Treatment for acute myeloid leukemia (AML) has not significantly changed in the last decades and new therapeutic approaches are needed to achieve prolonged survival rates. Leukemia stem cells (LSC) are responsible for the initiation and maintenance of AML due to their stem-cell properties. Differentiation therapies aim to abrogate the self-renewal capacity and diminish blast lifespan.An in silico screening was designed to search for FDA-approved small molecules that potentially induce differentiation of AML cells. Bromocriptine was identified and validated in an in vitro screening. Bromocriptine is an approved drug originally indicated for Parkinson’s disease, acromegaly, hyperprolactinemia and galactorrhoea, and recently repositioned for diabetes mellitus.Treatment with bromocriptine reduced cell viability of AML cells by activation of the apoptosis program and induction of myeloid differentiation. Moreover, the LSC-enriched primitive AML cell fraction was more sensitive to the presence of bromocriptine. In fact, bromocriptine decreased the clonogenic capacity of AML cells. Interestingly, a negligible effect is observed in healthy blood cells and hematopoietic stem/progenitor cells.Our results support the use of bromocriptine as an anti-AML drug in a repositioning setting and the further clinical validation of this preclinical study.The online version of this article (doi:10.1186/s12967-016-1007-5) contains supplementary material, which is available to authorized users. Acute myeloid leukemia (AML) is the most common acute leukemia in adults and the survival rate 5 years after diagnosis is below 50 % according to data collected from SEER Epidemiologic Datasets . StandarLeukemia stem cells (LSC) have been described in the majority of AML patients and constitute a cell fraction with self-renewal and differentiation properties . Due to By means of an in silico screening searching for approved small bioactive drugs that potentially induce differentiation of AML cells, bromocriptine, a drug originally indicated for Parkinson’s disease, acromegaly, hyperprolactinemia and galactorrhoea, and recently repositioned for diabetes mellitus , 8, was http://www.broadinstitute.org/cancer/software/genepattern/) and probe set with a differential expression of at least twofold of change and p value below 0.005 were chosen. The 481-top-ranking upregulated  . Indeed, viability of this primitive cell fraction was reduced about 50 % 24 h upon treatment with bromocriptine. Next, the sensitivity to bromocriptine was analyzed according to the prognostic cytogenetic category of AML samples  and secondary AML (sAML) . MDS invBromocriptine is described as a potent anti-leukemia drug that mainly targets leukemia stem cells,Bromocriptine treatment spared healthy mature blood cells and hematopoietic stem cells.


Taking advantage of this approach, we attempted dual observation of Ca2+ and Rho GTPase (RhoA and Cdc42) activities in astrocytes and found that ATP triggers both RhoA and Cdc42 activation. In early phase, while Cdc42 activity is independent of Ca2+ transient evoked by ATP, RhoA activity is Ca2+ dependent. Moreover, the transient Ca2+ upregulation triggers long-lasting Cdc42 and RhoA activities, thereby converting short-term Ca2+ signaling to long-term signaling. Thus, the new FRET pair should be useful for dual observation of intracellular biochemical reactions.Intracellular signal transduction involves a number of biochemical reactions, which largely consist of protein-protein interactions and protein conformational changes. Monitoring Förster resonance energy transfer (FRET) by fluorescence lifetime imaging microscopy (FLIM), called FLIM-FRET, is one of the best ways to visualize such protein dynamics. Here, we attempted to apply dark red fluorescent proteins with significantly smaller quantum yields. Application of the dark mCherry mutants to single-molecule FRET sensors revealed that these dark mCherry mutants are a good acceptor in a pair with mRuby2. Because the FRET measurement between mRuby2 and dark mCherry requires only the red region of wavelengths, it facilitates dual observation with other signaling sensors such as genetically encoded Ca Intracellular signal transduction involves a number of signaling proteins such as kinases3557resonance energy-accepting chromoprotein (REACh) was developed and applied to 2pFLIM2+ and Rho GTPase activity in astrocytes.A decade ago, a nonradiative yellow fluorescent protein called 2+ and the activities of cell division cycle 42 (Cdc42) or Ras homolog A (RhoA). These are members of the Rho family of small GTPases and are known to play pivotal roles in morphological changes and migration of cells by regulating actin polymerization2+ transient evoked by ATP, RhoA activity is dependent on Ca2+ transient. Moreover, the late-phase activities of both Cdc42 and RhoA are triggered by transient Ca2+ upregulation, converting the short-term signaling to the long-term signaling. Thus, an ATP-evoked calcium transient temporally regulates Cdc42 and RhoA activities in astrocytes.Astrocytes are the most abundant glial cell type in the central nervous system and play essential roles in maintaining the structure, metabolism, and synaptic functions of the neural network15I202Y and mCherryI202T). These mutations are the same as the previously reported mutations yielding longer-emission mutants19I202Y and mCherryI202T are half that of mCherry (I202Y (0.52 ns) and mCherryI202T (0.60 ns) are much shorter than that of mCherry , these values turned out to be much smaller than the lifetime of mRuby2 (2.52 ns)  . These r2.52 ns) . Next, w2.52 ns) 1121. TheI202Y or mCherryI202T was fused to the C terminus of mRuby2-CaMKIIα, creating Camuiα-mRmCI202Y or Camuiα-mRmCI202T: a configuration similar to previously reported formats2122I202Y or Camuiα-mRmCI202T, and compared the response signals by 2pFLIM521232+ activates calmodulin, and subsequent binding to CaMKII induces a structural change of CaMKII  differing in their acceptor fluorophores . mCherryf CaMKII 25, we us minutes . A compa signals . Next, w signals 2627. mRuRmCI202T . To evalRmCI202T . We founRmCI202T .2+ transient in astrocytes2829152+-related processes have remained poorly studied. To monitor the activity of Cdc42 and RhoA, we developed FRET sensors based on the mRuby2-and-mCherryI202Y pair using a previously reported strategy (I202Y rather than mCherryI202T because of the slightly wider dynamic range (I202Y was fused to an activated Cdc42 binding domain (CBD) derived from PAK3 as an acceptor, as described previouslyI202Y-CBD bound to the activated mRuby2-Cdc42, leading to FRET between mRuby2 and mCherryI202Y (2+ concentration simultaneously.Because the pair of mRuby2 and dark mCherry requires only a narrow range of wavelengths (550–650 nm) for observation , we nextstrategy  and 4a24ic range . For therryI202Y . For a RrryI202Y 24. We el2+ changes by means of 2pFLIM. After ATP stimulation, a cytosolic Ca2+ transient occurred within 30 s  in combination with removal of extracellular Ca2+. Imaging of Ca2+ confirmed strong inhibition of the ATP-evoked cytosolic Ca2+ upregulation  to stop the Ca2+ influx. This protocol effectively mimics the temporal pattern of the Ca2+ transient evoked by ATP  with astrocytes expressing the FRET sensor, and simultaneously monitored the GTPase activity and Cahin 30 s  and 4b,c30 s 2829 and 4c. gulation . It is nependent , RhoA acependent , suggesthibition  and 4g, onophore  and 4e,fd by ATP  and 4e. ly phase  but gradependent . In conte phases , suggestmulation . Furtheral cells , and fouly phase , implyinHere, we successfully demonstrated that the pair “mRuby2 and a dark red fluorescent protein” is suitable for FLIM-FRET measurement . Notably2+. We found that in early phase, while Cdc42 activity is independent of Ca2+ transient evoked by ATP, RhoA activity is Ca2+ dependent  by error-prone PCR using the Diversity PCR Random Mutagenesis Kit with a high error rate . Subsequently, the PCR fragments were digested with PstI and BsrGI and ligated into the mCherry-pRSET vector. Escherichia coli DH5α cells were transformed with the plasmid library by electroporation and grown for 18–20 h at 34 °C on Luria-Bertani (LB) agar plates supplemented with antibiotics.Random mutagenesis was performed by amplifying mRuby2 gene was amplified by PCR from the pcDNA3-AKAR2-CR plasmid  as a template and was inserted into the modified pEGFP-C1 vector by replacing EGFP. This vector was used in all mammalian-expression experiments in this study. For construction of tandem fluorescent protein plasmids, gene mCherryI202Y or mCherryI202T or the synthesized Ultramarine gene was inserted into the multiple cloning site in the FLAG-tagged mRuby2 plasmid (encoding amino acid residues 1–229 of mRuby2) with a linker encoding the peptide SRAQASNSAVDGTAGPGSG. For construction of mRuby2-based CaMKIIα FRET sensors, mRuby2-CaMKIIα with the linker peptide SGLRSRA was fused to mCherryI202Y, mCherryI202T, or Ultramarine with a linker peptide GSG. For construction of the mRuby2-based LOV2 FRET sensors, we fused mRuby2 (DNA sequence corresponding to amino acid residues 1–234) to the N terminus of the LOV2 domain (DNA sequence corresponding to amino acid residues 404–546 in phototropin 1) with a linker encoding the peptide ASM. Then, mCherryI202Y, mCherryI202T, or Ultramarine was subcloned into the C-terminal region of LOV2 with the linker peptide KLGNS. For construction of the RhoA FRET sensor, we fused mCherryI202Y to the binding domain of Rhotekin (amino acid residues 8–89) with the linker peptide SGLRS. Subsequently, mRuby2-RhoA with the linker peptide SGLRSRG was fused to the C terminus of the above protein via the P2A sequenceCMV-G-GECO1.1 was a gift from Robert Campbell (Addgene plasmid # 32445). For construction of the mRuby2 plasmid, the mRuby2, mCherry, mCherryI202Y, or mCherryI202T gene was inserted into the pRSET vector (Invitrogen). The respective proteins were overexpressed in E. coli DH5α cells and purified on a Ni+-nitrilotriacetate column  with subsequent dialysis against PBS. Absorption, excitation, and emission spectra of the fluorescent proteins were recorded on a UV-Vis spectrophotometer  or a fluorescence spectrophotometer . The molar concentration of the purified proteins was measured by the Bradford protein assay (Bio-Rad). The molar extinction coefficients were determined by dividing the peak optical density by the molar concentration determined by the alkaline denaturation method2039For purification of His-tagged proteins, 2. Then, the cells were transfected with the plasmids using Lipofectamine 3000 (Invitrogen), followed by incubation for 18–24 h. FLIM-FRET imaging was conducted in a solution containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid -buffered artificial cerebrospinal fluid  at room temperature.HeLa cells were cultured in the Dulbecco’s modified Eagle’s medium (DMEM)  at 37 °C in a humidified atmosphere containing 5% of CO2 culture flask . The culture medium was exchanged with the plating medium after 2 days in vitro and once a week. When the cultured cells reached confluence at 10–14 days in vitro, the culture flask was rinsed twice with HBSS as a conventional shake-off protocol to remove nonadherent cells. Adherent cells were trypsinized, and cDNA was transfected into these cells by means of an Amaxa Nucleofector . Transfected astrocytes were plated at the density of 1.2 × 106 cells/dish in 35-mm culture dishes. FLIM-FRET imaging was carried out in solution  at room temperature. BAPTA were used in a Ca2+-free solution .All animal procedures were approved by the National Institutes of Natural Sciences Animal Care and Use Committee and were performed in accordance with the relevant guidelines and regulations. Astrocytic culture was prepared from cerebral cortices of newborn (P0) C57BL/6 J mice  after deep anesthesia and decapitation. The tissues were dissociated in cold Hank’s Balanced Saline Solution  by trituration as described previously2+ imaging, G-GECO1.1 fluorescence was detected by means of a photomultiplier tube  placed after the emission filter . A fluorescence lifetime curve was recorded by means of a time-correlated single-photon-counting board  controlled with custom softwarem) in each pixel were translated into a color-coded imageA custom-made two-photon fluorescence lifetime imaging microscope was used for this analysis as described elsewherefree) is 2.52 ns, the same as that of free mRuby2; 2) mature mRuby2 fused to a mature acceptor fluorescent protein where FRET takes place and fluorescence lifetime of mRuby2 (τFRET) gets shorter. To measure τFRET, we used the following formula as described previouslyWe quantified free mRuby2 and mRuby2 undergoing FRET as described elsewhere0 is the time offset, σG is the standard deviation of the IRF, and τfree and τFRET are the decay time constants of free mRuby2 and mRuby2 with FRET, respectively. Pfree and PFRET are the coefficients of the free mRuby2 component and mRuby2 with FRET, respectively. Using this formula, we obtained τFRET for mRuby2-mCherryI202Y (0.80 ns), mRuby2-mCherryI202T (0.74 ns), and mRuby2-Ultramarine (0.84 ns). Using the obtained τfree and τFRET, we calculated the proportion of mRuby2 undergoing FRET in individual cells using the following formula as described elsewhere5In How to cite this article: Nakahata, Y. et al. Dual observation of the ATP-evoked small GTPase activation and Ca2+ transient in astrocytes using a dark red fluorescent protein. Sci. Rep.6, 39564; doi: 10.1038/srep39564 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Wistar rats were treated by 2 or 100 Hz EA at Zusanli (ST 36) and Xiajuxu (ST 39) bilaterally for 60 minutes. The accumulation of Na99mTcO4 in the gastric wall and the free ions, including Ca2+, K+, Na+, and Cl−, in the acupoints were measured every 60 minutes. The radioactivity uptake in the stomach was significantly increased during EA, reaching peak at 180 minutes after the EA. The concentration of extracellular ions was also significantly increased during EA. The Ca2+ level continued to rise until 60 minutes after EA, then started to decrease at 120 minutes post-EA. The results suggest this up-regulatory effect of EA on gastric activity might be triggered by the increase of the extracellular ion levels, this effect lasts longer than stimulating the release of transmembrane Ca2+ flow alone. This might aid in providing a better understanding of the long-lasting effect claimed in acupuncture treatment.Electroacupuncture (EA) is a therapeutic modality in which the electrical stimulation is integrated with concepts of acupuncture to treat diseases. This study was designed to evaluate the connection between the electro-acupuncture induced increase in Na Recent research suggested acupuncture may take effect by changing the cellular K+, Na+, and Ca2+ levels2. Experiments indicated the possibility of the biological modulation information of acupuncture encoded in the magnitude, frequency, and spatial organisation of the changes in the cellular-free Ca2+4. The latency and memory effects of Ca2+ oscillations that remained at 30 minutes or even 1.5 hours after the needle stimulation was turned off appeared to agree with the long-lasting healing claimed in acupuncture treatment5.Acupuncture, as an integral part of Traditional Chinese Medicine, has long been used to treat diseases6. Zusanli (ST 36) and Xiajuxu (ST 39), among others acupoints, are most extensively studied for their positive effects on various gastrointestinal disorders7. Although several categories of assessment methods were applied to investigate the modulation process on the gastric organs, the explanation of the mechanisms and effects of EA on those organs remains unclear8. Based on previous studies, we hypothesised that the EA-induced up-regulation of the stomach function, manifested by an increase in radiopharmaceutical 99mTc-sodium-pertechnetate (Na99mTcO4) uptake surrounding the fundus of the stomach, might be induced by the ionic molecule levels changing in the acupoints on the stomach meridian9.Electroacupuncture (EA) is a therapeutic modality in which the principles of electrical stimulation are integrated with traditional concepts of acupuncture10. It has been used widely in both animals and human subjects to investigate the changes experienced by extracellular molecules or medicine under certain physical or pathological conditions11. In our hypothesis-driven study, we set up an MD system for ionic molecule detection at the acupoints in rats undergoing EA, and checked the correlation between these free-molecule levels and the photon activity in the stomach. As far as we are aware, no reports are found to investigating this issue for acupuncture.Microdialysis (MD) is a specific, local sampling method to collect free molecules of interest from the extracellular fluidSixty healthy male Wistar rats, weighing 180 to 250 g, aged 4 to 5 months, were used in this study. They were housed at a room temperature (22 ± 3 °C) under standard 12-h light/dark cycles (lights on at 07:00) with unlimited access to food and water, all rats were acclimatized for one week. The experiments were performed according to the guidelines of the Institutional Animal Care Committee of the University. All experiments were approved by the Ethical Committee of The First Affiliated Hospital of Xian Jiaotong University . All efforts were made to minimize the number of animals used and their suffering. The rats were randomly chosen and divided into three groups (n = 20 each): G1, treated by 2Hz-EA at Zusanli (ST 36) and Xiajuxu (ST 39); G2, treated by 100 Hz-EA at ST 36 and ST 39 . Rats in the non-EA group were also anaesthetised in a similar fashion for 60 minutes as described12.Rats were anaesthetized with an intraperitoneal injection of 20% Urethane at a dose of 5 mg/kg. Rats were placed in a ventral decubitus position. EA was applied at Zusanli (ST 36) and Xiajuxu (ST 39) bilaterally as previously describedThe CMA 30 Linear Microdialysis Probe  to study the peripheral tissues  was used. The probe consists of inlet tubing, outlet tubing, and a middle part with a window where the membrane is located. The inlet of the probe was connected to a peristaltic pump , which would push the tube with normal saline (NS) at a rate of 2.0 µL/min. Before sample collection, the membrane part of the probe was immersed in distilled water for 30 minutes. Then, the probe was immersed in the tissue around ST 36 (depth of about 3 mm), and the dialysate was collected in the outlet part every 60 minutes for the following 4 hours . The dialysate was stored at −20 °C for further analysis.2+, Na+, K+, and Cl− concentrations by LABOSPECT008 (Hitachi Japan). Dialysate ions were calculated as13:We tested the target molecules, detecting efficiency of the probe with standard ion solutions. The MD probe was put into NS for 20 minutes for stabilisation and then was switched to standard ion solutions. The standard ion sampling was collected at 60-minute intervals. All diluted dialysates samples were analysed for Ca99mTcO4 SPECT/CT was performed 5 minutes after the injection of 7.4 MBq of 99mTcO4− via the caudal vein. The scanners were dual-head gamma cameras, using low-energy high-resolution collimators and a 20% energy window centred on 140 keV. The DICOM image files of each rat were saved on optic discs and transferred to a Symbia Evo Excel workstation  for centralized reconstruction, reading, and analysis.Na99mTcO4 SPECT/CT images were read and interpreted by the consensus of three experienced nuclear medicine physicians with reference to SPECT/CT fusion images. For semiquantitative analysis, gastric-to-background ratios of SPECT images were measured and calculated by the same person using a consistent standard14. The data are collected and compared with before EA, during EA, and after EA, to observe the adjusting effect of EA on gastric function.Nap-value < 0.05 was considered statistically significant.The data were analysed using SPSS 10.0 software . All results are expressed as the mean ± standard error (SEM). Two-way ANOVA and Duncan post hoc tests were performed to investigate the effects of electroacupuncture on the extracellular ions concentrations and on the gastric functions during each session, and a The data sets used and/or analyzed during this study are available from thecorresponding author upon reasonable request and have been approved by the institutional committee on scientific research.This study is approved by the ethics committee on biomedical study of the Xi’an Jiaotong University Health Science Center.2+, K+, Na+, and Cl− . The Ca2+ level increased gradually and reached the peak plateau at 60 minutes after 2Hz-EA (p = 0.75 vs EA), then started to decrease at 120 minutes post-EA (p = 0.04 vs EA). The concentrations of extracellular Na+ and Cl− were also significantly increased during EA as compared with pre-EA . After EA, they started to decrease. Their levels decreased to 71.81 ± 15.09 mmol/L and 57.42 ± 14.30 mmol/L 60 minutes after 2Hz-EA (p = 0.09 and p = 0.07 vs. EA). The level of extracellular K+ showed similar changes, although the comparisons showed no significance . Interestingly, the up-regulation increased gradually  and did not reach the peak until 180 minutes after the EA (T/B ratio 22.80 ± 1.40 mmol/L at 180 min after EA). Then the stomach uptake started to slowly decrease 3 hours after 2 Hz EA at ST 36  and Xiajuxu (ST 39)1. In traditional Chinese medicine, ST 36 and ST 39 are the points of the “Stomach Meridian of Foot-Yangming,” which is reported to be useful in treating GIT disorders such as stomach ache, abdominal pain and distension, constipation, diarrhoea, vomiting, dysentery, and indigestion2.Studies have shown functional gastrointestinal tract (GIT) disorders can be treated by acupuncture99mTcO4) is distributed throughout the vasculature and interstitial fluids by a slow diffusion mechanism, and the concentration varies between organs15. In the gastrointestinal tract, Na99mTcO4 is actively concentrated mainly in the gastric mucosa16. The bioavailability of Na99mTcO4 in the gastric wall can be changed in many circumstances, including the patient’s health status, disease states, invasive medical procedures, and complementary therapies such as acupuncture17. Despite that the specificity of acupuncture is still questioned, the actions of acupuncture at ST 36 have been shown to significantly increase the radioactivity uptake of Na99mTcO4 in the stomach9. In the current study, we utilized the Single positron emission tomography (SPECT/CT) as main testing modality to observe the gastric function under electroacupuncture (EA) in vivo. (SPECT/CT) could reproduce anatomical and physiological imaging by mapping GIT changes in vivo in response to acupuncture stimuli18. Our results suggest the up-regulation effect of EA on gastric wall Na99mTcO4 uptake. Previous studies have suggested that EA could produce an excitatory effect on the gastrointestinal motility of the rat9. These ST 36 acupuncture effects may be correlated with our data, which suggested that 2 Hz-EA could obviously increase the uptake of Na99mTcO4 in the stomach, whereas no significant differences were found between the 100Hz-EA group and the control group.The unbound form of pertechnetate anion  and 2 Hz (low frequency) are commonly used for EA research19. Our results showed that EA at both frequencies could induce increases in gastric function, similar to previous studies. Tian et al. suggested that EA at both low and high frequencies could induce a therapeutic effect on obesity20. Tseng et al. showed that EA in either frequency could induce a decrease in glucose, an increase in lactate metabolites, and a reduction in lactate/glucose ratios21. Studies of EA in which ST 36 has undergone electrostimulation at 2 or 100 Hz have suggested that the mechanism underlying the effect might be that EA could lead to the regulation of internal organ systems as a network22. EA stimulation could lead to effects on gastric myoelectrical activity, brain-gut peptides responses in the gastric mucosa, and the vagal and splanchnic nerve responses on the activity of gastric motility and emptying23.Compared with simple acupuncture, EA is a therapy in which the principles of electrical stimulation are integrated with traditional acupuncture+, Na+, Ca2+, and the content of neurotransmitters5. Among them, Ca2+ was supposed to play a key role5. As an important intracellular second messenger, Ca2+ participates in various physiological and biochemical processes of cells24. The proposed acoustic signaling paths, jing-luo, are distinctly and physically separated from the nervous system. This appears to be the missing “calcium” communication link proposed but not found in the frequency-encoded second messenger25. It has been suggested that calcium may be taken as the carrier of the biological modulation system, of which acupuncture has been implemented as a clinical application26. Moreover, because the intracellular Ca2+ concentration is controlled by the opening and closing of both voltage-dependent and receptor-dependent Ca2+ pathways, as well as by the Na+/Ca2+ exchanger and the activity of the Ca2+ pump, the concentrations of K+ and Na+ were reported to change with Ca2+ levels27.In recent years, more and more laboratory proof has accumulated that acupuncture can change the charge and potential of neurons, the concentrations of K2+ in the gastric smooth muscles1. The AM and FM schemes of calcium signaling in the acupuncture was proposed to cause cellular Ca2+ flux. The signal is broadcasted with a chevron-like pattern radiating from the needle like a radio antenna28. Our study showed that acupuncture at ST 36 and ST 39 could markedly enhance release of intracellular Ca2+ and, thus, significantly increased the extracellular Ca2+ levels. The extracellular Ca2+ level increase was correlated with gastric function upregulation. One potential testable hypothesis could be that the AM and FM schemes of calcium signaling were proposed based on the amplitude modulation of extracellular Ca2+ levels, and separated from the central nervous system, there might be a channel of cellular communications with calcium waves playing the role3. This proposed mechanism might explain our surprising finding that the increase of the gastric wall Na99mTcO4 uptake lasted longer than the enhancing of the release of intracellular Ca2+ to extracellular space observed in our study. Similar observations have been reported that Ca2+ oscillation remained at 30 minutes or even 1.5 hours after the needle stimulation was turned off5. The latency and memory effects appeared to agree with the long-lasting healing claimed in traditional acupuncture treatment29.The effects of acupuncture at the points of the Foot-Yangming meridian on gastric movement are related to the release of intracellular Ca30. Our experimental finding is that the when EA was applied at non-acupoints, the coupling of the increase of Ca2+ levels and gastric Na99mTcO4 uptake disappeared. There is a slight increase in the Ca2+ level, whereas no upregulation of the gastric function was observed (data not shown).In this experimental model, the authors did not include the “sham acupuncture” group as an “inactive” control, since needling non-acupoints (any cutaneous location of the body surface) could produce unpredictable somatovisceral reflex responses, and therefore could complicate the interpretation of the resultsIn the current study, EA at acupoints of the stomach meridian in rats showed that EA could upregulate gastric function, which is thought to be correlated with extracellular ions concentrations changes at the acupoints. Further rigorous experimental studies to examine their correlations need to be performed.


Continuous in vivo monitoring of intracellular Ca2+-dynamics in C. elegans pharynx muscle revealed surprisingly complex Ca2+ patterns. Despite the age-dependent decline of pharynx pumping, we observed unaltered fast Ca2+ oscillations both in young and old worms. In addition, sporadic prolonged Ca2+ increases lasting many seconds or minutes were often observed in between periods of fast Ca2+ oscillations. We attribute them to the inhibition of ATP-dependent Ca2+-pumps upon energy depletion. Accordingly, food deprivation largely augmented the frequency of prolonged [Ca2+] increases. However, paradoxically, prolonged [Ca2+] increases were more frequently observed in young worms than in older ones, and less frequently observed in energy-deficient mitochondrial respiratory chain nuo-6 mutants than in wild-type controls. We hypothesize that young animals are more susceptible to energy depletion due to their faster energy consumption rate, while nuo-6 mutants may keep better the energy balance by slowing energy consumption. Our data therefore suggest that the metabolic state of the pharynx during feeding stimulation depends mainly on the delicate balance between the instant rates of energy production and consumption. Thus, in vivo monitoring of muscle Ca2+ dynamics can be used as a novel tool to study cellular energy availability.Ca C. elegans is one of the leading model organisms for aging research. One of the main advantages of this animal is the availability of well-described mutant strains with shortened or extended life spans, which provides a unique tool to dissect the molecular pathways related to aging  increase is amplified by Ca2+ release from the sarcoplasmic reticulum through the ryanodine receptor c levels.tentials . Motoneunx cells , and thereceptor . Activat2+ dynamics in C. elegans pharynx muscle have been previously measured using Ca2+-sensitive fluorescent proteins [2+] oscillations in live worms during aging have never been studied. We were interested in obtaining detailed extended recordings of the pharynx muscle Ca2+ dynamics in worms at different times during the aging process. Our results show that, despite the obvious pharynx muscle decline observed during aging, Ca2+ oscillations do not undergo an age-dependent decline. In fact, similar fast Ca2+ oscillations could be obtained for both, young and physiologically active 2 day old worms as well as older and physiologically much less active 12 day old worms. Here we also report that the pattern of Ca2+ oscillations is strongly influenced by the availability of energy in the pharynx muscle cells, which in turn is determined by the balance between local energy production versus consumption. Our results suggest that imbalance due to excess energy consumption would lead to ATP depletion, which would result in the inhibition of Ca2+ pumps, leading to a sustained high [Ca2+]c. Thus, the new technique we show here to study long-lasting pharynx muscle Ca2+ dynamics may provide a novel methodology to in vivo monitor the energy status of the worm.Caproteins , 12. HowC. elegans pharynx is a neuromuscular organ that undergoes rhythmic contractions induced by oscillatory changes in cytosolic Ca2+ concentration ([Ca2+]c) [2+]c dynamics in the pharynx during pumping have been previously monitored with fluorescent Ca2+-sensitive protein probes targeted to the pharynx [in vivo measurements of the C. elegans pharynx [Ca2+]c dynamics, and studied the changes of the [Ca2+]c patterns at different time points during worm aging.The [Ca2+]c) , 12. The pharynx , 12. How2+]c oscillatory activity for 30 minutes and even longer. Even though the frame recording frequency (1.5Hz) may have cut off some of the high frequency [Ca2+]c changes, we were still able to detect a large variety of [Ca2+]c spikes and patterns of [Ca2+]c oscillation. We were interested in studying the [Ca2+]c dynamics at different times during the life of the worms, thus we performed Ca2+-recording experiments with young (2 day old), mid-age (5 and 8 day old), and old worms (12 day old). Figure 2+]c signaling in live worms. Among the Ca2+ patterns that we observed, we distinguished two major types of [Ca2+]c transients. The large majority of transients were comprised of relatively fast and repetitive [Ca2+]c spikes, with rapid up and down phases. However, a few of the [Ca2+]c transients were much more prolonged and had a “square-wave” shape, with a variable plateau phase of high [Ca2+]c between the up and down phases , which are known to be metabolically more active. In fact, it has been reported that the total C. elegans ATP reaches its maximum level when worms are 2-4 day old and then decreases progressively, so that only about 10% of the original ATP level is left by days 12-14 [2+]c transients was highest at day 2  [Ca2+]c recordings acquired from more than 20 worms at four different ages, made it necessary to implement a custom-made computer analysis. When applied to the [Ca2+] records obtained from each worm, the program was able to quantify the height and the width of each [Ca2+] peak, as well as the spike frequency (see Methods). Following this approach we first analyzed the distribution of the [Ca2+]c peak widths obtained from experiments performed with multiple individual worms of different ages, as we reasoned that the presence of the “square-wave” [Ca2+]c increases would be best detected in that way.The large number of [Ca2+] records of Figure 2+] spikes were involved in high-frequency [Ca2+] oscillations. Accordingly, about 50% of the [Ca2+] peaks at each age corresponded to fast spiking and had a narrow width, between 1s and 2s  . Instead, if the spiking activity continued for more than 30 minutes or it ended before but in the form of narrow spikes, we assumed that the end of activity was due to other reasons and classified it as “Non Energy Depleted” (NED). Figure If the presence of “square-wave” c transients, whereby [Ca2+]c is kept high for minutes at a time, can only be attributed to the blocking of the Ca2+ pumps responsible for clearing [Ca2+]c. We hypothesize that this phenomenon would be produced by the lack of ATP, which would prevent Ca2+ pumping activity.A second unexpected finding involves the formation of “square-wave” [Ca2+]c transients, we studied worms that were subjected to food deprivation. It has been reported that complete food deprivation extends C. elegans lifespan [ad libitum fed worms [2+]c transients was largely increased by this treatment not only in young adults but also during the entire life of the worms. Therefore, these data supports our hypothesis that the lack of energy may be a key determinant for the appearance of “square-wave” [Ca2+]c transients.To investigate whether ATP depletion was the cause of the “square-wave” c transients in wild type ad libitum fed worms was obtained when the nematodes were 2 days of age. This was a counterintuitive result as a priori we would not expect ATP depletion or cell deterioration for these young worms. Instead, the number of “square-wave” [Ca2+]c transients was largely decreased by day 12. It has been shown that the ATP content in wild-type C. elegans worms reaches its maximum when worms are 2-4 days of age, and then decreases progressively until day 12-14 [2+]c transients, it seems contradictory that they appear only in young wild-type adults and then progressively disappear as they get older.Paradoxically, the highest number of “square-wave”  oscillations lead to a fast pumping rate and repetitive contraction, which uses large amounts of ATP. For many instances, this leads to depletion of the cellular energy stores during the 30-min time of the experiment. As worms become older, the Ca2+ oscillatory activity remains constant, but the pharynx pumping rate decreases and so does the amount of energy dedicated to contraction. In fact, Ca2+ oscillatory activity remains nearly intact in 12-day old worms but these display minimal pharynx muscle contraction. We believe that the lack of contraction reduces substantially the amount of energy used by the pharynx muscle cells and this may allow the worm to maintain Ca2+ oscillations.Our interpretation of this paradox is based in the concept of energy balance. In the case of young adults, energy depletion could come from a very high energy use rate, which would overwhelm the energy production rate. In all our Caerotonin , 26. In  induced , 12. In nuo-6 mutant worms were also paradoxical, but could again be explained on the basis of the energy balance hypothesis. These mutant worms have a lower ATP content than wild-type worms [2+]c transients at any age. The pumping rate of these mutant worms has been shown to be much smaller than that of wild-type worms [nuo-6 mutants display a low pumping rate and slow energy-consuming processes in general [nuo-6 worms are less prone to reach energy depletion and thus show fewer “square-wave” [Ca2+]c transients.The data obtained in the pe worms , but shope worms , and thepe worms , 22. We  general , 22, the2+]c. This balance was again altered when nuo-6 worms were food deprived. Under these conditions, the lack of energy production caused by the lack of food rapidly leads to ATP depletion and to a significant increase in the number of “square-wave” [Ca2+]c transients at all ages. The lower ATP levels of nuo-6 mutant worms may also be the reason for the increase in the width of the fast spikes (mean peak width < 10s) of the mutant worms. These fast spikes mainly depend on the rate of Ca2+ pumping out of the cytosol, either out of the cell or into the sarcoplasmic reticulum. Accordingly, the mean peak width (< 10s) was further increased in food-deprived nuo-6 mutant worms.The balance between energy production and consumption becomes therefore essential to avoid energy depletion of pharynx muscle cells and the subsequent deleterious effects as consequence of the persistent increase in [Ca2+ dynamics allows to monitor in vivo the energy status of the worms and provides new information on the metabolic state of the pharynx muscle cells. We show that the balance between energy production and consumption determines the ability of the worm to cope with stress situations such as those induced by persistent feeding stimulation with serotonin. In young worms, their fast rate of energy use for both, muscle contraction as well as Ca2+-pumping, overwhelms their energy balance and leads them to energy depletion. In older worms the balance between energy production and consumption is restored despite their slower energy production and lower total energy available. Thus, pharynx muscle cells in older worms do not undergo full energy depletion.In conclusion, the new technique we show here to study long-lasting pharynx muscle Canuo-6 mutant worms (even in young worms) due to the lower rate of energy use of these mutants. Uncoupling of energy production and consumption through decreased energy utilization, the so-called reduced “rate of living”, has been proposed before [C. elegans mutants. Our data support the “rate of living” hypothesis and suggest that reduced energy consumption in nuo-6 mutant worms may allow them to survive under stress conditions that would otherwise generate energy depletion and persistent intracellular Ca2+ increase, which would in turn lead to mitochondrial Ca2+ overload and irreversible cell damage.The same phenomenon occurs in the energy-deficient mitochondrial respiratory chain d before , 27 as apmyo-2::YC2.1) [nuo-6 strains expressing the Ca2+-sensitive protein were obtained by crossing AQ2038 worms with nuo-6 (qm200) [Strains used were: AQ2038, integrated strain expressing cytosolic cameleon 2.1. (YC2.1) on pharynx due to the promoter sequence of the myo-2 gene (::YC2.1) -30, kind (qm200) , obtaineEscherichia coli (OP50) and all the strains were maintained at 20°C. Calcium imaging experiments were also conducted at 20°C. Experiments were carried out on synchronized worms. Eggs were obtained as described previously [E. coli (OP50) seeded NGM plates. Young adults (day 1) were transferred to E. coli (OP50) seeded NGM plates with 15μM FUDR to avoid progeny.Worms were maintained and handled as previously described . Hardeneeviously  and were2+ measurements were carried out at days 2, 5, 8 and 12 of worm life. Worms were first starved for 4 - 6 hours before the experiments. Then, the nematodes were glued  on an agar pad (2% agar in M9 buffer) and feeding behavior was stimulated by adding 5mg/ml of serotonin [Pharynx Caerotonin , 26. The2+ peaks in each experiment. The frequency was measured at each peak as 9 divided by the distance among the peak 4 positions before and the peak 4 positions after. The mean frequency was calculated as the mean of all the individual frequencies higher than 5 peaks/min obtained in worms of a given age and condition.Fluorescence records were analyzed using the Metafluor program . The traces shown were obtained as the ratio among the image obtained at 535nm emission and that obtained at 480nm emission. Ratio was only considered acceptable when mirror changes at both wavelengths were clearly present . Fluores


Components responsible for this increase are still not uncovered, although current models of plant immune signaling portray extracellular Ca2+ influx as paramount to flg activation of defense pathways. Work presented here provides new insights into cytosolic Ca2+ increase associated with flg-induced defense responses. We show that extracellular Ca2+ contributes more to immune responses evoked by plant elicitor peptide (Pep3) than that evoked by flg, indicating an intracellular Ca2+ source responsible for immune responses evoked by flg. Genetic impairment of the inositol polyphosphate (InsP) and G-protein signal associated with flg perception reduced flg-dependent immune responses. Previous work indicates that prior exposure of Arabidopsis plants to flg leads to an immune response reflected by less vigorous growth of a pathogenic microbe. We found that this immune response to flg was compromised in mutants lacking the ability to generate an InsP or G-protein signal. We conclude that the recruitment of intracellular Ca2+ stores by flg may involve InsP and G-protein signaling. We also found a notable difference in contribution of intracellular stores of Ca2+ to the immune signaling evoked by another PAMP, elf18 peptide, which had a very different response profile to impairment of InsP signaling. Although Ca2+ signaling is at the core of the innate immune as well as hypersensitive response to plant pathogens, it appears that the molecular mechanisms generating the Ca2+ signal in response to different PAMPs are different.Cytosolic Ca This Ca2+ elevation is critical to downstream signaling and initiation of defense responses that limit spread of the pathogen. One of the most well-characterized PAMP–PRR signaling pathways is the interaction of the bacterial motility organ protein flagellin (flg) with the flg receptor FLS2  is entirely or primarily dependent on influx of extracellular Ca2+ into the cytosol through plasma membrane-localized Ca2+-conducting ion channels. This conclusion is primarily based on experiments with Arabidopsis leaves or seedlings exposed to EGTA, which is known to chelate extracellular Ca2+. These studies also used Ca2+ channel blockers and other inhibitors.Detection of invading pathogens by plant cells is typically initiated upon binding of evolutionarily conserved essential components of the microbes, namely pathogen associated molecular patterns (PAMPs), to pattern recognition receptor (PRR) proteins localized on the plasma membrane. Pathogen perception (upon PAMP–PRR interaction) initiates a signal transduction cascade in the cytosol of plant cells that leads to basal immune responses, known as PAMP-triggered immunity (PTI). An early and paramount step of this immune signaling cascade is a transient elevation of cytosolic Cator FLS2 . Recent 2+ into the cytosol. Working with purified Arabidopsis leaf mesophyll protoplasts, we observed a maximal degree of Ca2+-dependent immune signaling initiated by exposure of protoplasts to flg22 in the presence or absence of Ca2+ in the external solution  generation and cytosolic Ca2+ elevations in response to external signals  and ips  null mutants led to increased susceptibility to different types of pathogens. Pseudomonas syringae pv tomato (Pst) DC 3000 led to an elevation in IP3 in Arabidopsis seedlings.Further support for this possibility was obtained using Arabidopsis plants expressing mammalian type I inositol polyphosphate 5-phosphatase (IP5-ptase); this enzyme specifically catabolizes inositol 1,4,5-trisphosphate (IP3) in animals  was first identified by ipk1 (SALK_065337C) mutant were obtained from the Arabidopsis Biological Resource Center and G-protein mutant seeds gpa1-4, agb1-2, xlg2-1, and xlg3-1  were obtained from the Sarah Assmann lab at Pennsylvania State University. ips1 and ips2 mutant seeds were obtained from the Alex M. Murphy lab at the University of Cambridge  phosphorylation with IP5-ptase-aeq plants (−2 s−1 illumination)/night cycle of 12/12 h at 25 °C. For the experiments involving measurement of cytosolic Ca2+, seeds were grown on square plates containing ½ MS medium for 10 d and the seedlings were used for measurement. For the NO experiment, seeds were transplanted to soil about 10 d after germination. Seedlings were transplanted into pots containing LP5 soil-less mix containing starter fertilizer (Sun Gro) at 12 h light (100 μmol m−2 s−1 illumination)/12 h dark (72% relative humidity) and 22 °C. Seeds were stratified at 4 °C in the dark for 3 d before use. During growth, plants were irrigated with Jack’s Professional Peat-Lite 20:10:20 (NP2O5-K2O) solution (at 2 g l−1) one to two times to provide supplementary fertilizer.All Arabidopsis lines used in the reported work are in the Columbia (Col) background. The 2+ elevation, and for monitoring gene expression. Effects of elf18 on ROS production used ~2-week-old seedlings and followed the luminol luminescence methods of The preponderance of prior studies examining flg22 signaling in Arabidopsis have used the PAMP at or near 1 µM to activate defense responses e.g. . Thus, f2+ chelating agents  seedlings were placed in a capless 2 ml centrifuge tube containing 300 μl distilled water. For each tube, 0.6 μl coelenterazine-cp  was added  ethanol). Seedlings were incubated at room temperature in the dark overnight to allow CTZ incorporation into plants. As the CTZ-cp is a light-sensitive reagent, all preparatory steps after adding the CTZ-cp were carried out in dark; tubes were covered with foil paper.The method of 2+ spikes. The luminescence level was measured every second and ligand (flg22) was added only after background luminescence of the leaves was stable; 300 μL flg22  was added to the tubes containing seedling by gently pipetting the solution against the interior wall of the centrifuge tube. For BAPTA and EGTA pre-treatment, 250 μl 40 mM BAPTA or EGTA, with a final concentration of 10 mM, was added to the solution and incubate for 30 min prior to adding flg22. After the incubation, 250 μl 80 nM flg22 or Pep3 was added to a final concentration of 20 nM. Results shown in the figures are mean values calculated from a minimum of at least three biological replicates. After recording luminescence from a treatment replicate, the remaining aeq (i.e. not bound to Ca2+) in an assay tube was discharged by adding 800 μl Ca2+ release buffer (2 M CaCl2.6H2O in 30% ethanol) with continued recording for ~10 min or until the instantaneous luminescence level dropped below 2000. Values obtained from aeq discharge were used to convert luminescence readings to cytosolic Ca2+ concentration for each treatment replicate using an algorithm as described by A single-tube luminometer  was used for measurement of the luminescence level. The centrifuge tubes were placed in the luminometer, and left for 2–3 min to allow seedlings to recover from touch-induced Caipk1 and IP5-ptase transgenic plants were detached from plants and used to make epidermal peels. Epidermal peels were incubated in buffer A  for 1–2 h. Peels were then placed in 3 ml buffer B  which contained 50 µM DAF-2DA to load cells with NO-specific dye. After 30 min incubation to load the NO-specific dye in the tissue, peels were washed three times in buffer A and then incubated in 3 ml buffer B containing ligand flg22 (1 µM) for 5 min. The epidermal peels were placed underneath a coverslip on a microscope slide with several drops of buffer B. NO-dependent fluorescence was monitored over time. Mean maximal fluorescence of guard cells (occurring ~5–10 min after ligand addition) in a peel was ascertained exactly as described previously  was adapted from –2 s–1) at 22 °C. flg22 (1 µM) was added directly to the growth medium and the seedlings were incubated for 30 min. Water was given to the seedlings for the control treatment. For BAPTA and EGTA pre-treatment, WT seedlings were fully submerged and incubated in 10 mM BAPTA or EGTA for 30 min, then flg22 or Pep3 was added to a final concentration of 20 nM, and the seedlings were treated for a further 30 min for gene induction. After incubation, the seedlings were collected and frozen immediately with liquid N2, and stored at −80 °C for future use. Total RNA was extracted using the Plant RNA Extraction Kit .Growth of Arabidopsis seedlings in half-strength MS liquid medium was performed according to WRKY29 (AT4G23550) and WRKY33 (AT2G38470) were examined. UBQ10 (AT4G05320) was used as an endogenous control. The primers used for these analyses were as follows: WRKY29 (forward) 5′-ATCCAACGGATCAAGAGCTG-3′, WKRY29 (reverse) 5′- GCGTCCGACAACAGATTCTC-3′; WRKY33 (forward) 5′-CCCGTTGGCTTCATACCG T-3′, WRKY33 (reverse) 5′-TTACAGCGAGTGTCAATTTGGC-3′; and UBQ10 (forward) 5′-CGGCTACCACATCCAAGGAA-3′, UBQ10 (reverse) 5′-GCTGGAATTACCGCGGCT-3′. We used the qPCR assay for the FRK1 gene (Bio-Rad).After extraction, 500 ng to 2 μg of total RNA was used for reverse transcription using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The synthesized cDNA was diluted 1:10 (v/v) in water, and 1 µl of the diluted cDNA was used for each qPCR reaction. qPCR assays were performed using the Applied Biosystems 7900 HT real-time PCR system. Treatment effects on the expression level of CT) values was used to generate standard errors of the means for control treatments  dye, and denatured by incubation at 95 °C for 5 min. The proteins were loaded onto a 12% SDS-PAGE gel. After electrophoresis, proteins were transferred from the gel to a nitrocellulose membrane. After being blocked with 5% bovine serum albumin , the membrane was incubated with the primary antibody anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204)  BSA, 1× Tris-buffered saline (TBS), 0.1% Tween 20) at 4 °C overnight. After incubation, the membrane was washed with 1× TBS with 0.1% Tween three times. The membrane was then incubated with secondary antibody, anti-rabbit IgG  BSA, 1× TBS, 0.1 % Tween 20) at room temperature for 1 h.Chemiluminescent horseradish peroxidase substrates  were used in the detection of western blots. The chemiluminescent substrate was added to the membrane and incubated for 1 min. The extent of MPK phosphorylation was detected by exposure of photographic X-ray film  to the transfer membranes for 5 min in darkness.ipk1 and gpa1agb1  plants 24 h prior to bacterial inoculation. Pst DC3000 cells were isolated from overnight liquid cultures and resuspended in sterile 10 mM MgCl2 to a concentration of 3 × 108 cfu. The resuspended bacteria were applied (in 0.01% (v/v) Silwet L-77 surfactant) by spraying on leaves previously treated with water or flg22 from 4- to 5-week-old plants. Leaf tissue was collected for measurement of bacterial titer 3 d post-inoculation. For each biological replicate, 0.7-cm-diameter leaf discs were cut from leaves of two different plants and ground together in 100 µl of 10 mM MgCl2 in a 1.5 ml microcentrifuge tube. The samples were thoroughly vortexed in 900 µl of water and diluted 1:10 serially. Then 10 µl of the samples  was spread on plates containing low-salt Luria-Bertani agar medium containing 100 mg l−1 rifampicin. Plates were left at 25°C for 3 d; after this incubation period, colonies on plates were counted.The following method for pre-exposure of plants to flg22 and subsequent inoculation of leaves with virulent pathogen was adapted from 2+ elevation, and protein phosphorylation. We use the well-studied  intact, whole ~2-week-old seedlings, (ii) epidermal peels of mature leaves, and (iii) discs cut from fully expanded, non-senescing leaves. The rationale for the use of these materials is as follows. Most studies of the immune response signal transduction pathway use seedlings  concentration . Exposure of seedlings to this same level of either immune-activating ligand flg22 or Pep3 in the absence of extracellular chelation agents (EGTA or BAPTA) caused similar levels of cytosolic Ca2+ elevation as shown in JXB online; the Ca2+ signal generated by 20 nM Pep3 was at least as great as that occurring in response to a similar concentration of flg22. Chelation of extracellular Ca2+ with either BAPTA  such as MPK3, and receptor-like kinases (RLKs) such as FRK1  on defense gene expression induced by flg22 and Pep3. In a fashion similar to the effects of extracellular Ca2+ on the generation of the Ca2+ signal, the signal transduction cascade downstream from flg22 and Pep3 leading to activation of defense gene expression was also affected differently. The Pep3-dependent expression of MPK3 and WRKY33 was inhibited to a much greater extent by application of EGTA or BAPTA  has been identified in plant cells. In order to study IP3 signaling in plants, we used transgenic plants expressing the animal IP5-ptase; as mentioned above, the IP5-ptase in these plants breaks down IP3 generated during signaling cascades in plant cells. Results shown in 2+ signal generated by flg22. The results in 2+ stores may contribute to the early and ‘vital’ Ca2+ signal generated upon flg22 perception during immune defense responses.IP3 is known to be involved in intracellular CaWRKY29 and WRKY33 to interrogate the involvement of InsP signaling with basal immunity signaling pathways. We found that InsP signaling was involved in the up-regulation of these transcription factors in response to flg22 perception. The increased expression of WRKY29 and WRKY33 that occurred when flg22 was applied to WT plants was impaired in IP5-ptase plants  has been known to be involved in the plant immune response for some time, both as an antimicrobial . Some wo2+ elevation  InsP in this signaling pathway. As mentioned above, we found that impairment of InsP signaling (in IP5-ptase-expressing plants) resulted in a reduction in flg22-dependent Calevation . In the 2+ currents from the vacuole lumen. Consistent with these patch clamp studies of Ca2+ currents across the tonoplast, release of caged IP3 . IP5-ptase hydrolyses IP3, and therefore prevents the activation of plant cell processes that respond to this cytosolic signal  points t of Ca2+ . The lacipk1 mutant as genetic tools to probe InsP signaling related to PAMP perception. As shown in ipk1 null mutants as compared with the response of wild type (WT) plants to flg22. The extent of impairment of NO generation (as compared with corresponding control plants) was about the same (approximately 50% reduction from levels found in WT plants) in the IP5-ptase and ipk1 genotypes  were similar to that shown in ipk1 null mutation).Additional results demonstrating involvement of PI-PLC in the signaling pathway linking PAMP perception with immune responses in plant cells are shown in tivation . We usedgpa1, agb1, xlg2, and xlg3 in some of our experiments. As shown in WRKY33 was significantly reduced in agb1 and xlg2 seedlings compared with WT, while gpa1 and xlg3 contained similar amount of WRKY33 transcripts as WT. Similarly, MPK3 expression was significantly decreased in agb1 seedlings compared with WT. The findings indicate that it is not GPA1 but AGB1 and XLG2 that are responsible for G-protein effects on flg22-induced defense gene expression. Recent reports have shown that GPA1 does not play a role in flg22-induced defense signaling  production were all impaired . Thus, ‘immunization’ of plant leaves by exposure to flg22 could be observed in the experimental results presented in  mutants . The res2+ reduces the Ca2+ signal and Ca2+-dependent immune signaling occurring in response to Pep3 to a greater extent than effects on flg22 signaling. These results suggest that intracellular Ca2+ stores contribute significantly to flg immune signaling. InsP signaling was linked to flg22-dependent generation of a cytosolic Ca2+ signal as well as immune defense responses downstream from the Ca2+ signal. The cytosolic InsP signaling cascades contributed to flg22-dependent NO generation, MPK activation, and increased expression of pathogen defense genes.The experimental results in this report provide new information about PAMP-triggered immunity, specifically with regard to the signaling cascade evoked upon perception of the PAMP flg. Chelation of apoplastic Ca2+ signal and an impairment of all of these downstream defense responses is well established in an extensive body of published literature  seedlings.Fig. S1. flg22- and Pep3-dependent cytosolic Ca2+ elevation is transitory.Fig. S2. The flg22-dependent cytosolic CaSupplementary Figures S1-S2Click here for additional data file.


BDNF, one of the neurotrophins, has various important roles in cell survival, neurite outgrowth, neuronal differentiation, synaptic plasticity and the maintenance of neural circuits in the CNS. Intracellular Ca2+ signaling is important for microglial functions including ramification, de-ramification, migration, phagocytosis and release of cytokines, NO and BDNF. BDNF induces a sustained intracellular Ca2+ elevation through the upregulation of the surface expression of canonical transient receptor potential 3 (TRPC3) channels in rodent microglia. BDNF might have an anti-inflammatory effect through the inhibition of microglial activation and TRPC3 could play important roles in not only inflammatory processes but also formation of synapse through the modulation of microglial phagocytic activity in the brain. This review article summarizes recent findings on emerging dual, inflammatory and non-inflammatory, roles of microglia in the brain and reinforces the importance of intracellular Ca2+ signaling for microglial functions in both normal neurodevelopment and their potential contributing to neurodevelopmental disorders such as ASDs.Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by deficits in social interaction, difficulties with language and repetitive/restricted behaviors. Microglia are resident innate immune cells which release many factors including proinflammatory cytokines, nitric oxide (NO) and brain-derived neurotrophic factor (BDNF) when they are activated in response to immunological stimuli. Recent  Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by deficits in social interaction, difficulties with language, and repetitive/restricted behaviors  and higher levels of macrophage inhibitory factor, leptin, interleukin 1β (IL-1β), IL-6, interferon-γ (IFN-γ) and IL-12 in various age groups of patients with ASDs  is cytotoxic, [Ca2+]i is tightly regulated by intrinsic gating processes mediated by voltage-gated calcium channels (VGCCs) and NMDA receptors  and Ca2+-dependent inactivation (CDI). Limpitikul et al.  led to both haplo-insufficiency and reduced BKCa activity  depends on a number of different types of voltage- or ligand-gated ion channels that are permeable to inorganic ions such as sodium, potassium, chloride and calcium. While the former three ions predominantly support the electrogenic roles, Cal et al.  recently2+]i is also important for the activation of microglia, including proliferation, migration, ramification, de-ramification and release of NO, proinflammatory cytokines and BDNF -activated microglia resulted in necrotic cell death  and mitochondria. The main route for the generation of intracellular Ca2+ signaling is associated with inositol 1,4,5-trisphosphate (InsP3) receptors on the ER membrane. Stimulation of G protein-coupled metabotropic or tyrosine kinase receptors results in the activation of the phospholipase C (PLC), production of two second messengers including the diacylglycerol (DAG) and the InsP3 and the release of Ca2+ from the ER. Importantly, the depletion of ER activates the store-operated Ca2+ entry (SOCE), known as a capacitative Ca2+ influx, mediated by plasmalemmal channels such as calcium release-activated Ca2+ (CRAC) channels and/or transient receptor potential (TRP) channels plasmic reticulum Ca2+-ATPases (SERCA). The concentration of Ca2+ in the ER is precisely controlled by SERCA. Recently, Schmunk et al.  identified Trpc3 as a target of MeCP2 transcriptional regulation. BDNF mRNA and protein levels are also lower in Mecp2 mutant hippocampus and dentate gyrus granule cells, which is reflected in impaired activity-dependent release of endogenous BDNF. These results identify the gene encoding TRPC3 channels as a MeCP2 target and suggest a potential therapeutic strategy to boost impaired BDNF signaling in RTT  receptor and induces the activation of intracellular signaling pathways, including PLC-γ, phosphatidylinositol 3-kinase (PI3K) and mitogen activated protein kinase-1/2 , resulting in activation of the PLC pathway and SOCE in rodent microglial cells. Sustained activation of SOCE occurred after a brief BDNF application and contributed to the maintenance of sustained [Ca2+]i elevation. Pretreatment with BDNF significantly suppressed the release of NO from activated microglia. Additionally, pretreatment of BDNF suppressed the IFN-γ-induced increase in [Ca2+]i, along with a rise in basal levels of [Ca2+]i in rodent microglial cells , neural cells derived from induced pluripotent stem cell (iPSCs) and mouse models, Griesi-Oliveira et al.  recently2+ is important for the activation of microglial cell functions, including proliferation, release of NO, cytokines and BDNF. It has been shown that alteration of intracellular Ca2+ signaling underlies the pathophysiology of neurodevelopmental disorders including ASDs. BDNF induces a sustained intracellular Ca2+ elevation through the upregulation of the surface expression of TRPC3 channels in rodent microglial cells. Microglial cells are able to respond to BDNF, which may be important for the regulation of inflammatory responses, and may also be involved in the normal development of CNS. BDNF is first synthesized as proBDNF protein. ProBDNF is then either proteolytically cleaved intracellularly or by extracellular proteases, such as metalloproteinases and plasmin, to mature BDNF. Interestingly, interaction of mature neurotrophins with Trk receptors leads to cell survival, whereas binding of proBDNF to p75NTR leads to apoptosis. In addition, mature BDNF and proBDNF facilitates long-term potentiation (LTP) and long-term depression (LTD) at the hippocampal CA1 synapses, respectively. Thus, Trk and p75NTR preferentially bind mature- and pro-neurotrophins, respectively, to elicit opposing biological responses in the CNS  is a pituitary neuropeptide hormone synthesized from the paraventricular and supra-optic nuclei within the hypothalamus. Like other neuropeptides, OT can modulate a wide range of neurotransmitter and neuromodulator activities. OT is secreted into the systemic circulation to act as a hormone, thereby influencing several body functions. OT plays a pivotal role in parturition, milk let-down and maternal behavior and has been demonstrated to be important in the formation of pair bonding between mother and infants as well as in mating pairs. Furthermore, OT has been proven to play a key role in the regulation of several behaviors associated with neuropsychiatric disorders, including social interactions, social memory response to social stimuli, decision-making in the context of social interactions, feeding behavior, emotional reactivity, etc. An increasing body of evidence suggests that dysregulation of the oxytocinergic system might be involved in the pathophysiology of neurodevelopmental disorders such as ASDs (Romano et al., 2+ signaling for microglial functions in both normal neurodevelopment and their potential contributing to neurodevelopmental disorders such as ASDs.There is increasing evidence suggesting that pathophysiology of neurodevelopmental disorders is related to the inflammatory responses mediated by microglial cells. In addition, recent advances in the understanding of microglial functions suggest an important role for these cells in the normal development of CNS in addition to their traditional role as immune cells of the brain. Dysregulation of normal microglial functions such as regulation of programmed cell death and/or synaptic pruning is supposed to be increasingly implicated in ASDs associated with cognitive deficits. These findings have resulted in a new model of the synapse as “tripartite,” recognizing the important role of not just neurons and astrocytes but also microglia in the normal physiological function of the brain. We now need to explore the emerging dual, inflammatory and non-inflammatory, roles of microglia in the brain and recent findings reinforce the importance of intracellular CaYM and AM wrote this article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ca2+ events were recorded for a period of 60 seconds (s) in control and after drug treatment. A heterogeneous network of cells was identified at the interface of the urothelium and lamina propria of postnatal rat pups, aged ≤ postnatal (P) day 21, with diverse morphology  and expressing platelet-derived growth factor receptor alpha (PDGFRα)- and TRPV4-immunoreactivity (IR). Ca2+ transients occurred at a slow frequency with an average interval of 30 ± 8.6 s. Waveform analyses of Ca2+ transients in cells in the lamina propria network revealed long duration Ca2+ events with slow upstrokes. We observed slow propagating waves of activity in the lamina propria network that displayed varying degrees of coupling. Application of the TRPV4 agonist, GSK1016790 (100 nM), increased the duration of Ca2+ events, the number of cells with Ca2+ events and the integrated Ca2+ activity corresponding to propagation of activity among cells in the lamina propria network. However, GSK2193874 (1 μM), a potent antagonist of TRPV4 channels, was without effect. ATP (1 μM) perfusion increased the number of cells in the lamina propria exhibiting Ca2+ events and produced tightly coupled network activity. These findings indicate that ATP and TRPV4 can activate cells in the laminar propria network, leading to the appearance of organized propagating wavefronts.The lamina propria contains a dense network of cells, including interstitial cells (ICs), that may play a role in bladder function by modulating communication between urothelium, nerve fibers and smooth muscle or acting as pacemakers. Transient receptor potential vanilloid 4 (TRPV4) channels allow cation influx and may be involved in sensing stretch or chemical irritation in urinary bladder. Urothelium was removed from rats (P0-Adult), cut into strips, and loaded with a Ca The micturition reflex undergoes marked changes during prenatal and postnatal development but the mechanisms underlying these changes are largely unknown. As the CNS matures during the postnatal period, reflex voiding is brought under voluntary control involving higher brain centers  has been extensively studied and found to be key to generating slow waves and coordinating motility as well as regulating neurotransmission  4 (TRPV4) channel is expressed in different cells of the urinary bladder and allows Na+ and Ca2+ influx into the cell. Measurements of ionic currents and Ca2+ events induced by agonists  or stretch have demonstrated functional expression of TRPV4 in urothelial cells  channels have been identified in the urinary bladder  of both sexes and various postnatal (P) ages (P0-Adult) were used in these studies. The University of Vermont Institutional Animal Care and Use Committee approved all experimental protocols involving animal use. Animal care was under the supervision of the University of Vermont's Office of Animal Care Management in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and National Institutes of Health guidelines. All efforts were made to minimize the potential for animal pain, stress or distress. Tissues were harvested after euthanasia by decapitation or isoflurane overdose followed by thoracotomy.n = 13) and rat pups  of both sexes and placed in cold HEPES solution consisting of (mM): 134 NaCl, 6 KCl, 10 glucose, 10 HEPES, 1 MgCl2, 2 CaCl2, 10 glucose and adjusted to pH 7.4 with NaOH. The urothelium was removed from the detrusor and carefully cleaned of lamina propria with sharpened forceps until all visible lamina propria was removed. The cleaned urothelial sheet was placed in buffered 4% paraformaldehyde (pH 7.4) for immunohistochemistry or placed in a special chamber (PSS) for Ca2+ imaging studies.Urinary bladders were removed from adult rats ; blocking peptide for PDGFRα, MyBioSource, Inc., ]. Use of the TRPV4 or PDGFRα blocking peptide eliminated immunostaining (data not shown) in tissue sheets. Specificity of TRPV4 expression was also confirmed in TRPV4 null mice  (data not shown)  containing 20%, 0.2% Triton X-100 for 2 hr at room temperature and the primary antibody was applied in PBS containing 4% normal goat serum, 0.2% Triton X-100 overnight at 4°C. The following primary antibodies were used: rabbit anti-TRPV4   with a Ca2+ sensitive fluorescent dye, either Fluo-2 AM leak resistant  (10 μM) or Cal 520  + pluronic acid (2.5 mg/ml) in HEPES solution. All experiments were conducted in physiological saline solution (PSS) (35–37°C) consisting of (mM): 119 NaCl, 4.7 KCl, 24 NaHCO3, 1.2 KH2PO4, 2.5 CaCl2, 1.2 MgSO4, 7 glucose and constantly bubbled with Biological Gas (5% CO2) to maintain pH at 7.4. The urothelium was visualized with a 60X water immersion (NA 1.2) fluorescent objective. Images were collected with a Noran Oz laser scanning confocal microscope or with a Yokogawa CSU-W1 spinning disk confocal microscope housed in the Imaging/Physiology Core (Larner College of Medicine at The University of Vermont), at a rate of 16–30 images/s. Fluo-2 or Cal 520 were excited at 488 nm, and the emitted fluorescence collected at >500 nm. Imaging fields were 133 × 133 μm (512 × 512 pixel). Ca2+ events were initially visualized offline using software developed in our laboratory by Dr. Adrian Bonev. For some experiments Tetramethylrhodamine  was incubated with fluo-2 AM leak resistant Ca2+ sensitive dye for 90 min (35–37°C). TRITC was taken up by urothelial cells and provided contrast to Fluo-2 loaded cells. The tissue was visualized using a Yokogawa CSU-W1 spinning disk confocal microscope housed in the Imaging/Physiology Core. TRITC was excited at 561 nm and emitted fluorescence was collected using a 525/50 filter.To detect Ca2+ events in lamina propria cells was made using custom-written software (Volumetry G8e/G9: Grant Hennig). To localize cells that had Ca2+ activity, movies were differentiated (Δt = ±1 s), then frame averaging (±0.15 s) and Gaussian smoothing (3 × 3: sd = 1.0) were applied. The processed movies were thresholded at the level at which noise particles (granular frame by frame fluctuations in intensity) occupied 1% of the total spatio-temporal volume  in total area were filtered out and calcium transient particle (PTCL) files were created. To filter spurious noise particles, the spatial overlap between particles on successive frames was tabulated, then an existence filter was applied that preserved particles that had a continuous spatial overlap for more than 0.25 s. Ca2+ transient particles that met or exceeded the above conditions were considered to be resolved Ca2+ transients. Initiation sites of Ca2+ transients were flagged by choosing the first appearance of a resolved Ca2+ transient.Detailed analysis of Ca2+ transient particles were summed throughout the movie to create a prevalence map of activity (as % of recording time a resolvable Ca2+ event was present). Ca2+ transient prevalence maps were thresholded (>0.5% activity during recording) and bitMasks created from which intensity traces were calculated in the original recording.The refined Ca2+ transients were calculated from intensity traces calculated within cell bitMasks. To measure the overall effect of drugs on cells in the lamina propria network, we integrated the area of resolvable Ca2+ transient PTCLs over the course of the recording period .The interval, duration, amplitude, and rise/decay times of lamina propria cells Ca2+ transients in lamina propria cells ranged from apparently random to well demarcated wavefronts. To quantify the degree of coordination of Ca2+ activity in lamina propria cells, the temporal order of every resolvable Ca2+ transient in cells within the FOV was determined, then for each sequential pair of events, the (i) time delay, (ii) distance of separation and (iii) angle of between sites was calculated. Any bias in the number of “next-to-fire” angles in a particular direction demonstrate a non-random pattern of activation and was used to gauge relationships between Ca2+ transients within cells in the lamina propria syncytium. Random patterns of activation have a little bias , where as a greater degree of bias  are indicative of a well-formed wave front.The pattern of CaImages were imported into Photoshop 7.0  or Powerpoint  where groups of mages were assembled and labeled.n rat pups. Statistical comparisons between groups were made using one-way analysis of variance (ANOVA). When F ratios exceeded the critical value (p ≤ 0.05), the Dunnett's post-hoc test was used to compare groups.Values are expressed as mean ± S.E.M. from N-((1S)-1-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide] and GSK2193874 [3--7-bromo-N-(1-phenylcyclopropyl)-2-[3-(trifluoromethyl)phenyl]-4-quinolinecarboxamide] were purchased from Sigma , dissolved in PSS, and superfused over the urothelial preparation.ATP, Cyclopiazonic acid (CPA), GSK1016790 [In the lamina propria, just below the junction with the urothelium lies a dense network of capillaries, small blood vessels and cells. When incubated with calcium reporter dyes, capillaries, blood vessels and cells readily loaded with the fluorescent dye Figures . Cells i2+transients could be observed in many cells from lamina propria networks in urothelium sheets from postnatal rats  Ca Figures . Ca2+ trs Figure , Video. s Figure  with slos Figure . Ca2+ trts Table .2+ transients , indicating lamina propria cell activity was not occurring randomly and had a defined direction of propagation regardless of whether the wavefront was loosely or tightly coupled. Spatio-temporal maps demonstrated varying degrees of coupling including tight, partial or loose organization of the lamina propria network are presented in Figures 2+ waves illustrate the ability of the lamina propria network to act as a functional syncytium.During continuous recordings from the lamina propria network, we observed slow propagating waves of activity in which numerous cells in the lamina propria displayed robust, prolonged Ca Figures . Spatio- Figures . In prep2+ events and the number of cells (p ≤ 0.05) with Ca2+ events in cells in the lamina propria  ATPase (SERCA) inhibitor cyclopiazonic acid . CPA significantly (p ≤ 0.01) reduced the overall duration of cell activation and the number of cells exhibiting Ca2+ events in the lamina propria network suggesting that Ca2+ transients are dependent on release of Ca2+ from internal stores  increased the number of cells in the lamina propria exhibiting Ca Figures  that eve Figures . This fi2+ transients that can be loosely or tightly coupled (wavefronts) between cells. In addition, these studies demonstrate several novel findings, including: (i) the predominance PDGFRα- and TRPV4-immunoreactivity in the lamina propria layer from early postnatal rat pups (P ≤ 21), (ii) ATP and a TRPV4 agonist activated and increased the number of lamina propria cells that exhibited active Ca2+ events, (iii) lamina propria cell activity was not random, with spatio-temporal maps and PTCL analysis demonstrating varying degrees of coupling  and (iv) the coupling of Ca2+ activity of cells in laminar propria network could be modified to generate organized, propagating bands of activity (wave fronts) with ATP. These findings are consistent with the hypothesis that lamina propria Ca2+ signaling facilitates communication through this syncytial network to other cell types or tissue layers of the urinary bladder; our study shows the spatio-temporal patterning of this potential communication that may affect sensory processing and/or detrusor contractility.The current studies confirm the presence of a heterogeneous cellular network in the lamina propria that exhibits spontaneous CaThe spinobulbospinal micturition reflex is triggered by tension receptor afferents in the bladder and begins to elicit voiding in the rat between P16-18  for each photodiode. With this system, mechanical or carbachol induced Ca2+ transients were found to originate near the urothelial-suburothelial interface with spread to the detrusor  increases lamina propria Ca2+ signaling and facilitates communication through this syncytial network to other cell types or tissue layers , GSK1016790A) or stretch  the ability of ATP and TRPV4 agonist to increase the rate of integrated Ca2+ activity corresponding to coupled lamina propria network events and the formation of propagating wavefronts. Thus, the lamina propria network may have an active role in sensing  and signaling, perhaps reciprocally, between bladder layers and cell types to achieve coordinated bladder function. The importance of the lamina propria network may depend upon the integrity and maturity of neural pathways that coordinate micturition reflex events.At the urothelial-lamina propria junction, the network of cells expressing PDGFRα- or TRPV4-IR was sparse and calcium events were not observed. In wholemount preparations from rat pups aged ≤ P21, we consistently identified a dense network of PDGFRα- or TRPV-IR cells in the lamina propria. The reasons underlying these observations are unknown but may reflect the continuing maturation of the micturition reflexes during the early postnatal period. It has previously been speculated that the functional syncytium between ICs and the detrusor smooth muscle is important for coordination of bladder emptying in the early postnatal period because of the absence of mature and functional neural input needed to coordinate the activities of the CNS and PNS including the urinary bladder  to increase the rate of integrated Ca2+ activity corresponding to coupled lamina propria network events could contribute to altered sensory processing  and altered urinary bladder function . There is precedent for plasticity in the expression of lamina propria cellular network. ICs in the bladder wall following SCI (5 weeks post injury) are decreased, target organ innervation is reduced and the smooth muscle is hypertrophied  and National Institutes of Health guidelines. All efforts were made to minimize the potential for animal pain, stress or distress.Analyzed the data, performed experiments, conceived, discussed and outlined the experimental design: TH, GH, and MV. Wrote the paper, drafted and revised paper: TH, GH, MN, and MV.The authors declare that the research described from the Vizzard laboratory were conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funding entity, NIH, had no role in the studies described including: design, data collection and analysis of studies performed in the Vizzard laboratory, decision to publish or preparation of the review. The contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH.


FLT3 internal tandem duplication (FLT3 ITD) is typically associated with an aggressive clinical course with rapid progression, relapse, and short overall survival in the absence of transplantation. We report here the first case of SR of FLT3 ITD mutant AML in the literature. Our patient was an elderly woman with relapsed NPM1 and FLT3 ITD mutant AML whose disease underwent SR for a brief duration without precipitating cause. We review the potential immune mechanisms underlying SR in AML and discuss the implications for novel immunotherapeutic approaches for FLT3 mutant AML.Spontaneous remission (SR) of acute myeloid leukemia (AML) is a very rare phenomenon. AML characterized by FLT3 ITD mutant AML whose disease underwent spontaneous complete remission without interim therapy.Spontaneous remission (SR) of acute myeloid leukemia (AML) is a very rare clinical phenomenon that was first described in 1878 by Eisenlohr , 3. AbouFLT3) gene, known as FLT3 mutant AML [ FLT3, leading to constitutive activation of FLT3 receptors and triggering of multiple downstream signaling pathways , which promote tumor growth [ FLT3 ITD mutation in AML connotes worse outcomes [ FLT3 ITD mutant AML experience significantly shortened remission duration, lower salvage rate in first remission, and decreased overall survival [ FLT3 ITD mutant AML patients remains allogeneic stem cell transplantation [AML is a biologically heterogeneous myeloid malignancy with diverse disease subsets defined by distinct cytogenetic and molecular features. Nearly 30% of AML cases are characterized by molecular abnormalities in the FMS-like tyrosine kinase-3  gene were detected (data not shown). Findings were consistent with NPM1-mutated AML of FAB M5 subtype.The patient was a 73-year-old woman with a prior medical history of hypertension who presented with three weeks of dyspnea, nausea, loose stools, and fatigue. Examination was unremarkable. However, complete blood count (CBC) revealed leukocytes of 240,000/9,000/μL . Periphe2 once daily × 7 days) and daunorubicin (45 mg/m2 once daily × 3 days) was initiated. The patient achieved a complete remission (CR) based on count recovery and repeat bone marrow aspirate showing no blasts or promonocytes. At the time of morphologic CR, no FLT3 mutation was identified by quantitative PCR in the marrow; however, exon 12 mutation of NPM1 was identified in a very small fraction of the cells . Allogeneic stem cell transplantation was offered given the high risk for disease progression, but patient declined. She subsequently received two cycles of high-dose cytarabine (1.5 g/m2 once daily × 6 days), which she tolerated well with complete count recovery.Induction chemotherapy with standard-dose cytarabine  using the AmpFLSTR® Profiler Plus® PCR Amplification Kit from Life Technologies. All of the samples had identical results at all nine loci, thus confirming that the specimens were from the same individual. Unfortunately the patient was not deemed a candidate for clinical trial of experimental FLT3 inhibitor due to the lack of overt morphological disease, which was an eligibility criterion. She elected to resume observant therapy.The patient was referred to a clinical trial of an experimental FLT3 inhibitor. She underwent screening bone marrow biopsy and aspirate  on the crocedure  was perfμL and 16% peripheral blasts. Marrow biopsy revealed 84% myeloblasts with del (15q) in all 20 analyzed cells and new del (16q) in 1 of 20 cells. Molecular analyses confirmed the same NPM1 and FLT3 ITD mutations with a high allelic ratio of 1.99. The patient decided to forego further treatment and died a few weeks later.Thirty-five days from first documented SR (BM-2), the patient presented with overt disease recurrence with leukocytes of 29,900/ FLT3 ITD mutant AML undergoing SR. Given the overall prevalence of FLT3 ITD in AML patients, it is more than likely that some of the prior published cases of SR in AML patients contained other cases of FLT3 ITD mutant disease which were not discerned due to the lack of available testing. Additionally, the immediate use of chemotherapy after initial diagnosis and at relapse and the innately worse outcomes rendered by mutant FLT3 in AML would also have likely contributed to the lack of any prior report of SR. Despite the spontaneous morphologic CR in our patient, it is clear that she had cytogenetic and molecular evidence of AML disease throughout her entire disease course. It is possible that many of the prior reported cases of SR in the literature would not survive the test of today's diagnostic standards. As an example, 21 of the 46 patients reported by Rashidi and Fisher as cases of SR did not even have baseline cytogenetic analysis [ NPM1 as a tool for MRD assessment in AML [ NPM1-mutated transcripts after two cycles of chemotherapy in standard-risk AML was shown to be a powerful independent prognostic factor for disease relapse and overall survival in one study [ NPM1-mutated transcripts had strong prognostic significance and predicted benefit from allogeneic stem cell transplant [ FLT3 ITD, only age, white blood cell count, and 4-log reduction in peripheral blood MRD, but not FLT3 ITD allelic ratio, were significantly associated with a higher cumulative incidence of relapse. Indeed, MRD may eventually be incorporated into routine practice as an important determinant of therapy response, modification, and need for transplant, similar to our current usage of AML cytogenetics and mutation information [Spontaneous remission of AML is a rare phenomenon documented in the medical literature. To our knowledge, we present here the first reported case oft in AML –25. The ne study . In anotansplant . In the ormation , 26. FLT3 ITD allelic ratio and then a worsening of these findings corresponding with frank relapse are highly indicative of true SR in this patient by current criteria. SR in AML patients are hypothesized to result from activation of innate host immune responses exerting direct antileukemic effects. Two factors resulting in immune activation, specifically infections and blood transfusion, have been repeatedly associated with the majority of SR cases. Unequivocal infections have been documented in 32 out of 45 (71.1%) SR patients, while 32 out of 39 (82.1%) SR patients had antecedent blood transfusion [ Pneumocystis jirovecii, and infectious mononucleosis have been described [α  and interferon-γ (IFN-gamma) has been shown to directly inhibit myeloblast proliferation [Our patient's disease recurrence likely occurred from the AML founding clone or one of its subclones . Howeverescribed –33. Immuferation , 35. Incferation –36. Hypeferation , 38. In Allogeneic blood product transfusion has also been linked to SR in AML. Because nonirradiated blood products contain potentially functional antileukemic lymphocytes, transfusion of such products can potentially induce antileukemic effects in a manner similar to graft-versus-leukemia , 40, 41. NPM1 mutant AML similar to our patient may also be predisposed towards SR [ NPM1 mutation, found in approximately 35% of AML cases, is known to confer favorable prognosis [ FLT3 ITD, the presence of NPM1 mutation may partly mitigate the poor prognostic effects of FLT3 ITD, particularly in patients with low FLT3 ITD mutant disease levels, for unknown reasons [Cases of SR unrelated to transfusions or infections, as in our case, have also been noted previously , 43. Potwards SR . The prerognosis , 46. Whe reasons , 47–50. In conclusion, SR in AML is a rare but real and well-documented clinical phenomenon resulting in clinical complete remissions lasting for an average of 5–7 months and ranging from as short as 2 weeks to in excess of 100 months , 51–54.