The assembly of the V1-VO holoenzyme on the vacuole is stabilized in part through interactions between the VO a-subunit ortholog Vph1 and the lipid phosphatidylinositol 3,5-bisphosphate P2). PIP2 also affects vacuolar Ca2+ release through the channel Yvc1 and uptake through the Ca2+ pump Pmc1. Here, we asked if H+ and Ca2+ transport activities were connected through PIP2. We found that overproduction of PIP2 by the hyperactive fab1T2250A mutant augmented vacuole acidification, whereas the kinase-inactive fab1EEE mutant attenuated the formation of a H+ gradient. Separately, we tested the effects of excess Ca2+ on vacuole acidification. Adding micromolar Ca2+ blocked vacuole acidification, whereas chelating Ca2+ accelerated acidification. The effect of adding Ca2+ on acidification was eliminated when the Ca2+/H+ antiporter Vcx1 was absent, indicating that the vacuolar H+ gradient can collapse during Ca2+ stress through Vcx1 activity. This, however, was independent of PIP2, suggesting that PIP2 plays a role in submicromolar Ca2+ flux but not under Ca2+ shock. To see if the link between Ca2+ and H+ transport was bidirectional, we examined Ca2+ transport when vacuole acidification was inhibited. We found that Ca2+ transport was inhibited by halting V-ATPase activity with Bafilomycin or neutralizing vacuolar pH with chloroquine. Together, these data show that Ca2+ transport and V-ATPase efficacy are connected but not necessarily through PIP2.Yeast vacuoles are acidified by the v-type H Others igericin .2 on H+ transport and how H+ and Ca2+ transport affect each other. We demonstrate that PIP2 overproduction augments vacuole acidification while the lack of the lipid reduces acidification. We also show that increasing extraluminal Ca2+ concentrations blocked vacuole acidification in a manner linked to Vcx1, while chelating Ca2+ accelerated acidification. Finally, we show that a fully functioning V-ATPase is needed for Ca2+ efflux. Together, this study shows that the transport of H+ and Ca2+ is interdependent and can be affected by the PIP2 composition of the membrane.In this study, we examined the role of PIP2 to purified yeast vacuoles augmented acidification P2 inhibited vacuole fusion (2 levels to affect Ca2+ transport across the vacuolar membrane (2 may affect V-ATPase activity through modulating Ca2+ efflux from the vacuole lumen.Others have shown that adding exogenous short chain dioctanoyl (C8) PIPfication . This wais lipid . Howevere fusion . This wamembrane . Based o2 and other C8 lipids and found that C8-PIP2 indeed enhanced acidification as measured by changes in acridine orange (AO) fluorescence; however, the changes were modest in our hands (not shown). This prompted us to ask whether changing endogenous production of the lipid would have a stronger effect. To do this, we performed AO fluorescence assays with vacuoles isolated from WT yeast as well as strains expressing the kinase-deficient fab1EEE or the hyperactive fab1T2250A mutations P2 by fab1T2250A led to a pronounced increase in acidification, as manifested by an augmented drop in AO fluorescence. To show that the increased H+ transport activity by fab1T2250A vacuoles was indeed due to the overproduction of PIP2, we added the PIKfyve/Fab1 inhibitor apilimod to reactions containing fab1T2250A vacuoles (fab1T2250A vacuole acidification to WT levels (B). Previously, we showed elevated levels of PIP2, whether added exogenously as C8-PIP2 or overproduce by fab1T2250A led to prolonged Ca2+ uptake by Pmc1 P2 may link Ca2+ and H+ transport on the yeast vacuole.To test our hypothesis, we started by recapitulating the published results using C8-PIPutations , 22. AO utations , 24. Thuvacuoles , 26, 27.T levels B. Previo by Pmc1 . Importa2 led to enhanced acidification, we next asked if the lack of PIP2 would prevent the spectral shift AO fluorescence. To test this, we used vacuoles harboring the kinase-deficient fab1EEE mutant (fab1EEE vacuoles had attenuated vacuole acidification (C and D), which is in keeping with the destabilization of the V1–VO complex when PIP2 is absent P2, we supplemented the reaction with C8-PIP2. Curiously, our data showed that supplementing fab1EEE vacuoles with C8-PIP2 did not restore acidification (D). In contrast, adding C8-PIP2 to WT vacuoles did augment acidification as shown by the Kane lab P2 restored Ca2+ transport by fab1EEE vacuoles to WT levels P2in vivo prior to vacuole isolation. For instance, we have shown that fab1EEE vacuoles have diminished levels of the ABC transporter Ycf1, which interacts with the V1 subunit Vma10 (fab1EEE vacuoles showed that they contained more of the polyphosphate synthase Vtc4 (not shown) P2 can activate TORC1 and its phosphorylation of the kinase Sch9, which in turn affects the assembly and function of V-ATPases P2. Nevertheless, it is evident that modulating endogenous PIP2 production can augment or dampen vacuole acidification. The lack of a complete inhibition of proton pumping by fab1EEE vacuoles is in accord with a study showing that fab1Δ vacuoles were able to acidify as measured with cDCFDA , an esterase dye that is pH sensitive, and pHluorin fluorescence PE mutant . This shfication , C and Ds absent . That safication D. In conKane lab . The inaT levels . The lacit Vma10 , 29. Whit shown) . Deletio-ATPases , 33, 34.rescence . On the m 6 to 7 . Still, m 6 to 7 .fab1 mutants on vacuole acidification, we looked at whole cells stained with FM4-64 to label the vacuole and quinacrine to label acidified compartments. Calcofluor White was used to stain the cell walls. As shown in Figure 1E, the vacuoles of WT cells stained brightly with quinacrine. In comparison, cells expressing fab1EEE only lightly stained with quinacrine, further indicating that cells lacking PIP2 are defective in vacuole acidification. The reduction in quinacrine fluorescence in fab1EEE relative to WT was starker that what was seen with AO fluorescence, suggesting that tracking AO fluorescence using a plate reader was more sensitive compared to quinacrine and fluorescence microscopy. Finally, we found that fab1T2250A vacuoles stained more brightly compared to WT, which was indicative of augmented acidification and agreed with what was seen with AO fluorescence. While examining fab1EEE is informative, the remainder of the study focuses on the overproduction of PIP2 by fab1T2250A.To confirm the effects of expressing fab1 mutations affected vacuole acidification; however, it was unclear whether PIP2 was directly involved. To test this, we sequestered PIP2 with a specific lipid binding protein. We used the N terminus (ML1N) of the endolysosomal TRP Mucolipin-1/TRPML1 Ca2+ channel (2 and inhibits vacuole fusion (2 (A and B). As a control, we used FYVE domain to bind PI3P at concentrations that inhibit vacuole fusion (C and D). We have also used the PI4P binding PH domain from FAPP1 at concentrations that inhibit vacuole fusion and saw no effect on acidification P2 production by Fab1 on isolated vacuoles (2 production had inhibited vacuole acidification (E and F). The dimethyl sulfoxide (DMSO) treatment had no effect, showing that the effect of apilimod was not due to its solvent.In the results aforementioned, we used Fab1 mutations to examine the effects of PIPvacuoles . Here, wfication , E and F2+ transport directly affected vacuole acidification. Isolated vacuoles rapidly take up Ca2+ from the medium upon adding ATP, after which they release Ca2+ during the docking stage of fusion in a SNARE-dependent manner . Because these treatments also inhibited the uptake of Ca2+, it suggested that maintaining elevated extraluminal Ca2+ could inhibit vacuole acidification . Adding DMSO had no effect on vacuole acidification, indicating that the effect was due to apilimod. Similarly, adding verapamil at 300 s led to a rapid deacidification. Prenylamine has similar effect (not shown). The loss of the H+ gradient was consistent with the rapid release of Ca2+ when vacuoles were treated with verapamil after 600 s of incubation . Full inhibition of acidification occurred with 250 μM Ca2+, which was nearly 10-fold less that what was has been shown to inhibit fusion . When cells were incubated with CaCl2, we observed that quinacrine staining was blocked. This is in accord with the AO fluorescence data in A and B.To test the connection between PIPr levels , A and Bt fusion . To verile cells . As expele cells C. When c2+ inhibited vacuole acidification, we also tested if it affected vacuole acidification after the H+ gradient has been established. To answer this, we added a dosage curve of Ca2+ after 300 s of incubation, which is enough time to acidify vacuoles. This showed a dose-dependent deacidification of vacuoles as shown by an increase in AO fluorescence at 520 nm (D and E). This suggests one of two possibilities. First, it could be that the addition of Ca2+ at this point in the assay uses the antiporter activity of Vcx1, which would expel H+ as it takes up the newly added Ca2+. Alternatively, it could indicate that the added Ca2+ altered V-ATPase efficiency, resulting in the accumulation of extraluminal H+ (E). This was similar to findings by Cagnac et al.; however, they added bafilomycin A1 before adding Ca2+, preventing a direct comparison (2+ was added late in the reaction (E), while addition at the beginning prevented the fluorescence shift of AO for the duration of the experiment (A). This suggests that once vacuoles have established a H+ gradient equilibrium, they are able to quickly recover from the Ca2+ spike and reestablish the H+ gradient.After determining that adding excess Cat 520 nm , D and Eminal H+ E. This wreaction E, while periment A. This s2+ gradient in vacuole acidification could also be tested by chelating extraluminal Ca2+ with EGTA. This would make a near instant gradient of free lumenal Ca2+ without the need of Pmc1 or Vcx1 function and accelerate vacuole acidification. As hypothesized, we found that EGTA enhanced the rate of acidification, suggesting that Ca2+ uptake precedes acidification (F and G). Based on the effects of adding Ca2+ late in the reaction, we asked if adding EGTA would further enhance H+ uptake when added late. Unlike the effects of excess Ca2+, the addition of EGTA at 300 s did not affect acidification (H). We attribute this to the relative absence of extraluminal Ca2+ after 300 s of incubation as Ca2+ uptake is typically completed between 300 and 500 s P2, and vacuole acidification can be split into two major tracks. The first track occurs under homeostatic conditions where Ca2+ is at normal nontoxic levels. Here, Ca2+ uptake into the vacuole is primarily through the high affinity pump Pmc1 . Similarly, the shift in AO fluorescence using vacuoles from yvc1Δ and pmc1Δ cells was inhibited by ≥250 μM Ca2+ . The results with yvc1Δ vacuoles were as predicted since the TRP channel is an exporter of luminal Ca2+. The sensitivity seen with pmc1Δ vacuoles suggests that Vcx1 mediated transport of Ca2+ is linked to vacuole deacidification. When vcx1Δ vacuoles were tested, it showed that they were indeed resistant to the Ca2+ all concentrations (D and E). Together, these data suggest that the excess added Ca2+ is taken up through Vcx1, leading to the expulsion of H+ through its antiporter activity. This is also in agreement with previous studies.Here, WT and Ca blocked , A and Etrations , D and E2+ on V-ATPase activity also occurred after vacuoles had acidified. We added Ca2+ after 400 s of incubation and continued measuring fluorescence for 200 additional seconds before the addition of FCCP. WT and pmc1Δ vacuoles showed that the late addition of Ca2+ resulted in a dose-dependent increase in AO fluorescence at 520 nm, illustrating that H+ was released . This was comparable to what was seen with the late addition of bafilomycin. When vcx1Δ vacuoles were tested, we again saw a resistance toward the added Ca2+ (C and D). This further indicates that vacuole deacidification in the presence of micromolar levels of Ca2+ was linked to Vcx1 antiporter activity that released H+ as the result of taking in Ca2+.We next examined whether the effects of Caded Ca2+ , C and D2+ to synaptic vesicles led to the release of H+ with varying degrees of intensity . Similar effects were observed with pmc1Δ vacuoles (D and F) and vcx1Δ vacuoles (G and I). When Zn2+ was tested, we found that there was no significant effect on any of the vacuole types . Together, these data indicate that Cd2+ and Zn2+ do not reproduce the effects seen by the addition of Ca2+. Because of the activating effect of Cd2+ on vacuole acidification, we next asked if it could reverse the effects of Ca2+. To do this, we added a fixed amount of Ca2+ (250 μM) along with a curve of Cd2+. This showed that Cd2+ was able to partially restore vacuole acidification in the presence of Ca2+ (J and L). Interestingly, Zn2+ had a similar effect on restoring the effects of Ca2+ on acidification, even though it had no effect on its own (K and L). While the mechanism is unknown, the effect of Cd2+ could be attributed to its role as a mimetic and directly competing with Ca2+ for binding sites while not having an inhibitory effect. This is especially important to consider since Cd2+ can affect calmodulin (CaM)-dependent kinase II (CaMK-II) function and alter cytoskeletal dynamics as well as apoptosis , which raises the pH of vacuoles . This was followed by a rise in fluorescence near the 15 min mark as a reporter for SNARE-dependent efflux. The anti-Sec17 IgG-treated vacuoles took up Ca2+ but did not release it later due to the lack of SNARE pairing. We found that Ca2+ transport was delayed with 250 μM CQ and completely blocked with higher concentrations. This indicated that acidified vacuoles were needed for optimal Ca2+ uptake. To confirm that the levels of CQ used sufficiently blocked vacuole acidification, we used it in the AO acidification assay. As expected, we observed that CQ inhibited vacuole acidification in a dose-dependent manner (C and D).To start, we used WT vacuoles in the Cal-520 fluorescence assay to track the effects of vacuole acidification on Cavacuoles , 67. Untrescence , A and Bt manner , C and D+K+/H+ antiporter Nhx1 . Similarly, adding the V-ATPase inhibitor concanamycin A to reactions at the beginning completely blocked Ca2+ uptake (not shown). To see how quickly stopping V-ATPase function affected Ca2+ retention, we added bafilomycin after 10 min of incubation. This caused a rapid release of Ca2+ that reached the starting levels as measured by Cal-520 fluorescence. The Ca2+ release was faster and more pronounced than the natural SNARE-dependent release. Again, adding DMSO at the 10 min mark had no effect on Ca2+ flux, demonstrating that the release in Ca2+ was due to bafilomycin.While CQ served as a tool for raising the overall pH of the vacuole lumen, it is not a specific inhibitor of V-ATPase activity. In fact, CQ has been used to lower the pH of hyperacidified vacuoles lacking the Nater Nhx1 , 68. To activity , A and B2+ after the initial uptake has been linked to trans-SNARE pairing, we wanted to see if the effects of bafilomycin were also SNARE dependent. To this end, we added bafilomycin to reactions that were pretreated with anti-Sec17 IgG. We found that it led to the release of Ca2+ with the same kinetics and magnitude as adding bafilomycin alone, indicating that the release was independent of SNARE complex formation (C and D). We should note that bafilomycin inhibits SNARE pairing on its own when added early in the pathway; however, the effects of adding it during docking/tether on already formed SNARE complexes is unknown . This also indicated that the Stv1 isoform of Vph1 does not replace Vph1 to form a functional V-ATPase. This is important to keep in mind because only the overexpression of Stv1 can partially restore vacuole quinacrine staining in cells lacking Vph1 . This further acknowledges the requirement for Vph1 in vacuole acidification.Finally, we asked how mutations of the V-ATPase complex would affect Caeviously  . Moreover, they were equally sensitive to CQ treatment. When we tested vph1Δ vacuoles in Ca2+ transport assays, we found that they were able to take up Ca2+ but were inhibited in Ca2+ efflux (G). This showed that a functional V-ATPase was not needed for Pmc1 and Vcx1 activity. That said, the uptake was significantly slower versus the WT. Vacuoles lacking Vph1 did not plateau in Ca2+ uptake until ∼20 min of incubation compared with the 10 min uptake seen with WT. The uptake of Ca2+ by vph1Δ vacuoles appears at first to contradict the effects of bafilomycin; however, we must remain cognizant that physical interactions occur between Pmc1 and Vph1. We previously found that Pmc1 and Vph1 exist in a complex with the R-SNARE Nyv1 . However, the duration of the release was short in comparison and total Ca2+ uptake was completed by the 10 min mark as seen with WT vacuoles. Also, similar to the Vph1 deletion, vph1E789Q vacuoles did not release Ca2+. These data suggest that the assembly of the V1–VO blocks the inhibitory effects of Vph1 on Pmc1, perhaps through inducing conformational changes.The lack of Vph1 is known to destabilize the Vdeletion . We usedvacuoles H. Howeve2+ transport and vacuole acidification are interdependent and that they can be affected by PIP2 under specific conditions. Elevating PIP2 levels through the hyperactive fab1T2250A mutation increases vacuole acidification, whereas the kinase-dead fab1EEE mutant reduces acidification. Similarly, the presence or absence of PIP2 has opposing effects on Ca2+ transport. In vitro Ca2+ transport assays show that Ca2+ is taken into the vacuole lumen upon the addition of ATP, after which a wave of Ca2+ is released from the organelle. While the uptake is independent of SNAREs, the release requires the formation of trans-SNARE complexes between paired vacuoles P2.In this study, we present data showing that Cavacuoles . In a prvesicles . On the + and Ca2+ transport by testing the effect of changing the concentration of free Ca2+ on the formation of an H+ gradient. First, we showed that increasing extraluminal Ca2+ blocked the shift in AO fluorescence, indicating that the H+ gradient had been broken. This was linked to the role of the Ca2+/H+ exchanger Vcx1. The converse was seen when extraluminal Ca2+ was chelated with EGTA. The lack of free Ca2+ augmented the rate of H+ uptake and formation of a proton gradient. Interestingly, the deletion of VCX1 does not replicate the accelerated H+ uptake seen with EGTA, suggesting that the difference was not due to the lack of H+ export by Vcx1. Thus, we can postulate that a separate Ca2+-dependent mechanism could modulate vacuole acidification.We further demonstrated the connection between H2+ transporters are not rare. For instance, L-type Ca2+ channels in murine cells physically interact with the G2 subunit of the V-ATPase, and inhibition of V-ATPase function leads to the mislocalization of the Ca2+ channel  of the VO complex interacts with Ca2+-loaded CaM to promote normal eye development  for this relationship remains to be elucidated, we can add that their interdependence on the vacuole could be associated with the production of PIP2. These connections begin to unveil a more complicated network of interactions that integrates the composition of the membrane with ion homeostasis.In conclusion, this study shows that vacuole acidification by the V-ATPase is regulated by Ca2  was purchased from Echelon Inc. ATP was purchased from RPI. Apilimod, bafilomycin A1, concanamycin A, and verapamil were from Cayman Chemical and dissolved in DMSO. AO, Calcofluor White solution, chloroquine, CoA, creatine kinase, EGTA, FCCP, and quinacrine were purchased from Sigma. Creatine phosphate was from Abcam. Cal-520 dextran conjugate molecular weight 10,000 was from AAT Bioquest. FM4-64 was purchased from ThermoFisher. Anti-Sec17 IgG  with 125 mM KCl, unless indicated otherwise. C8-PIPec17 IgG , GST-ML1ec17 IgG , and GSTec17 IgG  were pre2+ transport assays (STV1 was deleted by homologous recombination using PCR products amplified from pAG32 plasmid with primers 5′-STV1-KO (5’ – AGGCCCACGAAGGTGATTGGAAGTTCAGTGTTGAATCT GTTTAGCTTGCCTCGTCC – 3′) and 3′-STV1-KO (5′- GCAAACGTAGCGCATGCAACATTGCGTGGATGGCGGCGTTAGTATCGA – 3′), with homology flanking the STV1 coding sequence. The PCR product was transformed into chemically competent yeast by standard lithium acetate methods and plated on yeast extract–peptone–dextrose (YPD) containing hygromycin (200 μg/ml) to generate BJ3505 stv1::hyhMX4 (RFY108). RFY107 (vph1Δ) was transformed with pRS316-VPH1E789Q  to make RFY109. The proton pumping activity of isolated vacuoles was performed as described by others with some modifications (In vitro acidification reactions (60 μl) contained 20 μg vacuoles, reaction buffer , ATP-regenerating system , 10 μM CoA, 283 nM Pbi2 (inhibitor of protease 2), and 15 μM of AO. Reaction mixtures were loaded into a black, half-volume 96-well flat-bottom plate with nonbinding surface. ATP-regenerating system or buffer was added, and reactions were incubated at 27 °C while AO fluorescence was monitored. Samples were analyzed in a fluorescence plate reader  with the excitation filter at 485 nm and emission filter at 520 nm. Reactions were initiated with the addition of ATP-regenerating system following the initial measurement. After fluorescence quenching plateaus were reached, we added 30 μM FCCP to collapse the proton gradient and restore AO fluorescence.Vacuoles were isolated as described from BJ3505 genetic backgrounds and used for vacuole acidification and Cat assays   contained 20 μg vacuoles from BJ3505 backgrounds, reaction buffer, 10 μM CoA, 283 nM Pbi2, and 150 nM of the Ca2+ probe Cal-520 dextran conjugate molecular weight 10,000. Reaction mixtures were loaded into a black, half-volume 96-well flat-bottom plate with nonbinding surface. ATP-regenerating system was added, and reactions were incubated at 27 °C while Cal-520 fluorescence was monitored. Samples lacking ATP were used a negative control for Ca2+ uptake. Antibody against the SNARE cochaperone Sec17 was added as a negative control for SNARE-dependent Ca2+ efflux .Vacuolar Caescribed , 88, 89.+ efflux . SamplesIn vivo vacuole staining with quinacrine and FM4-64 (5 μM) was carried out as described , and an AxioCam CCD camera. Quinacrine was visualized using a 38 HE EGFP shift-free filter set, FM4-64 was visualized with a 42 HE CY 3 shift-free filter set, and Calcofluor White was visualized with a 49 4′,6-diamidino-2-phenylindole shift-free filter set.escribed , 90, 91.t test or one-way ANOVA for multiple comparisons using Prism 9 (GraphPad). Statistical significance is represented as follows: ∗p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.Results are expressed as the mean ± SD. Experimental replicates (n) are defined as the number of separate experiments. Statistical analysis was performed by Student’s unpaired two-tailed All primary data are available upon request. Additional data sharing information is not applicable to this study.The authors declare that they have no conflicts of interest with the contents of this article.


Results displayed that OPP with high (42.13%), middle (25.88%), and low (4.77%) DE values were successfully prepared by mild alkaline de-esterification, and their primary chemical structures  and molecular characteristics  were overall stable. Additionally, results showed that the notable decrease of DE value did not significantly affect antioxidant activities  of OPP, suggesting that the DE was not closely related to its antioxidant activity. In fact, the slight decrease of antioxidant activity of OPP after the alkaline de-esterification might be attributed to the slight decrease of uronic acid content. Nevertheless, the immunostimulatory effect of OPP was closely related to its DE, and a suitable degree of acetylation was beneficial to its in vitro immunostimulatory effect. Besides, the complete de-acetylation resulted in a remarkable reduction of immune response. The findings are beneficial to better understanding the effect of DE value on antioxidant and immunomodulatory activities of OPP, which also provide theoretical foundations for developing OPP as functional foods or health products.Pectic-polysaccharides are considered as one of the most abundant bioactive components in okra, which possess various promising health-promoting effects. However, the knowledge regarding the structure-bioactivity relationship of okra pectic-polysaccharides (OPP) is still limited. In this study, effects of various degrees of esterification (DEs) on  Pectic-polysaccharides are complex heteropolysaccharides existed in the primary cell walls of vegetables and fruits, which are predominantly composed of homogalacturonan (HG), rhamnogalacturonan I (RG-I), and rhamnogalacturonan II (RG-II) domains . RecentlAbelmoschus esculentus L. Moench, known as okra or lady’s finger, is a vital edible and medicinal plant in China. It is native to the Africa but can now be found throughout tropical and subtropical areas of the world -α-L-Rhap- (ABTS), sodium nitroprusside (SNP), vitamin C (Vc), griess reagent, lipopolysaccharide (LPS), and 3--2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich .Okra fruits of in vitro biological activities as previously reported  at the power of 520 W for 0.5 h  of DOPP in NaOH solution at basic pH values of 11.0 and 13.0 for 0.5 h under 4°C, respectively. At the end of reaction, the sample solution was acidified to pH = 6.0 by adding HCl (1 M). Then, after dialysis  and freeze-drying in turn, okra pectic-polysaccharides with a middle DE value (DOPP-MDE) and a low DE value (DOPP-LDE) were obtained. Indeed, the yields of DOPP-MDE and DOPP-LDE were measured to be 95.73 and 90.82%, respectively. Correspondingly, the original DOPP was named DOPP-HDE, which possessed a relatively high DE value.Furthermore, the preparation of DOPP with various DE values was carried out according to a previous study with a few modifications . In brieMw), molecular weight distribution (w/MnM), and radius of gyration (Rg) as well as rheological property of DOPP-HDE, DOPP-MDE, and DOPP-LDE were also measured as previously reported  as previously reported  as previously reported  and 1,600–1,640 cm–1 , which was estimated based on the following equation:Total polysaccharides, uronic acids, and proteins of OPP with various DE values were detected by colorimetric methods as previously reported . Moleculreported , 22. In reported . A C18 creported , 25. Furreported , 27. Ind50 values (mg/mL) of DOPP-HDE, DOPP-MDE, and DOPP-LDE for scavenging free radicals could be determined on the basis of a logarithmic regression curve. Additionally, for the determination of FRAP, 100 μL of each sample (2.0–10.0 mg/mL) was mixed with 100 μL of potassium ferricyanide  at 50°C for 20 min, and then 100 μL of trichloroacetic acid  was added and centrifugated. Finally, both distilled water (100 μL) and ferric chloride (20 μL) were added into the supernatant (100 μL). The absorbance of the mixture was recorded at 593 nm. Vc was used as a positive control in each experiment.The ferric reducing antioxidant power (FRAP), ABTS radical scavenging ability, and nitric oxide (NO) radical scavenging ability of OPP with various DE values were evaluated according to previously reported methods . In briein vitro model of RAW 264.7 macrophages according to a previously reported method , 1,4-α-D-GalAMep , 1,2-α-L-Rhap , 1,2,4-α-L-Rhap , 1,4-β-D-Galp , could be found in DOPP-HDE, DOPP-MDE, and DOPP-LDE. These results suggested that the RG-I backbone with galactan side chains existed as the main pectic-polysaccharides in DOPP-HDE, DOPP-MDE, and DOPP-LDE  degrade the molecular weight of OPP. This phenomenon was similar with previous studies that β-elimination reaction could cause the degradation of molecular mass . Furthermore, in terms of NO radical scavenging activity, the IC50 values of DOPP-HDE, DOPP-MDE, and DOPP-LDE were detected to be 8.43 ± 0.21, 9.02 ± 0.49, and 10.64 ± 0.38 mg/mL, respectively, which were also higher than that of Vc (0.62 ± 0.02 mg/mL). Surprisingly, results showed that DOPP-LDE with the lowest DE value (4.77%) among three samples exhibited the lowest antioxidant activities in the present study. Besides, although the DE value of DOPP-MDE (25.88%) was significantly lower than that of DOPP-HDE (42.13%), the in vitro antioxidant activities of DOPP-HDE and DOPP-MDE were similar. Therefore, these results indicated that the remarkable decrease of DE value (mainly degree of acetylation) of OPP by mild alkaline de-esterification did not obviously affect their antioxidant activity, suggesting that the DE was not closely correlated to the antioxidant activity of OPP. This phenomenon is quite different from previous studies that the lower DE of pectic-polysaccharides is closely related to their higher antioxidant activity (activity , 38. In activity , 40. Howactivity , which mactivity , 36, 41.activity .in vitro and in vivo immunostimulatory effects (Immunity refers to the protection effects of biological organisms against foreign bacteria, viruses, and other harmful substances. A large number of studies have demonstrated that dietary polysaccharides from edible and medicinal plants can maintain the human health by regulating the immune system , 43. Gen effects , 46–48.  effects . HoweverAsparagus officinalis L., and a relatively high DE value was associated with the relatively high immunostimulatory effect (Polygonatum cyrtonema might also benefit its immunostimulatory effect (in vitro immunostimulatory effect, and the complete de-acetylation in DOPP-LDE results in a remarkable reduction of immune response. Nevertheless, the precise structure-immunostimulatory activity relationship of OPP and related mechanism of action are required to be deeply uncovered in the future.Therefore, the RAW 264.7 macrophage was applied as a cell model for the determination of immunostimulatory effects of OPP with various DE values. y effect . Indeed,y effect . In addiy effect , 53, whiy effect , 55. Thein vitro antioxidant capacities and immunostimulatory activities of OPP were investigated. Results showed that the decrease of DE was mainly attributed to the de-acetylation of OPP according to the 1H NMR spectra analysis. In addition, results showed that the DE value was not related to the antioxidant activity of OPP. However, the immunostimulatory effect of OPP was closely related to its DE value, and a suitable DE value is beneficial to its in vitro immunostimulatory effect. Collectively, the findings are beneficial to revealing the effect of esterification degree on antioxidant activity and immunomodulatory activity of OPP. However, due to the limitations of in vitro models, it is necessary to evaluate the bioactivities of okra pectic-polysaccharide and its structure dependent relationships in animal models in the future.Pectic-polysaccharides are regarded as one of the most abundant bioactive components in okra. However, the knowledge about the precise structure-bioactivity relationships of OPP is still limited. Therefore, in order to further clarify the potential structure-bioactivity relationship of okra pectic-polysaccharides, effects of various degrees of esterification on The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.WL and JL: investigation, formal analysis, resources, software, and writing – original draft. JW: investigation, formal analysis, and validation. YH: investigation and validation. Y-CH: resources and software. D-TW: data curation, methodology, formal analysis, funding acquisition, and writing – review and editing. LZ: methodology, formal analysis, supervision, resources, and writing – review and editing. All authors contributed to the article and approved the submitted version.


Langerhans cell histiocytosis (LCH) and acute myeloid leukemia (AML) are distinct entities of blood neoplasms, and the exact developmental origin of both neoplasms are considered be heterogenous among patients. However, reports of concurrent LCH and AML are rare. Herein we report a novel case of concurrent LCH and AML which shared same the driver mutations, strongly suggesting a common clonal origin.An 84-year-old female presented with cervical lymphadenopathy and pruritic skin rash on the face and scalp. Laboratory tests revealed pancytopenia with 13% of blasts, elevated LDH and liver enzymes, in addition to generalised lymphadenopathy and splenomegaly by computed tomography. Bone marrow specimens showed massive infiltration of MPO-positive myeloblasts, whereas S-100 and CD1a positive atypical dendritic cell-like cells accounted for 10% of the atypical cells on bone marrow pathology, suggesting a mixture of LCH and AML. A biopsy specimen from a cervical lymph node and the skin demonstrated the accumulation of atypical cells which were positive for S-100 and CD1a. LCH was found in lymph nodes, skin and bone marrow; AML was found in peripheral blood and bone marrow (AML was predominant compared with LCH in the bone marrow).NRAS-G13D, IDH2-R140Q, and DNMT3A-F640fs/-I715fs), equally shared by both the lymph node and bone marrow, suggesting a common clonal origin for the concurrent LCH and AML. Prednisolone and vinblastine were initially given with partial response in LCH; peripheral blood blasts also disappeared for 3 months. Salvage chemotherapy with low dose cytarabine and aclarubicin were given for relapse, with partial response in both LCH and AML. She died from pneumonia and septicemia on day 384. Our case demonstrates a common cell of origin for LCH and AML with a common genetic mutation, providing evidence to support the proposal to classify histiocytosis, including LCH, as a myeloid/myeloproliferative malignancy.Next generation sequencing revealed four somatic driver mutations ( Langerhans cell histiocytosis (LCH) and acute myeloid leukemia (AML) are distinct entities of blood neoplasms, and the exact developmental origin of both neoplasms is considered to be heterogenous among patients –6. HowevAn 84 year-old female presented with cervical lymphadenopathy and pruritic skin rash on the face and scalp. She had no past history or family history of hematological or solid organ malignancies or other treatments. She had no fever but multiple lymph nodes were palpated bilaterally in the neck, axilla, and inguinal region. Laboratory tests revealed pancytopenia  of 10.9 g/dL, and a platelet count of 92,000/μL) and elevated LDH (604 U/L) and liver enzymes . Systemic lymphadenopathy and splenomegaly, but no other abnormal findings, were noted on computed tomography. Bone marrow specimens showing a massive infiltration of MPO-positive blasts including 53.5% of myeloblastic cells and 13.8% of monoblastic cells indicated the diagnosis of AML. Whereas, 9.1% of cellular components were occupied by MPO-negative atypical dendritic cell-like cells . Elevated liver enzymes and splenomegaly suggested the presence of hepatic and splenic lesions, but which tumor was responsible could not be determined.NRAS-G13D, IDH2-R140Q, and DNMT3A-F640fs/-I715fs), equally found in both the lymph node and bone marrow . The mutant allele frequencies were 24.6%-33.0% and 27.0%-38.5% in the lymph node and bone marrow, respectively. Assuming each mutant allele to be a single hit, 50-70% of tumor cells harbored each mutation, suggesting a common clonal origin of concurrent LCH and AML.Taken together, the patient was diagnosed with concurrent LCH and AML. Next generation sequencing with the TruSight Myeloid Sequencing Panel (Illumina) revealed four somatic driver mutations  than LCH, and, in combination with IDH2, DNMT3A and other mutations, contribute to the pathogenesis of AML   . In thisASXL1, IDH2, and STAG2 mutations  and AML (bone marrow), no genetic mutations present in only AML or LCH were detected, making it difficult to dissect the developmental process of LCH and AML. Thus, the present case provides important evidence that myeloid progenitors are the common origin of the two neoplasms, and reaffirms the importance of MAPK pathway activation in the pathogenesis of histiocytic disorders.Five cases of myeloid neoplasms have been suggested to have a common origin with LCH , thus, this is the third report of common gene mutation between LCH and AML. In the other two reports of shared mutation between LCH and AML, both adult males were diagnosed with LCH on skin biopsy and AML from bone marrow biopsy. One patient shared utations , and therisomy 8 . Of noteOn the other hand, a different biological mechanism by which leukemic progenitor cells are misdirected into LCH by environmental factors was considered. This disease is an inflammatory myeloid neoplasm with features of both abnormal reaction processes and neoplastic processes. De Graaf et al. reported that inflammatory cytokines are expressed in LCH lesions . KannourAccording to the 2016 WHO classification, histiocytic disorders, including LCH and ECD, are classified as lymphoid neoplasms . This clIn summary, our case demonstrates a common cell of origin for LCH and AML with a common genetic mutation, providing evidence to support the proposal to classify histiocytosis, including LCH, as a myeloid/myeloproliferative malignancy.The detailed clinical data and datasets presented in this article are not publicly available due to ethical and privacy restrictions. Requests to access the datasets should be directed to the corresponding authors.Written informed consent was obtained from the [individual(s) AND minor(s)’ legal guardian/next of kin] for the publication of any potentially identifiable images or data included in this article.SK, TU, HKa, HS, MS, II, YT, TD, YO, and HKo were involved in the description of clinical information. KY, NY, ES, RY, SI, SM, and AT were involved in the data analysis. All authors contributed to the article and approved the submitted version.www.editage.com) for English language editing.We would like to thank Editage (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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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The objective of this study was to determine whether (5S)-5--5,9-dihydro-8H-furo  naphtho  dioxol-6-one (JNC-1043), which is a novel chemical derivative of β-apopicropodophyllin, acts as a novel potential anticancer reagent and radiosensitizer in colorectal cancer (CRC) cells. Firstly, we used MTT assays to assess whether JNC-1043 could inhibit the cell proliferation of HCT116 and DLD-1 cells. The IC50 values of these cell lines were calculated as 114.5 and 157 nM, respectively, at 72 h of treatment. Using doses approximating the IC50 values, we tested whether JNC-1043 had a radiosensitizing effect in the CRC cell lines. Clonogenic assays revealed that the dose-enhancement ratios (DER) of HCT116 and DLD-1 cells were 1.53 and 1.25, respectively. Cell-counting assays showed that the combination of JNC-1043 and γ-ionizing radiation (IR) enhanced cell death. Treatment with JNC-1043 or IR alone induced cell death by 50~60%, whereas the combination of JNC-1043 and IR increased this cell death by more than 20~30%. Annexin V-propidium iodide assays showed that the combination of JNC-1043 and IR increased apoptosis by more 30~40% compared to that induced by JNC-1043 or IR alone. DCFDA- and MitoSOX-based assays revealed that mitochondrial ROS production was enhanced by the combination of JNC-1043 and IR. Finally, we found that suppression of ROS by N-acetylcysteine (NAC) blocked the apoptotic cell death induced by the combination of JNC-1043 and IR. The xenograft model also indicated that the combination of JNC-1043 and IR increased apoptotic cell death in tumor mass. These results collectively suggest that JNC-1043 acts as a radiosensitizer and exerts anticancer effects against CRC cells by promoting apoptosis mediated by mitochondrial ROS. Colorectal cancer (CRC), which is also known as bowel cancer, colon cancer, or rectal cancer, comprises cancer developed from the colon or rectum. CRC is the second most common cause of cancer death in the United States ,2. BetwePodophyllum peltatum Linnaeus. PPT is known to exert immunosuppressive activity and antiviral effects against herpes, measles, influenza, and venereal warts, and has also been shown to act against skin cancer  naphtho dioxol-6-one (JNC-1043), and explored its anticancer drug and radiosensitizer effects and the underlying molecular mechanisms in CRC cells [Podophyllotoxin (PPT) is a major material used in the development of novel anticancer drugs. PPT is an active plant ingredient that can be isolated as an abundant lignan from podophyllin, which is a type of resin produced by n cancer . Given tn cancer ,11,12. Wn cancer ,14. We an cancer ,16. The RC cells .JNC-1043 was synthesized from APP a. StructTo investigate the anticancer effect of JNC-1043, we examined its IC50 (50% inhibition of viability) values. HCT116 and DLD-1 cells were treated with various concentrations of JNC-1043 for 48 or 72 h, and MTT assays were performed. The IC50 values of JNC-1043 against HCT116 and DLD-1 cells at 48 or 72 h were 171.9 nM and 159.8 nM or 114.5 nM and 157 nM, respectively . The relTo begin examining the anticancer mechanism of JNC-1043, we used an immunoblot assay to assess the phosphorylation level of H2AX (γH2AX) c. Our reTo investigate whether JNC-1043 has a radiosensitizing effect on CRC cells, clonogenic, cell counting, and immunoblot assays were performed. Firstly, clonogenic assays were performed by pre-treating cells with JNC-1043 (110 nM JNC-1043 for HCT116 cells and 150 nM JNC-1043 for DLD-1 cells) for 24 h and then irradiating them with 1, 2, 3, or 4 Gy IR. The DER values were calculated as 1.528 for HCT116 cells and 1.25 for DLD-1 cells . These rL, cleaved caspase-3, cleaved caspase-9, and cleaved PARP, were performed to assess whether the apoptotic cell death mechanism is increased in CRC cells co-treated with JNC-1043 and IR. Indeed, the levels of Bcl-2 and Bcl-XL were decreased while those of cleaved caspase-3, caspase-9, and PARP were increased in cells co-treated with JNC-1043 and IR, relative to those in cells treated with JNC-1043 alone or IR alone  and IR compared to the levels seen in the JNC-1043-only and IR-only groups a. ImmunoIR alone b,b’. TheIR alone c. These Based on previous reports, we next explored whether JNC-1043 also could facilitate IR-induced cell cycle arrest. Cell cycle analysis was performed with HCT116 and DLD-1 cells co-treated with 110 (HCT-116) or 150 nM (DLD-1) of JNC-1043, respectively, and 1 Gy of IR for 4 (HCT-116) or 2 (DLD-1) h, respectively. After undergoing treatment with IR and JNC-1043, samples were fixed for 24 h, stained with PI/RNase staining buffer, and analyzed with a FACSort flow cytometer. Our results revealed that the population of CRC cells at the G2/M phase was increased by ~20% in samples co-treated with 1 Gy and 110 or 150 nM JNC-1043, respectively, compared to the level in the JNC-1043-only- treated and IR-only-treated groups . ImmunobSince cancer cells are more vulnerable than normal cells to disruption of the redox balance and mitochondrial function, oxidative stress triggered by disruption in ROS homeostasis is an important principle of many cancer therapies, including chemotherapy, radiotherapy, and combination therapy ,20. IndeThus, intracellular and mitochondrial ROS detection assays were performed to determine whether JNC-1043 and IR co-treatment could enhance ROS production. The results of an intracellular ROS detection assay performed using H2DCFDA showed that the combination of JNC-1043 and IR enhanced the production of ROS by more than 20~40% compared to the levels seen in the JNC-1043-only or IR-only groups a. A mitoΨm)  a. Our reΨm) b. TreatmΨm) c,d. AddiΨm) a,b. ThesL, the levels of cleaved caspases and PARP (Ψm) disruption followed by mitochondrial ROS accumulation.As reported above, the combination of JNC-1043 and IR increased ROS production and apoptotic cell death. Therefore, we hypothesized that the increase in ROS could be linked to cell death in the radiosensitizing effect of JNC-1043 and tested this using NAC. Cell counting and Annexin V-PI staining assays were performed to determine whether pre-treatment with NAC could affect the disruption of ROS homeostasis by the combination of JNC-1043 and IR a,b. Indeand PARP c,c’, andand PARP d. Taken Next, we tested whether the radiosensitization effect of JNC-1043 could be observed in an in vivo animal model. Xenograft model mice were prepared using HCT116 cells, and apoptotic cell death in xenografts was measured under various experimental conditions: mock control, IR only, JNC-1043 only, and the combination of JNC-1043/IR a. TUNEL Our results demonstrate that JNC-1043 has a radiosensitization effect against CRC cells via the induction of apoptosis, both in vitro and in an in vivo system. From our findings, we conclude that the combination of JNC-1043 and IR enhances apoptotic cell death via increases in mitochondrial ROS and DNA damage, followed by caspase activation. b.Radiosensitizers in cancer therapy are various materials that, when combined with IR, achieve greater tumor treatment effects than seen following the application of IR or the material alone. Radiosensitizers were historically characterized as: (1) suppressors of intracellular thiols or other endogenous radioprotective substances; (2) inducers of cytotoxic substances by intracellular radiolysis of the radiosensitizer; (3) inhibitors of repair mechanisms/molecules of biomolecules; (4) DNA-incorporating thymine analogs; and (5) oxygen mimics containing electrophilic activity . RecentlL, up-regulated cleaved caspase-3, caspase-9, and PARP, and enhance the release of cytochrome c. It is known that released cytochrome c can form a complex with procaspase 9 and apoptosis protease-activating factor 1 (APAF1), which can activate caspase 9. Caspase 9 then activates pro-caspase 3 and pro-caspase 7, resulting in cell death [When IR induces DNA damage to eukaryotic cells, cell death is triggered via the formation of DNA double-strand breaks (DSBs). DSB formation is immediately followed by histone H2AX phosphorylation (γH2AX) and the accumulation of numerous repair factors . The accll death ,29,30. Tll death ,32,33. Sll death . Our datll death ,36,37. Wll death ,38: ROS ll death . ROS can2 incubator. For harvesting, the cells were washed with cold PBS and scraped into lysis buffer. JNC-1043 was synthesized by J&C Sciences  as follows: a 60% dispersion of NaH in mineral oil  was added portion-wise to a solution of 4′-demethyl-β-apopicropodophyllin  in anhydrous DMF (100 mL) at 0 ℃. The mixture was stirred for 1 h at 0 ℃, benzyl bromide  was added slowly, and stirring was continued for 12 h at ambient temperature. Water (300 mL) was added slowly to quench the reaction, and the mixture was extracted with ethyl acetate (2 × 100 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified via column chromatography on SiO2 using ethyl acetate:n-hexane  to give JNC-1043  as a white solid.The HCT116, DLD-1, and CCD-18Co cell lines were purchased from the American Type Culture Collection . All three cell lines were cultured in RPMI medium containing 10% fetal bovine serum and 1% penicillin, at 37 °C in a 5% CO3 cells/well) and exposed to various concentrations of JNC-1043 for 48 h or 72 h at 37 °C. MTT -2,5-diphenyltetrazolium bromide) solution (20 μL of 2 mg/mL) was added to each well, and the cells were incubated for 1 h at 37 °C. The formazan crystals generated in living cells were dissolved in 100 μL of DMSO, and the absorbances of individual wells were measured at 545 nm using a microplate reader . The 50% inhibitory concentration (IC50) of JNC-1043 was calculated by performing a concentration–response analysis using the SoftMax Pro software Ver. 6.5 .HCT116, DLD-1, and CCD-18Co cells were seeded on 96-well plates  at different doses . The cells were incubated for at least 14 days until colonies formed, and colonies larger than 200 μm in diameter were stained with 1% methylene blue in methanol. Stained colonies were counted using a colony counter . The colony number of each dose was compensated with ratios that the maximum seeded cell number (1000 cells/dish) is divided by each seeded cell number. The dose-enhancement ratio (DER) value of each cell line was determined from the colony numbers using the Excel program .HCT116 and DLD-1 cells were seeded in 60-mm dishes at cell concentrations estimated to yield 20–100 colonies/dish . After 24 h of incubation, the cells were treated with or without 110 nM and 150 nM JNC-1043, respectively, for 24 h, and then irradiated with 5 cells/60-mm dish) were incubated with or without 110 nM and 150 nM JNC-1043, respectively, and exposed to 3 Gy IR. The cells were incubated for 72 h at 37 °C, collected by trypsinization, and washed twice with cold PBS, and equal volumes of cells were stained with trypan blue. The cells were counted using an EVETM Automated Cell Counter . These experiments were repeated in triplicate.HCT116 and DLD-1 cells (1 × 105 cells/60-mm dish) were incubated with or without 110 nM and 150 nM JNC-1043, respectively, and exposed to 1 Gy IR for 2 or 4 h, respectively. The cells were collected by centrifugation at 1200 rpm for 1 min, washed twice with ice-cold phosphate-buffered saline (PBS), and fixed with ice-cold 70% ethanol. The fixed cells were stained with PI/RNase Staining Buffer  for 15 min, and then analyzed with a FACSort flow cytometer . The percentages of cells in each phase of the cell cycle were determined using the Cell Quest software . These experiments were repeated in triplicate.HCT116 and DLD-1 cells  and a microplate reader (Molecular Devices). Twenty-milligram aliquots of proteins were heated at 95 °C for 1 min, resolved by SDS-PAGE, and transferred to a nitrocellulose membrane. After being blocked for 1 h at room temperature with 5% skim milk, the membranes were incubated at 4 °C with primary antibodies in 5% BSA solution. After 24 h of incubation, the membranes were washed and incubated with a secondary antibody for 1 h at room temperature. Bands were detected with the ClarityTM Western ECL Substrate (Bio-Rad) and visualized using an Amersham ImageQuant 800 . Primary antibodies were used against Bcl-2, Bcl-XL, pro-caspase-3, pro-caspase-9, cleaved caspase-3, cleaved caspase-9, pro-PARP, cleaved PARP, γ-H2AX , cyclin B1, and p21 . An anti-β-actin antibody  was used as a loading control. The relative immunoblot band densities were determined via densitometry using the ImageJ software , normalized to that of each control, and analyzed with GraphPad Prism Ver. 5 . All experiments were repeated in triplicate.HCT116 and DLD-1 cells were harvested and lysed with RIPA buffer , and the lysates were centrifuged at 14,000× 2, 0.25 M EGTA, and 1 M DTT), homogenized, and centrifuged at 12,000× g at 4 °C for 15 min. The supernatants were used as the cytosolic fraction and the pellets were resuspended with lysis buffer  and used as the mitochondrial fraction. The fractions were immunoblotted with a cytochrome c antibody . VDAC  was used as a loading control. These experiments were repeated in triplicate.HCT116 and DLD-1 cells were treated with 110 nM and 150 nM JNC-1043, respectively, irradiated 3 Gy IR, and incubated for 48 h at 37 °C. The cells were then incubated with trypsin-EDTA for 5 min, collected by centrifugation at 250 g for 1 min, resuspended with extraction buffer  were incubated with or without 110 nM and 150 nM JNC-1043, respectively, exposed to 3 Gy IR, and incubated for 72 h. The cells were then collected by trypsinization, washed twice with cold PBS, and stained with an FITC Annexin V apoptosis detection kit I  as described by the manufacturer. A FACSort flow cytometer  was used to measure the fraction of apoptotic cells (x-axis: FL1 channel and y-axis: FL-2 channel). These experiments were repeated in triplicate.HCT116 and DLD-1 cells (1 × 105 cells/60-mm dish) were incubated with or without 110 nM and 150 nM JNC-1043, respectively, and exposed to 3 Gy IR. After incubation for 24 h, the cells were stained with 25 mM H2DCFDA  for 5 min and trypsinized. The cells were harvested by centrifugation and resuspended with cold PBS. A FACSort flow cytometer  was used for the detection and analysis of intracellular ROS . These experiments were repeated in triplicate.HCT116 and DLD-1 cells (5 × 105 cells/60-mm dish) were incubated with or without 110 nM and 150 nM JNC-1043, respectively, and exposed to 3 Gy IR. After incubation for 48 h, the cells were treated with 5 µM MitoSOX  for 20 min and trypsinized. The cells were harvested by centrifugation and resuspended with ice-cold PBS. A FACSort flow cytometer  was used for the detection and analysis of mitochondrial ROS . These experiments were repeated in triplicate.HCT116 and DLD-1 cells (4 × 105 cells/60-mm dish) were incubated with or without 110 nM and 150 nM JNC-1043, respectively, and exposed to 3 Gy IR. After incubation for 48 h, the cells were treated with 10 µM JC-1  for 20 min and trypsinized. The cells were harvested by centrifugation and resuspended with ice-cold PBS. A FACSort flow cytometer (Becton Dickinson) was used for the detection and analysis of mitochondrial membrane potential . These experiments were repeated in triplicate.HCT116 and DLD-1 cells . The mice were divided into four groups (n = 3 mice/group): control (mock-treated), IR treatment only (IR only), JNC-1043 treatment only (JNC-1043 only), and co-treatment with JNC-1043 and IR (IR+JNC-1043). When the xenografts reached 100−120 mm3 in size, 40 mg/kg JNC-1043 in DMSO was intratumorally injected into mice of the JNC-1043-only and IR+JNC-1043 groups. The IR-only and control groups were intratumorally injected with an equal volume of DMSO (vehicle). At 6 h after the injection, mice in the IR-only and IR+JNC-1043 groups were locally irradiated with 1.5 Gy using a 60Co γ-ray source . Irradiation was repeated three times at 3- to 4-day intervals for a total of 12 days. Mice were sacrificed 30 days after the start of the experiment. Extracted tumors were fixed with formaldehyde, paraffin-embedded, and sliced. TUNEL assay for the detection of dUTP nicks was performed using Super Bio Chips . The TUNEL-positive cells were measured for each group, and the ratios of TUNEL-positive to total cells in each image were measured with the Image J software . Percentages were calculated relative to the value obtained from the control group.All animal experiments were performed using approved protocols of the Institutional Animal Care and Use Committee (KIRAMS 2021-0017). The in vivo radiosensitization effects of JNC-1043 were measured using a xenograft model constructed by subcutaneously injecting 1 × 10p-values < 0.05 were considered significant, and the individual p-values are denoted in the figures by asterisks . The number above each point or bar represents the mean results from three independent experiments and the error bars signify the standard deviation (SD).Data were analyzed using GraphPad Prism Ver. 5 . The significance of differences between experimental groups was determined using two-way ANOVA and Student’s t-test. Taken together, our present findings reveal the anticancer and radiosensitizer effects of JNC-1043. Our detailed intracellular mechanistic studies and the lower toxicity of JNC-1043 against normal cells demonstrate that JNC-1043 might be a candidate radiosensitizer for inducing apoptotic cell death via DNA damage/cell cycle arrest/ROS-triggered apoptosis in CRC cells.


To determine the impact of online mentoring sessions on the students during the pandemic time.The cross-sectional descriptive study was conducted at Bahria University Medical and Dental College, Karachi. The total study duration was 5 months from March 2021 to July 2021. Quantitative research design was used. Categorical data was scored on a three point Likert scale . Frequencies and percentages were calculated to determine the impact of online mentoring.nd year MBBS students were of the opinion that online mentoring was helpful as compared to 58% 1st year and 50% 3rd year students. Students were anxious while sharing their issues online. A total of 61.66% were eager to have classes on campus as compared to online as learning difficulties were felt in 70%, 77% and 81% of 1st, 2nd and 3rd year classes respectively. Of the 1st year 39%, 2nd year 46% and 3rd year 32% showed relief after the mentoring session but were in favor of face to face sessions. Technical issues were faced by 54% 1st year, 66% 2nd year and 64% 3rd year students.Sixty two percent of 2The study suggested that students were overall satisfied with the online mentoring sessions. They do have certain apprehensions like privacy and confidentiality issues but on the whole, they considered this medium as being a powerful one in times of the pandemic. The concept of mentoring dates back to approximately 3,000 years and it takes its origin from Ancient Greece. The idea first arose when Odysseus entrusted one of his close companions, Athena, with accompanying his son, Telemachus, during the Trojan War. Predominantly, mentoring is defined as a one on one relationship where an expert guides an inexperienced individual to help them gain professional, academic and personal maturity.1Due to the various unforeseen challenges in the Health Care Sector, there is an urgent need for medical professionals who can offer high quality services. As a result, there is an exponentially increasing pressure on the medical educators to produce doctors who are professional, highly analytical, social and have advanced decision making skills. Therefore, it is crucial for budding medical professionals to be assigned a mentor to help them navigate not only through medical school but also through the complications of hospital life.3Research in academia shows a correlation between mentoring and improved personal and professional development of students. A study was conducted in which students were divided into an experimental group that was given a mentor and a control group that received no mentorship. The results of the research showed that students who were under the guidance of a mentor showed higher retention rates as compared to non-mentored students with comparable pre-enrollment credentials.5Meta- analysis of existing research has evaluated the usefulness of mentoring in academic and workplace environments. Ingersoll and Strong analyzed 15 empirical studies examining the relation between mentoring and three factors among new teachers: retention, classroom practices and student success. Most of the studies showed a positive relation between mentoring and the three factors.6Mentoring in medical education has been around for several decades and has proven to be an essential part of the training. However, since the outbreak of COVID-19 there has been a dramatic shift in the way mentoring is executed as compared to traditional practices. The concept of online-mentoring has existed for several years but it has recently gained popularity due to the social distancing required to prevent the spread of COVID-19.7Online mentoring overcomes the barriers of physical distance by allowing mentors and mentees to interact over the internet. This has proven to be more convenient for both parties as they can schedule the meetings at any time that is suitable for them without being limited by the set time they are present at the institution they belong to. One of the major advantages of online mentoring is that it focuses more on the needs of the mentee as compared to conventional forms of mentoring.10The organizations should devise strategies to cope up with the challenges of the new environment (COVID-19) faced by the university students, so that students could be able to find their ways out of the hurdles. Therefore there is a need to carry on with the mentoring sessions through virtual medium and to continue with the fruitful activity in the computer-generated world. Keeping in mind the need, the present study was planned to determine the impact of online mentoring sessions on the students during the pandemic time. The study can aid the organization finding the loop holes in the online teaching methods, assist the institutes to design curriculum according to the new normal and also help to devise a roadmap according to which online teaching environment can be made students friendly.The cross sectional descriptive study was conducted at Bahria University Medical & Dental College, Karachi. The approval was taken from the Ethical Review Committee prior to the start of the research. The ERC approval letter number was ERC 80/2021. The sample size was 450; all three years students  were included in the research as they all were part of online mentoring program. The sampling technique was a non-probability convenience. The total study duration was from May 2021 to July 2021.The inclusion criterion was to include male and female MBBS students of Bahria University Medical & Dental College who were part of online mentoring program, Karachi. Students other than MBBS and those who were not part of online mentoring program were excluded from the research. The research project used quantitative research design. Categorical data was scored on a three point Likert scale . The questionnaire was developed using literature search mentioning medical students’ and its impact on mentees.nd year MBBS students were of the opinion that online mentoring was helpful as compared to 58% 1st year and 50% 3rd year students. All years of MBBS students in the study group were equally anxious while sharing their issues online, however 38% (1st year), 53% (2nd year) and 50% (3rd year) were sure that no recording was being done. Majority of the students showed satisfaction towards time spent towards the activity of mentoring (63%). Fifty-three percent of 1st year students, 55% of 2nd year and 50% of 3rd year agreed that mentoring was helpful. In peer mentoring it was determined that 87% of 2nd year students were facing similar issues as compared to 63% and 69% of 1st year & 3rd year students. Majority agreed that the attendance issue was addressed along with coverage of curriculum. The students of all years i.e. 59%, 68%, 58% of 1st, 2nd and 3rd year respectively agreed that exam strategies were discussed in detail. A total of 61.66% were eager to have classes on campus as compared to online as learning difficulties were felt in 70%, 77% and 81% of 1st, 2nd and 3rd year classes respectively. The students felt that individual mentoring was very helpful as they wanted to discuss personal issues and agreed that during the pandemic these sessions were the need of the hour. However, 30%, 28%, 37% of the respective years disagreed that online mentoring should be a regular feature and that some level of discomfort was felt which caused apprehension that someone who is not in view of the camera may be listening. Of the 1st year 39%, 2nd year 46% and 3rd year 32% showed relief after the mentoring session but disagreed that mentoring online was better than face to face mentoring. They were in favor of face to face sessions. It was seen that 54% 1st year, 66% 2nd year and 64% 3rd year students faced technical issues during the mentoring sessions.Sixty two percent of 2The emergence of COVID-19 pandemic has greatly affected the academic institutions and their strategies related to students.16The current study showed that students of all the three batches considered mentoring as being helpful. Ali et al reported analogous findings.13The present study suggested that students while discussing their issues online had apprehension as they were uncomfortable in sharing their personal issues in an online format. They had reservations as they were not face to face with their mentors and that’s why they had apprehensions that their information could be leaked to others. Parallel information regarding Zoom bombing was shared by Mc-Reynolds et al in their study. It was documented that there are many challenges associated with sharing of information on the online platform since internet meeting rooms are not reliable and lack privacy; the information shared by the students could be in reach of others they do not trust.17The current study documented that students felt satisfied towards time spent during the activity of mentoring. Temini et al. stated that online mentoring is the need of the day. In the age of uncertainty, adaptations like online mentoring serve as a shelter for the undergraduates to combat their inner dissatisfaction and to voice their grievances.22The present study showed that students were satisfied regarding the coverage of the curriculum. Another study also emphasized that virtual mentoring was used as a medium for the students to discuss their academic issues with their mentees. Although the mentees were not very satisfied by the e-mentoring process, it still fulfilled its purpose during the pandemic.17The current study implied that the scheduled online mentoring sessions were fruitful as they were a platform through which students were able to vent out their stress, be it academics related or personal. An Iranian study published parallel findings and documented that these days students have to cope with mental and emotional issues, including tension, nervousness and fear, which may necessitate significant psychological support. Therefore, it is important that the medical schools not only care about student mental health, but also implement strategies to support students’ understanding of crisis management; self-mental care and other important measures in order to strengthen the students’ coping skills and psychological preparedness.23It also showed that during online mentoring some level of discomfort was felt by the mentees and they were also apprehensive that someone who is not in view of the camera may be listening. Another study mentioned comparable results stating that the virtual mentoring system lacks privacy component which is the backbone in terms of trust building between the mentor and mentee.17The current study stated that the majority of the students believed that on-campus mentoring is better than the online meetings. They were in favor of face to face sessions. Comparable results were published in an article by Ziboid et al. which stated that although the mentoring sessions were fruitful to some extent but they cannot take the place of face to face sessions.13Kumar et al have identified several areas that can be taken under consideration while online mentoring is in process. Other than the mentors and mentees’ interaction and collaboration, institutional support is considered to be of prime importance.25As with other platforms, visual mentoring is also associated with certain difficulties and hurdles. Nevertheless, the platform is being used by many all over the globe as a medium to interact in periods of uncertainty like COVID-19 pandemic. A study conducted at Konkuk University has stressed the importance of certain strategies that could be helpful in carrying online mentoring sessions. Training of mentors, along with structured online mentoring activities and better communication system are the need of the day.14Our mentoring program which was started since the inception of the institute is one of its kind as it has proven itself to be useful over the years as suggested by the researches addressing its impact. During the pandemic, the online mentoring was introduced which was novel not only to our institute but also to our country. The online mentoring program had some limitations also. As the online teaching has some associated glitches, same is the case with online mentoring. During the sessions, some of the mentors and mentees had faced connectivity issues.Online mentoring is the need of the day but since it is a novel method there is a need to conduct future research to compare the outcomes of e-mentoring to face to face mentoring sessions. The mentors should also be included in the study projects so that their opinion regarding the web based mentoring sessions can also be included.The study suggested that students were overall satisfied with the online mentoring sessions. They do have certain apprehensions like privacy and confidentiality issues but on the whole, they considered this medium as being a powerful one in times of the pandemic.AU: Conceived the idea and designed the study She was responsible for the overall accuracy and integrity of the work, manuscript writing.MI: Literature search, manuscript writingQJ: Manuscript writing and revision, Data collectionJT: Data analysis


As a result, eukaryotic cells have developed a wide variety of mechanisms for controlling Ca2+ influx and efflux across the plasma membrane as well as Ca2+ release and uptake from intracellular stores. The STIM and Orai protein families comprising of STIM1, STIM2, Orai1, Orai2, and Orai3, are evolutionarily highly conserved proteins that are core components of all mammalian Ca2+ signaling systems. STIM1 and Orai1 are considered key players in the regulation of Store Operated Calcium Entry (SOCE), where release of Ca2+ from intracellular stores such as the Endoplasmic/Sarcoplasmic reticulum (ER/SR) triggers Ca2+ influx across the plasma membrane. SOCE, which has been widely characterized in non-excitable cells, plays a central role in Ca2+-dependent transcriptional regulation. In addition to their role in Ca2+ signaling, STIM1 and Orai1 have been shown to contribute to the regulation of metabolism and mitochondrial function. STIM and Orai proteins are also subject to redox modifications, which influence their activities. Considering their ubiquitous expression, there has been increasing interest in the roles of STIM and Orai proteins in excitable cells such as neurons and myocytes. While controversy remains as to the importance of SOCE in excitable cells, STIM1 and Orai1 are essential for cellular homeostasis and their disruption is linked to various diseases associated with aging such as cardiovascular disease and neurodegeneration. The recent identification of splice variants for most STIM and Orai isoforms while complicating our understanding of their function, may also provide insight into some of the current contradictions on their roles. Therefore, the goal of this review is to describe our current understanding of the molecular regulation of STIM and Orai proteins and their roles in normal physiology and diseases of aging, with a particular focus on heart disease and neurodegeneration.Tight spatiotemporal regulation of intracellular Ca Homologs of human plasma membrane Ca2+ channels have been found in organisms as distant as the protozoan Naegleria gruberi demonstrating that regulation of extracellular influx as a Ca2+ signaling mechanism has existed for over 1 billion years .It is universally recognized that tight spatiotemporal regulation of cytoplasmic Caon years . Eukaryo2+ from intracellular stores to subsequent influx of extracellular Ca2+ (2+ entry (SOCE), became increasingly well characterized over the following two decades -mediated activation of the IP3 receptor (IP3R). This was followed by the activation of a highly selective non-voltage gated, Ca2+ channel in the plasma membrane. In contrast to IP3-induced release of Ca2+ from intracellular stores, which results in transient increases in Ca2+ of the order of seconds or less, SOCE can remain active for minutes or longer  and nuclear factor kappa B (NF-κB)  . Howeverted SOCE .Since their identification, STIM1 and Orai1 have been widely accepted as being essential components of SOCE. As discussed below, the detailed molecular interactions between the two proteins required to facilitate SOCE have been elucidated; however, the role of their homologs STIM2, Orai2, and Orai3 remain poorly understood. To complicate matters further, several variants of STIM1, STIM2, Orai1, and Orai2 have also been identified . While tGOK that was predicted to encode a protein that contained a transmembrane helix  domain  connects the ER and cytosolic regions of STIM1. In the Ca2+ bound state, the EF-SAM domains on STIM1 are kept apart to prevent spontaneous activation.The domain structure of mammalian STIM1  is charaf the ER , is critve STIM1 . The cEF) domain . The com2+ levels, Huang et al., demonstrated that the cytoplasmic carboxy-terminal domain of STIM1 was sufficient to activate Ca2+ entry in the absence of store depletion (2+-release-activated Ca2+ (CRAC) activation domain (CAD) were identified in the CC domains , requires the ID domain although this domain itself is not the primary Ca2+ sensor for CDI of SOCE  the CC1× 10−4 M . The CC1ed state . In respve state . The imp+ influx . The sub of SOCE . Of noteing SOCE . The CC3ing SOCE ; the reging SOCE . The resepletion . While tepletion . Another of SOCE . Full CD for CDI ; however for CDI . Calmodund Orai1 .et al., identified STIM1 as an microtubule associated protein RP/EB family member 1 (EB1) interacting protein; however, the function of this interaction was unclear as loss of EB1 had no effect on SOCE (2+ overload (2+ depletion leads to phosphorylation of Tyr361 in the SOAR/CAD domain by proline rich kinase 2 (Pyk2) thereby facilitating SOCE  domain and the Proline/Serine rich region (P/S). In 2008 Grigoriev  on SOCE . More reoverload . EB1 is overload . STIM1 hoverload . Phosphooverload . Of noteoverload . On the overload . To dateoverload . Phosphoing SOCE . AMPK phing SOCE . PKA phoing SOCE  and dualing SOCE . A numbeing SOCE ; however2+ entry independent of Ca2+ store levels  than STIM1, which also helps facilitate STIM2 clustering with minimal changes in ER Ca2+ levels , most of which are in the cytosolic C-terminal region ; howeverted SOCE . In contunctions . Under b2+ entry .In 2015, there were two reports describing a novel STIM2 splice variant, STIM2β , which antagonized STIM1-Orai1 mediated SOCE . In diff2+ signaling defects in T-cells from a patient with severe combined immunodeficiency (SCID) . Two additional studies published in 2006 confirmed the essential role of Orai1 in SOCE and correctly predicted that it had 4 transmembrane domains with both C- and N-terminal regions in the cytosol  . Subsequy (SCID) , illustrentified ; a singl cytosol . While O cytosol . Howeverto occur .2 binding in the same region as AC8 has been reported to enhance Orai1-STIM1 interactions (residues 28–33) (2+ entry (2+ dependent inhibition (CDI) of SOCE (The domain structure of Orai1  consistss 28–33) . Adjacen2+ entry ; however2+ entry . The ETO of SOCE ; however of SOCE .et al., found that cholesterol depletion increased SOCE and identified a cholesterol binding motif in the region of the ETON domain that interacts with calmodulin (The interaction of cholesterol with Orai1 is complex with reports that it inhibits its activity and decreases SOCE  on the olmodulin . Others lmodulin . The rollmodulin . A caveolmodulin . Howeverlmodulin .2+ accumulating region (CAR), formed by aspartate residues, which increase local Ca2+ concentrations facilitating Ca2+ entry even when extracellular Ca2+ concentrations are low  contains a Ca are low . The TM22+ entry  and inte2+ entry . A cyste2+ entry . The sec the CAR . Loop3 oing SOCE . TM4 is ing SOCE . Residueing SOCE .2+ channels and are associated with various diseases including cancer  channels, suggesting the possibility of physiologically distinct roles for the two isoforms  channels (Icrac), it has been shown that TRP channel mediated Ca2+ entry is not dependent on either STIM or Orai proteins  or its metabolite leukotriene and is dependent on both STIM and Orai proteins (2+ (ARC) channel is responsible for a highly selective Ca2+ current, Iarc, which has distinct physiological roles from SOCE  and mutations in this region prevent STIM1 activation of Orai1 . The Ca2 cytosol . The pretivation . While ttivation . It is itivation .2+ channels are regulated by feedback inhibition by Ca2+, a process known as CDI; this is also true for Orai1-mediated SOCE. As noted earlier, STIM1 contains an inhibitory domain (IDSTIM) that is essential for CDI; surprisingly however, it is does not appear to be the primary Ca2+ sensor responsible for initiating CDI. Calmodulin, which binds to the N-terminal of Orai1, was thought to be the CDI sensor, but this turned out not to be the case , which induced CDI   . Severalll types ; howeverll types . While t protein ; consequ2+ entry . They de2+ entry . In subs2+ entry ; 2009.2+ channel by leukotrieneC4 (LTC4) that was also STIM1-dependent was reported to have very similar biophysical characteristics as the ARC channel (4 regulated Ca2+ (LRC) channel activation  and ryanodine receptor (RyR). The abnormal mitochondrial activity was the result of changes in the expression of several mitochondrial proteins which include mitochondrial transcription factor A (Tfam), peroxisome proliferator-activated receptor gamma coactivator (PGC1α), nuclear respiratory factor 1 (Nrf1), Sirtuin 1 (Sirt1), and mitochondrial fission 1 protein (Fis1) and an increase in apoptosis  and increased Dynamin related protein 1 (Drp-1) expression . Mitochoet al., , also shepletion . In suppepletion . Silva-Rpoptosis . These s2+ uptake is essential for regulating STIM1, Orai1, and SOCE (2+ uniporter (MCU) or uncoupling protein 2 (UCP2) resulted in slowed STIM1 oligomerization and reduced SOCE (3R) in lymphocytes has been shown to not only result in reductions in mitochondrial proteins such as MCU, but also these lymphocytes had altered mitochondrial metabolism dependent on cAMP response element-binding protein (CREB)  . Also, tpression  through pression , which spression .2+ uptake  led to significant Ca2+ overload and mitochondrial dysfunction, which was attenuated by an SOC inhibitor or a siRNA knockdown of STIM1 , Yan and colleagues found that ethanol increased intracellular Cat manner , and wast manner . In a mot manner . In neurof STIM1 . In a trll death , suggestet al. identified, that Orai1 was the main source for Ca2+ influx in prostate cancer cells  in a calcium/calmodulin-dependent protein kinase kinase (CAMKK) dependent manner. On the other hand, Medina et al., concluded that lysosomal calcium was responsible for activation of the Ca2+ dependent phosphatase calcineurin, the subsequent dephosphorylation of Transcription factor EB (TFEB) a master transcription factor for autophagy leading to its nuclear localization  and subsequent covalent attachment of NO to cysteine (Cys) thiols on proteins. Interestingly, it has been shown that neuronal nitric oxide synthase (nNOS), which generates NO, is expressed in the SR , where Set al., , that STses SOCE . In the handling . One wouet al.,  treatment inhibits Orai3-mediated SOCE but not Orai1 and Orai2-mediated SOCE. The authors determined that this difference was due to the presence of Cys226 and Cys232 in Orai3 both of which were absent in other Orai isoforms and were shown to mediate the reduction in SOCE in response to hydrogen sulfide (H2S)  . It is pIt is increasingly clear that STIM-Orai signaling is an important regulator of cardiovascular physiology and homeostasis as well as playing a significant role in cardiovascular disease processes, such as hypertrophy, ischemia/reperfusion (I/R), and heart failure. In this section, we will discuss the current knowledge concerning the canonical and non-canonical functions of STIM and Orai proteins in the heart during instances of cardiac pathology and during normal physiology.2+ signaling associated with cardiac hypertrophy and heart failure  current). Like the studies in KO mice, the responses to hypertrophic stimuli in these mice were significantly exacerbated. Given that it appears too much or too little cardiomyocyte STIM1 results in similar cardiovascular phenotypes, it is likely that STIM1 levels are tightly regulated and play significant roles in the precipitation of cardiovascular diseases.In addition, changes in STIM1, STIM1L, and Orai1 have been observed in a pulmonary hypertension model of monocrotaline-induced RV-hypertrophy in which STIM1 expression was reduced but the expression of both STIM1L and Orai1 were increased and this was associated with significant changes in Cahandling . Examinahandling  showed t2+ overload, in addition to reduced mPTP opening and reduced ROS generation  where it regulates SAN function through modulation of SOCE and LTCC and the regulation of heart rate and cholinergic responsiveness . The samet al., , have shthe LTCC . Recentlthe LTCC . It is let al. who used cardiomyocyte specific Orai1 mutant mice with a mutation in the Orai1 pore and found that inhibition of Orai1 activity during TAC resulted in preserved cardiac function and preserved Ca2+ handling  in endothelial cells where STIM1, STIM2, Orai1, Orai2, and Orai3 are all increased in addition to an increase in SOCE through calcineurin/NFAT-dependent mechanisms , and fibroblasts, where they have different effects on the functionality of each cell type. For example, it has been shown that Orai1-mediated SOCE is important for endothelial cell function since endothelial cells with Orai1 knockdown or inhibition have been shown to have reduced tube formation and migration . In thischanisms . On the chanisms . These set al., (2+ channels (LTCC) and a reciprocal upregulation of STIM1 and Orai1  . Althoug2+ homeostasis and intracellular signaling in the development of Alzheimer’s disease  gene. It is well acknowledged that overstimulation of glutamate receptors as well as the downstream effects on Ca disease . Studies disease . Of note disease . Upregul of SOCE . Efforts of SOCE . Importaine loss . Ongoingine loss . The samine loss . It is i2+ homeostasis play a large part in the process. Two studies by Singh and colleagues demonstrated the functional role of SOCE in dopaminergic neurons in the substantia nigra. Interestingly, however, they reported that SOCE in these neurons were mediated by STIM1 and TRPC1 rather than Orai1 (2+ current as well as the open probability of Cav1.3 were reduced (et al. found that in fibroblasts from idiopathic Parkinson’s disease patients SOCE was impaired, but there was no change in STIM1 or Orai1 protein levels; however, the levels of PLA2g6 a Ca2+ independent phospholipase, which activates SOCE were significantly reduced (Parkinson’s disease is currently the second most common neurodegenerative disease affecting more than 10 million people worldwide. Although currently the mechanisms for the loss of dopaminergic neurons in the substantia nigra remains unclear, it has been shown that mitochondrial dysfunction and alterations in Caan Orai1 . Dopamin reduced . They al reduced . Zhou et reduced . Interes reduced . This stCurrently, most studies on STIM/Orai in Huntington’s and Parkinson’s disease have originated from only very few research groups and clearly much remains to be learned about the role of STIM and Orai proteins these diseases. For example, it remains unclear why STIM2 appears to play a predominant role in Huntington’s, whereas the STIM1-TRPC1 interactions have been identified in models of Parkinson’s disease. Moreover, it is surprising that there have been no reports of potential roles of Orai proteins in these diseases, even though STIM-Orai interactions are more commonly recognized as mediating SOCE. Future studies on iPSCs from Huntington’s and Parkinson’s disease patients represent a potentially valuable area of future investigation.2+ homeostasis is a hallmark of many age-related diseases such as cardiovascular and neurodegenerative diseases, the effect of aging itself on Ca2+ homeostasis has not received extensive investigation (2+ signaling have been linked to dysfunction in aged lymphocytes. However, although the main Ca2+ signaling pathway in lymphocytes is mediated by STIM-activated Orai channels, little was known about the role during lymphocyte aging. Angenendt et al., recently reported reduced SOCE in quiescent and activated lymphocytes from 18 to 24-month-old mice compared to 3–6-month-old mice (ex vivo long term cell culture model designed to mimic aspects of aging, such as oxidative stress and DNA damage, the amplitude of SOCE was unchanged in aged lymphocytes although the Ca2+ dynamics following stimulation were altered (2+ signaling were potentially a consequence of increased thiol oxidation of STIM1.While dysregulation in Catigation . Progeritigation . Overexptigation , suggesttigation . Age-relold mice . This re altered . The aut2+ homeostasis and signaling in both VSMC as well as endothelial cells (2+ homeostasis and growth (Aging is known to lead to vascular dysfunction in part by altered Caal cells . Howeverd growth , little d growth . Interesd growth . In a mud growth . Of noted growth . DespiteIn skeletal muscle there have been some contradictory reports, where SOCE was maintained in muscles from aged mice despite reduced STIM1 levels  while in2+ signaling. Given the relative paucity of studies examining how STIM and Orai proteins change during normal healthy aging it is premature to draw any firm conclusions. Nevertheless, the data that is available suggests that this could be a rich and important area for future research.While there is some evidence that STIM1/Orai1 mediated SOCE is affected during normal aging the data are somewhat contradictory and gain or loss of function with aging appears to be tissue specific. It is also unknown whether aging leads to changes in the stoichiometry of STIM/Orai oligomers and if that would lead to changes in Ca2+ entry into the cell have been explored in detail. While the canonical roles of STIM1 and Orai1 are broadly accepted in non-excitable cells, arguably there is much less consensus regarding the roles of their isoforms, STIM2, Orai2 and 3; moreover, the identification of an increasing number of splice variants further complicates the picture. In excitable cells, such as neurons and cardiomyocytes, the function of all STIM and Orai isoforms is much less clear; however, the use of cell-specific knockout or mutant models have clearly shown that they play an essential role in maintaining cellular homeostasis. The observations that STIM and Orai isoforms are subject to redox regulation has broad implications into how they might contribute to cellular dysfunction in the setting of oxidative stress.In the years since STIM1 and Orai1 were identified as the essential mediators of SOCE, the molecular mechanisms by which they facilitate Ca2+ signaling pathway in all eukaryotes, numerous gaps in our knowledge remain. Such gaps include the understanding of the role of many post-translational modifications on the function (2+ signaling responses (2+ signaling observed across different tissues and cell types. A better consensus as to the specific stoichiometry of STIM and Orai isoforms will be critical in helping to understand their role in aging and age-related diseases.Despite the rapid growth in our understanding of the role of STIM and Orai and the widely accepted notion that they are core elements of a highly evolutionarily conserved Cafunction . In addifunction . While tesponses  are intr2+ meditated transcription pathways. In addition to metabolic and mitochondrial regulation and cell survival, a protein complex including STIM1 is responsible for trafficking of early and late endosomes (As we have described here, STIM and Orai proteins are increasing recognized as regulating cellular functions beyond the classic Candosomes , consistndosomes . Clearly