In HEK293T cells, GCaMP6fu revealed a 4-fold faster decay of ATP-evoked intracellular calcium transients than GCaMP6f. Stimulation of hippocampal CA1 pyramidal neurons with five action potentials fired at 100 Hz resulted in a single dendritic calcium transient with a 2-fold faster rise and 7-fold faster decay time (t1/2 of 40 ms) than GCaMP6f, indicating that tracking high frequency action potentials may be limited by calcium dynamics. We propose that the design strategy used for generating GCaMP6fu is applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators.Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6f A limiting factor in the application of GECI has been their high Ca2+ affinity and slow response kinetics23456789102+ binding to calmodulin (CaM) induces the formation of a complex with a target peptide which in turn restores the fluorescence of circularly permutated (cp) EGFP2+-induced binding of CaM to a target peptide allows the detection of Ca2+ signals also by GCaMP-related G-GECO-s1316172+ induced fluorescence enhancement to date are variants of a GCaMP6 generation termed GCaMP6 slow, GCaMP6 medium and GCaMP6 fast 2+ - CaM - RS20 peptide interaction limit achieving temporal fidelity. Typically, Ca2+ - CaM - RS20 interactions have a high affinity  as for GCaMP6s and GCaMP6f2+ dissociation rates and thus the signal decay rates are slow, 1–5 s−1 at 20 °C, comparable to that of GCaMP32+ - CaM - RS20 binding interface have produced GCaMP3- and GCaMP6f-derived probes with improved decay kinetics23t1/2 of 1 ms) and decay (t1/2 of 3 ms) kinetics (37 °C) so far is GCaMP3fastfast can serve as a template for generating fast-response CaM-based GECI.Genetically encoded Ca2+ - CaM - RS20 peptide interface to weaken their interactions. In CaM, Ca2+ binding sites are disabled by asingle-point mutation of the first Asp to Ala in each EF-hand Ca2+ binding loop. The EF-3 mutation denotes a mutant with the third Ca2+ site inactivated, EF-4 with the fourth site. A mutation termed RS-1 which substitutes the Trp residue in position 43 for Tyr in the RS20 peptide sequence, further weakens the Ca2+- CaM - peptide interaction24Kd) and fast kinetics. Although the lower brightness and dynamic range of the EF-3:4 double mutants requires improvement, kinetically they are thus promising for development of extracellular or intra-organelle Ca2+ probes. Finally, the combination of mutations EF-3 and RS-1 results in the fastest response kinetics and has given the to-date most kinetically improved probe mGCaMP3 RS-1 EF-3 (GCaMP3fast) which has Ca2+ rise and decay times of 1–3 msWe developed a strategy to generate fast-response GCaMP-type GECI by introducing specific mutations at the Cafast to the popular bright probes GCaMP6s and GCaMP6f. Here we test the hypothesis that the EF-3, EF-4 and RS-1 mutations and their combinations result in significantly faster kinetics when applied to CaM-based GECI. We have generated twelve mutant probes, six each for GCaMP6s and for GCaMP6fu) has 1 ms rise and 3 ms decay half times in vitro (37 °C). The temperature dependence of the fluorescence response kinetics of the probes is analyzed to enable predictions for physiological applications and for better understanding of the molecular mechanisms of GCaMP probes. A kinetic model is proposed for the processes through which fluorescence develops in CaM-based GCaMP-s. Faster response kinetics by GCaMP6fu compared to GCaMP6f are demonstrated in ATP-stimulated cells and in neurons stimulated by high frequency action potentials.We applied the strategy used to produce GCaMP3+Ca2+) measurements revealed that the EF-4 and RS-1 EF-4 mutations had little effect on the Φ+Ca2+ of GCaMP6s and GCaMP6f. Using the reported value of 0.59 at 20 °C for GCaMP6f+Ca2+ value of 0.58 for GCaMP6s and 0.46 for GCaMP6fu. With measured molar extinction coefficients εo(497 nm) of 50294 ± 259 M−1cm−1 for Ca2+-saturated GCaMP6f and 1564 ± 1140 M−1cm−1 for Ca2+-saturated GCaMP6fu, brightness values of 29673 and 7197 M−1cm−1 were obtained for GCaMP6f and GCaMP6fu, respectively. Similarly, Ca2+-induced fluorescence increases (Fr(+Ca2+)), expressed relative to apo-GCaMP6s (Fr(−Ca2+) of 1)) of 27 for GCaMP6s and 15 for GCaMP6fFr(+Ca2+)/Fr(−Ca2+) of GCaMP6s RS-1 EF-3 and GCaMP6fu were reduced to 18 and 5, respectively First the effects of the mutations on the fluorescence brightness and dynamic range of the probes were determined. Quantum yield (Φectively . Even thKd) values of the probes. Cooperativity for Ca2+ remained high, characterised by Hill coefficient (n) values of ~2–3. Comparison of affinities at 20 °C and 37 °C revealed that Kd-s decreased with increasing temperature: for GCaMP6f, from 220 nM to 88 nM, respectively  with n of 2 and low affinity (0.2–6 mM Kd) with n of 1. The high affinity Kd decreased with temperature while the low affinity Kd increased. Moreover, while 80% of the total fluorescence enhancement of mGCaMP6f RS-1 EF-4 occurred with high affinity at 20 °C, at 37 °C, a temperature closer to those in physiological applications, the lower affinity component dominated representing 70% of the signal ). The fastest rate was measured for GCaMP6fu which, responding in an all-or-none manner to [Ca2+] rise, had a single rise t1/2 of 5 ms at 20 °C  and at 20 °C represents 30% of the fluorescence enhancement. The rate of the fast phase is essentially independent of temperature in the range of 20 °C to 30 °C, however with a diminishing amplitude of only 10% at 30 °C and no longer measurable at 37 °C. The rate of the slow phase is ~2-fold increased from 38 s−1 to 66 s−1 over the temperature range of 20 °C to 37 °C, representing the only measurable rate for GCaMP6f at 37 °C. The apparent Ca2+ response of GCaMP6f is thus slower at 37 °C than 20 °C. In contrast, the single observed limiting rate for mGCaMP6f RS-1 EF-3 (GCaMP6fu) is ~4-fold increased, to an extrapolated 546 s−1 at 37 °C, from 142 s−1 at 20 °C giving a fluorescence rise time of 1 ms.Examination of the temperature dependence of the Caat 37 °C . The fas2+ rise kinetics of GCaMP6s and mGCaMP-s as well (t1/2 12 ms) represents 90% of the signal  with a decay time t1/2 of 9.4 ± 0.2 s. In comparison, GCaMP6fu and mGCaMP6f RS-1 EF-4 both showed lower dynamic ranges (ΔF/F0 of 0.2 ± 0.02) and significantly reduced, 2.3 ± 0.04 s decay time. Faster probes GCaMP6fu and mGCaMP6f RS-1 EF-4 thus revealed that ATP-induced intracellular Ca2+ release dynamics were more rapid than reported by GCaMP6f  were 5.9 ± 0.3, 0.8 ± 0.05 and 0.5 ± 0.04 for GCaMP6f, GCaMP6fu and mGCaMP6f RS-1 EF-4, respectively, consistent with small [Ca2+] changes. Notably, GCaMP6f achieved a significantly higher ΔF/F0 value throughout the 10–100 Hz range tested compared to GCaMP6fu and mGCaMP6f RS-1 EF-4. Due to its low Kd for Ca2+, only GCaMP6f was able to detect the low post-synaptic [Ca2+] elevation evoked by 5 AP-s fired at 10 Hz. For the same reason, however, the kinetics of GCaMP6f response were the most prolonged. With their higher Kd values GCaMP6fu and mGCaMP6f RS-1 EF-4 did not show a response to stimulation up to 20–40 Hz frequency and the dynamic range remained low. The kinetic advantage of GCaMP6fu and mGCaMP6f RS-1 EF-4 became evident at stimulation at 40 Hz or greater  which has a 1 ms rise and 3 ms decay t1/2 at 37 °Cfast in cardiac myocytes revealed faster Ca2+ transients then previously indicated by slow indicatorsThe aim of this work was to reveal generally applicable modifications for the generation of fast-response GCaMP-like GECI for more accurate time resolution of Cafast retains the brightness of GCaMP3, whereas GCaMP6fu is 4-fold decreased compared to GCaMP6f. Even though GCaMP6m is significantly brighter than GCaMP3, the alignment of their crystal structures (PDB codes 3SG3 and 3WLD) reveals only subtle differences in the position of helix I and the linker region to cpEGFP3fast and GCaMP6fu, prevents Ca2+ binding to the EF-3 site (flanked by helices V and VI), which thus, rather than being in an “open” conformation as in Ca2+-bound GCaMP-s  EF-3 (D395A) mutation pair has equal effects on the kinetics of GCaMP3 and GCaMP6f, they differently affect the brightness and dynamic range of their parent variants: GCaMP3Y EF-3 23t1/2-s of GCaMP3 with 147 ms and GCaMP6f with 71 ms. However GCaMP3fastu have the fastest decay with a t1/2 of 3 ms, at 37 °C.We observed that GCaMP-type GECI have a limiting response rate that is independent of  would make GCaMP-type probes well suited for spike detection, however the current fast variants have insufficient brightness. RCaMP2 and some mGCaMP3, mG-GECO and mGEM-GECO have slower kinetics and reduced cooperativity for Ca2+ with Hill coefficients of 1–1.7 162+] changes in cells.A commonly observed property of cpEGFP- and CaM-peptide interaction-based GECI is high cooperativity for CaKd to low μM, the EF-4 single and the EF-3:4 double mutations break up the affinity into two components, one with a Kd of low μM and one in the mM range with most of the fluorescence enhancement occurring in mM Ca2+. In the kinetic modeling only high affinity sites with μM Kd are considered. As there are only two Ca2+ binding sites in the EF-3:4 mutants, the low, mM affinity Ca2+ binding is likely attributable to the mutated EF-hand 3 (necessary for high brightness and dynamic range) which in the presence of the RS20 target peptide may acquire such affinity for Ca2+. EF-3:4 double EF-hand mutant probes with mM Kd-s may be for suitable for intra-organelle and extracellular applications, after further improvements to their brightness and dynamic range. mGCaMP6s EF-3:4 is the most promising low affinity probe with a 500 μM Kd and a 19-fold Ca2+-induced fluorescence enhancement (20 °C), comparing favourably with CEPIAWhile the EF-3 single mutation raises the 2+ dynamics requires that the detection range of the probe is appropriate for the size and time course of the [Ca2+] transient. In the conditions of ionomycin stimulation, the expected peak [Ca2+] was 0.4 to 0.8 μMKd of 110 nM, 5–10-fold lower than the peak of the [Ca2+] transient, was thus expected to be mostly saturated and thus signal such a transient with close to its maximum dynamic range. However, at resting [Ca2+] of ~ 50–80 nM, there is already considerable Ca2+ binding to such a probe. As a result of the two parameters the measured ΔF/F0 of 18 for GCaMP6s in ionomycin stimulated HEK293T cells is a reasonable value.The success of monitoring intracellular Ca2+ indicators, it is evident that low Kd probes with slow response report slower intracellular Ca2+ dynamics than higher Kd probes with fast responses. GCaMP6fu and mGCaMP6f RS-1 EF-4 both showed 4-fold faster Ca2+ decay in response to ATP stimulation than GCaMP6f. By its two-fold faster response rate, in cardiac myocytes, GCaMP3fast revealed removal of Ca2+ and return to resting intracellular levels between each beat, a phenomenon not clearly resolved by GCaMP6fRegarding the kinetics of Cau are not sufficient for the detection of the post-synaptic dendritic [Ca2+] elevation evoked by a single AP. Although the kinetic properties of GCaMP6fu would allow the separation of individual AP-s even at 100 Hz, the actual Ca2+ transient reveals an integrated Ca2+ response, raising the possibility that the Ca2+ dynamics associated with AP firing limit the use of Ca2+ as an indicator of neuronal activity.It transpires that the brightness and dynamic range of GCaMP6fu with its fast rise and decay kinetics may still be useful for the detection of repeated AP-s with between-train intervals of tens rather than hundreds of milliseconds. The Ca2+ induced fluorescence rise times of GCaMP3 RS09 and GCaMP6f RS09 remained slow in physiological experiments232+ rise rates following a train of 5 AP-s in organotypic hippocampal slices reported by the GCaMP6fu and mGCaMP6f RS-1 EF-4 probes were markedly faster than GCaMP6f. When comparing the novel variant GCaMP6fu to GCaMP3fastu which has a 3-fold lower Kd (0.89 μM) compared to GCaMP3fast (2.8 μM) is more sensitive to small Ca2+ concentration changes and hence more suitable for Ca2+ imaging in neurons. Improved brightness and fluorescence dynamic range would help make GCaMP6fu with its ultrafast kinetics a useful physiological tool.Although unable to detect single AP-s, GCaMP6fGCaMP6s and GCaMP6f plasmids were a gift from Douglas Kim E. Coli BL21(DE3) Gold cells. Cells were lysed by sonication and clarified lysates were purified by a single-step GST-chromatography  in 50 mM Na+-HEPES pH 7.5, 200 mM NaCl and 10 mM glutathione, at 4 °C. Purity of the eluted fractions was determined by SDS-PAGE (6.4%–20% acrylamide/bisacrylamide gradient). Protein concentrations were measured spectroscopically at 280 nm by a Nanodrop 1000 spectrophotometer (Thermo Scientific) using an εo value of 76585 M−1cm−1 calculated from the amino acid composition for GCaMP6s, GCaMP6f and their EF-hand mutants and εo of 70710 M−1cm−1 for the RS-1 mutantsThe strategy previously developed and described for the improvement of GCaMP3 probes was followed to design, generate and purify mutants of GCaMP6s and GCaMP6f probes+ pH 7.5, 100 mM KCl, 2 mM MgCl2 with either 5 mM EGTA or 1 mM CaCl2) and the fluorescence spectra were recorded on a Fluorolog3 spectrofluorimeter (Horiba Scientific) at 20 °C. The quantum yield measured for GCaMP6f in Ca2+-saturated buffer was used as a reference (Φ = 0.59)GCaMP6f and Sprotein, respectively. Quantum yields were obtained using the following equation: Φprotein = ΦGCaMP6f × (Sprotein/SGCaMP6s).Protein concentrations were adjusted such that the absorbance at the excitation wavelength (490 nm) was between 0.001 and 0.1. A series of dilutions was prepared in a buffered solution . The pH titrations were performed by recording fluorescence spectra of 1 μM protein in Ca2+-free  or Ca2+-saturated  conditions . BAPTA was chosen over EGTA as Ca2+ chelator because of its stable affinity for Ca2+ over the pH rangeTo determine the apparent p2+] were calculated using the Ca/Mg/ATP/EGTA Calculator v1 constants from Schoenmakers’ Chelator program for experiments at 20 °C and 37 °C concentrations (concentrations in the mixing chamber). For the determination of temperature dependence of Ca2+ association rates, protein samples at 1 μM concentration were mixed as above with saturating Ca2+ .The solution containing 1 μM protein in 50 mM K+-HEPES, 100 mM KCl, 2 mM MgCl2, pH 7.5 with saturating Ca2+ (0.5 mM) was rapidly mixed (1:1) with 50 mM K+-HEPES, 100 mM KCl, 2 mM MgCl2, 10 mM EGTA pH 7.5 (concentrations in the mixing chamber).The solution containing 1 μM protein in 50 mM Kegfp initially present in the pEGFP-N1 vector was replaced by the gcamp6 genes (without the GST tag).For expression in eukaryotic cells, the GCaMP6s and GCaMP6f mutant DNAs were subcloned from pET41a into pEGFP-N1 vectors by restriction-ligation using BglII and NotI restriction enzymes and T4 DNA ligase following manufacturer’s protocol. During this process, the 2.Cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C and 5% CO2, 10 mM glucose and 2 mM CaCl2. For determination of the fluorescence dynamic range, cells were imaged before and after stimulation by 10 μM ionomycin at 20 °C. Imaging was performed with a Leica SP1 confocal microscope using a HCX ApoL 40 × 0.8 NA water immersion objective, images were acquired at 512 × 512 pixel size. Experiments were performed at least in triplicates, elliptical regions of interest (ROI) were selected and data were analysed using Leica Application Suite.Transfection of cells was carried out on 10 mm diameter coverslips with 0.6 μg DNA and 2.5 μL Lipofectamine 2000 in 100 μL OptiMem® for 1 h at 37 °C. Cells were imaged 16–24 h post-transfection in 10 mM HEPES pH 7.5, 145 mM NaCl, 2.5 mM KCl, 1 mM MgClHEK293T cells were transfected with GCaMP6f variants using Fugene HD following the manufacturer’s recommendations. Imaging was performed 16–24 h post-transfection at 37 °C (OKO lab incubation chamber) in a 35-mm glass bottom dish (MatTek) with a 3i Marianas spinning-disk confocal microscope equipped with a Zeiss AxioObserver Z1, a 40 × 1.3 NA oil immersion objective and a 3i Laserstack as excitation light source (488 nm). Emitted light was collected through a single bandpass filter (Yokogawa CSU-X filter wheel) onto a CMOS camera . Cells were stimulated with 100 μM ATP and images were collected at 1 s intervals for 40 seconds. Elliptical ROI were stacked using ImageJ program. Data obtained from 14–24 cells was plotted and analysed on GraphPad Prism 6. Fluorescence decay times were determined from single exponential fits to the data.4, 4 mM CaCl2, 1.2 mM NaH2PO4 and 11 mM glucose) saturated with a gas mixture of 95% O2/5% CO2.Sprague Dawley rats were purchased from Charles River. All experiments were conducted in accordance with the Canadian Council on Animal Care standards and guidelines, with approval of the Dalhousie University Committee on Laboratory Animals. Transverse organotypic hippocampal slices 350  μm) were made from brains of rat pups aged 6 to 8 postnatal days humanely killed by Schedule 1 procedure (anesthetic (ketamine) overdose followed by decapitation) and cultured on Millicell CM membranes for 5 to 6 days0  μm werAn upright epifluorescence microscope (Olympus BX51WI) with a 60 × 1.0 NA IR water immersion objective (Olympus) and an MRC1024MP  laser scanner with nondescanned photomultiplier tube detectors were used to view slices. Two-photon imaging was carried out 1–4 days post-transfection using a MaiTai titanium sapphire laser) at 930 nm.F/F0 / corresponding to 5 AP-s evoked by brief depolarizing current injection at frequencies between 10–100 Hz via a Multiclamp 700B amplifier . Ca2+ decay kinetics of GCaMP6 variants were determined by monitoring fluorescence changes evoked by 5 APs at 100 Hz.Action potentials were evoked and recorded in expressing CA1 pyramidal neurons using a sharp intracellular electrode (60–80 MΩ) filled with 3 M KCl in 20 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid. Line scans taken at 2 ms interval across proximal apical dendrites were collected to determine the calcium transients measured as ΔElectrophysiological and imaging data were collected and analysed with software from AxoGraph , LaserSharp (Bio-Rad Microscience) and ImageJHow to cite this article: Helassa, N. et al. Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics. Sci. Rep.6, 38276; doi: 10.1038/srep38276 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The effects of increased Ca2+ on BDNF up-regulation and neurite outgrowth remain unclear. We showed here that ES increased phosphorylation of the cAMP-response element binding protein (CREB). Blockade of Ca2+ suppressed CREB phosphorylation and neurite outgrowth. Down-regulation of phosphorylated (p)-CREB reduced BDNF transcription and neurite outgrowth triggered by ES. Furthermore, blockade of calmodulin-dependent protein kinase II (CaMKII) using the inhibitors KN93 or KN62 reduced p-CREB, and specific knockdown of the CaMKIIα or CaMKIIβ subunit was sufficient to suppress p-CREB. Recombinant BDNF or hyperforin reversed the effects of Ca2+ blockade and CaMKII knockdown. Taken together, these data establish a potential signaling pathway of Ca2+-CaMKII-CREB in neuronal activation. To our knowledge, this is the first report of the mechanisms of Ca2+-dependent BDNF transcription and neurite outgrowth triggered by ES. These findings might help further investigation of complex molecular signaling networks in ES-triggered nerve regeneration in vivo.Electrical stimulation (ES)-triggered up-regulation of brain-derived neurotrophic factor (BDNF) and neurite outgrowth in cultured rat postnatal dorsal root ganglion neurons (DRGNs) is calcium (Ca The initial rise in [Ca2+]i activates downstream CaMKII, which subsequently triggers CREB phosphorylation, thereby promoting BDNF gene expression and neurite outgrowth (schematic shown in via a CREB-independent signaling pathway. Future studies will investigate the role of CaMKK in ES-induced neurite outgrowth in neuronal cells.The current findings together with a previous report  support


We have previously shown that the stimulation of PAR1 by thrombin promotes intracellular signaling leading to RPE cell transformation, proliferation, and migration which characterize fibroproliferative eye diseases leading to blindness. Within this context, the elucidation of the mechanisms involved in PAR1 inactivation is of utmost importance. Due to the irreversible nature of PAR1 activation, its inactivation must be efficiently regulated in order to terminate signaling. Using ARPE-19 human RPE cell line, we characterized thrombin-induced [Ca+2]i increase and demonstrated the calcium-dependent activation of μ-calpain mediated by PAR1. Calpains are a family of calcium-activated cysteine proteases involved in multiple cellular processes including the internalization of membrane proteins through clathrin-coated vesicles. We demonstrated that PAR1-induced calpain activation results in the degradation of α-spectrin by calpain, essential for receptor endocytosis, and the consequent decrease in PAR1 membrane expression. Collectively, the present results identify a novel μ-calpain-dependent mechanism for PAR1 inactivation following exposure to thrombin. The serine protease thrombin activates Protease-Activated Receptors (PARs), a family of G-protein-coupled receptors (GPCRs) activated by the proteolytic cleavage of their extracellular N-terminal domain. Four members of this family have been identified: PAR1–4. The activation of Protease-Activated Receptor 1(PAR1), the prototype of this receptor family, leads to an increase in intracellular Ca The retinal pigment epithelium (RPE) is a monolayer of differentiated, quiescent cells located between the neural retina and the choroid. The RPE is the predominant component of the outer blood-retina barrier (BRB) and plays an essential role in the maintenance of the functional and structural integrity of the neural retina required for visual function. Among its functions, the RPE is involved in the transepithelial transport of nutrients, the storage and metabolism of vitamin A derivatives, the renewal of photoreceptor outer segments, and the ionic homeostasis of the subretinal space .Under pathological conditions involving the alteration of the BRB due to ocular trauma, diabetic retinopathy, retinal detachment, retinal hemorrhage, or, importantly, retinal surgery, the RPE is exposed to serum-contained growth factors and proinflammatory agents including thrombin, generated by the activation of the coagulation cascade. Thrombin has been shown to promote the release of cytokines, chemokines, and growth factors by RPE cells . This re42SFLLRN47) that acts as a tethered ligand which binds intramolecularly to the second extracellular loop of the receptor and triggers signaling. Synthetic ligands corresponding to the cleaved N-terminus can displace the tethered ligand from the binding site and fully activate PAR1 in an intermolecular mode. The coupling of PARs to GPCR Gα subunits Gq11α, G12/13α, and Giα has been linked to a wide array of physiologic responses. Particularly, PAR1 coupling to Gq11α activates phospholipase C-β (PLC-β), which catalyzes the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), leading to an increase in intracellular Ca+2 concentration ([Ca+2]i) i within the low micromolar range induces μ-calpain activation by autoproteolysis of the N-terminus, which exposes the catalytic site i increase and demonstrated that the calcium-dependent activation of μ-calpain and the subsequent degradation of α-spectrin by calpain significantly decrease PAR1 expression at the cell membrane. These findings indicate that PAR1 inactivation by internalization is controlled by specific mechanisms, distinct from those promoting RPE cell transformation, and further support a role for thrombin in this process.Using human RPE-derived ARPE-19 cell line, we analyzed thrombin-induced i was determined as previously described i was determined using fluorescence microscopy, as described in the Methods. Although thrombin-induced increase in [Ca+2]i in RPE cells has been reported i increase in RPE cells, sustained for ~3 min. [Ca2+]i increase was found to be dose-dependent from 100 pM thrombin concentration with maximum stimulation attained at 10 nM thrombin treatment, equivalent to calcium elevation induced by FBS (positive control). The specificity of the effect was demonstrated by inhibition upon the addition of the thrombin catalytic inhibitor PPACK (25 μM)  over time (minutes). Results in μM) induced an increase in [Ca2+]i comparable to that elicited by 10 nM thrombin.  [Ca+2]i increase induced by thrombin.In order to identify the specific receptor mediating thrombin effect, we tested the effect of PAR-APs on [Ca+2]i increase, thrombin stimulation was carried in Ca+2 free medium containing 0.25 mM EGTA. As shown in  [Ca2+]i rise. In contrast, thrombin- and PAR1 AP-induced [Ca+2]i increase was inhibited by ~80% upon the inclusion of 2 μM thapsigargin, known to deplete ER calcium stores  completely prevented thrombin-induced calpain activation. Calpain activity was abolished by the calpain endogenous inhibitor calpastatin (1 μM) and by the synthetic inhibitor PD150606 (100 μM), thus confirming the specificity of the effect  and significantly inhibited by thapsigargin  suggests that Ca2+ entry through Orai-containing or voltage-gated Ca2+ channels could account for thrombin-induced extracellular Ca2+ entry ( [Ca2+]i increase, since PAR1-AP elicited [Ca2+]i increase equivalent to that induced by thrombin ( [Ca2+]i induced by PAR4 stimulation in ARPE-19 cells .–43. i- and calpain-independent mechanisms shown to regulate PAR1 membrane expression, the N-linked glycosylation of PAR-1 second extracellular loop is known to regulate the internalization of this receptor i, the activation of calpain by calcium, and the degradation of α-spectrin by calpain. Together with the present results, the future definition of additional thrombin-activated pathways involved in the regulation of PAR1 membrane expression may provide information aimed at preventing thrombin-mediated RPE cell transformation involved in proliferative retinopathies.We here identified a novel mechanism through which thrombin activation of PAR1 autoregulates its membrane expression through the increase in


Natural killer (NK) cells play an important role in the recognition of AML blasts through the interaction of the activating NKG2D receptor with its ligands (NKG2DL: MICA/B and ULBPs1-3). However, soluble NKG2DL (sNKG2DL) can be released from the cell surface, impairing immune recognition. Here, we examined whether hypomethylating agents modulate the release of sNKG2DL from AML cells. Results demonstrated that AZA- and DAC-treated AML cells reduce the release of sNKG2DL, preventing downregulation of NKG2D receptor on the cell surface and promoting immune recognition mediated by NKG2D-NKG2DL engagement. We show that the shedding of MICA, MICB and ULBP2 is inhibited by the increased expression of TIMP3, an ADAM17 inhibitor, after DAC treatment. The TIMP3 gene is highly methylated in AML cells lines and in AML patients (25.5%), in which it is significantly associated with an adverse cytogenetic prognosis of the disease. Overall, TIMP3 could be a target of the demethylating treatments in AML patients, leading to a decrease in MICA, MICB and ULBP2 shedding and the enhancement of the lytic activity of NK cells through the immune recognition mediated by the NKG2D receptor.Acute myeloid leukemia (AML) is a disease with great morphological and genetic heterogeneity, which complicates its prognosis and treatment. The hypomethylating agents azacitidine (Vidaza AML patpatients –6. Thesepatients . Thus, k+ T cells, NK cells, γδ T cells and NKT cells [MICA, ULBP1, ULBP2 and ULBP3 genes are aberrantly hypermethylated in AML cells, and that treatment with demethylating agents increases their expression promoting recognition and cytolysis by NK cells [Natural killer (NK) cells play an important role in the innate immune response to AML through a balance between activating and inhibiting signals . NKG2D iKT cells . NKG2D rKT cells , 11. TheKT cells , whilst KT cells , 14. In KT cells . In thisNK cells . MoreoveNK cells . Some NKNK cells –23. The NK cells . Thus, tNK cells .The tissue inhibitor of metalloproteinases-3 (TIMP3), a potent inhibitor of the MMP subfamily and some ADAMs, has been associated with MICA and MICB shedding , 27. TheTIMP3 gene may be responsible for the lower level of shedding of MICA, MICB and ULBP2 in AML cells; and (v) high TIMP3 DNA methylation levels in AML patients are associated with an adverse cytogenetic prognosis for the disease. Therefore, our study reveals that hypomethylating treatments in AML cells could modulate the shedding of MICA, MICB and ULBP2 in a TIMP3 demethylation-dependent manner.In this study, we analyze the effect of hypomethylating agents on the shedding of sNKG2DL in AML cells and their consequences for NK cell-mediated immune recognition. We show that (i) AZA and DAC limit the release of all NKG2DL in the supernatants of AML cell lines; (ii) decreased levels of sNKG2DL prevent the downregulation of the NKG2D receptor and favor the recognition and lysis of AML cells by NKL cells; (iii) ADAM17 is the sheddase involved in the release of sNKG2DL in AML cell lines; (iv) demethylation of in vitro experiments were carried out with DAC alone. Moreover, as shown in Figure We determined the effect of the AZA and DAC hypomethylating agents on the release of sNKG2DL  in two AML cell lines (KG1a and NB4) that showed high levels of these soluble molecules in their cellular supernatants at basal level. AML cells were treated with DAC or AZA (1 μM or 5 μM) for 48 hours, and the presence of sNKG2DL in the cell-free supernatants was quantified by ELISA. The levels of all sNKG2DL were significantly reduced after treatment with both demethylating drugs Figure . The dowp = 0.009 for KG1a and p = 0.002 for NB4) whilst the NKG2D expression was partially restored after incubation with DAC-treated supernatants (p = 0.023 for KG1a and p = 0.011 for NB4)  .The presence of NKG2DL in soluble forms can have a dual effect: on the one hand, it down-modulates the NKG2D receptor on the surface of NKG2D-positive effector cells, and on the other hand, it damages the immune recognition mediated by cytotoxic cells by blocking the NKG2D-binding site to their NKG2DL expressed on the surface of target cells. To explore those functional consequences after DAC treatment, we first analyzed the effect of sNKG2DL on the expression of NKG2D receptor in NKL cells before and after DAC treatment. NKG2D expression was significantly weaker in the presence of supernatants from untreated AML cells  Figure . AdditioThus, our results suggest that downregulation of sNKG2DL in AML cells after DAC treatment favors NKL cell-mediated immune recognition through NKG2D-NKG2DL engagement.Proteolytic shedding of NKG2DL is mediated by different proteases in which ADAM10 and ADAM17 play a key role [p = 0.01 for KG1a and p = 0.039 for NB4) when analyzed by a fluorometric assay  works as a natural inhibitor of ADAM17 and has also been shown to block the release of sNKG2DL . First, TIMP3 gene in AML cells. To find evidence to support this hypothesis, we determined the TIMP3 DNA methylation profile by pyrosequencing before and after treating KG1a and NB4 cells with DAC. The analyzed region contains seven CpG sites within a CpG island located between positions +579 bp and +637 bp. Results show that the TIMP3 gene is highly methylated in KG1a and NB4 cells , whereas these levels were partially reduced (50% in KG1a and 52.7% in NB4) after treatment with DAC . These data suggest that demethylation of TIMP3 could be associated with their increased expression levels in AML cells after DAC treatment.These findings led us to hypothesize that epigenetic mechanisms such as DNA methylation might regulate the expression of the p = 0.001), MICB (p = 0.031) and ULBP2 (p = 0.002)  Figure . However® and Dacogen®) are currently approved for the treatment of AML patients. Decitabine is indicated for those aged 65 years or more and who are not considered candidates for induction chemotherapy, while Vidaza® is administered to AML patients as an initial regimen and to those who have relapsed. For these reasons, Vidaza® is used more widely than decitabine in AML patients with high percentage of blasts (> 20%) or myelodysplastic related changes.Two hypomethylating agents (Vidaza®. All patients showed soluble levels of two or more NKG2DL. MICB and ULBP3 were the most commonly released NKG2DL (83.3% of the patients), followed by ULBP2 (66.7%), ULBP1 (58.3%) and MICA (50%) (data not shown). When we compared the soluble levels of each NKG2DL before and after treatment, we noted a clear tendency towards the reduction of sNKG2DL after treatment although not significant except for MICB (p = 0.022)  Figure . More th® treatment. There was a clear increase in the expression of some NKG2DL on the cell surface of blasts of three patients, whilst this increase was more discrete or nonexistent in the other two patients .We were only able to obtain blasts from the bone marrow of five patients before and after Vidazas Figure . Patientin vivo effect of the treatment with hypomethylating agents on immune recognition mediated by NK cells, we carried out a cytotoxicity assay using the NKL cell line as effector and C1R-MICA transfectants as target cells, at different E:T ratios. The co-culture assay was performed in the presence of serum from two independent AML patients obtained before and after Vidaza® treatment. The specific lysis of target cells was significantly inhibited in the presence of pre-Vidaza® sera (positive sNKG2DL) at all analyzed ratios (p = 0.006 and p = 0.01 at 2.1 E:T ratio for both patients respectively), and was partially restored after co-culture in the presence of post-Vidaza® sera   whilst AML patients show highly variable methylation rates for TIMP3  . Taking a DNA methylation rate greater than 20% to be anomalous, we found that 25.5% (23/90) of the AML patients exhibited aberrant DNA methylation for TIMP3.To confirm the results previously obtained in AML cell lines, the ) Figure . ComparaTIMP3 DNA methylation and the clinical characteristics of AML patients, we classified the patients as unmethylated (TIMP3 DNA methylation levels < 20%) and methylated (TIMP3 methylation rate ≥ 20%). No statistically significant association was found with age, sex, FAB classification, AML etiology (primary or secondary) or induction therapy (data not shown). In order to analyze the genetic prognosis, AML patients were divided into three groups  based on the abnormalities described by Yohe et al. [TIMP3 and cytogenetic prognosis (p = 0.001)  than did those with a favorable or intermediate prognosis (10% each). We also observed that 80% of AML patients with trisomy 11 and 66.7% with deletions linked to TP53 gene loss showed aberrant methylation for TIMP3 (Table p = 0.002)  Figure . AML pat p = 0.00 , and enhanced their expression on the surface of KG1a and NB4 cells. Cell-free supernatants obtained from untreated AML cell lines, which show high levels of soluble NKG2DL, downregulate the expression of the NKG2D receptor, diminishing recognition and lysis by NKL cells. By contrast, supernatants from AML cells treated with DAC have lower soluble levels of these ligands, increased expression on the cell surface and thereby a higher level of recognition by cytotoxic cells.® treatment confirmed that soluble levels of all NKG2DL are diminished after treatment. Preliminary data from this study suggest that NKG2DL expression on the cell surface of AML blasts could be enhanced in proportion to the number of cycles of hypomethylating agent received, but we are aware of the limitation of this analysis due to the small number of cases involved. This is the first demonstration of hypomethylating agents controlling the release of sNKG2DL in AML cells. However, further analysis with a larger cohort of patients and samples is needed to verify the functional in vivo consequences of these treatments on the release of NKG2DL from AML blasts.Analysis of soluble NKG2DL in serum from AML patients before and after VidazaThe ADAMs family sheddases, ADAM10 and ADAM17, have been implicated in the proteolytic cleavage of NKG2DL in several types of cancer cells , 32–33. It has been reported that ADAM17 is expressed by cell lines and primary AML cells . ConsistTIMP3 gene is hypermethylated in KG1a and NB4 cells, which show high levels of sNKG2DL, but is completely unmethylated in HELR cells, which do not release soluble ligands. Thus, the TIMP3 methylation rate could help modulate the release of soluble ligands in AML. Further, AML patients (25.5%) showed aberrant DNA methylation for TIMP3 in blasts obtained from their bone marrows. TIMP3 DNA methylation has been described in several epithelial cancers [Several studies have reported diminished expression of TIMP3, a natural inhibitor of ADAM17 activity, contributing to the invasion and migration of tumor cells of various types of epithelial cancers  –39. Cons cancers , 42, but cancers , 44. Mor cancers , 45.FLT3, NPM1 and CEBPA are customarily found in AML, others have emerged in recent years that allow patients to be categorized on the basis of their evolution [TIMP3 methylation was significantly associated with an adverse cytogenetic prognosis, whilst methylated TIMP3 was less prevalent in patients with a favorable or intermediate cytogenetic risk. FMS-like tyrosine kinase 3 (FLT3) is a member of the class III receptor tyrosine kinase (RTK) expressed in hematopoietic stem cells, which are involved in processes such as proliferation and cell activation [TIMP3 is higher in FLT3-ITD (75%) than in FLT3-D835 (12.5%) mutations. These results are consistent with the poor prognosis of patients with FLT3-ITD mutations [TIMP3 could be a new biomarker that helps establish the cytogenetic prognosis of the disease.Most AML patients show some chromosomal anomality or gene mutations . Althougvolution . Here, wtivation . We fountivation , 49. Theutations . Moreoveutations –53. ThusNowadays, therapies based on alloreactive NK cell transplantation have been proposed to strengthen the immunological response of AML patients . In otheTIMP3 methylation rate could be an additional useful new prognostic biomarker of disease in AML and open the door to new therapeutic approaches by potentiating NK cell anti-leukemic potential.In summary, our study demonstrates that treatment with hypomethylating agents diminishes the release of sNKG2DL, which in turn promotes NKG2D-NKG2DL engagement. This effect is mediated by TIMP3 demethylation, which inhibits the activity of ADAM17 sheddase, leading to a more limited release of sMICA, sMICB and sULBP2 from the cell surface of leukemic blasts. Although hypomethylating drugs promote the induction of several genes during oncogenesis, the association found between aberrant DNA methylation of TIMP3 and poor prognosis for patients with the disease suggests that the 2 incubator at 37°C. Cells were cultured at a density of 0.4 × 106 cells/ml with complete media supplemented with DAC , ADAM10 inhibitor  or ADAM17 inhibitors  for 48 hours. A dose-response curve was derived for all drugs to identify the doses that did not affect cell viability (data not shown). Cell viability was determined by staining with 7AAD .The human myeloid leukemia KG1a and NB4 cells lines were purchased from the American Type Culture Collection and the Leibniz-Institute DSMZ GmbH (Germany), respectively. Cells were maintained in RPMI-1640 medium containing 10% heat-inactivated fetal calf serum in a 5% CO® after diagnosis  at the Hospital Universitario Central de Asturias . NKG2DL expression on the cell surface of blasts before and after Vidaza® treatment at different cycles was studied in a cohort of five AML patients from Hospital Clínico San Carlos . All patients and healthy donors gave their written informed consent, in accordance with the Principles of the Declaration of Helsinki. The local hospitals’ ethics committees approved the study.To analyze TIMP3 DNA methylation, bone marrow samples were obtained from 25 healthy donors and 90 AML patients at diagnosis  in Hospital Clinico Universitario Virgen de la Arrixaca and Hospital General Universitario Santa Lucía de Cartagena . Patients had been diagnosed with AML during the previous 10 years (2006–2016) according to the morphological and cytochemical criteria of the French-American-British (FAB) classification. The clinical characteristics of the patients are summarized in Table AML cell lines (KG1a and NB4) were stained with the human-specific mAb for ADAM17  or each NKG2DL: MICA , MICB , ULBP1 , ULBP2  and ULBP3 . All mAbs were from R&D Systems . Subsequently, cells were stained with FITC-goat anti-mouse immunoglobulin G  and analyzed with a Gallios™ flow cytometer . In AML patients, blasts from bone marrow were stained with CD34-PE and CD45-APC antibodies (Biolegend). The specific fluorescence index was calculated by dividing the mean fluorescence obtained with the respective specific mAb by that of the secondary antibody.Soluble NKG2DL (sNKG2DL) was detected using sandwich ELISA. Plates were coated with a specific mAb (4 μg/ml) for each NKG2DL. Coating mAbs are the same clones that we used for the previously described flow cytometry. Subsequently, plates were blocked with 2% BSA-PBS. After blocking, 100 μl of sera or supernatant samples and recombinant human (rh) Fc chimera proteins specific for each NKG2DL  were added for 2 hours at RT. After washes, a specific biotinylated polyclonal antibody for each NKG2DL (0.4 μg/ml)  was added for 2 hours at RT. Afterwards, plates were washed and incubated with streptavidin-HRP . Plates were then washed and developed using TMB  and absorbance was measured at 450 nm. The detection threshold for each ELISA was less than 400 pg/ml and as a positive control we used the recombinant proteins specific for each ligand.X, Poland) according to the manufacturer's instructions. Reverse transcription of purified RNA was performed using the High-Capacity cDNA Reverse Transcription kit . Next, quantitative gene expression analysis was performed by real-time PCR on a MyiQTM Single Color Real-Time PCR Detection System (Bio-Rad) using SYBR® Green PCR Master Mix (Applied Biosystems). Data were normalized to GAPDH RNA expression and all samples were run in triplicate. Relative mRNA abundance was determined by comparison of threshold values and calculated by the 2-ΔCT method (ΔCt: Ct gene test - Ct endogenous control). The following specific primers were used; TIMP3: fwd: 5′-TCCCAGCGCAAGGGGCTGAA-3′, rev: 5′-GCCGGATGCAGGCGTAGTGTT-3′; MICA: fwd: 5´-AATGGAACCTACCAGACCTGGG-3′, rev: 5´-AC ATGGAATGTCTGCCAATGACT-3´ and GAPDH: fwd: 5′-TCGGAGTCAACGGATTTGGTCGT3′; rev: 5′- TGCC ATGGGTGGAATCATATTGGA-3′.Total cellular RNA was extracted with a GeneMATRIX Universal RNA purification kit (EUR® 520 (quencher) FRET peptide substrate. Upon cleavage of the FRET peptide by the active enzyme, the fluorescence of 5-FAM is recovered and monitored at an excitation of 490 nm and emission of 490/520 nm. Data are expressed as the mean fluorescence intensity per microgram of total protein.The activity of TACE (ADAM17) was determined with the Sensolyte 520 TACE Activity Assay Kit  using 20 μg of cell lysate proteins, according to the manufacturer's protocol. This assay uses a 5-FAM (fluorophore) and QXL6 cells were plated in 12-well plates and transfected with control or 200 nM of TIMP3-specific siRNA. Transfection was performed using the Cell Line Nucleofector® Solution V and the AMAXA nucleofector apparatus . NKG2DL shedding was analyzed by ELISA 48 hours after transfection.TIMP3 and control non-specific scrambled siRNA were purchased from Qiagen and Dharmacon , respectively. 2 × 10AML cell lines (KG1a and NB4) were lysed in RIPA buffer  supplemented with protease inhibitors cocktail (Sigma-Aldrich). Samples were clarified by centrifugation, and stored at −20°C. 20 μg of protein extract were separated on 15% polyacrylamide-SDS gels and blotted onto nitrocellulose membranes. After blocking, primary polyclonal goat anti-human TIMP3 and ß-actin  were incubated overnight at 4°C. Membranes were washed and subsequently incubated with horseradish peroxidase-conjugated rabbit anti-goat IgG  for 1 hour at room temperature. The blots were developed with the use of a chemiluminescent substrate .® Blood & Tissue kit . 1 μg of purified DNA was modified with bisulfite using a DNA Methylation-Gold™ Kit . Modified bisulfite DNA was amplified using specific biotinylated primers for TIMP3 designed by PyroMark Assay Design Software 2.0 (Qiagen). Primer sequences were as follows: fwd: 5′-TGGGTGGGTGTTAGTTGG-3′; rev: 5′-CCCCCTCAAACCAATAAC-3′; seq: 5′-ATTTA GTGGTTTAGGTGG-3′. Pyrosequencing was performed with the PyroMark™ Q24 System version 2.0.6, and methylation levels were quantified using PyroMark Q24 Software 2.0 following the manufacturer's instructions (Qiagen).Firstly, genomic DNA was extracted using a DNeasy® treatment were used. Cells were then stained with 5 μl of 7AAD and analyzed by flow cytometry. For gating strategy (Cytotoxicity was assayed by flow cytometry using the PKH67 Green Fluorescent Cell Linker kit (Sigma-Aldrich). We used the NKL cell line as the effector  and the strategy , target in vitro experiments were done at least three times with similar results. Differences between groups were examined using Student's paired-samples t-test and the Mann-Whitney U test. Comparison between the sNKG2DL levels from the AML patients sera before and after Vidaza treatment was analyzed using Wilconxon test. The chi-square test was used to compare the TIMP3 methylation status with the clinical characteristics in AML patients. In all cases, statistical significance was concluded for values of p < 0.05. Analyses were performed with SPSS version 15.0 .Data of soluble NKG2DL are expressed as mean ± standard error of the mean (SEM). All


Our detailed spectroscopic study reveals the simplest explanation for how GCaMP6m changes fluorescence in response to Ca2+ is with a four-state model, in which a Ca2+-dependent change of the chromophore protonation state, due to a shift in pKa, is the predominant factor. The pKa shift is quantitatively explained by a change in electrostatic potential around the chromophore due to the conformational changes that occur in the protein when calmodulin binds Ca2+ and interacts with the M13 peptide. The absolute pKa values for the Ca2+-free and Ca2+-saturated states of GCaMP6m are critical to its high signal-to-noise ratio. This mechanism has important implications for further improvements to GCaMP6m and potentially for other similarly designed biosensors.The goal of this work is to determine how GCaMP6m’s fluorescence is altered in response to Ca The newest generation GCaMP6 sensors  are brihe brain  and in ehe brain .2+ binding protein calmodulin, and its target peptide the M13 domain, are individually fused to the new C- and N-termini of the circularly-permuted GFP, respectively  of the chromophore in the Ca2+ sensor is different for the Ca2+-free and Ca2+-saturated states 2, where α is the polarizability of the chromophore in the ground state, and R is the distance between the center of chromophore and D380 side chain in the corresponding Ca2+-dependent state. Using the polarizability value for the GFP chromophore, α = 76 x 10−24 cm3 [a (ΔΔΔG = 2.9 kJ/mol).One explanation for this difference could be that we disregard the chromophore polarizability, i.e. the redistribution of electronic density that occurs when GCaMP3 binds Ca0−24 cm3 , and thea value between GCaMP3 and GCaMP5A (due to the mutation D380Y) approaches the experimental value. The calculated ΔΔpKa is consistent with the experimental ΔΔpKa once the atom potentials from the phenolate oxygen to the CB2 atom are set to zero .Large discrepancies between experimental and calculated values of electrostatic interaction energies have been observed for similar systems in which a charged amino acid moves relative to a fluorescent probe (tryptophan residue) at the protein surface, if only protein charges are considered . The disa  is not in the pocket for both Ca2+-free and Ca2+-saturated states, and all other mutated charged residues . There are approximately 9–10 contributing charged amino acid residues for both proteins  because the interactions of the rest of the residues almost cancel each other. This can explain why the point mutations E61K and E61G (GCaMP2 numbering) can reduce or completely eliminate the Ca2+-dependent change in fluorescence of GCaMP2 [2+-dependent fluorescence response have also been demonstrated by point mutagenesis [This approach reveals critical charged amino acids that affect the ΔΔG and the corresponding Cavidually . As befoproteins . Their tl values . The numf GCaMP2 . The impagenesis ,18.2+-saturated and Ca2+-free states at neutral pH. In both states, the chromophore exists as a mixture of neutral (N) and anionic (A) forms . Both forms of the chromophore are fluorescent within the same spectral region (green), have unique two-photon absorption spectra, and they contribute to the observed spectrum based on their relative concentrations, ρN = nN/(nN + nA) and ρA = nA/(nN + nA). Considering all of the above, the y-axis of 2 (λ) = σ2,N (λ) φN ρN + σ2,A (λ) φA ρA, where σ2(λ) is the wavelength-dependent two-photon excitation cross-section and φ is the fluorescence quantum yield.The GCaMP6 sensors are important tools for imaging activity in living brains, much of which is done with two-photon excitation –7. The t2+-saturated state, the spectrum is dominated by the anionic form, peaking at ~ 930 nm , we normalized the whole spectrum to the value of F2 = σ2,A(λ) φA ρA at 930 nm. Since the neutral form does not absorb at this wavelength, the latter was calculated as a product of independently measured σ2,A (930 nm) = 50 GM, φA = 0.63  = 130 GM, φN = 0.041 (N = 0.97 (obtained similarly to ρA above). The neutral form two-photon excitation peak position (790 nm) is very close to twice the wavelength of the corresponding one-photon absorption peak, and the anionic form peak position is blue shifted with respect to twice the one-photon absorption peak.In the Ca = 0.041 , and ρN 2+-free state to be σ2(930 nm) = 54 GM. This value is very close to what would be expected from the F2(930 nm) value, obtained directly from the spectrum  and quantum yields  should be the same for one- and two-photon excited fluorescence. However, the two-photon cross-section for the neutral form (σ2(790 nm) = 140 GM) is 2.6 times larger than the anionic form (54 GM) contrary to the ratio of extinction coefficients, where the neutral form is 2 times smaller than the anionic form  is very close to physiological pH. This means at pH 7.2, roughly 50% of the Ca2+-saturated GCaMP6m remains in the non-fluorescent (with ~470 nm for one-photon excitation or ~930 nm for two-photon excitation) neutral form of the chromophore. It also means that any small fluctuations of intracellular pH will affect the fluorescence signal of the Ca2+ sensor. At pH 7.2, a 0.2 pH unit fluctuation would result in a 17% change  in the Ca2+-saturated anionic chromophore fluorescence. If the pKa of the Ca2+-saturated state was shifted to a more acidic pH, it would reduce GCaMP6m pH sensitivity and increase ΔF/F0. Altering the GCaMP6m design to include positive residues that move closer to chromophore when calmodulin binds Ca2+ would produce this acidic pKa shift, increasing the ΔpKa and the ΔF signal. As shown in a properties [a values  or increased ΔpKa would provide more information regarding pKa and ΔpKa effects on Ca2+ sensor function, and could be beneficial for evolving GCaMP6m for increased ΔF without changing Ca2+-binding dynamic.The pK2+-dependent fluorescence changes observed for other GCaMPs [a shift reported for iGluSnFR is from 7.0 in the "free" state to 6.5 in the "bound" state, the relative concentrations of neutral and anionic forms change upon binding glutamate. FlinG2 and FlincG3 are cGMP sensors, also based on a GCaMP-like circularly-permuted GFP design, and report pKa shifts of 7.48 (free) / 7.33 (bound) and 7.94 (free) / 7.53 (bound), respectively [a is important to how these sensors function, but to what extent is unclear. Applying a four-state model for these sensors, or any other circularly-permuted fluorescent protein biosensor [The four-state model we have applied for GCaMP6m could also be applied to understand the Car GCaMPs ,19,28,42r GCaMPs ,48–57. iectively . The Fliiosensor ,49–55,57a (~7.1) [a of the pH-sensitive fluorescent protein, triggered by conformational changes in the CiVSP voltage sensing domain. Remarkably, there are very few measurements of the neutral form in these single GFP-based voltage sensors [a is certainly worth exploring, and will be a topic of our future investigation.There are several clues in the literature that indicate a four-state model may also provide some insight into how ArcLight  and simia (~7.1) ,61. A cha (~7.1) ,62. It ia (~7.1) ,62. Padoa (~7.1) . Pado isa (~7.1) . In altea (~7.1) , much li sensors , or biop sensors , that mi2+ sensor is dark at low Ca2+ concentrations, and only fluorescent in active cells, producing an excellent signal-to-noise ratio in complex tissues like the brain. Second, since the neutral form of the chromophore is not absorbing the 470 nm light, the Ca2+ sensor is only susceptible to bleaching during the brief periods that the cell is active. This is also true for two-photon excitation with 930 nm light. The Ca2+ dependent equilibrium of the GCaMP6m chromophore essentially helps the Ca2+ sensor "hide in the dark" when there is no activity to report, making these biosensors uniquely resistant to photobleaching. This will be a beneficial property to incorporate into any future biosensor designs, especially for applications involving high frame rates and increased excitation light intensity.The remarkable redistribution of the GCaMP chromophore from neutral to anionic forms has two important consequences. First, with 470 nm excitation, the Ca2. Transient transfection were done with lipofectamine following the manufacturer's protocol (Invitrogen). Cells were co-transfected with pGP-CMV-GCaMP6m  and an M1 muscarinic receptor in the CMV expression vector pUB2.1 . For imaging, DMEM was replaced with warmed Dulbecco's Phosphate Buffered Saline (DPBS) with Ca2+ and Mg2+. The wide-field imaging experiments were performed on an Olympus IX70 microscope fitted with Sutter filter wheels and a 20x dry lens, alternating excitation with 410/20 nm and 480/20 nm bandpass filters and collecting emission with a 515 nm long-pass filter and CCD camera (Hamamatsu). To elicit intracellular Ca2+ release, carbachol was added to the well to a final concentration of 50 μM.HEK293 cells were maintained in Dulbecco's Modified Eagle Medium, High Glucose (DMEM) and supplemented with 8% fetal bovine serum (FBS). Cells were plated in 24-well glass bottom plates coated with poly-D-lysine and kept in an incubator at 37°C with 5% CO2+-free and Ca2+-saturated samples for protein measurements, except for the Ca2+ titration experiments. All purified GCaMP6m Ca2+-free protein measurements for this work were carried out in 10 mM EGTA /100 mM KCl / 30 mM MOPS at pH 7.2. All purified GCaMP6m Ca2+-saturated protein measurements for this work were carried out in 10 mM CaEGTA /100 mM KCl /30 mM MOPS at pH 7.2.The coding region of GCaMP6m was moved into a constitutive bacterial expression vector , using ligation-independent cloning . E. coli2+ titration experiments for purified protein samples were done using Ca2+ Calibration Buffer Kit #1 (Life Technologies), following their protocol. We measured fluorescence intensity at the spectral peak (between 512 nm and 516 nm) as a function of Ca2+ concentration by varying CaEGTA concentrations of the 2 mL sample (0 to 10 mM CaEGTA). We used excitation at 470 nm to measure the Ca2+ dependent fluorescence of the anionic form, and 400 nm for the neutral form. The pH of the sample was measured at beginning and end of each titration series to ensure there was no shift in pH during titration. For the apparent Kd and Hill coefficient values from the 470 nm excited measurements, we averaged 3 separate Ca2+ titration measurements.Ca2SO4, whose corrected spectrum is published [2+-saturated at 513 nm; for Ca2+-free at 516 nm) upon its direct excitation at 470 nm was measured versus fluorescein in 1M NaOH (φ = 0.95) as a reference standard [2+-saturated at 512 nm, for Ca2+-free at 514 nm) was measured using excitation at 380 nm versus quinine sulfate in 1M H2SO4 (φ = 0.53) as a reference standard [2+-free form excited at 470 nm (which lacks this contribution) and normalized it to the maximum of the spectrum excited at 380 nm (see Fluorescence spectra and fluorescence excitation spectra of purified protein samples were collected using a PC1™ spectrofluorimeter (ISS). Fluorescence spectra were corrected for the detection spectral sensitivity using the quinine sulfate solution in 1M Hublished . Fluoresstandard . Fluoresstandard . The flu0 nm see . This no2+-free or Ca2+-saturated states) separately, we measured the changes of the corresponding absorption spectrum upon alkaline pH titration of the purified protein solutions  will give the straight line with a slope equal to:A and εN is obtained from the fact that the total concentration of chromophore before the titration (index 0) n(0) = n(0)A + n(0)N is equal to the concentration of chromophore in alkaline denatured form n(f)D  which absorbs at 447 nm and has an extinction coefficient of 44,100 M-1 cm-1 [A and εN. Combining Eqs . AbsorpM-1 cm-1 . TherefoODN=−εAεNAn addit+ODN(0)εNIn this 2+-saturated and Ca2+-free states. To calculate the pKa, the peak fluorescence intensity near 515 nm was plotted against pH to get a pH titration curve. The data were then fit using two-binding site model function in the range of pH 4.3 to 11.4 for Ca2+-free GCaMP6m, and using a single-binding site model function for Ca2+-saturated GCaMP6m in the range of pH 4.8 to 8.3.We independently measured the changes in fluorescence spectra for alkaline and acidic titration experiments with purified protein samples. To do this we used the same alkaline titration method as the extinction coefficient measurements, and for acidic titration method we gradually added small volumes of 1M HCl (1–3 μl) to the 2 mL sample, from pH 7.2 to 4.3. pH  and corresponding fluorescence spectra  were collected upon excitation of anionic form at 470 nm for the Ca2+-free and Ca2+-saturated purified GCaMP6m protein samples (5 x 10−5 M) were measured in 0.1 cm glass spectroscopy cuvettes (Starna cells). Two-photon excitation (TPE) spectra were measured using an MOM Sutter Instrument two-photon fluorescent microscope coupled with an Insight DeepSee (Newport) femtosecond laser tunable from 680 to 1300 nm in a similar approach as described in [2+-free GCaMP6m and 930 nm for the anionic form in the Ca2+-saturated and Ca2+-free GCaMP6m, using coumarin 485 in methanol (for 790 nm excitation) and fluorescein in NaOH aqueous solution (pH 11) (for 930 nm excitation) as reference standards [F, as a function of excitation power, P, was measured for both the sample and the reference in the same conditions through a 507/10 nm narrow-bandpass filter. From the fit of these dependences to a quadratic function, F = α P2, factors α values were obtained and then normalized to the concentrations  and differential quantum efficiencies at 507 nm .Caribed in . A Plan ribed in . The TPEtandards . FluoresS1 Fig2+-free  and Ca2+-saturated  states, excited at 400 nm (A) and 470 nm (B). Inset graphs: zoomed in view of Ca2+-dependent peak emission shift.A and B) Emission spectra for purified GCaMP6m protein in the Ca(TIF)Click here for additional data file.S2 Fig2+-free (dotted black trace) and Ca2+-saturated (solid black trace) states, fluorescence emission of the anionic form collected at 550 nm. The ratio of the Ca2+-saturated and Ca2+-free spectra illustrates the ΔF/F0 wavelength dependence (red trace).Excitation spectra for the anionic chromophore using purified GCaMP6m protein in the Ca(TIF)Click here for additional data file.S3 Fig2+-free GCaMP6m excited at 380 nm (black trace) and at 470 nm (red trace). The 470 nm excited spectrum was normalized to the maximum fluorescence intensity of the 380 nm excited spectrum.Fluorescence spectra of Ca(TIF)Click here for additional data file.S4 Fig2+-saturated anionic chromophore, 50 GM, is the absolute cross-section at 930 nm excitation. Light grey arrow (B) marks the weak shoulder at ~990 nm that corresponds to the pure electronic transition of the anionic form, which occurs at double the wavelength of the one-photon peak absorption. B) Two-photon excitation spectra for Ca2+-free GCaMP6m . The cross-section of the Ca2+-free neutral chromophore, 140 GM, is the absolute cross-section at 790 nm excitation, and the cross-section of the Ca2+-free anionic chromophore, 54 GM, is the absolute cross-section at 930 nm excitation. For both Ca2+-saturated (A) and Ca2+-free (B), the one-photon absorption is illustrated by the red solid trace .The cross-section of the Ca(TIF)Click here for additional data file.


We recently identified the human leukocyte immunoglobulin-like receptor B2 (LILRB2) and its mouse ortholog-paired Ig-like receptor (PirB) as receptors for several angiopoietin-like proteins (Angptls). We also demonstrated that PirB is important for the development of acute myeloid leukemia (AML), but exactly how an inhibitory receptor such as PirB can support cancer development is intriguing.Here, we showed that the activation of Ca (2+)/calmodulin-dependent protein kinases (CAMKs) is coupled with PirB signaling in AML cells. High expression of CAMKs is associated with a poor overall survival probability in patients with AML. Knockdown of CAMKI or CAMKIV decreased human acute leukemia development in vitro and in vivo. Mouse AML cells that are defective in PirB signaling had decreased activation of CAMKs, and the forced expression of CAMK partially rescued the PirB-defective phenotype in the MLL-AF9 AML mouse model. The inhibition of CAMK kinase activity or deletion of CAMKIV significantly slowed AML development and decreased the AML stem cell activity. We also found that CAMKIV acts through the phosphorylation of one of its well-known target (CREB) in AML cells.CAMKs are essential for the growth of human and mouse AML. The inhibition of CAMK signaling may become an effective strategy for treating leukemia.The online version of this article (10.1186/s13045-018-0574-8) contains supplementary material, which is available to authorized users. Recently, we identified the human leukocyte immunoglobulin-like receptor B2 (LILRB2) and its mouse ortholog-paired Ig-like receptor (PirB) as receptors for several angiopoietin-like proteins (Angptls) . LILRB2 LILRB2 contains four extracellular Ig-like domains and a cytoplasmic portion with three immunoreceptor tyrosine-based switch motifs (ITIMs) . The moucamk4-knockout mice have defects in HSC survival and proliferation [CAMKs are serine/threonine kinases that are activated by increases of intracellular calcium concentration and that mediate subsequent cellular activities. Few studies have shown that CAMKs play important roles in hematopoietic physiology and pathology. CAMKIV is required for HSC activity; feration . CAMKII feration . CREB isferation –13.In this study, we sought to investigate the function of CAMKs in AML cells and understand how CAMKs support PirB signaling in AML development. We found that the expression of several CAMKs inversely correlates with the overall survival of human AML patients. Knockdown of CAMKs decreased human myeloid leukemia development in vitro and in vivo. In the mouse MLL-AF9 primary AML cells, the defective PirB signaling decreased CAMK activation, and the forced expression of CAMKs partially rescued the PirB-defective phenotype. Importantly, the inhibition of CAMK kinase activity or deletion of CAMKIV significantly lowered the activity of AML stem cells. By rescue assay, we also identified the phosphorylation of CREB is critical downstream for CAMKIV signaling in AML cells. Our results indicate that the CAMK signaling supports self-renewal and inhibits apoptosis of AML cells./SCID-IL2RG (NSG) mice were purchased from the UT Southwestern Medical Center animal breeding core facility or Jackson Labs. The PirB TM mice [C57 BL/6 and NOD TM mice  and CaMK TM mice  were purAntibodies for flow cytometry, anti-Kit-APC, anti-Mac-1-APC, anti-Gr-1-PE, anti-CD3-APC, and anti-B220-PE, were purchased from BioLegend and used as described [− cells with retroviral supernatant in the presence of 8 μg/mL polybrene. After infection, these cells were incubated overnight in medium with 10% FBS, 20 ng/mL SCF, 10 ng/mL IL-3, and 10 ng/mL IL-6. Infected cells  were transplanted into lethally irradiated (1000 rad) WT mice by retro-orbital injection. For secondary transplantation, YFP+ bone marrow (BM) cells from primary transplanted mice were sorted and transplanted into mice with 200,000 normal BM cells as competitors.For retrovirus production, human embryonic kidney 293T cells were grown in DMEM with 10% fetal bovine serum (FBS) and transfected by MSCV-MLL-AF9-IRES-YFP, MSCV-CAMKIV-IRES-GFP, MSCV-CAMKI-IRES-GFP, or MSCV-CREB-IRES-GFP encoding plasmids and pCL-ECO to produce retroviruses. The infection of Linage-negative cells with retrovirus was performed as described previously , 16. BriFor lentivirus production, the lentiviral vector Pll3.7 was used to express shRNAs to target CAMKI  and CAMKIV . The lentivirus plasmid, pSPAX2, and pMD2.G (4:3:1) were transfected using PolyJet™ In Vitro DNA Transfection Reagent (SignaGen Laboratories) into 293T cells. The resulting virus supernatant was collected 48–72 h later.For Crisper-Cas9 system, we used pCW-Cas9 (gift from Eric Lander & David Sabatini (Addgene plasmid # 50661) ) to makeWe performed flow cytometry, immunohistochemistry, and cytospin as described previously , 15, 16.2 and the normal level of O2. Human leukemia cell lines were from ATCC. Xenografts were performed essentially as we described [6 human GFP+ viable cells per mouse for retro-orbital injection. One to 4 months after transplantation, the peripheral blood (PB), BM, spleen, and liver were assessed for engraftment by flow cytometry.Human leukemia cell lines were cultured in RPMI supplemented with 10% FBS at 37 °C in 5% COescribed , 18. Bri+Mac-1+Kit+ BM cells from AML mice were plated in colony-forming unit (CFU) medium  for CFU-GM assays, according to the manufacturer’s protocols [Five hundred YFProtocols . After 7https://tcga-data.nci.nih.gov/docs/publications/tcga/; accessed September 5, 2013), and levels were normalized to GADPH expression. Patients were divided into two groups based on whether their expression was above or below the median level of a gene.Data were obtained from the TCGA AML database (Cell lysates (100 μg samples) were separated by electrophoresis on a 4–12% SDS-polyacrylamide gel (BioRad), and the proteins were transferred onto a nitrocellulose membrane. The membrane was probed with indicated primary antibody for 1 hour at room temperature and then incubated with horseradish peroxidase-conjugated secondary antibody, which was detected with the chemiluminescence SuperSignal kit (Pierce).t test and differences were considered statistically significant if p < 0.05. The survival rates of the two groups were analyzed using a log-rank test, and differences were considered statistically significant if p < 0.05.Data are expressed as mean ± SEM. For all experiments except determination of survival, data were analyzed by Student’s PirB are deleted, were used to provide PirB-defective cells. PirBTM and control wild-type (WT) cells infected with MSCV-MLL-AF9-IRES-YFP retrovirus were transplanted to establish AML mice. We sought to determine whether the CAMK family decreased expression/activities in the PirB-defective MLL-AF9 AML mouse model. Compared to WT controls, PirBTM cells from MLL-AF9 AML mice had significantly decreased phosphorylation of CAMKI, CAMKII, and CAMKIV  cells , we usedn et al. )  activity. Primary transplants with lls Fig. a. In draCamk4-null and control AML BM cells. Overall, the MLL-AF9 BM AML cells had very low levels of early and late apoptosis  of that in control WT AML mice. This limiting dilution transplantation assay confirms that CAMKIV supports the activity of AML-SCs.To quantitate how CAMKIV deficiency affects the AML-SC frequency, we performed transplantations by limiting dilution of sorted YFPPreviously, we showed that the activation of LILRB2 induced CAMK activation. Similarly, based on our in silico analysis of human samples, the expression of LILRB2 and several CAMKs correlate inversely with the overall survival of AML patients Fig. a–d, and or CAMK4 expression in MV4-11 leukemia cells in transplanted NOD/SCID-IL2RG (NSG) mice. Both Camk1 or Camk4 knockdown significantly prolonged the survival of xenografted mice  The expression of CAMKs inversely correlates with the overall survival of AML patients. (2) Knockdown of CAMKI or CAMKIV inhibits growth of human AML cells in vitro and in vivo. (3) CAMKI and CAMKIV rescue PirB deficiency in a mouse AML model, and deletion of CAMKIV decreased the activity of AML-SCs. (4) CAMKIV supports the development of acute lymphoblastic leukemia in a mouse model (unpublished data). These results are in agreement with the previous reports that CAMKIV is required for HSC activity and CAMKII suppresses differentiation and stimulates proliferation of myeloid leukemia cells . BecauseBecause CAMKs network with many different signaling pathways, and the signaling of ITAM or ITIM receptors is rather divergent , it is pCamk4-knockout mice had only minor defects in HSCs [We demonstrated that CAMKs are important signaling molecules to support self-renewal and survival in AML cells. Our results thus revealed one mechanism by which the ITIM-containing inhibitory receptors support stem cell activity and AML development and also have important implications in development of new therapeutic strategies in leukemia treatment. It is known that deletion of PirB from normal hematopoiesis or adult HSCs does not have significant in vivo effects , and Cam in HSCs . By contAdditional file 1:Figure S1. Western blot showed expression level of CAMKI and CAMKIV in each samples of Fig. Figure S2. CaMKIV is highly expressed in AML-SC enriched population. (a-b) The expression of PirB (a) and CaMKIV (b) in different normal BM populations as determined by real-time RT-PCR (n = 3). (c) CaMKIV expression in total, YFP+Mac-1+Kit−, and YFP+Mac-1+Kit+ BM AML cells as determined by real-time RT-PCR (n = 3). *p < 0.05. Error bars, s.e.m. Figure S3. Phosphorylation of CAMKIV is greater in myeloid cell lines (U937 and MV4-11) than in ALL cell lines (KASUMI-2 and SUP-B15). Figure S4. MV4-11 cell growth was inhibited by three different gRNAs targeting camk1. Cell numbers were calculated four days post doxycycline (DOX 1μg/ml) inducement (a). Western blot showed silent effect of gRNAs targeting CAMK1 (b). (PDF 660 kb)


Genetically engineered murine models offer the opportunity to dissect these mechanisms in vivo. We generated a gene-targeted knock-in murine model of the autosomal dominant Arg141Trp (R141W) mutation in Tnnt2, which was first described in a human family with DCM. Mice heterozygous for the mutation (Tnnt2R141W/+) recapitulated the human phenotype, developing left ventricular dilation and reduced contractility. There was a gene dosage effect, so that the phenotype in Tnnt2R141W/+mice was attenuated by transgenic overexpression of wildtype Tnnt2 mRNA transcript. Male mice exhibited poorer survival than females. Biomechanical studies on skinned fibers from Tnnt2R141W/+ hearts showed a significant decrease in pCa50  relative to wildtype hearts, indicating Ca2+ desensitization. Optical mapping studies of Langendorff-perfused Tnnt2R141W/+ hearts showed marked increases in diastolic and peak systolic intracellular Ca2+ ([Ca2+]i), and prolonged systolic rise and diastolic fall of [Ca2+]i. Perfused Tnnt2R141W/+ hearts had slower intrinsic rates in sinus rhythm and reduced peak heart rates in response to isoproterenol. Tnnt2R141W/+ hearts exhibited a reduction in phosphorylated phospholamban relative to wildtype mice. However, crossing Tnnt2R141W/+ mice with phospholamban knockout (Pln-/-) mice, which exhibit increased Ca2+ transients and contractility, had no effect on the DCM phenotype. We conclude that the Tnnt2 R141W mutation causes a Ca2+ desensitization and mice adapt by increasing Ca2+-transient amplitudes, which impairs Ca2+ handling dynamics, metabolism and responses to β-adrenergic activation.Most studies of the mechanisms leading to hereditary dilated cardiomyopathy (DCM) have been performed in reconstituted  TNNT2) can lead to either DCM or HCM   2+ desenobserved , 7 have mutation –11, thersitivity . In contutations .in vitro systems. Genetically engineered murine models offer the opportunity to dissect the mechanisms leading from genotype to phenotype in vivo. We i), prolonged systolic rise and diastolic fall in [Ca2+]i, shortened action potential durations, and a lower peak heart rate with adrenergic stimulation. Furthermore, analysis of Ca2+ handling proteins shows a reduction in phospholamban phosphorylation. Crossing Tnnt2R141W/+ mice with phospholamban knockout (Pln-/-) mice   2+ desen protein  have docTnnt2R141W/+ mice that showed increased diastolic intracellular Ca2+ ([Ca2+]i), a prolonged systolic rise of [Ca2+]i, increased peak systolic [Ca2+]i, and a prolonged diastolic fall in [Ca2+]i. These findings suggest increased Ca2+ bioavailability to the sarcomere to compensate for decreased Ca2+ sensitivity. Furthermore, the increased Ca2+ bioavailability is associated with a reduction in phosphorylated phospholamban expression. The prolonged Ca2+ transients may limit the ability of Tnnt2R141W/+ hearts to increase heart rates and to respond to adrenergic stimulation. In fact, they are predisposed to arrhythmias when exposed to adrenergic stimulation. The reduction in phosphorylated phospholamban suggests greater inhibition of SERCA2, which would lead to the observed prolongation in diastolic fall in [Ca2+]i and the increased end diastolic [Ca2+]i. These observations may also be related to the decreased myofilament affinity for Ca2+, so that there is more free, unbound Ca2+. However, there are likely also other complex mechanisms at play that remain to be identified, because decreased phosphorylation of phospholamban is generally associated with decreased Ca2+ transient amplitudes rather than our observed increased Ca2+ transient amplitudes. Our findings in Tnnt2R141W/+ hearts are similar to those in Tnnt2K210Δ/+ hearts, which exhibited increased peak amplitude of the Ca2+ transient in cardiomyocytes [Tnnt2K210Δ/+ hearts was attenuated by a positive inotropic agent, pimobendan, which directly increases myofilament Ca2+ sensitivity [Pln, which increases the uptake, sequestration, and systolic release of Ca2+ in the sarcoplasmic reticulum, had no salutary effect on the phenotype of Tnnt2R141W/+ mice. We speculate that the increased Ca2+ bioavailability associated with Pln ablation had no additional benefit in Tnnt2R141W/+ mice in which sarcoplasmic Ca2+ was already increased. The R141W mutation is located within the tropomyosin binding domain of cTnT, and has been shown to increase the affinity of cTnT for tropomyosin [2+ sensitivity of the troponin complex.To understand the physiologic consequences of the R141W mutation, we performed optical mapping studies in myocytes . The abnsitivity . In contpomyosin . Thus, t2+ sensitivity. β-adrenergic stimulation leads to a PKA phosphorylation-dependent decrease in myofilament Ca2+ sensitivity, an associated increase in the rate of Ca2+ dissociation from TnC, and an increase in the rate of relaxation that facilitates more rapid heart rates and increased contractility. However, Marston and colleagues have documented uncoupling of Ca2+ sensitivity from phosphorylation status in several DCM mutations in vitro [in vivo [Decreases in adrenergic sensitivity are generally expected to increase Cain vitro , and a c[in vivo .Tnnt2 transcript was expressed at a level 1.5–2.0-fold greater than endogenous wildtype Tnnt2 transcript, a proportion that likely was greater than the approximate parity between mutant and wildtype Tnnt2 transcript in Tnnt2R141W/+ mice.  Transgenic mice exhibited similar LV dilation and impaired contractility, and exhibited some of the same changes in signaling pathways as Tnnt2R141W/+ mice. In contrast to Tnnt2R141W/+ mice, however, the transgenic mice exhibited a greater degree of interstitial fibrosis. Another transgenic mouse model with cardiac overexpression of R141W mutant human cTnT [2+-sensitivity of myofibrillar protein ATPase activity.A C57BL/6J transgenic mouse model with cardiac overexpression of R141W mutant human cTnT has been previously reported [man cTnT  also exhTNNT2 leading to DCM, and to describe the consequences of the mutation on gene dosage, sex-dependent survival, molecular remodeling pathways, electrophysiological function, and Ca2+ sensitivity, transients, and homeostasis. Our data point to a broad and central role of Ca2+ in the pathophysiology of DCM, with impaired Ca2+ sensitivity as possibly the initial direct consequence of the TNNT2 mutation. The decreased sensitivity leads to perturbations in Ca2+ transients and arrhythmogenesis. Combined with other studies, our findings are consistent with the paradigm that intracellular Ca2+ homeostasis is a major player in the pathogenesis of both DCM and HCM, with the distinct phenotypes resulting from divergent effects of genetic mutations on Ca2+ sensitivity.This report is one of only a few to describe a gene-targeted knock-in murine models of human mutations in


The purpose of this study was to identify chronic urticaria (CU) etiologies and treatment modalities in Ecuador. We propose that the sample distribution fits the expected one, and that there is an association between the etiology and its treatment.P values less than 0.05 were considered significant.We performed a retrospective study involving 112 patients diagnosed with CU using a Checklist for a complete chronic urticaria medical history. Demographic and clinical variables were collected. The etiology of CU was classified using the EAACI/GA2LEN/EDF/WAO guideline. Descriptive analyses were performed for demographical and clinical variables. Chi square tests were applied to analyze the fit of distribution and the independence of variables. p < 0.01).. On the other hand, 23.2% inducible urticarias (CIndU) were indentified; dermographism was the most common (10.7%) (p < 0.01).Among all the patients, 76.8% were diagnosed with chronic spontaneous urticaria (CSU), of which 22.3% had a known etiology or possible exacerbating condition. Food allergy was identified as the most common accompanying condition in patients with CSU (10.7%) (p < 0.01). Almost 48% of CSUs of unknown etiology were treated with any antihistamine plus another drug. In patients with known etiology, sg-antihistamines alone (44.0%) was the most common management. In addition, 53.8% of CIndUs were treated with sg-antihistamines alone. Though, these associations were not statistically significant.Regarding treatment regimens, sg-H1-antihistamines alone represented the highest proportion (44.6%). The combination of any H1-antihistamine plus other drug was a close second (42.0%) (CSU is the most frequent subtype of CU. Modern non-sedating antihistamines in licensed doses are the drug of choice. Nevertheless, a great proportion of patients require the addition of another type of medication. Urticaria is defined by the presence of hives that appear and resolve within 24 h. Urticarial lesions can be circumscribed, raised, erythematous plaques, with central pallor. They can adopt different shapes and sizes , and are characterized by three main features: swelling and erythema; itching/burning sensation; spontaneous resolution within 24 h .With regard to the duration of urticaria, it can be classified as “acute” (< 6 weeks) or “chronic” (> 6 weeks) . Among pAccording to its underlying etiology, chronic urticaria is classified in two main groups: (i) chronic spontaneous urticaria (formerly known as “chronic idiopathic urticaria”), and (ii) inducible urticaria  .Despite recent updates to the management guidelines for urticaria, it remains a challenge for healthcare providers to diagnose and identify each subtype of chronic urticaria due to the broad spectrum of clinical manifestations, and the possibility that several subtypes of the disease coexist in the same patient , 6. Amon2LEN/EDF/WAO  advocate a simplified three-step approach. Both guidelines agree on second-generation H1 antihistamines as the cornerstone and first-line therapy for chronic urticaria [With regard to treatment, two major research teams have published guidelines based on the available evidence and expert opinion , 6. The rticaria . Treatmerticaria , 9. Howerticaria , 11.Data regarding the prevalence, demographics, and clinical characteristics of patients with chronic urticaria in Latin America, specifically in Ecuador, is limited. We aimed to fill this knowledge gap by describing the most common features and treatment choices of patients with chronic urticaria in Guayaquil, Ecuador.2LEN/EDF/WAO [We carried out a retrospective study involving 112 patients diagnosed with chronic urticaria from 2005 to 2016 at Respiralab Research Center, Guayaquil-Ecuador. Demographic and clinical variables such as age, sex, years with the disease, type of urticarial, and medications were collected using medical records from the institution. The etiology of chronic urticaria was classified using EAACI/GAP < 0.05 was considered significant.Medications were subclassified into four groups: first-generation H1 antihistamine alone; second-generation H1 antihistamine alone; first- and second-generation H1 antihistamine combined; any H1 antihistamine with other types of medications . These therapeutic modalities apply only to treatment initiation; no follow up or treatment modifications are described in this study.Descriptive analyses  were carried out for demographic and clinical variables. The chi-square test was applied to analyze the fit of distribution as well as the independence of variables. All statistical analyses were carried out using SPSS v24.0 . Among the 112 patients studied, 69.6% were female and 30.4% were male, with the age ranging from 14 to 73  years. The mean duration of suffering from chronic urticaria was 1.6 (SD 2.2) years (Table p = 0.001)   Fig. .Fig. 1– p = 0.00) , followed by vibratory angioedema (5.4%), cholinergic urticaria 2.7%), with 1.8% for both delayed-pressure urticaria and cold urticaria, and 0.9% for solar urticarial  . The combination of any H1 antihistamine (regardless of generation) plus another type of treatment  was also used widely (42.0%). A combination of a first- and second-generation H1 antihistamine was used in 10.7% of cases, whereas only 2.7% were prescribed with a first-generation antihistamine alone (0) Table .Table 3–p = 0.76) . In addition, 53.8% of patients with chronic inducible urticaria were prescribed a second-generation antihistamine alone, and 30.8% required an antihistamine associated with another medication. However, this association between the type of urticaria and treatment was not statistically significant (76) Fig. .Fig. 2– Chronic urticaria is widely recognized as a relatively common disease that necessitates consultation with a dermatologist and/or allergist. Maurer and coworkers suggested that 66–93% of patients with chronic urticaria are diagnosed with the chronic spontaneous variant, 4–33% with physical urticaria, and 1–7% with cholinergic urticaria . Our resAmong chronic urticaria in Latin America, our demographic findings were comparable with the study by Gomez et al.; patients were were predominantly women, with a median age ranging in the 30s, and in average one and a half years with the disease (1.7 years in the Argentinian records) . Treatme2LEN/EDF/WAO classification, would suggest that the proportion of patients with chronic spontaneous urticaria has not changed from 2005 to the present day in our local setting.In a previous study conducted from 2002 to 2004 in Ecuador, 161 patients diagnosed with chronic urticaria were classified according to guidelines set by the British Society for Allergy and Clinical Immunology . Those fIt is difficult to determine the role of food as an allergen in the induction of chronic urticarias for four main reasons. First, identification of a food as a trigger depends on medical history, which is often unreliable. Second, the results of elimination diets are considered contradictory. Third, measuring the allergen-specific immunoglobulin (Ig)E level for food has a poor positive predictive value. Finally, the results of double-blind placebo-controlled oral food challenge are difficult to interpret . SimilarThe second most common etiology reported by our patients with chronic spontaneous urticaria was induced by drugs. Kozel et al. published a study with 220 adults diagnosed with urticaria, of which 9.0% of cases were caused by drug allergies . In the Confino-Cohen et al. found a strong association between chronic urticaria and major autoimmune diseases. They also reported thyroid diseases to be the most common autoimmune diseases in patients with chronic urticaria . To illuThere is a subgroup of patients with chronic spontaneous urticaria who have autoantibodies against IgE or its receptor: FcεRI . These aAutoimmunity has been reported to be an etiologic factor in 40–60% of cases of chronic spontaneous urticaria . In our Chronic inducible urticaria is characterized by itchy wheals, flare-type skin reactions, and/or angioedema induced by external physical factors. The latter can be mechanical , thermal  stimuli, or electromagnetic radiation (solar radiation) . AbajianThe most prevalent type of chronic inducible urticaria is symptomatic dermographism . The latIn the study by Sanchez et al., cold urticaria was reported to be the second most prevalent type of chronic inducible urticaria. However, we found vibratory angioedema to be the second most prevalent type of chronic inducible urticaria. In addition, Sanchez hypothesized that exposure to cold environments might protect against cold urticaria. Interestingly, the prevalence of cold urticaria in our sample was one of the lowest, even though our patients were not exposed to low temperatures. Thus, we agree with Sanchez et al. that temperature or altitude are not the only determining factors in the development of chronic inducible urticaria.Although urticarial vasculitis is not a subtype of chronic urticaria, it is a relevant syndrome to exclude in a patient whose chief complaint is an urticarial eruption accompanied by angioedema. In urticarial vasculitis, the lesions are painful, burning, and tender, with plaques lasting for > 24 h (sometimes ≤ 72 h). The wheals are associated with residual purpura or hyperpigmentation, and occasionally have a central dark-red or brown macule signifying underlying purpura and vasculitis. Other characteristics of urticarial vasculitis are swelling, residual bruising, and edema from focused pressure. Up to 81% of patients might present with extracutaneous symptoms, as reported in one retrospective study of 47 patients . Skin biIn our transversal study, we diagnosed 12 patients with urticarial vasculitis. If such patients were to be added to the sample of patients with chronic urticaria, it would represent 9.68% of all diagnoses. Hence, we consider it relevant to include urticarial vasculitis in the differential diagnosis of chronic urticaria. Thus, we highlight how such an entity must always be ruled out.2LEN/EDF/WAO guidelines for chronic urticaria, the management of chronic spontaneous urticaria differs among various parts of the world [Despite the recent update of the EAACI/GAhe world , 7, 40. he world . Patienthe world . Howeverhe world .In our study, a second-generation H1 antihistamine alone was the most common medication type prescribed, followed by antihistamines combined with other types of treatment . In a recent study, we found that only one-third of physicians reported using regular doses of second-generation H1 antihistamines as first-line treatment, and that specialists  prescribed them more frequently . ConversIncreasing the dose of second-generation H1 antihistamines up to fourfold is a relatively new recommendation. Nevertheless, physicians are not sufficiently confident to use this approach, in part because increasing the dose of antihistamines improves the control of pruritus significantly but does not reduce the number of wheals . MoreoveThe prevalence of combined use of first- and second-generation H1 antihistamines, or the use of a first-generation H1 antihistamine alone, were relatively low for our patients . In Ecuador, some physicians (particularly pediatricians) commonly use first-generation H1 antihistamines to treat chronic urticaria . Ferrer Chronic inducible urticaria appears to be more resistant to standard doses of antihistamines compared with chronic spontaneous urticaria, thus necessitating higher doses to achieve symptom control . In our During the last 14 years, 273 patients have been diagnosed with chronic urticaria at our center. No significant changes in the etiology of urticaria have been found. Even though urticarial vasculitis is not a type of urticaria, it was identified as the principal differential diagnosis in our cross sectional study.Our has study has some limitations. First, we didn’t assess severity of the disease using the Urticaria Activity Score (UAS) before and after treatment. By the time these patients were diagnosed and treated, an official Spanish version of UAS wasn’t available. Instead, we used a Visual Analog Scale (VAS) to assess the pruritus and wheals severity. Second, for clinical entities such as food and drug associated urticaria, we performed the diagnosis mostly through anamnesis. A careful clinical history is recommended for both etiologies, but it relies on the patient’s awareness and memory, which is a major downside of major concern for drug associated urticaria. For food allergy, we complemented diagnosis with specific IgE. Even though oral food challenge (OFC) is the gold standard for food allergy diagnosis, we didn’t perform such test. For drug associated urticaria, we interpreted symptoms resolution after offending drug removal as a confirmatory finding for diagnosis, though, more objective tests such as Prick test or in vitro testing could have been helpful. Third, we didn’t provide any data concerning follow up, severity, response or quality of life. This study focusses mainly on the distribution of urticaria etiology and first therapeutic regimen prescribed.Chronic spontaneous urticaria is one of the most prevalent subtypes of chronic urticaria. Non-sedating antihistamines in licensed doses are the drug of choice. Nevertheless, a considerable proportion of patients require the addition of another type of medication.There is a need to elucidate the other factors that contribute to the development of chronic urticaria and the the optimal management of this disease. Furthermore, we believe that urticarial vasculitis must be excluded as a differential diagnosis of any subtype of chronic urticaria.