To control Ca2+ signaling, these organelles are often observed close to influx and release sites and may be tethered near Ca2+ transporters. In this study, we used high-resolution, wide-field fluorescence imaging to investigate the regulation of Ca2+ signaling by mitochondria in large numbers of endothelial cells (∼50 per field) in intact arteries from rats. We observed that mitochondria were mostly spherical or short-rod structures and were distributed widely throughout the cytoplasm. The density of these organelles did not increase near contact sites with smooth muscle cells. However, local inositol trisphosphate (IP3)-mediated Ca2+ signaling predominated near these contact sites and required polarized mitochondria. Of note, mitochondrial control of Ca2+ signals occurred even when mitochondria were far from Ca2+ release sites. Indeed, the endothelial mitochondria were mobile and moved throughout the cytoplasm. Mitochondrial control of Ca2+ signaling was mediated by ATP production, which, when reduced by mitochondrial depolarization or ATP synthase inhibition, eliminated local IP3-mediated Ca2+ release events. ROS buffering did not significantly alter local Ca2+ release events. These results highlight the importance of mitochondrial ATP production in providing long-range control of endothelial signaling via IP3-evoked local Ca2+ release in intact endothelium.Endothelial cells are reported to be glycolytic and to minimally rely on mitochondria for ATP generation. Rather than providing energy, mitochondria in endothelial cells may act as signaling organelles that control cytosolic Ca Endothelial cells (ECs)1–versus oxidative phosphorylation may enable ECs to meet rapid changes in energy demands. As endothelial cells are required to grow into hypoxic surroundings during angiogenesis, a reliance on anaerobic metabolism may enable ECs to form new vessels. These observations have led to the proposal that endothelial mitochondria act primarily as essential signaling organelles rather than being energy providers , adhesion molecules (e.g. von Willebrand factor), and clotting factors, occur in a Ca2+-dependent manner. In various other cell types, Ca2+ signals are regulated by mitochondria. Uptake of the ion by mitochondria may promote Ca2+ release from IP3R  may cluster   Ca2+ release and mitochondrial Ca2+ uptake in RBL-2H3 cells. Conversely, disrupting the linkage by limited proteolysis decreases mitochondrial Ca2+ uptake  , demonst2+ release events arising from the ER may occur preferentially at sites of contact between endothelial cells and smooth muscle cells ) , rapid (∼0.25 s) events arising from IP3Rs and referred to as pulsars ) . Ca2+ si pulsars . MEPs thsynthase , IP3Rs . Localitivation , 46 and tivation , raising tissues .2+ release events in endothelial cells in intact blood vessels obtained from rats. We found that local endothelial Ca2+ signals preferentially initiate at contact sites with smooth muscle cells, and the Ca2+ signals share the pharmacological profile of Ca2+ pulsars. However, the local signals have a substantially different temporal profile from pulsars. The occurrence of local Ca2+ signals at MEPs requires polarized mitochondria. Inhibition of mitochondrial respiration eliminates Ca2+ activity at MEPs. Surprisingly, neither mitochondrial positioning nor density correlates with sites that give rise to cytosolic Ca2+ events, as might be predicted if tethering were required for mitochondrial control of Ca2+ release. Ca2+ event initiation sites are, on average, 0.9 μm from the nearest mitochondrion and on occasion up to 5 μm away. These results indicate that close coupling is not required for mitochondrial control of Ca2+ release in the endothelium and that control is exerted over distance. Inhibition of the ATP synthase eliminated local Ca2+ signaling events, whereas buffering reactive oxygen species had little effect. Thus, mitochondria exert long-range control of IP3-mediated intracellular Ca2+ signaling dynamics in native endothelial cells via ATP production.To address this issue, we examined spontaneous Ca2+ signaling was assessed in endothelial cells of intact second-order mesenteric arteries (∼150-μm diameter). Time-series image recordings (20 Hz) of the endothelium were obtained using a high-NA (1.3) ×100 microscope objective and a large-format EMCCD camera . In opened arteries (en face preparations), this experimental set-up provided a field of view of ∼17,720 μm2 with a pixel size of 130 × 130 nm projected onto the endothelium. On average, 52 ± 2 whole or partial endothelial cells were visualized in each field of view . Thus, we calculated the density of endothelial cells to be on the order of 2000 cells/mm2, in agreement with our previous estimates (Castimates .A and Movie S1) and baseline-corrected  fluorescence recordings revealed extensive Ca2+ activity in mesenteric artery endothelial cells. The signals formed a continuum of events that ranged from small, highly localized focal increases in Ca2+ (akin to Ca2+ puffs) to traveling spatial gradients (waves) that progressed completely or partly  through cells. Partial Ca2+ waves were the predominant form of Ca2+ activity. On average, 12.5 ± 1.5% of ECs exhibited spontaneous Ca2+ activity . Often, a single location would give rise to repetitive Ca2+ events, although the properties  of events that arose from single sites varied (C–E).In the absence of stimulation with pharmacological agents or mechanical forces, close visual inspection of raw  the initiation sites from which Ca2+ activity originated and applied an automated Ca2+ signal analysis algorithm. The algorithm was adapted from our previous work  For each initiation site, we extracted baseline-corrected Ca2+ signals  and then automatically identified Ca2+ events . The algorithm identifies Ca2+ events using peaks in the time derivative of the F/F0 signal. Ca2+ events were taken as changes in the time derivative of the F/F0 signal that exceeded a threshold value of 10 times the S.D. value of the baseline signal fluctuation. This threshold (10-fold baseline noise) was chosen empirically, as being large enough to exclude noise fluctuations but small enough to faithfully detect low-amplitude Ca2+ signals. A suitable baseline period was automatically identified for each trace . Following event extraction, we then used a least-squares optimization algorithm to fit an exponentially modified Gaussian function  to each Ca2+ peak to measure event parameters. Histograms  show that each measured parameter , rise time, fall time, and spatial spread) may be described by a continuous log normal distribution. The mean values of each were as follows: frequency ; amplitude ; FDHM ; rise time ; fall time , and spatial spread .To analyze spontaneous local endothelial Caous work  . Removal of external Ca2+ had no significant effect on either the density of Ca2+ event initiation sites  or the density of Ca2+ events , suggesting that the events arose via Ca2+ release from the internal store rather than Ca2+ influx . In support, the SERCA inhibitor, cyclopiazonic acid , abolished basal endothelial Ca2+ events . CPA reduced the initiation site density from 59 ± 7 to 5 ± 10 sites min−1 mm−2, whereas the event density was reduced from 115 ± 15 to 5 ± 2 events min−1 mm−2 . Ca2+ events that persisted after CPA incubation could still be described by an exponentially modified Gaussian function , inset). The broad-spectrum TRPV channel antagonist, ruthenium red (RuR) , was without effect on either the density of Ca2+ event initiation sites  or the density of Ca2+ events . We have previously shown that this concentration of RuR (5 μm) is sufficient to inhibit endothelial Ca2+ activity induced by the specific TRPV4 agonist, GSK1016790A  , also failed to inhibit basal endothelial Ca2+ activity .Spontaneous Ca1016790A . Similar2+ release from the endoplasmic reticulum may occur primarily via IP3Rs in the vascular endothelium . 2-APB reduced the Ca2+ event initiation site density from 36 ± 6 to 2 ± 0 sites min−1 mm−2 and the Ca2+ event density from 76 ± 21 to 3 ± 0 events min−1 mm−2 . Although 2-APB blocks IP3Rs in native endothelial cells, it may also inhibit Ca2+ entry pathways . Caffeine reduced the Ca2+ event initiation site density from 50 ± 11 to 4 ± 2 sites min−1 mm−2, and the Ca2+ event density from 86 ± 20 to 5 ± 2 events min−1 mm−2 . As a control, we also show that caffeine inhibited Ca2+ release induced by photolysis of caged IP3 .Caothelium . To invepathways . Therefoe 10 mm; , B and C2+ events did not arise from mechanisms involving voltage-activated Ca2+ channels located on smooth muscle cells. In support, spontaneous Ca2+ events were present in isolated endothelial patches (A). Furthermore, the Ca2+ channel blocker, nimodipine (10 μm), did not reduce the density of initiation sites  or indeed of events themselves  in intact arteries . This concentration of nimodipine (10 μm) is sufficient to fully block voltage-dependent Ca2+ currents in smooth muscle cells (–m K+) contraction of isolated arteries  occur extensively in unstimulated endothelium  and confined (spread of ∼15 μm2) events when compared with the Ca2+ signals observed in the present study   where MEPs occur.In murine mesenteric arteries, transient nonpropagating IPothelium . Pulsarsnt study . The diftensity) . Notwithdark regions in the fluorescence field . To highlight the location of these IEL holes, we smoothed, inverted, and colorized autofluorescence images so that IEL holes were shown as blue on a black background . IEL holes were distributed extensively across the IEL, with 819 ± 58 holes/mm2 corresponding to a fenestrated area of 7.0 ± 0.6% of the elastic lamina. This measurement is similar to the estimate in murine mesenteric arteries . A pooled analysis of all IEL holes observed in 27 fields of endothelial cells (n = 9) demonstrated the lognormal distribution of IEL hole area .The IEL of intact small mesenteric arteries was visualized using autofluorescent emission from the elastin layer upon UV illumination. In autofluorescence images, holes in the IEL appear as ce field A, left. ckground A, right.arteries  but subsarteries . The aveA shows a typical image obtained when Ca2+ event initiation sites from a field of endothelial cells (identified in F/F0 recordings) are overlaid on an image of the underlying IEL holes. In all 27 fields in which endothelial Ca2+ and the underlying IEL were investigated, Ca2+ events occurred close to IEL holes . Measuring the distance from each event initiation site to the nearest IEL hole revealed that, on average, ∼50% (n = 225 distinct event sites) of Ca2+ events initiated directly at an IEL hole location , and ∼85% initiated with 5 μm of an IEL hole.Fig. 72+ signals. We thus next investigated whether or not local Ca2+ events occurred more often at holes in the IEL than would be predicted from a random overlap of the two. To this end, two separate tests were performed to determine whether Ca2+ events were statistically more likely to occur close to an IEL hole than elsewhere in the cell.However, fenestration of the IEL is extensive, and the apparent co-localization may be expected from a random distribution of IEL holes and local CaB; p < 0.05, two-sample Kolgorov–Smirnov test). This result suggests that Ca2+ events are more likely to occur close to an IEL than elsewhere in the cell.First, we pooled all data to generate (and then compare) cumulative probability distributions for the distances between initiation sites and IEL holes and the distances between all locations and IEL holes. In this analysis, we found that the separation between IEL holes and initiation sites was significantly less than the separation between IEL holes and all other sites . This resulted in 1000 sets of random Ca2+ event locations, and for each 1000 permutations, the centroid–centroid distances were once again measured. The average minimum distance between an IEL hole and a randomly redistributed event initiation site  was significantly higher than that measured from the real data . Thus, spontaneous Ca2+ events occurred more often near IEL holes than would be expected if the Ca2+ events initiated randomly throughout the cytoplasm.Next, we performed Monte Carlo simulations (permutation tests) to generate random distributions of Ca00 times , C–E. Th2+ events described are IP3-mediated Ca2+ pulsars, albeit with a different kinetic profile from those occurring in murine mesenteric artery. These events occur at initiation sites that are closer to MEPs (IEL holes) than expected from a random distribution. However, the large mean distance between MEPs and initiation sites (2.4 μm) suggests that direct coupling of initiation sites to MEPs is unlikely. It may be that in rat mesenteric arteries, Ca2+ events manifest as local propagating waves to couple activity at the initiation site to effector proteins that are located within MEPs.Taken together, the results presented thus far suggest that the Ca2+ signaling in a variety of excitable and nonexcitable cell types  (240 nm) to visualize the organelles . This value is similar to that observed in native smooth muscle cells , we observed extensive movement of the majority of endothelial mitochondria (Movie S3). In recordings of shorter duration (5 min), mitochondrial motion was less pronounced but was observed nonetheless in two of six recordings (A–C). The period for Ca2+ imaging was 1 min. The findings suggest that mitochondria in native endothelial cells are largely mobile structures and so are unlikely to be tethered to Ca2+ release sites.As a first step in examining the relationship between the location of mitochondria and Cacordings , A–C. Th2+ release event initiation sites. Because of the possibility of mitochondrial movement, we dual loaded the endothelium with the Ca2+ indicator, Cal-520/AM (5 μm), and TMRE (240 nm) to enable us to record both Ca2+ activity and mitochondrial position from the same field of endothelial cells . However, the separation between IEL holes (where Ca2+ events occur) and mitochondria was not significantly different from the separation measured between IEL holes and all other sites . This result suggests that mitochondria are not coupled to Ca2+ release sites. Notably, a substantial percentage of Ca2+ release sites (9%) had no mitochondria located within a 2.5-μm radius, and ∼2% of Ca2+ release sites had no mitochondria within a 5-μm radius . The averaged separation between local Ca2+ signal initiation sites and nearest mitochondria was slightly lower  than expected from a random distribution of event initiation sites . However, there was no correlation between sites of Ca2+ event initiation and mitochondrial density . The large mean separation (0.9 μm) between mitochondria and Ca2+ event initiation sites strengthens the view that direct modulation of Ca2+ initiation site activity by mitochondria is unlikely.During short imaging sessions (1-min duration), we observed that mitochondria appeared to be positioned close to many Caon sites , A and B2+ signaling, we investigated the effects of the mitochondrial uncoupler, CCCP (5 μm), or the complex 1 inhibitor, rotenone (2 μm). CCCP reduced the initiation site density from 35 ± 8 to 3 ± 2 sites min−1 mm−2, whereas the event density was reduced from 48 ± 7 to 3 ± 2 events min−1 mm−2 . Rotenone reduced the Ca2+ event initiation site density from 39 ± 7 to 3 ± 3 sites min−1 mm−2 and the Ca2+ event density from 60 ± 17 to 17 ± 6 events min−1 mm−2 . When the proton gradient across the mitochondrial membrane is impaired by mitochondrial inhibitors, such as CCCP or rotenone, the ATP synthase may reverse and consume ATP. Therefore, in the next series of experiments, the effects of the mitochondrial ATP synthase inhibitor, oligomycin (6 μm), on the CCCP-induced decreases in basal endothelial Ca2+ activity were examined. Oligomycin by itself was without effect on the mitochondrial membrane potential, as assessed by TMRE fluorescence . In contrast, when CCCP was subsequently added, there was a rapid loss of punctate mitochondrial staining and a significant decrease in TMRE fluorescence . Rotenone also significantly depolarized mitochondria, as revealed by a reduction in TMRE fluorescence , consistent with its role as a mitochondrial complex 1 inhibitor . Nor did oligomycin prevent the reduction in Ca2+ activity seen when rotenone was applied by itself (B).When applied in combination with CCCP, oligomycin did not prevent the reduction in Cay itself A. Nor diy itself B.2+ activity caused by these mitochondrial toxins arose from depletion of intracellular Ca2+ stores, we examined the Ca2+ response to the ionophore, ionomycin, in a Ca2+-free physiological saline solution (PSS) . The ionomycin releasable store content was unaffected by rotenone (with oligomycin present) or oligomycin applied alone. However, the ionomycin-evoked Ca2+ increase was significantly reduced by CCCP (with oligomycin present). It is possible that CCCP may inhibit Ca2+ release from the store, as has been shown previously for other nonexcitable cells  (i.e. without simultaneously uncoupling mitochondria using CCCP or rotenone). Remarkably, we found that inhibition of the ATP synthase nearly eliminated basal Ca2+ dynamics (A); oligomycin reduced the density of initiation sites from 44 ± 6 to 1 ± 0 sites min−1 mm−2 and the density of events from 93 ± 23 to 1 ± 0 events min−1 mm−2 . Oligomycin also inhibited spontaneous Ca2+ activity in isolated endothelial cell patches .Taken together, these results suggest that polarized mitochondria are required for IPactivity . Howeverdynamics A; oligom patches B; n = 3.2+ event initiation sites  or the density of Ca2+ events , suggesting that ATP derived from glycolysis is not required to maintain basal endothelial Ca2+ dynamics . These results suggest that ATP produced by mitochondria, and not glycolysis, facilitates IP3-mediated Ca2+ signaling in native endothelial cells.Removal of external glucose had no significant effect on either the density of Ca3-mediated Ca2+ signaling in endothelial cells. To this end, we investigated the effects of the nontargeted, cell-permeable reactive oxygen species (ROS) scavenger, TEMPOL (100 μm), and the mitochondria-targeted ROS scavenger, mitoTEMPO (50 μm). We found that nontargeted ROS scavenging was ineffective in reducing the density of Ca2+ event initiation sites  or of Ca2+ events  in native endothelial cells (B). Targeting the mitochondrial site of oxidative stress resulted in a modest decrease (∼20%) in both the Ca2+ event initiation site density  and the Ca2+ event density  in native endothelial cells (C).In a final series of experiments, we investigated whether oxidative stress induces basal IPal cells B. Targetal cells C.3-mediated Ca2+ signals, in the endothelium of intact small arteries, initiate at sites that are close to MEPs and are controlled by polarized mitochondria. When the mitochondrial membrane potential is depolarized, spontaneous Ca2+ signals cease. However, in contrast to many other cell types, mitochondrial control of local signals does not require the organelles to be positioned close to the Ca2+ release site. Rather, mitochondria exert long-range control of Ca2+ signaling in the vascular endothelium. The most likely candidate for the control is mitochondrial ATP production , which is generated by proton pumps . Together, the membrane potential and the proton gradient form the transmembrane potential of hydrogen ions used to make ATP. Collapse of the mitochondrial membrane potential inhibits ATP production, prevents mitochondrial Ca2+ uptake, and alters the production of ROS. In the present study in native endothelial cells, the uncoupler, CCCP, or the complex I inhibitor, rotenone, each collapsed the mitochondrial membrane potential and inhibited IP3-mediated basal Ca2+ signaling. Each drug (CCCP or rotenone) was effective in inhibiting Ca2+ signals when applied alone or in combination with oligomycin to prevent reversal of the ATP synthase. The ATP synthase blocker oligomycin, applied alone (as expected), did not decrease ΔΨm and yet also inhibited local Ca2+ signals. These results suggest that ATP production may maintain local endothelial Ca2+ signals in intact arteries. IP3-induced Ca2+ release may be regulated by a variety of cofactors and processes, which include ATP  and limit a Ca2+-dependent feedback process that controls Ca2+ release . The close apposition exposes the organelles to a high local Ca2+ concentration as the ion is released from the store, which overcomes the low affinity of the uniporter for Ca2+ . The stpes –103–, 61, 62.2+ events. Previous studies have also demonstrated co-localization of Ca2+ signals and MEPs  Act 1986, United Kingdom), under UK Home Office regulations. All experiments used second- or third-order mesenteric arteries obtained from male Sprague–Dawley rats , euthanized by overdose of COm NaCl, 4.7 mm KCl, 2.0 mm MOPS, 1.2 mm NaH2PO4, 5.0 mm glucose, 2.0 mm pyruvate, 0.02 mm EDTA, 1.17 mm MgCl2, 2.0 mm CaCl2, adjusted to pH 7.4 with NaOH. Small mesenteric arteries were then cleaned of connective tissue and fat, removed from the mesenteric bed, cut open using microscissors, and pinned endothelial side-up on a Sylgard block. The endothelium was then preferentially loaded with the fluorescent Ca2+ indicator, Cal-520/AM (5 μm with 0.04% Pluronic F127 and 0.26% DMSO in PSS) at 37 °C for 30 min. Following incubation, preparations were gently washed in PSS and mounted in a custom chamber designed for use on an inverted microscope  and spatial resolution  using an inverted fluorescence microscope  equipped with a ×100 objective  and a large-format (1024 × 1024 13-μm pixels) EMCCD camera . Cal-520/AM was excited with 488-nm wide-field epifluorescence illumination provided by a monochromator . The resulting image field (∼133 × 133 μm) enabled us to visualize connected networks of ∼50 whole or partial endothelial cells. Following equilibration (30 min), Ca2+ activity was recorded at room temperature for periods of 60 s. In experiments investigating the effects of pharmacological intervention, Ca2+ recordings were obtained from at least three separate fields of view per animal. Each of these regions was imaged both before and after the intervention. In these experiments, drugs were added directly to the imaging chamber, incubated for 20 min (unless otherwise indicated), and remained present throughout recordings. In experiments using a glucose-free PSS, glucose was substituted with d-mannitol on an equimolar basis. In experiments using a Ca2+-free PSS, Ca2+ was substituted with Mg2+ on an equimolar basis, and 1 mm EGTA was included.Immediately following euthanasia, the mesenteric bed was removed and placed in PSS composed of 145 mcroscope , 114. En2+ indicator, Cal-520/AM, and loaded at 37 °C for 30 min. The digestion protocol was designed to leave intact sheets of endothelial cells, which could be identified by their morphology (A) and by their response to ACh (B).Cut open arteries were cut into three or four smaller segments, and the segments were transferred into a PSS solution containing collagenase (2 mg/ml). The segments were incubated in this solution for 20 min at 37 °C and then gently washed three times in PSS. Following the wash steps, endothelial sheets were dispersed by triturating with a fire-polished glass pipette. The suspension was then transferred to an imaging chamber, and isolated endothelial sheets were allowed to attach to the glass coverslip for 1 h. After this period of time, the solution was exchanged for one containing the Carphology A and by e to ACh B.2+ signals recordings were analyzed using a custom semiautomated Python-based analysis adapted from our previous work  preprocessing of Ca2+ imaging data; 2) identification of sites of Ca2+ activity; 3) extraction of Ca2+ signals from active sites; and 4) analysis of Ca2+ event parameters. Each step is described below.Local Caous work . The pro2+-imaging recordings were preprocessed in FIJI as described previously . The preprocessing resulted in an image stack where positive pixel values indicate an increase in fluorescence above the average. Ca2+ event initiation sites were then identified manually. To do this, ΔF/Favg image stacks were then scrolled through, and each Ca2+ event was marked by a circular ROI that was centered over the point of initiation. Events often occurred repeatedly at a single site. In these cases, only a single ROI was positioned over the active site. Each ROI was added to the FIJI ROI manager, and, after ensuring that all Ca2+ activity was marked, the center coordinates of the ROIs were saved for subsequent Ca2+ signal extraction, as described below.Caeviously . First, 2+ signals were extracted from the raw fluorescence intensity (F) image stacks, using 30-pixel (∼4-μm) diameter circular ROIs positioned at the initiation site of each Ca2+ event as described above. The signals were extracted and processed using a modification of our previously published algorithm for batch processing of two-dimensional Ca2+ data , third-order polynomial Savitzky–Golay filter, corrected for baseline drift using asymmetric least squares fitting,F) traces were expressed as fractional changes from baseline (F/F0) by dividing the fluorescence intensity trace by the average value of a 100-frame (5-s) baseline period (F0). The baseline period was automatically determined for each trace as the portion of signal exhibiting the lowest S.D. This was achieved by applying a rolling S.D. (100 frames) and a rolling summation (100 frames) to each trace. The minimum of the rolling summation corresponds to the center of the “quietest” portion of the F/F0 trace. Ca2+ events were then automatically identified (A) using a zero-crossing detector on the derivative F/F0 traces (2+ events from noise. A positive peak in the derivative function corresponds to the positive edge of a Ca2+ event. A negative peak (nadir) in the derivative function corresponds to the negative edge of a Ca2+ event. The zero-crossings associated with a peak and a preceding nadir  in the derivative trace indicate, respectively, the start, peak, and end of an event in the corresponding Ca2+ trace. The zero-crossing times were used to extract those parts of the original Ca2+ trace that contained Ca2+ events. Event parameters  were then extracted by fitting each detected Ca2+ event with a Gaussian function.Temporal Caa2+ data . For eacentified A using a0 traces . A thres2+ events and myoendothelial gap junctions, images of the internal elastic lamina underlying endothelial cells and Ca2+ imaging data were recorded. The IEL was visualized using autofluorescence from 390-nm excitation light and single images generated by averaging 100-frame recordings obtained at 10 Hz. In rat mesenteric arteries, “holes” in the IEL correlate with the presence of myoendothelial gap junctions and may present a low-resistance pathway for diffusible factors  initiation site to IEL hole separation was calculated.To determine whether the extent of co-localization of Caanalysis . First, m) (m) in all perfusion solutions. Images of TMRE fluorescence (excited at 555 nm) were acquired at 20 Hz using the TE2000U microscope system described above. In experiments assessing mitochondrial morphology, TMRE fluorescence was recorded (1-min periods) from at least three separate fields of view per animal. For each field of view, single images were generated by averaging 100 frames, and mitochondrial morphology was determined using MicroP , the same single field of endothelial cells was imaged throughout, and TMRE fluorescence was measured after performing a 10-pixel-wide background subtraction using FIJI. In experiments examining mitochondrial motility, mitochondria were imaged for periods of 5 or 30 min at 2 Hz. Mitochondrial movement tracks were generated using TrackMate  . TMRE wa nm . TMRrackMate .2+ event initiation sites, mitochondrial TMRE fluorescence and Ca2+ activity were each recorded in the same field of endothelial cells. Single images of mitochondria were generated by averaging 100 frames (5 s) of the full recording. To facilitate comparison of localization, mitochondria were contrast-enhanced by converting images to a binary form. This was achieved by applying (in sequence) an unsharp mask filter (2-pixel radius), a rolling ball (5-pixel diameter) background subtraction, a mean filter (1-pixel radius), a linear contrast enhancement, an adaptive local contrast enhancement, a median filter (1-pixel radius), and finally an intensity threshold (Otsu's automatic method). The extent of co-localization between mitochondria and the Ca2+ event initiation site was then determined as described above for IEL holes.To assess the extent of spatial coupling between mitochondria and Ca2+ signaling was examined in response to photolysis of caged IP3. The endothelium of en face arteries was dual-loaded with Cal-520/AM (5 μm), and a membrane-permeant caged IP3, caged IP3 4,5-dimethoxy-2-nitrobenzyl (10 μm), 0.02% Pluronic F-127, and 0.35% DMSO in PSS for 30 min at 37 °C. Endothelial Ca2+ imaging was then performed at 10 Hz, using an inverted fluorescence microscope  equipped with a ×40 objective  and a large-format (1024 × 1024 13-μm pixels) EMCCD camera  with a 325-nm projected pixel size at focal plane. Cal-520/AM was excited with 488-nm wide-field epifluorescence illumination provided by an LED illumination system . The EMCCD was configured to record from the central 512 × 512 pixels, resulting in a field of view of ∼166 × 166 μm . Photolysis of caged IP3 was achieved using a xenon flashlamp  attached directly to the TE300 microscope . To create probability distributions, data were pooled from all experimental animals within each treatment group. In general, summary data are presented graphically as individual data points  and in the text as the grand mean with the S.E. indicated. Non-Gaussian data (identified using the D'Agostino–Pearson omnibus test) were log normal. Log normal data were transformed (log10), and mean values for each experimental unit were calculated on the logarithmic scale and then back-transformed to their original scale for presentation. In the text, log normal data are presented back-transformed grand means with 95% confidence intervals provided for completeness.Except for probability distributions, the 2+ response to ionomycin or photolysis of caged IP3 or experiments utilizing isolated sheets of endothelial cells, Ca2+ imaging data were collected from at least three different fields of endothelial cells from three different arteries per rat. In Ca2+ experiments in which ionomycin or caged IP3 was used, a single field of endothelial cells was studied per animal. A single field of endothelial cells was also studied  in experiments examining the effects of CCCP/rotenone on the mitochondrial membrane potential. Data were analyzed using Student's t test or repeated measures analysis of variance, with Dunnett's multiple-comparison test, as indicated throughout. All statistical analysis was performed using GraphPad Prism version 6 . A p value of <0.05 was considered statistically significant. Data supporting the findings of this study are available from the corresponding authors on request.With the exception of experiments assessing the CaC. W. and J. G. M. developed the concept. C. W., M. D. L., H. R. H., X. Z., and C. B. performed the experiments. C. W. and C. D. S. wrote the analysis software. C. W., C. S., C. B., and J. G. M. analyzed the data. C. W. and J. G. M. drafted the manuscript. C. W., C. D. S., J. M. G., and J. G. M. revised and edited the manuscript. C. W., C. D. S., J. M. G., and J. G. M. sourced funding. All authors approved the final version of the manuscript.


The tandem mevalonate pathway and Ca2+ channels originated early in eukaryotic evolution, and has since been well conserved, “promoting” it as a ubiquitous player in Ca2+ homeostasis in all eukaryotes. This paper accentuates how this drastic change in thinking gained momentum after the discovery by Paroulek and Sláma that the huge amounts of JH I in male accessory glands of the Cecropia moth, are actually synthesized in these glands themselves and not in the corpora allata, the hitherto assumed unique synthesis site of such compounds. In addition, MAG-JHs have no hormonal- but an exocrine function. Here we hypothesize that MAG-JHs may function in protecting the spermatozoa against toxic Ca2+ concentrations, and in enabling their flagellum to undulate. They may do so by acting through membrane receptors. Our novel paradigm assigns to farnesol/JHs a function of flexible hydrophobic molecular valves for restricting untimely Ca2+-passage through some types of canonical Ca2+channels, using covalently bound farnesyl- or geranyl-geranyl group attachment as well as GPCRs-G proteins all containing a prenyl group. The high rotatable bond count, and their horseshoe-shape are instrumental to their valve function. In our paradigm, Met/Tai and Gce, to date generally thought to be the (only) functional (nuclear) receptors for JHs, are classified as probable Ca2+-sensitive transcription factors. Some theoretical and practical considerations for possible applications in a medical context will be discussed.Farnesol, the sesquiterpenoid precursor of insect juvenile hormones (JH) that itself has JH activity, existed already long before animals and their hormones came into being. Although it is omnipresent in all eukaryotes, this molecule remains a “noble unknown” in cell physiology. It is neither documented as a hormone nor as another type of signaling molecule. To date, its function as an intermediate in the synthesis of squalene-cholesterol-steroids in chordates/vertebrates, and of the insect/arthropod JHs, esters of farnesol, in the mevalonate biosynthetic pathway is assumed to be the only one. This assumption neglects that already two decades ago, farnesol has been shown to be a potent endogenous inhibitor of N-type voltage-gated Ca When the chemical nature of insect juvenile hormone was identified in 1968 by Röller and Dahm, the majority of the nowadays well-developed subdisciplines in endocrinology and evolutionary theory were still in their infancy. Comparing the endocrine system of insects with that of vertebrates was not yet an acceptable research strategy to many endocrinologists. Endocrine archaeology did not exist. The major focus was on uncovering its mode of action, a challenge that to date still has not yet met full success. When dealing with the mode of action of either juvenile hormone (JH) and/or ecdysteroids, the most accepted model focuses almost exclusively on the role of nuclear receptors and/or transcription factors, thereby minimalizing a possible role for membrane receptors . This neHyalophora cecropia, where it does not display any known hormonal function. It nevertheless was denominated “hormone” because it was highly active in typical bioassays where the hormonal activity of this compound could be demonstrated. The exact role of exocrine non-hormonal “MAG-JH I” (a contradictory wording) in the male reproductive system has so far not yet been clarified. However, the finding that farnesol, the precursor of all JH-isomers and itself a compound with moderate juvenile hormone activity -7-ethyl-9-[-3-ethyl-3-methyloxiran-2-yl]-3-methylnona-2,6-dienoate), namely “Cecropia juvenile hormone I (JH I)” or “C18 JH.” This compound had been extracted from male accessory glands (MAGs)  of adult males of the moth activity , acts aschannels ; §5.3 ma as well . These fmembrane receptors of which some reside in the plasma membrane, others in intracellular membranes.The second cause of confusion was that 50 years ago insect endocrinologists preferred the model of membrane receptors for farnesol/farnesol-like substances (FLS: JHs) and 20E involve the Ca2+-homeostasis systems of the target cells. Although it is such a basic fact in cell biology that Ca2+ is a ubiquitous intracellular messenger, which encodes information by temporal and spatial patterns of concentration . A first reason is that one did not consider the possibility that a so called “intermediate compound” in the biosynthesis of cholesterol and steroid hormones in vertebrates/mammals might have an important additional function on its own in Ca2+-homeostasis, in particular as a tool to restrict the gating of some types of Ca2+-channels  requires classical chemical chromatography, mass spectrometry and electrophysiology in addition to the molecular biological tools that are nowadays omnipresent in almost every life science laboratory. This list is not exhaustive. As a result, farnesol/FLS is indeed a “noble unknown” in vertebrate endocrinology as cited in the Introduction.Farnesol does not have the status of a hormone in vertebrates, neither of a control agent of Cachannels . Second,channels . An inbrchannels . Fourth,2+-homeostasis, an important issue in our model, requires some background information. Ca2+ is the most abundant toxic pollutant on earth. Its concentration in the extracellular aquatic environment of cells, blood e.g., amounts to about 2 millimolar on the average versus about 100 nanomolar in the cytoplasm of unstimulated/resting cells. This represents a gradient of about 20,000 fold. As a result, the passive entry of excess Ca2+ into the cytoplasm through channels in the plasma membrane that untimely open, is a continuous threat. 2+ gradient and the additional intracellular Ca2+ gradients that can be generated over the membranes of cell organelles in which various types of enzymes are anchored: Endoplasmic Reticulum (RER and SER), Golgi, mitochondria and  even the nucleus. In addition, there is also the risk that [Ca2+]i will rise for too long above a toxic level when Ca2+ is released from internal stores, e.g., by the common mechanism of Ca2+-induced Ca2+-release. As such, excess Ca2+ has to be removed as quickly as possible. The key tool for this purpose is the removal of Ca2+ by Ca2+-pumps located in the plasma membrane and in internal membranes lining the ER  and mitochondria, organelles that can act as temporary Ca2+ -storage sites. A second mechanism is to restrict as much as possible the passive entry of Ca2+ through all Ca2+-channels. For figures, see 2+-entry will be emphasized.The link between farnesol and CaHyalophora cecropia silkworms  activity. He found high activity in lipid extracts from abdomens of adult male, but not of female, ilkworms  . This is a common practice: hormones are usually named after their first detected biological activity . The assumption that no endocrine role is attributed to the MAGs of animals in general, the human prostate inclusive, seems to be the rule. This view did not change much since its original formulation by Cecropia seemed to be exceptional. In corast to most insect species in which MAGs start developing to full maturity much later, namely after adult eclosion, the MAGS of H. cecropia develop precociously during metamorphosis, enabling males to mate very soon after adult eclosion. Yet, in the mosquitoes Aedes aegypti, Culex nigripalpus, Anopheles rangeli, and Anopheles trinkae in which JH synthesis in male accessory glands has also been experimentally documented  are listed. This enzyme catalyzes farnesoate/juvenile hormone (JH) and JH bisepoxide in the corpora allata. The highest expression of the coding gene as assessed by microarray is in the head, in particular in the brain. The second highest expression is found in the male accessory glands. In addition there is also some expression in the crop, midgut, hindgut, salivary glands, and in the testis. In females, the highest expression is situated in mated spermatheca, while values for virgin spermathecal are lower. However, when assessed by RNA-Seq, the highest expression is found in the male accessory glands of 4 days mated males, which is 4 times higher than Jhamt expression in the testis and 20-fold that of the head, the CNS and the digestive system. No data are available on whether the mRNA is translated into the corresponding protein in all these tissues.In FlyBase that documents the genome of For various reasons, but especially because “exocrine JH” is an exotic (and contradicting itself!) concept in insect endocrinology, several questions beg for an answer.•H. cecropia contributes to optimizing the sperm’s fitness?CA-JH (endocrine) and MAG-JH (exocrine) are chemically identical, but do they also act in the same way, or alternatively do they act complementarily, or differently? For instance: Is it possible that the high concentration of JHs in the MAG-secretion of •2+ instrumental to such activity as in case of ciliated cells in general . 2+-channel-farnesol/FLS.This raised as yet unanswered questions as to the cell physiologic archaeology and ancient role of the tandem voltage gated Ca Unicellular organisms do not have hormones by definition. Does the presence of JHs in Cecropia MAGs still reflect the original function of farnesol in ancestral flagellate cells? Are some of the known effects and mode of action of farnesol/FLS as a hormone(s) also derived from the mode of action of the tandem?Farnesol, the precursor of all juvenile hormones (JHs) and itself a compound with moderate JH activity , has bee•no situation can be created in which farnesol/FLS is completely absent from the whole body. Such situation of total absence of farnesol and its esters occurs naturally at the onset of metamorphosis in the beginning of the last larval instar of all holometabolous insect species  and, in addition, to changes in the intracellular phosphorylation pathways  versus at high [Ca2+]i (= metamorphosis and reproductive state)In other words, could it be that the transcription factor complex Met/Tai is Ca? This mode of action is  compatible with the fact that the very fast effects of binding of JHs to their membrane receptor(s) precede the much slower effects via transcription. The younger generation of insect endocrinologists is probably not aware of the experimental work of Lezzi and Kroeger some 50 years ago : If the nuclear receptor named Met/Tai is a transcription factor complex of which the conformation and activity, along with that of chromatin , is senspathways  suffice Vachellia farnesiana, around 1900  cannot synthesize cholesterol, because they lack the gene coding for squalene synthase. Vertebrates have this gene, hence they can synthesize cholesterol and use it as an intermediate in the synthesis of vertebrate steroids, e.g., of the sex steroids testosterone and estradiol. Insects also have sex steroids, but they belong to the family of the ecdysteroids . These aeostasis .The 3D structure is important for understanding how farnesol binds to its receptors, and how it exerts its function(s) at the level of the plasma membrane. A parameter instrumental to the functioning of a ligand that is seldom mentioned is the “rotatable bond count” of the ligand (7 for farnesol and 10 for JH I: PubChem). The definition given in the “Molinspiration” website reads: “Rotatable bond is defined as any single non-ring bond, bounded to nonterminal heavy  atom. Amide C-N bonds are not considered because of their high rotational energy barrier.” This simple topological parameter is a measure of molecular flexibility.2+ channel  2+-induced apoptosisinduce Ca, among others required for metamorphosis in holometabolous insects. Thus the SERCA pump, in its uninhibited, normal state, may need farnesol/FLS to keep the intraluminal [Ca2+] high in the endoplasmic reticulum . Furthermore, given the high degree of molecular conservation among various types of Ca2+-channels, it may be possible that the ryanodine-sensitive Ca2+-channel which resides in membranes of the endoplasmic reticulum, may also need farnesol for keeping it in the closed state. Thus, a link of controlling the influx of Ca2+ concurrently at the level of the plasma membrane and the intracellular membrane systems is mandatory, otherwise the entire Ca2+-homeostasis would collapse. Several questions remain to be explored, e.g., how does the horseshoe-shape of farnesol, if it enters the nucleus, play a role in effects on transcription?This mode of action may not be restricted to the Roullet-Luft type channel. Farnesol is a sesquiterpenoid like the potent SERCA-Ca2+ channels , reported that farnesol acts as an inhibitor of a voltage-gated Ca2+-channel(s), more specifically as a selective, high affinity inhibitor of N-type Ca2+ channels. 2+ channel antagonist that targets the alpha 1C subunit, which represents the heart of the pore forming complex. The evolutionary roots of the four subunits of voltage-gated calcium channels have been well studied by Both 2+ channels are evolutionarily ancient: they date from before the split between plants and Opisthokonts and before animals with their excitable muscle- and nerve cells appeared on the scene  as one of its main targets.There is no reason to assume that the way farnesol acts in mammalian cells, would be much different in insects. Indeed, both the mevalonate pathway and voltage-gated Cahe scene . This suNoteworthy is that no membrane receptor for farnesol esters known as JHs has so far been identified by either classical biochemical- or by electrophysiological methods. However, a plausible extrapolation about its identity can be made from the electrophysiological 490 characterization of the membrane receptor of farnesol, the precursor of all JHs that itself has 491 moderate JH activity .Ras, which play a central role in the development of cancer, and the nuclear lamin having a role in Hutchinson-Gilford progeria in humans.Farnesol as a hormone starts acting at the extracellular side of cells, when blood contacts the plasma membrane. Yet, there is another equally important possible mechanism of action, namely at the border between the cytoplasm at the inner side of the plasma membrane with its numerous embedded helix bundle transmembrane proteins, in particular the G Protein-Coupled Receptors (GPCRs) and their associated G-proteins. Prenylation, which is also named as “lipidation,” is the likely mechanism involved. Prenylation is the covalent addition of hydrophobic molecules to a protein or chemical compound  Figure . Protein2+-channels triggered the hypothesis that farnesylation of G-proteins may, perhaps serve a similar function, but acting at the cytoplasmic side of the membrane. A re-analysis of the existing literature yielded the proposal that a farnesyl/prenyl group covalently attached to a G protein (α or γ), upon being inserted in the intramolecular microchannel that is present in any GPCR, may act as a possible “flip-flopping” 2+flexible molecular valve that can restrict the untimely influx of excess Ca through the microchannel inside the GPCRs. This supposed mode of action can be compared with screwing a moderately flexile cork into a wine bottle.The usual functions that are ascribed to prenylation are mediating protein–protein interactions and protein–membrane interactions (Wikipedia: Prenylation). In particular the finding that farnesol is an endogenous inhibitor of some types of voltage-gated Ca+ transport, and a covalently attached farnesyl-group likely functions as the functional substitute for microbial rhodopsin’s retinal. Like flip-flopping retinal acts as a valve for restricting H+ transmembrane transport in prokaryotic rhodopsins, so may a prenyl/farnesyl- do for restricting Ca2+ transmembrane transport of Ca2+.This has to be seen in a cell-physiologic archaeological context: the majority of GPCRs are thought to be the molecular progeny of microbial rhodopsins with a function in transmolecular HThe novel “flip-flop concept” that approaches the problem of ligand-associated allosteric changes in GPCR-signaling in an alternative way as compared to contemporary methods e.g., , will beGalleria bioassay for JH  has been found. Its effects are partially redundant in transducing JH action (Kr-h1). Both met and gce null mutants are fully viable, but the met-gce double mutant dies during the larval-pupal transition. Exogenous application of JH agonists rescued the JH-deficient animals but not the met(27)gce(2.5k) mutants  forms a dimer with ultraspiracle (USP) . The besH action . The MET mutants . Here it2+ -sensitive transcription factor complex that controls different sets of genes being transcribed at lower Ca2+ levels (= when the JH titre is high) versus at higher intranuclear Ca2+ concentrations ? This differential activity happens in concert with conformational changes in chromatin which are also known to be Ca2+-dependent.An unanswered key question whether Met/Tai is a Ca2+ signaling in the activation of these receptors was reported. However, both farnesol  forms a dimer with ultraspiracle (USP) , and Metfarnesol  and probignaling , 2016. Ie §5 and . Their t2+ concentrations stimulate the G-protein coupled receptor calcium-sensing receptor. This results in the induction of the expression of biologically active early growth factor response protein 1 (Egr-1), a zinc finger transcription factor. Two other transcription factors, Elk-1 and AP-1, were also upregulated by stimulation of the calcium sensing receptor. The intracellular signaling pathway includes the protein kinases Raf and ERK. The authors stated that regulation of gene transcription is an integral part of calcium-sensing receptor induced signaling. Comparable results were reported by 2+ into the cells, and that this rise in [Ca2+]i is essential for activating transcription factor AP-1.• 2+] on chromatin remodeling are also important. • Effects of i low. How this is achieved is largely unknown. An increase in cytoplasmic Ca2+ negatively influences the secretion of proteins synthesized by the RER. The main function of the RER is to remove excess Ca2+ from the cell by secreting 2+-binding proteinsCa. 2+ from the cell. Because farnesol is an endogenous blocker of some types of Ca2+-channels, a degrading mevalonate biosynthetic pathway will cause an increase in [Ca2+]. It may be worthwhile to investigate whether this process might contribute to initiating Alzheimer’s or other major Ca2+-dependent diseases.In an earlier papers  have sugFrom many experimental studies in insect development and endocrinology, it is known that farnesol and its JH esters are much more active if topically applied to the cuticle than if injected. In humans, topical application to the skin might be an effective slow release method. If nonetheless injected, they are only effective when mixed with an oil, and if no wetting agents/detergents are added . Oil actH. cecropia. Most contemporary researchers, in particular the younger ones, are either not aware that such questions are still open, or think that they are not worth pursuing because this type of research is too dated, or because such projects are currently unfundable from granting agencies. Yet, even to date, in the golden era of molecular biology , some “old-fashioned” experimental approaches, exceptional cases/situations, serendipity, a good historical insight in the development of successive ideas and working hypotheses on the mode of action of JH and ecdysteroids, may ultimately lead to a unifying concept on the endocrinology of the various hormone-dependent aspects of metamorphosis and disease control.Because of the perseverance of Paroulek and Sláma who, half a century after JH was chemically identified, engaged in trying to answer questions with respect to the hormonal status of JH-I in MAGs of H. cecropia seems to represent an exceptional situation in insects. Thus the fact that many decades ago Carroll Williams used this species for his experimental work on JH may have been a lucky coincidence, not to say a fluke. That does not negate that these results coincidental of “a fluke-like event” had truly major consequences for advancing both insect- and general endocrinology. Indeed, it enabled the chemical identification of juvenile hormone in 1968. Yet, it also left many questions unanswered. Molecular biological research methods have answered some, but not all.According to H. cecropia? It may ensure that the sperm cells get a package of farnesol as luggage that helps to keep Ca2+ channels, in particular the voltage-gated Ca2+ channels . Such anti-apoptosis role confirms the view that such role has also been postulated for explaining why the high titre of JH in larvae prevents metamorphosis (The functions of contemporary farnesol/FLS/JHs in the male reproductive system may still reflect evolutionarily ancient roles in the ancestral unicellular progenitor of all animals. A plausible suggestion is that they serve as an overlooked activating “whip to the flagellar undulating beating”, as well as an the overlooked key inhibitor of Caorphosis ,b. FinalADL and LS jointly conceived the model and wrote the manuscript.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.


Analysis of the proteome identified networks and processes that significantly affected transcription regulation including misexpression of 11 transcription factors with seven proteins not previously implicated in AML. Transcriptome analysis identified changes in 40 transcription factors but none of these were predictive of changes at the protein level. The highest differentially expressed protein in AML nuclei compared with normal CD34+ nuclei (not previously implicated in AML) was S100A4. In an extended cohort, we found that over-expression of nuclear S100A4 was highly prevalent in AML . Knock down of S100A4 in AML cell lines strongly impacted their survival whilst normal hemopoietic stem progenitor cells were unaffected. These data are the first analysis of the nuclear proteome in AML and have identified changes in transcription factor expression or regulation of transcription that would not have been seen at the mRNA level. These data also suggest that S100A4 is essential for AML survival and could be a therapeutic target in AML.Inappropriate localization of proteins can interfere with normal cellular function and drive tumor development. To understand how this contributes to the development of acute myeloid leukemia (AML), we compared the nuclear proteome and transcriptome of AML blasts with normal human CD34 Acute myeloid leukemia (AML) is a disorder arising from developmental arrest of cells of the myeloid lineage . The rea+ hemopoietic stem/progenitor cells. A parallel transcriptome analysis was performed to correlate the protein data with transcriptional changes. Using this approach, we identified over 110 commonly misexpressed nuclear proteins including known abnormalities (such as WT1 and CEBPA) and novel abnormalities such as NFIC, hnHRPs. The most strongly over-expressed (novel) protein in the nucleus of AML patients was S100A4 .Given that inappropriate localization of cancer-related proteins, including oncoproteins and tumor suppressor proteins may interfere with normal cellular function, we hypothesized that the developmental arrest, sustained proliferation, and prosurvival characteristics of AML blasts may in part be mediated through misexpression or mislocalization of proteins to the nucleus. The aim of this study was to identify new therapeutic targets in AML through examination of the abundance of proteins in the nuclei of AML blasts using MS proteomics. We used isobaric tags for relative and absolute quantification (iTRAQ) together with liquid chromatography-tandem MS (LC-MS/MS) to analyze the nuclear proteome of the minimally differentiated AML blasts in comparison with developmentally-matched human CD34S100 multigene family of calcium-binding proteins of the EF-hand type . They hopoiesis , 10. S10opoiesis  and in lopoiesis , 16. The+ cells were isolated as previously described [Diagnostic bone marrow or peripheral blood from AML patients and cord blood were collected with informed consent; patient clinical characteristics were outlined in escribed .TM  or ATCC  and cultured under recommended conditions. The genetic identity of the cell lines was confirmed by short tandem repeat (STR). Cells at passages greater than twenty were not used in the experiments performed in this study. Monitoring for Mycoplasma contamination was performed using the MycoAlert Detection Kit (Sigma). S100A4 harboring a nuclear localization sequence (NLS) was expressed utilizing retroviral and lentiviral vectors co-expressing GFP as a selectable marker  following manufacturer’s instructions. A fraction of AML cells were also lysed in Trizol® for comparative mRNA analysis  via the PRIDE partner repository with the data set identifier PXD002799.Detailed MS proteomic methods and data analysis are shown in Gene Expression workflow . Significant differences were determined by ANOVA and a >±1.5 fold changes in AML vs. CD34+. Data is available as supplementary material at https://www.ebi.ac.uk/arrayexpress/ under the following Accession Number: E-MTAB-3873.RNA isolation and GEP using Affymetrix Human Transcriptome Array 2.0 GeneChips® was performed as detailed in 5 cells/mL and incubated for up to 48 h post infection. Cultures were harvested and viable (propidium iodide (PI)-negative) cells were counted by flow cytometry. For apoptosis assays, Annexin V-APC in combination with PI staining was performed.Cells were seeded in triplicate in a 96-well flat-bottom tissue-culture plate in serum-replete growth media at 1.6–2 × 10TM C6 cytometer (B.D. UK). Data was analyzed using FCS Express® v6 . The threshold for GFP positivity was determined from the autofluorescence of GFP negative or mock transduced cells. Supplier and isotype matched control stained cells were used to determine background of labeled cells. Debris and ejected nuclei were excluded from the analysis of >10,000 events.Flow cytometric data were acquired using an AccuriU-Test. Data represent mean ±1 SD. Calculations were performed using Minitab® v16 (Minitab Inc. USA). Network and Pathway data analysis was performed using Key Pathway Advisor and MetacoreTM .Statistical significance of nonparametric data was analyzed by Mann–Whitney + cells; immunophenotypic analysis of these cells established all samples were >95% viable and 95% CD34+ , to minimize variability arising from developmental differences. AML blasts were >80% viable and did not express CD14 and CD15 Fig.   was significantly altered in AML blasts. We then identified frequently dysregulated proteins that changed in at least five AML patients (±>2 fold) from at least two independent MS runs. Where an AML patient sample was compared with two CD34+ controls we accepted a protein as ‘changed in AML’ if changes against both controls were coincident. This yielded 113 proteins of which 84 (75%) were designated nuclear proteins . Whilst we validated the role of the former  by analysis of the corresponding transcriptome, we were unable to achieve this with novel TF abnormalities (probably due to relatively poor database annotation for these proteins).+ cells  2 encodes a 45 kDa protein and forms a complex with the 90 kDa interleukin enhancer-binding factor 3 (ILF3). This has been shown to affect the redistribution of nuclear mRNA to the cytoplasm and to negatively regulate the microRNA processing pathway [trans-factors in regulating gene expression. Within the nucleus these proteins are involved in RNA splicing, 3′-end processing, transcriptional regulation, and immunoglobulin gene recombination [HNRNPK expression implicating the role of this protein in the development of AML [Although model systems strongly implicate TF dysregulation in this disease, in patient material only a few common transcription factor abnormalities are known  and these are mainly associated with particular cytogenetic subsets of AML . Therefotibodies . Given ttibodies , we rest pathway . TAR DNA pathway . hnRNPs bination . Recentlt of AML . NFIC hat of AML , 48.TM to examine whether the modulated genes are connected to TF. Several TF networks were identified but none of them correlated with dysregulated TF protein expression. Interestingly, 13 proteins involved in mRNA processing were shown to be dysregulated suggesting that posttranscriptional regulation of mRNAs and/or LncRNAs could play a critical role in modulating transcription. In support of this, analysis of our microarray data revealed significant differences between the transcriptome of AML cells and CD34+ cells particularly with the generation of mRNA transcripts through alternative splicing (data not shown).To correlate a transcriptome signature with TF expression we analyzed the transcriptome of AML cells and used the network-building algorithm on transcription regulation from MetaCoreS100 multigene family of calcium-binding proteins of the EF-hand type. These proteins are distributed into three main subgroups based on regulatory control within the extracellular or intracellular environments (or both). They have diverse roles in a variety of cellular processes including regulation of proliferation, cell cycle progression, apoptosis, differentiation, Ca2+ homeostasis, migration, adhesion and transcription [We next examined which proteins were the most significantly changed in AML based on frequently of detection coupled with highest fold changes in our cohort of patients. We further focused on novel abnormalities by excluding proteins with known published associations with AML Table . S100A4 cription , 49. S10cription , 50, 51.cription , particucription . Others cription ). S100A4cription . This iscription , 56. It cription . This wocription . In thiscription  and SP1 cription , 61, key+ cells and myeloid differentiated lineages express this protein in the cytosol. Knocking down expression of S100A4 in AML lines results in cell death through induction of apoptosis and hence is an attractive target for cancer therapy particularly in AML given that normal cells would be spared [TM analysis; though this analysis is dependent on the level of annotation in the Clarivate knowledgebase [Whilst S100A4 has been widely studied in solid tumors very little is known of its role in hematological malignancies. We show for the first time that normal CD34e spared . Increase spared –65; these spared –68. In oe spared . Nuclearedgebase . We are In summary, we report the first study to use iTRAQ proteomic analysis coupled with mRNA GEP to identify several proteins that are expressed or repressed in the nucleus of AML blasts. One of these proteins, S100A4, is essential for AML cell growth and survival suggesting that therapeutically targeting S100A4 would be an effective strategy while sparing normal hemopoietic cells.Supplemental MethodsSupplemental FiguresTable S1Table S2Table S3Table S4Table S5Table S6


The experimental factors influencing the synchronous fluorescence of the considered compounds were sensibly adjusted. The chromatographic analysis was executed on a Nucleodur MN-C18 column of dimensions; 250 × 4.6 mm i.d. and 5 µm particle size). The fluorimetric detection was time-programmed at λem = 440 nm for AML (0.0–7.5 min) and at λem = 400 nm for LOS (7.5–10 min) after excitation at λex = 245 nm. The mobile phase is a blend of acetonitrile with 0.02 M phosphate buffer in a proportion of 45 : 55, pH 4.0, pumped using a flow rate of 1 ml min−1. The calibration plots were established to be 0.1–4.0 µg ml−1 for both drugs in method I and 0.05–4.0 µg ml−1 for both drugs in method II. The study was extended to the evaluation of the two drugs in their co-formulated tablets.This study is concerned with two sensitive, fast and reproducible approaches; namely, second-derivative synchronous fluorimetry (method I) and reversed phase high-performance liquid chromatography with fluorimetric detection (method II) for synchronized evaluation of losartan (LOS) and amlodipine besylate (AML). Method I is based on measuring second-derivative synchronous fluorescence spectra of LOS and AML at Δ The. Then-buAmlodipine (AML) (3-ethyl-5-methyl (4RS)-2-[(2-aminoethoxy) methyl]-4-(2- chlorophenyl)-methyl-1-ihydropyridine-3, 5-dicarboxylate benzenesulfonate) is a long-term calcium channel blocker employed as a hypotensive drug [Review of the literature disclosed that spectrophotometric and chromatographic techniques have been used for evaluation of LOS –6, and AThe target of the current work is to set up and progress two new, sensitive and selective methods for synchronized evaluation of LOS and AML either in pure form or in pharmaceutical formulations employing derivative synchronous fluorescence spectroscopy (DSFS) (method I) and LC with fluorimetric detection (method II). In authors' information, neither synchronous spectrofluorimetry nor HPLC with fluorimetric detection were previously used for the synchronized analysis of LOS and AML in their co-formulated dosage forms.Synchronous fluorescence spectroscopy (SFS) has a few focal points over routine fluorescence spectroscopy, comprising simple spectra, low interference in addition to high selectivity . As a reLately, derivative synchronous fluorimetry is employed for estimation of many combinations in formulations and biological fluids. For example, binary mixtures of topiramate and levetiracetam , itoprid2.2.1.® tablets  considered to have 50 mg LOS and 5 mg AML in proportion 10 : 1 (Batch # 08455) were purchased from India.LOS and AML pure analytical standards were obtained from Sigma-Aldrich  and used as obtained. Losar-A2.2.All solvents and reagents were of analytical reagent grade. Methanol and acetonitrile (HPLC grade) were purchased from Merck, Darmstadt, Germany. Acetate buffer was prepared by blending suitable amounts of acetic acid (0.2 M) with sodium acetate (0.2 M) in pH range of 3.6–5.6. Borate buffer (pH 5.5–13) was prepared by blending suitable amounts of boric acid (0.02 M) with sodium hydroxide (0.2 M). Dipotassium hydrogen phosphate was purchased from Sigma-Aldrich, Seelze, Germany. Phosphoric acid was from Riedel-deHaën .2.3.Spectrofluorimetric data were measured using a luminescence spectrometer of Perkin-Elmer model LS 45 (UK), containing a xenon arc lamp of 150 W, grating monochromators for excitation and emission, and a recorder of Perkin-Elmer. The used slit width for both monochromators was 10 nm. A quartz cuvette of 1 cm was used. Fluorescence Data Manager (FLDM) software was used for recording second-derivative spectra using a number of points of 99. The fluorescence intensities of the second-derivative spectra were measured at 260 and 380 nm for LOS and AML, respectively.A Shimadzu Prominence HPLC system  with an LC-20 AD pump was used to carry out chromatographic analyses. DGU-20 A5 degasser, CBM-20A interface and fluorimetric detector (FXL-10A) with 20 µl injection loop were used. The column used was a Nucleodur MN-C18 column of dimensions 250 × 4.6 mm i.d. and particle size of 5 µm) purchased from Macherey-Nagel, Düren, Germany.pH adjustments were made using pH meter (Model pHS-3C) obtained from Shanghai Leici Instruments Factory, China. A sonicator BHA-180 T obtained from Abbotta Corporation in the USA was used.2.4.λem = 440 nm for AML (0.0–7.5 min) and at λem = 400 nm for LOS (7.5–10 min) after excitation at λex = 245 nm.The chromatographic analysis was carried out on a C18 column. The column was used at room temperature. The HPLC system was washed daily with 50 ml of 1 : 1 methanol : water mixture to eliminate the mobile phase; this led to reproducibility of the analytical column. A mobile phase consisted of a blend of acetonitrile with 0.02 M phosphate buffer at a proportion of 45 : 55 with pH 4.0 controlled using sodium hydroxide (0.2 M) or orthophosphoric acid (0.2 M), with sonication of the mobile phase for 30 min. Then filtration of the mobile phase was done through a membrane filter of pore size 0.45 μm purchased from Sartorius-Stedim, Goettingen, Germany. The separation was done using time-programmed fluorimetric detection at 2.5.Stock solutions of each of LOS and AML prepared by dissolving 10.0 mg of each compound in 100 ml volumetric flask and completed to the volume with methanol were additionally diluted with water (method I), and were additionally diluted by mobile phase (in method II) as suitable. The stability of standard solutions was found to be stable for 10 days if refrigerated.3.3.1.−1 for both LOS and AML) were conveyed into a sequence of 10 ml volumetric flasks, 2 ml of 1% sodium dodecyl sulfate (SDS) solution was added, then diluted with water and blended well. The SFS of the samples were performed at a constant difference of wavelength Δλ = 80 nm. The second-derivative fluorescence spectra (SDFS) of LOS and AML were calculated from standard synchronous spectra. The second-derivative spectra peak heights were determined at 260 and 380 nm for LOS and AML, correspondingly. A blank experimentation was executed in the same time. The second-derivative method peaks amplitude was graphed against the ultimate concentration of the studied compounds (µg ml−1) to obtain the calibration curve. On the other hand, the equivalent regression equations were inferred.Aliquots of LOS and AML standard solutions throughout the working range of concentration as mentioned in 3.2.−1 for each LOS and AML were set by successive dilutions of the stock solutions. To each flask, 2 μg ml−1 veratic acid was added as internal standard (IS), then mobile phase was added to dilute to final volume. Samples of 20 μl volume were injected in triplicate and chromatographed under prescribed chromatographic conditions. Peak area ratios (drug/IS) of each studied drug were plotted versus the concentrations of drug in μg ml−1. On the other hand, the equivalent regression equations were inferred.Working solutions containing 0.05–4.0 μg ml3.3.Aliquots of LOS and AML standard solutions in the therapeutic proportion of 10 : 1 were moved into a series of 10 ml volumetric flasks. Then, the steps described under Construction of calibration curves for the second-derivative synchronous fluorescence spectroscopy (SDSFS) method (method I) or for the HPLC method (method II) were performed. On the other hand, the equivalent regression equations were inferred.4.Ten tablets were weighed and finely powdered. A weighed amount of the powder corresponding to 50 mg LOS and 5 mg of AML (in the therapeutic proportion of 10 : 1) was moved to a 100 ml volumetric flask then extracted with methanol. After sonication for 30 min, filtration was done. Aliquots covering the working concentration range were transmitted to 10 ml volumetric flasks. The recommended procedures in §§3.1 and 3.2 were performed. The nominal content of the tablets were determined either from a previously plotted calibration graph or using the corresponding regression equation.5.LOS and AML show innate fluorescence with determined wavelengths of 355 and 440 nm, using excitation at 267 and 239 nm for LOS and AML, correspondingly. Overlapping of both spectra of excitation and emission of LOS and AML occurred . Thus, t−1). It is obvious that there is still an overlap in the SF spectra of both drugs. Hence, the second-derivative synchronous fluorescence spectroscopy (SDSFS) method was selected for concurrent estimation of LOS and AML together in their dosage forms. Spectra of LOS and AML were greatly differentiated using SDSFS with zero-crossing method . It was found that only SDS produced a substantial rise in the relative fluorescence intensity; therefore, SDS was chosen as the fluorescence promoter for studied drugs as shown in electronic supplementary material, figure S1.The impact of addition of different organized media on the synchronous fluorescence intensities of the two drugs was considered, using SDS as an anionic surfactant, CTAB as a cationic surfactant, Tween-80 as a non-ionic surfactant, methyl cellulose and HP-β-CD. To the aqueous solution of the drugs mixture, 2 ml of every surfactant (1.0% w/v) was added . Therefore, 2.0 ml of SDS solution of concentration 1% w/v was selected to be the optimal volume for each of LOS and AML.5.1.4.The effect of different concentration of SDS was studied (0.25–1.5% w/v). The results revealed that increasing SDS concentration led to increasing fluorescence intensities up to 1.0% SDS, after which additional rise in concentration resulted in no additional rise in RFI. So, 1.0% w/v SDS solution was chosen as the optimal concentration for both studied drugs.5.1.5.The impact of various diluting solvents on the RFI of LOS and AML in presence of SDS was considered using water, acetonitrile methanol, dimethyl sulfoxide and dimethyl formamide. The results obtained showed that water was the optimal diluting solvent due to giving the maximum RFI and the minimum blank reading. In the case of using methanol and acetonitrile, an obvious and sharp drop in the RFI occurred. This impact is ascribed to their denaturizing impact on micelles, as alcohols of short-chain like methanol are solubilized chiefly in the water phase and influence the micelle-formation procedure via altering the characteristics of the solvent. Furthermore, adding those organic solvents decreases the dimensions of micelles, but with an advanced collapse of the surfactant aggregate at very high concentrations . Both di5.1.6.The pH impact on the SF intensities of the binary mixture was considered using various buffers to cover all the pH range, where acetate buffer and borate buffer were used in the pH range of 3.5–13. No effect was observed on the synchronous fluorescence intensity of LOS and AML upon using different pH values. Therefore, no buffer was used in the study, as shown in electronic supplementary material, figure S3.5.2.tR = 9.07 min) and AML (tR = 6.01 min)). Furthermore, the suggested method shows good sensitivity, precision and accuracy with limit of detection (LOD) 0.018 µg ml−1 LOS and 0.048 µg ml−1 AML. The suggested method can be applied for examination of the quality of the compounds under study, either per se or in their co-formulations.The developed HPLC method allows the estimation of LOS and AML with good resolution in a reasonable time as shown in 5.2.1.The optimization of the mobile phase constituents was carried out to obtain a highly selective and sensitive method through a brief period of time in order to attain distinct regular peaks. 5.2.2.The fluorescence detector signals of LOS and AML were carefully considered and the top emission wavelengths were established at 400 and 440 nm after excitation at 245 nm for LOS and AML, correspondingly giving the best sensitivity. The programmed fluorescence detection was adjusted for allowing sensitive determination of both LOS and AML concurrently. For AML, it was recorded at 440 nm within 0–7.5 min, while LOS was recorded at 400 nm within 7.5–10 min.n-propanol and methanol were studied to choose the most suitable one for the study. Acetonitrile proved to be the best, giving the maximum number of theoretical plates and well-separated peaks within sensible retention times. The impact of varying the proportion of organic modifier was considered on the retention times and selectivity of the studied compounds.Different organic modifiers like acetonitrile, Mobile phases having ratios of acetonitrile: 0.02 M phosphate buffer (50 : 50), (45 : 55) and (40 : 60) were considered. The mobile phase less than 40% acetonitrile resulted in retardation of the peaks of both drugs, while mobile phase having ratios higher than 50% of acetonitrile resulted in low resolution. Hence, a ratio of mobile phase comprising acetonitrile : 0.02 M phosphate buffer at a proportion of 45 : 55 proved to be the greatest due to providing the optimal number of theoretical plates and sharp symmetrical peak.The pH impact of mobile phase on the retention times and the selectivity of the studied compounds were considered using mobile phases with different pH values which ranged from 3.0 to 5.0. The pH 4.0 was the optimum, giving reasonable separation, sharp peak and the maximum number of theoretical plates. The pH 3.0 resulted in broadening of peak; however, pH 5.0 produced low sensitivity for the studied drugs.For the purpose of choosing an appropriate internal standard, two different drugs were studied: veratic acid and ethopabate. Veratic acid proved to be the best one, producing a well-separated peak from each of the studied drugs.6.The proposed methods validity was studied in expressions of linearity, ranges, accuracy, precision, limits of detection and limits of quantification.6.1.−1 for the two compounds, LOS and AML in method I; whereas in method II, the concentration ranges for LOS and AML were found to be 0.05–4.0 µg ml−1.The linearity graphs of the proposed methods were obtained. The calibration graphs presented a linear response between  in method I or ratios of peak area in method II and concentrations of the drug. The concentration ranges were established to be 0.1–4.0 µg mlr > 0.9999) in addition to lowered intercept values designate low scattering points around the calibration curves. The statistical results of the data [The excellent estimate of correlation coefficients (the data  are pres6.2.The limit of quantification (LOQ) was determined by measuring the lowest concentration that can be measured according to ICH Q2(R1) recommendations , below w6.3.−1 and 5–50 µg ml−1 for LOS and AML, respectively. Statistical analysis of the results using Student's t-test and variance ratio F-test (The results obtained by the suggested methods were matched with the results obtained from comparison method . The proo F-test  revealedAccuracy and precision of inter-day and intra-day were evaluated for the proposed methods and the results are abridged in 6.4.t-test and variance ratio F-test.Also, the proposed methods were applied for the synchronized evaluation of LOS and AML in the co-formulated dosage form in ratio 10 : 1 for LOS and AML, respectively. There was no interfering from ordinary tablet excipients and additives. The results are shown in 7.Two accurate, sensitive, rapid and reproducible methods have been recognized for synchronized evaluation of LOS and AML in pure form and co-formulated tablets. The small values of RSD indicate the validity of the proposed methods; thus, the study allows their applications in quality control work.