Given that both cytosolic and mitochondrial Ca2+ dynamics strongly interplay with energy metabolism and nutrient-sensitive pathways, both of them involved in the aging process, we have studied the effect of SERCA inhibitors on lifespan in C. elegans. We have used thapsigargin and 2,5-Di-tert-butylhydroquinone  as SERCA inhibitors, and the inactive analog 2,6-Di-tert-butylhydroquinone  as a control for 2,5-BHQ. Every drug was administered to the worms either directly in the agar or via an inclusion compound with γ-cyclodextrin. The results show that 2,6-BHQ produced a small but significant increase in survival, perhaps because of its antioxidant properties. However, 2,5-BHQ produced in all the conditions a much higher increase in lifespan, and the potent and specific SERCA inhibitor thapsigargin also extended the lifespan. The effects of 2,5-BHQ and thapsigargin had a bell-shaped concentration dependence, with a maximum effect at a certain dose and smaller or even toxic effects at higher concentrations. Our data show therefore that submaximal inhibition of SERCA pumps has a pro-longevity effect, suggesting that Ca2+ signaling plays an important role in the aging process and that it could be a promising novel target pathway to act on aging.The sarco-endoplasmic reticulum Ca In recent years, evidence has accumulated suggesting a key role in aging for the so-called nutrient-sensitive signaling pathways: the insulin/IGF-1 pathway, the AMP kinase pathway (AMPK), the mTOR kinase pathway, and the sirtuin pathway . These p2+ signaling, but the specific mechanisms are not known in detail . In turn, AMPK inhibits mTORC1, so that this pathway provides an indirect link between the Ca2+ signal and mTOR. According to this mechanism, cytosolic [Ca2+] ([Ca2+]c) increase would inhibit mTOR, thus activating processes such as autophagy. In fact, there is a large body of evidence showing that [Ca2+]c signals from different sources can trigger autophagy by this “canonical” CaMKKβ-AMPK-mTORC1 pathway  necessary for the activation of mTOR , through an increase in the expression of the STIM1/Orai1 proteins responsible for this pathway , which releases Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR) and is able to transfer Ca2+ from the ER to the mitochondria through close contacts that connect both organelles. These contacts, called Mitochondrial Associated ER Membranes (MAMs), contain both IP3R and mitochondrial Ca2+ uniporters (MCU), the main channel responsible for mitochondrial Ca2+ uptake. In addition, it has been reported that mTORC2 is also an important component of MAMs, able to activate AKT kinase, which may then phosphorylate and inhibit IP3R  appears to be closely related to the activity of the nutrient sensitive pathways. In this work we have studied the effect of the inhibition of the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) on survival in C. elegans. The rationale of this treatment is that submaximal SERCA inhibition should decrease [Ca2+]ER, leading to a reduced Ca2+ release from the ER and a smaller Ca2+ transfer from ER to mitochondria. This effect should in theory activate AMPK, inhibit mTOR and have a pro-longevity effect. Our data show in fact that SERCA inhibitors induced a significant increase in C. elegans lifespan, suggesting that Ca2+ homeostasis plays an important role in the mechanisms responsible for aging and survival.In conclusion, there is little doubt that ER measured in HeLa cells expressing ER-targeted aequorin. Addition of 2,5-BHQ rapidly induced a decrease in [Ca2+]ER due to the inhibition of the SERCA pump. In contrast, addition of 2,6-BHQ hardly produced any effect.As a control for the activity of 2,5-BHQ, we have used 2,6-BHQ, a compound which is inactive on the SERCA (see E. coli OP50. Young adults (day 1) were then transferred to these plates to start the lifespan assay. In this way, worms ingest the inclusion compound by the pharynx together with the food, and the active 2,5-BHQ is then released directly into the digestive tube. Table 1 shows the results of a series of lifespan assays performed with three concentrations of either 2,5-BHQ or 2,6-BHQ. Figure 1 shows representative lifespan assays performed in each of the conditions . Although 2,6-BHQ produced small increases in survival, the pro-survival effect was much larger in the case of 2,5-BHQ, and its effect was concentration-dependent. The effect was maximum when 50 μg of the inclusion compound was added to the plate, and for this concentration all the lifespan assays were highly significant and produced increases in survival between 8.2 and 21.3% (mean 13.9 ± 2.4%). The effects on lifespan were smaller at concentrations of 2,5-BHQ below and above 50 μg. In contrast, for 2,6-BHQ, all the concentrations used produced smaller and similar increases in survival, and the effect showed a much higher variability.Because of the poor water solubility of 2,5-BHQ, we decided to use inclusion compounds for drug administration to the worms. 2,5-BHQ was enclosed in a chemical cover of γ-cyclodextrin, forming an inclusion compound , which wC. elegans lifespan of similar magnitude as that induced by 50 μg of the compound  showing that the effect is not dependent on the method of administration of the drug. Panel H shows a comparison of the mean increase in survival obtained in every condition.In spite of the low water solubility of BHQ, we have also tested the effect of two concentrations of 2,5-BHQ directly dissolved in the NGM agar after strong stirring, 250 and 350 μM. Both produced also an increase in the E. coli (OP50), and young adults (day 1) were then transferred to these plates to start the lifespan assay.We have then studied the effect of thapsigargin, a very potent and specific irreversible inhibitor of SERCA pumps, on the lifespan of the worms. For drug administration, we have used two different mechanisms, either inclusion compounds or direct addition to the plates. In the first method, the inclusion compound with thapsigargin was prepared and used as mentioned above. In the second method, as previously described , the druTable 2 shows the results of a series of lifespan assays performed with three concentrations of γ-cyclodextrin enclosed thapsigargin  and two concentrations of thapsigargin added directly to the plates (10 and 50 nM). Representative lifespan assays performed in each of the conditions are shown in Figures E, together with the mean increase in survival obtained from the different assays made at each thapsigargin concentration . Thapsigargin produced also an increase in survival which was dependent on the concentration. The effect was maximum at 1 μg of the inclusion compound (mean increase 8.0 ± 0.3%), and was smaller at concentrations below and above that. Also, when the compound was added directly to the plates, the increase in lifespan was obtained at 10 nM (mean increase 9.6 ± 2.1%), but disappeared at 50 nM.The same effects were obtained with both drug administration methods. eat-2 mutants. These mutants have a reduced rate of pharynx pumping and an increased lifespan, which has been shown to be due to caloric restriction  mutants. Representative plots of assays made at each of the conditions studied are shown in Figure 3. The results show that SERCA inhibitors increased also the lifespan of eat-2 (ad1113) worms with a similar magnitude to that found in wild-type worms. Therefore, this suggests that caloric restriction is not involved in the mechanism of the lifespan increase induced by these compounds.To exclude that the mechanism of the increased lifespan in the presence of the SERCA inhibitors could be related to caloric restriction, we have studied the effects of the SERCA inhibitors on the survival of triction . Table 32+ dynamics is a key controller of cellular metabolism. The rate of mitochondrial ATP synthesis depends on the [Ca2+] inside mitochondria ([Ca2+]M), which stimulates several key dehydrogenases responsible for NADH production  has been attributed to this effect  mutant worms, a model for caloric restriction because of the reduced rate of pharynx pumping (2+ signaling. The SERCA pump plays a key role in shaping the intracellular [Ca2+] transients. Its activity determines the level of filling with Ca2+ of the ER/SR, which is one of the factors conditioning the rate of Ca2+ release through either IP3R or ryanodine receptors. In addition, ER/SR Ca2+ reuptake after stimulation also depends on SERCA activity. Our hypothesis is that a decrease in SERCA activity would reduce [Ca2+]ER, with the subsequent decrease in Ca2+ release, Ca2+ transfer from ER to mitochondria and metabolic activity. It is remarkable that SERCA is one of the proteins that undergo a largest decrease in concentration during aging in C. elegans. In fact, sca-1 decreases in abundance nearly 10-fold from day 4 to day 10 of worm life (C. elegans, because a marked decrease in SERCA2a mRNA and activity during aging has also been reported in mammals (2+ signaling is negative for survival during aging. Young individuals have a higher SERCA content, and this may be optimal for rapid response to external stimuli. Older individuals have instead a much smaller SERCA content, and this may dampen Ca2+ signaling and favor survival. Submaximal SERCA inhibition may extend lifespan in this way. Further work will be necessary to clarify the molecular mechanism involved in the effect of SERCA inhibitors. Measuring the changes in [Ca2+]ER along the life of the worm, although challenging from a experimental point of view, would be a exciting approach. In addition, the decrease in [Ca2+]ER should produce changes in the cytosolic Ca2+ dynamics, which could be detected in live treated or untreated worms. Using other mutants with alterations in nutrient-sensitive pathways or with ER stress may also provide clues on the mechanism of the pro-survival effect of SERCA inhibitors.The molecular mechanisms linking SERCA inhibition and lifespan extension still await clarification. We have seen that the SERCA pump inhibitors increased also with a similar magnitude the lifespan of  pumping . This suorm life . This ph mammals . This deC. elegans. This indicates that Ca2+ signaling plays a role in the aging process and that reduction in [Ca2+]ER and ER-Ca2+ release has a pro-longevity effect. Our data also suggest that Ca2+ signaling could be a promising novel target pathway to act on aging.In summary, we have shown here that partial inhibition of SERCA pumps increases the lifespan in The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.MM and JA designed the project. PG-C performed most of the lifespan experiments. JA-d-V and PA-I joined in performing some of them. JA wrote the manuscript and RF and MM helped in discussing and editing the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


While such crystal internalization produces reactive oxygen species (ROS), cell membrane damage, and apoptosis; the upstream signaling events involving dysregulation of intracellular Ca2+ homeostasis and ER stress, remain largely unknown. We have recently described a transepithelial Ca2+ transport pathway regulated by receptor-operated Ca2+ entry (ROCE) in PT cells. Therefore, we examined the pathophysiological consequence of internalization of stone-forming calcium crystals such as calcium phosphate (CaP), calcium oxalate (CaOx), and CaP + CaOx (mixed) crystals on the regulation of intracellular Ca2+ signaling by measuring dynamic changes in Ca2+ transients in HK2, human PT cells, using pharmacological and siRNA inhibitors. The subsequent effect on ER stress was measured by changes in ER morphology, ER stress-related gene expression, endogenous ROS production, apoptosis, and necrosis. Interestingly, our data show that crystal internalization induced G-protein-coupled receptor-mediated sustained rise in intracellular Ca2+ concentration ([Ca2+]i) via store-operated Ca2+ entry (SOCE); suggesting that the mode of Ca2+ entry switches from ROCE to SOCE following crystal internalization. We found that SOCE components—stromal interacting molecules 1 and 2  and ORAI3 (SOCE) channel were upregulated in these crystal-internalized cells, which induced ER stress, ROS production, and cell death. Finally, silencing those SOCE genes protected crystal-internalized cells from prolonged [Ca2+]i rise and ER stress. Our data provide insight into the molecular mechanism of crystal-induced Ca2+ dysregulation, ER stress, and PT cell death and thus could have a translational role in treating crystal nephropathies including kidney stones. Taken together, modulation of Ca2+ signaling can be used as a tool to reverse the pathological consequence of crystal-induced conditions including cardiovascular calcification.Calcium crystal internalization into proximal tubular (PT) cells results in acute kidney injury, nephrocalcinosis, chronic kidney disease (CKD), and kidney-stone formation. Ca However, such elimination processes could be hindered, when the crystals adhere to the tubular lining3. Interestingly, several studies have proposed that the onset of nephrolithiasis lies within the nephron, moreso, renal biopsies of some stone-formers presented with crystals inside renal tubular cells5. Thereafter, many in vitro studies have shown that calcium crystals, such as CaOx monohydrate, are taken up by renal tubular cells and subsequently influence the process of kidney-stone formation6. The proximal tubular (PT) cells are more prone to crystal internalization than distal tubular (DT) cells, due to their greater crystal binding affinity7. Interestingly, PT cells are also much more susceptible to insult/injury from extracellular stimuli8. Also, crystal size and shape determine the extent of internalization in various tubular cell types9. Internalization of CaP and CaOx crystals into PT cells has been shown to induce upregulation of inflammatory mediators, cellular damage, apoptosis, and renal interstitial fibrosis10. However, the effect of mixed (CaP + CaOx) crystals, which are usually the most abundant in calcium nephrolithiasis, has not yet been studied. Further, this crystal-induced cytotoxicity as a downstream mechanism toward cell death has been extensively studied13, which could be linked to the upstream mechanisms involving endoplasmic reticular (ER) and plasma membrane (PM) signaling in crystal-induced condition.Crystals are often present in the renal tubular fluid of both stone-forming and nonstone-forming individuals2+ signaling is critically involved in a variety of deleterious physiological responses15. Moreover, dysregulation of intracellular Ca2+ concentration [(Ca2+)i] due to prolonged Ca2+ influx can elicit reactive oxygen species (ROS) dependent proapoptotic signals16. Furthermore, preformed CaP crystals induced ROS and subsequent cell damage, which results in the aggregation of calcium crystals17. Several studies show that crystal-induced effects promote inflammatory and apoptotic responses10. However, the ER-functional status and upstream PM pathway of Ca2+ signaling resulting from crystal internalization into the cells are unknown. Store-operated Ca2+ entry (SOCE), an ER-dependent Ca2+ entry mechanism, which is stimulated by a reduction in Ca2+ levels in the ER, has been shown to control ER-dependent downstream signaling effect18. SOCE serves as a major mechanism for triggering Ca2+ influx into the non‐excitable cells, which helps maintain [Ca2+]i, and to prevent any downstream deleterious event. SOCE comprises of two Ca2+ sensors STIM1/2 located on the surface of the ER and three structurally related pore‐forming subunits (ORAI1/2/3), CRAC channels, located in the PM20. However, we do not know whether the internalization of crystals can have consequence on the ER–PM Ca2+ signaling mechanism in PT cells. Therefore, we focused on the mechanism of SOCE-mediated Ca2+ signaling events in CaOx, CaP, and mixed crystal-induced human PT cells (HK2). We show here that the crystal internalization into PT cells results in a sustained [Ca2+]i rise via a STIM–ORAI3 SOCE-mediated mechanism, which drives the downstream ER stress response, ROS-induced cell damage, and apoptosis in HK2 cells. Blockade of such SOCE-mediated mechanism alleviates the deleterious effect of crystal internalization.It is well understood that ER–PM Ca2+ signaling mechanism. Thus, to examine such Ca2+ signaling, we tested the Ca2+-sensing receptor (CaSR) as G-protein-coupled receptor (GPCR)-mediated mechanism upon internalization of CaP, CaOx, and mixed crystals. We found that crystal internalization elicited an increase in Ca2+ release and produced a greater response in the Ca2+ rise due to Ca2+ entry compared with the noninternalized cells  manner in response to GPCR activation. Interestingly, [Ca2+]i release was increased between 8 and 80 µg/mL but not between 24 and 80 µg/mL mixed crystals  blocker, Pyr10 in our previous study to differentiate between the role SOCE vs. ROCE, respectively22, we introduced CaP, or mixed crystals (8 µg/mL) into HK2 cells, treated cells with Pyr6 (3 µM) or Pyr10 (3 µM) and performed Ca2+ imaging experiments to measure Ca2+ entry induced by neomycin. Our data show that Pyr10 significantly inhibited the [Ca2+]i rise in control cells induced by CaSR activation, indicating that the main Ca2+ signaling pathway under physiological condition is the ROCE. In contrast, Pyr6 decreased [Ca2+]i rise in crystal-internalized cells to a greater extent than Pyr10 23. To further investigate the difference in dynamics of Ca2+ mobilization among HK2 cells internalized with CaP, CaOx, and mixed crystals. We found that crystal-internalized cells demonstrated a greater [Ca2+]i rise compared with the noninternalized (control) HK2 cells  following internalization of CaP, CaOx, and mixed crystals. Similar to previous findings that ER stress induces dysregulation of ER morphology24, we found that crystal internalization induced swelling of the ER and formation of ER tracker positive vesicle-like ER structures  cells for all CaP, CaOx, and mixed crystal-internalized cells, whereas noninternalized cells did not show such response  cells  by siRNA inhibitors corresponded to the expression of SOCE components in crystal-internalized cells. Taken together, our results confirm that ORAI3 is the SOCE channel acting in combination with STIM1, STIM2 as ER sensors to drive Ca2+ response in crystal-internalized human PT cells.Since we found that SOCE is the major CaAI3 Fig. . We thenlls Fig. . Followinse Fig. . We alsolls Fig. . Remarka2+ entry and altered ER morphology, both of which indicate ER stress. Here we show that crystal internalization upregulates the expression of ER stress-related genes compared with the noncrystal condition 33. Furthermore, overexpression of ORAI1 has been shown in other kidney-related diabetic disease34. Since we observed that crystal internalization switches the mode of Ca2+ signaling from ROCE to SOCE, we examined the expression of STIM and ORAI and found that both STIM1, STIM2, and ORAI3 channel expression were upregulated following crystal internalization. These findings were significant because they are consistent with observed sustained Ca2+ entry and ER stress, revealing a link between Ca2+ supersaturation and dysregulated Ca2+ mobilization in human PT cells.STIM proteins are ER intraluminal Ca35, however, its involvement in kidney stones remains unclear. In our study, crystal introduction into human PT cells, upregulated the expression of ER stress genes—ERN1, GRP78, and CLDN1. Interestingly, knockdown of ER Ca2+ sensor STIM1, STIM2, and PM ORAI3 channel using specific siRNAs markedly attenuated crystal-induced expression of ER stress genes. Notably, our data provides further evidence that targeting ER–PM Ca2+ regulation may provide a novel avenue for the development of therapeutic agents not only for kidney-stone disease, but also for other crystal-related nephropathies. Of note, our in vitro inhibition of STIM and ORAI using siRNA may have transient effects, which should be confirmed in PT-specific conditional knockout murine models to understand the role of these players in crystal-related pathologies. Crystal deposition resulting from luminal supersaturation of Ca2+, is a mainstay of renal disease such as nephrocalcinosis, oxalosis, and CKD. Crystal-internalization induced ER morphological alteration and promoted ER stress-induced gene upregulation, evidence of ER stress. Inhibition of STIM-gated SOCE alleviated crystal-induced ER stress, indicating that inhibition of SOCE components may protect the kidney from diseases involving aberrant crystal deposition. Our study provides clues for the mechanism of cell death by dysregulated Ca2+ signaling mediated by crystal internalization. Significantly, the current work highlights a mechanism for the deleterious effects of crystal internalization in human PT cells and provides evidence for targeting SOCE as a therapeutic option for the prevention of kidney diseases such as acute kidney injury, CKD, and renal fibrosis.ER stress has a role in acute kidney injury, CKD, and renal fibrosisCalcium chloride, monosodium phosphate, disodium phosphate, sodium oxalate, tharpsigargin, Pyr6, Pyr10, neomycin, and other chemicals were purchased from Sigma-Aldrich . Fura-2AM was purchased from Invitrogen . ER tracker was purchased from Cell Signaling Technology .2.Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), antibiotics (penicillin and streptomycin), and glutamine were purchased from Invitrogen. Human kidney proximal tubule epithelial cells (HK2) were obtained from Lonza . HK2 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, and 1% penicillin/streptomycin at 37 °C in 5% CO6. Briefly, CaP crystals were prepared by mixing solutions of 2.4 mM CaCl2, 0.9 mM Na2HPO4, and 5.8 mM NaH2PO4. CaOx crystals were prepared by mixing solutions of 2.4 mM CaCl2, 1.0 mM Na2C2O4. Mixed crystals were prepared by combining CaP and CaOx solutions. Crystals were prepared in HBSS to a concentration of 800 µg/ml. Mixed solutions were agitated for 30 min at RT and then centrifuged for 5 min , the supernatant was discarded, and the crystals were washed with HBSS twice.CaP, CaOx, and mixed crystals were prepared as described previously2. After 4 h incubation, complete media (DMEM) with 10% FBS was added, and cells were incubated for a further 20 h.HK2 cells were seeded and grown to 80% confluency in complete media. For crystal induction, appropriate crystals  (800 µg/ml) were added in serum-free media and incubated for 4 h at 37 °C in 5% CO6. Briefly, following crystal induction, cells were rinsed with HBSS without phenol red, Ca2+, and Mg2+, fixed with 3% paraformaldehyde for 10 min at RT, then washed with HBSS three times. Crystals/cells were then incubated in 2% alizarin red solution (pH = 4.3 or 6.8) and incubated at 37 °C for 15 min. Stained images were obtained using Zeiss Axiovision microscope  staining was performed as previously described to detect the presence of CaP and/or CaOx crystalsHK2 cells were transfected with (10 nM) siRNA against STIM1 , STIM2 (Santa Cruz sc-76591), ORAI3 (Santa Cruz sc-76005), and scrambled siRNA-A (Santa Cruz sc-37007) as negative control. Transfection was performed using Lipofectamine-2000 Reagent  according to manufacturer’s instructions.2+]i were performed as described previously21. Briefly, Fura-2-loaded cells were placed on an IX81 motorized inverted microscope equipped with a IX2-UCB control box . For time-lapse fluorescence/ratiometric measurements, the IX81 microscope images were fed into a C9100-02 electron multiplier CCD camera with an AC adaptor A3472-07 . A Lambda-LS xenon arc lamp and 10–2 optical filter changer  were used as an illuminator capable of light output from 340 and 380 nm to a cutoff of 700 nm. All experiments were conducted in a microincubator with a constant temperature set at 37 °C and a gas mixture of 95% O2 and 5% CO2. Cells were bathed in Ca2+-free SES during the experiment. Ratiometric measurements of [Ca2+]i were obtained using digital microscopy imaging software . Fura-2 fluorescence was recorded at an emission peak absorbance at 500 nm wavelength with excitation peak absorbance that continuously shifted at wavelengths of 340 and 380 nm. Time lapse was set at 250–500-time points at 1 s intervals in 50–150 cells, selected as region of interest . Analysis was performed offline using SlidebookTM software and further analyzed using statistical analysis by Origin 6.1.Ratiometric (340/380) measurements of [Ca2O2 release from cells following crystal induction was assessed using H2O2 Cell-Based Assay Kit . Pre-assay and assay preparations were performed according to manufacturer’s instructions. Assay was performed in triplicate. H2O2 release was assessed by subtracting the background wavelength (540 nm) from the emission wavelength (590 nm).H15. Following crystal induction, cells were fixed with 3% paraformaldehyde, permeabilized with 0.01% Triton X-100 in 0.1% BSA for 2 min, washed with 1 × PBS once, and incubated with DAPI (1 μg/ml in 1 × PBS pH 7.4) in 500 µl for 10 min at room temperature in the dark. After staining, the DAPI solution was removed; cells were washed two times with 1 × PBS and visualized under fluorescence microscopy .Apoptotic nuclei were detected using DAPI. Cell culture and crystal induction followed the same procedure as described earlier6. Following crystal induction, cells were trypsinized and stained with toluidine blue stain. Live cells were counted using a hemocytometer and standard procedure, and cell viability was calculated relative to control  condition. Cell counts were performed in triplicates.HK2 cells were seeded and grown to 80% confluency. Cells were induced with crystals as described previouslyApoptotic and necrotic cells were assessed using Alexa Fluro 488 Annexin V/Dead Cell apoptosis kit (Thermo Fisher Scientific). Staining was performed according to manufacturer’s instructions.LDH release from crystal-induced HK2 cells was measured using Pierce LDH Cytotoxicity Assay kit (Thermo Fisher Scientific) according to manufacturer’s instructions. Briefly, following crystal induction, cell culture media was collected and transferred to a 96-well plate in triplicates. LDH release reactions were performed and assessed by the subtraction of the absorbance at 680 nm (background) from the absorbance at 490 nm.15. Subsequently, DNase treatments were performed, and RNA concentrations were measured using nanodrop spectrophotometer. Afterward, a cDNA synthesis kit  was used to reverse transcribe the RNA into the cDNA, which were amplified with gene specific primers  purchased from Invitrogen and Integrated DNA Technologies  using the master mix PCR amplification reagent (Promega). With a T100 Thermocycler , the following PCR conditions were used: one initial cycle at 95 °C for 3 min; 30–35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 45 s; an additional 5 min at 72 °C; and a final hold at 4 °C.Total RNA’s were isolated from HK2 cells using TRIzol as previously describedt-test (two-tailed), in Origin 6.1. Statistically significant comparisons were accepted at P value < 0.05.Experimental results are expressed as mean ± S.E.M. Statistical comparisons were performed using Student’s unpaired Supplementary Figure 1Supplemental Material File #1


Treatment with 5 mM AZC did not induce poly adenosine diphosphate ribose polymerase (PARP) cleavage while levels of binding immunoglobulin protein (BiP) and phosphorylated eukaryotic translation initiation factor 2α (eIF2α) increased and those of activating transcription factor 6 (ATF6) decreased, indicating activation of the protein kinase RNA-like ER kinase (PERK) and the ATF6 arms of the UPR but not of apoptosis. AZC treatment in combination with bafilomycin A1 (Baf A1) led to elevated levels of the lipidated form of the autophagy marker microtubule-associated protein light chain 3 (LC3), pointing to activation of autophagy. Using the specific PERK inhibitor AMG PERK 44, we could deduce that activation of the PERK branch is required for the AZC-induced lipidation of LC3. Moreover, both the levels of phospho-eIF2α and of lipidated LC3 were strongly reduced when cells were co-treated with the intracellular Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraaceticacid tetra(acetoxy-methyl) ester (BAPTA-AM) but not when co-treated with the Na+/K+ ATPase inhibitor ouabain, suggesting an essential role of Ca2+ in AZC-induced activation of the PERK arm of the UPR and LC3 lipidation. Finally, AZC did not trigger Ca2+ release from the ER though appeared to decrease the cytosolic Ca2+ rise induced by thapsigargin while also decreasing the time constant for Ca2+ clearance. The ER Ca2+ store content and mitochondrial Ca2+ uptake however remained unaffected.Accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) to reduce protein load and restore homeostasis, including via induction of autophagy. We used the proline analogue  Endoplasmic reticulum (ER) stress is a particular form of cellular stress that occurs when the ER machinery is overwhelmed by the amount of unfolded or misfolded proteins . To restAutophagy is an important, evolutionary conserved pro-survival process that promotes cellular homeostasis. Long-lived proteins and dysfunctional organelles are thereby engulfed in newly formed double-membrane vesicles called autophagosomes, which eventually will undergo fusion with lysosomes. In the resulting autolysosomes, the autophagosomes and their cargo are degraded and the components recycled .2+ signaling in the regulation of the autophagic process and especially whether the role is stimulatory or inhibitory as evidence for both mechanisms have been proposed .2+ handling [2+ ATPase (SERCA), leads to ER Ca2+ depletion and ER stress [2+ ions in the lumen of the ER [ER stress itself is intimately linked to intracellular Cahandling ,12,13,14R stress ,16,17,182+ was required for autophagic flux induced by either nutrient starvation [2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM), the inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor Xestospongin B and/or IP3R knockout all inhibited autophagy induction. Moreover, these results are fully in line with those obtained by other groups [2+ sensitivity of autophagy occurring after ER stress as part of the UPR has never been tested, and it was the aim of this study to investigate this point.Previous work by our own group had shown that intracellular Caarvation , rapamycarvation , or resvarvation . This cor groups ,26,27,282+, we could not use TG. Moreover, since autophagy and apoptosis influence each other [2+ release by the IP3R and mitochondrial Ca2+ overload [l-azetidine-2-carboxylic acid (AZC), a proline analog , GlutaMAX  and penicillin and streptomycin , as described before [HeLa cells were cultured at 37 °C and 5% COd before ,20,21. CReagents used were AZC , bafilomycin A1 , ethylene glycol tetraacetic acid , BAPTA-AM , TG , Fura-2 AM , staurosporine , and AMG PERK 44 .3R (Rbt475 [3R isoforms), anti-LC3 , anti-MCU , anti-PARP , anti-PMCA  recognizing all PMCA isoforms, anti-SERCA2B , and anti-vinculin .Primary antibodies used were anti-ATF6 , anti-BiP , anti-eIF2α , anti-phospho-eIF2α , anti-ERp57 , anti-ERp72 , anti-GAPDH , anti-IP , PhosSTOP phosphatase inhibitor ). Lysates were incubated on ice for 30 min and centrifuged for 5 min at 8000× g. Protein concentration of the supernatant was determined using a bicinchonic acid protein assay kit and bovine serum albumin standards . Proteins were then separated on 10–20% Tris-glycine gels (Thermo Fisher Scientific) using Tris-glycine running buffer . Proteins were transferred to polyvinylidene difluoride membrane in running buffer  containing 10% methanol and blocked in 5% non-fat milk in Tris-buffered saline (TBS) containing 0.1% Tween. Membranes were incubated with primary antibodies overnight at 4 °C, and horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Proteins were detected using Clarity Western Enhanced Chemiluminescence (ECL) Substrate  or ECL Western Blotting Substrate . Individual experiments were always performed in duplicate.Cells were washed with PBS and lysed with lysis buffer (150 mM Hepes (pH 7.5), 150 mM NaCl, 100 mM NaF, 10 mM ethylene diamine tetraacetic acid (EDTA), 10 mM Nag. RNA was extracted using the High Pure RNA Isolation kit  according to manufacturer’s protocol. cDNA was prepared using the High Capacity cDNA Reverse Transcription kit  according to manufacturer’s protocol. XBP1 mRNA was amplified using GoTaq Green master mix  and XBP1 specific primers  and separated on a 2.5% Ultrapure agarose  gel containing 0.005% EtBr . Individual experiments were always performed in duplicate.Cells were trypsinized and centrifuged for 5 min at 500× Cell death was assessed by PARP cleavage and by propidium iodide (PI) staining. PARP cleavage was assessed by Western blotting as described in 2, 11.6 mM Hepes (pH 7.3), 11.5 mM glucose, 1.5 mM CaCl2). Fluorescence was measured on a FlexStation 3 microplate reader  by alternate excitation at 340 nm and 380 nm and recording emission at 510 nm. Compounds were added as indicated. Individual experiments were always performed in triplicate.Cells were plated in 96-well plates and treated as indicated. During the last hour of treatment the cells were loaded for 30 min with 1.8 μM Fura-2 AM followed by 30 min de-esterification in modified Krebs solution  according to the manufacturer’s protocol. After 48 h single-cell measurements were performed on a Zeiss Axio Observer Z1 Inverted Microscope equipped with a 20× air objective and a high-speed digital camera . Extracellular Ca2+ was chelated with 3 mM ethylene glycol tetraacetic acid (EGTA) and one minute later the indicated compound was added. Changes in G-CEPIA1er fluorescence were followed after excitation at 480 nm and measurement of emission at 520 nm. Changes in R-GECO1mt fluorescence were followed after excitation at 377 nm and measurement of emission at 466 nm. The traces were normalized to baseline fluorescence (F/F0) where the baseline was calculated as the average of the first six time points. At least 99 cells per condition were measured in the course of six individual experiments.ER and mitochondrial Ca-GECO1mt  kindly pp < 0.05.Results are presented as mean ± standard error of the mean (SEM). Significance was tested using a one-way analysis of variance with Tukey post-hoc test. Results were considered significant when To analyze the induction of ER stress, HeLa cells were treated with 5 mM AZC for up to 9 h. The cell lysates were analyzed by Western blotting for the ER stress markers ATF6, binding immunoglobulin protein (BiP), ER protein (ERp) 72, ERp57, and total and phospho-eIF2α B,C. AZC Taken together, these results indicate that a 6 h treatment with AZC activates both the PERK and the ATF6 arm of the UPR, resulting in upregulated BiP protein expression and increased phosphorylation of eIF2α.Because it was important to study ER stress and subsequent autophagy in conditions where cell death was not induced, we assessed the levels of cleaved poly adenosine diphosphate ribose polymerase (PARP) upon AZC treatment during the same time period. While STS led to clear PARP cleavage, we observed virtually no cleavage upon AZC treatment A. The quFrom both the PARP-cleavage analysis and the PI staining, we can conclude that treatment with 5 mM AZC for 6 h did not lead to any significant cell death.To avoid interference of ER stress-induced cell death, we wanted to use conditions that allowed induction of the UPR and of autophagy in a time frame as short as possible. Based on the results presented in The larger increase of LC3-II levels in AZC-treated cells in the presence of Baf A1 as compared to the control conditions suggests that the upregulation of LC3-II levels does not depend on a block of autophagosome-lysosome fusion and/or of lysosomal degradative activity but is related to the induction of autophagy.To investigate the relation between the UPR and the increased LC3-II levels after application of AZC, we used the PERK-selective inhibitor AMG PERK 44 . AMG PERThese results indicate that activation of the PERK pathway is a prerequisite for the occurrence of the AZC-induced increase in LC3-II levels, and that the ATF6 branch of the UPR is not sufficient for obtaining this effect.2+ in the elevation of LC3-II levels triggered by AZC, we co-treated the cells with increasing concentrations of the intracellular Ca2+ chelator BAPTA-AM. This led to a dose-dependent reduction of the AZC-induced increase in LC3-II   C,D.2+ handling, though without directly affecting the ER Ca2+ leak, ER-mitochondrial Ca2+ transfer or the expression of IP3Rs, PMCAs, SERCA2B, and the MCU.Taken together, long-term treatment of cells with AZC (6 h) seems to modify cellular CaOne of the main functions of the ER is the proper folding of nascent proteins. To ensure this, chaperones assist and correct the process when necessary . Stress 2+ homeostasis. Therefore, we turned to AZC. This proline analogue contains a four-membered carbon ring in contrast to the five-membered ring in proline (Classic chemical ER stress inducers include dithiothreitol, tunicamycin, and TG. In our preliminary experiments however, these compounds also showed induction of apoptosis or they displayed an intrinsic effect on Ca proline A. Incorp proline . This is proline , which s2+ plays an important role in both ER stress and autophagy [2+ were described [2+ in intracellular processes like autophagy, treatment with the intracellular Ca2+ chelator BAPTA-AM is the favored technique [2+-binding molecules, including BAPTA-AM, can inhibit the Na+/K+ ATPase, independently of their Ca2+-binding affinity [+/K+ ATPase inhibitor than the other compounds tested, it readily accumulates in the cell and we therefore wanted to verify whether Na+/K+ ATPase inhibition by BAPTA-AM could affect the interpretation of our results. One way to discriminate between the Ca2+ chelating and the Na+/K+ ATPase inhibiting effect of BAPTA-AM, is to assess whether the effects provoked by BAPTA-AM are replicated by the well-known specific Na+/K+ ATPase inhibitor ouabain [+/K+ ATPase isozymes are characterized by a Kd for ouabain between 13 and 37 nM [+/K+ ATPase inhibition, we applied a range of concentrations of ouabain between 10 nM and 1 µM. However, at none of these concentrations did ouabain (2+ chelation rather than Na+/K+ ATPase inhibition and that intracellular Ca2+ is critical for both AZC-induced ER stress and elevation of LC3-II.Cautophagy ,9,14. Inescribed ,55. To aechnique . We theraffinity . Althoug ouabain . All humnd 37 nM . In orde ouabain  mimic th ouabain . Therefo2+ in AZC-induced elevation of phospho-eIF2α. Given that the PERK-arm of the UPR has been reported to be required for ER stress-mediated induction of LC3-II [2+ observed for the subsequent elevation of LC3-II levels. Whereas Ca2+ was recently shown to be required for non-UPR-related functions of PERK [2+ is also required for efficient eIF2α phosphorylation, and thus for transduction of PERK-arm mediated UPR signaling. On the other hand, Ca2+ did not seem to be required for AZC-mediated upregulation of BiP. The Ca2+ sensitivities of the different parts of the UPR signal transduction pathways remain to be determined, but it is already noteworthy that the BiP upregulation is insensitive to both the PERK inhibitor and to BAPTA-AM.Interestingly, our data indicate an essential role of Caf LC3-II ,59, this of PERK , our stu2+ released from the ER Ca2+ store with TG, but did not induce Ca2+ release itself (2+ release from the ER by itself although they also claim that the ER is not significantly depleted after pre-treatment for 4 h with 5 mM AZC [2+ signal was performed, and on the traces shown the agonist-induced Ca2+ release is anyway smaller than in control conditions.Finally, we found that 6 h of pre-treatment with 5–10 mM AZC reduced the amount of Cae itself A. These 5 mM AZC . However2+ rise after pre-incubation with AZC may be due to a reduced Ca2+ content in the ER and therefore a lower release of Ca2+ into the cytosol upon TG application, or due to an increased Ca2+ transfer from ER to mitochondria, thereby preventing detection of the Ca2+ in the cytosol. However, when measuring Ca2+ simultaneously in the ER and the mitochondria, we did neither observe a reduction in ER store content nor an increase in mitochondrial Ca2+ transfer in cells pretreated for 6 h with 5 or 10 mM AZC as compared to control cells (2+-transport systems outside the ER and mitochondrial compartments. For instance, it is possible that PMCA, responsible for exporting Ca2+ from the cytosol to the extracellular environment, is affected. We could exclude a modification in PMCA expression levels (2+ from the cytosol by PMCA may lead to an apparent reduction of the amount of cytosolic Ca2+ without an actual decrease of the ER Ca2+ store content, and thus explain the reduced τ value for the clearance of Ca2+ from the cytosol.We initially hypothesized that the reduction in TG-induced cytosolic Caol cells A,B. We tn levels C though 2+ in the process of eIF2α phosphorylation and LC3-II production upon AZC treatment. AZC however does not directly affect ER Ca2+ store content or ER-to-mitochondria Ca2+ transfer and the observed effects on intracellular Ca2+ handling are likely not related to its effect on UPR and autophagy.Taken together, our results indicate a critical role for Ca


However, because these Thy-1 effects occur exclusively in reactive astrocytes, we wondered whether by elevating calcium levels and promoting hemichannel opening we could prompt non-reactive astrocytes to respond to Thy-1. Cx43 immunoreactivity increased at juxta-membrane sites, where hemichannels (not gap junctions) participate in astrocyte polarization and migration stimulated by Thy-1. Also, intracellular Ca2+ increase, due to ionomycin treatment, induced hemichannel opening, but activated astrocyte migration only partially, and this limitation was overcome by pre-treatment with tumor necrosis factor (TNF) and Thy-1. Finally, αvβ3 integrin formed membrane clusters after TNF stimulation or overexpression of β3 integrin. We suggest that these microclusters are required for cells to respond to Thy-1 stimulation. Therefore, the large increase in intracellular Ca2+ and hemichannel opening induced by ionomycin are required, but not sufficient, to permit Thy-1-induced astrocyte migration. Thus, we suggest that proinflammatory stimuli prompt astrocytes to respond to migratory signals of neuronal cells.Under pro-inflammatory conditions, astrocytes become reactive and acquire a migratory phenotype. Our results show that hemichannels formed by connexin 43 (Cx43) play an important role in Thy-1-induced astrocyte migration. The neuronal protein Thy-1 binds to αvβ3 integrin in astrocytes, thereby leading to intricate signaling pathways that include calcium (Ca Astrocytes are the most abundant cell type in the central nervous system (CNS); however, their function has always been overshadowed by neurons. Astrocytes not only help and nourish neurons, but also participate in synapse function . In addi3R) activation, Ca2+ release from the endoplasmic reticulum, connexin (Cx)- and pannexin (Px)-hemichannel opening, ATP release, and finally P2X7 receptor (P2X7R) activation, which further increases intracellular Ca2+ concentrations ([Ca2+]i) i) ,5,6,7,8.[Ca2+]i) ,10.After inducing astrocyte reactivity in vitro, Cx43 is overexpressed and pharmacological inhibition precludes astrocyte migration ,11. In n2+]i induced by mechanical stimulation or by ATP, effects that are inhibited by a Cx43-blocking peptide i provoked by ionophores induces hemichannel opening in astroglioma cells [2+]i levels and an induction of the hemichannel opening prompt non-reactive astrocytes to respond to Thy-1.The increased levels of Cx43 during astrocyte reactivity help maintain the reactive phenotype of astrocytes and microglia by the release of molecules, such as ATP and glutamate . In this peptide . The incma cells ,17, as wHere, we show that Thy-1 enhanced cell surface Cx43 levels and induced hemichannel opening resulting in cell polarization. Additionally, TNF treatment or enhanced surface levels of αvβ3 integrin by β3 integrin overexpression promotes β3 integrin microcluster formation, clusters that are suggested here to be necessary for astrocytes to respond to neuronal migratory signals. Moreover, we found that ionomycin induces the hemichannel opening independently of the reactive phenotype of astrocytes, an effect that is potentiated by Thy-1. Ionomycin also promotes astrocyte migration; however, ionomycin and/or Thy-1 require TNF for effective cell migration.2+ entry, which are ultimately required for the adhesion, polarization, and migration of astrocyte cell lines, as well as primary rat neonatal astrocytes in culture i due to release from intracellular stores, as well as through Ca2+ influx, are necessary for astrocyte migration. The addition of Thy-1 enhances [Ca2+]i in astrocytes prior to hemichannel opening and cell migration i by activating P2X7R [2+]i increase in astrocytes by releasing Ca2+ from intracellular stores and by Ca2+ uptake through opening of P2X7R i increases. Migration stimulated by Thy-1 in ionomycin-treated cells was lower than that induced by Thy-1 in TNF-treated cells, suggesting that the increase of [Ca2+]i alone, is not sufficient to trigger Thy-1-induced cell migration. In astrocytomas, glutamate induces cell migration by regulating Ca2+ signals, and the addition of ionomycin leads to glutamate release [2+]i, glutamate release and cell migration. However, in our experiments, migration induced by Thy-1/ionomycin was notably inferior to that induced by TNF/Thy-1, suggesting that astrocyte reactivity and the changes involved in this process require more than just changes in [Ca2+]i. Other studies in astrocytes have reported that ionomycin induces NFAT translocation to the nucleus, which is a hallmark of TNF-induced reactive astrocytes i increase required for astrocyte migration can be partially replicated by the addition of ionomycin. However, Thy-1-induced migration in this case is lower than that observed with Thy-1 in TNF-pretreated astrocytes, an effect that relies on the formation of β3 integrin clusters at the plasma membrane, which are not related to focal adhesions, but rather exist in astrocytes that acquire the reactive phenotype.In summary, we describe here that Cx43 is a key regulator of astrocyte reactivity, polarization and migration. Cx43 hemichannels rapidly move to the plasma membrane by the addition of Thy-1, where they can regulate the release of molecules relevant for astrocyte reactivity, such as ATP. In this study, we show that the  octane. Samples were photographed using an oil-immersion (HCXAPO_63x/1.40 or 100x/1.40) objective, coupled to a TCS SP5 confocal microscope under sequential scanning mode, and a 2.0-fold digital magnification. Files were digitalized in a 1024 × 1024 pixels format file.n = 3 or more experiments as indicated in each figure legend. The results were analyzed using one-way ANOVA tests and Tukey post-tests. Statistical significance was set at p < 0.05.The results are shown as the means ± standard error of the mean (s.e.m.) for


The programmed intermittent epidural bolus (PIEB) technique offers multiple benefits over continuous epidural infusion (CEI), but controversy still exists when it is used in conjunction with a parturient-controlled epidural analgesia (PCEA) regimen. A systematic review and meta-analysis was thus conducted using the Medline, EMBASE, CENTRAL and Web of Science databases with the aim of identifying those randomized controlled trials (RCTs) that performed a comparison between PIEB and CEI in healthy parturients using a PCEA regimen with regard to the duration of labor, labor pain, anesthesia interventions, maternal satisfaction and main side effects. The data were analyzed using a random-effects model. Eleven eligible trials were included, in which 717 participants were allocated to the PIEB + PCEA group and 650 patients were allocated to the CEI + PCEA group. The rate of instrumental delivery, incidence of breakthrough pain, PCEA usage rates and local anesthetic usage were significantly reduced, the labor duration was statistically shorter, and the maternal satisfaction score was significantly improved in the PIEB + PCEA group compared with that in the CEI + PCEA group. There were no differences in the side effects between the two groups. The results of the present study suggest that the PIEB technique in conjunction with the PCEA regimen was more advantageous than CEI + PCEA, but additional studies should be conducted to consistently demonstrate an improvement in the maternal and fetal obstetric outcomes. The degree and relief of pain affects maternofetal physiology and neuropsychology, as well as maternal satisfaction2. It is thus necessary that feasible analgesia is used to improve maternal satisfaction and decrease the side effects on the mother and fetus3. It has been acknowledged that patient-controlled epidural analgesia (PCEA) is an effective method of labor analgesia that has been associated with superior maternal satisfaction and a lower incidence of adverse events compared with other analgesia techniques5; however, the PCEA regimen without a background infusion is not beneficial for decreasing pain scores of the parturient and the workload of the medical staff. Therefore, the continuous epidural infusion (CEI) technique added to the PCEA regimen has become a standard labor epidural analgesic regimen in North America and Europe in recent decades7, but CEI + PCEA could increase the risk of instrumental delivery and prolong the second stage of labor compared with PCEA-only epidural labor analgesia8, and it is controversial whether there is a decrease in local anesthetic (LA) usage and an improvement in analgesic efficacy with CEI + PCEA compared to PCEA alone8.Labor pain is one of the most painful experiences for a woman9, lower rate of requiring rescue boluses10, and greater patient satisfaction11. However, it has been demonstrated that the PIEB + PCEA mode has similar LA consumption, motor blockade, instrumental delivery and breakthrough pain rates compared with the PCEA or CEI + PCEA regimens14. Therefore, the aim of this systemic review and meta-analysis was to compare whether PIEB in conjunction with PCEA in healthy pregnancy improved delivery mode, labor analgesia, patient satisfaction, maternal and neonatal obstetric outcomes compared to the CEI + PCEA regimens.Compared with CEI + PCEA, the computer-integrated PCEA regimen comprising a PCEA regimen plus a programmed intermittent epidural bolus (PIEB), which automatically administers epidural solution at scheduled intervals, has resulted in a lower incidence of motor block and instrumental deliveryOur IRB did not require ethics approval because there were no data directly collected from patients. We evaluated and synthesized only data in published trials.The study was performed and reported in accordance with the recommended methods of the PRISMA checklist  Group17. The epidural LA dose per hour from the summarized data and milligram equivalents of ropivacaine per hour of anesthesia delivery were calculated as previously described16; ropivacaine and levobupivacaine were assumed to be approximately 60% as potent as bupivacaine20.The primary outcomes were the evaluation of the mode of delivery , duration of labor, efficacy of analgesia and anesthesia interventions, which was reflected by visual or verbal analog scale  scores, incidence of breakthrough pain, PCEA usage rate, time to first PCEA or breakthrough pain, LA dose delivered per hour, and patient satisfaction. The secondary outcomes included the degree of sensory blockade and motor blockade, the incidence of pruritus, hypotension, nausea and vomiting. Apgar scores were determined at 1 minute and 5 minutes. Studies were included if they reported any of the primary outcomes. Two investigators independently extracted the relevant data. All data were tabulated in Microsoft Excel. Any discrepancies regarding the inclusion of eligible trials were handled by discussion; if a consensus was not achieved, the opinion of the third investigator was sought. If the data were reported as medians, ranges, and confidence intervals (CIs), the mean and standard deviations were calculated as previously describedI2 statistic was used to assess the heterogeneity of the trials. P values < 0.1 were predefined as indicating statistically significant heterogeneity. I2 > 50% was viewed as indicating heterogeneity. It was decided a priori that the primary outcome would be subanalyzed according to the technique  used to initiate labor analgesia, if possible. Sensitivity analyses were performed using the leave-one-out method to explore the heterogeneity; if particular studies were methodologically different from the others, these studies were excluded. When meta-analytic methods were unavailable due to different types of data or significant heterogeneity in the outcome effect, a qualitative descriptive analysis was considered for these data. Publication bias was appraised with the Egger test using Stata version 12.0, and P < 0.05 indicated a significant publication bias.Pooled analysis was conducted with Review Manager Version 5.3 . An inverse variance random-effects model was applied due to clinical or methodological heterogeneity across studies. Continuous parameters were weighted using the mean difference (MD) with 95% CI, and binary variables were calculated using the odds ratios (OR) with 95% CI. The 27. The flow diagram is depicted in Fig. 27. The risk of bias is shown in Fig. A total of 646 records were initially searched, and 388 records were removed as duplicates. A total of 231 irrelevant records were excluded, and 27 articles remained for in-depth, full text review after screening titles or abstracts. Finally, eleven potentially eligible articles were retrieved for this systematic reviewet al.11 and Zhao et al.27, included parous parturients. The pooled results did not show a difference when these two trials were removed . There was no publication bias in the trials involving CD (P = 0.089) and instrumental delivery (P = 0.747).Ten trials, which included 1167 subjects, reported the mode of delivery. A significant difference in CD rate between the two groups was not present in these studies. The pooled analysis did not find a difference in the rate of CD , duration of the first stage of labor (P = 0.711) or duration of the second stage of labor (P = 0.764).All trials reported the duration of labor, and ten of the studies offered information on the total duration of labor. The overall analysis showed that there was a significantly shorter total duration of labor when the PIEB + PCEA regimen was available . Six trials reported the rate of PCEA usage and showed that fewer subjects administered a bolus through the PCEA regimen in the PIEB + PCEA group, and there was a significant decrease in the rate of PCEA usage in the PIEB + PCEA group compared with that in the CEI + PCEA group .There was significant heterogeneity among studies regarding the timing of the VAS scoring and the assessment of average or maximal VAS values. We therefore report the results qualitatively. Nine trials estimated the efficacy of labor analgesia with a VAS at different intervals, of which 6 reportsI2 = 57.8%; Fig. 27 including 784 participants, and the overall analysis showed that a significant decrease was observed with the PIEB + PCEA regimen compared with the CEI + PCEA regimen  and hourly (P = 0.129) LA delivered.The specific regimens and LA concentrations are summarized in Table 11. All trials, except for Sia et al.25 and Zhao et al.27, reported higher maternal satisfaction scores in the PIEB + PCEA group than in the CEI + PCEA group; the overall analysis showed that participants had a significantly higher maternal satisfaction score in the PIEB + PCA group than in the CEI + PCEA group  among these studies. A subgroup analysis was thus conducted. The subgroup of subarachnoid anesthesia initiation showed a significantly lower heterogeneity (I2 = 15%) among the trials, while a higher heterogeneity was still observed in the subgroup using epidural technique initiation (I2 = 99%). There was no publication bias in the trials reporting maternal satisfaction (P = 0.608).Nine trials reported overall maternal satisfaction with labor analgesia, and most studies used a verbal rating scale (VRS) in which 0 represented very dissatisfied and 10 or 100 represented extremely satisfied. Only one trial reported maternal satisfaction with a visual analog scale (VAS) score29 <6 or a traditional Bromage score28 >1, occurring at least once during labor (Table I2 = 54%). A sensitivity analysis suggested that Capogna et al.9 appeared to increase the heterogeneity and used the Breen-modified Bromage score29, which is different than the Bromage score used by other trials; after removing this trial, the overall results showed no significant difference between the two regimens.The incidence of motor block, assessed with the Breen-modified Bromage score27. There was no difference between the 2 groups in any trials. Eight trials reported the 5-minute Apgar scores, of which five trials gave the concrete scores27. There were no significant differences observed between the two regimens across all trials. Fang et al.21 and Wang et al.28 reported that the 5-minute Apgar score for all subjects exceeded 7. Sia et al.25 reported that 81% (17/21) of the subjects in the PIEB + PCEA group and 90% (19/21) of subjects in the CEI + PCEA group surpassed scores of seven. Significant differences were not observed between the two groups.Six trials reported 1-minute Apgar scoreset al.10 and Sia et al.24, and there was no difference between the two groups.Other maternal side effects  are listed in Table The important findings of this systematic review and meta-analysis study show that PIEB in conjunction with the PCEA regimen is of greater benefit to the parturient and fetus. In these healthy women requesting labor epidural analgesia, PIEB as the background infusion of the PCEA, compared with CEI plus PCEA, significantly reduced the risk of instrumental delivery, improved labor pain and pain relief, reduced LA consumption, resulted in higher maternal satisfaction, and statistically shortened the labor duration, but there were no differences with regard to the incidence of adverse events .8. CEI with or without PCEA boluses has been regarded as standard labor epidural analgesic regimens in many institutions in North America and Europe16. However, a higher risk of instrumental delivery exists when CEI was added to the PCEA regimen than for the PCEA-only regimen8. Multiple studies have found that the rate of instrumental delivery was decreased when PIEB was added to PCEA compared with that for CEI + PCEA30, but the 2013 systematic review by George et al.16 did not show a significant difference in the combined results. Our study showed that PIEB reduced the risk of instrumental delivery compared with CEI when pooling the data reported in the recent studies that conducted a comparison of PIEB and CEI being added to the PCEA regimen.PCEA allowed the parturient to self-administer a bolus and thereby reduce the time between the onset of pain and the administration of additional analgesia31. A lower incidence of motor blockade was observed with the PIEB + PCEA regimen than with the CEI + PCEA regimen. Motor blockade may influence the duration of labor, and the use of PCEA for maintenance of labor analgesia may have a noteworthy effect on the duration of the first stage and the total duration of labor. George et al.16 showed that the duration of the second stage was as much as 22 minutes shorter with IEB alone than with the CEI with or without PCEA, and when IEB added to PCEA was used for maintenance of labor, the duration of the first stage was longer (20 minutes). The total duration of labor was reduced (−4 minutes) compared with that for CEI, and thus, they speculated that IEB may enter the realm of clinical significance and positively affect labor progression. Our pooled results showed a statistically significant difference  in the length of total, first and second stage of labor, but such differences are hardly clinically significant.The incidence of instrumental delivery is associated with pelvic muscle tone, motor blockade and the ability to “bear down” during the second stage of labor33. A superior efficacy of labor analgesia for parturients with the PIEB + PCEA regimen was presented in our study, and there was a significant decrease in the incidence of breakthrough pain, which is a transitory exacerbation of pain that occurs once previously used labor analgesia becomes ineffective and often requires supplemental epidural medications using a PCEA regimen or manual bolus37. A lower rate of PCEA usage was also observed, and the time to first PCEA usage or first breakthrough pain was postponed in most included studies27. The relief of labor analgesia is partially contingent upon a greater LA consumption38. Our pooled results showed that the total or hourly dose of LA was markedly decreased in the PIEB + PCEA group compared with that in the CEI + PCEA group. Lower LA usage to achieve optimal analgesia was indeed disputed between the PIEB or PCEA regimen maintaining labor analgesia, likely due to lack of a standardized useful tool to assess the degree of pain. The VAS is one of the most commonly used methods39, but when an acceptable VAS pain score for parturients is a consistently controlled score of less than 340, a significant difference could not occur easily; once the scores were greater than 3, the parturients or anesthetists would take measures to relieve the pain, therefore a difference would not be observed.In fact, patients’ preferences about labor are focused on both pain relief and labor duration, and patients preferred lower intensity, longer duration to higher intensity, short duration labor pain; thus, direct management of labor pain is needed to meet patient expectations43. The meta-analysis and review by George et al.16 stated that PIEB with or without PCEA slightly improved maternal satisfaction. Our study showed that PIEB + PCEA resulted in significantly higher maternal satisfaction scores than CEI + PCEA, but the aggregated studies had significant heterogeneity (I2 = 98%). The use of a different neuraxial analgesia technique to initiate labor analgesia might have influenced maternal satisfaction. Our subgroup analysis suggested that heterogeneity was especially apparent when an epidural technique was used to initiate labor analgesia, likely due to the analgesia efficacy of the epidural technique itself being influenced by many factors . In fact, women’s self-diagnosis of the onset of labor and their perception of their labor duration when meeting their midwives has some impact on their admission to the labor ward and the timing of epidural analgesia44; thus, it is difficult to accurately measure maternal satisfaction at standard intervals or the same times with the standard assessment tools, all of which could cause heterogeneity, so the pooled results have to be interpreted with caution.The main goal of labor analgesia is to accomplish an ideal and desired level of pain relief and greater satisfaction of care provided to the patients. Maternal satisfaction, a multidimensional measure, is a common evaluation method that involves personal expectations, labor pain, perception of emotional control, communication skills of her caregiver and maternal involvement in decision making45.There are several limitations to this study. First, none of the included studies reported all of the relevant outcomes, thus limiting the statistical power of this systematic review. Second, labor analgesia was not evaluated in the same way; some trials recorded VAS scores during the overall labor progression at different time points, while several studies used the incidence of breakthrough pain to indirectly reflect labor analgesia. Third, most trials nearly consistently reported maternal satisfaction to evaluate the relief of labor pain, but significant heterogeneity was observed among studies. Fourth, only two trials recruited parous subjects; the remaining studies included uncomplicated nulliparous women, which likely limits the conclusions to be extrapolated to women presenting with multiple gestations and parous women. Fifth, despite only these trials directly comparing the PIEB + PCEA regimen with the CEI + PCEA regimen were included in this systematic review, significant clinical heterogeneity with respect to labor analgesia initiation, LA concentration or volume and drug delivery regimens still existed, which may affect maternal and obstetric outcomesIn conclusion, the PIEB + PCEA regimen, compared with the CEI + PCEA regimen, showed greater benefit for decreasing the risk of instrumental delivery, relieving labor pain, decreasing LA consumption, and improving maternal satisfaction, but future prospective and adequately powered studies should be conducted to confirm earlier findings and optimize the PIEB settings with respect to LA concentration, volume boluses, time intervals, etc. Given that women’s own perceptions and expectations regarding the onset and process of labor affect the evaluation of satisfaction, educational strategies for the implementation of these techniques should be taken to guide pump programming.PCEA PRISMA-Supplemental Appendix S1Supplemental Appendix S2


Acute myeloid leukemia (AML) represents 80% of adult leukemias and 15–20% of childhood leukemias. AML are characterized by the presence of 20% blasts or more in the bone marrow, or defining cytogenetic abnormalities. Laboratory diagnoses of myelodysplastic syndromes (MDS) depend on morphological changes based on dysplasia in peripheral blood and bone marrow, including peripheral blood smears, bone marrow aspirate smears, and bone marrow biopsies. As leukemic cells are not functional, the patient develops anemia, neutropenia, and thrombocytopenia, leading to fatigue, recurrent infections, and hemorrhage. The genetic background and associated mutations in AML blasts determine the clinical course of the disease. Over the last decade, non-coding RNAs transcripts that do not codify for proteins but play a role in regulation of functions have been shown to have multiple applications in the diagnosis, prognosis and therapeutic approach of various types of cancers, including myeloid malignancies. After a comprehensive review of current literature, we found reports of multiple long non-coding RNAs (lncRNAs) that can differentiate between AML types and how their exogenous modulation can dramatically change the behavior of AML cells. These lncRNAs include: H19, LINC00877, RP11-84C10, CRINDE, RP11848P1.3, ZNF667-AS1, AC111000.4-202, SFMBT2, LINC02082-201, MEG3, AC009495.2, PVT1, HOTTIP, SNHG5, and CCAT1. In addition, by performing an analysis on available AML data in The Cancer Genome Atlas (TCGA), we found 10 lncRNAs with significantly differential expression between patients in favorable, intermediate/normal, or poor cytogenetic risk categories. These are: DANCR, PRDM16-DT, SNHG6, OIP5-AS1, SNHG16, JPX, FTX, KCNQ1OT1, TP73-AS1, and GAS5. The identification of a molecular signature based on lncRNAs has the potential for have deep clinical significance, as it could potentially help better define the evolution from low-grade MDS to high-grade MDS to AML, changing the course of therapy. This would allow clinicians to provide a more personalized, patient-tailored therapeutic approach, moving from transfusion-based therapy, as is the case for low-grade MDS, to the introduction of azacytidine-based chemotherapy or allogeneic stem cell transplantation, which is the current treatment for high-grade MDS. Plurigenitors , 12.All cells of the BM, including progenitors, precursors, mature cells and stromal cells, interact in a complex manner through cytokines, messenger RNAs (mRNAs), microRNAs, and other non-coding RNAs (ncRNAs) –16, by tNon-coding RNAs are small RNA molecules that are not translated into a protein , and havMyeloid precursors are formed by at least four generations of cells between granulo-monocytic CFUs and the mature elements of the series. Young precursors have azurophil granulations, also called primary granulations. Lineage-specific granulations  appear at the myelocyte stage. All lineages have the same maturation steps, with the only difference occurring in lineage specific granulations –29.In normal conditions, the BM contains <5% blasts. When a mutation appears in a myeloid progenitor of the BM, the result is the development of a myelodysplastic syndrome (<20% blasts) or an acute leukemia (more than 20% BM blasts). Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells, characterized by cytopenia in the context of hyper or normocellular bone marrow, multilineage dysplasia, and frequent chromosomal abnormalities –33. The Recurrent cytogenetic abnormalities are considered diagnostic for myelodysplastic syndrome, in the presence of cytopenia of unknown etiology and in the absence of myelodysplastic changes. Morphologic changes that are characteristic for myelodysplastic syndrome are described in Depending on genetic background, MDS can either respond to standard therapies, or can evolve into an acute leukemia –43. AcutA common characteristic of acute leukemia is the hypercellularity of the bone marrow, with more than 20% blasts at the time of diagnosis. Blast subtype represents the principal diagnostic criteria between acute lymphoblastic leukemia and AML. WHO classification of AML divides them into four principal subtypes: AML with recurrent cytogenetic abnormalities, AML with dysplasia, AML associated with therapy and myelodysplastic syndrome, and the “not otherwise specified” AML .t  and inv(16), where a high percentage of patients achieve complete remission  and therapy-related AML (t-AML) are considered one entity in therapy-related myeloid malignancies because of their similar pathogenesis, rapid progression from t-MDS to t-AML, and their equally poor prognosis. A small percentage of patients present with favorable risk fusion genes, whereas 50% have adverse cytogenetics. The most frequent molecular aberration in t-AML and t-MDS affects TP53 (33%). The selection of pre-existing treatment-resistant hematopoietic stem cell clones with the TP53 mutation has been shown as an important mechanism in the development of t-MNs and explains the high frequency of TP53 mutations in these patients , 67.As previously mentioned, ncRNAs are transcripts that do not possess protein-coding activity, but regulate cell behavior through interactions established at the DNA, RNA, or protein levels. Due to their versatility, the ncRNAs are often proposed as diagnostic and prognostic tools , 69 or aIn vitro, the inhibition of SBF2-AS1 lncRNA causes leukemia cells to undergo apoptosis and cell cycle arrest. This lncRNA sponges the tumor suppressor miR-188-5p. The lncRNA DANCR is downregulated in undifferentiated hematopoietic progenitors, both malignant and BM-derived normal stem cells  and the PML-RARA fusion gene have increased co-expression of MALAT1 and NTRK3; patients with t exhibit an overexpression of MALAT1 and underexpression of NTRK3. As follows, in the clinical setting AML patients carrying the ETV6-NTRK3 gene fusion and associated MALAT1 overexpression can exhibit a better therapeutic response to NTRK3 inhibitor, AZ-23  , 116 andZEAS1 is upregulated in AML cell lines, and its knockdown lowers cell proliferation, induces apoptosis and cell cycle arrest in the G0/S phase . CCDC26 The search for lncRNAs biomarkers for AML can yield negative results. For instance, Sayad et al. have tried to analyze the expression level of FAS antisense RNA 1 (FAS-AS1) in the peripheral blood of AML patients vs. healthy controls. The study found no significant change in expression between the two groups, even though FAS has been found to play key roles in cellular apoptosis . In a sthttps://xenabrowser.net/datapages/).In the present manuscript we also present a TCGA data analysis on myeloid malignancies. The TCGA data on the clinical characteristics of the patients and RNASeq counts was downloaded from the GDC TCGA Acute Myeloid Leukemia (LAML) cohort, which included 151 patients , we downloaded the full set of human lncRNAs found in the Ensembl database. A total of 27.969 Ensemble ID codes for lncRNAs were retrieved and using a Venn diagram (http://bioinfogp.cnb.csic.es/tools/venny/), we identified the list of lncRNAs from the TCGA dataset, reaching a total of 275 lncRNAs. We ran a wide-range gene expression analysis with an online gene expression analysis tool based on the Jupyter Notebook in Python , we were able to construct three lncRNA-microRNA interaction networks that have shown common ceRNA activity between the lncRNAs with differential expression between the analyzed groups. To better illustrate the difference in expression between the three groups, we developed violin plots in the Jupyter Notebook. PRDM16-DT has a highly statistically significant (p <0.001) downregulated expression in the favorable risk category in comparison with normal or poor cytogenetic risk categories  revealed a significant clustering of lncRNA expression in the case of favorable vs. poor prognostic cytogenetic risk category . With thtegories .DANCR is upregulated in poor prognostic cytogenetic patients in comparison to the intermediate/normal risk category, while the difference between favorable and poor or normal risk is not significant .p < 0.01). The difference in expression between the other two categories is not significant   .There is published data on analysis made in AML cell lines after long-term exposure to chemotherapeutics or bone marrow analysis post-treatment. The lncRNA named urothelial carcinoma-associated 1 (UCA1) is generally overexpressed in AML and it was proven that acquired chemoresistance to adriamycin in HL60 cells is correlated with increased expression of UCA1. In adriamycin–resistant AML cells UCA1 sponges miR-125a thus releasing hexokinase 2 (HK2) and hypoxia-inducible factor 1α (HIF-1α) from its control. The overexpression of UCA1 in post-treatment AML cells was confirmed by clinical analysis done on pediatric patients . Linc002in vitro and tumor growth in vivo, thus proving the resensitizing process of AML cells . The in vivo experimental model, in which NOD-SCID mice were injected with either shRNA against ANRIL or scrambled shRNA, followed by flow cytometry analysis of their BM sample, validated the in vitro data and offered more reliability to the study (in vivo tumor formation capacity through the demethylation of the promoter region in the case of the tumor suppressor gene HOXA5 (in vitro silencing of the zinc finger antisense 1 (ZFAS1) lncRNA led to a higher percentage of AML apoptotic cells and a slower rate of proliferation of AML cells, while in vivo it resulted in the formation of smaller tumors, as compared to untreated mice (ANRIL is a lncRNA which has been reported to reprogram glucose metabolism in AML cells he study . The samhe study . HOTAIR ne HOXA5 . The in ted mice .+ cells in the bone marrow and in mononuclear cells from peripheral blood isolated from MDS patients, as compared with age-matched healthy individuals. We later discovered that the SNP rs6983267 overlapping CCAT2 is found in two allelic forms that contain either T or G and by using allele specific CCAT2 transgenic mice, confirming this data on patient samples from the US and Romania. In vivo, using transgenic mice after 7–9 months, both CCAT2-G mice and CCAT2-T mice had clinical symptoms of an underlying malignancy when compared to the wild-type siblings, having severe leukopenia and lymphocytopenia, moderate anemia and thrombocytosis (We have previously reported the potential role of the lncRNA CCAT2 in the highly amplified 8q24.21 region, with implications for both myeloid malignancies and digestive cancers , 155. Foocytosis , thus afocytosis , 135. Evocytosis –138, it cis regulatory mechanisms, it is extremely challenging to restore its function without expressing at the specific genomic locus (One of the limitations of the manuscript is the insufficient information presented regarding the potential use of lncRNAs as therapeutics. Very little information has been published thus far regarding the topic, as the mechanisms that underlie their function have yet to be fully elucidated. One direction would be to use these molecules as potential targets. If a long non-coding RNA functions via ic locus . Howeveric locus . To regaic locus . This coic locus , 143. WhStill, lncRNAs may represent interesting potential targets in AML therapy and monitoring, as they constitute most cellular transcripts and play pivotal roles in hematopoiesis , 145. FoThere are several lncRNAs that have been implicated in several roles in AML. ANRIL, RP11-732M18.3, LNC_104449, CASC15, CCDC26, DANCR, LINC00467, MIR9-3HG, AL035071.1, AP001042.1, RP11-395P13.6-001, FAM30A, H22954, LEF1-AS1, LINC00152, LINC00239, LINC00265, LINC00319, LINC00504, LINC00899, LINC00926, LNC177417, LRRC75A-AS1, MALAT1, NEAT1, PANDAR, PILNA, RP11-222k16.2, RP11-305O.6, SBF2-AS1, TUG1, UCA1, ZFAS1 have potential oncogenic roles in AML. RP11-704M14.1, SFMBT2-4:1, IRAIN, TET2 function as tumor suppressors in AML.There are two lncRNAs with both oncogenic and tumor suppressor roles in AML. MEG3 is upregulated in AML-M3, but in AML cell lines is downregulated. CRINDE is downregulated in AML-M3 and upregulated in AML bone marrow samples and cell lines.There are lncRNAs with specific expressions for each FAB AML subtype. H19 is upregulated in M1-2 AML. LINC00877, RP11-84C10.2, RP11-848P1.3, ZNF667-AS1 are downregulated in M3 AML, and LINC02082-201, AC009495.2 are upregulated in M3 AML. HOTTIP, SNHG5, CCAT1 are upregulated specifically in M4-5 AML. CCAT1, PVT1, and XLOC_109948 are overexpressed in AML patients bearing increased risk mutations. HOTAIR is upregulated only in stem cells from bone marrow samples of AML patients.Our original analysis of TCGA data on AML revealed that PRDM16-DT is downregulated, and DANCR is upregulated in poor cytogenetic prognostic AML samples compared with normal cytogenetic risk. OIP5-AS1 is downregulated in both poor and favorable prognostic compared with normal cytogenetic risk. SNHG6 is underexpressed in poor prognostic compared with intermediate cytogenetic risk and overexpressed in poor prognostic compared with favorable prognostic. SNHG16, JPX, FTX, KCNQ1OT1 are upregulated in AML samples with favorable risk as compared with normal risk. GAS5 is overexpressed in poor prognostic compared with favorable prognostic, whereas TP73-AS1 is upregulated specifically in poor prognostic patients, being overexpressed in comparison with favorable and normal cytogenetic risk. These lncRNAs have the potential of becoming future biomarkers of cytogenetic risk, however they still need further validation on bone marrow samples, peripheral blood from another cohort, along with additional gene expression analysis methods.The potential of lncRNAs in their use as biomarkers or therapeutic targets is still in its infancy. More knowledge has to be gained on their established molecular interactions, biological roles, standardization of isolation and gene expression analysis. As follows, more data is needed to fully introduce the idea of clinical uses of lncRNAs in the clinical evaluation or treatment of AML, especially in a large cohort non-interventional clinical trial that would be able to compare the sensitivity and specificity of currently applied diagnostic/prognostic methods.A-AZ did the bioinformatics analysis. CT and A-AZ wrote the manuscript. IS critically revised the manuscript. GC and IB-N contributed to the design of the manuscript and supervised the project.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.


Although evidence suggests that cigarette smoking affects inflammatory response via an increase in intracellular calcium, it remains unclear if the use of smokeless tobacco  elicits a similar response. In this study, we evaluated the effects of tobacco product preparations (TPPs), including total particulate matter (TPM) from 3R4F reference cigarettes, smokeless tobacco extract (STE) from 2S3 reference moist snuff, and nicotine alone on Ca2+ mobilization in HL60 cells. Treatment with TPM, but not STE or nicotine alone, significantly increased [Ca2+]i in a concentration-dependent manner in HL60 cells. Moreover, TPM-induced [Ca2+]i increase was not related to extracellular Ca2+ and did not require the activation of the IP3 pathway nor involved the transient receptor potential (TRP) channels. Our findings indicate that, in cells having either intact or depleted endoplasmic reticulum (ER) Ca2+ stores, TPM-mediated [Ca2+]i increase involves cytosolic Ca2+ pools other than thapsigargin-sensitive ER Ca2+ stores. These results, for the first time, demonstrate that TPM triggers [Ca2+]i increases, while significantly higher nicotine equivalent doses of STE or nicotine alone, did not affect [Ca2+]i under the experimental conditions. In summary, our study suggests that in contrast with STE or nicotine preparations, TPM activates Ca2+ signaling pathways in HL60 cells. The differential effect of combustible and non-combustible TPPs on Ca2+ mobilization could be a useful in vitro endpoint for tobacco product evaluation.Changes in the level of intracellular calcium ([Ca Chronic smoking is associated with the onset of chronic obstructive pulmonary disease and is a major risk factor for lung cancer and cardiovascular disease –5. CombuA number of studies have reported that the use of smokeless tobacco (ST) products is associated with a reduced risk of adverse health effects compared with combustible cigarette smoking –17. For Chronic smoking impacts several key immune functions, including adaptive and innate immune functions –25. Addi2+]i, which, in turn, activates diverse signal transduction pathways involved in regulating cell function. Two major mechanisms contribute to the elevation of [Ca2+]i are: (1) store-operated calcium entry (SOCE) from intracellular pools, including the endoplasmic reticulum (ER), mitochondria, and lysosomes and (2) receptor-operated calcium entry (ROCE) of extracellular Ca2+. Both processes often occur either simultaneously or sequentially. Additionally, in many excitable cell types, entry of Ca2+ through transient receptor potential (TRP) channels can be activated by membrane depolarization i in human leukemia HL60 cells. The objective of this study was to evaluate if combustible TPM differentially induces [Ca2+]i compared with STE and nicotine alone. We performed a series of experiments to investigate whether (1) [Ca2+]i induced by combustible TPM or STE was mediated through SOCE and/or ROCE and (2) the role of IP3 receptors and TRP channels using pharmacological inhibitors to better understand the effect of the TPPs on intracellular Ca2+ mobilization.In this study, we evaluated the effects of tobacco product TPM and smokeless tobacco extract (STE) from reference tobacco products as well as nicotine alone, on i concentration, cells were loaded with Fluo-3AM  calcium indicator dye. A Fluo-3AM stock solution of 1.25 mM was prepared in DMSO. Control and treated cells were pelleted and washed in warm MACS running Buffer (Miltenyi Biotec) at a density of 1 × 106 cells/mL. A 4.4 μM working solution of Fluo-3AM was prepared in warm MACS running Buffer and added to the pelleted cells and incubated at 37 °C for 30 min. Cells were washed three times with warm MACS running buffer and resuspended in 400 μL of 1 mM EGTA solution made with MACS running buffer to provide Ca2+ free cell suspension. The cell suspensions were then transferred to cluster tubes for acquisition on the flow cytometer .In order to measure the changes in i compared with the PB vehicle control  was significantly larger than the increase at the low 1 μg/mL dose or control. Thus, while the [Ca2+]i increase was slightly greater in the presence of extracellular CaCl2 at all treatments compared with absence of externally provided calcium, the difference was not statistically significant, indicating that TPM did not mobilize significant extracellular Ca2+ when intracellular ER stores were available. In contrast to the effects of CaCl2 following TPM exposure, addition of extracellular CaCl2 following exposure to different concentrations of STE plasmic reticulum calcium ATPase (SERCA). In order to determine the role of ER Ca2+ stores on the TPM-induced increase in [Ca2+]i, we depleted ER Ca2+ stores using the SERCA inhibitor TG [2+]i required ER calcium stores, their depletion by TG would impact the magnitude of the response from the test materials on [Ca2+]i.In lymphocytes and polymorphonuclear leukocytes, most of the intracellular calcium is stored within the ER. The intracellular calcium concentration is therefore mainly regulated by calcium release from the ER bitor TG , 42. We 2+ free media to TG (5 μM), preceding the application of TPM  or STE . To further test whether the TG-induced depletion of intracellular stores could trigger a capacitative Ca2+ entry through the plasma membrane, we continued the experiment by adding CaCl2 (0.5 mM) to the extracellular media, providing an extracellular calcium source to the cells.We exposed Fluo-3AM–labeled HL60 cells in Ca2+]i in a dose-dependent manner compared with the DMSO vehicle control (p < 0.05)  Fig. , indicat2+]i increase, we tested the role of TRP channels present on the plasma membrane. HL60 cells were maintained in Ca2+-free media together with non-selective TRP channel blockers, either ruthenium red  or SKF-96365  . DMSO was used as a negative vehicle control.To further investigate the mechanisms underlying TPM-induced [CaμM) Fig. . The Flu2+]i in the presence of extracellular CaCl2 tended to be lower at all TPM concentrations, compared with the response with no inhibitors. However, the differences were not statistically significant. Ru and SK had no effect on the DMSO vehicle control group , with a view to understanding if combustible and smokeless tobacco preparations demonstrate different impacts on intracellular calcium levels and, possibly, the underlying mechanisms of action. A key finding of the present study shows that TPM treatment increased [Ca2+]i in a dose-dependent manner, starting at very low equi-nicotine concentrations.In this study, we sought to elucidate the pathways mediating any TPP-induced [Ca2+]i mobilization [2+]i increases, even at equi-nicotine concentrations that are two orders of magnitude higher than the TPM tested in the same cell type used in this study. For example, TPM-mediated [Ca2+]i increase occurred at equi-nicotine concentrations as low as 1 μg/mL, whereas a higher equi-nicotine level of 250 μg/mL of STE had no effect. These results suggest that combustion-related toxicants may play a role in the mobilization of [Ca2+]i when compared with smokeless tobacco products in the HL60 leukemia cell line. In line with our findings, Sassano et al. (2017) assessed the impact of 13 individual tobacco smoke constituents on [Ca2+]i in HEK293T cells and found that 1-aminonaphthalene, formaldehyde, and nicotine-derived nitrosamine ketone (NNK) were able to elicit [Ca2+]i increases [Other studies have similarly reported that cigarette smoke extract triggers [Calization –49. In cncreases .2+]i. This finding is in apparent contrast with the findings of Sassano et al. (2017) who found that nicotine triggered increases in [Ca2+]i in both HEK293T and human bronchial epithelial cells (HBEC). However, their study did not compare equi-nicotine units of nicotine and TPPs. Hence, the relative impact of nicotine merits further characterization using different cell types.In our study, testing nicotine alone, even at concentrations two orders of magnitude higher (100 μg/mL) than those present in the TPM, did not induce changes in [Ca2+]i in the HL60 cell line, we evaluated capacitative Ca2+ mobilization by supplementation of external CaCl2. Our results indicated that TPM-induced mobilization of extracellular CaCl2 is not significant. Similarly, Rasmussen et al. (2014) reported that the vapor phase of cigarette smoke increased [Ca2+]i and that this increase was unaffected by extracellular Ca2+ chelation i increase independently of ER store depletion, suggesting that TPM-mediated [Ca2+]i increase occurred through TG-insensitive Ca2+ stores. This finding is in line with published work from Rasmussen et al. i and could therefore activate Ca2+-dependent signaling pathways, potentially leading to altered inflammatory responses in leukocytes. This study provides an understanding of the differential effects of different tobacco products on inflammatory responses in HL60 cells. Additional studies are warranted to identify the source of TPM-induced intracellular calcium mobilization.In this study, we evaluated the effects of TPPs from combustible and non-combustible tobacco products, as well as of nicotine alone, on cellular Ca