Duchenne muscular dystrophy (DMD). Being an X chromosome-linked recessive disease, it mostly affects boys, with a prevalence of 1 in 5000 male live births .Children with DMD will generally experience upper limb deterioration during their teens , which wSpinal muscular atrophy (SMA) is an inherited autosomal recessive neuromuscular disorder, which results in the loss of motor neurons. Motor neurons are responsible for communication between the brain and the skeletal muscles. Without them, the communication is severed, which leads to muscle atrophy and weakness. The estimated prevalence of SMA is around 1 per 10,000 live births .Children and adults with SMA experience progressive proximal muscle weakness and paralysis. Regarding the upper limb, the shoulder muscles are the most affected, impacting most of ADL. Assistance through gravity compensation could help them to increase their autonomy and independence.Arthrogryposis Multiplex Congenita (AMC) is not a specific diagnosis, but rather a clinical finding. AMC is used to describe a variety of conditions involving multiple congenital contractures that affect two or more different areas of the body. A contracture is a condition where the range of motion of a joint is limited. The prevalence of AMC is 1 in 3000 live births .Most contractures in AMC take place in the shoulder, elbow, wrist, and hand, which limits greatly the ability to perform ADL. While there is no cure for AMC, occupational and physical therapy can help to improve the range of motion of contractures. In some cases, surgical procedures can be realized to improve the range of motion. Nevertheless, people with AMC could benefit from exoskeletons to improve the functionality of their upper limb by targeting the joints affected by contractures.Brachial plexus palsy (BPP) is an injury of the brachial plexus nerves that occurs during labor. The estimated incidence rate of BPP varies between 0.1 and 8.1 per 1000 live births . BPP injWhile most infants will recover spontaneously from BPP, around 35% of them will experience lifelong residual shoulder weakness, contracture, or joint deformity . These iIt is important to acknowledge that the pediatric and adult health care systems are structured differently, and that the transition to the latter occurs at around 18 or 19 years old . The proNowadays, most children with one of the diagnoses presented in this section will survive into adulthood. For CP, 65% to 90% of children will reach adulthood . Life exIn this review, the term sensorless describes exoskeletons that do not contain any form of sensor. Conversely, the term sensor-based is used to describe exoskeletons that contain at least one type of sensor which can provide useful information to the user or therapist.Upper limb exoskeletons can be classified using several methods , such asEach exoskeleton was first categorized according to its application domain: rehabilitation or assistance. A rehabilitation exoskeleton’s primary purpose is to enhance motor function by allowing partial or full recovery of the impairment. An assistance exoskeleton’s primary purpose is to support the motor function by facilitating the movements of the upper limb. While assistance exoskeletons can also have a rehabilitation purpose, the targeted population for this type of device is mainly people with a condition that cannot be improved. A third category, known as augmentation exoskeletons, was not included in this review. The primary purpose of these exoskeletons is to improve human strength and endurance . TherefoRegarding the motorization solution, each device was categorized as active or passive. An active exoskeleton uses powered actuators to move the user joints. A passive exoskeleton generally uses gravity compensation mechanisms to reduce the effect of gravity on the user’s arm.The five diagnoses presented in The movements supported by upper limb exoskeletons can either be at the shoulder, the elbow, the wrist, the hand, or a combination of these.This section presents briefly the sensorless upper limb exoskeletons developed for pediatrics and listed in The Dynamic Orthosis  is a pseThe Elbow Flexion Assist Orthosis  is a 1 DPlayskin Lift  is an exPlayskin Air  is a sofThe WREX  and P-WRThis section briefly presents the sensor-based upper limb exoskeletons developed for pediatrics and listed in Armeo Spring ,62 is a ChARMin  is a 6 DThe CT-DEA-based exoskeleton  is stillThe Hand exoskeleton  is a 2 DThe isolated orthosis for thumb actuation (IOTA)  is a 2 DMyoPal  is a comPEXO  is a 3 DThis exoskeleton  is a sofA total of 14 different upper limb exoskeletons were identified for pediatric purposes. Of these exoskeletons, six of them are sensorless  while eiTo assist therapists and reduce their workload, robots are increasingly used in pediatric rehabilitation to support frequent and long training sessions. Indeed, rehabilitation with exoskeletons has multiple advantages over traditional interventions with a therapist.First, it allows for high-frequency and high-intensity sessions ,70 with A second advantage is that robot-aided rehabilitation allows the therapist to quantitatively monitor the progress of the patient throughout the rehabilitation process ,70. IndeIt is important to note that reaching and grasping function for ADLs may be challenged in different ways due to impairment in shoulder complex, elbow flexors, and/or wrist complex . TherefoOne drawback of rehabilitation exoskeletons, such as Armeo Spring and ChARMin, is that they do not allow at-home therapy since they are bulky and impossible to transport out of the rehabilitation center. Moreover, the high cost of rehabilitation exoskeletons makes it impossible for families to even consider buying one for home.The obligation to pursue therapy at the rehabilitation center makes it difficult for children to have natural interactions with their environment, which can have a negative impact on their development. Indeed, for children, interaction with objects and other children is important in early language, perceptual–motor, social, and cognitive development and is a precursor for future essential life skills such as dressing and feeding . While tIn brief, rehabilitation devices bring great advantages to traditional interventions since (1) they do not suffer from fatigue, which allows for high-intensity and high-frequency training; (2) they provide reliable quantitative data to monitor the patient’s progress; (3) they allow for single joint training to adapt to patients’ needs, and (4) they allow for task-based therapies. Rehabilitation exoskeletons are generally sensor-based, which is essential to follow and adapt to the patient’s progress. However, rehabilitation exoskeletons (1) do not allow at-home therapy, (2) limit natural interactions because of their bulkiness, and (3) are not optimal for diagnoses without potential of motor function recovery.As is the case for rehabilitation exoskeletons, assistance exoskeletons also allow for high-intensity and high-frequency training. The lower acquisition cost of these exoskeletons makes it possible to bring the device back home, hence reducing the travel time for therapy ,70. The The possibility of at-home therapy and using an assistance exoskeleton in ADL is of the utmost importance. Indeed, it is well accepted that the skills developed during in-clinic therapy  do not always translate well into ADLs such as inserting a key into a door lock, combing hair, buttoning clothes, eating, and drinking .The main objective of actual assistance exoskeletons is helping anti-gravity motions . This isAlthough the wearability of the assistance exoskeletons is a great advantage over the rehabilitation exoskeletons, they are often limited in the number of supported movements. Indeed, there is currently no assistance exoskeleton that supports more than two movements, while some rehabilitation exoskeletons can support up to six movements. When designing an assistance exoskeleton, it is also important to make sure that it is light and compact enough so that it does not impact negatively other ADLs, such as leading to an unsteady gait  or reducAn inconvenience of sensorless passive assistance exoskeletons is that it can be difficult to adjust the gravity compensation mechanism precisely to the children’s needs. Taking the WREX as an example, the final adjustments are made by adding or removing rubber bands. An incorrect number of bands will contribute to incorrect function and limit anti-gravity assistance . MoreoveAnother limitation of assistance exoskeletons is that they generally do not account for children’s growth. The focus on comfort, weight, and portability makes it difficult to design an exoskeleton that covers the range of patients from children to late adolescents .Research and development in upper limb exoskeletons in pediatrics are still recent. In comparison, the total number of exoskeletons for adults, based on the recent review of Gull et al. , is 69. Despite the numerous options for adults, it is unfortunately not possible to simply scale them down in size . The hetAs stated by Aubin et al. , developAdditional concerns towards the motivational aspect should be considered when designing an exoskeleton for children, since the level of motivation of children with an impairment during rehabilitation is generally difficult to maintain . To inciAnother challenge is that each diagnosis in pediatrics has specific requirements that give rise to different fitting, support, and control challenges, which require new technical methods and solutions . The ideIt is widely accepted that a user-centered design approach is preferred to guarantee better acceptability and continuous use of exoskeletons . This deThere exist a wide variety of sensors that can be integrated in pediatric exoskeletons. Nonetheless, the sensors used in upper limb exoskeletons should allow the measurement and processing of upper limb movement data  to adaptThe objective of this review was to present the principal diagnoses in pediatrics that causes upper limb impairments and identify the current trends and challenges for pediatric access to upper limb exoskeletons. The review highlighted that the most prevalent diagnoses in pediatrics do not allow for potential motor function improvements. Therefore, it is essential for these children to have access to an exoskeleton that can assist them in ADL. Assistance exoskeletons are better suited than rehabilitation exoskeletons in pediatrics. There exist both sensorless and sensor-based assistance exoskeletons. However, sensor-based exoskeletons are more promising since the additional data provided by the sensors allow better adjustment to the user’s needs. Nevertheless, the options in pediatrics are still limited when comparing to adults. This is mainly explained by additional challenges regarding children’s growth and wearability. New design methods, such as user-centered approaches, will help to tackle these challenges and improve the accessibility of pediatric exoskeletons. This is important to improve children’s participation in ADL and limit the risks of cognitive, social, and motor impairments during their development.


This research aimed to investigate the mechanisms of exercise-induced immunosuppression by measuring intracellular calcium levels, Ca2+-regulating gene expression, and agonist-evoked proliferation of murine splenic T lymphocytes. Mice were randomly assigned to the control, sedentary group (C), and three experimental groups, which performed a single bout of intensive and exhaustive treadmill exercise. Murine splenic lymphocytes were separated by density-gradient centrifugation immediately (E0), 3h (E3), and 24h after exercise (E24). Fura-2/AM was used to monitor cytoplasmic free Ca2+ concentration in living cells. The combined method of carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling and flow cytometry was used for the detection of T cell proliferation. The transcriptional level of Ca2+-regulating genes was quantified by using qPCR. Both basal intracellular Ca2+ levels and agonist -induced Ca2+ transients were significantly elevated at E3 group (p<0.05 vs. control). However, mitogen-induced cell proliferation was significantly decreased at E3 group (p<0.05 vs. control). In parallel, the transcriptional level of plasma membrane Ca2+-ATPases (PMCA), sarco/endoplasmic reticulum Ca2+-ATPases (SERCA), TRPC1, and P2X7 was significantly downregulated, and the transcriptional level of IP3R2 and RyR2 was significantly upregulated in E3 (p<0.01 vs. control). In summary, this study demonstrated that acute exercise affected intracellular calcium homeostasis, most likely by enhancing transmembrane Ca2+ influx into cells and by reducing expression of Ca2+-ATPases such as PMCA and SERCA. However, altered Ca2+ signals were not transduced into an enhanced T cell proliferation suggesting other pathways to be responsible for the transient exercise-associated immunosuppression.Ca Exercise can enhance marked transient physiological changes and has a profound influence on the human immune function. There are a number of studies showing that the effects of acute exhaustive exercise seem to be detrimental, while regular, moderate-intensity physical activity can improve immune defense functions. There is evidence that the transient “open-window” of immunosuppression might exist in the recovery from strenuous exercise as indicated by an impaired immunity around 3–72h after exercise when athletes seem to be more susceptible to infections. However, this consensus meaning has been disagreed recently due to novel findings demonstrating that exercise stimulates T cell redistribution within organs and tissues, enhances mobilization of hematopoietic stem cells as a result of apoptotic T cells, and reverses T cell immunosenescence . Therefo2+ is a key second messenger in the network of cellular signal transduction, which participates in many physiological and pathological processes. The stress of exercise may influence the intracellular Ca2+ dynamics of lymphocytes. By using Fura-2 AM dual-wavelength detection technology, our previous work reported that long-term physical training had a significant effect on intracellular calcium signal transduction of lymphocytes in mice , which were fed in the animal facilities at the Department of Sports Medicine of University of Münster and Justus-Liebig University (Germany), and kept under controlled conditions of temperature and humidity. This specie and model were chosen for being a homogeneous line of mice. Mice were housed collectively (4–6 per cage) and fed tap standard rodent diet and water at will, with a standard 12h day-night cycle. All procedures were performed following the approval of the local Animal Care and Use Committee. We exert our effort to minimize animal pain and discomfort, and this experiment was performed in accordance with the ARRIVE guidelines.2max) and the fastest speed as our group previously described , 3h (E3), and 24h (E24) after exercise for spleen removal.Animals were adapted to exercise for 1week before the exercise test, aiming to minimize the stress induced by the equipment. Each animal underwent an incremental exercise test to exhaustion to measure individual maximal oxygen uptake . And the mesh was put into a culture dish, which PBS was poured into. The spleen was gently squeezed with a 2-ml syringe plunger to generate single cell suspensions. Biocoll separating solution  was used, and lymphocytes are stratified after density gradient centrifugation as our group described before . The whi2+ level was assessed by the fluorescence intensity ratio of the calcium probe Fura-2 AM  as described our previous study . The fluorescence intensities were measured in the dual wavelength ratio mode in a spectrofluorometer  at 340 and 380nm (excitation filters) and 510nm (emission filter). According to the equation of 2+]i was calculated as follows: [Ca2+]i=(R-Rmin)/(Rmax-R)×Kd×F, with a Kd of Fura-2 for calcium of 220nM, and where R is the ratio of fluorescence of the sample at 340 and 380nm. Rmax and Rmin are the ratios for Fura-2 at these wavelengths in the presence of saturating Ca2+ (after application of 10mM digitonin) and during Ca2+-free conditions , respectively, and F is the ratio of fluorescence intensity at 380nm during Ca2+-free conditions to the fluorescence intensity at 380nm during Ca2+-saturating conditions. To remove the free fura-2AM, cells loaded with the dye were washed two times with PBS solution. The maximum peak and plateau level of the agonist-induced [Ca2+]i transient was quantified after the addition of mitogen, concanavalin A , the anti-CD3 antibodies , or thapsigargin . To estimate intracellular Ca2+ release and extracellular Ca2+ influx, experiments were performed either in absence or in presence of 0.8mmol/l Ca2+ in the measurement medium. For experiments performed in the absence of extracellular calcium, CaCl2 was replaced by 1mM EGTA in the buffer. The addition of Ca2+ allowed measuring transmembrane Ca2+ influx.Intracellular Caus study . Briefly2+ and Ca2+, can share common entry channels in cell membrane . Therefore, ∆slope was regarded as an index to access the rate of fluorescence quenching by extracellular Mn2+ influx into cells.Bivalent cation, Mnmembrane , and theechnique . The Mn26 cells/per culture dish and then left to incubate at 37°C in a humidified 5% CO2 atmosphere with additional doses of 2.5μg/ml Con A or PHA . After 72h of incubation, cells were harvested, stained with anti-CD3-PE antibody . Data analysis was performed using a flow cytometer with FlowJo software . AUC is defined as the area under the curve enclosed by the coordinate axis, of which the x-axis indicates cellular generations, and the y-axis shows the percentage of cells in each generation. △AUC as an index was used to evaluate cell proliferative ability.Lymphocytes were prepared as described above, and cell proliferation was monitored using the cell tracker dye carboxyfluorescein diacetate succinimidyl ester  according to the standard procedure provided by −Δ(ΔCt) method and expressed as fold change in comparison with controls.Murine CD3+ T cells were purified directly from splenic cells using an EasySep™ Mouse T Cell Isolation Kit  based on magnetic bead separation technique according to manufactures’ instructions. Total RNA was extracted from T lymphocytes using the RNA isolation kit , and a UV-Vis spectrophotometer  was used to determine the quantity and purity of the extracted RNA. To remove possible DNA contamination, on-column DNase digestion was applied by using the RNase-free DNase set  in the context of RNA isolation. cDNA was subsequently synthesized by using a cDNA synthesis kit  from RNA samples according to the manufacturer’s protocol. cDNA was obtained by on a PCR thermal cycler , and the product was used for qPCR. To determine mRNA expression in T cells, PCR was performed by using the iQ SYBR Green Supermix , and an iCycler  was applied to quantify the amplification products. The PCR primers used are listed in p<0.05 was set as statistical significance. SPSS 20.0 software was used throughout.Data are expressed as means±SEM. Shapiro–Wilkʼs test revealed that data were normally distributed; thus, statistical analysis was performed using one-way ANOVA for comparison between groups. 2+]i of T cells was determined to be 45.5±5.1nM. Immediately after exercise, the resting [Ca2+]i was 49.7±6.2nM, and no significant change was observed . After 3h of post-exercise recovery, the resting [Ca2+]i was highly significantly increased to 101.0±30.9nM , and the resting intracellular calcium level 24h after exercise (E24) was 46.0±15.2nM and unchanged to pre-exercise conditions . Representative tracings of intracellular Ca2+ signal after the administration of Con A in calcium containing or Ca2+-free buffer are shown in 2+ transient with a peak and a plateau phase in calcium containing buffer. After addition of Con A, a significant absolute and net increase (=Δ[Ca2+]I) of intracellular Ca2+ concentration ([Ca2+]i) in the E3 group could be observed during both peak . In 2+-free buffer. After an initial resting phase of 100s (=time 1), Con A was added (=time 2) followed by administration of CaCl2 (=time 3). By subtracting mean concentrations at time 1 from time 2 and at time 2 from time 3, the resulting differences should indicate Ca2+ release from intracellular stores (stage 1) and transmembrane Ca2+ influx (stage 2), respectively. After the administration of Con A, intracellular Ca2+ release wasnot affected in the E3 group , while an improved transmembrane Ca2+ influx into cells could be observed . Further stimulations were performed only up to 3h after exercise.Next, cells were stimulated with different agonists known to affect intracellular Caoth peak ,D and pl2+ signals in calcium containing or Ca2+-free buffer are shown in 2+ transients, however, was similar to Con A. In 2+]i was found in the E3 group . When cells were suspended first in Ca2+-free solution followed by addition of OKT3, the release of intracellular Ca2+ wasnot changed significantly in the E3 group . Upon addition of Ca2+, an improved transmembrane Ca2+ influx was shown after exercise , the representative tracings of intracellular Ca2+-ATPase, was used. After application of TG in the Ca2+-free buffer, a similar release of Ca2+ from intracellular stores could be observed suggesting an identical load of intracellular calcium stores. However, after addition of Ca2+ into the extracellular medium, an enhanced Ca2+ influx signal was found in the E3 group suggesting an improved entry of extracellular calcium , an inhibitor of sarco-endoplasmic Ca2+ entry, we used the Mn2+-quenching technique. As shown schematically in 2+. After the application of TG, we observed a larger deflection of the Fura-2 fluorescence quenching in the E3 group (bold line) compared to control group (light line), which indicates an enhanced transmembrane Mn2+ influx into cells after exercise , as shown in In order to confirm an exercise-associated enhanced Ca2+ release channels  were significantly enhanced in the E3 group compared to control group . The transcription level of ion channel, TRPM5, was significantly reduced, while transcription levels of intracellular Ca2+-regulating factors, IP3R2, ATP2C1, Cav2.3, and Kcnk5, were significantly increased in the E24 group compared to control group . The transcription levels of TRPC1 and Cav2.3 were significantly increased, while the transcription levels of intracellular Ca2+-regulating genes, TRPV6 and Hspa1a, were statistically reduced in the E24 group compared to E3 group .As shown in p<0.05/p<0.001 vs. control, n=5). The effect could be observed for both PHA and ConA, as shown in A single acute bout of exhaustive exercise significantly reduced the mitogen-induced cell proliferation of T cells in the E3 group compared to control group  in the current study points to another, more likely regulatory processes. The determination of Ca2+-handling proteins mRNA showed a significant reduced expression of both the plasma membrane Ca2+-ATPases (PMCA) and sarco/endoplasmic reticulum Ca2+-ATPases (SERCA). Ca2+-ATPase maintains the global basal free Ca2+ levels actively on the expense of ATP in order to transport free calcium ion against its electrochemical gradient either out of cells or into intracellular Ca2+ stores. The decreased expression of Ca2+-ATPases could mean a decreased enzyme activity and an impaired ability to regulate intracellular Ca2+ levels. The reduced Ca2+-ATPase expression and function should therefore result in a cytoplasmic Ca2+ buildup as a result of a new balance between Ca2+ increasing and decreasing processes. Previous studies showed also that the impairment in Ca2+ homeostasis just due to one of the Ca2+ extruding pathway is enough to interfere with calcium homeostasis and cell function. Therefore, defective Ca2+ extrusion due to decreased PMCA expression was responsible for an enhanced cell apoptosis , a key regulator of T cell activation, is followed by increased basal Ca2+ levels . In order to discriminate between the contribution of Ca2+ release from intracellular pools such as endoplasmic reticulum and Ca2+ influx via transmembrane Ca2+ channels such as store-operated calcium entry (SOCE), a unique plasma membrane Ca2+ entry mechanism, experiments were performed under Ca2+-free conditions. CRAC channels, which are formed by ORAI1-3, are the major SOCE channels in T cells; other channels, such as transient receptor potential (TRP) and voltage-dependent calcium (Cav) channels, also can participate in SOCE (2+ (Cav) channels are less characterized in T cells. The results suggest that after exercise the transmembrane Ca2+ influx into T cells is the major factor for the altered Ca2+ signals. Likewise, thapsigargin (TG) by inhibiting the SERCA pumps releases similar amounts of Ca2+ from intracellular Ca2+ pools suggesting that their load wasnot affected by exercise. Furthermore, the Fura-2-quenching experiments using Mn2+ as a surrogate permeable bivalent cation for Ca2+ after application of TG support that intensive exercise could improve the transmembrane Ca2+ entry during the early (3h) post-exercise recovery period. The capacity of extracellular Ca2+ influx is thereby directly proportional to the ER store depletion  that induces mitochondrial Ca2+ uptake, an event that drives mitochondrial respiration thereby causing the generation of further free radicals, which may amplify the cellular oxidative stress. A Ca2+ overload in mitochondrial matrix causes the opening of the permeability transition pore (PTP) followed by a collapse of mitochondrial potential, mitochondrial swelling and release of cell death inducing agents , AP-1, and NFAT (2+ signaling (Finally, acute exhaustive exercise can modulate the ability of T cell proliferation. Many studies have shown that an acute bout of strenuous exercise is followed by reduced immune cells functions in humans and mice . The resferation . These cferation . The Ca2esponses . On the and NFAT . T cellsignaling . NFAT coignaling . Furtherignaling .2+ influx into cells and an altered expression of cellular Ca2+ ATPases such as PMCA and SERCA as well as intracellular Ca2+ release channels such as IP3R and RyR. However, the alterations of Ca2+ signaling could not be related to the inhibition of cell proliferation in T cells making further studies on the role of additional signaling pathways such as transcription factor activation necessary.Thus, taken together, our findings demonstrated that acute exercise affects intracellular calcium homeostasis during resting and stimulated conditions in murine T cells. The changed calcium levels result from an enhanced transmembrane CaThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by the Animal Care and Use Committee of Giessen University.All authors have made substantial contributions to the conception or design of the work or the acquisition, analysis, or interpretation of data for the work, drafted the work or revised it critically for important intellectual content, approved the final version to be published, and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors contributed to the article and approved the submitted version.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.


T cell immunoglobulin and mucin protein 3 (Tim-3) is an immune checkpoint and plays a vital role in immune responses during acute myeloid leukemia (AML). Targeting Tim-3 kills two birds with one stone by balancing the immune system and eliminating leukemia stem cells (LSCs) in AML. These functions make Tim-3 a potential target for curing AML. This review mainly discusses the roles of Tim-3 in the immune system in AML and as an AML LSC marker, which sheds new light on the role of Tim-3 in AML immunotherapy. T cell immunoglobulin and mucin protein 3 (Tim-3) was first discovered in 2002 and is a type I membrane-bound glycoprotein . Tim-3 bTim-3 plays a vital role in immune tolerance. Tim-3 was originally identified as being expressed on IFN-γ-producing CD4+ and CD8+ T cells, and Tim-3 is now known to be expressed on Treg cells , natural+ T cells in human tumors could define the functional regulatory T cells that contribute to the immunosuppressive tumor micromilieu  and peritoneal mast cells (pMCs) without affecting degranulation . The pron vitro . Tim-3 cn vitro . AlthougFour relevant ligands have been reported to interact with different regions of the Tim-3 extracellular immunoglobulin V domain . These iGal-9 was identified as a natural ligand of Tim-3. Gal-9 contains two distinct carbohydrate receptor-binding domains  and can + DCs  is a key cytokine that contributes to the development of AML, and it is important to understand the mechanism underlying the interactions of Tim-3-mediated responses with SCF-induced signaling networks –46. ProkStudies relating to Tim-3 function in AML are limited, and some studies have also shown a correlation between Tim-3 and AML. Tim-3 levels were higher in AML than in other AML cytogenetic subgroups and associated with core-binding factor translocations or mutations in CEBPA . ResearcImmature leukemic blast cells are largely produced by leukemic progenitors, which are generated by self-renewing LSCs in the bone marrow –53. Apprin vitro and in vivo. A phase 1/2 trial in previously untreated older patients with AML investigated a single dose of cusatuzumab monotherapy with azacitidine (NCT03030612). The hematological responses in the 12 patients enrolled included 8 complete remission, 2 complete remission with incomplete blood count recovery and 2 partial remission, with 4 patients achieving minimal residual disease negativity. No dose-limiting toxicities were reported. Treatment with the CD70 antibody cusatuzumab substantially reduced the number of LSCs and triggered gene signatures related to myeloid differentiation and apoptosis. Sallman et al.  and/or MBG453 in combination with decitabine (a DNA hypomethylating agent) in patients with AML or high-risk MDS in 2017 (NCT03066648). MBG453 is a high-affinity, humanized anti-Tim-3 IgG4 monoclonal antibody that blocks the binding of Tim-3 to PtdSer. In 2019, Novartis Pharmaceuticals began another clinical trial in which HDM201 (an MDM2 inhibitor) was combined with MBG453 or venetoclax (a BCL2 inhibitor) in patients with AML or high-risk MDS (NCT03940352). Although these clinical trials have not yet published data, the continuation of these clinical trials indicates that the Tim-3 antibody strategy for AML is attractive. There will be promising results soon.via NK cell-dependent mechanisms. Ma et al.  and ADCC function, which could kill AML LSCs, dysfunctional immune cells with Tim-3 expression and differentiated monocytes. Elimination of dysfunctional Tim-3-expressing immune cells could be a new way to address immune tolerance. Although Tim-3 CAR-T and IgG1 antibodies could kill differentiated monocytes, they are not more toxic than chemotherapy in AML patients, and HSCs remain viable, supporting reconstitution of the immune system. Tim-3 CAR-T cells could be followed by hematopoietic stem cell transplantation (HSCT) and provide an opportunity for AML (relapse/refractory) treatment with HSCT. Due to the importance of Tim-3 in AML, the underlying mechanisms require further investigation.Thus, it is important to examine the mechanisms of Tim-3 in types of multiple immune cells, which might be a valid therapeutic strategy for cancer immunotherapy. However, there are more studies relating to the role of Tim-3 in solid tumors than in AML . The mecZW and JC drafted the manuscript. ZW and LY conceived the idea, and LY supervised the process. MW and LZ helped with the opinion and discussion in clinical. All authors contributed to the article and approved the submitted version.This work was supported by grants from the “Major New Drug Development Project” from Ministry of Science and Technology of China (2019ZX09201002003), the State Key Program of National Natural Science of China (82030076), the National Natural Science Foundation of China , the Beijing Natural Science Foundation (7202186), the Science and Technology Foundation of Shenzhen (JCYJ20200109113810154), the Shenzhen Science and Technology Investigation Project (JCYJ20190808163601776), and the Natural Science Foundation of Shenzhen University General Hospital (SUGH2020QD008).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.


A greater diversity of rabbit species occurs in North America compared with Europe.Archeological evidence demonstrates thousands of years of human–rabbit interactions in both Europe and North America, particularly at the ancient city of Teotihuacan (~AD 1–550) in central Mexico where several studies suggest practices of rabbit management by humans.Oryctolagus cuniculus) is the only lagomorph species to have been domesticated by humans.The European rabbit  were domesticated in southern France within the last 1500 yr  populations in both Europe and North America . Resulting from these relationships, European rabbits . We then review the archeological evidence for human–rabbit interactions in both the Old and New Worlds, with an emphasis on new data from the ancient Mexican city of Teotihuacan, where archeological and chemical evidence suggests the importance of rabbits in the local diet and economy. Finally, we consider the biological and behavioral characteristics of European rabbits and North American cottontails, emphasizing traits that probably influenced their differential domestication outcomes.Together with the pika family (Ochotonidae), the Leporidae belong to the order Lagomorpha. The evolution of the leporid family is complex, but the earliest fossils have been found in eastern Asia during the Early to Middle Eocene epoch (~60 to 40 mya) . LeporidLepus) with 32 individual species that are native to North America, Europe, Africa, and Asia. The colloquial term of “rabbits” includes 10 distinct genera and 31 species (O. cuniculus). This species includes two subspecies: O. cuniculus algirus and O. cuniculus cuniculus . It occurs from Canada to Venezuela. The two other genera of New World rabbits are the monotypic pygmy rabbits (Brachylagus idahoensis), which are found in the western United States, and the monotypic volcano rabbits (Romerolagus diazi), which are found in central Mexico.A greater diversity of rabbit species exists in the Americas than in Europe, and they occur in a broader range of environments. Containing 17 species, the most diverse rabbit genus of the New World is lvilagus . The mosOryctolagus cuniculus  likely followed the “directed pathway” to domestication, a process that implies the deliberate attempt by humans to domesticate the animal  indicate their use for food and fur since at least the terminal Pleistocene  of the Classic Teotihuacan fauna remains analyzed (Lepus/Sylvilagus ratios at Teotihuacan (0.47) indicate the greater prevalence of rabbits over hares  provides one of the best case studies to understand intensive human–leporid interactions in an urban landscape. The city extended over 20 kmompounds . Leporidanalyzed : Table 3analyzed , 2020. Ianalyzed , 2000, Ler hares . One poser hares , whereaser hares .Lepus, Romerolagus, and Sylvilagus) and six species , with the eastern cottontail (S. floridanus) being the most commonly represented  called Oztoyahualco . The arcCanis familaris, 11% MNI) and the turkey   . Both plEvidence of rabbits offered as food for sacrificed animals buried within the Moon and Sun Pyramids at Teotihuacan suggests that rabbits were utilized in state functions. Isotope data confirm that the rabbits found in the stomach contents of ritually sacrificed carnivores, including pumas and eagles, were also fed a diet high in C4 resources. In this way, rabbit production would provide a stable meat source to raise captive carnivores within the city . The higthe social structure of the organism, with favorable traits including large group size, a social hierarchy, and the presence of males within the group; 2) the sexual behavior of the organism, with favorable characteristics including a promiscuous mating system, males being dominant, and the signaling of sexual receptivity by females; 3) parent–young interactions, with favorable characteristics including social imprinting, females accepting young soon after birth, and precocial offspring; 4) the nature of the response to humans, with favorable characteristics including a short flight distance, low reactivity, and the ability to be readily habituated; and 5) the feeding behavior and habitat choice of the organism, with favorable characteristics including a generalist feeding strategy, a wide environmental tolerance, and nonshelter seeking. Here we briefly summarize the behavioral ecology of the European rabbit (O. cuniculus) and that of the eastern cottontail (S. floridanus), which is the most common rabbit of the Americas and was the most abundant species present at Teotihuacan. Because dietary practices, digestive strategies, habitat preference, and response to humans are similar for these species, we focus the discussion on the first three of these behavioral characteristics that “preadapt” an animal for domestication.Despite a far greater diversity of leporid species, over 10 millennia of human–rabbit interactions, and centuries of an intensive relationship at Teotihuacan, cottontail rabbits were not domesticated in North America as they were in Europe. Although European rabbits may have followed the directed pathway to domestication, North American rabbits likely embarked on the commensal or prey pathways, but never reached the final destination. Scholars have long noted that the social behavior of an animal is an important factor in the domestication process . In a suwarrens (O. cuniculus can be considered a gregarious species. In laboratory settings, rabbits raised in individual cages are generally more stressed, less healthy, and display more pathlogical behaviors, including fur pulling and bar biting, than do group-raised rabbits (The natural range of the European rabbit extends across the Iberian Peninsula and varies from woodland to open field habitat. It readily becomes accustomed to human presence and frequently inhabits areas near human settlements. The European rabbit is the only leporid species to form stable social groups under wild conditions . Groups warrens , which awarrens . Groups warrens . The pop rabbits .The mating system of the European rabbit is primarily polygamous, but the exact social arrangement varies from monogamous pairs, to promiscuity, to harem polygyny . These dO. cuniculus, but in an experimental setting, males are known to defend juveniles from antagonistic adult does (Females give birth to altricial young and nurse infants only for a few minutes once every 24 hr, and weaning is completed within 3 to 4 wk . Little ult does .The eastern cottontail is the most common and widely distributed rabbit species of the Americas, stretching from southern Canada to Venezuela . They inS. floridanus ranges from promiscuous to polygynous. A ridged and linear dominance hierarchy exists among males, principally resulting from male–male competition over access to receptive females with dominant males obtaining more successful copulations (S. floridanus; instead, males chase or dislodge lower ranking males when receptive females are present (The mating system of ulations . The est present . Althoug present . Male ho present . Althoug present . Males aEastern cottontail nests are created by females digging shallow and slanted burrows (~10 to 15 cm deep; O. cuniculus is a social animal that inhabits large communal warrens, whereas S. floridanus is a largely solitary animal. Indeed, Eastern cottontails are difficult to breed in captivity as they often fight when penned together, occasionally resulting in death (Although the European rabbit and the eastern cottontail are similar in many ways, including their diet, digestion, and the degree of parental investment for their altricial young, several key differences distinguish these rabbit species. The most significant of these differences concerns the degree of sociality or gregariousness of the rabbits. in death .Though both European and North American rabbits embarked on pathways to domestication, we suggest the behavioral qualities of European rabbits made them more susceptible to complete the path than eastern cottontails in two primary ways. First, the gregarious nature and ability to form stable social groups allowed European rabbits to be penned by humans and entire breeding populations could be managed within confined areas spaces with a minimal amount of inter-rabbit conflict. Enclosing eastern cottontails would have been more difficult due to their solitary nature and propensity to fight. Secondly, the natural tendency of European rabbits to form spatially clustered breeding groups centered on underground warren systems, would have allowed humans to easily locate, hunt, and eventually enclose and for managed breeding. New World cottontails, on the other hand, are solitary and more diffuse across a landscape making them harder to directly pen and manage.extensive than intensive relationships with rabbits, a pattern not conducive to domestication. The biodiversity of North American cottontails may have thus acted to discourage the domestication of any singular species, despite direct human provisioning and management, and in spite of the importance of rabbits to human nutrition and culture.In addition to their behavioral qualities, the overall diversity of rabbit species in North America may have served as a limiting factor for domestication. The fact that six different leporid species were found among the faunal bones at the Oztoyahualco compound of Teotihuacan indicates that residents practiced mixed acquisition and management strategies of diverse leporid populations rather than managing large breeding colonies of a singular species. Domestication requires a sustained multigenerational relationship with a specific animal population that has restricted gene flow with closely related wild populations . The divO. cuniculus and S. floridanus, we found that the social tendencies of these two species were the factors with the greatest divergence. Although O. cuniculus is gregarious and inhabits subterranean communal warrens, S. floridanus is solitary and their populations do not spatially cluster. Additionally, the biodiversity of rabbit species in North America encouraged humans to engage in extensive relationships with multiple leporid taxa rather than an intensive relationship with a singular rabbit species, as had occurred with O. cuniculus in Europe. We suggest that these factors made the European rabbit a more likely candidate for domestication than eastern cottontails.In this article, we attempted to explain why though both Old World and New World rabbits embarked on pathways to domestication, only Old World rabbits obtained this status. We reviewed the archeological and historical evidence for the antiquity and intensity of human–leporid interactions in both Europe and North America, with an emphasis on new data from the archaeological site of Teotihuacan. We demonstrated that rabbits were dietary staples across large portions of North America and the Iberian Peninsula for many thousands of years. After reviewing the differing behavioral strategies of Finally, the parallels observed in the human–rabbit relationships in Europe and North America challenge us to reconsider the utility of the concept of domestication itself. For thousands of years and in both Europe and North America humans hunted, traded, and managed rabbits in captivity, but one rabbit population ultimately became the focus of intensive directed breeding by humans in southern France. Although all domesticated breeds today can be traced back to this small population, the archeological record demonstrates the rich history of human–rabbit interactions in Europe and North America. The observation that humans were moving, managing, and feeding populations of wild rabbits in multiple areas around the globe prior to the domestication of the European rabbit suggests that the binary distinction between wild and domesticated may fail to capture the complexities of many human–animal relationships.


In VSMCs, Ca2+-dependent negative feedback regulation of myogenic tone is mediated by Ca2+-activated K+ (BKCa) channels and also Ca2+-dependent inactivation of voltage-gated Ca2+ channels (VGCC). Transient receptor potential subfamily M, member 4 channels (TRPM4); Ca2+-activated Cl− channels , Ca2+-dependent inhibition of voltage-gated K+ (KV) and ATP-sensitive K+ (KATP) channels; and Ca2+-induced-Ca2+ release through inositol 1,4,5-trisphosphate receptors (IP3Rs) participate in Ca2+-dependent positive-feedback regulation of myogenic tone. Calcium release from VSMC ryanodine receptors (RyRs) provide negative-feedback through Ca2+-spark-mediated control of BKCa channel activity, or positive-feedback regulation in cooperation with IP3Rs or CaCCs. In some arterioles, VSMC RyRs are silent. In ECs, transient receptor potential vanilloid subfamily, member 4 (TRPV4) channels produce Ca2+ sparklets that activate IP3Rs and intermediate and small conductance Ca2+ activated K+ (IKCa and sKCa) channels causing membrane hyperpolarization that is conducted to overlying VSMCs producing endothelium-dependent hyperpolarization and vasodilation. Endothelial IP3Rs produce Ca2+ pulsars, Ca2+ wavelets, Ca2+ waves and increased global Ca2+ levels activating EC sKCa and IKCa channels and causing Ca2+-dependent production of endothelial vasodilator autacoids such as NO, prostaglandin I2 and epoxides of arachidonic acid that mediate negative-feedback regulation of myogenic tone. Thus, Ca2+-dependent ion channels importantly contribute to many aspects of the regulation of myogenic tone in arterioles in the microcirculation.Arterioles in the peripheral microcirculation regulate blood flow to and within tissues and organs, control capillary blood pressure and microvascular fluid exchange, govern peripheral vascular resistance, and contribute to the regulation of blood pressure. These important microvessels display pressure-dependent myogenic tone, the steady state level of contractile activity of vascular smooth muscle cells (VSMCs) that sets resting arteriolar internal diameter such that arterioles can both dilate and constrict to meet the blood flow and pressure needs of the tissues and organs that they perfuse. This perspective will focus on the Ca Arterioles are prominent resistance vessels that regulate blood flow to and within tissues and organs; determine capillary blood pressure and fluid exchange in the microcirculation; and contribute to the regulation of systemic blood pressure . A defin2+ triggering contractile machinery activation and increased arteriolar tone (vasoconstriction). In endothelial cells (ECs), ion channels provide a key Ca2+source controlling EC autacoid production including prostacyclin (PGI2), nitric oxide (NO) and epoxides of arachidonic acid  which provide a major source of activator Ca2+ in VSMCs  channels provide negative-feedback regulation of myogenic tone. Ryanodine receptors (RyRs) can be both inhibitory (negative feedback) or excitatory (positive feedback) dependent on where in the ER they are expressed and with which ion channels they interact. Inositol 1,4,5-trisphosphate receptors (IP3Rs), transient-receptor potential melanostatin member 4 (TRPM4) channels, Ca2+-activated Cl− channels (CaCCs) and transient receptor potential polycystin-family member 1 [TRPP1 (PKD2)] channels are excitatory and contribute to the positive-feedback regulation of myogenic tone. In addition, VGCCs (+ (KV) channels (+ (KATP) channels  which have seven transmembrane spanning domains (2+ is sensed by two regulator of conductance of K+ (RCK) domains (RCK1 and RCK2) in the long, cytosolic C-terminus of the α-subunit  to open at negative, physiological membrane potentials  in arteriolar VSMCs may be due to lower expression of the β1-subunits , facilitating their negative feedback function (Ca channel sensitivity to activation by docosahexaenoic acid (DHA) also is increased by LRRCs : bursts of activity of small groups of BKCa channels coinciding with the RyR-based Ca2+ sparks  to be po −25mV, ] of VSMC in tone .− and coupled to RyR-mediated Ca2+ sparks or IP3-based Ca2+ waves have been reported  family of ion channels that contribute to myogenic tone in in response to agonists of EC Gαq-coupled receptors resulted from Ca2+ release from ER stores  expressed in these IP3R clusters has not been identified, they were demonstrated to be the sites of EC Ca2+ pulsars, localized IP3-dependent Ca2+ events arising from clusters of IP3Rs in the ER that extend into MEPs  leads to EC hyperpolarization, which can be conducted through MEGJs to overlying VSMCs, deactivating VGCCs, reducing VSMC Ca2+ influx and decreasing myogenic tone  that diffuse to overlying VSMCS and reduce myogenic tone  and IKCa  channels  channels, leading to EC hyperpolarization and conduction of this signal to overlying VSMCs. Hyperpolarization then deactivates VSMC VGCCs reducing myogenic tone  and colocalize with caveolin-1  reduces myogenic tone of mesenteric resistance arteries  myogenic tone in rat cerebral parenchymal arterioles  than sKCa and IKCa channels, do not require association with calmodulin, and display pharmacology distinct from sKCa and IKCa channels , initial studies did not report expression of TMEM16A in ECs in lung sections , activation of these CaCCs leads to membrane depolarization, counter to the effects of activation of EC sKCa and IKCa channels which produce robust EC hyperpolarization. Thus, it may be that CaCCs in ECs are part of a negative feedback mechanism to dampen membrane hyperpolarization induced by EC sKCa and IKCa channels when intracellular Ca2+ is elevated.The function of TMEM16A in arteriolar ECs related to regulation of myogenic tone is not clear. In murine capillary ECs, block of TMEM16A results in membrane hyperpolarization suggesting that in ECs, like in VSMCs , activation of IKCa channels and dampening of myogenic tone  is used.Endothelial TRPV4 channels mediate agonist-induced, endothelium-dependent vasodilation, particularly in arterioles where activation of these receptors leads to activation of IKdilation . In addiation  to form IP3 and DAG  initiate the myogenic response culminating in steady-state myogenic tone . Putativ and DAG .2+ influx through these channels  [Ca2+]in levels, leading to calmodulin-mediated myosin light-chain kinase (MLCK) activation, phosphorylation of the myosin light-chains (MLC), actin-myosin cross-bridge formation, cross bridge cycling and an increase in myogenic tone  blunts and limits depolarization-induced activation of VGCC  supporting the increase in tone by increasing the activity of TRPM4 channels (supporting depolarization) and VGCCs (promoting Ca2+ influx) while blunting the activity of several K+ channels  channels, limiting their activity and promoting depolarization  and by conduction of Ca2+-dependent membrane hyperpolarization from the endothelium to overlying VSMCs via MEGJs (3) and ionic currents (including Ca2+) to move between the cells. Pressure-induced increases in VSMC [Ca2+]in or IP3 can pass to endothelial cells leading to EC IP3R-induced Ca2+ signals (Ca2+ pulsars and wavelets) that can increase the production of Ca2+-dependent EC vasodilator autacoids that feedback to the VSMCs reducing myogenic tone (2+]in will activate EC sKCa and IKCa channels causing EC membrane hyperpolarization. Myoendothelial gap junctions allow this hyperpolarization to be passed from ECs to VSMCs, producing VSMC hyperpolarization, deactivation of VSMC VGCCs and reduced myogenic tone (2+-dependent ion channels in the endothelium including IP3Rs, TRPV4 channels, sKCa channels and IKCa channels (Endothelial cells lining resistance arteries and arterioles play a negative-feedback role, dampening myogenic tone both through the Caia MEGJs , 3. Endonic tone . In addinic tone . Thus, tchannels .2+-activated ion channels in both VSMCs and ECs contribute to the regulation of myogenic tone. However, there appears to be considerable heterogeneity in the specific details of their roles in this process among vessels in different vascular beds around the body. The mechanisms responsible for this heterogeneity remains to be established. It is also clear that there is a paucity of information about the cellular and molecular details surrounding which channels are expressed, their localization and their regulation relative to myogenic tone in arterioles around the body. Mesenteric and cerebral resistance artery ion channel expression and function has been well studied. However, we know relatively little about ion channel expression and function in the downstream arterioles in microcirculation, which is really the business end of the cardiovascular system. Future studies directed specifically at understanding control of ion channel expression and function in the microcirculation and how they vary among vascular beds in different organs is warranted.As outlined in this perspective, CaWJ conceived, wrote, and edited this manuscript.Supported by National Heart, Lung and Blood Institute grants HL-137694 and PO1-HL-070687.The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.The author declares 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. 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