Cortical SOCs were sensitive to the Orai blockers Pyr6 and GSK-7975A, as well as to EVP4593, zinc, copper, and gadolinium ions, the latter one being the most potent SOCs blocker tested (IC50 ∼10 nM). SOCs were insensitive to the TRPC channel blockers Pyr10 and SAR7334. In addition, preventing mitochondrial Ca2+ uptake inhibited SOCs which were unaffected by inhibitors of the Ca2+-independent phospholipase A2. Altogether, Orai2 channels are present at the beginning of the embryonic murine cortico-genesis and form the core component of native SOCs in the immature cortex. This Ca2+ route is likely to play a role in the formation of the brain cortex.In the murine brain, the first post-mitotic cortical neurons formed during embryogenesis express store-operated channels (SOCs) sensitive to Pyr3, initially proposed as a blocker of the transient receptor potential channel of C type 3 (TRPC3 channel). However, Pyr3 does not discriminate between Orai and TRPC3 channels, questioning the contribution of TRPC3 in SOCs. This study was undertaken to clarify the molecular identity and the pharmacological profile of native SOCs from E13 cortical neurons. The mRNA expression of STIM1-2 and Orai1-3 was assessed by quantitative reverse transcription polymerase chain reaction. E13 cortical neurons expressed STIM1-2 mRNAs, with STIM2 being the predominant isoform. Only transcripts of Orai2 were found but no Orai1 and Orai3 mRNAs. Blockers of Orai and TRPC channels  were used to further characterize the endogenous SOCs. Their activity was recorded using the fluorescent Ca SOCs mediate a prominent Ca2+ signaling process called store-operated Ca2+ entry (SOCE), initially named capacitative Ca2+ entry  and showed that the passive leakage of Ca2+ out of the ER induced by the sarco/endoplasmic Ca2+ ATPase (SERCA) inhibitor thapsigargin is associated with an influx of Ca2+ (Store-operated channels (SOCs) are Catry CCE; . Althoug of Ca2+ . Since t of Ca2+ , freshly of Ca2+ , culture of Ca2+ , and cor of Ca2+ .2+ sensors of the ER. To date, two STIM (STIM1-2) and three Orai (Orai1-3) isoforms are known , a family of Care known . But where known .2+ signaling plays a critical role in cortical development, it is important to understand how the first post-mitotic neurons acquire this essential cation. Ca2+ imaging experiments revealed that murine E13 cortical neurons display a SOCE .The C57Bl6/J mice used in this study were from the Jackson Laboratory (USA). They were housed in temperature-controlled rooms under a 12 h light – 12 h dark cycle with SAR7334 and GSK-7975A were kind gifts from Sanofi-Aventis Deutschland and Dr. Malcolm Begg (GlaxoSmithKline), respectively. All the reagents were from Sigma-Aldrich  except for Fluo-4/AM, Fura-2/AM, and tissue culture media from Molecular Probes, Invitrogen .2+- and Mg2+-free Hank’s solution supplemented with 33 mM glucose, 4.2 mM NaHCO3, 10 mM HEPES, and 1% penicillin/streptomycin. Cortical cells were dissociated by mechanical trituration using a sterile Pasteur pipette. The cell suspension was filtered through a 40 μm cell strainer and isolated cells were plated at a density of ∼1.5 × 105 cells/16 mm diameter glass coverslips before being placed into 35 mm diameter petri dishes containing 2 mL of a Neurobasal medium supplemented with B27 (2%) and glutamine  using primers at 200 nM. PCR reaction mixtures (10 μl) were placed in a Cfx96 instrument (Bio-Rad) where they underwent the following cycling program, optimized for a 96-well block: 95°C for 15 min, immediately followed by 40 cycles of 10 s at 95°C and 30 s at 60°C. At the end, PCR products were dissociated by incubating for 1 min at 95°C and then 30 s at 55°C, followed by a ramp up to 95°C. PCR quality and specificity were verified by analyzing the dissociation curve. qRT-PCR reactions were run in triplicate, and quantification was performed using comparative regression (Cq determination mode). Quantitative PCR data were comparatively analyzed according to RNA from cortex and isolated neuron cells were isolated using Absolutely RNA miniprep kit (Agilent # 400800). RNA concentration was determined using a NanoDrop spectro photometer (ND-1000). Reverse transcription was performed with the Affinity script qPCR cDNA synthesis kit (Agilent # 600559) according to the manufacturer’s instructions. Gene specific primers for the different STIM and Orai murine genes were designed according to the primer software Primer-Blast. The designed primers are given in 2, 1 MgCl2, 10 HEPES, 10 glucose, pH 7.4 (NaOH) supplemented with 1.25 μM Fluo-4/AM. After dye loading, cells were rinsed twice and kept 10 min at room temperature in the dark before recording Fluo-4 responses. To this aim, the baseline Fluo-4 fluorescence was recorded for ≥1 min and averaged (F0). The results are expressed as F/F0 as a function of time, with F being the Fluo-4 fluorescence intensity. Each solution was prepared fresh each day from 1000× stock solutions so that the final concentration of DMSO never exceeded 0.1%. Images were acquired at room temperature at a frequency of 0.2 Hz with a CCD CoolSnap HQ2 camera  placed on an inverted Axio Observer A1 microscope  having a Fluar 40× oil immersion objective lens . A DG-4 wavelength switcher  was used. The excitation light for Fluo-4 was filtered through a 470–495 nm excitation filter and the light was collected through a 525 nm filter. Data acquisition and off-line analysis were conducted with MetaFluor . All of the live cell Ca2+ imaging experiments were conducted on cultured neurons at 2–3 days in vitro.The experimental setup was as described in 2+]i in RBL-2H3 cells were monitored using the Fura-2 technique as previously described  supplemented with 2 μM Fura-2 AM for 40 min at room temperature in the dark. After the incubation period, cells were washed twice with saline and left to equilibrate for at least 20 min. The coverslip was then mounted in a perfusion chamber on an inverted microscope (Olympus IX71) and perfused with different solutions at room temperature. During the recordings using Live Acquisition 2.5 software , cells were excited alternately at 340 and 380 nm using an Oligochrome excitation system  and fluorescent images were captured at 510 nm every 1.2 s with an ORCA-03G digital CCD camera .Changes in  or iPLA2γ [with (R)-BEL] was not associated with any impairment of SOCE. Of note, BEL blocks Ca2+ entry through voltage-gated Ca2+ channels and TRPC1, TRPC5, and TRPC6 channels with IC50 values of 7–8 μM  can be employed to discriminate native Orai channels since they inhibit all types of Orai channels. Mitochondria are controlling SOCE which seems to develop without the requirement of iPLA2 activity. On the other hand, the passive entry of Ca2+ exhibit distinct pharmacological and kinetics profiles indicating that they deliver Ca2+ to neurons via channels distinct from SOCs. The presence of functional SOCs at the beginning of cortico-genesis suggests that this Ca2+ route is likely to play a role in the formation of the brain cortex.Our data indicate that Orai2 is a critical pore component of native neuronal SOCs in the immature cortex. They are sensitive to various Orai blockers like Pyr6 and GSK-7975A. These channels are also blocked by EVP4593, and the cations GdSC performed experiments and analyzed data. LJ performed experiments and analyzed data. NS performed experiments and analyzed data. MC performed experiments and analyzed data. KG contributed new reagents. AB designed the study, performed experiments, analyzed data and wrote the manuscript.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.


Mutant nucleophosmin-1, proposed as a possible marker for RA response, is the criterion for recruiting patients in three active RA phase 3 clinical trials. We tested the ability of RA alone or in combination with either bosutinib (B) or 6-formylindolo carbazole (F) to induce conversion of 12 de novo AML samples toward a more differentiated phenotype. We assessed levels of expression of cell surface markers associated with differentiation, aldehyde dehydrogenase activity, and glucose uptake activity. Colony formation capacity was reduced with the combined treatment of RA and B or F, and correlated with modulation of a c-Cbl/Lyn/c-Raf-centered signalsome. Combination treatment was in most cases more effective than RA alone. Based on their responses to the treatments, some primary leukemic samples cluster closer to HL-60 cells than to other primary samples, suggesting that they may represent a hitherto undefined AML subtype that is potentially responsive to RA in a combination differentiation therapy.Acute myeloid leukemia (AML) has high mortality rates, perhaps reflecting a lack of understanding of the molecular diversity in various subtypes and a lack of known actionable targets. There are currently 12 open clinical trials for AML using combination therapeutic modalities including all- Acute myeloid leukemia (AML) is the most frequently occurring adult leukemia (comprising about 30% of new leukemias). It is also is the most deadly (causing 43% of leukemia deaths yearly), with a five-year survival rate below 27%, whereas other leukemias have a survival rate over 66% , 2. SincThe poor five-year survival rate is due to very high heterogeneity in etiology, incomplete understanding of molecular signature seminal to different subtypes, and lack of actionable targets and precision therapies. Many AML cases present recurrent genetic abnormalities Table  4–22]. –22. 4–22in vitro models, which provide an experimentally tractable and simplified context for pathway analysis. We previously characterized an ensemble of proteins that form a putative complex that generates signaling seminal to RA-induced differentiation of a patient-derived non-APL AML cell model, the HL-60 cell line. The signaling complex embodies molecules historically found to drive mitogenesis upon stimulation by growth factors, but drive differentiation upon stimulation by RA. Prominent members include E3 ubiquitin-protein ligase CBL (c-Cbl) [Given the high heterogeneity of AML pathology, improving AML patient survival rates depends on characterizing the networks that link genetic defects to molecular signaling and molecular signaling to cellular functional outcomes. To gain mechanistic insights that reveal diagnostic markers and targets of therapeutic intervention, a longstanding approach has been analysis of  (c-Cbl) –26, Raf- (c-Cbl) , 27–29,  (c-Cbl) , 30, LYN (c-Cbl) , 28, and (c-Cbl) . We posi (c-Cbl) , 32–35 w (c-Cbl) , 35. The (c-Cbl)  or Src f (c-Cbl)  enhances (c-Cbl) . Consist (c-Cbl) , 39 and  (c-Cbl) . Hence AIn the present studies, we show first that in mut-NPM1 but not wt-NPM1 patients c-Cbl is a marker for discriminating AML response to cytotoxic chemotherapy. We then look at the correlation of NPM1 and markers of differentiation status. Using this data to select a limited number of samples that embodied a diversity of traits, we tested their response to RA or RA plus a small molecule targeting AhR, measuring their colony forming/growth capacity in methylcellulose. The AhR agonist was 6-formylindolo carbazole, i.e. FICZ (F). A SFK inhibitor bosutinib (B) was also tried but it was not as consistently effective in reducing the colony forming capacity. We then characterized the signaling signature associated with response to this combination of RA plus F to reveal a c-Cbl related signaling telltale predictive of response. Finally we determined if there was an AML subtype that embodied this signature.in vitro by the HL-60 cell line.From these studies, we report four main conclusions. 1. c-Cbl used as a prognostic indicator with NPM1 results in better disease-free survival (DFS) stratification than NPM1 alone; in particular, AML with mut-NPM1 and low c-Cbl expression performed better than high c-Cbl. 2. Low colony formation capacity occurred in samples where RA could regulate signaling, specifically c-Cbl, Lyn and c-Raf/phospho c-Raf. 3. RA-induced responses could be enhanced by combining RA with F or B. 4. The widely used patient–derived RA-responsive HL-60 AML cell line bore fidelity to a hitherto uncharacterized AML subtype with wt-NPM1, low CD34 where c-Cbl, Lyn and c-Raf/phospho c-Raf could be regulated by RA. We ergo put forth the conjecture that there is an AML subtype that can respond to RA in combination with other agents, and it is captured NPM1 [NPM1 is a known stratification marker for AML. The 2016 WHO classification includes a subtype of AML with recurrent genetic abnormalities consisting of mutations of NPM1 . The proNPM1 , 42. In NPM1 , 42 thatWe previously reported a signalsome that generates signaling seminal to RA-induced differentiation in a well-studied patient derived non-APL AML cell line, HL-60, that responds to RA and differentiates with cell cycle arrest. One of the members of the signalsome is the c-Cbl adaptor/E3-ligase, which has been implicated in myelodysplastic syndromes  and AML de novo non-APL AML blasts, based on expression of surface markers.The data presented in Figure de novo AML patient samples were assessed for their NPM1 status. Of the samples, 5 were wt-NPM1: AML 37; AML 61; AML 76; AML 94; AML 100; and 7 were mut-NPM1: AML 8; AML 34; AML 72; AML 74; AML 75; AML 95; and AML 99. HL-60 was also assessed and determined to be wt-NPM1. About 70% of the wt-NPM1 exhibit high expression of CD34 [12 Two wt-NPM1 samples (AML 100 and AML 61) have very high CD34 expression, and the response to treatment is greater in AML 100 than in AML 61. One wt-NPM1 sample (AML 94) has very low CD34 expression, lower than that of several mutants . HL-60 cells, which are wt-NPM1 cells, at two different passages (A and B) also have low CD34 expression. AML 94 and AML 100 exhibit diverse total CD34 expression. It is thus evident that NPM1 status per se by itself is not directing CD34 expression. This is consistent with the notion put forth above that NPM1 status reflects a heterogeneous population where NPM1 should be analyzed with other combinations of markers, e.g. c-Cbl.Next we added expression of surface differentiation markers to the evaluation. CD34, CD38, CD11b, CD14 and CD15 levels were assessed by flow cytometry. CD34 is a glycoprotein adhesion molecule taken as one of the molecular markers for stemness , 49; CD3Interestingly, when comparing the untreated AMLs, wt-NPM1 AML 94 clusters closely with HL-60 cells (passages designated A and B) and then with several mut-NPM1 AML samples, AML 8, AML 34 and AML 95, and more distantly with the two wt-NPM1 AML 61 and AML 100. It appears that the HL-60 cell line bears fidelity to an AML subtype represented by AML 94.We next analyzed the percent change with treatment for the same surface markers by flow cytometry, setting gates to exclude about 50% of the untreated cells. We then performed the clustering analysis. We observed that the wt-NPM1 AML 94 cells are again close to the wt-NPM1 HL-60 AML cell line and to the mut-NPM1 AML 99, AML 95, AML 74, and AML 75 cells  where they are not coupled. To evaluate this further, we tested the behavior of those two AML cell types, AML 100 with high CD34/low ALDH1/lower glucose and AML 94 with low CD34/high ALDH1/higher glucose, plus, to get an idea of potential dynamic range, an additional two more wt-NPM1 samples, AML 37, AML 76, for colony formation capacity in methylcellulose. We compared cultures that were untreated or treated with RA alone or in combination with either F or B.Although CD34, ALDH1 activity and glucose uptake are often coupled, we identified interesting cases of The response to RA alone or in combination varied among the four AML samples tested, and in all cases colony size decreased with treatment. Figure . There wSince we observed that the treatment decreases the number of large colonies and increases the number of smaller colonies, we next estimated the total number of cells per plate. We calculated this for each plate by adding the numbers obtained by multiplying the numbers of colonies in each category with the mean number of cells in that category. This parameter varies greatly among AML samples Figure , indicatAs FRA was effective in decreasing the number of colonies and the colony size in all samples tested, western blot analysis was performed for several signaling markers for untreated vs. FRA treated samples. Of these c-Cbl –26, c-RaSince AML 100 and AML 37 had high numbers of cells in methylcellulose whereas AML 94 and AML 76 were in stark contrast with low numbers – indicative of a potentially prominent proliferative difference - we explored their signalsome-related signaling attributes. Figure In AML 94 and AML 100, MMP9 and LCN2 are highly expressed, but expression decreased with FRA treatment, mirroring decreased number of colonies. Interestingly, in the samples with the highest number of colonies, AML 100 and AML 37, SFK pY416 levels are the highest, and they do not change with treatment. This suite of molecules thus may represent at least elements of a signature for response with prognostic significance.We next explored whether the ensemble of molecules implicated above betrayed an AML subgroup by a cluster analysis of the TARGET AML RNA-seq data based on the proteins we tested in the 4 wt-NPM1 patients Figure . It ergoThe present studies address the question of whether there is a subtype of non-APL AML that is potentially susceptible to differentiation therapy and if so what molecular signature it might have. We started with an analysis of the public domain TARGET data base for AML RNA-seq. That showed that as previously thought NPM1 wt vs mut provided a stratification showing better DFS for mut-NPM1. We refined that and showed that c-Cbl provided an additional criterion and found that low c-Cbl expression provided a further advantage in DFS. c-Cbl is known to associate with a suite of partners in a signalsome originating signaling that propels RA-induced differentiation and inhibition of proliferation. The expression of elements of this suite was found here to be co-regulated in AML cells. We have previously reported that the signalsome contains a module of interrelated kinases, including Lyn and c-Raf, contributing propulsion to RA-induced differentiation , 32, 35.de novo non-APL AML samples that were either untreated or treated with RA alone or in combination with F or B, which are ligands for signalsome AhR and SFK, for cell surface markers that have been used as indexes of differentiation/stemness and have been attributed with signaling capabilities governing proliferation/differentiation. We also assayed colony formation capacity, ALDH1 activity, and glucose uptake, and synthesized this data. It was found that the non-APL AML cell line, HL-60, bore fidelity to a subset of AML. In a set of samples restricted to expressing a range of expression in CD34, ALDH1 and glucose uptake, it was found that colony formation capacity correlated with ability of FRA to regulate a suite of signalsome-related entities, specifically c-Cbl, Lyn, c-Raf/phospho-c-Raf and VAV1. Significantly, the responses due to RA were enhanced by using RA in combination with F or B. Perhaps what is the most interesting is that there is a hitherto undefined subtype of non-APL AML, marked in part by a suit of responsive signalsome molecules that is susceptible to differentiation therapy using RA in combination with other agents that target components of a signalsome seminal to signaling propelling differentiation.Predicated on the emerging significance of the c-Cbl-centered signalsome, we interrogated RA differentiation therapy presents a significant reduction in toxicity compared with standard chemotherapy. However it is routinely applicable only in APL and not other cases of AML. Some clinical trials have been motivated by the hope of extending its therapeutic use to other subtypes of AML, targeting in particular mut-NPM1 non-APL AML cases. The mut-NPM1 cases are a minority of non-APL AML. The subtype identified by us and highlighted in Figure Primary samples were acquired in accordance with the Declaration of Helsinki and with Weill Cornell Medicine Institutional Review Board approval.et al. [Total RNA was isolated using the RNeasy extraction kit . NPM1 analysis was performed using a massively multiplexed assay that detects all NPM1 insertions in exon 12 at position 863 as previously described . ABL1 waet al. . Briefly6 cells/mL and primary sample cultures were initiated at 1 × 106 cells/mL. Cell collection for flow cytometry and total cell lysate collection for western blot was done at 72 h post treatment. For CFU assay in methylcellulose, 25000 primary cells were plated in 3 mL culture for 15 days. For treatments, all-trans retinoic acid (RA) (Sigma) was added from a 0.5 mM stock solution in ethanol to a final concentration of 1 μM in culture for HL-60 cells and to a final concentration of 150 nM in culture for primary cells, 6-formylindolo carbazole, i.e. FICZ (F) , was added from a 100 μM DMSO stock to a final concentration of 100 nM in culture, bosutinib (B) (Sigma) was added from a 5 mM stock solution in DMSO to a final concentration of 0.25 μM in culture. All reagents were purchased from Sigma unless otherwise indicated.The primary samples were thawed and cultured as previously described . The Hum6) were harvested by centrifugation, and pelleted cells were resuspended in 200 µL of PBS containing 2.5 µL of antibodies against CD11b (clone ICRF44), CD38 (clone HIT2), CD34 (clone 581), CD14 (clone M5E2), CD15 (clone HI98) conjugated either with allophycocyanin or phycoerythrin (BD Biosciences). Following incubation for 1 h at 37°C, cells were analyzed by flow cytometry . Gates to determine percent change in expression were set to exclude 50% of untreated cells. The mean expression per cell is derived from the entire population gated using FSC and SSC to include all cells and exclude debris.The primary and HL-60 cells (0.5 × 10ALDH1 enzymatic activity was measured using the Aldefluor kit (Stem Cell Technologies) by flow cytometry –65 as deGlucose uptake assay was performed using 6-NBDG amino)-6-Deoxyglucose) –69  was resolved by SDS-PAGE analysis  using 12https://ocg.cancer.gov/programs/target/data-matrix, May/18/2017). 284 patient samples from the BCCA cohort were used in this study. The processed FPKM (Fragments Per Kilobase of transcript per Million mapped reads) obtained directly from TARGET was converted to TPM (Transcripts Per Million) by exp(log(fpkm) - log(sum(fpkm)) + log(1e6)), and then log2 transformed for further analysis.TARGET RNA-Seq data set was downloaded from National Cancer Institute (NCI)’s data portal (http://www.r-project.org/). Survival analysis was used to compare disease free survival between wt-NPM1 and mut-NPM1 groups using the R package ‘survival’ . A log-rank test was used to compare survival distributions of different subgroups, with p < 0.05 considered statistically significant. Gene expression data was z-normalized for clustering analysis using the ‘hclust’ function in R, with Euclidean distance and complete-linkage method. The heatmap was generated using the ‘aheatmap’ function in R package ‘NMF’ (https://github.com/renozao/NMF). The mean expression of differentiation markers for de novo samples under each treatment were analyzed using the ‘heatmap.2’ function available in the ‘gplots’ package for R [All statistics were performed using R (version 3.3.3; ge for R .


While conventional indicators can improve individual aspects of indicator performance such as Kd, wavelength, and ratiometric measurements, the use of NP sensors can achieve combined benefits of addressing these issues simultaneously. The nanosensor incorporates highly calcium-selective ionophores and two fluorescence indicators that act as signal transducers to facilitate quantitative ratiometric imaging. For intracellular Ca2+ application, the sensors are fine-tuned to physiological sensing range, and live-cell imaging and quantification are demonstrated in HeLa cells loaded with nanosensors and their responsiveness to carbachol-evoked store release (~400 nM). The current nanosensor design thus provides a promising sensing platform for real-time detection and optical determination of intracellular ions.Optical nanoparticle (NP)-based sensors have been widely implemented as tools for detection of targeted ions and biomolecules. The NP sensing platform offer a modular design that can incorporate different sensing components for greater target specificity and the ability to tune the dynamic range, as well as encapsulation of multiple dyes to generate a ratiometric signal with varying spectra. Despite these advantages, demonstrating quantitative ion imaging for intracellular measurement still possess a major challenge. Here, we describe fundamentals that enable intracellular validation of this approach using ion-selective nanosensors for investigating calcium (Ca However, when applied to intracellular measurement, accurate determination of the intracellular concentration with these indicators can be complicated by a non-optimal dissociation constant (Kd), low signal, undesired compartmentalization, and interference with cellular components potentially leading to variability and inaccurate measurements. A number of fluorophores are commercially available to achieve quantitative ion imaging as demonstrated by single wavelength dyes in Ca2+ recordings10, as well as other approaches including fluorescence resonance energy transfer (FRET)-based sensors12. The accuracy of optical quantification is often improved by ratiometric methods which eliminates interference caused by fluctuations in excitation intensity, fluorophore concentration, and light scattering due to specimen thickness. Traditionally, quantitative Ca2+ imaging has been achieved with ratiometric indicator dyes such as Fura-213 and Indo-114, and fluorescence lifetime imaging (FLIM) methods15. However, ratiometric imaging in practice is limited to only a few indicators in the UV spectrum associated with excitation light-induced phototoxicity, higher tissue scattering, and most importantly, the requirement of structural modification to alter its physiochemical properties such as the sensing range for desired biological applications.Optical imaging and fluorescent probes provide key tools for exploring the spatiotemporal dynamics of ions in living systems. In particular, real-time detection of ion dynamics in the cellular environment requires designing optical probes with high target specificity and sensitivity. Major progress has been achieved with the advent of fluorescent calcium  brighter and photostable probes by integrating a high number of dyes; (ii) ability to fine-tune the sensor dynamic range; (iii) greater selectivity and range of analyte detection; (iv) adjustable emission spectra and ratiometric response; (v) reduced effects from light-induced cell damage23 by integrating longer wavelength dyes; and (vi) low cytoplasmic mobility27 to prevent dye diffusion, and protection from intracellular organelle sequestration29. Indeed, the recent advances in NP-based sensors from various nanomaterials have been widely implemented in cellular tracking and imaging applications22. For example, there are several nano- and micro-particle sensors for detection of ions reported for sodium32, potassium37, calcium39, magnesium41, zinc42, silver43, and chloride44. Other efforts include nanosensors termed PEBBLEs  developed by Kopelman and co-workers45. These sensors incorporate molecular indicators such as rhod-2 into a polymeric matrix, however, the flexibility to tune the dynamic range remains challenging with this mechanism46. While the development of NP-based sensors has been an increasingly growing field of research, the application for measurement of intracellular ions, and detailed procedure for quantitative ratiometric ion imaging has not been fully established in these sensors.Nanoparticle (NP)-based sensors2+ signaling, as widely exemplified by cellular processes ranging from gene expression, synaptic transmission, hormone secretion to cell survival and death50.To address this issue, we present a ratiometric sensor termed calcium-optode nanosensor (opCaNS) for quantitative fluorescence imaging of ions in living cells, and establish a procedure for estimating ionic concentration using ratiometric fluorescent signal. Calcium is selected as a model target ion to reliably assess our sensor characteristics to the extensive work established on the local control of Ca51, an optical counterpart to ion-selective electrodes (ISEs), were applied and miniaturized to nanoscale for intracellular investigation. In the ISO sensing scheme, three reagents are embedded within the NP matrix: a highly selective, optically silent ion carrier (ionophore)51 that is capable of reversibly binding ions; a fluorescent pH indicator dye (chromoionophore) which acts as a reporter to yield a fluorescence readout based on its protonation degree; and a lipophilic charge-carrying molecule (ion exchanger) to maintain electroneutrality within the NP. Hence, ion-specific optical change is facilitated by binding of calcium ions by the ionophores, which causes displacement of protons of the pH-indicator with a quantifiable fluorescence signal. Furthermore, we have incorporated a second pH-indicator [octadecyl rhodamine (R18)] and term it as reference dye, whereby fluorescence intensity moves in opposite direction to pH changes in comparison to the chromoionophore to achieve a highly sensitive sensor for ratiometric measurement of intracellular Ca2+. Most importantly, the sensing range of the probe is easily modified and tuned by changing the ratio of components, which provides flexibility to the user for specific imaging applications.In the nanosensor design, the ion-selective optodes (ISOs)2+ measurement using ratiometric approach with sensor characteristics tuned to exhibit physiological sensing range, high selectivity, fast response time, and a reversible profile. Herein, we demonstrate the performance of this class of ion-selective nanosensors for cellular applications, achieving real-time detection and quantitative ion measurements by live-cell imaging for the first time.Overall, the opCaNS provides a sensing platform suitable for intracellular Ca2+]i), we applied ISOs as the principle sensing scheme, and further developed into miniaturized nanoscale sensors for ratiometric fluorescence imaging. In this sensing platform, the nanosensors are fabricated by co-immobilizing hydrophobic optode components comprised of calcium ionophore (I), a fluorescent chromoionophore (C), ion exchanger (R), and reference fluorophore (Ref) within a biocompatible plasticizer nano-emulsion matrix, and stabilized by a biocompatible polyethylene glycol (PEG)-lipid as surfactant  to deprotonate to maintain charge neutrality within the sensor with an accompanying change in fluorescence. In this scheme, the opCaNS respond to Ca2+ in accordance with optode function  of the nanosensor and the ion activity is:To achieve nanosensors for real-time detection and measurement of intracellular free calcium ions . The emission spectra for individual fluorophores are shown in Supplementary Figure 53.For sensor calibration, the emission intensity ratios of two fluorophores   Fig. . The opC50. Response time of the nanosensor was assessed by a two-photon (2P) Ca2+ uncaging experiment . It is worth noting that the measured response time of the nanosensors here is limited by the imaging frame-rate, and the temporal resolution can be significantly improved by other imaging methods31.It is critical to determine the sensor response time to assess the ability of nanosensors to capture ionic transients which occur on the millisecond-to-second time scaleent Fig. . We firsent Fig. . Subsequ2+ release from the intracellular store due to its rise and decay characteristics50. The ratiometric response of the opCaNS exhibited a clear reversibility profile , which is indicative of excellent cycling capability of the nanosensors. The reversibility profile for individual fluorophores (CHIII and R18) is shown in Supplementary Figure Reversibility is also key to monitoring Caile Fig.  over rep2+ concentration by exhibiting nanomolar response range, high selectivity, fast response time, and reversible response.Collectively, the results show applicable sensor characteristics for reporting local cytoplasmic Ca2+ store release in HeLa cells (n = 3) which acts on inositol 1,4,5-triphosphate receptors (IP3Rs) on the endoplasmic reticulum (ER) membrane57. First, nanosensors were loaded into HeLa cells using pressure controlled microinjection  per cell  is less or comparable to those observed in previous studies using dye indicators and PEBBLE NP-sensors 75 in order to minimize physical perturbation of living cells. Lastly, we find that the nanosensors are dynamically fluorescent under 2P excitation, demonstrating their potential use in vivo.The main advantage of our sensor platform is the flexibility to tune the sensor to a given response range and selectivity for various applications by altering the sensing components. While molecular probes can improve individual aspects of indicator performance such as Kd, wavelength, and ratiometric measurements, NP-based sensors benefit from addressing these issues simultaneously. For example, sensor sensitivity can be tuned to a dynamic range of interest which can mitigate issues with indicator buffering and affinities that are either lower or higher relative to the magnitude of the Ca77, our nanosensor response is also subject to pH changes due to the encapsulation of pH-sensitive dyes. This is illustrated in Supplementary Figure 78, and the effect of pH is minimized. Also, the CHIII (ETH 5350) used in this work has a pKa of >1021, which is highly protonated under standard physiological conditions, and as such, the predominant sensor response is dictated by the changes in Ca2+ concentrations. Currently, there are developments of other efforts to circumvent such pH dependency79.Similar to the pH dependent nature of conventional dye indicators2+ dynamics using these sensors which has not been described previously. The current sensor platform combines the ease of synthesis and the flexibility to allow incorporation of different sensing components. As such, the sensor response can be tuned for specific dynamic range, selectivity, and wavelengths. In future work, detection of other physiologically important ions such as sodium, potassium, and chloride will be explored for intracellular signaling applications.This work expands on the continual development of optode-based nanosensors and highlights its importance as tools for monitoring physiological analytes. The current work introduces a ratiometric method for demonstrating quantitative determination of intracellular CaN,N,N′,N′-Tetra[cyclohexyl]diglycolic acid diamide), chromoionophore III -5-[(2-octyldecyl)imino]benzo[a]phenoxazine, ETH 5350), dichloromethane (DCM), ethylene glycol tetraacetic acid (EGTA), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), magnesium chloride (MgCl2), sodium tetrakis--borate (NaTFPB), and tetrahydrofuran (THF) were obtained from Sigma-Aldrich. 1--1,2-diaminoethane-N,N,N′,N′-tetraacetic acid (DMNP-EDTA), octadecyl rhodamine B chloride (R18), ionomycin, calcium calibration buffer kit (C3008MP), Fluo-4 AM, and all cell culture media and supplements were obtained from Thermo Fisher. Tris-base was obtained from Fisher BioReagents. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550]  (DSPE-mPEG550) was obtained from Avanti Polar Lipids, Inc.Bis(2-ethylhexyl) sebacate (DOS), calcium ionophore II  equipped with two Ti:Sa lasers (SpectraPhysics) and separate beam paths/galvanometer mirrors for independent imaging and uncaging. Laser power was controlled independently for each beam path using two Pockels cells (Conoptics).Fluorescent spectra for routine nanosensor calibration in solution and selectivity experiments were measured with Spectramax M3 plate reader (Molecular Devices). Sensor reversibility test and cell imaging were performed with Zeiss LSM700 inverted confocal laser scanning microscope (Carl Zeiss). Hydrodynamic diameter and zeta-potential measurements were determined using a 90 Plus particle size analyzer (Brookhaven Instruments Corporation). Ca2, 10 mM HEPES; pH adjusted to 7.2 with tris base). In a typical opCaNS fabrication, 50 μL of polymer-free optode was added to 70 μL DCM, and this mixture was sonicated with the 4 mL of HEPES buffer containing DSPE-mPEG550 at 10% intensity for 1 min . After sonication, the organic solvents were removed using a rotavap (Buhle) for 15 min at room temperature, and the resulting emulsion was filtered with a 100 nm syringe filter (Millipore).To prepare the opCaNS, the calcium-selective optode sensing components comprising CaI-II , CHIII , NaTFPB , and R18  were first reconstituted separately in 300 μL THF. DOS  was then added to the optode sensing components and vortexed briefly for 30 s to form a homogeneous solution. Just prior to nanosensor fabrication, DSPE-mPEG550 (250 μg) was dried in a glass scintillation vial and rehydrated in HEPES buffer  based on the intensity of scattered 640 nm light at a fixed angle of 90°. All particle size measurements were taken in triplicates with sample solutions diluted in 10 mM HEPES buffer, pH 7.2 adjusted to a detector count rate of 150–450 kcps. Zeta-potentials were measured in PALS mode collected over 25 cycles each 10 runs per cycle. Size and zeta-potential measurements were analyzed from three separate batches of nanosensors each from three discrete optode stock preparations.2+ EGTA buffers with controlled concentrations of free calcium ion . In order to determine the fraction protonated during measurements in Ca2+, acid and base standards were used as endpoints for fully protonated or deprotonated chromoionophore conditions, respectively.Nanosensors were calibrated in solution for fluorescent response to a series of CaEX: 639 nm, λEM: 670 nm) and R18  were measured with plate reader in bottom read mode through clear-bottom 96-well plates  was calculated as:R is the fluorescence ratio measured experimentally, and RP and RD is the ratio of fully protonated and deprotonated state of the chromoionophore, respectively. The effective Ca2+ concentration at half-maximal sensor response , for the nanosensors was calculated from Ca2+ concentrations and the corresponding values of alpha, α, was fitted according to the dose-response (Hill) equation:R(α) is intensity ratio of measured response, C, denotes Ca2+ concentration at which a ratio was measured, and Rmin, Rmax, and EC50 are parameters obtained from the fitted sigmoidal curve defined by the Hill equation.The fluorescence intensities for CHIII  and primary ion (Ca2+), respectively.Nanosensor selectivity was determined by measuring the ratiometric fluorescent response (CHIII:R18) to CaEX: 555 and 639 nm) and peak intensities were recorded at two emission channels (R18 λEM: 575 nm and CHII λEM: 670 nm) using diffraction grating set to split the emission at 620 nm. The 40× oil-immersion objective lens (NA = 1.3) was used with the pinhole aperture setting at 2 µm which remained constant throughout the entire experiment.For reversibility and cell imaging experiments (see below), confocal laser scanning microscope  was used to measure fluorescence intensity ratios (CHIII:R18), as described above. Briefly, simultaneous images were acquired from dual excitation  as described by Reinhard 2+, and the ends of the capillary were sealed with wax. Imaging was carried out at 875 nm excitation wavelength through a 20 ×/0.45 N.A. objective (Nikon). Images were collected via photomultiplier tube  after a 595/50 emission filter (Semrock), at a frame-rate of 6 Hz. For DMNP uncaging, the uncaging beam (730 nm) dwelled on the target for 500 ms. Images were analyzed using ImageJ to extract raw data at the site of uncaging pulse (20 × 20 pixels).Calcium caging agent  and opCaNS were loaded into 20 μL glass capillaries (Drummond) with 1 mM Ca2. The cells were plated on poly-d-lysine coated 50 mm glass bottom dishes at a density of 4–8 × 104 cells. For Ca2+ imaging studies, cells were cultured in serum-free medium for 12 h, washed with PBS, and subsequently imaged using Ca2+-free Hank’s Balanced Salt Solution  with 10 mM HEPES, adjusted to pH 7.2 (Ca2+-free imaging buffer).HeLa cells  were grown in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 1% non-essential amino acids (NEAA), penicillin (50 U/ml), and streptomycin (50 μg/ml) at 37 °C in a humidified atmosphere containing 5% COin situ cell calibration, nanosensor loaded cells were initially incubated with Ca2+-free imaging buffer containing 5 µM ionomycin for 15 min to equilibrate Ca2+ levels within the cytosolic environment to the extracellular space. Ca2+ calibration buffer with incremental levels of Ca2+ were subsequently added and fluorescence images from the confocal microscope were acquired after 10 min incubation at each calcium concentration. The values of alpha, α, and the EC50 was determined based on the same methods used to calibrate sensors in solution. There were batch-to-batch differences in sensor response and consequently, in solution and in situ cell calibrations were routinely performed to ensure the sensors maintain responsiveness to the intracellular Ca2+ levels.Nanosensors were delivered to cytosolic compartment of HeLa cells using a microinjection technique (Eppendorf InjectMen 4) with 1.0 mm/0.5 mm (OD/ID) borosilicate glass tubes pulled to a tip size 0.1–0.5 µm using P-97 micropipette puller (Sutter Instrument). For 2+]i) was carried out in opCaNS-loaded HeLa cells. Carbachol (1 mM) was used to invoke changes in [Ca2+]i by acting on IP3R on the endoplasmic reticulum (ER) membrane. Fluorescence confocal images were acquired at 1 s interval for 10 min after addition of carbachol to the 50 mm culture dish. Fluorescence intensities in the regions of interest (ROI) on each channel (575 nm and 670 nm) were first background subtracted by the intensities of nearby area that were void of cells. For individual regions of interest (ROI) in the cytosolic cellular region we subsequently used the corrected fluorescence intensities on both channels to calculate intensity ratio R (F670 /F575) normalized by the photobleaching profile. Changes in nanosensor fluorescence ratio were expressed as changes in Ca2+ concentration based on in situ calibrations as described above. Conversion of fluorescence ratio to Ca2+ concentration, C, involved empirically deriving from acquired fluorescence ratio using the dose-response (Hill) equation . Numerical values are represented as a mean ± standard deviation, and graphical representations contain error bars for the standard deviation values.Supporting Information


The vascular endothelial growth factor (VEGF) signaling pathway regulates acute myeloid leukemia (AML) cell proliferation, survival and chemotherapy resistance. We have shown that VEGF/VEGFR2 signaling regulates leukemia cell survival by autocrine internal and external loops, with induction of pro-survival pathways and inhibition of apoptosis downstream of VEGFR2 internal signaling , 2. MoreOur recent study  reveals With all this in mind, we reasoned that VEGFR2 signaling could counteract chemotherapy-response by regulating mitochondrial metabolism. Accordingly, we show that VEGFR2 inhibition restored mitochondrial biogenesis and function and, importantly, increased the cell-death response of chemoAML cells to chemotherapy, revealing that VEGFR2 signaling modulates the response of AML cells to chemotherapy by controlling mitochondrial mass and biogenesis. The development of resistance to anti-angiogenic drugs, targeting VEGFR among other receptors, is accompanied by an increase reliance on mitochondrial respiration that drives solid tumor survival . CollectThere is an increasing appreciation of the importance of targeting mitochondria in acute leukemia. Enhanced mitochondrial metabolism is believed to represent a liability due to the role of mitochondria in apoptosis and generation of reactive oxygen species. On the other hand, increased reliance on lipid metabolism and mitochondrial respiration has been described in leukemia relapses, highlighting the CD36-FAO-OXPHOS axis as a plausible target to eradicate therapy-resistant leukemia . In our PGC-1α, PGC-1β, ERRα, and NRF1, known regulators of oxidative metabolism and mitochondrial biogenesis. Importantly, we show that low levels of PGC-1α expression are associated with worst overall AML patient survival, and in chemotherapy-exposed AML cells, PGC- 1α depletion abolished the capacity of VEGFR2 inhibition to re-sensitize to chemotherapy treatment, highlighting PGC-1α as a molecular suppressive axis for VEGFR2- induced viability in therapy-resistant AML cells (Figure Mechanistically, we show that VEGFR2 inhibition elicited a transcriptional response in chemotherapy- exposed AML cells, resulting in increased expression of per se, this strategy may confer another layer of specificity by directly suppressing a leukemic-specific downstream target without interfering with VEGFR2 signaling in normal cells or AML-associated angiogenesis. It will be important to determine if PGC-1α activation is a viable therapeutic modality to increase the response to chemotherapy in multiple therapy-resistant AML subgroups or only for VEGFR2-dependent patients subsets. Further studies are warranted but a new avenue for combinatorial strategies to treat therapy-resistant leukemia is now open.A current challenge of designing new combinatorial therapies for AML is the diversity of genetic abnormalities found at diagnosis, making the identification of a common target among AML subgroups an urgent need. Our results lend support for the development of PGC-1α activators as a suitable alternative for new combinatorial therapies in treating relapsed AML. As opposed to blocking VEGFR2


TRPC4 assembles as homo or hetero-tetramer in the plasma membrane, allowing a non-selective Na+ and Ca2+ influx. Gαq protein-coupled receptor (GPCR) stimulation is known to increase TRPC4 channel activity and a TRPC4-mediated Ca2+ influx which has been regarded as ideal Ca2+ source for calcineurin and subsequent nuclear factor of activated T-cells (NFAT) activation. Functional properties of TRPC4 are also based on the expression of the TRPC4 splice variants TRPC4α and TRPC4β. Aim of the present study was to analyze cytosolic Ca2+ signals, signaling, hypertrophy and vitality of cardiomyocytes in dependence on the expression level of either TRPC4α or TRPC4β. The analysis of Ca2+ transients in neonatal rat cardiomyocytes (NRCs) showed that TRPC4α and TRPC4β affected Ca2+ cycling in beating cardiomyocytes with both splice variants inducing an elevation of the Ca2+ transient amplitude at baseline and TRPC4β increasing the Ca2+ peak during angiotensin II (Ang II) stimulation. NRCs infected with TRPC4β (Ad-C4β) also responded with a sustained Ca2+ influx when treated with Ang II under non-pacing conditions. Consistent with the Ca2+ data, NRCs infected with TRPC4α (Ad-C4α) showed an elevated calcineurin/NFAT activity and a baseline hypertrophic phenotype but did not further develop hypertrophy during chronic Ang II/phenylephrine stimulation. Down-regulation of endogenous TRPC4α reversed these effects, resulting in less hypertrophy of NRCs at baseline but a markedly increased hypertrophic enlargement after chronic agonist stimulation. Ad-C4β NRCs did not exhibit baseline calcineurin/NFAT activity or hypertrophy but responded with an increased calcineurin/NFAT activity after GPCR stimulation. However, this effect was not translated into an increased propensity towards hypertrophy but rather less hypertrophy during GPCR stimulation. Further analyses revealed that, although hypertrophy was preserved in Ad-C4α NRCs and even attenuated in Ad-C4β NRCs, cardiomyocytes had an increased apoptosis rate and thus were less viable after chronic GPCR stimulation. These findings suggest that TRPC4α and TRPC4β differentially affect Ca2+ signals, calcineurin/NFAT signaling and hypertrophy but similarly impair cardiomyocyte viability during GPCR stimulation.The Transient Receptor Potential Channel Subunit 4 (TRPC4) has been considered as a crucial Ca In ordemplitude  which wamplitude . In ordeII 1 μM; . The adm2+ transients at baseline, but does not affect Ca2+ transients during GPCR stimulation. The overexpression of TRPC4β on the other hand affects the Ca2+ transient amplitude at baseline and is connected with elevated Ca2+ transients during GPCR stimulation.These findings suggest that TRPC4α increases Ca2+ signals that activate the pro-hypertrophic calcineurin/NFAT signaling pathway [2+ signals were translated in an altered calcineurin activity. As surrogate of calcineurin activation, we assessed the translocation of the transcription factor NFATc1 from the cytosol into the nucleus [A series of studies has shown TRPC channel-mediated Ca pathway . Accordi nucleus . As show2+ cycling and signaling were reflected by changes in the hypertrophic response, we monitored the hypertrophic growth of cardiomyocytes in an agonist-mediated approach. Cardiomyocytes were infected with Ad-C4α, Ad-C4β or Ad-βgal, serum starved and stimulated with Ang II/PE or vehicle in serum free conditions for 24 h. Ang II/PE treatment resulted in a typical enlargement of the cell surface in Ad-βgal NRCs  as alternative Ca2+-dependent mechanism which might counteract a calcineurin/NFAT-mediated hypertrophic effect. To indirectly assess the activity of CamKII we examined the phosphorylation status of PLN (Thr 17) and HDAC4 (Ser 632) in cardiomyocytes lysates from TRPC4-infected cardiomyocytes . EBP50 0  have been found as crucial pro-death signaling proteins responding to high level Gαq activation [2+ entry to hypertrophy at baseline but to apoptotic signaling pathways under stimulatory conditions. In this context it is interesting to note that both apoptotic and hypertrophic functions have been described for calcineurin in cardiomyocytes [Regulatory properties of different TRPC4 isoforms might also be dependent on the species. For example, human TRPC4α has been found to be barely stimulated by GPCR agonists ,23, whicf TRPC4α . Our restivation  and coultivation . In TRPCmyocytes ,32.2+ influx and increased the Ca2+ transient amplitude at baseline. Paradoxically, the diastolic Ca2+ levels in both TRPC4α and TRPC4β over-expressing NRCs were not altered. It is possible that a TRPC4-mediated Ca2+ overload might be compensated by an enhanced Ca2+ uptake in the SR which would also explain the increased Ca2+ transient amplitudes in Ad-C4α and Ad-C4β NRCs.An increased apoptotic rate was also noticeable in cardiomyocytes overexpressing TRPC4β, although the preceding processes seemed to be different to Ad-C4α NRCs. Similar to TRPC4α, TRPC4β allowed an enhanced Ca2+ transient amplitude during Ang II stimulation. This might indicate that Ang II stimulation evokes a TRPC4β-mediated Ca2+ influx that contributes to elevated systolic Ca2+ peaks. The sensitivity of TRPC4β towards GPCR stimulation was also noticeable as a pronounced Ca2+ influx in quiescent Ad-C4β-infected cardiomyocytes. A GPCR-dependent regulation is a well-described characteristic of TRPC4β across different species [In contrast to TRPC4α, TRPC4β over-expression was also connected with a marked elevation of the Ca species , and acc2+ influx in the presence of the non-selective TRPC inhibitor BTP2. In NRCs of all three groups , the Ca2+ influx was blocked to a similar extent. However, it has to be kept in mind that BTP2 also blocks CRAC channels as suggested for other cell types [2+ responses after Ang II stimulation.We also analyzed the Ang II-dependent Call types ,33. Due ll types . Further2+ characteristics of TRPC4β overexpressing NRCs, we expected a pronounced hypertrophic cardiomyocyte growth under baseline as well as stimulatory conditions. However, it came as a surprise that the TRPC4β-mediated baseline Ca2+ influx and the elevated Ca2+ transients were not coupled to an increased calcineurin/NFAT activation and hypertrophic growth as found for TRPC4α. This discrepancy might be influenced by intrinsic structural and functional differences between TRPC4α and TRPC4β, as well as by distinct interactions with protein partners such as PLCβ1b [Given the Cas PLCβ1b  that reg2+ influx was coupled to an enhanced calcineurin/NFAT activity, however an attenuated hypertrophic cardiomyocyte growth. We instantly assumed that the inefficiency of TRPC4β to alter cardiomyocyte remodeling during GPCR stimulation might be simply based on a reduced activity of other Ca2+-dependent signaling proteins, such as CamKII. We found no obvious changes in the phosphorylation status of the CamKII-sensitive downstream targets, PLN or HDAC4, however, an enhanced caspase activity accompanied with a lower cardiomyocyte viability after chronic agonist treatment. Thus, during GPCR stimulation TRPC4β might activate calcineurin which is not necessarily connected with pro-hypertrophic but rather pro-apoptotic effects. Our findings also suggest that both TRPC4α and TRPC4β initiate apoptotic processes during chronic agonist stimulation in pathological conditions irrespective of their sensitivity to GPCR signaling.Even more puzzling was the fact that under stimulatory conditions, an elevated TRPC4β-mediated CaPro-apoptotic effects in cardiomyocytes have been described for other TRPC family members, such as TRPC7 which mediates apoptosis through the activation of calcineurin during Ang II stimulation . HoweverIn summary our findings indicate that, although TRPC4α and TRPC4β might assemble to ion channels with different regulatory characteristics, their activity converges into a common cardiomyocyte phenotype which is more vulnerable to apoptosis during GPCR stimulation. In a bigger context these results might show that an upregulation of TRPC4α and TRPC4β could be a critical alteration contributing to cardiac disease progression.S1 Fig2+ traces in neonatal rat cardiomyocytes (NRCs) infected with either Ad-βgal (A), Ad-TRPC4α (B) or Ad-TRPC4β (C). Quiescent cells were perfused with a normal Tyrode solution containing 1 mM Ca2+ + BTP2 (5 μM). Verapamil (10 μM) was included throughout the recordings. After 50 s, angiotensin II  was applied.Representative Fura-2 Ca(TIF)Click here for additional data file.


V1.3 Ca2+-channels, their tight coupling to synaptic vesicles (SVs) and fast replenishment of fusion competent SVs. Here we studied the role of RIM-binding protein 2 (RIM-BP2)—a multidomain cytomatrix protein known to directly interact with Rab3 interacting molecules (RIMs), bassoon and CaV1.3—that is present at the inner hair cell active zones. We combined confocal and stimulated emission depletion (STED) immunofluorescence microscopy, electron tomography, patch-clamp and confocal Ca2+-imaging, as well as auditory systems physiology to explore the morphological and functional effects of genetic RIM-BP2 disruption in constitutive RIM-BP2 knockout mice. We found that RIM-BP2 (1) positively regulates the number of synaptic CaV1.3 channels and thereby facilitates synaptic vesicle release and (2) supports fast synaptic vesicle recruitment after readily releasable pool (RRP) depletion. However, Ca2+-influx—exocytosis coupling seemed unaltered for readily releasable SVs. Recordings of auditory brainstem responses (ABR) and of single auditory nerve fiber firing showed that RIM-BP2 disruption results in a mild deficit of synaptic sound encoding.Ribbon synapses of inner hair cells (IHCs) mediate high rates of synchronous exocytosis to indefatigably track the stimulating sound with sub-millisecond precision. The sophisticated molecular machinery of the inner hair cell active zone realizes this impressive performance by enabling a large number of synaptic voltage-gated Ca V1.3 Ca2+-channels, RIMs and bassoon. We find evidence that RIM-BP2 (1) positively regulates the number of synaptic Ca2+-channels—the key regulators of synaptic vesicle exocytosis and (2) contributes to fast synaptic vesicle recruitment after depletion of the readily releasable pool (RRP) of vesicles. This way RIM-BP2 contributes to normal sound encoding at the inner hair cell synapse.The inner hair cell synapse provides great access for biophysical analysis of individual ribbon–type active zones. Here, we studied the role of RIM-binding protein 2 (RIM-BP2), a multi-domain protein that is part of the cytomatrix of the inner hair cell presynaptic active zone and was previously shown to directly interact with CaV1.3 Ca2+-channels. In fact, the release of each RRP SV seems to be under the control of only few Ca2+-channels in nanometer proximity: Ca2+-nanodomain-like control of exocytosis  to the auditory nerve is a fundamental step in hearing. It is this first synapse of the auditory pathway, where incoming sounds are encoded at high rates and with sub-millisecond precision . In neuronal synapses, bassoon, piccolo, liprin-α, Munc13s/CAPS, CASTs/ELKSs, Rab3 interacting molecules (RIMs) and their binding proteins (RIM-BPs) form the core of the CAZ and contribute to docking and priming of SVs  and their wild-type littermates RIM-BP2 (RIM-BP2+/+) . Systems physiology and extracellular recordings from single SGNs were carried out at the age of 8–10 weeks. All experiments complied with national animal care guidelines and were approved by the University of Göttingen board for animal welfare and the animal welfare office of the state of Lower Saxony.RIM-BP2+/+ and RIM-BP2−/− mice were either fixed with 4% formaldehyde (FA) in phosphate buffered saline (PBS) on ice for 10 min or with methanol at −20°C for 2 min. Thereafter, the tissue was washed 3 × 10 min in PBS and incubated for 1 h in goat serum dilution buffer  in a wet chamber at room temperature. Primary antibodies were dissolved in GSDB and applied overnight at 4°C in a wet chamber. After washing 3 × 10 min  the tissue was incubated with secondary antibodies in GSDB in a wet light-protected chamber for 1 h at room temperature. Then, the samples were washed 3 × 10 min in wash buffer and 1 × 10 min in 5 mM phosphate buffer, placed onto the glass microscopy slides with a drop of fluorescence mounting medium (Mowiol) and covered with thin glass coverslips. The following antibodies were used: mouse-IgG1-anti-CtBP2 , guinea pig-anti-Synapsin1/2 , rabbit-anti-CaV1.3 , guinea pig-anti-bassoon , rabbit-anti-GluA2/3 , rabbit-anti-RIM-BP2  as well as secondary AlexaFluor568- and AlexaFluor647-labeled goat-anti-mouse and goat-anti-rabbit antibodies  and STAR580-, and Star635P-labeled goat-anti-mouse, goat-anti-rabbit and goat-anti-guinea pig antibodies . Confocal images were acquired using a laser scanning confocal microscope  equipped with 561 and 633 nm lasers for excitation and a 63x oil immersion objective . STED images were acquired using a 2-color STED microscope  equipped with 561 and 640 nm excitation lasers, a 775 nm laser for STED (1.2 W) and a 100x oil immersion objective . Samples were treated in parallel and images were acquired in parallel, using same laser power, gain, and microscope settings. Images were analyzed using ImageJ, Igor Pro, OriginPro and Imaris software, and assembled for display in Adobe Illustrator software.Freshly dissected apical turns of the organs of Corti of 3 week old RIM-BP+/+ and RIM-BP−/− mice was performed as described previously  as described : 130 Cs-gluconate, 10 TEA-Cl, 10 4-AP, 10 HEPES, 1 MgCl2, amphotericin B (300 μg/ml), pH 7.2. For confocal Ca2+-imaging we performed ruptured-patch-clamp recordings with the solution described above excluding amphotericin but including 4 mM Mg-ATP, 0.3 mM Na-GTP, 1 mM EGTA, 0.4 mM Fluo-4FF (Invitrogen), and 0.01 mM carboxytetramethylrhodamine(TAMRA)-conjugated dimeric RIBEYE-binding peptide  controlled by “Patchmaster” software (HEKA) were used for measurements. All voltages were corrected for liquid junction potentials (14 mV). Currents were leak-corrected using a p/10 protocol. Current-voltage relationships (IVs) were calculated from the last 8 ms of currents evoked by step depolarizations to various potentials. From these, fractional activation curves were calculated by first calculating the conductance of the calcium current as V is the command potential and Vrev is the reversal potential of the current obtained by extrapolating a line fit from 6 to 26 mV. After normalizing these traces to the maximum conductance in the range of −20 to +10 mV, they were fit with a Boltzmann equation n,max, as the maximum conductance, V as the command potential, Vhalf as the voltage of half-maximal activation, and k as the slope factor. ICa inactivation kinetics were calculated from fitting a single exponential function to the last 195 ms of ICa traces during 200 ms depolarizations to −14 mV . For membrane capacitance (Cm) measurements, IHCs were stimulated by depolarizations to −14 mV with intervals of 60–120 s to allow for recovery of IHC exocytosis. Two pulse protocols were used: (i) single depolarizations of different durations and (ii) paired depolarizations with variable inter-pulse interval. We measured Cm changes (ΔCm) using the Lindau-Neher technique  of the dihydropyridine L-type Ca2+-channel antagonist isradipine (10 μM) in the extracellular solution (here using 5 mM CaCl2 and 104 mM NaCl). The exocytic ΔCm and corresponding ICa evoked by 20 ms depolarizations  were recorded while slowly reducing the number of unblocked Ca2+-channels and ΔCm to the Ca2+-current integral (QCa) for each individual IHC and fit by a power function ΔCm = A (QCa)m, where the power m describes the apparent Ca2+-cooperativity of synaptic vesicle exocytosis.The apparent Ca2+-imaging we used the low affinity Ca2+-indicator Fluo-4FF  and also loaded the IHCs with TAMRA-labeled RIBEYE-binding peptide for visualization of the ribbons  over the last 10 ms of depolarization. ΔF was normalized to F0, for which we used the fluorescence intensity of the same three peak-centered pixels before IHC depolarization.For confocal CaZenisek, . Line scABR, DPOAE, and extracellular recordings from single SGNs were performed as described before  was used to compare non-normal or data with unequal variances. Else, unpaired, two-tailed Student's t-test were used. For comparing the variance of the ΔF/F0 estimates for synaptic Ca2+-signals we used the Brown-Forsythe-modified Levene test. The distance of MP-SVs from the plasma membrane was compared between genotypes in the ranges of 0–25 and 26–50 nm by the Kolmogorov–Smirnov test. Data analysis for systems physiology was done using waveform-based spike detection using custom-written MATLAB software. Further statistical analysis was done in Igor Pro as described above for all parameters except spontaneous rate distributions, which were compared using Mann Whitney U-test in GraphPad Prism.Data analysis and statistical tests were done in Igor Pro for electrophysiology, CaRIM-BP2+/+ IHCs and was absent from the basal pole of IHCs of RIM-BP2−/− IHCs confirmed specificity of the immunolabeling .First, we studied the localization of RIM-BP2 at IHC AZs using immunolabeling and confocal microscopy of mouse IHCs. RIM-BP2 immunofluorescence at CtBP2/RIBEYE-marked ribbon-occupied AZs of g Figure . In addig Figure . This nog Figure  , we first addressed the afferent connectivity of IHCs using confocal microscopy and 3D analysis of immunolabeled synapses  and ribbon-occupied synapses (number of juxtaposed RIBEYE/CtBP2 and GluA2/3 spots) (p = 0.6) to be comparable in RIM-BP2+/+ and RIM-BP2−/− IHCs   Figure , Table 1n Figure , which wCa of all AZs and the extrasynaptic membrane, we sought to further analyze Ca2+-influx at the single AZ level using laser scanning confocal Ca2+-imaging  we expect the Fluo-4FF fluorescence changes (ΔF/F0) to be a good proxy for the synaptic Ca2+-influx  of the Fluo-4FF maximal fluorescence change (ΔFmax/F0) at AZs of RIM-BP2−/− IHCs, which exceeded the reduction of their whole-cell Ca2+-current   and RIM-BP2+/+ AZs (CV = 0.88). Hence, despite the reduced synaptic Ca2+-influx, presynaptic heterogeneity was unchanged at RIM-BP2−/− AZs  at RIM-BP2−/− AZs were not significantly different to that of RIM-BP2+/+ AZs  Figure . This sus Figure . FurtherV1.3 Ca2+-channels remained clustered at ribbon-occupied AZs in RIM-BP2−/− IHCs  assumed a “stripe-like” or “double stripe-like” arrangement of Ca2+-channels. Only 12% of Ca2+-channel clusters formed “round” and 22% “complex” cluster shapes. We note that we focused on synapses seemingly lying en face in the imaging plane, and refer to apparent shapes as we did not assess the clusters with 3D STED resolution. We found the classes of apparent shapes and their representation to be very similar in RIM-BP2−/− IHCs with a slight trend toward less stripe-shaped (62%) and more round (16%) appearance. We approximated the dimensions of “stripe-shaped” Ca2+-channel clusters by fitting a 2-dimensional (2D) Gaussian function to STED images of CaV1.3 immunofluorescence  and the cluster area  as well as the integral of the 2D Gaussian fit function  were significantly smaller in RIM-BP2−/− AZs . Consequently, there was a trend toward a smaller FWHM ratio of long over short axis, which, however, did not reach statistical significance (p = 0.1). We interpret the calculated area and integral of the 2D Gaussian fit as a measure of Ca2+-channel cluster size and the integrated CaV1.3 immunofluorescence as a proxy for the number of Ca2+-channels. However, we note that CaV1.3 immunofluorescence might not relate linearly to the number of Ca2+-channels. In summary, our morphological STED imaging indicates a “rounder” shape, a smaller size and a halved integrated CaV1.3 immunofluorescence of the Ca2+-channel clusters of RIM-BP2-deficient AZs, which is consistent with the 41% reduction of the ΔFmax/F0 found by Ca2+-imaging. The same analysis was performed for bassoon clusters  in response to voltage-gated Ca2+-influx in RIM-BP2+/+ and RIM-BP2−/− IHCs. We studied RRP and sustained exocytosis by probing ΔCm in response to depolarizations of various durations to the potential eliciting the maximum Ca2+-current . The sustained phase of SV exocytosis, probed by responses to stimuli longer than 20 ms, is thought to primarily reflect vesicle resupply to the RRP and subsequent fusion . In addition, a line was fit to the sustained phase of exocytosis (from 50 to 200 ms) to estimate the kinetics of SV exocytosis during ongoing depolarization  compared to RIM-BP2+/+ IHCs . However, we still observed a significant difference between the two genotypes regarding the sustained phase of exocytosis . This indicated that the reduction of presynaptic Ca2+-influx does not fully account for the impaired sustained exocytosis, suggesting a role of RIM-BP2 in SV-replenishment. When raising the extracellular Ca2+ concentration ([Ca2+]e) from 2 to 5 mM, thereby boosting single channel Ca2+-current, we observed a rescue of the exocytic response of RIM-BP2−/− IHCs that then was indistinguishable from RIM-BP2+/+ at 2 mM [Ca2+]e   Figure . This su2+-influx–exocytosis coupling by probing the apparent Ca2+-cooperativity m of RRP exocytosis m restricted to the range that did not show obvious saturation of ΔCm,20 ms  arguing for intact Ca2+-influx–exocytosis coupling for RRP SVs, at least after full RRP recovery. This, however, does not rule out a function of RIMB-BP2 in promoting the engagement of newcoming vesicles with Ca2+-channels. In fact, the reduction of sustained exocytosis as well as its increased Ca2+-dependence would be in line with such a scenario.Next, we studied Cas Figure . The estRIM-BP2−/− IHCs as evident from significantly smaller responses at an interval of 50 ms  were sufficient for normal RRP replenishment in RIM-BP2-deficient IHCs  to the first pulse Figure . RRP recs Figure . Likely,−/− and RIM-BP2+/+ mice using electron tomography . There was a tendency of more MP-SVs in RIM-BP2−/− AZs to populate the smaller distance bins . In addition, ABR wave I amplitude, reporting the compound action potential of the SGNs, was significantly reduced . As the presence of otoacoustic emissions with normal amplitudes   and distortion product otoacoustic emissions (DPOAE) in ) Figure  indicate) Figure . Potentiin vivo analysis of single AZ function as each SGN is thought to receive input from just one AZ of one IHC , which might result from the reduction in the Ca2+-channel abundance at the IHC ribbon synapses , while the adapted firing rates where indistinguishable between SGNs of both genotypes , but the trend toward an increased variance of first spike latency did not reach statistical significance  and significantly so for 16 ms (p = 0.04), but from the interval of 64 ms onwards, the recovered response was comparable to that of RIM-BP2+/+ SGNs, and the time constant for recovery from masking was unaltered. In summary, recordings from single SGNs indicated a mild impairment of sound onset coding and suggested that fast vesicle replenishment is slowed in the absence of RIM-BP2.Using extracellular recordings we studied sound encoding by single postsynaptic SGNs, which offers  Figures . We foun Figures  and soun Figures  of RIM-Bn Figure . We thenn Figure , which in Figure . The pean Figure . The pars Figure . The fire Figure . Togethee Figure . Therefoe Figure , which pe Figure . Probe r2+-channels and, in addition, (2) promotes fast SV-replenishment. STED microscopy indicated that RIM-BP2 is part of the typically stripe-like appearing presynaptic density at IHC AZs. Recordings of whole-cell and synaptic Ca2+-influx as well as STED microscopy of CaV1.3 immunofluorescence collectively demonstrated a reduced abundance of CaV1.3 Ca2+-channels at IHC AZs upon disruption of RIM-BP2. Sustained exocytosis was reduced. RRP exocytosis and its Ca2+-nanodomain-like control by Ca2+-channels seemed preserved when probed with long inter-stimulus intervals. RRP replenishment, however, was slowed in RIM-BP2-deficient IHCs. In addition, subtle morphological changes in the spatial organization of SVs at the plasma membrane were found by electron tomography. Finally, we found a mild impairment of sound onset coding in SGNs of RIM-BP2−/− mice that likely reflects the presynaptic impairment of Ca2+ signaling and SV-replenishment. In summary, we propose that RIM-BP2 promotes the clustering of CaV1.3 channels at the IHC AZs and supports fast RRP replenishment.In this study, we examined the expression of RIM-BP2 in mouse IHCs and studied the structural and functional effects of genetic RIM-BP2-deletion in IHC ribbon synapses. We demonstrate the presence of RIM-BP2 at the IHC AZs where it positively regulates (1) the number of synaptic Ca2+-influx and exocytosis enabling presynaptic studies of Ca2+-channels and their regulation of transmitter release. Ribbon-type AZs of IHCs, on average, are thought to cluster ~80–100 Ca2+-channels  , suggesting that AZs fail to cluster the available Ca2+-channels. Consistent with impaired synaptic clustering of Ca2+-channels we found a reduced length of the stripe-like clusters, which also showed lower integrated CaV1.3 immunofluorescence intensity. Importantly, the length of the presynaptic density, measured by STED microscopy of bassoon immunofluorescence, was normal for the AZs of RIM-BP2-deficient IHCs. Together, this suggests that the reduction of Ca2+-channel number occurs despite the presence of the ribbon and of a normal bassoon-containing presynaptic density, placing RIM-BP2's function in channel clustering downstream of bassoon. Both, the width and area of CaV1.3 clusters revealed by STED microscopy and the spread of the Ca2+ signal at the AZ appeared normal arguing against a mis-localization of Ca2+-channels to outside the area marked by the presynaptic density. Moreover, the biophysical properties of IHC Ca2+-channels, voltage-dependence of activation as well as extent and kinetics of inactivation, were unaltered. We cannot fully exclude changes in Ca2+-channel open probability due to delayed synapse maturation upon RIM-BP2 disruption. However, several findings argue against a developmental deficit in RIM-BP−/− IHC AZs: (i) Ca2+-current inactivation was unaltered and mature  and requires d-RIM-BP for sufficient Ca2+-influx and transmitter release  reduction of exocytosis beyond that of the Ca2+-influx due to additional disruption of Ca2+-nanodomain-like control of exocytosis and/or impaired vesicle replenishment. Emerging evidence shows for neuromuscular junction of drosophila larvae  reduction of exocytosis in proportion with the loss of presynaptic Ca2+-cooperativity m of RRP exocytosis for changes in Ca2+-influx brought about by successive dihydropyridine block of CaV1.3 Ca2+-channels. We had established by various experiments and modeling that the near linear m  for channel block, which contrasts an m of 3–4 during changes of single channel current, is an indicator for Ca2+-nanodomain-like control of exocytosis at the mature IHC AZ (Brandt et al., m of RRP exocytosis during dihydropyridine block with inter-pulse intervals of 60 s and more, in order to secure full RRP replenishment, we found no indication for a looser Ca2+-influx—exocytosis coupling in RIM-BP2-deficient IHCs. The mild effect of genetic deletion RIM-BP2 on RRP exocytosis and the intact Ca2+-nanodomain coupling are surprising given the findings of previous studies on RIM-BP function and might suggest that IHC ribbon-type AZs can use additional molecular mechanisms to establish tight coupling of Ca2+-channels and release sites in the absence of RIM-BP2. In this respect it is worth mentioning, that disruption of the RIM-BP interaction partners bassoon or of RIM2α did not lead to an obvious switch to Ca2+-microdomain control of RRP exocytosis at IHC synapses either (Frank et al., 2+-channels and release sites. Further, the observation of tight Ca2+-nanodomain-like control of exocytosis for fusion-competent SVs of the RRP in the absence of RIM-BP2 was made under the artificial conditions of the biophysical experiment, where the synapse was inactivated due to voltage-clamp to hyperpolarized potential for full RRP recovery. In physiology, on the other hand, IHC AZs mediate massive vesicle turnover, which requires rapid replenishment at rates of hundreds of vesicles per second (Pangršič et al., 2+-channel complexes needs to proceed at a similar pace (Cho et al., 2+-channels e.g., through Rab3—RIM—RIM-BP2—Ca2+-channel interaction (Hibino et al., 2+-channel after RRP depletion. This step was found to be rate-limiting as it converts slow releasing SVs into fast releasing SVs and was called “positional priming” (Neher and Sakaba, RIM-BP2−/− EM tomograms suggests a change in SV position with respect to release sites. The observed change was small, but likely, the recruitment process of SVs is so fast that it is not fully reflected in our EM data. Both paired-pulse experiments and forward masking point out a significant delay in RRP refilling for short inter-stimulus intervals, and thereby support the hypothesis that RIM-BP2 plays a role in fast RRP refilling. Additionally, the reduced number of synaptic Ca2+-channels in RIM-BP2 deficient AZs might enhance the delay during “positional priming,” since fewer channels are available for SVs to connect. Hence, SV turnover speed would be limited by a lower probability of SV—release site matching due to a reduced number of Ca2+-channels and possibly also through the slowed engagement of SVs with the remaining Ca2+-channels. Given the possibility of RIM-BP2 interaction with the ribbon anchor bassoon (Davydova et al., 2+-channels and the presynaptic density and thus, ribbon-tethered SV. Both, STED microscopy and Ca2+-imaging did not reveal changes in the width and area of CaV1.3 clusters and spread of the Ca2+ signal, arguing against major changes in Ca2+-channel localization. In addition, the distance of MP SVs to the PD was unaltered in RIM-BP2 deficient synapses. However, we cannot rule out that already a slight increase of the distance of Ca2+-channels from the PD could slow down the engagement of new incoming ribbon-tethered SVs with a Ca2+-channel during RRP replenishment. Interestingly, the reduced efficiency of Ca2+ influx to trigger exocytosis could be rescued by elevating extracellular [Ca2+]e in RIM-BP2−/− IHCs. Elevated extracellular [Ca2+]e could lead to accelerated Ca2+-dependent SV trafficking and replenishment which was reported for hair cells (Moser and Beutner, 2+ signaling due to the reduced number of synaptic Ca2+-channels in RIM-BP2 deficient AZs might therefore explain their slower SV replenishment. In addition, or alternatively, increasing Ca2+-influx through elevation of extracellular [Ca2+]e might recruit more distant release sites so that the exact spatial distribution and matching of Ca2+-channels and SVs is less critical (Fuchs et al., Drosophila NMJ (Müller et al., Here, we tested the apparent Ca2+-imaging, and contributed to EM analysis, TB performed auditory nerve fiber recordings, SJ performed patch-clamp capacitance measurements and CW performed electron microscopy. TM and SK prepared the manuscript.SK, CW, and TM designed the study. SK performed immunohistochemistry and confocal/STED microscopy, patch-clamp capacitance measurements and CaThe 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.


Myeloproliferative neoplasms (MPNs) are hematopoietic malignancies characterized by unchecked proliferation of differentiated myeloid cells. The most common BCR-ABL1-negative MPNs are polycythemia vera, essential thrombocythemia, and primary myelofibrosis. The discovery of JAK2 V617F mutation has improved our understanding of the molecular basis of MPN. The high frequency of JAK2 mutation in MPN makes JAK2 mutation testing an essential diagnostic tool and potential therapeutic target for MPN. Here, we present a rare case of a 34-year-old patient who was initially diagnosed with acute myeloid leukemia (AML) with mutated NPM1. After chemotherapy treatment followed by granulocyte colony stimulating factor administration, the patient achieved complete remission of AML. However, the bone marrow showed hypercellularity with granulocytic hyperplasia, markedly increased atypical megakaryocytes (50.2/HPF) with focal clustering, and reticulin fibrosis (3/4). JAK2 V617F mutation was also detected. Considering the possibility of AML transformed from a previous undiagnosed MPN, patient underwent peripheral blood allogenic stem cell transplant. This case illustrates the diagnostic challenges of firmly establishing a diagnosis between similar, but distinct, disease entities and an accurate clinicopathological differentiation is crucial. Myeloproliferative neoplasms (MPNs) are characterized by excessive production of terminally differentiated myeloid cells. The most common BCR-ABL1-negative MPNs are polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). In MPN, the JAK2 V617F mutation is the most prevalent mutation discovered in 2005 .MPNs have a risk of transforming to secondary acute myeloid leukemia (sAML). Leukemic transformation of ET, PV, and PMF occurs at rates of approximately 1%, 4%, and 20%, respectively, over the first decade from time of MPN chronic phase diagnosis. After leukemic transformation, patients have poor prognosis with an adverse outcome within a few months. The frequency of JAK2 mutation in de novo AML is about 1%. In contrast, the prevalence of JAK2 mutation in AML transformed from MPN is about 50% .Administration of granulocyte macrophage colony stimulating factor (GM-CSF) in combination with thrombopoietin (TPO) to patients who had received chemotherapy for acute myeloid leukemia (AML) has been found to cause bone marrow hypercellularity, marked megakaryocytic hyperplasia, megakaryocytic atypia, and reticulin fibrosis with a rapid resolution of the morphologic abnormalities after discontinuation of TPO. These findings, in combination with a possible peripheral blood leukoerythroblastic reaction, yield an overall picture that morphologically resembles that seen in patients with MPN , 9.Here, we present a rare case of a patient with a quite complicated clinical course and histopathologic picture of megakaryocytic hyperplasia following treatment and resolution of acute myeloid leukemia. This case illustrates the diagnostic challenges of firmly establishing a diagnosis between similar, but distinct, disease entities.Patient was a 34-year-old male with a medical history significant for gout, chronic pain secondary to a motor vehicle accident, and bipolar disorder. There was no documented prior history of childhood malignancy or radiation. He had a gout exacerbation which was followed by the development of fever, sweats, and sore throat. He presented to a local urgent care center where he was found to have moderate splenomegaly and significantly high white blood cell (WBC) count with circulating blasts in the peripheral blood . The firμL with a normal white count and hemoglobin level.The postinduction bone marrow biopsy was performed and showed 95% cellularity with granulocytic hyperplasia, markedly increased megakaryocytes (50.2/HPF), and increased reticulin fibrosis (3/4). No blasts were identified in the marrow. The megakaryocytes were large, hyperlobulated, and clustered . The perThe patient started consolidation chemotherapy with high dose intermittent ARA-C (HiDAC). The subsequent bone marrow biopsy revealed 80% cellular marrow with mild megakaryocytic hyperplasia (6.7/HPF) and complete resolution of marrow fibrosis with no blasts identified in the marrow . The megμL, and WBC count 2,000/μL with normal differentiation. The JAK2 V617F mutation was not detected. The patient was last seen in July 2016. He remained in complete remission with no clinical or laboratory evidence of relapse of AML and MPN.Considering the possibility of AML transformed from a previous undiagnosed MPN, patient underwent peripheral blood allogenic stem cell transplant from his HLA-match, ABO mismatch sister after myeloablation. Day 30 chimerism revealed 100% donor myeloid cells and 64% donor T cells and a day 60 evaluation for chimerism showed 100% donor myeloid cells and 99% donor T cells. The follow-up bone marrow biopsy revealed normocellular marrow with no overt phenotypic abnormalities. The megakaryocytes were mildly decreased in number and morphologically unremarkable without clustering. His hemoglobin level was 12.3 g/dL, platelet count 93,000/PV is characterized by unregulated red blood cell overproduction and ET by excessive platelet production, while PMF is a clonal stem cell disorder with a consequence of megakaryocytic and granulocytic proliferation, progressive marrow fibrosis, and extramedullary hematopoiesis –3.The JAK family proteins (JAKs) are cytoplasmic tyrosine kinases that play important roles in cytokine receptor superfamily signaling which are crucial for normal hematopoiesis. Aberrant hyperactivation of JAK signaling pathway has increasingly been implicated in the pathogenesis of hematopoietic malignancies , 4, 5. TAML is a clonal hematopoietic disorder involving clonal hematopoietic stem cell or a lineage-specific progenitor cell, resulting in the accumulation of blast cells associated with disrupted granulomonocytic differentiation in the bone marrow. AML accounts for approximately 80% of all adult leukemia cases with the majority of the cases arising de novo , 7. Thern = 77) demonstrated elevated levels of activated phospho-Jak2 (p-JAK2) in bone marrow samples which is associated with high white blood cell count, low platelet count, and shorter survival in patients with either de novo or secondary AML. In this study, JAK2 V617F mutation was detected in only 1 of the 77 patients, indicating that alternative mechanisms account for JAK2 signal activation and may represent a potential therapeutic target [JAK2 mutation has been found in approximately 1% of de novo AML patients, more commonly in t8; 21) AML. In addition, activating JAK2 gene fusions with the TEL ETV6) (TEL-JAK2) and PCM1 genes has been identified in leukemia patients. A recent study from a large cohort of patients with AML  and thrombopoietin (TPO) to patients treated with chemotherapy for AML has been found to cause bone marrow hypercellularity, marked megakaryocytic hyperplasia, megakaryocytic atypia, and reticulin fibrosis with a rapid resolution of the morphologic abnormalities after discontinuation of TPO , 9. In tIn this patient, the diagnosis of de novo AML with mutated NPM1 was made on his first bone marrow biopsy showing 70% CD13, CD33, CD117, HLA-DR(+), and CD34(−) myeloblasts with circulating blasts and marked leukocytosis. After induction therapy, the patient achieved complete remission of AML. However, the bone marrow showed hypercellularity with granulocytic hyperplasia, markedly increased atypical megakaryocytes (50.2/HPF) with clustering, and reticulin fibrosis (3/4). The peripheral platelet count was also dramatically elevated. The possibility of sAML transformed from a preexisting undiagnosed MPN was considered and supported by presence of persistent JAK2 V617F mutation, history of long standing gout, and marked thrombocytosis approximately 2 months before his AML presentation. As well documented, after leukemic transformation from MPN, patients have poor prognosis with an adverse outcome within a few months. In this patient, the absence of a complex cytogenetic abnormality, complete remission of AML after induction chemotherapy, and rapid normalization of bone marrow after consolidation all argue against the diagnosis of sAML transformation from preexisting MPN. It is less likely that this is a de novo AML with JAK2 mutation given the frequency of about 1% JAK2 mutation in de novo AML and about 50% in AML transformed from MPN. All these facts also raise the possibility that there might be two different hematopoietic clones present in the bone marrow with full-blown manifestations during the course of his disease. Quantitative analysis of the variant allele frequency for JAK2 and NPM1 mutation might be helpful to solve the diagnostic dilemma.This rare case demonstrates the challenges of firmly establishing a diagnosis between similar, but distinct, disease entities. Synthesized interpretation of available data and close clinical follow-up are required.


Chemical pulps are produced by chemical delignification of lignocelluloses such as wood or annual non-woody plants. After pulping , the remaining lignin is removed by bleaching to produce a high quality, bright paper. The goal of bleaching is to remove lignin from the pulp without a negative effect on the cellulose; for this reason, delignification should be performed in a highly selective manner. New environmentally-friendly alternatives to conventional chlorine-based bleaching technologies  have been suggested or implemented. In an attempt to find inorganic agents that mimic the action of highly selective lignin-degrading enzymes and that can be applicable in industrial conditions, the researchers have focused on polyoxometalates (POMs), used either as regenerable redox reagents (in anaerobic conditions) or as catalysts (in aerobic conditions) of oxidative delignification. The aim of this paper is to review the basic concepts of POM delignification in these two processes. The capability of these POMs to keep a nearly neutral pH within a small range has been demonstrated 5- and [SiVW11O40]5- is 1732 g/mol and 2741 g/mol, respectively), the required quantity of POMs on a mass basis is many times higher than the quantity of agents which are conventionally used in both chorine-based  and chlorine-free  bleaching technologies. Accordingly, to apply the two-step POM-based delignification on a commercial scale, an improvement of the process related to the efficiency of lignin oxidation with POMs is required. Elucidation of the reactions taking place during POM treatment of pulp and a better understanding of the desirable and undesirable modifications which lignin undergoes may help achieve this goal.The two-step POM delignification process offers numerous advantages compared to other TCF bleaching technologies. These include an acceptable selectivity of POM delignification performed in a radical-free environment (anaerobic conditions), which can be improved by conducting the process in less acid conditions (by selection of a POM which is more tolerant to an increase in pH); and an effective regeneration of POMs, which provides the basis for an effluent-free technology. Unfortunately, there are some disadvantages of this approach in the application of POMs for pulp bleaching. The disadvantages are primarily related to the high concentration of POMs used in lignin oxidation; most of the delignification trials have been accomplished by using 0.5 M POM at 8–10% pulp consistency. Due to the high molar mass of POMs  ,25,26,27β-Cγ bond cleavage; vanillin, vanillic acid, and syringaldehyde, which are the products of the Cα-Cβ bond cleavage; and 2,6-dimethoxy benzoquinone, which confirms the C1-Cα bond cleavage in lignin. The presence of 3,4-dimethoxy carbonyl and carboxyl aromatic compounds, veratraldehyde, and veratric acid, respectively, reveals a potential methylation reaction taking place with methanol released during the lignin demethylation reaction . Th. Th1-Cα In contrast to the POM-treated pulp, unbleached kraft pulp treated under the same conditions except without POMs, showed only a minimal decrease in kappa number; the kappa numbers of unbleached mixed-pine kraft pulp, pulp treated under the same conditions without POMs, and POM-treated kraft pulp were 33.6, 29.2, and 7.4, respectively . In addi1-Cα cleavage was indicated by the presence of the para-benzoquinone product ,8,376,8, product  - (n = 1–6), have been used. The most important properties of the HPA-n series of polyoxometalates are their simple re-oxidation with oxygen, a property which enables reactions (1) and (2) to occur in the same step, and their stability at pH 2.5–5, a property which requires acidic process conditions. The redox potential of the HPA-n series of POMs decreases with increasing n, and catalytic action in oxidative delignification is performed with HPA-5 in particular  /[Mn2+] ratio of 1.5, in the aerobic delignification of eucalyptus kraft pulp resulted in a slightly lower decrease in kappa number but higher viscosity of the pulp compared to HPA-5 . The higher selectivity of HPA-5-1.5MnII (designated HPA-5-MnII) compared to HPA-5 may be attributed to the formation of MnII complexes with lacunary HPA-5, which is more stable than HPA-5 and produces less VO2+ in acidic conditions [II/O2 delignification of eucalyptus kraft pulp was confirmed in pilot-scale experiments [3/g; kappa number and viscosity of oxygen-delignifed pulp, 7.7 and 990 cm3/g; kappa number and viscosity of HPA-5-MnII/O2-delignifed pulp 6.6 and 835 cm3/g, respectively). Additionally, in contrast to laboratory studies in which most of the lignin/LMCs were oxidized to CO2, the pilot scale experiments were less successful, with the catalyst solution having a COD of 16.6 kg/ton [II/O2 delignification system represents a promising alternative for chlorine-based bleaching processes, further development and scale-up optimization of the system is necessary.The delignification efficiency depends on the concentration of HPA-5 and was optimal at about 2 mM, whereas at higher concentrations delignification efficiency was reduced, probably due to increase in the ionic strength in the solution . To mitinditions . The feaeriments . In thes6 kg/ton ,41,42. T2 and HPA-5-MnII/O2 on lignin have been performed using hardwood  and softwood (spruce) species, monomeric and dimeric lignin model compounds, and dioxane lignin adsorbed on pulp [2+ released from the HPA-5 is demonstrated in the suppression of these lignin autooxidation reactions, and even more in the oxidation of non-phenolic structures for which the VO2+ ions were suggested to be the active catalysts [α-Cβ and C1-Cα bonds, demethylation, and formation of quinone structures. The comparative analysis of lignin before and after HPA-5/O2 treatment of dioxane lignin adsorbed on pulp showed that the content of the β-O-4, β-β, and β-5 bonds (Studies of the effect of HPA-5/O on pulp ,42,43,44 on pulp . They su on pulp  (Figure As laccase may be an efficient catalyst of re-oxidation of different kinds of POMs, biocatalytic re-oxidation of reduced POMs with laccase has been proposed as a method that will enhance re-oxidation efficiency in aerobic delignification . Re-oxidAs a promising alternative to chlorine-based bleaching, oxidative delignification with polyoxometalates has been suggested. Both of the approaches, one based on the anaerobic process in which polyoxometalates are used as oxidative delignification agents, and a second based on an aerobic process in which polyoxometalates are used as catalysts, have their advantages and disadvantages. Currently, both processes require further development to make implementation feasible.