Many of the molecular components and biochemical interactions required have been identified; the precise mechanistic steps they modulate and the biochemical interactions that need to occur across steps are still the subject of intense investigation. Particularly, although the involvement of phosphorylation in modulating exocytosis has been intensively investigated over the past three decades, it is unclear which phosphorylation events are a conserved part of the fundamental fusion mechanism and/or serve as part of the physiological fusion machine . Here, the homotypic fusion of cortical vesicles was monitored by utilizing new high-throughput, cost-effective assays to assess the influence of 17 small molecule phospho-modulators on docking/priming, Ca2+ sensitivity and membrane fusion. Specific phosphatases and casein kinase 2 are implicated in modulating the Ca2+ sensitivity of fusion, whereas sphingosine kinase is implicated in modulating the ability of vesicles to fuse. These results indicate the presence of multiple kinases and phosphatases on the vesicles and critical phosphorylation sites on vesicle membrane proteins and lipids that directly influence late steps of regulated exocytosis.Regulated exocytosis enables a range of physiological functions including neurotransmission, and the late steps (i.e., docking, priming and Ca This approach is fundamentally similar to one previously utilised by Vogel et al. free trigger a fraction of CV to fuse, and CV fusion vs. [Ca2+]free is best described by a sigmoidal Ca2+ activity curve [2+]free are indicative of changes to the Ca2+ sensitivity of the mechanism(s), while alterations in the response to saturating doses of [Ca2+]free (~1 mM) indicate changes to the capacity for vesicles to fuse . In this study, minimised versions of standard endpoint and settle Ca2+ activity assays were used to enable high-throughput screening of all the test compounds. Two sub-maximal doses of [Ca2+]free (38.8 ± 1.7 µM and 69.8 ± 4.8 µM [Ca2+]free) were used to trigger CV fusion and were designated as ‘low’ and ‘medium’ Ca2+, respectively, in the endpoint assay; 60.7 ± 1.5 µM [Ca2+]free and 91.2 ± 4.3 µM [Ca2+]free were designated as low and medium Ca2+, respectively, in the settle assay. In both assay formats, responses to 1 µM and ≥1 mM [Ca2+]free were defined as 0% and 100% fusion, respectively; as is standard for such Ca2+ activity assays, ≥1 mM [Ca2+]free was thus defined as ‘high’ Ca2+. Thus, here, monitoring changes in the range of [Ca2+]free corresponding to the rising phase of the standard sigmoidal Ca2+ activity curves enabled us to carry out broadly parallel assays with high throughput and thus scan and identify small molecules that altered the Ca2+ sensitivity of fusion. Sigmoidal curves were plotted using responses to low and medium doses of [Ca2+]free to estimate the transition phase of the sigmoid curve in order to obtain an initial estimate of the half maximum effective concentration (EC50). Treatments that were found to significantly alter CV fusion responses to both the low and medium doses of [Ca2+]free in the endpoint assay were further assessed using a range of [Ca2+]free in order to better define the EC50 from full Ca2+ activity curves. In all assays, [Ca2+]free was measured in parallel mock solutions using a Ca2+-sensitive electrode , calibrated using CALBUF Ca2+ standards  free in the endpoint assay are indicative of changes in Ca2+ sensitivity, whereas changes in the settle assay  are indicative of alterations to attachment/priming factors [2+]free (mM) in the endpoint assays is indicative of alterations to the membrane fusion machinery, as this assay is thought to bypass the need for those endogenous components required for priming and attachment. Small molecules found to alter CV fusion in response to low and medium doses of [Ca2+]free in the minimised endpoint assay were tested again using a more detailed standard Ca2+ activity assay (CV fusion versus [Ca2+]free) to best measure EC50.Seventeen small molecule modulators of phosphorylation  were scr factors ,9,27. In2+]free, suppressed the total extent of CV fusion by 8.2% ± 1.6%, but had no effect in the settle assay . 4,5,6,7-Tetrabromobenzotriazole also potentiated the extent of CV fusion in the settle assay in response to a medium dose of [Ca2+]free without significantly altering the EC50 , but suppressed total fusion extent by 5.0% ± 1.0%  caused 61.3% ± 10.4% inhibition of fusion in response to low doses of free in the settle assay only  inhibitor, GSK 429286, had no effect on the total extent of CV fusion  left-shifted to 33.6 ± 5.5 µM [Ca2+]free in the endpoint assay and from 82.1 ± 5.7 µM to 56.8 ± 6.9 µM [Ca2+]free in the settle assay (p < 0.05). The change in EC50 is thus comparable in both assay formats indicating that the potentiation of Ca2+ sensitivity by α-NP was less likely due to enhanced docking or priming since the endpoint assay is thought to bypass endogenous components essential to these early steps. Treatment with 5 mM α-NP also suppressed the extent of CV fusion in the endpoint assay by 10.1% ± 3.4% (p < 0.05) only in the settle assay, in response to low and medium [Ca2+]free , a broad-spectrum phosphatase inhibitor, CV fusion was potentiated by 60.0% ± 4.7% and 22.4% ± 8.8%, respectively, in response to low and medium doses of free in the endpoint assay, but had no effect on CV fusion in response to any other dose of [Ca2+]free tested in the endpoint and settle assays (50 (2+]free in the settle assay (2+]free (p < 0.001) in the endpoint and settle assays, respectively  to regulatory domains results in the activation of catalytic sites (containing the ATP binding domain) that bind and phosphorylate the substrate(s). Inhibitors that bind regulatory domains, such as pseudopeptides, tend to show a higher degree of specificity than those that interact with the ATP binding domain  free, but not that triggered by a slightly higher, but still submaximal dose of [Ca2+]free . Severa2+ binding domain of synaptotagmin 1 and its interaction with Munc18 free  and do not involve cell isolation from vertebrate, mammalian sources. The results indicate the presence of multiple phosphorylation sites on proteins and lipids, localised on the secretory vesicle membrane, serving as components of both the PFM and FFM, and this is consistent with a recent analysis of protein phosphosites in synaptosomes [2+ sensitivity, and sphingosine kinase activity is implicated more directly in enabling membrane fusion. While it is also possible that kinases and phosphatases in the local cytosol might also modulate components of the PFM and FFM, here we sought only to assess the effects of vesicle-associated enzymes. In any case, it would be difficult to assess local cytosol effects or components specifically near the site of docking and fusion. This study thus defines a modified, high-throughput fusion assay format to better capitalize on the well-established CV model system. The results provide a rationale for larger scale proteomic and lipidomic analyses of CV membranes to identify the CK2 and phosphatase substrates and the potentially wider lipid alterations resulting from sphingosine kinase inhibition, in order to further identify critical components and correlate molecular changes with alterations in specific steps underling triggered exocytotic release. The data also provide a rationale for examining the role of CK2 and sphingosine kinase in the modulation of regulated exocytosis in mammalian cells, using techniques including high temporal resolution patch clamp assays of membrane capacitance and fusion pore expansion and a range of imaging techniques  and stimulated emission depletion (STED) microscopy).The study of CV fusion has enabled targeted identification of components of the conserved molecular mechanism underlying the late Captosomes . Uniquel


FPD/AML) is characterized by germline RUNX1 mutations, thrombocytopaenia, platelet dysfunction and a risk of developing acute myeloid and in rare cases lymphoid T leukaemia. Here, we focus on a case of a man with a familial history of RUNX1R174Q mutation who developed at the age of 42 years a T2‐ALL and, 2 years after remission, an AML‐M0. Both AML‐M0 and T2‐ALL blast populations demonstrated a loss of 1p36.32‐23 and 17q11.2 regions as well as other small deletions, clonal rearrangements of both TCRγ and TCRδ and a presence of 18 variants at a frequency of more than 40%. Additional variants were identified only in T2‐ALL or in AML‐M0 evoking the existence of a common original clone, which gave rise to subclonal populations. Next generation sequencing (NGS) performed on peripheral blood‐derived CD34+ cells 5 years prior to T2‐ALL development revealed only the missense TET2P1962T mutation at a frequency of 1%, which increases to more than 40% in fully transformed leukaemic T2‐ALL and AML‐M0 clones. This result suggests that TET2P1962T mutation in association with germline RUNX1R174Q mutation leads to amplification of a haematopoietic clone susceptible to acquire other transforming alterations.Familial platelet disorder with predisposition to acute myeloid leukaemia ( RUNX1 are frequently associated with numerous myeloid malignancies, especially acute myeloid leukaemia (AML)  and/or AML. RUNX1 mutation on the second allele CDC25C and GATA2 have also been identified at a high frequency but only in a Japanese cohort FLT3, PHF6, KIT, KRAS, RAD21, BCOR, BCORL1, CBL, CEBPA, MPL, TP53, WT1, SRSF2, DNMT3A, TET2 and ASXL1 were described in patients who developed AML/MDS and mutations in PHF6, WT1, NOTCH1, FLT3 and ASXL1 in patients with T‐ALL Germline + cells, peripheral blood mononuclear and bone marrow total cells using kit Qiagen .Biologic samples of one FPD/AML pedigree were collected between 2006 and 2013 after informed consent, in accordance with the Declaration of Helsinki. Genomic DNA was extracted from fibroblasts, CD34versus normal‐matched commercial reference. CGH data and protocols have been submitted to ArrayExpress at the EBI with the accession number E‐MTAB‐4623.Comparative genomic hybridization (CGH) arrays were performed on human CGH 2x400K (G4448A)  by hybridization of sample Library preparation, capture, sequencing and variant detection have been carried out by IntegraGen. Exons were captured from blood DNA using Agilent SureSelect Human All Exon v5–70 Mb kit and sequenced on IlluminaHiSEQ 2000 instrument as previously described The mutated regions were amplified by PCR using primers listed in Table S1. PCR products were end‐repaired, extended with an ‘A’ base on the 3′ end, ligated with indexed paired‐end adaptors  using the Bravo Platform (Agilent) and amplified by PCR for four cycles. Amplicon libraries were sequenced in an IlluminaMiSeq flow cell using the onboard cluster method, as paired‐end sequencing (2 × 250 bp reads) .RUNX1R174Q mutation, previously referenced as AII‐1 ASXL1R693* mutation in the leukaemic clone. The patient was pre‐treated with corticosteroids and then treated according to the French GRAALL2003 protocol based on a polychemotherapy We focus on a man with a familial history of +, CD13+, HLA‐DR+, CD33low, CD7+ and CD56low) and absence of cytoplasmic and surface expression of CD3 and cyMPO . This phenotype was initially described by Suzuki as myeloid/NK‐cell precursor leukaemia Two years later, he was admitted to the hospital for relapse with 87% blasts in peripheral blood. The clinical course was aggressive and the patient died 1 month after hospitalization. The blasts exhibited basophilic cytoplasm and a lack of azurophilic granules  and were characterized by immature marker expression  and two possibly deleterious de novo variants  in genes that were also found with a frequency of >40% but had not previously been causally linked to AML/MDS. A second heterozygous NOTCH1K1488N mutation occurred only in the T2‐ALL  and relapsed (AML‐M0) leukaemic blasts originated from the same clone, we performed first aCGH on both blast populations. In both samples, aCGH revealed loss of 1p36.32‐23 and of 17q11.2 and other small deletions (Table S2). One additional deletion on chromosome 11 and five duplicated regions on chromosome 14 were found exclusively in T2‐ALL (Table S2). In the AML‐M0 blasts, the loss of the 1p36.32‐23 region was associated with two large amplifications at the same break point (1p36.33‐32 and 1p36.23‐1p31.2) surrounding the deleted region . Other additional deletions and duplications were identified (Table S2). These results suggested that T2‐ALL and AML‐M0 blasts originated from the same clone, but harboured also specific features acquired during disease progression. Furthermore, identical clonal rearrangements of TCRγ (Vγ9‐Jγ1.1) and TCRδ (Dδ2‐Jδ1 and Dδ2‐Jδ3)  were identified in both T2‐ALL and AML‐M0 blasts strongly supporting their clonal affiliation. Finally, analysis of WES revealed 34 variants in the T2‐ALL and 49 in the AML‐M0. Interestingly, 20 variants were common between these two cell populations  reaching more than 40% at leukaemic stages. These results suggest that a missense TET2P1962T mutation in association with germline RUNX1R174Q mutation led to amplification of an haematopoietic clone susceptible to acquire other transforming alterations  has been described in AML P1962T mutation leading to the amplification of a leukaemic clone.Here, we describe a unique case of one FPD/AML patient who developed first one T2‐ALL and less than 2 years later an AML‐M0. We show that these two leukaemic clones originate from the common subclone harbouring alterations in genes frequently mutated in both types of leukaemia. We cannot exclude that AML‐M0 was induced by the chemotherapy used for T2‐ALL treatment, as it involved anthracyclines and cyclophosphamide known to generate genomic instability. Interestingly, one of the variant RUNX1 mutation induces both alteration in haematopoiesis (more particularly increasing cycling) and a genetic instability TET2 mutation enhances self‐renewal of haematopoietic stem/progenitor cells Our results suggest the following model of transformation: the germline RUNX1 mutation and an acquired TET2 mutation leading to clonal haematopoiesis. During a five‐year follow‐up, the haematopoietic clone acquired other genetic alterations including mutations in 18 genes. These subclonal alterations led first to T2‐ALL development, and later to a T/Myeloid phenotype with an AML‐M0 development (Fig. In conclusion, this report presents a FPD/AML patient with a germline RUNX1 carriers under the age of 50 years was already reported Clonal haematopoiesis in asymptomatic The authors declare no competing financial interests. The online version of the article contains a data supplement.MVT, BH, ADI, AB, MG, DN and DMK performed genetic analysis and analysed mutational data. MVT, BH, VW, PI, ME, AV, FR, BV and RH conceptualized the idea, designed the research and analysed data. FR, PT, CTR and BV provided samples and data. MVT, BH, ADI, ME, AV, BV and RH wrote the manuscript, which was approved by all the authors.Figure S1 Morphology and phenotype of AML‐M0 blasts.Figure S2 Comparative genomic hybridization array on T2‐ALL and AML‐M0 blast populations.Figure S3 TCRδ and TCRγ rearrangement analysis.Click here for additional data file.Table S1 List of primers used for NGS.Table S2 Deletions and insertions found in AML‐M0 btasts and/or T2‐ALL blasts by CGH array.Table S3 Variants found in AML‐M0 blasts, T2‐ALL blasts and CD34+ cells by NGS.Click here for additional data file.


Drosophila adult gut, high levels of reactive oxygen species (ROS) has been detected with different types of tissue damage, and oxidative stress has been shown to be both necessary and sufficient to trigger intestinal stem cell (ISC) proliferation. However, the connection between oxidative stress and mitogenic signals remains obscure. In a screen for genes required for ISC proliferation in response to oxidative stress, we identified two regulators of cytosolic Ca2+ levels, transient receptor potential A1 (TRPA1) and ryanodine receptor (RyR). Characterization of TRPA1 and RyR demonstrates that Ca2+ signaling is required for oxidative stress-induced activation of the Ras/MAPK pathway, which in turns drives ISC proliferation. Our findings provide a link between redox regulation and Ca2+ signaling and reveal a novel mechanism by which ISCs detect stress signals.Precise regulation of stem cell activity is crucial for tissue homeostasis and necessary to prevent overproliferation. In the DOI:http://dx.doi.org/10.7554/eLife.22441.001 Drosophila midgut  are responsible for tissue homeostasis in the adult a midgut . Similara midgut . Althouga midgut , Hippo/Ya midgut , and JAKa midgut , play crDrosophila hematopoietic progenitors  is emerging as a critical microenvironment signal that regulates stem cell activity in neuronal and glial progenitors , as wellgenitors . In mostgenitors . In the e damage . Moreovee damage . Althouge damage , the con2+ homeostasis in ISCs and mediate increases in cytosolic Ca2+ in response to oxidative stress and ISC proliferation. Our results are consistent with a recent study  and ryanodine receptor (RyR), which are required for ISC self-renewal and damage-induced proliferation. We show that these channels are required for Cant study  showing From an RNAi screen for new transmembrane proteins required for ISCs proliferation, we identified TRPA1 and RyR as candidate hits required for ISC pool size maintenance and damage-induced ISCs proliferation . We confirmed that the lines producing a consistent phenotype  effectively knockdown the corresponding transcripts, while the lines that did not score as hits from our screen  cannot achieve efficient knockdown .2+-permeable cation influx channel -mediated Ca2+ entry into the cytosol (trpA1 or RyR in adult ISCs suppressed damage-induced proliferation (trpA1 RNAi is associated with a shortening in midgut length (trpA1 RNAi expression in the ISCs exhibit accelerated shortening following tissue damage. This damage-induced midgut shortening phenotype is reminiscent to the gut phenotype observed after depletion of the esg+ cell population when the cell death gene reaper (rpr) is expressed in ISCs for 4 days  (trpA1 RNAi can still differentiate into mature Pros+ enteroendocrine cells (EEs) or Pdm1+ ECs Gbe+ or Pros+ cells  . We alsoes (ECs) , which rdm1+ ECs . Furtherdm1+ ECs  are drams+ cells . The Nots+ cells . AltogettrpA1 gene is expressed as multiple mRNAs Gbe+ cells, also called enteroblasts (EBs)) or visceral muscles, blocks damage-induced proliferation  into the bath solution of oocytes sequentially to trigger the opening of the TRPA1-C or -D channel and compare their strength and dynamics. TRPA1-C barely displayed any response to paraquat  during larval stages and in adults ISCs, see (2+ concentrations in their ISCs. While the number of GFP+ (i.e. high cytosolic Ca2+ concentration) ISCs in wild-type midguts increases significantly 10 min after adding paraquat to a final concentration of 4 mM  not only reduced the basal levels of cytosolic Ca2+ in stem cells    to exami% to 63%  when fli feeding . Unlike 2+ signaling have been extensively studied in neurons, where it has been established that membrane depolarization and Ca2+ influx can activate Ras/MAPK , which is a measure of Drosophila Ras/MAPK activity , the Hippo pathway transcriptional factor required for damage-induced ISC proliferation (Yki RNAi could inhibit ISC proliferation caused by SERCA RNAi (Ras1 (Drosophila Ras) RNAi could block SERCA RNAi-induced MAPK activity  in ISCs triggers ectopic MAPK activation and proliferation , CREB-regulated transcriptional co-activator (CRTC), and cyclic-AMP response element binding protein B (CrebB), displayed an hyper-proliferation phenotype , constitutively active Raf (Rafgof), or secreted form of spi is epistatic to trpA1 RNAi for ISCs proliferation (RyR RNAi (trpA1 RNAi effectively blocked the mitogenic activity of constitutively active CanA1 (CanA1ca) (act)  , CRTC (KCanA1ca) , or actia) (act)   (act) . FurtherAi could , and CRTferation . Resultsferation , indicatEGFR RNAi completely inhibited ISCs proliferation caused by SERCA RNAi  EGFR is required for MAPK activity and ISC proliferation . We founRCA RNAi , indicatand pvf1  were supnduction . Altoget2+ signaling and ISC proliferation. Under homeostatic conditions, the basal activities of TRPA1 and RyR are required for maintaining cytosolic Ca2+ in ISCs to ensure their self-renewal activities and normal tissue turnover  and inositol-1,4,5-trisphosphate (InsP3). Interestingly, L-glutamate and mGluR RNAi mainly affected the frequency of Ca2+ oscillation in ISCs, while their influence on cytosolic Ca2+ concentration was very weak. Strikingly, the number of mitotic cells induced by L-glutamate  is far less than what has been observed in tissue damage conditions  , and none blocked the damage response in ISCs, suggesting that L-glutamate and mGluR do not play a major role in damage repair of the gut epithelium.Previously, Deng et al. identified L-glutamate as a signal that can activate metabotropic glutamate receptor (mGluR) in ISCs, which in turn modulates the cytosolic Ca damage) . Consist2+ increases through the redox- sensitive cation channels TRPA1 and RyR under damage conditions. In particular, we demonstrate using voltage-clamp experiments that the TRPA1-D isoform, which is expressed in the midgut, is sensitive to the oxidant agent paraquat. In addition, the results of previous studies have demonstrated the direct response of RyR to oxidants via single channel recording  mechanism. Interestingly, expression of oxidant- insensitive TRPA1-C isoform in the ISCs also exhibits a tendency to induce ISC proliferation , y v; attP2 , UAS-Luc RNAi (BL31603), UAS-trpA1 RNAi , UAS-RyR RNAi , UAS-SERCA RNAi , 1 (BL26504), UAS-trpA1-AtrpA1 , UAS-Ras1 RNAi (BL29319), UAS-Yki RNAi (BL31965), UAS-Src42A RNAi , UAS-EGFR RNAi , UAS-CanA1 RNAi , UAS-CRTC RNAi (BL28886), UAS-rpr (BL5823), caUAS-Hep (BL6406), caUAS-Src42A (BL6410), UAS-Src64B (BL8477), UAS-GCaMP6s (BL42749), UAS-GCaMP6f (BL42747), DBD, UAS-p65AD::CaM; UAS-p65AD::CaM, tubGal80tsUAS-RFP LexAop2-GFP; UAS-MKII::nlsLexA , tstubGal80 , ts[10]tubGal80 (BL7108), ts[20]tubGal80 (BL7019), ts[7]tubGal80 (BL7018), UAS-mCherry (BL52268), UAS-mCherryCAAX (BL59021), R14G09RyRGal4 , FRT19A (BL1744), FRT42D; FRT82B (BL8216), FRT42D w+ (BL1928), 605FRT42D; ry (BL1802), FRT2A (BL1997). Strains from Vienna Drosophila RNAi Center: yw; attP , UAS-trpA1 RNAi (v37249) (UAS-Ras1 RNAi (v106642), UAS-Yki RNAi (v104523). Strains from Perrimon lab stock: A, UAS-Rafgof, esgGal4 UAS-GFP tubGal80ts (EGT), esgGal4, tubGal4, 24BGal4, DaGal4, esgGFPw1118, UAS-Luciferase, UAS-nlsGFP, UAS-Ras1 (generated by David Doupé), Su(H)GbeGFP (generated by Li He), UAS-spi . 1 FRT2AtrpA1 and the stock with a deletion of trpA1, Df4415, were obtained from C. Montell lab, DlGal4 and Su(H)Gal4 from Steven Hou, UAS-Cas9.P2 from Fillip Port, UAS-2xEGFP [m5B29]; UAS-2xEGFP [m6B1] (UAS-ultraGFP) from Fabio Demontis, UAS-CncC RNAi from Dirk Bohmann, UAS-trpA1-C 10b) and UAS-trpA1-D 10a) (fb5UAS-CanA1 RNAi  and caUAS-CanA1 (constitutively active)  , UAS-Ras1(A)10a)  from Pau active)  from PattstubGal80 was achieved by maintaining flies at 18°C until 4–7 days after eclosion, and then shifting young adults to 29°C. For MARCM experiments, flies were maintained at 18°C until 3–5 days after eclosion, heat-shocked at 37°C for 1 hr, and then maintained back at 18°C before dissection and analysis.Flies were reared on standard cornmeal/agar medium in 12: 12 light: dark cycles at 25°C unless noted otherwise. Fly food was changed every 2 days to keep fresh. Conditional expression in adult flies using See Pseudomonas entomophila (from Tony IP) was grown in LB media at 29°C until reaching OD600 = 1, and Pseudomonas aeruginosa 14 (from Chrysoula Pitsouli) was grown in LB media at 37°C until reaching OD600 = 3. Flies were transferred to vials containing a cotton ball impregnated with 5 ml of water solution composed of 20% Pseudomonas culture media and 4% sucrose.Standard cornmeal/agar medium were melted at 70 ~ 80°C and mixed with a final concentration of 25 µg/ml bleomycin  or 2 mM paraquat (Sigma Aldrich #856177) to prepare food that can induce tissue damage in the fly midgut. For pathogen infection, yv; attP2 for TRiP stocks, v60100 for Vienna stocks, and w1118 for NIG stocks). In particular, we noticed that trpA1 RNAi (BL31504), trpA1 RNAi (v37249), and RyR RNAi (BL29445) reduced EGT>luciferase activity by 74%, 88%, and 80%, respectively under normal feeding condition, and blocked damage-induced EGT>luciferase expression. We validated the top candidates from the screen by fluorescence imaging.With a luciferase reporter that we have previously developed as a sensitive surrogate for stem cell pool size , we perfDrosophila midguts were dissected in PBS, fixed in 4% paraformaldehyde PBS solution for 30 min, rinsed and kept for 1 hr in PBST solution  containing 5% Normal Donkey Serum. Midguts were stained overnight at 4°C with the following primary antibodies in PBST solution: rabbit anti-pH3 , mouse anti-GFP , rabbit anti-GFP , rabbit anti-RFP , rabbit anti-β-galactosidase , rabbit anti-dpErk1/2 , rabbit anti-Pdm1 , rat anti-TRPA1 . Fly midguts were then washed three times in PBST and incubated with secondary antibodies and DAPI (1:2000) in PBST for 2 hr in the dark at room temperature. Secondary antibodies were donkey anti-rabbit, anti-mouse, and anti-rat IgGs conjugated to Alexa488, Alexa594 and Alexa647 . Fly guts were then washed three times in PBST, mounted in Vectashield (Vector Laboratories). For anti-TRPA1 staining, primary antibody incubation was done for 1 day and secondary antibody incubation overnight at 4°C. Images of posterior midgut area were captured with a Zeiss LSM780 confocal microscope equipped with 40x oil lens. A z-stack of 10–20 images covering the epithelium from the apical to the basal side were acquired and shown in max projection. All images were adjusted and assembled using NIH ImageJ.For counting mitotic cells or MARCM clone size, an epifluorescence microscope was used to examine the stained midguts. For the quantification of esg+, Pros+, Dl+ or Su(H)Gbe+ cell density, as well as GCamP+ cell number, images of the majority of posterior midgut region were acquired on a Keyence microscope.For measuring midgut length, images of the whole midgut were captured with an epifluorescence microscope. The middle line of the midgut was measured using NIH ImageJ, starting from the end of the proventriculus to the border between the posterior midgut and hindgut, where the malphighian tubules connect to the gut.For detection of ROS via DHE staining, we followed the previously described protocol . In brietrpA1 or RyR were designed using the ‘Find CRISPRs' online tool (http://www.flyrnai.org/crispr2/) (Two pairs of short guide RNAs (sgRNAs) targeting the coding region of rispr2/) , and clorispr2/)  for sitetrpA1: CACACGATCCACGTCACTGT & CTGTAGACTCCGTTGTAGAGU6-sgRyR: TTCGTGTTCCATCTGTACAA & ACAAGAACGTGCCGCCGGATU6-sgtrpA1 isoform typing) with GAPDH and rp49 as an internal control. RT-qPCR data were analyzed with Bio-Rad CFX Manager software. Primers used for RT-qPCR or regular PCR (for trpA1 isoform typing) are shown below:Total RNA was extracted from 15 to 20 midguts or 15 heads or 5 larvae  by TRIZOL reagent (Thermo Fisher), converted to cDNA template after DNase I treatment and purification by QIAGEN RNeasy kit. The cDNA was analyzed by real-time quantitative PCR using SYBR Green or by regular PCR (for trpA1-All_FW: AGACGCTCATTAAAGTGCTGAtrpA1-All_RV: AGAAGGACAAGTGGTTCGGTtrpA1-CD_FW: CTTCAGGATATTGCGGGCGtrpA1-AB_FW: GTGGACTATCTGGAGGCGGtrpA1-AB/CD_RV: CCTTCGCATTAAAGTCGCCAtrpA1-AD_FW: TGCCGGCTTTGAATACCATGtrpA1-BC_FW: CTTTGGCCGTGGTGAACAtrpA1-AD/BC_RV: GCCAGGTGAAAGTACTTGCCRyR_1_FW: GATCGGGACACAGGACATTGRyR_1_RV: GACAGCTTGTCGTTCGACGRyR_2_FW: TGCCTGACCATACCGAGTACARyR_2_RV: GCCACCAGTCCACTTGGTTGAPDH_FW: CCAATGTCTCCGTTGTGGAGAPDH_RV: TCGGTGTAGCCCAGGATTrp49_FW: ATCGGTTACGGATCGAACAArp49_RV: GACAATCTCCTTGCGCTTCTesg_FW: ATGAGCCGCAGGATTTGTGesg_RV: CCTCCTCGATGTGTTCATCATCTε-Trypsin_FW: TAGACACCGGGATACCTCACATCAε-Trypsin_RV: TCCAGCATTCGCGAGATCCGTATTPdm1_FW: AGCTGTCCTAACGAGTTCCGPdm1_RV: ACATCGCGCATATTTGTGTCAApros_FW: TTTGACCGGAGATGGTGACGpros_RV: GGTCGTTCCTGCCCAGTTTCspi_FW: CGCCCAAGAATGAAAGAGAGspi_RV: AGGTATGCTGCTGGTGGAACkrn_FW: CGTGTTTGGCAACAACAAGTkrn_RV: TGTGGCAATGCAGTTTAAGGvn_FW: AAGCATCACAAAAGACGTTCvn_RV: CCGCATCGGAGGAACTATTGApvf1_FW: GCGCAGCATCATGAAATCAACCGpvf1_RV: TGCACGCGGGCATATAGTAGTAGpvf2_FW: TCAGCGACGAAACGTGCAAGAGpvf2_RV: TTTGAATGCGGCGTCGTTCCpvf3_FW: TCGTGAAGAGCAGTAAGCATCGpvf3_RV: AGGTGCAACTCAGTATGGTGGdilp3_FW: GCAATGACCAAGAGAACTTTGGAdilp3_RV: GCAGGGAACGGTCTTCGAupd1_FW: CAGCGCACGTGAAATAGCATupd1_RV: CGAGTCCTGAGGTAAGGGGAupd2_FW: GACTCTTCTCCGGCAAATCAGAupd2_RV: TGCTATCGCTGAGGCTCTCGCRTC_FW: GCAGCAGTACGCAACAAACCCRTC_RV: TTCACGTCGCTATTGAAGCCCCanA1_FW: CCGCCAGTGGAAACAAACAGCanA1_RV: GCGGAAGTGGAACATCATCGXenopus laevis oocytes were recorded as previously described (Xenopus ovaries were dissected and treated with 1.5 mg/ml collagenase A (Roche) and 1 mg/ml trypsin inhibitor (Roche) in OR2 Ringers . The deflocculated oocytes were recovered at 18°C for 12 hr in OR3 medium (pH = 7.5): 50% Leibovitz’s media L-15 (Sigma L1518), 13 mM HEPES, 90 μg/ml gentamicin, 90 μg/ml Fungizone, 90 μg/ml penicillin/streptomycin. trpA1-C and trpA1-D  with 1.8 mM CaCl2. In each experiment, we used 4 mM paraquat to treat the oocytes expressing TRPA1-C or -D, and added 0.01% AITC near the end of the recordings as a positive control.TRPA1 currents in escribed . Xenopus trpA1-D  mRNAs we2+ in stem cells was monitored ex vivo using UAS-GCaMP6f . Average value and standard error of the mean (SEM) were calculated and shown in the figures. For comparison of data groups, p values were determined by Student’s two-tail t-test with unequal variances, except for the comparison before and after drug treatment in  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.2+ signaling in the Drosophila midgut" for consideration by eLife. Your article has been favorably evaluated by Jonathan Cooper (Senior Editor) and three reviewers, one of whom, Bruce Edgar (Reviewer #1), is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: W. Daniel Tracey (Reviewer #2).Thank you for submitting your article "Oxidative stress induces stem cell proliferation via TRPA1/RyR mediated CaThe reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.General assessment:Drosophila gut, and provide evidence that one of these effectors, the Ca2+ channel TRPA1, may be activated by reactive oxygen species (ROS). They present an extensive and coherent set of tests supporting a model in which ROS trigger Ca2+ release via TRPA1 in ISCs, and that the Ca2+ in turn activates Ras/Mapk signaling to promote intestinal stem cell (ISC) divisions. The reviewers agreed that the experiments were generally performed to a high standard and that most of the conclusions were convincingly supported. However, no mechanistic insight was offered as to how ROS might activate TRPA1, or how Ca2+ might activate Ras, though similar effects have been reported before in other cell types. Given that other studies of the fly gut have concluded that Ras/Mapk activation in ISCs is effected non-cell autonomously, via the EGFR and ligands produced by other cells, the authors' proposal that Ras is activated cell autonomously by Ca2+ is novel, interesting, and provocative.Xu et al. demonstrate that two conserved regulators of intracellular calcium, TrpA1 and RyR, are required for stem cell stress responses in the Essential revisions:2+ on ERK and ISC division is cell autonomous, is not well supported and runs counter to many previous publications suggesting that ERK is activated by stress-dependent EGFR ligands non-cell autonomously supplied from other cells in the epithelium. The authors should either provide additional data supporting their cell-autonomous mechanism and ruling out non-autonomous mechanisms, or they should alter the model they present in 1) Two reviewers noted that authors' proposal that the effect of ROS via Ca2+, and to support the authors proposal that ERK is the principal Ca2+ effector.2) Along the same lines, reviewer #3 notes that further data should be provided to rule out the role of CrebB and CanA as effectors of Ca3) Reviewer #2 notes a substantial body of relevant published work that was not cited or discussed (i.e. Du and Guntur papers). The manuscript should be revised to address these deficiencies.2+ release to promote ISC division. This might also address point #1 above.4) Two reviewers commented on the weakness of data showing the TRPA1 activation is sufficient to promote ISC divisions, and reviewer #2 suggests an experiment to test this using a heat-activatable TRPA1 variant. If this experiment is possible, the authors should try it, or present some other type of data further supporting the sufficiency of TRPA1 mediated Ca5) Reviewers 1and 3 discussed several issues with conclusions that could be clarified by counting ISCs and other cell types (EEs), from experiments that were already performed. This additional analysis should be provided if possible.Reviewer #1:Drosophila gut, and provide evidence that one of these effectors, the Ca2+ channel TRPA1, may be activated by reactive oxygen species (ROS). They present an extensive and coherent set of tests supporting a model in which ROS trigger Ca2+ release via TRPA1 in ISCs, and that the Ca2+ in turn activates Ras/Mapk signaling to promote ISC divisions. No mechanistic insight is offered as to how ROS might activate TRPA1, or how Ca2+ might activate Ras, though similar effects have been reported before in neurons . The results are consistent with a recent report in Nature from Jasper's group . Given that other studies of the fly gut have concluded that Ras/Mapk activation in ISCs is effected non-cell autonomously, via the EGFR and ligands produced by other cells, the authors' proposal  Subsection “TRPA1 and RyR are required for stem cell damage response and self-renewal”, third paragraph, 2+ cells after paraquat treatment in controls, but not in animals expressing RNAi against TRPA1.  Similarly, in 2+ is not explained mechanistically, and the authors' model that the effect of ROS via Ca2+ is autonomous to stem cells is based on correlations that don't rule out other explanations. Although TrpA1 and RyR are clearly necessary for ISC activation, and although other treatments that increase Ca2+ are sufficient to activate ISCs (e.g. SERCA-RNAi), this paper doesn't provide strong evidence that activation of TrpA1 or RyR (in this case by ROS) is sufficient to activate ISCs. Moreover the data on ROS activation of TrpA1, though extensive and essential to the model  As alluded to above, the regulation of Ras activity by Cahe model , is the 4) The dpERK stainings in 5) For most of the study, only TrpA1 is tested in detail. The manuscript would be better if the authors had carried through with a deeper analysis of RyR as wellReviewer #2:Drosophila TRPA1 channel and Ryanodine Receptor channels in Reactive Oxygen Species (ROS) induced proliferation of intestinal stem cells. The combined evidence in support of the role of these two channels in this biological process as presented in the study is very strong. With respect to dTRPA1, the study points to a specific isoform (dTRPA1-D) in the sensing of ROS and the study also finds that Ras/Mapk function downstream of Ca influx/release that are triggered by dTRPA1 and Ryr respectively. The strength of the advance in the study is that it ties calcium signaling to ISC proliferation specifically in the context of ROS triggered proliferation and the study also specifically ties the calcium to dTRPA1 and Ryr. The authors do discuss the fact that a prior study has tied calcium to ISC proliferation through mGluRs and dietary glutamate. But some of the prior literature on dTRPA1 channels  has not been adequately credited by the current study. For example, the dTRPA1-D isoform has been previously shown to be activated by ROS :) through a dual oxidase pathway in the ring gland. As well, a role for dTRPA1 in sensing ROS in the gut  has been previously reported: TrpA1 Regulates Defecation of Food-Borne Pathogens under the Control of the Duox Pathway. Du EJ, Ahn TJ, Kwon I, Lee JH, Park JH, Park SH, Kang TM, Cho H, Kim TJ, Kim HW, Jun Y, Lee HJ, Lee YS, Kwon JY, Kang K.PLoS Genet. 2016 Jan 4;12(1)). This latter study also finds that it is the dTRPA1-D isoform which is activated by ROS and the study has not been cited in the current manuscript. Although Guntur et al. is briefly cited in the current study, the analysis of ROS activation by dTRPA1in heterologous oocytes do not significantly extend those that were initially reported by the studies of Guntur and Du. As such the new heterologous expression analyses should thus be presented as important confirmatory findings  with a bit more credit/discussion given to the prior studies. The new biological role for this function- in promoting ISC proliferation in response to ROS- is what is important here, and this will be of great interest to the readers of Experimental points to be addressed:The anti-dTRPA1 staining does not provide a clear description of where dTRPA-C/D is actually expressed in the gut. Reporters for dTRPA1-C/D are available from several labs and these could be used to more clearly illustrate the expression pattern.Reviewer #3:Drosophila. They found that oxidative stress stimulates Ca2+ signaling by regulating TRPA1 and RyR. While the role of calcium signaling has previously been reported, this new study describes a different pathway causing changes in [Ca2+] and a different downstream effector cascade (Ras/ERK).In this manuscript, Xu et al. describe a novel mechanism controlling intestinal stem cell proliferation in Experiments reported in this paper are generally well designed and properly controlled. They address an important question in the field, regarding the mechanisms that allow the integration of multiple signaling pathways in ISCs to control proliferation rates in response to stress (oxidative stress and tissue damage).eLife.This study has the potential to be of interest for the large audience of In my opinion, however, a few important points would need to be addressed to fully support the authors' main conclusions:The authors show that TRPA1 and RyR knock-down results in a loss of Dl+ stem cells. Because they argue that these cells do not die of apoptosis and do not commit to the enteroblast lineage, I am confused about what the fate of these ISCs is, after these genetic manipulations. The authors should at least test whether it promotes enteroendocrine cells fate by monitoring the number of prospero-positive cell in these epithelia. Also, it is possible that the expression of Δ is lost in these stem cells? Other ISC markers such as Sanpodo would be useful to test this hypothesis. Finally, are the single cell trpA and RyR mutant clones Dl-positive?Altogether, these experiments would greatly help clarifying the fate of ISCs in which trpA or RyR activities is impaired.2+ signaling by ROS via a cell autonomous mechanism (including the control of TRPA1 activity), relatively simple experiments could be performed to rule out a non-cell autonomous mechanism following paraquat-induced tissue damage.The authors use paraquat exposure as an oxidative stress in most of their experiments. While the results are compatible with a regulation of Ca2+ signaling? Do ISC-specific genetic manipulations that have been shown to induce ROS production in ISCs and promote cell proliferation (such as ND42 RNAi) increase Ca2+ signaling?Does the expression of antioxidant proteins in ISCs prevent ROS-induced Ca2+ release, arguing that Ras/ERK is the main Ca2+ effector in ISCs. This is an important point as it has been previously published that these factors are part of the signaling pathway that regulate ISC proliferation in response to [Ca] changes, and this new study challenges this view. I believe that this conclusion needs to be supported by additional data.In the last part of this manuscript, the authors try to exclude the role of CanA1 and CrebB downstream of CaThe experiment in 2+ signaling in the Drosophila midgut" for further consideration at eLife. Your revised article has been favorably evaluated by Jonathan Cooper (Senior Editor), a Reviewing Editor and two reviewers.Thank you for resubmitting your work entitled "Oxidative stress induces stem cell proliferation via TRPA1/RyR mediated CaThe consensus is that the manuscript has been improved, and we appreciate the new data you've included as well as your editorial efforts. However, there are two remaining issues that are quite significant, and really need to be addressed before acceptance, as outlined below:2+] activates ERK. A key issue is whether this happens directly, cell-autonomously, or through non-autonomous interactions between different cells in the gut epithelium. The old and new data do not resolve this issue, despite requests from reviewers. The model figure tries to cover both  mechanisms and as such is a confusing conclusion – in fact the model as presented will only serve to obscure whatever the real mechanism is, not to resolve it. We would very much like to see a clear resolution of this issue, so that this paper can be seen as an obvious advance over the recent similar publication from Deng et al. A reviewer's specific comments on this point follow: "[…] the authors provide new data demonstrating that [Ca2+] controls the expression of Spitz and other RTK ligands. However, the authors appear to favor a model where [Ca2+] controls ERK activation directly through Src, but such activation is not demonstrated and the mechanism is unclear. As an example of this, the revised model still highlights a direct regulation of Ras/MAPK by Ca2+, and the authors discuss a scenario where Spitz induction is downstream of an initial direct activation of ERK rather than an integral part of the ERK activation mechanism. I think the two models (direct or autocrine/paracrine) could be easily be distinguished by testing whether EGFR and/or PVR are required for ISC proliferation induced by SERCARNAi and TRPA1-D overexpression, as suggested in the initial review. I believe that confirming that Ca2+ can activate ERK directly in ISCs  would greatly increase the impact of this study." Conversely, demonstrating the EGFR is essential for deregulated proliferation after Ca2+ signaling dysfunction would at least show that the mechanism is not simple and not cell autonomous.1) One reviewer and the reviewing editor still believe the manuscript still falls short of establishing how, mechanistically,  1) Subsection “TRPA1 and RyR are required for stem cell damage response and self-renewal”, third paragraph, The observation that anti-apoptotic p35 protein cannot rescue ISC proliferation defects ’-I’, and2+ cells after paraquat treatment in controls, but not in animals expressing RNAi against TRPA1.  Similarly, in  Ca2+, rather than to an increase in the fraction of ISCs with high Ca2+. This would not occur in the RNAi treated ISCs, since the RNAi blocks their division. To address this potentially confounding issue the authors should count total ISC numbers in these experiments, as well as numbers of high Ca2+cells, and display the fraction of ISCs that are Ca2+high.2+cells caused by paraquat treatment cannot be caused by expansion of cells with high Ca2+because the GCamP imaging lasted less than 20 min, not long enough for cell division. We have also stained flies used in The increases of high Ca3) As alluded to above, the regulation of Ras activity by Ca2+ is not explained mechanistically, and the authors' model that the effect of ROS via Ca2+ is autonomous to stem cells is based on correlations that don't rule out other explanations. Although TrpA1 and RyR are clearly necessary for ISC activation, and although other treatments that increase Ca2+ are sufficient to activate ISCs (e.g. SERCA-RNAi), this paper doesn't provide strong evidence that activation of TrpA1 or RyR (in this case by ROS) is sufficient to activate ISCs. Moreover the data on ROS activation of TrpA1, though extensive and essential to the model , is the 2+Ca might also be required for autocrine activation of EGFR/Ras/MAPK via spi, through or in parallel with the Src-mechanism that we have alluded to. On the other hand, it was reported that Ras activation causes increases in cytosolic 2+Ca level . We hypothesize that the 2+Ca and EGFR mechanisms are intertwined, amplify each other for activation, and turn off each other if one of them gets restricted. The hypothesis is reflected in the revised model in Mitosis quantification of midguts overexpressing TRPA1 isoforms C and D, as well as the heat-activatable TRPA1 isoform A  demonstr4) The dpERK stainings in We have added the imaging experiments as suggested . As cont5) For most of the study, only TrpA1 is tested in detail. The manuscript would be better if the authors had carried through with a deeper analysis of RyR as wellWe have added quantification of RyR RNAi effect on EE and EB population ; and addReviewer #2:The study of Xu et al. finds an important role for the Drosophila TRPA1 channel and Ryanodine Receptor channels in Reactive Oxygen Species (ROS) induced proliferation of intestinal stem cells. The combined evidence in support of the role of these two channels in this biological process as presented in the study is very strong. With respect to dTRPA1, the study points to a specific isoform (dTRPA1-D) in the sensing of ROS and the study also finds that Ras/Mapk function downstream of Ca influx/release that are triggered by dTRPA1 and Ryr respectively. The strength of the advance in the study is that it ties calcium signaling to ISC proliferation specifically in the context of ROS triggered proliferation and the study also specifically ties the calcium to dTRPA1 and Ryr. The authors do discuss the fact that a prior study has tied calcium to ISC proliferation through mGluRs and dietary glutamate. But some of the prior literature on dTRPA1 channels  has not been adequately credited by the current study. For example, the dTRPA1-D isoform has been previously shown to be activated by ROS :) through a dual oxidase pathway in the ring gland. As well, a role for dTRPA1 in sensing ROS in the gut  has been previously reported: TrpA1 Regulates Defecation of Food-Borne Pathogens under the Control of the Duox Pathway. Du EJ, Ahn TJ, Kwon I, Lee JH, Park JH, Park SH, Kang TM, Cho H, Kim TJ, Kim HW, Jun Y, Lee HJ, Lee YS, Kwon JY, Kang K.PLoS Genet. 2016 Jan 4;12(1)). This latter study also finds that it is the dTRPA1-D isoform which is activated by ROS and the study has not been cited in the current manuscript. Although Guntur et al. is briefly cited in the current study, the analysis of ROS activation by dTRPA1in heterologous oocytes do not significantly extend those that were initially reported by the studies of Guntur and Du. As such the new heterologous expression analyses should thus be presented as important confirmatory findings  with a bit more credit/discussion given to the prior studies. The new biological role for this function- in promoting ISC proliferation in response to ROS- is what is important here, and this will be of great interest to the readers of eLife.-induced current than TRPA1-C, it seems inconsistent that they could rescue uracil-dependent defecation defects in trpA1 mutant flies with TRPA1-C overexpression but not with TRPA1-D. We found that TRPA1-D, rather than TRPA1-C, exhibits response to paraquat . Guntur et al. found that TRPA1-C and TRPA1-D isoforms are H2O2-sensitive and that their endogenous expressions in the ring gland, or ectopic expression in other tissues, confer UV light sensitivity via sensing UV light-induced H2O2 production. Although Du et al., 2016 reported that TRPA1-D mediates much stronger NaOClparaquat , and thaparaquat .Experimental points to be addressed:The anti-dTRPA1 staining does not provide a clear description of where dTRPA-C/D is actually expressed in the gut. Reporters for dTRPA1-C/D are available from several labs and these could be used to more clearly illustrate the expression pattern.The signal to noise ratio is not ideal for immunostaining with anti-dTRPA1 antibody in the midgut . PersonaReviewer #3:[…] In my opinion, however, a few important points would need to be addressed to fully support the authors' main conclusions:The authors show that TRPA1 and RyR knock-down results in a loss of Dl+ stem cells. Because they argue that these cells do not die of apoptosis and do not commit to the enteroblast lineage, I am confused about what the fate of these ISCs is, after these genetic manipulations. The authors should at least test whether it promotes enteroendocrine cells fate by monitoring the number of prospero-positive cell in these epithelia. Also, it is possible that the expression of Δ is lost in these stem cells? Other ISC markers such as Sanpodo would be useful to test this hypothesis. Finally, are the single cell trpA and RyR mutant clones Dl-positive?Altogether, these experiments would greatly help clarifying the fate of ISCs in which trpA or RyR activities is impaired.A more careful quantification of esg+ , Su(H)Gb2+ signaling by ROS via a cell autonomous mechanism (including the control of TRPA1 activity), relatively simple experiments could be performed to rule out a non-cell autonomous mechanism following paraquat-induced tissue damage.The authors use paraquat exposure as an oxidative stress in most of their experiments. While the results are compatible with a regulation of Ca2+ signaling? Do ISC-specific genetic manipulations that have been shown to induce ROS production in ISCs and promote cell proliferation (such as ND42 RNAi) increase Ca2+ signaling?Does the expression of antioxidant proteins in ISCs prevent ROS-induced Ca2+ signaling mechanism we have identified. In the revised manuscript we have incorporated the known mechanism via EGFR into our model . We do not think the non-cell autonomous mechanisms are mutually exclusive with the Caur model . Expresson trpA1 .2+ release, arguing that Ras/ERK is the main Ca2+ effector in ISCs. This is an important point as it has been previously published that these factors are part of the signaling pathway that regulate ISC proliferation in response to [Ca] changes, and this new study challenges this view. I believe that this conclusion needs to be supported by additional data.In the last part of this manuscript, the authors try to exclude the role of CanA1 and CrebB downstream of CaThe experiment in As the reviewer suggested, we quantified mitosis  for genoThe consensus is that the manuscript has been improved, and we appreciate the new data you've included as well as your editorial efforts. However, there are two remaining issues that are quite significant, and really need to be addressed before acceptance, as outlined below:2+] controls the expression of Spitz and other RTK ligands. However, the authors appear to favor a model where [Ca2+] controls ERK activation directly through Src, but such activation is not demonstrated and the mechanism is unclear. As an example of this, the revised model still highlights a direct regulation of Ras/MAPK by Ca2+, and the authors discuss a scenario where Spitz induction is downstream of an initial direct activation of ERK rather than an integral part of the ERK activation mechanism. I think the two models (direct or autocrine/paracrine) could be easily be distinguished by testing whether EGFR and/or PVR are required for ISC proliferation induced by SERCARNAi and TRPA1-D overexpression, as suggested in the initial review. I believe that confirming that Ca++ can activate ERK directly in ISCs  would greatly increase the impact of this study." Conversely, demonstrating the EGFR is essential for deregulated proliferation after Ca++ signaling dysfunction would at least show that the mechanism is not simple and not cell autonomous.1) One reviewer and the reviewing editor still believe the manuscript still falls short of establishing how, mechanistically, [Ca++] activates ERK. A key issue is whether this happens directly, cell-autonomously, or through non-autonomous interactions between different cells in the gut epithelium. The old and new data do not resolve this issue, despite requests from reviewers. The model figure tries to cover both  mechanisms and as such is a confusing conclusion – in fact the model as presented will only serve to obscure whatever the real mechanism is, not to resolve it. We would very much like to see a clear resolution of this issue, so that this paper can be seen as an obvious advance over the recent similar publication from Deng et al. A reviewer's specific comments on this point follow: "[…] the authors provide new data demonstrating that  are required for MAPK activation and ISC proliferation, and autocrine Spi-EGFR signaling might participate in amplifying MAPK activity, downstream of [Ca2+] . We have added the mitosis quantification showing that Src is epistatic to trpA1 RNAi , in a process faster than any transcriptional response . […] These data suggest that Src might mediate the activation of Ras/MAPK by cytosolic Ca2+ in ISCs.”“In neurons, a common mechanism by which Ca2+ signaling.”“Previous studies have documented that receptor tyrosine kinase (RTK) EGFR is required for MAPK activity and ISC proliferation . […] Altogether, these results suggest that positive RTK signaling feedback loops may play a role in the activation of Ras/MAPK in the context of Ca2+ signaling and EGFR“Cross-talk between Caspi is induced by EGFR-Ras/MAPK signaling in Drosophila cells , and DNA binding mapping (DamID) analyses from our lab and others indicate that spi might be a direct target of transcriptional factors downstream of EGFR-Ras/MAPK in the ISCs , the autocrine ligand Spi might act as a positive feedback mechanism for EGFR-Ras/MAPK signaling in ISCs.”Prior to our study, it has been shown that paracrine ligands such as Vn from the visceral muscle, and autocrine ligands such as Spi and Pvf ligands from the stem cells, can stimulate ISC proliferation via RTK-Ras/MAPK signaling . […] As 2) A second, and related, issue is how your results and conclusions relate to the Deng et al. publication. We feel that you should show the relevant data you have and clarify how your conclusions are different from Deng's and why. On this issue, a reviewer commented: "some of the data required is not presented and some of the conflicting data not discussed. For example, the genetic interaction between SERCARNAi and CrebBRNAi (CrebBRNAi not being able to block SERCARNAi-induced proliferation) is still reported as "data not shown" in the revised version. This result is used as an important piece of data to support the authors' conclusion that ERK is the major effector of Ca++ in the control of ISC proliferation. Deng et al. reported that CrebBRNAi expression causes ISC loss: do the authors here find something different? Also, Deng et al. reported that dpERK levels are not affected by SERCARNAi expression. This inconsistency should be at least noted and discussed." Although we feel these issues are less pressing than #1 above, we would appreciate some revisions to make it clear how your results and conclusions differ from those presented by Deng et al.SERCA RNAi with CrebB RNAi and CRTC RNAi  and is non-autonomously induced in some ECs (2+ signaling.As requested by the reviewers, we have included the epistasis analysis of RTC RNAi . In addisome ECs , resultiSpecifically, to address the issue raised by the reviewer we have now added 2+ in the ISCs is before they enter the hyper-proliferative stage. Once ISCs start to expand massively following prolonged high Ca2+ induction, pErk returns to normal levels in many ISCs and is non-autonomously induced in some ECs, resulting in a diffusive activation pattern (“It should be noted that the best timing to detect autonomous activation of dpErk by high Ca pattern .”Ras1 RNAi could suppress SERCA RNAi-induced ISC proliferation, neither CanA1 RNAi nor CrebB RNAi could (CRTC RNAi could only confer moderate suppression of ISC hyper-proliferation (“Furthermore, while Ai could , and CRTferation .”


The correlation coefficients for most of the reactions against each of the five antigens and the inhibition of the anti-CA16 VP1 antibody response produced by the various antigens were higher in Shanghai blood donors compared to those in Shanxi individuals. VP11-297 and VP141-297 strongly inhibited the anti-CA16 VP1 response in serum samples from both populations, while VP145-58 and VP161-297 intermediately and weakly inhibited the anti-CA16 VP1 response, respectively, in only Shanghai group. A specific type of inhibition (anti-CA16 VP1 was completely inhibited by both VP11-60 and VP141-297) characterized by high neutralizing antibody titers was identified and accounted for 71.4% of the strongly reactive samples from the Shanghai group. These results indicate that the Shanghai blood donors exhibited a consistent and specific antibody response, while the Shanxi individuals showed an inconsistent and non-specific antibody response. These findings may improve the understanding of host humoral immunity against CA16 and help to identify an effective approach for seroepidemiological surveillance and specific diagnosis of CA16 infection based on normal and competitive ELISA.Coxsackievirus A16 (CA16) is one of the major causative agents of hand, foot, and mouth disease worldwide. The non-neutralizing antibody response that targets CA16 VP1 remains poorly elucidated. In the present study, antibody responses against CA16 VP1 in Shanghai blood donors and Shanxi individuals were analyzed by ELISA and inhibitory ELISA using five CA16 VP1 antigens: VP1 Picornaviridae family) has allowed their phylogenic classification into 12 species, namely, enterovirus A, B, C, D, E, F, G, H and J (EV A–J) and rhinovirus A, B and C. In addition to CA2-8, A10, A12, A14 and EV71, CA16 has been classified as an EV A species based on its genome sequence . The background absorbance was obtained in the absence of sample or HRP-LD5.To estimate the contributions of full-length VP1 and the various truncated VP1 proteins to the anti-CA16 VP1 reaction, a competitive inhibition ELISA was performed as previously described , 40, 41.2, followed by the addition of 100 μl of an RD cell suspension (1 x 105 cells per 0.1 ml). Infected cells and controls were incubated at 37°C in 5.0% CO2 and observed with an inverted microscope daily for seven days. The highest dilution that prevented the development of cytopathic effects in 50.0% of the wells was considered the antibody titer of the sample. A virus back-titration was also performed to determine the virus titer and this serve as a no-serum control.Neutralization tests were performed in RD cells using a TCID50-reduction assay , 42. SerStatistical analyses were performed using SPSS 17.0 and SAS 9.3 software. All experiments were performed in triplicate, and the values obtained from three replicate samples were averaged for each experiment. The statistical significance was tested using the Nemenyi non-parametric test, the Wilcoxon rank sum test or the independent samples t test. Differences between measurements were considered significant at p-values less than 0.05.45-58), N antigen (VP11-60) and C antigen (VP141-297), were proposed [1-297 (full antigen), VP141-297 (C antigen), VP11-60 (N antigen), and VP145-58 (core antigen), as well as a control antigen (VP161-297) without the CECRS, were designed and constructed (E. coli BL21 (DE3) cells and verified by SDS-PAGE. The size of each recombinant protein was in agreement with its expected molecular weight. pET21b-VP11-297, pET32a-VP141-297 and pET32a-VP161-297 were located in inclusion bodies and could be easily solubilized in 8 M urea and conveniently purified under denaturing conditions. pET32a-VP11-60 and pET32a-VP145-58 were found in the soluble fractions of bacterial lysates. These soluble proteins were purified under native conditions.Our previous study demonstrated that the host antibody response against EV71 VP1 mainly targets epitopes based on the common enterovirus cross-reactive sequence (CECRS), and three types of antigens, including core antigen  and VP141-297 (C antigen) and low reactivity against VP11-60 (N antigen), VP145-58 (core antigen) and VP161-297 (control antigen). In the Shanxi group, three reactivity levels were detected: high reactivity against VP11-297, intermediate reactivity against VP141-297 and VP161-297, and low reactivity against VP11-60 and VP145-58  exhibited stronger inhibition potency than VP161-297 in the Shanghai group  from 142 Shanghai blood donors and 95 Shanxi individuals respectively, were submitted to competitive inhibition ELISA to determine the inhibition potencies of VP1nhibited . Moreovexi group . These rai group , and VP1xi group , suggest1-297 and VP161-297), in the Shanghai blood donors. Moreover, the inhibition levels produced by each pair of the four CECRS-based antigens (Nos.1-6 in 61-297 and each of four CECRS-based antigens). Specifically, the inhibition levels produced by the pairs of VP11-297, VP141-297 and VP11-60 in the Shanghai group were very highly correlated, with correlation coefficients ranging from 0.914 to 0.974 (Nos.1-3 in 1-297 and VP141-297 and the pair of VP11-60 and VP145-58). These results consistently demonstrated that different antibody responses occurred between the two study populations, and that the antibody responses against CA16 VP1 had higher correlations in the Shanghai group than the Shanxi group.The correlation analysis of the CA16 VP1 inhibition produced by various VP1 antigens further demonstrated that the Shanghai and Shanxi groups showed different inhibition patterns. As shown in 1-60, the N antigen of VP1, exhibited the same strongest inhibition potency compared to VP141-297 (the C antigen) and VP11-297 (the full antigen) in Shanghai blood donors, whereas, in Shanxi individuals the inhibition potency of VP11-60 was significantly lower than that of VP141-297 or VP11-297 : 7) complete inhibition by the C antigen and weak inhibition by the N antigen; 8) complete inhibition by the C antigen and no inhibition by the N antigen; 9) strong inhibition by the C antigen and no inhibition by the N antigen; 10) weak inhibition by the C antigen and strong inhibition by the N antigen; and 11) no inhibition by the C antigen and weak inhibition by the N antigen . The maiTo determine whether the inhibition type 1 might represent specific antibody response, the strongly reactive serum samples from both groups were submitted to the CA16 neutralizing antibody (NtAb) titer assays. Due to the limited volumes of the serum samples, only 70 (58 samples from inhibition type 1 and 12 samples from inhibition types 2–6) and 43  serum samples that were strongly reactive against VP1 from the Shanghai and Shanxi groups, respectively, were utilized to detect anti-CA16 NtAb titers. For both groups, the samples with specific inhibition type showed significantly stronger reactivity compared to the samples with the other inhibition types . The NtA1-60 and VP161-297 and the levels of inhibition of anti-CA16 VP1 produced by VP11-60 and VP161-297 were also significantly correlated in the Shanghai group, despite that these two antigens share no overlapping amino acid sequences  could effectively inhibit interactions between specific antibodies and CECRS-containing VP1 antigens including VP11-297 and VP141-297. This inhibition would in turn prevent the binding of specific antibodies to all CECRS-based epitopes in VP1. Consistently, the core antigen (VP145-58) exhibited strong inhibition potency similar to that produced by VP11-60  to define 11 different inhibition profiles for this reaction  that infect humans with two or three amino acid mutations. Hence, CA16 VP1 could cross-react with antibodies elicited by enteroviruses from seven species that infects humans at different levels of prevalence. Consistently, 11 different inhibition profiles were found in the two study groups, with the Shanghai and Shanxi groups showing 6 and 9 types, respectively. These different inhibition profiles were assumed to arise from infections with different enteroviruses, suggesting that broad cross reactivity against CA16 VP1 can be produced from many CA16-related enteroviruses.We characterized the anti-CA16 VP1 reaction based on the degrees of inhibition produced by VP1reaction . The Shareaction , while treaction . These f41-297 contained a 257 amino acid sequence that overlapped with most of VP11-297, the correlations between the anti-VP141-297 and anti-VP11-297 reactions in the Shanghai and Shanxi groups varied greatly  showed significantly higher inhibition potency than VP161-297 in the samples from the Shanghai group , p values and the sample sizes (n) are shown.(TIF)Click here for additional data file.S2 FigThe correlation was assessed using Spearman’s correlation coefficient. Correlation coefficient values (r), p values and the sample sizes (n) are shown.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Emetine, a hit compound, was validated for its anti-leukemic effect in vitro, ex vivo and in vivo. Emetine, a second-line anti-protozoa drug, differentially reduced cell viability and clonogenic capacity of AML primary patient samples, sparing healthy blood cells. Emetine treatment markedly reduced AML burden in bone marrow of xenotransplanted mice and decreased self-renewal capacity of the remaining engrafted AML cells. Emetine also synergized with commonly used chemotherapeutic agents such as ara-C. At a molecular level, emetine treatment was followed by a reduction in HIF-1α protein levels. This study validated the anti-leukemiceffect of emetine in AML cell lines, a group of diverse AML primary samples, and in a human AML-transplanted murine model, sparing healthy blood cells. The selective anti-leukemic effect of emetine together with the safety of the dose range required to exert this effect support the development of this agent in clinical practice.Acute myeloid leukemia (AML) is an hematologic neoplasia characterized by the accumulation of transformed immature myeloid cells in bone marrow. Although the response rate to induction therapy is high, survival rate 5-year after diagnosis is still low, highlighting the necessity of new novel agents. To identify agents with the capability to abolish the self-renewal capacity of AML blasts, an  Although the outcome of therapy for leukemia has improved over the years, less than a third of adults with acute myeloid leukemia (AML) are cured by current treatment, a fact strengthening the need for new therapeutic approaches , 2. The Chemotherapy currently used for AML is composed by the combination of conventional antineoplastic agents with considerable myelotoxicity and extramedullary side effects that may be irreversible and limit their therapeutic potential . On the Using the gene expression signature associated with vitamin D3-induced differentiation of AML cells, we have identified Emetine as a potential anti-leukemia agent that differentially reduces cell viability and the clonogenic capacity of AML cells sparing healthy blood cells .in silico screening was performed to seek for FDA-approved small bioactive compounds that induce a similar gene expression pattern than our gene signature. Emetine, a natural product alkaloid obtained from Cephaelis ipecacuanha, displayed the highest score 2 D3, induces differentiation of normal and leukemic myeloid cells along the monocyte/macrophage lineage . This cost score .In order to study the cytotoxic effect of emetine on AML cells, a panel of 5 AML cell lines was treated for 24 h. The EC50 obtained among these cell lines ranged between 40 to 320 nM Figure . A concein silico screening was aimed to find drugs that were able to induce differentiation together with cell death. As described previously [in silico screening.The eviously , C/EBPα eviously . In the Emetine was originally described as a ribosomal-mediated protein  and DNA Combination chemotherapy has been the standard of care in cancer treatment since it is a multitargeted strategy that might result in an increase of both response and tolerability. Moreover, combination chemotherapy might prevent the emergence of treatment-related mutations. Currently, for most AML patients, frontline treatment regimen still involves high doses of chemotherapeutics such as the cell cycle-specific inhibitor cytarabine (ara-C) in combination with a cell cycle-unspecific inhibitor anthracycline such as daunorubicin or idarubicin . In ordeex vivo. As shown for AML cell lines, primary AML samples were sensitive to emetine treatment at the nanoMolar range  for 7 days, starting 7 days after transplantation when the leukemia was already established in the recipient mouse. At day 21, mice were analyzed for the presence of human AML cells in the bone marrow. Mice were left untreated 7 days after finishing the treatment regimen to allow AML cells to regenerate the disease. This treatment strategy was chosen to mimic clinic presentation of the disease Figure . A signis Figure . In facts Figure . Taken tin silico screening was performed searching for AML differentiation-inducing FDA-approved small bioactive molecules. Emetine was identified and validated in AML cell lines. Treatment with emetine induced apoptosis of AML cell lines and primary samples at the nanomolar range. The presence of emetine decreased the clonogenic capacity of AML ex vivo. The anti-leukemic effect of emetine was even more pronounced in AML samples harboring high-risk cytogenetics. Moreover, emetine was able to reduce AML burden in a xenotransplantation mouse model and even more the clonogenic capacity. Taken together, our results support further clinical studies of emetine as an anti-leukemia novel drug in combinatory therapies and highlight the importance of HIF-1α, emetine molecular target, in drug resistance.Although the majority of AML patients respond initially to induction therapy and enter remission, more than half of these patients will experience a relapse episode, highlighting the necessity of new novel agents . Here, ain vivo experiments shown here, of 1 mg/kg per day for 7 days, no significant side effects were observed in treated mice compared to the vehicle control group. According to the “Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers” published by the FDA 2005, the regimen used in the xenotransplantation mouse model corresponds to a 0.08 mg/kg dose in humans. Therefore, the anti-leukemic effect of emetine treatment described in this report is achieved at a concentration below toxicity.Emetine is a crystalline alkaloid derived from the ipecac root approved for the treatment of amoebiasis , althougex vivo and in vitro. Accordingly, emetine acts a bifunctional agent. Ideally, any therapy against AML should be aimed at inhibiting selectively the pivotal pathways of leukemogenesis without affecting normal hematopoietic cells, thus neutralizing leukemic cells and restoring normal hematopoiesis. Little effect on healthy mature peripheral and bone marrow blood cells was observed upon emetine treatment. Moreover, the clonogenic capacity of hematopoietic stem cells remained unaffected in the presence of emetine. Similarly, no bone marrow suppression has been reported in clinical trials where emetine has been evaluated [AML is characterized by a differentiation blockage together with an increase in proliferation capacity. Differentiation therapies aimed to force terminal differentiation of AML cells that will convert AML cells into non-dividing growth-arrested cells, more sensitive to chemotherapeutics and with a reduced life span are promising. The ideal drug in this context would induce simultaneously or sequentially differentiation and apoptosis . As showvaluated , suggestAlthough HIF-1α has been involved in hematopoietic malignancies, its specific role remains unclear. Several studies indicated that HIF-1α is elevated in LSCs and emerging evidence suggests that HIF-1α may be required for survival of LSCs . At a clDue to the heterogeneity and dynamics of AML biology, a single agent used as monotherapy is unlikely to be capable to completely eradicate the disease. Induction of sensitivity to chemotherapy would allow the reduction of the effective concentration of chemotherapy and, potentially, decrease the severity of side effects. Emetine and ara-C synergized to induce cytotoxicity in AML cells. In fact, the combination of 1/2 of their EC50 reduced cell viability by 50%. Our results suggest that emetine constitutes an AML chemosensitizer that can eliminate LSC function, supporting further clinical investigation to elucidate its therapeutic potential.in silico predicted effect of emetine as a differentiating AML agent in AML cell lines, a group of diverse AML primary samples, and in a mouse AML-transplanted model. The anti-leukemic effect of emetine was shown in terms of induction of cytotoxicity and apoptosis, as well as significant reduction of clonogenic activity, suggesting targeting of LSCs. Moreover, the selective anti-leukemic effect of emetine together with the safety of the dose range required to exert this effect support the development of this agent in clinical practice.In conclusion, this study validated the p value < 0.005 were chosen using GenePattern software. The 500 top-ranking downregulated and 500 top-ranking upregulated probes during vitamin D3 treatment were selected for in silico signature-based screening Connectivity Maps . Raw files (.cel) were normalized and probe sets with a differential expression of at least 2-fold of change and ity Maps  and 2 [1AML cell lines HL-60 (ACC-3) , KG-1 . AML diagnosis and classification was based on accepted WHO criteria. Main AML patient's characteristics are summarized in Emetine dihydrochloride hydrate and Ara-C were obtained from Sigma-Aldrich.5 cells per mL were cultured in 96-well plates in complete medium. Emetine and Ara-C were added at indicated concentrations. Cell viability was measured by 7-AAD (eBioscience) exclusion and Hoechst33342 (Sigma) positivity staining by flow cytometry; and cell count was obtained by volume in a FACSCantoII cytometer (BD). Statistical analysis and EC50 determination were calculated in GraphPad (Prism software). FlowJo software (TriStar) was used for flow cytometry analysis.7.5 × 10MonoMac-1 and Kasumi-1 cells were treated for 18h. Apoptosis was measured using the Annexin V apoptosis detection kit (BD) according to the manufacturer's instructions. Data were collected by flow cytometry  and analyzed in FlowJo software (Tristar).5 cells per mL were cultured in 96-well plates in complete medium. 24h after treatment, cells were harvested, washed, and fixed and permeabilized in 70% ethanol (Sigma) at 4°C. DNA content was stained with 7-AAD (eBioscience) and measured by flow cytometry .5 × 10RNA was isolated using the Total RNA purification kit (Norgen biotek) according to the manufacturer's instructions and reverse transcriptase PCR was performed using the qScript cDNA synthesis kit (Quanta Biosciences). Quantitative PCR was performed using a Step One Plus Real-Time PCR System (Applied Biosystems) with a Power SYBR Green PCR mastermix (Applied Biosystems) according to the manufacturer's instructions and using specific primers for each gene (summarized in AB - EA - EB + EA· EB). Results are expressed in percentage.Synergism was assessed by Excess Over Bliss Additivism (EOBA) . Fractio3 primary AML cells or 1 × 103 cells from AML cell lines were treated for 24 h with the indicated compound and mixed with 1 mL of MethoCult H4034 Optimum (StemCell Technologies). Colonies were screened based on morphology and cellularity at day 5 (HL-60), day 7 (KG-1) or day 14 (primary AML cells).50 × 106 cells) were incubated with the indicated compounds for 24 h. Cells were harvested and lysed in RIPA buffer. The protein extract was subjected to SDS-polyacrylamide gel (BioRad) electrophoresis and transferred to a nitrocellulose membrane (BioRad). Mouse anti-human HIF-1α and goat anti-β-actin antibodies were purchased from BD and Abcam respectively.HL-60 and KG-1 cells  clone C31E5E from Cell Signaling Technologies following manufacturers' recommendations. RPE-coupled anti-rabbit secondary antibodies (Life Technologies) were used and samples were acquired in a FACSCanto II (BD).scidIl2rgtm1Wjl/SzJ  mice were myeloablated by busulfan (3 mg/kg IP) at day −1. At day 0, 10 million HL-60 cells were injected IV and left untreated for one week. From day 7 to day 14, 8 mice were treated daily with emetine (1 mg/kg IP), while the other 9 mice were treated with saline vehicle. At day 21, mice were sacrificed and their bones  were harvested. Cells for cytometric analysis were stained with Hoechst 33342 and PE-Cy7 Anti-Human CD45 (BD Pharmingen) and acquired in a FacsCantoII (BD) cytometer. Live cells were gated based on their size, complexity, absence of 7-AAD staining and the expression of human CD45 on the surface.NOD.Cg-Prkdc


Then, the cyclic adenosine monophosphate (cAMP) concentration and protein kinase A (PKA) activity in primary neonatal rat ventricular cardiomyocytes were determined by ELISA assay. Furthermore, the effect of propranolol against the cardiotoxicity of TE was evaluated in Langendorff-perfused rat hearts and intact rats.Under extracellular Ca2+ fluorescence signal by TE was significantly attenuated and delayed when the extracellular Ca2+ was removed. The β adrenergic blockers, including propranolol, atenolol and esmolol, partially inhibited the increase of intracellular Ca2+ in the presence of 1.8 mM extracellular Ca2+ and completely abolished the Ca2+ increase under an extracellular Ca2+-free condition. Both cAMP concentration and PKA activity were stimulated by TE, and were inhibited by the β adrenergic blockers. Cardiomyocyte toxicity of TE was antagonized by β adrenergic blockers and the PKA inhibitor H89. Finally, the acute heart dysfuction by TE was antagonized by propranolol in Langendorff-perfused rat hearts and intact rats.The increase of intracellular Ca2+ overload through intracellular Ca2+ release by TE from the jellyfish C. capillata.Our findings indicate that β adrenergic receptor/cAMP/PKA signaling contributes to the intracellular Ca Jellyfish is one of the main groups in the marine biological species and ecosystem diversity. In recent years, the jellyfish explosion is no longer an unfamiliar topic, but became another marine ecological disaster after the red tides. Jellyfish outbreaks cause harm to fishery resources and marine ecosystem , 2, and 2+ into heart muscle cells is an important reason for cardiovascular collapse  × 100%, where AS is the absorbance of the samples, AC is the absorbance of the PBS, and AB is the absorbance of the background.NRVMs were treated with TE from the jellyfish 2 and 5% CO2, was adjusted to keep a constant flow rate of 12–16 ml/min, thus maintaining an initial perfusion pressure of 60–80 mmHg. A fluid-filled balloon was introduced into the left ventricle through a polyethylene cannula. The initial balloon volume was set to generate the Lvedp of 2–8 mmHg. After a 20-min stabilization period, the isolated perfused heart was pre-treated with 1 μmol/L propranolol followed by the TE administered in bolus (180 μg). Heart indexes, including heart rate (HR), left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP) and maximal ascending and descending derivative of left ventricle (± dP/dtmax), were recorded/calculated by the MPA-2000 bio-signal analysis system.Male SD rats (280–320 g) were anaesthetized with a mixture of urethane (1.2 g/kg i.p.) and heparin (400 IU/kg i.p.). The heart was rapidly excised and placed into an ice-cold Krebs solution. Then the heart was mounted onto a Langendorff apparatus and retrogradely perfused using a peristaltic pump  with warm Krebs solution . The Krebs solution, equilibrated with a mixture of 95% OEffects of the venom and propranolol on blood pressure and heart rate were evaluated in male SD rats  anaesthetized with urethane (1.2 g/kg i.p.). Heparinized polyethylene catheters were implanted into the left femoral artery and external jugular vein for mean arterial pressure (MAP) recording and venom injection, respectively. During all experiments, animals breathed spontaneously. 0.75 mg/kg propranolol was pre-administrated, followed by 0.5 mg/kg TE through the external jugular vein in intact rats. The mean blood pressure (BP) was recorded through a blood pressure transducer  and signals were processed using the MPA-2000.P < 0.05.All the quantitative data were expressed as mean ± SD. The graphs were built using GraphPad Prism 5 for each experiment. Analysis of variance (ANOVA) was used to analyze the effects of TE administration and the invention either by β blockers or H89. A statistical significance was indicated by 2+ entry plays a pivotal role in acute heart failure by TE from jellyfish C. capillata. In this study, fluorescent Ca2+ indicator Fluo-4 AM and frame scanning of confocal microscopy were used to firstly record the intracellular Ca2+ concentration affected by TE in adult mouse cardiomyocytes. Congruously, the signal of Ca2+ fluorescence started to rise at less than 1 min, and got to the maximum with a very bright vision at around 5 min after 60 μg/ml TE administration. The F/F0 got to and stayed at an approximate maximum value of 12 until cell death that normally occurred 15 min later. To exclude the influence of extracellular Ca2+ entry through either Ca2+ pore or Na+-Ca2+ exchanger, we repeated the experiments in a bath solution without Ca2+. Intracellular Ca2+ still increased after 60 μg/ml TE administration, just in a much slower and weaker manner. The time-to-maximum was at about 10 min after TE administration, with a peak F/F0 value very close to that induced by TE under 1.8 mM extracellular Ca2+ condition. In addition, the cardiomyocytes under Ca2+ free condition went to die at least 30 min later, or even without morphological changes and cell death  and Ca2+ uptake through sarcoplasmic reticulum Ca2+ pump, therefore enhancing both contraction and relaxation with fear, stress, exercise or excitation. Here β blockers, including propranolol, atenolol and esmolol, were used to check if TE has a pseudo adrenergic effect that stimulates the intracellular Ca2+ release in isolated adult mouse cardiomyocytes. Under the 1.8 mM extracellular Ca2+ condition, all the β blockers showed a partial inhibitory effect on TE-induced intracellular Ca2+ overload. In the β blocker groups, the elevation of intracellular Ca2+ was much slower with an obviously delayed time-to-peak value, and the maximal F/F0 of fluorescent Ca2+ signals was around 5, much smaller than that of the control. To our great surprise, all the three blockers almost abolished the elevation of intracellular Ca2+: we did not see any increase of intracellular Ca2+ within 10 min after TE administration  coupling, sympathetic excitation through β adrenergic signaling stimulates both CaThe concentration of the second messenger cAMP that is associated with the β adrenergic stimulation was tested by ELISA in primary neonatal rat cardiomyocytes. TE significantly increased the concentration of cAMP which reached 1.5 times of that in the control. All the β blockers, including propranolol, atenolol and esmolol, prevented the cAMP increase, and no difference was seen among the three groups Fig. , further2+ homeostasis during sympathetic excitation is to activate PKA activity. Similar with cAMP concentration, PKA activity in primary neonatal rat cardiomyocytes increased after TE administration, which was also antagonized by the β blockers propranolol, atenolol and esmolol  to 256 ± 26 bpm Fig. . LVDP dengly Fig.. All thengly Fig., indicatBlood pressure was determined in order to evaluate the antagonistic effect of propranolol on the TE-induced acute heart dysfunction in intact rats. Pre-administration of 0.5 mg/kg propranolol greatly recovered the TE-induced decrease of blood pressure Fig. , indicatCarukia barnesi [Chiropsalmus quadrigatus, produced an increase in contraction of isolated rat right atrial preparation in a concentration-dependent manner that was significantly inhibited by diltiazem [Chiropsalmus quadrigatus produced a dose-dependent hypertension and bradycardia immediately, followed by a significant depressive response of blood pressure and irreversible cardiac arrest in anaesthetized rats and rabbits, which were not affected by prazosin, atropine or BQ123 but attenuated by nicardipine [The cardiotoxicity of jellyfish venom showed great variations. Patients with the famous Irukandij syndrome presented with a significant and ongoing pain but with no or minimal skin markings, tachycardia, hypertension followed by hypotension and pulmonary oedema, indicating a significant cardiac dysfunction and life-threatening induced by the jellyfish  barnesi , 28. Veniltiazem . Intraveardipine , 31. Intardipine .C. capillata mainly displayed in three aspects, including HR decrease, systolic and diastolic dysfunction and imbalance between cardiac oxygen supply and demand. Both in vitro and in vivo investigation displayed that HR decreased dose-dependently that occurred immediately and partially recovered in 10 min after TE administration, indicating a blemish of the sinus node/conduction system [C. capillata is related to the direct injuries of sinus node/conduction system, ventricular working cells, as well as myocardial ischemia and hypoxia by coronary spasm [The manifestation of acute heart dysfunction reported by us from the TE of jellyfish n system , 33. Then system . Thus, try spasm .2+ overload induced by extracellular Ca2+ entry has been congruously recognized as an important mechanism underlying the cardiotoxicity of jellyfish venom. As early as 1973, Calton and Burnett found the effect of two jellyfish toxins on calcium ion transport [2+ antagonists verapamil, diltiazem and nifedipine were sequentially reported to antagonize cardiotoxicity of the venom from Chironex fleckeri [Physalia physalis) [Carybdea rastonii [C. capillata [2+ influx whilst La3+, a non-specific channel and pore blocker, inhibited the Ca2+ influx, consistent with the presence of a pore-forming toxin existing in the venoms which was demonstrated by transmission electron microscopy in the case of C. fleckeri [C. fleckeri caused extracellular Na+ entry and intracellular Ca2+ overload in cardiomyocytes that was not inhibited by blockers of Ca2+ or Na+ channels or by inhibitors of Na+/K+ ATPase or Na+/H+ exchange in the sarcoplasmic reticulum, but blocked by prior exposure to a solution which contained no Na+ and by Ni2+, indicating a possible mechanism of intracellular Ca2+ overload induced by jellyfish venom via the Na+/Ca2+ exchange [The exact mechanism of the acute heart dysfunction is not clear, however, intracellular Caransport . Since ffleckeri , Portuguhysalis) , Carybderastonii , 39 or Capillata . Howeverfleckeri . It was exchange .C. capillata led to an intracellular Ca2+ overload that was greatly weakened after removal of the Ca2+ in extracellular solution in this study, further supporting the importance of extracellular Ca2+ entry. Although the extracellular Ca2+ entry can be prevented by the L-type Ca2+ blockers, the mechanism of extracellular Ca2+ entry is now dominated by the existence of pore-forming complex in jellyfish venom that allows a non-specific translocation of Ca2+ across the cell membrane [C. rastoni, C. fleckeri, Cyanea nozakii Kishinouye and Chironex yamaguchii (as C. quadrigatus), respectively [2+ entry. However, this is not the only situation, comparative functional analysis of the two pairs of structurally similar hemolytic proteins from Chironex feckeri, CfTX-1/2 and CfTX-A/B, showed that CfTX-1/2 caused profound effects on the cardiovascular system of anaesthetized rats, whereas CfTX-A/B elicited only minor cardiovascular effects but displayed a hemolytic activity at least 30 times greater than that of CfTX-1/2, indicating that the hemolytic proteins of jellyfish venoms have structural diversification and functional selection during evolution [TE from membrane , 23. So ectively , 40, 41.C. rastonii [Alatina nr mordens was significantly inhibited by prazosin. However, these effects were not uniformed among different jellyfish species. The concentration dependent inotropic response in left atria induced by the crude venom of a newly described jellyfish Malo maxima causing Irukandji syndrome was significantly attenuated by propranolol but not by prazosin [Chiropsalmus sp. [C. fleckeri [C. fleckeri displayed that both propranolol and prazosin have no effect on the cardiovascular toxicity of the nematocyst venom [The involvement of adrenergic system in the cardiovascular toxicity of jellyfish venom was firstly drew by the partial inhibition of the α-adrenergic antagonist phentolamine, the central antiadrenergic reagent trifluoperazine as well as the phosphodiesterase inhibitor papaverine on the aorta contraction induced by the venom of the jellyfish rastonii . Furtherrastonii , 43. Simprazosin . Howeverlmus sp. . Prazosifleckeri . Anotherst venom . Therefo2+ overload through intracellular Ca2+ release that was induced by TE from the jellyfish C. capillata. This phenomenon was hindered by β adrenergic blockers propranolol, atenolol and esmolol. cAMP concentration and PKA activity were both increased correspondingly, implying the involvement of β adrenergic signaling in cardiotoxicity of jellyfish venom. We also forwarded our studies in Langendorff-perfused rat hearts, which have got the partial antagonistic effects of propranolol on the heart dysfunction. The reason for the negative results of adrenergic blockers from previous studies can be explained by the mask of significant extracellular Ca2+ entry through pore-performing components. In intact rats, we used a gentle dose of TE from C. capillata that only led to a BP decrease of approximate 20 mmHg, and thereby allowing to display the role of β adrenergic signaling as well as the antagonistic effect of propranolol. This result is consistent with the change of intracellular Ca2+, where propranolol only partially inhibited the intracellular Ca2+ overload with normal extracellular 1.8 mM Ca2+ while completely abolished the intracellular Ca2+ increase when the extracellular Ca2+ was removed from the bath solution.In this study, we are interested in the intracellular Ca2+ overload through intracellular Ca2+ release as well as the cardiotoxicity of jellyfish venom from the respects of fluorescence Ca2+ scanning, cardiomyocyte toxicity and acute heart dysfunction in Langendorff-perfused rat hearts and intact rats.Collectively, we have firstly confirmed the involvement of the β adrenergic signaling in the intracellular Ca


The Beuchet Chair is a powerful and highly popular optical illusion. The illusion involves two reasonably large pieces of apparatus: an oversized chair seat and four normal-sized chair legs. When properly arranged and viewed from a precise location, a person standing on the seat appears to be much smaller than they actually are. Although compelling, the illusion is relatively challenging and expensive to construct, requires a large amount of space to stage, and is not especially portable. Here, I outline a new version of the illusion that just involves a small piece of cardboard, a cloth, and a tripod. This new version costs almost nothing to create, is highly portable, and requires far less space than the original. Educational institutions and visitor attractions across the world regularly use the Beuchet Chair illusion to entertain and educate. Created by French psychologist Jean Those wishing to stage this powerful and compelling illusion currently face several practical challenges. First, constructing the two pieces of apparatus is far from easy as it involves building an oversized seat and cutting the tops of each of the chair legs at a precise angle. Second, the resulting apparatus is not especially portable and takes up a considerable amount of storage space. Finally, the illusion can only be staged in a relatively large room because of the required distances between the observer, legs, and seat.front leg of the template is attached to the top of the tripod (see I recently devised a very different way of creating the illusion that overcomes all of these difficulties. This new version of the chair illusion simply involves a 20-cm cardboard template of a chair, a 2.5 m × 1.5 m piece of brightly coloured cloth, and a 60-cm high tripod. The ipod see , and theipod see . The resipod see . An alteipod see , it can The method of creating the illusion is somewhat different to the one used in the original Beuchet Chair and the template proved challenging to design. First, all of the items involved in the original illusion are relatively large, and so a camera does not struggle to focus on the various elements. However, in the new version, the chair is much smaller and very close to the camera, which could result in focusing issues. Because of this, it was necessary to create a template that was large enough to create the required illusion, but not so large that it completely dominated the field of view and therefore caused the camera to focus solely on the chair. Second, unlike the Beuchet Chair, the person who appears to be on the chair cannot seem to stand in front of the chair back. Because of this, the seat of the chair had to be widened to allow them to stand to the right of the chair back. Initial attempts resulted in a chair seat that looked unnaturally wide, but it eventually proved possible to create a design that does not arouse suspicion. Third, whereas the legs of the Beuchet Chair simply rest on the floor, the template has to be supported by the tripod. This was made possible by extending the front leg of the chair and again involved creating a design in which an extended leg did not appear suspicious.This new version of the Beuchet chair costs very little to make, is extremely easy to construct and transport, takes up almost no storage space, and can be staged in a much smaller room than the traditional Beuchet chair. I briefly described the illusion in a recent blog post  and was