Our objectives were therefore to determine (i) if hypercholesterolaemia alters Ca2+ signalling in aortic endothelial cells before overt atherosclerotic lesions occur, (ii) how Ca2+ signals are affected in older plaque-containing mice, and (iii) whether Ca2+ signalling changes were translated into contractility differences. Using confocal microscopy we found agonist-specific Ca2+ changes in endothelial cells. ATP responses were unchanged in ApoE−/− cells and methyl-β-cyclodextrin, which lowers cholesterol, was without effect. In contrast, Ca2+ signals to carbachol were significantly increased in ApoE−/− cells, an effect methyl-β-cyclodextrin reversed. Ca2+ signals were more oscillatory and store-operated Ca2+ entry decreased as mice aged and plaques formed. Despite clearly increased Ca2+ signals, aortic rings pre-contracted with phenylephrine had impaired relaxation to carbachol. This functional deficit increased with age, was not related to ROS generation, and could be partially rescued by methyl-β-cyclodextrin. In conclusion, carbachol-induced calcium signalling and handling are significantly altered in endothelial cells of ApoE−/− mice before plaque development. We speculate that reduction in store-operated Ca2+ entry may result in less efficient activation of eNOS and thus explain the reduced relaxatory response to CCh, despite the enhanced Ca2+ response.Little is known about how hypercholesterolaemia affects Ca In addition, total plasma cholesterol levels are up to five times that of normal mice Atherosclerosis is a leading cause of illness and death in the Western world. While a large literature exists describing many aspects of the atherosclerotic disease process  were obtained from Charles River and a breeding colony established in-house. Control C57BL/6J mice were obtained from Charles River as required. All were maintained on a normal chow diet. Young (8–12 weeks) or old (28–44 weeks) male mice were used in experiments.2.22) and humanely killed by cervical dislocation in accordance with Schedule 1 of the UK Animals (Scientific Procedures) Act of 1986.Mice were anaesthetized : 5 g Sudan IV, 500 ml acetone, 500 ml 70% EtOH) and gently agitated for 15 min and then destained in 80% EtOH for a further 5 min. Aortae were rinsed in PBS, dissected open along the outer edge of the arch and pinned out in a dissection dish containing PBS. Images were obtained using a dissecting microscope mounted with a Leica camera. These are confirmatory experiments and the data are not shown.The timescale for the development of plaques in the ApoE2.44·7H2O 1.2, HEPES 10.9, glucose 8, and CaCl2 2 (adjusted to pH 7.4). The aorta was cleared of adhering tissue, incubated with 23 μM Fluo-4 AM for 2 h at room temperature in the presence of 0.25% of the non-ionic detergent Pluronic F-127 and subsequently cut into strips (∼1 mm × 6 mm). The tissue was then placed in physiological salt solution to allow de-esterification of the dye. Strips were mounted, endothelium face down, under a small amount of isometric tension between two fixed aluminium foil clips at the bottom of the chamber, on the stage of an Olympus inverted microscope. The chamber was perfused with physiological salt solution at a constant flow rate (1 ml/min) and maintained at 30 °C. Experiments were performed using an Ultraview LCI spinning (Nipkow) disc, widefield confocal microscope , equipped with an Orca ER cooled CCD camera  and either a 20× objective (N.A. 0.7) or a 60× water immersion objective (N.A. 1.20). Mean fluorescence intensity was measured on-line from regions of interest drawn over individual cells using UltraView software. Healthy-looking, well-loaded cells were chosen for analysis, with up to 16 individual cells analysed per tissue strip. Movement artefacts were rarely a problem when measuring from individual cells in a vessel under isometric tension. However, if substantial movement occurred, measurements were not made. The numerical data obtained were saved to an ASCII file for further analysis using Origin 7.0 software. The amplitude of the [Ca2+]i signal was expressed as a normalized pseudo ratio of Fluo-4 fluorescence (F/F0). Once a stable baseline signal was established, the agonists carbachol (CCh) and ATP were applied and the resulting Ca2+ signals measured in terms of the amplitude of the initial peak response, the amplitude of the secondary plateau and area under the curve (measured over a 50 s range from the start of the Ca2+ rise). Data collected from plaque-laden vessels consist of measurements taken from individual cells adjacent to and some distance from plaques since the Ca2+ responses observed were similar regardless of the location of the cells examined . Cyclopiazonic acid , a highly selective inhibitor of the Ca2+-ATPase of the intracellular Ca2+ stores 2+ in the endoplasmic reticulum (ER) and gadolinium  was used to block store-operated Ca2+ entry The heart, with thoracic aorta attached, was removed and placed into a physiological salt solution of composition (mM): NaCl 154, KCl 5.6, MgSO2.52 mm rings of thoracic aorta were mounted between 2 parallel stainless steel wires in 10 ml organ baths containing physiological salt solution (composition as above) maintained at 37 °C and continuously bubbled with oxygen. Rings were placed under an initial resting tension of 1.5 g and then allowed to stabilize for 30 min, during which time the organ bath fluid was replaced twice at 10 min intervals. Aortic rings were pre-contracted with 10 μM PE and once a stable contraction was achieved, cumulative concentration–response curves were obtained to the relaxatory agonist carbachol (CCh: 10 nM to 30 μM). In some experiments, single concentrations of agonist were applied instead . In additional contractility assays, 2 mm rings of aorta were mounted in a DMT dual wire myograph and maintained under the same conditions as described above. The rings were set to their normalized diameter according to the method of Mulvany and Halpern 2.6Membrane cholesterol was extracted using methyl-β-cyclodextrin (MCD), a cyclic oligosaccharide which sequesters cholesterol as previously described 2.73+), MgCl2 replaced MgSO4 in the physiological salt solution.Unless otherwise specified, chemicals were obtained from Sigma (UK). Pluronic-F127 and Fluo-4 AM were obtained from Invitrogen. All compounds were made in aqueous solution apart from SNAP which was dissolved in DMSO (100 mM stock). In experiments using gadolinium  or rings (organ bath studies), from a minimum of 3 aortae. The Student t test was used for statistical comparisons. A value of p < 0.05 was considered significant.Data are presented as mean ± SEM, where 3n = 3). In older ApoE−/− mice, all aortae exhibited clear atherosclerotic plaques . In young ApoE−/− mice, no plaques were visible by eye, and Sudan IV staining revealed either no plaque or barely visible tiny areas presumably indicating where plaques would later form. These data confirm the pre-established timescale of plaque development No WT mice, young or old, exhibited atherosclerotic plaques in the aorta. This was confirmed by staining with Sudan IV  and ATP (10 μM) were investigated in aortic endothelial cells. Both agonists produced a rapid upstroke of intracellular Ca2+ followed by a strongly sustained plateau response in WT and ApoE−/− cells .The effects of hypercholesterolaemia on intracellular Ca/− cells A and D. 2+ responses to CCh (10 μM) stimulation were all significantly larger in the ApoE−/− endothelial cells (n = 116 cells/15 mice) compared to WT control responses .In contrast, there were no changes in these parameters of the Ca3.2−/− aortic endothelial cells. When ApoE−/− aortic strips were incubated with 2% MCD for 10 min, the endothelial cell Ca2+ response  to CCh was significantly reduced . This was a selective effect, in that incubation with MCD did not alter the Ca2+ responses to CCh in the WT aortic endothelial cells . MCD did not alter the response to ATP in either WT or ApoE−/− aortic endothelial cells  to extract cholesterol from the cell membrane should reduce the CCh response in the ApoEal cells D and E.3.32+ responses are affected by hypercholesterolaemia, we evaluated (i) basal Ca2+ levels, (ii) the effect of SERCA inhibition on Ca2+ signalling and (iii) the store-operated Ca2+ entry (SOCE) component of the Ca2+ signals.To gain mechanistic insight into how CCh-mediated Ca2+ levels and compare them between ApoE−/− and WT cells, we measured the basal fluorescence in a large number of WT and ApoE−/− endothelial cells under identical experimental and recording conditions. No significant differences were observed (fluorescence (arbitrary units): 387 ± 4, n = 192 cells/10 mice versus 397 ± 4, n = 235 cells/13 mice; To determine basal Ca2+ signalling was determined by administering CPA (20 μM). CPA produced a significantly larger rise in peak and AUC intracellular Ca2+ signals in the young WT mice compared to the ApoE−/− mice , suggesting that it is SOCE, not ER content, that is altered in the knockout mice.The effect of SERCA inhibition on Ca−/− mice , and thi−/− mice . When th3+ (1 μM), a blocker of this form of Ca2+ entry and using a paired experiment protocol, compared Ca2+ responses to CCh in its absence and presence. The mean data are given in 2+ peak response was unaltered by Gd3+, however the size of the sustained plateau and area under the curve were greatly reduced. In ApoE−/− cells from young animals, Gd3+ produced a small but significant reduction of the initial Ca2+ peak . AUC was reduced to a similar degree in both WT and ApoE−/− cells.To assess SOCE we used Gda2+ peak B, and a 3.42+ response to CCh in 8–12 week old endothelial cells was characterized by an initial peak and a sustained secondary plateau. In most cases, this characteristic response was observed in both WT and ApoE−/− cells. However responses to CCh were more oscillatory in several preparations from young ApoE−/− mice , in older mice, the difference was larger and significant . After treatment with Gd3+, the number of old ApoE−/− endothelial cells that generated an oscillatory signal increased significantly , while no difference was observed in WT cells after Gd3+ treatment. These observations, together with the CPA and Gd3+ data, further suggest that Ca2+ handling is altered in the knockout animal.As mentioned above, the typical Ca−/− mice Aii and v3.52+ signalling differences in endothelial cells in the hypercholesterolaemic model, we were interested to determine whether these differences would be translated into functional differences in an aortic ring organ bath assay.Since we had observed clear Ca3.6−/− rings . However as the mice aged, the PE response became significantly smaller in the knockouts .Rings of thoracic aorta were pre-contracted with phenylephrine  and the relaxatory effects of carbachol determined. In young mice, the size of the PE response was not significantly different in WT and ApoE−/− animals, there was a clear deficit in the CCh-mediated relaxation. While the pEC50 remained unaltered (WT 6.16 ± 0.1 versus ApoE−/− 5.97 ± 0.2), a significantly smaller relaxation was observed to 1, 3, 10, and 30 μM CCh in ApoE−/− rings compared to WT (n = 12–14 rings from 5 to 7 mice). The response to CCh deteriorated further as the animals aged, with all concentrations of CCh (0.1–30 μM) now producing an attenuated relaxation in ApoE−/− mice compared to WT (n = 9–11 rings from 3 to 5 mice), with little change in the pEC50 (WT 6.58 ± 0.23 versus ApoE−/− 6.05 ± 0.24). Treatment with MCD to counteract the effects of the hypercholesterolaemia, led to a partial rescue of the CCh response . Neither 100 nor 300 U/ml SOD altered the CCh dose-response curves in WT or ApoE−/− rings (n = 6 rings/3 mice).It has been suggested that hypercholesterolaemia results in the formation of reactive oxygen species (ROS), which may further result in reduced NO bioavailability −/− rings to relax upon administration of SNAP, an NO donor. WT and ApoE−/− rings (8–12 weeks) responded with a rapid and complete relaxation to 20 μM SNAP . In older animals (20–25 weeks), the same ability to relax rapidly and completely to SNAP was observed .We also compared the ability of pre-contracted WT and ApoE−/− rings. When we compared the half-time of relaxation of the CCh responses (in young mice), it was clear that this parameter was significantly slower in the knockouts compared to WT . In old animals (20–25 weeks), the half-time of relaxation for both CCh  and SNAP  were not significantly different in WT and ApoE−/− mice.We have already seen that there is a reduced maximal relaxation to CCh in ApoEn = 6 rings/3 mice). Rings from ApoE−/− mice behaved identically . Relaxations to CCh were observed in all tissues that failed to relax to ATP stimulation, indicating that the endothelium was indeed intact and functional.When pre-contracted rings from WT mice were exposed to ATP (0.3 μM to 1 mM), no relaxation was observed, instead a small contractile response was obtained on top of the pre-contraction  no evidence for a rise of basal Ca2+, (ii) no evidence for altered ER releasable Ca2+, (iii) a reduction in SOCE, (iv) no alteration in the Ca2+ response to ATP but an exaggerated response to CCh and (v) evidence for an increasingly oscillatory Ca2+ response to CCh as disease progresses. These data suggest an adaptation to the hypercholesteremic environment is occurring in these cells. Consistent with previous reports of impaired vasorelaxation in atherosclerosis, we found impaired relaxation of the vessels to CCh, despite the increased Ca2+ signal. As discussed below, this may be a consequence of the reduced SOCE in the knockout endothelial cells. These findings give novel insight into the aetiology of altered vascular function occurring at the earliest stages of atherosclerotic disease.Confocal microscopy confers several advantages compared to other techniques to measure Ca2+ response to CCh, but not ATP. This is the first reported example of an altered response to muscarinic receptor activation in young ApoE−/− animals. This alteration, being present in young mice, indicates that it is a very early change that pre-dates the development of plaques and is related to the hypercholesterolaemic state, since modulation of cholesterol levels with MCD reversed the changes. Some previous studies in endothelial cells had shown ATP responses to be modified by experimental manipulation of cholesterol 2+ response 2+ signalling and handling, or muscarinic receptor functional coupling and caveolar localization. These suggestions will be discussed further. Our finding of agonist-specific alterations in Ca2+ signalling suggests selective pathways are affected by dyslipidaemia, and that there is not a general downstream effect on all receptor-mediated responses. This agonist specificity has been observed previously after cholesterol manipulation We observed a significantly elevated intracellular Ca+/Ca2+ exchangers (NCX) and SERCA pumps all function to remove Ca2+ from the cell −/− mice, then elevated intracellular Ca2+ levels would occur. However, we are confident this does not explain the elevated response to CCh seen here, as basal Ca2+ levels remain unaltered in ApoE−/− mice and such changes would be expected to alter responses to both CCh and ATP, which we did not observe. Additionally, removal of cholesterol using MCD, did not affect the ATP response, all of which argues against the activity of these transporters being modified in the ApoE−/− endothelial cells.Plasma membrane calcium ATPases (PMCA), Na2+ signals in ApoE−/− endothelial cells, CPA generated a reduced Ca2+ response in these cells, without a change in the amount of Ca2+ released from the internal store, suggesting a reduced role for SOCE in knockout cells. This was confirmed by the SOCE blocker Gd3+ having a significantly smaller inhibitory action on CCh-mediated responses in ApoE−/− endothelial cells compared to WT. Interestingly, Van Assche et al. demonstrated that IP3-mediated SOCE was upregulated in aortic smooth muscle cells from these ApoE−/− mice In addition to the specific increase in CCh-mediated Ca2+ regulation may be altered when caveolar function is modified. We and others have shown that cav-1 and cholesterol content of the plasma membrane play a role in Ca2+ regulation and SOCE 2+ entry pathways. Such alterations could explain why SOCE is reduced and why responses to carbachol are increased in young ApoE−/− aortic preparations, and suggests that the purinergic receptors may not couple to this pathway, as responses to ATP were not altered. There is existing evidence to support caveolae facilitating muscarinic receptor-induced Ca2+ mobilization. For example in airway smooth muscle, muscarinic M3R and Galpha(q/11) cofractionate with caveolin-1-rich membranes, and MCD in tracheal strips reduced the sensitivity to ACh 2+-sensitive PKC isoforms. Thus caveolae can influence contraction and signalling not only by affecting Ca2+ regulation through co-localization of channel subunits and Ca2+ pumps, but also through modifying the receptor-evoked signalling pathways in endothelial cells and therefore fine tuning the responses to muscarinic agonists. This functional organization of caveolae provides for agonists that signal through common Ca2+ pathways, still giving rise to disparate responses.Caveolae are distinct lipid microdomains involved in the compartmentalization of many signalling molecules, including eNOS, and facilitating signal transduction. Caveolin-1 (cav-1) is abundantly expressed in the vasculature, binds cholesterol and is necessary for caveolae formation. Several components of Ca2+ signalling in areas adjacent to fatty streaks, became augmented in the second week of hypercholesterolaemia, but not in animals on a normal diet, and when the entire aorta was covered with fatty streaks in the fourth week of hypercholesterolaemia, endothelial calcium signalling was no longer elevated. Furthermore they also reported no changes in basal Ca2+ signals, as we found in the ApoE−/− mice endothelial cells. Impaired muscarinic-mediated relaxation was also observed in the dyslipidaemic WHHL rabbit aorta 2+ mechanisms appear to function normally, it is carbachol-induced Ca2+ responses that consistently show alteration in the face of chronic hypercholesterolaemia. It should be noted that Kaiser et al. Consistent with our CCh findings are data from Chen et al. 2+ signals lead to production of nitric oxide, which not only causes vasorelaxation but also has powerful anti-inflammatory and anti-thrombotic properties −/− mice, the protective role of NO will fail. Our studies showed that in the aortae of ApoE−/−-deficient mice, endothelium-dependent relaxation becomes progressively retarded with age. We did not find however normal relaxation in young ApoE−/− aortic preparations, despite the increased level of Ca2+ signalling. The relaxatory response was reduced and the half-time of relaxation for CCh was slower in knockout mice, whereas the relaxatory response to SNAP  was unaltered. This may be because the compensation is inadequate in the face of other damaging changes. However our data also point to a role of altered Ca2+ signals.Caveolae, and hence cholesterol levels, are intimately connected to NO signalling. Enhanced coupling of cav-1 with eNOS is associated with diminished production of NO in chronic hypoxia 2+ that enters the cell via SOCE than Ca2+ released from the internal store. The reduced SOCE that we observed in both young and old knockout mice may therefore result in less efficient activation of eNOS and thus explain the reduced relaxatory response to CCh, despite an enhanced Ca2+ response. Our experiments with SNAP show that if NO is supplied by this route, then the vessels from the ApoE−/− mice relaxed without impairment. This strongly suggests that impairment in NO generation is a key change provoking poor relaxation in hypercholesterolaemic environments. In studies of human vessels, decreased function is also reported prior to plaque formation −/− aortic rings suggests this is not the case in our study. Hypercholesterolaemia has also been shown to reduce the expression of gap junction connexins . Removal of extracellular Ca2+ reduced the peak Ca2+ response to ATP by about 34% in WT and 39% in ApoE−/− mice. For CCh these respective figures were 28% in WT but 50% in ApoE−/− mice. If this can be equated to the CCh response in ApoE−/− mice being comprised more from Ca2+ entry, then the reduction in SOCE may have more impact on CCh than ATP.Our data with ATP are challenging, as we found no ATP-mediated relaxatory effect in ApoE2+ responses in ApoE−/− endothelial cells using confocal microscopy and we have identified specific and discrete Ca2+ signalling and handling changes, even before overt plaque development. As a result of the chronic hypercholesterolaemia, carbachol-mediated Ca2+ responses are enhanced, while ATP-mediated responses are unchanged. A reduction in SOCE has also been observed, which we speculate may lead to poorer activation of eNOS and thus explain the reduced vasorelaxation in ApoE−/− tissues despite an elevated Ca2+ response. Thus our data add to those of others suggesting how cholesterol levels and caveolae may affect the regulation of specific receptor pathways and Ca2+ signalling processes. These changes, the earliest derangements of Ca2+ signalling in dyslipidaemia may be key to atherosclerotic progression.In conclusion, this is the first study to measure Ca


In addition to regulating cytoplasmic Ca2+ signals, these responses also affect the concentration of Ca2+ within the ER and mitochondria. Single fluorescent protein-based Ca2+ indicators, such as the GCaMP series based on GFP, are powerful tools for imaging changes in the concentration of Ca2+ associated with intracellular signalling pathways. Most GCaMP-type indicators have dissociation constants (Kd) for Ca2+ in the high nanomolar to low micromolar range and are therefore optimal for measuring cytoplasmic [Ca2+], but poorly suited for use in mitochondria and ER where [Ca2+] can reach concentrations of several hundred micromolar. We now report GCaMP-type low-affinity red fluorescent genetically encoded Ca2+ indicators for optical imaging (LAR-GECO), engineered to have Kd values of 24 μM (LAR-GECO1) and 12 μM (LAR-GECO1.2). We demonstrate that these indicators can be used to image mitochondrial and ER Ca2+ dynamics in several cell types. In addition, we perform two-colour imaging of intracellular Ca2+ dynamics in cells expressing both cytoplasmic GCaMP and ER-targeted LAR-GECO1. The development of these low-affinity intensiometric red fluorescent Ca2+ indicators enables monitoring of ER and mitochondrial Ca2+ in combination with GFP-based reporters.Ca 2+ indicators with low affinities. These indicators enable monitoring of mitochondrial and ER Ca2+ dynamics in combination with a green fluorescent indicator.This paper describes the engineering of two red fluorescent genetically encoded Ca Spectra mCherry  was usedeviously . For Ca22. Cells were seeded on fibronectin-coated, glass-bottomed culture dishes and transiently transfected after 24 h with plasmids using Transit-LT1 (Mirus Bio). Cells were imaged 24–48 h after transfection, using an Olympus IX83 microscope with a ×100/1.49 numerical apesture (NA) objective, at 20°C in Hepes-buffered saline (HBS). HBS had the following composition: 132 mM NaCl, 5 mM KCl, 2 mM MgCl2, 10 mM D-glucose, 10 mM Hepes and 2 mM CaCl2, pH 7.2. Ca2+ was omitted from nominally Ca2+-free HBS. Cells were illuminated alternately, via a 405/488/561/647-nm quad band dichroic/emitter , with a 559-nm light-emitting diode (LED)  for visualizing ER-LAR-GECO1 and a 488-nm diode laser (Coherent) for visualizing ER-GFP and CatchER. Ratiometric measurements of ER-GAP1 fluorescence were achieved by illuminating cells alternately with a 395-nm LED  or a 488-nm laser and measuring fluorescence emission at 520–550 nm using a 535/30 nm emission filter (Chroma Technology). With the filters used there was no significant cross-talk between blue, green and red channels. Images were acquired with an iXon Ultra electron multiplying charge-coupled device (EMCCD) camera (512×512 pixels) (Andor Technology) and images were processed using MetaMorph (Molecular Devices). All records were corrected for background fluorescence determined from regions outside cells. Fluorescence changes from regions of interest were expressed as F/F0, where F0 and F denote the average fluorescence at the start of the experiment (F0) and at each time point (F), respectively. Ratios of the ER-GAP1 fluorescence with alternating excitation at 488 nm and 395 nm were expressed as R (fluorescence intensity with 488-nm excitation/fluorescence intensity with 395-nm excitation), and R0 was defined as the average value of R at the start of the experiment.HeLa, human embryonic kidney (HEK)-293 and U2-OS cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (Life Technologies) supplemented with FBS  at 37°C in air containing 5% CO2 flask, split with PBS, and plated in 100-mm tissue culture dishes at ~10% confluence for 18–20 h before transfection with cDNA encoding D1ER or ER-LAR-GECO1 using the calcium phosphate precipitation method. After 24 h, the growth medium was changed to an induction medium containing 1 μg/ml tetracycline (Sigma). After induction for ~22 h, the cells were perfused continuously with KRH buffer  containing various concentrations of CaCl2  and tetracaine (1 mM) for estimating the store capacity or caffeine (20 mM) for estimating the minimum store level by depleting the ER Ca2+ stores at room temperature (23°C). For D1ER recordings, images were captured with Compix Simple PCI 6 software every 2 s using an inverted microscope (Nikon TE2000-S) equipped with an S-Fluor ×20/0.75 NA objective. The filters used for D1ER imaging were excitation, 436±20 nm for CFP, and 500±20 nm for YFP; and emission, 465±30 nm for CFP, and 535±30 nm for YFP, with a dichroic mirror (500 nm). FRET was determined from the ratio of the light emission at 535 nm and 465 nm. For ER-LAR-GECO1 recordings, images were captured using a Nikon A1R confocal system. The cells were excited using a diode laser (561 nm) and fluorescence emission was detected at 570–620 nm.HEK-293 cells stably expressing wild-type murine type 2 ryanodine receptors (RyR2) (GenBank® accession number: AF295105) were grown to 95% confluence in a 75-cm2+ concentration within mitochondria ([Ca2+]mt) and in the cytosol ([Ca2+]i). Cells were then placed in a chamber for flowthrough perfusion and mounted on an inverted IX-71 microscope (Olympus). Cells were perfused with Hepes-buffered Hanks balanced salt solution (HBSS) composed of 140 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 0.4 mM MgSO4, 0.5 mM MgCl2, 0.4 mM KH2PO4, 0.6 mM NaHPO4, 3 mM NaHCO3, 10 mM glucose and 10 mM Hepes, pH 7.4 with NaOH (310 mOsm/kg with sucrose). Fluorescence was excited sequentially at 340 nm (12 nm bandpass) 380 nm (12 nm bandpass), and 550 nm (12 nm bandpass) using a Polychrome V monochromator (TILL Photonics) and focused on the cells via a ×40/1.35 NA oil-immersion objective (Olympus). Fluorescence emission was separated from excitation by using a dual fluorophore beamsplitter FF493/574-Di01 (Semrock) and collected with a dual band emission filter FF01-512/630 (Semrock) using an IMAGO charge-coupled device (CCD) camera . 2×2 binning was used for acquisition (1 pixel ~500 nm). A series of 340 nm, 380 nm and 550 nm images was acquired every 2 s. [Ca2+]i was calculated from the fluorescence ratio (R=F340/F380) using the formula [Ca2+]i=Kdβ(R−Rmin)/(Rmax−R), where Kd is dissociation constant and β is the scaling factor. Rmin, Rmax and β were calculated by application of 10 μM ionomycin in either Ca2+-free buffer (1 mM EGTA) or in HBSS (1.3 mM Ca2+). [Ca2+]mt changes were quantified as ΔF/F0=(F−F0)/F0×100% where F is the fluorescence intensity at each time point (λex=550 nm) and F0 is the fluorescence intensity in the resting cell before experimental manipulations. [Ca2+]i data were analysed using TILLvisION (TILL Photonics) software. To stimulate neurons by depolarization, the extracellular KCl concentration was elevated by replacing equimolar amounts of extracellular NaCl with KCl.Primary dorsal root ganglion (DRG) neuron cultures were prepared from adult C57BL/6J mice (8–12 weeks of age) and transfected with cDNA plasmids using an Amaxa nucleofection system (Lonza) as described previously ,28. Prim2. Prior to imaging, culture medium was changed to HBSS with 25 mM Hepes. Widefield imaging was performed on an inverted Nikon Eclipse Ti microscope equipped with a 200 W metal halide lamp (PRIOR Lumen), ×20/0.45 and ×60/1.40 NA objectives (Nikon), and a 16-bit QuantEM 512SC EMCCD camera (Photometrics). A filter set of 470/40 nm (excitation), 525/50 nm (emission), and 495 nm (dichroic) was used for Cyto-GCaMP3. Another filter set of 545/30 nm (excitation), 620/60 nm (emission) and 570 nm (dichroic) was used for ER-LAR-GECO1. For time-lapse imaging, HeLa cells were treated with 100 μM histamine.For dual-colour imaging of ER-LAR-GECO1 and Cyto-GCaMP3, HeLa cells  were cultured on collagen-coated 35-mm glass-bottomed dishes (Matsunami) until they reached 40–60% confluency. Transfection was performed by incubating HeLa cells with a mixture of 1 μg of plasmid DNA and 3 μl of Lipofectamine 2000 (Life Technologies) for 2 h. After incubation, the medium was changed to DMEM supplemented with FBS (10%), GlutaMAX™ , penicillin-G potassium salt (50 units/ml) and streptomycin sulfate (50 μg/ml). The cells were incubated for 24 h at 37°C in air containing 5% CO2+-free HBS. Cells were illuminated with 488 nm (for Cyto-G-GECO1.1) or 561 nm (for ER-LAR-GECO1) diode lasers (Olympus), using U-MNIBA  and LF561A  filters, respectively. With the filters used there was no significant cross-talk between green and red channels. Images were acquired with an iXon 897 EMCCD camera (Andor Technology) and processed using cellR software (Olympus). All records were corrected for background fluorescence determined from regions outside cells.For characterizing ER-LAR-GECO1 with Cyto-G-GECO1.1 in cultured human cells, cells were seeded on fibronectin-coated glass-bottomed culture dishes and transiently transfected after 24 h with plasmids using Transit-LT1 (Mirus Bio). Cells were imaged 24–48 h after transfection using an Olympus IX81 microscope with a ×60/1.45 NA objective, at 20°C in HBS or nominally Ca2+ sensors with decreased affinity, we initially considered two single RFP-based Ca2+ indicators, R-GECO1 (Kd=0.48 μM) mt during neuronal activity mt and the cytosolic Ca2+ concentration [Ca2+]i, respectively. The results showed that depolarization evoked coincident and rapidly reversible increases in [Ca2+]mt and [Ca2+]i , slowed the recovery of the increase in [Ca2+]mt, and this effect was reversible . In most cells, the fluorescence intensity of ER-LAR-GECO1 declined transiently and then recovered, mirroring the time course of the cytosolic Ca2+ signals reported by Cyto-G-GECO1.1 . The recovery of the fluorescence intensity of ER-LAR-GECO1 was observed in nominally Ca2+-free medium  and it was inhibited by thapsigargin . These observations are consistent with re-uptake of Ca2+ from the cytosol occurring via the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA).To explore the possibility of using LAR-GECO1 and a green fluorescent indicator for dual-colour imaging of Ca, GCaMP3  (hencefo-GECO1.1  (hencefo2+ indicators, LAR-GECO1 and LAR-GECO1.2, with Kd values for binding to Ca2+ of 24 μM and 12 μM respectively. These indicators enable robust detection of Ca2+ dynamics in organelles containing relatively high Ca2+ concentrations, as we demonstrate for the mitochondria in primary neurons and the ER in cultured human cells. Furthermore, comparisons of LAR-GECO1 with other FP-based low-affinity Ca2+ indicators demonstrate that LAR-GECO1 is a sensitive single-wavelength Ca2+ indicator for detecting Ca2+ dynamics within the ER of mammalian cells. In addition, LAR-GECO1 enables simultaneous dual-colour imaging with GFP-based fluorescent probes.By utilizing rational design and directed evolution, we have developed two low-affinity red fluorescent Ca


In this study, differentiated U937 cells, as a macrophage-like cell model, was used to characterize the fMLF response using electrophysiological and Ca2+ imaging techniques. We demonstrate by means of pharmacological and molecular biology tools that fMLF induces a Ca2+-dependent hyperpolarization via activation of the K+ channel KCa3.1 and thus, enhancing fMLF-induced intracellular Ca2+ increase through an amplification of the driving force for Ca2+ entry. Consequently, enhanced Ca2+ influx would in turn lengthen the hyperpolarization, operating as a positive feedback mechanism for fMLF-induced Ca2+ signaling.Formylated peptides are chemotactic agents generated by pathogens. The most relevant peptide is fMLF (formyl-Met-Leu-Phe) which participates in several immune functions, such as chemotaxis, phagocytosis, cytokine release and generation of reactive oxygen species. In macrophages fMLF-dependent responses are dependent on both, an increase in intracellular calcium concentration and on a hyperpolarization of the membrane potential. However, the molecular entity underlying this hyperpolarization remains unknown and it is not clear whether changes in membrane potential are linked to the increase in intracellular Ca Escherichia coli i) [m) [2+]i by activating IP3 receptors causing Ca2+ release from intracellular reservoirs [m (-15 to -60 mV) in macrophages [2+-activated K+ channel [m increases further [Ca2+]i by modulating the driving force for Ca2+ entry.fMLF-dependent effects in macrophages and neutrophils are mediated by an increase in intracellular calcium concentration ([Ca2+]i) –9 and by+]i) i leading to an hyperpolarization of the Vm [Ca3.1-dependent hyperpolarization triggered by external ATP and mediated by an increase in [Ca2+]i has been previously described , EGTA or control conditions for approximately 5 min before starting the fluorescence measurements. fMLF was perfused throughout the experiments as shown in each figure. Fluorescence was measured using three different systems depending on the type of experiment and Ca2+ indicator used. Sinergy 2 multi-mode microplate reader  was used to measured fluorescence changes in 50–100x103 U937 cells for each condition and acquisition was made using Gene5 Data Analysis software (BioTek). Disk Spinning Confocal Unit  was used to quantify fluorescence changes in the cytoplasm of U937 cells and Cell v2.8 software (Olympus Corp.) was used for acquisition. Two fluorometers were used to measured fluorescence changes in U937 cells incubated with Indo-1 AM and acquisition was performed with AxoScope 8.2 software (Molecular Devices). For Fluo-3 and Fluo-4 the cells were excited at 485 nm and emission was detected at 520 nm. Fluorescence changes were expressed as (Ft-F0)/F0, where Ft was the fluorescence at any time and F0 was the basal fluorescence during 2 min before stimulus. For Indo-1 cells were excited at 340 nm and the acquisition was made at 405 nm and 485 nm. Fluorescence changes were expressed as the ratio 405/485 and the results were expressed as R1/R0, where R1 was the ratio at any time and R0 was the basal ratio during 2 min before stimulus.Intracellular CaAll reagents were of analytical grade and were purchased from Sigma  and Merck . fMLF and dibutyryl cAMP were purchased from Sigma  and dissolved in DMSO.t test was used to compare between two groups. For comparing three or more groups, one-way analysis of variance (ANOVA) followed by Bonferroni's post-hoc test was used. Statistical significance was set at p < 0.05.Data are represented as mean ± SEM. Unpaired Student’s 2+]i induced by fMLF, differentiated U937 cells were loaded with Fluo-3. As depicted in 2+ induced a rapid increase in intracellular Ca2+ followed by a slower decay phase. Under 0 external Ca2+ conditions (no Ca2+ added + 5 mM EGTA) the slower decay phase disappears. As shown in 2+]i in the absence of external Ca2+ was not different to that observed with a solution containing 2 mM Ca2+. However, the normalized area under the curve (AUC) for Ca2+ increase was 94% reduced in the absence of external Ca2+ (To monitor changes in i and hyperpolarization followed a similar temporal course, suggesting that both cellular responses might be linked by a common molecular mechanism.To determine whether the i, by modifying the driving force for Ca2+ influx. 2+ monitoring experiments showing the effect of TRAM-34 and external high-K+ on fMLF-induced Ca2+i dynamics. 2+i time course (left panel) and the normalized AUC (right panel) in normal K+ (5 mM) and high-K+  external solutions. As shown, fMLF-induced Ca2+i increase is reduced (~49%) in high-K+. On the other hand, 1 μM TRAM-34 inhibited (~29%) fMLF-induced Ca2+i increase . In addition, when [K+]e is increased to 65 mM, which changes EK+ to -20 mV, the magnitude of the hyperpolarization is significantly decreased. We also found that this K+ conductance corresponds to a Ca2+-dependent K+ channel because the activation of the current follows the same temporal course of the intracellular Ca2+ increase. Also, absence of Ca2+ influx and inhibition of Ca2+ release from intracellular reservoirs hindered the activation of the fMLF-induced K+ conductance. Finally, based on the pharmacological profile (inhibition by TRAM-34 and potentiation by DC-EBIO) i followed by a sustained phase dependent on Ca2+ influx. Although we did not explore the molecular entities underlying fMLF-induced Ca2+ entry, it is interesting to note that clotrimazole is almost 3-fold more effective in reducing fMLF-induced Ca2+ increase than TRAM-34, a value similar to that observed under 0 external Ca2+ plus EGTA. The robust effect of clotrimazole on the Ca2+ increase  was found to reduce the increase of Ca2+i triggered by extracellular UTP in approximately 30% [Immune cell function depends on the magnitude and duration of the increase in the [Ca2+]i , 33. Thee by 29% . Interestely 30% .Ca3.1 has a role in the Ca2+ signaling induced by fMLF. Thus, the maximal effect of the KCa3.1 blocker on Ca2+ signaling should be observed at a concentration sufficient to abolish fMLF-dependent hyperpolarization. However, we observed that to block the fMLF-induced hyperpolarization 0.1 μM TRAM-34 was enough, while the increase in the [Ca2+]i triggered by fMLF was only significantly diminished with 1 μM TRAM-34. Despite the inconsistency regarding the concentration required to block fMLF-induced hyperpolarization and Ca2+ increase, our results do support a relevant role of KCa3.1. It should be noted that fMLF-induced Ca2+ signaling was diminished by 49% when hyperpolarization was prevented by increasing [K+]e, which confirms that half of the increase in the [Ca2+]i is mediated by a K+-dependent hyperpolarization. Also, although TRAM-34 at 1 μM is still considered a specific blocker of KCa3.1, at this concentration TRAM-34 could block other voltage-gated K+ channels, which have an IC50 of approximately 5 μM [2+ signaling is mediated by KCa3.1, although we can not discard the role of other K+ channels.As mentioned above Kely 5 μM . TherefoCa3.1 plays several roles in the immune response [Ca3.1 facilitates mast cell degranulation in mice [2+i increase by boosting the driving force for Ca2+ entry. Therefore, our study suggests that KCa3.1 might enhance chemotaxis, phagocytosis, reactive oxygen species as well as cytokine production in monocytes and macrophages upon stimulation with chemotactic peptides.It has been previously shown that Kresponse –36. For  in mice , migrati in mice  and promCa3.1 is the molecular entity responsible for the hyperpolarization observed upon fMLF exposure in differentiated U937 cells, thereby controlling the driving force for Ca2+ entry and thus, modulating Ca2+i signaling in these cells through a positive feedback mechanism.In summary, our data indicate that KS1 FigA and B. Representative Indo-1 (A) and Vm (B) recordings during fMLF treatment in non-differentiated U937 cells, 100 nM fMLF and 5 μM ionomycin (ION) were applied during the time indicated by the horizontal bars.(EPS)Click here for additional data file.S2 Figm was recorded in a differentiated U937 cell in the presence of 20 mM TEA in the bath and 140 mM Cs+ in the pipette. Horizontal bar indicates exposure to 100 nM fMLF (n = 4).V(EPS)Click here for additional data file.S3 Figm recording obtained form a differentiated U937 cell in the presence of 100 nM TRAM-34 in the bath. Horizontal bar indicates exposure to 100 nM fMLF (n = 4).V(EPS)Click here for additional data file.S4 FigA. Average of Indo-1 records performed in control conditions (●) or in the presence of 10 μM clotrimazole (○) in differentiated U937 cells. In both conditions cells were treated with 100 nM fMLF during the time indicated by the horizontal bar. B. AUC of fMLF-induced Ca2+ increase in control (black) or clotrimazole (empty) treatment .(EPS)Click here for additional data file.


We found that, in MPAL patients, there were high proportions of blast cells in bone marrow and incidence of hepatosplenomegaly, lymphadenopathy, and Philadelphia chromosome. The overall survival (OS) and relapse-free survival (RFS) in MPAL patients were significantly shorter than those in Ly+AML and Ly−AML. With regard to the patients with normal karyotype only, the OS and RFS of MPAL were significantly lower than those of the Ly+AML and Ly−AML; but there were no significant differences in OS and RFS among the patients with complex karyotype. The OS rates of 3 groups with complex karyotype were lower than those of patients with normal karyotype. In Cox multivariate analysis, complex karyotype was an independent pejorative factor for both OS and RFS. Therefore, MPAL is confirmed to be a poor-risk disease while Ly+AML does not impact prognosis. Complex karyotype is an unfavorable prognosis factor in AML patients with different immunophenotype. Mixed immunophenotype and complex karyotype increase the adverse risk when they coexist.Mixed phenotype acute leukemia (MPAL) is a complex entity expressing both lymphoid and myeloid immunophenotyping. In the present study, 47 MPAL, 60 lymphoid antigen-positive acute myeloid leukemia (Ly However, close to 2 to 5% of cases , 2, the +AML patients have led to inconsistent conclusions and there is lack of reports comparing clinic characteristic and outcome in MPAL with Ly+AML. As the result, the therapy for this subtype of leukemia is not consistent. The goal of present study was to analyze the molecular genetic, cytogenetic, and immunophenotypic features of patients with MPAL according to WHO 2008 classification and the relationship between clinic outcomes and laboratory characteristics.Hitherto, although lack of the MPAL cases research according to the WHO 2008 classification was reported, it is well known that MPAL appears to be a complex entity with different biological characters  and low +AML), and 90 were with acute myeloid leukemia with common myeloid immunophenotype (Ly−AML). They were matched based on the subtypes, gender, and age (α-positive were excluded. Cases were classified by morphology and simultaneously by cytochemistry for myeloperoxidase (POX), sodium fluoride sensitive naphthalene acetic acid esterase (NAE/NAF), and glycogen (PAS). Study protocol underwent thorough review and approval process at the hospital's ethics committee. Informed consent was obtained from all patients included in the study.A total of 197 patients with acute myeloid leukemia diagnosed according to WHO 2008 criteria [ and age . AML aftFCM immunophenotyping was performed on isolated bone marrow mononuclear cells by flow cytometry after collection according to standard procedure. All cases were profiled by a panel of leukocyte-associated markers, including cMPO, CD117, CD13, CD33, CD14, CD15, CD64, and CD11b/c for the myeloid lineage; cyCD3, CD2, CD5, CD7, and CD8 for T-cell lineage, cyCD79a, CD22, CD19, CD10, CD22, and CD20 for B-cell linage; and CD34 and HLA-DR for the stem/progenitor cell clone. A marker was considered positive when more than 20% of the blasts showed a positive signal.β/MYHll, BCR/ABL, and MLL rearrangements as well as P53, Del(5q), Del(7q), Del(20q), and +8 were performed according to manufacturer's instructions.Conventional cytogenetic analysis was carried out on direct preparations or 24 h unstimulated culture of bone marrow cells according to standard technique of G-banding. Meanwhile, the abnormal cloning was defined based on the criteria from International System for Cytogenetic Nomenclature (ISCN). FISH studies aimed at detecting AML1/ETO, PML/RARa, CBFPatients who received at least 2 circles of chemotherapy were involved in current study in order to analyze clinic outcome. All patients received induction and maintenance treatment according to guidelines set by the Hematological Society of the Chinese Medical Association. The induction treatments were as follows: ① combined AML/ALL regimens (DOALP and DOAP), ② ALL-type induction therapy (VDLP and VDCP), and ③ AML-type induction therapy (DA/DAE/HA/TA). Meanwhile, they received postremission therapy as follows: ④ 3 circles of consolidation therapy and less than 3 circles of MD-Ara-C/HD-MTX, ⑤ equal to or more than 3 circles of MD-Ara-C/HD-MTX or more than 3 circles of Hyper-CVAD-A/B or autotransplantation after 5 median circles of standard-dose induction therapy, which ranges from 3 to 10 circles, and ⑥ allogeneic hematopoietic stem cell transplantation after 3 to 6 circles of consolidation or intensive therapy. The distribution of cases of induction regimen and consolidation therapy was summarized in χ2 tests were used for the analysis of qualitative data. All analyses were done with SPSS version 17.0, and P value of less than 0.05 was considered to be associated with statistical significance.The two independent samples nonparametric tests were used for comparison of quantitative data. Kaplan-Meier method was used to estimate the long survival of patients. Cox univariate and multivariate analyses were performed to identify risk factors related to OS and RFS. The Pearson +AML or Ly−AML patients, MPAL patients displayed a higher ratio of lymphadenopathy and hepatosplenomegaly (63.8%) and higher percentage of blasts in bone marrow (67.6%). However, there were no statistical differences between the two groups of Ly+AML and Ly−AML.Patient characteristics were summarized in +AML (81.7%) subtypes was significantly higher than in Ly−AML group (47.8%). On the contrary, a marker of differentiated cells, CD117, was significantly lower in MPAL cases (8.5%) than Ly+AML (61.7%) and Ly−AML (51.1%).Immunophenotype data showed that the positive incidence of CD34 in MPAL (76.6%) and Ly+AML  and Ly−AML . However, it seemed that MPAL patients had the lowest incidence of normal karyotype (27.0%) in the three groups (P = 0.003), and there were no statistical differences between the two groups of Ly+AML and Ly−AML. Philadelphia chromosome and fused BCR-ABL gene incidence were highest in MPAL group of patients (15 out of 37 MPAL patients with available karyotypic data (40.5%) had complex karyotype. Complex karyotype that frequently appeared in MPAL was observed in lower incidence in Lypatients . Fluores+AML, and 79 Ly−AML patients according to WHO 2008 criteria were shown in P = 0.022 and P = 0.014). By applying the results of paired comparison, we found that the OS in MPAL was significantly shorter than that in Ly+AML (P = 0.046) and Ly−AML patients (P = 0.006). Meanwhile, the RFS in MPAL was also significantly shorter than that in Ly+AML (P = 0.019) and Ly−AML group (P = 0.007). However, the OS and RFS were not significantly different between Ly+AML and Ly−AML groups  and relapse-free survival (RFS) rates for 35 MPAL, 55 LyL groups .+AML and Ly−AML groups with normal karyotype, complete remission (CR) rates were 88.5% (23/26) and 80.4% (37/46) after a second induction regimen, which was higher than the cases with complex karyotype (P = 0.013 and P = 0.029). However, in MPAL group, there was no difference in CR rate between normal karyotype and complex karyotype patients . Meanwhile, the overall final complete remission rate after two cycles of chemotherapy of AML patients with different immunophenotype  had no statistical differences in the same genetic background. Details were shown in In Ly+AML (29 months) and Ly−AML (46 months) patients. There was significant difference in overall and relapse-free survival rates among MPAL, Ly+AML, and Ly−AML patients with normal karyotype (P = 0.002 and P = 0.004). By applying the results of paired comparison, the OS rate of MPAL patients was significantly lower than that of the Ly+AML (P = 0.008) and Ly−AML (P = 0.001), but no statistical differences were found between Ly+AML and Ly−AML patients. The RFS in MPAL with normal karyotype was significantly lower than that in the Ly+AML (P = 0.02) and Ly−AML (P = 0.002), but there was no statistical difference between the latter two groups, as shown in + chromosome or BCR/ABL fusion gene), there were no statistical differences among the 3 groups in OS and RFS. The median survival time of patients with complex karyotype in three groups was 7.5 months in MPAL, 9.4 months in Ly+AML, and 15 months in Ly−AML, respectively  compared with Ly months c+AML, and Ly−AML patients with either normal or complex karyotype were shown, respectively, in Figures +AML group, the RFS rates of MPAL and Ly−AML patients with complex karyotype were lower than those in patients with normal karyotype (P = 0.05 and P = 0.04). There were statistical differences of OS between MPAL patients with normal and complex karyotype (P = 0.005). Meanwhile, MPAL patients with normal karyotype had distinctly longer median survival time than those with complex karyotype (median 18 months versus 6 months). Furthermore, statistical differences were also found in the Ly+AML and Ly−AML patients between normal and complex karyotype (P = 0.027 and P = 0.038). In the two groups (Ly+AML and Ly−AML), median survival time was 37.4 and 46 months for patients with normal karyotype versus 7.3 and 15 months for patients with complex karyotype  and complex karyotype (P < 0.01) in univariate analysis and complex karyotype (P < 0.01) in multivariate analysis were also poor prognostic factors on RFS  to attempt identifying risk factors relating to OS. Age, peripheral white blood cells, MPAL, complex karyotype, and abnormal karyotype were shown to display adverse prognostic significance on OS . HoweverS Tables  and 5. SAML is a genetically heterogeneous disease with widely different treatment outcomes and disparate survivals. Thus, it is important to propose prognostic model effectively for subtype of AML subtypes diagnosis and evaluations of outcome. Now, the most important prognostic factors for AML are age and chromosome karyotype , 10. How+AML and Ly−AML groups. And, in the current study, the median age of 34 years at diagnosis and a slight predominance of the male population (63.8%) is similar to that reported by Yan et al. [According to the WHO 2008 classification of leukemia  and the n et al. . As repon et al.  and Bachn et al. , we also−AML, and Ly+AML. However, there were no significant differences in OS and RFS between Ly−AML and Ly+AML patients. These results demonstrated that MPAL may be a poor prognostic indicator in AML. However, it needed to be taken with caution because of the high incidence of complex karyotype in MPAL patients included. Therefore, all patients with normal karyotype were considered only, and it was found that the survival of MPAL patients was also worse than that in Ly+AML and Ly−AML. Meanwhile, factors relating to survival were evaluated with the Cox proportional-hazards regression model. As a result, in multivariate analysis, only patients with complex karyotype predicted a shorter OS and RFS.The previous studies showed that survival in biphenotypic acute leukemia was shorter than in other acute leukemia patients whatever adults or children , 15–17. +AML and Ly−AML groups. These findings demonstrated that complex karyotype was strongly associated with adverse outcome in AML, which was in harmony with several early studies [+AML and Ly−AML patients with complex karyotype present similar survival time with MPAL? A possible explanation could be that complex karyotype has a more significant impact on clinical outcome in AML patients with no matter which subtype of immunophenotype. Several studies also demonstrated that complex karyotype is a poor prognosis factor [Notably, within the MPAL group, patients with complex karyotype had shorter survival compared with normal karyotype patients. Similar phenomena were also found on OS in Ly studies , 19. It s factor , 19, and+AML and Ly−AML, which was consistent with those of Casasnovas et al. [+AML patients.In multivariate analysis for OS, in addition to complex karyotype, peripheral white blood cells (WBC) were also found as risk factor as it was also established in report by Mikulic et al. . Howevers et al. . To our +AML does not impact prognosis. Complex karyotype is an unfavorable prognostic factor in AML patients with different immunophenotype. Moreover, mixed immunophenotype and complex karyotype increase the adverse risk when they coexist.In conclusion, MPAL is confirmed to be a poor-risk acute leukemia subtype with normal chromosome karyotype, while Ly


Localized Ca2+ signals (LCSs) in skeletal muscle fibers of mice have been mainly studied as Ca2+ release events from the sarcoplasmic reticulum. Their location with regard to cell nuclei has not been investigated. Our study is based on the hypothesis that LCSs associated with nuclei are present in skeletal muscle fibers of adult mice. Therefore, we carried out experiments addressing this question and we found novel Ca2+ signals associated with nuclei of skeletal muscle fibers (with possibly attached satellite cells). We measured localized nuclear and perinuclear Ca2+ signals (NLCSs and PLCSs) alongside cytosolic localized Ca2+ signals (CLCSs) during a hypertonic treatment. We also observed NLCSs under isotonic conditions. The NLCSs and PLCSs are Ca2+ signals in the range of micrometer . Additionally, global nuclear Ca2+ signals (NGCSs) were observed. To investigate which type of Ca2+ channels contribute to the Ca2+ signals associated with nuclei in skeletal muscle fibers, we performed measurements with the RyR blocker dantrolene, the DHPR blocker nifedipine or the IP3R blocker Xestospongin C. We observed Ca2+ signals associated with nuclei in the presence of each blocker. Nifedipine and dantrolene had an inhibitory effect on the fraction of fibers with PLCSs. The situation for the fraction of fibers with NLCSs is more complex indicating that RyR is less important for the generation of NLCSs compared to the generation of PLCSs. The fraction of fibers with NLCSs and PLCSs is not reduced in the presence of Xestospongin C. The localized perinuclear and intranuclear Ca2+ signals may be a powerful tool for the cell to regulate adaptive processes as gene expression. The intranuclear Ca2+ signals may be particularly interesting in this respect.Nuclear Ca Skeletal muscle fibers with possibly attached satellite cells from the interossei muscles of the toe were enzymatically isolated using collagenase following a protocol similar to that described in an earlier study  and the DNA dye HCS NuclearMask Deep Red stain  for 15–30 min at 37°C or with Fluo-4 AM (10 μmol/l) and the membrane dye di-8-ANEPPS  for 15–20 min at 37°C.For the measurements of LCSs, the skeletal muscle fibers were stained with the Ca2  for 30–60 min before the hypertonic dantrolene solution was added.For measurements with Xestospongin C , the skeletal muscle fibers were preincubated with Xestospongin C for 30–40 min and the hypertonic solution containing Xestospongin C was added.2+ indicator Rhod-2 AM  and the DNA dye HCS NuclearMask Deep Red stain (volume ratio 1/3000) were added.For the identification of the satellite cells, the skeletal muscle fiber preparations were exposed to anti-CD34-FITC  in a volume ratio 1/200 for 3 h at 37°C as described by Liu and Schneider . During An inverted microscope , a confocal laser scanning unit , a 63X water immersion objective , the laser line 488 nm of an Argon laser and the laser lines 543 and 633 nm of a HeNe laser were used. The fluorescence was detected on a descanned light path by a photomultiplier tube .XYT images were saved as 8-bit images and contain 512 ∗ 512 pixels with pixel sizes between 0.186 and 0.232 μm. Each image was recorded within 0.82 s.XT 8-bit images were recorded with a time resolution of 800 lines per second or 1000 lines per second. The pixel size is 0.063 μm and the images contain 512 pixels in x-direction.The images shown in the figures were converted from 8 bit to RGB.The dye-loaded skeletal muscle fibers were placed on Poly-L-lysin (Sigma Aldrich) coated coverslips. The different combinations of dyes, the different laser lines used for each combination, the detected spectral ranges and the maximal deviations from the stated detected ranges are summarized in Table In a few experiments with Fluo-4 AM and HCS NuclearMask Deep Red stain only the laser line 488 nm was used for the excitation of both dyes. We performed these experiments to exclude the possibility of chromatic errors of the optical system because of the different wavelengths used for excitation. Even in conditions when the DNA dye was excited first with 633 nm and then with 488 nm we could not observe a dislocation of the nucleus. In all figures presented here the DNA dye was excited with 633 nm.Microspheres (see next chapter) were excited with 488 nm and the spectral ranges 500–600 and 650–800 nm were simultaneously detected and no shifts could be observed. These observations show that if the same laser line was used for excitation no chromatic shifts are detectable. Small lateral shifts  could be observed in some experiments when the microspheres were excited with 488 nm detecting the spectral range 500–600 nm and then excited with 633 nm detecting the spectral range 650–800 nm. Such shifts could not be observed as described above when the outlines of nuclei were compared using 488 nm  and 633 nm  for excitation.2+ signal were measured simultaneously. After this measurement the same structural measurement as at the beginning was recorded and this measurement is again an average of eight images. This image was compared with the first image and the structural images recorded simultaneously with the Ca2+ signal to determine if there was a displacement of the fiber during the measurement. In some cases, the signals of the DNA dye or the membrane dye recorded simultaneously with the Ca2+ measurements were too noisy. If not specifically mentioned the DNA dye signal or the di-8-ANEPPS signal shown in the figures are the signals recorded before or after the measurement of the Ca2+ signal.For most measurements, after the addition of the hypertonic solution or the hypertonic solution containing sorbitol first a structural measurement of the DNA dye signal or the di-8-ANEPPS signal was recorded taking averages of eight images. Then the image sequence of the structural signal and the Ca2+ signal was recorded simultaneously with the structural measurements at the beginning and at the end. These records were performed for structural purposes since some nuclei could be observed in the Fluo-4 AM signal due to elevated fluorescence in the nuclear envelope.In some measurements an average of eight images of the CaFor one of the four recorded NGCSs, the signal of the DNA dye was solely recorded before and after the measurement, and not simultaneously.All measurements were performed not later than 70 min after the addition of the hypertonic solution.We used microspheres with a diameter of 0.1 μm  diluted in water to determine the lateral and axial resolution of the optical system.For the determination of the lateral and axial resolution z stacks of the microspheres were performed and the Fiji plugin MetroloJ  μm and rlateral;488;500−600 = (0.48± 0.03) μm  and raxial;488;500−600 = (1.67± 0.16) μm and rlateral;488;500−600 = (0.44± 0.02) μm . For the record of the DNA dye signal the following settings were used: excitation 633 nm, detection 650–800 nm and the axial and lateral resolutions for those settings are: raxial;633;650−800 = (1.78± 0.08) μm and rlateral;633;650−800 = (0.48± 0.02) μm,  and raxial;633;650−800 = (1.62± 0.12) μm and rlateral;633;650−800 = (0.44± 0.05) μm . The minimal distance dm for two dyes with two different axial resolution limits is: dm = 1∕2. For our settings dm is 2.09 μm for a pinhole diameter of 70 μm (3.15 Airy units) and 1.79 μm for a pinhole diameter of 50 μm (2.24 Airy units).After the measurements of the LCSs, z stack images of the nuclei were taken as due to the limited axial resolution of the optical setup, it is possible that the measured LCSs within the nucleus in the two dimensional records could have been originated outside of the nucleus. To demonstrate that the LCS is indeed inside the nucleus, a signal of the DNA dye should be recorded inside the region of the LCS in a plane above and under the plane of the Caaxial and dm) compared to the true axial resolution of the system . The NLCSs and CLCSs, used to determine the properties of the NLCSs and CLCSs  and the nuclear and cytosolic frequencies of LCSs, were identified by the algorithm and visually confirmed by the experimenter. Signals identified solely by the algorithm or visually by the experimenter are not included.2+ signals (INCSs: NLCSs and NGCSs) used to determine the fraction of responding fibers and nuclei were only identified by the experimenter as the occurrence of “false negatives” was more critical for this analysis and several NLCSs were restricted by the boundaries of the nucleus providing further challenge for the detection with the algorithm. When LCSs were solely identified by the experimenter, they are referred as NLCSs if they are located inside the nucleus in the two dimensional image. Additionally, the fraction of NLCSs is stated in Table CLCSs and intranuclear CaNLCSs marked in the graphs were identified by the algorithm and visually by the experimenter.LCSs are referred as PLCSs if the distance between their centroid and the nucleus is ≤ 2 μm (for records in xyt images). PLCSs were identified by the algorithm and also visually by the experimenter. The dataset used for the analysis of PLCSs is somewhat smaller than the dataset used for the analysis of INCSs since datasets with small displacement during the measurement (for instance during the addition of the hypertonic solution) could not be analyzed with the algorithm. In xt images PLCSs were identified by the experimenter.2+ indicator Fluo-4 AM and the membrane dye di-8-ANEPPS and found LCSs in nuclear shaped compartments of the fiber /F0 is 0.55 ± 0.05. When the NLCS measured under isotonic conditions is excluded from the statistics, the properties of the 36 NLCSs recorded after the addition of the hypertonic solution are: the mean FWHM is (2.80 ± 0.27) μm and the mean amplitude (F – F0)/F0 is 0.51 ± 0.04. The properties of the 878 LCSs (seven fibers with NLCSs) detected in the cytosol of fibers with NLCSs during the hypertonic treatment are the mean FWHM: (2.31 ± 0.04) μm and the mean amplitude (F – F0)/F0: (0.79 ± 0.02). Those results are summarized in Figures Thirty-seven NLCSs in eight fibers  out of 41 fibers  were identified by the algorithm and visually by the experimenter. Ten NLCSs in two fibers were identified when using di-8-ANEPPS. Twenty-seven NLCSs in six fibers were identified when using the DNA dye to visualize the nuclei. One NLCS was measured under isotonic conditions and 36 NLCSs after the addition of the hypertonic solution. The mean full width at half maximum (FWHM) is (2.75 ± 0.27) μm and the mean amplitude (F) of LCSs in the nucleoplasm and in the cytosol of fibers and nuclei with NLCSs are: FCytosol: (0.233 ± 0.093) LCSs/(1000 μm2* s) and FNucleus: (0.500 ± 0.186) LCSs/(1000 μm2* s)  by the experimenter but not by the algorithm.2+ signal is presented. We observed NGCSs in nuclei with a similar location (at the periphery of the muscle fiber) in two further fibers.We also recorded NGCSs in four skeletal muscle fibers out of 41 fibers during the hypertonic treatment. In Figure 2+ signal , likely to be satellite cells, we observed global cellular CaWe determined the fraction of fibers and nuclei responding with CLCSs (only for the fibers), INCSs, NLCSs and NGCSs under isotonic conditions and during the hypertonic treatment Table . Not incUsually NLCSs were observed in nuclei at the periphery of skeletal muscle fibers. In one skeletal muscle fiber one NLCS  and in a further skeletal muscle fiber from a different animal NLCSs  were observed in central nuclei. Using z-stacks we showed that those are central nuclei.2+ signals associated with nuclei were observed. (4 ± 1) nuclei per fiber were investigated. In nine out of 11 fibers with NLCSs we observed NLCSs only in one nucleus in each fiber. In two fibers, we observed NLCSs in two nuclei of each fiber. (5 ± 1) nuclei per fiber were investigated.We identified INCSs in 15 fibers. In four fibers with NGCSs, we observed in each fiber only in one nucleus NGCSs and no other CaUnder hypertonic treatment, we observed PLCSs in 10 skeletal muscle fibers  out of 32 fibers . Twenty-five nuclei out of 121 nuclei were associated with PLCSs /F0 = (0.99 ± 0.06).The properties of the 58 detected PLCSs are: FWHM (2.55 ± 0.17) μm and  of cellular LCSs in skeletal muscle fibers after the addition of the hypertonic solution to 1.000 ± 0.369. In the presence of dantrolene the relative frequency of LCSs was reduced to 0.205 ± 0.107. The effect of nifedipine was even stronger and reduced the relative frequency of LCSs to 0.038 ± 0.006. The results are summarized in Figure We normalized the frequency  is slightly decreased  in nuclei of satellite cells attached to skeletal muscle fibers.It cannot be excluded that the nuclear Ca2+ indicator Rhod-2 AM . The measurement shows that one of the nuclei belongs to a satellite cell.Therefore we used a marker for the satellite cells, as recently described by Liu and Schneider  out of 23 fibers .In one skeletal muscle fiber, we observed two LCSs located in close proximity to the nucleus. One of those signals is shown in Figure 2+ signal in three fibers  out of four fibers  and this signal may mask the LCSs during the first minutes. Similar observations have been described in skeletal muscle fibers after the addition of sucrose or mannitol containing hypertonic solutions  in a significant number but they may become more abundant (and their properties may change) during the stressing stimulus so they become easier to detect. Interestingly, we observed also NLCSs in skeletal muscle fibers under isotonic conditions. But we could not observe PLCSs or NGCSs under isotonic conditions. Additionally, we observed LCSs in two fibers in close proximity to the nucleus under hypertonic treatment with a solution containing sorbitol, leading to a less stressful condition and keeping the physiological Ca2+ gradient between the cytosol and the extracellular solution. The observations obtained under isotonic conditions and with the sorbitol containing hypertonic solution suggest that the high concentration of Ca2+ is not essential as a stimulus for the recording of LCSs associated with the nucleus. However, we mainly studied LCSs with a high Ca2+ concentration in the extracellular solution and this is not a physiological condition. Interpretations and conclusions of results obtained in this condition have therefore limitations when transferred to more physiological states of the fiber. It may be noted that under a hypotonic condition the frequency of cellular LCSs is markedly lower compared to the hypertonic condition with a high Ca2+ concentration  may be located inside the nucleus. It is also possible that Ca2+ described by Gerasimenko and Gerasimenko  identified simultaneously by the algorithm and by the experimenter and only by the experimenter under isotonic and hypertonic conditions (without blocker) is ~73%. The results presented in Table 2+ signals may occur in the satellite cells  or in nuclei of the satellite cells  instead of the nuclei of skeletal muscle fibers. Recently, it has been shown that FGF2 increases the Ca2+ signals in satellite cells attached to the skeletal muscle fibers  global cellular Ca2+ signals in cells in close proximity to skeletal muscle fibers  and they are likely to be satellite cells. On the other hand similar NGCSs were already observed in nuclei of myotubes  (RHAF), Landesforschungsschwerpunkt Baden Württemberg (RHAF), and Chile PIA-ACT1111 (EJ). TG received a Ph.D. scholarship from the Heidelberg Medical School and a scholarship from the German Academic Exchange Service (DAAD Exzellenz II Heidelberg Chile).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.


Early detection of fibrosis is important in identifying individuals at risk for advanced liver disease in non-alcoholic fatty liver disease (NAFLD). We tested whether second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy, detecting fibrillar collagen and fat in a label-free manner, might allow automated and sensitive quantification of early fibrosis in NAFLD.We analyzed 32 surgical biopsies from patients covering histological fibrosis stages 0–4, using multimodal label-free microscopy. Native samples were visualized by SHG and CARS imaging for detecting fibrillar collagen and fat. Furthermore, we developed a method for quantitative assessment of early fibrosis using automated analysis of SHG signals.We found that the SHG mean signal intensity correlated well with fibrosis stage and the mean CARS signal intensity with liver fat. Little overlap in SHG signal intensities between fibrosis stages 0 and 1 was observed. A specific fibrillar SHG signal was detected in the liver parenchyma outside portal areas in all samples histologically classified as having no fibrosis. This signal correlated with immunohistochemical location of fibrillar collagens I and III.This study demonstrates that label-free SHG imaging detects fibrillar collagen deposition in NAFLD more sensitively than routine histological staging and enables observer-independent quantification of early fibrosis in NAFLD with continuous grading. The subset of patients with NAFLD who have fibrosis (any stage) are at increased risk of liver-related death ,2. While2 hydro-carbon bonds [Nonlinear imaging entails several optical microscopy techniques that enable sensitive detection of native structures in cells and tissues without exogenous labels. In particular, second-harmonic generation (SHG) is a non-resonant process that takes advantage of a coherent optical signal generated by non-centrosymmetric structures, such as fibrillar collagen . Coherenon bonds ,8. Thus,on bonds ,10. In Gon bonds ,10.In NAFLD, fat initially accumulates around the central vein in zone 3. In this area, which has lower levels of oxygen than the periportal area , hepatocUntil now, there are no data available on the utility of SHG imaging in NAFLD. In the present proof-of concept study, we wished to determine whether it is possible to develop an automated analysis of liver fibrosis in NAFLD and whether the use of SHG imaging might be able to detect early, subtle signs of fibrosis better than routine histopathology.The patients underwent a metabolic study for clinical characterization approximately one week prior to the liver biopsy, which was taken during bariatric surgery. Subjects were eligible if they met the following criteria: (a) age 18 to 75 years; (b) no known acute or chronic disease except for obesity or type 2 diabetes on the basis of medical history, physical examination and standard laboratory tests ; (c) alcohol consumption less than 20 g per day and less than 30 g for men; (d) no clinical or laboratory evidence of other liver disease. Patients were excluded if they were pregnant. The study protocol was approved by the ethics committee of the Helsinki University Central Hospital and follows the 1975 Declaration of Helsinki guidelines. Each participant provided written informed consent. At the metabolic visit, a blood sample was taken after an overnight fast for the screening laboratory tests (vide supra) as well as for measurement of fasting plasma glucose, serum insulin, fS-LDL cholesterol, total serum cholesterol, fS-HDL cholesterol, fS-triglycerides, fS-AST, fS-ALT, and fS-GGT concentrations as described . Body weWedge biopsies of the liver (50–150 mg) were taken at laparoscopic surgery. Approximately one-half of the liver sample was fixed in formalin and sent to the pathologist, who was unaware of the SHG-scoring data, for histopathological assessment, whereas the rest was immediately frozen and stored in liquid nitrogen. The sample frozen in liquid nitrogen was embedded in optimal cutting temperature (OCT) compound and cryosectioned at 20μm thickness. Slices were fixed with 4% paraformaldehyde for 60 min and a coverslip added. The prepared slides were stored at -20°C for no longer than a week prior to examination by non-linear microscopy.NASH was defined according to the scoring system proposed by Kleiner et al. . For his-1. The pulses from the two sources were temporally and spatially overlapped on the focal plane of the microscope. Up to 100 mW of average power from both the pump and the Stokes source was delivered to the sample. No signs of photodamage as assessed in [2 hydro-carbon bonds at 2845 cm-1. Images were acquired from unstained slides using a 25x water immersion objective (Leica HCX IR APO L 25X/0.95 W). To cover the area of the entire biopsy specimen (about 4 x 4 mm), multi-tile scanning (up to 25x25 tiles) was performed. All images were recorded using the Leica Application Suite Advanced Fluorescence (LAS AF) software.Images were acquired with a commercial Leica TCS SP8 CARS confocal microscope. The instrument consists of an inverted microscope equipped with an ultra-short pulsed light source  that produces the two synchronous beams needed for CARS microscopy. The Stokes beam at 1064 nm was emitted from a neodymium-doped yttrium orthovanadate (Nd:YVO4) laser while a tunable pump/probe beam at 780–940 nm was generated by an optical parametric oscillator (OPO). The pulse width was 5–7 ps with a repetition rate of 80MHz corresponding to the Raman line width of 2–3 cmessed in  were obsThe images were processed and analyzed using Fiji . BackgroDetermining sample borders: The signals acquired from the SHG and CARS channels were overlayed to highlight the sample area. The image was then auto-thresholded by the percentile method  and convDetermining portal areas: The SHG channel was extracted from the image. To remove background, the 32-bit image was auto-thresholded to mean intensity. Next, iterations of filtering were used to harmonize the highly fibrillar structure of collagen fibrils . Finally, the image was thresholded (1.17% of maximum pixel intensity) for particle analysis. Recognized particles were filtered with loose size and roundness parameters.Measuring signal intensities: The background signal of the original image was first removed, by subtracting the mean signal intensity outside of the sample (recognized in step A). Mean SHG- and CARS-signal intensities were then measured after excluding the portal areas and the capsule.Human tissue samples were analyzed using a basic immunofluorescence protocol for formalin-fixed and paraffin-embedded tissue sections. In brief, 5-μm sections underwent xylene/ethanol rehydration series and were blocked with 1% BSA in PBS (pH 7.2) for 1h, incubated with a primary antibody overnight at 4°C and with the secondary antibody for 1 hour at room temperature. All washes were performed with PBS. The following primary antibodies were used with the dilutions indicated: collagen I  and collagen III . Before staining, epitope retrieval was performed by a heat-mediated method for 15 min with sodium citrate buffer (pH 6.0). Anti-rabbit Cy3  was used as a secondary antibody. For negative control stainings, the primary antibodies were omitted and replaced by PBS. Immunostaining with different antibodies was performed on parallel sections.For the patient data, continuous variables were tested for normality using the Kolmogorov-Smirnov test. Normally distributed data were reported in means ± SEM while non-normally distributed were reported in medians and interquartile ranges. For the microscopy data, the Mann-Whitney-U test was used to estimate statistical significance. Inter-rater agreement was evaluated using the weighted kappa score. Statistical analyses were performed using IBM SPSS Statistics 22.0.0.0 version .Table 1. In liver histology, the median proportion of hepatocytes with macrovesicular steatosis averaged 20 (10–39) %. The distribution of fibrosis stage (0/1/2/3/4) among the subjects was: 12 (37.5%), 11 (34.4%), 5 (15.6%), 3 (9.4%) and 1 (0.03%); and for steatosis grade (0/1/2/3): 19 (59.4%), 8 (25%), 3 (9.4%) and 2 (6.3%).A total of 32 patients were studied, of whom 11 were men and 21 were women. The mean BMI of the subjects was 47.2 ± 1.0 kg/m2 and the median age 47.5 (41.5–58.0) years. Their key laboratory findings are summarized in Fig 1A. Freshly frozen biopsies were cryosectioned, followed by immediate mild fixation of the sections in 4% paraformaldehyde, to minimize sample manipulation. Imaging was performed on a confocal microscope capable of both SHG and CARS imaging, using near-infrared laser excitation at 816.4 nm and 1064.5 nm, and taking advantage of the characteristic changes in the photon energy states . The entire biopsy  was imaged, with up to 25 x 25 overlapping squares and the images stitched together . An example of a mosaic image of a biopsy after simultaneous recording of SHG and CARS signals and reconstruction of the data set to display the entire specimen is shown in Fig 1D.An overview of the preparation of biopsies for SHG and CARS imaging and the timeline is shown in Fig 2A. In stages 1 and 2, the signal from the portal areas typically predominated, while in stages 3 and 4, marked signals were also observed in septal regions, in agreement with the expected distribution of fibrosis in these stages. Moreover, there was a good overall correlation between the mean intensity of the SHG signal and fibrosis stage in all samples . In the same samples, the mean signal intensity of the CARS signal correlated positively with histologically determined macroscopic steatosis .To analyze if the SHG signal intensity correlated with histological fibrosis staging, we analyzed 32 samples, from which parallel samples had been histologically evaluated by a pathologist. As the patients were undergoing bariatric surgery, advanced stages of fibrosis (3 and 4) were rare in this material. Examples of SHG images of stage 0–4 fibrosis are shown in S1 Fig.We next developed an automated image analysis method to evaluate and quantify early liver fibrosis from the biopsies using SHG. To this end, we excluded SHG signals derived from the capsule and from portal areas. This is because these regions generate a prominent SHG signal even in a normal liver, due to their physiologically high collagen content, and this masks any weaker signal from elsewhere in the parenchyma. Furthermore, the area occupied by portal regions and the capsule varied considerably between biopsies, affecting signal mean intensity. The automatic procedure developed for exclusion of these areas involved background subtraction and thresholding, filtering, particle identification and exclusion, prior to SHG and CARS intensity measurements in the remaining area, is illustrated in Fig 3). Remarkably, we found that already stage 0 NAFLD samples showed specific, albeit weak, fibrillar SHG signals. These were typically relatively short (20–200 micron length) wavy fibrils or narrow bundles of fibrils . In stage 1, the signal was essentially similar in character but these thin threads were more densely distributed between cords of hepatocytes, resulting in an overall higher SHG signal . The fibrils often appeared to bridge between portal areas , reminiscent of early septal fibrosis ( insets 2 and 3). The fibrils were interspersed between heterogeneously sized lipid deposits as visualized by the CARS signal. This signal derives from lipid-laden hepatocytes.The SHG signal derived from the liver parenchyma and included in the analysis was fibrillar in nature . The collagen III signal concentrated in the same areas but was weaker, presumably due to lower abundance of collagen III and/or weaker antibody affinity. Importantly, both collagen signals overlapped with the SHG signal in the same areas , demonstrating specificity of the SHG signal. Moreover, SHG imaging revealed multiple thin threads that emanated from the central vein but were not highlighted by the antibody stainings , suggesting that SHG microscopy is more sensitive than collagen immunostaining in detecting early fibrosis.To assess if the SHG signal in early fibrosis is specific and derives from collagens, we performed indirect immunofluorescence stainings of stage 0 fibrosis NAFLD samples using anti-collagen antibodies. The samples were imaged using both fluorescence and SHG channels. Due to solvent exposure during processing, lipids were extracted in these samples and lipid-rich areas appeared as holes in the specimen. We found specific immunoreactivity for the major fibrillar collagens I and III, with prominent labelling of collagen I in the extracellular matrix surrounding the central vein and the portal area , indicating that SHG imaging can differentiate between these stages of fibrosis. SHG imaging showed a roughly 5-fold difference in signal mean intensity within the stage 1 samples . Three stage 0 samples (independently scored as stage 0 fibrosis by two pathologists) had higher SHG intensities than the lowest stage 1 sample, and one of them had a higher intensity than stage 1 samples on average . The average SHG signal intensity in stage 0 fibrosis samples was approximately 8-fold above background . This suggests that SHG imaging can detect early fibrosis in NAFLD more sensitively than routine histological staging.We examined whether the automated SHG imaging system can be used for detection of early fibrosis and whether it correlates with the stage of fibrosis as determined by histology. For this, we compared the SHG signal from 24 NAFLD samples with the degree of fibrosis (stage 0 or 1). This analysis showed that on average, stage 1 samples had significantly higher SHG intensities than stage 0 samples Click here for additional data file.


Long non-coding RNAs (lncRNAs) are deregulated in several tumors, although their role in acute myeloid leukemia (AML) is mostly unknown.HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) in 241 AML patients. We have correlated HOTAIRM1 expression with a miRNA expression profile. We have also analyzed the prognostic value of HOTAIRM1 expression in 215 intermediate-risk AML (IR-AML) patients.We have examined the expression of the lncRNA P < 0.001) and the highest in t AML (P = 0.005). In 215 IR-AML patients, high HOTAIRM1 expression was independently associated with shorter overall survival , shorter leukemia-free survival  and a higher cumulative incidence of relapse . Moreover, HOTAIRM1 maintained its independent prognostic value within the favorable molecular subgroup . Interestingly, HOTAIRM1 was overexpressed in NPM1-mutated AML (P < 0.001) and within this group retained its prognostic value . Moreover, HOTAIRM1 expression was associated with a specific 33- microRNA signature that included miR-196b (P < 0.001). miR-196b is located in the HOX genomic region and has previously been reported to have an independent prognostic value in AML. miR-196b and HOTAIRM1 in combination as a prognostic factor can classify patients as high-, intermediate-, or low-risk .The lowest expression level was observed in acute promyelocytic leukemia (HOTAIRM1 level at diagnosis provided relevant prognostic information in IR-AML and allowed refinement of risk stratification based on common molecular markers. The prognostic information provided by HOTAIRM1 was strengthened when combined with miR-196b expression. Furthermore, HOTAIRM1 correlated with a 33-miRNA signature.Determination of  NPM1, internal tandem duplication of FLT3 (FLT3-ITD), and CEBPA [Acute myeloid leukemia (AML) is a highly heterogeneous disease, with diverse biological, phenotypic and prognostic behaviors and strikingly different outcomes to standard therapy. This prognostic diversity is most evident in the cytogenetic intermediate-risk AML subgroup (IR-AML), which is now commonly subdivided into molecular prognostic categories according to the mutational status of In recent years, several studies of prognostic markers have focused on the role of RNA molecules that lack protein coding potential, known as non-coding RNAs (ncRNAs). Until recently, the majority of studies of ncRNAs in AML focused on microRNAs (miRNAs) and their roles in pathogenesis and prognosis –7, whileHOX genes play a key role in hematopoiesis [HOX expression patterns is under complex epigenetic regulation and numerous ncRNAs, including lncRNAs, may participate in the regulation of HOX expression [HOTAIR, a lncRNA located in the genomic region of HOXC, regulates the expression of several HOX genes [HOTAIR cooperates with miR-196a  to drive malignancy in gastric tumors [HOTTIP, located in the region adjacent to the HOXA locus, is essential for the coordination of several 5′ HOXA genes [HOTTIP and HOXA13 have been associated with progression in hepatocarcinoma [opoiesis  and leukopoiesis –13 and topoiesis –16. Mainpression . For exaOX genes . HOTAIR c tumors . The lncarcinoma .HOTAIRM1 (HOX antisense intergenic RNA myeloid 1) is a lincRNA located in the HOXA genomic cluster, on chromosome band 7p15 between HOXA1 and HOXA2. HOTAIRM1 is transcribed antisense by RNA polymerase II. HOTAIRM1 expression is induced by retinoic acid and modulates the expression of genes involved in myeloid differentiation. It interacts with proteins of the Polycomb repressive complex2 in mouse embryonic stem cells [HOTAIRM1 is overexpressed in myeloid leukemia cell lines and mature leukocytes compared to hematopoietic stem and progenitor cells [HOTAIRM1 expression and its prognostic impact in AML patients have not yet been investigated.lncRNAs transcribed in the intergenic regions, known as long intergenic non-coding RNAs (lincRNAs) , are dynem cells . HOTAIRMor cells . HoweverHOTAIRM1 expression in a large cohort of AML patients (n = 241) with several cytogenetic subtypes. We have also explored its potential prognostic value in a subset of 215 IR-AML patients included in intensive therapy protocols for younger patients. Finally, we have examined the potential association between HOTAIRM1 and a miRNA profile.In the present study, we have analyzed HOTAIRM1 expression level was observed among the five AML genetic subgroups included in the study and the healthy controls (ANOVA P < 0.001)   . This information is shown in Figure NPM1 mutations (NPM1mut patients) (P < 0.001)  or any clinical features, including age, WBC, bone marrow blast proportion, or FAB subtype.A significant difference in ) Figure . The low) Figure , whereas) Figure . HOTAIRM) Figure . HOTAIRMhttp://www.leukemia-gene-atlas.org/LGAtlas), an in silico analysis of HOTAIRM1 levels in different AML subgroups showed that in other cohorts [HOTAIRM1 was also expressed at the lowest levels in APL and at the highest in t AML  AML . Similarpatients .HOTAIRM1 expression within the IR-AML as identified by the MaxStat package. Patients with higher HOTAIRM1 levels had shorter OS and LFS , and a higher CIR  compared to patients with lower levels  performed in first CR (CR1) did not differ according to HOTAIRM1 expression levels .With a median follow-up of 7.6 years (range: 15–196 months) among patients alive at last follow-up, the 215 IR-AML patients had a CR rate of 80% and a 5-year OS, LFS, and CIR of 42 ± 6%, 42 ± 7%, and 44 ± 7%, respectively. Figure  Figures . HOTAIRMHOTAIRM1 expression as an independent prognostic factor for OS , LFS  and CIR , in addition to age , gender (LFS), WBC count at diagnosis (OS), presence of FLT3-ITD , and NPM1 mutations  . Among the 99 NPM1mut patients, those with high levels of HOTAIRM1 had shorter OS  and LFS  and a higher CIR  , LFS  and CIR  , WBC at diagnosis (OS), and FLT3-ITD .Since s Figure , we anal) Figure . In the HOTAIRM1 expression on outcome in the molecularly defined subgroups (FAVmol and UNFAVmol), among the 68 patients in the FAVmol subgroup, high HOTAIRM1 expression identified a subset of patients with shorter OS  and LFS  and a higher CIR  , LFS , and CIR     but not HOTAIRM1 expression , miR-10a , and miR-10a* . In addition, the signature included other miRNAs involved in hematopoiesis and leukemogenesis, such as miR-9 , miR-222  and miR-424    Figure .HOTAIRM1 expression identified in the present study, and the correlation between miR-196b and HOTAIRM1, we performed a multivariate analysis for OS including miR-196b and HOTAIRM1 expression, as well as other recognized prognostic markers . Both miR-196b  and HOTAIRM1  retained their independent prognostic significance.Given the prognostic value of miR-196b expression identified in our previous work [HOTAIRM1 expression were defined as high-risk factors. Patients were classified into three groups according to the number of high-risk factors: low-risk group, 0 factors; intermediate-risk group, 1 factor; and high-risk group, 2 factors. Five-year OS for the low-, intermediate-, and high-risk groups was 70 ± 19%, 42 ± 8%, and 24 ± 12%, respectively (P = 0.004)  (P = 0.08) (We then constructed a risk score based on the effect of these two factors in combination in order to identify prognostic subgroups of IR-AML patients. High miR-196b and high ) Figure . Five-ye< 0.001) . A non-s = 0.08) .P = 0.016)   . No asso= 0.001) .NPM1mut patients, only two were classified as low-risk and were not included in the analysis. However, when high-risk patients were compared with intermediate-risk patients, high-risk patients had a shorter 5-year OS , shorter 5-year LFS , and a higher 5-year CIR  (Among the < 0.001) .NPM1mut patients, and in the FAVmol subgroup  AML. Among IR-AML patients, HOTAIRM1 expression was significantly higher in NPM1mut patients. When we validated these results using arrays from the Leukemia-gene Atlas repository (http://www.leukemia-gene-atlas.org/LGAtlas), an in silico analysis of HOTAIRM1 levels in different AML subgroups showed that in other cohorts [HOTAIRM1 was also expressed at the lowest levels in APL and at the highest in t AML  AML . Similarpatients .HOX clusters have a specific pattern of lineage-restricted expression, where HOXA genes are predominantly expressed in myeloid cells [HOXA genes are deregulated in several AML subtypes. HOXA gene cluster is downregulated in APL [HOTAIRM1, and in CEBPA-mutated patients [HOXA cluster has been observed in MLL-AML [NPM1mut AML [HOTAIRM1 expression in our study parallels that reported for some HOXA genes in previous studies in APL and NPM1mut AML, lending support to the proposal by Sessa et al that the intergenic non-coding transcription of the HOX genomic regions may be crucial to maintaining the active state of HOX clusters [id cells . Moreoved in APL , which ipatients . In cont MLL-AML , MYST3-C MLL-AML , NUP98-f MLL-AML , 35, and1mut AML , 36, 37,clusters .HOTAIRM1 expression were found in t  DEK/NUP214 AML. HOXA gene upregulation has previously been described in SET-NUP 214 leukemia [NUP214-fusion gene leukemia, through binding to HOXA promoters. This binding facilitates the recruitment of DOTL1, which in turn activates the transcription of some members of the HOXA cluster [DEK-NUP214 and SET-NUP214 fusion proteins, both of which retain the CC and GLFG domains of NUP214 and also exhibit a similar gene-expression profile [HOXA genes, which ultimately results in HOTAIRM1 overexpression.In the present study, the highest levels of leukemia , another cluster . Given t profile , it is lHOTAIRM1 was independently associated with worse outcome in the entire series of 215 IR-AML patients, with shorter OS (P = 0.001) and LFS (P < 0.001) and higher CIR (P = 0.04). Moreover, we confirmed the prognostic value of HOTAIRM1 expression in patients in the FAVmol subgroup ; AML-99 (n = 31) (NCT01716793); AML-03 (n = 174) (NCT01723657). Briefly, the induction regimen of AML-94 was ICE , while in AML-99 and AML-03 it consisted of one or two courses of IDICE , with or without priming with G-CSF. All patients achieving complete remission (CR) received an additional course of chemotherapy with mitoxantrone and high-dose cytarabine, and then a transplant decision was made. In protocols AML-99 and AML-03, an autologous hematopoietic stem-cell transplantation (autoHSCT) was planned for patients harboring a normal karyotype without additional risk factors, whereas an allogeneic HSCT  in first CR (CR1) was recommended for the remaining patients with an available donor. Risk factors considered for risk assignment were the need for two induction courses to achieve CR, detectable minimal residual disease (MRD) by flow cytometry (AML-03), and presence of FLT3-ITD (AML-03). In AML-94, post-remission strategy  depended exclusively on the availability of an HLA-identical sibling.All 215 IR-AML patients were treated from 1994 to 2009 in three consecutive CETLAM trials of intensive chemotherapy for fit patients: AML-94 (Samples were obtained from bone marrow aspirates in 232 (96%) patients and from peripheral blood, with a minimum blast infiltration of 80%, in the remaining 9 patients. Mononuclear cells were purified by Ficoll density gradient centrifugation and total RNA was isolated using Trizol reagent according to manufacturer's protocol .HOTAIRM1 (Hs03296533_g1) and GUSB(Hs00939627_m1). All gene expression determinations were run in triplicate. Relative quantification was calculated using the 2−ΔCt method using GUSB as housekeeping gene.cDNA was synthesized from 1000 nanograms of total RNA using TaqMan Reverse Transcription Reagent Kit (Applied Biosystems). Real-time PCR was performed in the ABI Prism 7500 Sequence Detection System (Applied Biosystems) using TaqMan Gene expression assays (Applied Biosystems) to determine mRNA levels of HOTAIRM1 level was correlated with miRNA expression data obtained in previous studies by our group [® Array Human MicroRNA Set Cards v2.0 (AB), we selected a group of ten miRNAs with potential prognostic value . These miRNAs were subsequently analyzed by individual assays.ur group , 6. BrieFLT3-ITD, NPM1 and biallelic CEBPA mutations have a strong prognostic impact in patients with IR-AML [NPM1 mutation or the biallelic CEBPA mutation but without the FLT3-ITD mutation, when associated to normal cytogenetics, comprise a favorable genetic group – with better prognosis, while patients with the FLT3-ITD mutation and/or without the NPM1 and the biallelic CEBPA mutation comprise the intermediate-I and intermediate-II genetic groups. In the present study, we have classified all IR-AML patients with NPM1 mutations or biallelic CEBPA mutations but without FLT3-ITD mutations as the favorable molecular (FAVmol) subgroup and all remaining IR-AML patients as the unfavorable molecular (UNFAVmol) subgroup.The presence or absence of 2 test and Fisher's exact test, when applicable, for categorical variables, and the t-test for continuous variables. Multivariate analyses for OS and LFS were performed using the Cox proportional hazards model including age , gender, white blood cell count  at diagnosis, mutational status of NPM1 and FLT3-ITD, and HOTAIRM1 expression level. A multivariate analysis for CIR was performed using the subdistribution regression model of Fine and Gray [HOTAIRM1 expression predicted to show a survival risk either above or below average risk, using the cutoff points of HOTAIRM1 expression levels identified by MaxStat package of R software. All analyses were performed with SPSS v.20 or R software version 2.12.2. Significance was set at ≤ 0.05. For the identification of miRNAs significantly correlated with HOTAIRM1 expression, we used data from our previously identified miRNA profile [Overall survival (OS) was calculated from diagnosis to death and leukemia-free survival (LFS) from CR to relapse or death. Both OS and LFS were estimated with the Kaplan-Meier method and comparisons among subgroups of patients were performed using the log-rank test. Relapse risk (RR) was calculated from CR to relapse and estimated using the cumulative incidence of relapse (CIR) method computed with the cmprsk package for R 2.12 software. The competing event in the RR analysis was death without relapse. Comparison of RR between groups of patients was performed using the Gray test . Charactand Gray  with the profile  with adj