Importantly, we find that the frequency of sCaLTs markedly increases as OLs undergo extensive process outgrowth and branching. We further show that sCaLTs are primarily generated through a combination of Ca2+ influx through store-operated Ca2+ entry (SOCE) and Ca2+ release from internal Ca2+ stores. Inhibition of sCaLTs impairs the elaboration and branching of OL processes, as well as substantially reduces the formation of large myelin sheets in culture. Together, our findings identify an important role for spontaneous local Ca2+ signaling in OL development.Oligodendrocytes (OLs) insulate axonal fibers for fast conduction of nerve impulses by wrapping axons of the CNS with compact myelin membranes. Differentiating OLs undergo drastic chances in cell morphology. Bipolar oligodendroglial precursor cells (OPCs) transform into highly ramified multipolar OLs, which then expand myelin membranes that enwrap axons. While significant progress has been made in understanding the molecular and genetic mechanisms underlying CNS myelination and its disruption in diseases, the cellular mechanisms that regulate OL differentiation are not fully understood. Here, we report that developing rat OLs in culture exhibit spontaneous Ca This study identifies a novel form of Ca2+ signaling, spontaneous Ca2+ local transients (sCaLTs) that play an important role in oligodendroglial development. In addition, this work reveals a new role for store-operated Ca2+ entry (SOCE) and release in generating sCaLTs and Ca2+ signaling in oligodendrocytes (OLs). Together, these findings establish a novel Ca2+ mechanism underlying oligodendroglial development.While CaMyelination of axons by oligodendrocytes (OLs) and Schwann cells in the central and peripheral nervous systems is essential for rapid conduction of nerve impulses . InflammDuring development, oligodendroglial precursor cells (OPCs) undergo drastic changes in their morphology. Multiple processes emerge from the cell body followed by extensive branching and outgrowth. These highly branched OL processes seek out target axons and enwrap them. From these branched OL processes, myelin membranes emerge and expand, finally compacting into lipid-rich myelin sheathes that insulate axons for fast nerve conduction. Each OL is capable of myelinating over 50 different axons , meaning2+ is a key second messenger that regulates a diverse array of cellular activities, ranging from cell motility to gene transcription. The intracellular Ca2+ concentration ([Ca2+]i) of most cells at the resting level is maintained in the low nanomolar range. Spatiotemporally restricted fluctuations in [Ca2+]i are the triggers for various cellular reactions and responsible for translating extracellular stimuli to specific cell responses. Ca2+ signaling is well studied in neurons for its essential role in synaptic transmission and the regulation of many, if not all aspects of neural development  are expressed in OLs and demonstrated the elevation of [Ca2+]i in response to ligand stimulation, neuronal activity, or injury  that are largely restricted to discrete sites in branched OL processes. In particular, these sCaLTs are observed in purified rat OL cultures devoid of neurons. Importantly, the frequency of sCaLTs peaks in highly ramified OLs just before the formation of flat myelin sheets. Mechanistically, sCaLTs depend on store-operated Ca2+ entry (SOCE) and internal Ca2+ release. Finally, we present evidence that sCaLTs play an important role in OL development, especially in the elaboration of highly branched processes and the formation of myelin basic protein (MBP)-positive membranes in culture. Thus, these findings indicate an important role for spontaneous Ca2+ signals in OL development.In this study, we report that developing OLs in culture exhibit spontaneous Ca2 tissue culture flasks with NM12 medium [high-glucose DMEM with 12% fetal bovine serum (FBS)]. The medium was fully replaced on days 3 and 7. At day 10, the flasks were shaken in a 37°C incubator for 45 minutes (min) at 50 rpm to remove loosely attached microglial cells. The flasks were then shaken overnight at 210 rpm in a 37°C incubator to dislodge OPCs from the astrocyte monolayer. The next morning, non-adherent cells were plated on an uncoated 10-cm tissue culture dish for 10 min to permit adherence of any residual microglia, while the loosely adherent OPCs were dislodged by gentle manual shaking. This step was repeated two more times to remove all microglia. The final supernatant from these shaken cultures contained ∼85–90% OPCs . To express exogenous proteins in OLs, we used Lipofectamine 2000 (Invitrogen), following the manufacturer’s standard protocol, to transfect mixed glial cultures in 75-cm2 culture flasks 4 h before the overnight shaking step. We found that the transfection efficiency is much higher in OPCs grown in NM12 medium than in purified OLs grown in Super Sato medium.DNA constructs encoding pN1-Lck-GCaMP3 was a gift from Baljit Khakh , Rabbit anti-β3-tubulin , mouse anti-MBP , goat anti-Olig2 , and mouse anti-O4 IgM . Alexa Fluor-conjugated secondary antibodies were all purchased from Thermo Fisher Scientific with a dilution of 1:500. nifedipine, verapamil, diltiazem, NNC 55-0396, ω-conotoxin GVIA (ω-CTX), ω-agatoxin IVA, SKF96365, cyclopiazonic acid (CPA), thapsigargin (Tg), and ryanodine (Ry) were all purchased from Sigma.The following antibodies and chemicals were used for this study: rabbit anti-α-tubulin , progesterone (20 nm), selenium (30 nm), biotin (10 ng/ml), PDGFAA (10 ng/ml), and bFGF (10 ng/ml). Differentiation of CG4 cells was induced by switching the cells from the proliferation medium to a differentiation medium containing DMEM, insulin (5 μg/ml), transferring (50 μg/ml), tri-iodothyronine (50 nm), and 0.5% FBS as previously described  was performed using iScript cDNA Synthesis kit (Bio-Rad), followed by PCR and agarose gel analysis. The primers used for the PCR were reported previously  and are Cells were grown on coverslips, fixed in 4% paraformaldehyde for 20 min, rinsed with PBS, permeabilized with 0.1% Triton X-100 for 5 min, blocked with 3% bovine serum albumin (BSA) for 1 h, incubated with primary antibodies diluted in 3% BSA overnight at 4°C, and then incubated with fluorophore-conjugated secondary antibodies at room temperature for 45 min.All imaging experiments were performed on an inverted Nikon Eclipse Ti-E microscope equipped with an automated z-drive with Perfect Focus and NIS-Elements software (Nikon). A 20× objective  was used for imaging fixed slices, and a 60× objective  was used for live-cell imaging. Two digital cameras were used for imaging acquisition: QuantEM 512SC CCD (Tyledyne Photometrics) and Orca-Flash four v.2 sCMOS (Hamamatsu). The QuantEM CCD offers the highest sensitivity for weak fluorescence but with only 512 × 512 resolution, whereas Flash4 sCMOS offers a balance between sensitivity and resolution. Both cameras offer a wide dynamic range with 16-bit digitization. For all the experiments, imaging settings were optimized such that the fluorescence signals are in the lower half of the camera dynamic range without any saturation. Presentation of the 16-bit digital images is done by using specific look up tables (LUTs) to illustrate the structures of interest.m fluo-4-AM (Sigma) in Krebs–Ringer’s saline  and Orca-Flash4 camera were used. Time-lapse DIC images were acquired every 5 s. To measure the size of each OL from the DIC images, we connected the farthest tips of all the protruding branches, and then measured the total area of the resulting polygon.2 culture flasks 4 h before shaking, but otherwise OLs were differentiated and cultured as described above. OLs on coverslips were mounted in a 35-mm chamber and maintained at 37°C with a heated stage adaptor. A 60× Apo TIRF objective (NA 1.49) and Flash4 camera were used. Time-lapse fluorescence images were acquired every 2 s for Lck-GCaMP3 and 10 s for Ruby-Lifeact.Mixed glial cells at day 10 were co-transfected with Lck-GCaMP3 and Ruby-Lifeact using Lipofectamine 2000 on 75-cm0 time-lapse sequence using ImageJ. Here, ΔF was generated using the “δ F up” plugin in ImageJ and F0 was the first fluorescent image without sCaLTs in the sequence. A maximal intensity projection of this ΔF/F0 time-lapse sequence was used to identify the discrete sites of sCaLTs, as well as to define the regions of interest (ROIs) by thresholding above the signal of sCaLT free regions. These ROIs were then used for frame-by-frame analyses of the original F sequence to generate ΔF/F0 for each ROI. To eliminate the possibility of encountering sCaLTs in the first frame, we selected F0 from the frame with the lowest signal within the first 1 min imaging for each ROI followed by background subtraction. We scored sCaLTs as increases of ΔF/F0 equal to or >20% of the baseline. We defined the frequency of sCaLTs as the number of sCaLTs observed in a 2-min period and measured the amplitude as the ΔF/F0 changes over the baseline. Data from all different regions of one cell was pooled together to generate one data point, at least four dishes from different batches of cultures were pooled together and analyzed for statistically significant differences using one-way ANOVA with post hoc tests. Compiled data are expressed and graphed as mean ± SEM, with n denoting the number of cells studied.To quantify individual sCaLTs, each time-lapse fluorescence (F) sequence was processed to generate a ΔF/FTo better depict the spatiotemporal dynamics of sCaLTs in individual OLs, we generated a temporal-color coded image from a ΔF time-lapse sequence using ImageJ . In this temporal-color coded image, sCaLTs occurring at different time points are visualized in distinct colors as indicated on the color-coded scale bar, thus providing a visual summary of all the sCaLTs from the sequence.in vitro or in vivo  or mean ± standard error of the mean (SEM) as stated in text and in figure legends.All data from this study were collected from at least three replicates of independently prepared samples. We first assessed the normality of our data using histogram combined with Shapiro–Wilk test. Data following a normal distribution with only two conditions were analyzed using a two-tailed unpaired Student’s 2+ in axon myelination by OLs  sCaLTs per cell over the 2-min period (n = 16) and 84.67 ± 39.20  sCaLTs per cell, respectively (n = 12). Since multiple sCaLTs were observed at each local site, we also calculated the number of sCaLTs per site over the 2-min window (referred to as the frequency/site). We found no difference in sCaLT frequency/site among the different groups except for the mature OLs, which showed a slight but significant reduction  of the pretreatment period and the amplitude of remaining sCaLTs was reduced to 52.04 ± 7.41% (mean ± SEM) of the pretreatment period  in our primary OL culture, we elected to use a rat CNS glial precursor cell line (CG-4 cell) to obtain a large quantity of differentiating OLs (m), verapamil (100 μm), or nifedipine (10 μm); N-type: ω-CTX (1 μm); T-type: NNC 55-0396 (2 μm); and P/Q-type: ω-agatoxin IVA (100 nm). As summarized in Since VGCCs have been implicated in OL development , we examting OLs  for RT-Pting OLs A. Of the2+ signaling in a variety of cells  Ca2+ ATPase, to deplete the internal Ca2+ store. Consistent with previous studies, exposure of OLs to Tg elicited a large and transient elevation in cell body [Ca2+]i due to the Ca2+ release from the internal store (hereafter referred as peak #1), which is followed by the resting [Ca2+]i dropping to a lower level than that of the control due to the lack of Ca2+ in the extracellular space (m Ca2+ to the extracellular buffer elicited another large and transient elevation of [Ca2+]i (hereafter referred to as peak #2), which, as previously described  was very effective in inhibiting the elevation in [Ca2+]i (peak #2) in response to the depletion and reintroduction of extracellular Ca2+  of the control values, respectively (m), or Tg (1 μm), to inhibit the ER Ca2+ pumps and found that both inhibitors abolished the sCaLTs in OLs, even in the presence of extracellular Ca2+ (m) also significantly reduced the frequency of sCaLTs. Together, these results indicate that Ca2+ release from the internal Ca2+ store coupled with SOCE underlie the development of sCaLTs in cultured OLs.It should be noted that, in the presence of SFK96365, sCaLTs in the OL branches were largely diminished even when extracellular Caectively A. We nexlar Ca2+ B. Furthein vitro (DIV)1] were treated with SKF96365, Tg, or nifedipine for 4 d and fixed on DIV5 for immunolabeling with specific OL markers Olig2, O4, and MBP (m) and Tg (100 nm) markedly reduced the percentage of cells expressing these OL lineage markers (p = 1.2E-06 and p = 1.1E-13). Of the inhibitors used, Tg appears to have the most profound effect on OL maturation, indicating the importance of Ca2+ internal stores in OL development. Importantly, we did not observe any changes in the overall number of cells, suggesting that cell death was not induced by these pharmacological treatments  or Tg (100 nm) displayed a lower D than control cells, indicating a reduction in the morphologic complexity of these OLs  analysis as described previously . Hence, hese OLs D. The rehese OLs E. Exposut not O4 B. Howevebor size C–E, whic2+ using the calcium-free solution for 24 h resulted in a substantial reduction in OL branches (m) for 2 h and then imaged for 20 min using DIC. We found that OLs treated with SFK96365 showed essentially no extension of their processes during the 20-min live cell recording (m) or calcium-free buffer  that is spontaneously generated, spatiotemporally restricted in discrete regions of OL processes, and independent of neuronal input. Significantly, our study has identified SOCE as a major contributor to sCaLTs, thus shedding new light on Ca2+ signaling mechanisms underlying oligodendroglial development.Several previous studies have shown the expression of VGCCs in OLs as well as the elevation of i can elicit local actin-based filopodia protrusions (2+ signals in the late stage of OL development, such as during myelination. Given that Ca2+ is known to play a critical role in myelination vivo (2+ signals that regulate the myelination process. The absence of neurons and the cell culture nature of our system do represent the limitation of our study. Nonetheless, our study in culture has enabled us to identify a novel Ca2+ mechanism involving spontaneous Ca2+ signals from SOCE and Ca2+ stores that regulate OL development. Future studies are needed to investigate the role of spontaneous Ca2+ signals in OL development in vivo.It remains to be determined how sCaLTs may function to regulate the development and differentiation of OLs in culture. Morphologically, the occurrence of sCaLTs reaches its peak at the same time that OLs undergoes extensive process outgrowth and branching. Previous studies in neurons have shown that local elevation in [Catrusions . It is ptrusions . Previoutrusions . We specion vivo , neurona


Ishige okamurae affects calcium levels, resulting in enhanced vasodilation. We examined calcium modulation with the well-known receptors, acetylcholine receptor (AchR) and vascular endothelial growth factor 2 (VEGFR2), which are related to NO formation, and further confirmed the vasodilatory effect of DPHC. We confirmed that DPHC stimulated NO production by increasing calcium levels and endothelial nitric oxide synthase (eNOS) expression. DPHC affected AchR and VEGFR2 expression, thereby influencing transient calcium intake. Specific antagonists, atropine and SU5416, were used to verify our findings. Furthermore, based on the results of in vivo experiments, we treated Tg(flk:EGFP) transgenic zebrafish with DPHC to confirm its vasodilatory effect. In conclusion, the present study showed that DPHC modulated calcium transit through AchR and VEGFR2, increasing endothelial-dependent NO production. Thus, DPHC, a natural marine component, can efficiently ameliorate cardiovascular diseases by improving vascular function.Nitric oxide (NO) is released by endothelial cells in the blood vessel wall to enhance vasodilation. Marine polyphenols are known to have protective effects against vascular dysfunction and hypertension. The present study is the first to investigate how diphlorethohydroxycarmalol (DPHC) isolated from  Hypertension is a medical condition characterized by persistently high pressure on the blood vessels . The hig2+] concentration and eNOS-derived NO levels can contribute to hypertension ER level represented calcium stored in the endoplasmic reticulum (ER). To investigate whether DPHC could upregulate [Ca2+] levels in endothelial cells, [Ca2+]ER and [Ca2+]cytol levels were measured separately. After treating the cells with 60 μM of DPHC, [Ca2+]ER levels were elevated after 20 s (2+]ER levels by measuring the area under the curve (AUC). We found that 60 μM of DPHC significantly raised [Ca2+]ER levels in EA.hy926 cells (p < 0.001). Moreover, [Ca2+]cytol levels were dramatically increased starting from 30 s after the treatment, with a peak detected around 60 s. At 60 s, [Ca2+]cytol levels were 1.75 times higher than that in the untreated control .In the present study, the [Cater 20 s a. We qua control c. Quanti2+] levels can be influenced by the activation of VEGFR2 and AchR [2+]cytol levels was only observed in the DPHC treatment group, with a maximal calcium concentration observed at about 30 s after treatment, followed by a gradual decline. As expected, [Ca2+]cytol levels were remarkably decreased when cells were treated with atropine and SU5416 compared to cells treated with DPHC. Additionally, there was no significant difference in [Ca2+]cytol levels between the antagonist-treated and control groups (p < 0.001). Thus, these findings indicate that the modulation of [Ca2+] levels by DPHC was closely related to AchR and VEGFR2 activation. levels could be regulated by AchR and VEGFR2 activation, we further hypothesized that [Ca2+] levels would deeply influence NO production. To prove our hypothesis, cells were pretreated with atropine or SU5416 and incubated for 2 h, followed by the addition of 60 μΜ of DPHC. After 24 h, intracellular NO concentrations were measured using 4 amino-5-methylamino-2′, 7′-difluorescein diacetate (DAF-FM DA). We observed that both antagonists significantly suppressed NO formation upon DPHC stimulation (2+]cytol levels by AchR and VEGFR2 activation was sufficient and necessary for NO formation.Having found that /calmodulin [2+] is bound to calmodulin, the [Ca2+] signal transduction pathway is activated [2+]ER level would activate the calmodulin-bound domain of eNOS, resulting in NO production [2+]cytol levels led to NO formation, which was shown in human umbilical vein endothelial cells [2+] signaling under DPHC administration. After adding 60 μM of DPHC, it was possible to detect a remarkable increase in [Ca2+]cytol and [Ca2+]ER levels after 30 s, characterized by a 60 s oscillation peak phase. This increase was completely prevented by different antagonists (VEGFR2 and AchR antagonists). Theoretically, after the peak phase, a stabilization declining phase, also characterized by the presence of less intense and less frequent oscillations, would follow [2+]cytol and [Ca2+]ER levels . The increase in [Ca2+]cytol level is precipitous and is followed by a decline in [Ca2+] levels, maintained by the homeostatic regulation of [Ca2+] concentration [2+]cytol and [Ca2+]ER levels, we further focused on investigating how DPHC affects calcium regulation via cell membrane receptors (VEGFR2 and AchR). The muscarinic AchR family is divided into 5 subtypes: M1, M2, M3, M4, and M5 cytol. In addition, we observed that the level of NO was obviously suppressed by atropine. Therefore, we concluded that DPHC-induced rise in [Ca2+]cytol and NO formation could proceed with AchR activation in EA.hy926 cells. Another potential regulator of vasodilation is VEGFR2, mainly expressed in the vascular endothelium [2+]cytol levels or NO production in EA.hy926 cells pretreated with SU5416. Therefore, we hypothesize that DPHC triggered an increase in [Ca2+]cytol levels by regulating AchR and VEGFR2 activation, resulting in NO production.When the cells are stimulated, such as during membrane depolarization, extracellular signaling molecules or intracellular messengers enhance -2-(2-amino-5-methylphenoxy) ethane-N, N, N′, N′-tetraacetic acid, pentaacetoxymethyl ester) . Atropine, a specific AchR antagonist, was purchased from Sigma Aldrich. SU5416, a VEGFR2 inhibitor, was obtained from Tocris Bioscience .n-hexane, EtOAc, MeOH, and H2O. Further purification was performed using an HPLC system  equipped with a YMC-Pack ODS-A column . The sample was eluted using an isocratic solvent system.DPHC isolation was performed as previously described . In briev/v) CO2. Cells at 2–5 passages were used for all experiments.Human cardiovascular endothelial cell line EA.hy926 was acquired from the American Type Culture Collection  and cultured in DMEM supplemented with 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10% FBS. Cells were grown in a humidified incubator at 37 °C, in an atmosphere with 5% (5 cells/well (180 μL of cell suspension) were seeded in a 96-well plate and incubated at 37 °C for 24 h. Cells were treated with different concentrations  of DPHC for another 24 h. Then, 100 μL (concentration: 2 mg/mL) of MTT was added to the wells, and cells were incubated for 2 h. After 2 h, the medium was replaced with 150 μL of DMSO. The supernatant was collected, and the absorbance was measured using a microplate reader at 540 nm .EA.hy926 cell viability was assessed by an MTT assay. A total of 1 × 10DAF-FM DA is a fluorescent probe used to detect intracellular NO. DAF-FM DA is a cell-permeable deacetylated form of DAF-FM and is hydrolyzed by intracellular esterases to form a cell-impermeable DAF-FM that reacts with NO. Therefore, the fluorescence intensity in cells was used to evaluate intracellular NO levels . EA.hy922+ levels correspond to the concentration of calcium ions in the ER ([Ca2+]ER), while the extracellular Ca2+ levels refer to the concentration of calcium ions in the cytosol ([Ca2+]cytol). Physiological salt solution (PSS)  was used as the basal reagent. To measure [Ca2+]cytol levels, the Ca2+ -sensitive Fluo-4 probe was dissolved in PSS. On the contrary, to measure [Ca2+]ER levels, the Fluo-4 probe was dissolved in a modified version of PSS, without CaCl2. EA.hy926 cells were seeded in 96-well plates overnight when they reached approximately 80% confluence. Next, 1× Fluo-4 was added, and cells were incubated for 30 min at 37 °C in the dark. The cells were rinsed twice with 1× phosphate-buffered saline (PBS), and 50 μL of 1× PBS was added to the wells. Fluorescence intensity was measured for 10 s at 1-s intervals. The cells were then treated with 1× PSS (Control) or DPHC at different concentrations  dissolved in 0.1% bovine serum albumin (BSA). After the treatment, fluorescence was detected for another 50 s at 1-s intervals. To investigate the relationship between DPHC and either AchR or VEGFR2 activation, cells were pretreated for 2 h with 60 μΜ of atropine, an AchR antagonist, and 100 μM of SU5416, a VEGFR2 antagonist. The 1× Fluo-4 fluorescent dye was added, and cells were incubated for another 30 min at 37 °C in the dark. Calcium levels were then measured as described earlier. The box plot data represent the mean value of the AUC calculated from 0 to 60 s. The error bars indicate the maximum and minimum values.By definition, intracellular Cap-AKT , anti-p-eNOS , and anti-p-PI3K  dissolved in 5% skim milk. Immunoblots were incubated for another 2 h at room temperature with specific secondary antibodies (1:3000). Protein bands were ultimately developed and photographed using the FUSION SOLO Vilber Lourmat system, Paris, France. The Image J 1.50i software  was used for quantifying the band intensities.EA.hy926 cells were treated with different concentrations  of DPHC. After 24 h, cells were washed and harvested with ice-cold PBS and lysed with a lysis buffer on ice for 1 h. Lysates were centrifuged at 12,000 rpm for 20 min, and the protein concentration in the supernatant was evaluated with a BSA protein assay kit . Next, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with 10% gel. Proteins were transferred onto a nitrocellulose membrane. Membranes were incubated at 4 °C overnight with the following primary antibodies added separately: anti-β-actin , anti-Tg(flk:EGFP) zebrafish model was used to investigate the behavior of endothelial cells in vivo. Fish were housed in 3-L tanks  containing buffered water (pH 7.5) maintained at 28.5 °C. Fertilized eggs were collected from the bottom of the tank in water  containing methylene blue and were placed in Petri dishes after being washed thoroughly with water and transferred to the incubator. For all the experimental procedures, larvae were maintained in 24-well plates containing egg water . Larvae were treated with 0, 0.06, 0.2, and 0.6 μM of DPHC at 3 dpf. After six days of treatment, larvae were photographed using a fluorescence microscope at 4× magnification to capture blood vessels in the whole body. Fluorescence intensity signals of the whole body were measured using the Gen5 3.04 software (Bioteck). Fluorescence images were imported into the Image J software to calculate the corrected total object fluorescence (CTOF), according to the following formula:The zebrafish experiment received approval from the Animal Care and Use Committee of Jeju National University . The p-values were considered statistically significant, and they have been illustrated with asterisks in all figures: * p < 0.05, ** p < 0.01, and *** p < 0.001.All experiments were conducted in triplicates, and data are shown as mean ± standard deviation. Statistical analysis was performed using the one-way analysis of variance (ANOVA) with Dunnett’s post hoc test with the Prism 5.0 software . The following In this study, we demonstrated the molecular mechanisms underlying the vasodilatory effect of the marine polyphenol, DPHC. DPHC promoted endothelium-dependent vasodilation by modulating calcium concentration through AchR and VEGFR2 activation, thereby triggering NO synthesis through activation of the PI3K/Akt/eNOS pathway. Therefore, DPHC is a potential novel natural compound for vasodilation and the prevention of endothelium-dependent cardiovascular diseases.


One example are [Ca2+] increases in the stroma of chloroplasts during light‐to‐dark transitions; however, the function and mechanisms responsible are unknown, in part because there is a disagreement in the literature concerning whether corresponding dark‐induced changes in cytosolic [Ca2+] ([Ca2+]cyt) can be detected.Changes in the spatiotemporal concentration of free Ca2+]cyt upon darkness in addition to the already known dark‐induced increases in [Ca2+]stroma in the aerial part of the Arabidopsis thaliana plant.We have measured changes in [Ca2+]cyt transients depend on the photoperiod and time of day, peaking at anticipated dusk, and are superimposed on daily 24 h oscillations in [Ca2+]cyt. We also find that the magnitude of the dark‐induced increases in Ca2+ in both the cytosol and chloroplasts are gated by the nuclear circadian oscillator.These [Ca2+]stroma and [Ca2+]cyt by transcriptional regulators in the nucleus that are part of the circadian oscillator demonstrates a new role for the circadian system in subcellular Ca2+ signalling, in addition to its role in driving circadian oscillations of [Ca2+] in the cytosol and chloroplasts.The modulation of the magnitude of dark‐induced increases in [Ca Seeds were germinated and entrained in growth cabinets  with a constant temperature of 19°C and 100 μmol m−2 s−1 cool white light from fluorescent tubes under 12 h : 12 h, light : dark cycles, unless otherwise stated.Arabidopsis seeds were surface‐sterilized with 10% (v/v) NaClO and 0.1% (v/v) Triton X‐100 for 3 min and rinsed three times with sterile dH−2 s−1) at the desired photoperiod and was cooled to 19–20°C. When just red or blue light was used, the intensity was 50 μmol m−2 s−1. Luminescence was recorded from clusters of seven to 12 seedlings grown as described, and the data for one experiment were obtained as the sum of the signal of all the cluster together. Image analysis was done with Photek ifS32 software.Photon counting was performed in a light‐tight box using an ICCD225 photon‐counting camera system  mounted above the seedlings. The camera chambers supplied equal amounts of red (630 nm)/blue (470 nm) LED light in a mixed array  for 20 min in the dark the night before going into the camera box when they were 11–12 d old.2+]cyt was performed as described by Love et al.  to carry out fast Fourier transform nonlinear least‐squares analysis  containing 1 ml of 0.8% (w/v) bactoagar media with ½MS and incubated with 100 μl of 20 µM coelenterazine (Prolume) for 20 min in the dark. At the end of the photoperiod, bioluminescence was measured using a photon‐counting luminometer (photomultiplier tube 9899A) cooled to −20ºC with a FACT50 housing   ethanol). Measurements were made until the detected luminescence reached 10% of the first peak after discharge injection. [Ca2+] concentrations were determined according to Fricker et al. , suggesting that it is the transfer from light to dark that results in the change of aequorin luminescence. No signal was detected from nontransgenic Col‐0 plants  Fig. . The darnce Figs . Statistnts Figs  grown an2+]cyt signal measured using the cytosolic aequorin in plants that were grown in 12 h : 12 h, light : dark cycles differs from the dark‐induced signal from aequorin targeted to the stroma  arrays because the discharge of available aequorin with excess Ca2+ to perform a normalization saturated pixels on the CCD detector. To obtain an estimate of the magnitude of the dark‐induced increase in [Ca2+]cyt that occurs at 3.5 min after darkness, when Chl fluorescence has dissipated, we revisited the use of luminometry in the same experimental conditions, with three plants per sample to avoid the leaves covering each other  plants ; Ler, 8135 ± 906; cry2, 4676 ± 132 ). These analyses indicated that the dark‐induced [Ca2+]cyt changes were still present in these mutant lines, suggesting that the remaining photoreceptor systems in the mutants were able to respond to the transition into darkness. In Fig. phyA and phyB on the dark‐induced [Ca2+]cyt changes. The absence of PHYB did not affect the response ). However, we found that in the phyA mutant, the dark‐induced [Ca2+]cyt was absent ), suggesting that PHYA, at least, is directly involved in the light signalling pathway that leads to the dark‐induced cytosolic‐free calcium increases.To test further the involvement of the red and blue photoreceptors, we performed a light‐to‐dark transition using nts Fig. b resulte2+]cyt after darkness was derived from internal stores or from the extracellular space. First, we decided to evaluate whether Ca2+ influx to the cytosol might be across the plasma membrane using two inhibitors of plasma membrane‐mediated influx of Ca2+, 1 mM GdCl3 and 10 mM LaCl3 ; control, 12 844 ± 642, La3+, 16 816 ± 7296 )  is a Ca2+‐signalling molecule synthesized by ADP‐ribosyl cyclase (ADPRc) which can release Ca2+ into the cytosol from the endoplasmic reticulum (ER) and vacuole ; control, 12 844 ± 642; cPTIO, 5953 ± 49 ) , suggesting that photosynthesis may affect the [Ca2+]cyt signal upon darkness  and short‐day cycles . The [Ca2+] response in the cytosol was affected by the length of day . Data were recorded every 2 h over 1 d in 12 h: 12 h conditions as shown in Fig. 2+]cyt upon darkness were modulated by the time of day, being higher at the end of the photoperiod and absent during the night when there was no transition from light to darkness  with dark interruptions, a protocol standard for circadian luminescence such as promoter::luciferase fusions measurements, because the short dark breaks do not interfere with functioning of the circadian oscillator  in the LD cycle, there was no increase in [Ca2+]cyt  and the last 800 s of each integration  to determine if we could detect basal circadian [Ca2+]cyt oscillations in addition to the dark‐induced signal. In both, the first 1500 s  and the last 800 s , [Ca2+]cyt increased during the day, peaked several hours after dawn and then decreased, reaching the minimum value during the night , a condition that does not have a light‐to‐dark transition during measurement but is complicated by the dampening of circadian rhythms of [Ca2+]cyt in prolonged DD in Arabidopsis   Figs d, S10, ints Figs d, S10, d2+]cyt changes upon transition to darkness have not been detected using either the luminescent reporter protein aequorin   and BICAT2  should be directed to the Click here for additional data file.


Panax ginseng C. A. Mey., Panax notoginseng (Burk.) F. H. Chen, Aralia chinensis L., cardioprotection, and other Chinese herbal medicines have been used to treat patients with coronary heart disease, angina pectoris, and other cardiovascular diseases. Studies have shown that saponins are the main active substances in TCMs containing Panax ginseng C. A. Mey., Panax notoginseng (Burk.) F. H. Chen, Aralia chinensis L., and Radix astragali. In the present review, we sorted the saponin components with anti-MIRI effects and their regulatory mechanisms. Each saponin can play a cardioprotective role via multiple mechanisms, and the signaling pathways involved in different saponins are not the same. We found that more active saponins in Panax ginseng C. A. Mey. are mainly dammar-type structures and have a strong regulatory effect on energy metabolism. The highly active saponin components of Aralia chinensis L. are oleanolic acid structures, which have significant regulatory effects on calcium homeostasis. Therefore, saponins in Chinese herbal medicine provide a broad application prospect for the development of highly effective and low-toxicity anti-MIRI drugs.Myocardial ischemia is a high-risk disease among middle-aged and senior individuals. After thrombolytic therapy, heart tissue can potentially suffer further damage, which is called myocardial ischemia-reperfusion injury (MIRI). At present, the treatment methods and drugs for MIRI are scarce and cannot meet the current clinical needs. The mechanism of MIRI involves the interaction of multiple factors, and the current research hotspots mainly include oxidative stress, inflammation, calcium overload, energy metabolism disorders, pyroptosis, and ferroptosis. Traditional Chinese medicine (TCM) has multiple targets and few toxic side effects; clinical preparations containing  To date, revascularization, such as thrombolysis, is an effective method for the treatment of ischemic cardiomyopathy in patients with acute myocardial infarction . HoweverPanax ginseng C. A. Mey., Panax notoginseng (Burk.) F.H. Chen, Panax quinquefolium L., Aralia chinensis L., and Radix astragali  has a holistic treatment concept, and has the advantages of multiple targets, multiple links, and multiple approaches. In the field of TCM, the etiology of MIRI involves deficiency of qi, blood stasis, and phlegm. The clinical treatment is often based on TCM and compound preparations with the effects of replenishing qi and nourishing yin, warming the heart, promoting blood circulation and removing blood stasis, and expelling phlegm . TCM constragali . In receammation . Saponinammation . Each saIn this review, we discuss the classic mechanisms of MIRI and a few emerging regulatory mechanisms . Based o2−), hydroxyl radical (OH−), and hydrogen peroxide (H2O2), which are the culprits responsible for inducing oxidative stress in the vascular wall, are mainly produced through xanthine oxidase, NADPH oxidase, endothelial nitric oxide synthase, and other enzyme systems  are the main driving forces for reperfusion injury . ROS and systems . In othe systems . Therefo systems , which aThe inflammatory response activates during myocardial ischemia and is significantly aggravated during reperfusion . The adhEndothelial cells are closely related to inflammation, and endothelial dysfunction and microcirculation injury are important bases of MIRI. Studies have found that endothelial cells are more vulnerable to damage than cardiomyocytes during reperfusion, whereas cardiomyocytes are more vulnerable to damage during the ischemic phase . Therefo2+ can maintain cardiomyocyte function, proliferation, division, energy metabolism and other important processes (2+ ATPase (SERCA) activity , and a large amount of Ca2+ is released from the sarcoplasmic reticulum (SR) through ryanodine receptor 2 (RyR2). Then, calcium and troponin C combine to cause myocardial cell contraction /Parkin, BNIP3/NIX, and FUNDC1 signaling pathway . ModeratIn recent years, the application of microRNA (miRNA) and long noncoding RNAs (lncRNA) in MIRI treatment has increasingly become a research focus. miRNA is an endogenous, single-stranded, non-coding, small regulatory RNA in a variety of eukaryotic cells . StudiesPyroptosis is a new form of programmed cell death that is accompanied by inflammatory reactions . It is cFerroptosis is a new form of cell death caused by MIRI . It is cDue to ischemia and hypoxia of myocardial tissue, energy metabolism disorder is the initiation of MIRI . In addiPanax ginseng C. A. Mey., Panax notoginseng (Burk.) F. H. Chen, and Panax quinquefolium L., as well as Cucurbitaceae plants such as Gynostemma pentaphyllum (Thunb) Makino (Panax ginseng C. A. Mey. and Panax notoginseng (Burk.) F. H. Chen are the two main plant sources of ginsenosides. Among them, authentic Panax ginseng C. A. Mey. is mainly distributed in Southwest China, East Asia and North America, and authentic Panax notoginseng (Burk.) F. H. Chen is mainly distributed in Southwest China. Although the proportion of ginsenosides in these Chinese herbal medicines varies, all of them are excellent TCMs for anti-tumor, anti-oxidation, anti-aging, anti-fatigue, regulating blood sugar balance, improving cardiovascular and cerebrovascular, and enhancing immunity  by regulating autophagy and apoptosis via the HIF-1α/BNIP3 and PI3K/Akt pathways  Makino . Panax gimmunity . Xuesait vessels . Studies vessels . Ginseng vessels . Panax npathways   F. H. Chen, and Panax quinquefolium L., has an excellent therap effect on MIRI in vivo and ex vivo, mainly by inhibiting apoptosis pathways and activating mTOR phosphorylation  Seem. and Aralia taibaiensis  Seem. is mainly distribution in Northeast China, North Korea, Japan and Russia, and Aralia taibaiensis is distribution in Midwest China. It contains several triterpene saponins, flavonoids, coumarins, and alkaloids, of which saponins are its main active ingredient  Seem. protects against MIRI by suppressing ERS-related apoptosis and calcium overload via the PKCε-dependent signaling pathway  and HR (in vitro), and the protective mechanism was associated with AMPK pathway-related apoptosis , the National Natural Science Foundation of China (No. 81973514 by GS), the Drug Innovation Major Project (No. 2018ZX09711001-009 by XS) and National Natural Science Foundation of China (No. 81891012 by XS).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.


If these two parameters are simultaneously present, the probability for AFFs occurrence increases dramatically. Our scientific report, which is based on the current evidence about AFFs, is that if both femoral bowing angle and femoral neck-shaft angle are evaluated before BPs administration, this intervention may reduce the incidence of AFFs. Thus, in cases with excessive lateral femoral shaft bowing or very small femoral neck-shaft angle, the prescription of another anti-osteoporotic treatment than BPs should be recommended. If, however, BPs can’t be avoided, clinicians should be aware of the fact that long-term administration may be implicated with AFFs occurrence. In these cases, short term BPs administration with timely drug holiday between three and five years may be reasonable. Finally, roentgenographic evaluation of both femurs every six months and medical reference in case of any emerging thigh pain are also logical interventions to prevent and reduce AFFs.Bisphosphonates (BPs) are the mainstay of osteoporosis treatment due to their safety and efficacy. There is evidence that BPs medication may be complicated by atypical femoral fractures (AFFs). Prolonged administration of BPs is even more strongly associated with AFFs. AFF is a relatively rare complication of BPs when taking into account the huge population worldwide that benefits from this pharmacotherapy. AFF is, however, a serious complication of BPs treatment, which includes prolonged healing time and high revision rate when operative treatment is required. Less frequently, AFFs occur even without BPs administration, while these fractures have all the characteristics of “stress” or “insufficiency” fractures. The critical point of view in AFFs pathogenesis seems to be not only the biology of cortical bone, but also the mechanical issue. It has been proven that BPs, glucocorticoids and proton pump inhibitors (PPIs) can cause bone turnover suppression and affect the biological parameter of AFFs pathogenesis. Specific mechanical femoral bone properties predispose to AFFs pathogenesis. Several studies have already reported that increased femoral bowing > 5.25 Atypical femoral fractures (AFFs) are often associated with bisphosphonates (BPs) administration for osteoporosis treatment . As BPs As the prevalence of osteoporosis increases dramatically, AFF is a devastating complication of BPs usage, our scientific report, that is based on current evidence about AFFs, is that if both femoral bowing angle and femoral neck-shaft angle are evaluated before BPs administration it may reduce the incidence of AFFs. Thus, in cases with excessive lateral femoral shaft bowing or very small femoral neck-shaft angle, prescription of another anti-osteoporotic treatment other than BPs should be recommended. If, however, BPs can’t be avoided, clinicians should be aware of the fact that long-term administration may be implicated with AFFs occurrence. In these cases, short term BPs administration with timely drug holiday between three and five years may be reasonable. Patients with osteoporosis and increased BMI (body mass index)  should aAFFs belong to the category of insufficiency fractures as they present almost all the essential elements of them . The meco and 7o [o) [In cases with atypical femoral shaft fractures, the lateral femoral bowing plays a significant role in pathogenesis of these fractures . AFF inco and 7o ,12. Clino and 7o . The diso and 7o . This had 7o [o) .It is worth noting that patients who develop AFFs are somewhat younger than those who develop osteoporotic proximal femoral fractures, with a mean age of 74-77 years old (AFFs group) ,14.As the population is getting older, bone mineral density decreases and prevalence of osteoporosis increases. BPs are the mainstay of osteoporosis treatment, so evaluation of femoral bowing and femoral neck-shaft angle can contribute to the reduction of AFFs either by no initiation in extreme cases, or by administration of BPs for a relatively shorter period of time than usually, introducing drug holiday timely.To support our scientific report, we appose an example of a female patient, 85 years old with senile osteoporosis with intertrochanteric hip fracture and excessive lateral femoral bowing Figure .o). If, in this case, we prescribe BPs for osteoporosis treatment, we also affect the biological parameter and we simultaneously increased the possibility for AFF occurrence. In this particular case, the administration of an anabolic agent plus calcium and vitamin D is recommended [After fixation of the fracture, we should treat our patient for osteoporosis. As it has already been highlighted, AFF pathogenesis is associated with both mechanical and biological mechanisms of cortical bone . In thisommended . Furtherommended .It is worth noting that the incidence of atypical fractures is less significant than the benefit of BPs administration . BPs admo or the femoral neck-shaft angle is < 125o, there is an increased risk for diaphyseal and sub-trochanteric atypical femoral fracture respectively, regardless of BPs usage [o) or small femoral neck-shaft angle (<125o), if BPs administration can’t be avoided, clinicians should be aware of the fact that long term therapy may be implicated with AFFs occurrence. In these cases, short term BPs administration with timely drug holiday between 3 and 5 years is reasonable. Thus, BPs administration for more than 5 years should be avoided and these patients should be counseled properly and followed up regularly. Few studies have shown that there is a tendency for AFFs to occur bilaterally [The critical point of view is that AFFs have all the characteristics of stress or insufficiency fractures, and the pathogenesis of these fractures is affected by mechanical stresses and by cracks self-healing bone potency . In casePs usage . So, if aterally , so follTo the best of our knowledge, proposal for femur radiographic evaluation before anti-osteoporotic treatment prescription has never been reported in the current literature, to reduce AFFs. Considering the large population benefiting from BPs pharmacotherapy, the incidence of this fracture entity is rather low. Despite that, we believe that with this scientific report, AFFs can be further reduced, although more research is urgently needed is in this area, to confirm this.


Thus, to avoid or slowdown the development of late stages of AMD (nAMD or GA), new therapies targeting iAMD are needed such as crocetin obtained through hydrolysis of crocin, an important component of saffron (Crocus sativus L.), and norbixin derived from bixin extracted from Bixa orellana seeds. We have shown that these apocarotenoids preserved more effectively RPE cells against apoptosis following blue light exposure in the presence of A2E than lutein and zeaxanthin. In this review, we will discuss the potential use of apocarotenoids to slowdown the progression of iAMD, to reduce the incidence of both forms of late AMD.Age-related macular degeneration (AMD) is the commonest cause of severe visual loss and blindness in developed countries among individuals aged 60 and older. AMD slowly progresses from early AMD to intermediate AMD (iAMD) and ultimately late-stage AMD. Late AMD encompasses either neovascular AMD (nAMD) or geographic atrophy (GA). nAMD is defined by choroidal neovascularization (CNV) and hemorrhage in the subretinal space at the level of the macula. This induces a rapid visual impairment caused by the death of photoreceptor cells. Intravitreal injection of anti-vascular endothelial growth factor (VEGF) antibodies is the standard treatment of nAMD but adds to the burden of patient care. GA is characterized by slowly expanding photoreceptor, and retinal pigment epithelium (RPE) degeneration patches progressively leading to blindness. There is currently no therapy to cure GA. Late AMD continues to be an unmet medical need representing a major health problem with millions of patients worldwide. Oxidative stress and inflammation are recognized as some of the main risk factors to developing late AMD. The antioxidant formulation AREDS (Age-Related Eye Disease Studies), contains  Age-related macular degeneration (AMD) is the main cause of blindness in the industrialized world with over 30 million people suffering from this disease . In the Neovascular AMD is defined by choroidal neovascularization (CNV) and hemorrhage in the subretinal space at the level of the macula. This induces the rapid death of photoreceptor cells and then rapid loss of vision . CurrentNCT02503332 testing Pegcetacoplan® (APL-2) from Apellis Pharmaceuticals Inc., . IA. IA44]. β-carotene  are known for their antioxidant properties. In the organism of mammals, carotenoids originate exclusively from the diet . Cr. CrCrocute light . Preventactivity . Similaractivity . In paraell line . Pretreaell line . Crocetiell line . The neuexamined . Both cral cells . Interesmyloid-β . In a simyloid-β . These amyloid-β . These rreatment . In the reatment . These ated mice . The out000 lux) . Interesrecently , 91. In recently . A subserecently  reportedrecently  supporticis-Norbixin is a 6,6′-di-apo-carotenoid extracted from annatto (Bixa orellana) seeds. Norbixin  model) protect retinal tissues and photoreceptor cells [−/−/Rdh8−/− mice with a diet containing norbixin also prevented the reduction of rod and cone photoreceptor electrical activity measured by scotopic and photopic ERG amplitude, respectively [in vitro [in vivo administration of norbixin reduces the ocular accumulation of A2E in ABCA4−/− and Rdh8−/− mice [in vitro that norbixin significantly reduced the production of VEGF and of several inflammatory cytokines, such as IL-6 and IL-8, in porcine RPE cells cultivated in the presence of A2E  [in vitro data confirm the anti-inflammatory effects of norbixin observed in human studies in vivo [In vivo, consumption of a high-fat meal induces the production of the proinflammatory cytokines IL-1, TNF-α, and IL-6 in human plasma, which can be reduced by norbixin treatment [in vivo [9′-Norbixin  structurmination . Interescrocetin . However similar . In vivoor cells . Similaror cells . In addiectively , 92. Thiin vitro . A2E acc−/− mice , suggestbioRxiv) . These i in vivo , 100. Inreatment . Interesreatment . Convers[in vivo . Altoget[in vivo , 101. Du[in vivo , this obThere is no effective therapeutic strategy for the late form of dry AMD, and intraocular treatments of nAMD are costly and do not prevent long-term loss of vision. As both GA and nAMD originate from iAMD, treating patients at this early stage of the disease could potentially prevent the development of late AMD. Since oxidative stress and inflammation appear to play an essential role in iAMD, the use of therapeutic strategies is aimed at reducing oxidative stress, and inflammation is potentially attractive. At present, prescribing the antioxidant AREDS/AREDS formulations to iAMD patients is the only available therapeutic strategy to reduce late AMD incidence, but its effectiveness appears limited. Here, we described the potential use of apocarotenoids such as crocin, crocetin, and norbixin, for long-term therapy to slowdown the progression of iAMD towards late stages of the disease. Developing such new and more effective treatments for patients with iAMD could drastically reduce the incidence of late forms of AMD in the general population and then could reduce the burden for society as a whole. Indeed, limiting the development of GA and nAMD will not only benefit patients by improving their quality of life but also provide “peace of mind” to the healthy and caregivers. It is also expected that development of such oral drugs will reduce costs to healthcare providers if it becomes the preferred treatment of patients with the intermediate form of AMD.


In humans, the uniporter functions as a holocomplex consisting of MCU, EMRE, MICU1 and MICU2, among which MCU and EMRE form a subcomplex and function as the conductive channel while MICU1 and MICU2 are EF-hand proteins that regulate the channel activity in a Ca2+-dependent manner. Here, we present the EM structures of the human mitochondrial calcium uniporter holocomplex (uniplex) in the presence and absence of Ca2+, revealing distinct Ca2+ dependent assembly of the uniplex. Our structural observations suggest that Ca2+ changes the dimerization interaction between MICU1 and MICU2, which in turn determines how the MICU1-MICU2 subcomplex interacts with the MCU-EMRE channel and, consequently, changes the distribution of the uniplex assemblies between the blocked and unblocked states.Mitochondrial Ca To obtain the uniplex samples in the Ca2+-bound and Ca2+-free state for cryo-EM studies, the buffers used in the final SEC purification step contained either 2 mM Ca2+ or 2 mM EGTA without Ca2+, respectively. The SEC elution volume of the purified uniplex indicates a size much larger than the dimeric form of the MCU-EMRE subcomplex, suggesting that the uniplex also forms a dimer in solution  are relatively parallel in the apo state but swing apart and become more perpendicular to each other in the Ca2+-bound state  competent cells were freshly transformed, plated onto LB- kanamycin (LB-Kan) agar, and grown overnight. A single colony was picked from LB-Kan plate to seed LB media with 50 μg/mL kanamycin and grown in shaker flasks at 37°C in an orbital shaker until the cells reached an OD600 = 1.0 ~ 1.2 at which point they were chilled on icy water for half an hour before protein expression was induced by addition of IPTG to a final concentration of 0.3 mM. The cells were then grown at 16°C for 20 hr before being harvested. In general, 6 L of E. coli culture were used for each purification of MICU1 or MICU2.Full-length human MICU1 (NCBI: NM_001195518.2) and MICU2 (NCBI: NM_152726.3) without mitochondrial targeting sequence (MTS) were PCR amplified from human cDNA library  and sub-cloned into pET28a vectors with a C-terminal 8 × His tag using the restriction sites Xba I-Not I. E. coli cell pellets from a 6-L culture were re-suspended in 100 mL of lysis buffer  and lysed by sonication followed by centrifugation at 40,000 x g for an hour. The supernatant was incubated with 3 ml Ni-NTA resins (QIAGEN) with gentle agitation at 4°C for an hour. The resin was then collected on a disposable gravity column and washed with 10 column volumes of lysis buffer. The bound protein was eluted with three column volumes of elution buffer . The protein eluent was concentrated to 500 µL using an Amicon centrifugal concentrator  and loaded onto a Superdex 200 10/300 GL column  pre-equilibrated with GF buffer A  for further purification. Peak eluent containing MICU1 or MICU2 was collected for further reconstitution.The same purification protocol was used for both MICU1 and MICU2. Human MCU-EMRE subcomplex was purified and reconstituted into nanodisc as was described previously . Briefly2+-free state) or 2 mM CaCl2 (for Ca2+-bound state)] at 4°C overnight. The sample after dialysis was further purified by Superose 6 Increase 10/300 GL column  pre-equilibrated with GF buffer C. The MCU-EMRE-MICU1-MICU2 holocomplex in nanodisc eluted around 12.5 mL and the protein components of the purified holocomplex were verified by SDS-PAGE analysis. The purified uniplex sample was immediately concentrated to 1.6 mg/mL for cryo-EM grid preparation.The purified MICU1, MICU2 and HsMCU-EMRE in nanodisc were mixed with excess amounts of MICU1 and MICU2 based on SDS-PAGE estimation. The mixture was concentrated to 500 µl using an Amicon centrifugal concentrator  followed by dialysis against 1 L GF buffer C  is extremely low (~ 1.72e-11 M) and we don’t believe the formation of the bridged apo complex is caused by incomplete Ca2+ chelation. As we mentioned in the Discussion, our apo bridged holocomplex is very similar to the structure by Zhuo et al. , which is also obtained in the absence of Ca2+.We have expanded our discussion on the bridging and competing forms of the apo complex (in the presence of EGTA) in the main text as suggested. In the presence of 2 mM EGTA, the free [Ca


Resuscitative thoracotomy is a damage control procedure with an established role in the immediate treatment of patients in extremis or cardiac arrest secondary to cardiac tamponade however Its role in resuscitation of patients with abdominal exsanguination is uncertain.The primary objective of this systematic review was to estimate mortality based on survival to discharge in patients with exsanguinating haemorrhage from abdominal trauma in cardiac arrest or a peri arrest clinical condition following a resuscitative thoracotomy.A systematic literature search was performed to identify original research that reported outcomes in resuscitative thoracotomy either in the emergency department or pre-hospital environment in patients suffering or suspected of suffering from intra-abdominal injuries. The primary outcome was to assess survival to discharge. The secondary outcomes assessed were neurological function post procedure and the role of timing of intervention on survival.Seventeen retrospective case series were reviewed by a single author which described 584 patients with isolated abdominal trauma and an additional 1745 suffering from polytrauma including abdominal injuries. Isolated abdominal trauma survival to discharge ranged from 0 to 18% with polytrauma survival of 0–9.7% with the majority below 1%. Survival following a thoracotomy for abdominal trauma varied between studies and with no comparison non-intervention group no definitive conclusions could be drawn.Timing of thoracotomy was important with improved mortality in patients not in cardiac arrest or having the procedure performed just after a loss of signs of life. Normal neurological function at discharge ranged from 100 to 28.5% with the presence of a head injury having a negative impact on both survival and long-term morbidity.Pre-theatre thoracotomy may have a role in peri-arrest or arrested patient with abdominal trauma. The best outcomes are achieved with patients not in cardiac arrest or who have recently arrested and with no head injury present. The earlier the intervention can be performed, the better the outcome for patients, with survival figures of up to 18% following a resuscitative thoracotomy. More high-quality evidence is required to demonstrate a definitive mortality benefit for patients. The management of the peri-arrest or arrested patient following major trauma is controversial with poor outcomes in both civilian and military practice , 2. CurrA thoracotomy is referred to by a variety of names in peri-arrest or arrested patients depending on the physical location it is performed and the physiological status of the patient  , 5. It iAlthough patient’s with abdominal trauma presenting to UK hospitals have benefited from improved mortality following the introduction of major trauma networks and centralisation of specialist services, survival in this subset of peri arrest patients continue to be poor . It has Timing of resuscitative thoracotomy is important as there is a demonstrated correlation between early intervention and improved mortality . ProtocoAlthough survival from a procedure is the ultimate end point, the quality of life for patients afterwards is also essential to consider. Neurological outcomes following resuscitative thoracotomy are variable. In patient undergoing the procedure in the pre-hospital environment following penetrating trauma after a short period of time in cardiac arrest outcomes were described as good however, in some cases patients never recover normal neurological function –19.Comparing the outcomes of a thoracotomy to no thoracotomy in abdominal trauma is difficult as little data is available. The Eastern Association for the Surgery of Trauma have made estimates of survival based on expert opinion . SurvivaIn blunt trauma the expert group estimated that patient survival was much lower when suffering from an extra thoracic pattern of injury when compared to penetrating trauma but still felt emergency thoracotomy made a difference to survival. In those arriving with a pulse, survival following a thoracotomy was 4.6% compared to 0.5% without. Patient arriving without a pulse following blunt trauma had an estimated survival of 0.7% with a thoracotomy compared to 0.001% without.The aim of this systematic review was to estimate the overall change in mortality for adults with clinically diagnosed exsanguinating abdominal haemorrhage treated with resuscitative thoracotomy before entering theatre, based on available evidence and then compare this with the expert group estimates of survival.Resuscitative thoracotomy does not improve mortality, quantified as survival to discharge, in blunt or penetrating abdominal trauma in contrast to expert opinion estimating outcomes following current conventional management.Identify current use of pre-hospital and emergency department resuscitative thoracotomy in intra-abdominal trauma (either isolated or part of multiple injuries)Evaluate overall outcomes in intervention group and focus specifically on survival to discharge with secondary assessment of timing of intervention and neurological outcome in survivors.Contrast outcomes of patient’s undergoing resuscitative thoracotomy with expert opinion of survival with patient’s suffering similar injuries but not having the intervention.P Patient with suspected haemorrhagic shock secondary to intra-abdominal trauma (either isolated or as a combination of injuries) in a peri-arrest or arrest clinical condition requiring immediate resuscitation and intervention.I Resuscitative thoracotomy (before entering operating theatre).C Survival based on estimated opinion of standard care .Mortality , role of timing of intervention, neurological outcome.S A systematic review of current literature evaluating resuscitative thoracotomy  for patient with intra-abdominal injuries.This systematic review was conducted in line with the Preferred Reporting Items for systematic reviews and Meta-Analysis statement .Pre-hospitalEmergency departmentAmbulanceParamedicEMSOut of hospitalAbdominal traumaBluntPenetratingWoundInjur*ThoracotomyResuscitative thoracotomyEmergency thoracotomyTraumaA comprehensive literature review was conducted by a single author (M.H) which was completed on 24/1/2018. A search of the electronic databases Medline and Embase was conducted using medical subject heading (MESH) terms shown below with the Cochrane database and PROSPERO also being used to identify any completed or ongoing systematic reviews on the subject matter . FinallyPapers were screened and reviewed based on the eligibility criteria outlined below by a single reviewer. Title of paper, abstract and if required full paper review was used to assess for inclusion. All references in papers were followed up and included where relevant and authors were contacted if any information was unclear.All types of studies were included apart from expert opinion, single case reports and review articles . Studies before 1987 were excluded as it was felt that data from more than 30 years ago would not be relevant to current medical practice. Foreign language papers were also excluded as no interpreter was available to ensure any translations were accurate and precise. There was no exclusion on age and therefore any paediatric data was included.Table A single author (MH) evaluated each paper using the CASP checklists and the Cochrane handbook for systematic reviews to assess for validity and reliability , 24.If aThe following data was extracted for each study which is summarised in Table Figure Seventeen retrospective case studies were included in the review. No meta-analysis was attempted due to the heterogenicity of data both methodological and clinical. Clinically some institutions had clear protocols for performing a thoracotomy but not all. Recording of the timing of intervention as well as the patient state at the start of the procedure (e.g. what constituted a loss of signs of life) and clear details of survival to discharge were also variable between studies.There was wide variation in the indications for resuscitative thoracotomy with no pre-hospital thoracotomies being included in the review. The indications, exclusion and inclusion criteria for each study are summarised in Additional file The range of survival to discharge for patients undergoing a resuscitative thoracotomy before entering theatre in all studies was 0–16%. This increased to 18% of 22 patients if only an isolated iliac injury was present . Table 2The widest variation in survival to discharge rates was found in patients with blunt trauma, although It was difficult in several studies to differentiate whether this was isolated abdominal trauma or combined with pelvic trauma and injuries to other locations.None of the studies included looked specifically at timing of intervention from onset of cardiac arrest or injury and the effect on mortality. Several commented on the improved mortality of those who’s arrest was witnessed or occurred within the emergency department, but none were tested and found to be statistically significant. On-going CPR was generally a negative predictor of survival in both penetrating and blunt trauma . SeveralNeurological outcome in patients undergoing a pre-theatre thoracotomy was explored in three studies with a wide variety of outcomes. The first study of 252 patients had 4 survivors none of which had any neurological deficit after undergoing pre-theatre thoracotomy for penetrating abdominal and pelvic traumatic injuries . The mosUnfortunately, there was a small number of studies regarding the use of resuscitative thoracotomy for abdominal trauma and the overall quality of the evidence was poor. The initial PICO question focused on contrasting the current management of the peri-arrest or recently arrest trauma patient with abdominal trauma with that of the information gathered from the systematic review. The only information that could be found on outcomes following best current management of abdominal trauma was expert opinion which it is well documented is open to a bias. A systematic review and meta-analysis on the use of emergency department thoracotomy in blunt trauma concluded 1.5% of patients survived this intervention following a loss of signs of life with the majority having poor neurological outcome at discharge but did not look at specific location of injury . This stAssuming the expert opinion is accurate then the best probability of survival to discharge for patients with abdominal injuries was in the group who were suffering from unrelenting shock but with a pulse present following penetrating trauma. Based on current management, without a resuscitative thoracotomy before entering an operating theatre survival was 1.7%. All other patterns of injury and worsening states of shock including cardiac arrest had even lower survival to discharge figures without a resuscitative thoracotomy. This is in a population predominantly of young, fit individuals with trauma being the commonest cause of death in the UK within the ages of 1–40 . This caThe use of pre-laparotomy thoracotomy in theatre was strongly advocated by several studies as a method of improving overall survival and was proven to be statistically significant , 38. ThiA literature review including 640 patient’s with isolated abdominal injuries and 590 patient’s suffering from polytrauma undergoing a resuscitative thoracotomy before theatre suggested similar findings to those in the systematic review . It repoThe presence and loss of signs of life was explored in most studies suggesting the importance of the patients clinical condition at the time of intervention. Although there was wide variety of practice in the indications and use of resuscitative thoracotomy, the majority were performed once a patient had lost cardiac output. This clearly has a bearing on the interpretation of the results as the majority of patients suffering a traumatic cardiac arrest are highly unlikely to survive making even small improvement in mortality significant. Other large studies looking at resuscitative thoracotomy for blunt trauma have suggested the lack of electrical activity is a negative predictor of mortality and no intervention should be performed if cardiac electrical activity is absent .There is no unifying definition of a resuscitative thoracotomy with varying terms for thoracotomies performed within the emergency department making the initial literature search problematic leaving the potential for some studies to have been omitted, thus affected the overall reliability and validity of the study. Another consideration not assessed was clinicians discretion to perform intervention based on other factors such as a patients co-morbidities. Although the use of protocols may aid decision making the final decision whether to perform an intervention lies with the physician and thus will be open to individual interpretation.The longer patients went without intervention the worse the outcome was, in the studies that analysed this however no statistically significant finding about the exact time was suggested in any of the papers. A large literature review of resuscitative thoracotomy found that outcomes were improved if the patient arrested in front of the physician performing the thoracotomy or in the recent past  . This fiOnly 3 studies investigated or commented on the neurological outcome of survivors to discharge , 37, 46.The main weakness in this study was the lack of a control group for comparison with the intervention group. The outcomes of a patient with abdominal trauma and unrelenting shock either in, or close to, cardiac arrest not undergoing a resuscitative thoracotomy is largely unknown and thus expert opinion was used to provide comparison. The study therefore relies on this information being accurate to try and draw comparisons and thus conclusions, which, given the known bias associated with expert opinion questions the overall validity of the study. All the papers included were retrospective cohort studies with no randomisation and thus the potential for selection bias. This was potentially further compounded as the investigators were not blinded and therefore gives a risk of measurement bias. Although this is unlikely to be a problem when assessing 30 day mortality with the secondary outcomes of the study this may have had some bearing. The majority of the data collection occurred at large level 1 trauma centre’s within the united states. It is not unreasonable to question not only performance bias when compared to studies published outside the large trauma centre’s but also the applicability to the UK patient population and practices. Ideally a more controlled study should be performed with a clear indication for intervention with randomisation to groups with blinding of researchers to increase overall validity of the findings. This however would be a difficult if not impossible task. Practically, in the UK, a pre-alert from the ambulance service may be forth coming with accurate information about the patients en route to hospital however this is often not the case with key details that affect decision making being omitted . In the This study has a number of limitations when looking specifically at the research question. Although the primary aim of the study was to look at outcomes following abdominal trauma, patients suffering with polytrauma were also included. This decreases the overall validity of the findings particularly guiding application within the clinical environment when faced with a patient with isolated abdominal trauma. The exclusion of foreign language papers also has the potential to lead to missing data which when dealing with the small numbers described could have an influence on the conclusions drawn.The use of resuscitative thoracotomy has been documented for many years however new technologies are in development. One of the aims of a thoracotomy in non-thoracic trauma is to cross clamp the descending aorta and achieve haemorrhage control. Resuscitative endovascular balloon occlusion of the aorta (REBOA) aims to achieve control of haemorrhage with a less invasive approach. Although it is in the early stage of development is has already been used in the pre-hospital environment as well as trauma centres within the UK . HoweverResuscitative thoracotomy for abdominal trauma is and will remain a controversial and divisive procedure. Although the evidence is of low quality it is difficult to see how higher levels of evidence, such as randomised control trials, will ever be put into place given the ethical implications and controversy surrounding practice. With experts suggesting a survival rate, with current management strategies, at best 1.7%. In the population in which trauma has the highest prevalence, namely young fit individuals, this is unacceptable when compared to medical cardiac arrest survival of 8.6%. Newer, more targeted interventions are in development however the current evidence is sparse and suggests at best equal outcomes when compared to thoracotomy.Although a highly controversial, invasive and arguably last-ditch effort procedure, pre-theatre thoracotomy should be considered in the peri-arrest or arrested patient with abdominal trauma either in isolation or as part of polytrauma. The best outcomes are achieved with patients not in cardiac arrest or who have recently arrested and who do not have a head injury present. The earlier the intervention can be performed, the better the outcome for patients, with survival figures of up to 18% with intervention compared to 0.001% without, however more high quality evidence is require to demonstrate a definite mortality benefit for patients.Additional file 1: Appendix’s: Outcomes following resuscitative thoracotomy for abdominal exsanguination, a systematic review


However, the specific mechanisms of action are still under debate and intense effort is ongoing to improve our understanding of the crucial steps involved in the mechanisms of AβOs toxicity. We report the development of a mathematical model describing a proposed mechanism by which stimulation of Phospholipase C (PLC) by iAβO, triggers production of IP3 with consequent abnormal release of Ca2+ from the endoplasmic reticulum (ER) through activation of IP3 receptor (IP3R) Ca2+ channels. After validating the model using experimental data, we quantify the effects of intracellular rise in iAβOs on model solutions. Our model validates a dose-dependent influence of iAβOs on IP3-mediated Ca2+ signaling. We investigate Ca2+ signaling patterns for small and large iAβOs doses and study the role of various parameters on Ca2+ signals. Uncertainty quantification and partial rank correlation coefficients are used to better understand how the model behaves under various parameter regimes. Our model predicts that iAβO alter IP3R sensitivity to IP3 for large doses. Our analysis also shows that the upstream production of IP3 can influence Aβ-driven solution patterns in a dose-dependent manner. Model results illustrate and confirm the detrimental impact of iAβOs on IP3 signaling.Alzheimer’s disease (AD) is a devastating illness affecting over 40 million people worldwide. Intraneuronal rise of amyloid beta in its oligomeric forms (iA In AD, iAβOs are now believed to play a major role in the early phase of the disease as their intracellular rise correlates well with the symptoms of AD [βOs have been found to be predictive of cognitive status at death among patients with AD [2+ elevation with the concomitant increase of iAβOs observed in neurons during the progression of the AD [βOs on the normal functioning of the IP3-signaling pathway has been indicated as a potential mechanism responsible for alteration of the Ca2+ homeostasis in AD neurons.Ca neurons . Accordiechanism . Althougms of AD . More ge with AD . Variousf the AD . Among tβ42Os is responsible for the overproduction of IP3 and consequent rise of cytosolic Ca2+ observed in cells exposed to iAβ42Os [β may cause cytosolic Ca2+ rise by a mixed mechanisms of PLC-dependent and independent manner [β42Os on intracellular Ca2+ fluxes have previously been investigated by developing a computational model to study important intracellular Ca2+ pathways in normal and in iAβ42Os affected conditions [3 production processes were incorporated in the model. Here, we have illustrated a possible mechanistic way for how iAβ42Os triggers IP3 overproduction with consequent rise in cytosolic Ca2+ by including some mechanisms of upstream IP3 production in the model. Specifically, we pinpoint two main possible sites of action for iAβ42Os to interact in the cascade of events resulting from stimulation of G-protein in the plasma membrane to the release of Ca2+ from the ER.We and others have suggested that a G-protein mediated activation of PLC by iA iAβ42Os , 18. Mort manner , 16, 55.nditions . Howeverβ42Os act on IP3Rs in the generation of Aβ-induced Ca2+ signaling events. The results of the model are consistent with this for the small doses parameters. However, the model also suggests that iAβOs may in-fact be directly affecting the IP3Rs when large doses are introduced. The analysis illustrated in 2+ signaling in the presence of iAβ42Os as changes to IP3Rs occur. The persistent increase of iAβ42Os may alter the sensitivity of IP3Rs to Ca2+ over time. For large doses of iAβ42Os, IP3Rs may become more sensitive to low- or sub-threshold IP3 levels and in turn trigger local and global Ca2+ signaling events. The fact that the parameter k−4 appears to play a major role in the decay of observed Ca2+ signals singles out the potential that iAβ42Os do act on the IP3R itself, at least for large doses. Our model suggests the need for further investigation on the relationship between iAβ42Os and the sensitivity of IP3Rs to IP3 levels.In our previous study , we arguβ42Os on IP3 signaling mechanisms that does not necessarily depend on the choice of the IP3R model. When a saturating binding rate model for the IP3R model is used  2+ signaling, our model also provides a way to control for and test various therapeutic strategies in a modeling environment. For example, to mimic the intrinsically slow accumulation of Aβ seen in the pathology of AD, Aβ can be introduced very slowly into the model and solutions simulated accordingly. We can then introduce artificial agonists or antagonists that affect G-protein activation and PLC function to see how they affect Ca2+ signals over various timescales. Using the model to better understand what happens to Ca2+ regulation in these simulations can directly influence and suggest how one could control Ca2+ signaling in the presence of Aβ, and more generally, various AD environments.Because of the complex cross-talk nature of Ca2+ signaling in the presence of increasing concentrations of iAβ42Os. In in vivo environments, it may be the case that in the early phase of AD, slowly accumulating levels of iAβOs remain relatively small. Under such conditions, the small doses model may be better suited than the large doses model. Regardless, our model development and analysis suggests that increasing the amount of iAβ42Os present in the cell can have a pervasive impact on numerous cellular mechanisms.The results of this study suggest the need for two different dose-dependent models to incorporate changes in cellular CaBuilding computational models can help provide a better understanding for the complex cross-talk between various signaling mechanisms within neurons, something difficult to establish with current experimental capabilities. Through further analysis and development, researchers can use the model to formulate novel experimental procedures and eventually suggest new therapies for treating AD.2+ concentration are associated with changes in fluorescence through the equationKD is a dissociation constant, fmax is the intensity of the dye at maximum Ca2+ concentration, Rf = fmax/fmin is the indicator’s dynamic range with fmin being the intensity at minimum Ca2+ concentration, δfmax is the saturation of the Ca2+ indicator, fm = fmax/f0, and c0 is the resting Ca2+ concentration. The values of KD and Rf are associated with attributes of the indicator in a particular cellular environment [KD and the dynamic range of Rf may vary batch to batch and should be estimated using a similar protocol and cellular cytoplasmic domain [According to Maravall et al. (2000) , changesironment  and as sironment . Howeverc domain , 59.δfmax is the key parameter needed for establishing the conversion from fluorescence to Ca2+ concentrations. When we fix the initial Ca2+ concentration, c0, we can estimate δfmax usingKD and Rf are known. In practice, δfmax can be used to estimate the unknown resting Ca2+ concentration by inverting the relationship in In attained  and KD a2+ concentrations only requires obtaining values for fm, Rf, and δfmax during experimental procedure. However, these values are often not reported in favor of the traditional δf fluorescence measurements and extracting them from reported changes in fluorescence ratio, or establishing their values a posteriori, can be challenging. As such, in order to complete a conversion for data given in terms of δf we approximate a number of parameters. Since both KD and Rf depend on indicator properties, they can be approximated for a variety of indicators. Based on the experiment in [KD ≈ 0.2−0.5 μM and that Rf has a dynamic range Rf ≈ 85−100 and note that uncertainties in Rf have minimal affect on δfmax is plotted as a function of KD and Rf when c0 = 0.01 μM and c0 = 0.05 μM in A and B, respectively.With Eqs iment in , we assuRf has little effect on the value of δfmax. This is consistent with the idea that for indicators with a large dynamic range, the exact value of Rf is insignificant [KD is often assumed to be between 0.25 and 0.5 μM but some studies suggest that KD may have much greater range [As can be seen from nificant . For inder range , 61.2+ concentration of c0 = 0.05 μM and set KD = 0.3 μM and Rf = 100. Because we have no previous knowledge for the value of fm, we consider a range fm ≈ 1−100 where the exact value depends on the ratio of the maximal intensity and the resting intensity. Using these values, we plot the corresponding Ca2+ concentrations from the fluorescence data in [fm. β at concentrations of a = 3 μg/ml, a = 10 μg/ml, and a = 30 μg/ml, respectively. In fm ranging from fm = 1 to fm = 100 (black) with n = 11 . The maximum value is also highlighted for each conversion plot (circle) and provides the peak time for the three Aβ levels.Without loss of generality, here we consider a basal Ca data in  for vari2+ concentrations, fm and the range of δfmax between 6 and 20. These three dimensional plots allow us to better understand the impact of the conversion parameters on the maximum values of the fluorescence data in [fm or c0, a true conversion from fluorescence to concentration is elusive. However, in all the profiles illustrated, each conversion does capture the changes in amplitude and latency to peak time observed experimentally as levels of Aβ are increased.To study the impact of the conversion to Ca data in . Again,


All-trans retinoic acid (atRA) has a dramatic impact on the survival of patients with acute promyelocytic leukemia, but its therapeutic value in other types of acute myeloid leukemia (AML) has so far remained unclear. Given that AML is a stem cell-driven disease, recent studies have addressed the effects of atRA on leukemic stem cells (LSCs). atRA promoted stemness of MLL-AF9-driven AML in an Evi1-dependent manner but had the opposite effect in Flt3-ITD/Nup98-Hoxd13-driven AML. Overexpression of the stem cell-associated transcription factor EVI1 predicts a poor prognosis in AML, and is observed in different genetic subtypes, including cytogenetically normal AML. Here, we therefore investigated the effects of Evi1 in a mouse model for cytogenetically normal AML, which rests on the combined activity of Flt3-ITD and Npm1c mutations. Experimental expression of Evi1 on this background strongly promoted disease aggressiveness. atRA inhibited leukemia cell viability and stem cell-related properties, and these effects were counteracted by overexpression of Evi1. These data further underscore the complexity of the responsiveness of AML LSCs to atRA and point out the need for additional investigations which may lay a foundation for a precision medicine-based use of retinoids in AML. Even LT3-ITD) ,5,6,9,10LT3-ITD) ,11,12,13LT3-ITD) ,12,13,14 decades ,16,17. D decades ,25,26,27 decades ,29,30,31 decades .Nup98-Hoxd13 fusion gene together with an FLT3-ITD [AML1-ETO-expressing murine BM cells [MLL-AF9-driven mouse model of AML, atRA augmented stem cell abundance, quiescence, and activity in a manner that was dependent on the expression of Evi1 [EVI1 and atRA also collaborated to promote stem cell-related properties in human AML cell lines and primary samples [Both normal and leukemic hematopoiesis are organized in a hierarchical manner, and emanate from mostly quiescent stem cells  that reside in a specialized niche in the bone marrow (BM) ,34,35. TFLT3-ITD . By contBM cells . Similar of Evi1 . EVI1 an samples .EVI1 would also interact with atRA to alter LSC-related properties on the background of different genetic driver lesions. Since 21% of EVI1-overexpressing AMLs are cytogenetically normal [FLT3 and NPM1 genes  [Flt3-ITD and an Npm1c allele develop an aggressive, AML-like disease [Flt3-ITD/Npm1c model was therefore used in the current study. We found that atRA inhibited leukemia cell (LC) viability as well as LSC-related properties in Evi1lowFlt3-ITD/Npm1c-driven AML, but these effects were counteracted by experimental Evi1 expression.In the present study, we asked whether y normal , we soug“NPM1c”) ,5,6, and disease . The FltAnimal experiments were approved by the Animal Ethics Committee of the Medical University of Vienna and the Austrian Federal Ministry of Education, Science, and Research . Federation of European Laboratory Animal Science Associations and Austrian guidelines to minimize animal distress and suffering were followed.Flt3-ITD/Npm1c-transformed hematopoietic cells [high and Evi1low variants of Flt3-ITD/Npm1c-driven AML, these cells were transduced either with a vector encoding an epitope-tagged version of murine Evi1  or with empty vector as a control. In brief, vectors were transfected into Platinum-E cells, along with the ecotropic packaging plasmid psi2  using a standard calcium chloride protocol. Virus-containing supernatants were harvested after 48–96 h, filtered (0.45 µm pore size), and supplemented with polybrene (4 μg/mL). Cells were spinoculated with retroviral supernatant for 60 min at 1300 rpm and 34 °C. The process was repeated with fresh retroviral supernatant after 24 and 48 h. Five days after the last transduction, Flt3-ITD/Npm1c_Evi1 and Flt3-ITD/Npm1c_vec cells were sorted for GFP positivity and expanded in the above-described medium. For transplantation, 6–8-week-old female C57BL/6 recipient mice were sub-lethally irradiated (5 Gy). On the next day, mice were anesthetized by i.p. injection of 100 µL Ketasol/Rompun solution , 1.5 mg/mL Rompun , and 0.9% sodium chloride ) followed by retro-orbital injection of 400,000 Flt3-ITD/Npm1c_Evi1 or Flt3-ITD/Npm1c_vec cells. Mice were monitored for signs of disease , and sacrificed when terminally ill. Their BM and spleen cells were collected and vitally frozen. The GFP-positive fractions of these cells were considered as LCs, and are referred to as LCFlt3-ITD/Npm1c_Evi1 and LCFlt3-ITD/Npm1c_vec, respectively. For ex vivo experiments, cells were thawed and maintained in the medium described at the beginning of this chapter.Spleen cells from C57BL/6 mice that had succumbed to AML following transplantation with ic cells  were culFlt3-ITD/Npm1c_vec and LCFlt3-ITD/Npm1c_Evi1 were seeded at 200 cells/µL and incubated with various concentrations of atRA  or with solvent for 48 h. Metabolic activity as a proxy for cell viability was determined in white-walled 96-well-plates  using the CellTiter-Glo® Luminescent Cell Viability Assay . Luminescence was measured using the Varioskan LUX microplate reader with SkanIt Software for Microplate Readers RE, Version 5.0.0.42. (Thermo Fisher Scientific).For cell viability and apoptosis assays, BM LC2 (Sigma-Aldrich)) for 15 min at room temperature, and analyzed by flow cytometry . Annexin V− cells were classified as viable and Annexin V+ as apoptotic.Annexin V assays were performed to quantify the proportions of apoptotic cells after drug treatment. Thus, cells were stained with 2 µL of Annexin V-APC  in 100 µL Annexin V binding buffer (10 mM HEPES (Sigma-Aldrich), pH 7.4, 140 mM NaCl , and 2.5 mM CaClTo analyze myeloid differentiation, BM cells from leukemic mice were treated with 1 µM atRA (Sigma-Aldrich) or the corresponding amount of solvent for 72 h. Afterwards, 500,000 cells per sample were washed once with PBS and stained with 1 µL of CD11b  and Gr-1  antibodies in 100 µL 2% fetal bovine serum/PBS for 30 min. Cells were washed again and analyzed by flow cytometry . Analyses were restricted to leukemic cells by gating on the GFP-positive population.Flt3-ITD/Npm1c_vec and LCFlt3-ITD/Npm1c_Evi1 were treated with 1 µM atRA or the corresponding amount of solvent for 72 h. Thereafter, 3000 cells were plated per well of a six-well plate in MethoCult GF M3434 . Every seven days, the numbers of colonies were quantified, and 3000 cells were used for replating.For serial replating assays, BM LCEvi1 , ß-2-microglobulin . Assays were performed in triplicate, and Evi1 expression was normalized to ß-2-microglobulin expression using the ΔΔCT method [Total RNA was extracted using Trizol (Thermo Fisher Scientific) and reverse-transcribed using random hexamer primers (Thermo Fisher Scientific) and M-MLV reverse transcriptase (Thermo Fisher Scientific). Quantitative RT-PCR (qRT-PCR) was performed on a Step One Plus Real Time PCR system (Thermo Fisher Scientific) using GoTaq qPCR Master Mix (Promega) and the following primers: T method .Preparation of protein lysates from spleen LCs, SDS-PAGE, transfer to PVDF membranes , and antibody incubations were performed using standard procedures. The following antibodies were used: anti-FLAG  and anti-GAPDH . Blots were developed using SuperSignal West Femto or Pico Chemiluminescent Substrate (both from Thermo Fisher Scientific) and scanned using a ChemiDoc Touch Imaging System .Flt3-ITD/Npm1c_vec -atRA in each round of plating; significance was assessed using the one-sample t-test for comparisons with the control and Student’s t-test for other comparisons. The log-rank test was used to evaluate survival differences between groups of mice. Two-sided p-values < 0.05 were considered statistically significant. Analyses were performed using GraphPad Prism 6  software.Significance of differences between multiple groups was determined by 2-way ANOVA followed by Bonferroni’s post hoc test. In serial replating assays, numbers of colonies were expressed relative to LCEVI1 or not [Evi1 in Flt3-ITD/Npm1c-driven murine AML [Evi1 mRNA levels in BM from Flt3-ITD/Npm1c mice were comparable to those in normal mouse BM, and substantially lower than in BM from mice with MLL-AF9-driven AML (Flt3-ITD/Npm1c mice were transduced with a vector encoding an epitope-tagged version of murine Evi1 (pMYs_FLAG-Evi1_IRES_GFP) or with empty vector as a control, and sorted for GFP positivity to yield Flt3-ITD/Npm1c_Evi1 and Flt3-ITD/Npm1c_vec cells, respectively. These were transplanted into sub-lethally irradiated C57BL/6 recipient mice . The presence of the EVI1 protein in LCFlt3-ITD/Npm1c_Evi1, but not LCFlt3-ITD/Npm1c_vec, obtained from terminally ill recipient mice was confirmed by immunoblot analysis using a FLAG antibody  cells within the myeloid (CD11b+) compartment than LCFlt3-ITD/Npm1c_vec  was not confirmed in subsequent studies [www.clinicaltrials.gov).atRA in combination with chemotherapy and, more recently, arsenic trioxide has greatly improved the outcome of APL ,16,17. I studies ,30,31,32 studies ,26,27,36 studies ,44 and tAML1-ETO-expressing murine BM cells, atRA increased serial replating ability and led to the formation of larger and more immature colonies [MLL-AF9-driven mouse model of AML in a manner dependent on the expression of Evi1 [MLL rearrangements are frequently associated with EVI1 overexpression in human AML [MLL-AF9 leukemic mice, atRA promoted the abundance and quiescence of an immunophenotypically defined LSC-enriched cell population and enhanced the activity of LSCs as determined by serial replating and in vivo limiting dilution assays. These effects of atRA were abolished by knock-down of Evi1 [Evi1-dependent manner in vitro and in vivo, and prolonged survival of both of primary treated and secondary recipient mice with Evi1high, MLL-AF9-driven AML. Confirmatory results were obtained with both human AML cell lines and with primary AML samples [Surprisingly, even though AML is well established as a stem cell-driven disease , to datecolonies , suggest of Evi1 . MLL reauman AML ,46, and uman AML ,47. In B of Evi1 . Corresp samples .AML1-ETO and MLL-AF9 models, atRA inhibited LSC activity in an AML mouse model driven by an Flt3-ITD in combination with a Nup98-Hoxd13 fusion gene [Flt3-ITD—which in this case was combined with an Npm1 mutation in order to reveal its leukemogenic potential—renders AML LSCs sensitive to the inhibitory effects of atRA. Furthermore, we show that overexpression of Evi1 abolishes the inhibitory effect of atRA on Flt3-ITD/Npm1c-bearing LSCs. Together, these data suggest that complex genetic and molecular interactions determine the response of AML LSCs to atRA. Further underscoring this, and in sharp contrast to its role in AML, on the background of the APL-typical PML-RARA fusion, the Flt3-ITD mutation reduced the inhibitory effect of atRA on the ability to initiate leukemia in secondary recipients [In sharp contrast to the observations with the ion gene . Treatmeion gene . The precipients .AML1-ETO, with complex results: only the combination of a RARA and a RARG agonist reproduced the LSC-promoting effects of atRA [The molecular and genetic complexity determining the atRA responsiveness of AML LSCs may explain, to some extent, why it has been difficult to identify patient groups benefitting from atRA in non-APL AML. Other aspects that warrant consideration are the identity of the drugs used in conjunction with atRA, as well as the timing and duration of treatment. Different trials assessing the efficacy of atRA in AML were based on different chemotherapeutics. atRA was initiated at different time points relative to the start of chemotherapy, and was included in the maintenance therapy in some studies but not others . Prolong of atRA .Further research is needed to carefully dissect which patient subgroups may benefit from which type of retinoid, and how retinoids can be combined with other drugs to maximize anti-leukemic effects. An enhanced understanding of the effects of retinoids on LSCs may ultimately facilitate the development of precision medicine-based retinoid therapy for certain subgroups of AML.