Ca2+ release through RyR2 can be either stimulated by an action potential (AP) or spontaneous. The latter is often associated with triggered afterdepolarizations, which in turn may lead to sustained arrhythmias. It is believed that some synchronization mechanism exists for afterdepolarizations and APs in neighboring myocytes, possibly a similarly timed recovery of RyR2 from refractoriness, which enables RyR2s to reach the threshold for spontaneous Ca2+ release simultaneously. To investigate this synchronization mechanism in absence of genetic factors that predispose arrhythmia, we examined the generation of triggered activity in multicellular cardiac preparations. In myocardial trabeculae from the rat, we demonstrated that in the presence of both isoproterenol and caffeine, neighboring myocytes within the cardiac trabeculae were able to synchronize their diastolic spontaneous SR Ca2+ release. Using confocal Ca2+ imaging, we could visualize Ca2+ waves in the multicellular preparation, while these waves were not always present in every myocyte within the trabeculae, we observed that, over time, the Ca2+ waves can synchronize in multiple myocytes. This synchronized activity was sufficiently strong that it could trigger a synchronized, propagated contraction in the whole trabecula encompassing even previously quiescent myocytes. The detection of Ca2+ dynamics in individual myocytes in their in situ setting at the multicellular level exposed a synchronization mechanism that could induce local triggered activity in the heart in the absence of global Ca2+ dysregulation.In myocardial tissue, Ca The RyR2 has numerous binding sites for regulatory molecules and accessory proteins with the capability to modulate RyR2 function. Ca2+ release from the SR is a key determinant of cardiac contractility and both the cytoplasmic as well as the luminal Ca2+ concentration can regulate the RyR2. In the SR lumen, in particular, there is a substantial amount of Ca2+ buffering, and, according to several studies, approximately 50–90% of the total Ca2+ is bound to SR Ca2+ binding proteins, such as calsequestrin (CASQ2) (2+ in the lumen of the SR (2+ present in the SR is the driving force for SR Ca2+ release (The sarcoplasmic reticulum (SR) Ca (CASQ2) . Therefof the SR , and, ul release .2+ release is diminished, the open probability of the RyR2 decreases. The temporary, unresponsive state that the RyR2 enters after SR Ca2+ release is important to the mechanical refractoriness that is required for effective relaxation and refilling of the ventricles , and there is an increase in the open probability. This enhanced RyR2 open probability can result in an increased Ca2+ release during systole; however, in diastole, it can cause an inadvertent SR Ca2+ leak . The Ca2+ release from the SR increases the Ca2+ concentration in the cytoplasm, allowing for the activation of the Na+/Ca2+ exchange (NCX), which pumps the Ca2+ that leaked from the SR out of the myocyte. Since the NCX ratio is three Na+ ions to one Ca2+ ion, the membrane potential becomes more positive and can produce transient depolarizations causing DADs (Triggered arrhythmias can originate from delayed afterdepolarizations (DADs) and early afterdepolarizations (EADs), and they are initiated by variations in the membrane potential . DADs aring DADs .2+ release simultaneously. We recently showed that such a mechanism can occur in genetically mutated murine myocardium  in a single myocyte, the transient current must be large enough to reach the threshold for AP generation. However, this situation is much more complicated at the whole heart level. Since myocytes are electrically coupled by gap junctions, any depolarization that arises in one myocyte would most likely be dampened due to dissipation of the current in the neighboring myocytes; therefore, the likelihood of triggering an ectopic AP is low. It has been proposed that a DAD would have to arise in approximately 1000 myocytes for an ectopic beat to take place . Severalocardium , but it All protocols were approved by the institutional laboratory animal care and use committee.2PO4, 1.2 MgSO4, 20 NaHCO3, 0.25 CaCl2, and 10 Glucose. 2,3-Butanedione monoxime  was added to the KH buffer to minimize the cutting injury  were anesthetized using an intraperitoneal injection of euthasol . Heparin (1000 U) was injected at the apex of the left ventricle after the chest had been opened by bilateral thoracotomy. The heart was quickly removed and placed in a Krebs–Henseleit (KH) buffer containing (in mM) 137 NaCl, 5 KCl, 1.2 NaHg injury , as expog injury . Hearts escribed . The musThe trabeculae were stretched to a length where further stretching raised diastolic and systolic force proportionally . It has 2+ imaging (F/F0 [(F-F0)/F0], where F is the fluorescence at time t and F0 represents the background signal.Isolated rat trabeculae were loaded with 40 μM Rhod-2 AM, which was dissolved in 2% pluronic (20% w/v) in 1 mL KH buffer with the addition of TPEN (4.3 mg/L) and cremophor (5 mg/L), in a bath for 45 min at room temperature . The ele imaging . In orde2+ transients are typically measured. In order to confirm that the line-scan represented five to seven myocytes in the center part of the preparation, trabeculae were loaded with di-4-ANEPPS to identify myocytes borders, as shown in Figure 1.In separate experiments, we performed 2D scanning of the entire muscle at the same depth the CaData were collected and analyzed using LabView, ImageJ, and Origin software. All data are presented as mean ± SEM, unless otherwise stated. All experiments were performed using protocols approved by The Ohio State University Laboratory Animal Care and Use Committee.Figure 2A); however, after 1 min of perfusion with 100 nM isoproterenol and 0.5 mM caffeine the trabeculae displayed ESCs between successive twitches . Upon application of both isoproterenol and caffeine, the developed force remained unchanged in the muscles as compared with control, most likely due to the diastolic Ca2+ release diminishing the SR Ca2+ store. Thus, ESCs in the trabeculae occurred in a highly synchronized manner leading to a spontaneous contraction with characteristics similar to those of the stimulated twitch in both amplitude and relaxation timing . We found that not only does the frequency of ESCs increase with time but that the amplitude is altered as well.To investigate the effect of both isoproterenol and caffeine on the incidence of ESCs in trabeculae, we monitored the contractile activity in these muscles while electrically pacing at 1 Hz. As expected, the control, untreated muscle showed no signs of ESCs following relaxation  have an amplitude of less than 40% of the developed force of stimulated contractions. However, 24% of the ESCs showed an amplitude between 40 and 80%, while 14% of ESCs showed and amplitude 80% or higher. Notably, the majority of the ESCs with the highest amplitude occurred within the first ESC group. Although we were not able to simultaneously measure the membrane potential, we hypothesize that many of these high-amplitude ESCs would synchronize and then could result in extra-systolic APs triggered in the trabeculae. In a previously published study by our group . The latency distribution shows a period of refractoriness after the stimulated twitch, when no ESC occurred, followed by the presence of the first ESC at 310 ± 4 ms . The second ESC occurred at 578 ± 10 ms  while the third ESC appeared at 702 ± 10 ms . Therefore, these results show that the ESCs were highly temporally aligned, which is consistent with a synchronized recruitment of myocytes in the multicellular preparation. The number of waves, and amplitude, varied from muscle-to-muscle, likely due to the various sizes of cross-sectional areas of muscle used, and also increased with time of exposure.We calculated both the ESC amplitude and the time from the stimulated contraction to the start of the ESC, or latency period from recordings from ur group , perform2+ release, we loaded cardiac trabeculae with the Ca2+ indicator Rhod-2. Figure 5 shows a line-scan image of Ca2+ changes in a single trabecula. As expected, the electrical stimulation triggers SR Ca2+ release simultaneously across each of the myocytes in the field of view. Following the addition of isoproterenol and caffeine, the trabecula shows spontaneous Ca2+ waves that occur between the stimulated Ca2+ release. These Ca2+ waves have a lower amplitude than the induced Ca2+ transients and, while they occur in several of the cells, they do not occur simultaneously across each of the myocytes. However, after 5 min the Ca2+ waves synchronize as the Ca2+ release occurs much more uniformly in the myocytes. Figure 5 also shows the line-scans and corresponding Ca2+ fluorescence plot profiles for each of the individual myocytes in the field of view. The fluorescent plot profiles in the top panel demonstrate that each of the diastolic Ca2+ release events in the cells behaves differently, as the fluorescence signal from the Ca2+ waves peaks throughout the entire diastolic period. As the Ca2+ release becomes synchronized, the fluorescence peaks at the same time in each of the myocytes. Consistent with our force experiments, these results indicate that there is a synchronized diastolic Ca2+ release that occurs across each of the myocytes in the field of view . In Figure 6A, we show a typical example of the time of onset of the largest ESC at 1 min, and after 5 min in six adjacent cells. This shows that over time, the adjacent cells seem to synchronize their ESC, i.e., onset of ESC grows closer and closer in all adjacent cells. Still, after 5 min, there is remains some little variation in time of onset (up to 32 ms in this example) in the adjacent cells, indicating synchronization that is not directly due to an AP. In Figure 6B, it can be seen that a non-externally stimulated AP causes the adjacent cells (four in this example), to all start the non-externally stimulated event at identical timepoint (0–1 ms variation among the four cells).To further examine the synchronization of CaFigure 7). At 0.2 mM caffeine, the distribution of the latency of the first Ca2+ wave was shifted slightly rightward, compared to the distribution at 0.5 mM caffeine. The distribution of all waves had a similar behavior. In addition, the number of waves per cycle decreased significantly at the 0.2 mM caffeine concentration.We next investigated the myocytes response to a lower dose of caffeine . Immediately following the last stimulated Ca2+ transient, there was a non-externally triggered Ca2+ release event that occurred uniformly throughout the myocytes; however, over time, this Ca2+ release decreased in both amplitude and temporal homogeneity among myocytes until there were only sporadic and spontaneous Ca2+ waves. Conversely, on occasion, there was a synchronized SR Ca2+ release event long after the stimulation had been removed . Before this synchronized release, the myocytes in this line-scan were completely quiescent, but the amplitude of this Ca2+ transient was as large as in stimulated ones. This indicated that the Ca2+-transients in these myocytes were likely triggered by a propagating AP that originated elsewhere in this preparation.To investigate the role of systolic Ca2+ dysregulation, i.e., in the presence of both isoproterenol and caffeine, diastolic SR Ca2+ release occurs with increasing synchronization among the myocytes in situ in isolated cardiac muscles in otherwise healthy rat myocardium. The Ca2+ dysregulation causes ESCs, and the amplitude of these ESCs ranged up to 100% of the amplitude of the developed force in electrically stimulated contractions. This latter finding suggests that all the myocytes were activated during some of these ESCs, which is in close agreement with the findings of our previous study regarding synchronization leading to non-externally stimulated APs in genetically manipulated murine myocardium  within the line-scan, indicating it was  triggered rather than spontaneous. As the fluorescence profiles of the individual cells show, when the (spontaneous) Ca2+ release was asynchronous, the corresponding fluorescence signal peaked at different time points throughout diastole. However, when there was a synchronous Ca2+ release across all of the myocytes, the fluorescence peaked around the same time point in each cell. Consistent with the force experiments, this finding shows that with the addition of isoproterenol and caffeine diastolic Ca2+ release events do, indeed, occur, and these Ca2+ release events were able to synchronize across multiple myocytes in all the trabeculae examined. Gap junctions connect myocytes, and possibly calcium diffusion across these intercellular junctions can help contribute to a reduction in the threshold for a diastolic calcium release event. When a sufficient number of adjacent, coupled myocytes experience this reduction in threshold, it would increase the chance for the generation of a propagating AP.To further examine the synchronization of Ca2+ release in the synchronization of Ca2+ waves, we observed that after the electrical stimulation had been stopped, a Ca2+ release event occurred uniformly throughout the myocytes. Over time, Ca2+ release decreased in both amplitude and temporal homogeneity until only sporadic Ca2+ waves occurred. Occasionally, a non-externally triggered whole-preparation synchronized SR Ca2+ release happened long after the stimulation had stopped. Since in the monitored area of the muscle there were no Ca2+ waves leading up to this uniform Ca2+ release, we conclude that it was the result of a triggering event that occurred from a distant cluster of myocytes and propagated to the field of view.When investigating the role of systolic Ca2+ handling is primarily focused on individual myocytes; however, DADs and triggered arrhythmias in myocytes cannot directly be extrapolated to the intact heart, where the electrical coupling of myocytes acts as a “sink” for depolarizing currents (2+ release was able to trigger a full-sized ESC in the entire preparation, including in parts of the muscle that were virtually completely quiescent, and were thus at least partially acting as an electrical sink. Anatomically, these trabeculae typically insert into the more solid ventricular mid-myocardium in wafer-like shapes, where the number of myocytes in the cross-section slowly increases as the trabecula inserts into the free wall. Many of the smaller muscles insert first into larger ones that, in turn, insert into the ventricular wall. We postulate that it is possible that this gradual change in the electrical sink capacity is not sufficient to quench the propagation of DADs, and may thus allow the trigger of an ESC. Future investigations will be needed to address this hypothesis.The vast majority of research in cardiomyocyte Cacurrents . On avercurrents ; when thcurrents , while ocurrents . This nu2+ release can become synchronized in multicellular trabeculae. Assessment of the developed force and intracellular Ca2+ suggests that the addition of both isoproterenol and caffeine can result in ESCs, and these events were able to spatially and temporally synchronize across multiple myocytes. Given the anatomy of the endocardial surface, it is feasible that triggered ventricular ESCs can be caused by a small, specifically localized group of myocytes, and may not necessarily require the much larger amounts of myocytes predicted by previously conducted modeling studies.The present study demonstrates that diastolic SR CaAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer KL and handling Editor declared their shared affiliation.


We expanded a previously published mathematical model of water and solute transport in the rat PT  that did not include Ca2+. Our results indicate that Ca2+ reabsorption in the PT is primarily driven by the transepithelial Ca2+ concentration gradient that stems from water reabsorption, which is itself coupled to Na+ reabsorption. Simulated variations in permeability or transporter activity elicit opposite changes in paracellular and transcellular Ca2+ fluxes, whereas a simulated decrease in filtration rate lowers both fluxes. The model predicts that PTH-mediated inhibition of the apical Na+/H+ exchanger NHE3 reduces Na+ and Ca2+ transport to a similar extent. It also suggests that acetazolamide- and SGLT2 inhibitor-induced calciuria at least partly stems from reduced Ca2+ reabsorption in the PT. In addition, backleak of phosphate (PO4) across tight junctions is predicted to reduce net PO4 reabsorption by ~20% under normal conditions. When transcellular PO4 transport is substantially reduced by PTH, paracellular PO4 flux is reversed and contributes significantly to PO4 reabsorption. Furthermore, PTH is predicted to exert an indirect impact on PO4 reabsorption via its inhibitory action on NHE3. This model thus provides greater insight into the mechanisms that modulate Ca2+ and PO4 reabsorption in the PT.The objective of this study was to examine theoretically how Ca There is some evidence , acetazolamide, and inhibitors of Na+-glucose transporters (SGLTs). We expanded a model of water and solute transport in the PT that we published previously . In plasma, PO4 is mostly present as 4 is reabsorbed by the PT (4 entry into the cell is mediated by three types of Na+-phosphate cotransporters: NaPi-IIa (SLC34A1), NaPi-IIc (SLC34A3), and PiT-2 (SLC20A2)  . NaPi-II+ and + at a given perfusion rate.The PT model incorporates flow-dependent transport, i.e., the observation that high flow velocity augments transepithelial fluxes by increasing transporter membrane abundance . Du et alleagues , the abu+ and K+ equals approximately two-thirds of the filtered load.The mathematical model of transport along the PT of a male rat is based on conservation equations, which are solved at steady state. The PT consists of two cortical (S1–S2) segments (with a combined length taken as 0.97 cm) and a medullary (S3) segment (0.13 cm). We assume that all segments of the PT express the same types of channels, pumps, and cotransporters, with the exception of glucose transporters . Membran2+ in the lumen, cells, and intercellular space is written as follows:V represents the luminal flow rate, SNM is the surface area per unit length at the interface between compartments N and M, and NMJ is the flux across that interface. Note that at steady state the net flux of Ca2+ from intracellular stores to the cytosol and the net rate of Ca2+ binding to Ca2+ buffers in the cytosol and the sarcoplasmic reticulum are zero  is computed as follows:JV is the volume flux (superscripts are omitted for simplicity), Ca is the reflection coefficient of the membrane to Ca2+; σCa is set to 1 for cell membranes, 0.89 for the tight junction (TJ)  , and 0 f2+ is taken as 20 × 10−5 cm/s based on the compilation in Ref. 2+ is computed based on the permeability to Na+, given a Ca2+-to-Na+ free diffusivity ratio of 7.93:13.3  at rate given by2+  increases linearly from 1.25 mM at the corticomedullary junction to 1.60 mM at the end of the S3 segment and to 2.50 mM at the junction between the outer medulla (OM) and inner medulla (IM). The single-nephron glomerular filtration rate (SNGFR) is taken as 30 nl/min, and the filtered load of Ca2+ is 37.5 pmol/min per nephron.Interstitial fluid concentrations are prescribed in this model: they are equal to plasma concentrations in the cortex and increase linearly along the corticomedullary axis in the medulla. Based on our macroscopic model of Carstitium , which d4 transporter was represented as a 1 Na+:1 4 entry into PT cells is mediated by the cotransporters NaPi-IIa, NaPi-IIc, and PiT-2 Ca] increases from 1.0 to 1.3, in accordance with reported measurements  segment, owing to the interstitial osmolality gradient, so do [Ca2+] in the lumen and (TF/GF)Ca Ca .JCa denotes the local or average Ca2+ flux (in pmol·min−1·mm−1) and TCa denotes the rate of Ca2+ reabsorption along the entire PT (in pmol/min). The base-case TCa is computed as 26.0 pmol/min, 85% of which is paracellular. Equivalently, the average JCa equals 2.36 pmol·min−1·mm−1 . As water reabsorption raises [Ca2+] in the lumen above that in the lateral interspace and the interstitium  is zero; moreover, there is significant water reabsorption from the cell into the lateral interspace, so that the water flux across the BM is larger than that across the TJ. The rapid convective transport of Ca2+ into the interstitium lowers [Ca2+] in the lateral interspace below that in the interstitium along parts of the tubule . The electric potential in the lumen is predicted to rise from −0.22 mV at the PT inlet to a maximum of 1.31 mV about halfway through the tubule and to decrease to +0.94 mV at the outlet. The electric potential in the lateral interspace remains between −0.06 and +0.01 mV in the cortex and the medulla. As ΔΨ across the TJ changes sign and becomes lumen-positive, its contribution to the electrodiffusive flux increases. Nevertheless, our computations suggest that, overall, ΔΨ exerts a lower driving force than the lumen-to-lateral interspace [Ca2+] gradient.Note that electrodiffusion is governed by both the transmembrane  gradient is predicted to enhance the diffusion of Ca2+ across the paracellular pathway in the S3 segment and augment its overall reabsorption . More specifically, as the filtered load of Ca2+ is raised from 37.5 to 45.0 pmol/min per nephron, TCa increases from 26.0 to 32.9 pmol/min, and the length-averaged JCa increases from 2.36 to 2.99 pmol·min−1·mm−1. Ca2+ transport is elevated across both pathways: transcellular JCa increases owing to enhanced membrane abundance of Ca2+ transporters, and paracellular JCa increases because the higher rate of water reabsorption augments the lumen-to-interstitium [Ca2+] gradient.To examine the coupling between the transport of Caransport , 34, 62.+, and Ca2+ is predicted to then decrease by 5–7% (from 69.3% to 62.7% for Ca2+). With a Ca2+ filtered load of 30.0 pmol/min per nephron, TCa equals 18.8 pmol/min, and the length-averaged JCa equals 1.71 pmol·min−1·mm−1 , Na+-PO4 cotransporters, and the basolateral Na+-K+-ATPase pump , thereby augmenting basolateral Na+ secretion via the Na+-dependent Cl−/+-K+-ATPase activity partly counterbalances these effects. Moreover, as transcellular Na+ transport decreases, less water is reabsorbed, so that luminal flow decreases less rapidly; the higher microvillous torque then recruits more transporters to the membrane, which conversely enhances transcellular transport. In the following simulations, Na+ reabsorption was reduced by 30%, while intracellular [Na+] was maintained at >10 mM, by lowering the basal (without torque-mediated effects) expression of NHE3, Na+-PO4 transporters, and Na+-K+-ATPase by 75%, 75%, and 25%, respectively. Owing to the compensatory and torque-modulated effects described above, the Na+ fluxes across NHE3, Na+-PO4 transporters, and Na+-K+-ATPase were equal to 79%, 57%, and 92%, respectively, of their base-case values.PTH acts to augment Ca in dogs . This maase pump ; the res+ transport is predicted to reduce TCa also by 30%, in accordance with experimental observations . Overall, these results indicate that PTH indeed reduces TCa indirectly, via its effects on Na+ transport.As summarized in rvations . As wate+-PO4 transporters, and Na+-K+-ATPase by 75%, 75%, and 25%, respectively. As shown in + transporter activity on Ca2+ transport are almost additive; fractional Ca2+ reabsorption is computed as 47.0% vs. 69.3% in the base case.PTH is also known to reduce glomerular filtration rate (GFR), perhaps via tubuloglomerular feedback . Thus, i2+  are sufficient to overcome the effects of the permeability reduction. Nevertheless, overall TCa decreases by >10% if There is some evidence that PTH may also decrease paracellular permeability in the PT , 31. To  effects . Decreas however . Moreoveor of >2 .2+] and, therefore, in transcellular TCa. Furthermore, paracellular JCa becomes lower than in the base case beyond a certain point along the PT  increases in Increasing g the PT , owing t2+-sensing-receptor (CaSR) or CaSR-like molecules may be expressed in the PT  expression of NHE3 by 40%. Expression of Na+-K+-ATPase was raised concomitantly, albeit to a lesser extent (10%), so as to maintain intracellular [Na+] below 25 mM. The basal expression of basolateral Na+-+-Several studies have suggested that the Can the PT , 22, 43,f debate . Capassoxtrusion . Togethe studies . To examn the PT , 59, 61.+ flux across NHE3 raises intracellular [Na+], which then decreases Na+ entry via Na+-PO4 cotransporters. It also increases water reabsorption, thus reducing the luminal flow and microvillous torque, such that fewer transporters are recruited to the membrane. Specifically, upregulating Na+ transporter activity as described above is predicted to raise fractional Na+ reabsorption from 70.1% to 73.5%, which in turn elevates fractional Ca2+ reabsorption from 69.3% to 72.8% . As previously described , raises urinary Caxcretion , 38. To escribed , luminale by 23% ; an iden+-HCO3 transporter NBC1 , and delivery of Ca2+ to the loop of Henle increases from 11.5 to 14.7 pmol/min per nephron.Inhibitors of SGLT2 are increasingly used to treat diabetes, but they have been linked to bone loss or increased risk of fracture, possibly as a result of altered Catabolism . SLGT2 iand mice , 35, buts by 12% , fractio2+ reabsorption decreased even further (to 56.6%), and the distal delivery of Ca2+ remained higher than in the base case (13.8 pmol/min per nephron). Together, these simulations suggest that SGLT2 blockers may significantly affect renal handling of Ca2+ (see below).By significantly reducing proximal reabsorption, SGLT2 blockers activate tubuloglomerular feedback, which in turn reduces SNGFR. Chronic SGLT2 blockade was found to lower GFR by 15% in diabetic rats . When we4 reabsorption in the PT PO4] is <1.0 and is comparable to micropuncture measurements in rats and dogs  is 61.8 pmol/min, with 78.5 pmol/min reabsorbed via transcellular routes and 16.7 pmol/min secreted via paracellular routes. The average PO4 flux (JPO4) is computed as 5.6 pmol·min−1·mm−1.If it is assumed that the TJ permeability to PO0−5 cm/s , the mods the TJ . Net PO44 reabsorption in mice is thought to be ~70%, based on gene knockout studies (+-PO4 transporter in turn. Results are summarized in 4 reabsorption by 26% (to 45.7 pmol/min) and net Na+ reabsorption by 3%. The lower PO4 transport rate means that (TF/GF)PO4 remains above 1.0, backleak across the TJ into the lumen is abolished, and the paracellular pathway mediates PO4 reabsorption instead of secretion; it is predicted to represent 16% of the total flux. The contribution of NaPi-IIc to TPO4 then exceeds that of PiT-2  gradient still favors paracellular PO4 secretion, as in the base case, but the PO4 flux across the TJ is nevertheless reduced. As a result of both effects, net TPO4 is predicted to decrease only slightly, by 4% , transcellular JPO4 remains higher, and paracellular PO4 reabsorption is significantly lower . When combined with PTH-mediated inhibition of Na+-PO4 transporters and NHE3, fractional PO4 reabsorption is further reduced . If it is assumed that the reflection coefficient of the TJ to Ca2+ is equal to 0.89 . Conversely, reducing transcellular Ca2+ transport elicits a compensatory increase in paracellular Ca2+ transport, such that the overall TCa also does not vary much , the reflection coefficient of the TJ to Ca2+, and the permeability of the BM to Ca2+. Our results suggest that the latter three parameters have only a moderate impact on TCa  by the same factor according to our simulations. PTH is also known to affect GFR. Per se, a PTH-induced decrease in GFR reduces the absolute transport rates of Na+ and Ca2+, but fractional reabsorption is somewhat maintained by flow-dependent (torque-mediated) transport mechanisms. Since the largest proportion (nearly two-thirds) of the filtered load of Ca2+ is reabsorbed in the PT, small variations in transepithelial Ca2+ transport in the PT may have a considerable impact on final calciuria, even if Ca2+ reabsorption increases downstream via tubular cross talk between segments.Whereas PTH augments Can the PT . This re2+ may also lower TCa substantially  changes in paracellular and transcellular Ca2+ fluxes (2+ transport increases (or decreases) both across and between cells, as described above.In addition, the model predicts that a (putative) inhibitory action of PTH on TJ permeability to Caantially ; indeed,+ fluxes . One exc2+] has a demonstrable impact on water and Na+ fluxes in the PT  via two mechanisms: not only is the activity of Na-PO4 cotransporters stimulated by the decrease in intracellular [Na+], but their membrane abundance (and that of other transcellular transporters) is also upregulated in response to the higher luminal flow. Nevertheless, even a small (5%) SGLT2 inhibition-induced decrease in SNGFR more than counterbalances these effects and lowers TPO4 below its baseline value. If it is assumed that blocking SGLT2 lowers SNGFR by 15%  is increased threefold, from 0.13 to 0.40%  can be sufficiently upregulated to maintain urinary Ca2+ excretion at ∼1%. This suggests that the higher FECa observed in claudin-2 KO mice might only be an indirect effect, the consequence of reduced Na+ and water reabsorption in the PT. A model of transport along the entire nephron would help fully elucidate this question. Experiments designed to block Ca2+ reabsorption specifically in the thick ascending limb and/or the distal convoluted tubule of claudin-2 knockout mice would also yield a better understanding of the relative contribution of these segments in compensating for the loss of Ca2+ in the PT due to the absence of claudin-2.A 10-fold decrease in the paracellular permeability to Na+-PO4 transporter in the PT. In mice, but not in humans, the contribution of NaPi-IIa predominates  and Swiss National Foundation Grant 310030-163340.No conflicts of interest, financial or otherwise, are declared by the authors.A.E. and O.B. conceived and designed research; A.E. performed experiments; A.E. analyzed data; A.E. and O.B. interpreted results of experiments; A.E. prepared figures; A.E. drafted manuscript; A.E. and O.B. edited and revised manuscript; A.E. and O.B. approved final version of manuscript.


Nitellopsis obtusa, obtained in artificial pond water (APW) and under osmotic stress of 90 or 180 mM sorbitol APW or saline stress of 50 or 100 mM NaCl APW, were simulated by the Thiel-Beilby model  mediating the opening of Ca2+ channels on internal stores. In plants the IP3 receptors have not been identified, so other second messengers might translate the threshold plasma membrane depolarization to Ca2+ release. The increased Ca2+ concentration in the cytoplasm activates Cl− channels, which lead to the depolarizing phase of the AP. The repolarization to normal resting potential difference (PD) results from the Ca2+ being re-sequestered by the Ca2+ pumps, the closure of the Cl− channels, efflux of K+ through the depolarization-activated outward rectifier channels and the continuing activity of the proton pump. The Nitellopsis AP form is longer in APW compared to that of Chara, with more gradual repolarization. The tonoplast component of the AP is larger than that in Chara australis. The plasma membrane AP is prolonged by the exposure to saline to a “rectangular” shape, similar to that in Chara. However, the changes are more gradual, allowing more insight into the mechanism of the process. It is possible that the cells recover the original AP form after prolonged exposure to brackish conditions. Some cells experience tonoplast APs only. As in Chara, the proton pump is transiently inhibited by the high cytoplasmic Ca2+ and gradually declines in saline media. However, if the cells are very hyperpolarized at the start of the experiment, the pump inhibition both by the AP and by the saline medium is mitigated. The model parameters and their changes with salinity are comparable to those in Chara.Action potentials (AP) of characean cells were the first electrical transients identified in plants. APs provide information about plethora of environmental cues. Salinity stress is critical for plants and impacts on excitability. The AP of brackish Characeae  Nitellopsis obtusa is a slightly salt tolerant Characeae that can grow at salinities of ~50 mM NaCl and survives transient exposures to 100 mM NaCl or more  Cl− and K+, as seen in Chara longifolia  form of d et al.  found th2+]cyt changes and the voltage clamp Cl− currents at the time of excitation   can be phosphorylated in plants to form IP6 and phosphatidic acid, which may also act as second messengers  of Nitellopsis plants in brackish media. We also plan to compare the results to salt tolerant turgor regulator Lamprothamnium, which does not show spontaneous excitation upon salinity increase  J. Groves algae were collected from Lithuania lakes during autumn months and maintained at the room temperature in glass aquariums under daylight conditions (9.5 ± 0.19 μmolm−2s−1) with light/dark photo regime of 12/12 h. The internodal cells (second or third below the tip), separated from neighboring cells were used in experiments. The internodes were kept at least overnight in buffered artificial pond water (APW) in standard light conditions.Solutions are shown in At the time of the experiment the cells were placed in borosilicate chamber consisting of three compartments filled with APW and were continuously bathed in a flowing solution of APW or test solution at a rate of ~1 ml/min using perfusion system Scientifica PPS (UK).2 electrodes placed in each compartment. The current clamp mode was employed to record the membrane potential difference and AP. Universal computer I/O and data acquisition system: Voltage/Current Clamp amplifier TEC-10CX (USA), Digitizer Digidata 1440A, A/D board controlled by pCLAMP 10.2 software . The clamped region of the cell in the central compartment was 5 mm long. Microelectrode impalement, using Micromanipulator: Eppendorf PatchMan (Germany), into the cell vacuole or cytoplasm was followed by 1.5 h resting period. The position of internal electrode was determined from the AP shape and the peak potential  were not fully reset to resting state. More detailed exploration of this short time response will be used in future experiments. After 30 min of resting period different solutions were applied.Intracellular and reference electrodes were placed in the central compartment of 0.5 cm length which was isolated from adjacent compartments with vaseline and constantly perfused. Calomel (Hg/HgCl) half-cells saturated with KCl were used in our experiments. Intracellular microelectrodes with 1 μm tip diameter were made from borosilicate glass capillaries , using high-quality micropipette puller SUTTER INSTRUMENTS P-97 (USA). The microelectrodes were filled with 3 M KCl. Reference electrode, immersed in the vicinity of the cell, was plugged with glass capillary filled with 3 M KCl in agar-agar. Current was injected and APs were evoked by external direct current using separate Ag/AgCltial see . Only th2+ channels on the internal stores having four possible states: unbound (R), bound to IP3 (RI), bound to IP3 and activating Ca2+ molecule (RIC+), bound to IP3, and second inactivating Ca2+ molecule (RIC+C−). The channel conducts in RIC+ state and the refractory period might be caused by the long lifetime of the last state.Wacke et al.  based th2+ cytoplasmic concentration, C, and fractions of channels in each state: x2 (R), x3 (RI), x4 (RIC+), and x5 (RIC+C−):The rate equations for change in Ca3, I0, into the cytoplasm. The IP3 concentration, I, then decays with time t and the time constant determined experimentally by repetitive stimulations  volume to cytoplasmic volume.C0 = permeability of Ca2+ store in absence of IP3, γ1 = density of IP3 activated channels, λ = 1 + νr. The ratio in the Equation (4) is the Hill function, which describes the calcium conductance of the Ca2+ pump with scaled Hill coefficients γ Othmer, . These ci  are the rate constants for forward and backward transitions between the channel states. Some of these are scaled by C0: k2 = k'2C0 and k3 = k'3C0. With the scaling, all xi range between 0 and 1. As the channels have to be in one of the four states, conservation condition applies:ki values are also adjusted to fit the data. The rate Equations (4–8) in xi are numerically integrated to obtain the change in Ca2+ concentration in the cytoplasm  equation, multiplied by the Boltzmann distribution of open probabilities, Po− and Po+, to make the PD-dependence stronger  were adjusted to fit the initial resting PD of the data and, in e AP see , 8, 9, woi, which was set to simulate the cell pre-excitation Resting PD, the best results were obtained by linear decrease (and sometimes subsequent increase) of the parameter producing small changes in the membrane PD. At the peak of the AP the chloride current is much larger than the pump current, so the shape of the AP was only affected toward the end of excitation. The example of such manipulation can be seen in 3 was held at 0, so that the model excitation did not occur, showing how these small changes in κoi, propagate through the integration process of the AP. Unfortunately, there is no such technique in the experiments, to reveal the true time-course of the pump contribution to membrane PD. More information can be gathered in future by following the Resting PD and plasma membrane conductance for some minutes after the AP.Taking the initial κTRP, Ca approximates an initial fast transient inflow of Ca2+ across plasma membrane due to activation of transient receptor potential (TRP)—like channels, possibly activated by another second messenger diacylglycerol (DAG) formed at the same time as IP3. The ITRP, Ca was simulated by ICa = GCa(V–ECa), turned on as a square pulse of Ca2+, with the timing adjusted to fit the data . As it is not known how much of this Ca2+ inflow reaches the store channels, a small fraction is added to x1 in the same time interval to increase the rate of depolarization to match the data . One of these transiently activates a channel in the plasma membrane that allows a brief inflow of Ca2+, while the other opens Ca2+ channels on internal stores. The increase of cytoplasmic Ca2+ activates plasma membrane Cl− channels and Cl− outflow depolarizes membrane PD. A further increase of cytoplasmic Ca2+ concentration closes Ca2+ channels on the stores, while Ca2+ pumps are restoring low cytoplasmic Ca2+, and membrane PD is repolarized by depolarization-activated outward rectifier channels, by diminishing Cl− conductance and by the action of the proton pump, which is only slightly diminished by the transient high cytoplasmic Ca2+.To sum up the model: depolarization to threshold membrane PD produces two second messengers  similarity to the response of salt-sensitive Chara (b) small differences that might contribute to Nitellopsis survival in brackish media.We calculated the average visually  and in tvisually . Initialembranes . The tonChara australis . To achieve the wider Nitellopsis AP form, several parameters were changed (Nitellopsis column). Most parameter values were comparable to those of Chara model, but k−3, that controls the return of the Ca2+ channel from inactivated state , was an order of magnitude larger. However, good fit was only obtained when Hill coefficient n was reduced from 2 to 1 (see section Discussion). The average AP was fitted with the Thiel-Beilby model  showing a transition from slightly extended “normal” AP to a typical rectangular saline AP.Chara. The spontaneous generation of the APs revealed sharp prompt spike at the beginning of each AP (this feature is difficult to resolve from the imposed stimulating depolarization). The spike is quite prominent in the more detailed plot of However, Cell 3, which was also treated overnight in 50 mM NaCl APW, exhibited different AP shape . This ce2+ concentration in the cytoplasm (Methods and Discussion). The best agreement with the data was obtained by linearly decreasing (and sometimes subsequently increasing) pump parameter κoi. The decrease was started between 0.5 and 5 s of the AP and stopped and sometimes reversed between 9 and 12 s of the data , p2′ (decrease), and k−3 (decrease) (Cell 6 exhibited depolarized Resting PD in APW and the AP shape responded much more strongly to exposure to 100 mM NaCl APW . The celecrease) . Cell 6 ecrease) . Interesecrease) , 9. HoweNitellopsis obtusa is native to deep lakes of Europe and Asia, but recently has been colonizing more polluted and shallow water habitats and became invasive in the Great Lakes of North America . The Hill coefficient n provides a measure of substrate co-operativity in binding to the pump protein. For n > 1, there might be more than one substrate binding sites on the protein (two or more binding sites in a protein monomer or assembly of multiple subunits with a single substrate binding site) and binding of one substrate facilitates the binding of another substrate to the protein. For n = 1 the Hill equation becomes the Michaelis-Menten equation with no cooperativity. Chara AP was modeled using Hill equation with n = 2, resulting in a steep repolarization  appear more at random, sometime exhibiting wide AP upon saline exposure and narrow APs after many minutes in saline . It is possible that the cells recover the original AP form after prolonged exposure to brackish conditions. Some cells experience tonoplast APs only, where the Cl− might be easier to recover by the vacuole, thus providing adaptation to brackish media. As in Chara, the proton pump is transiently inhibited by the high cytoplasmic Ca2+ and gradually declines in saline media. However, if the cells are very hyperpolarized at the start of the experiment, the pump inhibition both by the AP and by the saline medium is mitigated. Future experiments will investigate electrophysiological characteristics of long-term survival and growth of Nitellopsis in brackish media with 20–50 mM NaCl to resolve in detail this partial step toward salt tolerance.The AP form in Mimosa or Dionaea. However, APs are now detected in many land plants, including food crops , as well as in the model plant Arabidopsis. AP propagation affects vital plant functions, such as photosynthesis, gene expression, production of phytohormones, or stomatal movements. APs signal many types of biotic and abiotic stress (Yan et al., Is our research relevant to land plants? In recent years APs became important as multifunctional signals in excitable tissues of higher plants (Sukhov and Vodeneev, 1′, p2′ and k−3 for the Cells 1 - 6 with different treatments have been graphed on MJB website http://newt.phys.unsw.edu.au/~mjb/APproj.htmlThe trends of parameters pVK, IL, and VP performed the experiments, while MB analyzed the data and fitted the Thiel-Beilby model. All authors listed contributed to writing the paper and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Trypanosoma brucei (T. brucei), a protozoan parasite that causes sleeping sickness in humans and nagana in cattle in sub‐Saharan Africa. In the present study, we identified that centrin5 of T. brucei (TbCentrin5) is localized throughout the cytosol and nucleus and enriched in the flagellum. We further identified that TbCentrin5 binds Ca2+ ions with a high affinity and constructed a model of TbCentrin5 bound by Ca2+ ions. Meanwhile, we observed that TbCentrin5 interacts with TbCentrin1, TbCentrin3, and TbCentrin4 and that the interactions are Ca2+‐dependent, suggesting that TbCentrin5 is able to form different complexes with other TbCentrins to participate in relevant cellular processes. Our study provides a foundation for better understanding of the biological roles of TbCentrin5.Centrin is an evolutionarily conserved EF‐hand‐containing protein, which is present in eukaryotic organisms as diverse as algae, yeast, and humans. Centrins are associated with the microtubule‐organizing center and with centrosome‐related structures, such as basal bodies in flagellar and ciliated cells, and the spindle pole body in yeast. Five centrin genes have been identified in  CaMcalmodulinCTDC‐terminal domainDAPI4',6‐diamidino‐2‐phenylindoleITCisothermal titration calorimetryNTDN‐terminal domainRT‐PCRreverse transcription‐PCRTnCtroponin2+‐sensitive contracting fibers. Subsequently, homologous centrin proteins were observed in the microtubule‐organizing center 2+ ions 2+‐dependent cell motility. Target Sfi1 in yeast and its homologous protein (hSfi1) in humans were identified to interact centrins and responsible for SPB duplication Centrins are a member of the EF‐hand calmodulin (CaM) superfamily, which are highly conserved in eukaryotic cells. Centrins were initially identified in unicellular green algae Mus musculus Centrin1 (MmCentrin1) Chlamydomonas reinhardtii centrin (CrCentrin) 2+ ions. The highly conserved CTD shows a higher affinity with Ca2+ ions and other partners 2+ ions and its partners The overall topology of centrins or centrin/target peptide complexes reported by NMR spectroscopy or X‐ray crystallography Trypanosoma brucei (T. brucei), a protozoan parasite that causes sleeping sickness in humans and nagana in cattle in sub‐Saharan Africa, five centrin isoforms have been identified T. brucei  is still unknown.In T. brucei and identified the interactions between TbCentrin5 and Ca2+ ions. We further identified that TbCentrin5 is able to interact with other TbCentrins and the interactions are Ca2+‐dependent. The work will provide a basis for better understanding of the biological functions of TbCentrin5.Here, we investigated the localization of TbCentrin5 in T. brucei were obtained from TriTrypDB (https://tritrypdb.org/tritrypdb/). All other centrin protein sequences were obtained from NCBI protein database (https://www.ncbi.nlm.nih.gov/protein) and UniProt (https://www.uniprot.org/). Sequences were aligned using clustalxmega7The centrin protein sequences of −1 G418 and 50 μg·mL−1 hygromycin.The wild‐type procyclic Lister 427 strain was cultivated at 28 °C in Cunningham's medium supplemented with 10% FBS. The procyclic 29‐13 strain T. brucei procyclic forms from 427 strain. The transfection by electroporation was carried out as follows: Briefly, 108 cells were harvested and washed twice with cytomix buffer  and suspended in 0.45 mL of cytomix buffer containing 30 μg of the linearized vectors. Electroporation was carried out in a 2‐mm cuvette  using the Gene Pulser  with parameters set as follows: 2.0 kV voltages, 25 μF capacitance, and 200 Ω resistance. The transfected cells were immediately transferred into 10 mL of fresh Cunningham’s medium. Transfectants were selected with 10 μg·mL−1 zeocin and were induced with 1.0 μg·mL−1 tetracycline.RNA interference (RNAi) of TbCentrin5 was performed using the RNAi vector pZJM. Recombinant pZJM vector containing segment (nucleotide number 121–420) of TbCentrin5 was linearized and electroporated into in situ tagging of TbCentrin5, the cDNA segment (nucleotide number 121–558) was cloned into pC‐EYFP‐NEO vector T. brucei procyclic forms from 427 strain. The transfection conditions were the same described as above. Successful transfectants were selected under 2.5 μg·mL−1 G418. Expression of TbCentrin5‐EYFP fusion protein was verified by western blotting.For imagej .Cells stably expressing TbCentrin5‐EYFP were harvested and washed twice with PBS. Resuspended cells were fixed with 4% paraformaldehyde and washed with PBS. Fixed cells were settled on the coverslip at room temperature for 30 min. The cells were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) and examined with an inverted microscope . Images were analyzed by T. brucei and cloned into a modifier vector pET‐28a (+) , which provided a cut site of TEV protease at the N terminus to remove His‐tag. The recombinant vector was transformed into Escherichia coli BL21 (DE3). The transformed cells were cultured in LB at 37 °C until OD600 reached 0.8 and induced by 0.5 mm IPTG at 16 °C for 20 h. The cells were harvested and purified in lysis buffer containing 20 mm Tris and 500 mm NaCl at pH 7.0. The eluted protein was digested by TEV protease and further purified by gel filtration column Sephadex G‐75 .Full‐length gene encoding TbCentrin5 was amplified by PCR from the genomic DNA of 2+ ions was carried out using SWISS‐MODEL based on homology modeling techniques The 3D structure of TbCentrin5 or the complex of TbCentrin5 and Ca2+‐binding capacity and the interactions between TbCentrin5 and TbCentrin1/2/3/4. Samples of TbCentrin5 titrated with Ca2+ ions were mixed with EGTA to remove potential Ca2+ ions and then further purified by gel filtration column Sephadex G‐75 . TbCentrin5 and calcium ions were equilibrated in the same buffer containing 20 mm Tris/HCl (pH 7.0) and 100 mm NaCl. 0.1 mm TbCentrin5 in cell was titrated against 2.0 mm CaCl2 from syringe. Two microliter of CaCl2 was injected into 0.2 mL TbCentrin5 at 120 s intervals. Samples of TbCentrin1/2/3/4 titrated with TbCentrin5 were purified and equilibrated as above expect mixed with EGTA or not. Fifty micromolar TbCentrin1/2/3/4 in the cell was titrated against 0.5 mm TbCentrin5 from the syringe with the same sample volume and rotate speed. The data collected were analyzed by microcal LLC ITC software .Isothermal titration calorimetry (ITC) measurements were performed on iTC200  at 16 °C to investigate the Ca6 cells·mL−1 in fresh medium. Each cell line was cultured in two cuvettes and incubated at 26 °C and was measured by optical density (600 nm) every 2 h. To monitor the OD600, one cuvette was resuspended for monitoring cell growth while the other cuvette was not disturbed for monitoring sedimentation. The change in the OD600 (ΔOD600) was calculated by subtracting the OD600 of the resuspended sample from that of the undisturbed sample. The experiment was repeated three times.Sedimentation assay was carried out according to the procedures published previously T. brucei are on different branches with centrins from higher animal, but closer to centrins from fungi and other protest, especially to centrins from Leishmania major (L. major) which belong to the same family of Trypanosomatidae as T. brucei. TbCentrin5 and TbCentrin2 were on utterly different branches with other centrins but same as LmCentrin4 and LmCentrin3, suggesting that TbCentrin5 and TbCentrin2 are far from other centrins but closer to LmCentrin4 and LmCentrin3. The phylogenetic analysis indicated the position of TbCentrin5 and its relationship with other centrins, which is helpful for us to determine the location of Trypanosoma genera in the evolution.To further understand the position of TbCentrin5 in evolution and its relationship with other centrins that have been identified in other organisms, a phylogenetic analysis was performed using a Neighbor‐Joining method Fig. A. Becausin vivo  of the cell cycle, TbCentrin5 was slightly distributed in nucleus. As the cell cycle progressed (1N2K and 2N2K), the distribution of TbCentrin5 in nucleus became stronger. The results suggested that TbCentrin5 might be involved in the karyokinesis.To determine the subcellular localization of TbCentrin5, TbLa was endogenously tagged with EYFP at the C terminus. The level of TbCentrin5‐EYFP fusion protein was examined by western blot with GFP probe, which indicated the successful expression of TbCentrin5‐EYFP ivo Fig. B. Fluoreivo Fig. C. IntrigT. brucei, L. major and Homo sapiens using clustalw2 and espript 3.0 The sequence of TbCentrin5 was aligned with other centrins from 5d43), HsCentrin2 (PDB: 2ggm), SdCentrin (PDB: 3kf9), and CrCentrin (PDB: 3qrx) with respective sequence identity of 43%, 42%, 41%, and 39% were selected to build model  in an exothermic mode and one molecule of TbCentrin5 is able to bind two Ca2+ ions (N = 2).As an important second messenger, Ca2+‐binding mode of TbCentrin5 is similar to that of TbCentrin4 2+ ions. In other Ca2+‐binding modes, one centrin molecule binds more than two Ca2+ ions 2+ ions 2+‐binding site of TbCentrin5 should be located in the CTD containing EF‐hand III and EF‐hand IV. The structure model of TbCentrin5 bound by Ca2+ ions was built using SWISS‐MODEL  in the loop of EF‐hand III may provide five coordinated bonds for Ca2+ ions binding  in the loop of EF‐hand IV may provide six coordinated bonds for Ca2+ ions binding  of TbCentrin5 binding to TbCentrin1, TbCentrin3, and TbCentrin4 are 105, 52, and 12 μm, respectively.To further investigate the binding affinity of TbCentrin5 with TbCentrin1, TbCentrin3, and TbCentrin4, ITC analysis of TbCentrin1‐4 titrated with TbCentrin5 was carried out Fig. B. The re2+ ions in TbCentrins. In the protein expression in LB culture medium and purification procedures, Ca2+ ions may be copurified with TbCentrins. Therefore, it is necessary to ensure complete removal of Ca2+ ions by treatment of EGTA to investigate the effect of Ca2+ ions on the interactions between TbCentrin5 and other TbCentrins. ITC analysis of TbCentrin1, 2, 3, 4 titrated with TbCentrin5 was then performed without Ca2+ ions. In the absence of Ca2+ ions, TbCentrin5 is not able to interact with TbCentrin1, 2, 3, 4  monitored that the mRNA level of TbCentrin5 in the RNAi cells decreased by ~ 80% compared with that in the noninduced control cells after tetracycline induction for 2 days Fig. A. The re600 values among wild‐type 29‐13 cell line, the noninduced control cells, and TbCentrin5 RNAi cells showed no significant distinction (Fig. TbCentrin5 has the same flagellum localization as TbCentrin3, and the effect of TbCentrin5 RNAi on cell growth is also similar to that of TbCentrin3 RNAi ion Fig. B. Under 2+ ions with a high affinity and built the model of TbCentrin5 bound by Ca2+ ions. Besides, we demonstrated that TbCentrin5 interacts with TbCentrin1, TbCentrin3, and TbCentrin4 depending on Ca2+ ions binding, suggesting TbCentrin5 might be able to form different complexes with other TbCentrins depending on cellular Ca2+ ions to participate in the relevant biological processes. Our study will provide a basic information for better understanding the biological functions of TbCentrin5.In conclusion, we identified that TbCentrin5 is localized at the cytosol and nucleus and enriched in the flagellum. We further identified that TbCentrin5 binds CaThe authors declare no conflicts of interest.FS and XT designed the research. FS, XY, YD, and HM performed the experiments. FS, XY, and XT analyzed the data. FS and XT wrote the paper. XY, YD, and HM discussed and gave suggestions on the manuscript.


AML) patients have a poor response to conventional chemotherapy. The survival of chemoresistant cells is thought to depend on leukemia‐bone marrow (BM) microenvironment interactions, which are not well understood. The CXCL12/CXCR4 axis has been proposed to support AML growth but was not studied at the single AML cell level. We recently showed that T‐cell acute lymphoblastic leukemia (T‐ALL) cells are highly motile in the BM; however, the characteristics of AML cell migration within the BM remain undefined. Here, we characterize the in vivo migratory behavior of AML cells and their response to chemotherapy and CXCR4 antagonism, using high‐resolution 2‐photon and confocal intravital microscopy of mouse calvarium BM and the well‐established MLL‐AF9‐driven AML mouse model. We used the Notch1‐driven T‐ALL model as a benchmark comparison and AMD3100 for CXCR4 antagonism experiments. We show that AML cells are migratory, and in contrast with T‐ALL, chemoresistant AML cells become less motile. Moreover, and in contrast with T‐ALL, the in vivo exploratory behavior of expanding and chemoresistant AML cells is unaffected by AMD3100. These results expand our understanding of AML cells‐BM microenvironment interactions, highlighting unique traits of leukemia of different lineages.The majority of acute myeloid leukemia ( We, and others, have reported AML to be associated with endosteal nichesAcute myeloid leukemias (AML) are aggressive myeloid lineage leukemias with a poor prognosis. This is due to poor responses to current chemotherapy regimens.et al.et al.CXCL12 is abundantly secreted in the marrow stroma and binds to the receptor CXCR4. CXCL12 is fundamental for the retention of CXCR4‐expressing cells in the BM. We previously showed that up‐regulation of CXCR4 is associated with increased engraftment and motility of hematopoietic stem cells within the BM microenvironment.in vivo prior to and following chemotherapy, (2) expression of CXCR4 of early infiltrating and chemoresistant cells, and (3) the role of CXCR4 inhibition on the biology of AML within the BM.To address the questions outlined above, we used intravital microscopy of calvarium BM to study the biology of AML in the BM using the well‐established preclinical model of MLL‐AF9‐driven AML.+ or YFP+ granulocyte‐macrophage progenitors with retrovirus encoding the MLL‐AF9 oncogene and T‐ALL was generated by transducing fetal liver cells with DsRed‐Notch‐ICN retrovirus. Preleukemic cells were transplanted into sublethally irradiated recipients. Primary leukemias were then isolated and subsequently transplanted for intravital imaging experiments. We analyzed the motility of single AML and T‐ALL cells during disease establishment, when cells were found either as single, isolated cells or small clusters in the BM (‘seeding’ stage), or following treatment . Similar to T‐ALL,P < 0.0001), . Intravital microscopy and image analysis were performed as described.The AML and T‐ALL experimental models were as described.  Click here for additional data file.  Click here for additional data file.  Click here for additional data file.  Click here for additional data file.  Click here for additional data file.  Click here for additional data file.   Click here for additional data file.


Bellevalia saviczii is a medicinal plant used as anti-rheumatic and anti-inflammatory herbal remedy in Iraqi-Kurdistan. The aim of this study was to evaluate the anti-inflammatory activity of its extract and the isolated homoisoflavonoid (Dracol) by studying the Ca2+-dependent NF-kB pathway. Nuclear translocation of p65 NF-kB subunit, as parameter of NF-kB activation, was visualized in human leukemic monocytes by immunofluorescence and Western blot analyses, after cell treatment with B. saviczii root extract or Dracol followed by Lipopolysaccharide stimulation. In parallel, Ca2+ signals responsible for NF-kB activation and levels of inflammatory cytokines were investigated. LPS-induced p65 translocation was evident in monocytes and both treatments, in particular that with Dracol, were able to counteract this activation. Intracellular Ca2+ oscillations were halted and the cytokine release reduced. These results confirm the traditional anti-inflammatory efficacy of B. saviczii and identify one of the molecules in the extract which appears to be responsible of this action. Bellevalia saviczii, a perennial plant belonging to the family of Asparagaceae, represents a plant genus particularly rich in homoisoflavonoids, a special subclass of flavonoids, rarely found in nature but responsible for the biological activity of Bellevalia species used in traditional medicine by people from Mediterranean to Central Asia benzopyr-an-4-one, for which the trivial name Dracol has been proposed, is a natural compound isolated for the first time by Hernandez et al. in 2006 from genus Dracaena draco together with another compound namely icodeside i), which recruit the Ca2+-dependent decoders calcineurin and calmodulin to promote IkB degradation i in the 54.1 ± 13.8 % (n = 377) of THP1 cells loaded with the Ca2+-sensitive fluorochrome, Fura-2/AM. This Ca2+ signal consisted either in a single Ca2+ spike (59.5 ± 17.7%) (2+ oscillations that persisted throughout the period of recording (40.5 ± 17.7%)  A or in r± 17.7%) B. 2+]i were detected in the majority of THP1 cells pre-treated with 1 µg/mL LPS for 4 h (2+ transient during the period of recording (not shown). These repetitive Ca2+ spikes were similar to those recorded in mouse pulmonary artery endothelial cells previously exposed to 1 µg/mL LPS for 16 h -8-methyl-4H-[1]-benzopyran-4-one). Pale yellow amorphous solid, yield: (9.17 mg), Molecular formula C19H20O6 deduced from the negative-ion ESIMS- spectrum ; UV (MeOH) λmax 290.5, 375.5 nm; 1H-NMR  3.99 , 4.04 , 6.15 , 2.92 , 1.95 , 3.76 , 3.87 , 6.82 , 7.14 , 7.14 , and 6.82 .13C-NMR : δC 14.51 (C-8 Me), 61.7 (OMe), 61.9 (OMe), 70.9 (C-2), 62.9 (C-3), 201.5 (C-4), 146.9 (C-5), 105.16 (C-6), 149.7 (C-7), 130.3 (C-8), 33.1 (C-9), 135.5 (C-4a), 151.72 (C-8a), 121.8 (C-1′), 131.9 (C-2′), 116.7 (C-3′), 157.6 (C-4′), 116.7 (C-5′) and 131.5 (C-6′). The 1H and 13C-NMR spectra were in agreement with the literature i was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when exciting alternatively at 340 and 380 nm [Ratio (F340/F380)]. An increase in [Ca2+]i causes an increase in the ratio [After washing in PSS, the coverslips were fixed to the bottom of a Petri dish and the cells observed by an upright epifluorescence Axiolab microscope , usually equipped with a Zeiss ×40 Achroplan objective . The cells were excited alternately at 340 and 380 nm, and the emitted light was detected at 510 nm. A first neutral density filter  reduced the overall intensity of the excitation light and a second neutral density filter  was coupled to the 380 nm filter to approach the intensity of the 340 nm light. A round diaphragm was used to increase the contrast. The excitation filters were mounted on a filter wheel . Custom software, working in the LINUX environment, was used to drive the camera  and the filter wheel, and to measure and plot on-line the fluorescence from 30–45 rectangular “regions of interest” (ROI) enclosing 20–30 single cells. Each ROI was identified by a number. Adjacent ROIs never superimposed. [Cahe ratio . Ratio mt-test where only probability values * p ≤ 0.05, ** p ≤ 0.01 were considered to be statistically significant. Intracellular Ca2+ oscillations were analyzed by measuring the percentage of oscillating cells for each condition , the amplitude of the 1st spike and the frequency of Ca2+ over 1000 s. Pooled data are presented as means ± SE and the number of analyzed cells is reported above or within column bars. Each data is representative of four different coverslips. Statistical significance (p ≤ 0.05) was evaluated by the Student’s t-test for unpaired observations.Data are presented as means ± SD. Statistical analysis was performed by using the Student’s B. saviczii, which has been traditionally used in Iraqi-Kurdistan as a medicinal plant, is a valuable source of active compounds, especially homoisoflavonoids. Among them, Dracol has been identified for the first time in this plant and, in our experimental conditions, it is able to prevent the NF-kB activation induced in human monocytes by LPS treatment. Very few studies have considered the biological activity of this compounds, which deserves further investigation, particularly regarding their use in treating inflammatory diseases. The observed decrease in nuclear translocation of p65 NF-kB subunit, associated with TNFα release reduction, strongly depend on the modification of calcium signaling. The present study demonstrated that the root of


It has been suggested that oxidative stress may be involved in this process. We and others have reported that in cardiomyopathies the NADPH oxidase (NOX)-derived superoxide is increased, with a negative impact on [Ca2+]i and contractility. We tested the hypothesis that in the aged heart, [Ca2+]i handling and contractility are disturbed by NOX-derived superoxide. For this we used adults (≈5 month-old) and aged (20–24 month-old) rats. Contractility was evaluated in isolated hearts, challenged with isoproterenol. To assess [Ca2+]i, isolated cardiac myocytes were field-stimulated and [Ca2+]i was monitored with fura-2. Cardiac concentration-response to isoproterenol was depressed in aged compared to adults hearts (p < 0.005), but was restored by NOX inhibitors apocynin and VAS2870. In isolated cardiomyocytes, apocynin increased the amplitude of [Ca2+]i in aged myocytes (p < 0.05). Time-50 [Ca2+]i decay was increased in aged myocytes (p < 0.05) and reduced towards normal by NOX inhibition. In addition, we found that myofilaments Ca2+ sensitivity was reduced in aged myocytes (p < 0.05), and was further reduced by apocynin. NOX2 expression along with NADPH oxidase activity was increased in aged hearts. Phospholamban phosphorylation (Ser16/Thr17) after isoproterenol treatment was reduced in aged hearts compared to adults and was restored by apocynin treatment (p < 0.05). In conclusion, β-adrenergic-induced contractility was depressed in aged hearts, and NOX inhibition restored back to normal. Moreover, altered Ca2+ handling in aged myocytes was also improved by NOX inhibition. These results suggest a NOX-dependent effect in aged myocytes at the level of Ca2+ handling proteins and myofilaments.Cardiac aging is characterized by alterations in contractility and intracellular calcium ([Ca The aged population is increasing worldwide. Cardiovascular diseases are the leading cause of death worldwide and this becomes even more pronounced in the elderly population. In the heart, aging increases the risk of ischemic disease, atrial fibrillation, and heart failure. Cardiac aging involves a progressive degeneration and decline in function in absence of major cardiovascular risks, which makes the heart more vulnerable to stress and contributes to increased cardiovascular mortality, and morbidity. Cardiac aging includes hypertrophy, fibrosis, and apoptosis of myocytes  and at tIn the heart, the aging process is associated with an increase in the production of reactive oxygen species (ROS) . Traditi2− and H2O2) through electron transfer from NADPH to molecular oxygen. Seven family members of these oxidases are known, each based on a distinct catalytic subunit . This oxidase is a multi-sub unit enzyme composed of a membrane-bound heterodimeric subunit (p22phox and gp91phox) and several cytoplasmic regulatory sub units including Rac1, p47phox, and p67phox. NADPH oxidases are crucial regulators of redox signaling through tightly regulated production of low ROS levels in the vicinity of target proteins. In addition, NOX activity has been hypothesized to contribute to cardiovascular aging i regulation is directly responsible for the attenuated contractility and relaxation observed in aged myocardium, which correlated with impairment of both cell shortening and relengthening i to obtain the hysteresis loops  from 1 to 4 Hz, which was more pronounced in the aged groups (either in the absence or presence of apocynin) compared to the adult group . This confirms that use of the NOX inhibitor reduced the cardiac oxidative stress. To verify the NADPH oxidase origin of this oxidative stress, we evaluated NADPH oxidase activity indirectly, by the cytochrome c reduction method (p < 0.001).NOX-derived oxidative stress was investigated by two methods . First, n method B. Aged hp < 0.05). In the case of NOX3, in our hands it was barely detected and was not different between groups. For the case for NOX4, no differences between groups were found. These results suggest that NOX2 is the origin of increased superoxide in the aged hearts.To gain further insight into the origin of the superoxide source in aged hearts, we examined the levels of NOX isoforms of the different groups in our study. For this purpose we analyzed the levels of NOX2, NOX3, and NOX4 in cardiac homogenates by Western blotting . NOX2 wa2+ release and reuptake were altered in aged myocytes, we decided to look into the levels of Ca2+ handling proteins in cardiac homogenates (p < 0.05). SERCA2 levels were reduced in aged hearts, and were not changed by apocynin treatment: 0.72 ± 0.08 in adults, 0.38 ± 0.07 in aged hearts, and 0.40 ± 0.045 ratio of SERCA/GAPDH levels in aged hearts treated with apocynin (p < 0.05). Consequently, the ratio of SERCA to PLB was decreased in aged hearts (p < 0.05). This suggests that in the aged rat myocardium, the capacity for Ca2+ reuptake is diminished by reduced levels of SERCA2, and upon adrenergic stimulation, PLN phosphorylation is impaired.Finally, since the kinetic parameters of Caogenates . SERCA2 In the aged myocardium, increased ROS are associated with a decrease in function, as well as on ventricular remodeling. This ROS production has been ascribed traditionally to the mitochondria.In the heart, both NOX2 and NOX4 are expressed. NOX2 is located in the sarcolemma while NOX4 is located in the mitochondrial matrix . It is p2+ transients and reduced myofilaments Ca2+ sensitivity. Apocynin, a NOX inhibitor increased the amplitude of these transients. Our results suggest that this agent improved the function of the RyR2 and SERCA2, improving the Ca2+ cycling, although the molecular mechanism remains to be determined.Here we observed that the aged heart presents a diminished response to β-adrenergic stimulation compared to adult heart and for the first time show that acute NOX inhibition is able to restore this fundamental mechanism of cardiac inotropism. At the subcellular level, we observed that aged myocytes presented a slightly increase in amplitude of Ca2+ is reduced i) was determined by exciting with light from a Xenon lamp passed alternately through 365 and 380 nm filters. The emission fluorescence was reflected through a barrier filter (510 ± 15 nm) to a photomultiplier tube. The fura-2 emission ratio at 360/380 nm represents an non-calibrated signal of [Ca2+]i,Cardiomyocytes were loaded with 1 μM fura 2-AM  in the dark for 15 min, and fluorescence measurements were recorder with a dual excitation fluorescence photomultiplier system (Ionoptix). Cardiomyocytes were placed in a Lucite chamber on the stage on a Nikon Eclipse E200 inverted microscope and imaged through a Fluor 40× oil objective. Intracellular Ca2+]i in systole and diastole: [Ca2+]i = 100 × (F − F0)/F0). The time course of Ca2+ fluorescence signal decay (the duration at which Ca2+ transient decays 50%) was calculated to determine [Ca2+]i clearing rate. The time constant (τ) for myocyte relengthening and the Ca2+ transient decay is calculated by fitting the decay of the transients to the exponential curve: Y(t) = a·e−λt − b. Where a = twitch amplitude or Ca2+ transient; b = baseline value; λ is calculated and τ = 1/λ.This was determined as the percentage of change between the levels of [Ca2+ were assessed using the steady-state relation between cell length and [Ca2+]i in intact single cardiac myocytes tetanized by high-frequency (40 Hz) stimulation after exposure to thapsigargin (0.2 µmol/L for 15 min), as described previously i can be reversibly clamped near peak systolic levels during the tetanic contracture for 10 to 20 s and then rapidly returned to resting levels on cessation of electrical stimulation via normal Na/Ca exchange mechanisms. Thus, changes in the degree of cell shortening between tetani clamped at the same [Ca2+]i level can be attributed to changes in the relative myofilament responsiveness to Ca2+.Changes in myofilament responsiveness to Caeviously . Thapsig2+]i against its corresponding value for sarcomere shortening, to obtain information of the activating and the relaxation phases of the myofilaments, as has been previosly reported [2+]i relationships were determined experimentally and fitted to the Hill equation to obtain Sarc Shtering mx, or maximal [Ca2+]I activating shortening and ECa50 and the Hill slope as follow.Phase loop analysis. In addition, the myofilaments dynamics was studied by plotting the levels of i transients amplitude and improved Ca2+ kinetics. Aged rat hearts presented lower levels of SERCA2 and reduced phospholamban phosphorylation upon adrenergic stimulation that was reversed by NOX inhibition.Aged rat hearts presented a diminished response to isoproterenol, which was restored upon pharmacological NOX inhibition. In aged myocytes, twitch contractions, presented slowed kinetics, and reduced myofilaments CaUnderstanding how NOX activity influences the aged myocardium function at the cellular level may help to develop strategies for the prevention and treatment of cardiovascular disease in this population. Pharmacological targeting of NOX in aging may help to improve cardiac function.


The Hedgehog (Hh) pathway, containing the Patched (PTCH) and Smoothened (SMO) multitransmembrane proteins, is the main regulator of vertebrate embryonic development. A non-canonical Hh pathway was recently observed in numerous types of solid cancers and hematological malignancies. Although acute myeloid leukemia (AML) is a common and lethal myeloid malignancy, the chemotherapy for AML has not changed in the last three decades. The Hh pathway and other intracellular signaling pathways are important for the tumor cells’ cycle or therapeutic resistance of AML cells. In this article, we will review the current trends in Hh pathway inhibitors for treating AML. Intracellular signaling pathways are considered as therapeutic targets for acute myeloid leukemia (AML). AML is a highly complex and heterogeneous disorder; conventional chemotherapy for patients with AML is partially effective, but not in all patients. The treatment for AML depends on the characteristics of each patient, such as age, performance status, and cytogenetic/molecular abnormalities. Systemic combination chemotherapy includes two phases: the induction phase and the consolidation of remission phase . Older oIn the 1990s, Dick et al. showed that CD34+ CD38-AML-initiating cells can migrate to the bone marrow (BM) and reconstitute human AML in immunodeficient mice . This waSome healthy elderly patients have clonal hematopoiesis of indeterminate potential (CHIP), which involves a mutation in a leukemia-associated gene at a variant allele frequency of >0.02 and an annual risk of developing a myeloid neoplasm ,12. ThesIn the following sections, we will describe the Hh-pathway activation and dysregulation in AML. We will summarize the various drugs used as Hh-pathway inhibitors, which are under clinical trials. We hope this review will be helpful for researchers in this field.The Hh pathway was first discovered by Wieschaus and Nusslein-Volhard . In the Although the expression of GLI1 reflects Hh pathway activity, several other proteins, such as RAS and transforming growth factor β, are the factors regulating GLI activity, regardless of the Hh pathway ,27 Figu. AbnormaThe Hh pathway plays a role in the survival of AML patients. This pathway is related to the resistance of AML cells to drugs and radiotherapy, resulting in poor outcomes. GLI1 and the UDP-glucuronosyl transferase (UGT1A) family of enzymes are elevated in blasts with resistance to Ara-C . UGT1As Resistance to radiotherapy is another clinical problem. Total body irradiation with chemotherapy is the myeloablative regimen for allogeneic stem-cell transplantation, and radiotherapy is the part of an effective procedure in conditioning. The expression of Hh signaling proteins, including SMO and GLI1 in radiation-resistance myeloid leukemia cells, are higher than that observed in radiation-sensitive myeloid leukemia cells, suggesting that Hh pathway activation is related to radiotherapy resistance . AnotherFrom the perspective of clinical outcomes, GLI2 expression has a negative influence on event-free survival (EFS), relapse-free survival, and overall survival (OS) and is correlated with the FLT3 mutation status. A single genetic mutation in FLT3-ITD causes gradual myeloid cell expansion as in a myeloproliferative neoplasm (MPN), but does not cause the full development of AML . AlthougAs for MDS, the expression of DHH and SHH proteins was higher in the MDS cells than in the non-MDS cells  . The expThe SMO inhibitor, Vismodegib GDC-0449), showed efficacy and was approved by the US Food and Drug Administration (FDA) for treating other non-hematological malignancies, such as basal cell carcinoma and basal cell nevus syndrome ,42. Beca9, showedLDE225 is a specific SMO inhibitor and spreads to other multi-pathway through Hh-mediated regulation of target genes, which can inhibit insulin-like growth 1 receptor (IGF-1R), phosphoinositide 3-kinase (PI3K), Akt, and multidrug-resistance protein 1 (MRP1) pathway. In refractory primary AML cells and adriamycin-resistant AML cells, LDE225 increased cell apoptosis and the efficacy of adriamycin against tumor cells and lowered the expression of the targeted protein. For Adriamycin-treated AML cells, LDE225 suppressed tumor cell proliferation in an AML xenograft mouse model . Using a2 Ara-C on days 1–7 and 60 mg/m2 daunorubicin (DNR) on days 1–3 in a 28-day cycle) for untreated and fit AML or high-risk MDS patients revealed that 46.4% of patients achieved CR [PF-04449913 (Glasdegib) is a selective SMO inhibitor and decreased the leukemia-initiation potential in AML cells in a serial transplantation mouse model . To evalieved CR . In all GDC-0449 was first discovered from a library of small-molecule compounds and appeared to cause an objective response for locally advanced or metastatic basal-cell carcinoma . GDC-004BMS-833923 has a high-binding affinity to SMO receptors . In a phGANT-61 blocks GLI1 function, decreases proliferation and clonogenic potential, and causes growth arrest and apoptosis in AML cells ,56,57. GWe will show the representative clinical trials and their details of Hh-pathway-targeted agents in AML below . As mentIn the last decade, numerous new agents for treating patients with AML, such as Hh pathway inhibitors, monoclonal antibodies, or other intracellular pathway inhibitors, have become available or are under clinical trials. No Hh pathway inhibitors other than glasdegib for AML or high-risk MDS have been approved, and their long-term toxicity profiles are unclear. In a dose escalation trial, the most frequent non-hematological AEs were associated with the gastrointestinal system, followed by alopecia and prolonged QTc. Among healthy donors treated with proton-pomp inhibitors, the same AEs were observed following the administration of 100 mg glasdegib daily . SeveralA programmed death receptor-1 (PD-1) pathway inhibitor with CD8+ cytotoxic T-cell adoptive infusion can reduce the levels of human AML cells in an AML mouse model . In gastAML is a heterogeneous hematological malignancy. Numerous pathways including Hh pathway and genetic or epigenetic abnormalities may be correlated to the occurrence of AML, its pathological condition, and drug resistance. Future studies will provide an improvement of the outcomes of AML patients by using Hh-pathway inhibitors in combination with other novel agents.


Therefore, developing new bronchodilators is essential. Herbal plants were extracted and the extracts’ effect on airway smooth muscle (ASM) precontraction was assessed. The ethyl alcohol extract of semen cassiae (EESC) was extracted from Semen cassia. The effects of EESC on the ACh- and 80 mM K+-induced sustained precontraction in mouse and human ASM were evaluated. Ca2+ permeant ion channel currents and intracellular Ca2+ concentration were measured. HPLC analysis was employed to determine which compound was responsible for the EESC-induced relaxation. The EESC reversibly inhibited the ACh- and 80 mM K+-induced precontraction. The sustained precontraction depends on Ca2+ influx, and it was mediated by voltage-dependent L-type Ca2+ channels (LVDCCs), store-operated channels (SOCs), TRPC3/STIM/Orai channels. These channels were inhibited by aurantio-obtusin, one component of EESC. When aurantio-obtusin removed, EESC’s action disappeared. In addition, aurantio-obtusin inhibited the precontraction of mouse and human ASM and intracellular Ca2+ increases. These results indicate that Semen cassia-contained aurantio-obtusin inhibits sustained precontraction of ASM via inhibiting Ca2+-permeant ion channels, thereby, which could be used to develop new bronchodilators.β Approximately 300 million and 328.6 million people suffer from asthma and COPD worldwide . BronchoSemen cassiae is the seed of Cassua obtusifolia L. and Cassia tora L., which are globally distributed and used as a medicine and a drink  inhibits ASM contraction via inhibiting Ca2+ permeant ion channels.We attempted to screen herbal compounds and develop new bronchodilators.  a drink . Accorditoxicity , diabetitoxicity , Alzheimtoxicity , allergytoxicity , and hyptoxicity . Its hypSix-weeks-old male BALB/c mice were purchased from the Hubei Provincial Center for Disease Control and Prevention  and were housed in a standard animal facility. All animal use and experimental procedures were approved by the Institutional Animal Ethics Committee of the South-Central University for Nationalities. The license number is 2016-SCUEC-AEC-0030.Human bronchial ASMs were from lung tissue samples of transplant donors and recipients and respected tissue from subjects with lung carcinoma. The experiments adhered to guidelines and protocols approved by the Ethics Committee of the South-Central University for Nationalities, and the subjects provided signed, written informed consent.Aurantio-obtusin was purchased from Yuanye Bio-Technology . Nifedipine, Pyr3 and fluo-4 AM were dissolved in DMSO .Nifedipine, acetylcholine chloride (ACh), pyrazole 3 (Pyr3), YM-58483, and agarose were purchased from Sigma . Fluo-4 AM was purchased from Invitrogen . Semen cassiae was purchased from Beijing TongrenTang  and was authenticated by Professor Ding-Rong Wan . A voucher specimen was deposited at the Herbarium of the College of Pharmacy, South-Central University for Nationalities, China. Semen cassiae was powdered and then extracted with 70% aqueous ethyl alcohol twice. The extracts were boiled for 1.5 h, and the supernatants were collected and evaporated under reduced pressure. The dried ethyl alcohol extract of semen cassiae (EESC) was dissolved in DMSO.-1. The detection wavelength was set at 285 nm.HPLC analysis was performed using an Ultimate 3000 HPLC system . The separations were conducted using a ReproSil 100C18 column  as the stationary phase and methanol-0.1% aqueous phosphoric acid solution (60:40) as the mobile phase. The flow rate was 1.0 mL⋅min2PO3 in H2O and methanol (the ratio is 30:70). A flow rate of 5 mL/min was used and the chromatograms were monitored at 285 nm. Fractions under the peak area of 25.540 min were separated and removed, the rests were collected .Semi-preparative HPLC was performed on Agilent 1200 system equipped with a chromatographic column . The mobile phases consisted of 0.1%Haurantio-obtusin, which was removed using the semi-preparative HPLC technique . TRs (∼5 mm) were cut from the bottom of the tracheae. Epithelium removal was achieved by gentle mechanical rubbing of the luminal surface with cotton under microscope. TRs were mounted in 10 mL organ baths containing PSS bubbled with 95% O2 and 5% CO2 at 37°C. A preload of 0.3 g was added. The TRs were equilibrated for 60 min and then contracted with 10-4 M ACh three times with an interval of 30 min. After resting for 30 min, experiments were performed. Contraction in human bronchial transverse ASM strips was, similarly, measured, except for the preload was set at 0.3–0.8 g depending on the tissue size.Contraction of mouse tracheal ASM was measured in epithelium-intact and -denuded tracheal rings (TRs) as previously described, with some modifications . In brie2, 5%CO2, 37°C) for 2 h before experiments. The slices were then placed in a chamber and held with a fine nylon mesh, respectively. The chamber was placed under an LSM 700 laser confocal microscope  and perfused with Hanks’ balanced salt solution (HBSS) at a rate of ∼800 μL/min at room temperature. HBSS contained (in mM) 20 mM HEPES, NaCl 137.93, KCl 5.33, NaHCO3 4.17, CaCl2 1.26, MgCl2 0.493, MgSO4 0.407, KH2PO4 0.4414, Na2HPO4 0.338, and D-glucose 5.56 and was adjusted to a pH of 7.4. Images under a 10× objective were acquired at a rate of 30 frames/min. The airway lumen areas were measured using Zen 2010 software .Contraction of mouse bronchial ASM was also measured in mouse lung slices . Briefly2 in a 37°C incubator. TRs were washed with PPS and ACh- and 80 mM K+-induced contractions were measured.Mouse TRs were transferred in Dulbecco’s Modified Eagle Medium  supplemented with 10% (w/v) fetal bovine serum , 100 units/mL penicillin and 100 mg/mL streptomycin, and cultured with or without EESC for 24 h at 5% CO2PO4, 4.16 mM NaHCO3, 10 mM glucose, 10 mM HEPES, 0.34 mM NaHPO4⋅12H2O . The trachealis tissues were isolated and minced and incubated for 22 min at 35°C in the above solution supplemented with 2 mg/mL papain, 1 mg/mL dithioerythritol, and 1 mg/mL bovine serum albumin (BSA). The tissues were transferred to the above solution supplemented with 1 mg/mL collagenase H, 0.15 mg/mL dithiothreitol, and 1 mg/mL BSA and continued to incubate for 8 min. The tissues were washed and gently triturated in the above solution to release single smooth muscle cells. Cells were stored at room temperature and used for experiments.Single mouse tracheal smooth muscle cells were enzymatically isolated as previously described with some modifications . Briefly2+ was used as the charge carrier of LVDCC currents. The intracellular solution contained (in mM) CsCl 130, EGTA 10, MgCl2 4, Mg-ATP 4, HEPES 10, and TEA 10 . The extracellular solution contained (in mM) NaCl 105, CsCl 6, BaCl2 27.5, glucose 11, HEPES 10, TEA 10, and NA 0.1 . The holding potential was -70 mV, and currents were recorded every 10 s using 500 ms steps from -70 to 40 mV in 10 mV increments. For ACh-activated current measurements, the intracellular solution contained (in mM) CsCl 18, cesium acetate 108, MgCl2 1.2, HEPES 10, EGTA 3, and CaCl2 1 . The free Ca2+ concentration was approximately 70 nM as calculated using the WEBMAXC standard program1. The extracellular solution was PSS without K+ containing 10 μM nifedipine, 100 μM NA, and 10 mM TEA to block Ca2+, Cl-, and K+ channel-mediated currents, respectively. Cells were patched in the classical whole-cell configuration and held at -50 mV. Currents were measured using a 500 ms ramp from -80 to 60 mV, and the values at -70 mV were used to represent the current.Currents mediated by LVDCCs and ACh-activated channels were recorded using an EPC-10 patch-clamp amplifier  at room temperature as previously described with some modifications . Ba2+ wa2+ was measured and analyzed as previously described using an LSM 700 laser scanning confocal microscope   . The bact-tests between two groups were performed, and statistical significance was established as P < 0.05.The results are expressed as the means ± standard error of the mean (SEM). Student’s 50 value and maximal inhibition were calculated as 0.51 ± 0.05 mg/mL and 93.93 ± 1.66% (n = 8), respectively. In addition, EESC inhibited the 80 mM K+-induced contraction . These data indicate that EESC almost completely blocks ACh- and high K+-induced ASM contraction.We first tested whether EESC affects the contraction of tracheal smooth muscle. As shown in Figure n Figure . The IC550 value and maximal inhibition were 0.52 ± 0.04 mg/mL and 104.0 ± 8.7% (n = 4), and 0.36 ± 0.01 mg/mL and 102.3 ± 1.1% (n = 4) for the precontraction induced by ACh and high K+, respectively. These results demonstrate that EESC-evoked relaxation is not mediated by epithelia. Thereby, the following experiments were conducted in epithelia-intact TRs.We then assessed whether the EESC-induced relaxation was affected by epithelia. The following experiments were performed in epithelia-denuded TRs Figures . The IC5We next studied whether EESC inhibits contraction in bronchial ASM. As shown in Figure To investigate the underlying mechanism of EESC-induced inhibition on precontraction, we first did two experiments as shown in Figure + induces activation of LVDCCs, which mediate Ca2+ influx and trigger contraction. Thus, we studied whether the EESC-induced relaxation is due to inactivation of LVDCCs. As shown in Figure + was used to open LVDCCs inducing contraction, however, 80 mM K+ did not induce a contraction in TRs in Ca2+-free conditions (0 mM Ca2+ and 0.5 mM EGTA) but was able to induce a contraction after the restoration of the Ca2+ concentration to 2 mM  by nifedipine and YM-58483. In addition, in the presence of nifedipine and Ca2+-free conditions (0 Ca2+ and 0.5 mM EGTA), ACh induced a transient contraction, however, a sustained contraction occurred after addition of 2 mM Ca2+ and was blocked by EESC  overlapped with that of the standard for aurantio-obtusin (Rt = 25.500). These data indicate that aurantio-obtusin is present in EESC. We found that the standard aurantio-obtusin blocked the contraction of ASM induced by ACh and high K+ in mouse TRs , by YM-58483 from -10.1 ± 2.4 pA to -0.8 ± 0.3 pA , and inhibited by Pyr3 from -11.2 ± 3.0 pA to -4.7 ± 2.6 pA  and further blocked by EESC .We finally observed whether EESC and aurantio-obtusin) of Semen cassia reversibly reduced both ACh- and high K+-induced precontractions in mouse TRs and lung slices. The ACh-induced precontraction was inhibited by the blocker of LVDCCs, SOCs and TRPC3/STIM/Orai channels, respectively. These channel-mediated currents were inhibited by aurantio-obtusin, which is one component of EESC. Aurantio-obtusin also blocked ACh- and high K+-induced precontraction. When aurantio-obtusin was removed from EESC using semi-preparative HPLC, EESC failed to induce relaxation. All these results together indicate that EESC-induced relaxant effects resulted from aurantio-obtusin.In this study, we found that the ingredients (i.e., EESC and Semen cassiae contains such ingredients. Its extract, EESC, potently inhibited ACh- and/or high K+-induced contraction of mouse ASM from large and small airways (Figures The purpose of this study was to screen for active herbal constituents that inhibit ASM contraction. We found that  Figures , 2. Thes Figures , suggestWhat is the mechanism underlying EESC-induced relaxation? Firstly, this relaxation results neither from the cytotoxicity of EESC, because its inhibition is reversible Figure  nor from+ is known to induce activation of LVDCCs, which then mediate Ca2+ influx, subsequently triggering contraction. Our results show that this pathway was blocked by EESC and then resulted in ASM relaxation (Figure aurantio-obtusin, which is one component of EESC (Figure High Kn Figure . MoreoveC Figure  and blocC Figure  and currC Figure .aurantio-obtusin (Figure LVDCCs also involve in ACh-induced precontraction, based on that a selective blocker of LVDCCs, nifedipine, inhibited the contractions Figure , consistn Figure . Therefon Figure .2+ store, which will then activate SOCs mediating Ca2+ influx. The results show that SOC-mediated Ca2+ influx plays an important role, because YM-58483 almost blocked the nifedipine-insensitive contraction (Figure 2+ influx pathway can also be blocked by EESC based on the Ca2+ restoration experiments (Figures aurantio-obtusin, because it blocked ACh-induced contractions (Figures To define the other pathways, we assessed the effect of the selective blocker of SOCs YM-58483 on ACh-induced contraction, since that ACh induces a depletion of Can Figure . This YM Figures . Moreove Figures  and SOC- Figures .It has been reported that Pyr3 sensitive channels play a role in ACh-induced ASM contraction . TherefoSemen cassiae contains > 70 chemical compounds including aurantio-obtusin. Its aqueous and ethanol extracts have hypotensive effects via inhibiting nervous reflex and iNOS/NO-related receptor-controlled calcium channel-mediated Ca2+ influx (aurantio-obtusin, based on our HPLC results (Figure 2+ influx and relaxation (aurantio-obtusin induced relaxation in rat mesenteric arteries, but not pulmonary arteries, through endothelial intracellular PI3K/Akt/eNOS/NO signaling pathway (It has been reported that + influx . The eths Figure , thus, ilaxation . This li pathway , althoug pathway .aurantio-obtusin induces ASM relaxation via inhibiting increased intracellular Ca2+ by blocking ACh-activated Ca2+-permeant ion channels, as aurantio-obtusin significantly decreased ACh-mediated increasing of intracellular Ca2+ (Figure 2+ is a critical modulatory factor for ASM tension and airway inflammation that is a characterized feature of asthma (2+ dynamics is a remarkable therapeutic target for developing bronchodilators. More recently, a clinical study showed that in patients with severe asthma, LVDCC inhibition attenuated airway remodeling that is another characteristic of asthma (aurantio-obtusin both exhibited the excellent capability of modulating Ca2+ dynamics in ASM. Moreover, both had the similar inhibitory action on human ASM contraction (Figure We further showed that + Figure , the res+ Figure . Ca2+ isf asthma . Hence, f asthma . In our n Figure . These raurantio-obtusin inhibits ASM contraction through inhibiting Ca2+-permeable ion channels. These findings suggest that aurantio-obtusin may be used to develop novel antiasthmatic drugs.In summary, our study demonstrates that EESC-contained Y-SS, L-QM, and Q-HL designed the experiments. Y-SS, L-QM, Q-HL, B-BL, W-JZ, J-YQ, M-YL, XL, QW, HX, D-AZ, YYC, X-XZ, LC, JS, Y-BP, PZ, LX, M-FY, WC, XN, CS, ShuC, ShaC, GQ, JD, and JC performed the experiments, analyzed the data. Y-SS, L-QM, and Q-HL wrote the paper.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.


We present a case report of a patient with acute myeloid leukemia (AML) characterized by the simultaneous presence of nucleophosmin 1 (NPM1) mutation and the breakpoint cluster region-Abelson (BCR-ABL) fusion oncogene. Our findings emphasize the importance of routinely including BCR-ABL in the diagnostic workup of AML in order to offer to the patients the most appropriate risk category and treatment options. NPM1 gene are the most frequent genetic abnormalities occurring in AML and are highly specific for de novo AML [de novo AML cases from CML-blastic phase (CML-BP).Mutations in the novo AML , 3. In BBCR-ABL fusion gene and NPM1 mutations in de novo AML has been reported in only few cases [NPM1 gene.To the best of our knowledge, the co-occurrence of ew cases . Herein,9/L (150–450 × 109/L), a WBC of 62 × 109 (4.30–10.80 × 109/L), basophils <2% (0–1.5%), and with 50% of blasts. The coagulation profile showed INR 1.5 (0.8–1.2), fibrinogen 69 mg/dL (200–400 mg/dL), ATIII 77% (75–128%), and D-dimer 10757 ng/mL (0–500 ng/mL), suggesting a disseminated intravascular coagulopathy (DIC). Bone marrow aspirate showed infiltration by 89% of hypergranular leukemic blasts , Plts 35 × 10Immunophenotyping of the leukemic population showed positivity for CD45, CD33, CD117, and MPO and negativity for CD34, HLA-DR, CD13, and CD56, compatible with a diagnosis of AML. Clinical examination showed mild splenomegaly (14 cm) and multiple thick and erythematous skin lesions localized on the back. A biopsy of one such lesion followed by histologic examination was consistent with extramedullary localization of AML.Conventional karyotyping  and FISHNPM1 gene [4/ABL [4/ABL [The p210 BCR-ABL fusion transcript was detected by standard RT-PCR, which allowed to identify both b3a2 and b2a2 transcript isoforms . NowadayPM1 gene  and the e [4/ABL  and NPM1L [4/ABL .The CT scan showed hepatic lesions suggestive for extramedullary involvement and disseminated venous thrombosis localized in the sovrahepatic veins and the right frontal sinus. Lumbar puncture showed no central nervous system involvement. The patient was started on initial cytoreductive treatment with hydroxyurea for ten days and was subsequently treated with second-generation tyrosine kinase inhibitor (TKI) dasatinib 140 mg PO daily. Dasatinib was preferred over imatinib, a first generation TKI, due to high risk of the central nervous system involvement, extramedullary localization , 10 and,In December 2014, due to trilineage pancytopenia, research for point mutations in BCR/ABL fusion gene was detected and showed, unfortunately after patient died, T315I mutation.In January 2015, three months after the start of dasatinib, he was hospitalized for neutropenic fever and was diagnosed with pneumonia. He died of sepsis few days later.BCR-ABL rearrangement and NPM1 mutation have been reported so far [de novo BCR-ABL +ve AML and CML-BP is still challenging [NPM1 mutation) were more suggestive of a de novo AML rather than of CML-BP. In previous reports of simultaneous NPM1 mutation and BCR-ABL rearrangement, the p210 and p190 transcripts were detected in 2 and 4 cases, respectively. However, the concomitant presence of b3a2 and b2a2 BCR-ABL p210 isoforms combined to simultaneous NPM1 mutation, as observed in the present report, has been reported to date in only one case [To the best of our knowledge, only few cases of AML with concomitant d so far . In clinllenging , 13. In llenging ) and molone case . An addiBCR-ABL seems to cooperate with several AML-specific aberrations, including CBFB-MYH11 and NPM1, although the precise molecular interaction among the altered proteins remains poorly understood [derstood .BCR-ABL lesion was present at the subclonal level [In our case, RQ-PCR analysis of NPM1 and BCR-ABL detected in both instances a high transcript copy number. Together with the elevated number of metaphases harboring the Philadelphia chromosome (15/15), these findings suggest that the two molecular aberrations were present within the same leukemic clone rather than occurring in two separate clones. However, in recently reported patients with Ph-positive AML, targeted treatment with TKIs was able to abrogate the BCR-ABL +ve clone but did not lead to complete hematologic response, indicating that the al level . This hiBCR-ABL and NPM1 mutation are considered distinct entities in the WHO classification of AML with recurrent genetic abnormalities. According to the updated ELN criteria, the presence of BCR-ABL fusion gene classifies AML in the adverse risk category while NPM1 mutation is generally regarded as a favorable prognostic in absence of the FLT3-ITD aberration. This case report highlights the possibility to concomitantly detect these two alterations in rare cases of AML and emphasizes the importance of including BCR-ABL screening in routine AML diagnostic panel in order to better assign the patient to the correct risk category and targeted treatment.At the moment, there is no standardized treatment for patients with Ph-positive AML although few case reports suggested a favorable response to TKIs . In ligh