The minimum acoustic energy required for sufficient evocation was 1.7 μJ/mm2. With the same acoustic energy, sonoporation, by which calcein and propidium iodide would become permeated, was not observed. It was found that intracellular Ca2+ increases evoked by LESW irradiations do not require sonoporation. In the intracellular Ca2+ increase, actin cytoskeletons and stretch-activated Ca2+ channels were involved; however, microtubules were not. In addition, with Ca2+ influx through the Ca2+ channels, the Ca2+ release through the PLC-IP3-IP3R cascade contributed to the intracellular Ca2+ increase. These results demonstrate that LESW irradiations can evoke cellular responses independently of sonoporation. Rather, LESW irradiations evoke cellular responses through mechanosensing.Low-energy shock waves (LESWs) accelerate the healing of a broad range of tissue injuries, including angiogenesis and bone fractures. In cells, LESW irradiations enhance gene expression and protein synthesis. One probable mechanism underlying the enhancements is mechanosensing. Shock waves also can induce sonoporation. Thus, sonoporation is another probable mechanism underlying the enhancements. It remains elusive whether LESWs require sonoporation to evoke cellular responses. An intracellular Ca Recently, shock waves have been further applied to heal wounds, burns, and ischemia8. The acoustic energies of shock waves that are irradiated for tissue healing are limited to a tenth of the energies of shock waves used for ESWT7. Therefore, shock waves for tissue healing are termed low-energy shock waves (LESWs). LESW irradiations enhance gene expression and protein synthesis in cells12. Mechanosensors such as VE-cadherin, caveolin, and integrin are involved in the enhancements. Thus, mechanosensing is supposed to be involved in cellular signals evoked by LESW irradiations.Shock waves were first applied in medicine as extracorporeal shock wave therapies (ESWT) in the early 1980 s. A decade later, shock waves were found to affect treatments for musculoskeletal disorders13. Foreign genes and drugs are transmitted into cytosol through transient pores on plasma membrane. Irradiating shock waves cause sonoporation in cells15. Through the transient pores formed by shock waves, intracellular biomolecules could be released into extracellular space. Those released biomolecules can evoke cellular signals in pore-formed cells themselves or in neighboring cells. From this point of view, sonoporation is expected to be another possible mechanism underlying cellular signals induced by LESW irradiations10. However, whether sonoporation is involved in evoking cellular signals remains elusive.Sonoporation is a formation of transient pores on plasma membrane caused by the application of acoustic energies on cells2+ increase is a second messenger that regulates broad cellular fates17. This second messenger is often evoked by various mechanical stimulations including mechanical stretch and shear stress22. Fluorescence imaging reveals the intracellular Ca2+ increase in single cells. This single-cell detection enables the detection of subtle cellular responses in a limited number of cells. The mechanical stimulation required to evoke such a subtle cellular response should be weaker than that required to evoke a large number of cells. In previous studies, cellular responses those were evoked by LESW irradiations were detected as a result of enhancements of gene expression and protein syntheses. Those detections require large numbers of cells. By detecting an intracellular Ca2+ increase, it is expected that the acoustic energy of shock waves required to detect a cellular response could be suppressed. When acoustic energy is suppressed, sonoporation is also suppressed15. With suppressed acoustic energy, an intracellular Ca2+ increase is expected to be evoked under conditions in which sonoporation is suppressed or excluded.Intracellular Ca2+ increases were observed in endothelial cells on which LESWs were irradiated with spatially uniform acoustic energies. The acoustic energy necessary to evoke intracellular Ca2+ increases was quantified. Sonoporation was detected in cells on which LESWs were irradiated with the minimum acoustic energy in order to understand whether the minimum acoustic energy is also sufficient for sonoporation. In cells in which an intracellular Ca2+ increase was evoked with the minimum acoustic energy, cascades underlying the evocation were further investigated.In this study, intracellular CaShock waves generated by high-voltage discharge in water spread spherically. The spread shock waves were refocused with a stainless reflector to irradiate the shock waves with sufficient acoustic energies Fig. . Around 2+ increase was observed in bovine aortic endothelial cells (BAECs) positioned at the refocus point. Endothelial cells are one of mechanosensitive cell types22. The imaging region for fluorescence imaging was 0.46 × 0.35 mm . Irradiation of single-shot shock waves on BAECs with the uniform acoustic energy evoked intracellular Ca2+ increases .Intracellular Ca mm Fig. . This wases Fig. . Among t2+ increase without inducing sonoporation. The acoustic energy of shock waves was suppressed by inserting an acrylic plate between the stainless reflector and the refocus point . When the shot number was increased to 3 and 5, the fraction of evoked cells increased to 63 ± 5 and 76 ± 5%, respectively  influx. The Stokes radii of calcein and PI are 0.6–0.7 nm24. When cells were irradiated with the minimal acoustic energy, neither calcein leakage nor propidium iodide influx was observed. When the acoustic energy was increased to 4.4 or 15.8 μJ/mm2, both calcein leakage and PI influx were observed. However, the fraction was limited to less than 2% of total cells  and blebbistatin, respectively. With the inhibitions, the intracellular Ca2+ increase was inhibited . Thus, LESWs are supposed to evoke an intracellular Ca2+ increase under the threshold for sonoporation.The threshold for sonoporation by shock waves has been discussedmm2 Fig. . The imp2+ increase remain elusive. Shock waves largely deform cells during their tensile phase, not during the compression phase37. In the present study, although the compression phase was dominant, the tensile phase was also slightly observed . For each BAEC, 0.66 nJ of energy flux transmitted. Thus, the attenuated acoustic energy per single-shot LESW is estimated to be 0.035 pJ. This level of energy is analogous to that required to dissociate 2% of crosslinkers40.Another possible mechanism is the attenuation of acoustic energies by actin cytoskeleton2+ increase evoked in astrocytes by blast shock waves was investigated46. The peak pressure of the blast shock waves was relatively comparable: around 1 MPa for blast shock waves and typically 3.8 MPa for shock waves in this study  was inserted between the foci.Between two electrodes , high voltages of 3–5 kV were applied by a voltage power supply (Sparkling Photon)Pressure profiles were measured with a polyvinylidene difluoride (PVDF) needle hydrophone with a sensitive diameter of 0.5 mm . The point of origin was set at the second focus of the spheroid reflector. The Z-axis was set along the direction of shock wave propagation. Parallel to the electrodes, the Y-axis was set. The X-axis was set so that it crossed the Z- and Y- axes orthogonally Fig. , S1A. To6 kg/m2s and comparable to that of water, 1.5 × 106 kg/m2s. This similarity was expected to transmit LESWs into cell chambers with high efficiency. At the second focus, a cell chamber was set for shock wave irradiations.Polystyrene sheets, 200 µm thick and 10 × 10 mm in area, were sonicated with 99.5% ethanol for 5 min and dried under a nitrogen flow. A silicone rubber wall, 6 mm in inner diameter and 1 mm thick, was attached to the polystyrene plate and treated with oxygen plasma  for 30 sec. After sterilization with 70% ethanol under UV irradiation for 20 min, the cell chambers were incubated with collagen solution  for 1 h and washed with PBS three times. The acoustic impedance of polystyrene is 2.4 × 102. Cells were washed with HEPES buffered saline solution (HBSS (in mM): NaCl 130, KCl 5.4, CaCl2 1.8, MgCl2 0.8, glucose 5.5, HEPES 20; pH adjusted to 7.4 with NaOH) and loaded with Fluo-4AM  for 30 min at room temperature. The cells were washed three times and stabilized at room temperature for 30 min. After the cell chamber was covered with a polyvinylidene chloride film 11 μm thick, the chamber was set on a plane at Z = −0.5 mm close to the second focus of the stainless reflector  were cultured in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin (Gibco), and 2 mM L-glutamine (Sigma). After the cells reached confluence, they were detached and seeded in the cell chambers. For 1–2 days before the experiments, the cells were cultured in an incubator at 37 °C with 100% humidity of 5% COWith an upright microscope , excitation light having a wavelength of 490 nm was irradiated, and emission light having a wavelength of 520 nm was corrected through an objective lens . Fluorescence images were obtained with a digital 3CCD camera  with a sampling rate of 0.3–0.5 seconds. When shock waves were irradiated on cells, the objective lens was displaced out of the water for 3–5 seconds to prevent shock wave reflection at the lens surface. During shock wave irradiations, cell chambers were maintained at 37 °C.The fluorescence intensity in each cell was quantified with image analysis software . The time courses of fluorescence intensities were divided by the fluorescence intensities before the shock wave irradiations. When the ratio exceeded 1.1, it was considered to have reacted. Cellular regions were calculated by using the particle analyze command in ImageJ (NIH).2+ release, either thapsigargin , U73122 , or 2-APB  was loaded for 30 min. To inhibit Ca2+ influx, extracellular Ca2+ was depleted with Ca2+-free HBSS 21 for 30 min. To inhibit SAChs, either Gd3+  or GsMTx-4  was loaded. To inhibit cytoskeletons, either cytochalasin D , blebbistatin , or nocodazole  was loaded for 30 min. Without washing away the inhibitors, the cells were irradiated with shock waves.After loading Fluo-4AM onto cells, the cells were loaded with inhibitors. To inhibit Caσ were calculated in each image. Plasma membranes were determined to be permeabilized if the normalized fluorescents were less than Cell membrane permeabilization was analyzed by measuring calcein leakage from cytosol. Cells were loaded with calcein AM  for 30 min and stabilized at room temperature for 30 min. Fluorescence images were obtained with a 3CCD camera 0.3–0.5 sec intervals. Plasma membrane permeabilization was detected by fluorescent decay. Fluorescence intensities in single cells at 6 min after shock wave irradiation were normalized with those from before shock wave irradiation. From the normalized fluorescence intensities, average intensity Data are expressed as the means ± SEMs (standard error of the mean). Differences between groups were calculated by paired Student’s t-test. Statistical significance is indicated by the symbol “*” when p is below 0.05 and “**” when p is below 0.01.Supplemental Information


Hepatic angiomyolipoma (AML) is a rare mesenchymal tumour with an undetermined malignant potential. Clinical symptoms are non-specific. The radiological hallmarks are high vascularization of lesion and presence of macroscopic fat. The proportion of fatty tissue varies significantly and discrepancies between pre-operative imaging and histological findings are observed in more than 50% of cases. Visualization of the draining vein may aid in differentiation between AML and hepatocellular carcinoma with abundant fatty component. Biopsy is indicated in ambiguous cases. Presence of clinical symptoms warrants surgical treatment. We present a clinical case of giant hepatic AML, discuss its typical features and treatment options. Vishnevsky National Medical Research Center of Surgery for diagnostic evaluation and treatment. Ultrasound examination at her local institution had visualized a right liver lobe mass. Biochemical blood assay revealed increased levels of hepatic transaminases.Sonography showed a large heterogeneous hyperechoic lesion with irregular contours and moderate degree of vascularization in the right liver lobe (segments V-VIII) .CT and MRI  confirmeNo pseudocapsule was detected.Due to tumour presentation, high rupture risk and possible malignant nature with differential diagnosis of hepatocellular adenoma and fat-abundant hepatocellular carcinoma, the patient underwent curative surgery. Intraoperatively soft yellow-coloured mass up to 9 cm in diameter was observed. Right liver lobe resection was performed . The posMicroscopically, the tumour was comprised of mature fatty tissue and large polygonal cells with abundant eosinophilic granular cytoplasm. . Large mHMB-45 —cytoplasmic (+++);Melan A —cytoplasmic (+/++);aSMA —cytoplasmic (+/++);S100 —cytoplasmic (+/++);Ki-67 <1%.6 5 months after resection, tumour recurred presenting with multiple liver metastases and portal vein thrombosis. Flemming et al described recurrent hepatic tumours 3 years after operation. The authors also suggested that a proliferation index exceeding 3% and multicentric growth indicate a propensity for recurrence. Deng et al presented a case report of malignant hepatic AML with marked cell atypia, vascular invasion and proliferation index >30%.9In the past, hepatic AML was considered a benign tumour to be managed conservatively. In 2000, Damme et al published first case report of malignant liver AML.1In most cases, hepatic AML is asymptomatic. In a review by Nonomura et al, clinical symptoms included upper abdominal pain, weight loss, malaise, and periodic increase in body temperature.10 The percentage of adipose tissue in AML varies from 5 to 90%.11 Therefore, differential diagnosis of AML and other hypervascular liver lesions, in particular HCC with abundant fatty components, is difficult. In a recent retrospective study, all hypervascular AML were misdiagnosed as fat-containing HCC by CT or MRI.12 According to the literature, discrepancies between pre-operative and histological findings are noted in more than half of cases.13,14 In a study by Jeon et al, venous drainage to hepatic vein was noted in 80% of cases with AML and only in 7% of cases with HCC.15 Thus, visualization of draining vein has a high negative predictive value for HCC with fatty components. In our case, the draining vein was not well visualized, likely due to thick 5 mm slices in the available outpatient CT examination. It was also noted that the majority of tumour vessels in hepatic AML were venous structures connecting with an early draining vein, while most of the pathologic vessels inside HCC were abnormal arteries.15 Wang et al have also noted that it is difficult to differentiate hepatic AML from fat-containing HCC on MRI as (1) the lesion-to-liver intensity of the two tumour types at the T1- and T2-weighted images was various, and (2) most of the tumours were well-defined, heterogeneous hypervascular enhancing during the arterial phase.16The classic radiological criteria of AML are as follows: (1) high vascularization of solid tumour, (2) presence of macroscopic fatty components.16 The enhancement pattern may be divided into two subtypes: (1) “washout” in lesions with abundant central vessels and “retention” in lesions with small or no vessels. The latter subtype reflects mesenchymal nature of AML.13On the other hand, the presence of a pseudocapsule is useful for diagnosing HCC, as this feature is absent in 95– 96% cases of AML.17 The frequency of rupture is still unclear, with only handful of cases reported in the world literature.17–19The treatment options of hepatic AML remain controversial. Some authors, emphasizing the benign nature of the tumour, suggest a wait-and-see approach. Surgical intervention may be entertained if imaging and laboratory studies are equivocal. Ding et al proposed indications for resection as follows: symptomatic patients, tumours greater than 6 cm in size, tumours showing extrahepatic growth and risk of rupture, tumours showing a tendency to grow and equivocal findings at diagnostic imaging and/or biopsy, when a definitive diagnosis cannot be established.2. If clinical symptoms are present or tumour growth is observed resection is indicated. Radiofrequency ablation may be used if the tumour measures less than 5 cm.20 Tumour regression is extremely rare.21Annual follow-up is advised in hepatitis-free patients with high compliance after histological verification if the tumour size is less than 5 cmHepatic angiomyolipoma (AML) is a rare mesenchymal tumour with an undetermined malignant potential. Clinical symptoms are nonspecific.The radiological hallmarks are high vascularization of lesion and presence of macroscopic fat. The proportion of fatty tissue varies significantly and discrepancy between preoperative and histological findings is observed in more than 50% of cases.Visualization of the draining and pseudocapsule vein may aid in differentiation between AML and HCC with abundant fatty component. Biopsy is indicated in ambiguous cases.Presence of clinical symptoms warrants surgical treatment.


Because electron microscopy cannot be utilized for an assessment of dynamics of mitochondrial morphology in intact cells, most studies were performed using wide-field or laser confocal fluorescence microscopies that, due to limitations of their spatial resolution, do not allow investigating sub-mitochondrial structures. Accordingly, our understanding of the dynamics of substructures of mitochondria is quite limited. Here, we present a robust super-resolution method to quantify the dynamics of mitochondrial cristae, the main substructures of the inner mitochondrial membrane, exploiting structured illumination microscopy (SIM). We observed that knockdown of the dynamin-like 120-kDa protein, which is encoded by the OPA1 gene, specifically reduces the dynamics of the mitochondrial cristae membranes (CM), while the inner boundary membrane (IBM) remained flexible. We further used dual color SIM to quantify the dynamics of CM in the junction between mitochondria and the endoplasmic reticulum . Intracellular Ca2+ release spatially reduced CM-dynamics in MAMs. Moreover, CM-dynamics was independent from matrix Ca2+ signal. Our data suggest that local Ca2+ signals specifically control CM-dynamics and structure to facilitate a well-balanced functional (Ca2+) interplay between mitochondria and the ER.Mitochondria are multifunctional organelles that essentially contribute to cell signaling by sophisticated mechanisms of communications. Live cell imaging studies showed that mitochondria are dynamic and complex structures that form ramified networks by directed movements, fission, and fusion events. There is emerging evidence that the morphology of mitochondria determines cellular functions and vice versa. Several intracellular signaling pathways and messengers including CaThe online version of this article (10.1007/s00424-018-2133-0) contains supplementary material, which is available to authorized users. In addition, most mitochondria move within cells mainly via motor proteins attached to the cytoskeleton , 23, 36.membrane .3R-Grp75-VDAC complex [2+ transfer upon IP3-mediated ER Ca2+ release to mitochondria [2+ signaling [Mitochondria are often in close proximity to the endoplasmic reticulum (ER) and interorganellar tethers are established by various proteins like mitofusin 2, the inositol 1,4,5-trisphosphate receptors (IP complex ), or VDA complex , 30. Thichondria . Contactignaling , 16.Until recently, an accurate visualization and quantification of these important organelle contact sides and the ultrastructure of mitochondria was only feasible in fixed cells using electron microscopy approaches. However, recently, various super-resolution fluorescence imaging approaches substantially surpass the diffraction limit and allow a far better visualization of organelles in living cells. In this study, we utilized structured illumination microscopy (SIM) that uses a shifting and rotating grid pattern to illuminate the sample plane at various optical angles. This allows the analysis and subtraction of the moiré pattern that surpasses the Abbe diffraction limit by about twofold , 29. RecThe N-SIM-setup  used is equipped with 405-, 488-, 515-, 532-, and 561-nm excitation lasers introduced at the back focal plane inside the SIM box with a multimodal optical fiber. For super-resolution, a CFI SR Apochromat TIRF 100×-oil (NA 1.49) objective was mounted on a Nikon-Structured Illumination Microscopy (N-SIM®) System with standard wide field and SIM filter sets and equipped with an Andor iXon3® EMCCD camera. For calibration and reconstruction of SIM images, the Nikon software Nis-Elements was used. Reconstruction was always done with the same robust setting to avoid artifact generation and ensures reproducibility with a small loss of resolution of 10% compared to most sensitive and rigorous reconstruction settings. The general obtained resolution  was approximately 120 to 130 nm measured with fluorescent 100-nm Tetraspec beads .l-lysine and incubated on the 1.5H high-precision glass coverslips  for 20 min. Afterwards, the plate was washed once and transferred to the live cell camber containing 1 ml of buffer (2CaB) containing the following (in mM): 2 CaCl2, 140 NaCl, 5 KCl, 1 MgCl2, 1 HEPES, and 10 D-Glucose; pH was adjusted to 7.4 . Ring correction was done as follows: 3D-stacks of beads were acquired to verify the point spread function of the system. Potential asymmetric point spread function was corrected using the objective correction ring. Subsequently, grating block adjustment was performed to find the perfect focus for the laser beam interference at the focal plane. This process was automatically carried out by the Nikon software.Prior to each measurement, laser adjustment was checked by projecting the laser beam through the objective at the top cover of the bright field arm of the microscope. Lasers were aligned and focused using the interlock system screws at the N-SIM box to ensure appropriate illumination of the sample. The 100-nm Tetraspec beads  were diluted 1:200 in 1 ml 0.01% poly-2/95% air).HeLa cells (ATCC® CCL-2.2™) were seeded on 1.5H high-precision glass coverslips (Marienfeld-Superior) and cultured in DMEM  containing 10% FCS , penicillin (100 U/ml), streptomycin (100 μg/ml), and amphotericin B (1.25 μg/ml)  in a humidified incubator , 0.1% vitamins, 0.2% essential amino acids, and 1% penicillin/streptomycin (Gibco™) at pH 7.4). Cells were incubated in loading-buffer containing 0.5 μM MTG for 40 min. Thereafter, cells were washed once with loading buffer and imaged in 2CaB.Cells were washed once with loading-buffer containing the following (in mM): 2 CaCl2, 135 NaCl, 5 KCl, 1 MgClHeLa cells were grown under standard culture conditions until 50% confluence was reached and transfected in DMEM (without FCS and antibiotics) with 1.5 μg/ml plasmids and/or 100 nM of the respective siRNA with 2.5 μg/ml TransFast™ transfection reagent . After 24 h, the medium was replaced with DMEM containing 10% FCS and penicillin and streptomycin and kept for further 24 h prior the experiments. The specific siRNAs (Microsynth) used in this study are listed in Supplementary Table Live SIM imaging sequences of 30 frames with a frame rate of 1 Hz of MTG-stained HeLa cells were recorded. The open-source program ImageJ (Fiji) was used to analyze the CM-kinetics. First, time-lapse acquisitions were background corrected using a histogram-based background subtractor (Mosaic group). Further, a time-dependent bleach correction using an exponential fitting method was applied to correct for intensity decrease over time caused by photo bleaching. The corrected time lapse was auto thresholded twice. A global Otsu auto threshold  was usedHeLa cells transfected with ER-RFP and stained with MTG were recorded with live dual color SIM imaging. Three repeats of one frame ER-targeted red fluorescence protein (ER-RFP) and 10 frames MTG at a frequency of 1 Hz were recorded. To quantify the inner mitochondrial membrane kinetic in correlation to the sub mitochondrial localization, the IMM-kinetics were measured like described above. Additionally, ER-RFP was globally Otzu thresholded. The overlap of both labels was generated by framewise multiplication and defined as mitochondrial-associated membrane (MAM). These regions were enlarged by stepwise dilatation  to increase the area. By framewise multiplication of these differently enlarged MAM regions with the CM-inter-frame changes, the IMM-kinetic in close proximity to the ER could be defined. Further, the auto Otzu thresholded MTG area was reduced by multiplication with the differently enlarged MAM regions to get the proper size for normalization.Total cellular RNA was isolated from HeLa cells using the PEQLAB total RNA isolation kit , followed by reverse transcription to cDNA, performed in a thermal cycler (Peqlab) using the high-capacity cDNA reverse transcription kit . The qPCR reaction was set up with the GoTag® qPCR Master Mix  together with gene-specific primers . Experiments were performed on a LightCycler 480 . Relative expression of specific genes was normalized to human GAPDH, as a reference gene. Primer sequences are as follows: Opa1 for: 5’-GGTTGGAGATCAGAGTGCTGG-3′, Opa1 rev: 5’-GGACCTTCACTCAGAGTCACC-3′, MCU for: 5’-TTCCTGGCAGAATTTGGGAG-3′, MCU rev: 5’-AGAGATAGGCTTGAGTGTGAAC-3′, GAPDH ., Cell Signaling Technology) and β-actin . HRP labeled anti-mouse  and anti-rabbit  were used as secondary antibodies. For visualization, the SuperSignal West Pico PLUS kit (Fisher Scientific) was used and detection was conducted on the ChemiDoc System .Western blots were performed according to standard protocols. Briefly, cell lysis was conducted with RIPA buffer (Bio-Rad formulation) supplemented with protease inhibitor cocktail , followed by sonication . Samples were denatured in 1 × Laemmli sample buffer and resolved on a 7.5% or 12.5% SDS-PAGE gel together with PageRuler™ Plus Prestained Protein Ladder . Blots were blocked and antibodies diluted in 5% BSA (Sigma Aldrich) in TBS-T. The following antibodies were used: MCU , Opa1  or Microsoft Excel. Analysis of variance and t test were used for evaluation of the statistical significance. P values are indicated within the figures, and P < 0.05 was considered to be significant.Each exact All source data for the preparation of this manuscript are available from the authors on request.Initially, we verified the feasibility of our attempt to determine and quantify the dynamics of the IMM by utilizing SIM. Therefore, HeLa cells were loaded with MitoTracker Green® (MTG) and directly imaged at a frequency of 1 Hz with SIM. Live SIM time-lapse imaging allowed a detailed analysis of the dynamic changes of mitochondrial membranes First, images out of a time-lapse acquisition were binarized applying locally auto thresholding. Second, to extract the individual kinetics of the IMM and CM, a closing and fill holes algorithm was applied and the delta intensity of subsequent frames were detected and determined as mitochondrial IBM-changes. Third, the delta intensities of subsequent frames without applying the closing and fill holes algorithm were detected and determined as mitochondrial IMM-changes. Fourth, IBM-changes were subtracted for IMM-changes revealing the changes within the CM . Fifth, the overall pixel intensities of CM-changes were framewise normalized to the global threshold area to get the percentage of moving mitochondrial area inside the mitochondria.To determine the cristae dynamics under various conditions, we developed an analysis strategy to filter and quantify the dynamic movement of the CM separately to that of the IBM. This strategy consists of the following five steps Fig. b: First, influence the spatial IMM kinetics in their neighborhood, HeLa cells were transfected with ER-targeted red fluorescence protein (ER-RFP) and stained with MTG. The co-localization of both auto thresholded labels obtained in the SIM was defined as MAMs. Spatial IMM-kinetics were measured within distinct areas with a given proximity to MAM structures experimentally defined as incremental dilatations of the defined MAM area Fig. a. Expres2+ release on CM-dynamics, cells were transfected with ER-RFP and stained with MTG. Ionomycin (200 nM) was used to evoke global Ca2+ release from the ER [2+ release, the kinetics of CM decreased with the proximity  to MAMs  and histamine influenced mitochondrial branching  ion channels and transporters, like UCP4, were found across the CJ in the IBM . As the 2+ influx from the intermembrane space into the inner cristae. Such Ca2+ accumulation inside the cristae is known to accompany altered cristae shape [2+-independent manner by stabilizing CJ [The critical importance of an optimized mitochondrial ultrastructure for the organelle’s fate is exemplarily illustrated by OPA1, which determines the width of the CM due to its oligomerization of membrane-bound and soluble forms , 14, 35.ae shape , 14, impae shape , and incae shape . Our pre2+ hotspots by BAPTA normalized CM-dynamics in proximity of MAMs to that of the global mitochondria indicates an involvement of spatially localized Ca2+ in the regulation of CM-dynamics within the MAMs even under resting conditions. Hence, we investigated the impact of global and local ER Ca2+ release as potential regulator of CM-dynamics. Our findings unveil that ER membrane permeabilization for Ca2+ with ionomycin [2+ mobilization with either histamine or ATP, two physiological IP3-generating agonists, yielded a deceleration of CM. This effect strictly correlated with the proximity to MAM structures. Accordingly, we speculate that Ca2+ hotspots in MAMs [2+ elevations do not affect CM-kinetics. At this stage, we do not know whether the Ca2+ hotspots, similar to OPA1 knock-down, widen the cristae structure for more efficient mitochondrial Ca2+ uptake and thereby reducing the local IMM-dynamics. However, it is also possible that the phenomenon of spatial CM deceleration is mediated by the physical coupling between the ER and the OMM. This junction might be further strengthened by a Ca2+-dependent mechanism [2+-dependent mechanism opening the cristae structure within the MAMs and thereby reducing the local IMM-dynamics might be important for establishing efficient mitochondrial Ca2+ uptake. Hence, Ca2+-triggered widening of the CJ within the MAMs might also contribute to the increased fragmentation of mitochondria by facilitating fission events in addition to other already described mechanisms [Furthermore, we were able to measure the CM-dynamics in relation to the proximity to MAMs. Our findings that CM-dynamics was reduced in the proximity of MAMs and chelation of Caonomycin  reduce C in MAMs  spatiallechanism  and, subchanisms , 34.2+ hotspots within the MAMs decelerate CM-dynamics, at least in part, by widening the CJ is further supported by our findings that in cells depleted from OPA1, which per se yields CJ opening [2+-induced deceleration of the dynamics of MAM-close CMs were found. These findings also point to a possible, so far underestimated, importance of a dynamic regulation of the width of CJ that facilitates (ion) signaling within the MAMs upon ER Ca2+ release. The importance of intra-junctional Ca2+ as spatial regulator for CM-dynamics, especially in the MAMs, is further supported by our findings that the deceleration of the dynamics of MAM-associated CMs remains unaffected in cells that were depleted from MCU. These data suggest that elevations in mitochondrial matrix Ca2+ is not involved in the vibrant regulation of CM-dynamics and further supports the role of MAM Ca2+ elevation in facilitating inter-organelle (Ca2+) communications.Our hypothesis that Ca opening , no furt3-mediated intracellular Ca2+ release. Furthermore, a spatial regulation of the CJ by a Ca2+-sensitive and OPA1-independent mechanisms as a so far unknown part of the mitochondrial cristae organization system (MICOS) [Overall, our present data point to a spatial deceleration of the dynamics of CM specifically within the MAMs upon IP (MICOS)  can be pESM 1(PDF 456 kb)


By measuring the changes of [Ca2+]i levels, critical physiologic functions can be characterized and aberrant pathologic conditions or drug responses can be efficiently monitored. We developed a protocol for assessment of Ca2+ signaling in the acinar units of submandibular glands isolated from C57BL/6 mice, using benchtop, multi-mode, high throughput plate reader (FlexStation 3). This method represents a powerful tool for unlimited in vitro studies to monitor changes in receptor-mediated Ca2+ responses while retaining functional and morphological features of a native setting.Calcium ions (Ca Acetylcholine is the parasympathetic postganglionic transmitter that mainly acts on the acinar M3 muscarinic receptors to increase intracellular calcium [Ca2+]i and stimulate ﬂuid secretion i is proportional to the ratio of fluorescence at 340/380.Intracellular calcium concentration variations at the nanomolar scale can be detected in live cells with either dyes or biosensors. Fura-2 is by far the most commonly used ratiometric fluorescent dye for quantitative [Caurements . Fura-2-ind Ca2+  t 510 nm . Therefo2+ mobilization assays in a 96-well format. The FlexStation 3 has been used for studying the function of Ca2+-permeable ion channels and G-protein coupled receptors in the HTC4 rat hepatoma cell line [2+]i using single cell fluorescent microscopy provides sensitivity and temporal resolution, and it is also exhaustive and time consuming.Multi-mode, automated, microplate readers with integrated fluidics transfer capabilities can measure time-resolved changes in fluorescence and are suitable for performing intracellular Caell line . In addiell line  and P2 rell line . To our 2+]i kinetics with the FlexStation 3 can be more favorable, mainly because of its cost-effectiveness, speed and the automated fluidics system. Furthermore, the use of this instrument for measurements of intracellular Ca2+ in salivary gland cells or any acutely isolated cells from other tissues provides two overlapping advantages. First, the possibility of automated screening of multiple wells at the same time, overcomes the delay that can be anticipated with linear systems measuring one well at a time. Reasonably, for real-time measurements of several minutes, linear systems introduce an undesirably long delay between measurement of the first and last well and the kinetic Ca2+ measurements of only a few minutes/well will introduce a delay of several hours between the first and the last measurement in a 96-well plate. During this delay, cells maintained in suboptimal conditions, may show deterioration of function and viability. In addition, this could result in dye leakage as well as dye sequestration in intracellular organelles, e.g., mitochondria i responses, which can directly cause cell dysfunction and increase the rates of cell damage i in virtually all cell types, including salivary acinar cells, via globally depleting Ca2+ from all intracellular stores i levels. Prompt [Ca2+]i mobilization from the acinar ER stores in response to CCh was reflected as a fluorescence signal spike, that peaked for 12 s after the secretagogue application. This peak was then followed by a plateau phase, remarkably higher than the resting [Ca2+]i, which usually had Ca2+ oscillations superimposed on it . The rapid initial increase in [Ca2+]i due to the activation of muscarinic receptors by agonists has been shown to be mediated through the release of IP3 and the subsequent mobilization of Ca2+ from internal stores i which bypass the receptor-operated mechanisms ivia two pathways in a concentration-dependent manner. One is the Ca2+/H+ exchange, which is mediated by a high concentration of IM, such as 5 µM. The other is the store-operated Ca2+ entry channels (SOCs), which are activated by a low concentration of IM, such as 1 µM [The Cachanisms . As a po as 1 µM .2+ in order to determine the responsiveness of salivary gland acini to changing concentrations of autonomimetics. This approach allows the study of cells in their native conformation, avoiding the problems associated with single cell dispersions and immortalized cell lines. However, a limitation of acutely isolated cells of this type cannot be used over an extended period of time and experiments should be performed on the day of preparation. Future studies could be undertaken in order to determine the applicability of this protocol to other exocrine glands. Furthermore, changes to the tissue preparation protocol might enable use of the technique to organ cultures of embryonic salivary glands or salivary tissue grown in vitro on different scaffolds. Table 3 provides the most common problems that can encountered during performance of this protocol.The present study has shown that a multimode plate reader with integrated fluidics (Flexstation 3) can be used to make multiple and repeated measurements of intracellular Ca


Toxoplasma gondii and Plasmodium spp. manufacture a complex arsenal of secreted proteins used to interact with and manipulate their host environment. These proteins are organised into three principle exocytotic compartment types according to their functions: micronemes for extracellular attachment and motility, rhoptries for host cell penetration, and dense granules for subsequent manipulation of the host intracellular environment. The order and timing of these events during the parasite's invasion cycle dictates when exocytosis from each compartment occurs. Tight control of compartment secretion is, therefore, an integral part of apicomplexan biology. Control of microneme exocytosis is best understood, where cytosolic intermediate molecular messengers cGMP and Ca2+ act as positive signals. The mechanisms for controlling secretion from rhoptries and dense granules, however, are virtually unknown. Here, we present evidence that dense granule exocytosis is negatively regulated by cytosolic Ca2+, and we show that this Ca2+‐mediated response is contingent on the function of calcium‐dependent protein kinases TgCDPK1 and TgCDPK3. Reciprocal control of micronemes and dense granules provides an elegant solution to the mutually exclusive functions of these exocytotic compartments in parasite invasion cycles and further demonstrates the central role that Ca2+ signalling plays in the invasion biology of apicomplexan parasites.Apicomplexan parasites including  The phylum embraces at least 6,000 species with global distribution infecting animals and even other protists  levels by release from intracellular stores signals release of MICs to the extracellular environment ‐trisphosphate (IP3) from diacylglycerol (DAG), and IP3 releases Ca2+ stores in many systems  was observed with increasing concentrations of both ionomycin and A23187 from 1 to 5μM , which is believed responsible for recharging sequestered Ca2+ pools. Thapsigargin treatment, thus, leads to cytosolic accumulation of Ca2+. BAPTA‐AM treatment resulted in loss of constitutive secretion from micronemes, and no change to GRA secretion, consistent with a role of elevated Ca2+ in both of these processes  1 and 3 in Toxoplasma are involved in controlling cell processes relevant to invasion and egress, including microneme exocytosis, where elevated Ca2+ triggers activation of these processes   treatment , levels of MIC secretion were strongly reduced in both constitutive and A23187‐treated states compared with the −ATc controls. The suppression of GRA secretion with A23187 treatment was also reduced in the TgCDPK1‐depleted cells  is specific to TgCDPK1 in T. gondii  MIC secretion was lost with 3‐MB‐PP1 treatment, consistent with inhibition of TgCDPK1  to independently test if dense granule exocytosis control is regulated directly by Ca2+ rather than cGMP. TgRNG2‐depleted cells showed normal levels of Ca2+‐induced dense granule exocytosis inhibition and concurrent elevation of microneme secretion , no change in dense granule secretion was seen compared with untreated cells  cells as previously described .4.2g at 15°C for 10 minutes. Pellets were aspirated and washed with 3 ml of invasion buffer  and pelleted as before. Supernatants were aspirated and pellets resuspended in invasion buffer at 2.5 × 108 cells.ml−1.Fifty microlitres of parasite suspension was then mixed with an equal volume of invasion buffer containing 2× the final concentration of agonist or vehicle (DMSO) equivalent. Parasite samples were incubated at 37°C for 20 min to allow secretion and quenched on ice for 2 min to stop secretion. Cells were then separated from supernatants by centrifugation , 85 μl of supernatant removed, and this was centrifuged a second time to remove any remaining cells, with a final volume of 75 μl carefully aspirated. The cell pellets were washed with 1× PBS, repelleted as before, and the supernatant was aspirated. Supernatant and cell pellet proteins were solubilised in SDS sample buffer, separated by SDS‐PAGE, transferred to nitrocellulose membranes, and GRA, MIC, or mitochondrial proteins immuno‐detected. Western blot detection was performed with horseradish peroxidase conjugated secondary antibodies detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce). Signal strength was quantified using a BioRad Chemidoc imager and ImageLab software, and replicate assays signals were normalised to their respective cell pellet TOM40 signal to control for any minor cell number variation. Standard error of the means (SEM) were calculated for replicate data as a measure of experimental consistency, however, due to the non‐linear nature of chemiluminescent detection, statistical analyses of these data are not appropriate. Plaque assays were performed on cells recovered after the 2‐min ice quenching. Cells were pelleted, washed, and then resuspended in 1 ml of growth medium, and finally inoculated into HFFs at equal parasite densities.Parasite cultures were preincubated for at least 48 hr with or without ATc. Parasites were harvested after multiple rounds of parasite replication within the PV and with approximately 50–80% of vacuoles intact prior to natural egress. Cultures were scraped, host cells disrupted by syringe passing through a 26 gauge needle, filtered through a 3‐μm polycarbonate filter to remove host debris, and parasites pelleted at 1000x


Further evaluation of diagnostic leukemic cell gene-expression identified 19 SNP-gene pairs with a concordant triad of associations: i)SNP genotype with cytarabine sensitivity (p<0.0001), ii) gene-expression with cytarabine sensitivity (p<0.05), and iii) genotype with gene-expression (p<0.1). Two genes from SNP-gene pairs, rs1376041-GPR56 and rs75400242-IGF1R, were functionally validated by siRNA knockdown in AML cell lines. Consistent with association of rs1376041 and gene-expression in AML patients siRNA mediated knock-down of GPR56 increased cytarabine sensitivity of AML cell lines. Similarly for IGF1R, knockdown increased the cytarabine sensitivity of AML cell lines consistent with results in AML patients. Given both IGF1R and GPR56 are promising drug-targets in AML, our results on SNPs driving the expression/function of these genes will not only enhance our understanding of cytarabine resistance but also hold promise in personalizing AML for targeted therapies.Cytarabine has been an integral part of acute myeloid leukemia (AML) chemotherapy for over four decades. However, development of resistance and high rates of relapse is a significant impediment in successfully treating AML. We performed a genome-wide association analysis (GWAS) and identified 113 (83 after adjusting for Linkage Disequilibrium) SNPs associated with  The difficulty of obtaining bone marrow samples and the in vitro cytarabine chemosensitivity in primary AML cells is probably the reason for the lack of such studies, especially in pediatric patients.Acute myeloid leukemia (AML) is the second most common pediatric leukemia with poor outcomes indicated by 5-year survival rates of 50-60%, 2. Cytain vitro leukemic cell cytarabine chemosensitivity. Of these 65 patients, 50 (including 40 white patients) had leukemic cells with in vitro cytarabine with IC50 < 5 ng/μL and 15 (including 10 white patients) had leukemic cells that were resistant to cytarabine (IC50 not achieved). The baseline demographics of the patients included in the study are summarized in Table The current study included 65 patients enrolled in the multi-center St. Jude AML02 clinical trial with specimens available for determination of -8; Figure After rigorous quality control (QC) as described in the methods below, we evaluated 1,317,106 variants in 50 white patients. With this limited sample size, none of the SNPs reached genome-wide significance at the Bonferroni threshold for the familywise error rate (P<5 × 10-7). It is located on chromosome 12 downstream of NEDD1 and co-localized within a long intergenic non-coding RNA (LincRNA) region. A few other potentially interesting SNPs localized within or close to the genes of potential relevance in myeloid malignancies. A synonymous SNP, rs1376041 G>A (P=1.42x10-5), is located within a conserved region close to exon/intron splice junction within the G protein-coupled receptor 56 (GPR56) gene. Presence of the minor allele A for rs1376041 was associated with cytarabine resistance. Our results on this SNP are very exciting given that GPR56 contributes to AML development and its expression has been associated with inferior outcome in AML.[-5) SNP is located in a region upstream of IGF1R (Insulin like growth factor receptor) a gene expressed in human leukemia cell lines with pathological significance in AML [IGF1R is among one of the significantly phosphorylated receptor tyrosine kinase in AML that has been associated with activation of PI3K/AKT signaling and thus cell growth and survival in AML [The top GWAS SNP associated with cytarabine resistance is rs721947  is located in a LincRNA ENSG00000233574 and is roughly 495 kb downstream of the transcriptional co-activator Meningioma1 (MN1) proto-oncogene. MN1 overexpression has been reported to be an important step in the development of inv(16) AML, and is predictive of poor outcome in AML patients with normal cytogenetics.[-5), in opioid binding protein/cell adhesion molecule like (OPCML) occurs in high LD  with rs540923, which is an eQTL for Homeobox A10 (HOXA10), a well-known myeloid leukemia gene. rs7565278 is present around 26kb 3’ of the adducin 2 beta (ADD2) gene and approximately 85kb 5’ of the transforming growth factor alpha (TGFA) gene, which is expressed in neoplastic myeloblasts.rs56837868 G>A  and genes involved death receptor signaling pathways (HTRA2 and ZC3HAV1a PARP family member). SNPs with in or close to other biologically interesting genes included: CXC3R1, a chemokine receptor which is expressed at higher levels in AML [DLK1, a member of the NOTCH signally pathway with a potentially oncogenic role in myeloid dysplastic syndrome [VEGFC , for which higher expression has been previously associated with chemo-resistance and adverse prognosis in AML [Several SNPs were located in or near cell adhesion genes . Furthermore, IGF1R mRNA expression level within diagnostic leukemic cells was significantly higher in cytarabine resistant cases  association of SNPs with cytarabine in vitro sensitivity (p< 0.0001), (2) association of gene expression with cytarabine in vitro sensitivity (p<0.05) and (3) association of SNP with gene expression in a consistent direction (p < 0.1). Although this approach highlights significance of some SNPs, it is possible that other significant SNPs impact gene function by mechanisms other than regulating gene expression. Figure in vitro validation. Consistent with the SNP and gene-expression results, siRNA mediated knockdown of these genes increased cellular susceptibility to cytarabine. GPR56, also commonly referred to as ADGRG1, encodes for G protein-coupled adhesion molecule and is highly expressed in leukemic stem cells and has been implicated in development of AML.[et al. showed greater GPR56 expression in AML samples with high ecotopic viral integration site-1 expression (EVI1high), which is considered a refractory type with poor prognosis. Further, GPR56 knockdown in mice has been shown to result in an increase in cell migration and a decrease in cellular adhesion to the extracellular matrix, resulting in a reduction in cell growth rate and enhanced apoptosis.[Development of resistance to cytarabine adversely impacts treatment outcome, making it very critical to understand the molecular mechanisms underlying cytarabine resistance. Past efforts to understand factors influencing cytarabine chemosensitivity have focused on the use of lymphoblast cell lines (LCLs) derived from healthy population as a model system or comparing gene-expression differences between parental and cytarabine resistant AML cells lines.–12 Howevt of AML. We have t of AML., Consistt of AML. GPR56 ist of AML. Saito etpoptosis..GPR56 rs1376041 SNP was associated with cytarabine resistance as well as with higher mRNA expression levels of GPR56 gene, which in turn was associated with greater cytarabine resistance. This result indicates that rs1376041 SNP’s contribution to cytarabine in vitro sensitivity might actually be through its regulation of GPR56 gene expression levels. This adds a unique level of evidence indicating GPR56 rs1376041 SNP to be predictive of its expression and cytarabine chemosensitivity of patient leukemia cells. Further, GPR56 knockdown in AML cell lines increased cytarabine sensitivity, suggesting its potential role in drug resistance and thus inferior outcome. To the best of our knowledge, even though recent data suggests significant role of GPR56 in AML, SNPs in GPR56 have not been studied in context of developing AML or resistance to treatment response.Our results show that the A allele of the IGF1R, a gene encoding a transmembrane receptor protein having tyrosine kinase activity.[IGF1R, can lead to upregulation of phosphoinositide 3 kinase/protein kinase B (PI3K/Akt) signaling, promoting growth and survival of AML cells.[IGF1R receptor-mediated activation of the PI3K/Akt signaling pathway has been reported previously [IGF1R to be associated with cytarabine in vitro chemosensitivity and in conjunction with reports of specific targeting of IGF1/IGF1R signaling pathway having potent anti-leukemic activity in AML cells with constitutive PI3K activation, makes it a likely drug target.The second signal, the rs75400242 SNP, is a G>A intronic SNP located 360 kb upstream of activity., 34 Uponactivity. ActivateML cells., 36 Celleviously , 37, 38,in vitro drug sensitivity data from primary leukemia cells. To the best of our knowledge, even though limited by the relatively small sample size as compared to other GWAS studies, our study is the largest data set with in vitro cytarabine chemosensitivity in leukemic cells from pediatric AML patients.Our study clearly adds another level of evidence by mapping a genetic variation that is predictive of leukemic cell cytarabine resistance. Although further studies are warranted to both validate the associations of SNPs identified in our study and to understand the functional underpinnings of the mechanisms contributing to cytarabine resistance, this is difficult due to the lack of such data sets owing to the practical challenges associated with obtaining In conclusion, although future work in larger clinical cohorts is warranted, our results identified SNPs, mapping to genes of relevance in AML.ClinicalTrials.gov identifier: NCT00136084). AML02 patients with t[FLT3-ITD mutation, t[in vitro cytotoxicity study. The mononuclear cells were isolated using Ficoll-Hypaque density-gradient centrifugation within 24 h. Cells were resuspended in modified RPMI-1640 medium supplemented with 20% fetal calf serum, penicillin (100 IU/mL), streptomycin (100 μg/mL) and Fungizone™ , as well as ITS medium supplement containing insulin (5 ng/mL), transferrin (5 μg/mL), and sodium selenite (5 ng/mL), as previously described.[in vitro using the 4-day 3--2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay.[This study included patients that were treated under the multicenter AML02 clinical trial  at Hussman Institute for Human Genomics, University of Miami, Miami FL, USA. Genotype calling was performed using Illumina's Genome Studio software V2011.1 .The mRNA expression levels in diagnostic leukemic blasts from the patient samples were previously determined using the Affymetrix U133A microarray data. All geneQC steps were performed to obtain a high quality dataset for use in statistical analysis. QC is crucial since the capability of a GWAS to reveal true associations and the utility of those GWAS results is dependent on the quality of the data with significant impact on downstream analyses and replication studies.The initi-8 [-4 for follow-up with gene expression data. All GWAS was conducted using PLINK 1.9.[Given the phenotype of interest, namely cytarabine IC50, was not normally distributed , the pat-8  and a suLINK 1.9., 44.cis eQTL analysis was conducted on candidate SNP-gene pairs identified in the GWAS and differential gene expression analysis above using Wilcoxon and Kruskal-Wallis tests, depending on how many genotype groups are available for each SNP. These analyses were done using R statistical software version 3.4.0.[Differential gene expression for genes within +/-500kb of the significant/suggestive GWAS SNPs was conducted using Wilcoxon test to detect genes differentially expressed between the sensitive and resistant groups. on 3.4.0..In vitro validation of two genes identified in GWAS and validated in gene expression analysis was performed using AML cell lines: THP-1, K562 . The cell lines were maintained in RPMI-1640  supplemented with 10% FBS  at 37°C in a humidified atmosphere containing 5% CO2. The cells were passaged every 2–3 days in order to maintain them in logarithmic growth phase. siRNAs targeting GPR56 and IGF1R as well as scrambled siRNA were procured from Dharmacon . Just before transfection, the cells were grown in RPMI-1640-medium free of antibiotics. Delivery of siRNA at a final concentration of 50 nM was performed using RNAi Max reagent  according to the manufacturer's instructions. To monitor the effect of siRNA on gene silencing, transfection (5 × 105 cells/well) was done in 6-well plates for 48 hours. RNA levels of target genes GPR56 and IGF1R as well as house-keeping gene GAPDH was then measured by Taqman gene expression assays . Transfected cells harvested and exposed to cytarabine at 5 μM and 200 μM final concentration for 48 hours. Cells were stained with acridine orange (AO) and propidium iodide (PI) for viability assays and fluorescein isothiocyanate-conjugated Annexin V for apoptosis assays followed by manufacturer’s procedure, . Cell imaging was undertaken by the Cellometer Vision CBA and FCS Express-6 software. In order to measure threshold in the software, it was set to 0% to quantify total fluorescence of each counted cell from the captured images. Based on fluorescence intensities, the results were calculated and converted to FCS file for analysis in the De Novo Software.55.


The more than 30-year-old Calcium hypothesis postulates that dysregulation in calcium dependent processes in the aging brain contributes to its increased vulnerability and this concept has been extended to Alzheimer’s disease and Parkinson’s disease. Central to the hypothesis is that increased levels of intracellular calcium develop and contributes to neuronal demise. We have studied the impact on cells encountering a gradual build-up of aggregated α-synuclein, which is a central process to Parkinson’s disease and other synucleinopathies. Surprisingly, we observed a yet unrecognized phase characterized by a reduced cytosolic calcium in cellular and neuronal models of Parkinson’s disease, caused by α-synuclein aggregates activating the endoplasmic calcium ATPase, SERCA. Counteracting the initial phase with low calcium rescues the subsequent degenerative phase with increased calcium and cell death – and demonstrates this early phase initiates decisive degenerative signals. In this review, we discuss our findings in relation to literature on calcium dysregulation in Parkinson’s disease and dementia. First, the autonomous firing of these dopaminergic (DA) neurons along with their large terminal fields causes a continuous and large influx of Ca2+ that in order to maintain low normal Ca2+ levels has to be balanced by active transport processes at the risk of oxidative stress , dopaminergic neurons in the substantia nigra pars compacta have been the focus of interest for decades due to their disease-associated loss that gives rise to the striatal dopamine deficiency and motoric symptoms. An intense interest in the cytosolic Cae stress . Second,symptoms . We receD Figure . In contgregates . This ph2+ levels in neurons are crucial for their development and function  and the extracellular compartment where Ca2+ concentrations are in the 1 mM range (2+ ATPases (SERCA), plasma membrane ATPases (PMCA), and Na+/Ca2+ exchangers (NCX).The average 150 nM cytosolic CamM range  and thes2, per default will increase cytosolic Ca2+ and ultimately cause cell death  mutations within the SNCA gene encoding α-synuclein was identified as causative for autosomal dominant PD in rare families , (ii) agy CryoEM  and soliy CryoEM . The abitibodies  that alltibodies  or cellstibodies .2+ in both mitotic OLN-93 and a non-mitotic human neuroblastoma cell model (SH-SY5Y) of α-synuclein aggregate stress, and in primary cultures of mouse hippocampal neurons expressing human α-synuclein from the Thy1-a-Syn Line 61  leading to reduced cell stress and increased viability in the OLN-93 model, SH-SY5Y model, in the hippocampal neurons, and an in vivo C. elegans model, UA44  expressing AS and GFP in their dopaminergic neurons. The protective CPA dose did not negatively affect the viability of control cells. The abnormal complexes between α-synuclein aggregates and SERCA could be demonstrated by co-immunoprecipitation of SERCA from brain homogenates using in vitro formed soluble aggregates as bait and in situ in SH-SY5Y cells using anovel α-synuclein/SERCA proximity ligation assay. The proximity ligation assay also revealed positive signals in human brain tissue affected by dementia with Lewy bodies, but not control tissue corroboration this novel mechanism of α-synuclein aggregates engaging with SERCA is relevant to human pathology. Hence, the degenerative phase with reduced cytosolic Ca2+ may well be active in human brain. It should be stressed that the mechanisms, whereby α-synuclein aggregates activates SERCA is not restricted to neurons but is generic and will occur in all cells experiencing intracellular α-synuclein aggregates, since SERCA is ubiquitously expressed  encountering a progressive build-up of soluble α-synuclein aggregates , 2014. T Line 61 . The earxpressed . SERCA ixpressed . The expxpressed . It willxpressed  as modelxpressed . In PD, xpressed  but it dxpressed .2+ have not been appreciated before? Firstly, the experimental focus has been on cell models to generate inclusion or pathology that resembles Lewy bodies e.g., phosphorylation of Ser129 on cellular α-synuclein or insoluble α-synuclein species. These phenomena likely require a significant build-up of α-synuclein aggregates and may occur in the late degenerative stage with increased cytosolic Ca2+ and enhanced cell death. Secondly, methodologically it is not trivial to demonstrate a 20% decrease in basal cytosolic Ca2+ of 150 nM if it is not the aim of the experiment. The genetically encoded Ca2+ sensors, like GCAMP6, are designed to demonstrate increases in Ca2+ as readouts for neuronal activity. To obtain quantitative measurement of reductions in cytosolic Ca2+, we used the ratiometric Ca2+-sensor Fura-2 that is exited at 340 and 380 nm and has its emission quantified at 510 nm, which upon calibration allows quantitative measurements. In our study, we chose a region of interest to measure the Fura-2 signal in the cell body located outside the nucleus that may reflect neurons average cytosolic Ca2+ in the somatodendritic compartment.One may ask why this phase with reduced cytosolic Ca2+ and a degree of Ca2+ overload in their ER. This phase is not characterized by degenerative death promoting processes, but slowly developing dysfunctions in critical Ca2+ dependent processes that compromise the cells ability to contribute to its normal circuitries and thereby brain functions. We propose a neuron-centric mechanisms where Ca2+ dysfunctions initiates in neurons that contain the α-synuclein aggregate but one can speculate if it also affect the tripartite synapse coupling neuron and glia   . Maintaie stress . The CaVs firing .2+ channel blockers of the dihydropyridine type (DiCCBs) used to treat hypertension reduce the risk of developing PD and its associated mortality, but did not reduce the risk of dementia in PD patients  rather than having a long-term impact on neurodegeneration”  of Ca2+ across the plasma membrane into the ER  in the outer mitochondrial membrane and through the mitochondrial Ca2+ uniporter (MCU) in the inner mitochondrial membrane (2+-rich ER by means of mitochondrion-associated membrane (MAM) domains  and VDAC1 in the mitochondria by the action of the chaperone GRP75 and this facilitates the flux of Ca2+ from ER into mitochondria. Mitochondria are rich in the lipid cardiolipin that facilitates their α-synuclein binding , expression of ryanodine receptors (RyR), and inositol trisphosphate receptors (IP3R)  . This beynuclein .2+ dysfunctions will open for novel mechanisms to counter the dysfunction associated to PD and other synucleinopathies.Aggregation of α-synuclein in neurons is a progressive process sculpted by the local concentration of α-synuclein, presence of prion-like α-synuclein seeds, and proteostatic balances. When early soluble aggregated α-synuclein species are formed, they will bind to and activate the SERCA pump in the ER. Blocking the α-synuclein aggregate dependent activation by SERCA antagonists protected the affected neurons, thus demonstrating the existence of pivotal downstream signaling pathways. The ER is a dynamic organelle that permeates neurons from nerve terminals through axons, cell bodies into dendritic spines  and thisCB and PJ designed and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The infected layer 2/3 pyramidal cells were identified in rat cortical slices using Venus fluorescence. Analysis of short-term synaptic plasticity using paired patch clamp recordings between layer 2/3 pyramidal cells (presynaptic cell) and fast-spiking (FS) interneurons (post-synaptic cell) showed that calretinin expression in the pyramidal cells did not change the failure rate in this synapse but did decrease synaptic delay. Analysis of the parameters of short-term synaptic plasticity showed that the amplitude of the first EPSP in the train was not affected by calretinin, however, calretinin strongly enhanced short-term depression. In addition, we found that the effect of calretinin depended on the presynaptic firing frequency: an increase in frequency resulted in enhancement of synaptic depression.Analysis of the effects of various proteins on short-term synaptic plasticity is a difficult task, which may require the use of knockout animals. Here, we propose an alternative experimental approach for studying the roles of desired proteins in synaptic plasticity. We packed the Ca These proteins differ by Ca2+-affinity, expression pattern, and binding kinetics, which determine their effect on synaptic efficacy and plasticity. Analysis of their functional role may be quite challenging because some of these proteins are predominantly expressed in interneurons constituting a relatively small neuronal population (<20%), which is further subdivided into numerous subtypes. Each subtype has its own unique connectivity pattern, synaptic properties, and Ca2+-endogenous buffer expression profile. It would not be an exaggeration to say that for the vast majority of interneurons most of these parameters are not well studied when compared to the excitatory synapses formed by cortical glutamatergic neurons. The second complication arises from the classical investigation approach of the function of specific gene products by comparing WT and knockout animals. In the case of endogenous Ca2+ buffers, this should be done on connections where the identities of both pre- and post-synaptic neurons are constant in all experiments, this requirement greatly narrows experimental conditions. Moreover, it has been shown that the removal of some Ca2+-binding proteins has more global consequences than simply reduction of endogenous buffer capacity. For instance, parvalbumin knockout leads to changes in short-term plasticity from depression to facilitation  in a similar way as it does in WT calretitin-positive interneurons. Pyramidal cells in layer 2/3 of the rat somatosensory cortex have low endogenous Ca2+ capacity and some of the excitatory connections formed by these neurons are very well characterized  extraction and converted to cDNA with the reverse transcription reaction using a mixture of random oligonucleotides (N6) and 20-mer oligo(dT) primers._ORF of the calretinin precursor was amplified from cDNA with a pair of primers: ATGGATCCTTACACGGGGGGCTCACTGC and ATGCTAGCGCCGCCACCATGGCTGGCCCGCAGCAG. The PCR fragment was cloned into a pBluescript vector and sequenced from both ends. Clones confirmed with the proper sequence were used for further cloning; the fragment was excised with NheI and BamHI restriction enzymes and cloned into the pAAV-Syn-IRES-Venus plasmid .An AAV cassette carrying mouse calretinin-IRES-Venus under a synapsin promoter was packed into AAV using a mix of pDp1 and pDp2 packing vectors . The infected layer 2/3 pyramidal cells were identified using Venus fluorescence and IR-DIC imaging. In paired recordings, postsynaptic putative FS basket cells in layer 2/3 were identified by location, morphology, and firing pattern  peak and the onset of the corresponding postsynaptic EPSP, then the values obtained for the individual cell pairs were averaged to get the final estimate.n = 52 cells).After electrophysiological recordings, slices were fixed in 4% paraformaldehyde and stained using anti-calretinin antibodies . t-test for pairwise comparison  and 15 virally modified calretinin-positive (CR+) neurons are summarized in n = 12) and 1.98 mV (n = 12) in WT and CR+ cell pairs respectively,  and, in CR+ terminals, 7.5%  and 0.6 (n = 9) milliseconds at WT and CR+ synapses, respectively  and their firing pattern. Calretinin-expressing pyramidal cells were recognized by the presence of a fluorescence signal. Postsynaptic responses in the FS cells were triggered by 5 and 10 Hz trains of APs evoked in the presynaptic pyramidal neurons. At least 50 sweeps were collected in each neuronal pair for subsequent analysis . First, 2+ ion to calretinin strongly increases the affinity of the remaining unoccupied binding sites converting calretinin from a slow calcium buffer to a fast calcium buffer. It has been shown that at layer 2/3 pyramidal to FS cell synapses slow buffers (EGTA) do not have a strong impact on short-term depression; this is in contrast with the fast buffer BAPTA which strongly reduces the amplitude of the first response and degree of depression  of the second and the first EPSPs evoked by 5 Hz trains of three APs was 0.71 (n = 17), however, PPR measured at CR+ synapses was significantly smaller, 0.52 . Thus, in contrast to another fast-endogenous buffer, calbindin which has the main influence on the synaptic release during the first AP in the train and after partial saturation with Ca2+ can cause synaptic facilitation, the calretinin effect on the first response is rather weak. However, “sensitization” of calretinin by Ca2+ during the first AP increases its binding properties and therefore leads to the suppression of synaptic release.At WT layer 2/3 pyramidal to FS cell synapses, a change in stimulation frequency from 5 to 10 Hz results in moderate and often not significant enhancement of both paired-pulse and steady state depression  and subsequent vesicle fusion is triggered by Ca2+ binding to the release calcium sensor. Synaptic release occurs at active zones in the close vicinity of presynaptic VGCC where [Ca2+]i rapidly rises reaching levels sufficient for activation of exocytosis in a very short time. This spatially and temporally restricted elevation of [Ca2+]i is known as a Ca2+-concentration microdomain. Conversion of Ca2+ entry into characteristic release properties  at individual types of synapses crucially depends on diffusional distance, as well as on endogenous buffer capacity and buffering kinetics. It is commonly accepted that release probability (Pr) is negatively related to the distance between VGCCs and Ca2+ vesicular release sensors  can bind Ca2+ before it reaches vesicular sensors, and therefore, significantly reduce Pr at both types of terminals. The effect of a given buffer concentration is several fold stronger at low-Pr compared to high-Pr synapses. However, during repetitive high frequency presynaptic activity, fast buffers undergo partial saturation with Ca2+ which attenuates the suppressant effect on release and leads to synaptic facilitation or a significant reduction in paired pulse depression . However, when the first pair of binding sites is occupied with Ca2+, kon of the second pair drastically increases to 310 μM−1s−1  can partially saturate the first binding site of calretinin shifting its Ca2+ binding affinity from “slow calretinin” to the “fast 1Ca2+-calretinin”. “Fast” calretinin in turn can more effectively prevent Ca2+ binding to the Ca2+ sensor release suppressing vesicle fusion more efficiently during the second AP. Upon long lasting repetitive stimulation, the rate of “partial saturation” and, therefore, the ratio between “slow” and “fast” calretinins will depend on Koff of 1Ca2+-calretinin and 2Ca2+-calretinin, calretinin diffusion rates, and most probably, on residual [Ca2+]i and the Ca2+ extrusion rate. There is evidence suggesting that residual [Ca2+]i (>1 μM) remains in the terminals for hundreds of milliseconds after AP (Koester and Sakmann, 2+]i can accelerate a transition from the “slow” calretinin to the “fast” 1Ca2+-calretinin resulting in stronger depression. However, in this case, the total Ca2+-binding capacity of the buffer in the terminal should considerably exceed the amount of Ca2+ coming with each AP. Thus, upon high frequency stimulation calretinin has a moderate effect on synaptic transmission during the first AP, but can greatly reduce information flow throughout the rest of the train.In contrast to calbindin, at low [CaAll datasets generated for this study are included in the manuscript.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Recently, it was shown that intracellular Ca2+ signaling also drives vasculogenesis by stimulation proliferation, tube formation and neovessel formation in endothelial progenitor cells. Herein, we survey how growth factors, chemokines and angiogenic modulators use endothelial Ca2+ signaling to regulate angiogenesis and vasculogenesis. The endothelial Ca2+ response to pro-angiogenic cues may adopt different waveforms, ranging from Ca2+ transients or biphasic Ca2+ signals to repetitive Ca2+ oscillations, and is mainly driven by endogenous Ca2+ release through inositol-1,4,5-trisphosphate receptors and by store-operated Ca2+ entry through Orai1 channels. Lysosomal Ca2+ release through nicotinic acid adenine dinucleotide phosphate-gated two-pore channels is, however, emerging as a crucial pro-angiogenic pathway, which sustains intracellular Ca2+ mobilization. Understanding how endothelial Ca2+ signaling regulates angiogenesis and vasculogenesis could shed light on alternative strategies to induce therapeutic angiogenesis or interfere with the aberrant vascularization featuring cancer and intraocular disorders. It has long been known that endothelial Ca It has been estimated that, if lined end-to-end, the ≈10 trillion (1013) interconnected endothelial cells that form the lumen of blood vessels would wrap more than four times around the circumference of the earth i) within vascular endothelial cells plays a crucial role in angiogenesis i has long been recognized as a key pro-angiogenic pathway that, lying at the intersection of multiple signaling cascades, is recruited by distinct mitogens to promote and modulate endothelial cell fate [2+]i that stimulates endothelial cell proliferation, adhesion, migration and bidimensional tube formation i by recruiting, respectively, phospholipase C-γ (PLCγ1-2) and phospholipase C-β (PLCβ1-4)  and Ghormones ,54,55. RPLCβ1-4) . Althoug signals . It has  GTP-γ-S ,61. The of PLCβ1  and PLCβof PLCβ1 . In addiof PLCβ1 . As a coas P2Y12 ,65, sphias P2Y12 , and C-Xas P2Y12 .2), a minor membrane phospholipid, into the two intracellular second messengers, InsP3 and diacylglycerol (DAG)   42]. As. As2), asiveness . Surprisby PLCβ3 ,71. Furtby PLCβ3 , an endoby PLCβ3 , may recby PLCβ3 . PLC actth InsP3 ,69,70,73th InsP3 ,76,77,782+ store in vascular endothelial cells by containing approximately 75% of the intracellular Ca2+ reservoir (2+ concentration ([Ca2+]ER) is maintained at around 100-500 μM by SERCA2b . Th. Th2+ st SERCA2b ,49,50,80capacity . Vasculacapacity ,82, howecapacity . In addit SERCA3 . 3 evokes endogenous Ca2+ release by binding to the non-selective cation channels, InsP3R i quickly raises from around 100 nM up to 1 μM i, while InsP3R3 rather instigates monophasic Ca2+ signals i in human cerebrovascular endothelial cells, in which InsP3R3 is the most abundant isoform . T. T122]. channels ,122,123. (ICRAC) . The ICR> 1.000) . An ICRAepletion ,128 and,2+ entry and ER Ca2+ loading by regulating resting Ca2+ permeability . I. I3-syntd by DAG . TRPC chand Ca2+ ,146. Qui current  or a lesand ISOC ,147,148.ductance ,150,151.ductance ,153. Theby STIM1 , which cby STIM1 , and by by STIM1 ,143. In  current . While t2+ toolkit described in Paragraph 2 may be differently recruited by distinct growth factors and cytokines to induce spatio-temporal Ca2+ signals, which are precisely tailored to regulate specific phases of the angiogenic process. The endothelial Ca2+ response to pro-angiogenic cues usually consists of a transient or biphasic increase in [Ca2+]i or in repetitive Ca2+ spikes. These Ca2+ signatures are mainly driven by endogenous Ca2+ release and SOCE, although the contribution of TRPC channels has been reported. The pro-angiogenic Ca2+ signal can be modulated by concomitant changes in the endothelial membrane potential (VM) due to the recruitment of Ca2+-dependent conductances. Novel high-resolution and high-speed imaging techniques confirmed that VEGF-induced endothelial Ca2+ signals arise in vivo and drive endothelial cell sprouting and migration. The Ca121, VEGF-A145, VEGF-A165, VEGF-A189), VEGF-B, VEGF-C, VEGF-D, VEGF-E (encoded by Orf virus), VEGF-F , and placenta growth factor (PlGF) i is regarded as a crucial signal whereby VEGF stimulates vascular endothelial cells to undergo cell fate specification, proliferation, migration, tubulogenesis and neovessel formation i in several types of endothelial cells, including HUVEC i as originally reported in . A. A3-depeof PLCγ1 . In addihampered . VEGF-iny InsP3R  and requ toolkit ,167,168  toolkit , but the2+ signals have been widely characterized in peripheral and pulmonary circulation. For instance, endothelial Ca2+ signaling drives VEGF-induced retinal angiogenesis, whose dysregulation could lead to several sight-threatening diseases, including diabetic retinopathy, age-related macular degeneration, and retinopathy of prematurity [2+]i in bovine retinal endothelial cells (BREC). As reported in HUVEC, the initial Ca2+ peak is patterned by InsP3-mediated ER Ca2+ release, whereas the plateau phase depends on Ca2+ entry through a yet to be defined Ca2+-permeable route. The pharmacological inhibition of InsP3-dependent Ca2+ mobilization prevents VEGF-induced BREC proliferation, migration, tubulogenesis and sprout formation . VE. VE2+ si in MAEC . In addial cells . Finallyin HDMEC . 2+ toolkit shapes the pro-angiogenic Ca2+ response to VEGF is provided by computational modeling. A rule-based modeling approach utilizing the programming language BioNetGent recently confirmed that the extent of VEGF-induced Ca2+ entry during the plateau phase is finely tuned by the ICRAC. Likewise, a reduction in the rate of Ca2+ clearing through either SERCA or PMCA could further sustain the plateau amplitude i [The Cabranches . Angiostbranches . Similarbranches . Early w in BAEC  192]. I. I2+ resct tasks ,194. Themulation . In addidostatin . It woulignaling . Thromboignaling . In silipathways . FinallySprouty4 , a membr [Ca2+]i .3 signaling axis, many other growth factors can induce pro-angiogenic Ca2+ signals in vascular endothelial cells [2+]i in a variety of vascular endothelial cells, including HUVEC i in HUVEC [2+ oscillations in rat cardiac microvascular endothelial cells (CMEC) [2+]i in ovine uterine endothelial cells i, a biphasic Ca2+ signal or repetitive Ca2+ spikes i, although it has been shown that extracellular Ca2+ entry is involved in the onset of the signal and in SDF-1α -induced migration in HRMEC . O. O2+ sig signals , is not ogenesis . Future 2+]i, multiple Ca2+-dependent decoders translate endothelial Ca2+ signals into a pro-angiogenic outcome. These include, but are not limited to (2+/CaM-dependent protein kinase II (CaMKII) i stimulates the Ca2+-dependent PKCβ2 (cPKCβ2), which in turn recruits the downstream RAF1–MEK–ERK1/2 cascade more efficiently than Ras , the lipid second messenger responsible for Akt activation. However, VEGF may engage the PI3K/Akt pathway also through an increase in [Ca2+]i, as demonstrated in HUVEC i could directly recruit the PI3K class II α-isoform i to, respectively, induce long-term potentiation and regulate cardiac contractility. Emerging evidence has postulated a key role for endothelial CaMKII in the control of endothelial cell proliferation, migration and permeability  i displaces Cav1 from eNOS, thereby relieving this tonic inhibition and inducing NO release  located within the autoinhibitory reductase domain, which prevents eNOS activation in the absence of a Ca2+ rise . A numbe2+ rise ,270,271. and PKC ,226. TheKC [2+]i . In addi release ,226,272.ugh TPC2  and by Sugh TPC2 . 2+ signaling pathway plays a pivotal role during early development by controlling cell proliferation, migration, polarity and cell fate specification. In addition, dysregulation of the Wnt/Ca2+ signaling pathway may also lead to neoplastic transformation [q/11PCR able to recruit PLCβ and induce InsP3-dependent Ca2+ release [2+]i stimulates calcineurin and CaMKII to, respectively, engage NFAT and NF-κB, thereby inducing gene expression i plays a crucial role also in vasculogenesis i in ECFC and to remove cytosolic Ca2+ upon chemical stimulation is similar to that described in vascular endothelial cells  i is necessary to instigate UCB-derived ECFC to migrate and to adhere to a fibronectin matrix in vitro. Furthermore, IGF-2-evoked Ca2+ signaling drives UCB-derived ECFC homing to damaged vessels and engraftment into nascent vasculature in vivo, thereby promoting vascular reconstruction in a murine model of hindlimb ischemia [2+]i in circulating ECFC i in circulating [2+ oscillations in circulating ECFC are shaped by rhythmic ER Ca2+ release through InsP3Rs followed by SERCA-mediated Ca2+ sequestration into ER lumen. SOCE is required to sustain the spiking response by refilling the ER with Ca2+ during prolonged stimulation [2+ oscillations are more robust and frequent in UCB-derived ECFC [2+ entry triggers the dynamic interplay between InsP3 and SOCE -derived ECFC due to the reduction in [Ca2+]ER and InsP3R transcripts i are down-regulated and do not stimulate either proliferation or tube formation also in breast cancer (BC)-derived ECFC i regulates angiogenesis and vasculogenesis. The available evidence hints at InsP3Rs and SOCE as the major driver of pro-angiogenic Ca2+ signals in both vascular endothelial cells and ECFC/MAC. Nevertheless, the role of NAADP and TPC1-2 is rapidly emerging and it remains to understand whether extracellular Ca2+ entry through DAG-gated channels, i.e., TRPC3 and TRPC6, during the plateau phase observed in some cell types, such as HUVEC, is merely redundant or is required to recruit Ca2+-sensitive decoders that are not targeted by SOCE. An additional feature that should be taken into account is the dose-response relationship of each growth factor as the Ca2+ signature and Ca2+-dependent pro-angiogenic activity could vary depending on the dose, as reported for VEGF in PAEC. Endothelial Ca2+ signaling controls angiogenesis and vasculogenesis is indispensable to design alternative strategies to induce therapeutic angiogenesis in ischemic disorders and interfere with the vascular network in solid malignancies. For instance, it has been demonstrated that genetic or pharmacological manipulation of the endothelial Ca2+ toolkit improves angiogenesis in vivo. VEGF-induced extracellular Ca2+ influx was enhanced by engineering HDMEC with an adenovirus encoding for TRPC6, thereby enhancing their proliferation rate [2+ signalling to stimulate therapeutic angiogenesis. Of note, it has been suggested that autologous or endogenous ECFCs, which represent the most suitable EPC subtype for regenerative therapy of ischemic disorders, could be redirected towards a more reparative phenotype by intervening on their Ca2+ toolkit. It has been suggested that ECFCs’ angiogenic activity could be improved by lentivirus-mediated expression of TRPC3, subsequent expansion in vitro and reinoculation into the ischemic tissue [2+ oscillations in resident ECFC in a murine model of AMI [2+ signalling in resident ECFC. An alternative, but not mutually exclusive mechanism, consists in taking advantage from small molecule drug to stimulate angiogenesis by enhancing endothelial Ca2+ signaling. For instance, inhibiting PMCA4 with the small molecule aurintricarboxylic acid enhanced VEGF-induced calcineurin activation, thereby boosting HUVEC migration and tube formation as well as neovessel formation in a murine model of hindlimb ischemia [2+ signaling could be exploited to favor normalization of tumor vasculature [2+-dependent manner [Understanding how endothelial Caion rate . Similarion rate . These pc tissue ,319. Morl of AMI . This evischemia . Conversischemia  and tumoischemia , has beeischemia ,312. Altischemia ,321. Altculature , therebyt manner .


Double strand DNA breaks (DSBs) are dangerous events that can result from various causes including environmental assaults or the collapse of DNA replication. While the efficient and precise repair of DSBs is essential for cell survival, faulty repair can lead to genetic instability, making the choice of DSB repair an important step. Here we report that inverted DNA repeats (IRs) placed near a DSB can channel its repair from an accurate pathway that leads to gene conversion to instead a break-induced replication (BIR) pathway that leads to genetic instabilities. The effect of IRs is explained by their ability to form unusual DNA structures when present in ssDNA that is formed by DSB resection. We demonstrate that IRs can form two types of unusual DNA structures, and the choice between these structures depends on the length of the spacer separating IRs. In particular, IRs separated by a long (1-kb) spacer are predominantly involved in inter-molecular single-strand annealing (SSA) leading to the formation of inverted dimers; IRs separated by a short (12-bp) spacer participate in intra-molecular SSA, leading to the formation of fold-back (FB) structures. Both of these structures interfere with an accurate DSB repair by gene conversion and channel DSB repair into BIR, which promotes genomic destabilization. We also report that different protein complexes participate in the processing of FBs containing short (12-bp) versus long (1-kb) ssDNA loops. Specifically, FBs with short loops are processed by the MRX-Sae2 complex, whereas the Rad1-Rad10 complex is responsible for the processing of long loops. Overall, our studies uncover the mechanisms of genomic destabilization resulting from re-routing DSB repair into unusual pathways by IRs. Given the high abundance of IRs in the human genome, our findings may contribute to the understanding of IR-mediated genomic destabilization associated with human disease. Efficient and accurate repair of double-strand DNA breaks (DSBs), resulting from the exposure of cells to ionizing radiation or various chemicals, is crucial for cell survival. Conversely, faulty DSB repair can generate genomic instability that can lead to birth defects or cancer in humans. Here we demonstrate that inverted DNA repeats (IRs) placed in the vicinity of a DSB, interfere with the accurate repair of DSBs and promote genomic rearrangements and chromosome loss. This results from annealing between inverted repeats, located either in different DNA molecules or in the same molecule. In addition, we describe a new role for the Rad1-Rad10 protein complex in processing fold-back (FB) structures formed by intra-molecular annealing involving IRs separated by long spacers. In contrast, FBs with short spacers are processed by the Mre11-Rad50-Xrs2/-Sae2 complex. Overall, we describe several pathways of DSB promoted interaction between IRs that can lead to genomic instability. Given the large number of IRs in the human genome, our findings are relevant to the mechanisms driving genomic destabilization in humans contributing to the development of cancer and other diseases. Double strand breaks (DSBs) in DNA result from the interactions of DNA with environmental agents or cellular metabolites, and are a major source of genetic instability . TheThe two main types of DSB repair pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR), are conserved from yeast to humans , 6. NHEJin cis to HO-induced DSB affected its repair ).outcomes  from IR-outcomes . Furthermilar to , 17) , EmAccording to our model , 5’- to The predominance of intra-molecular SSA for IRs separated by short (12-bp) rather than long (1-kb) spacer might be explained by a higher probability of interaction for IRs in close proximity to each other as compared to those located further away. Another possible contributing factor is thermodynamic stability of FB structures, which was shown to be inversely proportional to the length of the spacer separating IRs , 56. ImpWe propose that when FBs are formed, they can be processed by two different protein complexes. Specifically, FBs containing short single-strand loops are processed by MRX-Sae2 complex Here we characterized two pathways of DSB repair involving annealing between IRs that can result in genomic destabilization. IRs are especially frequent in the 17% of the human genome comprised of LINEs and Alu repeat elements that commonly form clusters, where individual repeats often have opposite orientations , 67. AluWith IRs being abundant in the human genome In the future, it will be important to compare the structural properties of inverted DNA repeats promoting different disease-associated rearrangements, since it may reveal that different IR-mediated SSA pathways are involved in their formation. It is also important to characterize the respective roles of human homologs of Rad1-Rad10 and MRX-Sae2 complexes in the formation of DSB and IR-promoted chromosomal rearrangements since it may shed light on the specific mechanisms of chromosomal rearrangements leading to various pathologies in humans.PHO87 sequences in inverted orientation to each other, separated by a 1-kb-long spacer, and located 3-kb centromere proximal to MATa. AM1102 was derived from JKM111 . Lower-case letters correspond to the sequences specific to the pGSKU cassette  calculated based on a minimum or 3 experiments are indicated above each strain. (B) The schematics of AvrII digest of Chr III (OF) in IR-1000 and its DSB-derivatives, CF, ID, FB. The location of probe P-3 (specific to BUD5 sequence) is indicated by green box. (C) Southern blot analysis of DSB repair in IR-1000-rad1Δ following AvrII digestion and hybridization with probe P-3. (D) Efficiency of FB formation (%) calculated by dividing the normalized intensity of FB band formed at 6-hrs following DSB induction by the intensity of cut-fragment generated at 0.5-hr following DSB in various strains. The median of FB formation and the range [in the brackets] calculated based on minimum of 3 experiments are indicated above each strain. The cut-fragment band (0.5-hr time-point post DSB induction) was used for calculation because it represented the actual amount of chromosomes that were broken following DSB induction and therefore could be involved in DSB repair. In addition, the Southern transfer of the original (uncut) chromosome fragment (OF) at 0-hr time-point (before DSB) was inefficient and therefore was unreliable for calculations.(TIF)Click here for additional data file.S2 FigA) The schematic of AvrII digest of Chr III (OF) in IR- λ1000 strain (two 2-kb IRs separated by 1000-bp spacer DNA derived from λphage-DNA) and its DSB-derivatives including: CF, ID, and FB. (B) Southern blot analysis of DSB repair in RAD1 and rad1Δ derivatives of IR- λ1000 strain following hybridization to probe P-1. (C) The schematic of AvrII digest of Chr III (OF) in IR- λ1500 strain (two 2-kb IR separated by 1500-bp spacer DNA derived from λphage-DNA) and its DSB-derivatives including: CF, ID, and FB. (D) Southern blot analysis of DSB repair in RAD1 and rad1Δ derivatives of IR- λ1500 strain following hybridization to probe P-1.((TIF)Click here for additional data file.


Rhodiola rosea L., has shown various pharmacological functions, including antioxidant effects, but the signal transduction pathway of its antioxidant effects is not very clear. In this study, we found that salidroside could attenuate hydrogen peroxide (H2O2)-induced HL-7702 cell damage, inhibit H2O2-induced cytosolic free Ca2+ ([Ca2+]i) elevation, scavenge reactive oxygen species (ROS) and increase 3’-5’-cyclic adenosine monophosphate (cAMP) level in a dose-dependent manner, but it couldn’t influence 3’-5’-cyclic guanosine monophosphate (cGMP) levels. Therefore, these results indicated that the antioxidant effects of salidroside were associated with down-regulation of [Ca2+]i, ROS occur via a cAMP-dependent pathway.Salidroside, a major component of  Rhodiola rosea L. has been widely used for a long time in Traditional Chinese Medicine. Salidroside , as shown in 2+]i and NO signaling pathways i and ROS levels in HL-7702 cells to determine whether the cAMP/cGMP pathway participate in the antioxidant effects of salidroside. Our results would be benefitial for development of salidroside as a potential agent for treatment of oxidative stress-related diseases.Oxidative damage, mediated by reactive oxygen species (ROS) has been thought as a major cause of atherosclerosis, cancer, liver disorder and the aging process . In prevpathways ,19, thus2O2-induced cell damage as determined by the MTT assay is shown in 2O2(0.5 mM) for 100 min induced significant cell damage. Pretreatment with salidroside  could attenuate H2O2-induced cell damage in a dose-dependent manner (p < 0.05), though it couldn’t restore the damaged cells totally. Data are expressed as mean ± S.D. (n = 4). # p < 0.05 and ## p < 0.01 compared with Control.The protective effect of salidroside on H2+]i. We found that incubating with H2O2(0.5 mM) for 100 min induced a significant [Ca2+]i elevation, while pre-incubation with salidroside dose dependently suppressed the elevation of [Ca2+]i  for 100 min induced ROS elevation significantly, while pre-incubation with salidroside dose dependently suppressed the elevation of ROS  could attenuate H2O2-induced damage in a dose-dependent manner. It is suggested that salidroside at dose of 0.03, 0.05, 0.1 μg/mL could efficiently protect cells from oxidative damage. In this paper, we used H2+ homeostasis [2O2 induced an [Ca2+]i rise which could still be evoked upon [Ca2+]i withdrawal and mitochondrial uncoupling [2O2 successfully increased the level of [Ca2+]i in HL-7702 cells. [Ca2+]i levels are critical to cell life and changes in [Ca2+]i will affect matrix synthesis, as well as other functions [2O2-induced [Ca2+]i and ROS level and might be used in Ca2+ overloading disease.There is a mutual relationship between calcium and ROS signaling, which affects the local Caeostasis . H2O2 incoupling . In our unctions . In this2+]i and [cAMP]i are often interrelated and linked via adenylate cyclase or phosphodiesterase [2+]i, and our result is consistent with this. The increased level of cAMP participates in activating PKA and consequently this enzyme phosphorylates its substrate proteins. Thus, the increased level of cAMP is linked to the activation of PKA i through direct inhibition of L-type Ca2+ channels i elevation, scavenged ROS at low dose through cAMP-dependent pathway. These findings suggested that antioxidant protection of salidroside was associated with the cAMP-dependent pathway and decrease of [Ca2+]i and ROS levels.In the experiment, we found that salidroside attenuated H2DCFDA) was obtained from Invitrogen (Molecular Probe), USA. RPMI 1640 was obtained from Gibco (USA). Human cyclic guanosine monophosphate ELISA kit and Human cyclic adenosine monophosphate ELISA kit were obtained from Blue Gene . FBS was obtained from Hangzhou (China). All other reagents were from commercial suppliers and of standard biochemical quality.Salidroside  was purchased from the National Institute for the Control of Pharmaceutical and Biological Products. 3--2,5-diphenyl-2-tetrazolium bromide (MTT) was purchased from Sigma (USA). Fluo-3/AM was obtained from Dojin Laboratories (Japan). 5-(and-6)-Chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate, acetyl ester  for 100 min, the MTT solution  was added into each well and made the final concentration 0.5 mg/mL, and then the plates were incubated for an additional 2 h. After the medium was removed, DMSO (100 μL) was added into each well before reading the microplates at 570 nm. Cell viability was expressed as absorbance density (OD).The human hepatocyte HL-7702 was obtained from SGST (China). The protective effect of salidroside was assessed by the MTT assay. Briefly, cells seeded on 96-well culture plates at 5 × 102+]i was determined using Fluo-3/acetoxymethyl ester (Fluo-3/AM), described previously with slight modification i, ROS and at least partly via cAMP-dependent pathway.The data reported here firstly suggested that the antioxidant effects of salidroside were associated with down-regulation of [Ca