This form of Ca2+ influx occurs at discrete loci where peripheral endoplasmic reticulum juxtaposes the plasma membrane. Stimulation evokes numerous STIM1-Orai1 clusters but whether distinct signal transduction pathways require different cluster numbers is unknown. Here, we show that two Ca2+-dependent transcription factors, NFAT1 and c-fos, have different requirements for the number of STIM1-Orai1 clusters and on the Ca2+ flux through them. NFAT activation requires fewer clusters and is more robustly activated than c-fos by low concentrations of agonist. For similar cluster numbers, transcription factor recruitment occurs sequentially, arising from intrinsic differences in Ca2+ sensitivities. Variations in the number of STIM1-Orai1 clusters and Ca2+ flux through them regulate the robustness of signalling to the nucleus whilst imparting a mechanism for selective recruitment of different Ca2+-dependent transcription factors.Store-operated Ca Endoplasmic reticulum Ca2+ sensing STIM proteins and plasma membrane Orai1 channels together regulate Ca2+ influx in eukaryotic cells. Here authors show that two Ca2+-dependent transcription factors, NFAT1 and c-fos, have different requirements for the number of STIM1-Orai1 clusters and on the Ca2+ flux through the clusters. Architecturally simpler cells are also punctuated with membrane signalling complexes, suggesting these clusters have a fundamental role in signal transduction.Formation of signalling protein hubs physically associated with membranes is an evolutionarily conserved mechanism throughout the taxonomic ranks that serves to increase the rate and efficiency of transduction pathways. In geometrically complex or polarized cells such as neurons and pancreatic acini, signalling complexes maintain asymmetric vectorial ion transport and compartmentalised responses2+ release-activated Ca2+ (CRAC) channels represent a striking example of membrane protein clustering. CRAC channels activate upon a loss of Ca2+ from within the lumen of the endoplasmic reticulum (ER), which occurs following stimulation of cell-surface receptors that generate the Ca2+-releasing second messenger inositol trisphosphate2. The fall in ER Ca2+ is directly sensed by STIM proteins4, which then oligomerise6 and translocate to within 20 nm of the plasma membrane. At these ER-plasma membrane junctions, clusters of STIM protein tether to and gate the plasma membrane protein Orai1, the pore-forming subunit of the CRAC channel9. The ensuing Ca2+ flux controls a salmagundi of responses, including exocytosis, metabolism, motility, gene expression and cell growth and differentiation10.Store-operated Ca2+ microdomains near open CRAC channels activate downstream signalling pathways, including certain isoforms of adenylyl cyclase11, plasma membrane Ca2+ ATPase pumps12, Ca2+-dependent phospholipase A213 and endothelial NO synthase14. These enzymes are located close to the CRAC channel and, at least for adenylyl cyclase isoform type 8, direct binding to Orai1 has been reported11. Ca2+ microdomains near CRAC channels also increase gene expression through activation of Ca2+-dependent transcription factors c-fos and nuclear factor of activated T cells (NFAT). Transcription of the immediate early gene c-fos is stimulated following activation of the non-receptor tyrosine kinase Syk by local Ca2+ entry through CRAC channels15. Co-immunoprecipitation and immunocytochemical studies demonstrate that Syk associates with Orai116. Syk phosphorylates the transcription factor STAT515, which dimerises and migrates to the nucleus to regulate c-fos transcription17. NFAT is extensively phosphorylated at rest and trapped within the cytosol18. Dephosphorylation by the Ca2+-activated phosphatase calcineurin exposes a nuclear localisation sequence, enabling NFAT migration into the nucleus18. A fraction of cellular NFAT and calcineurin are held at the plasma membrane through association with the scaffolding protein AKAP-7920. Upon store depletion, AKAP-79 interacts with Orai1, resulting in activation of calcineurin by Ca2+ microdomains near the open channels.Ca2+, are fewer puncta each with large Ca2+ influx equally effective in activating signalling pathways as many puncta with reduced Ca2+ flux? Previous work has shown that, for a similar number of functional CRAC channels, channel clustering leads to more robust activation of NFAT and c-fos transcription factors16. Here, we address the second question. We find, for the same bulk Ca2+ rise, reduced Ca2+ flux through a large number of STIM-Orai1 puncta is considerably more effective in activating NFAT1 than larger flux through fewer puncta. Additionally, for similar number of puncta, larger Ca2+ flux is required to stimulate c-fos. Our data reveal that different signalling pathways can be recruited by variations in the number of channel clusters formed and, for similar numbers of clusters, by the extent of Ca2+ flux through the channels.The autonomous assembly of functional CRAC channels from STIM1 and Orai1 components into numerous yet discrete puncta raises two important questions. First, is there a signalling advantage conferred by channel clustering over a similar number of dispersed channels? Second, for the same rise in bulk cytosolic Ca2+ content falls below a threshold21, STIM1 redistribution and CRAC current increase as a function of store depletion  pump24. Strong store depletion with a high dose of thapsigargin leads to formation of numerous STIM1 puncta below the plasma membrane whereas weaker store depletion evokes less accumulation of peripheral STIM122. The electrical driving force can be manipulated by varying the external K+ concentration, since RBL cells express numerous inwardly rectifying K+ channels25. In standard K+ (2.8 mM) solution, the membrane potential is ~ −80 mV but depolarises close to ~0 mV in high K+ (100 mM) solution. Therefore, a high dose of thapsigargin in high K+ solution will induce numerous STIM1-Orai1 puncta but unitary Ca2+ flux will be low. Conversely, in the presence of a low thapsigargin concentration in standard external K+ solution, fewer puncta will form but the unitary Ca2+ flux will be relatively large. Careful selection of appropriate thapsigargin concentrations should, therefore, enable comparison of the signalling power of few versus many puncta, with both producing the same rise in bulk cytosolic Ca2+  with cells exposed to 30 nM thapsigargin for the same time but in the continuous presence of Ca2+-free solution . Two micromolar or 100 nM thapsigargin elicited relatively large and prolonged elevations in cytosolic Ca2+ whereas Ca2+ signals to 30 nM thapsigargin in standard K+ solution or 100 nM thapsigargin in high K+ external solution were significantly smaller . Following stimulation with thapsigargin in the presence of external Caity Fig. .+ solution led to significantly more NFAT nuclear accumulation  led to robust nuclear accumulation within 40 min Fig. . Applicaion Fig. .Fig. 3Ef+ solution with 100 nM thapsigargin in high K+ solution showed a striking difference in pattern , only NFAT was activated.We measured c-fos expression using quantitative PCR (qPCR), stimulating cells with thapsigargin for the same duration as used for NFAT migration. Although 100 nM thapsigargin evoked a significant increase in c-fos mRNA Fig. , very smion Fig. . We alsoels Fig. , but neiase Fig. . ActivatFAT Fig. . Althoug+ solution, whereas c-fos is not. As we measured NFAT1 and c-fos activities using different techniques (fluorescence microscopy versus qPCR), one explanation for our results could reflect a possible difference in sensitivity between the two methods. To address this, we compared c-fos and NFAT activities using the same experimental techniques.The preceding data suggest that NFAT1 is robustly activated by 100 nM thapsigargin in high K+ solution with 30 nM thapsigargin in standard K+ solution using qPCR. In RBL cells, Ca2+ entry through CRAC channels increases interleukin-5 (IL-5) expression through a pathway absolutely dependent on NFAT activity27, although the increase in IL-5 mRNA is modest. Pre-treatment with the phorbol ester phorbol 12-myristate 13-acetate (PMA) increases Ca2+-dependent IL-5 expression considerably28. To increase the bandwidth of IL-5 detection, we stimulated cells with thapsigargin following exposure to phorbol ester. Phorbol ester alone failed to increase IL-5 levels . We surmise that this reflects, at least in part, that qPCR measurements of IL-5 transcription reflect the combination of responders and non-responders in the population, as well as slower NFAT nuclear dynamics to 30 nM thapsigargin16.First, we compared c-fos and NFAT-driven gene expression to 100 nM thapsigargin in high Kion Fig. . We repeion Fig. , consistion Fig. . Therefoeus Fig.  and subseus Fig. . Howevereus Fig. , this fa+ solution failed to increase nuclear levels of STAT5-GFP, compared with non-stimulated cells . We co-transfected YFP (under a c-fos promoter) and RFP (under an NFAT promoter). Little expression of either protein occurred under resting conditions Fig. , when coion Fig.  but led ion Fig. . The medYFP Fig. . By contYFP Fig. . Whereas+ solution activates NFAT-dependent gene expression more robustly than c-fos in the same cells and that 100 nM thapsigargin in high K+ solution is a stronger stimulus for NFAT activation than 30 nM thapsigargin in standard K+ solution. In these experiments, we measured protein expression ~12 h after stimulation. We considered the possibility that c-fos expression might have already declined considerably by this time point. We therefore measured c-fos-YFP and NFAT-dependent RFP protein expression over different times. Both proteins had increased significantly by 6 h  suggest that the NFAT activation pathway has a higher sensitivity to local Ca2+ than c-fos, despite both being dependent on Ca2+ microdomains near CRAC channels. A prediction would, therefore, be that NFAT should be activated more strongly than c-fos in the presence of lower external Ca2+ concentrations, since the latter condition produces Ca2+ microdomains of smaller size29. We applied different external Ca2+ concentrations (0.1–2 mM) to cells pre-stimulated with 2 μM thapsigargin in Ca2+-free solution , confocal images showed that nuclear accumulation of NFAT1-GFP increased following stimulation with 2 μM thapsigargin in 0.1 mM external Ca2+ and this became considerably stronger in 0.5 mM Ca2+ and 2 mM Ca2+ . Histograms are shown for the NFAT1 nuclear/cytosolic ratio in unstimulated resting cells  in external Ca2+ after cells had been challenged with thapsigargin/0Ca2+/La3+ increased NFAT translocation . Stimulation with sub-maximal concentrations of LTC4 elicited a series of cytosolic Ca2+ oscillations, which run down in Ca2+-free solution . The relationship between agonist concentration and the rate of Ca2+ entry, shown in Fig. 50 of 10 nM and a Hill coefficient of ~1.Based on the preceding data, we reasoned that lower concentrations of agonist, which induce fewer Caion Fig. . Readmis4 evoked dose-dependent nuclear accumulation of NFAT1-GFP  was also dose-dependent , NFAT migration distributed into either non-responders or responders, with the latter exhibiting responses that were quantitatively similar to those induced by maximally effective stimuli. The population average showed nuclear/cytosolic ratios that were intermediate between those seen in unstimulated cells and in maximally stimulated cells, but this reflected the ratio of non- and maximal responders rather than a graded range of responding cells. Store-operated Ca2+-free solution containing La3+. Although the distribution of NFAT between nucleus and cytosol in thapsigargin/0Ca2+/La3+ was qualitatively similar to that evoked by maximal thapsigargin concentration, the mean response was ~50% of the latter despite being unimodal. One possible explanation is that the bulk Ca2+ rise induced by thapsigargin/0Ca2+/La3+ raises local Ca2+ uniformly in the vicinity of AKAP79/Orai1 but to a lesser extent than that seen when Ca2+ microdomains near CRAC channels are activated by thapsigargin in the presence of external Ca2+.NFAT activation was unimodal following stimulation with high concentrations of thapsigargin, reflecting strong responses from all cells in the population. A somewhat milder response was obtained in response to challenge with thapsigargin in CaE. coli switch between different phenotypes in a stochastic manner. For those bacteria with the lac operon, a switch from a bimodal to a unimodal distribution has been found in a manner dependent on inducer intensity36. Our results demonstrating a switch from bimodal to unimodal distribution depending on stimulus strength extend the findings of Choi et al.36 to a mammalian system.Genetically identical 2+ flux through the channels, as dictated by their relative Ca2+ sensitivities. Variations in puncta number are determined by stimulus intensity. Flux through Orai1 is governed by channel pore properties that define unitary conductance and Ca2+ selectivity, and the prevailing electrical gradient for Ca2+ entry. Ca2+ selectivity of Orai1 is not an immutable feature of the channels but is tuned by STIM1 binding37, Single channel optical recordings using a Ca2+ indicator tethered to the Orai1 channel have revealed multiple channel open states, imparting variable Ca2+ flux through individual channels38. The electrical driving force for Ca2+ entry will depend on the activities of other open ion channels that set the membrane potential. Large fluctuations in membrane potential through Ca2+-dependent activation of non-selective cation channels have been described in T cells39 and pancreatic acini40, providing a mechanism to dynamically regulate Ca2+ flux through Orai1. Therefore, multiple regulatory mechanisms converging on the number of STIM1-Orai1 puncta that form, as well as Ca2+ flux through the channels enable the same agonist to couple Ca2+ microdomains to selective downstream signalling pathways.Our results show that transcription factors respond to different numbers of STIM1-Orai1 puncta and, for similar numbers of puncta, different transcription factors require different amounts of Ca2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were split using Trypsin-EDTA and plated onto glass coverslips for use 24–48 h later.RBL-2H3 cells were purchased from ATCC (via UK supplier LGC) and were cultured at 37 °C with 5% COSTIM1-YFP , NFAT1-GFP  and both the EGFP-based and RFP-based reporter plasmids containing an NFAT promoter  were transfected into RBL-2H3 cells using the Amaxa system. pOTTC589-pAAV c-fos Nuc-eYFP  was obtained from Addgene, deposited by Dr. Brandon Harvey.2+ measurements were carried out at room temperature using the IMAGO charged-coupled device camera-based system from TiLL photonics, as described16. Cells were loaded with Fura-2/AM in the dark for 40 min, washed and left for 15 min for further de-esterification. Cells were excited at 356 and 380 nm (20 ms exposures at 0.5 Hz) and emission was collected > 505 nm. External solution was composed of 145 mM NaCl, 2.8 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM d-glucose, 10 mM HEPES, pH 7.4 with NaOH. Ca2+-free solution was composed of 145 mM NaCl, 2.8 mM KCl, 2 mM MgCl2, 10 mM d-glucose, 10 mM HEPES, 0.1 mM EGTA, pH 7.4 with NaOH. High K+-containing external solution was composed of 100 mM KCl, 45 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 10 mM d-glucose, pH 7.4 with KOH. Ca2+ signals are plotted as R, which denotes the 356/380 nm ratio.Cytosolic Ca4 for 40 min and then fixed in 4% paraformaldehyde at room temperature. Resting (control) cells were treated identically but received no stimulus. Images were obtained using an Olympus FV1000 confocal microscope and nuclear and cytosolic distribution, obtained by drawing regions of interest of identical size, were analysed using Image J. Nuclei were co-stained with DAPI. In some experiments  or anti-c-fos antibody  overnight at 4 °C. Coverslips were then washed three times in PBS and incubated with anti-mouse Alexa 488 secondary antibody (ThermoFisher Scientific) for STIM1 (1:200) or anti-rabbit Alexa Fluor 568 (1:200) antibody (Thermo Fisher Scientific) for 1.5 h at room temperature. Coverslips were then washed in PBS, then preserved in mounting medium (Vector Laboratories). Images were taken with an Olympus FV1000 confocal microscope.After appropriate stimulation (see text), coverslips were washed twice in ice cold PBS and then fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with PBS/5% BSA/0.3% Triton. Cells were stained with anti-STIM1 antibody  and pOTTC589- pAAV c-fos Nuc-eYFP using the Amaxa system. Twenty-four hours after transfection, cells were treated with different concentrations of thapsigargin (as stated in the text) for 40 min and then washed several times in medium without stimulus. After 12 h incubation, cells were fixed with 4% paraformaldehyde and washed three times with PBS. The YFP and RFP expression in individual cells were detected using a BD X-20 flow cytometer. Data were analysed using FlowJo software.4 for 40 min (to match the measured NFAT1-GFP translocation time) in external Ca2+-containing solution. RNA was then extracted as described below. For measurement of IL-5 and c-fos from the same cell preparations, cells were pretreated with PMA (50ng/ml) for 5 min, exposed to thapsigargin and PMA for 40 min and then washed and maintained in fresh medium for a further 50 min. Thereafter, RNA was extracted using an RNeasy Mini Kit (Qiagen), as described16. RNA was quantified spectrophotometrically by absorbance at 260 nm. Total RNA (1 μg) was reverse-transcribed using the iScriptTM cDNA synthesis kit (Bio-Rad), according to the manufacturer’s instructions. Quantitative real-time RT-PCR was performed with cDNA, Taqman Universal PCR Master Mix (Applied Biosystems), H2O and specific primers for Taqman Gene expression assays . The samples were loaded into 96-well plates and analysed by the ABI Prism 7000 Sequence Detection System software (Applied Biosystems). The qPCR conditions were as follows: 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. For quantification, the relative quantities of samples were calculated according the comparative ΔCt method and normalized to β-actin.For measurement of c-fos transcription, RBL-2H3 cells were stimulated with thapsigargin or LTC21. Pipettes, pulled from borosilicate glass, Sylgard-coated and then fire-polished had resistances of 3–6 MOhms when filled with internal solution containing: 145 Cs+-glutamate, 8 mM NaCl, 2 mM MgCl2, 10 mM HEPES, 2 mM Mg-ATP, 0.3 mM EGTA, 2 mM pyruvic acid, 2 mM K-malate, 1 mM KH2PO4, pH 7.2 (CsOH). Pyruvic acid, malate and KH2PO4 were included to ensure mitochondria remained energised and therefore were able to maintain Ca2+ buffering in whole-cell recording41. External solution contained 145 mM NaCl, 2.8 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM CsCl, 10 mM HEPES, 10 mM D-glucose, pH 7.4 with NaOH. The CRAC current was measured by applying voltage ramps (−100 to + 100 mV in 50 ms) at 0.5 Hz from a holding potential of 0 mV. Currents were filtered using an 8-pole Bessel filter at 2.5 kHz and digitised at 100 μs. Capacitative currents were compensated before each ramp or step by using the automatic compensation of the EPC 9–2 amplifier. Leak currents were subtracted by averaging 3–5 ramp currents obtained shortly after break-in.Patch clamp experiments were conducted in the tight seal whole-cell configuration at room temperature (20–24 °C) as previously described+-glutamate, 8 mM NaCl, 2 mM MgCl2, 10 mM HEPES, 2 mM Mg-ATP, 0.1 mM EGTA, pH 7.2 (KOH). External solution contained 145 mM NaCl, 2.8 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 10 mM d-glucose, pH 7.4 with NaOH. High K+-containing external solution contained 100 mM KCl, 45 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 10 mM d-glucose, pH 7.4 with KOH. Membrane potential was measured over the first few seconds after break-in.Membrane potential was measured using the current clamp mode. Pipettes were pulled from borosilicate glass, Sylgard-coated and fire-polished. Pipette resistances were 4–5 Mohms when filled with a pipette solution containing 145 Kn, p); with the same value of ‘n’ (number of agents) and different values of ‘p’ (probability of nuclear NFAT). In our setting, rather than numbers of molecules, we hypothesise that a number of ‘NFAT activating units’ are involved . In a low 30 nM dose of thapsigargin, a single binomial distribution was not a good fit . We then found that a binomial distribution fitted all the unimodal histograms well Fig. . This diH’ denotes the absolute fluorescence, ‘N’ indicates a normal distribution, the Rest data gave a mean of μ = 2.06 and a standard deviation of σ = 0.21.For modelling of the FACS data Fig. , for theα represents the proportion of non-responding cells, the subscript ‘n’ represents the non-responders, and ‘r’ represents the responders. With 30 nM thapsigargin the non-responders accounted for 38% of the population (α = 0.38) and the responders 62%. The fluorescence distributions of non-responders were given by μn = 2.37, σn = 0.17 and responders by μr = 3.61, σr = 0.75. Corresponding distributions for cells challenged with 100 nM thapsigargin in high K+-containing external solution were 31% non-responders (α = 0.31) with a fluorescence distribution of μn = 2.29, σn = 0.17 and 69% responders with a distribution of μr = 4.07, σr = 0.67.For cells stimulated with thapsigargin, the experimental data were well described by fitting two normal distributions, as shown in Fig. +-containing external solution group were 19214.0 (mean) and 4420.2 (median).The log of the data was normally distributed, but in original fluorescence units the data were very skewed. The reason the means of FACS data were somewhat counter-intuitively above the means obtained from the fitted normal distributions was because of this skew. This was reflected in the difference between mean and median values from each group. The mean of FACS Rest group was 137.9 and the median was 114.2, in good agreement. However, the mean of FACS 30 nM thapsigargin group was 8994.9, whereas the median was 857.0, and corresponding values for the 100 nM thapsigargin/high Kt-test. In all graphs, * and ** denotes p-values < 0.05, 0.01, respectively and n.s. denotes not significant.Data are expressed as means ± SEM. No specific randomization or blinding protocols were used. Different groups were compared using a two-tailed paired or unpaired Student Supplementary InformationPeer Review FileSource Data File


Calcium dysregulation events can have a dramatic effect on the proper functioning of the pancreatic β-cells. The current review discusses the role of calcium signaling in health and disease in pancreatic β-cells and provides an in-depth look into the potential role of alterations in β-cell Ca2+ homeostasis and signaling in the development of diabetes and highlights recent work that introduced the current theories on the connection between calcium and the onset of diabetes.Pancreatic beta (β) cell dysfunction results in compromised insulin release and, thus, failed regulation of blood glucose levels. This forms the backbone of the development of diabetes mellitus (DM), a disease that affects a significant portion of the global adult population. Physiological calcium (Ca Under physiological conditions, blood glucose levels only transiently increase following intake of food . The obsThe islets of Langerhans are structures found throughout the exocrine pancreas, and together they form the endocrine part of this gland. They contain primarily β, alpha (α), gamma (γ), and delta (δ) cells. Each of these cell types secretes a separate hormonal product  and they are evident in different proportions across species . These cKey to the role of insulin in maintaining homeostatic blood glucose levels is the sensitivity of β-cells to the glucose itself . For exaPathophysiological conditions arise with insulin dysregulation; declines in either insulin’s release or its actual production by β-cells characterizes the main features of diabetes mellitus (DM) development. Diabetes is a significant health problem: according to the International Diabetes Federation close to 10% of the worldwide adult population is affected by this disease . Type 1 2+) has a significant role to play in physiological insulin release from islet β-cells. Glucose, acting as the main stimulus of these cells, actually serves to control Ca2+ concentrations across many compartments of the cell, including the endoplasmic reticulum (ER), mitochondria, nucleus, and cytosol, among others. Ca2+ dysregulation in β-cells also has been observed in disease states, including type 2 diabetes. The present review discusses previously known, as well as recently emerged aspects of Ca2 dynamics in pancreatic β-cells, and how this new information may relate to the pathophysiological development of T2DM.Calcium , an electrical change that functions to activate L-type Ca2+ channels, which allows an influx of Ca2+ into the cell. Finally, this wave of Ca2+ triggers the release of secretory granules containing insulin, to be released from the cell by exocytosis  and mitochondrial calcium uniporter (MCU) proteins is necessary for the physiological function of β-cells i is not a steady-state increase of the ion, but rather consists of periodic Ca2+ oscillations [2+ waves are synchronized with β-cell bursting electrical activity [2+ oscillations necessary for driving insulin secretion [2+-sensitive K+-channels and cyclic cytoplasmic Na+ changes are responsible for the fast and the slow oscillations, respectively. The ATP/ADP ratio and the ER Ca2+ levels are supposed to serve as pacemakers for fast bursts and cytosolic Ca2+ oscillations [2+ oscillations within β-cells should be mentioned. As these Ca2+ oscillations are the basis for the fluctuating plasma insulin signals, a down-regulation of peripheral receptors is prevented. This is substantiated by the finding that the recycling time for these receptors is shorter than the periods for plasma insulin pulses [2+ pulses prevent the desensitization of the secretory machinery of the β-cells. Jones et al. [2+ concentrations in permeabilized β-cells makes the machinery refractory to Ca2+ signals. A steady-state rise of cytosolic Ca2+ concentrations in stimulated β-cells could be responsible for the loss of the pulsatile insulin signals, a process occurring at the onset of diabetes [2+ rise may activate autolytic processes by stimulating phospholipases, proteases and endonucleases [The cycling variations of circulating insulin are reflected by the pulsatile release of the hormone from the pancreas .These inllations ,85. Thesactivity . The basecretion . Controlecretion . Diverseecretion . Slow acllations . The totllations . Severaln pulses . Furthers et al.  could shdiabetes ,83. Sustucleases  deteriorucleases .2+ in the process of (biphasic) insulin secretion is unchallenged but early studies on its contribution showed a great extent of controversy: Wollheim et al. [2+ but crucially depends on intracellular Ca2+ handling. Contrary findings were presented by Henquin et al. [2+ or inhibition of Ca2+ influx abolishes glucose-stimulated insulin secretion postulating that extracellular Ca2+ influx plays an essential role for insulin secretion. The current model of Ca2+ signaling in insulin secretion attributes important functions to both theories. Ca2+ influx through voltage-dependent Ca2+ channels is a prerequisite for proper insulin exocytosis [2+ flux is necessary for physiological insulin secretion. The latter recently has been outlined by our group as we demonstrated a pivotal engagement of a β-cell-specific permanent presenilin-1-mediated ER-Ca2+ leak in the initiation of first-phase insulin secretion. Inhibition of presenilin-1 activity or reduction of its expression or in other words a reduction of the ER Ca2+ leak leads to abrogation of the first phase of insulin release [Nowadays, it is well established that insulin secretion follows a biphasic pattern which was clearly described for the first time in perfused rat pancreas in 1968  and one m et al.  stated tn et al.  showing ocytosis  but also release .2+ homeostasis in β-cells is tightly regulated by a complex interplay between Ca2+ uptake and extrusion across the plasma membrane as well as uptake and release into/from internal Ca2+ stores. Since alterations in this equilibrium result in drastic changes of Ca2+ signaling in β-cells ultimately affecting insulin secretion, it is of crucial importance to keep cellular Ca2+ fluxes balanced. In the next paragraph, the most important contributors to β-cell Ca2+ homeostasis are discussed.Cytoplasmic Ca2+ transporters regulating Ca2+ extrusion from the cell—such as NCX and PMCA—located within the plasma membrane, function as essential players in maintaining a balanced Ca2+ homeostasis in β-cells. Notably, the activity of the forward-mode  of the NCX in the pancreatic β-beta-cell is sensitive to long-chain acyl-coenzyme and alterations in intracellular acyl-CoA levels may cause negatively control Ca2+-mediated exocytosis and insulin secretion [2+ in/3Na+ out) in most other cell types. Consequently, the pharmacological inhibition of the NCX1 with KB-R7943 results in a considerable augmentation in glucose-stimulated increases in cytosolic Ca2+ concentrations and insulin secretion in mouse and human β-cell islets, while KB-R7943 was without effect under non-stimulated conditions. [2+ accumulation and to a 1.5-fold increase in glucose-induced insulin release. Furthermore, PMCA2 inhibition results in increased β-cell mass, proliferation, and viability [As described above, Caecretion . Moreoveecretion  that ratditions. . Heteroziability .2+ concentrations regulate oxidative metabolism whereas the Ca2+ concentration needed for proper stimulation of the downstream processes ranges from 0.1–10 µM. The citric acid cycle  is the major energy-producing metabolic pathway in mitochondria and generally in cells, providing enormous amounts of reduction-equivalents which subsequently are oxidized in the electron transport chain to yield ATP. Ca2+ was shown to regulate three key dehydrogenases of the citric acid cycle i.e., NAD+-isocitrate dehydrogenase (NAD-ICDH), 2-oxoglutarate dehydrogenase (OGDH) and pyruvate dehydrogenase (PDH) [2+ increase is known to serve as a key trigger for insulin release in β-cells [2+ is involved. In fact, the current concept suggests that mitochondrial Ca2+ increase occurs upon Ca2+ entry due to the opening of the L-type Ca2+ channels, thus, already downstream from mitochondrial activation by glycolysis-derived pyruvate and ATP production. However, increases in mitochondrial Ca2+ are thought to be essential for stimulation of Ca2+-dependent dehydrogenases of the citric acid cycle in order to establish suitable enzyme activity to handle the substrate overflow upon elevated blood D-glucose [Cellular energy demand and mitochondrial activation are tightly linked especially in states of energy-consuming processes. Increased energy demand especially observed during stimulation of a cell—comes along with enhanced ATP hydrolysis which must be compensated for by accelerated ATP synthesis. Examples for such an increased energy demand where mitochondria are involved in balancing the energetic homeostasis are increased workload of the heart , activatse (PDH) ,118,119.se (PDH) , mitocho β-cells . This of-glucose . In part-glucose . Data of-glucose . These d-glucose .2+ homeostasis in β-cells plays an important role in β-cell physiology and insulin secretion. ER stress caused by ER Ca2+ depletion, has been shown to contribute to β-cell dysfunction and the development of type 2 diabetes [2+-sensor protein sorcin, which is a 22 kDa protein of the penta EF-hand family, is involved in maintaining ER Ca2+ stores by inhibiting RyR-activity and playing a role in the termination of CICR [2+ stores and inhibits GSIS [2+ levels lead to a conformational change of the sorcin protein, a subsequent translocation from the cytoplasm to membranes (amongst others to the ER) where it interacts with target proteins including RYR [The endoplasmic reticulum as preserver of Cadiabetes . The Ca2 of CICR . Sorcin  of CICR . Knock-dits GSIS . Elevateding RYR , therebyding RYR ,129 and ding RYR .2+ levels due to inhibition of the ER-located SERCA. In β-cells the SERCA2 and SERCA3 are expressed [db/db mice [Binding of insulin to the insulin receptor at the cell surface leads to autophosphorylation of tyrosyl residues of the IR-β-subunit and the phosphorylation of insulin receptor substrates 1 and 2 (IRS1 and 2). The subsequent cascade initiates the translocation of the insulin-responsive glucose transporter GLUT4 in muscle cells and adipocytes . In β-cexpressed . Loss of/db mice . SERCA3 /db mice .2+ depletion and subsequent ER Ca2+ stress can be explained—at least partially—by the existence of ER Ca2+ leak channels. Cassel and Ducreux [2+ leak in murine MIN6 insulinoma β-cells and human islets is influencing lipotoxicity. Furthermore, translocon inhibition resulted in reduced ER stress and a restoration of insulin secretion [2+ leak crucially contributes to β-cell physiology and insulin secretion. The presenilin-1-mediated ER Ca2+ leak is directly sequestered by mitochondria, leading to increased basal matrix Ca2+ levels that yield enhanced resting activity of mitochondria in the pancreatic β-cells due to pre-stimulation the Ca2+-dependent dehydrogenases of the citric acid cycle. Upon elevation of glucose, glucose is metabolized and the pre-activated citric cycle in the mitochondria efficiently converts glucose metabolism to activation of the respiratory chain (OXPHOS) and, subsequently, fast ATP production, thus, ensuring a fast, initial insulin secretion within 10 min of exposure to elevated glucose [The above-mentioned ER Ca Ducreux  could shecretion . Two rec glucose ,107.2+ storage important for a balanced Ca2+ homeostasis in mammalian cells and also in β-cells is the golgi apparatus. IP3 receptors are expressed at the surface of the golgi apparatus, mediating Ca2+ release from these IP3-sensitive pools [2+ demonstrate that upon cellular stimulation with IP3-generating agonists such as histamine, the golgi Ca2+ concentration rapidly decreases, presenting the golgi apparatus as IP3-sensitive Ca2+ pool [2+ extrusion of the golgi apparatus is mediated by these receptors [2+ pump responsible for fueling the golgi apparatus with Ca2+ from the cytoplasm is the secretory pathway Ca2+-ATPase Ca2+ pump (SPCA1) [2+ pump exist i.e., SPCA1 and SPCA2, whereas they show a tissue-specific expression. In mammals, SPCA1 is expressed in all tissues [2+ homeostasis [2+ levels and significantly reduced GSIS (bone), highlighting the importance of the Ca2+ homeostatic function of the golgi apparatus.Another intracellular Cave pools . Early ma2+ pool . Howevereceptors . The ATP (SPCA1) . Two mai tissues  whereas  tissues . SPCA1 heostasis , which ieostasis . Bone eteostasis  demonstr2+ is a crucial factor for β-cell survival, proliferation and function as well as for a proper insulin secretion on the one hand and is tightly regulated among diverse intracellular compartments within β-cells on the other hand. Therefore, an association with the development and progression of diabetes is obvious. Furthermore, deregulated Ca2+ signaling has been associated with the development of one of the key characteristics of T2DM i.e., insulin resistance [2+ homeostasis has been implicated in a vast range of disease conditions. The case is not different when considering T2DM. In fact, it seems likely that there can be multiple degrees of separation between an original Ca2+ dysregulation and the eventual development of T2DM. In this section some issues of how Ca2+ deregulation can contribute to diabetes pathophysiology by highlighting some Ca2+-associated aspects on the cellular level but also in the human body.As described in parts three and four of this review, Casistance ,142,143.2+ dysregulation. Decades ago, Levy and colleagues [2+ concentrations often accompanied by decreased Ca2+-ATPase activity. These findings highlight that deregulated Ca2+ homeostasis is a fundamental disorder in the diabetic state.Problems with pancreatic β-cell function and a loss of sensitivity to insulin are often significant factors in the development of T2DM . Whetherlleagues  stated t2+, taken either alone or in consideration with other important components of a healthy diet, such as magnesium (Mg2+) or vitamin D [2+ intake, for example, was found to correlate with a decreased risk of T2DM in subjects, while increased consumption of Ca2+ appeared not to show any association with risk [2+ homeostasis, thus promoting the healthy mineralization of bone. Recently, however, vitamin D and Ca2+ together have begun to be considered as factors affecting an individual’s risk of diabetes [2+-dependent process [2+, improved glycemic status [2+ and reactive oxygen species (ROS) [Much of the literature investigating a potential role for the ion in diabetes focuses on dietary Caitamin D ,149,150.ith risk . It is w process , appear es (ROS) —both faces (ROS) ,153,154—es (ROS) .2+ is involved in the pathophysiology of T2DM, then it is not a matter of maintaining a certain level of dietary intake of the mineral, but rather has to do with an ingrained physiological signaling role of the ion. Indeed, it has been determined that normal Ca2+ homeostasis is impaired in patients with T2DM. Irregularities have been observed in a range of cell types, including platelets, cardiomyocytes, and pancreatic β-cells, among many others [2+ levels indicates that this serves as a consistent factor in diabetic conditions.Such research implies, therefore, that if Cay others ,157,158.2+’s proper functioning across multiple signaling pathways is vital in all cells but is also important in some circumstances due to a tissue-specific role. Also important is the tight interconnectivity of Ca2+ signals across different cellular pathways and mechanisms, wherein a disturbance in one could affect a host of others. A dysregulation in such a signaling web oftentimes makes diagnoses of the original problem difficult, if not impossible. Undeniably, multiple changes to normal Ca2+ signals and regulation have been described in diabetes patients and animal models, hitting all of the main players in Ca2+ movement, such as SERCA and NCX, across tissues [2+ homeostasis in diabetes appears to be an increase in steady-state cytosolic Ca2+ levels across different cell types [Ca tissues ,156. Thull types .2+ dysregulation evident in diabetes is so far better characterized in animal and cell-level models of the disease. Changes to normal Ca2+ homeostasis include increased voltage-dependent Ca2+ channel activity in diabetic rodents as well as decreased SERCA activity [2+, including glycolysis, gluconeogenesis, and others [2+ dysregulation event occurs, which could be the end result of changes to normal Ca2+ channel or pump activity. For example, as mentioned, an increased intracellular Ca2+ concentration is evident in diabetes which results in the inhibition of glycogen synthase, ultimately causing, through a disrupted series of activations and phosphorylation events, glucose resistance [More specifically, in β-cells, the Caactivity ,159. Cond others . These vsistance .2+ leak for proper β-cell function [As the vigilant reader of the manuscript might have already realized, a new and for some maybe pugnacious finding concerning β-cell responsiveness and regulation of insulin secretion is presented in several sections of the manuscript—namely, the crucial role of a presenilin-1-mediated ER Cafunction ,107, dat2+ leak by either knocking down presenilin-1 or inhibiting its activity by blocking the PS1-regulatory kinase GSK3β causes several important effects in β-cells: i. reduction of mitochondrial respiration; ii. decreased mitochondrial ATP levels; iii. reduction of glucose-stimulated Ca2+ oscillation and delayed onset of these oscillations; iv. abrogation of the first-phase of insulin secretion. Several studies have already demonstrated that a loss of the first phase insulin response [2+ transfer mediated by presenilin-1 is pivotal for an adequate insulin response [Our work demonstrates that this leak is regulating many steps in the cascade ultimately leading to insulin secretion. Reducing the ER-Caresponse  which isresponse ,163,164 response . Thereforesponse ,167, higresponse ,107.2+ are known contributors to disease development namely T2DM and Alzheimer’s disease (AD). Interestingly, there is a profound epidemiologic and experimental correlation between these two devastating diseases. Several studies show that T2DM may lead to cognitive impairment [2+ comes into play: the more than 219 AD-associated mutations in the presenilin-1 gene [2+ handling [2+ handling. Examples, therefore, are the following: ΔE9 affects y-secretase function, M146V disturbs ER Ca2+ handling, L166P affects both [2+ homeostasis and β-amyloid toxicity due to mutations of presenilin-1 might act as independent causative factors for developing FAD. Importantly, the known crucial involvement of presenilin-1 in physiological amyloid degradation [2+ homeostasis due to alterations in presenilin-1 function could be the missing link for the causative factors of diabetes and AD development and could shed some light into the correlation of these two diseases. The exact regulation as well as additional mechanisms still need to be further elucidated but these data justify more intensive studies on this topic.In this section, we would like to draw the attention to a circumstantiated but sometimes underestimated correlation of two diseases where both presenilin-1 and Capairment ,169. Untpairment ,171,172.pairment . At thispairment ,175 resppairment . New dispairment ,177,178 pairment ,182,183 pairment . Moreovepairment ,186 and pairment ,188,189,n-1 gene  are the handling ,192,193.cts both . Accordiradation  and its radation ,107 migh2+ signaling and other machinery of β-cells is required to truly gain insight into how they relate to the pathophysiology of diabetes. Researchers are constantly innovating new techniques to accomplish exactly this. For example, the concept of synthetic cells has been around for decades [Considering the implications of the intracellular changes discussed here, it is clear that further investigation into the Ca decades ,197, but decades ,199 to m decades  have rec2+ to such processes. Despite now knowing of the intricate roles that this ion plays in pancreatic cell-specific function, there is a significant knowledge gap between early questions posed on Ca2+’s importance to the development of diabetes and current understanding of the same query.While promising concepts are constantly emerging, it is vital to continue diabetes investigation with the tried and tested cellular and animal models available to all. As we have outlined here, recent work has helped identify specific mechanisms of intra- and inter-cellular communication that allow for the complicated insulin-secreting function of β-cells, particularly considering the importance of CaThe research discussed here reveals a significant phenomenon in pancreatic β-cells that sheds further light on the mechanism by which these cells exhibit a response to increased glucose levels. Dysregulation of this mechanism could therefore potentially contribute to the pathophysiological development of T2DM or could be the missing link to other diseases.


Acute myeloid leukemia (AML) is a heterogeneous disease, and communication between leukemic cells and their neighboring leukemia-supporting normal cells is involved in leukemogenesis. The bone marrow cytokine network is therefore important, and the mediator release profile seems more important than single mediators. It is not known whether the characterization of primary AML cell proteomes reflects the heterogeneity of the broad and dynamic constitutive mediator release profile by these cells. To address this, we compared the intracellular levels of 41 proteins in 19 AML patients with the constitutive extracellular release during in vitro culture, including chemokines, growth factors, proteases, and protease regulators. The constitutive release of most mediators showed a wide variation (up to 2000-fold differences) between patients. Detectable intracellular levels were seen for 10 of 41 mediators, but for most of these 10 mediators we could not detect significant correlations between the constitutive release during in vitro culture and their intracellular levels. Intracellular protein levels in primary human AML cells do not reflect the dynamics, capacity, and variation between patients in constitutive mediator release profiles. Measurements of these profiles thus add complementary information to proteomic detection/quantification regarding the heterogeneity of the AML cell contributions to the bone marrow cytokine network. Acute myeloid leukemia (AML) is an aggressive malignancy characterized by the infiltration of immature leukemia blasts in the bone marrow . SeveralAML is a very heterogeneous disease , and pro9/L) and/or percentage (>80%) of circulating AML blasts, and a highly enriched blast population could thereby be prepared by density gradient separation alone [The study was approved by the local Ethics Committee , and samples were collected after written informed consent. AML blasts were derived from 19 unselected patients admitted to our hospital for treatment of acute myeloid leukemia . The present study included all patients from a defined geographical area and during a defined time period who were below 70 years of age, had circulating AML blasts in the peripheral blood, and were regarded to be fit for intensive  antileukemic treatment. The patient characteristics are summarized in on alone ,15,16. TPrimary AML cells from all 19 patients were thawed and immediately processed for proteomic analysis/quantification according to the sample preparation protocol previously described in detail ,11. Briem/z 375–1500) were acquired in profile mode with a resolution of R = 120,000 at 200 m/z, an automatic gain control (AGC) target value of 3 × 106 and maximum injection time (IT) of 100 ms. The top 12 precursors (above the intensity threshold of 5 × 104 counts) were sequentially isolated for MS/MS. The scans were acquired in centroid mode with a resolution of R = 30,000, an AGC target value of 1 × 105 and a maximum IT of 110 ms. Normalized collision energy was set to 28%, the isolation window was 1.6 m/z (offset 0.3 m/z) and the dynamic exclusion lasted for 25 s. Furthermore, for spray and ion-source parameters, the ion spray voltage was at 1800 V, no sheath and auxiliary gas flow, and the capillary temperature was at 260 °C.The QExactive HF was operated in data-dependent acquisition (DDA) mode, thus switching between survey full scan MS and MS/MS acquisition. The full scans (from The raw MS files were searched in MaxQuant (v.1.5.6.0)  using de6 cells per mL; Nunclon, Roskilde, Denmark). The levels of 41 mediators were analyzed either by Luminex assays or ELISA analyses, as described previously [Our in vitro model for investigation of constitutive mediator release has been previously described in detail ,13,20. Beviously ,13,21. TSpearman’s test was used to assess the correlation between the intracellular and extracellular levels of the mediators .AML is a bone marrow disease, and the constitutive release of soluble mediators by leukemic cells is important for the communication between these cells and their neighboring AML-supporting cells in the common bone marrow microenvironment ,22,23,24r-values >0.49 and p-values < 0.04). For 13 of these 19 mediators the r-values were >0.60. We investigated the constitutive release of 41 soluble mediators by in vitro cultured primary human AML cells derived from 19 patients; for 38 of them, detectable release was seen for a majority of patients, while ADAM12 had undetectable levels in all patients. The overall results are summarized in We identified 5790 protein groups in our proteomic data after removing reverse hits and proteins only identified by site. The number of quantified proteins per patient varied from 3823 to 5102. Of these, 5777 proteins had a quantitative value, and only 10 of the 41 mediators of interest were identified/quantified , indicatProteomic analysis of the AML cells allows identification and quantification of the proteins and their relative abundance in a particular compartment at a specific time. This analysis therefore represents a static snapshot of ongoing biological processes. By exploring the correlation of intracellular protein abundances with the concentration released into the media after 48 h in vitro culturing, we intended to investigate the dynamics of mediator release in the 19 AML patients . p = 0.0078, r = 0.65) whereas only borderline significance was reached for CCL5  and CFD . For the seven other mediators, the p-values did not obtain statistical significance, and r-values were generally <0.25  correlation of intracellular and extracellular levels, indicating a steady relationship between protein synthesis and mediator release; (2) high intracellular levels and low constitutive release of soluble mediators, indicating mainly intracellular protein functions; or (3) low intracellular levels and high constitutive release of soluble mediators, indicating rapid mediator release for extracellular functions. A correlation between the in vitro culture and ex vivo proteomic levels was only seen for CCL5, CFD, and cystatin C/CST3; a highly significant correlation was seen for cystatin C/CST3 (ly <0.25 . Interesly <0.25 . By contWe hypothesize that the lack of correlation between intracellular and released extracellular levels for most mediators indicates that the intracellular levels, at least for certain mediators, reflect additional intracellular functions rather than the capacity of extracellular release. This may also be the explanation for the similar lack of correlation between intracellular and extracellular levels of osteopontin . At the To decrease the risk of inducing biological alterations in the leukemic cells by more extensive cell separation procedures ,16, we oIt has been suggested by others that cryopreserved AML cells should be cultured in vitro for a short period before proteomic analysis to reduce the effects of the cryopreservation-induced stress on the leukemic cells . HoweverWe have previously shown that cryopreservation and thawing of primary human AML cells will alter the proteome and phosphoproteome ,39, inclOur previous studies suggest that the capacity of constitutive mediator release is associated with a favorable prognosis , and it Communication between malignant and nonmalignant neighboring cells through the cytokine network is important in AML. AML cells show constitutive release of a wide range of soluble mediators, and the overall release profile or combined effects of several mediators may then be more important than the effects of single mediators. Our study shows that for most mediators, the ability of constitutive extracellular release by primary human AML is best evaluated by in vitro culture, and not by analyzing intracellular levels as a static representation of the potential secretome. However, several soluble mediators demonstrate a wide variation between patients both intracellularly and when constitutively released, thus reflecting the large heterogeneity in AML. A combination of different methodological strategies may be essential to capture this heterogeneity and for a deeper understanding of leukemogenesis and AML cells’ communication with the microenvironment.


The effect of SKCa inward rectification on the excitability of these neurons is unknown. Furthermore, studies on the role of KCa channels in repetitive firing and their contributions to different types of afterhyperpolarization (AHP) in these neurons are lacking. In order to study these phenomena, we first constructed and validated a biophysically detailed single compartment model of bladder small DRG neuron soma constrained by physiological data. The model includes twenty-two major known membrane mechanisms along with intracellular Ca2+ dynamics comprising Ca2+ diffusion, cytoplasmic buffering, and endoplasmic reticulum (ER) and mitochondrial mechanisms. Using modelling studies, we show that inward rectification of SKCa is an important parameter regulating neuronal repetitive firing and that its absence reduces action potential (AP) firing frequency. We also show that SKCa is more potent in reducing AP spiking than the large-conductance KCa channel (BKCa) in these neurons. Moreover, BKCa was found to contribute to the fast AHP (fAHP) and SKCa to the medium-duration (mAHP) and slow AHP (sAHP). We also report that the slow inactivating A-type K+ channel (slow KA) current in these neurons is composed of 2 components: an initial fast inactivating (time constant ∼ 25-100 ms) and a slow inactivating (time constant ∼ 200-800 ms) current. We discuss the implications of our findings, and how our detailed model can help further our understanding of the role of C-fibre afferents in the physiology of urinary bladder as well as in certain disorders.Bladder small DRG neurons, which are putative nociceptors pivotal to urinary bladder function, express more than a dozen different ionic membrane mechanisms: ion channels, pumps and exchangers. Small-conductance Ca 2+ dynamics. One of the channels, the small-conductance Ca2+-activated K+ (SKCa) channel present in these neurons which was earlier thought to be activated solely by intracellular Ca2+ concentration was recently shown to exhibit inward rectification with respect to the neuron’s membrane potential. It was found that SKCa inward rectification can alter the repetitive firing of the bladder small DRG neurons. We also delineated the role SKCa and BKCa (large-conductance Ca2+-activated K+) channels to the complex afterhyperpolarization (AHP) of the neuron’s action potentials, shedding light on the mechanisms that govern repetitive firing in these sensory neurons. Our findings have implications for the normal biophysics and dysfunctional states of urinary bladder arising from changes in sensory axon function, and these are discussed.The small dorsal root ganglion (DRG) neurons of the bladder carry information related to pain from the bladder to the spinal cord. The level of electrical activity of these neurons is generally low but rises in abnormal conditions such as bladder inflammation and spinal cord injury. In order to investigate the reasons for this hyperactivity, we constructed a model of the bladder small DRG neuron soma including its membrane ion channels and intracellular Ca The sensory component of the reflex pathway of the bladder is formed by the dorsal root ganglion (DRG) neurons. Afferent signals such as bladder pressure, volume, temperature, pH, presence of irritants and pain are transmitted to the spinal cord via these neurons. Urinary incontinence—the involuntary voiding of urine from the body—is a major group of disorders including overactive bladder in some of which sensory neuron pathophysiology has been implicated –6. Biophδ fibre neurons. In rats, the C-fibre neurons have small soma diameter (∼24 μm) and are therefore called small DRG neurons while the A-δ fibres have a somewhat larger soma diameter range (30-35 μm) and are referred to as medium-sized or medium-diameter DRG neurons Delayed-r2+-activated K+ (KCa) channelsCa. KCa channels are activated by intracellular Ca2+ concentration ([Ca]i) and/or membrane potential (Vm), and are an important regulator of excitability in some neurons and muscle cells. The Ca2+ influx from voltage-gated ion channels, activation of ligand-gated receptors and intracellular Ca2+ release mechanisms can modulate the activity of KCa channels. There are 3 types of KCa channels: the large-conductance (BKCa) channels, the intermediate-conductance (IKCa) channels and small-conductance (SKCa) channels. These are classified on the basis of their single channel conductances: For SKCa conductances range between 5-20 pS, IKCa have a value of 10-60 pS whereas BKCa single channel conductance >100 pS [Ca channels are activated by membrane voltage and [Ca]i while SKCa and IKCa are activated by [Ca]i only [Ca channel exhibit inward rectification with respect to Vm [Ca channels have been shown to modulate the amplitude and duration of afterhyperpolarization (AHP) in some primary afferent neurons [Ca channel was included in the bladder small DRG neuron model along with SKCa channel. There was insufficient data for modelling IKCa channels for bladder DRG neurons and were not included in the model. >100 pS . BKCa cha]i only . Howeverct to Vm , 26, 28. neurons . BKCa ch2+-activated K+ (BKCa) channelsLarge-conductance Ca. Shieh et al. i and Vm. It was modelled using a Boltzmann equation in which the half activation value and slope factor are made a function of [Ca]i. The open channel probability data reported in [n∞. The open channel probability (Popen) versus Vm measured for different [Ca]i curves was taken as equivalent to the n∞ versus Vm  were calculated from the BKCa current curves for cutaneous DRG neurons i i = [K]o) as well as physiological K+ concentrations  [SKby [Ca]i . Soh andby [Ca]i , howeverfication . Recentlfication  reportedk et al.  and Hougk et al.  also sho = 4 mM) .Ca channel is modelled with two parameters: ‘o’ which defines the Ca2+-dependent activation, and ‘m’ which codes for voltage-dependent inward rectification. ‘m’ was modelled using a Boltzmann equation. Its half activation, V1/2 and slope factor, sf parameters were also found to vary with [Ca]i. Hence, ‘m’ was modelled as a function of both [Ca]i and Vm  was added to the equation for ‘m’ (see below) to account for parameter shifts due to changing K+ concentrations. Data for modelling was obtained from [V1/2 and sf found from the experimental I-V curves in [The SKi and Vm . An addined from  and 55]Ca channeurves in , 55 coulurves in .+ channels added to the model, KCNQ/M channel, Na+-activated K+ (KNa) channel, are described in Other K2+ (Cav) channels are classified into two groups: low voltage-activated (LVA) and high voltage-activated (HVA), based on the activation thresholds. The T-type LVA channels start activating at Vm more depolarized than −70 mV hence contribute to resting state of the cell i and in creating separate Ca2+ concentration pools in the neuron [2+ concentration ([Ca]i), endoplasmic reticulum (ER) Ca2+ concentration ([Ca]ER), and mitochondrial Ca2+ concentration ([Ca]MT) representing a separate pool for each component in each shell (see i transient in the outermost (towards plasma membrane) shell.The soma was divided into 12 concentric shells for encoding Cae neuron . Each shhell see  Figs. Mihell see . The num2+ concentrations in the outermost shell were considered [Ca]i, [Ca]MT and [Ca]ER, respectively of the bladder small DRG neuron soma model as the outermost shell receives Ca2+ from plasma membrane mechanisms and will have the maximum change in the concentrations. The outermost [Ca]i concentration defines the electrochemical gradient for movement of Ca2+ ions across the cell membrane via the Ca2+ permeable channels and also activates the membrane Ca2+-activated K+ channels (BKCa and SKCa), plasma membrane NCX and plasma membrane Ca2+-ATPase (PMCA) pump. Ca2+-activated Cl- channels(CaCC) are activated by Vm and by local Ca2+ concentration release from inositol triphosphate receptors (IP3Rs) present on ER i. For shells below the outermost shell, JMembrane and JPMCA are absent.The rate of change of [Ca]ER was modelled as:βER is the buffering component of ER, JSERCA is the flux through sarco-endoplasmic reticulum Ca2+ ATPase pump, JIP3R is the flux due to IP3 receptor, JCICR is the Ca2+ induced Ca2+ release (CICR) flux via the ryanodine receptors and JLeak is the flux through leak channels on the ER. A negative flux sign denotes the decrease in [Ca]ER with time as result of release of Ca2+ ions into cytoplasm and vice versa. Individual components of ER are discussed ahead.The changes in [Ca]Ca]MT were modelled as:MCU is the mitochondria uniporter flux and JMNCX is the flux due to mitochondrial Na+/Ca2+ exchanger(MNCX) and βMT is the Ca2+ buffering component of mitochondria and was added to prevent the depletion of mitochondrial Ca2+ during high neuron firing. MNCX being a mitochondrial release mechanism, JMNCX is negative and signifies a decrease in mitochondrial Ca2+.The changes in the  is either [Ca]i, [Ca]ER or [Ca]MT, KB is the dissociation constant of the buffer and [B]tot is the total buffer concentration i or the Ca2+ influx via SERCA and MCU for ER and mitochondria, respectively decreases due to buffer action.Endogenous Cantration . It givei, [Ca]ER and [Ca]MT are given in Tables β = 370 reported for rat small DRG neurons [Ca]i buffering whereas βER and βMT were calculated for [Ca]ER and [Ca]MT buffering using The parameters used for [Ca] neurons  for [Ca]i resulting from the soma membrane Ca2+ currents was taken into account by the following equation:ICa is the Ca2+ current due to membrane mechanisms, z = 2 is the valence of Ca2+, F is the Faraday’s constant and v is the volume of the outermost shell [The change in [Ca]st shell .2+ across shells was modelled using Fick’s first law of diffusion. The flux from one shell to the adjacent is given by:DCa is the diffusion coefficient of Ca2+ in the cytoplasm, β is the cytoplasmic endogenous buffer binding ratio [Ca]i is the concentration difference between shells and Δr is the distance between shell centres o is the extracellular Ca2+ concentration, K1, K2, K3 and K4 are reaction rate constants. JPMCA is given as the difference of forward and backward flux of the second reaction  is the Ca2+ concentration in the shell and n = 2 is the Hill constant.Like PMCA, these are low capacity, high-affinity Ca2+ pumps . It help neurons , 102. It neurons , 98:JSEi. The production of IP3 occurs via the activation of G-protein coupled receptors on the plasma membrane of the soma such as purinergic (P2Y) receptors and bradykinin receptors. The Ca2+ release from IP3Rs was based on a simplified model for IP3R  and [Ca] is the concentration of IP3 and Ca2+ in the shell, KIP3 is the dissociation constant for IP3 binding to the IP3R, Kactip3 is the dissociation constant for Ca2+ binding to activation site on the receptor, [Ca]ER is the ER Ca2+ concentration in the shell and h is the probability of the inhibition site on IP3R being unoccupied and is given by the equation:konip3 and kinhip3 are the rate of Ca2+ binding and dissociation to the inhibition site. The initial value of h is given by kinhip3/([Ca]+ kinhip3). The IP3 molecules undergo diffusion between the shells, and degradation to the resting concentration [IP3]0 at rate of kdegrip3. The change in IP3 concentration in shells is given by the equation:DIP3 is the diffusion coefficient of IP3 in the cytoplasm, A is the area of the partition between shells, Δ[IP3] is the concentration difference between shells and Δr is the distance between shell centres for IP3R  used by for IP3R . It was  centres .2+-induced Ca2+ release (CICR) in small DRG neurons [2+ released on the nearby ER membrane via IP3Rs and other RYRs and also from the Ca2+ coming from the membrane mechanisms (Ca2+ channels). The RYR model presented here was adapted from CICR model of Purkinje cell [JCICR) is defined by the equation:Ca] is the Ca2+ concentration in the shell and KCICR is the dissociation constant and KTCICR is the activation threshold for RYR. The value of KCICR was found by fitting the data on open probability of rat DRG neuron RyRs versus [Ca]i given in  is the Ca2+ concentration in the shell, LER leakage factor which is the sum of JSERCA, JIP3R and JCICR at resting state.An ER leak channels were added to the model such that there is no net flux from the ER at rest :JER,Leai and Ca2+ transients in small DRG neurons [2+ uptake and mitochondrial Na+/Ca2+ exchanger (MNCX) for release of [Ca]MT to cytoplasm. The MCU and MNCX fluxes cancel each other at steady state i [Mitochondria play an important role in regulating physiological [Ca] neurons , 107. Twdy state . The resn, [Ca]i , 108.MT lost via release through MNCX. It was modelled using data from  is the intracellular Ca2+ concentration in the shell and n is the Hill constant, which is 2.3 for MCU. The parameter values were obtained from [MCU helps to replenish [Ca]ata from . Flux thned from  for smal+/Ca2+ exchanger (see +/Ca2+ Exchanger). The parameter values were adapted from [Ca]MT,s represents mitochondrial Ca2+ concentration in shell and [Na]i is Na+ concentration in the cytoplasm, VMNCX is maximal NCX activity, KNa and KCa are constants for Ca2+ and Na+, respectively.MNCX was modelled using a modified equation for plasma membrane Nated from . The MNCCalcium indicator dyes used for imaging intracellular/ER/mitochondrial calcium concentrations can act as calcium buffers themselves and alter the amplitude and decay phase of the calcium transient , 110. ThCa]i are assumed to be always at equilibrium and follow the rapid buffer approximation. Hence, Ca]i was modified for an imaging dye with buffer biding ratio, βdye as:The calcium imaging dye and i validation where normalized simulated [Ca]i was compared with that from imaging data tot = 5 μM [KB = 224 nM [Ddye = 0.1 μm2/s (for free dye diffusion) [βdye was calculated by Ca]i and was stabilized by tuning the initial PMCA pump density (pump0) 4.08*10−13 mol/cm2.For i transient elicited by 1 AP, Indo-1 calcium indicator dye was used by [B]tot = 100 μM [KB = 250 nM [Ddye = 0.1 μm2/s (for free dye diffusion) [pump0 was tuned to 4.0815*10−13 mol/cm2 for stabilizing the initial [Ca]i.For i in experiments [B]tot = 200 μM [KB = 5500 nM [Ddye = 0.075 μm2/s (assumed). The changes in [Ca]MT of the small DRG neurons presynaptic terminals were monitored by using mtPericam overexpressed in the mitochondria [Ca]MT was modified as:βdye for mtPericam in simulations are: [B]tot = 15 μM (tuned) and KB = 1700 nm o in bladder small DRG neurons generated in experiments [i and change in [Ca]i for AP firing in small DRG neurons. Change in [Ca]i of ∼20 ± 3 nM was reported for a single AP. Model was tuned to generate an increase (∼18 nM) during an AP as low-voltage activated T-type calcium channel have only a small contribution to calcium current in bladder small DRG neurons [i rises from its base value of 136 nM [eriments  and by oeriments  reported neurons . Therefof 136 nM  to 154 n2+ concentrations ([Ca]MT) for bladder small DRG neurons, we used the data for small DRG neuron presynaptic terminals reported by [i and [Ca]MT from experiments [i and [Ca]MT were evoked by 20 action potentials fired at 10 Hz identical to the experimental stimulus. The comparison shows that the model is able to capture the rising and falling phase of the [Ca]i and [Ca]MT and their time courses closely. Effects of calcium dye buffering have been incorporated into the model to compare simulation results with experimental data. See “Effects of calcium indicator dyes” in Due to absence of data on mitochondrial Caorted by . Fig 12 eriments  and corr2+ dynamics on the one hand and corresponding model outputs on the other help repose confidence in the model, including the interaction of electrical and Ca2+ dynamics components.The match between experimentally observed cytoplasmic and mitochondrial Ca2+ removal mechanisms in small DRG neurons izations , 117, duCa with [Ca]i-dependent activation only and no inward rectification. Such an approach will clearly result in inaccurate model outputs, both for the SKCa conductance itself and for other model parameters that are influenced by it. We therefore, set out to model the voltage-dependent inward rectification along with [Ca]i-dependence and also validated our model against experimental data  was minimal. Comparison of APs from the two models did not show much difference, probably because of the almost linear I-V relationship for small increases in [Ca]i (20-30 nM for 1 AP) of the models. Hence, we tested the effect of an elevated value of SKCa conductance. A higher than normal SKCa conductance (or current) in DRG neurons could represent conditions during application of SKCa channel openers and positive modulators such as 1-EBIO and NS4591 i behaves similarly as the voltage-dependent inactivation parameter (h) of the Hodgkin-Huxley sodium conductance. During the depolarizing phase of the AP, there is a decrease in the value of IR parameter, m (g curve) and thus, results in a decrease in the conductance of the channels (Ca channel (SKCa w/o IR), g follows the same trend as the calcium-dependent activation parameter, o (g dynamics of the channel. The 3 colours in Ca channels: black = 9*10−4 S/cm2 (control), green = 2.7*10−3 S/cm2 and red = 4.5*10−3 S/cm2. Increasing o, m and g which were amplified version of the trend observed for the respective parameters in control conditions  for both the models (SKCa with IR and SKCa w/o IR).The presence of the IR parameter, meter, m  till thechannels . In contmeter, o . We alsoWe next checked how these changes due to IR translate into changes in repetitive firing in our model.+, KA and KDR channels i.We wished to study the sensitivity of the model spike firing to the inward rectification of SKchannels , 33, 119Ca IR on firing frequency, we used the above described tonic firing model with a slightly longer duration (1000 ms) rectangular current clamp of amplitude 0.12 nA which generated 20 APs (∼ 20 Hz). The Ca was altered between 9*10−4 S/cm2 and 4.5*10−3 S/cm2 in steps of 4.5*10−4 S/cm2. We measured the change in spike count and the peak [Ca]i reached during the protocol. A spike was counted whenever Vm crossed 0 mV from a negative to a positive Vm. A decrease in number of spikes with increasing conductance was observed for both the models (Ca with IR and SKCa w/o IR) as the outward current though SKCa increases. A similar downward trend is seen in the peak [Ca]i attained for both the models with increasing conductance. The outward current via inward rectifying SKCa is much less compared to SKCa lacking inward rectification. Thus, the probability of failure of APs is comparatively smaller for the SKCa with IR model which is also evident from our results , 2.7*10−3 S/cm2 and 4.5*10−3 S/cm2 for both the models: SKCa with IR  of the SKCa w/o IR  due to Ca and SKCa channels from the control value of 9*10−4 (for both BKCa and SKCa) to 4.5*10−3 S/cm2 in multiples of 4.5*10−4 S/cm2. As expected from the blocking studies, an elevated BKCa conductance resulted in increase in AHP Amplitude  and the slow AHP (lasting a few seconds) in bladder DRG neurons.BKmplitude  than SKCCa channels, we altered the conductance of channels individually in the range of 9*10−4 (control) to 4.5*10−3 S/cm2 to a model that shows inflammatory repetitive firing for a long duration rectangular current clamp of amplitude 0.12 nA and 1000 ms duration, as used in i. Spikes were counted whenever Vm crossed 0 mV from a negative to a positive Vm.In order to test the sensitivity of AP firing to the KCa channel is more likely to change the firing rate than the BKCa channel when channel conductance is elevated. A decremental trend is seen in maximum [Ca]i . AHP80%, which indicates the recovery time from AHP towards RMP, is relatively higher at larger conductances for SKCa channel than BKCa channels. Hence, a larger depolarization is required to bring Vm from slow AHP potential to the AP threshold. This will reduce the firing frequency of the neuron.The graphs in um [Ca]i . The dec2+ pump, Na+/Ca2+ exchanger as well as all essential components of Ca2+ dynamics, namely cytoplasmic diffusion and buffering, endoplasmic reticulum (ER) mechanisms and mitochondrial mechanisms. Hence, our model represents a physiologically realistic model constrained by available biophysical data.The computational model presented here describes a comprehensive electrophysiological model of the urinary bladder small DRG neurons validated against experimental data for these neurons. The model includes all the major known plasma membrane mechanisms viz. ion channels, CaCa channels increases the excitability of bladder small DRG neurons, (2) BKCa may contribute chiefly to the fAHP of the spike while SKCa may contribute to chiefly to the mAHP and sAHP, (3) SKCa channels are more potent in suppressing AP firing than BKCa channels, (4) the slow KA currents are composed of 2 inactivation components: a fast component and a slower component which in turn could be the result of 2 different molecular constituents of the channel. We discuss below the implications of these findings for various facets of bladder small DRG neuron functioning and, on a broader level, for urinary bladder function.Using our elaborate, validated model of bladder small DRG neuron soma, we addressed certain outstanding questions regarding the factors that govern the functioning of these neurons and showed the following: (1) the inward rectifying property of SKCa channel presented here incorporates both inward rectification and [Ca]i-dependence. Our working hypothesis was that since rectification gives rise to smaller outward currents over the relevant range of membrane voltage i-dependence  and glutamate which could be involved in intercellular communication between neighbouring somata via the satellite glial cells surrounding them ER caused by release from ryanodine receptors (RYR) which are activated by increase in cytoplasmic Ca2+; IP3R (IP3 receptors) which open on activation by IP3 molecules and Ca2+ ions; and the ER leak channel helps to maintain a steady resting state [Ca]ER. Ca2+ ions entering the ER as well as mitochondria is also buffered. Mitochondrial calcium entry occurs via mitochondrial uniporter (MCU) and calcium is released by mitochondrial sodium-calcium exchanger (MNCX). See Ca2+ dynamics in In each shell, 81% of the total volume is cytoplasm, 12% is occupied by endoplasmic reticulum (ER) and mitochondria make 7% of the total volume. Ca(TIF)Click here for additional data file.S3 Fig2+-activated Cl-s (CaCCs) are activated both by membrane potential and intracellular Ca2+. They are more potently gated by IP3R Ca2+ release in the outermost shell than by Ca2+ influx from voltage-gated Ca2+ channels on the membrane. The store-operated Ca2+ channels (SOCCs) are activated when there is a depletion of Ca2+ in the ER. The Orai1 and STIM1 proteins are responsible for store-operated Ca2+ entry in small DRG neurons. See The Ca(TIF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


To determine immunophenotypic pattern in newly diagnosed cases of acute myeloid leukaemia by flow cytometry and its correlation with morphological findings.This study was conducted at Haematology (Pathology) department, Army Medical College, in collaboration with Immunology Department Armed Forces Institute of Pathology, Rawalpindi from 16 November 2016 to 16 November 2017. One hundred and six patients of both genders and all age groups diagnosed as acute myeloid leukaemia were included in the study. Demographic data was noted. Complete blood counts, bone marrow examination and cytochemical stains were carried out and evaluated microscopically for blast percentage and morphology. Immunophenotyping was performed by flow cytometry using standard panel on peripheral blood or bone marrow samples. The surface and cytoplasmic antigens of interest were analysed and correlated with morphological findings.The most commonly expressed antigens were CD13, CD33, CD45 and HLA-DR. Almost all blasts expressed CD45 with no remarkable difference among the subtypes of AML. The mean positivity for CD13 among all AML subtypes was 57% and for CD33 was 67%. Aberrant expression of CD7 and CD19 were expressed in 26.4% and 1.1% of all cases respectively. There was concordance rate of 90% between morphology and FCM in our study.Flow cytometric analysis of acute leukaemia done by a combination of patterns and intensity of antigen expression improves diagnostic yield in AML. CD13, CD33 and CD45 are the most frequently expressed antigens in AML. Our findings suggest a 90% concordance between morphology and flow cytometry. It is pertinent to conclude that flow cytometry results interpreted with morphology are complementary. Acute Myeloid Leukemia (AML) is a clonal disorder of haemopoietic stem cells. It is characterised by inhibition of differentiation resulting in accumulation of cells at various stages of incomplete maturation. There is a decreased production of mature haemopoietic elements.3Diagnostic methods for acute leukemias include immature cell count, morphology, cytochemistry, immunophenotyping, cytogenetics and histochemistry in correlation with clinical features. All these diagnostic methods are complementary. Cell morphology remains the basic diagnostic tool to assess the number and morphology of blasts. Immunophenotyping by flow cytometry is a powerful adjuvant tool in delineating cell surface and cytoplasmic markers in AML. The expression of characteristic myeloid lineage markers CD13, CD33 and CD117 allows the distinction of AML from other types of leukaemias.4In the diagnosis of acute leukemia concordance between experienced observers increases from 70 to 99% when morphologic criteria are supplemented by cytochemical and immunophenotypic information.7Immunophenotypic markers shown in various studies implicating adverse outcomes are CD7, CD9, CD11b, CD13, CD14, CD33, CD34, CD56 and terminal deoxynucleotidyl transferase.9Aberrant phenotype is expression of lymphoid-associated markers in myeloblasts or that of myeloid associated markers in lymphoblasts.This descriptive cross sectional study was carried out in Haematology Department Army Medical College in collaboration with Haematology and Immunology department of Armed forces institute of pathology, Rawalpindi from 16 November 2016 to 16 November 2017. The sampling technique used was non probability convenient sampling Approval was taken from ethical review board and institutional review board. A total of 106 patients of all ages and both genders who were newly diagnosed with AML were included. Relapsed cases of AML and those evolving from myelodysplasia (MDS) or receiving treatment for AML were excluded.The samples were analysed for blast percentage and morphology on leishman stained peripheral blood and bone marrow smears followed by cytochemicalstains (Sudan Black B). Immunophenotyping was done using three colour flow cytometer. This included samples of peripheral blood or bone marrow aspirate from patients of De novo AML.Two and a half ml of venous blood in Ethylene Diamine Tetra Acetic Acid (EDTA) tube was collected for Complete Blood Count (CBC) and peripheral blood film examination, under aseptic conditions. Bone marrow aspiration was done after written informed consent, following standard guidelines. Complete blood counts were generated using automated haematology analyzer Sysmex KX-21. Leishman stained smears from peripheral blood and bone marrow aspirates were examined under microscope for morphology and percentage of blasts.11Labelling of each test tube  was done properly and placed in sequence. Ten µ| of monoclonal antibody was added into each tube. 50µl of whole blood / diluted bone marrow in each tube was added, thoroughly mixed and incubated in dark for 30 minutes at room temperature. Fluorescent activated cell sorter lysing solution (FACSLyse) in distilled water was prepared in 1:10 dilution. In each test tube 2 ml of diluted FACSLyse was added and incubated in dark for five minutes at room temperature. It was centrifuged at 300 g for five minutes at room temperature. The supernatant was discarded and the remaining 50µ| fluid was shaken for resuspension of cells. In each tube 2ml of phosphate buffered saline (PBS) was added. Centrifuge at 300 g for five minutes and supernatant was discarded. The remaining fluid was shaken and 0.5 ml of 3.3% formalin was added to each test tube. The samples were kept at 4ºC till analysis on flow cytometer.0 and where results of primary panel were inconclusive. The populations were considered positive if at least 20% of cells within gate showed the expression of a particular antigen. For cytoplasmic antigen expression, the threshold was 10%.Flow cytometric analysis was made by a top FACScan flow cytometer (Becton & Dickenson).The flourochromes FITC, PE and PerCP were used. The primary panel of monoclonal antibodies (mAbs) used were CD45 (for blast identification), CD13, CD33, CD34, HLA-DR and for AML M4/M5 CD14, for AML M6 anti-glycophorin A and for AML-M7, CD61, CD41 and CD42 were used. The extended panel included CD117 and Anti-MPO was used in cases where the morphology suggested AML-MIsotype control (antimouse IgG1FITC/Ig2αPE) is used as negative control (should not react with human blood/bone marrow cells). If it happens, the test/reagent should be rechecked.Data was analyzed using SPSS version 21. Frequencies and percentages were calculated for variables like gender. For quantitative data analysis, mean along with standard deviation were used to assess the values for monoclonal antibodies Correlation tests were applied using Pearson correlation coefficient. The age of the patients at the time of diagnosis ranged from one year to 80 years (mean age = 31.81±19.972 years). Males constituted 58.5% and females were 41.50%. The male to female ratio was 1.4:1.9 /L and highest being 456.77x109/L both extremes seen in AML-M1.The mean of platelet count was 58.23±81.29 × 109 /L. The highest count was 1000 ×109 /L seen in AML-M2 and lowest being 659 × 109 seen in AML-M1.The mean haemoglobin levels were 8.25±2.16 g/dl. The lowest haemoglobin level was 3g/dl seen in AML-M2 and highest levels in AML-M3 of 13.50 g/dl. The mean TLC was 57.46±79.39, the lowest being 0.99 x 10O (75%±33.42) and weakest positivity in AML-M3 (12%±23.84). CD34 showed mean positivity of 38% ± 30.84 among all AML subtypes, strongest being in AML-MO (74% ± 21.79) and weakest seen in AML-M6 (1.33% ± 2.31). In our study there were 18 cases (n =106) of AML-M3 of which only 1 case of AML-M3 showed positivity for HLA-DR and 3 cases (n=18) were positive for CD34, however none of them were positive for both CD34 and HLA-DR. CD13 was studied among the blast population with mean positivity of 57% ±27.53 among all subtypes with strongest positivity in AML-M4 (72% ± 20.01) and weakest in AML-M6 (22% ± 1.73).The least number of blasts were 22(22%) seen in AML-M2 and AML-M6 and highest abnormal promyelocytes with reniform nuclei were 98 %, found in AML-M3. Out of 106 cases analyzed, AML-M2 was the most frequent subtype constituting 49 (47.2%) cases followed by AML-M3 . The least common being AML-M6  as shown in 0 (r=0.717).CD33 showed mean positivity of 67% ± 27.56, strongest being in AML-M4 (87% ±9.02) weakest in AML-M6 (30% ±11.14).CD13 and CD33 were also studied for correlation using Pearson correlation and was found to be statistically significant among all subtypes. The strongest correlation being in AML-M6 (r= 0.962) and weakest being in AML-MThe concordance rate between morphology and FCM is seen in 102 (90%) cases with confidence interval of 95% in our study. While partial discordance was seen in 4(3.77%) cases where lineage was correctly identified with difference in defining subtypes only.r =-.321, .360 .297 and -.585 respectively) and insignificant correlation with CD33, CD117 and CD19 .The aberrant expression of CD7 showed positivity in 28 (26.4%) cases, most frequently expressed in AML-M2 and CD19 was expressed in 1(1.1%) case. The results of correlational analysis showed that CD 13, CD 34, CD7 and CD14 had a significant correlation with immunophenotyping .19In our study AML-M2 (47.2%) was the most frequent subtype similar to Gosh study AML-M2=34%).4%.3,5,14CD7, a T-cell antigen known to show aberrant expression was most commonly expressed in our study 26.4%) followed by CD19 (1.2%). The same trend was observed in most of the international studies where CD7 was most commonly expressed aberrant antigen followed by CD19.6.4% foll21The combined use of CD34 and HLA-DR was more helpful in distinguishing Acute Promyelocytic Leukemia (APL) from non-Acute Promyelocytic Leukemia (non-APL) none of the eighteen cases of APL were positive for both CD34 and HLA-DR whereas 64.77% of non-APL (83%) were positive for both CD34 and HLA-DR. While out of eighty-eight non-APL, two (2.27%) cases were negative for both HLA-DR and CD34 as compared to 77.87% of APL. Thus the negativity for these two antigens doesn’t refer to APL diagnosis exclusively.For more than 20 years, the FAB classification for acute leukemia has been the major system of classification. This classification enabled the diagnosis of a variety of morphologic and cytochemical subtypes of acute leukemia through a structured criterion. However, studies are indicative of failure of most of categories of FAB system in delineating significant disease groups based on morphology and cytochemistry in terms of survival of patients.In case 1, morphological diagnosis was that of AML-M3 while on FCM; HLA-DR and CD34 were positive and was reported as AML-M2. In case 2, morphology suggested AML-M5 but FCM showed negative expression for HLA-DR, CD34, and CD14, reported as AML-M3. Morphological diagnosis of case 3 was AML-M6 but FCM showed negativity for anti-glycophorin A and was reported as AML-M2. Morphology in case 4 was suggestive of AML-M2. However, on FCM positivity of CD14 suggested AML-M4. This discordance was resolved on cytogenetic and molecular analysis. Morphological analysis along with cytochemical stain (SBB) rendered the diagnosis in >80% of our AML cases. Similar study was carried out by Berlukar et al.24One of the limitations was that this study was conducted on a relatively small sample size. the specific markers for AML were used but there was limited use of markers like MPO and CD117. Both these markers are one of the most sensitive markers of myeloid lineage. These were used in only a few cases. Moreover this study would improve if parallel use of molecular analysis was done. However, there were only very few cases with molecular analysis.It is concluded that immunophenotyping is an essential tool in diagnosis and classification of AML. Our findings suggest a 90% concordance between morphology and flow cytometry However, it depends upon the availability of facility for broad panel of primary and secondary markers, which is available only in specialized centres. Interpretation of immunophenotyping should be done in close correlation with morphological findings as both these complement each other. It is need of the hour to extend facility of immunophenotyping at regional levels in the country so that access to these sophisticated facilities is available to patients of leukemias to avoid long distance travel by members of this diseased community.Our findings suggest complementary use of immunophenotyping with morphology improves outcome in the diagnosis of acute leukemias. Further research with use of extended panel of monoclonal antibodies is recommended. Additionally, development of complementary diagnostic techniques such as cytogenetics and molecular analysis should be developed for more precise diagnosis of AML.MB: Did data collection, statistical analysis & and manuscript writing.SAK: Conceived, designed, review and final approval of manuscript.ND: Did statistical analysis, review & editing of manuscript.DA: Did statistical analysis & review of manuscript.