Once such a precise regulation is unbalanced, however, neuronal functions and viability are severely compromised. Accordingly, disturbed Ca2+ metabolism has often been claimed as a major contributor to different neurodegenerative disorders, such as amyotrophic lateral sclerosis that is characterised by selective motor neuron (MN) damage. This notion highlights the need for probes for the specific and precise analysis of local Ca2+ dynamics in MNs. Here, we generated and functionally validated adeno-associated viral vectors for the expression of gene-encoded fluorescent Ca2+ indicators targeted to different cell domains, under the transcriptional control of a MN-specific promoter. We demonstrated that the probes are specifically expressed, and allow reliable local Ca2+ measurements, in MNs from murine primary spinal cord cultures, and can also be expressed in spinal cord MNs in vivo, upon systemic administration to newborn mice. Preliminary analyses using these novel vectors have shown larger cytosolic Ca2+ responses following stimulation of AMPA receptors in the cytosol of primary cultured MNs from a murine genetic model of ALS compared to the healthy counterpart.A finely tuned Ca Therefore, a precise control of Ca2+ homeostasis in the cytosol and other cell compartments/organelles – performed by several systems like pumps, channels, transporters and Ca2+-buffering proteins – is essential for cell physiology. In neurons, local Ca2+ fluctuations have a key physiologic impact, by controlling the spatiotemporal pattern of electrochemical signals, neurite outgrowth, synaptic plasticity and the patterning of dendritic spines, which is of key importance in the neurochemical/neuroanatomical establishment of learning and memory, and cell survival. Not surprisingly, small changes in Ca2+ dynamics can lead to pathological conditions1, and derangement of Ca2+ homeostasis has been frequently indicated as a major contributor to the onset of various neurodegenerative disorders, such as Alzheimer’s, Parkinson’s, and Huntington’s disease2.Ca2+ overloads because they possess low levels of Ca2+-buffering proteins4, and express high levels of Ca2+-permeable α-amino-5-methyl-3- hydroxisoxazolone-4-propionate (AMPA) receptors that lack the GluR2 subunit. Such an architecture makes AMPA receptors more vulnerable to dysregulation of intracellular Ca2+ homeostasis and excitotoxicity5. Not surprisingly, perturbed Ca2+ homeostasis has been claimed as a major actor in the pathogenesis of different MN disorders, such as amyotrophic lateral sclerosis (ALS)7 and spinal muscular atrophy8. The above notions highlight the need for molecular tools, such as suitable Ca2+ indicators, for the specific and precise analysis of Ca2+ movements in different sub-cellular compartments of MNs.Among neurons, motor neurons (MNs) are particularly sensitive to noxious cell Ca2+ indicators: chemical probes and gene-encoded calcium indicators (GECI). The first ones are hybridizations of small Ca2+-chelating molecules and a fluorophore. This class of probes include fura-2, quin-2, indo-1 and fluo-3, which were the first indicators used for Ca2+ measurements in cells9. A second class of probes is that of GECI, which includes different types of engineered proteins such as green fluorescent protein (GFP)-derived fluorescent probes, bioluminescent probes and fluorescence (or Förster) resonance energy transfer (FRET)-based indicators. A clear advantage of these probes is that – at difference from chemical indicators – they can be genetically targeted to different cell domains/compartments for local Ca2+ measurements. On the other hand, they suffer from the difficulty to be efficiently expressed in cells, in particular in primary neurons. Such a drawback, however, can be now easily overcome by use of viral expression vectors12.There are two major classes of Ca2+-sensing domain, consisting of calmodulin (CaM) and a CaM-binding peptide derived from the myosin light chain kinase M13. Upon Ca2+ binding to this domain, the FRET donor and acceptor are brought in close spatial proximity, thereby allowing FRET to occur. To date, the most used GFP-modified proteins in cameleons are the yellow fluorescent protein  and the cyan fluorescent protein . In last years, several studies based on site-directed mutagenesis have led to the generation of improved cameleon Ca2+ probes, by increasing the brightness of the FRET acceptor in response to Ca2+ fluctuations, reducing the possible competitive effect of endogenous CaM, and extending the affinity of cameleons for Ca2+ to render the probes suitable for Ca2+ measurements in different cell districts. In particular, different kinds of cameleon exist  covering a wide Kd range for Ca2+ that allow scientists to choose the best probe for the specific cell compartment under study15.Cameleon probes are based on FRET, a well-known quantum mechanical process consisting in the energy transfer from an exited fluorescent donor to an acceptor fluorophore placed in close proximity. Therefore, when FRET occurs, an increased fluorescence of the acceptor, together with a decreased fluorescence of the donor can be observed and quantified. In cameleons, the FRET donor and acceptor are GFP-derived fluorescent proteins separated by a Ca2+ measurements in the target cell compartment. Pilot experiments also demonstrate altered Ca2+ homeostasis in the cytosol of primary MNs from a genetic model of ALS, and that the generated AAV expression vectors can be suited for the in vivo expression of cameleons in spinal cord MNs in mice.In this work, we have generated and functionally validated adeno-associated viral (AAV)-based vectors for the expression of cameleons targeted to different cell domains  under the transcriptional control of a MN-specific promoter. We demonstrate that such probes are specifically expressed in MNs (and not in other cell types) in primary cell cultures from the mouse spinal cord, and that they allow reliable Ca2+ homeostasis in the pathogenesis of MN diseases8, in this study we sought to generate and validate expression systems for GECI that were suited for the assessment of local Ca2+ fluctuations in MNs. To this purpose, we have engineered AAV plasmids (pAAV) for the expression of cameleon probes targeted to the cytosol (pAAV-[Hb9_AB]-D1cpv), the mitochondrial matrix (pAAV-[Hb9_AB]-4mtD3cpv) and the ER lumen (pAAV-[Hb9_AB]-D4ER), under the control of a MN-specific, homeobox Hb9-derived, promoter . As described in Fig. 2+ probes is specific for MNs . In addition, the transduction efficiency of MNs was rather high, given that in all cases more than 70% of SMI32-positive cells were also positive for the expression of the cameleon probes  SOD1 and the healthy counterpart (hSOD1(WT)-expressing mice)].ALS is a fatal neurodegenerative disorder that occur on sporadic or genetic grounds, leading to a selective and progressive loss of MNs in the spinal cord, brainstem and cerebral cortex, thereby resulting in severe motor deficits and – eventually – to death2+ concentrations, which, however, could be determined by suitable calibration procedures13. Instead, we simply reported the ratio between the acceptor and the donor fluorescence of the FRET-based probes, which is, nevertheless, sufficient for comparative analyses. Such experiments demonstrated that all cameleon probes were functional, and indicated that hSOD1(G93A)-expressing MNs have larger cytosolic Ca2+ transients following AMPA stimulation, compared to SOD1(WT) MNs  we evaluated the expression of the probes in spinal cord sections. By use of a fluorescence stereo-microscope, we observed an intense and diffuse signal in tissue samples of mice transduced with either the mitochondrial , we have chosen a genetically engineered minimal promoter of the Hb9 gene (Hb9_AB), which is about 550 bp in length and has already been demonstrated to specifically drive the expression of a transgene into MNs, both in vitro and in vivo29. For the preparation of the viral particles, we have chosen the AAV9 serotype because of its broad tropism, including the capacity to target neurons28. We have demonstrated that the engineered AAV vectors are suitable for the specific expression of the cameleons targeted to the cytosol, the mitochondrial matrix and the ER lumen of MNs from spinal cord primary cultures of mice, and allow measuring Ca2+ responses to proper stimuli in such cellular districts. These vectors have several advantages with respect to other expression systems or the use of ubiquitous promoters. Indeed, they allow Ca2+ measurements in single primary MNs with no need for difficult and time-consuming MN purification steps. In addition, MN Ca2+ measurements can be performed in a more physiologic environment, containing glial cells and other neuronal types, compared to pure MN cultures.In this work, we have generated pAAV plasmids encoding cameleon Ca2+ probes at an expression level that easily allowed single cell recordings of Ca2+ dynamics.Although SMI32-positive MNs were less than 5% of total cells in our primary spinal cord cultures, the transduction efficiency ( >70%) of MNs by the AAV-mediated infection system was largely sufficient for single cell analyses. Indeed, the transduction rate was much higher than that achieved by transfection with plasmidic vectors, with which cameleon-expressing MNs were rarely found in each preparation, also when using a constitutive and ubiquitous , or the pan-neuronal human synapsin1 (hSyn), promoter (data not shown). In addition, in our experimental setting, a substantial number of SMI32-positive cells contained the Ca2+ concentration values upon proper calibration procedures13. The ratiometric nature of these Ca2+ indicators, as in the case of chemical probes such as Fura-2, prevents any differences due to level of expression or cell thickness. In addition, engineering of mutant cameleons have provided researcher with several cameleons displaying a wide range of Kd for Ca2+, thereby allowing reliable measurements of basal Ca2+ levels and evoked Ca2+ movements depending on the target cell compartment, the cell type and the desired stimulation protocol19.Among GECI, the ratiometric cameleon probes have several advantages, including low risk of artifacts, high sensitivity, good dynamic range, and the possibility to provide absolute Ca2+ measurements we have used the archetypal D1-based probe30, which allowed us to measure cytosolic fluctuations of the ion in MNs following AMPA stimulation.For cytosolic Ca2+ interplay is essential for cell physiology. On the one hand, the fine regulation of mitochondrial Ca2+ controls ATP production, because different mitochondrial enzymes rely on local Ca2+ concentration for their activity. On the other hand, mitochondria are a major Ca2+-buffering system avoiding noxious cytosolic Ca2+ overloads. While it is assumed that resting mitochondrial Ca2+ concentration is in the order of 10−1 μM, upon stimulation promoting Ca2+ entry from the extracellular space or its release from the ER, Ca2+ levels in the mitochondrial matrix may reach up to 102 μM, depending on the cell type and the stimulus31. Therefore, we have selected the 4mtD3cpv cameleon that, for its Kd and dynamic range, has been proved useful for measuring mitochondrial Ca2+ dynamics13.Mitochondria-Ca2+ homeostasis in the ER lumen, we have chosen the recently generated D4ER probe14 that has been proved useful for comparative Ca2+ measurements in an experimental setting of neurodegenerative disorders32.Finally, for the measurements of Ca34, which are particularly vulnerable to (AMPA-mediated) excitotoxic challenge and ALS-related damage7. Thus, to prove the efficacy of the probes to monitor local Ca2+ fluctuations in MNs, we have compared the response of MNs from mice expressing the ALS-related hSOD1(G93A) mutant, or the hSOD(WT)-expressing counterpart, to selected stimuli. In particular, because MNs express high levels of AMPA receptors through which they process strong glutamatergic inputs, and because excitoxicity has been proposed to contribute crucially to MN injury in ALS, probably as a consequence of glutamate-triggered Ca2+ overload35, we investigated Ca2+ responses to AMPA stimulation. The cytosolic and mitochondrial probes allowed the assessment of AMPA-mediated cell Ca2+ responses, thereby providing proof-of-principle for the functional application of these tools. Such conclusion is further corroborated by the finding that Fura-2-based measurements provided very similar results for cytosolic Ca2+ responses to AMPA stimulation. In addition, both cameleon- and Fura-2-based strategies underscored a larger cytosolic Ca2+ response in ALS MNs compared to the healthy controls supporting the idea that Ca2+ overloads may be involved in ALS MN damage36. Surprisingly, in spite of increased cytosolic Ca2+ load, no difference was observed in the mitochondrial matrix, suggesting a possible deficit of mitochondrial Ca2+ upload in ALS MNs that deserves further investigation. With this respect, it must be noted that, to the best of our knowledge, no direct measuring of mitochondrial Ca2+ fluctuations under our experimental settings has been previously reported, and that mitochondrial Ca2+ handling deficits could be stage-dependent during ALS disease course37. We also provided evidence that the ER cameleon was properly functioning and monitored the ER Ca2+ discharge promoted by caffeine38, although no difference was observed between the two SOD1 genotypes. These notions further highlight the importance of the here-presented tools for deepening our understanding of disease-associated local Ca2+ alterations in MNs, in in vitro and in vivo models.Notably, cameleons are mostly expressed in SMI32-positive α-MNs, enriched in the anterior ventral horn of the spinal cord2+ fluctuations in sub-cellular compartments of primary mouse MNs, thereby providing valuable and versatile tools for expanding our knowledge on Ca2+-related pathogenic routes in ALS and other neuromuscular diseases. With this respect, it is worth underlining that other GECI-encoding sequences can be easily cloned into the generated MN promoter-containing pAAV scaffold, thus further increasing the toolset of Ca2+ probes with different properties  for suitable Ca2+ measurements under different experimental protocols in MNs.In conclusion, the novel vectors we generated in this work allowed for the first time to directly monitoring local Cain vivo expression of the Ca2+ probes in MNs. This result was achieved by systemic administration of the virus, which is by far less invasive and arduous than local injection in the spinal cord of live mice. Thus, by means of suitable fluorescence microscopy equipment , this system could be used for Ca2+ measurements in more physiologic environments than primary cultures, such as tissue slices or even live animals.Importantly, we also provided evidence that the AAV vectors can drive the selective 2+ probes targeted to different cellular compartment  derived from the (MN)-specific promoter of the Hb9 gene29, or the pan-neuronal hSyn promoter.Multiple pAAVs have been generated, for the expression of the FRET-based cameleon Ca19, previously isolated by BamHI-EcoRI cleavage of a pcDNA3-D1cpv plasmid 19, was inserted into the BamHI-EcoRI sites of the pAAV-[hSyn]-ChR2/EYFP vector  14 was amplified by PCR using the pcDNA3-D4ER plasmid  as template, and the primers ER-KpnI-F and ER-HindIII-R (sequences are reported in the Supplementary Information). The PCR product was digested with KpnI-HindIII, and purified DNA fragments were ligated into the KpnI-HindIII sites of the pAAV-[hSyn]-ChR2/EYFP as indicated above, generating the pAAV-[hSyn]-D4ER plasmid  as template, and primers AB-MluI-F and AB-KpnI-R (sequences are reported in the Supplementary Information). The PCR product, subjected to MluI-KpnI cleavage and purified, was then ligated into both pAAV-[hSyn]-D1cpv and pAAV-[hSyn]-D4ERcpv plasmidic fragments digested with the same enzymes.Subsequently, the [hSyn] promoter in the obtained plasmids was substituted with the sequence coding for the ∼550 bp MN specific promoter [Hb9_AB], which was previously amplified by PCR using the Bg.Hb9_AB_GFP plasmid13 cameleon was obtained by replacing the [hSyn] promoter sequence of the pAAV-[hSyn]-4mtD3cpv plasmid  with the [Hb9_AB] sequence, by using the MluI and KpnI restriction sites.Concurrently, the pAAV vector encoding the mitochondrial matrix-targeted 4mtD3cpvE. coli TOP10 cells with the generated constructs and selection of positive clones, recombinant plasmids were amplified and validated by sequencing. All enzymes were from New England Biolabs, excepted the high-fidelity DNA polymerase (KAPA Biosystems), and DNA primers (Life Technologies). DNA manipulations have been performed accordingly to standard methods39 and to manufacturer’s instructions. Detailed maps and plasmids sequences are all available upon request to the corresponding author.After transformation of 13 to 10 × 1013 GC/ml (see Supplementary Information).AAV viral particles (AAV9 serotype) were produced by Vigene Biosciences, providing viral titers ranging from 2 × 10OPBA). All efforts were made to minimize animal suffering and reduce the number of animals used in the experiments.Tg mice expressing WT, or the ALS-related G93A mutant of hSOD1, (B6SJL(Tg-SOD1)2Gur/J and B6SJL(Tg-SOD1*G93A)1Gur/J mice, respectively) were purchased from The Jackson Laboratories. The colonies were maintained by breeding hemizygote (Tg) males to wild-type B6SJLF1/J hybrid females. Embryos and newborns were genotyped (as described in Supplementary Information), and used for subsequent experiments. All aspects of animal care and experimentation were performed in compliance with European and Italian (D.L. 26/2014) laws concerning the care and use of laboratory animals. All experimental procedures and animal care protocols were approved by the Italian Ministry of Health (authorization N. 305/2017-PR), and by the Ethical Committee for animal care and use of the University of Padova 40. Briefly, spinal cords were dissected from individual embryos in Hybernate medium (Gibco) added with 2% (v/v) B27 supplement (Gibco), and tissue was cut (<1 mm pieces) and incubated  in a buffer containing NaCl (124 mM), KCl (5.4 mM), NaH2PO4 (1 mM), glucose (3.6 mM), HEPES , added with BSA [0.3% (w/v)] and trypsin [0.025% (w/v)].Primary MN cultures were established from E12.5 mouse embryos according to the Henderson’s protocol  supplemented with sodium bicarbonate [7.5% (w/v)], horse serum , glucose [7.2% (w/v)], progesterone [0.1% (w/v)], insulin [1% (w/v)], putrescine [1% (w/v)], conalbumin [1% (w/v)], sodium selenite [0.1% (w/v)], BSA [0.4% (w/v)] and DNase I [0.1 mg/ml].2O, 2 h, RT) and then with laminin , and grown (37 °C in a 5% CO2 atmosphere).The cell suspension was then centrifuged  over a cushion of BSA [4% (w/v)] (in the above medium), after which cells were recovered from the bottom of the tube, resuspended in a MN culture medium consisting of Neurobasal medium (Gibco) supplemented with HS [2% (v/v)], B27 [2% (v/v)], L-glutamine (0.05 mM), glutamate (25 µM), mercaptoethanol (25 µM), BDNF (10 ng/ml), GDNF (10 ng/ml), CNTF (10 ng/ml), penicillin (100 U/ml), and streptomycin (100 µg/ml). Cells were then plated at a density of approximately 500,000 cells/well in 12-wells culture plates containing (18 mm diameter) glass coverslips coated with poly-D-ornithine ].Primary cultures of cerebellar granule and cortical neurons were prepared as described in De Mario et al.42. Once the cultures reached the confluence, they were re-plated at the optimal density 80,000 cells/well in 24-well plates containing glass coverslips and maintained in minimal essential medium , supplemented with FBS [10% (v/v)], L-glutamine (2 mM), glucose [0.3% (w/v)] in phosphate buffered saline (PBS), penicillin (100 U/ml]), and streptomycin (100 µg/ml).Primary cultures of spinal astrocytes were isolated from newborn mice and cultured as described previously in Martorana On day 2 from plating, all primary cultures were transduced with the AAV particles and used in experiments at different times depending on the cell type. For the transfection of cortical neurons and spinal astrocytes with plasmidic vectors, the Lipofectamine-2000 reagent (Gibco) was used, following the manufacturer’s instructions.For immunocytochemical analysis, cells were firstly washed in ice-cold PBS, and fixed  in paraformaldehyde  in PBS. After washing in PBS, cells were permeabilized in PBS containing Triton X-100  and then incubated  with the following primary antibodies : anti-SMI32 mouse monoclonal (m) Ab ; anti-glial fibrillary acidic protein (GFAP) rabbit polyclonal (p) Ab ; anti-microtubule-associated protein-2 (MAP-2) chicken pAb ; anti-calreticulin rabbit pAb ; anti-GAPDH rabbit pAb ; anti-Tom20 rabbit pAb . After extensive washings in PBS, cells were incubated  with the following secondary antibodies, depending on the used primary Ab: Alexa Fluor 555-conjugated anti-mouse IgG ; rhodamine-conjugated  or Alexa Fluor 568-conjugated  anti-rabbit IgG; Alexa Fluor 568-conjugated anti-chicken IgG . Cell nuclei were counter-stained with Hoechst 33342 , and coverslips were finally washed in PBS, mounted in montage solution [8% Mowiol 40–88 (Sigma) in glycerol and PBS (1:3 (v/v)], and observed with an inverted fluorescence microscope (Leica CTR6000) equipped with a computer-assisted charge-coupled camera , or with a Leica TSC SP5 inverted confocal microscope system, equipped with a HCX PL APO 63× or 100× magnification, numerical aperture 1.40, oil immersion objective, which also allowed the acquisition and analysis of digital images by a dedicated software (Leica AS). When indicated in the figure legends, cell images were reported as the merge of a complete z-stack of the observed field.2+ imaging with the cameleon probes, 12 days after plating cells were mounted into an open-topped chamber and maintained under perfusion with a Krebs-Ringer buffer  containing CaCl2 (2 mM), through a temperature-controlled (37 °C) instrument . Cells were stimulated with AMPA  for cytosolic and mitochondrial Ca2+ measurements, or with 100 μM caffeine for ER Ca2+ measurements (both stimuli were carried out in KRB).For Ca−1) of the ratio (R) between the acceptor and the donor fluorescence intensity, which was taken as a relative measurement of Ca2+ concentration. Peak values of Ca2+ transients were reported as the difference between the peak and the baseline R value (ΔR = Rpeak − Rbaseline).Cell were analysed using a DM6000 inverted microscope (Leica) with a 40× oil objective . Excitation light produced by a led  was filtered at the appropriate wavelength (425 nm) through a band-pass filter, and the emitted light was collected through a dichroic mirror , and a beam-splitter (OES) with emission filters (Chroma Technologies) HQ 480/40 M (for CFP) and HQ 535/30 M (for YFP). The beam-splitter permits the collection of the two emitted wavelengths at the same time, thus preventing any artefact due to uncontrolled movement of the recording chamber and/or intracellular organelles. Images were acquired using an IM 1.4 C cooled CCD  attached to a 12-bit frame grabber. Synchronization of the excitation source and the CCD was performed through a control unit ran by a custom-made software package , which was also used for image acquisition, with an exposure time of 100 ms. The software recorded with-time variations -expressing mice (P1) received 50 μl of viral suspension containing 3 × 1011 GC/ml of AAV9-Hb9_AB-4mtD3cpv or AAV9-Hb9_AB-D4ER, or vehicle (PBS), into the temporal vein using a Hamilton syringe with a 32-gauge needle. The injections were performed in a Biosafety Level 2 (BL2) laboratory.For 2 before killing by cervical dislocation, and spinal cords were collected and successively fixed in PFA . Fixed tissues were transferred to sucrose  for cryoprotection. 20 μm-thick sections were serially cut on a cryostat (Leica Microsystems), immunostained for the MN marker SMI32 and counter-stained with the nuclear dye Hoechst 33342, as described previously for immunocytochemical analyses. Finally, slices were mounted in montage solution on a glass coverslip, and observed with a fluorescence stereo-microscope (Leica M205-FA), or with the above described inverted confocal microscope system.Four weeks after the injection, animals were anesthetized by CO535/F480) and reported as %. All the data are representative of at least n (indicated in the figures and/or figure legends) independent experiments, and are reported as mean ± standard error of the mean (SEM). Statistics were performed by unpaired Student’s t test, with a p value < 0.05 being considered statistically significant.Off-line analysis of FRET data was performed with the ImageJ software. YFP and CFP images were subtracted for the respective background signals, and distinctly analysed after choosing proper regions of interest on selected cells. Subsequently, the ratio between YFP and CFP emission fluorescence intensity (F) was calculated (R = F2+ measurements, see Supplementary Information.For mouse genotyping, NSC-34 cell culturing, and methods for Western blot analysis and Fura-2-based CaSupplementary Information


Endoplasmic reticulum (ER) represents the major intracellular Ca2+ store and the free Ca2+ concentration ([Ca2+]) within its lumen ([Ca2+]ER) can reach levels higher than 1 mM. Several genetically-encoded ER-targeted Ca2+ sensors have been developed over the last years. However, most of them are non-ratiometric and, thus, their signal is difficult to calibrate in live cells and is affected by shifts in the focal plane and artifactual movements of the sample. On the other hand, existing ratiometric Ca2+ probes are plagued by different drawbacks, such as a double dissociation constant (Kd) for Ca2+, low dynamic range, and an affinity for the cation that is too high for the levels of [Ca2+] in the ER lumen. Here, we report the characterization of a recently generated ER-targeted, Förster resonance energy transfer (FRET)-based, Cameleon probe, named D4ER, characterized by suitable Ca2+ affinity and dynamic range for monitoring [Ca2+] variations within the ER. As an example, resting [Ca2+]ER have been evaluated in a known paradigm of altered ER Ca2+ homeostasis, i.e., in cells expressing a mutated form of the familial Alzheimer’s Disease-linked protein Presenilin 2 (PS2). The lower Ca2+ affinity of the D4ER probe, compared to that of the previously generated D1ER, allowed the detection of a conspicuous, more clear-cut, reduction in ER Ca2+ content in cells expressing mutated PS2, compared to controls.Calcium ion (Ca In resting conditions, cells are able to maintain a large [Ca2+] gradient between the cytosol ([Ca2+]~100 nM) and the extracellular medium ([Ca2+]~1.5–2 mM). Maintenance of this gradient is ensured by the activity of exchangers, pumps and buffers and by Ca2+ compartmentalization in organelles [2+], caused by its influx from the extracellular medium or release from intracellular stores, modulate a plethora of key cellular events, ranging from exocytosis to muscle contraction, adenosine triphosphate (ATP) production and transcription activation. Cell survival relies on complex signal pathways shaped by dynamic changes, both in time and space, of second messengers’ concentration. Among them, Ca2+ stores are represented by the ER and the Golgi apparatus (GA), while the role of acidic compartments  in cellular Ca2+ handling is still debated since the [Ca2+] measured varies depending on the cell type and the experimental conditions. The luminal [Ca2+] of the ER is rather high (estimates vary from 200 μM to over 1 mM) [2+] within the ER is of crucial importance for cell viability and the steady state level depends on the equilibrium between Ca2+ leak and uptake. In turn, Ca2+ accumulation depends on the activity of Sarco/Endoplasmic Reticulum Calcium ATPases (SERCAs), a family of transmembrane proteins that pump two Ca2+ ions into the ER lumen per molecule of hydrolysed ATP  inside the ER lumen ([Ca2+]ER) is of crucial importance. The first ER-targeted genetically-encoded Ca2+ indicator (GECI) was an aequorin construct specifically targeted to the ER lumen that, however, has been used exclusively for measurements in cell populations  measurements independently from probe expression levels, shifts in the focal plane, and movements of the sample. Only few ratiometric probes have been targeted to the ER; one of them, named GAP3-aequorin, is a dual-excitation ratiometric probe, based on the molecular pair aequorin/GFP ER, the binding interface between CaM and M13 was redesigned to decrease its affinity for Ca2+. This effort led to the development of a Ca2+ indicator (D1ER) with a relatively low Ca2+ affinity (Kds = 0.81 and 60 μM measured in vitro) that has been used to monitor [Ca2+] directly within the ER  of individual living cells ER changes reported were expressed as emission fluorescence ratios (540/475) [2+]ER changes in transfected mice cortical neurons [2+]ER that were not reported by a CRT-D1ER probe, suggesting an improvement of the newly generated sensor in comparison to the previous one  resting level in BHK and HeLa cell lines, whereas D1ER measurements are close to the probe’s saturating [Ca2+]; (ii) D4ER is able to report with much higher sensitivity (compared to its predecessor D1ER) changes in ER Ca2+ levels caused by the expression of a FAD-linked mutant form of PS2.Here we report the characterization of the previously generated ,29, ubiqConstructs: D4ER generation is reported in  was estimated using the MaxChelator2.5 software and checked by fluorimetric measurements with Fura-2.For Caeriments F, a Ca2+2 concentration (see below). Values are plotted as a function of time or, for the probe calibration curve, of [Ca2+].The reported ratio % values (hereafter named R%) represent 535/480-nm fluorescence emission intensities expressed as a % of the maximal ratio obtained in permeabilized cells upon perfusion of saturating CaClMaterials: Restriction and modification enzymes were purchased from NEB  or Thermo Scientific . CPA, digitonin, bradykinin, histamine, ATP-Na, and dextran were purchased from Sigma-Aldrich . All other materials were analytical or of the highest available grade.Statistical analysis: Off-line analysis of FRET experiments was performed with ImageJ software. Citrine and ECFP images were subtracted of background signals and distinctly analysed after selecting proper regions of interest (ROIs) on each cell; subsequently, the ratio between citrine and ECFP mean emission intensities at 535 and 480 nm (R = F535/F480), respectively, were calculated. Data are presented as R%, where R% = (R − Rmin)/(Rmax − Rmin) × 100 and Rmax and Rmin are the R values in permeabilized cells at saturating [Ca2+] and in the absence of Ca2+, respectively. For D4ER, the [Ca2+] was finally calculated as reported in  was calculated as reported in , N represents the number of independent transfections.All the presented data are mean values of at least three independent transfections. Calibration curve fitting was performed using Origin 8 SR5 (OriginLab Corporation). Averages are expressed as mean ± s.e.m.  was substituted with D4 c) in the very same cell co-expressing an organelle-targeted Ca2+ probe c (and thus of [Ca2+]n). Interestingly, the kinetics of the [Ca2+] changes in the two compartments were substantially different: the peak in nuclear signal occurred within 10–20 s, while the decrease in ER signal was maximal after 60–120 s. Thus, the maximal increase in cytosolic and nuclear [Ca2+] are reached when only a fraction (20%–30%) of the maximal decrease in ER signal has occurred. At the end of the experiment, permeabilized cells have been perfused with intracellular-like medium containing 600 µM EGTA, to get minimum R% value, and 3 mM CaCl2, to get the maximum R%.The functionality of the probe was then assessed in live BHK cells. Simultaneous recording of  variations in more physiological conditions, i.e., in cells bathed by a medium mimicking extracellular [Ca2+] (1 mM) and in the absence of SERCA inhibitors. The application of an IP3-generating stimulus  induced a transient ER Ca2+ release, followed by the ER Ca2+ refilling  and ATP to fuel SERCA pumps. The D4ER signal started to increase as soon as exposed to ATP and a new steady state level, close to that observed in the intact live cell, was rapidly gained. Finally, 3 mM CaCl2 was perfused and this was followed by a further substantial R% increase. No further rise was observed upon perfusion of CaCl2 to 4 mM, while after about 60 s the signal started to decrease as the probe was lost from the cells (likely due to the toxicity of Ca2+ at such high level in permeabilized cells).The response of D4ER to , ranging from 0.4 μM to 5 mM, assuming that luminal [Ca2+] in the ER should, under these conditions, passively equilibrate with the Ca2+ of the perfused medium . Moreovd medium A. R% vals 321 μM B. The saigitonin  µM in anreported ,24. As sectively C.d and lower affinity for Ca2+ compared to its predecessor D1ER, i.e., with a Ca2+ affinity more suitable for dynamically following Ca2+ variations within the organelle in live cells. To the best of our knowledge, the only two ER targeted GECIs available with a lower Ca2+ affinity, compared to D4ER, are the single fluorophore-based Ca2+ sensor CEPIA1er, with a reported Kd of 368 μM , the measured mean R% value correspond to 697.8 ± 16.1 µM . D1ER R% levels at rest were 80.6% ± 2.8% of Rmax  , a value not very different from that obtained with D4ER. However, the calculated D1ER value is subjected to larger errors because: (i) as shown in 2+] is subjected to larger variability; and (ii) D1 shows a biphasic Kd, making the calculation of [Ca2+] based on R% values more difficult.In order to evaluate the entity of this improvement, we evaluated resting ER CaR probes A, employ N = 19) B. In oth2+ probe was strengthened by measuring resting [Ca2+] level in cells with reported higher [Ca2+]ER [max  . The result obtained fits with what previously reported [max   D. In terreported  using a  N = 26) D. The ca2+] has been previously reported. In particular, by using aequorin-based Ca2+ probes in cell population measurements, we have previously shown that different FAD-PS2 mutants reduce ER and GA Ca2+ levels, mainly by inhibiting SERCA activity [2+] variations, BHK cells expressing one of the most effective PS2 mutant , to obtain minimum R values. Maximum R was obtained by the further addition of CaCl2  or 697.8 ± 16.1 µM  measured in control cells by D1ER or D4ER probe, respectively, to 336.0 ± 81.3 µM  or 318.1 ± 82.1 µM  measured in PS2-T122R expressing cells, by D1ER or D4ER probe, respectively , or traER probe . After 1controls B, this rcontrols E. In terectively C,F.2+ and, thus, it appears less prone to saturation in high Ca2+-containing organelles. Moreover, D4ER is characterized by a single Kd compared to the double Kd reported for D1ER. The latter aspect simplifies quantitative Ca2+ evaluation in intact live cells. Using the D4ER probe, the specific effect on intracellular Ca2+ stores due to the expression of the FAD-linked PS-T122R mutant was also evaluated. The data presented confirm previous results from our laboratory, showing that expression of FAD-PS2 reduces ER Ca2+ levels [2+].In this study, we characterized the recently generated, ubiquitously-expressible, ER-targeted D4ER Cameleon. This probe displays similar DR to that shown by the previously generated D1ER, but lower affinity for Ca+ levels ,31 and h


Confocal imaging of mouse small intestines from animals expressing GCaMP3 in ICC were used to investigate inhibitory neural regulation of ICC in the deep muscular plexus (ICC-DMP). We hypothesized that Ca2+ signaling in ICC-DMP can be modulated by inhibitory enteric neural input. ICC-DMP lie in close proximity to the varicosities of motor neurons and generate ongoing Ca2+ transients that underlie activation of Ca2+-dependent Cl− channels and regulate the excitability of SMCs in the SIP syncytium. Electrical field stimulation (EFS) caused inhibition of Ca2+ for the first 2–3 s of stimulation, and then Ca2+ transients escaped from inhibition. The NO donor (DEA-NONOate) inhibited Ca2+ transients and Nω-Nitro-L-arginine (L-NNA) or a guanylate cyclase inhibitor (ODQ) blocked inhibition induced by EFS. Purinergic neurotransmission did not affect Ca2+ transients in ICC-DMP. Purinergic neurotransmission elicits hyperpolarization of the SIP syncytium by activation of K+ channels in PDGFRα+ cells. Generalized hyperpolarization of SIP cells by pinacidil (KATP agonist) or MRS2365 (P2Y1 agonist) also had no effect on Ca2+ transients in ICC-DMP. Peptidergic transmitter receptors (VIP and PACAP) are expressed in ICC and can modulate ICC-DMP Ca2+ transients. In summary Ca2+ transients in ICC-DMP are blocked by enteric inhibitory neurotransmission. ICC-DMP lack a voltage-dependent mechanism for regulating Ca2+ release, and this protects Ca2+ handling in ICC-DMP from membrane potential changes in other SIP cells.Gastrointestinal motility is coordinated by enteric neurons. Both inhibitory and excitatory motor neurons innervate the syncytium consisting of smooth muscle cells (SMCs) interstitial cells of Cajal (ICC) and PDGFRα In fact this level of motor control is due not only to the functions of smooth muscle cells (SMCs), because the behavior of SMCs is modulated by interstitial cells . ICC and PDGFRα+ cells are electrically coupled to SMCs  generate pacemaker activity that develops into electrical slow waves  expressed specifically in murine ICC to investigate the responses of ICC-DMP to enteric inhibitory neurotransmission. Our results show that enteric inhibitory neurotransmitters exert powerful inhibitory effects on Ca2+ release, which would be expected to reduce activation of Ano1 and development of spontaneous transient inward currents (STICs) and reduce the excitatory drive exerted upon the SIP syncytium by ICC-DMP.The precise mechanisms through which ICC transduce inputs from motor neurons are poorly understood, largely because past studies have relied upon Gt(ROSA)26Sortm38(CAG−GCaMP3)Hze/J) and their corresponding wild-type siblings (C57BL/6) were purchased from Jackson Laboratories  and subsequently crossed with Kit-Cre mice (c-Kit+/Cre−ERT2) provided by Dr. Dieter Saur . Kit-Cre-GCaMP3 mice underwent treatment with tamoxifen at 6–8 weeks of age (2 mg for 3 consecutive days), as previously described . The jejunal segments opened along the mesenteric border, and intra-luminal contents were removed by washing with KRB. Mucosal and sub-mucosal layers were removed by sharp dissection, and the remaining 3, 15.5; NaH2PO4, 1.2; MgCl2, 1.2; CaCl2, 2.5; and glucose, 11.5. The KRB was bubbled with a mixture of 97% O2–3% CO2 and warmed to 37 ± 0.2°C. All drugs were purchased from Tocris Bioscience  and dissolved in solvents recommended by the manufacturer to create appropriate stock solutions. Final concentration used for experiments were obtained by diluting with KRB. All work was performed according to biosafety level II regulations.Tissues were maintained by perfusing with KRB containing (mmol/L): NaCl, 120.35; KCl, 5.9; NaHCONeural responses were elicited by electrical field stimulation  generated by a Grass stimulator Grass S48 stimulator  and delivered via two platinum electrodes placed on either side of muscle strips. Responses evoked by EFS were completely abolished by tetrodotoxin .+/copGFP mice as previously described . The PCR primers used and their GenBank accession numbers are listed in Table Jejunal ICC were dispersed from Kit2+ imaging experiments were performed in the presence of nicardipine (100 nM) to minimize movement artifacts resulting from contractions.For imaging studies, the muscles were equilibrated with continuous perfusion of warmed KRB solution at 37°C for 1 h. Imaging was performed with a spinning-disk confocal microscope  mounted to an upright Nikon Eclipse FN1 microscope equipped with a 60x 1.0 NA CFI Fluor lens . GCaMP3, expressed in ICC within the jejunal muscles, was excited at 488 nm using a laser coupled to a Borealis system . Emitted fluorescence (>515 nm) was captured using a high-speed EMCCD Camera . Image sequences were acquired at 33 fps using MetaMorph software . In some experiments images were acquired with an Eclipse E600FN microscope  equipped with a 60x 1.0 CFI Fluor lens . In this system, GCaMP3 was excited at 488 nm , as previously described  images and imported into custom software  for initial pre-processing analysis. Tissue movement was stabilized to ensure accurate measurement of Ca2+ transients from ICC-DMP. Whole cell ROIs were used to generate spatio-temporal (ST) maps of Ca2+ activity in individual ICC-DMP recorded in situ. ST maps were then imported as TIFF files into Image J  for post-hoc quantification analysis of Ca2+ events.Analysis of Ca2+ event frequency in ICC-DMP was expressed as the number of events fired per cell per second (s−1). Ca2+ event amplitude was expressed as ΔF/F0, the duration of Ca2+ events was expressed as full duration at half maximum amplitude (FDHM), and Ca2+ event spatial spread was expressed as μm of cell propagated per Ca2+ event. Unless otherwise stated, data is represented as mean ± standard error (S.E.M.). Statistical analysis was performed using either a student's t-test or with an ANOVA test where appropriate . In all statistical analyses, P < 0.05 was taken as significant. P < 0.05 are represented by a single asterisk (*), P < 0.01 are represented by two asterisks (**), P < 0.001 are represented by three asterisks (***) and P < 0.0001 are represented by four asterisks (****). When describing data throughout the text, n refers to the number of animals used in that dataset while c refers to the numbers of cells used in that same data set.Ca2+ transients in ICC-DMP were ongoing and stochastic in nature, as previously reported  resulted in multiphasic responses (inhibition of Ca2+ transients followed by enhancement of these events), as shown in a representative spatiotemporal map (ST map) and traces of Ca2+ transients . During EFS (5 s), an initial inhibitory period was observed in which Ca2+ transients ceased for ~2 s. The firing of Ca2+ transients escaped from inhibition during the final 3 s of EFS, and a robust increase in Ca2+ transient firing was apparent during this period and after cessation of EFS . The amplitude of Ca2+ transients decreased from 0.8 ± 0.07 ΔF/F0 prior to EFS to 0.14 ± 0.05 ΔF/F0 during the inhibitory phase , duration decreased from 189 ± 7 ms before EFS to 27 ± 9.7 ms , and the spatial spread of Ca2+ transients decreased from 11.1 ± 0.7 to 2 ± 0.9 μm . The number of active firing sites also decreased during the inhibitory phase of EFS from 5.2 ± 0.4 to 0.3 ± 0.09 . Lastly, the propagation velocity of Ca2+ signals decreased from 71.4 ± 5.6 μm/s prior EFS to 11.4 ± 4.4 μm/s during the first 2 s of EFS .In this study we focused our analysis on the inhibitory responses imposed on ICC-DMP by nerve stimulation, so Ca Figures . During 2+ firing sites in ICC-DMP, from a single site to 13 sites . The nature of Ca2+ transients during sustained EFS (5 s) was examined in more detail by the analysis described in Figure 2+ transients ceased during the initial 2 s of EFS, as shown in the ST map . The delay periods describing the escape from inhibition are plotted as a summary histogram in Figure n = 19, c = 48).In this and a previous study we noted a range in the number of Cap Figure . During s Figure . Ca2+ tre Figure . A greate Figure . The inh2+ transients in these cells are displayed in ST maps in Figures 2+ firing site active in cell 2 before anything occurred in cell 1  incubated for 15 min, increased the firing frequency of Ca2+ transients in ICC-DMP from 1.5 ± 0.3 s−1 in control to 2.5 ± 0.5 s−1, as shown in Figures P = 0.0042, n = 5, c = 11). The duration of Ca2+ transients was also increased by L-NNA from 232 ± 11 to 250 ± 6.5 ms . However, neither the amplitude  nor the spatial spread  of Ca2+ transients was affected significantly by L-NNA.The nitric oxide synthase (NOS) inhibitor 2+ transients were dramatically inhibited by this compound Figure 2+ transients during control conditions was 0.9 ± 0.2 s−1, and was reduced to 0.14 ± 0.09 s−1 by DEA-NONOate . The amplitude of Ca2+ transients was reduced from 0.7 ± 0.14 ΔF/F0 to 0.2 ± 0.13 ΔF/F0 in DEA-NONOate , duration was decreased by DEA-NONOate from 242 ± 21.2 ms to 50 ± 33.6 ms , and spatial spread was reduced from 8.9 ± 8 μm to 1.8 ± 1.2 μm . These results suggest that NO was released tonically from enteric nerves, however at lower levels than was achieved by addition of DEA-NONOate (10 μM), and NO modulates the basal level of firing of Ca2+ transients in ICC-DMP.The NO donor, DEA-NONOate (10 μM) caused effects opposite of L-NNA and Ca2+ transients for about ~2 s after the onset of stimulation . L-NNA also increased the amplitude of Ca2+ transients from 0.2 ± 0.1 ΔF/F0 to 1 ± 0.1 ΔF/F0 , the duration from 42 ± 14 ms to 216 ± 14.9 ms , and the spatial spread from 1.3 ± 0.7 μm to 11.3 ± 1.3 μm .As above, EFS evoked inhibition of Can Figure . L-NNA , amplitude (P = 0.91), duration (P = 0.48), or spatial spread (P = 0.86) . MRS 2500 (1 μM) also failed to affect Ca2+ transients in ICC-DMP during EFS, as shown in Figures 2+ transients were also unaffected by MRS 2500 during the initial 2s of EFS .As several studies have reported, enteric inhibitory signaling by purines is mediated by P2Y1 receptors in post-junctional cells . The amplitude of Ca2+ transients increased during this period from 0.1 ± 0.07 ΔF/F0 to 0.7 ± 0.08 ΔF/F0 , Ca2+ transient duration increased from 30 ± 16.2 ms to 201 ± 17.83 ms , and the spatial spread of Ca2+ transients increased from 0.9 ± 0.5 μm to 10.5 ± 1.3 μm .While the P2Y1 receptor antagonist had no resolvable effect on the inhibition of Ca2+-activated K+ channels  in GI muscles cause significant hyperpolarization due to opening of apamin-sensitive, small conductance Ca2+ transients in ICC-DMP. In the presence of TTX, ATP (100 μM) also had no significant effects on Ca2+ transient frequency (P = 0.85), amplitude (P = 0.31), duration (P = 0.1) or spatial spread . Similarly, the selective P2Y1 receptor agonist MRS 2365 (1 μM) had no effect on ICC-DMP Ca2+ transient frequency (P = 0.25), amplitude (P = 0.62), duration (P = 0.62), or spatial spread . Hyperpolarization of the SIP syncytium with the KATP channel agonist, pinacidil , amplitude (P = 0.36), duration (P = 0.26), or spatial spread . These data suggest that membrane potential transients have no effect on Ca2+ tranisents in ICC-DMP, and purines which exert their hyperpolarizing effects on SIP cells other than ICC-DMP do not affect the Ca2+ transients in ICC-DMP.Application of purinergic agonists had no significant effects on CaGucy1a1 and Gucy1b1, protein kinase cGMP-dependent type 1: Prkg1, and inositol-1,4,5 triphosphate receptor I-associated G kinase substrate (IRAG) (aka murine retrovirus integration site 1 homolog): Mrvi1 transcripts. We noted elevated expression in all of these downstream mediators of nitrergic responses in ICC relative to unsorted cells . Gucy1a1 transcripts were higher in ICC in comparison to unsorted cells , duration (P = 0.33), and spatial spread (P = 0.5) were not significantly different after ODQ . The role of sGC in mediating nitrergic responses in ICC-DMP was also shown by an activator of sGC. Bay 58-2667 (1 μM), reduced firing frequency of Ca2+ transients , amplitude from 0.3 ± 0.06 ΔF/F0 to 0.1 ± 0.04 ΔF/F0 , duration from 215 ± 8.2 ms to 72 ± 26.9 ms , and spatial spread from 7.6 ± 0.5 μm to 2.7 ± 1.2 μm .Similar to L-NNA, ODQ (10 μM), an inhibitor of sGC, significantly increased basal Ca Figures  from 1.62+ transients during the first 2 s of EFS was 0.6 ± 0.3 s−1 in control and increased to 4 ± 0.5 s−1 in the presence of ODQ . ODQ also increased Ca2+ transient amplitude during EFS from 0.4 ± 0.15 ΔF/F0 to 1.1 ± 0.2 ΔF/F0 , duration from 99 ± 37.2 ms to 252 ± 7.8 ms , and spatial spread from 2.8 ± 1.3 μm to 11.3 ± 1.4 μm .ODQ (10 μM) also blocked the inhibitory effects of EFS on ICC-DMP during the initial 2 s of stimulation Figures . In thes2+ transients in ICC-DMP. We tested the effects of two PKG inhibitors, KT 5823 (1 μM) and Rp-8-pCPT-cGMPS (10 μM). However, Neither of these compounds had any effect on the basal activity of Ca2+ transients in ICC-DMP , amplitude , duration  or spatial spread , n = 4, c = 11. Rp-8-pCPT-cGMPS also failed to exert any significant changes in Ca2+ transient frequency , amplitude , duration  or spatial spread , n = 8, c = 21. We next tested if inhibition of PKG relieved the EFS evoked inhibitory response in ICC-DMP. KT 5823 and Rp-8-pCPT-cGMPS both had no significant effects on Ca2+ transients during the initial 2 s period of EFS. Across all cells tested, there was no significant change in the firing frequency (P = 0.57), amplitude (P = 0.49), duration (0.96) or spatial spread (0.58) of Ca2+ transients during the initial 2 s period of EFS in the presence of KT 5823 . Also in the presence of Rp-8-pCPT-cGMPS (10 μM) there was no significant change in the firing frequency (P = 0.67), amplitude (P = 0.37), duration (0.92) or spatial spread (0.81) of Ca2+ transients during the initial 2 s period of EFS in the presence of the drug .We next sought to evaluate the role of cGMP dependent protein kinase (PKG) on basal firing of Ca Figures . KT 5823We also tested the PKG inhibitors seeking a positive control for their poor performance against nitrergic effects in ICC. Contractile experiments were performed on muscles of the colon, aorta and corpus cavernosum. Neither KT 5823 nor Rp-8-pCPT-cGMPS reduced the inhibitory effects of sodium nitroprusside  or DEA NONOate (10 μM) on muscles pre-contracted with norepinephrine  or carbachol  (data not shown). Taken together, the lack of effects of commercial PKG inhibitors on smooth muscles, in general, might be due to poor penetration of the drugs into cells in intact tissues or targets besides PKG contributing to inhibitory responses. This is a question in need of further evaluation.Vipr1 and Vipr2) and PACAP receptor (Adcyap1r1). We noted elevated expression in all of peptidergic receptors in ICC relative to unsorted cells . Vipr1 transcripts were higher in ICC in comparison to unsorted cells  are also released from nerve terminals and might modulate ICC-DMP activity. Therefore, we examined the expression profile of peptide receptors in sorted ICC from small intestinal muscles and characterized expression of VIP receptors . Similarly, the amplitude of Ca2+ transients was reduced from 0.5 ± 0.06 ΔF/F0 to 0.2 ± 0.08 ΔF/F0 by VIP . The duration of Ca2+ transients was decreased from 224 ± 9.8 ms to 66 ± 28 ms by VIP . Finally, VIP also significantly decreased the spatial spread of Ca2+ transients from 9.7 ± 1.4 μm to 5 ± 2.3 μm . VIP 6-28 (10 μM) caused a significant increase in basal Ca2+ transient firing in a similar manner to blocking nitrergic input with application of L-NNA . The amplitude (P = 0.38), duration (P = 0.67) and spatial spread (P = 0.24) of Ca2+ transients was not significantly affected by VIP 6-28, n = 5, c = 23 , amplitude (P = 0.16), or spatial spread (P = 0.58) of Ca2+ transients was significantly affected during the initial 2 s period of EFS VIP 6–28 (10 μM) . However, the duration of Ca2+ transients in the initial 2 s period of EFS was significantly increased from 59 ± 26.9 ms in control to 110 ± 29.5 ms in the presence of VIP 6-28 . It is possible that multiple receptors mediate responses to inhibitory peptides in ICC.While VIP 6-28 increased the basal level of CaA Figure , VIP 6-2A Figure . Neither2+ transients in ICC-DMP, the intramuscular class of ICC in the small intestine. Ca2+ handling mechanisms in ICC are of interest because Ca2+ release from stores couples to activation of Ano1 channels and the generation of STICs  in which ongoing Ca2+ transients in ICC-DMP were largely abolished. Shutting off of STICs during the initial phase of EFS may explain a portion of the hyperpolarization responses to nerve stimulation seen in electrophysiological recordings  leading to non-linear changes in voltage-dependent conductances, membrane potential and excitation-contraction coupling. At present there are no organ-specific, unified models of the many responses in GI muscles that might be initiated or repressed by stimulation of enteric neurons.It is difficult to reconcile generalized tissue level responses to EFS  with events occurring in a single type of ICC. Neurotransmission may affect conductances and Ca2+ firing sites within ICC-DMP escaped from inhibition. In our experiments this occurred within about 2 s from the onset of stimulation. However, the Ca2+ release sites escaped inhibition with different temporal characteristics. The heterogeneity in the periods before escape from inhibition occurred is further demonstration of the independence of Ca2+ release sites, even within individual cells, and supports the conclusion that there is no cellular or multi-cellular correlation between Ca2+ release sites within the ICC-DMP network . We showed that Ca2+ release events were virtually blocked if neurokinin receptor antagonists were present . Thus, ICC have the receptor and the molecular apparatus to transduce nitrergic signals and produce cGMP. Others have found that knock down of Gucy1b1 in ICC, using Cre-LoxP technology, abolished nitrergic inhibitory junction potentials (IJP) in gastric fundus and reduced IJPs in the colon, but these authors also reported that knockdown of Gucy1b1 in SMCs also either reduced the amplitude or shortened IJPs  is expressed in ICC and is the main receptor/transducer of the inhibitory effects of NO in the GI tract  is the principal downstream signaling molecule mediating nitrergic responses, and Prkg1 is expressed in ICC in the small intestine -associated cGMP-kinase substrate , and this gene is also expressed in ICC of the small intestine. IRAG co-precipitates with IP3R and was found to be indispensable for cGMP regulation of Ca2+ release in model cells or cultured human colonic SMCs  responsible for these effects are complex. Ca2+ transients initiate inward currents in ICC-DMP that are conducted to other cells in the SIP syncytium. Thus, suppression of Ca2+ transients in ICC-DMP by inhibitory neural inputs would tend to reduce SMC excitability and reduce contractile force in the small intestine.In summary, CaSB, BD, and KS: Conception and design of the experiments; SB, BD, CC, KK, and KS: Collection, analysis, and interpretation of data; SB, BD, and KS: Drafting the article and revising it critically for intellectual content. All authors read and approved the manuscript before submission.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. The reviewer MC and handling Editor declared their shared affiliation.


RAS mutations has been systematically investigated in acute myeloid leukemia (AML). However, clinical significance of RAS expressions in AML remains poorly determined. To explore the clinical significance, we analyzed KRAS and NRAS expressions in 143 de novo AML patients by real-time quantitative PCR. KRAS and NRAS expressions were significantly up-regulated in AML patients. KRAS and NRAS mutations were identified in 4% (6/143) and 8% (12/143) of these patients, respectively. However, no significant association was observed between RAS mutations and expressions. High KRAS expression was associated with older age, higher white blood cells, and a tendency of higher platelets, whereas high NRAS expression was only correlated with older age. Complete remission (CR) rate and overall survival of AML patients were adversely affected by KRAS overexpression, but not NRAS overexpression. Multivariate analysis revealed that KRAS acted as an independent prognostic predictor in cytogenetically normal AML (CN-AML). Moreover, the prognostic value of KRAS expression was validated using the published data from Gene Expression Omnibus datasets. In the follow-up patients, KRAS expression rather than NRAS expression in CR time tended to decrease compared to newly diagnosis time, and both KRAS and NRAS expressions were significantly increased when in relapse time. Our findings revealed that RAS overexpression and mutations were common events in AML with potential therapeutic target value. KRAS overexpression independent of RAS mutations conferred an adverse prognosis in CN-AML.The prognostic value of  CEBPA, NPM1, FLT3-ITD, C-KIT, DNMT3A, and IDH1/2, and changes in gene expression, such as overexpression of BAALC, ERG, EVI1, and MN1, have been discovered to strongly affect clinical outcome of CN-AML patients . Among CN-AML, KRAShigh and NRAShigh patients also presented an obvious tendency of lower CR rate .Follow-up data was available in 135 AML patients after receiving induction chemotherapy . In whole-cohort AML, KRAShigh was associated with shorter overall survival (OS) time among whole-cohort AML, non-APL AML, and CN-AML patients  databases were set as the independent validation cohort. Through the online tools GenomicScape, e Figure  and 3B, d Figure  and 3D.RAS abnormalities (overexpression and mutation) on prognosis. We divided patients into groups regarding RAS expressions and mutations including KRAS normal (without mutation and overexpression) vs KRAS abnormal (with mutation or overexpression), NRAS normal vs NRAS abnormal, and RAS normal vs RAS abnormal. All the results were presented in Figure Lastly, we further analyzed the impact of RAS expressions in AML patients of different clinical stages, we further detected RAS expressions in 51 patients who achieved CR after induction therapy and 21 relapsed patients. As was shown in Figure KRAS expression rather than NRAS expression in CR time tended to decrease compared to newly diagnosis time, and both KRAS and NRAS expressions were significantly increased when in relapse time.To observe the dynamic change of RAS are identified in -30% of human cancers especially in pancreatic cancer, lung cancer, and colon cancer [RAS mutations, usually in KRAS and NRAS, are also frequent events in myeloid malignancies, and have been detected in 3% to 40% of myelodysplastic syndrome (MDS) and AML [KRAS and NRAS mutations were identified in 4% and 8% AML patients, which showed lower percentage compared to previous study. Possible reasons were that we only detected hot-spot mutations in RAS, or the differences in ethnics. Moreover, the prognostic value of RAS mutations in AML remains controversial. Several investigators reported RAS mutations emerged as significant predictors for improved clinical outcome [NRAS mutation was associated with adverse prognosis and increased risk of leukemia transformation in MDS [RAS mutations were associated with distinct cytogenetic subgroups, usually M4, but not correlated with prognosis in AML patients [RAS mutations in de novo AML, but did not have an independent effect on prognosis [FLT3, NPM1 and CEBPA mutations on prognosis, which might be due to the small size of patients with mutations in our cohort.The signal switch molecules RAS proteins play crucial roles in relaying growth-promoting signals from cell surface receptors –7. Oncogn cancer –7. Signi and AML –7. In th outcome . Meanwhin in MDS . Converspatients , 12. OurRAS expressions in AML, and found that both KRAS and NRAS overexpression were common events more frequently than RAS mutations in de novo AML patients. Notably, our study did not find significant association of RAS overexpression with RAS mutations. Similarly, a recent report also showed KRAS mutation did not correlate with mRNA expression [RAS overexpression and mutations were independent events contributing to the pathogenesis of AML. Moreover, RAS overexpression rather than RAS mutations might play more critical roles in leukemogenesis. The underlying mechanism during leukemogenesis caused by RAS overexpression needed further studies. Interestingly, we observed that KRAS overexpression was associated with U2AF1 mutations, whereas NRAS overexpression was associated with IDH1/2 mutations. As is well known, RNA splicing factor gene U2AF1 and isocitrate dehydrogenase gene IDH1/2 mutations are recurrent in de novo AML especially in CN-AML, and have a prognostic impact on assessing treatment outcome [RAS overexpression with these gene mutations. No functional studies were found to verify our results. In addition, due to the limited cases of RAS mutations in our cohort, perspective studies in a larger cohort of AML patients are required to confirm these findings, and further reveal the underlying molecular mechanism.In the present study, we mainly focused on pression . These r outcome , 15. HowRAS expressions on clinical outcome of AML. Firstly, a negative effect of KRAS overexpression was observed among whole-cohort AML and CN-AML patients. The results indicated that KRAS activation may be associated with chemoresistance in the induction therapy of AML. Although there were no functional studies to validate the hypothesis in AML, several investigations revealed the oncogenic role of KRAS abnormalities (overexpression or mutation) were associated with resistant to anticancer drug treatments phenomenally and/or mechanically [KRAS overexpression was a prognostically adverse predictor in CN-AML patients. More importantly, KRAS expression may increase the power in predicting prognosis when combined with other molecular alterations such as NRAS expression or NRAS/KRAS mutations. The prognostic value of KRAS expression has been determined in various cancers. For instance, Chen et al disclosed that KRAS overexpression predicted poor prognosis in patients with colorectal cancer [RAS expressions as an independent indicator of patient outcomes in lung cancer treated with bevacizumab plus chemotherapy [RAS expressions could be used as a biomarker for monitoring disease treatment and recurrence in AML. Collectively, these above results emphasized a more crucial role of KRAS from RAS family in the process of leukemogenesis, and could act as a potential therapeutic target for designing cancer gene therapy.Importantly, our study revealed the impact of anically –18. Secol cancer . Moreoveotherapy . ThirdlyRAS overexpression and mutations are common events in AML with potential therapeutic target value. KRAS overexpression independent of RAS mutations confers an adverse prognosis in CN-AML.Taken together, our findings reveal that de novo AML patients at newly diagnosis time, 51 AML patients at CR time, and 21 AML patients at relapse time. The diagnosis and classification of AML patients were established according to the 2008 World Health Organization (WHO) criteria. BM samples from 30 healthy donors were collected as controls. The separation of BM mononuclear cells (BMMNCs) was performed using Lymphocyte Separation Medium  and washed twice with PBS.This study was approved by the Institutional Review Board of the Affiliated People’s Hospital of Jiangsu University. After written informed consents were obtained from all participants, bone marrow (BM) was collected from 143 All the AML patients received chemotherapy including induction therapy and subsequent consolidation treatment as reported in our previous literature , 22. ForKaryotypes were analyzed at the newly diagnosis time by conventional R-banding method and karyotype risk was classified according to reported previously .Total RNA was extracted from the BMMNCs using Trizol reagent . The synthesis of cDNA was performed by reverse transcription as reported .KRAS and NRAS expressions were detected by real-time quantitative PCR (RQ-PCR) using AceQ qPCR SYBR Green Master Mix . The primers of KRAS and NRAS expressions were used as reported [ABL was used to calculate the abundance of KRAS and NRAS mRNA. The detection of ABL expression was performed with primers as reported [2O) were included in each assay. Relative KRAS and NRAS expressions levels were calculated using 2-∆∆CT method.reported , 26. PCRreported . Both poKRAS and NRAS were screened using high-resolution melting analysis (HRMA) as reported [NPM1, C-KIT, DNMT3A, IDH1, IDH2, and U2AF1 were also detected by HRMA [FLT3-ITD and CEBPA mutations were examined by DNA sequencing  [Genomic DNA was isolated from BMMNCs using genomic DNA purification kit . The hot-spot mutations  of reported . All pos by HRMA –33, wher, China) , 35.http://www.ncbi.nlm.nih.gov/geo/; accession number GSE12417) were applied to analyze the prognostic impact of KRAS and NRAS expressions using the online web tool Genomicscape  [Two independent cohorts of CN-AML patients (78 and 162 patients) from GEO data  , 37.KRAS expression and NRAS expression was analyzed by Spearman test. The impact of KRAS and NRAS expressions on prognosis was determined by Kaplan-Meier and Cox regression analyses. All the statistical analyses were performed through SPSS 20.0 software package. For all analyses, a two-tailed P value less than 0.05 was determined as statistically significant.Mann-Whitney’s U test and Pearson Chi-square/Fisher exact test were employed to compare the difference of continuous and categorical variables between two groups. The relationship between


We identified a heterozygous mutation c.772_773delGTinsA in exon 4 resulting in the substitution of amino acid valine  with amino acid arginine (Arg) and the premature pause of the translation 46 amino acids later . Functional assay showed that cells transfected with the mutant CaSR had a significantly poorer response to extracellular Ca2+ stimulation compared with the WT. We have shown that the c.772_773delGTinsA mutation causes a significant alteration of CaSR function leading to features of FHH in an affected young infant since the first months of life.Familial hypocalciuric hypercalcemia (FHH) is an autosomal dominant disorder, associated with inactivating mutations of the calcium-sensing receptor (CaSR). To evaluate the functional significance of a CaSR mutation, identified in a young infant who presented with hypercalcemia and hypocalciuria. The CaSR gene coding sequences were analyzed by polymerase chain reaction amplification and direct sequencing analysis. The mutation identified was introduced by site-directed mutagenesis into a wild-type (WT) CaSR plasmid, and human embryonic kidney 293 T cells were transfected with either the WT or mutant CaSR. The function of the mutated CaSR protein was analyzed by evaluating the free intracellular calcium [(Ca Familial hypocalciuric hypercalcemia (FHH) is associated with inactivating mutations of calcium-sensing receptor (CaSR) gene. FHH, in most of the cases, is a benign condition featured by asymptomatic hypercalcemia, however, diagnosis is essential in order to avoid unnecessary parathyroidectomy.We describe the identification of c.772_773delGTinsA mutation of CaSR and the impact on the clinical phenotype in a young infant harboring the mutation. Our functional analysis shows that c.772_773delGTinsA mutation is associated with significant impairment of CaSR function leading subsequently to the phenotype of FHH.2+) levels within a narrow range. Ca2+ is a mineral that plays a pivotal role in bone mineralization as well as in other physiological cell functions like intracellular signal transduction, hormone secretion, neurotransmitter release, and muscle cell contraction. The receptor recognizes any fluctuations in extracellular Ca2+ concentrations and finally modulates the parathyroid hormone (PTH) synthesis/secretion and Ca2+ renal reabsorption  is a G protein-coupled cell surface receptor expressed abundantly in parathyroid chief cells  and renasorption .The human CaSR gene located in chromosome 3q13.3-21 contains 7 exons and encodes for a 1078 amino acid sequence (NM_000388). Exon 1 is untranslated, while exons 2-6 encode for a large extracellular N-terminal domain (ECD) and exon 7 for the seven transmembrane domains (TMDs) and the intracellular carboxy-terminal domain (ICD) . Loss-of2+, shifting the set point of Ca2+-dependent PTH secretion to the right , accompanied by normal phosphorus , alkaline phosphatase , and albumin levels , while magnesium levels were slightly elevated . The 24 h urinary Ca2+ excretion was reduced with a significantly low Ca2+ to creatinine clearance ratio (CCCR) . PTH levels were normal , while vitamin D levels were at the lowest range {25-hydroxyvitamin D [25(OH)D]: 21 ng/mL; normal range: 10-80 ng/mL}. Her mother’s laboratory evaluation revealed severe hypovitaminosis D [25(OH)D: 4 ng/mL] with normal Ca2+ , P , ALP , and PTH levels . It is interesting to note that the infant was exclusively breastfed and did not receive any vitamin D supplementation. Her father’s Ca2+ levels were at the upper normal range (Ca2+: 10.3 mg/dL), with normal P (2.66 mg/dL) and ALP (85 U/L). With the suspicion of FHH, mutational analysis of the CaSR gene was performed.A 4.5-month-old female infant of Greek origin was referred to our department in December of 2011 for evaluation of hypercalcemia detected in laboratory investigations performed during her hospital admission for urinary tract infection. She was born at term via normal vaginal delivery to healthy unrelated parents. She was growing well, her weight was at the 50Genomic DNA was isolated from whole blood using the Purelink Genomic DNA kit . Exons 2-7 of CaSR gene (NM_000388) and their respective flanking regions were amplified, and polymerase chain reaction (PCR) products were sequenced on ABI 310 . Primers and PCR conditions are available upon request. Informed consent was obtained from the parents of the patient in order to pursuit the genetic analysis.The human CaSR cDNA cloned in the pCR 3.1 plasmid (pCR3.1/hCaSR) (Invitrogen) was kindly provided by Dr. Lia Baldini, University of Limoges, France. The GTinsA mutation was directly introduced to the wild-type (WT) CaSR plasmid to generate the mutated receptor using the QuickChange Site-Directed Mutagenesis kit  according to the manufacturer’s instructions. The set of primers overlapping the target regions of the WT cDNΑ were: 5’-CAGCATGTGGTAGAGAGATTCAAAATTCCAC-3’ and 5’- GTGGAATTTTGAATCTCTCTACCACATGCTG-3’.2. The cells were transiently transfected with CaSR cDNA (12 µg) in 10 cm tissue culture plates, using calcium phosphate DNA precipitates. Ca2+ measurements were performed 60 h post transfection.Human embryonic kidney (HEK) 293 T cells were cultured in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 µg/mL), and 2 mM L-glutamine in a humidified incubator at 37 oC and a steady supply of 5% CO2PO4, 1.2 mM MgSO4, 2 mM CaCI2, 6 mM D-glucose, and 25 mM HEPES-NaOH (pH 7.4)] containing 2% FBS counted in a hemocytometer and loaded with Fura 2-AM dye (5.0 µM), for 30 min at RT in the dark. Following loading, the cells were washed twice with CaCl2- deprived KRH and re-suspended in CaCl2-free KRH supplemented with 250 mM sulfinpyrazone to prevent dye leakage. Cell aliquots (1.5x106) were transferred to a thermostatted cuvette (37 oC), maintained under continuous stirring, and analyzed in a Perkin-Elmer LS-55. The basal levels of (Ca2+)i were measured upon addition of 1 mM EGTA and presented as nM. To investigate alterations in CaSR function, CaCl2 (3 mM) was reintroduced into the medium, and (Ca2+)i increase was recorded.Sixty hours post transfection, cells were loaded with Fura 2-AM as already described (10). Cell pellets were suspended in Krebs-Ringer-HEPES (KRH) buffer i increase was recorded as the net nM difference between the peak of (Ca+2)i and the base-line (Ca+2)i measurements. In 2+)i of 118.6±5.590 nM. The cells harboring the c.772_773delGTinsA mutation exhibit significantly lower (Ca2+)i (71.42±3.977 nM), p<0.01. Moreover, stimulation with exogenous addition of CaCl2 resulted in a significantly poorer response of the c.772_773delGTinsA over-expressing cells (17.33±0.7028 ΔnM) in comparison to WT (27.77±3.870 ΔnM), p<0.05.As already described, the WT and the mutant CaSR were transiently expressed in cultured HEK 293 T cells. The induction of  , however2+)i influx after stimulation with exogenous Ca2+ compared with the WT. According to the known intracellular signaling pathway of CaSR, in response to binding of ligands to the receptor, the TMDs couple to G proteins and elicit several intracellular signaling cascades (2+ concentration stimulates the release of (Ca2+)i from the intracellular stores which acts as a secondary messenger with different cell-dependent actions . To our knowledge, 8 nonsense CaSR mutations leading to a shorter CaSR protein have been reported in the literature. These mutations have been described in exons 2 (1 mutation), 4  either primary, secondary, or tertiary. The majority of cases of HPT are sporadic (95%), while only 5% are associated with a hereditary syndrome. Inheritable disorders include NSHPT, isolated familial HPT, multiple endocrine neoplasia syndromes , HPT-jaw tumor syndrome, familial hypercalciuric hypercalcemia, and FHH (2+ levels (Disorders of Ca and FHH . In the  and FHH . A recen and FHH . In part+ levels . Our pat+ levels . Further+ levels ,25,26 thHence, genetic evolution and the identification of mutations of the CaSR gene helped clinicians to distinguish FHH from other causes of HPT as reliable distinction is not always possible on clinical grounds. The aforementioned consensus report indicates that the diagnosis of hereditary HPT should be confirmed by genetic analysis and followed by genetic counseling of both patient and relatives. The diagnosis of FHH should be excluded in all patients before scheduling parathyroidectomy . HoweverNevertheless, it is worth mentioning that CaSR mutations account for only 65% of FHH . Recentl2+ leading to typical features of FHH. This is the second family of FHH reported from Greece and, interestingly, both families bear the same mutation. Molecular analysis of the CaSR gene, especially in patients who present early in life with hypocalciuric hypercalcemia, can establish the diagnosis of FHH and facilitate the subsequent clinical management.In the present study, we showed that the Val258ArgfsTer47 mutation in the CaSR gene identified in a young patient with hypercalcemia and her father resulted in a significant reduction of CaSR sensitization to extracellular CaInformed Consent: Informed consent was obtained from the parents of the patient in order to pursuit the genetic analysis.Peer-review: Externally peer-reviewed.


Treatment of CLL mice harboring TCL1-Tg:−/−p53 genotype with PEITC significantly prolonged the median survival time of the animals. Our study identifies a vulnerability of p53-null CLL cells with high sensitivity to ROS-generating agents, and suggests that PEITC may potentially be useful for clinical treatment of CLL with 17p deletion and p53 mutations.Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries. Loss of p53 function in CLL cells due to chromosome 17p deletion or p53 mutations often leads to a more malignant disease phenotype and is associated with drug resistance and poor clinical outcome. Thus, development of novel therapeutic strategies to effectively target CLL cells with p53 deficiency is clinically important. Here we showed that p53-null CLL cells were highly sensitive to ROS-mediated cell killing due to their intrinsic ROS stress. We further demonstrated that a natural compound phenethyl isothiocyanate (PEITC) was able to effectively kill CLL cells with loss of p53, even under the protection of stromal cells. In p53-defficient CLL cells, PEITC induced a rapid depletion of glutathione and a severe accumulation of ROS, leading to massive leukemia cell death in the stromal microenvironment. The drug-induced cell death was associated with a significant decrease of in MCL-1 survival molecule. We further showed that ROS-mediated cell death was the key mechanism by which PEITC induced cytotoxicity, since such cell death could be prevented by addition of antioxidant NAC. Importantly,  In the miR-16-1 .Considering the important role of loss of p53 in cancer development and drug resistance in CLL cells, it is important to develop new therapeutic strategies that are effective in eliminating p53-null CLL cells based on their biological properties. One noticeable biochemical feature of CLL cells is their intrinsic high ROS stress –13, whicin vitro assay with primary leukemia cells isolated from CLL patients with 17p-deletion and in vivo test in a CLL mouse model with TCL1-Tg:p53−/− genotype [In vivo, PEITC exhibited significant therapeutic effects and significantly improved the overall survival of the p53−/− CLL mice.Based on the above observations, we hypothesized that CLL cells with loss of p53 function might be more vulnerable to further oxidative stress, and targeting ROS stress might be an attractive therapeutic strategy for treatment of CLL with 17p-deletion and/or p53 mutations. The main goal of this study was to test the possibility to use PEITC as a potential agent to effectively eliminate CLL cells with loss of p53, using both genotype . We demoin vitro study) and Oxaliplatin, were used for comparison with PEITC. As shown in Figure We first tested the cytotoxic effect of PEITC against primary CLL cells with 17p deletion isolated from the peripheral blood samples of CLL patients. Two standard anti-CLL drugs, Fludarabine . As shown in Figure + CLL cells with 17p deletion were relatively resistant to F-ara-A and Oxaliplatin , but remained highly sensitive to PEITC .We also used purified CD19+ CLL cells form patient samples to further confirm the above observations. CD19+ B-cells were isolated from three 17p- CLL blood samples, using CD19 antibody-coated microbeads. The purified CLL cells were then incubated with F-ara-A, Oxaliplatin, or PEITC in co-culture with stromal cells. As shown in Figure We also tested if PEITC could induce autophagy, which could play a role protecting cells under certain stress conditions. CLL cells isolated from four different CLL patient samples were incubated with various concentrations (1–20 μM) of PEITC for 24 h, and Western blotting was performed to analyze the expression of autophagy marker LC3. As shown in TCL1-Tg:p53−/− genotype to further test the ability of PEITC to kill leukemia cells with defined p53 deletion in vitro and in vivo. We previously showed that this mouse model resembles human CLL patients with 17p-deletion with aggressive disease progression and drug-resistant phenotype [TCL1-Tg:p53−/− mice (age 4-month) and co-cultured with mouse stromal cells (Kusa-H1 cells). The cells were treated with 5 μM PEITC for 24 h, 10 μM of F-ara-A for 48 h, or 10 μM Oxaliplatin for 48 h, and then analyzed by cell viability assay. We found that these cells were resistant to F-ara-A treatment (76% viable cells compared to 80% viable cells in the untreated control sample) and partially resistant to Oxaliplatin (42% viable cells). They were also resistant to Bendamustine  assay. As shown in Figure in vivo.To test the ability of PEITC to induce leukemia cell death TCL1-Tg:p53−/− mice, and incubated them with PEITC. Shortly after drug incubation, intracellular ROS was measured by flow cytometry, which revealed a substantial increase of cellular ROS in the PEITC-treated cells 90 minutes after PEITC treatment. This was consistently observed in both human primary CLL cells , PEITC, or their combination. NAC consistently prevented PEITC-induced ROS accumulation Figure , GSH depTCL1-Tg:p53−/− mice [Because MCL-1 is a ROS-sensitive anti-apoptotic protein that plays an important role in the survival of CLL cells  and is u−/− mice , we test−/− mice . In contin vitro and in leukemic splenocytes in CLL mice, we further evaluated the in vivo therapeutic activity of PEITC in CLL mice with TCL1-Tg:p53−/− genotype. PEITC was first formulated in a stable nanoparticle formulation using the pluronic F127 as the carrier as described in Methods. To ensure that such formulation did not cause loss of anticancer activity, we first compared the in vitro activity of the original PEITC and the formulated PEITC for their ability to kill both primary human CLL and mouse leukemic cells. As shown in Figure in vitro.Based on the observations that PEITC was able to induce significant CLL cell death in primary human CLL cells in vivo antitumor activity of the formulated PEITC in CLL mice with TCL1-Tg:p53−/− genotype. These mice developed leukemia with aggressive disease progression, resembling human CLL disease with 17p deletion [TCL1-Tg:p53−/− mice with a median survival time of 5.3 months (data not shown). These results together strongly suggest that PEITC was highly effective in killing p53-deficient CLL cells in vitro, and exhibited promising therapeutic activity in vivo in the TCL1-Tg:p53−/− mouse model.We then evaluated the deletion . WithoutTCL1-Tg:p53−/− genotype as experimental models. PEITC was used to induce ROS stress through its ability to cause rapid glutathione depletion, which is known to effectively kill K-Ras transformed ovarian cancer cells and F-ara-A-resistant CLL cells through a ROS-mediated mechanism [Metabolic alterations including altered nutrient metabolism and elevated ROS generation are among the hallmarks of cancer . Geneticechanism , 15, 21.in vitro and in vivo. We further confirmed that the key mechanism of PEITC action against p53-null CLL cells was mediated by induction of a rapid depletion of GSH and severe accumulation of ROS. This was evidenced by direct measurement of GSH  was purchased from Sigma (USA). Fludarabine used in the survival experiment was purchased from the Pharmacy of MD Anderson Cancer Center. PEITC was dissolved in Dimethyl Sulfoxide (DMSO) to make a 10 mM stock solution and PEITC working solution was freshly prepared by diluting the stock solution in culture medium. Ficoll-lite Lympho H was from Atlanta Biological . CD19 microbeads were purchased from MACS Miltenyi Biotech Inc. . ACK lysis buffer and Annexin V-FITC were from BD Biosciences . CM-H2DCF-DA was purchased from Invitrogen . Terminal deoxynucleotidyl transferase deoxyuridine-triphosphatase nick-end labeling (TUNEL) staining kit was purchased from Roche Applied Science . A glutathione assay kit was purchased from Cayman Chemical . Anti-MCL-1 was from Santa Cruz Biotechnology . Anti-LC3 was purchased from EMD Millipore Corporation . Anti-β-actin was purchased from Cell Signaling Technology Inc. .PEITC, F-ara-A, N-acetylcysteine (NAC), Oxaliplatin, Propidium Iodide (PI) and Ethyl Acetate (Et Ac) were purchased from Sigma-Aldrich . PluronicTCL1-Tg:p53−/− mice were treated with (100 mg/kg) formulated PEITC twice a week through tail vein injection (i.v.). Treatment began when the mice reached the age of 8 weeks.PEITC (20 mg) was dissolved in EtAc (1 ml) to form PEITC solution, which was introduced into 2 mL of pluronic F127 water solution at the concentration of 5% (w/w) under stirring by T10 homogenizer  in ice bath. About 30 min later, well-mixed oil in water (O/W) emulsion was formed. Then, EtAc was evaporated in rotator evaporator  at 4°C for 4–6 hours and the PEITC nanoparticles were obtained. The PEITC nanoparticles slurry was lyophilized and the powder was stored at 4°C for future use. All the procedures were performed in the environment direct light exposure. TCL1-Tg:p53−/− mice, and red blood cells were removed from the splenocytes by ACK lysis buffer. After lysis of red blood cells, the splenocytes were washed with RPMI1640 culture medium and PBS. On the same day of isolation, the mouse splenocytes were co-cultured with murine stromal cells (Kusa-H1) and treated with F-ara-A or Oxaliplatin for 48 h or PEITC for 24 h. Cell viability and cellular sensitivity to drug treatment in vitro were determined by flow cytometry after double staining of 1 × 106 cells with annexinV-FITC and PI as previously described [Primary leukemia cells (white blood cells) were isolated from the peripheral blood samples of CLL patients diagnosed according to the NCI criteria . Proper escribed .TCL1-Tg:p53−/− mice were generated and maintained as described [In Situ Cell Death Detection kit (Roche) according to manufacturer's instruction, and visualized using a fluorescent microscope.escribed . Mouse sMCL-1 protein levels were determined by Western blotting as described .5 cells) with 1 μM CM-H2DCF-DA for 60 minutes and analyzed by flow cytometry as described [Cellular ROS contents were measured by incubating CLL cells  was used to measure cellular glutathione. After preparing cell extracts by sonication and deproteination, GSH was determined as described .t test was used for testing the statistical difference between two groups of samples. Mouse survival curves (Kaplan-Meier plots) were generated by Graphpad Prism software  as described [p value of less than 0.05 was considered statistically significant.Student's escribed , and the


WT1 rs16754 polymorphism was also significantly associated with RFS in AML . In the subgroup analyses of age, race, and subtype of AML, WT1 rs16754 polymorphism was a independent favorable-risk marker. In conclusion, WT1 rs16754 polymorphism is associated with better survival of AML. It could be used as a cost-effective prognostic biomarker for AML.Although a number of studies suggested that WT1 rs16754 polymorphism might be related to decreased relapse free survival (RFS) and overall survival (OS). The results remain controversial. Published reports were searched in PubMed, EMBASE, and Google Scholar. Twelve publications with 3903 patients had met the inclusion criteria and were subjected to further examination. We found WT1 rs16754 polymorphism was significantly associated with OS in AML (OR = 0.62; 95% CI 0.52 − 0.75;  Acute myeloid leukemia (AML) is a serious disease of the hematopoietic system characterized by de-differentiation and uncontrolled proliferation of immature hematopoietic precursor cells in the bone marrow . It is uThe Wilms’ tumor gene 1 (WT1), located at chromosome 11p13, was firstly cloned in 1990 as a suppressor in Wilms’ tumor . Since tAlthough a number of studies suggested that WT1 rs16754 polymorphism might be related to decreased relapse free survival (RFS) and overall survival (OS). The results remain controversial . In thisThe flow chart in Figure p < 0.00001; Figure p = 0.004; and OR = 0.60; 95% CI 0.44 − 0.81; p = 0.001). In addition, both de novo AML and CN-AML showed better OS in AML . In a further stratified analysis by age, both children and adults showed better OS in AML . The results were showed in Table When all eligible studies were pooled into one dataset for the meta-analysis, we found WT1 rs16754 polymorphism was significantly associated with OS in AML . In terms of AML subtype, both de novo AML and CN-AML displayed better RFS in AML . In the stratified analysis by age, a significantly better RFS was found among children , and was also found among adults . The results were listed in Table As for RFS of AML, WT1 rs16754 polymorphism was found to be significantly associated with RFS  study design was prospective or retrospective cohort study; (2) the exposure of interest was WT1 rs16754 polymorphism; (3) the studied reported relative risks (RRs), hazard ratios (HRs), or odds ratios (ORs) with corresponding 95% confidence intervals (CIs); and (4) the outcome was RFS or OS. If the same cohort was used in more than one publication, we included the publication that reported the results in greater detail or, if similar, the one with the largest number of cases. Data published only in abstract form were excluded. Case reports, review articles and commentary articles were also excluded.The following data including: the first author, year, study design, ethnicity of the study population, age, sex, subtype, number of patients included in analysis, adjusted factors, and OR with its 95% CI for OS and RFS were extracted from each eligible study by two investigators.The strength of association between WT1 rs16754 polymorphism and RFS and OS of AML was evaluated by calculating the OR and 95% CI. To assess the significance of OR, we conducted the Z test and we regarded it as significant difference when P value less than 0.05 was detected. Moreover, X2 based Q and I2 test were performed to evaluate the between-study heterogeneity and P<0.1 was defined as statistical significance. The random-effects was used to calculated the OR if significant heterogeneity existed. Otherwise, the fixed-effects model was applied. Furthermore, we also conducted subgroup analyses stratified by ethnicity (Caucasian and Asian), age (child and adult), and subtype of AML. Publication bias was assessed by asymmetry of funnel plots. In sensitivity analysis, we sequentially excluded each study on the software to evaluate the stability of the results. We conducted all the analyses by using software Review Manager 5.1  and STATA 11.0 software .