Recently, the United States Food and Drug Administration approved a new oral dosage preparation of amlodipine besylate (AML) and celecoxib (CEL) for the management of hypertension and osteoarthritis. However, no simultaneous estimation procedures for these two analytes have been described. Hence, two simple, accurate, and precise ultraviolet spectroscopic procedures that manipulated the ratio spectra were established for concurrent quantification of AML and CEL using ethanol as a solvent. The first method involves determining the peak-to-trough amplitude difference of the ratio spectra of AML and CEL. The second method involves determining the peak amplitude of the ratio first derivative (Δλ 4 nm) spectra of AML and CEL at 334.2 nm and 254.2 nm, correspondingly. Both methods showed linearity in the range of 1–6 μg mL-1 for AML and 5–40 μg mL-1 for CEL with an excellent correlation coefficient (<0.999). The proposed procedures were validated by following the International Conference on Harmonization guidelines for accuracy, precision, selectivity, recovery, and stability studies. It is evident from the low %RSD and %RE that both analytical procedures were found to be accurate and precise, respectively. The percent recovery of AML and CEL from the formulation was found to be 99.79% and 99.34% using the ratio-difference method and 100.13% and 99.70% using the ratio first-derivative method, with a low percent relative standard deviation. Further, the proposed techniques permit concurrent quantification of AML and CEL in different concentration ratios without interference from each other; hence, these techniques can be adopted for regular quality-control studies. Middle-aged and older adults are suffering from many health problems, such as hypertension, diabetes, and osteoarthritis due to modern lifestyles and stress. Calcium-channel blockers are the most extensively prescribed medication in the management of hypertension due to its long duration of action. Amlodipine besylate AML; , a dihydOsteoarthritis is a degenerative disorder of the articular cartilage that leads to structural modifications of the bone.  OsteoartDifferent analytical techniques were described in the public domain for the quantification of AML from pharmaceutical preparations and plasma. AML alone has been estimated by spectrophotometric, , 10 fluoFew spectroscopic procedures have been described in the estimation of CEL alone and various drug combinations. –28 Many Instrumentation: A Shimadzu UV-Vis spectrophotometer  associated with the workstation was used for the development of the analytical methods. Then, 10 mm quartz cuvettes were used to measure solution absorbance. The slit width and scan speed were adjusted to 1 nm and medium, respectively. The manipulation of scanned spectra was performed using Shimadzu software . Analytes and formulations were weighed using a digital balance.Chemicals: Pure samples of AML and CEL were purchased from Sigma-Aldrich Co. . Analytical-grade ethanol was procured from Sigma Aldrich Co. Pure water was prepared using the Milli Q  water purification system, which was used throughout the experiments. AML (10 mg/tablet) and CEL (200 mg/capsule) were obtained from the pharmacy.The tablets were prepared in a laboratory and included 200 mg CEL, 10 mg AML / 5 mg AML, 10 mg sodium starch glycolate type A, 12.5 mg talc, 10mg microcrystalline cellulose, 5 mg magnesium stearate and 8 mg lactose per tabletTo prepare the standard stock solutions of AML and CEL, 100 mg of AML and CEL were accurately weighed into two 100 mL measuring flasks, separately. Both analytes were dissolved using 60 mL of ethanol, and the final volumetric capacity reached 100 mL with the addition of ethanol. Standard solutions were kept in the refrigerator (4°C) until use. Standard solutions were used to prepare the calibration curve and for validation purposes, and laboratory-prepared solutions were arranged by transferring the required amount of standard solutions into 10 mL volumetric flasks.AML and CEL combination tablets were not available in Saudi Arabia; hence, AML (10 mg) tablets and CEL (200 mg) capsules were used to prepare the samples. The weight of 20 AML tablets was determined; the average weight was computed, and the tablets were crushed into a smooth powder. Similarly, the powder of 20 CEL capsules was collected and the average weight was computed. AML powder corresponding to 50 mg of AML, as well as CEL capsule contents corresponding to 1,000 mg of CEL, were transferred and mixed thoroughly. Then, the powder mixture, corresponding to 10 mg of AML and 200 mg of CEL, was moved into a 100 mL measuring flask consisting of 60 mL of ethanol. The contents were dissolved with the help of ultrasonication (10 minutes). The solution was filtered using Whatman filter paper in a 100 mL measuring flask. The filter paper and remaining solution were rinsed with fresh ethanol; the final volume was adjusted up to the level with ethanol. The required amount of sample fraction was transferred into a 10 ml measuring flask and the ethanol was added to obtain the analyte quantity, which fell in the calibration range.Laboratory-prepared tablets were powdered, and powder equivalent to 10 mg of AML and 200 mg of CEL, as well as 5 mg of AML and 200 mg of CEL, was transferred separately into two conical flasks. Analytes were extracted by adding ethanol (30 ml x3). Each time solutions were sonicated for 10 min, filtered into 100 ml measuring flasks and the residue was washed with fresh ethanol. The final volume of both solutions were adjusted to 100 mL with ethanol. Further, solutions were diluted with ethanol to obtain a concentration in the range of the calibration curve.-1  of AML and 5–40 μg mL-1  of CEL were prepared by diluting the standard solutions with ethanol. AML and CEL solutions with a concentration 2 μg mL-1 and 10 μg mL-1, respectively, were arranged independently. UV absorption spectra were recorded for all solutions in the UV range of 200–400 nm using ethanol as a blank; the spectra were stored on a computer. Then, the ratio spectra were constructed by dividing the stored spectra by the CEL spectra of the 10 μg mL-1 solution; the ratio spectra were stored to perform the ratio-difference method. Further, the ratio spectra were converted into first-derivative spectra using 4 nm as Δλ. Similarly, the ratio spectra for CEL were constructed by dividing the combined spectra of the AML and CEL solutions by the AML spectra of the 2 μg mL-1 solution. Further, these spectra were converted into first-derivative spectra using 4 nm as Δλ. For the ratio-difference method, the peak amplitude difference was calculated by subtracting the amplitude of the trough at 345.6 nm from the amplitude of the peak at 336.4 nm for AML, and 236.5 nm from 266.8 nm for CEL. Then, the calibration curves were constructed by plotting a graph between the difference in peak amplitude with the corresponding concentrations of AML and CEL. Alternatively, regression equations were developed from the calibration curve.The six working standard solutions comprising of 1–6 μg mLFor the first-ratio-derivative method, peak heights were determined at 334.2 nm for AML and 254.2 nm for CEL. Then, the calibration curves were created by drawing a graph between the peak amplitude and its relevant concentration. Alternatively, regression equations were developed from the calibration curve.-1, respectively. Normal UV spectra were recorded for all solutions in the UV range of 200–400 nm; these spectra were converted into ratio spectra and the first-derivative of ratio spectra. Amplitudes were determined by adopting the above procedure; concentrations were determined by the regression equation.Nine mixed solutions were prepared in the laboratory by transferring the aliquot of AML and CEL stock solutions to obtain concentrations of 1:10, 2:10, 3:10, 1:20, 2:20, 3:20, 1:30, 2:30, and 3:30 μg mLNormal absorption spectra for AML and CEL  showed cAn˚ = εn˚ Cn˚], which results in Eq ; Eq  can be s1m and P2m represent the peak amplitudes of the ratio spectra at two wavelengths, λ1 and λ2, respectively.Constant K can be eliminated by determining the difference between the peak amplitude (ΔP) at two different wavelength points (λ1 and λ2), according to Eq :ΔP=PM1−Hence, the component n is canceled; the peak amplitude difference represents only component m. Component m can be determined from a mixture of components by constructing the calibration curve between the difference in peak amplitudes at two different wavelengths of the ratio spectra against the concentration of m. Similarly, following the same process, another component n can be determined in the presence of component m.-1; -1) solution  in Eq  and provAlso, a comparison of the first derivative of the ratio spectra for the combined AML and CEL, as well as the pure standard AML, showed the same amplitude at 334.2 nm,  and wherValidation of the proposed manipulation of the UV-ratio spectroscopic methods was carried out in terms of linearity, the limit of detection, the limit of quantification, accuracy, precision, recovery studies, selectivity, and stability studies following International Conference on Harmonisation (ICH) guidelines.-1 and 5–40 μg mL-1, correspondingly  when compared to the three steps required for the ratio-derivative method . Further, both methods are simple, fast, sensitive, and specific; they work without applying complicated equations or separation procedures. Hence, these techniques can be utilized to perform regular quality control studies of solid dosage forms that have different ratios of AML and CEL.S1 FigCalibration curve for ratio difference method AML (A), CEL (B) and ratio first derivative method AML (C), CEL (D).(DOCX)Click here for additional data file.S2 Fig-1 using 2 μg mL-1 solution spectra of AML. (B) First derivative of Ratio spectra of CEL 20,30,40 μg ML-1 using 2 μg mL-1 solution spectra of AML.(C) Ratio spectra of AML 1, 1.5, 2 μg mL-1 using 10 μg mL-1 solution spectra of CEL.(D) First Derivative of ratio spectra of AML 1, 1.5, 2 μg mL-1 for precision and accuracystudies using 10 μg mL-1 solution spectra of CEL for accuracy and precision studies.(A) Ratio spectra of CEL 20,30,40 μg ML(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig-1 using AML 2 μg ml-1. (B) First derivative (Δλ 4 nm) of ratio spectra of CEL 20, 25, 30 35 μg ml-1 using AML 2 μg ml-1. (C) Ratio spectra of AML 1, 1.5, 2, 2.5 μg ml-1 using CEL 10 μg ml-1. (D) First derivative (Δλ 4 nm) of ratio spectra of AML 1, 1.5, 2, 2.5 μg ml-1 using CEL 10 μg ml-1 for recovery studies.(A) Ratio spectra of CEL 20, 25, 30 35 μg ml(DOCX)Click here for additional data file.S5 Fig-1 tablet solution using AML 2 μg ml-1 as divisor, Ratio spectra (C) and First derivative (Δλ 4 nm) of ratio spectra (D) of AML: CEL 1:20 AND 1:40 μg ml-1 tablet solution using CEL 10 μg ml-1 as divisor.Ratio spectra (A) and First derivative (Δλ 4 nm) of ratio spectra (B) of AML: CEL 1:20 and 1:40 μg ml(DOCX)Click here for additional data file.


We compare here the effects of TRPM8 activation on VEGF-induced transactivation of TRPV1 in an UM cell line (92.1) with those in normal primary porcine melanocytes (PM) since TRPM8 is upregulated in melanoma. Fluorescence Ca2+-imaging and planar patch-clamping characterized functional channel activities. CAP (20 μM) induced Ca2+ transients and increased whole-cell currents in both the UM cell line and PM whereas TRPM8 agonists, 100 μM menthol and 20 μM icilin, blunted such responses in the UM cells. VEGF (10 ng/ml) elicited Ca2+ transients and augmented whole-cell currents, which were blocked by capsazepine  but not by a highly selective TRPM8 blocker, AMTB (20 μM). The VEGF-induced current increases were not augmented by CAP. Both 3-T1AM (1 μM) and menthol (100 μM) increased the whole-cell currents, whereas 20 μM AMTB blocked them. 3-T1AM exposure suppressed both VEGF-induced Ca2+ transients and increases in underlying whole-cell currents. Taken together, functional TRPM8 upregulation in UM 92.1 cells suggests that TRPM8 is a potential drug target for suppressing VEGF induced increases in neovascularization and UM tumor growth since TRPM8 activation blocked VEGF transactivation of TRPV1.In human uveal melanoma (UM), tumor enlargement is associated with increases in aqueous humor vascular endothelial growth factor-A (VEGF-A) content that induce neovascularization. 3-Iodothyronamine (3-T Among all cancers of the eye, uveal melanoma (UM) is the most frequent form in adults. Notably, UM is mostly found in the choroid  and in ciliary body (15%), but it rarely occurs in the retina  namely the canonical receptor type 4 or 6 (TRPC4 or TRPC6) in human microvascular endothelial cells  are overexpressed in prostate tumor cells  is a decarboxylated thyroid hormone (T3 and T4) metabolite, which activates G protein-coupled receptors (GPCRs) especially the trace amine associated receptor 1 (TAAR1). It also induces a dose-dependent reversible 10°C decrease in mice body temperature . CPZ and icilin were procured from Cayman Chemical Company . Medium and supplements for cell culture were ordered from Life Technologies Invitrogen  or Biochrom AG . Melanocyte Growth Medium M2 was obtained from Promocell . Dispase II was ordered from Boehringer  and accutase was provided by PAA Laboratories . Unless otherwise stated, all other reagents were procured from Sigma .2 atmosphere and 80% humidity  was kindly provided by Martine Jager and colleagues   is an index of relative intracellular Ca2+ ([Ca2+]i) levels . Cutoff filters isolated alternative fluorescence excitation at 340 and 380 nm every 5 s wavelengths provided by a LED light source . Fura-2 fluorescence was alternately excited at 340 and 380 nm and emission was detected at 510 nm (250 ms−3.8 s exposure time). For image acquisition and data evaluation, the Life Science imaging software cellSens was used . Results are shown as mean traces of the f340nm/f380nm ratio ± SEM (error bars in both directions) with n-values indicating the number of experiments per data point. The Ca2+ data presented from many cells in several experiments were normalized  and averaged (with error bars). The time delay of 1–2 min in inducing a Ca2+ transient stems from exposing the cells to a stationary bath rather than a flow through superfusion. When drugs were solubilized in dimethyl sulfoxide (DMSO) solution, their working concentration did not exceed 0.1%, which did not alter the Ca2+ base line.Semi confluent cells (≈80%) were loaded with fura-2/AM (2 μM) at 37°C. After about 40 min, the cells were washed with a Ringer-like (control) solution containing (mM): 150 NaCl, 6 CsCl, 1 MgCl©; Nanion, Munich, Germany) in connection with an EPC 10 patch-clamp amplifier  and the PatchMaster software  . The external solution contained (mM): 140 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 5 D-glucose monohydrate and 10 HEPES, pH ≈ 7.4, and osmolarity ≈ 298 mOsM. At first, 5–10 μl of a single cell suspension were placed onto a microchip containing the aforementioned external solution. A software-controlled pump (Nanion) provided a connection between a single cell and the electrical system . The mean membrane capacitance was 10 pF ± 1 pF (n = 88) and mean access resistance was 25 ± 3 MΩ (n = 88). Series resistances as well as fast and slow capacitative transients were compensated by the PatchMaster software. The liquid junction potential was calculated . If these tests failed, non-parametric Wilcoxon matched pairs were used. The Student's t-test was also used for unpaired data, if the data also passed the aforementioned normality tests. If these tests failed, the non-parametric Mann-Whitney-U test was performed. Welch's correction was applied if data variance of the two groups were too different. Probabilities of p < 0.05 [indicated by asterisks (*) and hash tags (#)] were considered to be significant. The number of repeats is shown in each case in brackets, near the traces or bars. All values are means ± SEM (error bars in both directions). All plots were generated with SigmaPlot software version 12.5 for Windows  and with GraphPad Prism version 5.00 for Windows , respectively.The paired two-tailed Student's 2+ transients induced by the highly selective TRPV1 agonist, capsaicin . Interestingly, CAP evoked a biphasic or delayed effect on intracellular Ca2+ increase in PM, which was absent in UM 92.1 tumorous cells. In UM 92.1 cells, CAP instead increased the f340nm/f380nm ratio more promptly but to the same level; namely, from 1.199 ± 0.001 (80 s) to 1.215 ± 0.002 after 590 s  CPZ for TRPV1 . Subsequently CAP failed to significantly enhance the whole-cell currents, which stabilized at − 35 ± 7 pA/pF and 154 ± 14 pA/pF respectively  to −25 ± 9 pA/pF  to 142 ± 40 pA/pF, which AMTB suppressed to 112 ± 45 pA/pF   Figure . Similar1AM and BCTC increased and inhibited Ca2+transients, respectively  induced a [Ca2+]i transient  to 0.2094 ± 0.0013 (300 s)  whereas a washout did not reduce the Ca2+ level. With 10 ng/mg VEGF instead of CAP  and a washout did not augment this response Ca2+ transient  and to 0.2187 ± 0.0034 at 600 s   to 0.2212 ± 0.0021 (250 s)    Figure . In cont2+]i in UM 92.1 cells. WIN 55,212-2 induced a Ca2+ transient at a different cell passage compared to our previous studies . This validates CB1 involvement in Ca2+ regulation in UM 92.1 cells   augmented this rise induced by 3-T1AM. The transient reached with 3-T1AM by itself was 0.2155 ± 0.0014 (n = 13) at 400 s only whereas with AM251 (10 μM) it rose to 0.2446 ± 0.0037 at the same time    is expressed in uveal melanoma cells  was used in this study, variations may occur in gene expression profiles between UM primary tumors and their derived cell lines. Nevertheless, there were only moderate modifications in the gene products . However, with 1 μM 3-T1AM VEGF-induced rises in Ca2+ influx also declined with a time course that was similar to that obtained with CPZ (Figures 1AM is membrane delimited rather than causing release of Ca2+ from intracellular stores. Perhaps, 1 μM 3-T1AM is at a high enough concentration for it to gain access to cytosolic intracellular TRPV1 binding sites with which CPZ also binds? If this supposition is proven to be correct, 3-T1AM may have dual effects that include activating TRPM8 and at higher concentrations also suppressing TRPV1 activation induced by VEGF. As the effects of modulators of TRP channel activity on Ca2+ influx mirrored those on whole-cell currents (Figures 1AM has a relevant role in regulating VEGF-mediated Ca2+ regulation in tumor cells. In addition, we could demonstrate that TRPM8 activation by menthol or icilin mimic the 3-T1AM effect and is able to suppress increases in TRPV1 activity by VEGF (Figures 2+ signal transduction pathways activated by 3-T1AM as a specific activator of TRPM8 may be more complex as suggested in Figure The 3-T Figures , 7E, 11D Figures  and now 1AM also regulates beta-adrenergic receptors, trace amine-associated receptor 2, muscarinic receptors, and K+ channels (Scanlan et al., 1AM has also been described as an antagonist of muscarinic type 3 receptor (Laurino et al., + channel activity have been implicated in modulating progression of melanoma (Oppitz et al., 1AM may not be directly or solely targeting TRPM8 (Figure There are numerous studies showing that 3-T8 Figure .1AM is a multitarget ligand (Zucchi et al., 1AM interacts also with the cannabinoid receptor 1 CB1. CB1 activation by WIN 55,212-2 induced Ca2+transients, which were larger in a Ca2+ free conditions than with 2 mM external Ca2+ (Figures 1AM failed to elicit a Ca2+ transient in a Ca2+ free medium (Figure 2+ in the medium 3-T1AM-induced Ca2+ transients that were larger in the presence of the CB1 blocker than in its absence (Figures 1AM modulation of TRPV1 channels may also involves contributions by other receptors such as CB1 and/or its coupled G-protein mediators.It has been suggested that 3-T Figures . On the m Figure  whereas  Figures . Since C Figures , there m1AM is a potential therapeutic agent for suppressing UM expansion as already indicated in other studies demonstrating that this thyroid hormone metabolite reduces cancer cell growth and viability (Rogowski et al., 2+ influx in endothelial cells, which is a requisite for driving angiogenesis and promotes UM proliferation and metastasis. Since TRPM8 activation has an opposing effect on TRPV1 activity, targeting TRPM8 may provide an effective alternative to suppress metastatic melanoma progression without side effects. Such an approach appears to be tenable based on the fact that functional TRPM8 expression is only detectable in the UM cells rather than PM cells. There is an urgent need for further assessment of the validity of this option since there are no measures currently available to reverse melanoma metastasis.3-TLW, CB, SM, and PR designed the study, analyzed the data, wrote and edited the manuscript. LW and CB contributed equally to the work. JK contributed with his expertise on thyroid hormone metabolites, discussed data and their interpretation and helped edit the manuscript. LW, CB, AB, PD, NL, MS, HvdW, IR, and SM performed calcium measurements and planar patch-clamp recordings as well as plot analyses. LW, CB, AB, PD, MS, HvdW, and SM created diagrams. SM created the schematic drawing.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 dynamic symmetry reduction by the moving order parameter allows us to separate electron dynamics from spin dynamics. As transient symmetry reductions are common to coherent excitations, we have a general tool for tracking the ultrafast motion of an AFM order parameter.The unique functionalities of antiferromagnets offer promising routes to advance information technology. Their compensated magnetic order leads to spin resonances in the THz-regime, which suggest the possibility to coherently control antiferromagnetic (AFM) devices orders of magnitude faster than traditional electronics. However, the required time resolution, complex sublattice interactions and the relative inaccessibility of the AFM order parameter pose serious challenges to studying AFM spin dynamics. Here, we reveal the temporal evolution of an AFM order parameter directly in the time domain. We modulate the AFM order in hexagonal YMnO 3 by combining time resolved measurements of Faraday rotation and magneto-optical second harmonic generation.Understanding antiferromagnetic dynamics enables future information technologies, but the detection remains challenging. Here, the authors show the capability of tracking the three dimensional spin motions in YMnO This illustrates the general usefulness of antiferromagnets for future applications4, yet the dynamics occurring during the switching process remain uncertain as time-resolved (TR) access to the AFM order parameter is notoriously difficult. Consequently, detecting its motion in the time domain often relies on measuring one spin component, such as a transiently occuring uncompensated magnetic moment, and extrapolating the full order-parameter dynamics from models1. A detailed study of all spin components during a ferromagnetic spin precession, however, revealed unexpected insights6. Thus, a dependable analysis of the non-trivial spin dynamics during switching events in antiferromagnets necessitates direct and TR experimental access to all the order-parameter components.Recently, the appealing properties of antiferromagnets culminated in the demonstration of both optically9. Here, optical second-harmonic generation (SHG), i.e., frequency doubling of a light wave in a material, is a powerful, symmetry-sensitive technique that can couple linearly to the AFM order parameter, thus maintaining its directional information11. The leading SHG contribution is given by E is the electric field of the incoming light wave, P is the induced polarisation oscillating at twice the frequency ω of the incident light12. Owing to the direct coupling of 14. Despite this and despite the instantaneous nature of the SHG process, which allows probing ultrafast processes, TR SHG studies of optically induced dynamics are scarce. The methodical difficulty to discriminate between genuine magnetic-order-parameter dynamics, affecting ω and 2ω, hampers the interpretation of TR SHG measurements15. In fact, distinguishing between order-parameter and electron dynamics is commonly believed to be a “practical impossibility”16. This is particularly true for thermal excitations, where redistribution of resonantly excited charge carriers leads to substantial changes in the linear optical properties20.While the uncompensated component is optically accessible by the Faraday effect, detection of the compensated part of the order parameter usually depends on more indirect processes, like magnetic linear birefringence, which is quadratic in the sublattice magnetisation. This leads to a loss of directionality and, thereby, of the ability to distinguish between spin-reversed domain states16, where a circularly polarised laser pulse generates a longitudinal effective magnetic field HIFE. This field exists for the laser pulse duration only. The impulsive, non-resonant excitation results in a well-defined initial phase of the spin precession. This process is also effective in fully compensated antiferromagnets26. Such a simple fundamental excitation is an ideal test case for us. Its well-understood dynamics allow us to demonstrate how to separate thermal electron dynamics from non-thermal spin dynamics and how to obtain the full order-parameter trajectory.An optical excitation, however, can also act as a non-thermal stimulus to the magnetic order. A well-known example is the inverse Faraday effect (IFE)3. We find that the coherent spin precession transiently reduces the point-group symmetry, which leads to the emergence of new SHG tensor components. We conclude this from a periodic modulation of the SHG anisotropy. In contrast to the Faraday rotation, which is sensitive to the out-of-plane spin component, we show that the SHG modulation reflects the in-plane spin component. Moreover, we quantify both the in-plane and out-of-plane spin-canting angles, where we find a pronounced ellipticity of the spin precession. With this proof-of-concept demonstration, we show that TR SHG is a valuable tool for the study of AFM spin dynamics providing complementary information that is inaccessible by other techniques.Here, we use a combination of Faraday rotation and SHG to track the order-parameter motion during a coherent spin precession in antiferromagnetic YMnO3 as a model system. The material is ferroelectric below TC ≈ 1250 K27. The three Mn3+ sublattices order antiferromagnetically below the Néel temperature TN ≈ 70 K and form a quasi-two dimensional triangular lattice with a fully compensated spin structure. The spins in the sublattices point along the local x axes11. The magnetic space group of the ground state is 10 .Our setup is sketched in Fig. π = 95.2 ± 0.3 GHz. The initial phase changes by π upon changing the pump helicity, as is expected for a Z-mode excitation via the IFE23. In parallel to the Faraday rotation, we measured the SHG response η for a probe polarisation enclosing an angle of ϕ ≈15° with respect to a crystallographic x axis. We find a coherent modulation of η on an incoherent background. By taking (η− − η+)/2 and (η− + η+)/2, with the sign indicating the pump–pulse helicity, we can separate helicity-dependent and helicity-independent contributions, respectively  symmetry-changing dynamics leading to the appearance of new components 39 and AFM switching processes1, this also includes transversal spin dynamics, where precessional spin motion causes a redistribution among the The helicity-dependent SHG difference in Fig. 3+ spins oriented along equivalent crystallographic x axis. The anisotropy exhibits six lobes of amplitude A spaced 60° each. We define their orientation as θ = 0°.We distinguish the two cases by measuring the angle-dependent anisotropy of the SHG signal before and after the excitation. In contrast to Fig. A and the orientation θ. The results are shown in Fig. The time-dependent SHG intensities at four representative angular components are visualised in Fig. A exhibits an exponential decrease of the order of 10%. The change of A cannot be caused by changes of the optical properties as the slight changes of the transmittivity at ω and 2ω  = 0.198° ± 0.007° and, hence, α(0) = 1.0° with a statistical and systematic uncertainty of 0.035° and 0.5°, respectively. This, in turn, allows us to quantify the effective magnetic field of the IFE as α(0) = γμ0HIFEτ, where 1° corresponds to μ0HIFE ≈760 mT for τ = 130 fs . Note that the optically induced spin canting by non-resonant excitation via the IFE is comparable to resonant excitation via the magnetic field component of a strong THz pulse43.We can use the SHG measurements to quantify the spin deflection angle 28.In addition to the quantification of the basal-plane spin-canting angle, we can use the TR Faraday rotation to estimate the maximum out-of-plane spin-canting angle as 2.4 mdeg -oriented surfaces for experiments in transmission geometry. The sample was mounted in a cryostat (Janis SVT-400).Our YMnO3 is low. A residual absorption coefficient of ≈30 cm−1 at the pump photon energy and ≈40 cm−1 at the probe photon energy31 results in an absorption of ≈6% and ≈8%, respectively. The pump fluence was approximately 60 mJ cm−2. Linearly polarised probe pulses at 1.22 eV allow for the detection of Faraday rotation and SHG. The pump and probe spots exhibit Gaussian profiles with diameters of approximately 180 and 150 μm, respectively. In order to observe the SHG modulation in Fig. The fundamental light source of our setup is a regeneratively amplified Ti:Sapphire laser  producing 130 fs laser pulses at 1 kHz with a central photon energy of 1.55 eV. For our measurements, we used the output of two optical parametric amplifiers providing pump and probe pulses, respectively. To ensure excitation by the IFE, the circularly polarised pump pulses were tuned to 0.97 eV, where the absorption of YMnOA/A and orientation Δθ were extracted from a fit according toAll measurements were performed in near-normal incidence. The polarisation dependence of the SHG anisotropy was obtained by keeping the sample fixed and rotating the light polarisation of the incoming probe laser pulse with a half-wave plate. The transmission direction of a Glan-Laser prism inserted between the dichroic mirror and the photomultiplier tube , Iave(±15°) and Iave(−30°).Figure Supplementary InformationPeer Review File


Urocortin (UCN) is a neuropeptide that belongs to the corticotrophin-releasing hormone family and is expressed byeutopic and ectopic human endometria. The past years, this expression has been thoroughly investigated in the fieldof endometriosis. The objective of this systematic review is to accumulate current evidence related to the expressionof UCN in tissue and blood samples of patients suffering from endometriosis. Literature search was designed accord-ing to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and primarilyconducted using the Medline (1966-2018), Scopus (2004-2018), EMBASE (1947-2018) and Clinicaltrials.gov (2008-2018) databases, along with the reference lists of electronically retrieved full-text papers. Overall, eight studies wereretrieved. Current evidence suggests that the expression of UCN is increased in patients with ovarian endometriomasand that its levels may correlate with the severity of the disease. The diagnostic efficacy of UCN1 plasma levels wasevaluated in three studies. Two of them suggested that the sensitivity and specificity of the method may reach, andeven exceed, 80%. However, the wide variation in outcome reporting and outcome reporting measures in endome-triosis among the included studies precludes meta-analysis of available data. Therefore, although UCN seems to be apromising biomarker for the identification and follow-up of patients that suffer from endometriosis, more studies areneeded to reach firm conclusions with respect to its predictive accuracy.  Endometriosis is a benign inflammatory gynecological disease that manifests in women of reproductive age and is defined as the presence of stromal and viable endometrial glands outside the uterine cavity. It has an estimated prevalence of up to 10% in the general population and is associated with chronic pelvic pain, dysmenorrhea and infertility . The patDuring the past few years, several novel serum biomarkers have been proposed for the early diagnosis of endometriosis. The most consistently studied molecule is cancer antigen 125 (CA-125), a glycoprotein that has been established as a biomarker for the follow-up of patients with epithelial ovarian cancer. Similarly, in endometriosis, CA-125 can only be used as a prognostic rather than a diagnostic marker as it is accompanied by a significant amount of false negative results  has been extensively investigated in the field of endometriosis. UCN is a neuropeptide that belongs to the corticotrophin-releasing hormone (CRH) family and is expressed by eutopic and ectopic human endometria and is thought to play a role during decidualization , 7. BecaTo date, it remains unclear whether UCN may be used as a screening and/or prognostic biomarker of endometriosis. The objective of this systematic review is to accumulate current evidence related to the expression of UCN in tissue and blood samples of patients suffering from endometriosis and provide directions for future research.The present systematic review was designed accordingto the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . EligibiLiterature search was primarily conducted using the Medline (1966-2018), Scopus (2004-2018), EMBASE (1947-2018) and Clinicaltrials.gov (2008-2018) databases, along with the reference lists of electronically retrieved full-text papers. Additional sources were identified through the Google Scholar (2004-2018) database. Our last search was on the 04 February 2018. The search strategy included the words “endometriosis and urocortin” and is schematically presented in the PRISMA flow diagram .Overall, 8 studies were included in the present systematic review and outcomes from a total of 567 women were assessed -18. The UCN in biopsies from 10 patients with endometriosis and 3 patients that did not have endometriosis. Although they did not perform statistical analysis on the observed differences, they found that endometriotic lesions had increased UCN expression compared with healthy peritoneum and normal endometrium. Two studies evaluated UCN transcript expression (UCN1 and UCN2 transcript expression in eutopic endometrium and in endometriotic lesions. While UCN2 expression did not seem to differ between eutopic endometrium and endometriomas (UCN and UCN3 in the endometriotic foci (ectopic lesions) were significantly higher compared with those in eutopic endometrium of the same women (UCN transcripts when ectopic and eutopic endometrium samples were compared (0.9566 ± 0.136 a.u vs. 0.2826 ± 0.075 a.u respectively).Kempuraj et al.  investigpression  and one etriomas , the expme women , 18. VerUCN transcript levels. Specifically, Carrarelli et al. (UCN than ovarian endometriomas (OMA)  . UCN, UCN2 and UCN3 gene expression in eutopic endometrium of healthy women and women with endometriosis during the menstrual cycle. In women suffering from endometriosis, the expression of UCN, UCN2 and UCN3 transcripts was the same in the secretory and the proliferative phases, whereas in healthy women UCN levels differed between the two phases. Specifically, UCN2 expression had peak values during the early proliferative phase, while UCN3 expression was at its maximum in the secretory phase (UCN2 and UCN3 expression levels were significantly lower in women with endometriosis when compared with healthy women (Novembri et al.  studied ry phase , 16. BoDecidualization is a process of endometrial remodeling that occurs in the secretory phase of the menstrual cycle and is essential for early pregnancy. Current knowledge suggests that this process is initiated by progesterone and is mediated by various molecules such as UCN. Novembri et al.  showed tTwo studies evaluated preoperative plasma levels of UCN as a diagnostic factor that would help differentiate patients with endometriosis from patients with non-endometriotic, benign ovarian cysts , 17. SpeChmaj-Wierzchowska et al.  comparedMaia et al.  studied UCN transcripts is significantly higher in endometriotic lesions compared with eutopic endometrium of endometriotic women (Current evidence suggests that UCN may be a promising factor for the identification and follow-up of patients that suffer from endometriosis. However, the methodological heterogeneity of these studies in terms of the reported measures precludes firm conclusions. The expression of UCN has been investigated post-transcription both at the transcription and protein levels. Three studies suggested that the expression of ic women , 16, 18.ic women . On the ic women , 14, wheic women , 17. MaiDespite the fact that our study is based on a meticulous review of current literature, certain limitations preclude definitive conclusions. Firstly, the wide variation in outcome reporting and outcome reporting measures of UCN expression in endometriosis precludes meta-analysis of current data. Furthermore, the correlation between the expression of UCN in endometriotic lesions and peripheral blood remains to be investigated. Its actual value as a minimally invasive diagnostic method therefore, remains to be elucidated. Future studies should also clarify whether UCN levels are elevated not only in endometriomas but also in non-ovarian endometriosis and in early stage disease. The presence of a biomarker with high sensitivity and specificity in these cases is particularly important for the differential diagnosis since, to date, minimally invasive methods are non-existent . MoreoveHowever, this interesting point deserves further investigation. Moreover, previous studies have also shown that UCN is produced by the liver and kidney of healthy animals  but evidCurrent evidence suggests that UCN may be a promising factor for diagnosis and/or prognosis of patients that suffer from endometriosis. However, available data are scarce and the findings of currently published studies remain to be validated. Specifically, future studies should examine whether plasma and tissue levels of UCN correlate in patients. Moreover, evaluation of the predictive accuracy of UCN with the use of pre-specified cut-off values, mentioned in the present systematic review, is needed to assess the reproducibility of previous findings in the field.


We noticed that two of Vincent Torre's affiliations were missing:“Cixi Institute of Biomedical Engineering, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Zhejiang, China; Center of Systems Medicine, Chinese Academy of Medical Sciences, Suzhou Institute of Systems Medicine, Suzhou Industrial Park, Suzhou, China.”Furthermore, there are two minor errors in the text:A correction has been made to Results, “Expression of Piezo1 Channels in NG108-15 Cells and MCS Inhibition”:“Ca2+ transient almost completely: in the presence of GsMTx-4 the amplitude of Ca2+ transient DF/”has been changed to:2+ transient almost completely: in the presence of GsMTx-4 the amplitude of Ca2+ transient DF/F”.“Ca2+.In “Discussion paragraphs 1 and 2,” Ca2+ has been changed to CaNone of these irregularities affect the original meaning of the article.The original article has been updated.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.


Nerve cell stimulation evoked Ca2+ signals in acinar but not in stellate cells.Ca2+ signals in response to membrane depolarization.Stellate cells are not electrically excitable as they, like acinar cells, did not generate CaThe responsiveness of the stellate cells to bradykinin was markedly reduced in experimental alcohol‐related acute pancreatitis, but they became sensitive to stimulation with trypsin.2+ signals in the stellate cells, which then in turn damage more acinar cells causing further trypsin liberation.Our results provide fresh evidence for an important role of stellate cells in acute pancreatitis. They seem to be a critical element in a vicious circle promoting necrotic acinar cell death. Initial trypsin release from a few dying acinar cells generates Ca2+ signals in pancreatic acinar cells control fluid and enzyme secretion, whereas excessive Ca2+ signals induced by pathological agents induce destructive processes leading to acute pancreatitis. Ca2+ signals in the peri‐acinar stellate cells may also play a role in the development of acute pancreatitis. In this study, we explored Ca2+ signalling in the different cell types in the acinar environment of the pancreatic tissue. We have, for the first time, recorded depolarization‐evoked Ca2+ signals in pancreatic nerves and shown that whereas acinar cells receive a functional cholinergic innervation, there is no evidence for functional innervation of the stellate cells. The stellate, like the acinar, cells are not electrically excitable as they do not generate Ca2+ signals in response to membrane depolarization. The principal agent evoking Ca2+ signals in the stellate cells is bradykinin, but in experimental alcohol‐related acute pancreatitis, these cells become much less responsive to bradykinin and then acquire sensitivity to trypsin. Our new findings have implications for our understanding of the development of acute pancreatitis and we propose a scheme in which Ca2+ signals in stellate cells provide an amplification loop promoting acinar cell death. Initial release of the proteases kallikrein and trypsin from dying acinar cells can, via bradykinin generation and protease‐activated receptors, induce Ca2+ signals in stellate cells which can then, possibly via nitric oxide generation, damage more acinar cells and thereby cause additional release of proteases, generating a vicious circle.Physiological Ca 2+ signalling in different cell types in exocrine pancreatic lobules was monitored simultaneously and signalling responses to various stimuli were directly compared.Ca2+ signals evoked by K+‐induced depolarization were recorded from pancreatic nerve cells. Nerve cell stimulation evoked Ca2+ signals in acinar but not in stellate cells.Ca2+ signals in response to membrane depolarization.Stellate cells are not electrically excitable as they, like acinar cells, did not generate CaThe responsiveness of the stellate cells to bradykinin was markedly reduced in experimental alcohol‐related acute pancreatitis, but they became sensitive to stimulation with trypsin.2+ signals in the stellate cells, which then in turn damage more acinar cells causing further trypsin liberation.Our results provide fresh evidence for an important role of stellate cells in acute pancreatitis. They seem to be a critical element in a vicious circle promoting necrotic acinar cell death. Initial trypsin release from a few dying acinar cells generates Ca All animals were killed humanely according to the Schedule 1 protocol by cervical dislocation. Before and throughout the experiment, mice were maintained in plastic cages with corn cob bedding; tap water and commercial pelleted diet were freely provided. The mice were killed before the removal of the pancreas according to Schedule 1 of the UK Animals Act. The investigators understand the ethical principles under which −1) and palmitoleic acid (POA) (150 mg kg−1), at 1 h intervals, preceded by injection of PBS, as previously described , they received two intraperitoneal injections of ethanol .Pancreatic tissue was fixed in 4% formaldehyde and embedded in paraffin, and histological assessment was performed after haematoxylin and eosin staining of fixed pancreatic slices (4 μm thickness). Evaluation was performed on ≥10 random fields (magnification: ×200) by two blinded independent investigators grading  oedema, inflammatory cell infiltration and acinar necrosis as previously described : NaCl, 140; KCl, 4.8; Hepes (KOH), 10; MgCl2, 1; CaCl2, 1; glucose, 10; pH 7.3. In experiments where the effects of omitting extracellular Ca2+ were investigated, CaCl2 was left out of the standard solution. In experiments where the effects of membrane depolarization were investigated, the medium contained 100 mm KCl and the NaCl concentration was reduced to 44.8 mm. Pancreatic lobules were then incubated with fluorescent dye following the manufacturer's description. All experiments on normal pancreatic lobules were carried out with fresh preparations attached to the coverslip of a perfusion chamber at room temperature (∼23°C). In experiments on lobules in which the effects of exposure to fatty acids and ethanol were investigated, the lobules were exposed to a medium containing POA (20 μm) and ethanol (12 mm) for 2.5 h before starting the experiments.Pancreatic lobules were isolated from the pancreas of adult normal mice , for 20 min at room temperature. The tissue was transferred into a flow chamber and superfused with the Na+‐Hepes‐based extracellular solution as described above. Cells were visualized using a Leica SP5 MPII two‐photon confocal microscope, with an ×63 1.3 NA objective lens. Fluo‐4 was excited with a 488 nm argon laser, at 1–4% power, and emitted light was collected at 500–580 nm. Generally, a series of images was recorded at 512 × 512 pixels resolution (at the speed of 1 frame s–1), and analysed using Leica Confocal Software . Fluorescence signals were plotted as F/F0 . In many experiments three‐dimensional recording in time have been conducted (2–3 images per time point). Statistical analysis was performed using ANOVA or Student's t‐test.Pancreatic lobules were loaded with 5 μet al. 2+‐sensitive fluorescent probes much more avidly than PACs, so the initial assumption – looking at the fluorescence intensity levels in the resting situation  – was that the bright cells represent PSCs. To check whether nerve cells were present and, if so, to test whether nerve stimulation could elicit Ca2+ signals in PACs or other cells, a solution with a high (100 mm) K+ concentration was introduced. As seen in Fig. Aiii, this caused a rise in [Ca2+]i in several relatively large cells, which must be the always quantitatively dominant PACs. Importantly, there was no rise in [Ca2+]i in the PSCs, but in one cell – partly ‘hidden’ by a PSC – there was a large Ca2+ signal. This cell is most likely a neuron (PN). The apparently unprovoked short‐lasting Ca2+ signal in this cell occurring later in the experiment may be due to a spontaneous action potential or a short burst of action potentials. The assumption that the bright cells seen in Fig. Aii were PSCs was confirmed when these cells became significantly brighter, indicating rises in [Ca2+]i, after stimulation with BK (1 nm) . Finally, the lobule was stimulated by ATP (100 μm), which caused a rise in [Ca2+]i in the PACs and in a cell  that had not reacted to high K+ or BK exposure. The nature of this cell is unclear and it is therefore labelled X.Our general aim was to simultaneously study signalling in the various cell types to be found in the acinar environment in a live pancreatic lobule preparation. Figure ion Fig. Aii – wah a high 00 mm K+ h a high 00 mm K+ h a high 00 mm K+ h a high 00 mm K+ 2+ signalling .As mentioned in the Methods, the pancreas contains insulin‐secreting β‐cells, in addition to the quantitatively dominant exocrine cells. It has long been known that PACs possess insulin receptors and that insulin can affect PAC functions including Ca2+ signals. One possibility is that they are PNs and we therefore tested this hypothesis. The fluorescent dye FluoroGold has been demonstrated to undergo retrograde axonal transport and stains nerve cells  in which a FluoroGold‐labelled cell  responded to membrane depolarization, elicited by a high‐K+ (100 mm) solution, with an increase in [Ca2+]i . A non‐FluoroGold‐labelled PAC also produced a rise in [Ca2+]i, presumably due to the action of ACh released from nerve endings (see below), whereas a PSC failed to respond .It is clear from experiments of the type shown in Fig. ment n =  in whichment n =  in whichment n =  in whichment n =  in whichn = 14) we could observe Ca2+ signal propagation in PNs along the bodies and elongated parts of PSCs as a pathway through the lobules .Many of the pancreatic cells that have neuron‐like properties are located close to PSCs see Fig. . In seve2+]i in PNs, elicited by K+‐induced depolarization , in agreement with the well‐established cholinergic innervation of PACs . ATP did not have any effects on PNs , whereas this agent could, in several cases, produce Ca2+ signals in PACs and PSCs  . Ca2+ signals elicited by a high‐K+ solution in PNs were not inhibited by the non‐selective purinergic antagonist suramin .The rise in Ca+i in PNsion Figs , 4, 5 copine n = 2, in agr2+ signals in PNs. Adrenaline (20 μm) evoked signals in 21 out of 35 PNs tested . This response was mediated by α‐ rather than β‐receptors as the β‐adrenergic agonist isoprenaline had no effect (n = 10) whereas the α‐receptor agonists cirazoline (50 μm) and UK 14.304 (50 μm) could mimic the effect of adrenaline (n = 8). Ca2+ signals in PNs were also elicited by Substance P (10 μm) in 4 out of 7 neurons . Bombesin, which is known to elicit Ca2+ signals in PACs by interaction with receptors that are distinct from the CCK receptors . On the other hand, PNs did not respond to BK  or VIP (n = 4).Several neurotransmitters elicited Caet al. et al. et al. m) for a prolonged period (15–30 min) before exposure to 10 mm glucose. As seen in Fig. C, a PN in which a high‐K+ solution, as well as Substance P, elicited Ca2+ signals failed to respond to stimulation with 10 mm glucose (n = 13).In the hypothalamus there are neurons responsive to glucose . Figure E shows the result of an experiment in which a high‐K+ solution elicited Ca2+ signals in both a PN and a PAC without evoking a response from two PSCs, which both subsequently generated Ca2+ signals when stimulated by BK.As previously described, PSCs consistently generate Caet al. EF), but did not always  stimulation. Bombesin (1 μm) elicited Ca2+ signals in some PSCs  and VIP (100 nm) could evoke Ca2+ signals in 37 out of the 78 PSCs tested .As previously shown . In these ATP‐sensitive X‐cells, high‐K+ stimulation could in many, but not all, cases evoke short‐lasting Ca2+ signals , but once these cells had been challenged with a high‐K+ pulse, they needed a long recovery time (>30 min) before they could respond again (n = 9). In these cells, adrenaline (20 μm) could evoke Ca2+ signals , but X‐cells never responded to BK (n > 100) . We also tested whether the X‐cells were glucose‐sensitive. We used the same protocol as for the similar experiments testing glucose sensitivity in PNs  . In five experiments, X‐cells that responded to ATP stimulation with Ca2+ signals failed to generate any increase in [Ca2+]i in response to 10 mm glucose  Fig. D. We alPNs Fig. C  we confirmed that PSCs respond to BK (1 nm) stimulation by generating substantial Ca2+ signals and also confirmed the previously reported result that trypsin does not elicit Ca2+ signals  of trypsin that had failed to elicit signals in the control preparations . The proportion of PSCs responding to trypsin in the AP lobules was markedly reduced by including the CRAC channel inhibitor GSK‐7975A in the POA/ethanol solution used to generate AP . Because CRAC channel inhibition has been shown to reduce store‐operated Ca2+ influx in both PACs and PSCs, this supports the idea previously proposed , hardly responded to 1 nm BK, but in a number of PSCs Ca2+ signals in response to trypsin (10 nm) were observed . Control PSCs did not respond to trypsin Fig. G), as also previously reported , the actions of this enzyme in this series of experiments  were also acute and reversible, indicating a receptor‐mediated (protease‐activated receptor) effect rather than a consequence of cell damage. Although many PSCs may have been destroyed by the actions of POA/ethanol in vivo, clearly those responding to trypsin were intact. In a few cases (n = 3 out of 59 cells tested), thrombin (5 mU ml−1), another protease, evoked Ca2+ signals in PSCs in lobules from FAEE‐AP mice, whereas this was not observed in control tissue, as also previously reported  would appear to represent a specific desensitization of the type 2 receptors. This is supported by the finding that although PSCs in the FAEE‐AP tissue failed to respond to a BK concentration (1 nm) that elicited a maximal Ca2+ signal in the control tissue, some PSCs from FAEE‐AP lobules could produce Ca2+ signals when stimulated with a high concentration (1 μm) of a BK agonist specific for type 1 BK receptors (S‐BK) . In contrast, S‐BK only evoked a Ca2+ signal in one PSC out of 118 tested in lobules from control mice. Figure N summarizes the results of the experiments comparing the responsiveness of PSCs to S‐BK, thrombin and trypsin in control and FAEE‐AP.In the res Fig. B, the ants Fig. H and N 2+ signal generation in control and AP . The results show that the concentration–response curve was shifted markedly to the right in AP, as compared to the control values. Thus a BK concentration of 1 nm, which evokes a near‐maximal Ca2+ signal in control PSCs , hardly evoked any change in [Ca2+]i in the PSCs from the AP lobules .We evaluated quantitatively the reduced responsiveness of the PSCs to BK in AP by comparing concentration–response curves for BK‐elicited Ca AP Fig. K–M. The2+ signalling in the peri‐acinar environment of the exocrine pancreas, we have for the first time been able to record Ca2+ signals from PNs, and demonstrated that PSCs are not electrically excitable and, in contrast to the PACs, do not appear to be functionally innervated. We have also discovered a hitherto unknown cell type (X) which is very responsive to stimulation with ATP . This desensitization is probably due to BK liberation from bradykininogen, as a result of kallikrein release from PACs undergoing necrosis . Given that the concentration of the type 1 agonist that is required to evoke a signal is very high, this may not in itself have any functional significance, but the reduced sensitivity to type 2 receptor activation and increase in sensitivity to type 1 activation is a general feature of inflammatory diseases , whereas induction of FAEE‐AP over 48 h in vivo produces a clear reduction in responsiveness to BK , showing that CRAC channel inhibition largely prevents the increased responsiveness of PSCs to trypsin that occurs in AP‐like conditions, provides fresh evidence in favour of CRAC channel inhibition as a potentially attractive treatment for AP.We have previously shown that CRAC channel inhibition markedly reduces the prolonged [CaThe authors declare no competing interests.All experiments were carried out in the School of Biosciences at Cardiff University. All authors jointly conceived the project. OG and JVG carried out the experiments with help from SP, and these were then also analysed by OVG. JVG created the figures and OHP wrote the paper with significant intellectual input from all the other authors. All authors have approved the final version of the mansucript.The work was supported by grants from the Medical Research Council (UK) (MR/J002771/1 and G19/22/2).2+ signals in the stellate cells which then, probably via formation of the diffusible gas nitric oxide in these cells, causes more acinar necrosis which, via release of proteases from the further damaged acinar cells, causes additional stellate cell stimulation, thereby generating a vicious circle. These findings have potential therapeutic implications, as they indicate that interventions that would break this vicious circle could be helpful in the treatment of acute pancreatitis, a disease for which there is currently no authorized rational therapy. The key elements in the vicious circle promoting acinar necrosis would appear to be the actions of bradykinin, generated by the action of kallikrein, and trypsin on specific stellate cell receptors. These actions cause rises in the cytosolic Ca2+ concentration in the stellate cells, which activate the Ca2+‐sensitive enzyme nitric oxide synthase, producing the very diffusible nitric oxide, which in this situation appears to be toxic for the acinar cells. There are clear pharmacological interventions that could prove effective. Bradykinin receptor antagonists, antagonists of protease‐activated receptors and inhibitors of nitric oxide synthase could all be helpful. Given that excessive Ca2+ signal generation in both stellate and acinar cells are critical elements, our previous proposal of treating acute pancreatitis with inhibitors of store‐operated Ca2+ entry via the so‐called CRAC channels, which has received further support from our new results, remains valid. However, it may well turn out that combination therapy with Ca2+ channel inhibitors and, for example, bradykinin receptor antagonists, could be particularly helpful and would allow relatively low doses of these agents to be used, thereby minimizing potential side effects.Our new data indicate that the pancreatic stellate cells play an important role in acute pancreatitis. They are key amplifying elements in a process resulting in necrotic acinar cell death. Initial release of proteases–including trypsin and kallikrein–from a small proportion of dying acinar cells generates Ca


A comparison of intrinsic SR Ca2+ cycling revealed that, at similar physiological Ca2+ concentrations, LCRs are large and rhythmic in permeabilized SANC, but small and random in permeabilized ventricular myocytes (VM). Permeabilized SANC spontaneously released more Ca2+ from SR than VM, despite comparable SR Ca2+ content in both cell types. In this review we discuss specific patterns of expression and distribution of SR Ca2+ cycling proteins (SR Ca2+ ATPase (SERCA2), phospholamban (PLB) and ryanodine receptors (RyR)) in SANC and ventricular myocytes. We link ability of SANC to generate larger and rhythmic LCRs with increased abundance of SERCA2, reduced abundance of the SERCA inhibitor PLB. In addition, an increase in intracellular [Ca2+] increases phosphorylation of both PLB and RyR exclusively in SANC. The differences in SR Ca2+ cycling protein expression between SANC and VM provide insights into diverse regulation of intrinsic SR Ca2+ cycling that drives automaticity of SANC.Spontaneous beating of the heart pacemaker, the sinoatrial node, is generated by sinoatrial node cells (SANC) and caused by gradual change of the membrane potential called diastolic depolarization (DD). Submembrane local Ca Sinoatrial node cells (SANC) are able to generate spontaneous action potentials because of the gradual depolarization of the membrane potential during diastole, called diastolic depolarization. The ensemble of sarcolemmal ion channels and electrogenic ion transporters , SR Ca2+ release channels, creating Ca2+-induced Ca2+ release (CICR) f and the kinetics of Ca2+ pumping into and release from the SR. A comparison of intracellular Ca by Ca2+ ,2,3. Cel in SANC ,35 or ve in SANC ,64,65,66 in SANC . In perm2+]f spontaneous LCRs in permeabilized SANC were large and roughly periodic events ), while no periodic events were observed in ventricular myocytes up to 150 nmol/L [Ca2+]f. At higher 150–250 nmol/L cytosolic [Ca2+]f, the total Ca2+ signal mass released by SANC was ~2-fold larger compared to ventricular myocytes  cytosolic [Ca2+]f concentrations  revealed that Ca2+ release from j-SR had larger Ca2+ spark amplitudes, but smaller SR Ca2+ depletions (Ca2+ blinks) compared to releases from the central non-junctional SR (nj-SR) i might rapidly reach substantial levels. Since the same amount of Ca2+ released in the cell center is not confined by surrounding membranes, it could rapidly dissipate via Ca2+ buffering or repumping Ca2+ into SR and not reach comparable peak levels. Another hypothesis put forward in the same study was that there could be different pools of Ca2+ for j-SR and nj-SR or j-SR and nj-SR CRUs have different Ca2+ release termination mechanisms f is 150–250 nmol/L  to a maximal suppression of ~50%  SERCA, which could provide an increased Ca2+ pumping into the SR required to sustain long-lasting elevated RyR Ca2+ releases. The PLB to SERCA ratio considered to be a key factor of SR Ca2+ cycling and cardiac contractility regulation i transient was larger and decayed faster compared to WT mice i transient and reduces duration of Ca2+ sparks [16 site was substantially higher in SANC compared to ventricular myocytes  CaMKII in rabbit SANC exceeded that in ventricular myocytes by ~2-fold E and thimyocytes F 39]. T. T17 sitrcolemma ,121. 2+ leak and Ca2+ sparks in cardiac myocytes [2+ sensitivity and open probability of RyR [2815 site  by addition of exogenous cAMP  associated with the expression of proteins comprising ion channels or proteins regulating Ca


In the original version of our article , insuffiThe corrected author list and author contributions are provided below:1,2,†, Neelakshi Gohain 1,2,†, Peter T. Pham 1,2, Christopher Hamlin 1,2,‡, Hongshuo Song 1,2, Eric Sanders-Buell 1,2, Meera Bose 1,2, Leigh A. Eller 1,2, Swati Jain 3, Gherman Uritskiy 3, Venigalla B. Rao 3, Sodsai Tovanabutra 1,2, Nelson L. Michael 1, Merlin L. Robb 1,2, M. Gordon Joyce 1,2,* and Mangala Rao 1,*Hung V. Trinh 1 htrinh@hivresearch.org (H.V.T.); ngohain@hivresearch.org (N.G.); ppham@hivresearch.org (P.T.P.); christopher.hamlin@nih.gov (C.H.); hsong@hivresearch.org (H.S.); esandersbuell@hivresearch.org (E.S.-B.); mbose@hivresearch.org (M.B.); leller@hivresearch.org (L.A.E.); Stovanabutra@hivresearch.org (S.T.); nmichael@hivresearch.org (N.L.M.); mrobb@hivresearch.org (M.L.R.)U.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA; 2 Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., Bethesda, MD 20817, USA3 80jain@cua.edu (S.J.); guritsk1@jhu.edu (G.U.); rao@cua.edu (V.B.R.)Department of Biology, The Catholic University of America, Washington, DC 20064, USA; * gjoyce@hivresearch.org (M.G.J.); mrao@hivresearch.org (M.R.); Tel.: +1-(301)-319-7528 (M.G.J.); +1-(301)-319-7699 (M.R.)Correspondence: † These authors contributed equally to this work.‡ Current address: DAIDS, National Institute of Allergy and Infectious Diseases, Rockville, MD 20852, USA.


PMCA exists in four main isoforms and PMCA2 and PMCA3 are highly expressed in the brain. The overall effects of impaired calcium extrusion due to age-dependent decline of PMCA function seem to accumulate with age, increasing the susceptibility to neurotoxic insults. To analyze the PMCA role in neuronal cells, we have developed stable transfected differentiated PC12 lines with down-regulated PMCA2 or PMCA3 isoforms to mimic age-related changes. The resting Ca2+ increased in both PMCA-deficient lines affecting the expression of several Ca2+-associated proteins, i.e., sarco/endoplasmic Ca2+-ATPase (SERCA), calmodulin, calcineurin, GAP43, CCR5, IP3Rs, and certain types of voltage-gated Ca2+ channels (VGCCs). Functional studies also demonstrated profound changes in intracellular pH regulation and mitochondrial metabolism. Moreover, modification of PMCAs membrane composition triggered some adaptive processes to counterbalance calcium overload, but the reduction of PMCA2 appeared to be more detrimental to the cells than PMCA3. The aging process is a physiological phenomenon associated with progressive changes in metabolism, genes expression, and cellular resistance to stress. In neurons, one of the hallmarks of senescence is a disturbance of calcium homeostasis that may have far-reaching detrimental consequences on neuronal physiology and function. Among several proteins involved in calcium handling, plasma membrane Ca Due to high buffering capacity of mitochondria, they are also an important component for maintaining calcium homeostasis and regulation of intracellular Caignaling ,13. The ignaling . It is wignaling . In oxidignaling . Accordiignaling . This thignaling  implicat2+-ATPase, can produce diverse defects in brain function. This posits disturbed calcium homeostasis in the center of several age-associated brain pathologies and should be considered as an important risk factor for the development of neurodegenerative diseases. The brain is especially vulnerable to oxidative damage but due to its high complexity, it is difficult to define precisely all the factors that trigger the aging process. It is now well established that deficits in ATP supply, which is critical for reestablishment of ion balance by the plasma membrane Ca2+ concentration is attributed to the plasma membrane Ca2+-ATPase, the most sensitive cellular calcium detector. There are four main isoforms of PMCA (PMCA1–PMCA4), that are similar in structure, despite low homology at the amino acid level c raises are generated mainly by the voltage-gated Ca2+ channels (VGCCs), of which four types: N, L, P/Q, and T are present in PC12 cells i. It has been widely documented that during aging, ion channel dysfunctions participate in the generation of ionic dyshomeostasis, alteration of membrane potential as well as signal transduction pathways, which can eventually modify cellular physiology . Earlierf L-VGCC ,65. Moreth aging ,67,68,69th aging . An inteferation . An impoferation . Downregferation . It was feration . Therefo2+]c rises during depolarization reflects the presence of more functional calcium channels in the plasma membrane. Activation of individual channels and coupling their functions with downstream cellular signaling pathways, can induce a different response e.g., by modulation of specific genes expression. In addition, changed VGCC profile in response to manipulations in PMCA expression strongly suggests a transcriptional link between these two opposing systems of Ca2+ transport.Based on this data, it can be assumed that higher amplitude of i by mitochondria increases when cells age, as was demonstrated in peripheral sympathetic neurons and intestinal smooth muscle cells [2+]I by mitochondria seems to be especially relevant in neuronal cells, in which local Ca2+ uptake by neighboring mitochondria regulates the duration of cytosolic Ca2+ fluxes generated by the influx through plasma membrane calcium channels [2+ homeostasis might explain mitochondrial impairments in senescent cells arise from calcium hypothesis of neuronal aging [2+ transporting systems over a long time period should affect [Ca2+]i. To confirm it, several research groups showed elevated resting [Ca2+]i and reductions in the amplitude of stimulation-induced [Ca2+]i signal in aged neurons i may directly reflect a partial loss of calcium clearing potency and sensitize mitochondria to buffer cytosolic calcium transients even in a condition of moderate increases in [Ca2+]i. One of the most important regulators of mitochondrial function in aging is the second messenger Cale cells ,77. The channels . The posal aging . Accordi neurons ,81. A si2+ by mitochondria will stimulate Ca2+-dependent dehydrogenases of the TCA cycle, providing reducing equivalents to boost the activity of respiratory chain and thereby, ATP production via oxidative phosphorylation (OXPHOS) i. Whether mPTP is involved in the physiological aging process is still under debate, but the latest data demonstrated reduced mitochondrial calcium buffering capacity and increased sensitivity to mPTP formation in putamen of aged monkeys [An enhanced susceptibility to mPTP opening and the loss in PMCA activity in synaptic membrane are both reported in the aged brain . In contold rats . Therefo monkeys . 2+]m) is to boost energy production. During respiration, electrons from reducing equivalents (NADH and FADH2) are transferred through ETC complexes (complex I-V) and H+ are pumped out to intermembrane space, creating an electrical (ΔΨm) and pH (ΔpH) gradients, both thermodynamically equivalent to drive ATP synthesis. Formation of ΔΨm is fundamental for Ca2+ uptake to mitochondria and adequate mitochondrial function, mainly for ATP synthesis by the F0F1ATP synthase c, thus protecting mitochondria from Ca2+ overload [2+]c above 2–3 µM are expected to trigger channel opening and promote cooperative increase in Ca2+ uptake as described in earlier studies c but inhibited the uptake rate when [Ca2+]c increased c. Therefore, MICU3 is considered to act as a highly potent cooperative MCU activator with no gatekeeping function. Patron and colleagues [2+ cycling in the steady-state conditions whereas MICU1/MICU3 dimers activated MCU-dependent Ca2+ uptake even in the low [Ca2+]c. MICU3, when silenced, was also the only MICU isoform that effectively decreased mitochondrial Ca2+ transients evoked by synaptic activity c rises, as shown by Patron et al. m is expected to coordinate upregulation of OXPHOS machinery resulting in faster respiration and ATP production H level accompanied by increased activity of proton-pumping ETC complexes . These findings are in agreement with the boosting effect of Ca2+ on mitochondrial metabolism and suggest that the rate of OXPHOS in PMCA2-deficient cells may be controlled at the activity of ETC proteins. Interestingly, knockdown of PMCA3 isoform did not change the rate of OXPHOS. However, enhanced lactate release and greater sensitivity of the ATP level to glucose withdrawal in the presence of pyruvate suggests the reliance on anaerobic glycolysis. This brings up an intriguing question about preferential fueling of PMCA isoforms by different energy-generating pathways. It is tempting to hypothesize that the loss of fast acting PMCA2 will stimulate other ATP-dependent Ca2+ extruding proteins to restore [Ca2+]i, thereby increasing cellular demand for ATP. When PMCA3 was depleted, [Ca2+]c rise was only moderate, so the local synthesis of ATP by glycolysis may be sufficient to provide adequate amount of energy . Because pHcyto was also higher in PMCA-deficient cells, but still lower than pHmito, which is consistent with chemiosmotic coupling hypothesis, pH gradient across the inner mitochondrial membrane (ΔpH= pHmito − pHcyto) was increased in these cells. Based on the PMCA/pH relationship, we anticipate that knockdown of neuro-specific PMCA isoforms, which are considered as fast reacting, dramatically limits the number of H+ entering cytosol leading to pHmito increase. In these circumstances, the rise in pHmito could result from compensation of Ca2+ charge by moving H+ down the ETC complexes. It is therefore attractive to propose that increased [Ca2+]c observed in PMCA2-deficient cells, and hence higher [Ca2+]m, is a main trigger for increased proton motive force to drive higher rate of ATP synthesis. To support this, application of FCCP which collapses the inner mitochondrial membrane potential, evoked significant rise in [Ca2+]c in PMCA-deficient lines suggesting higher ΔΨm-dependent mitochondrial Ca2+ loading, especially when PMCA2 was knockdown. The phenomenon of higher magnitude of [Ca2+]c rises in response to FCCP was also demonstrated by Behringer and colleagues in old microvascular endothelium, suggesting greater capacity of mitochondria for Ca2+ during aging c and [ATP]m and concomitant stable rise in ΔΨm upon histamine stimulation. This suggested that increased [ATP]m reflected long-lasting elevation of the mitochondrial energy state. Similarly, increase in both ΔΨm and ΔpH components of the proton motive force was seen after stimulation with vasopressin [2+]c transients that are relayed to mitochondria. The authors suggested that respiratory chain activity may be regulated by a long-lasting mitochondrial signal generated by [Ca2+]m transients and thus, partially by [Ca2+]c levels, which is coherent with our data from PMCA2-depleted line. These findings also fit the mitochondrial memory mechanism proposed by Jouaville et al. [2+]m. This allows mitochondria to meet the energy needs without the risk of organelle Ca2+ overload. Although, the mechanistic explanation of such “memory” is unknown, it plausibly involves the changes in ΔΨm, as observed by us and/or Ca2+-dependent activation of F0F1ATP synthase. Indeed, several in vitro and in vivo studies demonstrated that the rate of ATP synthesis can be controlled directly at the level of F0F1ATP synthase, independently from the respiratory rate and ΔΨm c response to FCCP and mitochondrial membrane hyperpolarization. Instead, FCCP produced massive loss of ΔΨm and slight hyperpolarization of plasma membrane suggesting that the plasma membrane potential is not created by proton pump and the protonophore equilibrates pH across the plasma membrane by carrying H+ from cytoplasm to the extracellular milieu. It has been demonstrated that lack of protons in mitochondrial matrix under the excess of electron donors strongly promotes ROS generation c rises. The prevalence in activation of certain CaM downstream signaling pathways may therefore sensitize neurons to aging-related physiological and pathological stimuli. Like PMCA, CaM shows functional defects in the aged brain, mainly due to intensive oxidation of multiple methionines i, more significantly in the PC12_2 line. Early studies on PC12 cells suggested the inversed relationship between CaM level and neurite elongation in response to a differentiation signal . In our 2+ concentration, while phosphorylation by PKC at Ser 41 liberates CaM due to significant reduction in GAP43 affinity. Dephosphorylation by e.g., calcineurin (CaN), reverses this process and decreases available free CaM i. Some data suggested the relationship between CaN/NFAT-dependent regulation of PMCA and brain aging, along with the onset of neurodegenerative diseases c due to specific PMCAs depletion produced distinct cell responses, which clearly depended on PMCA isoforms composition and isoform-specific regulatory mechanisms. When induced changes are adaptive, the cells can effectively counteract Ca2+-dependent destructive processes and retain the ability to survive. However, these protective actions in PMCA2 down-regulated cells were found to be much less efficient. One can assume that not only changes in global [Ca2+]I concentration, but also spatially and temporally limited modifications in transmission of calcium signal played a decisive role in the cell response, and the detrimental effects over time were not sufficiently counterbalanced by these adaptive mechanisms.Taken together, changes in PC12 cell lines initiated by moderate, but prolonged increase in c activates CaN, which dephosphorylates NFAT causing its translocation to the nucleus, where it becomes transcriptionally active. A return of [Ca2+]c to the resting level initiates re-phosphorylation and rapid relocation of NFAT back to the cytosol. Our results showed that moderately increased basal [Ca2+]c in PMCA-deficient lines sufficiently induced its nuclear translocation and DNA binding activity. Moreover, we found that a sequence for NFATc2 binding is located in the promoter region of Calm2 and Calm3 genes, but not in Calm1. Chromatin immunoprecipitation assay revealed higher promoter occupancy by NFATc2 in PMCA-modified cell lines resulting in diminished transcription of both CaM genes. In addition, overexpression of the constitutively active form of CaN increased NFATc2 nuclear accumulation and its promoter activity, which potentiated Calm2 and Calm3 repression. This strongly indicated the NFATc2 repressive role toward CaM gene expression. Further experiments with NFATc2 silencing, using selective siRNA, showed a partial rescue of the expression of Calm2 in both lines and Calm3 in PC12_2 cells, and confirmed that the activation of the CaN/NFAT pathway may repress CaM genes, but to various extent in each of the PMCA-deficient lines. Looking for a mechanism(s) that could underlie the diminished expression of CaM genes, a microarray screening of fundamental transcription factors was performed . Among hcineurin ,180. In Calm 2 and Calm3 genes. It was reported that CaN interacts with PMCA2 and PMCA4 which resulted in inhibition of its phosphatase activity c by the uptake into endoplasmic reticulum, IP3R and RyR act as channels releasing calcium from the ER following physiological stimulation. In PMCA-reduced cells, an increased level of SERCA2 and SERCA3 coexisted with higher Ca2+ accumulation in the ER, although the relationship between PMCA and SERCA expression has not been elucidated. More effective Ca2+ transport to the ER may decrease [Ca2+]c to its safe level, but also more Ca2+ could be released by activation of IP3R and RyR c rises was detected in PC12_2 cells. In addition, there were significant differences between lines with respect to the rate of Ca2+ clearance and the duration of the prolonged calcium signal upon chemokine stimulation. The return of Ca2+ to its new steady-state level was 3 times longer in PC12_2 and about 2 times longer in PC12_3 than in control cells. The differences in kinetic parameters could reflect actual rate of Ca2+ release from the ER and its extrusion to extracellular milieu. It is worth to emphasize that PMCA3-deficient cells still possess very active PMCA2, but also higher protein level of PMCA4, thus could operate more effectively to control [Ca2+]i. Despite that, the efficiency of this adaptation is still lower than in control PC12 cells with a full set of PMCA isoforms. All three receptors were detected in our experimental model cells, but only CCR5 was localized in the plasma membrane which is required for CCL5 action. Interestingly, in each of the PMCA down-regulated PC12 lines, CCR5 expression increased by nearly 50%. Functional assays in Ca2+, but downstream long-time effects seemed to be specifically linked to the function of particular isoforms. Some of the induced adaptive mechanisms were sufficient for protection of cell viability as observed in PMCA3-deficient cells (Structural and functional changes in PMCA-deficient PC12 cell lines were initiated by moderately increased cytosolic Cant cells  in compant cells . This un2+ extruding systems and aberrant regulation of downstream effectors could disturb the function of mitochondria and endoplasmic reticulum leading to cell death, as was widely documented in aging cells [Aging, as a physiological process, involves the perpetual adaptation to varying internal and external conditions by generation of the complex protective mechanisms. This new adaptive state enables the cells to continuously make short-term adjustments for optimal functioning, but because of the multifactorial nature of aging, the efficiency of these adaptations depends on the intensity of senescent signaling ,212. Calng cells ,213. 2+, allowed to track unique functions that each of these isoforms may potentially perform in aging. The most important findings demonstrate that the altered composition of PMCA isoforms is linked to:intracellular pH regulation and changes in the mitochondrial bioenergetic processes,2+ transport systems to the ER,adaptive functional changes in the VGCC and in Ca2+/CaN-dependent complex formation with GAP43,regulation of CaM availability by CaCalm2 and Calm3 genes by NFATc2,reduced CaM level and repressive regulation of altered expression of CCL5-sensitive receptors—CCR1, CCR3, and CCR5,3 receptors.altered expression of IPStudies performed on our PC12 cell model of senescent neurons revealed an apparent contribution of PMCA2 and PMCA3 isoforms to the aging process. A controlled deficiency of PMCA2 or PMCA3 and resulting permanent increase in intracellular CaComparison between PC12_2 and PC12_3 lines clearly indicates that down regulation of PMCA2, apart from several adaptive or compensatory processes, triggers more potentially damaging events that are also observed during neuronal aging, including higher sensitivity to inflammation. A declining potency to maintain cellular homeostasis as a potential contributor to age-dependent neuronal senescence markedly increases the risk for neurodegenerative processes. Although differentiated PC12 cells can only partially mimic the physiological processes in the functional neuron, the changes observed in our model might represent the age-related mechanisms common in functionally different cell types.


Some depolarizing agents trigger conducted vasoconstriction while others only evoke a local depolarization. Here we use a novel technique to investigate the temporal and spatial relationship between intercellular Ca2+ signals generated by smooth muscle action potentials (APs) and vasoconstriction in mesenteric resistance arteries (MA). Pulses of exogenous KCl to depolarize the downstream end (T1) of a 3 mm long artery increased intracellular Ca2+ associated with vasoconstriction. The spatial spread and amplitude of both depended on the duration of the pulse, with only a restricted non-conducting vasoconstriction to a 1 s pulse. While blocking smooth muscle cell (SMC) K+ channels with TEA and activating L-type voltage-gated Ca2+ channels (VGCCs) with BayK 8644 spread was dramatically facilitated, so the 1 s pulse evoked intercellular Ca2+ waves and vasoconstriction that spread along an entire artery segment 3000 μm long. Ca2+ waves spread as nifedipine-sensitive Ca2+ spikes due to SMC action potentials, and evoked vasoconstriction. Both intercellular Ca2+ and vasoconstriction spread at circa 3 mm s−1 and were independent of the endothelium. The spread but not the generation of Ca2+ spikes was reversibly blocked by the gap junction inhibitor 18β-GA. Thus, smooth muscle gap junctions enable depolarization to spread along resistance arteries, and once regenerative Ca2+-based APs occur, spread along the entire length of an artery followed by widespread vasoconstriction.The role of vascular gap junctions in the conduction of intercellular Ca However2+ -mediated APs can propagate via gap junctions to generate regenerative intercellular Ca2+ waves similar to those observed in visceral smooth muscles i in the vicinity of the application area (∼200 μm) of 4.9 ± 0.4% of peak Ca2+ . The small, localized rise in intracellular Ca2+ was associated with vasoconstriction of 2.7 ± 0.2% .5 s pulse of KCl: increased SMC Ca2+ signal by 93.4 ± 3.4% at 0 μm (n=5) and significant increases were now evident at 500 μm and 1000 μm upstream of the T1, with average increases of 44.4 ± 5.2%, 2.7 ± 0.1%, but with no detectable signal by 1500 μm, respectively , with a maximal (100%) Ca2+ signal at 0 μm .20 s pulse of KCl: was followed by detectable increases in Ca2+ at 1500 μm distance from T1. At both 0 μm and 500 μm there was a maximal (100%) increase in Ca2+  end of the artery  the presence of TEA and BayK 8644 allowed axial propagation of intercellular Ca2+ waves evoked by localized 1 s KCl pulses at the distal end of the arcade (T1) . Ca2+ change was measured at 5 ROIs in 1500 μm increments (a). T1 stimulation in the presence of 1 μM BayK 8644 only produced a transient local Ca2+ signal , however in the additional presence of 10 mM TEA a regenerative Ca2+ intercellular wave propagated along the entire length of the arcade as a Ca2+ spike(s) . The L-type VGCC blocker nifedipine (10 μM) fully blocked these propagating intercellular Ca2+ waves  n=3, Aa. Ca2+ crements Aa. T1 st3.42+ and vasoconstriction was measured at both downstream and upstream ends of denuded arteries , with Ca2+ change measured in five ROIs: 0 μm, 500 μm, 1000 μm, 1500 μm, and 3000 μm (a).Ca 3000 μm Aa.Fig. 5BayK 8644 alone did not enable propagating responses to KCl. In the presence of 1 μM BayK 8644, a 1 s KCl pulse raised local [Ca2+]i by 18.6 ± 2.1% and vasoconstriction by 7.4 ± 0.4% (n=7) . Ca2+ waves propagated at 2.8 ± 0.2 mm s−1  from T1 to T2 with constant amplitude . The amplitude of the Ca2+ wave and phasic contraction in the presence of TEA and BayK 8644 at T2  was not different from T1 .s−1 n=7, Cc from T to 15.4 ± 1.3% and vasoconstriction of 3.6 ± 1.1% (n=5). The subsequent addition of 1 μM Bay K 8644, as above, led to constant amplitude spreading responses at 2.8 ± 0.1 mm s−1 and 2.8 ± 0.2 mm s−1, for [Ca2+]i and vasoconstriction from T1 to T2 (n=6), respectively. Thus, the ability of arteries to spread intercellular Ca2+ waves and phasic contractions was independent of the endothelium.3.52+ waves and vasoconstriction, which started at any point along the artery (2+ spread at 2.6 ± 0.3 mm s−1 with constant amplitude (b–d and B top panel) and was accompanied by phasic vasoconstriction spreading at 2.5 ± 0.3 mm s-1 at both ends of the artery . Spontaneous intercellular Ca2+ waves and vasoconstriction in the presence of 10 mM TEA and 1 μM BayK 8644 were observed in denuded arteries as well.In the presence of 10 mM TEA and 1 μM BayK 8644, over half the arteries studied developed spontaneous intercellular Caery n=5, B–Cab. Sp3.62+ waves and vasoconstriction in denuded arteries exposed to TEA and BayK 8644, but prevented spread , so synchronous Ca2+ waves now appeared randomly with associated vasoconstriction .The gap junction uncoupler 18β-GA (20 μM) did not prevent the appearance of spontaneous propagating intercellular Ca44.12+ - based APs could propagate and as a result generate regenerative intercellular Ca2+ waves in vascular SMCs, in a similar manner to visceral smooth muscle. In the latter, Ca2+ influx via L-type VGCCs causes spike-like APs, which then give rise to propagating intercellular Ca2+ waves, which effectively synchronises contraction in a large group of SMCs [+ channels blocker TEA and L-type VGCC agonist BayK 8644, the propagating SMC Ca2+ transients accompanied by spreading vasoconstriction appears to be mediated by the conduction of APs through gap junctions. The generation and propagation of arterial spike-like APs is facilitated by the regenerative nature of L-type VGCCs.Arterial SMCs are generally quiescent, requiring agonist stimulation to evoke depolarization and vasoconstriction. In the presence of TEA (5–10 mM) arterial SMCs generate spike-like APs  sensitiv of SMCs ,20. In t2+ current in myocytes isolated from rat MA [+ channels are blocked with TEA (5–10 mM). The APs establish propagating spikes of Ca2+ associated with spreading vasoconstriction, which can be blocked by the presence of the selective L-type Ca2+ channel blocker, nifedipine. Thus, L-type VGCC are essential for coupling between APs and intercellular Ca2+ waves, which then cause propagating vasoconstriction. In over half the arteries studied, propagating Ca2+ and mechanical waves occurred spontaneously in the presence of TEA and BayK 8644, and in all cases could be evoked by a brief (1 s) local application of 60 mM KCl. In each case, intercellular Ca2+ wave could propagate in a regenerative manner from the point of initiation, at a speed of around 3 mm s−1 and with constant amplitude (around 50% of peak Ca2+). The intercellular spread of Ca2+ appeared as a Ca2+ spike and was accompanied by vasoconstriction, also of constant amplitude (about 30% of peak force) and at a similar speed. Interestingly, neither was altered in the absence of the endothelium, these parameters were not significantly different. Indeed, in denuded arteries intercellular Ca2+ wave could propagate in a regenerative manner at similar speed 2.8 + 0.1 mm s−1 accompanied by vasoconstriction spreading at 2.8 + 0.2 mm s−1. This contrasts with skeletal muscle feed arteries, in which the spread of vasoconstriction initiated by depolarization to KCl was abolished when the endothelium was removed [2+ could occur in either direction across the entire length of MA segments, and suggests gap junctions allow the transmission of AP, by cell-to-cell communication and without rectification. This required an active regenerative mechanism, as the Ca2+ signal caused by depolarization that did not initiate an AP decayed rapidly with distance. The regenerative propagating AP mediated by L-type VGCCs could be evoked provided K+ channels were blocked. Similar findings were observed in visceral SM e.g. guinea pig urinary bladder [2+ wave propagation in guinea pig urinary bladder was around 1.6 mm s−1 [−1).BayK 8644 affects a 10-fold augmentation in inward Cam rat MA . We show removed . The rap bladder  and rat  bladder . The spe6 mm s−1 , compara4.22+ transients, indicating that propagation was entirely due to the spread of APs via gap junctions. In visceral smooth muscle, the propagation of AP-mediated Ca2+ waves were also disrupted by gap junction blockers . In int network . Our dat network ,42. Barl network  also sugcellular  and myoecellular ,44 gap jcellular ,30,31, acellular ,22,26,29cellular ,27,28,32cellular  detected+ channels enabled the generation of regenerative Ca2+-based APs. These events could spread rapidly in either direction along the artery causing vasoconstriction. Homocellular gap-junctions between smooth muscle in the mesenteric artery enable AP spread without detriment and with no apparent detriment due to myoendothelial gap-junctions.In conclusion, while increasing the pulse duration of KCl enhanced the spread of depolarization and associated vasoconstriction, the spread was limited to less than 1500 μm. Block of current dissipation through KThis work was supported by the British Heart Foundation (BHF) .


SF3B1, SRSF2) and/or chromatin-related genes . Mutations in these gene categories frequently co-occur in AML, in which case they result in additive adverse prognostic effects. In particular, AML with ASXL1 and SRSF2 mutations  has been associated with a dismal prognosis, similar to AML with high-risk cytogenetic features.1 However, there is very little published literature regarding additional clinical or pathologic features of this AML subgroup.In recent years, the genetic profile of acute myeloid leukemia (AML) has been extensively characterized, and multiple prognostically distinct subgroups have emerged. One subgroup arising in elderly patients harbors somatic mutations in RNA splicing factor genes ,4 a myelodysplastic (MDS)/myeloproliferative neoplasm (MPN) clinically characterized by peripheral monocytosis and frequent transformation to acute leukemia.5 To date, the immunophenotype of ASXL1/SRSF2 co-mutated AML has not been characterized. However, in AML arising from antecedent CMML, leukemic blasts tend to retain monocytic differentiation.6 As such, we speculated that ASXL1/SRSF2 co-mutation may also drive monocytic differentiation in AML, potentially in addition to other clinicopathologic similarities with CMML.After ASXL1/SRSF2 co-mutation identified in diagnostic myeloid sequencing panel analysis performed at our institution over a 3-year time period . One-hundred and fifty consecutive non-co-mutated AML  from April, 2016 to April, 2018 were collected for comparison . Concomitant hematopathology reports were reviewed to confirm diagnoses in all cases. Patients with sequencing data available only from recurrent/relapsed disease were excluded, as were cases of acute promyelocytic leukemia.To address this possibility, we retrospectively identified adult AML patients (≥18 years) with Clinical sequencing and flow cytometric analysis (FCA) methods are detailed in the Supplemental Methods . Briefly, next generation sequencing was performed on DNA collected from diagnostic bone marrow or peripheral blood samples using the Illumina® TruSight Myeloid 54-gene sequencing panel . Cases where immunophenotypic FCA was performed on blood or bone marrow aspirate specimens within three months of sequencing without intervening curative therapy were included.ASXL1/SRSF2 co-mutated AML were identified during the study period. All patients with ASXL1/SRSF2 co-mutated AML were over 50 years old at diagnosis and significantly older than patients with non-co-mutated AML  classified as de novo AML who had available complete blood counts prior to diagnosis had an occult peripheral blood monocytosis, documented between 7 and 36 months before AML onset (Table S1). This observation raises the possibility that a subset of these AML cases may have evolved from unrecognized CMML.Sixteen patients with ASXL1/SRSF2 co-mutated AML showed overt evidence of monocytic differentiation. Specifically, 80% (12/15) of ASXL1/SRSF2 co-mutated AML cases with available FCA data expressed 2 or more of the monocytic lineage-defining markers CD11c, CD14, or CD64, compared to 47% of non-co-mutated AML , and the leukemic blasts of another patient expressed one monocytic marker and gated in the monocyte region by FCA , supporting monocytic differentiation.All but one 8 were more commonly expressed in ASXL1/SRSF2 co-mutated AML than in non-co-mutated cases . In addition, the myeloblast markers CD34 and CD117, which are not typically expressed in monocytic precursors, were less commonly expressed in ASXL1/SRSF2 co-mutated AML than in non-co-mutated AML . A representative flow cytometry plot from a patient with ASXL1/SRSF2 co-mutated AML is shown in Figure S2.Several additional immunophenotypic features provided further evidence of monocytic differentiation . CD56 and CD2, which are frequently aberrantly expressed on monocytes in CMML,ASXL1/SRSF2 co-mutated AML tends to exhibit evidence of monocytic differentiation, we next sought to determine if this association was dependent on co-mutation or whether similar trends existed for cases of AML with mutation in either ASXL1 or SRSF2. Compared to AML cases with either ASXL1 or SRSF2 mutation but not both , ASXL1/SRSF2 co-mutated AML was significantly more likely to express 2 or more monocytic markers , more likely to express aberrant CD2 and CD56 , and significantly less likely to express the myeloblast markers CD34 and CD117  . In all cases, SRSF2 mutations were identified at a VAF of ∼50%, suggesting the presence of a heterozygous activating mutation in the majority of cells. By comparison, VAF of ASXL1 mutation was more variable, ranging from 13% to 47%, but was >25% in 13/16 co-mutated cases . These results suggest that the majority of cells harbored concomitant ASXL1 and SRSF2 mutations in most cases.Next, we evaluated the mutation landscape of TET2, the most commonly mutated gene in CMML, was more likely to be mutated in ASXL1/SRSF2 co-mutated cases than in non-co-mutated cases . Other mutations more commonly seen in ASXL1//SRSF2 co-mutated AML compared to non-co-mutated AML included STAG2 and IDH1/IDH2 . Notably, mutations in these genes are often enriched for in AML with mutated chromatin-related or RNA splicing factor genes.1 No other genes on our 54-gene panel were mutated at statistically significant differences between cohorts (data not shown). However, after excluding the presence of ASXL1 and SRSF2 mutations, we noted that ASXL1/SRSF2 co-mutated AML harbored more total variants (median 3 per patient) than ASXL1wtSRSF2wt AML and all non-co-mutated AML .ASXL1/SRSF2 co-mutated AML cases had a normal karyotype at diagnosis, and none harbored AML-defining recurrent cytogenetic abnormalities (Table S1).1 Three patients (19%) with ASXL1/SRSF2 co-mutated AML had trisomy 8, the most common cytogenetic abnormality identified in CMML.9 Although cytogenetic abnormalities were uncommon in ASXL1/SRSF2 co-mutated AML, morphologic dysplasia in 2 or more myeloid lineages was seen in 8/11 (73%) evaluable ASXL1/SRSF2 co-mutated AML cases, including 4/6 (67%) cases with history of a prior myeloid neoplasm and 4/5 (80%) de novo cases (Table S1).In terms of cytogenetic features, half of ASXL1/SRSF2 co-mutated AML. However, overall survival (OS) was generally poor, as nearly all patients died within 2 years of AML diagnosis (Table S1). The apparently poor outcomes associated with this genotype may be related to disease ontogeny, as ASXL1/SRSF2 co-mutation occurred more often in patients with a prior myeloid neoplasm . These results are in keeping with findings from earlier studies which identified mutational signatures of secondary AML.10 Finally, while we did confirm a trend towards inferior OS in patients with co-mutated ASXL1/SRSF2 compared to ASXL1wtSRSF2wt AML patients (P = 0.099), no pairwise comparisons of OS between study groups reached statistical significance (Table Given the relatively small sample sizes in this cohort, it is not possible to make definitive conclusions regarding patient outcomes in NPM1-mutated AML with FLT3-internal tandem duplication.11 Moreover, in patients with chronic myeloid neoplasms with dysplasia, SRSF2 and TET2 mutations have been frequently associated with monocytosis and myelomonocytic phenotype, particularly when both are mutated concurrently.12 For instance, Awada et al13 showed that the presence of 2 or more TET2 mutations with VAF sum >55% most commonly occur in MDS/MPN, particularly CMML and other myeloid neoplasms associated with monocytosis and marrow myeloid dysplasia. SRSF2 mutations also frequently co-occurred with multiple TET2 mutations, further supporting a potential relationship between SRSF2 mutation and monocytic differentiation.13The association between certain co-occurring mutations and distinct monocytic phenotypes is well-established. For instance, monocytic cytomorphology is frequently seen in ASXL1/SRSF2 co-mutated AML shows evidence of monocytic differentiation and has genetic overlap with CMML, including the frequent presence of TET2 mutations. In addition, ASXL1/SRSF2 co-mutated AML frequently arises secondarily in patients with antecedent myeloid malignancy, and even when these mutations apparently occur “de novo,” patients may have unrecognized peripheral monocytosis prior to leukemic onset. Whether ASXL1/SRSF2 co-mutation is a biological driver of monocytic differentiation remains uncertain. In addition, it will be important to confirm these observations in larger, multi-institutional studies. Nonetheless, our findings raise the possibility that this subset of AML may often arise as a secondary AML from an occult CMML-like MDS or MDS/MPN even in the absence of a known prior myeloid neoplasm.In conclusion, we show that