We also develop intermittent quantification, a technique that combines absolute aquisitions with fast fluorescence acquisitions to deliver fast but fully quantitative measurements. We also show how the photochromism-based measurements can be expanded to situations where the absolute illumination intensities are unknown. In principle, PEAQ biosensing can be applied to other biosensors with photochromic properties, thereby expanding the possibilities for fully quantitative measurements in complex and dynamic systems.Genetically-encoded biosensors based on a single fluorescent protein are widely used to visualize analyte levels or enzymatic activities in cells, though usually to monitor relative changes rather than absolute values. We report photochromism-enabled absolute quantification (PEAQ) biosensing, a method that leverages the photochromic properties of biosensors to provide an absolute measure of the analyte concentration or activity. We develop proof-of-concept photochromic variants of the popular GCaMP family of Ca Biosensors often report relative rather than absolute values. Here the authors report a method that utilises the photochromic properties of biosensors to provide an absolute measure of the analyte concentration or activity: photochromism-enabled absolute quantification (PEAQ) biosensing. Most of the genetically encoded biosensors can be divided into two classes, depending on whether their signal is read out as a change in Förster resonance energy transfer (FRET) between donor and acceptor fluorophores, or as a change in the fluorescence intensity of a single fluorophore3. These fluorophores are usually derived from fluorescent proteins (FPs), though other systems have also been used4. A key advantage of FRET-based probes is that they can provide absolute quantification of the analyte concentration using ratiometric readout of the FRET efficiency7. However, they suffer from complications such as incomplete FP maturation8, take up considerable spectral bandwidth unless lifetime imaging or anistropy measurements can be used11, and result in relatively large genetic constructs, which complicate genome-engineering methods such as viral gene transduction. Single FP-based biosensors, on the other hand, provide fast and straightforward recording and take up less of the visible spectrum, facilitating multiplexing with other fluorescent probes or optogenetic modulators such as channelrhodopsins12. They are also encoded with considerably smaller genes. However, the absolute determination of the analyte concentration or activity is usually difficult to perform with these biosensors since their emission is also affected by the local concentration of the probe and the illumination intensity, which depend on the nature of the sample and the depth of the imaging. Furthermore, these measurements provide no way to detect saturation of the biosensor response. This complicates the development of the in situ quantitative biological models that are ultimately needed to understand and predict cell behavior.Genetically encoded biosensors have revolutionized our understanding of the inner workings of the cell, by reporting on in situ enzymatic activities or the presence of a wide range of molecules such as ions, metabolites, and messengers13 and organic dyes14. However, not all single-FP biosensors have proven amenable to ratiometric operation, while organic dyes present additional challenges for the labeling of more complex samples and when performing long-term experiments15. Fortunately, a range of other light-induced behaviors provide perspectives for innovative biosensor designs16. The development of photoactivatable and photoconvertible biosensors, such as CaMPARI17 and CaMPARI218, has enabled light-induced highlighting of specific features of a sample20. Other strategies have built on the use of photochromic FPs, which can be reversibly switched between a fluorescent and a non-fluorescent state through irradiation with light. Their photochromism has enabled powerful technologies such as dynamic labeling and sub-diffraction imaging30. Mechanistically, this process occurs via a light-driven cis/trans isomerization of the chromophore, followed by spontaneous (de-) protonation on account of the different pKa’s of the cis and the trans state31. Within the field of biosensing, photochromism has been used to enable more accurate FRET quantification33, to switch between different FRET pairs in a single molecule or complex34, to enable fluorescence lifetime imaging microscopy (FLIM)-like readout of the FRET activity using a conventional microscope35, as an alternative readout of homo-FRET36, to multiplex spectrally-overlapping biosensors37, and to acquire activity images with a sub-diffraction resolution39. So far, however, less work has been done on the use of these properties for biosensors based on single FPs.Intriguingly, a variety of previous work has shown that additional information can be provided by the photophysical and photochemical properties of the fluorophore. Ratiometric excitation and/or emission biosensors, for example, provide quantitative signals where the activity is encoded as the ratio between two excitation or emission bands, and have been reported both for biosensors based on FPs2+ is a prime target for biosensing as it is a crucial secondary messenger in many cellular processes such as neuronal signaling, muscle contraction, and the regulation of cell fate. The desire to record its spatiotemporally dynamic levels in situ41 has led to the development of genetically encoded Ca2+ indicators (GECIs) based on single FPs, which translate the intracellular Ca2+ concentration into a fluorescent signal42. Using these tools, Ca2+ concentrations can be monitored in a variety of systems, ranging from single cells to the brain43. However, these studies are often focused on relative changes in local Ca2+ levels, as the quantitative assessment of these signals is limited by the intensiometric nature of single-FP GECIs, though a number of ratiometric variants have also been developed, including Ratiometric-Pericam44, GEM-GECO and GEX-GECO45, as well as quantitative sensors based on the measurement of the excited-state lifetime46. Alternative ratiometric systems have also been pursued by fusing a second, red-shifted fluorescent protein to an intensiometric GECI, but this results in a construct that shares the limitations associated with FRET-based GECIs15. A range of organic dyes, such as the Fura and Indo families47 also achieve ratiometric readout.Ca48. Recent work also showed that it is possible to engineer Ca2+ sensitivity into an existing photochromic protein to obtain an erasable integrator of neuronal activity49. We reasoned that photochromism might form a more general mechanism to obtain additional information from genetically encoded biosensors.Even though single FP-based biosensors are often treated as exhibiting fluorescence that is solely dependent on the free analyte concentration, previous investigations reported that the emissive signal can also depend on the previous light irradiation2+ concentration. We then demonstrate that this dependence can be used to determine the absolute Ca2+ concentration in cells in a way that is largely insensitive to photobleaching and the intensity of excitation light. By delivering an additional strategy to obtain fully quantitative information using single FP-based biosensors, we expect that our approach can be used to perform quantitative biosensing across a variety of systems. In addition to this readout methodology, we also develop intermittent quantification, a fully generic method that can combine quantitative measurements with fast fluorescence acquisitions to deliver a fully quantitative readout with increased temporal resolution and/or reduced light exposure.In this work, we set out to develop a strategy for determining absolute analyte activities using photochromic single FP-based biosensors. First, we developed proof-of-concept GECIs that display photochromism that is highly dependent on the local Ca2+ biosensors50. They consist of a circularly permutated variant of EGFP and a Ca2+-sensitive moiety made up of a calmodulin (CaM) domain and RS20, a CaM-binding peptide. Binding of Ca2+ ions by CaM leads to an interaction between CaM and RS20, resulting in a fluorescence increase that is largely due to a change in the pKa of the chromophore53. Well-performing photochromic variants of EGFP were previously reported56, which suggested that photochromic behavior might be introduced in the GCaMP biosensor design while retaining its sensitivity to Ca2+.The GCaMP family is the most widely used class of single FP-based Ca57, GCaMP6 58, GCaMP7s50, and GECO1.1 and GECO-1.245 as the starting scaffolds, and introduced mutations that were shown to induce photochromism in EGFP, in particular T65A, Q69L, V150A, V/A163S, and S205N (numbering according to EGFP)54. The resulting proteins were expressed in E. coli and screened for brightness and reversible photochromism with cyan and violet light  that were previously shown to increase the expression levels of EGFP59 and GCaMP variants61. However, these so-called superfolder mutations did not appear to influence the overall brightness in bacterial lysates and had a negative impact on the photochromism and Ca2+ sensitivity  and Ca2+-free states (apo), normalized by the fluorescence of the sat-form, resulting in Ca2+ sensitivities that are in line with those reported in literature58  state. A more detailed investigation of the dynamics of photochromic sensors was recently published in Adelizzi et al.62.The photochromism of each probe was then assayed in 11 different free Ca2+ concentrations for GCaMP3, GCaMP6s, GCaMP6s-Q, and GCaMP6s-T , and negative photochromism at higher Ca2+ concentrations (cyan excitation light leads to a decrease in fluorescence). This change in photochromism modalities likely results from changes in the pKa’s of the fluorescent (cis) and non-fluorescent (trans) chromophore states due to Ca2+ binding, since these pKa’s determine the directionality of the photochromism63.Interestingly, the mentclass2pt{minim65. Successful photochromism-based sensing using such labels thus requires that an initial photochromism cycle is always performed at the beginning of an experiment, and discarded from the analysis. A few more cycles may need to be discarded if the photochromism cycles are incomplete due to lower-intensity or short illumination, which can be determined by inspecting when the photochromism reaches a stable level. Left in the dark, the samples recover back to the initial state following the spontaneous thermal recovery pathway, which usually takes on the order of minutes or longer  biosensing. In principle, the PEAQ methodology can be combined with any biosensor where the presence of the sensed stimulus results in a change in photochromic properties. The low brightness of cells expressing GCaMP6s-Q restricted our analysis to regions-of-interest consisting of an entire cell, though the methodology could be applied to smaller regions or even in a pixelwise manner when combined with sufficiently bright signals. Since it is based on the quantification of fluorescence intensities, our method is sensitive to the presence of background signal, though this sensitivity is highly similar to that observed for FRET-based quantification , where the duration between photochromism-based acquisitions should be sufficiently short that changes in the local probe concentration are small or negligible. However, photochromic sensors that show different dissociation constants for photochromism and intensiometric measurements may limit the analyte concentration window over which the calibration can be performed successfully. Furthermore, any changes in analyte concentration during a calibration measurement can lead to distortions in the determined concentration, as is inherent to any method where the acquisition takes a finite amount of time, which can then propagate to the dependent intensity-based acquisitions.2+ levels can be extracted from cells showing very different cell brightnesses. This procedure allowed us to reduce the cyan light exposure by 80% while still delivering direct measurements of the absolute Ca2+ concentration.We verified the feasibility of this approach on HeLa cells expressing GCaMP6s-Q stimulated with histamine. Figure More generally, our intermittent quantification strategy can in theory be combined with any sensor where the sensing mechanism is (indirectly) associated with a change in fluorescence brightness. The calibration measurements could be performed using ratiometric excitation/emission measurements, by measuring the excited state lifetime, or analogous methodologies, and the intermittent measurements could be directly performed using just a single fluorescence acquisition. Such a strategy would likewise speed up the measurement and reduce the light dose, while taking full advantage of the benefits associated with the quantitative readout of choice. A combination with FRET-based sensors would also be possible, by acquiring e.g. only the donor or acceptor channel during an intermittent measurement.2+ concentration, but also on the intensity and duration of the cyan light. In many systems the exact excitation intensity at the sample may not be known precisely, such as when performing measurements deeper in tissues, where part of the light may be absorbed or scattered before reaching the region of interest. Such conditions also preclude the quantitative determination of the activity using purely intensiometric measurements, even if the probe concentration were known. We therefore investigated whether our method could be used reliably when the absolute excitation intensity is unknown.The extent of the sensor photochromism depends not only on the Ca2+ concentrations in a representative HeLa cell at 47 mW and 24 mW excitation power  biosensing. Starting from the GCaMP family of Ca2+ concentration not only affected the overall fluorescence brightness but also altered the photochromic properties of the biosensor. We quantified the extent of the photochromism by measuring the fluorescence before and after illumination with cyan light, providing a metric that is conceptually straightforward and easily measured. In vitro calibration of 2+ in solution.The Ca2+ concentrations in live HeLa cells stimulated with histamine, enabling us to follow a diverse range of Ca2+ dynamics. Our approach not only allowed the measurement of absolute Ca2+ concentrations, but also made it possible to determine when these results are less reliable due to saturation of the biosensor, and is independent of changes in local probe concentration that may occur through sample dynamics or photodestruction. We did find that GCaMP6s-Q displayed poor brightness when expressed in cells, indicating that further probe development will be needed to achieve robust performance across a variety of systems, or in systems characterized by a potentially reduced expression such as stable cell lines, unless the local probe concentration is increased via e.g. subcellular targeting. Conventional ratiometric or FRET-based probes will currently yield a higher performance for quantitative Ca2+ imaging.We then demonstrated the measurement of intracellular Ca2+ concentration. In principle, a single photochromism-based reference measurement is sufficient to enable fast quantitative measurements for the entire duration of the experiment, though in practice periodic photochromism-based measurements are likely required to adapt to the dynamics of the sample. The ability to introduce a non-invasive reference measurement at any time is a key advantage of our method. An iPEAQ-like procedure could also readily be used in other situations where an orthogonal readout is available, as would be the case when measuring the analyte using e.g. conventional ratiometric or fluorescence lifetime imaging.A possible downside of PEAQ biosensing is that inducing the photochromism involves more irradiation and is slower compared to the acquisition of a single fluorescence image, increasing the chance of phototoxic effects and making it more difficult to resolve fast dynamics. In response, we developed iPEAQ, a scheme in which quantitative photochromism-based measurements are alternated with one or more regular fluorescence acquisitions. Ordinarily, such intensity-based acquisitions do not permit the quantitative determination of the biosensor activity. However, we showed that absolute quantification can be achieved for both types of acquisitions, by leveraging the dependence of both the photochromism and the fluorescence brightness on the CaMany experimental scenarios require measurements in more complex samples such as tissues, where the absolute intensity of the excitation light is typically not known due to the absorption and scattering. We find that our method does not require knowledge of the absolute excitation intensity, provided that the measurements are performed in such a way that the photochromism is allowed to go to a level where the on- and off-switching are in equilibrium, or to a level where this equilibrium can be estimated. This can be readily verified, even if the absolute intensity is unknown, by monitoring the fluorescence response to the illumination. This approach is also fully compatible with the reduced light doses afforded by our alternating photochromism- and intensity-based measurements, and should allow our method to scale to a broad variety of samples and settings.As it utilizes a single FP-based biosensor and ratiometric readout, our method is also less sensitive to issues such as limited FP maturation or photodestruction, while also being readily amenable to the multiplexing of multiple biosensors emitting in different spectral bands. We also expect that our methodology can be applied to any FP-based biosensor that displays, or can be engineered to display, photochromism, including the many GCaMP-like biosensors that have been developed. Overall, our work readily extends the possibilities for fully quantitative measurements inside complex systems.E. coli, GCaMP3, 6s, 6m, 6f, 7s, GECO1.1 and GECO1.2 were cloned in a pRSET B vector and mutations were introduced using a modified QuikChange protocol70. Eukaryotic expression vectors were created by inserting PCR-amplified GECI genes between BamHI and EcoRI restriction sites of a pcDNA3 vector. GCaMP6s-Q was also cloned from pcDNA3 into the chicken beta-actin promotor vector (which appears to have increased protein expression)9. Primers were ordered at Integrated DNA Technologies and all constructs were verified by DNA sequencing (LGC Genomics).For expression in g for 20 min at 4 °C, frozen, thawed, resuspended in 500 μL lysis buffer , and subsequently incubated at 30 °C for 1 h while shaking. Cell debris was pelleted by centrifugation at 3000 × g, and 95 μL clarified lysate was transferred to a 96-well fluorescence microplate (Greiner). The cell lysate samples were mixed with EGTA to a final concentration of 1 mM, and fluorescence was measured at 490 nm excitation and 530 nm emission ). Subsequently, CaCl2  was added to the lysates and the fluorescence was measured again.The GECIs were transformed into JM109(DE3) competent cells (Promega) and incubated at 37 °C. The next morning, colonies were inoculated into 1.2 mL of LB media supplemented with ampicillin in 96 deep-well blocks. Cultures were grown at 30 °C for 36 h, then harvested by centrifugation at 3000 × 2) and switched back on in 30 steps of 50 ms with violet light . After each step an image was acquired with a camera exposure time of 20 ms and 10 mW cyan excitation light .The photochromism was measured on an Olympus IX71 inverted microscope equipped with a Spectra X Light Engine (Lumencor), a 10 × UplanSApo objective (Olympus), a ZT488RDC dichroic mirror and ET525/30 nm emission filter (both Chroma), and an ORCA-Flash4.0 LT+ sCMOS camera (Hamamatsu). Lysates were switched off in 30 steps of 250 ms with cyan light , followed by a Gibson Assembly (New England Biolabs) reaction with the PCR product and an XhoI/HindIII-digested pBAD/His-B vector (Thermo Fisher Scientific). Using pBAD-GCaMP6s as the template, five separate primers, each encoding a mutation from rsEGFP or rsEGFP2 , were pooled together and used to perform multi-site mutagenesis using the QuikChange Multi site-directed mutagenesis kit (Agilent). Due to imperfect reaction efficiency, this procedure was expected to introduce all five single mutations and all possible combinations of multiple mutations. The resulting plasmid library was used to transform DH10B electro-competent cells (Thermo Fisher Scientific). Transformed bacteria were plated on LB-agar plates and incubated overnight at 37 °C. A total of 48 green fluorescent colonies were picked from the plates and cultured in 5 mL LB media with ampicillin and arabinose at 37 °C overnight. Bacteria were pelleted by centrifugation and soluble cell lysates were extracted using Bacterial Protein Extraction Reagent . Lysates were then screened for photochromism. Briefly, bacterial lysate samples in clear PCR tubes were illuminated for 10 s using a custom-built bacterial colony fluorescence imaging macroscope71 to either switch-on  or switch-off  the protein fluorescence. The fluorescence change was assessed by visual inspection, and samples exhibiting photochromic behavior were immediately transferred to a multi-well plate to record the fluorescence excitation and emission spectrum using a fluorescence plate reader (Tecan). Of the 48 colonies screened, two photochromic variants were identified: GCaMP6s-T and GCaMP6s-Q. Later effort focused on the GCaMP6s-Q mutant due to its more pronounced photochromism contrast.The sensors with the T65A mutation were discovered during an independent and parallel effort. This effort aimed to produce a sensor by grafting the photochromism-inducing mutations of rsEGFP and rsEGFP255. The purified proteins were diluted in 30 mM MOPS, 100 mM KCl, pH 7.2  containing either 10 mM CaEGTA  or 10 mM EGTA . Fluorescence excitation and emission spectra were measured on an Edinburgh 920FLS fluorimeter with 1.5 nm slits. Excitation spectra were taken from 300 to 515 nm, with emission at 520 nm. Emission spectra were taken from 485 to 700 nm, with excitation at 480 nm. Spectra were normalized to the calcium-saturated excitation and emission maxima, respectively. Absorption spectra were taken on a UV/VIS spectrometer , and normalized using the absorbance at 280 nm.Proteins were purified largely following our previously described method2+ affinity assays were performed by employing a reciprocal dilution method with the Calcium Calibration Buffer Kit #1, according to the manufacturer’s instructions . A small aliquot of the stock protein solution was added to 2 mL of the Zero Free Ca2+ Buffer  to create the “zero Ca2+ sample”. The “high Ca2+ sample” was prepared by diluting exactly three times as much protein into 6 mL of 39 μM Free Ca2+ Buffer . The initial 0 mM CaEGTA/GCaMP sample was loaded in the cuvette, and absorbance and emission spectra were recorded. Consecutively, the measured sample was used to prepare the next solution by removing a given volume from the sample and replacing this with an equal aliquot of the “high Ca2+ sample”.Ca2+ titration, the same small amount of the stock protein was added to 1.2 mL of the Zero Free Ca2+ Buffer and to 1.2 mL of the 39 μM Ca2+ Buffer. The two buffer solutions were mixed in a 96-well plate to a final volume of 200 μL in order to obtain the desired EGTA concentrations from 10 mM to 0 mM. The plate was stored at 4 °C and measured the next morning on the Olympus IX71 microscope  at room temperature to evaluate the Ca2+ dependence of F0 − Fend)/F0 with F0 the fluorescence read at the first frame of the second off-switching cycle and Fend the last frame of the second off-switching cycle  and switched off in 10 image acquisition steps of 100 ms exposure time with cyan light .For the in vitro Ca2+ concentration.The in vitro titrations for the analysis of the PEAQ and iPEAQ measurements were measured with the same irradiation procedure and parameters as used during the actual measurements. The amount of cycles was reduced to 15 cycles per Caapo) or presence (sat) of free Ca2+. Further spectral analysis (absorbance and emission) of Ca2+ titrations, and respectively, photochromism, thermal recovery, and the quantum yield of fluorescence, all in apo and sat, were measured on a home-built setup described elsewhere72.Spectroscopic properties of the sensor variants were investigated in buffer solutions in the absence (2), and switched back on with one 100 ms violet irradiation . This was repeated every 2 s for 1000 cycles to determine the decrease in on-state fluorescence (F0) and photochromism contrast The GCaMP6s-Q photochromism in vitro fatigue was measured on the Olympus IX71 microscope . Purified GCaMP6s-Q protein was diluted in 39 μM Free Calcium Buffer and Zero Free Calcium Buffer and transferred to a 96-well plate. The well plate was covered to prevent evaporation. The protein was switched off with ten acquisitions , and switched back on in 30 steps of 10 ms violet irradiation . This cycle was repeated 3 times. Images were recorded after each irradiation with 9.6 mW cyan excitation  and 100 ms exposure time.For the in vitro kinetics, the well plate was prepared as described before for the fatigue measurements and measured on the Olympus IX71 microscope . The protein was completely switched off in 30 steps of 200 ms cyan irradiation  were determined according to Ward’s method73 using the literature values of EGFP, GCaMP3 and -6s as references. Quantum yield (QY) was determined relative to EGFP, having a QY of 0.674. Molecular brightness was defined as the product of ϵsat and QY, scaled to a value of 100 for EGFP.Extinction coefficients  or 5 mM EGTA . Absorbance spectra were measured on a Varioskan LUX Multimode Microplate Reader at pH values ranging from 4.5 to 10.5 and a plot of the absorption maxima of the anionic form in function of the pH was fitted to a sigmoid curve to yield the pKa.pH titrations were performed in a universal buffer consisting of 50 mM citrate, 50 mM Tris, 50 mM glycine, 100 mM NaCl, and either 5 mM CaCl2. Before transfection, cells were seeded in 35-mm glass bottom dishes (MatTek), and allowed to settle between 4 h and 24 h. Cells were transfected using the FuGENE 6 Transfection Reagent (Promega) at a ratio of 1 μg DNA/3 μL FuGENE 6 according to the manufacturer’s protocol. Cells were supplied with fresh growth medium after 24 h, and imaged 48 h after transfection. The estimated transfection efficiency was around 80%. The HeLa (ATCC-CCL-2) cells were acquired at ATCC and were regularly replaced from frozen stocks. No authentication or mycoplasma contamination tests were performed.HeLa cells were cultured in DMEM supplemented with 10% FBS, glutaMAX, and 0.1% gentamicin  at 37 °C and 5% COFor the co-culture of GCamp6sQ and Yellow Cameleon YC2.6, Hela cells were independently transfected in two flasks (25 mL) using the aforementioned transfection protocol. After 30 h, the flasks were rinsed twice with PBS and then the cells were detached using 500 μL of PBS with 0.05% trypsin. After 5 min of incubation at 37 °C, 4.5 mL of DMEM supplemented with FBS and glutaMAX was added. Cells transfected with GCamp6sQ and Yellow Cameleon YC2.6 were mixed in a 3:1 ratio. Then 600 μL of the resulting cell mixture was added to 35-mm dishes containing 1 mL of DMEM supplemented with FBS and glutaMAX.d-glucose at pH 7.4 (HHBSS). Comparable solutions were made without Ca2+ and Mg2+, but with all other components (HHBSS(−)). All imaging experiments were performed on the previously described Olympus IX71 microscope  at room temperature.For imaging, cells were rinsed twice and maintained in Hanks’ Balanced Salt Solution , supplied with 20 mM HEPES and 2 g/L 2+ titration in HeLa cells, the cells were washed twice and kept in HHBSS(−). Subsequently, a 2 × stock solution of EGTA/ionomycin was added to the cells to a final concentration of 3 mM EGTA and 5 μM ionomycin, followed by incubation at room temperature for 10 min. Then, the buffer was replaced with ionomycin- (5 μM) and saponin- (0.005%) supplemented Zero Free Ca2+ Buffer, and acquisition was started immediately. Three photochromism cycles were recorded every 90 seconds. The medium’s Ca2+ concentration was changed every 180 s through a reciprocal dilution of 10 mM CaEGTA buffer (supplemented with 5 μM ionomycin and 0.005% saponin) into the Zero Free Ca2+ Buffer, according to the manufacturer’s protocol . The proteins were switched on using a single 50 ms pulse of violet light , while off-switching was achieved by acquiring 10 images with 46.7 mW of cyan excitation light  and a camera exposure time of 100 ms. For the cell titrations, multiple 35-mm glass-bottom dish samples were imaged at two sample positions each.For the Ca2+ and incubated for 10 min at room temperature with Zero Free Ca2+ buffer supplemented with 5 μM of ionomycin. The buffer was replaced with the same solution with 0.005% saponin for the acquisition of the apo state fatigue measurement or the 39 μM Ca2+ buffer supplemented with 5 μM ionomycin and 0.005% saponin for the sat state. The in cell fatigue measurement was conducted with the aforementioned in vitro imaging parameters.For the in cell fatigue measurement, cells were rinsed twice with HHBSS without Ca2+ buffers supplemented with 10 μM rotenone, 5 μM cyclopiazonic acid, 1.8 μM 2-deoxy-d-glucose and 10 μM 4-bromo-A23187 according to the protocal described in75. The measurements were repeated multiple times with the same dish at different sample positions.The cell kinetics measurements were performed with the same imaging parameters as described in the in vitro spectroscopic characterization. The cells were permeabilized using the Zero Free or 39 μM Free Ca2+ dynamics, cells were imaged for a duration of 15 minutes during which the GCaMP6s-Q was switched on  and off  every 2 s. Cells were washed twice with regular HBSS, and approximately 15 s after the start of the experiment, histamine (50 μL) was added to the cells to a final concentration of 25 μM. For each independent histamine stimulation experiment, a 35-mm glass-bottom dish sample was imaged at one sample position. Each experiment was repeated with multiple dishes over multiple days. To determine For the imaging of histamine-induced Ca2, 395/25 nm) to switch GCaMP6s-Q on, followed by one image acquisition with a 100 ms cyan light pulse . These intensiometric acquisitions were repeated every 2 s.The intermittent PEAQ (iPEAQ) measurements were conducted analogously to the PEAQ experiments as described in the previous paragraph. PEAQ acquisitions were alternated with eight fluorescence acquisitions. The PEAQ experimental parameters can be found in the previous paragraph. The eight fluorescence acquisitions were performed with a 100 ms pulse of 59.3 mW violet light  for the donor (Chroma ET480/40 nm emission filter) and acceptor (Chroma ET545/40 nm emission filter) were acquired, as well as a detection with teal light  to register the acceptor bleaching (Chroma ET545/40 nm emission filter). The combined PEAQ and FRET measurement scheme was repeated every 10 s. To determine 2+].For the resting HeLa co-culture experiments with GCaMP6s-Q (cloned into chicken beta-actin promotor vector) and Yellow Cameleon YC2.60, the described PEAQ measurement protocol for histamine-induced Ca2, 337 mW/cm2, 501 mW/cm2, and 654 mW/cm2.Measurements in vitro and in HeLa cells were performed as described previously. Cyan powers used for excitation were 10.9 mW, 23.8 mW, 35.4 mW, and 46.2 mW as measured at the top of the objective. The measured powers correspond to power densities of 154 mW/cmFdiff is the difference between the the minimal and maximal value, h is the Hill coefficient, and Kd is the dissociation constant.All data analysis was done using IgorPro 8.0 (Wavemetrics). Titration curves were fitted with the following Hill equation:A background correction was applied to HeLa cell data, by subtracting the average intensity trace of a sample area containing no cells.2+ titration in HeLa cells: F0 and Fend were obtained from background-corrected average intensity traces of selected cells, and were used to calculate the Analysis of the Ca2+-oscillations: F0 and Fend were obtained from background-corrected average intensity traces of selected cells, and were used to calculate F0, and the red curve corresponds to Analysis of the histamine-induced Ca2+ titrations acquired with the aforementioned iPEAQ measurement protocol. Two separate titrations can be extracted for the PEAQ measurements  are extracted from the background-corrected cell fluorescence traces. The corresponding F0 rather than a full photochromism cycle. At each such point we can then recover the local Ca2+ concentration using2+ concentrations at the intermittent points where no photochromism is measured. In principle, the A values should be constant at each calibration measurement point if the probe concentration does not change in time. In practice, we found that these showed deviations in time due to the low brightness of our probe in cells , and interpolated the A value used to determine the local Ca2+ concentration in Eq. (2+ traces.At the intermittent measurement points we record only Further information on research design is available in the Supplementary informationReporting Summary


Acute myeloid leukaemia (AML) stem cells (LSCs) cause disease relapse. The CD47 “don’t eat me signal” is upregulated on LSCs and contributes to immune evasion by inhibiting phagocytosis through interacting with myeloid-specific signal regulatory protein alpha (SIRPα). Activation of macrophages by blocking CD47 has been successful, but the ubiquitous expression of CD47 on healthy cells poses potential limitations for such therapies. In contrast, CD123 is a well-known LSC-specific surface marker utilized as a therapeutic target. Here, we report the development of SIRPα-αCD123 fusion antibodies that localize the disruption of CD47/SIRPα signalling to AML while specifically enhancing LSC clearance.SIRPα-αCD123 antibodies were generated by fusing the extracellular domain of SIRPα to an αCD123 antibody. The binding properties of the antibodies were analysed by flow cytometry and surface plasmon resonance. The functional characteristics of the fusion antibodies were determined by antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity assays using primary AML patient cells. Finally, an in vivo engraftment assay was utilized to assess LSC targeting.+ CD47+ AML cells even in the presence of CD47+ healthy cells. Furthermore, SIRPα-αCD123 fusion antibodies confined disruption of the CD47-SIRPα axis locally to AML cells. In vitro experiments demonstrated that SIRPα-αCD123 antibodies greatly enhanced AML cell phagocytosis mediated by allogeneic and autologous macrophages. Moreover, SIRPα-αCD123 fusion antibodies efficiently targeted LSCs with in vivo engraftment potential.SIRPα-αCD123 fusion antibodies exhibited increased binding and preferential targeting of CD123SIRPα-αCD123 antibodies combine local CD47 blockade with specific LSC targeting in a single molecule, minimize the risk of targeting healthy cells and efficiently eliminate AML LSCs. These results validate SIRPα-αCD123 antibodies as promising therapeutic interventions for AML.The online version contains supplementary material available at 10.1186/s13045-021-01163-6. Therapeutic options for acute myeloid leukaemia (AML) are limited, and the majority of patients relapse due to persistent chemorefractory LSCs –3. TargeNevertheless, CD47 is ubiquitously expressed on healthy cells as well, which generates an antigen sink lowering the effective dose and comprising a potential site of toxicity for αCD47 therapies , 15. ComSimilar to CD47, the interleukin-3 receptor alpha chain (CD123) is upregulated on AML LSCs and is associated with increased proliferation of AML cells and a poor prognosis –21. Furt+ CD47+ cells and stimulated efficient natural killer (NK) cell-mediated lysis of AML compared to the conventional αCD123 antibody in vitro. Importantly, SIRPα-αCD123 fusion antibodies blocked CD47 locally on CD123+ cells and induced phagocytosis of primary AML cells by allogeneic and autologous macrophages in vitro. Finally, the 2 × SIRPα-αCD123 antibody targeted LSCs that are capable of engrafting and reinitiating AML in an in vivo model.To improve AML LSC targeting and clearance, we fused an αCD123 antibody with the endogenous N-terminal SIRPα immunoglobulin V-like domains and generated 1 × SIRPα-αCD123 and 2 × SIRPα-αCD123 fusion antibodies. Both of our antibodies exhibited improved binding to CD123L) and variable heavy (VH) sequences of talacotuzumab , whereas treatment with the αCD123 antibody slightly delayed the time to positive engraftment  , We used the extreme limiting dilution analysis algorithm to determine whether the nearly absent engraftment in the 2 × SIRPα-αCD123 condition was due to specific LIC targeting or a lower number of injected residual cells . Even thThe ubiquitously expressed surface marker CD47 interacts with the SIRPα receptor to inhibit myeloid cell-mediated phagocytosis of autologous cells , 15. BloThe most serious side effects reported from CD47-targeting agents in clinical trials are anaemia and thrombocytopenia , 46–49. Despite the low-affinity binding of the SIRPα domains, SIRPα-αCD123 fusion antibodies were able to induce the same or even higher phagocytosis than high affinity αCD47 either alone or in combination with αCD123. This is in line with the well-known synergy between CD47-SIRPa axis disruption and prophagocytic signals , 50–52 aAML LSCs reside in specific niches in the bone marrow . Antibod+ LSCs at diagnosis is correlated with a better response to treatment and survival [+ LSCs [+ LSCs might lead to more durable responses and prolonged survival. We show here that compared to αCD123, SIRPα-αCD123 antibodies exhibit increased targeting efficacy of CD123+ CD47+ AML cells due to avidity-dependent binding to both antigens. Our fusion antibodies could take advantage of the high expression of both CD123 and CD47 on LSCs and effectively address this population. Indeed, we observed an extreme reduction in the engraftment of AML after an ex vivo ADCC assay with the 2 × SIRPα-αCD123 antibody, as our antibodies stimulated NK cell-mediated cytotoxic lysis of AML LSCs. The increased avidity of SIRPα-αCD123 antibodies thus provides the opportunity to preferentially target and eliminate AML LSCs.Because chemorefractory LSCs build a reservoir for relapse, elimination of these cells is essential for AML treatment , 2. In ysurvival . Similar [+ LSCs . TherefoBecause of avidity-dependent binding to CD123 and CD47, SIRPα-αCD123 antibodies could further target malignant LSCs cells over healthy haematopoietic stem cells, which express low levels of CD47 and minimal amounts of CD123 , 21, 64.+ cells when the αCD47 antibody Hu5F9-G4 was used. A growing body of evidence indicates that adaptive immunity, especially the activation of CD8+ T cells, further contributes to the effects observed in response to CD47-SIRPα inhibition [While we are the first to combine CD123 and CD47 targeting, other therapeutic molecules have been developed against CD123 alone –67. Talahibition –75. As SIn summary, we demonstrated that SIRPα-αCD123 antibodies specifically target LSCs, mediate their efficient clearance and stimulate phagocytosis of AML while restricting CD47-related on-target off-leukaemia toxicity. These encouraging results establish SIRPα-αCD123 antibodies as a promising approach for LSC targeting for prolonged remission in AML patients. Future in vivo studies using an appropriate AML mouse model are necessary for the translation of this approach into a clinical setting.Additional file 1. Supplementary tables and figures.


CEBPA, FLT3, GATA2, NPM1, PTPN11, TET2, and WT1 and a lower prevalence of mutated KIT, KRAS, and NRAS compared with AK-AML. Mutated CEBPA often co-occurred with mutated GATA2, whereas mutated FLT3 co-occurred with mutated WT1 and NPM1. In multivariate regression analysis, we identified younger age, WBC count ≥50 × 109/L, FLT3-internal tandem duplications, and mutated WT1 as independent predictors of adverse prognosis and mutated NPM1 and GATA2 as independent predictors of favorable prognosis in NK-AML. In conclusion, NK-AML in children is characterized by a unique mutational landscape which impacts the disease outcome.Normal karyotype acute myeloid leukemia (NK-AML) constitutes 20–25% of pediatric AML and detailed molecular analysis is essential to unravel the genetic background of this group. Using publicly available sequencing data from the TARGET-AML initiative, we investigated the mutational landscape of NK-AML in comparison with abnormal karyotype AML (AK-AML). In 164 (97.6%) of 168 independent NK-AML samples, at least one somatic protein-coding mutation was identified using whole-genome or targeted capture sequencing. We identified a unique mutational landscape of NK-AML characterized by a higher prevalence of mutated  FLT3-internal tandem duplication [ITD]) [Acute myeloid leukemia (AML) is a heterogenous malignant disease of the bone marrow resulting from the accumulation of acquired somatic genetic lesions in myeloid progenitor cells . Overalln [ITD]) .FLT3, NPM1, and CEBPA is useful in these cases to identify prognostically important mutations [FLT3-ITD positive AML [Importantly, ~20–25% of pediatric AML show no cytogenetic abnormalities following routine cytogenetic examination, and this group is referred to as normal karyotype or cytogenetically normal AML (NK-AML) ,8,9,10. utations ,12,13,14utations ,16 inclutive AML .From the publicly available data from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative AML study (TARGET-AML) , we herehttps://target-data.nci.nih.gov/Public/AML/ (accessed on 24 January 2021).The present study was based on the publicly available data from the TARGET-AML initiative . The oriThe AML patient samples in the TARGET-AML study were obtained from the Children’s Oncology Group protocols AAML0531 , AAML03PCytogenetic data including International System for Human Cytogenetic Nomenclature (ISCN) karyotypes used for disease classification were obtained from the clinical data files.FLT3, NPM1, CEBPA, and WT1 provided in the clinical data. For mutational burden of single nucleotide variants (SNVs) and insertions and deletions (indels) in each sample, we used the spreadsheet reporting variant calls from Strelka and SAMtools mpileup variant callers  and TCS (n = 684) (KMT2A (MLL) rearranged AML, another abnormal karyotype AML and NK-AML. The first three groups are collectively referred to as abnormal karyotype AML (AK-AML) in this study. The assigned categorization based on ISCN karyotypes remained throughout all analyses in this study.Based on the cytogenetic information provided in the clinical data, samples were categorized as core-binding factor AML (CBF-AML) including t and inv16(p13.1q22), Recurrently mutated genes (mutated in ≥2 samples) were identified in NK-AML. The SNV/indel mutational burden in NK-AML vs AK-AML was plotted and tested for equal distributions using the Wilcoxon rank sum test. The SNV/indel mutational landscapes of NK-AML vs AK-AML were visualized using two-level doughnut charts , conside2 test for categorical variables including mutation status. Event-free survival  and overall survival (OS) with 95% confidence intervals (CIs) were computed using the Kaplan-Meier estimator. Cumulative incidence of relapse (CIR) was estimated using the pseudo values method with death as competing risk [FLT3-internal tandem duplication (ITD), and mutational status of other co-occurring genes as relevant. Stratified analyses included cytogenetic group for the entire study cohort and for the NK-AML cohort, baseline characteristics and mutational status for genes mutated in ≥10 samples with wildtype NK-AML patients as reference. Stem cell transplantation was excluded from the analysis due to no time-points provided in the clinical data necessary for its inclusion in the model as a time-dependent variable. The assumption of proportional hazards was verified using Schoenfeld residuals test and we checked for multicollinearity computing variance inflation factor and tolerance values. Significance level was set at p < 0.05.Baseline patient characteristics and mutation frequencies were summarized using descriptive statistics. For comparing NK-AML vs AK-AML characteristics Wilcoxon rank sum test was used for continuous variables and χing risk . Log ranR (v3.6.2) using the Maftools library (v.2.2.10).Data management and analysis of the genetic and clinical data were performed using Stata Statistical Software Package . Analysis of co-occurrence and mutual exclusivity was performed in KMT2A rearranged, n = 175; other AK-AML, n = 242). p < 0.001) which has previously been reported [p < 0.001), and a high frequency of stem cell transplantation (SCT) in first complete remission (CR1) (p < 0.001) compared to AK-AML. No significant differences were seen in 5-year EFS and OS. However, 5-year CIR in NK-AML was lower compared with KMT2A rearranged and other AK-AML , aged 20 years or above (n = 16), French-American-British classification (FAB) M3 morphology (n = 2), and no cytogenetic information (n = 44), to obtain the final study cohort 873 patients in this analysis. We identified 208 (24%) NK-AML and 665 (76%) AK-AML patients . This may reflect a higher amount of bone marrow aspirate acquired from older patients, needed for sequencing studies.AML samples from 661 of 873 (76%) patients were analyzed by WGS and/or TCS including samples from 168 of 208 (81%) NK-AML patients . Of the KMT2A-MLLT4, KMT2A-MLLT3, HNRNPH1-ERG, NUP98-NSD1, CBFA2T3-GLIS2, and SEPT6-KMT2A. CNVs were identified in samples from eight patients with ZEB2 as the most commonly impacted gene (n = 4)  and gene fusions affecting 135 different genes were identified in samples from 164/168 (97.6%) patients (see  (n = 4) . In fourp = 0.0001) with a median of 3 called SNVs/indels in NK-AML , NPM1 , WT1 , NRAS , and CEBPA  as the top five most commonly mutated genes in NK-AML. We found significantly higher prevalence proportions of FLT3 (p < 0.001), NPM1 (p < 0.001), WT1 (p < 0.001), CEBPA (p < 0.001), GATA2 (p < 0.001), PTPN11 (p = 0.01), and TET2 (p = 0.006) mutants in NK-AML compared with AK-AML, whereas mutated NRAS (p = 0.05), KIT (p < 0.001), and KRAS (p < 0.001) were more rarely detected in NK-AML. Mutated NPM1 was associated with older age  in NK-AML.NPM1 (14%), tumor suppressors/WT1 (10%), and DNA methylation (7%). In comparison, the AK-AML mutational landscape mostly included genes involved in activated signaling (63%), chromatin modification (11%), and transcription factors (8%).CEBPA/GATA2 (p = 1.3 × 10−7), FLT3/WT1 (p = 0.03), and FLT3/NPM1 (p = 0.045) as commonly co-occurring events, whereas mutated CEBPA was likely to be mutually exclusive of both mutated NPM1 (p = 3.0 × 10−7) and FLT3 (p = 2.9 × 10−5).Next, we investigated the mutual exclusive and co-occurring gene pairs in NK-AML and AK-AML, considering genes mutated in ≥2% of samples in their respective groups C,D. Of tKMT2A rearranged AML  and other AK-AML   and other AK-AML  (Median follow-up time for non-deceased NK-AML patients was 5.5 years (range 0.3–10.9). We investigated EFS using Kaplan-Meier curves stratified by selected variables with log rank test results . Additio 32–44%) A. Five-y 55–69%) .p < 0.001; 9/L was also associated with inferior EFS , co-occurrence of a FLT3-ITD of low AR (<0.5) with mutated NPM1 was associated with a favorable outcome  whereas FLT3-ITD of high AR (≥0.5) with mutated NPM1 was associated with intermediate risk  and mutated GATA2  were associated with favorable prognosis  , PTPN11 , nor TET2 , were associated with any significant differences in prognosis  F. Of therognosis .FLT3-ITDs were predictive of inferior EFS  and OS  adjusted for NPM1 status  and OS , and this effect was further strengthened by co-occurrence of FLT3-ITD  irrespective of FLT3-ITD allelic ratio  and OS , including in co-occurrence with FLT3-ITDlow . In the unadjusted analysis, mutated CEBPA was associated with better EFS and OS; however, after adjustment including GATA2 status the effect was obliterated  adjusted for CEBPA status. In line with the 5-year survival estimates, mutated NRAS, PTPN11, and TET2 were not predictive of disease outcome in the multivariate analysis.In a multivariate analysis stratified by mutational status, 1 status . Mutatedic ratio . Mutatediterated . In contIn this study, we exploited the publicly available data from the TARGET-AML study  to invesIn 164 of 168 NK-AML patients not found to harbor any chromosomal aberrations from routine testing, the detailed sequencing analyses by WGS and TCS detected at least one protein-coding somatic mutation, demonstrating the utility of molecular characterization using these methods. Only four NK-AML patients were without any mutations identified.CEBPA, FLT3, GATA2, NPM1, PTPN11, TET2, and WT1 as well as a lower prevalence of mutated KIT, KRAS, and NRAS compared with AK-AML . Of the commonly mutated genes in NK-AML, CEBPA/GATA2, FLT3/WT1, and FLT3/NPM1 were identified as frequently co-occurring events meanwhile mutated CEBPA was mutually exclusive of mutated NPM1 and FLT3. This is of importance to consider when investigating the clinical implications of mutations in these genes.Based on the results from WGS and TCS analysis we identified a unique mutational landscape in pediatric NK-AML characterized by a higher prevalence of mutated h AK-AML A,B. The CBFA2T3-GLIS2) [NUP98 fusions) [Notably, six gene fusions not reported in the cytogenetic data were detected by WGS in NK-AML. This highlights the relevance of genomic sequencing to be implemented as clinical cytogenomic analyses as recently suggested ,28. Cert3-GLIS2)  and rearfusions) . For thefusions) ,28,30,31FLT3-ITD, and mutated WT1 as independent predictors of a poor outcome, and mutated NPM1 and GATA2 as independent predictors of favorable outcome in NK-AML. To our knowledge, younger age has not previously been highlighted as a risk factor in NK-AML. Possible confounders should be considered for this association, especially cryptic CBFA2T3-GLIS2 fusions associated with poor prognosis, infancy, and FAB-M7 morphology (4 of 10 with known FAB class within the 0–1 year age group were M7 in this study) [FLT3-ITD with an inferior EFS is well established in the literature [WT1 have been reported [NPM1 irrespective of FLT3-ITD status has also previously been reported [CEBPA has previously been reported to be an independent predictor of favorable outcome [GATA2 status. In contrast, we identified mutated GATA2 as an independent predictor of improved EFS adjusted for CEBPA status.In our multivariate analysis, we identified younger age, WBC count ≥50, s study) ,33. Noneterature ,12 and areported ,35. The reported . Mutated outcome ; howeverNPM1, did show some age dependency. Additionally, it should be noted that not all samples were analyzed using the same application. Hence, only 39 of 168 NK-AML were analyzed by WGS which resulted in an ascertainment bias in terms of limited detection of structural variation . Finally, the data used for this study did not allow us to adjust for the effect of stem cell transplantation in the multivariate analysis as stated in Some limitations must be considered interpreting these results. The complete dependency on the reported results of the study by Bolouri et al. may represent a limitation since we did not have any influence on the variant prioritization and a reanalysis of their raw data potentially could lead to the identification of previously unreported variants. Of note, we identified an older age among WGS/TCS analyzed subjects compared to non-sequenced subjects in the TARGET-AML cohort, possibly explained by the lower amount of bone marrow aspirate acquired from infant patients hindering inclusion for sequence analysis. This selection bias may have impacted the distribution of the mutational landscape of NK-AML as the prevalence of certain gene mutations, for instance, FLT3-mutated AML currently undergoing a phase II trial [IDH1, such as ivosedinib that is Food and Drug Administration (FDA) approved for relapsed/refractory AML in children ≥12 years (NCT03245424).The TARGET-AML study  has provII trial , and inhFuture molecular studies of pediatric AML will continue to be important to support this development. In this analysis, we only considered the genomics of NK-AML. However, to be able to obtain a more comprehensive understanding of the disease biology, multi-omics approaches investigating both the epigenome, transcriptome, microRNome, and proteome are needed.FLT3-ITD, and mutated WT1 as independent predictors of a poor outcome and mutated NPM1 and GATA2 as independent predictors of favorable outcome.Normal karyotype AML in children represents a unique group within pediatric AML with its own distinct genetic background and biology compared with abnormal karyotype AML. We identified younger age, WBC count ≥50,


In the present study, hypoxia exposure of rat's model was established. Two‐pore segment channels (TPCs) silencing was achieved in rats' models by injecting Lsh‐TPC1 or Lsh‐TPC2. The effects of TPC1/2 silencing on PAH were evaluated by H&E staining detecting pulmonary artery wall thickness and ELISA assay kit detecting NAADP concentrations in lung tissues. TPC1/2 silencing was achieved in PASMCs and PAECs, and cell proliferation was detected by MTT and BrdU incorporation assays. As the results shown, NAADP‐activated [Ca2+]i shows to be mediated via two‐pore segment channels (TPCs) in PASMCs, with TPC1 being the dominant subtype. NAADP generation and TPC1/2 mRNA and protein levels were elevated in the hypoxia‐induced rat PAH model; NAADP was positively correlated with TPC1 and TPC2 expression, respectively. In vivo, Lsh‐TPC1 or Lsh‐TPC2 infection significantly improved the mean pulmonary artery pressure and PAH morphology. In vitro, TPC1 silencing inhibited NAADP‐AM‐induced PASMC proliferation and [Ca2+]i in PASMCs, whereas TPC2 silencing had minor effects during this process; TPC2 silencing attenuated NAADP‐AM‐ induced [Ca2+]i and ECM in endothelial cells, whereas TPC1 silencing barely ensued any physiological changes. In conclusion, TPC1/2 might provide a unifying mechanism within pulmonary arterial hypertension, which can potentially be regarded as a therapeutic target.Pulmonary arterial hypertension (PAH) is a form of obstructive vascular disease. Chronic hypoxic exposure leads to excessive proliferation of pulmonary arterial smooth muscle cells and pulmonary arterial endothelial cells. This condition can potentially be aggravated by [Ca PAH is caused by morphological changes in the precapillary pulmonary vessels.An array of factors, including aberrant cell proliferation in the vascular wall, the loss of endothelial cell function and vascular remodelling accompanied with extracellular matrix (ECM) overdeposition in endothelial cells, can potentially contribute to PAH progression.2+ mobilization plays a crucial role in pulmonary vascular remodelling caused by chronic hypoxia.2+ signalling is enhanced in patients with idiopathic PAH. The elevated concentration of cytosolic free calcium within PASMCs can be regarded as a main inducing factor of pulmonary vasoconstriction and trigger calcium‐sensitive signalling cascades through calmodulin elicit an increase in the proliferating ability of cells.2+ entry) via SOC (store‐operated Ca2+ channels) is considered as a critical mechanism for enhancing cytosolic free [Ca2+].2+‐induced elevation in protein (AP)‐1 binding activity within PAEC is an essential process in AP‐1‐responsive gene up‐regulation, pulmonary vascular cell proliferation induction and the vascular remodelling of the lungs of hypoxia‐induced PAH patients.Cytoplasmic Ca2+ release can selectively be recruited to induce the Ca2+ response to specific cues in circulating ECFCs.2+ ]i in an extracellular calcium‐independent way, which could be disrupted via an NAADP antagonist Ned‐19, suggesting the release of calcium from acidic endolysosomal calcium stores.2+ mobilization, PASMC proliferation and PAEC angiogenesis are unknown as of yet.The increase in cytoplasmic calcium is controlled by calcium‐mobilizing messengers. For example, the application of NAADP (nicotinic acid adenine dinucleotide phosphate), a known calcium‐mobilizing molecule, to microsomes of aortic SMC (smooth muscle cells) induced calcium release.2+ with or without NAADP in PASMCs or PAECs, respectively, and on the proliferation of PASMCs or angiogenesis of PAECs were evaluated. This is regarded as a potential target in the treatment of PAH.In this study, a hypoxia‐induced rat PAH model was established; PASMCs and PAECs were isolated, and generation of NAADP and TPC1/2 was examined. Furthermore, the effects of TPC1/2 on PAH, on Ca22.12 inhalation was used to achieve euthanasia. The aforementioned mentioned process was carried out under the administration of an analgesic. One week following exposure, all rats were injected with lentivirus containing sh‐NC, sh1‐TPC1, or sh2‐TPC1 or sh1‐TPC2 or sh2‐TPC2. A further two weeks later, the pressure of the right ventricle (RV) and left ventricle (LV) was measured; the pulmonary tissues were harvested for additional assays. The pulmonary artery was infused with normal saline to cleanse the blood off of the pulmonary vessels. For histological analysis, the pulmonary veins were subsequently ligated. The pulmonary artery and trachea were perfused with 10% paraformaldehyde at constant pressure (100 cm H2O for the pulmonary artery and 25 cm H2O for the trachea) to fully distend the pulmonary blood vessels and airway, respectively. Peripheral lung specimens were then excised, fixed in 10% paraformaldehyde, paraffin‐embedded and sectioned for morphometric analysis of pulmonary arterioles. All vivisection experiments were performed as per National Institutes of Health (NIH) guidelines and were approved by the institutional standing committee on animals. The flow chart of animal model building and further experiments is depicted in Figure A total of 30 adult male Wistar rats weighing 190‐230 g were exposed to normobaric hypoxia (10% oxygen environment) in a ventilated hypoxia chamber 6 hours daily over 3 weeks and were compared to a control of 30 rats treated in normoxic conditions. To establish a state of hypoxia, the chamber was flushed with a gas mixture of room air and nitrogen from a liquid nitrogen reservoir. The age‐ and weight‐matched control 30 rats were maintained at an oxygen saturation of 21%. The anaesthetic used was sodium pentobarbital at a dosage of 40‐60 mg/kg. CO2.2Frozen sections were warmed for 60 minutes at room temperature, fixed for 15 minutes in the precooled acetone at 4°C and rinsed for 3 minutes with distilled water. The sample was stained for 5 minutes in the haematoxylin staining solution  and then rinsed once with water before being placed in 95% ethanol for 5 seconds and stained for approximately 1 minutes in the eosin solution (Beijing Beifang Biologic Technology Research Institute). The sample was then immersed in 95% ethanol for 2 minutes, transferred to xylene, mounted with central gum and observed under a microscope . The nucleus was stained in blue, whereas the cytoplasm was red or lighter hues of red. Photographic analysis was carried out with an inverted microscope. The wall thickness was quantified and analysed using the following formula: ratio = ‐inner diameter (ID))/ED2.3The NAADP concentrations in lung tissues were determined through the enzyme‐linked immunosorbent (ELISA) assay kit purchased from MyBioSource . The operation was carried out as per the instructions manual of the ELISA kit. The 96‐well plates were briefly covered with 100 μl/well of capture antibodies for each cytokine, as per the manufacturer's instructions (Peprotech Inc). The plates were incubated overnight at room temperature and washed with 0.05% Tween‐20 in PBS thrice. Then, 300 μl of blocking buffer 1% BSA in PBS was added to each well. Plates were incubated for 1 hour, and 100 μl of the samples (diluted 1:2) or standard solutions was added. The plates were incubated at room temperature for 2 hours. 100 μl of detection antibody and 100 μl of a conjugated avidin solution were added to each well. After 20 minutes, 100 μl of ABTS liquid substrate solution was added. The plates were maintained at ambient temperature, and the colorimetric reaction was read in a spectrophotometer at a wavelength of 405 nm with a correction of 650 nm.2.42, 5% CO2 and 92.5% N2 for 24 hours before the study.The isolation of rat PASMCs was in accordance with previously carried out studies.2.52, 5% CO2 and 92.5% N2 for 24 hours before study.The isolation of rat PAECs was according to the previous studies.2.6−ΔΔCt method.Total RNA was extracted from lung tissues and primary cells using the RNeasy mini kit (Qiagen) following standard procedures. Real‐time PCR was performed on triplicate samples using a miScript SYBR Green PCR kit (Qiagen) on the ABI 7500 Real‐time PCR System  following the 22.78 TU/ml) on the 7th day after hypoxia treatment. A fortnight later, the contents of TPC1/2 mRNA and TPC1/2 protein in lung tissue were detected through real‐time PCR and Western blot, respectively. For cell transfection, rat PASMCs or PAECs were plated at a concentration of 2 × 105 cells/ml and incubated for 16 hours. For infection, 1.5 ml/well viral supernatant was used to replace the medium. Cells were incubated at 37°C for 10 hours, and then, the fresh media was used to replace the viral supernatant. 48 hours after the infection, the infection efficiency was verified using Western blot or real‐time PCR assays.An effective short hairpin RNA targeting rat TPC1 or TPC2 was constructed and subcloned into the plasmid of lentivirus vectors, pLKO.1‐EGFP. Both the pLKO.1‐EGFP‐TPC1/2 and Lentivector package plasmid mixes were transferred into the 293T cells . The culture supernatant was harvested, and the virus titre was detected 48 hours following transfer. Rats were randomly divided into 2 groups: the normoxia group and hypoxia group. The rats in both hypoxia and normoxia group underwent venous injection of Lsh‐NC (negative control), Lsh1‐TPC1 or Lsh2‐TPC1 or Lsh1‐TPC2 or Lsh2‐TPC2 lentivirus  were used for Ca2.9g for 5 minutes at 4°C, the resulting supernatant was subsequently collected, and the protein concentration was estimated using a BCA assay. 20 μg of protein sample was resolved on an 8% SDS‐polyacrylamide gel and electro‐transferred onto a PVDF membrane (Millipore). The membrane was blocked with 5% (w/v) nonfat dry milk in PBS containing 0.02% Tween‐20 for 1 hours at room temperature, followed by overnight incubation at 4°C with a specific primary antibody. The following antibodies were used: anti‐TPC1 , anti‐TPC2 , anti‐CaMKII , anti‐calcineurin  and anti‐GAPDH . The GAPDH level was also determined and used as a loading control. The membrane was washed and incubated with the peroxidase‐conjugated goat anti‐rabbit secondary antibody (Bio‐Rad) at 1:2500 dilution at room temperature for 1 hours. Protein bands were detected by enhanced chemiluminescence (Pierce) and photographed using a Gel Logic 200 image system (Kodak).Primary cultures of rat PASMCs and PAECs were washed with phosphate‐buffered saline. The homogenate was centrifuged at 1000 × 2.102+ ([Ca2+] i) determination, PASMCs or PAECs cultured on 35‐mm dishes were incubated in a culture medium containing 3.5 μmol/L FURA‐2‐AM (Invitrogen) for 1 hours at 37°C and subsequently rinsed with HBSS (Sigma). Each dish was placed into a culture chamber at 37°C on the stage of an inverted fluorescence microscope (NikonTE2000E), connected to a cooled CCD camera . Samples were illuminated alternately at 340 and 380 nm using a random‐access monochromator , and emission was detected using a 510 nm emission filter. Images were acquired (1 ratio image per second) using Metafluor software .For intracellular Ca2.114 per well and cultured for 24 hours, followed by infection of Lsh‐TPC1 in the absence or presence of NAADP . The MTT (Sigma) was added until a final concentration of 0.5 mg/ml, and the cells were incubated for 4 hours at 37°C. The absorbance at 490 nm was measured by a microplate reader (Bio‐Rad). Each experiment was repeated at least thrice in triplicate.Cells were plated in a 96‐well plate at 2 × 102.12By measuring 5‐bromo‐2‐deoxyuridine (BrdU) incorporation, the DNA synthesis in proliferating cells was determined. Cells were seeded in 96‐well culture plates at a density of 2 × 10 cells/well and infected with Lsh‐TPC1 in the absence or presence of NAADP. Following a period of 24 hours or 48 hours, cells were incubated with a final concentration of 10 μmol/L BrdU (BD Pharmingen) for 2 hours. Then, the medium was removed, the cells were incubated with peroxidase‐coupled anti‐BrdU antibody (Sigma‐Aldrich) for 60 minutes at RT, washed three times with PBS, incubated with peroxidase substrate (tetramethylbenzidine) for 30 minutes, and the 450 nm absorbance values were measured for each well. Background BrdU immunofluorescence was determined in cells not exposed to BrdU but stained with the BrdU antibody.2.13P < .05) of the changes was assessed by paired or unpaired Student's t tests.Data are expressed as means ± SD Statistical significance (33.1P < .01)  . H&E staining results showed that following hypoxia exposure, the pulmonary arterial wall was thickening while narrowing its lumen, manifested as the up‐regulation of the external diameter ratio (ED): inner diameter (ID) by hypoxia stress. Figure ; the mea) Figure . The RS/) Figure . The afo3.2To explore the possible role of TPC1/2 in the pathogenic process of hypoxic exposure‐induced PAH, the NAADP concentration, TPC1/2 mRNA and protein expression within PAH rat lung tissues in each group were assessed and their correlations were analysed. ELISA results showed that NAADP concentration in the hypoxic group was significantly up‐regulated when compared to the normoxia group Figure . ConsistIn order to further explore the possible functional roles of TPC1/2 in PAH, a lentiviral vector expressing shRNA targeting TPC1/2 was constructed, namely  and the negative control lentiviral vector (Lsh‐NC containing the coding sequence for nonsense siRNA). Rats in hypoxia and normoxia groups were injected with the above lentivirus, respectively. Real‐time PCR was performed to verify the inhibitory efficiency of TPC1/2 mRNA expression in PAH rat lung tissues, which demonstrated that Lsh1‐TPC1/ Lsh2‐TPC1 significantly down‐regulated TPC1 mRNA expression Figure , and LshTo further explore the possible role of TPC2 in PAH, the changes in pulmonary morphology in peripheral lung sections from the hypoxic group and normoxic control animals were examined by H&E staining Figure . ResultsP < .01). Interestingly, TPC1/2 knockdown has not induced any apparent reversal in pulmonary artery pressure and RS/LV + S under normoxic conditions, suggesting that TPC1/2 exert their effect under hypoxic stress. The above data prove that the TPC1/2 knockdown could partially improve hypoxia stress‐caused changes during PAH.To confirm the possible role of TPC1/2 in PAH, the mean pulmonary artery pressure and the RS/LV + S from each group were calculated. The result proved that both the mPAP (mean pulmonary artery pressure) Figure  and the 3.32+ signals in PASMCs between endolysosomes and the sarcoplasmic reticulum modulate vascular reactivity and other cell functions. Herein, it is revealed that TPC1/2 knockdown proved the mean pulmonary artery pressure and PAH morphology. To further investigate the mechanism by which TPC1/2 might affect PAH development, the effects of TPC1/2 on [Ca2+]i in PASMCs were subsequently evaluated. Firstly, the impact of hypoxia on NAADP‐mediated Ca2+ signals was validated in PASMCs and PAECs. Figure 2+]i, and Ca2+ signal markers were enhanced in PAECs and PASMCs under hypoxia, compared with normoxic group. Moreover, under nigericin (50 μmol/L) treatment, which disrupted the lysosomal Ca2+ store,2+]i was increased in PAECs and PASMCs in response to hypoxia, indicating that the lysosomal Ca2+ content was increased in the hypoxic group  exerts a significant effect on controlling the formation of blood vessels.e Figure . These d3.7Herein, it is confirmed that TPC1/2 plays essential roles in NAADP‐mediated calcium release within PAH rat models. Within PASMCs, TPC1 plays a dominant role by regulating NAADP‐mediated calcium release in PASMCs and PASMC proliferation; within PAECs, TPC2 plays a dominant role through the regulation of NAADP‐mediated calcium release within PAECs and PAEC angiogenesis Figure .42+ signalling transduction.Pulmonary arterial hypertension induced by hypoxia is a life‐threatening progressive disorder characterized by abnormal blood pressure levels in pulmonary arteries. Thorough studies provided evidence that excessive proliferation of PASMC and the angiogenesis of PAECs could be correlated with the enhanced activation of Ca2+ mobilizer stored within cells.TPC gene family encodes a newly identified protein involved in the endolysosome‐targeted calcium release gated by NAADP, a Ca2+ release in PAH, whether TPC knockdown could act on [Ca2+]i in PASMCs was initially investigated. Upon NAADP‐AM stimulation, [Ca2+]i in PASMCs immediately presented a marked increase; after TPC1/2 knockdown by Lsh‐TPC1/2 infection, [Ca2+]i was reduced; however, TPC1/2 knockdown exhibited different inhibitory efficiency. Compared to the Lsh‐NC group, TPC1 knockdown sharply down‐regulated [Ca2+]i, whereas TPC2 knockdown only caused a slight down‐regulation on [Ca2+]i, suggesting the dominant effect of TPC1 on regulating [Ca2+]i within PASMCs. To further confirm our hypothesis, the mRNA and protein level changes of Ca2+ signalling‐related factors were subsequently monitored CaMKII and calcineurin in PASMCs. Consistent with [Ca2+]i determination, NAADP‐AM markedly increased CaMKII and calcineurin mRNA and protein expression within PASMCs. After TPCs knockdown, different results were yielded. TPC2 knockdown caused a slight reduction on CaMKII and calcineurin mRNA and proteins, whereas TPC1 knockdown or TPC blocker sharply suppressed CaMKII and calcineurin mRNA and proteins. As mentioned earlier, excessive proliferation of PASMC and PAEC could potentially trigger vascular remodelling, which alters the pulmonary artery structures along with its biochemical and functional phenotypes.2+]i within PASMCs and PASMC proliferation.Given the essential roles of Ca2+ entry and linked to lysosomal functions such as alkalinizing lysosomal pH, endolysosomal morphology and cell pigmentation.2+ signalling plays a crucial role in VEGF‐mediated proliferation and angiogenesis in vitro and in vivo.2+]i and PAEC angiogenesis. However, its potential role in PAH has not yet been determined as of yet. The aforementioned assays in PAECs were performed to evaluate TPC1/2 functions. Consistent with in PASMCs, NAADP‐AM also induced [Ca2+]i and Ca2+‐related CaMKII and calcineurin expression in PAECs. As expected, TPC2 knockdown or TPC blocker sharply reversed NAADP‐induced [Ca2+]i, as well as NAADP‐AM‐induced CaMKII and calcineurin expression, whereas TPC1 knockdown only resulted in slight changes of the indicated indexes in PAECs, suggesting the dominant effect of TPC2 on regulating [Ca2+]i within PAECs. Also, the effect of TPC2 on regulating PAEC angiogenesis was monitored. As expected, TPC2 knockdown significantly inhibited VEGF‐induced angiogenesis of PAEC, further confirming the dominant role of TPC2 in PAECs.In addition to TPC1, the calcium release from acidic compartments via TPC2 activation evoked by NAADP could be observed in different cell types such as megakaryoblastic cell lines, pulmonary arterial myocytes, gastric smooth muscle cells and mouse embryonic stem cells.2+] i in PASMCs during pulmonary artery hypertension. Meanwhile, TPC2 exerts a crucial effect on controlling excessive PAEC angiogenesis by regulating [Ca2+]i in PAECs. A theoretical basis of TPC1/2 serving as potential targets for new gene treatments of chronic hypoxic‐related lung diseases, including PAH, was provided. However, as for NAADP‐induced TPC activation, it is possible that NAADP might target a secondary protein which either closely binds to TPCs or translocates to TPCs after NAADP binding to form part of the TPC complex other than direct binding to TPCs,TPC1 has an essential role in controlling excessive PASMC proliferation through regulation of [CaNot applicable.The authors declare that they have no conflict of interests.Wen Hu: Conceptualization ; Data curation ; Methodology ; Writing‐original draft . Fei Zhao: Resources ; Software ; Visualization . Ling Chen: Supervision ; Validation . Jiamin Ni: Resources ; Software ; Validation . Yongliang Jiang: Conceptualization ; Funding acquisition ; Project administration ; Writing‐review & editing .The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Hunan Provincial People's Hospital. Prior consent was obtained from each patient enrolled.Fig S1‐5Click here for additional data file.


Acute myeloid leukemia (AML) remains an incurable malignancy despite recent advances in treatment. Recently a number of new therapies have emerged for the treatment of AML which target BCL-2 or the membrane receptor CD38. Here, we show that treatment with Venetoclax and Daratumumab combination resulted in a slower tumor progression and a reduced leukemia growth both in vitro and in vivo. These data provide evidence for clinical evaluation of Venetoclax and Daratumumab combination in the treatment of AML.The online version contains supplementary material available at 10.1186/s40364-021-00291-y. To the editor,Acute Myeloid Leukemia (AML) is associated with poor prognosis and high mortality causing around 85,000 global deaths per year . CurrentRecently numerous novel therapies have emerged for treatment of AML. The BCL-2 family proteins are important in regulating intrinsic apoptotic processes , 4. ReceUnfortunately, AML resistance to Venetoclax is common, therefore, numerous studies combining Venetoclax with other therapies are being trialed , 7. Our in vitro coculture system, AML was cultured with MSC with either Venetoclax alone, Daratumumab alone, or Venetoclax and Daratumumab combination for 24 h. AML cells were then stained with AnnexinV-FITC/PI and analyzed using flow cytometry. Cells treated with Venetoclax and Daratumumab combination had significantly higher apoptosis compared to untreated AML cells , Daratumumab alone on day 7 and 14 by intraperitoneal injection (5 mg/kg), Venetoclax alone by oral gavage (100 mg/kg/day) for 10 days or combination Daratumumab (5 mg/kg) and Venetoclax (100 mg/kg/day)  xenograft mouse model of AML whereby on day 1 we transplanted 0.5 × 10in vitro and in vivo. Our data opens a potential avenue for novel Venetoclax drug combination with Daratumumab and supports further clinical investigation as a therapeutic approach in the treatment AML.Here, Venetoclax and Daratumumab combination treatment resulted in a reduced leukemia growth Additional file 1: Additional file 2:


While most Ca2+ handling protein expression and/or function has been shown to be altered in many models of experimental HF, in this review, we focus in the sarcoplasmic reticulum (SR) Ca2+ release channel, the type 2 ryanodine receptor (RyR2). Various modifications of this channel inducing alterations in its function have been reported. The first was the fact that RyR2 is less responsive to activation by Ca2+ entry through the L-Type calcium channel, which is the functional result of an ultrastructural remodeling of the ventricular cardiomyocyte, with fewer and disorganized transverse (T) tubules. HF is associated with an elevated sympathetic tone and in an oxidant environment. In this line, enhanced RyR2 phosphorylation and oxidation have been shown in human and experimental HF. After several controversies, it is now generally accepted that phosphorylation of RyR2 at the Calmodulin Kinase II site (S2814) is involved in both the depressed contractile function and the enhanced arrhythmic susceptibility of the failing heart. Diminished expression of the FK506 binding protein, FKBP12.6, may also contribute. While these alterations have been mostly studied in the left ventricle of HF with reduced ejection fraction, recent studies are looking at HF with preserved ejection fraction. Moreover, alterations in the RyR2 in HF may also contribute to supraventricular defects associated with HF such as sinus node dysfunction and atrial fibrillation.Heart Failure (HF) is defined as the inability of the heart to efficiently pump out enough blood to maintain the body's needs, first at exercise and then also at rest. Alterations in Ca It is characterized by the failure of the cardiac pump to maintain a sufficient blood flow to oxygenize and carry nutrients to the whole body. According to left ventricular systolic function, HF has been divided into two major groups: HF with reduced ejection fraction (HFrEF) and HF with preserved ejection fraction (HFpEF). HFrEF generally occurs after cardiac injury  or under chronic stress (hypertension), leading to the alteration of contractile function of the heart. It is now well established that alteration of cardiomyocyte Ca2+ plays a key role in cardiomyocyte contraction. In each heartbeat, the membrane depolarization during an action potential (AP) activates L-type Ca2+ channels (LTCC), which are located in the sarcolemma and are more concentrated at the transverse (T) tubules  located in the neighborhood, on the junctional sarcoplasmic reticulum (SR), resulting in the coordinated and global increase in the cell [Ca2+]i, by the Ca2+-induced Ca2+-release (CICR) mechanism, activating contractile myofibrils. Relaxation happens when the cytosolic [Ca2+]i decreases, mainly by Ca2+ re-uptake into the SR through the SarcoEndoplasmic Reticulum Ca2+ ATPase (SERCA) and extrusion out of the cell through the Na+/Ca2+ exchanger (NCX). Other systems, such as the plasmalemmal Ca2+ pump and the mitochondrial Ca2+ uniporter, play a minor role in cytosolic Ca2+ removal. The RyR2 are not so sensitive to Ca2+ so their activation probability depends on their proximity to the LTCCs, which determines the local Ca2+ concentration , brief (~30 ms), and local (~1.5 μm) elevations in cytosolic Ca2+, named Ca2+ sparks , Ca2+ release units (CRU) formed by a cluster of RyR2s are activated by the LTCCs located in the T tubules in front of them, which open during the AP. The spatial and temporal summation of these coordinated Ca2+ sparks results in the whole [Ca2+]i transient. The efficacy of the LTCCs to activate CRUs has been referenced as CICR gain, and it has been studied in HF combining patch-clamp to measure L-type Ca2+ current (ICa) and confocal microscopy to visualize changes in ≪Cai2+.During diastole, some Ca2+ cycling. Alterations have been reported in several cardiomyocyte Ca2+ cycling elements, such as SERCA, ratio phosphlamban (PLN)/SERCA, and single LTCCs, during both experimental and human HF. In this review, we will focus on alterations in the RyR2 function on left ventricular HF, although alterations in Ca2+ handling have also been found in right ventricular failure . This decrease in CICR gain was observed in experimental HF, due to chronic hypertension  and metabolic changes (that may curse with lower ATP levels) tend to reduce its activity  from the RyR2 leading to an increase in the channel open probability  phosphorylation of S2814 (2815) has also been highlighted for its role in the increase in SR Ca2+ leak through RyR2 in HF. CaMKII is activated by catecholamine and by oxidation, which is high during HF  , which together with more active RyR2 due to post-translational modifications, resulting in a drop of Ca2+ stored in the SR. As a result, and despite normal ICa density and prolonged AP, the triggered [Ca2+]i transient is of lower amplitude, contributing to diminished cell contraction. During diastole, the higher RyR2 activity favors the probability of producing Ca2+ waves, generating an inward current through the NCX, when extruding Ca2+, which produces DADs. When DADs reach the threshold, a triggered AP is generated, which may initiate arrhythmias.2+ release. A lack of knowledge is probably related to the difficulty in generating accurate experimental models or the diversity of associated comorbidities. As described previously, in HFrEF, cardiac systolic and diastolic changes are linked to Ca2+ mishandling. In HFpEF, Ca2+ alterations are not as clearly defined and seem to differ depending on the associated comorbidity. In a rat model in HFpEF with chronic pressure overload, a significant increase in diastolic SR Ca2+ release through RyR2s leading, as in HFrEF, to higher diastolic [Ca2+] and irregular [Ca2+]i transients during pacing has been observed   decrease and is associated with a drop of SR Ca2+ load and [Ca2+]i transient without affecting systolic function  has been identified as a subtype of HF in addition to HFrEF. HFpEF is more frequently found in older patients with comorbidities such as hypertension, obesity, diabetes, chronic kidney diseases , diastolic dysfunction is associated with an increase in SR Ca2+ release of the pacemaker cardiomyocytes of the heart, located at the sinus node (SAN), contributes to heart automaticity. This observation was initiated in the late 80s by the finding that ryanodine slows down the final phase of diastolic depolarization, resulting in a significant increase in cellular pacemaker cycle length in cats , are responsible for a net depolarizing current mediated by the NCX. This process drives pacemaker depolarization, which is referred to as the “Ca2+ clock,” jointly with the “membrane or voltage clock” mediated by cyclic activation and deactivation of voltage-sensitive membrane ion channels i transient as compared to ventricular myocytes  has been shown to play a role in the pathophysiology of paroxysmal/persistent AF  and NIH (2R01HL055438-22).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.