MST1, a key kinase of the Hippo signaling pathway, is often presented as a substrate of caspases, and its cleavage by caspases potentiates its activity. Several studies have focused on the involvement of MST1 in caspase activation and also reported several defects in the immune system function caused by MST1 deficiency. Here, we show the rapid activation of the MEK-ERK-MST1 axis together with the cleavage and activation of caspase-3, -6, -7, -8, and -9 after PI3K signaling blockade by the selective inhibitor GDC-0941 in Jurkat T cells. We determined the phosphorylation pattern of MST1 using a phosphoproteomic approach and identified two amino acid residues phosphorylated in an ERK-dependent manner after GDC-0941 treatment together with a novel phosphorylation site at S21 residue, which was extensively phosphorylated in an ERK-independent manner during PI3K signaling blockade. Using caspase inhibitors and the inhibition of MST1 expression using siRNA, we identified an exclusive role of the MEK-ERK-MST1 axis in the activation of initiator caspase-8, which in turn activates executive caspase-3/-7 that finally potentiate MST1 proteolytic cleavage. This mechanism forms a positive feed-back loop that amplifies the activation of MST1 together with apoptotic response in Jurkat T cells during PI3K inhibition. Altogether, we propose a novel MEK-ERK-MST1-CASP8-CASP3/7 apoptotic pathway in Jurkat T cells and believe that the regulation of this pathway can open novel possibilities in systemic and cancer therapies.The discrete activation of individual caspases is essential during T-cell development, activation, and apoptosis. Humans carrying nonfunctional caspase-8 and caspase-8 conditional knockout mice exhibit several defects in the progression of naive CD4 Programmed cell death (apoptosis) is a crucial process that is essential for the survival of nearly all eukaryotic organisms. Apoptosis drives the development of multicellular organisms, regulates immune system functions, and controls the disposal of damaged cells in tissues . It alsoBCL2, cIAPs, or BCL-xL) mainly through YAP/TAZ co-activator inhibition and stimulates the transcription of several pro-apoptotic genes  through the activation of transcription factors such as FOXOs [MST1 kinase forms a part of the central kinase cassette of the Hippo signaling pathway, which acts as a strong tumor-suppressor due to its ability to induce apoptosis in eukaryotic cells . MST1 unas FOXOs ,9,10. Pras FOXOs ,12,13. Oas FOXOs ,15,16. Tas FOXOs ,18. Moreas FOXOs . Unfortuas FOXOs ,21.We confirmed the abrogation of AKT kinase phosphorylation at S473 followed by the fast activation of MST1 and caspase-3, -6, -7, -8, and -9 in a model of Jurkat T cells after treatment with the selective PI3K inhibitor GDC-0941. The inhibition of AKT kinase activity by GDC-0941 was confirmed as a decrease in the phosphorylation of downstream P70S6K kinase at S424 residue and increase in caspase-9 activity. We also observed an increase in the phosphorylation of MEK1/2 at S217/S221 residues and ERK1/2 kinase phosphorylation at T202/Y204 residues, although the phosphorylation of c-RAF at S338 residue was decreased. ERK1/2 kinase activation was confirmed by an increase in the phosphorylation of P90RSK at S380 residue which represents the established target of EKR1/2 signaling. Then, we determined the phosphorylation status of MST1 in control cells, GDC-0941-treated cells and cells treated with a combination of GDC-0941 and the ERK1/2 inhibitor, SCH-772984, with the aim to test the potential impact of MEK-ERK signaling on MST1 phosphorylation. We characterized several phosphorylated amino acid residues of MST1 and identified the ERK-dependent phosphorylation sites of MST1 at T177 and T387 residues together with a novel ERK-independent phosphorylation site at S21 residue. Using a bioinformatic approach, we predicted PLK1, NEK2 and GSK3B signaling to have a potential impact on MST1 phosphorylation. To clarify the role of individual caspases during the proteolytic activation of MST1, we investigated the effects of the selected caspase activity inhibitors on MST1 cleavage. Pre-treatment of cells with the Z-DEVD-FMK inhibitor completely blocked the proteolytic activation and activity of caspase-3, -6, -7, and -9 after GDC-0941 treatment. However, the Z-DEVD-FMK inhibitor was insufficient to block the proteolytic activation of caspase-8 and MST1 which were significantly blocked by the same concentration of the general Z-VAD-FMK inhibitor. Moreover, MST1 inhibition by siRNA significantly reduced the proteolytic activation of caspase-3, -7, and -8 together with proteolytic activity against the Ac-DEVD-pNA substrate during GDC-0941 treatment. These results demonstrate the MEK-ERK-MST1 axis as a novel regulator of the extrinsic apoptotic pathway in Jurkat T cells.MST1 kinase phosphorylation by AKT kinase inhibits its activity; therefore, we tested the effect of the selective PI3K inhibitor GDC-0941 on the phosphorylation levels and activity of these two kinases. We observed a significant decrease in AKT phosphorylation at S473 residue together with the proteolytic activation of MST1 after 2 h of GDC-0941 treatment A. DuringP70S6K represents kinase downstream of AKT and a decrease in its phosphorylation is a marker of the inhibition of AKT activity by GDC-0941. On the other hand, MEK1/2 represents the well-known target of c-RAF but its phosphorylation was increased, suggesting the non-canonical activation of MEK-ERK signaling during GDC-0941 treatment. The activation of MEK-ERK signaling during GDC-0941 treatment was confirmed by an increase in the phosphorylation of P90RSK, a well-established target of ERK1/2 B.Since we observed an increase in ERK1/2 phosphorylation during GDC-0941 treatment in Jurkat T cells, we tested the potential effect of ERK1/2 on the proteolytic activation of MST1. Pre-treatment of cells with the selective inhibitor of ERK1/2 signaling SCH-772984 resulted in a significant decrease in the phosphorylation of c-RAF, ERK1/2 and P90RSK together with an increase in the phosphorylation of MEK1/2 after GDC-0941 treatment B. ERK1/2To clarify the effect of PI3K, ERK1/2 signaling inhibition and their combination on MST1 phosphorylation, we performed MST1 pull-down in Jurkat T cells, followed by the identification of individual phosphorylated residues and quantification of the phosphorylation levels using mass spectrometry (MS). We were able to quantify the phosphorylation levels of eight individual AA residues of MST1 protein . The phosphorylation of seven residues was previously identified and was described in the PhosphoSite database but phosphorylation at S21 was newly identified in our study C,D. The We matched sequences around the phosphorylated sites of MST1 against the database of motifs recognized by individual kinases using the Eukaryotic Linear Motif (ELM) resource. This resource utilizes information on primary, secondary, and tertiary structures to determine the active regulatory motifs of individual proteins. We found that the sequence around S438 looks like a motif recognized by NEK2 kinase, the sequence around S21 looks like a motif recognized by PLK1 kinase, and that the sequence around S414 looks like a motif recognized by GSK3B kinase B–D. The We determined the activity of individual caspases after 2 h of GDC-0941 treatment using the Ac-DEVD-pNA substrate which is specific to caspase-3/-7, the Ac-LEHD-pNA substrate which is specific to caspase-9, the Ac-VEID-pNA substrate which is specific to caspase-6, and the Ac-YVAD-pNA substrate which is specific to caspase-1. We found a six-fold increase in caspase-3/-7 activities toward the Ac-DEVD-pNA substrate, two-fold increase in caspase-9 activity toward the Ac-LEHD-pNA substrate, insignificant increase in caspase-6 activity toward the Ac-VEID-pNA substrate, and no effect on caspase-1 activity toward the Ac-YVAD-pNA substrate A. This oUsing immunoblotting, we confirmed the generation of cleaved forms of caspase-3, -7, and, -8 and a decrease in the non-cleaved forms of caspase-3, -6, -7, and -8 after 2 h of GDC-0941 treatment. We could not detect the generation of the cleaved form of caspase-6 . Pre-treWe tested the effect of selected concentrations of individual caspase inhibitors on the proteolytic activation of MST1 by immunoblotting. We found that 2 µmol/L Z-DEVD-FMK, which effectively inhibited the generation of cleaved forms of caspase-3/-7 and decreased the levels of the full-length forms of caspase-3, -7, and -6, was insufficient to inhibit the generation of the cleaved form of MST1 and to decrease the levels of the full-length form of MST1. Significant inhibition of the proteolytic activation of MST1 during GDC-0941 treatment was achieved using a higher Z-DEVD-FMK concentration  control siRNA. We confirmed the inhibition of the proteolytic activation of caspase-3, -7, and -8 in cells transfected with MST1-specific siRNA compared with that in cells transfected with NT control siRNA after GDC-0941 treatment B. MoreovMST1 kinase was recently identified as a key regulator of apoptosis in immune cells ,23,24. IWe confirmed the complete inhibition of AKT phosphorylation at S473 residue after 2 h of GDC-0941 treatment A. ConsisSpecific ERK1/2 signaling inhibition decreased the proteolytic activation of MST1 during GDC-0941 treatment in Jurkat T cells. To characterize the impact of PI3K and ERK signaling on MST1, we determined the phosphorylation levels of individual AA residues in control cells and cells treated with GDC-0941, SCH-772984, and their combination. Using MS analysis, we determined eight AA residues that change in the phosphorylation levels between the tested conditions C. PhosphSeveral studies have highlighted the interplay between the activation of MST1 and cellular caspases, but this interplay shows a bidirectional character. Several studies have demonstrated the caspase-dependent proteolytic activation of MST1, whereas several others have shown the caspase-independent proteolytic activation of MST1 and the MST1-dependent activation of caspases. To clarify this Hippo–caspase concatenate, we determined the activity of caspase-1, -3, -6, -7, and -9 using colorimetric substrates specific for individual caspases. We observed a weak increase in the activity of caspase-6/-9 and a significant increase in the activity of caspase-3/-7 after 2 h of GDC-0941 treatment A. Using 2 in RPMI1640 medium supplemented with L-glutamine , 10% fetal bovine serum , 100-U/mL penicillin, and 100-µg/mL streptomycin (Thermo Fisher Scientific). The cells were seeded at a density of 2.5 × 105 cells/mL in 15 mL of RPMI1640 medium, grown overnight in 75-cm2 cell culture flasks  under standard cultivation conditions, and treated with GDC-0941  at a final concentration of 10 µmol/L or SCH-772984  at a final concentration of 1 µmol/L for 2 h. Alternatively, cells were pre-treated with SCH-772984 at a final concentration of 1 µmol/L concentration (effective concentration for Jurkat T cells according to the manufacturer) for 1 h and then treated with GDC-0941 at a final concentration of 10 µmol/L for 2 h  and cultured at 37 °C under 5% COT cells; ). GDC-09Precast gradient TGX gels  were loaded with 40 µg of proteins isolated from individual samples per lane. A Protean III apparatus  with a constant voltage of 100 V was used to run the SDS-PAGE protein samples. Separated proteins were blotted onto a nitrocellulose membrane  using a Trans-Blot™ SD Semi-Dry apparatus . Protein blots were blocked for 1 h in TBS supplemented with 5% non-fat milk  and 0.05% Tween-20 (Sigma-Aldrich). Membranes were washed with TBS containing 0.05% Tween-20 and incubated with the respective primary and secondary antibodies as per the manufacturer’s protocols. The following antibodies were used for immunostaining: MST1, AKT, p-AKT (S473), ERK1/2, p-ERK1/2 (T202/Y204), p-RAF (S338), p-MEK1/2 (S217/S221), p-P90RSK (S380), p-MSK1 (T581), caspase-3, caspase-6, caspase-7, and caspase-8 antibodies ; p-P70S6K (S424) and actin antibodies ; and HRP-conjugated secondary antibodies against rabbit and goat IgG . WestPico ECL substrate (Thermo Fisher Scientific) and ChemiDoc CCD system  were used for chemiluminescence signal detection and visualization. A representative image of each immunoblot was selected from at least three independent experiments.p-nitroanilide group (pNA). Each reaction contained 100 µg of proteins from cytosolic fractions resuspended in 50 µL of hypotonic buffer, 50 µL of 2× caspase assay buffer , and 5 µL of specific substrate . The following substrates were used: Ac-LEHD-pNA (Sigma-Aldrich), Ac-VEID-pNA, Ac-YVAD-pNA, and Ac-DEVD-pNA . Samples were pre-incubated in 96-well plates at 37 °C for 15 min, and absorbance at 405-nm wavelength (A405) was recorded using a plate reader . The background was subtracted from all A405 values, and the resulting A405 values from two technical replicates (mean only) were plotted in a time-dependent manner. The best-fit regression line was then superimposed using GraphPad software. Each graph shows the results of one representative measurement in a technical duplicate selected from at least three independent experiments.Caspase activities in cytosolic fractions were determined using specific substrates conjugated with the After overnight cultivation, cells were pre-treated with the selected concentrations of individual inhibitors conjugated with the fluoromethylketone group (FMK) instead of the pNA group. Stock solutions  of the following inhibitors were used: Z-DEVD-FMK and Z-VAD-FMK . Cells were pre-treated with individual inhibitors for 1 h, treated with GDC-0941 for 2 h, and processed as per the standard protocols.MST1 expression was inhibited by transfection with a pool of four MST1-specific siRNAs (Thermo Fisher Scientific) into Jurkat T cells using the DharmaFECT4 reagent (Thermo Fisher Scientific). A non-targeting pool of four individual siRNAs was used as a control. MST1 protein levels were determined by immunoblotting 72 h after transfection. Transfected cells were treated and processed as per the standard protocols.MST1 antibody  was used for co-immunoprecipitation experiments using the Dynabeads Co-Immunoprecipitation Kit (Thermo Fisher Scientific). For this purpose, 5 µg of MST1 antibody per mg of Dynabeads  was coupled as per the manufacturer’s protocol.5 cells/mL in 150 mL of RPMI1640 medium in 300-cm2 culture flasks  and cultivated under standard conditions for 1 h. Cells were then treated with the selected compounds as per the standard protocol. Cells were collected by centrifuging, washed once with PBS, and lysed by the detergent lysis method. Cells were resuspended in a 1:9 ratio of cell mass to extraction buffer with protease inhibitors , incubated on ice for 15 min, and then centrifuged at 2600× g for 5 min. The obtained supernatant was immediately used for co-IP. After co-IP, the precipitated proteins were eluted in 1000 µL of HPH EB buffer. We saved 100 µL of eluates for the MS identification of co-precipitated proteins and separated lyophilized eluates using SDS-PAGE followed by Coomassie staining for visualization.Cells were seeded at a density of 7 × 10g and 4 °C for 20 min. The supernatant was removed, and cell pellets were resuspended in 100 mM TEAB containing 2% SDC, followed by boiling at 95 °C for 5 min. Cysteines were reduced with TCEP at a final concentration of 5 mM (60 °C for 60 min) and blocked with MMTS at a final concentration of 10 mM (room temperature for 10 min). Samples were digested with trypsin  at 37 °C overnight. After digestion, samples were acidified with TFA at a final concentration of 1%. SDC was removed by extraction with ethyl acetate and the peptides were desalted in a Michrom C18 column. Dried peptides were resuspended in 25 µL of water containing 2% acetonitrile (ACN) and 0.1% trifluoroacetic acid. For analysis, 12 µL of sample was injected [Eluates from immunoprecipitation were precipitated by adding four volumes of ice-cold acetone, kept at −20 °C for 30 min, and centrifuged at 16,000× injected .Individual bands containing proteins of interest were excised from the Coomassie-stained SDS-PAGE gel using a razor blade and cut into small pieces (approximately 1 mm × 1 mm). Bands were destained by sonication for 30 min in 50% ACN and 50 mM ammonium bicarbonate (ABC). After destaining, the solution was removed and gels were dried in ACN. Disulfide bonds were reduced using 10 mm DTT in 100 mM ABC, at 60 °C, for 30 min. Subsequently, samples were re-dried with ACN, and free cysteine residues were blocked using 55 mM iodoacetamide in 100 mM ABC in the dark, at room temperature for 10 min. Samples were dried thoroughly, and digestion buffer  was added to cover gel pieces. Proteins were digested at 37 °C overnight. After digestion, 150 µL of 50% ACN with 0.5% formic acid was added, followed by sonication for 30 min. The supernatant containing peptides was added to a new microcentrifuge tube, another 150 µL of elution solution was added to the supernatant, and this solution was sonicated for 30 min. The solution was then removed, combined with the previous solution, and dried using Speedvac. Dried peptides were reconstituted in 2% ACN with 0.1% TFA and injected into Ultimate 3000 Nano LC coupled to Orbitrap Fusion.m/z were performed at 120K resolution (at 200 m/z) with a 5 × 105 ion count target. Tandem MS was performed by isolation at 1.5 Th with the quadrupole, HCD fragmentation with normalized collision energy of 30, and rapid scan MS analysis in the ion trap. The MS2 ion count target was set to 104, and the max injection time was set to 35 ms. Precursors with a charge state of 2–6 were sampled for MS2. The dynamic exclusion duration was set to 45 s with a 10 ppm tolerance around the selected precursor and its isotopes. Monoisotopic precursor selection was turned on. The instrument was run in the top speed mode with 2 s cycles.A nano reversed-phase column  was used for LC–MS analysis. Mobile phase buffer A was composed of water and 0.1% formic acid. Mobile phase buffer B was composed of ACN and 0.1% formic acid. Samples were loaded onto the trap column  at a flow rate of 15 μL/min. Loading buffer was composed of water, 2% ACN, and 0.1% trifluoroacetic acid. Peptides were eluted with buffer B gradient from 4% to 35% over 60 min at a flow rate of 300 nL/min. Eluting peptide cations were converted to gas-phase ions by electrospray ionization and analyzed on a Thermo Orbitrap Fusion . Survey scans of peptide precursors from 350 to 1400 All data were analyzed and quantified using MaxQuant software (version 1.6.1.0) . The fal


IL‐UAE) was developed to extract and separate the isochlorogenic acid C (ICGA) from a cultivar of Chrysanthemum morifolium (Chrysanthemummorifolium Ra Tnat.). The influencing parameters, including IL concentration, liquid‐to‐solid ratio, and ultrasonic time, were optimized using response surface methodology. Of the ILs studied, 1‐butyl‐3‐methylimidazolium bromide [(Bmim)Br] exhibited the best extraction ability. The optimized conditions included liquid‐to‐solid ratio of 23.44:1, ultrasonic time of 48.99 min, and IL concentration of 0.65 mol/L. Under the optimal conditions, the extraction yield of ICGA could reach to 4.20 mg/g. An aqueous two‐phase system was applied for purification and separation of ICGA. The maximum extraction efficiency of 98.18% was obtained under the conditions of (NH4)2SO4 of 4.5 g, pH of 3.0, and a temperature of 20°C at aqueous solution. Furthermore, the thermodynamic parameters showed that the purification of ICGA from salt‐rich phase to IL‐rich phase was a spontaneous and exothermic process. The results indicated that the proposed system is simple, rapid, and effective to serve as a viable and sustainable platform for the extraction and purification of ICGA from Chrysanthemum morifolium flowers.Ionic liquid‐based ultrasonic‐assisted extraction ( It has been known for its medical values such as cold scattering, liver clamping, toxicity clearing, and eyes brightening , which is usually composed of polymer/polymer, polymer/salt, or salt/salt, has been recognized as an economical and efficient processing method , which is developed by Gutowski et al. , is usuaIn this work, an optimized single‐step procedure of IL‐UAE‐ATPS was studied to extract ICGA from a cultivar of Chrysanthemum morifolium. The studied IL was (Bmim)Br (1‐butyl‐3‐methylimidazolium bromide), the chemical structure of which is presented in Figure 22.14)2SO4, K3PO4, NaH2PO4, K2CO3, methanol, and ethanol were of analytical grade and were acquired from Sinopharm Chemical Reagent Co., Ltd. . Other reagents were all of analytical grade and used without further treatment. Deionized water was obtained with a Milli‐Q water purification system (Millipore) to prepare the sample solutions. The HPLC equipment used was Agilent 1260 HPLC system .Chrysanthemum morifolium from Anhui Province was purchased from Shanghai Dedatang Sinopharm Co., Ltd. . The standard of ICGA (purity >98% by HPLC) was purchased from Chengdu Biopurify Phytochemicals Ltd. . (Bmim)Br (1‐butyl‐3‐methylimidazolium bromide) was purchased from Shanghai Cheng Jie Chemical Co., Ltd. . (NH2.2m is the mass (mg) of ICGA determined and M is the mass (g) of dried sample powder.Dried flowers were pulverized into a homogeneous size by a grinder, sieved through a 60 mesh sieve, and dried at 45°C in the oven for 6 hr. Powders and IL solutions were mixed with a certain ratio in a centrifuge tube. The blend was placed in an ultrasonic bath at 25°C for some time to ensure the dissolution of the active ingredients. The extract solution was centrifuged at 12,000 g for 10 min, and the supernatant was filtrated through a 0.45‐μm microporous membrane and then injected into HPLC for analysis. The extraction yield of ICGA by ionic liquid‐based ultrasonic‐assisted extraction (IL‐UAE) was calculated according to Equation 2.3Ten milliliters extraction solution and a given amount of salt were added to a 25‐ml centrifuge tube. The mixture was vortexed for 3 min to ensure the complete dissolution of the salt. Due to the incompatibility of IL solution and salt solution, and the low viscosity in the system, the phase separation can be achieved in a few seconds. The top IL‐rich phase was mainly composed of IL and ICGA occupying a small volume, and the bottom phase was salt‐phase solution containing the aqueous impurities occupying a large volume. The volume of each phase was noted down.2.4The two phases were separately withdrawn using pipettes for determining the concentration of ICGA by HPLC. The residues accumulated at the interface between two phases were discarded. All experiments were carried out at 25°C. The parameters are defined as follows.R), partition coefficient (K), and extraction efficiency (E) were evaluated. The parameters were defined as follows:Vt and Vb are the volumes of IL‐rich phase and salt‐rich phase, respectively, and Ct and Cb denote the measured ICGA concentration (mg/ml) in the IL‐rich phase and salt‐rich phase, respectively.The phase ratio . The injection volume was 5.0 μl, the temperature of the column oven was controlled at 30°C, and the flow rate of the mobile phase was maintained at 1 ml/min. The mobile phase consisted of 0.1% formic acid water (A) and 0.2% formic acid ACN (B) using a gradient elution of 10%–19% A at 0–16 min, 19%–20% A at 16–24 min, 10%–24% A at 24–48 min, 24%–50% A at 48–55 min, and 50%–10% A at 55–60 min. The UV wavelength was set at 384 nm. The data acquisition and analysis were performed by Agilent ChemStation software.Chromatographic analyses were performed using an Agilent 1260 series HPLC system equipped with an Agilent Extend‐C2.6In order to further compare the extraction yield of conventional heat reflux extraction (HRE) and UAE, the extractions of ICGA based on different solvents  were performed. All the experiments were performed at the same conditions. Briefly, 0.42 g sample powder was mixed with 10 ml methanol, ethanol, or water ultrasonic treatment for 48.99 min. The extraction time for HRE was 2 hr at 80°C. The subsequent steps were the same as those in IL‐UAE.33.1Ionic liquid is usually composed of an organic cation in combination with an inorganic anion. The structure of IL has a significant impact on the extraction yield, and different structures may lead to a different extraction yield of analytes. According to Yang et al. , the effStructurally, the π‐bond of the heterocyclic structure in ILs is rich in numerous electrons, which can produce intermolecular binding through n–π or π–π stacking and promote the dissolution of effective substances in the samples  liquid‐to‐solid ratio, 10:1–30:1; (B) ultrasonic time, 40–60 min; and (C) IL concentration, 0.25–0.75 mol/L. Table F‐value of 22.49 implied that the model was significant and there was only a 0.02% probability that a “model F‐value” of this large could occur due to statistical noise. Values greater than 0.1 indicated that the model terms were not significant and those less than 0.05 represented that the model terms were significant. Thus, the model explained the response adequately.As shown in Table 2 and C2) were highly significant (p < 0.01), and B2 (p < 0.05) was also significant. Among the interaction terms, the interaction between AB (p < 0.01) was very significant with regard to the extraction yield of ICGA. The others were not significant (p > 0.05). The “lack‐of‐fit F‐value” of 31.21 implied that the “lack of fit” was significantly relative to the pure error and that there was a 0.31% chance that a “lack‐of‐fit F‐value” this large could occur due to statistical noise. It was clear that this model could be used to navigate the design space. The final predicted regression model between the extraction yield and these factors was given by the following Equation Y was the extraction yield of ICGA and A, B, and C, respectively, corresponded to the coded values of the three independent variables solid‐to‐liquid ratio, ultrasonic time, and IL concentration.The magnitude of the absolute value of each coefficient in the equation reflected the degree of influence of each factor on the response value, and the positive and negative of the coefficient reflected the direction of influence. According to the significance of the regression coefficients of the quadratic polynomial model, IL concentration was found to be the most significant factor to affect the extraction yield of ICGA, followed by solid‐to‐liquid ratio and ultrasonic time. The quadratic terms , a comparison was made between the proposed IL‐UAE and conventional UAE and HRE methods. The results are summarized in Figure 3.4Following the extraction of ICGA by IL‐UAE, it was further purified by ATPS. The type and amount of inorganic salt, temperature, and pH can be considered as the prominent factors influencing the purification efficiency of ICGA by ATPS.3.52HPO4, (NH4)2SO4, NaHSO4, and Na2CO3) were considered. Among these salts, NaHSO4 and Na2HPO4 could not form ATPSs, and Bmim[Br]/(NH4)2SO4 and Bmim[Br]/Na2CO3 could observe the phenomenon of phase separation in just a few minutes. However, the Bmim[Br]/Na2CO3 system showed a significant change in color as the formation of phase separation. This may be due to the reason that there is a chemical reaction between alkaline Na2CO3 and acidic ICGA. Therefore, Bmim[Br]/(NH4)2SO4 system was selected for further studying the purification of ICGA.In order to choose an optimal inorganic salt for the enrichment of ICGA from Chrysanthemum morifolium, four different types of inorganic salt (Na4)2SO4 added to form Bmim[Br]/(NH4)2SO4, the range of (NH4)2SO4 (from 4.5 to 6.5 g) was investigated. When the amount of (NH4)2SO4 was too low, ATPS could not be formed and the system was a homogeneous solution. With the amount of salt gradually increased, the solution was cloudy. Until the amount reached to 4.5 g, phase separation was formed. The upper phase was formed by the more hydrophobic IL solution, and the lower phase was consisted of the more hydrophilic salt solution. The amount of salt also directly affected the volume of the upper phase, thereby affecting the purification effect. As shown in Figure 4)2SO4 increased, there was a slight decrease in the extraction efficiency of ICGA. This phenomenon may be due to the enhancement of salting‐out effect as the concentration increased in the bottom phase, and more free water molecules enter into the bottom phase, which leads to the increased viscosity and decreased contact area of the upper phase. Thus, the entry of target compound into the IL‐rich phase becomes more difficult, and the extraction efficiency decreased. Based on economic factors and extraction efficiency, 4.5 g of (NH4)2SO4 was selected to be the suitable amount.To optimize the amount of 2SO4 of 4.5 g, pH of 3.0, and extraction temperature of 20°C. By calculating the peak area of the HPLC chromatogram in Figure A maximum extraction yield of 4.2065 mg/g and a extraction efficiency of 98.12% were obtained under the optimal conditions of IL‐UAE‐ATPS with the liquid‐to‐solid ratio of 23.44, ultrasonic time of 48.99 min, IL concentration of 0.65 mol/L, (NH3.8G), from one phase to another, may be associated with its partition coefficients by Equation R is the universal gas constant and thermodynamic temperature (T) and distribution coefficient (K) were known.The parameters of thermodynamics during purification process were investigated to study the transfer process of ICGA from salt‐rich phase to IL phase. The Gibbs energy of transfer  was constant and the entropy change (∆S) also was calculated according to the intercept of relation curve. The detailed values of T∆S, ∆G, and ∆H are shown in Table As shown in Equation 4)2SO4 are listed in Table ∆G values were negative (∆G < 0), which indicated that the reaction of purification proceeded spontaneously from salt‐rich phase to IL‐rich phase.The thermodynamic parameters of the [Bmim]Br‐2SO4 was added to the system with the pH of 3.0 and the temperature of 20°C. Besides, the thermodynamics was explored simultaneously in the purification procedure for the first time, which could clearly explain the extraction mechanisms of IL/salt (Bmim[Br]/(NH4)2SO4) ATPS. Therefore, the IL‐UAE‐ATPS system is a sustainable, simple, and effective approach for the extraction and purification of ICGA from Chrysanthemum morifolium.In this work, IL‐UAE coupled with the IL‐ATPS could be effectively applied in the extraction and purification of ICGA from Chrysanthemum morifolium. The optimized conditions of UAE were identified as a liquid‐to‐solid ratio of 23.44:1, ultrasonic time of 48.99 min, and IL (Bmim[Br]) concentration of 0.65 mol/L. Under the optimal conditions, the extraction yield of ICGA could reach to 4.20 mg/g. The maximum extraction efficiency reached 98.18% when 4.5 g (NHThe authors declare that they do not have any conflict of interest.This study does not involve any human or animal testing.


Here, using a combination of in vitro binding assays and in situ structural and functional assays in ventricular trabeculae of rat heart and the expressed C1mC2 region of cMyBP-C, containing the m-domain flanked by domains C1 and C2, we tested whether these substitutions do in fact mimic the effects of phosphorylation. In situ changes in thin and thick filament structure were determined from changes in polarized fluorescence from bifunctional probes attached to troponin C or myosin regulatory light chain, respectively. We show that both the action of exogenous C1mC2 to activate contraction in the absence of calcium and the accompanying change in thin filament structure are abolished by tris-phosphorylation of the m-domain, but unaffected by the corresponding Ser–Asp substitutions. The latter produced an intermediate change in thick filament structure. Both tris-phosphorylation and Ser–Asp substitutions abolished the interaction between C1mC2 and myosin sub-fragment 2 (myosin S2) in vitro, but yielded different effects on thin filament binding. These results suggest that some previous inferences from the effects of Ser–Asp substitutions in cMyBP-C should be reconsidered and that the distinct effects of tris-phosphorylation and Ser–Asp substitutions on cMyBP-C may provide a useful basis for future studies.Myosin-binding protein-C (cMyBP-C) is a key regulator of contractility in heart muscle, and its regulatory function is controlled in turn by phosphorylation of multiple serines in its m-domain. The structural and functional effects of m-domain phosphorylation have often been inferred from those of the corresponding serine-to-aspartate (Ser–Asp) substitutions, in both  Ph. Phin vivivo (A) . Interacvivo (A) , 6. In cvivo (A) , 7. Phosin vivo in transgenic animal models  substitutions of serine residues within the m-motif have often been used as a proxy for phosphorylation, l models , 9, in sle cells , and in proteins , 12. Thein vitro, to determine whether Ser–Asp substitutions reproduce the structural and functional effects of phosphorylation.In this study, we used bifunctional fluorescent probes on myosin regulatory light chain (RLC) or troponin C (TnC) to monitor structural changes in the thick and thin filaments, respectively, in rat heart muscle cells. We induced activation of the muscle cells using exogenous rat cardiac C1mC2 fragments that were native, tris-phosphorylated, or carrying three Ser–Asp substitutions. We combined these structural measurements with measurements of active isometric force and with biochemical characterization of the interaction between C1mC2 and myosin S2, and C1mC2 and native thin filaments (NTF) 2+, and tris-phosphorylation of C1mC2 by protein kinase A (PKA) abolishes this effect (pCa 9) containing 40 μmol/liter unphosphorylated C1mC2, PKA tris-phosphorylated C1mC2 , or C1mC2 with three phosphorylatable serines mutated to aspartate (C1mC2–3SD) (n = 13) of that during full Ca2+ activation (B), whereas C1mC2–3P produced no activation, as described previously (n = 13). In complete contrast to phosphoserines, the Ser–Asp substitutions have no effect on the activating effect of C1mC2.N-terminal fragments of rat cardiac myosin-binding protein-C containing the Ig-domains C1 and C2 and the intervening m-domain (C1mC2) are sufficient to activate contraction of demembranated rat ventricular trabeculae in the absence of Cas effect . This asmC2–3SD) . Incubattivation B, whereaeviously . StrikinFig. S2) to monitor structural changes in the thin and thick filaments, respectively, in this assay. The cTnC E-helix is part of the core domain of troponin containing troponin I and troponin T (IT-arm of troponin), and its orientation is coupled to the position of tropomyosin on the surface of the thin filament. The order parameter 〈P2〉 describing the orientation of BR–cTnC-E decreases on calcium activation, suggesting a more perpendicular orientation of the E-helix (C). Incubation of trabeculae in relaxing solution (pCa 9) containing 40 μmol/liter C1mC2 induced a change in thin filament structure reported by the TnC E-helix probe that is significantly larger than that during full calcium activation alone, as reported previously (C). Thus, in full agreement with the functional effects described above, the Ser–Asp substitutions do not reproduce the effect of tris-phosphorylation of C1mC2 on thin filament structure.We used bifunctional (sulfo)rhodamine probes attached to either the E-helix of cardiac troponin C (BR–cTnC-E) or cross-linking helices B and C of cardiac myosin regulatory light chain (BSR–cRLC-BC) . Incubation of BSR–cRLC–BC exchanged trabeculae in 40 μmol/liter C1mC2 induced a change in BSR–cRLC–BC probe orientation of ∼70% of that for calcium activation, whereas C1mC2–3P produced no significant change in probe orientation. C1mC2–3SD had an intermediate effect.The RLC BC-helix probe is mainly sensitive to the regulatory state of the thick filament , and 〈P2tivation D. IncubaThe above results show that the Ser–Asp substitutions in C1mC2 do not mimic the effects of tris-phosphorylation for both active force and accompanying effects on thin and thick filament structure. For active force and thin filament structure, the action of C1mC2–3SD was indistinguishable from that of the native fragment. For thick filament structure, the effect was intermediate, suggesting the possibility that Ser–Asp substitutions in the m-domain might mimic phosphorylation of these residues with respect to the direct action of C1mC2 on thick but not thin filament structure, in the light of previous evidence for coupling between the regulatory states of the thin and thick filaments , 14.dK of 23 ± 7 μmol/liter  and a Bmax of 0.81 ± 0.10 mol/mol, suggesting roughly stoichiometric binding of C1mC2 to actin (A). PKA tris-phosphorylation of C1mC2 increased dK to 57 ± 5 μmol/liter  and reduced Bmax to 0.67 ± 0.06 mol/mol, indicating reduced binding to NTFs in vitro, in agreement with previously published results  binds NTFs with a dK of 16 ± 5 μmol/liter , similar to that of unphosphorylated C1mC2, but with a significantly lower Bmax, 0.51 ± 0.06 mol/mol.We used high-velocity co-sedimentation to determine the effects of phosphorylation and Ser–Asp substitutions in C1mC2 on binding to isolated NTF. The unphosphorylated C1mC2 binds NTFs in a saturable manner with a to actin A. PKA tr results . C1mC2–3 results A, purpledK was ∼100 μmol/liter, but at 30 °C it was reduced to ∼20 μmol/liter, in agreement with previous results .Next, we tested whether Ser–Asp substitutions mimic the effect of phosphorylation of m-domain serines on the binding of C1mC2 to myosin S2Δ, the first 126 amino acids of myosin S2 , by micrFig. S3;  16). Mo. MoKd wai.e. with the m-motif deleted , Fig. 2B; . The m-mC; Increasing ionic strength greatly reduced the affinity of C1mC2 for myosin S2Δ Fig. 2C; , suggestD,Fig. S4, and Both PKA tris-phosphorylation and replacement of the three phosphorylatable serines by aspartate abolished the high-affinity interaction between C1mC2 and myosin S2Δ , stabilizing their ON and OFF states, respectively; cMyBP-C phosphorylation abolishes both regulatory interactions, so that myosin heads are released from the thick filament backbone but the thin filament is OFF (B); and substitution of the phosphorylatable serines by aspartates releases the myosin heads from the thick filament backbone without affecting the regulatory interaction with the thin filament (C), thereby stabilizing the ON state of both thin and thick filaments. It follows that it could be quite misleading to interpret the effects of Ser–Asp substitutions in cMyBP-C as reproducing the structural or functional effects of tris-phosphorylation of cMyBP-C in vitro or in vivo. However, the differential effects of Ser–Asp substitutions and tris-phosphorylation on the thin and thick filament interactions of cMyBP-C may provide a useful approach to dissect the effects of those interactions in future studies.These results in combination with previous work therefore suggest the following: unphosphorylated native cMyBP-C binds to both the thin and thick filaments A, stabilt is OFF B; and sufilament C, therebWistar rats  were sacrificed by cervical dislocation , and demembranated right ventricular trabeculae were prepared as described previously  was incubated with 2000 units/ml of the catalytic subunit of PKA for 30–60 min at 30 °C. The reaction was followed by PhostagTM–SDS-PAGE and ESI-MS . The mainly tris-phosphorylated C1mC2 was further purified by IEC on Mono S, and homogeneity was determined by PhostagTM–SDS-PAGE and ESI-MS to be >95%. Prolonged incubation of C1mC2–3P with demembranated trabeculae did not result in any significant protein dephosphorylation . For experiments, aliquots of proteins were thawed on ice, gel-filtered, and dialyzed against new buffer prior to each experiment.All proteins were cloned from a rat cDNA library (BioChain) into a modified pET6a vector fused to an N-terminal hexa-histidine tag followed by TEV cleavage site. C1mC2 Ser–Asp mutations were introduced according to previously published protein primary sequences  and intrP2〉 that describes the dipole orientations in the trabeculae . Isometric force and steady-state fluorescence polarization values were measured once steady force had been established.Activation protocols and composition of experimental solutions were identical to those described previously for fluorescence polarization experiments . Polarizabeculae . The sarNative thin filaments (NTF) were prepared according to previously published protocols , and co-Microscale thermophoresis experiments were performed on a Monolith NT.115 instrument  in interaction buffer containing 20 mmol/liter MOPS, pH 7, 1 mmol/liter EDTA, 50 mmol/liter KCl, 1 mmol/liter DTT, and 0.05% (v/v) Tween 20. Myosin S2Δ was labeled with Alexa 647–NHS  according to the manufacturer's instructions, and dye incorporation (efficiency of ∼80%) was confirmed by HPLC and ESI-MS. All proteins were either gel-filtered into and/or extensively dialyzed against interaction buffer. Titration experiments were performed with a fixed Alexa 647–myosin S2Δ concentration of 100 nmol/liter in standard treated capillaries. For experiments with isolated m-motif, the pH of the MST buffer was adjusted to 6.2, and experiments were performed in enhanced gradient standard capillaries.Statistical significance of the difference between groups was assessed with a one-way ANOVA followed by Tukey's post hoc test. Details of significance levels are shown in the figure legends.T. K. and M. I. conceptualization; T. K. and S. P. data curation; T. K. and M. I. formal analysis; T. K. and S. P. investigation; T. K. visualization; T. K. writing-original draft; Y.-B. S. and I. S. resources; Y.-B. S. and I. S. methodology; I. S. and M. I. writing-review and editing.


Niche breadth is predicted to correlate with environmental heterogeneity, such that generalists will evolve in heterogeneous environments and specialists will evolve in environments that vary less over space and time. We tested the hypothesis that lizards in a heterogeneous environment were generalists compared to lizards in a homogeneous environment. We compared niche breadths of greater short‐horned lizards by quantifying resource selection in terms of two different niche axes, diet (prey items and trophic level), and microhabitat (ground cover and shade cover) between two populations occurring at different elevations. We assessed the heterogeneity of dietary and microhabitat resources within each population's environment by quantifying the availability of prey items, ground cover, and shade cover in each environment. Overall, our results demonstrate that despite differences in resource heterogeneity between elevations, resource selection did not consistently differ between populations. Moreover, environmental heterogeneity was not associated with generalization of resource use. The low‐elevation site had a broader range of available prey items, yet lizards at the high‐elevation site demonstrated more generalization in diet. In contrast, the high‐elevation site had a broader range of available microhabitats, but the lizard populations at both sites were similarly generalized for shade cover selection and were similarly specialized for ground cover selection. Our results demonstrate that environmental heterogeneity of a particular resource does not necessarily predict the degree to which organisms specialize on that resource. ResourcWhere an organism falls along the specialist–generalist continuum varies among populations, and even among individuals within a population. For example, populations that have undergone ecological release, particularly as a result of colonizing new habitat, often demonstrate a wider niche breadth than their ancestral counterparts  to test the hypothesis that a population in an environment with greater heterogeneity for a particular resource would exhibit a wider niche breadth  for that resource, compared to a population in an environment more homogeneous for the same resource, which should exhibit a narrow niche breadth  for that resource. Phrynosoma hernandesi is a high‐elevation species occurring in montane communities of the U.S. Great Basin, Colorado Plateau, and Mexican Highlands. Throughout its range, it is a common inhabitant of sagebrush scrublands and piñon‐juniper forest  would exhibit a broader dietary niche breadth  than lizards from the site with a narrower range of available prey items. Similarly, we predicted that lizards from the site with a wider range of microhabitats  would exhibit broader selection of microhabitat types  than lizards from the site with a narrower range of available microhabitats.We measured 22.1P. hernandesi at two sites on North Peak in the Abajo Mountains of southeastern Utah. The low‐elevation site was 2,080 m in elevation and was primarily a sagebrush scrubland surrounded by piñon‐juniper forest, while the high‐elevation site was 2,550 m and was primarily piñon‐juniper shrubland interspersed with sagebrush patches. Both sites were on east‐ or northeast‐facing slopes. Individual home range sizes average 1,218 m2 at the low‐elevation site and 643 m2 at the high‐elevation site, and individuals do not travel between sites even when reciprocally transplanted  and nitrogen (δ15N) stable isotope analysis. Stable isotopes provide a longer‐term assessment of diet, whereas stomach samples are a snapshot assessment of diet over the previous few days , this time from a height of 1.5 m above the ground and pointing downward, to record ground cover at the site used by the lizard. To assess the environmental heterogeneity in microhabitat at each study site, we quantified the ground cover types and amount of shade cover available in each population's environment. Following each radio‐location of a lizard, we walked 10 m in a random direction from the lizard's location and took a second set of photographs as described above to record ground cover and shade cover at an unused but available location in the environment. Throughout this study, we followed ASIH guidelines for use of live reptiles in Field Research (American Society of Ichthyologists and Herpetologists (ASIH) We compared microhabitat niche breadth between low‐ and high‐elevation lizards by comparing the ground cover types and amount of shade cover at sites used by lizards to ground cover and shade cover available in the landscape to determine which microhabitats lizards selected or avoided at each site during the sampling period. Ground cover was used here as an analog to “perch type,” which is commonly used to characterize microhabitat use in more arboreal lizards , to determine whether lizards overall were using prey taxa randomly , or selecting them nonrandomly at each study site. We also used a log‐linear model to determine whether lizards selected prey taxa differently at the low‐ versus high‐elevation site. Finally, we used the invertebrates collected in pitfall traps to calculate the Shannon diversity index ; δ15Nlizard was measured directly for each individual lizard; and Δn = 3.4‰ following averages determined from food chains in comparable lizard studies , to determine whether lizards used ground cover types randomly  or selected among ground cover types nonrandomly at each study site. We also used a log‐linear model to determine whether lizards selected ground cover types differently at the low‐ versus high‐elevation site. Finally, we used availability of the different ground cover types at unused locations to calculate the Shannon diversity index , to determine whether lizards used shade cover randomly or selected it nonrandomly at each site, as described above. We used a log‐linear model to determine whether lizards at each site selected shade cover differently, and we calculated Shannon diversity indices for available shade cover at each site, as described above.We quantified the percent shade cover over each used and available location from the hemispherical photographs using Gap Light Analyzer  made up the highest percentage of items found in stomach contents , with a small proportion of stomach contents at both sites also including beetles (Coleoptera) and flies  and high‐elevation  sites. Prey selection also differed between the two sites when controlling for differences in availability of prey items between the sites . At both sites, lizards selected for ants; however, at the high‐elevation site, lizards also selected for beetles, whereas at the low‐elevation site they selected against beetles when accounting for beetles’ availability in the environment.Lizards selected prey items nonrandomly at both the low‐ , but δ13C was higher for lizards from the low‐elevation site compared to the high‐elevation site , indicating differences in the composition of the primary producers between the two sites. Variances in δ15N and δ13C did not differ between sites . Lizards at both sites fed at a similar trophic level  and random (available) locations at the low‐elevation site and 100 pairs of locations at the high‐elevation site. At both sites, bare ground made up the highest percentage of available ground cover types , followed by grass and forbs at the low‐elevation site (6%) and rocks at the high‐elevation site (8%). The percentage of sampled available locations at each site that was sagebrush was only 10%. However, sagebrush constituted 46% and 44% of ground cover types used by lizards at the low‐ and high‐elevation sites, respectively Figure . Overalldf = 6, χ2 = 28.43, p < 0.001) and high‐elevation  sites. However, ground cover selection did not differ between the two sites after controlling for differences in availability of ground cover types between the sites . At both sites, lizards showed selection for sagebrush and against all other ground cover types.Lizards selected ground cover types non‐randomly at both the low‐ (3.4There was a wider range of shade cover available at the high‐elevation site than at the low‐elevation site. Locations with 21%–40% shade cover made up the highest proportion of sampled available locations at the low‐elevation site (41%), followed by locations with 0%–20% shade cover (34%). No locations at the low‐elevation site had 81%–100% shade cover, and locations with 61%–80% shade cover made up only 4% of sampled available points at the low‐elevation site. In contrast, at the high‐elevation site, the highest proportion of sampled available points had 41%–60% shade cover (32%), followed by 21%–40% shade cover (28%). Higher amounts of shade cover were more commonly available at the high‐elevation site, with 21% of sampled available points including >60% shade cover, including 2% of locations with 81%–100% shade cover . Lizards at both sites tended to select for greater amounts of shade cover, with lizards at the low‐elevation site selecting for 61%–80% shade cover and against other amounts; high‐elevation lizards selected for 41%–60% and 81%–100% shade cover, were neutral with respect to 21%–40% shade, and selected against 0%–20% and 61%–80% shade. Overall, lizard selection of shade cover did not differ between the low‐ and high‐elevation sites . In contrast, the high‐elevation site had a broader range of available microhabitats, in terms of both ground cover types and range of shade cover, and therefore was a more heterogeneous environment for microhabitat, than the low‐elevation site. However, the two lizard populations demonstrated a similar degree of generalization for amount of shade cover and a similar degree of specialization for ground cover type , as indicated by the populations’ similar niche breadths for both axes of microhabitat.P. hernandesi climbing up into low branches of sagebrush plants to bask (Refsnider et al., P. hernandesi in other shrub or bush species, likely because their lowest branches are too high off the ground for horned lizards, a primarily terrestrial clade, to access. In contrast to lizards’ strong selection for a particular ground cover type, we found that lizards at both the low‐ and high‐elevation sites used shade cover in accordance with its availability. Thus, lizards did not exhibit specialization for a particular range of shade cover at either study site. Previous research has demonstrated that lizards thermoregulate more effectively when their preferred microhabitats are dispersed rather than clumped in space (Sears et al., While we found greater heterogeneity in ground cover types at the high‐elevation site, lizards in both populations showed strong selection for sagebrush, demonstrating specialization for this particular type of ground cover. At a very fine spatial scale, sagebrush provides a variety of microhabitats: the stiff and dense branches provide cover and protection against predators, while small open patches between branches allow lizards to bask in full sun while still sheltering within the bush itself. We have occasionally observed It is not uncommon for populations that have undergone ecological release to evolve greater generalization in resource use, often resulting from relaxation of competition and/or predation pressure (e.g., Bolnick et al., Paramecium raised under fluctuating temperature environments evolved into generalists, and produced more offspring, than the specialist lineages that evolved under constant temperature environments (Duncan, Fellous, Quillery, & Kaltz, In support of the hypothesis that generalists are favored over specialists in spatially or temporally variable habitats, several studies have found that more generalist populations that evolved under fluctuating environmental conditions had reproductive success equal to or greater than specialists under a wide range of conditions (Condon et al., Niche theory predicts that heterogeneous environments should favor generalists, while more homogeneous environments will favor specialists. We found that, despite differences in prey and microhabitat heterogeneity at two different sites, prey and microhabitat selection by horned lizards did not consistently differ between populations at those sites. Moreover, environmental heterogeneity was not associated with generalization of lizards’ resource use. Our results demonstrate that environmental heterogeneity of a particular resource does not necessarily predict the degree to which organisms specialize on that resource. Future research should consider whether magnitude of plasticity for resource use, rather than resource use itself, is correlated with environmental heterogeneity.None declared.JMR conceived the study. JMR, SEC, GRK, ADS, and HMS carried out field data collection. JMR, SEC, and ADS analyzed the data. JMR wrote the manuscript.


Introduction: Cardiac resynchronization therapy (CRT) has emerged as one of the few effective treatments for heart failure. However, up to 50% of patients derive no benefit. Suboptimal left ventricle (LV) lead position is a potential cause of poor outcomes while targeted lead deployment has been associated with enhanced response rates. Image-fusion guidance systems represent a novel approach to CRT delivery, allowing physicians to both accurately track and target a specific location during LV lead deployment.Areas covered: This review will provide a comprehensive evaluation of how to define the optimal pacing site. We will evaluate the evidence for delivering targeted LV stimulation at sites displaying favorable viability or advantageous mechanical or electrical properties. Finally, we will evaluate several emerging image-fusion guidance systems which aim to facilitate optimal site selection during CRT.Expert commentary: Targeted LV lead deployment is associated with reductions in morbidity and mortality. Assessment of tissue characterization and electrical latency are critical and can be achieved in a number of ways. Ultimately, the constraints of coronary sinus anatomy have forced the exploration of novel means of delivering CRT including endocardial pacing which hold promise for the future of CRT delivery. Howev2.max), from a baseline control state . GrOther non-invasive imaging techniques which may become increasingly adopted are ECGI and Holographic imaging systems. ECGI allows an assessment of tissue characterization and can be used to locate areas exhibiting late electrical activation. Refinement of body surface mapping systems now means that instead of multiple electrode vests, a more straightforward ECG belt  can deriTransvenous, epicardial CRT remains an excellent therapy for those patients with right substrate and the right anatomy allowing this substrate to be corrected.The constraints of CS anatomy have forced the exploration of novel means of performing CRT and of these endocardial CRT holds the most promise.Endocardial CRT has several advantages over epicardial CRT, but the optimal endocardial site displays marked variability, and in order to maximize benefits, site selection is critical.Assessment of tissue characterization is essential as viable myocardium should always be targeted. This can be achieved in a number of different ways.There is conflicting evidence as to whether an assessment of mechanical dyssynchrony can aid in patient selection for CRT over and above current guideline indications, however, targeting sites exhibiting late mechanical activation appears useful.Targeting late electrical activation also appears a promising strategy. Q-LV and LVLED appear very useful when looking to identify the optimal site. The site of latest activation is not consistently associated with the optimal hemodynamic improvement. Instead, a position which displays late but not excessively late activation appears the most beneficial.Narrowing of the QRS during pacing appears a less reliable marker of the optimal pacing site.Optimizing the RV pacing site may be of benefit, especially when revising a previously implanted system where the position of the LV lead is relatively fixed.LV-only pacing appears promising, particularly in those with preserved LV function. New delivery technology means both of these systems can be implanted transvenously.Site selection is only as good as the adjunctive guidance system. Guidance systems which integrate pre-procedural imaging with peri-procedural CS balloon venography are unable to determine whether a suitable tributary of the CS subtends the target segment until the procedure has begun. This is a limitation of the transvenous epicardial approach. Systems which can directly visualize the CS during pre-procedural planning allow decisions to be made regarding the most suitable method of targeting this area.


In combination, the breakaway of icebergs  and submarine melting at marine-terminating glaciers account for between one third and one half of the mass annually discharged from the Greenland Ice Sheet into the ocean. These ice losses are increasing due to glacier acceleration and retreat, largely in response to increased heat flux from the oceans. Behaviour of Greenland’s marine-terminating (‘tidewater’) glaciers is strongly influenced by fjord bathymetry, particularly the presence of ‘pinning points’  and over-deepened basins (that encourage rapid retreat). Despite the importance of calving and submarine melting and significant advances in monitoring and understanding key processes, it is not yet possible to predict the tidewater glacier response to climatic and oceanic forcing with any confidence. The simple calving laws required for ice-sheet models do not adequately represent the complexity of calving processes. New detailed process models, however, are increasing our understanding of the key processes and are guiding the design of improved calving laws. There is thus some prospect of reaching the elusive goal of accurately predicting future tidewater glacier behaviour and associated rates of sea-level rise. Calvingablation  and are ablation ), but itice loss .Submarine melting and calving processes are not independent and are not simply additive. Melting of the submerged portion of an ice front can trigger calving events, which might be several times larger than the original melted cavity , 6. In aIn recent years, there has been significant progress in understanding processes of frontal ablation and their environmental controls. Major advances have been made in observations of ice dynamics, fjord-water characteristics, processes and patterns of submarine melting, and the physics of iceberg calving . The incDynamic ice loss is a major factor in the mass balance of the Greenland Ice Sheet. In 2000, the total discharge from all of Greenland’s tidewater outlet glaciers was 462 ± 6 Gt . FifteenTaken together, dynamic ice losses and runoff of surface meltwater exceed snow accumulation over the Greenland Ice Sheet, so the overall mass balance of the ice sheet is negative. Analysis of surface elevation and mass changes from CryoSat-2 and GRACE satellite data indicates that the Greenland Ice Sheet had a net annual mass balance of − 269 ± 51 Gt/year from Jan 2011–Dec 2014 [It has only recently become possible to quantify the dynamic contribution to mass loss from the whole Greenland Ice Sheet, and it is unwise to infer long-term trends from short-term data. However, long-term records of iceberg losses can be determined from monthly iceberg counts by the US Coastguard’s International Ice Patrol, which record all bergs greater than 5 m in above-water length south of a line extending along 48° N from the Newfoundland coast to approximately 40° W. The data (beginning in 1900) display large inter-annual variability, but clearly show much higher overall iceberg frequencies since the late twentieth century than in earlier periods , 16.Increasing dynamic ice loss from the Greenland Ice Sheet reflects a combination of increased ice velocities and ice-front retreat , 17–19. The overall trend of glacier acceleration and retreat masks considerable regional variation . Greenlafloating ice tongues, while others rest on the sea floor forming grounded ice fronts. Both of these conditions may occur on different parts of the same glacier front, while other glaciers may undergo temporal transitions between grounded and buoyant states. Research into processes of frontal ablation in Greenland has focused on two main mechanisms: submarine melting of ice fronts  and buoyancy-driven calving.Greenland tidewater glaciers have variable characteristics, with important implications for processes of frontal ablation. Some glacier termini are fully buoyant forming Observations have revealed the presence of warm subsurface waters of Atlantic origin in the upper reaches of Greenland’s deep fjords , 34, 35,Recently, new insights into spatial variability in submarine melting have emerged from sonar imaging of several glacier calving fronts in West Greenland , 44, 45.These findings indicate that melt-undercutting is sensitive to ocean temperature, glacier runoff, and the morphology of the subglacial hydrological system. Runoff input from a single channel produces a small zone of highly concentrated melting, whereas distributing runoff more evenly across the width of the glacier decreases the maximum local melt rates but can increase the total submarine melting of the calving front by a factor of 5 , 52. ThiWhile it is clear that melting below the waterline preconditions calving events, the precise relationship between melt-undercutting and calving remains unclear. Undercutting of grounded glacier fronts removes support from the overlying ice, potentially triggering calving events that may be smaller or larger than the submarine cavity. If calving events are consistently smaller than the cavity, long-term frontal ablation rates will simply be determined by the submarine melt rate. On the other hand, stress migration effects may trigger calving events larger than the submarine cavity, in which case long-term frontal ablation rates will be greater than the submarine melt rate , 6. In a2 in area, respectively. In the years preceding the first event, the floating ice tongue thinned by 5 m/year, largely because of basal melting [The impact of submarine melting also depends on glaciological factors such as the degree of buoyancy and rates of ice flow. In general, the impact of melt-undercutting should be greatest on well-grounded glaciers, because glacier fronts close to buoyancy are largely supported by water pressure and should be less affected by the loss of ice at the base. However, submarine melting is clearly implicated in calving and break-up of floating glacier tongues in Greenland. For example, the loss of the 15 km floating ice tongue of Jakobshavn Isbrae after 2001 was preceded by a 1 °C increase in ocean temperatures, enough to increase submarine melting by 25% , 57. At  melting . This th melting  noted th melting .If other factors are equal, melt-undercutting will likely have a larger impact on glacier evolution if submarine melt rates exceed rates of ice flow. However, even fast-flowing glaciers can be significantly impacted by submarine melting if, for example, loss of a floating ice tongue forces the glacier into a different dynamical regime .Buoyancy-driven calving is an important process on large, fast-flowing outlet glaciers [glaciers –64. Rapiglaciers , 61. Geoglaciers , 60, 64.glaciers –67. Detaglaciers  show thaBoth buoyancy-driven calving and melt-undercutting can occur on different parts of the same glacier front. Analysis of Extreme Ice Survey time-lapse camera data on Rink Glacier by Medrzycka et al.  has showIce-front behaviour is also strongly influenced by the presence of mélange, a mixture of calved icebergs and sea ice. In winter, when sea ice is well frozen, mélange forms a strong, rigid mass, whereas in summer, calved bergs have greater freedom to move relative to one another. Howat et al.  and Moonlocation based on theoretical crevasse penetration depths or yield criteria [rates from empirical functions . Ni. Nilocatns , vertically integrated models struggle to capture the effects of processes that may be key controls of tidewater glacier behaviour. For example, the effects of melt-undercutting on frontal ablation cannot be represented explicitly in vertically integrated models and must be parameterized by applying uniform melt rates to the glacier front e.g. , 29, 79), 7929, 7These problems have been overcome by the development of efficient calving routines in the ‘full-Stokes’ finite element model Elmer/Ice in both two and three dimensions , 77. Thediscrete element models to modelling frontal ablation processes [Another major breakthrough is the application of rocesses –83. Discrocesses . Such morocesses , 84. By These high-resolution models are not currently practical at the ice-sheet scale or for long-term simulations, due to their very high computational cost. However, they offer valuable insights into frontal ablation processes, which can guide the development of the simple calving parameterizations required for ice-sheet models . ExperimIn the last few years, significant advances have been made in understanding frontal ablation of Greenland tidewater glaciers, reflecting both theoretical advances and intensive development of new monitoring and modelling techniques e.g. , 81, 85), 8581, 8The main frontal ablation processes are now reasonably well known, at least in outline, but continued research effort will be required to obtain a comprehensive, fully quantitative understanding of the key processes and their controls. In particular, concurrent data on fjord circulation and heat flux, glacier hydrology and plume dynamics, mélange characteristics, melt-undercutting, and calving are needed to establish the links between ocean forcing and glacier response. The difficulty of obtaining representative data for model initialization and validation is exacerbated by the high spatial and temporal variability of both glaciological and oceanographic conditions around Greenland. Thanks to major projects such as Operation Icebridge, Oceans Melting Greenland (OMG), and other programs e.g. , 29, 88), 8829, 8In terms of numerical modelling, the current research effort is focused on two complementary problems: (1) the refinement and application of detailed process models  and (2) High-fidelity process models are very different in conception from the simple calving laws needed for ice-sheet models. Convergence of these two contrasting approaches, however, offers the prospect of reaching the elusive goal of accurately predicting future tidewater glacier behaviour and associated rates of sea-level rise.


Analysis of calcium sparks in cardiomyocytes can provide valuable information about functional changes of calcium handling in health and disease. As a part of the calcium sparks analysis, sparks detection and characterization is necessary. Here, we describe a new open-source platform for automatic calcium sparks detection from line scan confocal images. The developed software is tailored for detecting only calcium sparks, allowing us to design a graphical user interface specifically for this task. The software enables detecting sparks automatically as well as adding, removing, or adjusting regions of interest marking each spark. The results of the analysis are stored in an SQL database, allowing simple integration with statistical tools. We have analyzed the performance of the algorithm using a large set of synthetic images with varying spark sizes and noise levels and also compared the analysis results with results obtained by software established in the field. The use of our software is illustrated by an analysis of the effect of isoprenaline (ISO) on spark frequency, amplitude, and spatial and temporal characteristics. For that, cardiomyocytes from C57BL/6 mice were used. We demonstrated an increase in spark frequency, tendency of having larger spark amplitudes, sparks with a longer duration, and occurrence of multiple sparks from the same site in the presence of ISO. We also show that the duration and the width of sparks with the same amplitude were similar in the absence and presence of ISO. The software was released as an open source repository and is available for free use and collaborative development. This change is mainly caused by the opening of L-type Can the SR . Ca2+ reted cell . These ed signal . It has iewed in . Therefo2+ sparks per second per cell  specifically for this task . Depending on the studied cells, expected spark sizes, the distance between knots can be changed by the user. Note that the distance between knots should be considerably larger than the expected spark duration to avoid interpolation of the background influencing the spark parameters.During each iteration, intensity changes in the regions without sparks were fitted by a third order one-dimensional spline with the user-prescribed knots placement, 5 s apart by default, one spline fit per each location in the cell . With the spark regions detected, the image without them was used to make the next F0 estimation. The iteration continued until convergence or for a given number of iterations.For spark detection, the image was corrected by subtracting sses see . This cosses see . After cAfter estimation of the background, sparks are automatically detected using the smoothed image with the corrected mean and SD using the criteria specified above. The automatic detection determines the regions of interest (ROI) and the spark peak location, one per spark. Fluorescence changes, as on the image with the subtracted background, within each ROI, are analyzed by fitting temporal and spatial changes in fluorescence for data crossing the peak by a spline. Each automatically detected spark is assumed to be acceptable by default. The program, however, allows the user to go through all sparks and visually inspect each ROI as well as the quality of the fluorescence changes fits. In some cases, spline parameters, ROI location, and dimensions have to be changed to obtain a better fit. In addition, the user may deem necessary to delete an automatically detected spark. Such a review of the fit is an iterative process for each spark until the user is satisfied with the fit and detected sparks.The analysis results are stored in an SQL database. The software supports storing data in a central (PostgreSQL) or a local database (SQLite). From the local database, users can either transfer the data into some central database, analyze using local database tools, or export in spreadsheet form.The database schema was designed for simple integration into larger laboratory databases by allowing to link sparks recordings with the cardiomyocyte preparation, animals used, and other data. In our practice, we link the experiments with the description of cardiomyocyte isolation data allowing us to study sparks properties in relation to the animal properties. Each spark is marked by a unique ID, allowing to store the data from all experiments in the same database.https://gitlab.com/iocbio/sparks). The program is written in Python 3, using, among others, the following packages: numpy , τr and τd are the time constants ). For all sparks, spatial and time constants were the same: ξ = 1.5 μm, τr = 7.0 ms, and τd = 18.0 ms. For the amplitude R, we used values from range 0.05–0.8 with the step of 0.05 and, additionally, values 1.0, 1.25, 1.5, 2.0 .F0 levels . In the experiments, sparks were added to an experiment at random locations. Due to the quantum nature of light, the number of detected photons in a confocal microscope is a Poisson process  and were performed for ∼60 s  with the spark frequency of 1.5 sparks/(s · 100 μm) resulting 61 sparks per a synthetic experiment. Experiments were performed at different  process , and theFor 8-bit synthetic image preparation, the synthetic data was multiplied by a gain factorIn the analysis, we used images with the different signal-to-noise ratio (SNR). Here, SNR was defined as SNR of the background and calculated as a mean value of the background divided by the standard deviation of the background.For each synthetic image, we stored spark locations in a database as a “ground truth” reference. After that, the same algorithm, as for real experiments, was applied to detect sparks from the synthetic data. From the comparison of the spark locations ground truth with the detected spark locations, we could find the sensitivity of spark detection, the rate of the false positive detection, and the accuracy of spark morphology estimation for different conditions.For comparison of spark morphology estimation done by our program with the results of SparkMaster, we generated a reduced set of synthetic experiments. Due to a large amount of manual processing involved when performing analysis by SparkMaster and the lack of direct connection to the database, the experimental images were generated with the fixed spark amplitude allowing us to process SparkMaster results easier. For each studied condition (SNR and spark amplitude), five images were generated with a total of ∼300 sparks analyzed per each condition.C57BL/6J Ola Hsd mice were obtained from Envigo RMS B.V. (The Netherlands) at the age of 6–8 weeks. To acclimatize, they were kept for several weeks at our local animal facility with free access to water and food , an ambient temperature of 22–22.8 °C, and a 12:12 h light:dark cycle.± 6 g and a tibial length of 2.22 ± 0.07 cm.Cardiomyocytes were successfully isolated from five C57BL/six mice  208–215 days old. They had a body weight of 29 All animal procedures were approved by the Estonian National Committee for Ethics in Animal Experimentation .2O. The initial pressure and the initial flow were recorded. After the heart was washed free of blood, the perfusion was switched to a constant flow  with a digestion solution containing 0.25 mg/ml Liberase DL . The pressure, which was monitored continuously throughout the protocol, usually increased transiently. When the pressure had decreased back to the initial value, 1.36 mg/ml of dispase II (Roche) was added to the digestion solution. As the pressure decreased further, the flow was carefully increased up to 3 ml/min. The digestion was continued until the pressure had decreased to 30–40% of the initial value and the heart was soft. After perfusion, the ventricles were cut into smaller pieces, transferred to a beaker with digestion solution and incubated further at 37 °C with gentle shaking until the tissue started to dissociate. As a result, the cell suspension was a mix of isolated cardiomyocytes from the left and right ventricles. Cells were harvested with a pasteur pipette several times during this post-digestion and filtered through a 100 μm cell strainer  into a vial with sedimentation solution. Each time, additional digestion solution was added to the undigested cells. The viable cells were separated by sedimentation or by centrifugation for 2 min at 300 rpm/12 g in an Eppendorf 5810R centrifuge with an F-34-6-38 rotor . First, extracellular Ca2+ was gradually increased to two mM to ensure Ca2+ tolerance of the cells. After this, extracellular Ca2+ was washed out again by rinsing the cells three times with five ml of sedimentation solution. The isolated cells were stored in this solution at room temperature until use.Cardiomyocytes were isolated using a slightly modified version of a method described previously . The mic2PO4, 4.4 NaHCO3, 1.7 MgCl2, 21 HEPES, 20 taurine, 11.7 glucose and 10 2,3-butanedione monoxime. pH was adjusted to 7.4 with NaOH.The wash solution consisted of (in mM) 117 NaCl, 5.7 KCl, 1.5 KHFor the digestion solution, 0.25 mg/ml Liberase DL (Roche) and 1.36 mg/ml Dispase II (Roche) were added to 20 ml of the wash solution.For the sedimentation solution, two mM pyruvate, 10 μM leupeptin (Roche), two μM soybean trypsin inhibitor, and three mg/ml BSA (Roche) were added to 40 ml of the wash solution.2PO4, 1 MgCl2, 1.13 CaCl2, 10 glucose and 10 HEPES. pH was adjusted to 7.4 with NaOH.For the microscopy experiments following solution was used (in mM): 150 NaCl, 5.4 KCl, 0.33 NaHAll chemicals were obtained from Sigma-Aldrich  if not mentioned otherwise.The experiments were performed by initially pacing cardiomyocytes at one Hz either in the presence (50 nM) or absence of isoprenaline (ISO) for about 5 min at room temperature of 25 °C. A contracting cardiomyocyte, responding to each stimulation pulse and without spontaneous activation, was selected. Before experiment on the selected cardiomyocyte, an image was recorded with the background to obtain background intensity estimate. Then, confocal imaging was started while the cell was still being paced. After a few seconds of imaging, pacing was stopped and the occurrence of sparks was recorded. For each experiment, a linescan image was acquired. After an experiment on one (80% of cases) or two cells, the cells were replaced with a new batch.Fluorescent labeling was started 15–20 min before experiments, each batch separately labeled just before placing the cells into the measurement chamber under the microscope. For labeling, five μM of Fluo-4 AM  was used. One mM Fluo-4 AM stock solution was prepared by diluting 50 μg in DMSO containing 20% (w/v) of Pluronic F-127 .xt images were made to cover a line of ∼80 μm  along the cell during ∼90 s . The imaging was performed in RC-21BRFS chamber  with a coverslip on the bottom only. The chamber was connected with Stimulator C Type 224  to pace the cells at one Hz using square pulses . Fluo-4 was excited by Argon laser at 488 nm (about 1% of the power) with the fluorescence collected through a long-pass 505 nm filter after passing 488 nm dichroic.Confocal images were acquired on an inverted confocal microscope Zeiss LSM510 Duo built around Axio Observer Z1 with a 63×water-immersion objective (1.2 NA) at room temperature. The signal was acquired via high-voltage single PMT using 8-bit mode; the pinhole was set to one Airy disk. Line scans were performed unidirectionally. ± SD. Statistical tests were performed using JASP  images. Pixel dimensions in space (x) and time (t) coordinates are scaled separately, allowing the user to get an overview of the experiment as well as zoom into a region of interest through scrolling mouse wheel on the overall image or its axes. Average intensity changes in time are shown on the top together with intensity histograms for the whole experiment and the region selected for sparks detection, allowing a quick assessment of the experiment.The GUI gives an overview of the experiment as well as allows to examine each spark individually . At the The analysis of the experiment starts with the definition of experiment stages. The stages are defined by the time interval and cover the whole image in the spatial coordinate. The first stage is the one that will be analyzed for sparks. The other stages can be defined by the user for the assistance in statistical analysis of the experiment. In our case, we field-stimulated the cells in the first few seconds of the experiment allowing us to follow calcium transients during normal EC coupling. For each user-defined stage, mean, minimal, maximal, and values corresponding to 0.1% and 99.9% percentiles were found. The percentile values are useful to estimate the range of the signal changes without being overly sensitive to one spurious data-point, as minimal and maximal values are.When analyzing new experiment, the user has to define the experimental stage in which sparks are detected and, under “Image analysis parameters” subsection of “Parameters,” define the background intensity. The background intensity corresponds to image counts recorded in the absence of fluorescence. The background intensity is determined by the detector only and should be measured with the same detector settings, such as gain, pixel time, and offset, as during the experiments in the absence of illumination. In practice, we recorded a separate image before performing the sparks experiment and used it to obtain the background intensity. As soon as the sparks detection experiment stage and background intensity are defined, the user can start the automatic detection process by pressing “Detect.” At first, the image is normalized by finding slowly changing background fluorescence for each point of the cell. The calculated background fluorescence is available under “F0” tab in the image view. Next, the variability of the noise induced by uneven background fluorescence is accounted for with the result shown under “Corrected” image tab, the corrected image blurred (“Medblu” image tab), and, sparks detected (“Filtered” image tab). Example images corresponding to the different stages of the analysis are shown in With the sparks regions detected, whether earlier and loaded from the database or after automatic detection, properties of each spark are calculated. The user can examine each spark by either selecting it in the full experiment overview by hovering a cursor over it or using a list of sparks on the bottom left. The user can adjust the region for each spark by either moving it or changing the size in the experiment overview. In practice, we are zooming in into the region with the spark, changing the region, and zooming out. The regions can also be removed by the right click of the mouse either on the sparks list or experiment overview. To create a new sparks region, the user can click and hold the right mouse button and select it via dragging the pointer in the experiment overview.2+ release, for example. This can be done when selecting the corresponding spark region, changing the corresponding parameter, and pressing Enter on the keyboard to initiate a new fit.The default parameters for sparks intensity fitting can be provided by the user under “Parameters” in the “Spark analysis parameters” sub-section. Sometimes, one has to adjust the fitting parameters for some individual spark region, due to the longer duration of CaWhen analyzing an individual spark by selecting its region, the corresponding original image, corrected, and blurred corrected images at the bottom left of the GUI are shown. These images also show the detected spark surrounded by red contour and the main axes used to determine spatial and temporal characteristics of the spark. Below, the temporal and spatial variation of intensity along the principal axes is shown together with the spline fits allowing the user to access the quality of the fit with the current fit parameters  to 1 × F0 (SNR = 1.4).Similar to the effect observed in F0 to 0.6 × F0. Note that between these amplitudes, the cumulative frequency of sparks below given amplitude changes the most for simulations with SNR equal to 2  and related it to the estimated spark amplitude . This isr2 = 0.82). Most of the sparks found by only one of the programs were with the small amplitude, as shown in We compared the performance of our software with SparkMaster’s  analysis± 0.08 spark/(s · 100 μm), mean ± sem (n = 5). For comparison, our algorithm at the same conditions, had a false positives detection rate below 0.07 spark/(s · 100 μm)  . Thus, iUsing the same synthetic data as for evaluation of spark detection algorithm, we looked into estimated spark amplitude and its temporal (FDHM) and spatial (FWHM) characteristics. As shown in With the increase of noise level, the estimated spark amplitude is somewhat higher than the “true” value. This is a side effect induced by the freedom of the algorithm to select the maximum from detected spark region as a spark amplitude. Indeed, in the noisy image, one can imagine that there could be regional increases in fluorescence in the neighborhood of the spark maximum. If such region exists, it will be selected as a spark maximum and the amplitude will be estimated using it. As a result, a bias towards higher amplitudes is an expected outcome of the algorithm and plays a bigger role with the increase of the noise.Full width at half maximum and FDHM follow the trends that are similar for the both of them. Estimation of these parameters has a better precision for sparks with the larger amplitudes and measured in the conditions of lower noise. Increase of the noise and reduction of the amplitude lead to underestimation of the spatial and temporal spread of the spark.F0). Whether it is due to overestimation of the image background or some other reason is not clear to us.For comparison, we analyzed a smaller set of synthetic data by SparkMaster. The results are shown in F0 was 2.6 ± 0.9 (n = 16 cells), in the presence of ISO, the amplitude was significantly larger at 7.4 ± 1.8 . However, note that F0 could correspond to the different basal calcium values in control when compared with the measurements in the presence of ISO. So, one should be aware of this normalization while making the comparisons.To illustrate the use of the software, we analyzed the effect of ISO in the solution surrounding the cardiomyocytes. As expected, in the presence of ISO, the calcium transient during external pacing was much larger, as evidenced by changes in Fluo-4 labeled fluorescence during each beat. While in control, the amplitude of fluorescence change normalized to F0, we found the frequency of sparks with the amplitude larger than 1 × F0. As shown in F0-normalized amplitudes of 0.5, 0.75, 1.0, 1.25, and 1.5 were compared using classical repeated measures ANOVA, the difference was statistically significant (p < 0.001). Very strong evidence for the statistical difference between control and ISO cases was confirmed by Bayesian repeated measures ANOVA, with Bayes Factor BF10 > 70, very strongly suggesting effects of ISO (BF10 > 70) and its interaction with the cut-off amplitude (BF10 > 100). For comparison, the sparks detected by SparkMaster were analyzed in the same manner and we observed strong effect of ISO on increasing the spark frequency  and interaction between ISO and the cut-off amplitude .As shown in the representative experiments , sparks p < 0.005). The strong evidence for the difference between ISO and control cases (BF10 > 100) was attributed to the effect of ISO (BF10 > 15) and interaction between amplitude and ISO treatment (BF10 > 100).The sparks with longer duration were analyzed by comparing the frequencies at different cut-off amplitudes. Here, we looked at the sparks with the FDHM that was larger than 25 ms. A total of 25 ms was selected as a representative longer spark duration, as indicated by the analysis of FDHM for larger sparks (described below). As shown in p < 0.05) and had a tendency toward larger frequencies in occurrence of three-spark groups in the presence of ISO when compared to control (p = 0.1). When analyzed using Bayesian repeated measures ANOVA, the very strong evidence for the difference between frequencies of two-spark groups was found, with BF10 > 100, attributed to the interaction between amplitude and ISO treatment (BF10 > 100). Thus, our analysis confirmed the higher chance of occurrence of multiple sparks in the vicinity of the same location.As we observed sparks at the same location occurring close to each other in the presence of ISO, we analyzed the frequency of spark groups consisting of at least two or three sparks within given temporal and spatial limitations: less than 2 s between consecutive sparks and within one μm of the location of the first spark. The frequencies were higher in the presence of ISO as shown in n = 1,063) than in control , as tested by Welch test (p < 0.001). A same statistically significant increase in average spark amplitude was observed when the sparks detected by SparkMaster were analyzed .From the analysis of the overall distribution of spark amplitudes , it is cF0 to 1.2 × F0. When comparing the probability densities for FDHM and FWHM between sparks detected in control and in the presence of ISO, we observed that the probability densities were very similar . The performance of our software depended strongly on used spark profile fitting splines, with the monotonic splines, while fitting data better, were considerably slower than non-monotonic ones.The software was designed to assist researchers in the analysis of sparks recording experiments. All the steps required for the primary analysis of the data are easily available. For control over the overall quality of the experiment, the full experiment overview is presented to quickly determine such aspects as a movement of the cell during an experiment. As a part of the overall experiment quality assessment, the displayed intensity profile allows to check whether the overall calcium concentration was stable during an experiment, and, through shown histograms, whether imaging parameters were selected correctly. The experiment analysis is streamlined and has been partitioned into overall image normalization, spark detection, and spark analysis stages. Therefore, the researcher has full control over each of the analysis steps ensuring the image is appropriately normalized, sparks are detected, and, for each spark, the temporal and spatial fits have been obtained.By developing a dedicated program, we were not restricted in a selection of user interface elements and interaction between them. As a result, we could link overview of the experiment, spark regions in the overview images, and spark analysis data, allowing the researcher to assess the spark measurements in isolation or together with the surroundings. This all should assist the analysis and, as a result, lead to a better quality of the results.As an analysis results storage media, we selected SQL database. Through SQL database, it is easy to access and analyze the data as well as export it into spreadsheet format, if needed. At present, there are two databases supported: PostgreSQL and SQLite. For the research teams that are not routinely using databases for data analysis, SQLite database does not require any installation and will be created by the program on the first run. Data can be shared then either in the form of the database file or through spreadsheets. When the central PostgreSQL database is available, the data analysis results can be directly stored into it. Such storage in the central database, combined with the central storage of experiment images, has many advantages and we would like to recommend this approach to others. In particular, by storing the data centrally, the experiments and the analysis is accessible to everyone in the research team, as set by the database permissions. Since each experiment and each spark is assigned a unique ID, the spark analysis results can be related to all other experiments done on the same cardiomyocyte preparation, or animal phenotype, or any other aspect, such as details of cardiomyocyte isolation. With the development of the large set of data exploration tools, the analysis of such relationships could lead to discoveries on the basis of recorded data.The software was released together with its source code under an open-source license—version 3 of the GNU General Public License. This license clearly defines the usage of the software as well as its further development. The license has been selected to allow interested research teams to develop the software further and to be ensured that their work will be kept as open. In addition to the open-source license for our software, we selected only open-source software as a requirement for it. This allows using the software without any external environment with the limited availability, such as MATLAB or IDL, platforms of choice for some other spark analysis programs . As a reThe software was developed in an open manner with the code released through the version control repository management system. This makes all changes in the code documented and traceable, an important requirement in ensuring reproducibility of the research results obtained with this software.There are some limitations of the current version of the software, and the most important ones are addressed below.Sometimes the same spark is detected multiple times. This happens when a smaller spark is detected next to the larger one with the corresponding regions of the secondary analysis overlapping each other. This is a drawback of the used algorithm for the secondary analysis of the detected spark and can be handled either by the user through correction of the region of analysis for the smaller spark or removing the region of analysis. The latter approach would reduce the number of detected sparks, but, taking into account the relatively high frequency of smaller sparks, should not significantly impact the data analysis results.The algorithm was developed and tested for spark detection in the part of the recordings were the cells were unpaced. To extend it to the paced cells, we expect that the background subtraction algorithm would have to be enhanced. At present, with the fast changes in overall calcium-induced fluorescence during pacing, the estimated background tends to fit the data relatively poorly, probably due to the spline used for the background estimation.2+ waves occur during the experiments. Considering that the algorithm is developed to take into account a slowly changing background, the same limits, as mentioned above regarding pacing phase, are imposed. However, it is possible to handle occasional Ca2+ waves in the experiments by using relatively large interval between nodes of the spline approximating the image background. In this case, recordings during Ca2+ wave will be not used for the background estimation and will be detected as a large spark or ignored if the wave extends to at least one of the edges of the recordings. Since the software includes an opportunity to discard the incorrectly determined sparks, the area corresponding to the wave can be removed when reviewing the experimental analysis. Note that using a spline with a small interval between nodal points (with the hope of removing Ca2+ wave induced fluorescence as a background) frequently leads to the situation where the background estimation fails due to the used spline. For reference, we use 5–10 s interval between nodal points in these cases.While the cell is not paced, it is possible that CaAt present, all calculations are done through a single working thread. This is a current limitation that is enforced by the packages used to develop the software. However, the software architecture allows overcoming these limitations through separation of calculation and user interface parts by partitioning currently single-program into separate processes and utilizing one of the intra-process messaging libraries to communicate between the parts. Such development is planned for the future versions of the software and should speed up calculations notably.As an alternative to implementing parallelism in the analysis of a single experiment, one can also run multiple instances of the analysis software to analyze multiple experiments in parallel. This requires that the analysis data is stored in the database supporting parallel access well, with PostgreSQL recommended for such usage. In our practice, we have been analyzing about 6–10 experiments simultaneously which made the analysis time-limited from analysis speed by software to inserting and reviewing of the data by a researcher.As an advantage of the used open-source software license, the software limitations can be also addressed by community members and not rely only on one research team to resolve them all. We expect that such an open model would also allow using the developed software for testing other approaches for calcium sparks detection that would advance the field further.We have tested the performance of our software with the experiments done on mouse cardiomyocytes. This is a model that is established in our laboratory and, in agreement with the replacement, reduction, and refinement of animal experiments, we supplemented the tests against experimental data with the tests on synthetic images. Since the performance of software and the test results were similar to the results obtained by others , we expeAs an example test case, we looked into effect of β-adrenergic stimulation on the sparks. In agreement with others , we haveIn summary, we have developed and released an open-source calcium sparks detection program. The program was designed to streamline the primary analysis of experiments and, by the usage of the database backend, to the follow-up analysis of the data. Through the use of only open-source components, the usage of the program and its further development does not require any additional commercial licenses. By targeting only calcium spark detection and through open sourcing our work, we hope that many research teams in the field will find it useful.10.7717/peerj.6652/supp-1Supplemental Information 1Sparks and their characteristics detected for experiments performed in the control conditions.Click here for additional data file.10.7717/peerj.6652/supp-2Supplemental Information 2Sparks and their characteristics detected for experiments performed in the presence of isoprenaline.Click here for additional data file.