The advent of new immunotherapeutic agents in clinical practice has revolutionized cancer treatment in the past decade, both in oncology and hematology. The transfer of the immunotherapeutic concepts to the treatment of acute myeloid leukemia (AML) is hampered by various characteristics of the disease, including non-leukemia-restricted target antigen expression profile, low endogenous immune responses, and intrinsic resistance mechanisms of the leukemic blasts against immune responses. However, considerable progress has been made in this field in the past few years.Within this manuscript, we review the recent developments and the current status of the five currently most prominent immunotherapeutic concepts: (1) antibody-drug conjugates, (2) T cell-recruiting antibody constructs, (3) chimeric antigen receptor (CAR) T cells, (4) checkpoint inhibitors, and (5) dendritic cell vaccination. We focus on the clinical data that has been published so far, both for newly diagnosed and refractory/relapsed AML, but omitting immunotherapeutic concepts in conjunction with hematopoietic stem cell transplantation. Besides, we have included important clinical trials that are currently running or have recently been completed but are still lacking full publication of their results.While each of the concepts has its particular merits and inherent problems, the field of immunotherapy of AML seems to have taken some significant steps forward. Results of currently running trials will reveal the direction of further development including approaches combining two or more of these concepts. Advances in immunotherapy have revolutionized cancer therapy in the past few years. Novel immunotherapeutic approaches are entering the mainstream of oncology. In hematology, progress has primarily been made in the field of B-lymphoproliferative diseases including acute lymphoblastic leukemia (ALL). In acute myeloid leukemia (AML), novel strategies utilizing the immune system to eliminate leukemic cells have only recently approached clinical application , 2. ThisIn ALL, several antibody-based approaches have already entered standard treatment or are at the verge of approval. Rituximab, an anti-CD20 directed antibody has been shown to be beneficial as an additive to conventional chemotherapeutic agents . InotuzuHowever, targeted immunotherapy relies on a suitable target antigen to avoid unwanted on-target off-tumor toxicity. In ALL, the restricted expression profile of CD19 and CD20 allows to target these B cell-associated antigens. In AML, it is more difficult to choose an appropriate target antigen due to a more ubiquitous expression pattern overlapping with healthy hematopoiesis. Various potential target antigens are studied for each of the immunotherapeutic strategies , 12. StiOther immunotherapeutic concepts rely on the enhancement of endogenous or the priming of new immune responses. Checkpoint inhibitors have been successfully approved in several solid organ malignancies and are now entering the treatment of hematological diseases . And theIn this review, we will present recent advances made in the aforementioned fields of immunotherapy of AML. HSCT and immunotherapeutic strategies for relapse after HSCT constitute a review topic on their own and have been excluded. As published data from clinical trials is still scarce for the majority of immunotherapeutic approaches, we will integrate currently running clinical trials to point out upcoming directions in this field.Compared to conventional antibody formats Fig. , antibodCD33 (SIGLEC-3) is the antigen that has been most commonly targeted so far in AML. The first and most prominent ADC in clinical application was gemtuzumab ozogamicin , a humanized anti-CD33 IgG4 antibody conjugated to calicheamicin. Promising clinical results lead to an accelerated approval of the antibody by the Food and Drug Administration (FDA) in 2000 . Safety In order to further improve the clinical results with GO, several clinical trials have been performed evaluating GO in combination with HMAs. A regimen consisting of hydroxyurea, azacitidine, and GO was tested in a phase II trial for 142 older patients with newly diagnosed AML. The predefined goals concerning efficacy and safety were met for the poor-risk cohort (age ≥70 years and performance status 2 or 3), but not for the good-risk group . GO in cAs CD33 is expressed on >30% of healthy bone marrow cells, on-target off-leukemia toxicity is inevitable –25. HoweSGN-CD123A is a similar ADC with the antibody directed at CD123 instead of CD33. CD123 is more restrictively expressed in the healthy hematopoietic compartment, which might decrease on-target off-leukemia toxicities , 33. ThiFLT-ITD mutation [ImmunoGen developed IMGN779, a CD33-directed monoclonal antibody conjugated to the novel DNA-alkylating molecule DGN462. Preclinical data demonstrated highly specific in vitro and in vivo cytotoxicity against primary AML cells, especially in samples with an mutation , 35. Themutation . A clinimutation , 38.Taken together, the field of ADCs finally seems to recover from the huge setback it originally suffered after the voluntary withdrawal of GO in 2010. A lot of effort has been put into the optimization of the ADC technology, and clinical results from early trials demonstrate promising response rates. Results of randomized phase III trials are eagerly awaited in order to estimate the risk-benefit ratio between a potential increase in response rates and the discussed side effects due to on-target off-leukemia toxicities and toxin-induced hepatic toxicity. In order to increase target cell specificity of the therapy, alternative target antigens are being evaluated in preclinical  –41 and eT cell-recruiting antibody constructs are a novel class of molecules composed of the single-chain variable fragments (scFv) of two antibodies of different specificity connected by a short peptide linker Fig. . Through+/CD38− leukemia-initiating cells (LICs) vs. AML bulk cells, but expression was still significantly higher compared to their healthy counterparts . In preclinical studies, the preincubation of AML cells with AMG 330 and T cells prevented the subsequent engraftment of AML in NOD/SCID gamma null (NSG) mice. This suggests that the CD33 expression level of LICs is sufficient for elimination with T cell-recruiting constructs. Besides, it has been demonstrated in vitro that the CD33 expression level mainly influences kinetics of cytotoxicity, but not necessarily the response rate [n = 50) was initiated (NCT02520427), but data are not yet available. Several other CD33-targeting antibody constructs that differ from AMG 330 in their molecular structure are currently evaluated in preclinical settings [In AML, several T cell-recruiting antibody constructs are under preclinical and early clinical development Table . Similarnse rate , 48. Recsettings , 49, 50.A-VHB-VLB-VHA), which are stabilized through an additional C-terminal bridge [To reduce on-target off-leukemia toxicity, alternative AML-associated targets are being explored. CD123 has a lower level of expression on healthy hematopoietic cells compared to CD33 , 33. Thel bridge , 52. Encl bridge  and safel bridge  paved th+ cells from the bone marrow as well as from the periphery [XmAb14045, developed by Xencor, is a structurally distinct anti-CD123 T cell-recruiting antibody construct in early clinical development. The XmAb technology ensures structural stability and an extended serum half-life through the retention of an inactive Fc part. Preclinical studies in cynomolgus monkeys showed rapid clearance of CD123eriphery . These sn = 60, NCT02715011). Currently, the study is on hold because of the occurrence of undisclosed adverse events.JNJ-63709178, a CD123/CD3 humanized IgG4 antibody has been developed by Genmab using their DuoBody technology. Preclinical studies in vitro and in vivo showed highly specific T cell activation and targeting of primary AML cells , 57, whin = 50) [CLL-1 is a novel target antigen in AML characterized by its high expression on AML bulk cells as well as LICs , 59. Recn = 50) .Results of the ongoing trials are awaited to see if the success in ALL will translate into the setting of AML. A potential future strategy could be to use the evolving antibody technology to simultaneously target two different AML-associated antigens in order to increase specificity . Apart fIndependently of considerations about the optimal target antigen, we are only at the beginning of understanding the exact mechanism of action of those antibody constructs and resistance mechanisms that potentially evolve upon T cell activation. Despite the promising response rate of 43% using blinatumomab in heavily pre-treated ALL patients, reasons for resistance in the remaining patients have not been resolved. Only few biomarkers for response have been determined so far, e.g., in case of the blinatumomab studies, the percentage of blasts in the bone marrow and the degree of T cell expansion , 64. PD-Circumventing T cell exhaustion, anergy and senescence, CAR T cells take the technology of T cell-recruiting antibody constructs one step further and have already shown promising clinical results in various hematologic malignancies. CARs are genetically engineered cell membrane-bound receptors that combine extracellular antibody binding and intracellular effector cell signaling, thereby enabling both MHC-independent antigen binding and highly potent cytotoxic effector cell function Fig. . Since tTo date, the most prominent target antigen for CAR T cell therapy is CD19, due to its restrictive expression pattern and good safety profile. Groundbreaking early clinical trial results could be achieved for various B cell malignancies. In r/r B-ALL, treatment with anti-CD19 4-1BB-costimulatory CAR T cells achieved MRD-negative CR rates of 86% for 29 patients . These aDespite these promising early results and the rapidly expanding number of anti-CD19 CAR T cell trials, this novel drug format is still incompletely understood and cannot generally be considered safe. In March 2017, Juno announced to shut down development of anti-CD19 CD28-costimulatory JCAR015 CAR T cells and to close their phase II ROCKET trial in r/r adult ALL, after five treatment-related deaths had occurred due to CAR T cell-mediated neurotoxicity . As “livTranslating CAR T cell therapy to AML is complicated again by the non-restricted expression of AML-associated antigens. Given that current CAR T cell constructs can persist beyond 4 years in the human body , severalH and VL chains from different monoclonal antibodies could reduce myelotoxicity in an AML mouse model [CD33 is the most prominent target antigen for CAR T cells in preclinical trials due to its high and persistent expression in the majority of AML patients , 73. In se model . This coOther potential target antigens identified in preclinical studies include CD44v6 , CLL1 8, FLT3 8, FRβ 8484, LeY , NKG2D , and PR1To date, only one very small trial evaluating anti-LeY CAR T cells (CTX08-0002) in r/r AML has been completed. None of the four treated patients developed grade 3 or 4 toxicity, and infused CAR T cells persisted for up to 10 months. One patient with active leukemia responded with transient reduction in blast count before progression 1 month later. All patients relapsed 28 days to 23 months after adoptive CAR T cell transfer . CurrentNovel CAR designs are explored to increase the specificity and to improve safety profiles. In preclinical in vivo models, dual-targeting approaches targeting two independent leukemia-associated antigens were shown to provide increased specificity accompanied by reduced off-leukemia toxicity  and to pIn contrast to the immunotherapeutic concepts discussed so far, monoclonal antibodies against checkpoint molecules are applied with the idea to unleash pre-existing anti-tumor T cell responses Fig. . Within Only one clinical study applying a checkpoint antibody as a monotherapy to AML patients has been published so far. Eighteen patients with various hematologic malignancies, including eight patients with AML, were treated with the anti-PD-1 antibody pidilizumab within a phase I study. The antibody was shown to be safe and well tolerable, and one of the AML patients showed a minimal response manifested by a decrease in peripheral blasts from 50 to 5% . A phaseWhile the results of these studies have to be awaited to judge the potential of checkpoint inhibitors as a monotherapy for AML, various combination therapies are already tested in clinical trials. A phase II study is combining lymphodepletion with a fludarabine/melphalane regimen followed by autologous stem cell transplantation with anti-PD-1 therapy with the goal to reduce relapse rates in non-favorable AML patients in remission (NCT02771197). The combination of standard high-dose cytarabine with anti-PD-1 therapy is tested as a salvage therapy in a phase II study planned to recruit 37 patients with r/r AML (NCT02768792). And a phase I/II study analyzes the maximal tolerable dose of an anti-PD-1 antibody in addition to idarubicin and cytarabine for induction of de novo AML NCT02464657). No results for any of these studies have been reported so far. The combination of a PD-1 antibody with a vaccination strategy based on AML DC hybridoma is described in the DC chapter below , and vaccinations were well tolerated with induction of multi-functional immune responses, resulting in the preparation of a multi-center phase II study. However, there is no full publication of the study results available at present. To our knowledge, no other clinical trial is currently recruiting patients for vaccination concepts with DC-like cells, as a study based on a fusion concept has been terminated early due to slow accrual (NCT00100971), and two studies using modified leukemic blasts  have been completed, but their results have not been published  uses AML apoptotic corpses to load DCs (NCT01146262). A group in Belgium that has already completed a phase I/II study on vaccination with WT1 mRNA-loaded DCs for 10 AML patients in remission with high risk of relapse demonstrating immunological as well as clinical responses [WT1 mRNA-loaded DCs for AML as well as chronic myeloid leukemia and multiple myeloma patients (NCT 00965224). Besides, the same group also conducts a large  randomized phase II study on AML patients in CR/CRi with WT1 overexpression with the goal to determine clinical effects of DC vaccination in terms of relapse rate, disease-free survival, and overall survival (NCT01686334). Results of this study are eagerly awaited, but are not to be expected before 2020.Monocyte-derived DCs loaded with various antigens are the most commonly used source for DC vaccination trials. Five clinical studies are currently active or recruiting. A small French study  [Our group in Munich has developed a protocol for the generation of DCs by the use of a TLR7/8 agonist , 104. Th1734304) . Prelimi1734304) . A very Besides current clinical studies, a few interesting new developments in the field of DCs in the context of AML immunotherapy have been described in the past 2 years. In an effort to further optimize the immunostimulatory capacities of monocyte-derived DCs, electroporation of mRNA encoding both for IL-15 and for IL-15 receptor alpha was shown to result in enhanced NK cell activation . BesidesIn conclusion, current data suggests that DC vaccination is particularly successful at inducing novel immune responses. Combining this approach with checkpoint inhibition or immunomodulating agents including HMAs in order to further enhance the immune responses seems an interesting way to follow.Immunotherapy of cancer has made unprecedented progress in the past few years. While novel immunotherapeutic strategies have already moved into standard clinical practice for various solid cancers as well as selected hematological neoplasms including ALL, a similar development is lagging behind for the treatment of AML. However, different immunotherapeutic concepts are currently being evaluated in clinical trials, with some promising results already published and a lot more of interesting studies expected to be completed within the next couple of years.The lack of an appropriate target antigen with a restricted expression pattern similar to CD19 or CD20 for B cell neoplasms is a major obstacle for the application of targeted immunotherapy in AML. This problem is shared by ADCs, T cell-recruiting antibody constructs and CAR T cell constructs, where promising leukemia-specific responses seen in early clinical trials are often accompanied by severe on-target off-leukemia toxicity to the myeloid compartment. CD33 and CD123 are the major target antigens of constructs in clinical development so far. Results of the ongoing clinical trials need to be awaited in order to weigh potential benefits vs. side effects. In order to prospectively reduce on-target off-leukemia toxicities, several strategies are followed: The identification of novel leukemia-associated antigens could provide more specific targets. Comprehensive transcriptomic and proteomic analysis is ongoing to fully characterize the AML surfaceome . AlternaADCs have already proven their therapeutic potential in AML. Results of currently running clinical trials will help to identify the optimal clinical setting and to better estimate the risk-benefit ratio. In contrast, T cell-recruiting antibodies and CAR T cell constructs are still in the early phase of clinical development for the therapy of AML, with several currently running phase I trials studying the feasibility and toxicity of their application. Activation of endogenous T cell responses through checkpoint blockade and/or DC vaccines appears to be safe, but has yet to demonstrate its clinical potency when used as a monotherapy for the treatment of AML. Different combinations including HMAs to modulate immune responsiveness appear suitable and are increasingly being tested.While immunotherapy in AML is complicated by different characteristics including lack of an AML-specific target antigen, low mutational burden resulting in low endogenous immune responses and intrinsic resistance mechanisms of the leukemic blasts against immune responses, remarkable progress has been made with different strategies in the past few years. Hope is high that alternative immunotherapeutic strategies with less treatment-related morbidity and mortality compared to allogeneic HSCT will move into clinical practice within the coming years. Still, many further steps have to be taken before the vision of an individualized immunotherapy for each AML patient based on risk factors and biomarkers can become clinical reality.


In legumes, nuclear and perinuclear-localized Ca2+ oscillations have emerged as key signatures preceding downstream symbiotic signaling responses. Förster resonance energy transfer (FRET) yellow-based Ca2+ cameleon probes have been successfully exploited to measure the spatiotemporal dynamics of symbiotic Ca2+ signaling in legumes. Although providing cellular resolution, these sensors were restricted to measuring Ca2+ changes in single subcellular compartments. In this study, we have explored the potential of single fluorescent protein-based Ca2+ sensors, the GECOs, for multicolor and simultaneous imaging of the spatiotemporal dynamics of cytoplasmic and nuclear Ca2+ signaling in root cells. Single and dual fluorescence nuclear and cytoplasmic-localized GECOs expressed in transgenic Medicago truncatula roots and Arabidopsis thaliana were used to successfully monitor Ca2+ responses to microbial biotic and abiotic elicitors. In M. truncatula, we demonstrate that GECOs detect symbiosis-related Ca2+ spiking variations with higher sensitivity than the yellow FRET-based sensors previously used. Additionally, in both M. truncatula and A. thaliana, the dual sensor is now able to resolve in a single root cell the coordinated spatiotemporal dynamics of nuclear and cytoplasmic Ca2+ signaling in vivo. The GECO-based sensors presented here therefore represent powerful tools to monitor Ca2+ signaling dynamics in vivo in response to different stimuli in multi-subcellular compartments of plant cells.Spatiotemporal changes in cellular calcium (Ca Compared to fluorescent dyes, GECIs have the advantage of being accessible to any cell type and offer the possibility of measuring Ca2+ variations in subcellular compartments, by specific targeting of the sensor. The most popular GECI sensors are the ratiometric Förster Resonance Energy Transfer (FRET)-based Yellow Cameleons (YCs) composed of a fluorescent donor-acceptor pair, respectively, CFP and YFP, linked by a calmodulin (CaM) and the CaM-binding peptide of myosin light-chain kinase M13  have been engineered and successfully used in plants , consisting of lipo-chitooligosaccharides, and AM fungi chitin oligomers (COs), both induce Ca2+ spiking, essential for the transduction of downstream symbiotic signaling in the nucleus   revealed that both the NR-GECO1 and the two cameleon sensors were targeted to the nuclear compartment of M. truncatula root cells  Figure , and was) Figure  and NLS-) Figure  to geners Figure . NR-GECOs Figure .2+ responses were assessed in the nucleus of growing root hair cells using CLSM   with tetra-chitooligosaccharides (CO4) previously shown to elicit nuclear Ca2+ oscillations . Taken together, NR-GECO1 can efficiently measure symbiosis-related nuclear Ca2+ spiking responses in M. truncatula roots.To evaluate whether NR-GECO1 was suitable for detecting other Cas Figure . As prevs Figure , the NF 2+ spiking with a greater sensitivity than the cameleon sensors. Our results confirm the previously described higher sensitivity of the cytoplasmic R-GECO1 in comparison to the YC3.6, in response to the ATP-induced Ca2+ signals in A. thaliana roots  , by calculating the signal-to-noise ratio (SNR) values for each individual probe. The fractional fluorescence changes (ΔF/F) for NR-GECO1 and the fractional ratio changes (ΔR/R) for the cameleon sensors were measured as described Figure . However) Figure , S2. Thu2+ signal dynamics simultaneously in the cytoplasm and the nuclear compartments of the same root hair cell. We therefore combined the nuclear-tagged red GECO1 and the cytoplasmic green GECO1 in a single construct  for a large number of individual root hairs  initiated primarily and significantly in the nuclear compartment, while only 2% initiated first in the cytosol. In 30% of the cases, it was not possible to clearly distinguish differences between the Ca2+ spiking initiation sites.The dual sensor made it possible to simultaneously visualize highly coordinated Cae Figure . The sele Figure . In both Figures . These a Figures . To furts Figure  of indivs Figure . These a2+ spiking in the nuclear compartment  for the initiation of a spike between the nuclear and cytoplasmic compartments was similar for both dual sensors . Using the dual NGCR-GECO1 sensor we confirmed the preferential initiation of Cat Figure . The tims Figure , with mes Movies , S4 and M. truncatula, we stably transformed A. thaliana with the NRCG-GECO1.2 sensor , NaCl, ATP and cold stress, we focused our study on the root epidermal cells of the elongation zone. Treating 5 day-old roots with CO8 to a final concentration of 10−7 M elicited an increase in cytoplasmic Ca2+ within 2 min of application in 73% of the plants tested (8/11 plants)   Figure . In cont Figures , S6. Eac Figures . Howevere Figure . In cont2+ sensors, notably with the development of single fluorescent protein GECIs, the GECOs  and R-GECO (600–643 nm) to simultaneously assess Ca2+ dynamics in those contiguous cellular compartments. By targeting each of them to either the cytoplasm or the nucleoplasm, we developed the new dual sensors, NRCG-GECO1, NGCR-GECO1, and NRCG-GECO1.2 that allowed the simultaneous monitoring of nuclear and cytoplasmic Ca2+ dynamics in M. truncatula and A. thaliana root cells in response to biotic and abiotic stimuli. Despite the different association rates (Kon) of R-GECO and G-GECO , that assures downstream transcriptional regulation of endosymbiotic programs  can generate, within the same cell type, diverse Ca2+ responses in the cytoplasmic and nucleoplasmic compartments. Notably, CO8 and ATP trigger cytoplasmic-associated oscillatory Ca2+ releases that do not diffuse to the nucleoplasm, suggesting that Ca2+ buffering proteins or Ca2+ uptake mechanisms are rapidly recruited or activated, respectively, to avoid Ca2+ diffusion into the nucleus. Interestingly, the ATP-induced Ca2+ signals in root cells of the elongation zone differ from a previous study showing dual cytoplasmic and nuclear Ca2+ releases  than the cold-induced Ca2+ signal (within 25 s), following the cytoplasmic Ca2+ release. Moreover, The NaCl-induced nucleoplasmic Ca2+ signal had a higher amplitude than the cytoplasmic Ca2+ signal, which contrasts with the cold-induced Ca2+ signal, smaller in amplitude. These results support the induction of nuclear Ca2+ signals independently of the cytoplasmic Ca2+ release, as implied by a recent study regarding the NaCl-induced cytoplasmic/nuclear Ca2+ dynamics  and the 35S terminator in the Golden Gate compatible vector pCambiaCR1 ΔDsRed  and validated by sequencing before being used in Golden Gate assembly reactions . Arabidopsis seeds were surface-sterilized in 1.5% bleach for 15 min, followed by five washes in sterile water, and then plated in Murashige and Skoog (MS) solid medium (1% sucrose). After 3–5 days at 4°C, plates were moved to a growth cabinet  for germination and growth. The Escherichia coli DH5α strain was used for plasmid propagation, the A. rhizogenes Arqua1 strain . Plates were partially sealed with parafilm to allow crucial gas exchanges and placed in a 20°C growth chamber (16-h photoperiod and a light intensity of 70 μE·m−2·s−1) for 1 week before transferring to 25°C. About 2 weeks-post-inoculation, kanamycin-resistant composite plants were transferred to a beaker containing sterile water supplemented with 200 mg·L−1 augmentin , and incubated for at least 30 min, in order to reduce the growth of A. rhizogenes. Plants were rinsed twice before being transferred to square plates containing a nitrogen-free modified Fahraeus medium with 0.5% [w/v] phytagel (Sigma-Aldrich) and supplemented with 50 nM 2-amino-ethoxyvinyl-Gly (AVG), adapted for in vivo microscopic observations as described in Fournier et al.  and a Zeiss Axiophot epifluorescent microscope. Selection of transgenic roots was done using filters adapted to detect yellow/green YFP/GFP  or red mApple  fluorescent proteins from the different cameleon or GECO1 sensors. Only composite plants with fluorescent transgenic roots were transferred to plates containing nitrogen-free modified 0.5% [w/v] phytagel Fahraeus medium for imaging 3–5 days later. Transgenic roots lying on the medium and covered by a gas-permeable film were treated with 1 to 2 mL of a freshly diluted aqueous solution of purified S. meliloti NFs (10−9 M) or CO4 (10−5 M or 10−7 M) that were added to plates between the plastic film and the medium with the help of a micropipette. In the case of ROCs, imaging was performed with excised root sections that were mounted between a slide and a cover slip. Confocal imaging was performed before treatments to assess the background fluorescence levels, and then initiated about 5 min after treatment for a total period of up to 1 h.Kanamycin-resistant 2+ responses with the NR-GECO1 sensor in order to avoid photo bleaching of the red fluorescence during long acquisitions. Confocal imaging of transgenic roots expressing the dual NRCG-GECO1 and NGCR-GECO1 sensors was done using a beam-splitter Leica TCS SP8 AOBS confocal laser-scanning microscope equipped with a 25x water-immersion objective. The 488 nm argon laser was used to excite the G-GECO1 and the 561 nm diode to excite the R-GECO1. Fluorescent images were recorded simultaneously for NRCG-GECO1 and NGCR-GECO1, using emission windows of 500–550 nm and 600–643 nm for G-GECO1 and R-GECO1 signals, respectively. Fluorescence emission ranges were specifically set for each fluorescent protein in order to avoid overlap between the G-GECO1 and R-GECO1 signals, as detailed in Figure The comparison between the nuclear NR-GECO1, the NUP-YC2.1 and the NLS-YC3.6 Yellow Cameleon sensors was performed using a Leica TCS SP2 AOBS confocal laser-scanning microscope equipped with a long-distance 40x water-immersion objective (HCX Apo L 0.80). The 458 nm argon laser was used to excite the CFP of the cameleon sensors (pinhole diameter at 4 Airy units). Fluorescence emission was collected in the 470–500 nm range for CFP and 530–570 nm range for YFP. Images were acquired at 5 s intervals from 5 to 60 min either before or after treatment with a 512 × 512 pixels resolution. The 561 nm diode was used to excite the mApple red fluorescence protein of the NR-GECO1 sensor (pinhole diameter at 3 Airy units) and fluorescence was visualized in the specific 580 to 643 nm emission window. The laser intensity used for the cameleon probes  or non-perfusing chamber. Perfusion of cold media was performed using a RC-21BR chamber (Warner Instruments). For ATP, CO8, and NaCl treatments, the elicitors were applied at a 10x concentration to an open chamber made with cover glass and sealed with vacuum grease. After mounting in liquid MS (1% sucrose), samples were incubated at room temperature for at least 15 min before imaging. Ca2+ imaging was performed using a Nikon ECLIPSE FN1. The fluorophores were excited at a wavelength of 470 ± 24 nm. Emitted fluorescence was separated by an image splitter and passed through an emission filter of 520 ± 40 nm for G-GECO1.2, and 632 ± 60 nm for R-GECO1.2 . Images were collected every 2 or 3 s.The subcellular localization of the GECO1.2 Ca2+ spiking amplitudes, the average fluorescence for each ROI was calculated and used to determine the SNR of individual spikes collected from independent roots and biological experiments. The SNR of a given spike measures the difference between the fluorescence baseline and the maximum fluorescence peak value. The SNR values of individual spikes were obtained by calculating the ΔF/F (Fmax-Fmin/Fmin) or ΔR/R (Rmax-Rmin/Rmin), in which Fmax and Rmax correspond to the maximum fluorescence value or ratio change of a spike and Fmin and Rmin correspond to the data point before the onset of a spike.Processing, final cropping, and mounting of the Medicago images were performed using Fiji. The fluorescence, merged images and Supplementary time-lapse movies were obtained after applying a median filter of 2 pixel radius to fluorescence image series. Intensity data was calculated from selected ROI, delimited in individual root hairs within the nuclear region  or tailored in the perinuclear, middle or root hair tip regions (for cytoplasmic sensors). To quantify relative Cahttp://bradbusse.net/sciencedownloads.html), and application of a lookup table. Image data were obtained from processed images using Time Series Analyzer V3_2 (https://imagej.nih.gov/ij/plugins/time-series.html). Normalized datasets (ΔF/F) were calculated as (F–F0)/F0, where F0 represent the average of at least 2 min of baseline values (before treatment).For image processing of the Arabidopsis data the following steps were conducted using ImageJ 1.48v: background subtraction, registration using MultiStackReg v1.45 (http://r-project.org). Data were evaluated for normality with the Shapiro-Wilk test and homogeneity of variance with Levenne or Bartlett tests. Parametric (ANOVA) or non-parametric  statistical tests were used, respectively, for normally-distributed data and non normally-distributed data. Signal periodicity values of calcium sensors were first transformed into normality using Box-Cox (λ = 0.0606), before statistical analysis using one-way ANOVA followed by Tukey honest difference (HSD) tests. Statistical analysis of signal-to-baseline ratio was done using the non-parametrical Kruskal-Wallis test. For the dual GECO sensors, statistical analyses of the start of a calcium spike was done using a chi-square test (p < 0.05) to (i) compare the frequency of observations in each category to an expected frequency of 1/3 per category if the distribution was random and (ii) to compare the number of observations per category for each sensor grouped in a contingency table.Statistical analyses were performed using R (M. truncatula and NL: the A. thaliana experiments; MiC: contributed to the NLS-R-GECO1 in ROCs experiments; AK and NL: prepared the figures; AK, NL, MyC, and FdC-N: wrote the manuscript; MyC and FdC-N: are co-responsible for the work.MyC and FdC-N: conceived the project; AK, NL, MyC, and FdC-N: designed the experiments; AK: performed the 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.


Acute Myeloid Leukemia (AML) is a highly heterogeneous and poor prognosis disease with few available therapeutic options. Novel advances are urgently needed, however effective models to test experimental therapeutics have been lacking. Recently, NOD/SCID/IL2rγnull (NSG) mice were shown to engraft primary human AML in a manner that recapitulated the natural disease and its progression. Additionally, integrated genomic profiling was used to refine risk stratification of AML. In this study, we demonstrated the engraftment of molecularly defined primary AML in NSG mice. We showed that AML that express DNMT3A mutations, which predict for adverse outcome, engrafted with exceptional efficacy. Lastly, we demonstrated that human AML-engrafted NSG mice can be effectively used to study novel ceramide-based therapeutics. Ceramide is a bioactive sphingolipid that has been implicated as an inducer of apoptosis. Elevation in cancer cell ceramide levels either via exogenous delivery or by provoking intracellular ceramide generation is the goal of ceramide-based therapeutics. In this study, we used the human AML-engrafted NSG mouse model to evaluate nanoliposomal short-chain C6-ceramide and a nanoliposomal formulation of the ceramide-inducer tamoxifen. Altogether, the NSG model is likely to prove invaluable in the study of novel agents, sushc as ceramide-based therapeutics, with the ability to define therapeutic activity against specific molecularly defined and risk stratified AML. Acute Myeloid Leukemia (AML) is a growing public health problem in the United States with few available therapeutic options. AML is a highly heterogeneous disease or group of diseases. The 2008 WHO classification is accepted for clinical AML classification and incorporates known karyotypic data of prognostic importance but fails to predict outcome for many cytogenetically normal AML patients ,2. RecenNovel advances for the treatment of AML are urgently needed, however appropriate models to test experimental therapeutics have been lacking. The translation of laboratory studies to clinical studies is inherently dependent on the usefulness of preclinical animal models. In the case of AML, murine models have proved disappointing for therapeutic development due primarily to the inability to replicate the human disease. The engraftment of human AML stem cells within the bone marrow of sublethal-irradiated NOD/SCID/IL2rγnull (NSG) mice has recently been reported, with these mice developing a more efficient disease compared with other immunodeficient strains –9. Of paHuman AML samples were collected with informed consent and approval from the Penn State College of Medicine Institutional Review Board. Mononuclear cells were prepared by Ficoll separation, and cryopreserved prior to use. Genomic DNA was collected by extraction using a Qiagen DNeasy Kit according to the manufacture’s recommendations. Mutational profiling was performed as previously described in detail . BrieflyNanoliposomes were prepared as previously described –21. BrieBriefly, adult NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice (8–10 wks old) received 250 cGy of sublethal total body irradiation, followed by tail vein injection of primary human AML cells. Cryopreserved cells were injected intravenously at doses of 0.5–10×106 viable mononuclear cells containing 80–90% leukemic blasts. Engraftment was monitored in by flow cytometry in 20 μl samples of tail vein peripheral blood with anti-human CD13, CD33, HLA-DR, and CD45 antibodies at 1–4 week intervals. Engraftment was defined as >0.5% cells expressing human CD45 and one other human marker. For subsequent transplantation, mice were euthanized and bone marrow mononuclear cells were harvested from tibiae and femurs and injected into recipients. For therapeutic evaluation, C6-ceramide nanoliposomes or tamoxifen nanoliposomes were administered via tail vein injection as previously described three times per week for up to two months –20. TreaHerein, we report that AML isolated from the peripheral blood of human patients was successfully engrafted into sublethally irradiated NSG mice. Engraftment of human AML cells in NSG mice was defined by the prevalence of cells with both CD13 and CD33, or either in combination with one other human marker  when monitored by flow cytometry of peripheral blood at various time points or bone marrow or spleen at necropsy . We haveWe subsequently transplanted several cases from primary recipients into secondary and tertiary recipients. Subsequent engraftments were highly efficacious (near 100%), but most importantly allowed for expansion of human AML for experimental therapeutic evaluation. For primary engraftments, we confirm better engraftment of otherwise indeterminate cases which have a mutant FLT3 (88%), compared with other cases (71%). We used mutational profiling to further define the risk stratification for the human AML samples that we engrafted. This additional profiling revealed that samples with a mutation of DNMT3A engrafted at a rate of 100% . We can compare the engraftment of DNMT3A mutant cases with all others based on the integrated genetic risk profile by assigning values of 1–4 for good, intermediate, poor, and DNMT3A mutants, respectively . In thisThe utility of NSG mice engrafted with primary human AML has been limited. Initially, Saito et al. used Ara-C to define a chemotherapy resistant AML stem cell population , and recVarious therapeutics induce ceramide generation, a bioactive sphingolipid that regulates cellular stress and death, while metabolic pathways to eliminate ceramide have gained notoriety as mechanisms of therapy resistance ,23,24. W


This nanoscale nolinear optical converter that simultaneously owns high efficiency and broad bandwidth may open a new way for application in imaging, bio-sensing and on-chip all-optical signal processing operations.In this paper, we investigate second harmonic generation in a single hexagonal GaAs nanowire. An excellent frequency converter based on this nanowire excited using a femtosecond laser is demonstrated to operate over a range from 730 nm to 1960 nm, which is wider than previously reported ranges for nanowires in the literature. The converter always operates with a high conversion efficiency of ~10 SHG is well known to be a forbidden process in materials that have a center of inversion symmetry, but it can occur in noncentrosymmetric materials or at the surface of a material with any symmetry group1. As the first nonlinear optical effect to be found after the invention of the laser, SHG was first demonstrated in crystalline quartz by Franken et al. in 19612. Since then, SHG has been a subject of intense theoretical and experimental study in many optical materials, including potassium dihydrogen phosphate crystals3, nematic liquid crystals4, GaP photonic crystal waveguides5, magnetic metamaterials7, left-handed metamaterials8, AlGaAs microdisks9, quantum dots10, and biological tissues11.Second harmonic generation (SHG) is a second-order nonlinear optical process in which an optical wave with frequency 12, optical correlator13, integrated nanophotonic components14, highly localised excitation15, crystal structure identification17, and nonlinear optical microscopy20. As previously reported in the literature, NWs composed of several different materials, including alkaline niobates21, ZnO24, ZnTe20, ZnSe25, ZnS16, CdS26, GaAs27, GaN18, GaP29, InP30, perovskite Na0.5Bi0.5TiO331, graphene32 and Pt33, can be excited to generate second harmonic light. Among these materials, GaAs NWs are excellent candidate materials for SHG because of the high second-order nonlinear optical susceptibility of GaAs.Recently, SHG in nanowires (NWs), which are one-dimensional nanostructures with a thickness or diameter of tens of nanometres or less and unconstrained length, has attracted increasing attention because it has potential for use in a wide variety of applications, including generation of nanoscale coherent light sourceset al. demonstrated that a vertical free-standing GaAs-NW array could generate broadband SHG signals when excited using femtosecond (fs) lasers and briefly mentioned that an isolated single NW is also capable of producing broadband SHG signals34. However, the conversion efficiency of the broadband SHG signals that were generated, which is very important for SHG in practice26 was not given. In this work, we systematically investigated SHG using a single hexagonal GaAs NW. We found that a single GaAs NW can be excited using a fs laser to generate second harmonic light over a broad wavelength range from 730 to 1960 nm, which is wider than the ranges reported for NWs in the literature such as that of ref. −5 W−1, which is three orders of magnitude higher than that which occurs at the surface of bulk GaAs.In 2013 He We present SHG from a single hexagonal GaAs NW that was measured using a homemade confocal microscope system. The experimental setup is illustrated schematically in Fig. 2 substrate with a patterned gold grid that was used for spatial separation and identification. The SHG signal that was excited from the NW was subsequently imported into a charge-coupled device (CCD) or spectrometer. The occurrence of coupling effects between different GaAs NWs is excluded because of the focused laser spot size of ~1.5 μm  input. Here the polarization angle φ is defined as the angle of the electric field (E-field) direction of the SHG with respect to the long axis of the NW. The SHG intensity follows a cos2φ shape where the peak is always polarized along the long axis of the NW. More detailed theoretical calculations are provided in the Supplementary Information section. The intensity characteristic thus allows the NW’s long axis to be identified, and we also investigated the SHG intensity as a function of the FW laser polarization angle θ, with results as shown in Fig. θ is defined as the angle of the E-filed direction of the FW laser with respect to the long axis of the NW. The polarization direction is along the long axis of the NW , which indicates that the anisotropy of the SHG can be attributed to the intrinsic permutation symmetry of the GaAs crystal lattice.The polarization of the SHG signals was also studied. Figure 20.To illustrate the origin of the SHG in the nanowires, we then studied the SHG signals at different wavelengths under TM excitation. As shown in Fig. ηω2 of the SHG signal, which is defined in this work as Pω2 and Pω are the SHG signal and FW laser powers, respectively. The method used to measure the conversion efficiency can be found in the Methods section. It was previously established that broadband SHG often comes at the cost of reduced conversion efficiency36. High-efficiency SHG depends critically on the PM conditions. However, the PM requirement limits significantly the bandwidth of the SHG processes37. Thus, the high-efficiency broadband SHG has never been really obtained to the best of our knowledge. Herein we demonstrated that the broadband SHG observed in the single hexagonal GaAs NW was simply a high-efficiency SHG process. We found that the SHG conversion efficiency of the single GaAs NW maintained a high value over a wide FW wavelength range. Figure −5 W−1. The high SHG conversion efficiency of the single GaAs NW probably stems from its high surface-to-volume ratio. This ratio breaks the crystal symmetry and induces the dipoles to oscillate with the E-field of the FW laser. The second-order nonlinearity coefficient of the NW is therefore enhanced. Additionally, the NW structure offers confinement and surface enhancement effects to the laser E-field. The frequency conversion in the single GaAs NW is therefore more efficient than that which occurs at the surface of bulk GaAs34.To confirm these results, we also investigated the conversion efficiency As an additional trial, a fs-scale optical parametric amplifier  with a pulse width of <100 fs, a tuning range of 190 to 20000 nm, and a repetition rate of 1 kHz was used to excite the single hexagonal GaAs NW. The experimental setup was almost the same as that shown in Fig. −5 W−1, which is ~103 times higher than that obtained from the surface of bulk GaAs. This high-efficiency broadband SHG procedure based on a single NW may find applications in imaging, bio-sensing and on-chip all-optical signal and processing.In summary, we have investigated SHG in a single hexagonal GaAs NW in detail. We found that excitation of this NW can generate second harmonic light that has not been simultaneously accompanied by defect-related photoluminescence emission or two-photon excited fluorescence to date. Additionally, the generated SHG is highly coherent and has very good polarization properties. We observed SHG over a broad wavelength range from 700 nm to 1960 nm, which is the widest wavelength range to be demonstrated in NWs to the best of our knowledge. The SHG conversion efficiency is also very high and can reach the order of ~10We performed a series of experiments to measure SHG in a single GaAs NW using a homemade confocal microscope system. The experimental setup is illustrated schematically in Fig. 2/Ga flux ratio of 12.5; a 10 min interruption was then introduced under a high arsenic atmosphere to crystallize the gallium droplets on the tip and induce lateral growth of the nanowire. The deposition rate  substrate) of these GaAs NWs was set at 0.7 μm/h.The GaAs NWs under study were synthesized on GaAs (100) substrates using the Veeco Mod Gen-II MBE system and the VLS mechanism. The substrates were first coated by sputtering of a 15-nm-thick silicon dioxide layer and were then dipped for 2 s in a 10% HF aqueous solution. Prior to growth, the substrates were degassed at 700 °C for 10 min and growth was then initiated using 1 nm Ga droplets as a catalyst at 620 °C in the absence of arsenic overpressure. The GaAs core was grown at 620 °C for 30 min at an Asηω2, we first detected the power Pω of the FW laser using a power meter. Second, we recorded number of photons N of the SHG signal using the spectrometer and calculated the power Pω2 of the SHG signal using the formula h is Planck’s constant, c is the speed of light and λ is the wavelength of the SHG signal. We then calculated the SHG conversion efficiency ηω2, based on the definition 26.To obtain accurate measurements of the SHG conversion efficiency High-efficiency broadband second harmonic generation in single hexagonal GaAs nanowire


Such microglia activation results in increased phagocytic activity, activation of their inflammasome and release of cytokines to sustain neuroinflammatory . ATP-induced activation of ionotropic P2X4 and P2X7 receptors differentially induces receptor-operated Ca2+ entry (ROCE). Although store-operated Ca2+ entry (SOCE) was identified to modulate ROCE in primary microglia, its existence and role in one of the most common murine microglia cell line, BV2, is unknown. To dissect SOCE from ROCE in BV2 cells, we applied high-resolution multiphoton Ca2+ imaging. After depleting internal Ca2+ stores, SOCE was clearly detectable. High ATP concentrations (1 mM) elicited sustained increases in intracellular [Ca2+]i whereas lower concentrations (≤100 μM) also induced Ca2+ oscillations. These differential responses were assigned to P2X7 and P2X4 activation, respectively. Pharmacologically inhibiting P2Y and P2X responses did not affect SOCE, and in fact, P2Y-responses were barely detectable in BV2 cells. STIM1S content was significantly upregulated by 1 mM ATP. As P2X-mediated Ca2+ oscillations were rare events in single cells, we implemented a high-content screening approach that allows to record Ca2+ signal patterns from a large number of individual cells at lower optical resolution. Using automated classifier analysis, several drugs  were tested on their profile to act on Ca2+ oscillations (P2X4) and sustained [Ca2+]i increases. We demonstrate specific drug effects on purinergic Ca2+ pathways and provide new pharmacological insights into Ca2+ oscillations in BV2 cells. For example, minocycline inhibits both P2X7- and P2X4-mediated Ca2+-responses, and this may explain its anti-inflammatory action in neuroinflammatory disease. As a technical result, our novel automated bio-screening approach provides a biomedical engineering platform to allow high-content drug library screens to study neuro-inflammation in vitro.Microglia activation is a neuroinflammatory response to parenchymal damage with release of intracellular metabolites, e.g., purines, and signaling molecules from damaged cells. Extracellular purines can elicit Ca For instance, sepsis can reverse the transcriptomic profile for P2X4 and P2X7 receptors, the major two P2X isoforms expressed on murine BV2 microglia cells  are important in discriminating microglia responses via different purinergic receptors and signaling cascades, other sources of Ca2+ entry have also become of interest. In addition to receptor-operated Ca2+ entry (ROCE) in microglia cells, store-operated Ca2+ entry (SOCE) was also recently identified as an integral part of purinergic activation in primary mouse microglia  Ca2+ contents to plasmalemmal Ca2+ entry via the sensory right-handed EF protein Stim , and a ‘Ca2+ release-activated Ca2+ channel’ (CRAC), called Orai, that is assembled on the sarcolemma when ER Ca2+ is low.Immune-modulatory microglia cells constitute about one fifth of the brain glia population and screen the brain parenchyma for tissue damage. During brain injury, purines  released by damaged brain cells activate microglia by stimulating purinergic receptors, either metabotropic G protein-coupled P2Y  or ionot2+ entry . Fine-tuia cells . Both ioia cells . Intrigutrations . In parttrations . While Cicroglia . SOCE is2+ fluorescence recordings to study different Ca2+-dependent pathways in individual primary microglia cells can become exhaustive as exigent preparation and culturing usually limits their use in high-throughput/content assays. The murine BV2 cell line, an immortalized microglial cell line, is frequently used as a reliable alternative model system for primary microglia cultures, and it also reproduces complex cell–cell interaction studies  to provide evidence for the presence of SOCE and the differential pharmacokinetics of purinergic Ca2+ signaling in BV2 cells, and (2) to establish a high-content screening platform suitable for large-scale pathway screening in BV2 cells.Ca studies . However studies , and how2ATP, AZ106 (AZ10606120) and minocycline were from Tocris Bioscience . Thapsigargin, U73122, U73343, LY294  and wortmannin were from Sigma–Aldrich . Drugs were added to final solutions from frozen stock aliquots.Na2) in DMEM, supplemented with 10% fetal bovine serum and penicillin (100U/ml)/streptomycin (100 mg/ml) (Invitrogen) and passaged every 2–3 days (60–80% confluence). For high-content functional imaging, ∼5,000 BV2 cells were plated into each well of a 384 multi-well plate  and incubated for 24 h. Wells typically contained 7.5–10 × 103 cells at the time of experiments. For fluo-4 two-photon recordings, BV2-seeded round cover-slips were directly used for staining and imaging.BV2 cells  were cultured  were loaded onto 26 well, 4–15% Criterion TGX Stain Free gels  and run for 4 min at 200 V. Protein was transferred to nitrocellulose membrane at 100 V for 30 min, incubated in Pierce Miser solution  and blocked. Primary antibodies Stim1S  and Stim2  were diluted in 1% BSA-PBS with 0.025% Tween and membranes incubated overnight at 4°C and 2 h at RT. After incubating with a secondary antibody , the membrane was coated with chemiluminescent substrate . Each gel-lane was normalized to the total protein of that sample, determined from Stain Free gel, then to the three point calibration curve thereby providing a quantitative approach to western blotting . Data fr® Ti:Sa laser was used. BV2 cells were loaded with 5 μM fluo-4 AM  in normal saline  for 20 min at 37°C and washed. Before recordings, cells were either flushed with Ca2+ free medium for 5 min or kept in normal saline, depending on the experiment. Two-photon excitation was done at 900 nm, and backscattered fluorescence recorded in de-scanned mode. Fluorescence intensity was recorded with a photomultiplier tube after passing through a 525/50 bandpass filter. Time lapse 512 pixels × 512 pixels recordings (XYT) were performed at 0.937 fps. Laser power was <3%. All settings were kept constant between recordings. Chemicals were added from stock solutions to the bath medium to obtain the final concentrations indicated. XYT image stacks were manually analyzed, assigning ROIs to individual BV2 cells and extracting temporal fluo-4 profiles. Individual traces were normalized to the average fluorescence within the first 10 s under initial resting conditions and converted to relative changes ΔF/F0 for group analysis. Individual data traces are mostly presented as raw fluorescence intensities.A Nikon A1 multiphoton microscope equipped with a Coherent® Micro XLS System  and cells were imaged with a 10× objective. A mercury arc lamp, passing through a GFP dichroic mirror, was used to excite fluo-4 fluorescence and imaged with a CCD camera. The image resolution after 2 × 2 binning was 696 pixels × 520 pixels. The experimental protocol involved manual addition of 10 μl agonist or 30 μl agonist/drug solution at a rate of ∼0.5 ml/min, then imaging of each well 380 times at a rate of 2 Hz (300 ms exposure time).One and half hours before experiments, culture medium was removed from 384-well plates and replaced by normal saline (control solution), supplemented with 2 μM pluronic acid and 2 μM fluo 4-AM (Molecular Probes). After staining, solution was replaced by control solution. For receptor stimulation, control solution contained 1, 10, 100, or 1,000 μM final ATP concentration ([ATP]). For drug screening, control and agonist (ATP) solutions were supplemented with minocycline , AZ106 (10 μM) U73122 (5 μM), U73343 (5 μM), LY294002 (5 μM) or wortmannin (100 nM), respectively. Three hundred and eighty-four-well plates were placed onto the motorized stage of an ImageXpress® software  as:Fluorescence images of cells were segmented and quantitated using a modified version of DetecTIFFsoftware . Brieflyelongation factor of one represents a perfectly round cell, indicating a strong effect on cellular fitness or viability. A high value indicates normal morphology and unaffected viability. The drug effect was calculated as the arithmetic mean of the elongation factors of all cells. Individual [Ca2+]i responses were reconstructed from image series of the same cells exposed to a defined ATP concentration or a combination of ATP and a drug. Each image typically contained 100–500 fluorescent cells for each tested well. For functional analysis of time-resolved [Ca2+]i responses and subsequent classification and phenotyping of cell populations, a set of measures was taken from single cell-derived data. Responses were fitted with the LabView VI Curve Fitting Express VI using polynomial model type. The function finds the set of polynomial fit coefficients that best represents the input data. Each response was fitted twice with polynomial orders set to a value of 8 and 25. Responses fitted with polynomial order 8 were further evaluated using the LabView VI Peak Detector VI to identify a sustained calcium signal, indicating macro-pore formation, and activation of BV2 microglia cells. Responses fitted with polynomial order value 25 were analyzed for identification of ‘intermediate [Ca2+]ioscillations’ occurring (i) between a transient [Ca2+]i signal and sustained [Ca2+]i increase, or (ii) sporadically during the entire span of the measurement using the residual of the fit. In addition, the fast-Fourier-transform (FFT) power spectrum of [Ca2+]i responses was calculated for each cell using the LabView VI FFT Power Spectrum and PSD VI. Data obtained from curve fitting, peak and threshold detection and spectral analysis were used to calculate a total of 27 features (Table 1). Those data were used for classification and phenotyping of cell populations.An 2+]i responses was manually assigned to one of three classes using a custom-written LabView-based software. The classes were named and characterized as follows:To identify distinct functional phenotypes, a three-stage procedure was applied. First, a training set comprising a total of 1,709 single cell-derived i oscillations,’ followed by sustained [Ca2+]i increase  indicating macro-pore formation and activation of BV2 microglia•Peak (n = 536); response displays sustained [Ca2+]i increase only  indicating macro-pore formation and activation of BV2 cells•Other (n = 840); response displays none of the above listed characteristics, but any other type of [Ca2+]i signal .Table 2. Finally, all single cell-based ATP-induced [Ca2+]i responses recorded in the presence of different ATP concentrations and ATP/drug combinations were analyzed based on the computed classification model.Next, a phenotype classification model was computed by a 10-fold, cross-validation of the training set using J48 decision tree algorithm available in WEKA 3.6 software . We accupost hoc tests (Dunn or Bonferroni method). Asterisks in the graphs indicate significance levels; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001.Data were processed using MS Office 2010, SigmaPlot, Origin 7G, ImageJ and IrfanView. Biochemical protein data was analyzed using Prism GraphPad. Statistical analysis was done based on one-way and two-way ANOVA tests, checking for data normality and performing 2+ entry from intracellular ER Ca2+ release, BV2 cells were incubated in Ca2+-free media and internal stores emptied by 5 μM thapsigargin (TG), while two-photon excited fluo-4 fluorescence was recorded. Two-photon microscopy has the advantage of an excitation volume in the μl3 range, which substantially reduces dye bleaching and allows cellular long-term fluo-4 recordings. Figure 1A shows recordings from three different BV2 dishes. There was a small transient increase in fluo-4 fluorescence after TG application. Adding 2 mM external Ca2+ elicited a robust Ca2+ transient, while Ca2+ stores remained emptied. This is a hallmark of store-operated Ca2+ entry (SOCE). Applying ATP after an almost complete decline of the SOCE transient evoked a sustained increase in intracellular fluo-4 fluorescence in a concentration-dependent manner. This was mediated by purinergic receptor-operated Ca2+ entry (ROCE) to almost maximum Ca2+ permeability for ATP concentrations starting from 0.1 mM, as determined from subsequent Ca2+ ionophoresis (2 μM ionomycin). We confirmed this in several dishes in more than 20 cells for each condition . Lower ATP concentrations (50 μM) evoked significantly smaller peak ΔF/F0 levels than concentrations of 0.1 and 1 mM ATP, for which levels were similar to those in ionomycin-induced results. Not emptying internal stores and adding external Ca2+ again did not induce SOCE , but did reproduce the behavior of purinergic ROCE with empty stores, albeit at a somewhat lower level . One explanation could be that an ongoing blockade of SERCA with TG resulted in a small residual component of SOCE to the resulting Ca2+ entry (mainly ROCE). Figure 1B shows that also in the case of sole purinergic ROCE activation (without prior store depletion), the sustained intracellular Ca2+ increase was almost complete. This is because the subsequent unspecific breakdown of the sarcolemma by triton X-100 evoked only a brief minor increase before all of the dye dissipated from the cells, resulting in a loss of fluorescence. In other experiments, we addressed the question of whether purinergic receptor activation without external Ca2+, but with internal stores not emptied, would induce PLC-mediated (P2Y) signaling to empty internal stores and transiently increase fluo-4 fluorescence. Figure 1B shows a representative Ca2+ response demonstrating that this did not occur. Purinergic ROCE was only detected once external Ca2+ was re-introduced. Again, ATP-induced Ca2+ entry was almost complete compared with subsequent Ca2+ ionophoresis.To distinguish purinergic receptor-mediated Ca2+ fluctuations and the sustained slow Ca2+ increase. Both patterns were immediately initiated after ATP addition. Figure 2A shows a representative immediate oscillatory pattern followed by the sustained increase in intracellular Ca2+ due to purinergic ROCE (stores not emptied) over several minutes. Such oscillation patterns were rarely observed in response to 0.1 mM ATP in multiphoton experiments. They were sometimes observed with 50 μM ATP, still at a low frequency (∼15% of experiments). Figure 2B shows the concentration-response relationship for the three ATP concentrations tested in the scenario of recording purinergic ROCE with untouched internal Ca2+ stores, and in the presence of 2 mM Ca2+ . ΔF/F0 was significantly larger at 1 mM ATP than at lower concentrations. Due to rare detection in highly resolved two-photon experiments, we further studied Ca2+ oscillations in a setting of low-resolution, high-content screening (see below).When we further differentiated early purinergic ROCE responses in BV2 cells in highly spatio-temporally resolved multiphoton recordings, two distinct patterns were observed: oscillatory Ca2+ homeostasis, we performed two-photon Ca2+ fluorescence experiments in the presence of 0.1 mM minocycline and varying ATP concentrations. Pre-incubating BV2 cells with minocycline completely abolished the high [ATP]-induced response (1 mM), but not the ROCE oscillatory responses to lower concentrations (0.1 mM) . However, even at the lower concentration, ATP-evoked ROCE ΔF/F0 levels were already significantly reduced compared to the Ca2+ responses seen at 0.1 mM ATP under control conditions (no inhibitors present) and also remained well below the levels observed after Ca2+ permeabilisation with ionomycin . Minocycline alone did not induce any Ca2+ responses . To address potential PLC pathway activation, we tested the PLC inhibitor U73122. The Ca2+ response in BV2 cells to either low or high ATP concentrations were also inhibited by incubation with 5 μM U73122 (U73343 not tested here) as compared to controls without inhibitor present, but were not significantly different under low- or high-ATP conditions . To test whether minocycline or U73122 affected SOCE, we conducted similar experiments to those outlined in Figure 1. As shown in a representative recording in several BV2 cells, neither minocycline nor U73122 blocked SOCE in BV2 cells .The broad-spectrum tetracycline antibiotic, minocycline, is a known microglia activation inhibitor , with inFigure 4A shows a representative gel and corresponding blots probed for Stim1S and Stim2: both are present in control BV2 cells, and 1 mM ATP stimulation appears to increase the amount of Stim1S. Compared to controls, only 1 mM ATP significantly upregulated Stim 1S protein content, and only minor or no changes were observed with 0.1 mM ATP or LPS, respectively . Also, pre-treatment with minocycline blunted the upregulation of Stim1S. None of the microglia activation treatments  produced any upregulation in protein content for Stim 2 .To verify the effect of microglia activation on recruitment of SOCE at the protein level, quantitative Western blot analyses were carried out on BV2 cell lysates after differential treatment with microglia activators  and M1 polarization (minocycline) inhibitors. 2+ homeostasis in single BV2 cells, in particular less frequent event patterns, like Ca2+ oscillations seen during ATP BV2 activation, are difficult to statistically evaluate. To address this and to enable a high-content screening for Ca2+ responses, we established an assay for single cell-based, high-content, functional screening of ATP-induced [Ca2+]i responses.Although the above shown two-photon excitation approach provides a high-resolution metrology to study Ca2+]i oscillations’, BV2 cells were prepared for screening experiments in 384-well plates. Figure 5A shows a selection of representative [Ca2+]i responses of the three classes Oscillation & Peak (upper panel), Peak (middle panel), and Other (lower panel). Figure 5B shows the fraction statistics of BV2 cells exposed to 0 (n = 3 replica), 1 (n = 10), 10 (n = 10), 100 (n = 11), and 1,000 μM ATP (n = 12), assigned to one of the three classes. The average fraction of cells assigned to Other decreases with increasing ATP concentration, whereas the average fractions of cells assigned to Oscillation & Peak and Peak increase with increasing ATP concentration. These data clearly demonstrate that the established screening assay enables parallel recordings of ATP-induced [Ca2+]i responses in many BV2 cells and functional phenotyping and quantification of the fraction of cells displaying ‘intermediate oscillations.’ This high-content assay is thus, more robust in quantifying low probability intermediate oscillations as compared to the high-resolution but low-content, two-photon experiments when recording from less cells.To evaluate the fraction and concentration-dependence of ATP-induced ‘intermediate [Ca2+]i oscillations’ as a readout for microglia activation-targeted compound screening, BV2 cells were incubated with one of six selected drugs  for at least 30 min. [Ca2+]i responses were recorded after application of the agonist ATP (1 mM). Figures 6–8 are representative images of BV2 cells shortly (∼10 s) after ATP application. They illustrate the effect of drug pre-incubation on Fluo-4 intensity and elongation of cells exposed to control solution. Respective drug concentrations are also shown in the bar graphs. Recorded time-resolved fluo-4 signals were automatically assigned to one Oscillation & Peak, Peak or Other classes and phenotype fractions were calculated from four independent experiments each. Averaged values (±SD) as shown in Figures 8 are listed in Table 3.To assess the applicability of the established screening assay for employing ATP-induced ‘intermediate i increase which involves PLC to release Ca2+ from intracellular stores  confirms this assumption. As shown in Figure 7C, the fraction of cells assigned to the Peak class is significantly reduced compared with controls. Interestingly, inhibiting PLC-pathways with U73122 and blocking P2X7 with minocycline did not affect SOCE in BV2 cells . The former is consistent with our finding that probably, P2Y-activated, PLC-mediated pathways are not, or at least to a negligible degree, present in BV2 cells , in contrast to primary microglia cells , the data indicate no effect on ATP-induced Ca2+ signaling compared to controls. However, the presence of wortmannin resulted in reduced overall Ca2+ signals as compared to data with LY294 and AZ106 which is in line with a previous report on the suppressing effect of wortmannin on P2X4 in BV2 cells -induced NO release . BesidesV2 cells .Figure 8 suggest perturbation of ATP-induced Ca2+ signaling in cells displaying a sustained [Ca2+]i increase only, but not in cells showing ‘2+ oscillationsintermediate Ca’ plus sustained [Ca2+]i increase.LY294 is a morpholine-containing chemical compound and potent phosphoinositide 3-kinase inhibitor . The dat2+ signaling , and their involvement in downstream pro-inflammatory signaling and inhibitors used in this study is given in 2+ signaling to overcome constraints of cell-to-cell variability in single cell high-resolution experiments. It will be useful for identifying compounds modulating microglial activation in the context of neuro-inflammation. To further develop our high-content screening platform to understand mechanisms of different purinergic phenotypes, future studies will include expression systems with defined receptor expression profiles, BV2 cells and primary cells, for comparison.In summary, our study shows the existence and crucial contribution of SOCE to ROCE in immortalized murine BV2 microglia. Interestingly, ATP appears to more selectively activate P2X than P2Y receptor responses in BV2 cells. Our ‘low-resolution’ high-content platform provides a more robust representation of purinergic CaAlthough there are numerous examples of cell signaling studies carried out on BV2 cells alone, it is important to mention that some data obtained with cell lines may be prone to artifacts due to the immortalization procedure and may differ from data observed in primary cells. Thus, data reported in this paper are confined to BV2 cell and transferability has to be considered preliminary until cross-validation in primary microglia cells.DA, OF, MS, LS, and DG designed research. OF, DG, RM, EZ, MS, KK, and AB performed research. OF, DG, RM, and AB analyzed data. OF, DG, and DA wrote the paper. All authors agreed on the final version of 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.


DNA double-strand breaks (DSBs) pose a serious threat to genome stability andcell survival. Cells possess mechanisms that recognize DSBs and promote theirrepair through either homologous recombination (HR) or non-homologous endjoining (NHEJ). The evolutionarily conserved Mre11-Rad50-Xrs2 (MRX) complexplays a central role in the cellular response to DSBs, as it is implicated incontrolling end resection and in maintaining the DSB ends tethered to eachother. Furthermore, it is responsible for DSB signaling by activating thecheckpoint kinase Tel1 that, in turn, supports MRX function in a positivefeedback loop. The present review focuses mainly on recent works in the buddingyeast Saccharomyces cerevisiae to highlight structure and regulation of MRX aswell as its interplays with Tel1. DNA double-strand breaks (DSBs) are cytotoxic lesions that threaten genomicintegrity. Misrepair of DSBs often leads to chromosome rearrangements and loss ofgenetic information that can result in cell death or oncogenic transformation. DSBscan arise spontaneously in eukaryotic cells either during DNA replication or asintermediates in programmed recombination events, such as meiosis and immune systemdevelopment. They can also be induced by exposure to DNA-damaging agents used incancer therapies, including ionizing radiation and topoisomerase poisons.Cells have evolved two main mechanisms to repair DSBs: non-homologous end joining(NHEJ) and homologous recombination (HR). NHEJ directly religates the two broken DNAends with no or minimal base pairing at the junction 242S. cerevisiae, where the most mechanistic information isavailable.The highly conserved MRX/MRN complex  is among the first protein complexes that are recruited at DSBs 45The Mre11 subunit of MRX contains five conserved phosphoesterase motifs that areessential for both 3’-5’ double-stranded DNA (dsDNA) exonuclease and ssDNAendonuclease activities 101112131415181920212324252728293031While Mre11 and Rad50 are conserved in bacteria and archaea, only eukaryotes possessXrs2 , which is responsible for nuclear localization of Mre11 34The MRX complex plays a central role in signaling, processing and repairing of DSBs.Structural studies have shown that these diverse functions are regulated by the ATPbinding and hydrolysis activity of Rad50 that induce conformational changes of bothRad50 and Mre11. In the presence of ATP, Mre11 and Rad50 adopt a “closed”conformation, in which Rad50 head domains dimerize and occlude the nuclease activesite of Mre11  2423S. cerevisiae, MRXis known to interact with Rif2, which is recruited to telomeric DNA ends by Rap1 andnegatively regulates telomerase action 394041rif2∆ cells show an increased efficiencyof both end-tethering and NHEJ compared to wild type cells, indicating that Rif2counteracts end-tethering and DSB repair by NHEJ Rad50 has a slow ATP hydrolysis rate S. cerevisiae mutants lackingSae2 or any component of the MRX/MRN complex delay resection of anendonuclease-induced DSB by acting in the same epistasis group 4546474950sae2∆, mre11 nuclease defective mutantsand a class of separation-of-function rad50 andmre11 alleles, named rad50s andmre11s, allow formation of meiotic DSBs but prevent their resection1152535455The first step in HR is the degradation of the 5’ DNA strands on either side of theDSB through a process termed resection 4sae2∆ andmre11 nuclease mutants are defective in the removal of covalentadducts, such as Spo11  60616249506465However, Mre11 has a 3’-5’ dsDNA exonuclease activity, whose polarity is opposite tothat required to generate the 3’-ended ssDNA at the DSB ends 121657mre11∆ cells show a resection defect moresevere than sae2∆ or mre11 nuclease defectivemutants. In any case, mre11∆ cells are severely defective in DSBresection when the break is present in the G2 phase of the cell cycle, whereas theyslow down resection only of two fold when the break occurs when they areexponentially growing 454647These nucleases comprise the 5’-3’ exonucleases Exo1 and the endonuclease Dna2, whichcontrol two partially overlapping pathways 6769707273Both Sae2 and Dna2 have been shown to be targets of cyclin-dependent kinase (Cdk1 inyeast)-Clb complexes 77797676et al.Altogether, these data support a model, where MRX is recruited to the DSB ends in theATP-bound state. In this configuration, MRX maintains the DSB ends tethered to eachother to allow DSB repair by NHEJ . Upon ATP hydrolysis by Rad50 andCdk1-mediated phosphorylation events, MRX and Sae2 provide an initial incision ofthe 5’ strand at a certain distance from the DSB end. As first proposed by GarciaIn addition to promoting end-resection and to maintaining the DSB ends tethered toeach other, MRX/MRN is required to activate Tel1, which is a member of theevolutionary conserved phosphoinositide 3-kinase-related protein kinase (PIKK)family and whose mammalian ortholog is called ATM (Ataxia Telangiectasia Mutated)MRE11 gene causes a genetic syndrome, calledataxia-telangiectasia-like disease (ATLD), whose clinical phenotypes are nearlyindistinguishable from AT 90Tel1/ATM is a master regulator of the DNA damage response in both yeast and mammals,where it coordinates checkpoint activation and DNA repair in response to DNA DSBsand oxidative stress 858788Subsequent studies have revealed that MRX/MRN drives the localization of Tel1/ATM tothe site of damage through direct interaction between Tel1/ATM with Xrs2/Nbs1 3592939697S. cerevisiae by screen for genesinvolved in telomere length maintenance 9910010439TEL1 (TEL1-hy909) is capable to overcome theinhibitory activity of Rif2 on MRX in vitro and in vivo4142Tel1 was originally identified in S. cerevisiae, Tel1 signaling activity is disrupted when the DSBends are subjected to 5’-3’ exonucleolytic degradation 47In Attenuation of Tel1/ATM signaling by nuclease-mediated DSB resection is accompaniedby activation of the checkpoint kinase Mec1 , which is anothermember of the phosphoinositide 3-kinase-related protein kinase family 111112113S.cerevisiae mutants that require Tel1 to survive to genotoxic treatmentsrecently showed that a mutation in Rad50 (rad50-V1269M) makestel1∆ cells hypersensitive to DNA damaging agents rad50-V1269M mutationimpairs MRX association at DSBs that is further reduced by the lack of Tel1,indicating that Tel1 promotes/stabilizes MRX association to the DSB. Interestingly,Tel1 exerts this function independently of its kinase activity Recent data have shown that Tel1, once recruited to DSBs by MRX, supports MRXfunction in a positive feedback loop . In fact, a screen for rad50-V1269M mutation resides in the globular domain ofRad50, rad50-V1269Mtel1∆ cells are severely defective in the maintenance of the DSBends tethered to each other Although the tel1∆rad50-V1269M cells affect DSB repair not only by NHEJ, but alsoby HR 116117tel1∆rad50-V1269M cells are specifically impaired in SDSA Defects in maintaining the DSB ends tethered to each other in Overall, the MRX complex emerges as a multifunctional enzyme involved in a number ofactivities that include sensing and processing of the DSB ends. Furthermore, itsassociation with Tel1 links DSB sensing with signaling by the checkpoint machineryto coordinate DSB repair with cell cycle progression. These functions are regulatedby ATP binding and hydrolysis activities of Rad50 that provide intrinsic dynamicsand flexibility properties. Furthermore, the Tel1 protein itself, once recruited atDNA ends by MRX, supports the end-tethering activity of the MRX complex byfacilitating its proper association to DNA ends. These observations reveal thecomplex architecture that characterizes the activity of MRX and Tel1 in DNA-damageresponse, maintenance of genetic stability and cell cycle regulation.


Na+-K+-ATPase-rich cells (NaRCs), the specialized ionocytes in the embryonic skin and adult gills, play a dominant role in Ca2+ uptake in zebrafish. Transepithelial Ca2+ transport in NaRC, through apical epithelial Ca2+ channels (ECaC), basolateral plasma membrane Ca2+-ATPase (PMCA), and Na+/Ca2+ exchanger (NCX), is analogous to mammalian renal and intestinal Ca2+-absorption cells. Several hormones were demonstrated to differentially regulate Ca2+ uptake through modulating the expression of Ca2+ transporters and/or the proliferation/differentiation of NaRC in zebrafish. In addition, the counterbalance among these hormones is associated with the maintenance of body fluid Ca2+ homeostasis. Calcium-sensing receptor (CaSR) is expressed in several hormone-secreting tissues in zebrafish, and activated CaSR differentially controls calciotropic hormones. The major principles of Ca2+ transport and the hormonal control appear to be conserved from zebrafish to other vertebrates including mammals. The new knowledge gained from zebrafish studies provides new insights into the related issues in vertebrates.Zebrafish is an emerging model for the research of body fluid ionic homeostasis. In this review, we focus on current progress on the regulation of Ca In. In2+ wa2 was reported to inhibit PTH1 mRNA expression in zebrafish embryos [2+ level [2+ absorption and ECaC mRNA expression in zebrafish embryos based on PTH1 gain- and loss-of-function experiments [2+ absorption and ECaC mRNA expression in zebrafish  in zebrafish were not changed by PTH2 gain- or loss-of-function [2+ uptake mechanism in zebrafish.PTH is expressed and secreted from the parathyroid gland (PTG) in mammals . The PTG embryos . On the 2+ level ,26,68. Periments . In addiebrafish . In a re2+ level ,68. Suppfunction . Therefo2+ homeostasis, and this issue remains to be explored in the future.There are three types of PTH receptors, PTH1R, PTH2R, and PTH3R, in zebrafish, and zebrafish PTH1 was capable of activating the PTH1R and PTH3R in vitro study . PTH1R a2+ uptake [STC-1 is a homodimeric glycoprotein hormone and was first identified in fish . In mamm+ uptake .2+ water treatment, and knockdown of STC-1 enhanced zebrafish Ca2+ uptake [2+ uptake function by downregulating ECaC mRNA expression. STC-1 was recently demonstrated to decrease the capacity of Ca2+ uptake through negatively regulating the number of zebrafish NaRCs, which is probably mediated by the transcription factor of Foxi3a [2+ uptake. STC receptor has not been identified in animals so far, and this is a limitation to further understand the action of STC-1 on Ca2+ regulation in mammals.STC-1 expression in zebrafish was significantly stimulated by high-Ca+ uptake . ECaC, b+ uptake ,24. To r+ uptake ,35. Thus2+ absorption by stimulating NCX activity in the distal tubules of rabbit nephron [2+ reabsorption [Calcitonin (CT) is a type of small peptide produced in parafollicular C cells of the thyroid gland and may function as a hypocalcemic hormone in mammals . However nephron . In humasorption . As suchsorption . In fishsorption . Hypocalsorption ,79,80.2+ water treatment. The treatment of high-Ca2+ water also increased the mRNA expression of CT receptor (CTR) in embryos and adult gills [2+ absorption probably through directly or indirectly (via STC-1) downregulating ECaC expression. Notably, overexpression of CT induced a short-term hypocalcemia initially and a long-term hypercalcemia subsequently in zebrafish embryos [2+ homeostasis in vertebrates.In zebrafish, the mRNA expression of CT in embryos and adult UBG was significantly stimulated by high-Calt gills . Overexplt gills . Therefo embryos . The inc embryos . These f2+ uptake function through increasing ionocytes differentiation and proliferation. In zebrafish, isotocin receptors were identified and the expression was detected in gills [2+ uptake provides some clues to explore novel functions of mammalian oxytocin in terms of body fluid ionic homeostasis.Isotocin is the teleost homologue of mammalian oxytocin gene ,82. Oxytin gills . Mammaliin gills . The dis2S is a gaseous transmitter and there are two vital cytosolic enzymes, cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS), for the production of endogenous H2S [2S is involved in many physiological activities including cardiovascular regulation, cytoprotection, inflammation, and cell energy production [2S donors increased the intracellular Ca2+ level in rat microglial cells, rat aorta endothelial cells, and HeLa cells [2+ water, the treatment of H2S donors increased whole-body Ca2+ uptake and/or calcium content [2S on body fluid Ca2+ homeostasis. Both CBSb and CSE are expressed in zebrafish NaRCs, but only CBSb mRNA expression is stimulated by low Ca2+ water treatment [2+ absorption of zebrafish in low Ca2+ water. Supporting this loss-of-function result, only the pharmacological inhibition of CBS activity resulted in decreased Ca2+ uptake in zebrafish [2S, generated by CBSb in NaRCs, may activate ECaC activity through the PKA-cAMP pathway and then upregulate Ca2+ absorption in zebrafish based on the pharmacological experiments with the relevant inhibitors.Hnous H2S ,89,90. TLa cells ,92,93. I content , suggestreatment . Knockdoebrafish . There aebrafish ,95. Like2+ absorption in the kidneys through increasing vitamin D synthesis, and the physiological actions of IGF-1 are mediated by the IGF-1 receptor (IGF-1R) [2+ uptake requires further clarification [2+ water treatment increased the number of phospho-Akt signals and NaRCs [2+ absorption through stimulating NaRC proliferation [2+ homeostasis in vertebrates.In mammals, IGF-1, secretion from the liver and/or the local cells, regulates Ca(IGF-1R) ,97. Mammfication ,101,102.fication ,104. Injfication . IGF-1 ification . In zebrfication , and lownd NaRCs . Subsequferation . These f2+ level. CaSR, a G protein-coupled receptor, senses the change in extracellular Ca2+ level and is activated by increased Ca2+ levels in mammals. Activated CaSR differently modulates the calciotropic hormones  for the maintenance of body fluid Ca2+ homeostasis [2+ sensor and is activated by the increased extracellular Ca2+ level. In in vitro study, the activation of tilapia CaSR was enhanced within 5 min when the extracellular Ca2+ level was raised from 0.5 to 2 or 3 mM [2+ absorption, skeletal development, and the gene expression of calciotropic hormones and ECaC [2+ water situation [casr morphants [The expression and/or secretion of calciotropic hormones are dependent on extracellular Caeostasis . In fish or 3 mM ,108. Add or 3 mM ,109,110.and ECaC ,26,111. and ECaC ,26. CaSRand ECaC ,26. CaSRituation ,26, indiorphants . Pharmacorphants . CT mRNAorphants .Taken al2+ reabsorption, in the kidney, and activated CaSR is able to directly regulate TRPV5 activity in mammals [Several studies have revealed the role of CaSR in regulating cell proliferation in mammals ,114,115. mammals . The eff2+ homeostasis is tightly regulated by the crosstalk of calciotropic hormones in mammals. In zebrafish, the interplay between calcitropic hormones is a rising topic which has been recently discussed in a few studies. In a study by Lin et al., knockdown of CaSR caused a short-term decrease and a subsequent recovery/upregulation in STC-1 mRNA expression in zebrafish embryos; however, the PTH1 mRNA expression was continuously stimulated in zebrafish casr morphants [casr morphants, the long-term expression of STC-1 mRNA was no longer upregulated. Furthermore, PTH1 gain-of-function in zebrafish stimulated STC-1 mRNA expression [casr morphants may be eventually abolished by the increased PTH1. Reinforcing this notion, STC-1 gain-of-function in zebrafish enhanced PTH1 mRNA expression [2+ homeostasis [Body fluid Caorphants . As PTH1pression . Therefopression . Based oeostasis .Cortisol via GR positively regulates the gene expression of the receptor and the synthesis-enzyme of vitamin D in zebrafish . Exogeno2+ transport, being carried out through apical ECaC or TRPV5/6 and basolateral PMCA and NCX, is conserved between zebrafish NaRCs and the specific cells of mammalian kidney and intestine. As discussed in the present review, zebrafish has been an emerging animal model to explore the mechanisms associated with Ca2+ uptake and hormonal control, and the findings in zebrafish provide new insight into the related issues in vertebrates. Hormones via their receptor(s) regulate Ca2+ uptake through direct (via regulating Ca2+ transporter) and/or indirect  routes  routes . However