We unveiled alterations of SMPGs by transient CJ openings when Ca2+ binds to MICU1 resulting in spatial cristae depolarization. This Ca2+/MICU1-mediated plasticity of the CJ further provides the mechanistic bedrock of the biphasic mitochondrial Ca2+ uptake kinetics via the mitochondrial Ca2+ uniporter (MCU) during intracellular Ca2+ release: Initially, high Ca2+ opens CJ via Ca2+/MICU1 and allows instant Ca2+ uptake across the CM through constantly active MCU. Second, MCU disseminates into the IBM, thus establishing Ca2+ uptake across the IBM that circumvents the CM. Under the condition of MICU1 methylation by PRMT1 in aging or cancer, UCP2 that binds to methylated MICU1 destabilizes CJ, disrupts SMPGs, and facilitates fast Ca2+ uptake via the CM.Mitochondrial ultrastructure represents a pinnacle of form and function, with the inner mitochondrial membrane (IMM) forming isolated pockets of cristae membrane (CM), separated from the inner-boundary membrane (IBM) by cristae junctions (CJ). Applying structured illumination and electron microscopy, a novel and fundamental function of MICU1 in mediating Ca 2+ control over spatial membrane potential gradients (SMPGs), with alterations in SMPGs following Ca2+ binding to MICU1 resulting in spatial cristae depolarization.Within mitochondrial substructures, MICU1 is shown to mediate Ca Morphologically, the cristae junctions (CJ), bottleneck membrane structures with a diameter of approximately 17 nm3, separate IBM and CM from each other. The mitochondrial contact site and cristae organizing system (MICOS) proteins and the scaffold protein optic atrophy 1 (OPA1) stabilize the CJ4.The ultrastructure of the inner mitochondrial membrane (IMM) is separated into two parts, the inner boundary membrane (IBM) representing the interface of the IMM with the outer mitochondrial membrane (OMM), and the cristae membrane (CM), forming the membrane invaginations into the mitochondrial matrix5. The CM contains the four complexes of the respiratory chain6. Of those, complex I, III, and IV exhibit proton pump activity and, thus, contribute to the mitochondrial membrane potential (ΔΨm) across the IMM. The ΔΨm represents the energetic force for the production of ATP by the F0F1 ATPase that is also localized to the CM7. By knowing the spatial restriction of complexes I, III and IV to the CM, experimentally determined differences in pH of CM (pH 7.0)8 and mathematically estimated pH values at the IBM (pH 7.4)9 become comprehensible. The CJ, representing a physical diffusion barrier, constrains the diffusion of the protons to the cristae lumen. Rieger et al. showed that a pH gradient is even established between the Complex IV and the F0F1 ATPase in the isolated CM compartment8. The proton motive force towards the matrix is the main component contributing to establish the ΔΨm10. Wolf et al. showed recently that individual cristae are isolated insulators restricting the membrane potential generated at the CM by the CJ. This study demonstrated two distinct membrane potentials at the CM (ΔΨCM) and the IBM (ΔΨIBM)11.All three IMM compartments CJ, IBM, and CM differ in morphology and composition of characteristic proteins according to their spatio-specific functions. In particular, the IBM hosts the mitochondrial protein uptake complex TIM (translocase of the inner membrane) that interacts with the TOM (translocase of the outer membrane) complex in the OMM to translocate nuclear-encoded mitochondrial proteins into the mitochondrial matrix or mitochondrial membranes2+ uptake, which highly depends on ΔΨ, is essential to activate Ca2+-dependent dehydrogenases of the TCA cycle and increase oxidative phosphorylation12. Mitochondrial Ca2+ Uniporter (MCU) complex-mediated Ca2+ uptake is driven by the ΔΨm across the IMM, yet it was never elaborated if the different CM (ΔΨCM) and the IBM (ΔΨIBM) potentials influence the Ca2+ uptake on the level of mitochondrial ultrastructure.Mitochondrial Ca2+ uptake 1 (MICU1), is localized at the IBM, stabilizes the CJ, and interacts with the MICOS complex13. Under basal conditions, MICU1 exists as a hexamer or oligomer, which in case of Ca2+ binding via EF-hands of MICU1 disassembles into dimers with an EC50 of 3.8 μM Ca2+15. Methylation of MICU1 by protein arginine methyltransferase 1 (PRMT1) was shown to reduce the Ca2+-binding affinity of MICU1 to approximately 18.5 µM and inhibit thereby the Ca2+ induced dissociation into dimers. Contrary, the specific binding of uncoupling protein 2 (UCP2) to methylated MICU1 counteracts the methylation effect and resensitizes MICU1 towards Ca2+ (EC50 = 4.0)15. Intracellular ER Ca2+ release from the endoplasmic reticulum (ER) leads to OMM located Ca2+ hotspots (up to 16.42 µM) in close proximity to mitochondrial-associated ER membranes (MAM), while mitochondrial regions without direct contact with the ER are facing much lower Ca2+ concentration of approximately 2.94 µM16. The precise regulation of MICU1 by PRMT1 and UCP2 is important for cell metabolism and viability17. Especially cancer cells show a clear increase in UCP2 but also PRMT1 expression and both proteins when highly expressed are prognostic markers for lung carcinoma patients18.The main regulator of MCU, mitochondrial Ca2+ hotspots with changes in its quaternary structure paired with the observation that MICU1 stabilizes CJ led us to hypothesize that the opening and stability of the CJ are directly regulated by high Ca2+ 19. Strong opening of the CJ is associated with a loss of membrane potential11, impaired oxidative phosphorylation and ATP production21, and induction of apoptosis22. Accordingly, we used structured illumination microscopy, differential membrane potential measurements of the CM and IBM, electron microscopy, and mitochondria-targeted genetically encoded biosensors for Ca2+ to elaborate the involvement of MICU1 in CJ dynamics, effects on mitochondrial Ca2+ uptake, and ΔΨm homeostasis between IBM and CM.The ability of MICU1 to respond to CaCM) and the IBM (ΔΨIBM), we established a reliable and robust method using super-resolution microscopy. This approach builds on the assumption that the fluorescent potentiometric dye tetramethylrhodamine methyl ester (TMRM) is incorporated into the IMM25 according to local ΔΨ, thus, the individual fluorescent intensities reflect distinct ΔΨIBM and ΔΨCM. While at low and moderate concentrations TMRM binds to the IMM in a linear correlation with the ΔΨm, saturation effects have been reported at high TMRM concentrations21. Further, we could show that TMRM concentrations in the range of 1.35–81 nM show a slight saturation effect  between the CM  and stained with 81 nM TMRM were treated with 100 µM ATP to induce mitochondrial swelling. A clear separation of TMRM cristae labeling and matrix mt-sfGFP fluorescence became obvious, showing specific labeling of the IMM by TMRM . At low TMRM concentrations (≤5.4 nM) the overlay of TMRM with mt-sfGFP shows an exclusive TMRM staining of the cristae membrane Fig. . At TMRM(TMRM − mt-sfGFP)) illustrate the concentration-dependent TMRM expansion 2, which correlates the TMRM fluorescence intensities of the central cristae membrane (CM) with the IBM  and AIIBM, a sigmoidal correlation between a TMRM accumulation in the mitochondrial periphery  and increasing TMRM concentration was found, indicating different membrane potentials at the CM and IBM  The full width of half maxima (FWHM) of mitochondria for the TMRM and mt-sfGFP  channels were measured and their differences  TheIBM Fig. . For botIBM Fig. .11. MICU12 and OPA128 are known to contribute directly to the stability of the CJ. Accordingly, the contribution of these proteins to the isolation of the different mitochondrial membrane potentials (ΔΨm) in CM (ΔΨCM) and IBM (ΔΨIBM) was investigated. Knockdown of either MICU129   and AIIBM, knockdown of UCP231 results in the accumulation of TMRM in the CM for all TMRM concentrations applied . Both, MICU1 and OPA1 depletion result in a focused elevation of ρCM in the mitochondrial center  and IBM association index , and the respective fluorescence intensities and distributions were recorded upon stimulation with histamine  instead of histamine showed no response in −∆/+∆TMRM and basal −∆/+∆TMRM ratio did not change by silencing either MICU1 or UCP2 compared to control cells  after ER Ca2+ depletion, which leads to a homogeneous cytosolic Ca2+ elevation, was used to analyze −∆/+∆TMRM alterations . Therefore, we tested the sub-compartmental Ca2+ kinetics in mitochondria upon IP3-induced ER Ca2+ release and SOCE in MICU1, and UCP2 siRNA treated cells using red and green fluorescent genetically encoded Ca2+ biosensors targeted either to the IMS and mitochondrial matrix, CL and mitochondrial matrix, or IMS and CL, respectively. The temporal delay of Ca2+ propagation within the respective sub-compartment of mitochondria was quantified as the delta of areas under the curve (∆AUC) for normalized IP3- and SOCE-induced Ca2+ signals in the respective sub-compartment , the optic atrophy 1 (OPA1)32 and MICU113.In this study, we combined dual-color SIM, TEM, and sophisticated live-cell imaging with mitochondrial sub-compartmental targeted genetically encoded biosensors to investigate the fundamental function of MICU1, OPA1, and UCP2 in the dynamic regulation of spatial membrane potential gradients (SMPGs) between IBM and CM. Moreover, the individual role of the two distinct IMM compartments (i.e. IBM and CM) in the orchestration of mitochondrial Ca11, we herein demonstrate that the membrane potential of the IMM (ΔΨIMM) is separated by the CJ into two distinct potentials, that of the IBM (ΔΨIBM) and the CM (ΔΨCM). Notably, our data show that silencing of MICU1 leads to a strong shift of ΔΨIBM/ΔΨCM towards the IBM. This disruption of SMPGs points to a fundamental role of MICU1 in CJ integrity, thus, reemphasizing our previous results showing a CJ opening and clear reduction of the overall ΔΨIMM for MICU1 knockdown conditions2. Moreover, our new data point to an alternative model of mitochondrial uncoupling that builds on the erosion of SMPGs by means of CJ-controlled ΔΨCM. Opening of the CJ and the dissemination of the protons from the CL into the IMS yields depolarization of ΔΨCM that mimics a mitochondrial uncoupling across the IMM.Confirming recent results from ref. 2. We assume that a destabilization of the CJ leads to IMM constrictions which were shown to precede mitochondrial fission in ER close regions33. Our findings in this study suggest that mitochondrial fragmentation observed in MICU1 and OPA1 silenced cells is caused by destabilized CJ in ER close regions leading to IMM constriction and mitochondrial fission. Contrary reduction of UCP2 leads to a more stable CJ configuration19 and thus to elongated mitochondria.We found in our recent study that the CJ widened through silencing of OPA1 or MICU12+cycling within the MAMs achieves rare activation of MICU1 and, thus, increase in H+-permeability of the CJ. Besides the process of Ca2+ cycling, CJ H+-permeability is further influenced by the methylation status of MICU1 by PRMT1. Methylation of MICU1 lowers the Ca2+ binding affinity of MICU1 (EC50 for Ca2+ increases from 3.8 to 18.5 µM)16 resulting in an enhanced stability/impermeability of the CJ. However, UCP2 binds exclusively to methylated MICU1, restores Ca2+ sensitivity of methylated MICU1 (EC50 = 4.0 µM)15, and pseudo-normalizes basal CJ H+-permeability. Consistently, in the present work, depletion of UCP2 resulted in an increased CJ stability due to the low Ca2+ binding affinity of methylated MICU1 that cannot be activated by basal ER-originated Ca2+ cycling within the MAMs in the absence of UCP2. In line with our findings, UCP2 knockdown is reported to increase ΔΨm34, a phenomenon that, based on our present data, can be attributed to a stabilization of the CJ.Because silencing of MICU1 destabilizes CJ under basal conditions and yields disruption of SMPGs, we speculate that even under resting condition, basal ER-originated Ca3-induced intracellular Ca2+ release, high Ca2+ at the mitochondrial surface binds to MICU1 yielding its deoligomerization, subsequent opening of the CJ, and disruption of individual SMPGs. Notably, the Ca2+ concentrations achieved within the Ca2+ hotspots in MAMs (up to 16.4 µM) during cell stimulation16, have the potential to disassemble methylated MICU1 (EC50 for Ca2+ 18.5 µM)16 even in the absence of UCP2. Studies focusing on cristae dynamics revealed that CJ opening is predominantly restricted to MAMs after ER Ca2+ release30 and that CJs are closely associated with MAMs35. In line with this assumption, Gerencer and Vizi showed that initial mitochondrial Ca2+ uptake after IP3-induced ER Ca2+ release originates from local foci into the whole mitochondria36.Under the condition of cell stimulation that triggers IP2+ inside the CL in response to IP3-induced Ca2+ release26. Further, the disruption of the CJ by knockdown of MICU1 or OPA1 increases basal Ca2+ inside the mitochondrial matrix2. Considering both findings, one may conclude that MCU is constantly active in the CM. On the other hand, MICU1 homo and MICU1/MICU2 heterodimers were shown to increase MCU conductance38 but do not occlude the MCU channel38. Further, three different quaternary structures including MICU1  are known : MICU1 hexamers or oligomers that disassemble Ca2+ dependent into MICU1 dimers39 and the MCU super complex involving MCU, EMRE, MICU1 and MICU242. MICU1 was shown to potentiate MCU channel activity by direct interaction and MCU is not occluded by MICU138, raising the question of how MICU1 reduces mitochondrial Ca2+ uptake at low cytosolic Ca2+ concentrations. These findings raise the question how MICU1 is regulating both, Ca2+ availability in the CM and amplification of the conductance of MCU? Accordingly, based on the present findings, a 2-phase model of the orchestration of MCU-mediated mitochondrial Ca2+ uptake that meets all reported features is proposed  that does not generate Ca2+ hotspots in MAMs. Accordingly, SOCE-originated mitochondrial Ca2+ uptake always occurs independently from UCP244.Phase 2.2. Secondary to the fast transfer of ER-derived Ca2+-triggered opening of the CJ and biphasic mitochondrial Ca2+ uptake ensures full mitochondrial functionality even in phases of high Ca2+ challenges or to achieve uptake of low Ca2+. Due to local and temporal isolated CM depolarization, the mitochondria maintain general stability during Ca2+ uptake while increasing the metabolic output. By the local regulation of CJ opening, not only Ca2+ uptake is regulated but also the overall ΔΨCM, mostly distanced from MAMs, is protected, and only affected in a minor way. This mechanism ensures mitochondrial stability under physiological Ca2+ signaling.Such sophisticated organization of the Ca2+ uptake regulation by MICU1 at the structural level of the CJ  objective was mounted on a Nikon-Structured Illumination Microscopy  System with standard wide field and SIM filter sets and equipped with two Andor iXon3® EMCCD cameras mounted to a Two Camera Imaging Adapter . At the bottom port, a third CCD camera  is mounted for wide-field imaging. For calibration and reconstruction of SIM images, the Nikon software  was used. Reconstruction was permanently performed with the same robust setting to avoid artifact generation and ensures reproducibility with a small loss of resolution of 10% compared to the most sensitive and rigorous reconstruction settings. Microscopy setup adjustments were done as described elsewhere2/95% air). The origin of cells was confirmed via STR-profiling by the cell culture facility of the Center of Medical Research .HeLa (ATCC-CCL-2.2TM) cells were seeded on 1.5H high precision glass cover slips  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  with 1.5 µg/ml plasmids or 100 nM siRNA using 2.5 µg/ml TransFast™ transfection reagent . After 24 h, the medium was replaced with DMEM containing 10% FCS and 1% penicillin/streptomycin and kept for a further 24 h prior to experiments. The specific siRNAs  used in this study are listed in Supplementary Table 2, 135 NaCl, 5 KCl, 1 MgCl2, 1 HEPES, 2.6 NaHCO3, 0.44 KH2PO4, 0.34 Na2HPO4, 10 d-glucose , 0.1% vitamins, 0.2% essential amino acids and 1% penicillin/streptomycin at pH 7.4. Cells were incubated in loading buffer containing 1000, 500, 200, 81, 40.5, 13.5, 5.4, 2.7, or 1.35 nM TMRM  for 30 min. As TMRM might degrade over time in storage, TMRM concentrations were measured regularly. Therefore, after TMRM was dissolved in methanol the absorption at 550 nm was measured and the concentration was calculated using Eq. c is the molar concentration of TMRM, A the absorption of TMRM at 550 nm, ε is the specific extinction coefficient of TMRM at 550 nm in methanol, and l is the pathlength the light has to path through the TMRM solution.Cells were washed once with loading buffer containing in mM: 2 CaCl15. TMRM was excited at 550 nm, and the emission was collected at 600 nm. A full disruption of the mitochondrial membrane potential was done by the application of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) . In the case of the non-quenching mode of TMRM, a direct drop of mitochondrial fluorescence followed FCCP treatment. In the case of quenching mode, a clear increase in TMRM fluorescence after FCCP addition was observed, followed by a drop in intensity. During the entire experiment the respective TMRM concentrations were present within the imaging buffers and prior recording the cells were equilibrated in the perfusion chamber for a further 5 min. Mitochondrial TMRM signals of single cells were background corrected using a background ROI.HeLa cells seeded on glass slides were labeled for 30 min with 1000, 500, 200, 81, 13.5, or 1.35 nM TMRM and transferred into a perfusion chamber. During the measurements cells were continuously perfused using a gravity-based perfusion system  Measurements were performed on an inverted wide-field microscope  as described previouslyHeLa cells seeded on glass slides were labeled for 30 min with 81, 40.5, 13.5, 5.4, 2.7, or 1.35 nM TMRM and transferred into a perfusion chamber containing the same concentration of TMRM. Per coverslip, 40 cells were imaged randomly and the average cellular TMRM fluorescence was measured.mt-sfGFP from FWHMTMRM results in ∆FWHM.HeLa cells transfected with mt-sfGFP were stained with different concentration of TMRM ranging from 1.35 to 81 nM. After the staining procedure the cells were kept in loading buffer containing the respective concentrations of TMRM and imaged with dual-color 3D-SIM. To compensate for intensity differences for TMRM acquisition the laser power was adjusted to match image intensity histograms of different TMRM concentrations. Post imaging, the recorded data were background corrected using an ImageJ Plugin  with a sliding rectangle diameter of 50 pixel. Intensity line plots of mitochondrial mt-sfGPF and TMRM fluorescence were manually measured with a width of 50 pixels (1.6 µm). The FWHM of mt-sfGFP and TMRM fluorescence distributions was measured using linear interpolation to gain subpixel information. Subtraction of FWHM2. In short, images were subjected to background subtraction  with a sliding rectangle diameter of 50 pixels. The reference channel  was Otsu45 auto thresholded and further dilated and eroded in two independent subsets. One erosion and two dilation iterations were used. Pixel-wise subtraction of the erosion reference of the dilated reference image yields a hollow structure, used as a mask to measure the mean intensity in the mitochondrial periphery or IBM-related area in the object channel. The erosion reference served as a mask to measure the bulk or cristae mean fluorescence intensity. The ratio of IBM/CM mean intensity is a value to estimate changes in the object label distribution inside a mitochondrion, which is referred to as the IBM association index. The higher the ratio value the higher the distribution of protein labels in the IBM. For image analysis the freeware program ImageJ was used.The IBM association factor of TMRM or MTG was calculated as described elsewhere45 auto threshold. The ImageJ particle analyzer was used to extract the mitochondrial count (c), area (a), perimeter (p), minor (x), and major (y) axes of the mitochondria. Aspect ratio (AR) was determined as3D-SIM and time-lapsed images of EMRE-mCherry, MTR, MTG, or MICU1-YFP were used for morphological analysis. Images were background corrected with an ImageJ Plugin  and the subsequent binarization was done using an OtsuThe Form Factor (FF) was determined as followed:46. The cells were dehydrated in an ascending ethanol series, embedded in TAAB embedding resin, and sectioned on a Leica Ultracut 7 ultramicrotome using a Diatome diamond knife. The sections were counter-stained using platinum blue  and lead citrate  and visualized in an FEI Tecnai 20 transmission electron microscope. They were photographed at ×2000 magnification with a Gatan ultrascan 1000 camera.Cells were incubated in a loading buffer with 81, 13.5, or 1.35 nM TMRM for 30 min, washed with PBS, fixed with 2.5% glutardialdehyde and 2% formaldehyde in a buffered solution, and postfixed in either 2% osmium tetroxide or 1% osmium tetroxide that had been reduced with 1% potassium hexacyanoferrateIn a first step, mitochondria were segmented inside the micrographs manually by free hand selection. Afterwards, the segmented mitochondria were isolated and the cristae inside each mitochondrion was segmented by free hand selection in ImageJ. The process was semi-automated using ImageJ macros. Mitochondrial and cristae area and perimeter were measured. The perimeter of mitochondria divided by the cristae perimeter gives an indication of membrane alteration of the cristae. The ratio of cristae perimeter divided by mitochondrial area gives a representation of cristae density.For analysis of the spatial cristae density distribution, segmented mitochondria and respective cristae were selected. Segmented mitochondria were binarized and used as a mask for binarized cristae membranes to determine the percentage coverage of cristae perimeter inside the mitochondria area. Iteratively, the mitochondrial mask was eroded homogeneously in small increments of 2-pixel width (5.88 nm) and the respective coverage of cristae was measured. The result of these measurements represented a cristae density in circular segments starting from the outer mitochondrial membrane towards the mitochondrial center. As mitochondrial size and shape vary that results in different numbers of ring segments, the cristae densities were normalized by linear interpolation to 100 segments.48) as a marker for the ER and stained with 13.5 nM TMRM in loading buffer for 30 min. TMRM was present during the entire experiment. Cells were afterwards transferred to the live cell chamber containing 0.8 ml loading buffer and imaged over time with a frequency of 0.25 Hz. 8 s after image sequence initialization 0.8 ml of loading buffer with or without 200 µM histamine was added to a final concentration of 100 µM histamine.Cells were transfected with an insensitive ER targeted ATP probe ERAT4.03 NA  to empty ER-Ca2+ stores and imaged using the widefield setup with 380 nm illumination and widefield acquisition with the bottom port CCD camera. Afterward, the same cells were imaged over time with a frequency of 0.25 Hz using the dual-cam SIM setup. 8 s after image sequence initialization Ca2+ was added to a final concentration of 2 mM.To analyze whether local drops of membrane potential appear under SOCE, cells were incubated with 2 µM Fura-2AM  and 13.5 nM TMRM in loading buffer for 30 min. TMRM was present during the entire experiment. Cells were challenged in Ca2+ release. The analysis was separated into several steps, (1) definition of MAMs, (2) identification of drops in the mitochondrial membrane potential, and (3) spatial assignment of the drops to MAM and non-MAM mitochondrial areas.M) and the ERAT4.03 NA channel (further referred to as CE) were background subtracted  with a sliding rectangle diameter of 50 pixels. Both channels (CM and CE) were bleaching corrected using the histogram matching function, which is implemented in Fiji. An Otsu auto threshold was used to binarize CM and CE. The overlap of both channels was eroded  to remove small structures and subsequently dilated  and used as a mask for CM to assign areas of mitochondria with close proximity to MAMs.After acquisition and reconstruction using NIS-Elements AR the mitochondrial TMRM channel . Additionally, Otsu auto thresholded CM of both images involved for the calculation of ∆CM was used as a mask for ∆CM to remove the mitochondrial movement from ∆CM. Negative changes from one frame to the next (−∆CM) as well as positive ones (+∆CM) were subjected to a threshold of 35% of the original intensity of the first frame. Thresholded −∆CM and +∆CM were eroded  and subsequently dilated  to remove remaining halos around mitochondria still originating form mitochondrial movement. Finally, the area of −∆CM and +∆CM containing segments were quantified and the ratio of −∆CM/+∆CM was calculated. For more easy interpretation, −∆CM and +∆CM were replaced with −∆TMRM and +∆TMRM, respectively. The area of −∆CM quantifies distinct local losses of TMRM signal, which can be induced by direct loss of membrane potential or by the movement of the cristae membrane itself. Shifts in the lateral or axial direction within mitochondria can lead to a loss of TMRM fluorescence. Nevertheless, cristae membrane movement should account for increases in TMRM signals in the same quantity. A ratio of −∆/+∆TMRM = 1 states that equal amounts of distinct changes, increase or loss of TMRM signal, are present and can be referred to a steady state in which movement of cristae membrane is the main contribution to −∆TMRM and +∆TMRM. −∆/+∆TMRM < 1 shows more increases in TMRM signal indicating the generation of mitochondrial membrane potential. −∆ /+∆TMRM > 1 on the other side implies a local high loss of membrane potential.Background subtracted  and bleaching corrected (histogram matching) CA custom-made ImageJ macro was used to analyze spatial drops in mitochondrial membrane potential in dependency of spatial proximity to ER-related mitochondrial-associated membranes (MAM) and ER CaThe whole mitochondrial area derived from auto Otsu binarized CM as well as MAMs determined in (1) were used as a mask for −∆TMRM and +∆TMRM areas to differentiate −∆/+∆TMRM-ratios in regard to the proximity to MAMs.M) of subsequent frames was calculated and thresholded by ± 35% of the fluorescence intensity change per frame and per pixel and classified them into positive (+∆TMRM) and negative (−∆TMRM) area partitions. Accordingly, the ratio of −∆/+∆TMRM compensates for errors due to CM movements and quantifies local variations of membrane potential of the IMM. The ER-marker was used to identify mitochondrial structures in close proximity to the ER (MAMs)  Fig. . The rat2+ imaging was performed as described elsewhere49. In brief, Ca2+ was measured on a digital wide-field microscope, the iMIC  equipped with a 40-objective  and an ultrafast switching monochromator, the Polychrome V (Till Photonics). Illumination of GEM-GeCO1 targeted sensors was performed at 430 nm excitation and emissions were collected with a dichrotome dual emission filter set (dichroic 535dcxr). For dual recordings, GEM- and CAR-GeCO1 targeted sensors were alternately excited for 500 ms each at 430 and 575 nm. Emissions derived from both sensors were taken in 3 s intervals. During the measurements, cells were continuously perfused using a gravity-based perfusion system  and images were recorded with a charge-coupled device (CCD) camera . Data acquisition and control of the digital fluorescence microscope were performed using the live acquisition software version 2.0.0.12 (Till Photonics). For analysis, fluorescent traces of single cells were background corrected using a background ROI, and bleaching was corrected using an exponential decay function in a costume made excel macro. To compare the kinetics of two mitochondrial compartments, the fluorescent traces were normalized to the minimum and maximum of the IP3 or SOCE-induced mitochondrial Ca2+ elevations. The area under the curve was determined for both compartments using the trapezium method. The delta of both AUCs was calculated. The starting point of the ∆AUC was set by the point at which the Ca2+ signal in one of both compartments surpasses the three-fold of the standard deviation of the basal signal. The end of the ∆AUC was set to the time point at which one of the signals reached the maximum and crosses with the intensity of the opposite Ca2+ trace from the other sub-mitochondrial compartment.CaWestern 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: MICU1 , and Histone H3 . 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 .n value and the number of independent experiments are indicated in each figure legend. Statistical analysis was performed using the GraphPad Prism software version 5.04  or Microsoft Excel (Microsoft Office 2013). Analysis of variance (ANOVA) with Bonferroni post hoc test and t test were used for evaluation of the statistical significance. All box plots show minimum to maximum values if not otherwise indicated. The central line is the median with boxes extending to 25 and 75% and the whiskers encompass all data. P < 0.05 was defined to be significant. At least three experiments on 3 different days were performed for each experimental set-up.Each exact Further information on research design is available in the Peer Review FileSupplemental MaterialDescription of Additional Supplementary FilesSupplementary DataReporting Summary


Despite recent, rapid drug development success for patients with acute myeloid leukemia, distinct molecular and genetic aberrations still confer a poor prognosis. In this review, we explore the preclinical and early clinical development of two promising approaches: disrupting menin signaling leading to cell differentiation or blocking CD47 to unlock the innate immune system. These two approaches may improve treatment for patients with high unmet needs today.FLT3), B-cell lymphoma 2 and hedgehog pathways. Yet outcomes remain variable; especially in defined molecular and genetic subgroups such as NPM1 (Nucleophosmin 1) mutations, 11q23/KMT2A rearranged and TP53 mutations. Emerging therapies seek to address these unmet needs, and all three of these subgroups have promising new therapeutic approaches. Here, we will discuss the normal biological roles of menin in acute leukemia, notably in KMT2A translocations and NPM1 mutation, as well as current drug development. We will also explore how CD47 inhibition may move immunotherapy into front-line settings and unlock new treatment strategies in TP53 mutated disease. We will then consider how these new therapeutic advances may change the management of AML overall.After forty years of essentially unchanged treatment in acute myeloid leukemia (AML), innovation over the past five years has been rapid, with nine drug approvals from 2016 to 2021. Increased understanding of the molecular changes and genetic ontology of disease have led to targeting mutations in isocitrate dehydrogenase, FMS-like tyrosine kinase 3 ( Every year there are 20,000 new cases of AML diagnosed in the United States  and 119,FLT3 and IDH mutations have therapeutic agents approved for use in AML, but many patients lack these lesions and could benefit from development of novel therapeutic approaches. Several subsets, ranging from the common NPM1 mutation (~33% of AML) to the rarer lysine methyltransferase 2A (KMT2A) (5–10% of AML) rearrangements , may have a common sensitivity to disruption of the protein menin as these two subsets share a common, targetable final pathway. NPM1 mutated and KMT2A rearrangements are often mutually exclusive, models of both demonstrate common gene expression patterns, preclinical testing shows activity in both settings and clinical trials are underway to see if both groups may benefit. As reviewed elsewhere in this series, [TP53 mutant AML remains a largely incurable disease. Although TP53 modulating agents are being studied, [Molecular subsets such as patients with  series,  TP53 mutNPM1 mutations. 11q23/KMT2A rearrangements occur in 5–10% of patients, [NPM1 mutated AML [NPM1 mutation occurs in approximately 35% of all cases and up to 60% of patients with normal karyotype AML [FLT3-internal tandem duplication (FLT3-ITD). NPM1 and FLT3 mutations are now routinely screened at diagnosis with NPM1 mutation and wildtype FLT3 carrying a favorable prognosis [NPM1 mutation with FLT-3 ITD carrying a worse prognosis. Patients with both a FLT3-ITD and NPM1 mutation have poorer prognosis and may undergo transplantation in first remission when possible [Extensive work has been done characterizing the KMT2A epigenetic complex and its large group of interacting proteins  and we watients,  are assoatients, ,9 and matype AML ,11 and prognosis  and NPM1possible . DevelopMEN1 syndrome and sporadic parathyroid adenomas, insulinomas, gastrinomas and lung carcinoid tumors [KMT2A, is the human homolog of the drosophila gene trithorax which is essential for murine mid-gestational development [HOX) genes. Chromosomal translocations frequently disrupt transcription factors and lead to leukemogenesis. These translocations can form fusion proteins with novel functions such as new binding partners or changes in interactions with prior binding partners. These changes impact epigenetic regulation and affect hematopoietic differentiation. Most KMT2A rearrangements are balanced translocations resulting in the production of in-frame gain-of-function fusion oncoproteins. Importantly, these chromosomal rearrangements alone may be sufficient to generate a full leukemic phenotype, [KMT2A chromosomal alterations have been extensively studied in mixed lineage leukemia for more than twenty years [KMT2A encoded wildtype (wt)-KMT2A requires interaction with menin to maintain HOX expression and self-renewal properties of hematopoietic progenitors [FLT3, [PBX3, [HOXA9 and MEIS1 [KMT2A-fusion protein binding to menin [Menin, a nuclear protein comprised of 610 amino acid residues, is ubiquitously expressed in various organs. Menin helps regulate tissue-specific gene expression via interaction with chromatin regulators and transcription factors. Chromatin immunoprecipitation studies show menin interacts with thousands of human gene promoters. By linking transcription-factor function to histone-modification pathways, menin can act as a global regulator of transcription with different functions in different tissues ,15. Intrd tumors . Howeverd tumors . Endogenelopment . The wilelopment ,20. Bothelopment ,22,23 anelopment ,25 play enotype,  as seen enotype, ,28,29. Kty years . KMT2A egenitors ,32,33,34genitors ,36,37. As [FLT3, ,39 PBX3,, [PBX3,  HOXA9 annd MEIS1 ,41. Trannd MEIS1  and is cto menin ,43.KMT2Ar—or CBFB-MYH11, RUNX1T1 and PML-RARA—are often mutually exclusive with NPM1 mutations [NPM1 mutated AML commonly have co-mutated DNMT3A and/or FLT3-ITD and are associated with a characteristic gene expression profile including downregulation of CD34 and overexpression of HOXA cluster genes [HOXA 7/9/10 and MEIS1 gene overexpression is also seen in KMT2A rearrangements. The Armstrong lab demonstrated that NPM1 mutant leukemia is dependent on menin binding to wild type KMT2A and loss of KMT2A-menin abrogates the leukemogenic effect of NPM1 mutations [KMT2A fusion proteins and those with NPM1 mutations. There may be a common, shared, sufficient AML pathway vulnerable to menin disruption.Balanced translocations that initiate AML, like utations ,45. Patier genes . As noteutations . These oKMT2A-rearranged (KMT2Ar) AML [KMT2Ar models using a CRISPR/cas9 model system [HOXa/b targets, including MEIS1, and lead to differentiation in NPM1/DNMT3a mutant knock-in mice and leukemia cell lines [KMT2Ar and NPM1 mutated leukemias.Small molecules disrupting KMT2A and menin interactions show substantial antileukemic efficacy in cellular and murine models of 2Ar) AML ,48. The 2Ar) AML ,50. Small system . Menin ill lines ,52. BaseKMT2Ar fusion proteins, the wildtype complex or both . There were no discontinuations due to adverse events and grade 3 or higher adverse events included elevated lipase, pancreatitis, neutropenia, tumor lysis syndrome and deep vein thrombosis each occurring in 1 patient each (8.3%) while grade 1 or 2 adverse events included nausea, rash and diarrhea [NPM1 mutation the ORR was 30% . Patients lacking either KMT2Ar or NPM1 mutations did not respond [Preliminary data from the phase I/II KOMET-001 trial (NCT04067336) has been presented. Among patients in four dosing cohorts, median age was 67 years-old, a median of 3 prior lines of therapy had been received and 2 of 12 achieved a complete response α, an inhibitory receptor expressed on myeloid cells. SIRPα is a transmembrane protein whose cytoplasmic domain contains immunoreceptor tyrosine-based inhibition motifs which when phosphorylated recruit phosphatases and inhibits the activity of non-muscle myosin IIa. This decreases the ability of prophagocytic signals on tumor cells to trigger phagocytosis by macrophages [Blocking cluster of differentiation (CD) 47 may provide a path forward. CD47 is a nearly ubiquitous expressed cell surface protein belonging to the immunoglobulin family. It is a heavily glycosylated 50Kd cell surface protein with an extracellular rophages . As a cerophages . For exarophages  and exprrophages . By interophages ,78,79,80rophages ,82,83,84rophages ,86,87,88rophages ,90. ThesPreclinical studies have shown that CD47 blockade is not sufficient for phagocytosis of tumor cells . For antFLT3-ITD mutations. High CD47 expression has been shown to be an independent prognostic factor for poor overall survival in AML patient cohorts [CD47 is highly expressed in AML cells including leukemic stem cells  with app cohorts . Thus CDTP53 mutant patients have been shown to have an immune anergic profile, immunosuppressive tumor microenvironment and PD-L1 is upregulated on leukemic stem cells in TP53 mutant AML and MDS [TP53 mutational status may also be critical within the microenvironment itself. In p53 null mice, the size of tumor xenografts are greatly increased and correlate with the presence of myeloid derived suppressor cells, regulatory T cells, and a loss of effector function [TP53 mutations reshape the local innate and adaptive immune response.Blocking CD47 may be most effective in certain subsets of AML, where the balance of pro-phagocytic and anti-phagocytic signals is altered. For example,  and MDS ,98. Furt and MDS . TP53 mufunction ,101. CD4Multiple groups established monoclonal antibodies against CD47 that enabled phagocytosis of AML leukemic stem cells in vitro and inhibited their growth in leukemia cell lines and mice models ,96. TherMagrolimab, as the first-in-class clinical trial candidate, has provided early lessons. In a Phase 1 monotherapy trial, magrolimab failed to show any CR or CR with incomplete count recovery (CRi) in patients with relapsed and/or refractory AML . AnotherTP53 mutated AML CR was 33% and CRi was 8% with median time to CR of 3 months. 45% (14/31) of evaluable patients with any level of response achieved negative MRD by flow cytometry [TP53 AML to conventional treatment is poor, these clinical trial results have lead some investigators to focus anti-CD47 trials on patients with TP53 mutant AML. Whether TP53 mutation status will be a robust biomarker remains to be determined. Magrolimab has since moved into phase III trials. Other CD47 directed agents are not as far along in clinical development but reflect strategies to either enhance immune activity by coupling CD47 blockade with an activating signal or decrease the risk of hemolysis and hemagglutination.Magrolimab has since shown promising overall response rates in combination with azacitidine for AML and MDS. In a phase Ib trial of untreated AML, among 34 evaluable patients overall response rate was 65% 1 pathway has been disappointing in late-stage AML perhaps combination therapy could be feasible. Preclinical evidence is strongest for the combination of azacitidine and anti-CD-47 antibodies, with the combination leading to higher phagocytosis of AML cells in vitro, induction of proposed pro-phagocytic signals including increased calreticulin, long-term remission in AML xenograft mice models . There aTP53-mutated AML’s altered immune profile, spanning both innate and adaptive immunity, a trial of atezolizumab, an anti-PD-L1 antibody, and magrolimab will assess activity in relapsed or refractory AML patients (NCT03922477). Additionally CD-47 targeting antibodies could be combined with other monoclonal antibodies such as gemtuzumab ozogamicin. Alternatively, combination with targeted therapy might be feasible if toxicities are not overlapping. Although VIALE-A showed no statistically significant survival benefit for venetoclax and azacitadine versus azacitidine in TP53 mutated patients, [Clinical results may very well start to return before robust preclinical evidence. Given atients,  synergisatients, .TP53 mutations. Magrolimab in combination with azacitidine has provided proof of concept, it remains to be seen if mutational profiling will be sufficient to identify patients who will respond to therapy or if immunological biomarkers will have to be incorporated into trials. Additionally, novel combinations have entered clinical trials and may further increase efficacy. Ongoing large trials will determine if early signals of hemolysis and hemagglutination can be overcome with dose titration or if instead earlier pipeline agents will be needed to overcome this hurdle.CD47 blockade remains a promising immune checkpoint in AML, and therapies may offer new approaches for hard-to-treat patient populations like those with KMT2A rearranged AML but may hold additional potential in NPM1 mutated AML and even FLT3-ITD patients. Early clinical trials have confirmed an efficacy signal even as a monotherapy and beyond QTc prolongation no other major safety signals have yet emerged. On the other hand, CD47 blockade may finally bring immunotherapy to the AML treatment landscape. To date, this strategy appears to be meeting the major challenge of targeting a marker restricted to leukemia cells. Larger trials will attempt to confirm the promise of improved combination therapy efficacy while sparing hematopoietic cells and erythrocytes. These two treatment approaches may reshape management of intermediate and high-risk AML.As noted above and in other articles in this series, AML therapy has advanced significantly in the last few years but remains challenging. Ongoing basic studies continue to both elucidate the basic biology of leukemic transformation and the immunology of the immune escape of AML cells leading to more targeted and less toxic approaches to AML therapy. The days of high dose induction or no therapy are thankfully behind us. Targeting CD47 and menin inhibitors, if successful clinically, would have a major impact on subsets of AML patients with poor outcomes. Menin disruption represents more than twenty years of preclinical work and appears to have the strongest rationale in


Ca2+ is closely associated with diverse cellular functions. While ER Ca2+ homeostasis depends on different Ca2+ receptors, channels and transporters, the role of PKD2 within the ER remains controversial. Whether and how PKD2-mediated ER Ca2+ leak relates to ADPKD pathogenesis is not well understood. Here, we reviewed current knowledge about the biophysical and physiological properties of PKD2 and how PKD2 contributes to ER Ca2+ homeostasis.Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the PKD1 or PKD2 gene which encodes membrane receptor PKD1 and cation channel PKD2, respectively. PKD2, also called transient receptor potential polycystin-2 (TRPP2), is a Ca PKD2 wamembrane . Perhapsmembrane  rather tmembrane . In 2016membrane . In compmembrane . Despitemembrane .2+ pool plays critical roles in the synthesis, modification, folding, quality control and secretion of proteins , inositol 1,4,5-trisphosphate receptor (IP3R), PKD2, and translocon complex Sec61 . Relevance of PKD2 to ER stress and ADPKD will also be covered.ER as the most important intracellular Cah 800 μM , which iex Sec61  leak Ca2PKD2 is an ion channel that can be localized on different cellular membranes including those of the cell surface, primary cilia and ER . The mem2+ dependent manner  inhibit its plasma membrane targeting  is similar as that of the impermeant N-methyl-d-glucamine (NMDG) (PNMDG/PNa = 0.04) determined by patch clamping in primary cilia, suggesting low Ca2+ permeability of PKD2  was found to play an important role in this bell-shape dependence of the PKD2 function on the cytoplasmic [Ca2+]  PKD2. It is thus plausible to state that [Ca2+] regulates PKD2 trafficking to ER and plasma membranes because the latter was found to be dependent on phosphorylation at S812  was proposed to act as a Cal gating  . This regulation of IP3-dependent Ca2+ signals by PKD2 should be mediated by their physical interaction because PKD2 pathogenic mutant R742X, which has no physical interaction with IP3R, lost its regulatory ability. Interestingly, overexpressed PKD2 C-terminus also prolongs IP3-induced Ca2+ transients, indicating that PKD2-mediated ER Ca2+ release is not part of the IP3-dependent Ca2+ transients. Reciprocally, the IP3-dependent Ca2+ release increases the local cytoplasmic Ca2+ concentration thereby activating PKD2, for which the PKD2/IP3R binding is thought to be mandatory . In Xenterminus . Subsequterminus . Overexpandatory . In T ly release . Consist2+-induced Ca2+ release ion channel located on the sarcoplasmic reticulum (SR) mediating the sarcoplasmic release of stored Ca2+  , allowing external Ca2+ to enter the cell  . As a sithe cell . Althougthe cell . Subsequthe cell . Thus, w2+ homeostasis, lipid and protein synthesis, protein folding, post-translational modification, and regulation of gene expression  and they associate with each other on the ER membrane but not on primary cilia of the renal proximal convoluted tubule (PCT) cells (As a CaT) cells . The PKDT) cells . RecentlT) cells . The cha2+ permeabilities depending on its subcellular localization and regulation by its binding partners such as TMEM33, IP3R, TACAN, and PKD1. Reversely, PKD2 also acts as a modulator molecule of its partner proteins including IP3R, RYR2, PERK, eIF2α, and STIM1. The channel complex PKD1/PKD2 localized on the plasma membrane and primary cilia of renal epithelial cells is known to be relevant to ADPKD but so far there is no convincing evidence that defective ER PKD2 function can result in any cystic phenotype. The function and regulation of the PKD1/PKD2 channel complex deserve thorough investigations to provide molecular basis for design of drugs that are specific and effective. Similarly, although ER PKD2 acts as a Ca2+ release channel either by itself or through partnership with other ER proteins such as IP3R, STIM1, and TMEM33, its physiological and pathological implications have yet to be further determined. Further, based on its crosstalk with factors involved in ER stress and UPR, PKD2 also acts as a fundamental molecule in coping with stress conditions, but leaving an open question as to whether its channel function is essential for fulfilling the role. For this, one would test some loss-of-function point mutants of PKD2 for their ability in dealing with stress conditions. PKD2 is also known to regulate several other cellular functions, such as maintaining cell differentiation and proliferation, induction of autophagy, and developmental signaling in the kidney, heart and reproductive system (PKD2 is a nonselective cation channel with distinct Cae system . Whether


Pasteurella (P.) multocida. Transmission of the infection to humans results from scratches, licks, and bites but it also can occur from the inhalation of air particles containing the microorganism. Immunocompromised people or persons with pulmonary disorders are particularly susceptible to the infection. Infected rabbits may carry P. multocida with or without clinical signs. In this paper, the sensitivity to antibiotics and the invasiveness ability of P. multocida identified in a farm of pet rabbits affected by severe pasteurellosis were investigated. The strain was P. multocida belonging to capsular type A which is the type most often detected in humans. The identified strain was susceptible to the tested antibiotics, but it appeared equipped with several virulence genes which are responsible for fimbriae production, adhesion processes to host cells, enzyme production, and are involved in iron acquisition processes. These findings are of particular interest because rabbits recovered from pasteurellosis very often become carriers of the bacteria. Therefore, we suggest considering P. multocida screening in the routine medical checks of rabbits, especially if they are meant to be companion animals for children and elder people, given that the transmission of the pathogen cannot be excluded.The number of pets such as dogs, cats, rabbits, and parrots has increased in European families. Social benefits to owners such as decreasing feelings of loneliness and anxiety are provided by pets which are also used in Animal-assisted Therapy (AAT). Nevertheless, human-animal interactions are also associated with health problems including allergies, asthma, and zoonosis. Rabbits may carry potential pathogens for humans. One of the most common bacteria that colonizes the oro-pharynx and the upper respiratory tract of rabbits is  In recent years, the presence of pets in European families has increased and their number nowadays exceeds 300 million . After dChlamydia (C.) psittaci can be present in 40% of pet birds, most prevalently parrots but also other species such as canary [C. psittaci by excreta, nasal, and ocular secretions. The transmission of C. psittaci to humans occurs through the inhalation of the pathogen spread in the air and dust contaminated by respiratory secretions and feces from infected birds. In addition, transmission via bird bites and transmission beak to mouth have been reported [C. psittaci can cause asymptomatic infections or acute infections characterized by flu-like illness, mild to severe atypical pneumonia, and systemic disease, which can be fatal in untreated patients [Salmonella [Campylobacter jejuni [Mycobacterium avium subs. avium [Psittacosis is the most known zoonosis transmitted by pet birds. s canary ,7,8. Infreported . In humapatients . Pet birlmonella , Campylor jejuni , and Mycs. avium  to humanTrichophyton mentagrophytes, Microsporum (M.) canis, and M. gypseum which represent the most common agents of zoonosis in rabbits [Encephalitozoon cuniculi [Cryptosporidium spp. [Giardia spp. [Tricostrongylus spp. [Zoonoses are also associated with rabbits. Dermatophytosis in rabbits is caused by  rabbits ,14. Rabbcuniculi , Cryptosium spp. , Giardiadia spp. , and Trilus spp.  which caRabbits may also host viruses and bacteria which can be potential pathogens for humans. The hepatitis E virus has been recently identified in rabbits . In FranMycobacterium avium subsp. hominissuis has been reported in domestic rabbits [Bartonella (B.) species, B. vinsonii subsp. berkhoffii, B. alsatica, and B. rochalimae, were identified in fleas from rabbits, although they were wild animals [Pasteurella (P.) multocida which colonizes the upper respiratory tract and the oro-pharynx. P. multocida is a small coccobacillus, pleomorphic, Gram-negative, facultative aerobic-anaerobic, asporigenous, immobile bacterium with a fermentative metabolism, belonging to the Pasteurellaceae family. P. multocida is characterized by considerable genetic and antigenic variability. The strains are classified into 5 capsular types or serogroups , based on the antigenic characteristics of the capsule polysaccharides, which are further classified into 16 somatic serotypes, based on lipopolysaccharide antigens, using serological techniques. Infected rabbits may carry P. multocida without showing clinical signs or they may be affected by heavy clinical syndromes. Although the symptoms frequently develop when immunosuppression, stress, or adverse environmental conditions occur, the pathogenicity of P. multocida is influenced by the presence of several virulence factors (VFs) prevalently identified in capsule proteins and lipopolysaccharides [P. multocida include fimbriae, adhesion, and colonization factors , iron regulation factors and protein acquisition , superoxide dismutase (sodA and sodC), dermonecrotic toxins (toxA), a variety of Outer Membrane Proteins (OMPs) as protective factors , and neuraminidase (nanB and nanH) [P. multocida to humans is usually due to bites, scratches, and licks by companion animals with the development of local inflammation and occasional abscess formation and, sometimes, ascending infection [P. multocida infections in humans are associated with respiratory symptoms, such as pneumonia, tracheobronchitis, lung abscesses, and pulmonary emphysema [The detection of  rabbits . Moreove animals . One of charides ,25. Manynd nanH) ,27,28,29nd nanH) . Transminfection ,32. Wounnfection ,34. Immunfection . Additionfection ,36. Thermphysema .Pasteurella multocida infection in rabbits as well. The aim of this work is to achieve more information on P. multocida from pet rabbits starting from a severe outbreak of pasteurellosis that occurred on a farm of pet rabbits routinely sold as companion animals and to discuss a preventive approach in order to reduce the spread of P. multocida among the animals.The significant increase in the number of families who choose a rabbit as a companion animal has led to giving more attention to The flock of pet rabbits was evaluated because they were suffering from respiratory syndrome cyclically for about two years. The flock was in Corato, BA, southern Italy, and consisted of about 100 rabbits, 90 females and 10 males, all reared to breed. The animals were Lion’s head and lop rabbits. They were housed in a shed where a climate control system ensured an ambient temperature of 15–18 °C and relative humidity of approximately 65%. The rabbits were fed with pellets, fresh vegetables, and hay. Each rabbit was housed in a single cage equipped with a drinking bottle and a feeder. Cages and sheds were regularly cleaned. The reproductive activity in the flock was scheduled throughout the year except for July and August, for the lacking demand for bunnies by customers in that period. The female animals were usually introduced in the cages of male rabbits and left for three days to allow natural mating and pregnancy. The rate of fertility was about 95%. The bunnies were regularly sold to private individuals and pet shops.The respiratory syndrome occurred in both bunnies and adults. Conjunctivitis was the most evident clinical sign. This symptom was particularly observed in bunnies , and it Conjunctivitis started with catarrhal discharge, but the ocular discharge quickly became muco-purulent and occluded the eyelids .Purulent rhinitis was frequently observed  and was Affected bunnies were often undersized. The number of bunnies involved in the syndrome in each litter was variable. In fact, all the litter showed clinical signs in some cases, while only some bunnies were symptomatic in others. Sometimes, all the litter appeared healthy, but respiratory syndrome often appeared later, at the weaning time or after the sale of the bunnies. Breeders showed chronic rhinitis with muco-purulent discharge, but mothers of affected litters were not always symptomatic. Subcutaneous abscesses and torticollis were rarely observed only in adults. Moreover, some cases of late abortus were reported in lop rabbits. The animals were treated locally with ophthalmic ointments (chloramphenicol and chlortetracycline) by the owner which, cyclically, also administered trimethoprim/sulfamethoxazole via drinking water without improvements in the clinical conditions. Nasal and conjunctival swabs were collected from twelve symptomatic rabbits for laboratory investigations.Pasteurella multocida, Bordetella bronchiseptica, and Staphylococcus aureus) or not associated with .Nasal and conjunctival swabs were plated on blood agar  and incubated at 37 °C for 24 h. Blood agar allows the growth of several different bacteria which can be associated with respiratory disorders (such as Pasteurella multocida (P. multocida) and contextually to define the capsular type. Briefly, DNA extraction was carried out by boiling for 10 min. The primer set used in the PCR reaction is reported in Three to five colonies from each plate were tested by Multiplex PCR according to  with somP. multocida was investigated starting from different studies focused on pathogenicity [The presence of 15 different genes encoding for virulence factors of genicity ,27,39,40The PCR mixture for Multiplex-PCRs consisted of 12.5 µL of 1X Platinum Mastermix , 0.5 µL of each primer pair in a 50 pmol/µL primary concentration, and ultra-pure nuclease-free water  until a final volume of 25 µL.Pasteurella multocida identification paragraph (2.1.) were used for A, B, and C Multiplex-PCRs. All PCR products were visualized by agarose gel electrophoresis (1.5%) which was colored with ethidium bromide (0.5 μg/mL), using TBE (Tris-Borato-EDTA) as conductor. PCR results were read with Gel Doc-It (UVP) image analyzer.The thermal conditions previously reported in The susceptibility of the identified bacterium to the antibiotics more frequently used in veterinary and human medicine to treat respiratory diseases and wounds from animals was evaluated. Therefore, the susceptibility to ciprofloxacin (CIP 5 μg/mL), sulfamethoxazole-trimethoprim (SXT 25 μg/mL), enrofloxacin (ENR 5 μg/mL), ampicillin (AMP 10 μg/mL), tetracycline (TE 30 μg/mL), nalidixic acid (NA 30 μg/mL), gentamicin (CN 10 μg/mL), amoxicillin-clavulanic acid (AMC 30 μg/mL), imipenem (IPM 10 μg/mL), cefotaxime (CTX 30 μg/mL), tilmicosin (TIL 15 μg/mL), amoxicillin (AX 25 μg/mL), doxycycline (DO 30 μg/mL), meropenem (MEM 10 μg/mL), azithromycin (AZM 15 μg/mL), and erythromycin (E 15 μg/mL) was determined. The standard Kirby–Bauer disk diffusion method on Muller–Hinton agar supplemented with 5% horse blood in aerobic and microaerophilic conditions was used according to European Committee on Antimicrobial Susceptibility Testing  and ClinPasteurella spp.   . No otheP. multocida belonging to capsular type A  for 15 days, obtaining the remission of rhinitis and conjunctivitis in bunnies and the improvement of symptoms in adults. One month after the end of the therapy, an inactive commercial vaccine was used to immunize the rabbits, obtaining a complete remission of the clinical form in the following six months.Pasteurella multocida was identified as responsible for the syndrome observed in a flock of pet rabbits. P. multocida can be spread by affected animals mainly through nasal discharge. The effectiveness of the therapy is crucial to reducing the clinical symptoms and decreasing the spread of the bacteria involved in the disease process.The correct diagnosis in veterinary medicine is of relevance for both animal and human health, considering that some pathogens can be transmitted to humans. P. multocida infections in rabbits, its in vivo efficacy is known to be lower than its in vitro efficacy. Therefore, the antibiotic treatment led only to the improvement of clinical conditions of the most heavily affected or chronically infected rabbits. Additionally, enrofloxacin was administrated by drinking water due to the high number of animals to be treated. Drinking water treatment can lead to non-uniform dosages because of different water intake by sick animals. In addition, after the antibiotic treatment, clinically healed rabbits often become carriers of P. multocida, with the possibility that the clinical signs will reappear later when adverse conditions occur [In vitro, the identified strain was susceptible to the antibiotics more frequently used in veterinary and human medicine, but the applied therapy appeared only partially efficacious in rabbits. Although enrofloxacin is the most suitable drug for treatments against ns occur .Pasteurella infection from injuries caused by pets, but also by direct exposure to the bacteria [P. canis and P. dagmatis may be found in bites from dogs and cats, P. multocida is the most prevalent species associated with human infections due to bites from companion animals [These events could lead to possible risks for owners that may acquire bacteria ,9,31,32. animals .P. multocida capsular type A was identified in the affected rabbits in this study. Type A is the serogroup prevalently associated with pasteurellosis in rabbits [P. multocida infection, which seems to affect them independent of gender and age. The clinical presentation of pasteurellosis in rabbits includes rhinitis, sinusitis, conjunctivitis, dacryocystitis, and pleuropneumonia, as well as otitis, encephalitis, and abscesses of the subcutaneous tissues or internal organs, bones, joints, and genitalia [ rabbits  and, int rabbits ,45, even rabbits , has bee rabbits ,45. Rabbenitalia ,48. Sympenitalia ,50,51 anenitalia .P. multocida such as fimbriae, adhesion and colonization factors , iron regulation factors and protein acquisition , superoxide dismutase (sodA and sodC), dermonecrotic toxins (toxA), a variety of Outer Membrane Proteins (OMPs) as protective factors , and neuraminidase (nanB and nanH) have been proposed as possible markers of virulence [P. multocida strains identified as responsible for disease in humans and other species [fim4, fimA, and tadD, responsible for fimbriae production and adhesion processes to host cells; sodA, sodC, nanB1, and nanH1, responsible for enzyme production, and hgbB, exbB, oma87, and fur, involved in iron acquisition processes. This finding seems to explain the evidence of symptoms observed in the affected rabbits as well as some difficulties related to achieving the complete resolution of the syndrome that was obtained in the rabbit flock about 6 months after using the vaccine. The use of vaccines to immunize the rabbits against P. multocida, in particular, is recommended to minimize the risk of outcomes of the clinical form, but also to reduce the spread of bacteria by the infected animals. Therefore, rabbits should be routinely vaccinated against P. multocida when considering that they are reared as household pets and the transmission of the pathogen to humans cannot be excluded. Nevertheless, the vaccine is not able to prevent the infection, and bunnies that are sold to individuals and pet shops may appear healthy, even when they carry the bacterium. Therefore, screening for P. multocida should be considered as a part of the routine medical check of rabbits, particularly when they are purchased as companion animals for children and elder people. Additionally, typing the detected strains of the bacteria is important to assess their pathogenic potential and, consequently, to increase hygienic measures in the management of pet rabbits.Many virulence genes involved in the pathogenicity of irulence ,27,28,29 species ,44, the Pasteurella multocida detected in pet rabbits. We suggest the routine use of vaccines in pet rabbits and screening for P. multocida in the routine veterinary examination of rabbits, particularly when they will be used for pet therapy or kept as companion animals for children and elder people, considering that the transmission of the pathogen to humans cannot be excluded.This study provides more knowledge about


FOS supplementation in rabbits enhanced growth carcass characteristics, significantly improving hematological parameters and antioxidant status, and minimized pathogenic Escherichia coli bacteria (from 3.45 in control groups to 2.89 and 2.24 (Log10 CFU g−1) in 0.5 mL and 1 mL FOS-treated rabbits, respectively.Rapidly rising incomes are dependent on animal protein production and the worldwide demand for livestock. It is expected that moving towards more intensive production systems to sustain this increased demand will depend on growth promoters. Some growth promoters, such as prebiotics, might be considered alternative non-antibiotic feed supplementation as they enhance performance without any side effects on the consumer’s health. The present study inspected the influence of supplementation of β-fructanEscherichiacoli population were considerably low, with a substantial increase in the number of Lactobacilli supplemented by FOS. In conclusion, FOS supplementation enhanced growth and carcass traits by improving the hematobiochemical parameters and antioxidant status and reducing cecal pathogenic bacteria in both breeds.The present study examined the effects of fructooligosaccharide (FOS) supplementation in drinking water on the growth performance, carcass characteristics, hematological and biochemical parameters, antioxidant status, and cecal microbiota of New Zealand White (NZW) and APRI rabbits. A total of 180 male NZW and APRI rabbits  were divided into six groups  in a two × three factorial arrangement. Rabbits of each breed were randomly assigned to one of three treatments of FOS . Results showed that rabbits’ final body weight, FBWG, and carcass traits were considerably enhanced compared to those in the control group. The interaction effect of the supplement with the rabbit breed increased the growth, carcass traits, and hematobiochemical and antioxidant parameters with increasing FOS levels. In the cecum of both rabbit breeds, the total bacterial count and  Saccharomyces cerevisiae, and has been reported to possess the ability to improve growth performance, decrease pathogenic bacterial count, and enhance immunity in two different rabbit breeds  [Improving animal productivity and boosting immunity using natural substances is a primary goal in animal breeding ,4,5,6,7.rabbits) .Lactobacillus and Bifidobacterium, and reduce the accumulation of pathogenic bacteria, such as Clostridium perfringens and Escherichia coli [FOS may accelerate the gut fermentation of beneficial microorganisms, such as hia coli ,17,18,19hia coli ,17. Therhia coli . Dietaryhia coli ,22,23.®, a commercial FOS) in drinking water on the productive performance, carcass characteristics, hematobiochemical parameters, oxidative stress, and cecal microbiota of New Zealand White (NZW) and APRI rabbits. We hypothesized that oral FOS supplementation in combination with rabbit breed would enhance growth, improve blood biochemistry and antioxidant status, and improve microflora population diversity to alleviate the weaning stress of the rabbits.To our knowledge, there are no reports on adding FOSs to the drinking water of growing rabbits. Hence, this study was conducted to detect the possible effects of adding FOS .®). The control group was not treated with FOS, and the first and second groups were supplemented with FOS-0.5 mL and FOS-0.1 mL, respectively. The experimental groups received 0.5 and 1.0 mL β-fructan  per liter of drinking water for three sequential days per week . Each rabbit in the 0.5 mL β-fructan-treated group was supplemented with 349.8 mg of β-fructan during the eight-weeks experimental period, while in the 1 mL β-fructan-treated group, each rabbit was supplemented with 699.75 mg of β-fructan. Rabbits were housed in galvanized wire batteries with standard dimensions (60 × 35 × 35 cm). All cages were supplied with galvanized steel feeding hoppers and automatic drinkers (nipples). Plastic ear tags identified rabbits. Freshwater was provided ad libitum, and a standard pelleted ration was provided ad libitum twice daily at 8 am and 2 pm. The pellets measured 1 cm in length and 0.4 cm in diameter. Rabbit cages were regularly cleaned and disinfected. Urine and feces dropped beneath the batteries were removed every morning.APRI rabbit was produced by crossing Baladi Red bucks with a V line to create F1 (½B½V) stock, and it was continued for two generations of intersex mating to attain performing constancy. A total of 180 weaned APRI and NZW rabbits  were collected and allocated to six groups (30 rabbits per group), and each group was divided into five replicates, each with six rabbits. The rabbits were assigned at random using a two × three completely factorial design . The rabbit’s daily feed consumption was calculated every week to evaluate the feed conversion ratio (FCR). Final body weight (FBW), body weight gain (BWG), and total feed consumption (TFC) were determined. Fifteen rabbits from each group were randomly selected to evaluate carcass characteristics at the end of the experiment (13th week). Rabbits were fasted for 12 h before being slaughtered. After removing the skin and bones, the carcasses were measured individually to evaluate the weight and percentage of the dressed animals. The offal weight includes blood, viscera, lungs, skin, arms, and tail. The obtained results were presented as the % of live weight . The dreg) at 15–24 °C for plasma separation and stored at −20 °C until analysis. Total protein, albumin, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine levels were measured in plasma using commercial kits. Moreover, the levels of glutathione peroxidase (GPX), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC) were evaluated using the colorimetric method .Two blood samples were collected from the lateral ear vein (30 rabbits) during the slaughter. One sample contained an anticoagulant and was used to determine the count of white blood cells (WBCs), red blood cells (RBCs), lymphocytes, monocytes, and mean corpuscular hemoglobin (MCH), red cell distribution width (RDW), platelet count, hematocrit %, and hemoglobin concentration . The othE. coli, and lactobacilli were all counted using the ring-plate method in the rabbit cecum sample [Total bacteria, m sample ,30.®  [ijk = μ + Si + Ej + SEij + eijK, where μ = observed mean for the concerned treatment, Si = breed effect, Ej = treatment effect, SEij = interaction effect of breed and treatment, and eijk = the error related to individual observation using Duncan’s multiple range test [p ≤ 0.05.The attained results were statistically analyzed with the general linear model procedure of SASNC, USA) . HomogenNC, USA) . The anange test . Accordinge test , the perp < 0.001) the amount of feed consumed in both breeds. The NZW rabbits that consumed water supplemented with 1% and 0.5% FOS and the APRI rabbits that consumed 1% FOS-supplemented water showed the lowest (p < 0.001) FCR compared with the other groups  compared with the control groups  were found in the two breeds in hematobiochemical and antioxidant parameters , with a substantial increase in the Lactobacillus population compared with the control groups. Rabbits that consumed 1% FOS showed the most significant count of beneficial bacteria and a lower count of pathogenic ones compared with the other treatments.In both rabbit breeds that consumed FOS-supplemented water, the cecum, total bacterial, and ulations  were conThis study investigated the possible effects of adding FOS in drinking water on the growth performance, carcass characteristics, hematobiochemical parameters, oxidative stress biomarkers, and cecal microbiota of NZW and APRI rabbits. Our results showed that supplementing water with FOS significantly enhanced the growth performance traits of the two rabbit breeds.The beneficial effects of adding FOS to the drinking water of growing rabbits may be due to the augmentation of feed efficiency and absorption, which improves anabolic metabolism, enhances the intestinal response to pathogens, and increases serum protein levels, thereby encouraging rabbit growth ,6,7. PreBacillus subtilis and FOS with a more significant average daily BWG than the control [® (Propionibacterium acnes and coli lipopolysaccharides) as a prebiotic in the drinking water of fattening NZW rabbits resulted in an enhancement of BW at marketing, BWG, and FCR [S.cerevisiae and probiotics accelerated the BWG and FCR of NZW rabbits [S.cerevisiae did not affect rabbits’ BW, BWG, and FCR. Additionally, Zarei et al. [Consistent with our findings, the rabbits’ growth was enhanced with  control . In addi and FCR . Compara rabbits ,35. By c rabbits ,37 foundi et al.  reportedi et al.  conclude®/litter. However, Rotolo et al. [Regarding carcass characteristics, our findings were consistent with those observed by Abd El-Aziz et al. , Mahroseo et al.  found noo et al.  concludeo et al. ,7.Hematological measurements are valuable indicators for evaluating the animals’ health statuses . In our Lactobacillus profile, which shows diverse patterns for dendritic cell activation [−1 dietary oligofructose. The high leukocyte count may increase activity and improve defense mechanisms during feeding. Leukocytes are imperative cells that stimulate the immune responses of fish. They produce antibodies and may exhibit macrophage activities [In a study on birds, FOS supplementation resulted in low heterophil counts, indicating that FOS may reduce stress reactions and alleviate the possible damaging consequences on growth performance . Moreovetivation ,42. The tivation  mentionetivities . Saha ettivities  obtainedThe total protein and globulin levels were increased in the experimental groups treated with varying levels of FOS in their diets, indicating a more robust innate immune response. Globulin is believed to be the main protein that plays a significant role in immune response . MoreoveInterestingly, our findings showed an increase in SOD, GPX, and T-AOC values in the supplemented groups of the two rabbit breeds. These results suggest that the FOS-supplemented drinking water could alleviate oxidative stress in the two breeds of growing rabbits and maintain their healthy. The first line of antioxidant enzymatic defense is believed to involve GPX, SOD, and T-AOC , which aLactobacillus). Moreover, FOS supplementation decreased the total bacterial count and harmful or potential pathogens (E. coli) in the two rabbit breeds. Our results are consistent with those reported by Xu et al. [Bifidobacterium and Lactobacillus, with an immediate reduction of E. coli growth in the broilers’ gastrointestinal tract. Saminathan et al. [Lactobacillus species from the gastrointestinal tract of fowls. The in vitro data revealed that Lactobacillus species utilized FOS more competently than mannan oligosaccharides. The increased availability of FOS may be related to particular enzymatic actions and the oligosaccharide conveyance technique of Lactobacillus species. Nevertheless, broilers’ intestinal microbiota is further complicated than in vitro examinations. Prebiotics may be fermented not only by Lactobacillus species but also by other microbes in the gastrointestinal tracts of animals [In the present study, FOS supplementation in drinking water caused a stimulatory impact on the growth of health-supporting bacterial species (u et al.  who examn et al.  evaluate animals .FOS supplementation in the drinking water of rabbits improved most growth performance parameters, carcass characteristics, hematobiochemical parameters, antioxidant status, and cecal microbiota in NZW and APRI rabbits. Moreover, the response of NZW rabbits to FOS supplementation was more significant than that of APRI rabbits.


The lack of calcium signals selectively observed in PTEC, in which BzATP still retains its functional effect, suggests variability of intracellular signaling among TEC. These findings provide novel insights into the role of extracellular ATP as a multisensory regulator of migratory potential in tumoral endothelium.Extracellular ATP is highly concentrated in tumor stroma. In this study, we investigated the effects of the synthetic ATP analog Benzoylbenzoyl-ATP, 2′(3′)-O-(4-Benzoylbenzoyl)adenosine 5′-triphosphate (BzATP), an agonist for P2X receptors, on tumor-derived endothelial cells (TEC) obtained from three different human tumors . Treatment with high BzATP concentrations (100 µM) significantly reduced migration of all TEC types, resulting ineffective on human normal microvascular endothelium (HMEC); intriguingly, both the functional effect and associated calcium signals are sensitive to some key biological parameters of tumor stroma that include pH, Ca2+) and calcium (Ca2+). We previously reported the ability of some purinergic agonists to exert an anti-migratory activity on tumor-derived human endothelial cells (TEC) only when applied at a high concentration. They also trigger calcium signals associated with release from intracellular stores and calcium entry from the external medium. Here, we provide evidence that high concentrations of BzATP (100 µM), a potent agonist of P2X receptors, decrease migration in TEC from different tumors, but not in normal microvascular ECs (HMEC). The same agonist evokes a calcium increase in TEC from the breast and kidney, as well as in HMEC, but not in TEC from the prostate, suggesting that the intracellular pathways responsible for the P2X-induced impairment of TEC migration could vary among different tumors. The calcium signal is mainly due to a long-lasting calcium entry from outside and is strictly dependent on the presence of the receptor occupancy. Low pH, as well as high extracellular Zn2+ and Ca2+, interfere with the response, a distinctive feature typically found in some P2X purinergic receptors. This study reveals that a BzATP-sensitive pathway impairs the migration of endothelial cells from different tumors through mechanisms finely tuned by environmental factors.The tumoral microenvironment often displays peculiar features, including accumulation of extracellular ATP, hypoxia, low pH-acidosis, as well as an imbalance in zinc (Zn Extracellular purines and pyrimidines, including ATP, ADP, UTP and UDP, play important roles in a variety of physiological and pathological processes ,2. Their2+) c measurements, cells were loaded with 2 μM Fura–2AM  for 30 min at 37 °C and fluorescence was acquired by Nikon Eclipse TE-2000S  inverted microscope and Metafluor Imaging System . The sample was then excited at 340 nm and 380 nm alternatively, and the [Ca2+]c was expressed as a ratio (R) of emitted fluorescence at 510 nm corresponding to two excitation wavelengths.Cells were seeded at a density of 5000 cells/cmFor each experiment, at least 20 regions of interest (ROIs) were selected, each corresponding to a single cell in the chosen image field, and images were acquired every 3 s. In order to limit noise, real-time back-subtraction was applied.2+, the area underlying the Ca2+ influx during the sustained phase was evaluated at 300 s after the onset of the response. The total area under Ca2+ spikes in 0 Ca2+out was measured using the Event Detection protocol in Clampfit 11.1. Calcium imaging analysis was performed by peak amplitude and area quantification using Clampfit 11.1  and GraphPad Prism 6  for statistical analysis. Peak amplitude was calculated as the highest value of R within 300 s following the treatment. In the presence of extracellular Ca® GmbH, Planegg, Germany), with a density of 5 × 106 cells/mL. Cells were maintained in an incubator  until confluence within the 70 µL chambers was reached. Cell monolayers were starved for 2 h in EndoGRO 0% FBS, and then the inserts were removed. A wash with PBS w/Ca2+ and w/Mg2+ solution was completed before adding the treatments in duplicate. EndoGRO 5% FBS w/oVEGF was used as positive control (CTR).In vitro wound healing culture-insert assay. Cells were seeded in EndoGRO 5% FCS on 12-well culture plates using 3-well silicone culture inserts  [Images were acquired using a Nikon Eclipse Ti-E microscope with a 10× objective. Cells were kept at 37 °C and 5% COCA, USA) ,44. MigrCA, USA) ,46. At l2 on 12-well culture plates coated with 1% gelatin in EndoGRO 5% FCS. Cells were then washed with PBS w/Ca2+ and w/Mg2+ solution and treatments were added in duplicate. EndoGRO 5% FCS w/o VEGF was used as control (CTR). The results represent normalized values against the corresponding CTR obtained in each of the three independent experiments carried out for each experimental condition.Random Migration Assay. BTEC were seeded at a density of 4000 cells/cmExperiments were performed using the same set-up described above but using a Nikon Plan 20X objective. Images were acquired for 10 h every 10 min using MetaMorph® Software. Image stacks were then analyzed with ImageJ 1.8.0, and at least 250 cells/condition were tracked. Migration rate (µm/min) was obtained by measuring the distance covered by cells between two subsequent time points after conversion of pixels to micrometers. At least five fields for each condition were analyzed in each independent experiment. The results represent normalized values against the corresponding CTR obtained in each of the three independent experiments carried out for each experimental condition.t-test (or the non-parametric Mann–Whitney test) or the one-way ANOVA . A p-value < 0.05 was considered significant.Data were analyzed with GraphPad Prism 6 . In order to check the normal distributions of each dataset, a preliminary Shapiro–Wilk test was performed and accordingly to that, and to the number of datasets to be compared, statistical analysis was performed by using the Student’s The first set of experiments was conducted in order to verify and extend what was previously shown in BTEC to RTEC and PTEC. The application of 100 µM ATP significantly reduced migration in all three cell types; on the other hand, a lower concentration (1 µM) was ineffective A.The application of 100 µM BzATP impaired wound healing in BTEC starting from 8 h, with a relative effect being even more marked at 16 h B. ConverPre-incubation with 100 µM pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), a broadly used non-selective P2X antagonist, prevented this effect on all the TEC types E–G. Simi2+ increase was strictly dependent on the presence of the agonist, as revealed by the rapid recovery to the resting Ca2+ levels following agonist removal  completely prevented the response to BzATP E. Taken 2+ and Ca2+. In particular, their increased concentration inhibits P2X7 pore activation. For this reason, we investigated the effects of the Ca2+ response to 100 µM BzATP.A remarkable feature of P2XRs is their sensitivity to different environmental ions, including protons, Zn2+ increase triggered by BzATP in both BTEC and RTEC (Pre-incubation with an acidic medium (pH 6.4) strongly reduced the Caand RTEC A,E, as cand RTEC B–D,F–H.2 (but not 10 µM) or 10 mM CaCl2 in BTEC   and on sHere, we report that high BzATP concentrations reduce the migratory potential of all TEC used but not of normal microvascular ECs (HMEC).2+ signal is long-lasting and strictly dependent on the presence of the agonist and receptor occupancy: this feature fits well with the involvement of slow-inactivating P2X ionotropic receptors.The same stimulation evoked an acute cytosolic calcium increase in BTEC, RTEC and HMEC. The Ca2+ signals confers an acid-sensitivity to the purinergic signaling, a property that could be highly relevant in the tumor microenvironment usually associated with strong acidosis associated with hypoxia. Direct pH sensitivity is compatible with the recruitment of some P2XR isoforms; moreover, the depression of Ca2+ signals and the recovery of TEC migration by high Zn2+ concentration (50 µM) nicely agrees with the contribution of P2XRs, as well as the modulation by increased extracellular Ca2+ concentration [The ability of low pH to abolish Cantration 0 µM nicentration ,45.2+, Ca2+ and low pH. In addition, heteromeric P2XR complexes could be expressed in TEC, with peculiar pharmacology, already found in normal vascular endothelial cells, including P2X4/7 [Different P2XRs could account for the strong sensitivity to extracellular Zng P2X4/7 ,40. In ag P2X4/7 . Some P22+ and Ca2+ on other cellular targets.However, our data cannot rule out indirect interference of pH, Zn2+ signals, clearly points to the involvement of P2X ionotropic receptors.In addition to the modulation by inorganic ions, the sensitivity to suramin and PPADS, together with the long-lasting receptor-dependent Ca2+-dependent events and the alteration of migratory potential in TEC treated with BzATP, their causal relationship remains unclear. Indeed, here, we show that BzATP fails to exert any detectable Ca2+ signals in PTEC but retains the ability to inhibit migration; in this particular cell type, the purinergic stimulation could be mediated by non-canonical functions of P2XRs, independently of their ion conductivity. This observation agrees with recent evidence provided by our lab showing that UTP triggers strong Ca2+ increases  without any interference with BTEC migratory potential [Although the apparent correlation between Caotential . The intIn conclusion, the evidence supports the hypothesis of a key role of P2X receptors in the selective control of TEC migration. This event is modulated by a number of key environmental factors usually found in tumor stroma.The data provided in this paper are promising but deserve further investigation, particularly concerning two aspects. Firstly, the detailed definition of the molecular composition of the P2X complexes gathered at the TEC plasma membrane, their interactome and the intracellular machinery responsible for the regulation of endothelial migration in tumors. These mechanistic advancements are required for the set-up of new pharmacological approaches aimed at interfering with tumor vascularization. A second remarkable issue is related to the biological meaning of the antimigratory activity exerted by eATP accumulation in the broad context of in vivo vascular remodeling and angiogenesis.High concentrations of the ATP analog, BzATP, a P2XR agonist, are anti-migratory for different human TECs  but not for normal microvascular ECs.2+ and Zn2+ ions, all established hallmarks of tumoral stroma, together with abnormally high levels of eATP. While some features are shared by all TEC tested, intracellular signaling pathways are variable and deserve further mechanistic insight.The functional effect of BzATP is modulated by extracellular pH, Ca