S. aureus and P. aeruginosa for disinfectant efficacy testing and (ii) investigate the effect of drying times and temperatures on DSB development. We hypothesized that a minimum six log10 density of DSB could be achieved on glass coupons by desiccating wet surface biofilms near room temperatures. We also hypothesized that a DSB produced by the model in this study will be encased in extracellular polymeric substances (EPS).Bacterial biofilms persistent on dry environmental surfaces in healthcare facilities play an important role in the occurrence of healthcare associated infections (HAI). Compared to wet surface biofilms and planktonic bacteria, dry surface biofilms (DSB) are more tolerant to disinfection. However, there is no official method for developing DSB for in vitro disinfectant efficacy testing. The objectives of this study were to (i) develop an in vitro model of DSB of S. aureus ATCC-6538 and P. aeruginosa ATCC-15442 wet surface biofilms were grown on glass coupons following EPA MLB SOP MB-19. A DSB model was developed by drying coupons in an incubator and viable bacteria were recovered following a modified version of EPA MLB SOP MB-20. Scanning electron microscopy was used to confirm the EPS presence on DSB.10 densities of DSB for disinfectant efficacy were recovered per coupon after drying at different temperatures and drying times. Regardless of strain, temperature and dry time, 86% of coupons with DSB were confirmed to have EPS.Overall, a minimum of six mean logA rapid model for developing DSB with characteristic EPS was developed for disinfectant efficacy testing against DSB. Healthcare Associated Infections (HAIs) are a major cause of morbidity and mortality in the United States , 2. HAIsStaphylococcus aureus and Pseudomonas aeruginosa are some of the most common causes of HAIs [ of HAIs , 8. Thes of HAIs , 10. The of HAIs , 11, 12. of HAIs ; they ar of HAIs .S. aureus under low shear conditions is limited [P. aeruginosa [S. aureus [S. aureus can be dispersed to other surfaces even though they are dehydrated [S. aureus and P. aeruginosa DSB for disinfectant efficacy testing. In this study, we investigated the effect of different drying times and temperatures on the development of DSB of S. aureus and P. aeruginosa. We hypothesized that we would achieve a minimum six log10 bacterial density on DSB per test coupon for disinfectant efficacy testing by subjecting wet surface biofilms to desiccation at near room temperatures. We also hypothesized that DSB of S. aureus and P. aeruginosa produced by the model in this study will be encased in EPS similar to those of wet surface biofilms to mimic associated disinfection challenges.Environmental surfaces   and mobi limited . Once fo limited , 23. The limited , 25 as t limited . Althoug limited , 26. At ruginosa  and S. a. aureus  wet surf. aureus , 13, 18.hydrated . Howeverhydrated . The usehydrated , 30. Stuhydrated –33. In ahydrated . The tolhydrated , 36. Comhydrated . Howeverhydrated , a compahydrated . Followihydrated , a two-hhydrated . The objS. aureus ATTC-6538 and P. aeruginosa ATCC-15442 biofilms were developed on borosilicate glass coupons . These are standard strains for EPA disinfectant efficacy testing on wet surface biofilms and for disinfectant registration for use in healthcare facilities [cilities .S. aureus or P. aeruginosa overnight culture were transferred into 500 mL tryptic soy broth  batch culture medium (three TSB/L for S. aureus and 300 mg TSB/L for P. aeruginosa) in a CDC biofilm reactor . The CDC biofilm reactor was mounted on a magnetic hot plate stirrer  to initiate the growth of biofilms for 24 ± 2 h. The hot plate stirrer was set at a rotational speed of 60 ± 5 rpm at 36 ± 1 °C for S. aureus. The rotational speed and hot plate stirrer temperature for P. aeruginosa was 125 ± 5 rpm and 21 ± 2 °C, respectively, following EPA MLB SOP MB-19. The Continuous Stir Tank Reactor (CSTR) phase immediately followed the batch phase and involved a continuous flow of CSTR medium for 24 ± 2 h. A total of 500 mL or 50 mL of CSTR medium was added to 20 L of sterile distilled water in a carboy for S. aureus or P. aeruginosa respectively. This resulted in a final growth medium concentration of 1 g/L TSB for S. aureus and 100 mg/L TSB for P. aeruginosa. The 20 L CSTR growth medium in the carboys were preheated to 36 ± 1 °C S. aureus and maintained at 21 °C for P. aeruginosa before continuously pumping the medium through the CDC biofilm reactor for 24 ± 2 h. The CSTR growth medium was pumped  at a rate of 30 ± 2 min residence time for S. aureus and P. aeruginosa.Wet surface biofilms were established on coupons for 48 h following EPA MLB SOP MB-19-for growing biofilms using a CDC biofilm reactor . Wet surS. aureus and P. aeruginosa were continuously dried in a standard laboratory incubator to develop DSB while still contained to the rod. The dry times for both S. aureus and P. aeruginosa coupons were 24 h, 48 h, 72 h, 96 h, and 120 h. Bacteria recovery from DSB was done within 120 h as the CDC recommends routine disinfection of healthcare surfaces at least daily or every other day (approximately 48 h) [S. aureus to mimic near and elevated room temperatures and 16 °C and 21 °C as guided by preliminary studies conducted by our research group to establish dry surface P. aeruginosa biofilms. Incubators were disinfected with 70% ethanol prior to drying coupons. Incubator doors were also kept closed during drying.Polypropylene rods holding coupons were rinsed in 25 ml phosphate buffered saline (PBS) for 30 s to remove planktonic bacteria cells following EPA MLB SOP MB-20  before dly 48 h) . Drying S. aureus; 16 °C and 21 °C for P. aeruginosa) and at each dry time (24–120 h), three coupons with DSB  were harvested from the rods in the incubator and transferred into three 50 mL sterile conical tubes . Coupons were harvested successively at 24 h, 48 h, 72 h, 96 h and 120 h and processed to establish mean log10 bacterial densities per coupon. Three control coupons with wet surface biofilms were also processed to detect viable bacteria from biofilms before dehydration. A control coupon was also harvested before drying, at each dry time, and at each drying temperature for scanning electron microscopy (SEM) imaging.Bacterial cells from DSB were recovered following a modified version of EPA MLB SOP MB-20 for testing the efficacy of disinfectants against bacterial biofilms . At ever2O + 5.2 g Difco neutralizing buffer; Becton, Dickinson and Company Sparks, MD) was added. Bacterial biofilms were released from coupons into solution through alternate cycles of vortexing and sonication [S. aureus and P. aeruginosa from coupons were quantified after serial dilution with PBS, and ten mL aliquots vacuum-filtered onto sterile filter membranes  and incubated following EPA MLB SOP MB-20.Five biological replicates were conducted for each dry temperature. Each batch of three control coupons with wet surface biofilms and 15 technical replicates with DSB (N = 18 coupons) made up one biological replicate.Viable bacterial cells were recovered from coupons in 50 mL sterile conical tubes with four mL of PBS. After one min, 36 mL of neutralizing buffer  and fixed for SEM imaging following the step-wise sample fixation procedure used by the Life Sciences Core facility at Purdue University and as described by Murtey and Ramasamy [2/coupon.Coupons for SEM imaging were stored in an aldehyde fixative  at 4 °C before sample preparation for imaging. Bacteria cells on control coupons (wet surface biofilms) and DSB coupons were fixed for SEM imaging to confirm the presence of Ramasamy  with mod10 densities were calculated based on CFU counts from control and DSB coupons. Average bacterial log10 densities for each dry time and temperature were compared for significant statistical differences using the least squares method of Proc Glm to fit linear models . The interaction between each dry time and temperature were also analyzed using the same procedure. Significant differences between group means of bacterial log10 densities per dry time and temperature were compared pair-wise using Tukey adjustments SAS version 9.4  for all statistical analysis. Multiple logistic regression analysis was used to determine the relationship between the EPS presence and mean log10 densities per coupon.Mean bacterial logS. aureus mean log10 density per coupon ranged from 6.64 ± 0.08  after 24 h of drying to 6.01 ± 0.38 (CoV = 6.32%) at a dry time of 120 h. Specifically, mean log10 densities of 6.69 ± 0.45 (CoV = 6.73%) and 6.58 ± 0.60 (CoV = 9.12%) S. aureus DSB were recovered per coupon after drying for 24 h at 25 °C and 30 °C, respectively. Statistically, the bacterial mean log10 densities per coupon after 24 h of drying were not significantly higher than those achieved after 48 h and 72 h dry times at 25 °C or 30 °C . However, mean log10 densities after 24 h of drying were significantly higher than those achieved after 96 h and 120 h of drying (P < 0.05). Overall, there were no significant differences in the average log10 densities of DSB per coupon between different dry times  or temperatures (25 °C and 30 °C) in our method. (P ≥ 0.05).For the purpose of this study, we defined DSB as “hydrated” bacterial biofilms that remained viable despite prolonged dehydration 24 h – 120 h) under low nutrient availability and were confirmed by SEM imaging to be encased by EPS. For both drying temperatures (25 °C or 30 °C), the overall 20 h unde10 density of 7.85 ± 0.08 per coupon, and regardless of the dry time and temperature, mean log10 densities of dry surface S. aureus biofilms per coupon were significantly lower . On average, the exposure of wet surface biofilms to dehydration resulted in a 1.56 ± 0.30 log10 reduction per coupon.Compared to control coupons with wet surface biofilms that had an average mean log10 densities of P. aeruginosa DSB were achieved after drying at 16 °C compared to 21 °C  per coupon after drying at 24 h to 7.78 ± 0.72 (CoV = 9.25%) at 120 h when dried at 16 °C. However, when dried at 21 °C, the average P. aeruginosa log10 DSB density per coupon reduced to 7.37 ± 0.78 (CoV = 10.58%) at a dry time of 24 h and 6.30 ± 0.94 (CoV = 14.92%) after 120 h.On average, statistically higher logP. aeruginosa biofilm mean log10 densities achieved after drying at 16 °C and 25 °C for 24 h, 48 h, 72 h, 96 h and 120 h, as well as for log10 densities achieved from wet surface biofilms on control coupons  with DSB of S. aureus had detectable EPS. Specifically, 92% (23/25) and 76% (19/25) of all coupons with S. aureus DSB developed at 25 °C and 30 °C, respectively, had visible EPS through SEM imaging  of coupons harvested between 48 h and 120 h at 25 °C and 30 °C. At 25 °C EPS was identified in 100% (15/15) of S. aureus DSB dried for 24 h, 48 h and 72 h  and dehydration times 24 h, 48 h, 72 h, 96 h and 120 h. We found that overall, there were no statistically significant differences in the mean log10 densities of DSB per coupon when the drying temperature was 25 °C or 30 °C. For P. aeruginosa DSB developed on coupons, we found significant differences in mean log10 densities per coupon between drying temperatures of 16 °C or 21 °C. For S. aureus and P. aeruginosa, the average log10 density of DSB met and exceeded the US Environmental Protection Agency (EPA) minimum standard of six log10 [In this study, we developed a rapid in vitro model for establishing DSB of products . Specifiix log10  required10 standard of wet surface biofilms as our model validation standard. A six log10 density of DSB was achieved for S. aureus and P. aeruginosa. Our findings are similar to those of Almatroudi et al. who established an average log10 density of 7.13 ± 0.04 of partially dehydrated dry surface S. aureus per coupon using a 12-day biofilm development model [10 CFU of S. aureus DSB per coupon [S. aureus had similar average log10 densities of DSB per coupon (6.69 ± 0.45 and 6.58 ± 0.60 per coupon) after a 24 h complete desiccation step at 25 °C or 30 °C, respectively. Further desiccation from 48 to 120 h did not result in a significant decline in mean log10 densities of DSB per coupon, suggesting the consistency of our rapid method and the persistence of DSB of S. aureus on environmental surfaces under adverse conditions. In a recent review by Onyango and Alreshidi, the authors reported that Staphylococci could be persistent in nutrient–deficient environments in healthcare settings and contribute significantly in the incidence of HAI [S. aureus included repeated cycles of hydration and dehydration [10 densities of DSB per coupon. Once formed, DSB may stay dehydrated and may only rehydrate when surface disinfection occurs [While there is currently no standard for testing bactericidal efficacy of disinfectants against DSB , we elecnt model . Chowdhunt model  and achir coupon . The rapn occurs . The CDCn occurs . Conside10 densities after dehydrating wet surface biofilms of P. aeruginosa for 24 h compared to S. aureus.. However, there were no significant reductions in the mean long10 densities of P. aeruginosa DSB per coupon with prolonged drying from 48 to 120 h. This suggests rapid maturation of P. aeruginosa DSB during early drying stages. Meesilp and Mesil suggested that within a two-hour period, S. aureus and P. aeruginosa inoculated on stainless steel surfaces could develop into mature biofilms [10 densities were achieved on all dried coupons compared to controls. This finding is similar to those of Meesilp and Mesil who suggested that although mature, the average density of biofilms on stainless steel surfaces in vitro reduced after 24 h of growth [10 densities per coupons expected from wet biofilm preparation following standard EPA methods (8.0–9.5 log10 CFU/coupon for P. aeruginosa and 7.5–9.0 log10 CFU/coupon for S. aureus) [10 biofilm density required for disinfectant efficacy claims against biofilms.We achieved higher mean logbiofilms . As expef growth . While w aureus) , our rap10 density of viable bacteria cells recovered from DSB of S. aureus and P. aeruginosa and the presence of EPS on sampled coupons. Specifically, we found that although relatively lower mean log10 CFU of DSB per coupon were recorded at near room temperatures , more DSB developed at these temperatures after 24 h, 48 h and 72 h of drying were encased within EPS. This possibly explains why it was more difficult to recover higher mean log10 CFU per coupon at these temperatures than at 16 °C and 30 °C. Previous authors reported that the EPS encases underlying bacterial cells, preventing direct contact with solutions such as disinfectants [S. aureus and Pseudomonas spp. were among the most prevalent pathogens on the studied surfaces, with SEM imaging confirming the presence of EPS clusters [S. aureus had multi-layered biofilm matrices similar to those on dry sterile supply boxes in a hospital [S. aureus and Pseudomonas spp. biofilms are more likely to be encased within an EPS. Specifically, DSB survive drying through EPS production, and the expression of inherent resistance traits against environmental stress factors [S. aureus and P. aeruginosa quickly recover in media as exposure to one form of stress may induce protection against other stress factors [Overall, we found a significant relationship between the mean logfectants , 11, 12.clusters . Almatrohospital . These p factors . This ha factors , 51, whi factors .S. aureus and P. aeruginosa biofilms at near room temperatures could provide a more robust and challenging scenario for disinfectant efficacy testing and subsequent product registration with the EPA. Moreover, DSB are more tolerant to disinfection than wet surface biofilms [With regards to disinfectant efficacy testing against DSB, developing biofilms , 37. Whi10 densities of DSB significant for disinfectant efficacy can be developed at near room temperatures. We also found that despite prolonged drying, the bacterial density of DSB per coupon does not significantly reduce over time. Further, DSB developed at near room temperatures are predominantly encased within EPS. It is therefore critical to patient safety for the EPA to establish an efficacy method for testing disinfectants against DSB as recent studies have called into question the validity of using planktonic testing methods to represent a real world environment, where bacteria are generally encased in a biofilm on surfaces [We found that mean logsurfaces , 30. As


Mouse AM in controls exhibited active movement in vitro, and TUG-891 significantly restrained AM movement. Meanwhile, TUG-891 stimulated a quick increase in [Ca2+]i in AM, which was blocked separately by the Gq protein inhibitor YM-254890, the phospholipase C (PLC) inhibitor U73122, or depletion of endoplasmic reticulum (ER) Ca2+ store by thapsigargin. The inhibition of AM movement by TUG-891 was eliminated by YM-254890, U73122, thapsigargin, and chelation of cytosolic Ca2+ by BAPTA. Moreover, TUG-891 inhibited AM phagocytosis of fluorescent microspheres, which was also blocked by YM-254890, U73122, thapsigargin, and BAPTA. In conclusion, GPR120 activation in mouse AM increases [Ca2+]i but inhibits the motility and phagocytosis via Gq protein/PLC-mediated Ca2+ release from ER Ca2+ store.Movement and phagocytosis characterize the fundamental actions of macrophages. Although it is known that the free fatty acid receptor GPR120 is expressed in macrophages and regulates cytokine expression to exert anti-inflammatory activities, the effects of GPR120 activation on the motility and phagocytosis of macrophages are not clear. In this study, mouse alveolar macrophages (AM) were stimulated with the GPR120 agonist TUG-891, and the changes in cell motility, intracellular Ca The regoduction .2+ [2+-permeable ion channels that are located either on organelles such as endoplasmic reticulum (ER) or on the plasma membrane [2+ channel opening and increase the intracellular Ca2+ concentration ([Ca2+]i) in macrophages [2+]i increase in macrophages [2+]i is linked to macrophage activities and regulates a variety of functions such as cytokine production, inflammasome formation, and phagocytosis [2+]i to stimulate glucagon-like peptide-1 (GLP-1) secretion in enteroendocrine cells [2+]i and [Ca2+]i is related to motility and phagocytosis of macrophages, it is proposed that GPR120 activation and the following PLC-stimulated signaling may regulate the motility and phagocytosis of macrophages. It is necessary to clarify the effects of GPR120 activation on the motility and phagocytosis of macrophages for the comprehensive understanding of the role of GPR120 in immune regulation.Macrophages not only release cytokines but also possess the actions of movement and phagocytosis. The motility and phagocytosis of macrophages guarantee them to remove foreign pathogens and ensure body innate immunity . Phagocy2+ , 11. Macmembrane . Gq protrophages , 14. Calrophages –17. i according to previous reports i of macrophages. The extracellular solution used in [Ca2+]i measurement was the same as that used in movement measurement.Fluo-3 was used for the measurement of i in AM (μmol/L), the inhibitor of PLC, also significantly suppressed TUG-891-stimulated increase in [Ca2+]i in AM (μmol/L for 30 min) that inhibits the calcium pump in the ER [2+ leakage and depletion of Ca2+ in ER Ca2+ store also significantly suppressed TUG-891-stimulated increase in [Ca2+]i in AM . The statistical analysis of the effects of the inhibitor is shown in P < 0.01vs. control, n = 20). To clarify the special effect of TUG-891 on [Ca2+]i in AM, mouse blood lymphocytes were collected to observe the effects of TUG-891. GPR120 is not expressed in lymphocytes, and TUG-891 did not have effects on [Ca2+]i in lymphocytes  incubation significantly suppressed the inhibitory effects of TUG-891 (10 μmol/L) on macrophage movement compared with the placebo control, which was indicated by the changes in the cell area and the variation of the cell area . Treatment with U73122 (1 μmol/L for 10 min) and thapsigargin (5 μmol/L for 30 min) also significantly suppressed TUG-891-induced inhibition of the movement of AM as indicated by cell areas and the variation of the cell area . Chelation of cytosolic Ca2+ by BAPTA-AM (5 μmol/L for 30 min) also significantly suppressed TUG-891-inhibited movement of macrophages . The changes in the cell area and the variation of the cell area were analyzed for each group  and low phagocytic macrophages with the mean fluorescence intensity of 684.08 ± 46.61 . The percentage of highly phagocytic macrophages was decreased to 41.99% ± 9.15% of total cells in the TUG-891 treatment group, which was significantly lower than that of the control . In contrast, the percentage of low phagocytic macrophages increased to 55.48% ± 9.32%, showing a significant difference compared with the control . The mean fluorescence intensities of the total macrophages significantly decreased after TUG-891 treatment compared with those of the control . YM-254890 (1 μmol/L), U73122 (1 μmol/L for 10 min), thapsigargin (5 μmol/L for 30 min), and BAPTA (5 μmol/L for 30 min) all significantly suppressed the inhibitory effects of TUG-891 on phagocytosis of the macrophages as indicated by the decrease in low phagocytic macrophages and the increase in mean fluorescence intensities of the total macrophages .Mouse AM in the placebo control phagocytized the fluorescent microspheres and showed increased fluorescence as measured by flow cytometry . In cont2+]i in mouse AM but inhibits the movement and phagocytosis of mouse AM. It has been known that GPR120 activation suppresses the expression of proinflammatory cytokines and promotes the expression of anti-inflammatory cytokines in macrophages, which indicates the role of GPR120 in anti-inflammation i are related to pseudopod stretching and chemotactic migration of macrophages, and it is generally recognized that an appropriate fluctuation of [Ca2+]i is required for the movement of macrophages [2+]i with specific movement of macrophages and its mechanism remains to be clarified. In this study, AM contracted to the round shape with a significant decrease in the formation of pseudopods in parallel with the increase in [Ca2+]i when they were stimulated with TUG-891. Therefore, TUG-891-induced increase in [Ca2+]i did not promote the movement of macrophages, but it unexpectedly inhibited the movement of macrophages. The concurrence between [Ca2+]i increase and pseudopod retraction with cell area reduction was also observed in another type of macrophages, the osteoclast, when it was stimulated with a platelet-activating factor [2+]i are not clear. An appropriate rise of [Ca2+]i with local oscillation in a certain pattern ensured the normal movement and phagocytosis of the macrophages. It is proposed that the intense rising of [Ca2+]i that was stimulated with TUG-891 led to continuous strong contraction of skeleton proteins in macrophages, which causes their inability to free stretch and consequently restrained AM movement.Cell movement is a dynamic process in which the skeleton proteins play a vital role . The act2+ . [Ca2+]irophages , 40. Howg factor , 42. The3 signaling molecules, which stimulated Ca2+ release from the ER and lead to PKC activation. This signaling pathway was related to GPR120 agonist-stimulated secretion of GLP-1 from enteroendocrine cells [2+]i as well as the inhibition of macrophage movement that was induced by TUG-891. It indicates that TUG-891 stimulates the increase in [Ca2+]i in AM through Gq protein to activate PLC and the following phosphatidylinositol signaling pathway. In addition, the depletion of intracellular Ca2+ store of ER by thapsigargin eliminated TUG-891-stimulated increase in [Ca2+]i in macrophages. Therefore, this study indicates that TUG-891 stimulated the increase in [Ca2+]i through Gq protein and Ca2+ mobilization of intracellular Ca2+ stores in mouse AM. On the other hand, removal of extracellular Ca2+ attenuated TUG-891-stimulated increase in [Ca2+]i in macrophages. It is suggested that Ca2+ release triggered Ca2+ influx through the plasma membrane. Ca2+ influx through the plasma membrane is mediated by variable Ca2+-permeable ion channels, and the specific ion channels need to be investigated in the future study.Previous studies showed that GPR120 acts through Gq protein to activate PLC and the following DAG/IPne cells . In this2+ by BAPTA. It is suggested that TUG-891 activated Gq/PLC-DAG/IP3-Ca2+ signaling to inhibit phagocytosis of AM.As shown by the phagocytosis of fluorescent microspheres, AM in this study exhibited phagocytic ability, which is exactly consistent with previous reports , 44. TUGTUG-891 was used in this study to activate GPR120 for two reasons. First, TUG-891 is a more selective and potent agonist for GPR120 than GW9508 and NCG21 , 46. SecGPR120 activation-induced inhibition of AM phagocytosis possibly is involved in maintaining a nonactivating state of macrophages to avoid tissue inflammatory damage. It was found that lipopolysaccharide, the indicator of bacterial infection, inhibited GPR120 expression in macrophages . Here, w


Activation of this channel is mediated via binding intracellular Ca2+ to the channel with a relatively high apparent affinity, roughly in the sub-μM to low μM concentration range. Recently available high-resolution structures of TMEM16 molecules reveal that the high-affinity Ca2+ activation sites are formed by several acidic amino acids, using their negatively charged sidechain carboxylates to coordinate the bound Ca2+. In this study, we examine the interaction of TMEM16A with a divalent cation, Co2+, which by itself cannot activate current in TMEM16A. This divalent cation, however, has two effects when applied intracellularly. It inhibits the Ca2+-induced TMEM16A current by competing with Ca2+ for the aforementioned high-affinity activation sites. In addition, Co2+ also potentiates the Ca2+-induced current with a low affinity. This potentiation effect requires high concentration (mM) of Co2+, similar to our previous findings that high concentrations (mM) of intracellular Ca2+ ([Ca2+]i) can induce more TMEM16A current after the Ca2+-activation sites are saturated by tens of μM [Ca2+]i. The degrees of potentiation by Co2+ and Ca2+ also roughly correlate with each other. Interestingly, mutating a pore residue of TMEM16A, Y589, alters the degree of potentiation in that the smaller the sidechain of the replaced residue, the larger the potentiation induced by divalent cations. We suggest that the Co2+ potentiation and the Ca2+ potentiation share a similar mechanism by increasing Cl- flux through the channel pore, perhaps due to an increase of positive pore potential after the binding of divalent cations to phospholipids in the pore. A smaller sidechain of a pore residue may allow the pore to accommodate more phospholipids, thus enhancing the current potentiation caused by high concentrations of divalent cations.TMEM16A, a Ca It is highly expressed in the apical membrane of ductal epithelial cells, and one well-documented physiological function of this channel known for decades is its critical roles in transepithelial Cl- transport i). The other type of TMEM16 family members, such as fungus nhTMEM16, afTMEM16 and mammalian TMEM16F, are phospholipid scramblases. One documented physiological role of TMEM16F is to scramble phospholipids in platelet cell membranes and thus expose phosphatidylserine to the extracellular environment, an important step in the signaling cascade for blood coagulation [2+]i not only leads to scrambling of membrane phospholipids but also causes ionic conduction across lipid membranes. The physiological roles of the current conduction in these phospholipid scramblases remain to be identified.The TMEM16 gene family consists of two types of transmembrane proteins with distinct molecular functions: ion channels and phospholipid scramblases . TMEM16A neurons , 8. Physgulation –11. A degulation , 12. IntAlthough the physiological functions of ion channels and phospholipid scramblases appear to be quite different, the structures of these two types of TMEM16 proteins are similar to each other –18. Thes2+-activation sites have also been identified in the high-resolution structures of TMEM16 molecules ) in activating the channel—mutation of a single glutamate or aspartate residue increases the intracellular [Ca2+] ([Ca2+]i) required for channel activation by three orders of magnitude i. Yet, if [Ca2+]i is increased to hundreds of μM or to mM concentrations, TMEM16A current can be further increased—the current induced by 20 mM [Ca2+]i was ~30–40% larger than that activated by 20 μM [Ca2+]i [2+-activation sites have already been saturated by 20 μM [Ca2+]i, what is the mechanism responsible for the current induced by mM [Ca2+]i? Is the current induced by mM [Ca2+]i mediated by different Ca2+-activation sites or is the mechanism of this low affinity Ca2+ effect mediated by the high affinity Ca2+-binding sites formed by the aforementioned negatively-charged residues? To further examine the properties of TMEM16A, we initially aimed at using cobalt ions (Co2+), to interact with the channel activation sites on this Cl- channel. We found that intracellular Co2+ is a competitive inhibitor for the Ca2+ activation of TMEM16A. The experiments also lead to a serendipity finding that high concentrations (hundreds of μM or above) of intracellular Co2+ ([Co2+]i) potentiate the Ca2+-induced TMEM16A current. The results suggest that the TMEM16A current potentiation by mM [Co2+]i and that by mM [Ca2+]i described above may come from the same mechanism, thus explaining the biphasic dose-dependent activation curve of TMEM16A. Interestingly, mutating a pore residue of TMEM16A affects the degree of potentiation, suggesting that the relatively low affinity potentiation site(s) for these two cations may reside in or near the anion transport pathway.While the structural features reasonably explain some of the molecular properties of the TMEM16 proteins, the mechanisms of other functional phenomena of TMEM16 molecules remain unclear. For example, the biphasic dose-response activation curve for the activation of TMEM16A by Capuzzling , 29. It o low μM , 30–32.  [Ca2+]i , 29. If a” alternative splice variant i, EGTA was not included because of two considerations. First, including a Ca2+ buffer (such as EGTA) in solutions containing both Ca2+ and Co2+ would alter the free concentration of each ion in a complicated manner. Second, most of the solutions used in this study contained [Ca2+]i in μM to mM range, which is way beyond the buffering range of EGTA. Without the added EGTA, the free [Ca2+]i was assumed to be equal to the total added [Ca2+]i. Nonetheless, it should be noticed that when the indicated [Ca2+]i was low (such as 2 or 5 μM), the real [Ca2+]i was likely slightly higher because of the contaminating Ca2+. Based on the current of a TMEM16F mutant induced by an intraceullar solution containing neither EGTA nor Ca2+, the contaminating [Ca2+]i in this nominal zero-Ca2+ solution was less than 1 μM  and had a final [Cl-] of 140 mM. In the experiments of assessing Co2+ effects, the intracellular solutions contained Co2+ up to 20 mM. Because the source of Co2+ was from CoCl2, the concentration of NaCl in the intracellular solution was reduced according to the extra [Cl-] from the added [CoCl2]. For example, the solution containing 20 mM [Co2+] included 100 mM [NaCl] and 20 mM [CoCl2]. In experiments using high [Ca2+]i, the intracellular [NaCl] was also adjusted accordingly if the total [CaCl2] in the solution was more than 0.5 mM. These adjustments, likely would alter the ionic strength of the solution, and therefore, the measurements obtained with high divalent cation concentrations (such as 5–20 mM) may be less accurate.Twenty-four to forty-eight hours after transfection, patch-clamp recordings were conducted on HEK293 cells with green fluorescence. All experiments were from excised inside-out membrane patches. The pipette (extracellular) solution contained 140 mM NaCl, 10 mM HEPES, and 0.1 mM EGTA (pH 7.4). This solution was also used as the intracellular solution containing “zero Ca1 μM see . Such a 2+ solution. [Ca2+]i was then applied to open the TMEM16A channel, followed by the application of a particular [Co2+]i (with the same [Ca2+]i) for 6 sec, which allowed the Co2+ effect to reach a steady state. Co2+ was then removed by changing the solution back to the one before applying [Co2+]i. Finally, the intracellular Ca2+ was removed using the EGTA-containing zero-Ca2+ solution. In evaluating the kinetics of the current activation or deactivation upon applying or removing [Ca2+]i  using a PP830 electrode puller (Narishige). The electrode tip was ~1–2 μm in diameter, and the electrode resistance was between ~1.5 MΩ and ~3 MΩ when filled with the extracellular solution. Voltage clamp experiments were conducted using the Axopatch 200B amplifier (Molecular Devices), and the current was digitized via a Digidata1440 analog-digital signal-converting board controlled by the pClamp10 software (Molecular Devices). Exchanging solutions on the intracellular side of the excised inside-out patch was achieved using the SF-77 solution exchanger (Warner Instruments). Except where indicated, the recording was initiated by stepping the membrane voltage to ±20 mV in the EGTA-containing zero-Ca2+]i, we employed a three-pulse protocol described previously [2+]i, a test solution containing [Ca2+]i from 50 μM to 20 mM, and then again the 20 μM [Ca2+]i solution. We used this experimental protocol to minimize the effect of current rundown so that a dose-response effect of the test [Ca2+]i can be more precisely evaluated. The current induced by the test [Ca2+] was then normalized to the average of the two currents obtained in 20 μM [Ca2+], and the normalized values were used for generating dose-response curves.To assess the current potentiation by high [Caeviously . The cyt2+] was first subtracted from the current obtained in the presence of [Ca2+]i. Upon the application of [Co2+]i, the Ca2+-induced current of TMEM16A was potentiated almost immediately followed by a slower process of inhibition. We named I0 as the control current immediately before the application of [Co2+]i. The peak of the current potentiation was defined as Ipeak, while the current measured at the end of the 6-sec Co2+ application was defined as ICo. We performed the same experiments on the wild-type (WT) TMEM16A channel as well as on more than 10 point mutants of residue Y589. To evaluate the degrees of potentiation and inhibition, a Co2+ potentiation coefficient was calculated by dividing Ipeak by I0, while a Co2+ inhibition coefficient was calculated by ICo/Ipeak. All averaged results are presented as mean ± S.E.M.We analyzed the experimental data using the combination of pClamp (Molecular Devices) and Origin  software. For data analyses, the background leak current in the absence of [Ca2+], the values of ICo/Ipeak were plotted against [Co2+]i, and the dose-dependent Co2+ inhibitions were fitted to a Langmuir equation to evaluate the apparent Co2+ affinity:1/2 is the fitted half-inhibition concentration. For the current potentiation, because the potentiation effect appeared to be voltage dependent and because the effect of the highest [Co2+]i (20 mM) at +20 mV appeared unsaturated, we did not fit the [Co2+]i-dependent potentiation to any type of binding isotherm equations. The data points were connected by line segments.To analyze the dependence of the degree of inhibition on i was fit to a single-exponential decay function:peak and ICo are as defined above. The time constant of the current reduction process (τoff) was used to correlate with the degrees of Co2+ inhibition or Co2+ potentiation. Two other parameters, the sidechain hydrophobicity and the molecular volume of the amino acid placed at position 589, were also used to correlate with the Co2+ inhibition or the Co2+ potentiation. The values of the sidechain hydrophobicity and the molecular volume of the amino acid were obtained, respectively, from Kyte and Doolittle i. Upon applying [Co2+]i (in the presence of 30 μM [Ca2+]i), the currents were first potentiated, then inhibited, although the potentiation of the current by 50 μM Co2+ was not observed. It appears that the degrees of the current potentiation and inhibition depend on [Co2+]i.Examples of the intracellular Co2+ effects on TMEM16A, we first examined the mechanism of inhibition. 2+ inhibition on the TMEM16A current activated by different saturating concentrations of [Ca2+]i (from 10–300 μM). Although [Co2+]i in all these recordings is the same (2 mM), the degree of inhibition decreases with an increase of [Ca2+]i used for activating the current. 1/2 of [Co2+]i-dependent inhibition  and +20 mV (lower panel), respectively. The Y589A mutant has been reported to have a greater apparent affinity for Ca2+ activation than WT channels [2+]i removal in this mutant is significantly slower (τoff of the current reduction process is larger) than in the WT channel  after removing [Ca2+]i near the end of the recordings. For example, the current reduction upon washout of [Ca2+]i appears slower in Y589G, Y589S, Y589V, and Y589C than in Y589W, Y589H, Y589F and Y589K  are shown in 2+ inhibition (ICo/Ipeak) against the value of τoff confirms that the potency of Co2+ inhibition decreases with the increase of τoff; namely, the slower the Ca2+ dissociation rate, the weaker the Co2+ inhibition. We also correlated the Co2+ inhibition (ICo/Ipeak) obtained at +20 mV  and -20 mV (black traces), and the concentration-dependent Co2+ potentiation was shown in 2+]i. At the highest [Co2+]i (20 mM), the potentiation was ~10% and ~35–45% of the control current at -20 mV and +20 mV, respectively. The results reveal a voltage dependence of Co2+ potentiation: the degree of potentiation at +20 mV is significantly larger than that at -20 mV, with this voltage-dependent difference most clearly observed at [Co2+]i = 20 mM. It is also clear from these results that the affinity of Co2+ for the potentiation effect is low—the potentiation effect at +20 mV was not saturated even at 20 mM [Co2+]i! At -20 mV, the dose-dependent curve appears to have an apparent half-effective concentration of several mM.To study the Co2+]i is required for potentiation is reminiscent of the finding that [Ca2+]i in the mM concentration range induces more TMEM16A current even though the channel opening has already been saturated by low μM [Ca2+]i [2+ activation of TMEM16A thus appeared as biphasic. 2+]i (2 or 20 mM) with that by 20 μM [Ca2+]i, a concentration thought to already saturate the high-affinity Ca2+-activation sites [2+]i is significantly larger than the current induced by 20 μM [Ca2+]i. Furthermore, the current reduction process upon removing mM [Ca2+]i shows two exponential decays, and the remaining current after the first exponential decay matches the amplitude of the current induced by 20 μM [Ca2+]i. These recording traces thus indicate that the current induced by mM [Ca2+]i likely consists of two different components. We suspected that the extra current induced by mM [Ca2+]i may have the same underlying mechanism for the Co2+ potentiation as both potentiation effects are mediated by relatively low-affinity binding of these two divalent cations (mM concentrations). Accordingly, we examined the high [Ca2+]i-induced current in various Y589 mutants more closely. The exemplary recording traces shown in 2+]i also consists of two components: a fast and a slow current-decaying component. The fraction of the fast current-decaying component (the low-affinity component) is larger than that in the WT channel, consistent with a larger Co2+ potentiation in Y589A than in the WT channel. The dose-response curves of Ca2+ potentiation in the WT channel and in various Y589 mutants obtained with high [Ca2+]i (from 50 μM to 20 mM) are depicted in 2+ and Co2+, we plot the potentiation by 20 mM [Ca2+]i (relative to the current induced by 20 μM [Ca2+]i) against the potentiation by 20 mM [Co2+]i (the current was activated by 300 μM [Ca2+]i) for all the Y589 mutants we have created  to the excited state (sub μM to low μM) of cells.TMEM16A is expressed in various tissues and plays many physiological roles, including mediating transepithelial anion transport , 37, modng sites –18, thuson curve , suggest twofold . It is n2+, can also interact with TMEM16A, although intracellular Co2+ up to 20 mM cannot induce current in WT TMEM16A  upon removing [Ca2+]i, the weaker the Co2+ inhibition  roughly correlates with the degree of 20 mM [Ca2+]i-induced potentiation (relative to the 20 μM [Ca2+]i-activated current), except perhaps in two mutants, Y589W and Y589F (off) of current deactivation upon removing [Ca2+]i, so their affinity for Ca2+ activation are low. Perhaps the 20 μM [Ca2+]i used to induce the current in these two mutants was not a saturating concentration. Therefore, the current potentiation by 20 mM [Ca2+]i (compared to the current induced by 20 μM [Ca2+]i) may include a further opening of the channel by a more saturating [Ca2+]i. A second possibility for the disproportionally higher Ca2+ potentiation than the Co2+ potentiation in Y589W and Y589F may be a true difference of the potentiation due to, for example, a different binding of these two divalent cations to mutants with an aromatic sidechain. The less than perfect correlation between Ca2+ and Co2+ potentiation in Y589F and Y589W does not undermine the observations that the affinities for the Ca2+ and Co2+ potentiation are low, and the degrees of the potentiation by these two cations are similar to each other.Previous experiments have shown a biphasic Ca TMEM16A , 29. Nam induced , 29. Recnd Y589F . The les2+ and Co2+ potentiation. The fact that the mutants Y589K, Y589L, and Y589M exhibit a similar degree of potentiation indicates that sidechain charge plays little role in the potentiation. The correlation between sidechain hydrophobicity and the degree of potentiation is also weak. Rather, the potentiation appears to best correlate with the sidechain volume of the amino acid at position 589. We suspect that the sidechain of residue 589 probably does not directly interact with Ca2+ or Co2+. The potentiation requiring hundreds of μM or mM of Ca2+ or Co2+ further indicates that binding of these two divalent cations to generate the effect is of low affinity, and therefore could be non-specific. Recent experiments from our laboratory have revealed that introducing an aromatic residue at the Q559 position of TMEM16F, which corresponds to K584 of TMEM16A (a pore residue), significantly reduces the rundown of TMEM16F  via an electrostatic effect. The degree of the Co2+ potentiation best correlates with the volume of the amino acid placed at position 589—a larger Co2+ potentiation appears in the mutants with a smaller sidechain (Torpedo CLC-0 Cl- channel where the sidechain volume of a residue deep in the pore (E166 of CLC-0) affects the blocking affinity of amphiphlic pore blockers such as parachlorophenoxy acetate or octanoate i. We suggest that this potentiation may occur via the binding of divalent cations near or within the pore because of the voltage dependence of the potentiation and because pore residue mutations affect this potentiation. We suspect this potentiation effect may be related to the phospholipids near the intracellular pore region. It will require further experiments to refute or further support this conjecture that membrane phospholipids indeed involve in the Ca2+ and Co2+ potentiation of the TMEM16A current.In summary, we have shown that intracellular CoS1 Fig2+]i using a three-pulse protocol. The dash line represents zero-current level. Vm = +40 mV. [Ca2+]i in the first and the third pulse was 2 mM, a saturating concentration. For the second pulse in the top trace, a solution containing neither EGTA nor any added [Ca2+]i  was used, while a solution containing a calculated free [Ca2+]i of ~0.5 μM was used for the second pulse in the lower trace. The nominal zero-Ca2+ solution used in the top trace contains only 140 mM NaCl and 10 mM HEPES (pH = 7.4 adjusted with NaOH), while the solution used for the second pulse in the lower trace contains extra 100 μM EGTA and an added [Ca2+]i of 89.8 μM. Free [Ca2+]i in the solution was calculated using the MaxChelator program (http://maxchelator.stanford.edu/CaEGTA-NIST.htm). (B) Normalized currents of a TMEM16F mutant, Q559W, activated by various [Ca2+]i. The amplitudes of all recorded currents were normalized to that induced by 2 mM [Ca2+]i. Solid black circles represent the data from using solutions containing calculated [Ca2+]i of 0.5 μM, 2.0 μM, and 5.1 μM free [Ca2+]i (n = 4–11), and the data match the dose-response curve for TMEM16F Q559W mutant reported in Nguyen et al. [2+ solution containing only the contaminating [Ca2+]i (n = 11).(A) Recording traces of the currents of the TMEM16F Q559W mutant induced by various Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't KnowReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: No**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The submitted manuscript presents an in-dept investigation on the ion conductivity changes in a membrane Ca-activated chloride channel upon competitive binding of Co2+ ions. This protein is a transmembrane homodimer, each subunit having two high affinity binding sites for calcium. Correspondingly, the interpretation of just the current recordings upon the displacement of one type of metal by another type of metal is challenging. The authors have accumulated a respectable volume of current recordings in diverse conditions (including 10 mutant proteins). It would have definitely helped if the stability constants for cobalt ions were measured for all possible binding sites (and in the mutants). However, the complexity of lipid environment adds a whole extra layer of uncertainty. So, it is understandable that the data, as presented, do not make a completed picture.Long story short, there are a number of questionable parts in the authors’ treatment of data, plus the English of the article is very heterogeneous. All that let me only to recommend a major revision of the manuscript. I have compiled the specific comments below.Major comments1. Ln. 101, 104: “…alter the affinity of Ca2+ in activating the channel…” — the affinity and channel activation should be discussed separately.2. Ln. 169: “…the concentration of NaCl in the intracellular solution was reduced according to the extra [Cl-] from the added [CoCl2].” — such adjustments would have altered the ionic strength of the solution. Few words should be added on the possible impact on the observations.3. “Data analysis” section is very poorly written, hard to read .4. Ln. 209: “was fit to a single-exponential function” — What kind of function is this? Apparently, it is valid only for the chosen concentration of EGTA?5. The selection of a Langmuir-type equation for the data analysis is highly questionable. Why not employ standard competitive binding models?6. Ln. 321-323, 340-341: “the correlation of Co2+ inhibition with the sidechain hydrophobicity is weak . Inhibition appears to correlate with the molecular volume of the amino acid placed at position 589…” — I don’t see any correlations in these cases.7. Fig. 8: The inverse correlation between the mutant residue volume and the current, could it be interpreted as a mechanical obstruction of the channel?Minor and technical comments1. Throughout : ‘carboxylate’ is an anion of a carboxylic acid; the group is carboxyl (or ‘ionized carboxyl’ if one prefers).2. Ln. 41: “…mutating a pore residue…” — in the abstract it’s preferable to be specific about which residue was mutated.3. Ln. 64: “…conduction of ionic currents across lipid membranes. The physiological roles of the current conduction…” — it’s either ‘ion current’ or ‘ionic conduction’.4. Ln. 68: “…are dimeric proteins consisting of two identical subunits…” — a proper term is ‘homodimeric’.5. Ln. 96: “…each protein subunit consists of a set of Ca2+-binding sites…” — it doesn’t consist, but has exactly two binding sites, as follows from the published structures.6. Ln. 106 : “…the binding alters the rectification of the Cl- flux through the channel pore by reducing the negative charge from these Ca2+-coordinating residues…” — not clear, should be rephrased.7. Ln. 210: Fig 5C is cited not in sequence .8. Ln. 514: What is CLC-0?9. Ln. 521: Should be “vestibule”.Reviewer #2: TMEM16A is a Ca2+-activated Cl- channel that plays key roles in diverse physiological processes including transepithelial Cl- transport. TMEM16A functional studies have been instrumental in deciphering the structure-function of the channel. For example, studies have shown that plotting Ca2+ activation of TMEM16A currents requires a Hill coefficient greater than 1, thereby consistent with multiple Ca2+ binding to activate TMEM16A currents. Moreover, functional studies demonstrated that the channel has two independent Cl- conducting pores.Manuscript synopsis: Here, the authors explored TMEM16A gating using the divalent Co2+. Nguyen et al. report that Co2+ is a competitive inhibitor for the Ca2+ activation of TMEM16A. They also found that at high concentrations, Co2+ can potentiate Ca2+-evoked Cl- currents. These results were supported by inside-out patch experiments using intracellular Ca2+ application to activate TMEM16A, and application Co2+ to inhibit or potentiate these Ca2+-evoked Cl- currents. By varying concentrations of Ca2+ and Co2+ that were applied, the authors further characterize how Ca2+ and Co2+ regulate TMEM16A conducted Cl- currents. Additional experiments include mutations of the pore residue Y589 which were used to describe the relationship of Co2+ inhibition to amino hydrophobicity, volume, as well as the deactivation kinetics of the TMEM16A channel.The experiments presented in this manuscript clearly demonstrate inhibition and potentiation Co2+ on TMEM16A Ca2+-evoked Cl- currents. Additionally, these experiments showing the effect of Co2+ on TMEM16A Ca2+-evoked Cl- current are well described and easy to understand. However, this is not as true for the Y589, whose interpretation in the manuscript somewhat confusing. The data included in this manuscript are interesting and should be published. However, improving the writing – specifically motivating the experiments presented in figures 6-10 and discussing the interpretation of these data - will create a more impactful manuscript.Major points:1) The motivation for the experiments depicted in figures 6-10 is not easily accessible to the reader. Even though understanding the characteristics of TMEM16A itself is important for the field, the specific angle for why we need to learn more about the biphasic dose response of TMEM16A is not stated. Moreover, reasons why the Y589 mutant was chosen for these experiments, or why this residue was targeted for multiple mutations, even why the amino acids used for these mutations were chosen should be clarified. Perhaps addressing how these data fit with current understanding of TMEM16A gating, or will direct future experimental directions will help the reader.2) The manuscript alludes to the importance of amino acid carboxylates (line 97) in coordinating Ca2+ but does not address this concept in the context of the Co2+ findings and the Y589 mutants. The importance of these Y589 experiments in addressing the biphasic dose-response is also not clear. Perhaps further discussion on how these mutants address the main question could help.3) Lines 71-84 and Figure 1 highlight TMEM16F and its structural properties. It is not clear how this information adds to or sets up the motivation for the question being addressed in this manuscript.Minor points:1) Lines 42-43 refer to Ca2+ as potentiating TMEM16A and not activating. Are the data suggesting that Ca2+ plays two roles?2) Correlations described in Figures 7 and 8 are not apparent expect for �off. Are there patterns or features of the selected amino acids that should be described to help with the interpretation? The data are difficult to appreciate as described currently. Additionally, how these data relate to the overall question is quite unclear.3) The manuscript is written as though it is a follow up to another study in a way that makes the current form of this one difficult to grasp and appreciate. The data are interesting and present new information about TMEM16A, and providing more rationale for the experiments conducted will enhance the value of the data.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  30 Mar 2020See information in the "Response to Reviewers" file.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 2 Apr 2020Cobalt Ion Interaction with TMEM16A Calcium-Activated Chloride Channel: Inhibition and PotentiationPONE-D-20-03301R1Dear Dr. Tsung-Yu Chen,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Eugene A. Permyakov, Ph.D., Dr.Sci.Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 6 Apr 2020PONE-D-20-03301R1 Cobalt Ion Interaction with TMEM16A Calcium-Activated Chloride Channel: Inhibition and Potentiation Dear Dr. Chen:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofProf. Eugene A. Permyakov Academic EditorPLOS ONE


Despite continuous improvements of AML therapy, the prognosis of AML patients remains unsatisfactory. Recently, lncRNAs have been reported to participate in the development of AML. Our data demonstrated that MMP15 and LINC00963 were upregulated and miR-608 was decreased in AML cells  compared to HS-5 cells. RT-qPCR results showed that LINC00963 levels were higher in the serum and bone marrow of AML cases than in controls. Moreover, overexpression of LINC00963 promoted AML cell growth and EMT progression in both THP-1 and HL-60 cells. Furthermore, miR-608 levels were downregulated in the serum and bone marrow of AML cases compared with controls, and Pearson’s correlation analysis indicated that LINC00963 was negatively correlated with miR-608 in the serum and bone marrow of AML samples. In addition, we demonstrated that LINC00963 sponged miR-608 expression and that MMP-15 was a target of miR-608 in AML cells. Finally, rescue experiments indicated that ectopic expression of LINC00963 accelerated cell growth and EMT development by modulating MMP-15. These data demonstrated that LINC00963 acted as an oncogene and may be a potential target for AML treatment. Acute myeloid leukemia (AML) remains the most prevalent hematological tumor –3. AML iLncRNAs are widely found in mammals and are a group of noncoding RNA transcripts longer than 200 nt with little or no protein-coding ability –18. GrowWe first measured LINC00963 in AML cells, and RT-qPCR analysis demonstrated that LINC00963 was upregulated in AML cells  compared to HS-5 cells. RT-qPCR analysis showed that LINC00963 levels were higher in the serum and bone marrow of AML cases than in controls. Furthermore, overexpression of LINC00963 promoted AML cell growth and EMT progression in both THP-1 and HL-60 cells.Frist, RT-qPCR analysis was utilized to determine MMP15, LINC00963 and miR-608 levels in AML cells. MMP15 was overexpressed in AML cells  compared with HS-5 cells . LINC009Then, we measured LINC00963 in the serum and bone marrow of AML cases and controls. The data illustrated that LINC00963 levels were higher in the bone marrow of AML cases than in controls . MoreoveFurthermore, we measured miR-608 in the serum and bone marrow of AML cases and controls. The results demonstrated that miR-608 levels were lower in the bone marrow of AML cases than in controls . PearsonThe level of LINC00963 was significantly upregulated in both AML cell lines THP-1 and HL-60 after treatment with pcDNA-LINC00963 . The CCKAn online database (starBase) predicted that miR-608 was a potential target of LINC00963 . The levAn online database (TargetScan) predicted that MMP-15 was a potential target of miR-608 . LuciferTo further study the function of LINC00963 and MMP-15 in AML development, we treated LINC00963-overexpressing THP-1 cells with an siRNA-MMP-15 plasmid. MMP-15 expression was downregulated in THP-1 cells after treatment with the siRNA-MMP-15 plasmid, as demonstrated by RT-qPCR assay . MMP-15 Despite continuous advances in AML therapy, the prognosis of AML patients remains unsatisfactory –40. ReceRecently, LINC00963, a novel lncRNA, has been reported to be involved in the development of several tumors, such as breast cancer, ovarian tumors, esophageal tumors, cutaneous carcinoma, melanoma, hepatocellular carcinoma and prostate cancer –37. Wu eA growing number of lncRNAs have emerged as ceRNAs that protect mRNAs from targeting by miRNAs , 47. A pOur results illustrated that LINC00963 was upregulated in AML cells and that LINC00963 levels were higher in the serum and bone marrow of AML cases than in controls. Ectopic expression of LINC00963 accelerated cell growth and EMT development by modulating the miR-608/MMP-15 axis.TM2000.Serum and bone marrow samples were obtained from AML patients and control cases at the Affiliated Cancer Hospital of Zhengzhou University. Our research was approved by the Clinical Ethics Committees Board of the Affiliated Cancer Hospital of Zhengzhou University, and written informed consent was obtained from each participant. HS-5 and AML cells  were obtained from ATCC and cultured in RPMI-1640 medium supplemented with antibiotics and FBS. miR-608 mimic, siRNA-MMP-15, pcDNA-LINC00963 and controls were purchased from Genema and transfected using LipofectamineCell growth was determined with a CCK-8 assay . Cells were cultured in 96-well plates and measured at 0, 1, 2 and 3 days. The cell number was determined from the optical density (OD) at 450 nm.-ΔΔCt method. U6 and GAPDH were used as controls. The primer sequences were as follows: LINC00963, 5’-GGTAA ATCGA GGCCC AGAGAT-3’ (F), 5’-ACGTG GATGA CAGCG TGTGA-3’ (R); GAPDH, 5’-CATGA GAAGT ATGAC AACAG CCT-3’ (F), 5’- AGTCC TTCCA CGATAC CAAAGT-3’ (R); miR-608 5’-AGGGG TGGTG TTGGG ACAGC-3’; and U6, 5’-GCGCG TCGTG AAGCG TTC-3’ (F), 5’- GTGCA GGGTC CGAGG -3’ (R).Total RNA from all specimens and cells was isolated with a TRIzol kit . For mRNA, miRNA and lncRNA expression, RT-qPCR was used on an ABI7500 PCR system  using a SYBR kit . The fold change of these genes was determined with the 2For the luciferase reporter assay, wild-type LINC00963 and wild-type MMP-15 (LINC00963-wt and MMP-15-wt) or sequences with mutant binding sites (LINC00963-mut and MMP-15-mut) were cloned downstream of the Renilla luciferase gene in the pRLTK vector . Each vector was cotransfected into cells with miR-608 mimic or control . Luciferase analysis was carried out with a dual luciferase detection reagent , and the relative luciferase value was calculated from Renilla luciferase activity.The data values are presented as the mean ± SD. Analysis of statistical differences was assessed with Student’s t-test using SPSS 18.0 . The significance level was set at P<0.05. Spearman’s correlation analysis was performed to study the correlation.


Durable responses and persistence of SAR-transduced T cells could also be demonstrated in AML xenograft models. Together these results warrant further translation of this novel platform for AML treatment.Targeted T cell therapy is highly effective in disease settings where tumor antigens are uniformly expressed on malignant cells and where off-tumor on-target-associated toxicity is manageable. Although acute myeloid leukemia (AML) has in principle been shown to be a T cell-sensitive disease by the graft-versus-leukemia activity of allogeneic stem cell transplantation, T cell therapy has so far failed in this setting. This is largely due to the lack of target structures both sufficiently selective and uniformly expressed on AML, causing unacceptable myeloid cell toxicity. To address this, we developed a modular and controllable MHC-unrestricted adoptive T cell therapy platform tailored to AML. This platform combines synthetic agonistic receptor (SAR) -transduced T cells with AML-targeting tandem single chain variable fragment (scFv) constructs. Construct exchange allows SAR T cells to be redirected toward alternative targets, a process enabled by the short half-life and controllability of these antibody fragments. Combining SAR-transduced T cells with the scFv constructs resulted in selective killing of CD33 Modular platform enabling controlled targeting of AML by SAR-transduced T cells in combination with tandem scFv constructs.Efficient lysis of primary AML blasts in vitro and strong antitumoral effects and T cell persistence in xenograft models. With high relapse rates and few targeted therapeutic options, there is a need develop novel solutions for the treatment of acute myeloid leukemia (AML). While standard therapy (induction chemo- and consolidation therapy) does offer a curative first-line therapy to those eligible , leukemi+ leukemic cells have been shown to be an effective approach, and are now part of the standard-of-care [Immunotherapy that promotes the killing of tumor cells by cytotoxic T lymphocytes has entered clinical routine for hematological malignancies in recent years –6. In ac-of-care . Similar-of-care –10. The -of-care . In the -of-care , 12. In -of-care . Thus, t+ CD33+), thus LSCs that acquired one or more of their transforming events following commitment to the myeloid compartment are targetable [+ CD38- LSCs of AML patient blasts [CD33 is an antigen expressed in more than 99% of AML cases, therefore offering a targeted therapeutic modality with the potential to induce remission , 15. CD3rgetable . Moreovet blasts , 18. Alot blasts .The pan-T cell-CD33-targeting bispecific T cell engager (BiTE) AMG330 recently showed encouraging results in a phase I trial . In addiAn alternative approach is to transduce T cells with a synthetic agonistic receptor (SAR) composed of an inert extracellular domain (EGFRvIII -referred to as E3) acting as a unique antigen receptor fused to intracellular T cell-activating domains that can be specifically activated by an engineered BiAb . Because4S)4 linker. These constructs have dual specificities: one to target AML (via binding CD33 or CD123), and the other to target the SAR-expressing T cell (via binding E3 – which is the inert extracellular domain of the T cell-activating SAR).To enable better control over T cell activity for AML and ALL indications, we reasoned that we could replace the BiAb (IgG) with tandem scFv (taFv) constructs as these would be more controllable and safer due to their shorter half-life , 24. Her+ or CD123+) and the taFv molecule. We demonstrate that, unlike a conventional BiTE (anti-CD33–anti-CD3), which activates pan-T cells, our E3-specific constructs activate only SAR-transduced T cells – giving additional control over effector cell modifications, phenotype and dosage. Importantly, we highlight substantial activity of the platform in primary AML-blast cultures and in different AML-xenograft models, underpinning the translational potential of the approach.We could show that T cells expressing the SAR construct can, in a reversible manner, be selectively activated in the presence of AML cells . Prior to treatment mice were randomized according to tumor burden. Endpoints were registered by an observer blinded to the treatment groups as previously defined [4-week-old female NSG mice (NOD.Cg-Prkdcscid Il2rgtm1WjI/SzJ) were purchased from Charles River . MV4-11-LUC-GFP and THP-1-LUC-GFP xenograft models were established by intravenously injecting 2 ×10 defined . More thD) were measured by calibrated flow cytometry on a Guava easyCyte 6HT instrument  with 3.0 to 3.4 μm Rainbow Calibration particles  as calibration control [Apparent dissociation constants . The E.G7-OVA cell line was modified to express full-length human EGFRvIII , resulting in E.G7-EGFRvIII cells. Luciferase-eGFP (LUC-GFP) overexpressing cell lines PL-21-LUC-GFP, THP-1-LUC-GFP and MV4-11-LUC-GFP were generated according to previously described protocols . AntigenT cells were incubated with tumor cell lines and taFvs at indicated effector-to-target ratios and concentrations. Following a 24 h coculture, the BioGlo Assay  system was used according to the manufacturer’s protocol.Blinded confocal imaging and conjugate quantification were carried out following the selection of 10 representative areas of each slide. Cells in or out of conjugate within each area were quantified and a ratio thereof subsequently determined. For each conjugate, the position of the microtubule organizing center (MTOC) was observed, and its polarization to the immune synapse, or lack thereof, was noted. The ratio of polarized to nonpolarized MTOCs was used to determine the ratio of functional synapses out of all conjugates formed.Flow cytometry was carried out according to previously published protocols . For celSAR construct generation was previously described . SAR-traHuman T cell stimulation assays were set up at indicated concentrations and effector-to-target ratios. IFN-γ was quantified by ELISA (BD Bioscience).AML blasts were cultivated for 3 days before coculture. Allogeneic healthy donor T cells were incubated with patient-derived AML blasts at indicated effector-to-target ratios and concentrations. Untransduced T cells were used to control for allogeneic effect. Patient blasts were otherwise cultured according to the previously described protocol .After written informed consent in accordance with the Declaration of Helsinki and approval by the Institutional Review Board of the Ludwig-Maximilians-Universität , peripheral blood (PB) or bone marrow (BM) samples were collected from healthy donors and AML patients. At initial diagnosis or relapse, samples were analyzed at the Laboratory for Leukemia Diagnostics of the Klinikum der Universität München as previously described , 32. PatStatistical evaluation was performed using GraphPad Prism software V8.3.1 . Differences between experimental conditions were analysed as described in figure P values < 0.05 were considered to be significant. Data are shown as mean values SEM of a minimum of three biological replicates or independent experiments, as indicated. For in vitro experimentation with healthy donor or patient samples, no statistical methods were used to predetermine sample size. These were chosen based on prior experience with this experimental design and patient sample availability. For in vivo experimentation sample sizes were used in accordance with prior experience with the models used.Based on our previous results, we hypothesized that the SAR platform could be developed specifically for AML targeting and treatment . We begaD = 19.5 nM), and a lower affinity for the T cells (E3 KD = 235.8 nM) so that aggregates could form more easily on the target cells. The binding properties and apparent dissociation constants of the anti-E3–anti-CD33 molecule to both its targets were analyzed by flow cytometry    and THP-1 (p = 0.010) models  treatment group in both MV4-11 , whereas Lck and LFA-1 organization patterns were comparable to those reported for the IS of CAR T cells . This taDespite its similarly broad expression on myeloid progenitors and some normal B cell and activated T cell populations, CD33 remains a valuable antigen for the targeting of AML due to its overexpression on blasts in all AML , 44. LowTo date, anti-AML CAR T cells have shown limited efficacy in the clinic , 49, witMany approaches have emerged attempting to make CAR T cells more modular and controllable. These include but are not restricted to the SUPRA CAR (with tunable availability and affinity) , CAR T cFurthermore, its applicability and tailoring toward specific disease settings, such as AML, is yet to be shown . BispeciFurthermore, many of the modular CAR approaches (such as switchable CAR T cells) rely on the introduction of a neoepitope for selective targeting , which cPerhaps the greatest challenge hindering the success of adoptive T cell therapy in AML is target specificity, which results from disease heterogeneity and diverse antigen expression on LSCs. A recent AML proteomic and transcriptomic study revealed a series of differentially expressed AML-specific antigens, out of which came the rationale that systematic therapeutic combinations would be ideal in the context of AML therapy . This apA strength of our platform—its modularity, stands to benefit from the significant research that has already been carried out on many AML targets as stand-alone targeted therapies , 46, 62.Collectively, we could comprehensively demonstrate that AML-specific taFvs can be effectively used to target AML in a controllable manner. While further development and more extensive testing are required before its application in a clinical setting, the SAR platform undoubtedly offers new solutions to the ever-challenging AML disease setting.Supplementary Table 2Supplementary materialSupplementary figure 1Supplementary figure 2Supplementary figure 3Supplementary figure 4Supplementary figure 5Supplementary Table 1


Therefore, the regulation of Ca2+ homeostasis is one of the most important processes underlying the basic cellular viability and function of the neuron. Multiple components, including intracellular organelles and plasma membrane Ca2+-ATPase, are involved in neuronal Ca2+ control, and recent studies have focused on investigating the roles of mitochondria in synaptic function. Numerous mitochondrial Ca2+ regulatory proteins have been identified in the past decade, with studies demonstrating the tissue- or cell-type-specific function of each component. The mitochondrial calcium uniporter and its binding subunits are major inner mitochondrial membrane proteins contributing to mitochondrial Ca2+ uptake, whereas the mitochondrial Na+/Ca2+ exchanger (NCLX) and mitochondrial permeability transition pore (mPTP) are well-studied proteins involved in Ca2+ extrusion. The level of cytosolic Ca2+ and the resulting characteristics of synaptic vesicle release properties are controlled via mitochondrial Ca2+ uptake and release at presynaptic sites, while in dendrites, mitochondrial Ca2+ regulation affects synaptic plasticity. During brain aging and the progress of neurodegenerative disease, mitochondrial Ca2+ mishandling has been observed using various techniques, including live imaging of Ca2+ dynamics. Furthermore, Ca2+ dysregulation not only disrupts synaptic transmission but also causes neuronal cell death. Therefore, understanding the detailed pathophysiological mechanisms affecting the recently discovered mitochondrial Ca2+ regulatory machineries will help to identify novel therapeutic targets. Here, we discuss current research into mitochondrial Ca2+ regulatory machineries and how mitochondrial Ca2+ dysregulation contributes to brain aging and neurodegenerative disease.Calcium ions (Ca Recent studies of mitochondria-dependent Ca2+ handling have revealed the molecular identities of Ca2+-control components, including the mitochondrial calcium uniporter (MCU) and its auxiliary subunits  have allowed direct monitoring of mitochondrial Ca2+ dynamics . The ER imports Ca2+ through sarco/endoplasmic Ca2+ ATPase and releases it via ryanodine receptors or IP3 receptors (IP3Rs) (2+ transfer (2+ dynamics will be discussed here.Cytosolic Ca (IP3Rs) . ER is ptransfer . The invtransfer . Therefo2+ regulatory proteins have been identified in the outer and inner mitochondrial membranes. VDACs located in the outer mitochondrial membrane (OMM) are responsible for importing various ions and metabolites , with the interference of fission impairing mitochondrial Ca2+ uptake and LTP  is another MCU complex protein that bridges MCU and MICU1 and regulates the level of Ca opening . The MCU opening . Mitocho opening .+/Ca2+ exchanger (NCLX) is one of the primary Ca2+ efflux units in mitochondria , is activated by Ca2+ overload and ROS, leading to apoptosis or necrosis  peptide, which is produced by abnormal proteolytic cleavage of amyloid precursor protein (APP), as well as by the formation of neurofibrillary tangles composed of tau protein and by neuronal loss, leading to learning and memory impairment . Mutatio2+. In the past two decades, multiple underlying mechanisms have been suggested, including mitochondrial Ca2+ dysregulation (In-vivo Ca2+ imaging with mitochondria-targeted Förster resonance energy transfer (FRET)-based GECI has directly demonstrated an Aβ-dependent mitochondrial Ca2+ increase in mouse cortex. This upregulation was observed prior to neuronal death, with blockade of MCU restoring the mitochondrial Ca2+ level in the APP/PS1 mutant mouse model . Mutant APP-expressing-N2a cells also show decreased NCLX expression, resulting in impaired mitochondrial Caathology . Anotherd memory .2+ overload in AD can also result from impaired ER–mitochondria communication. Previous studies suggest that ER–mitochondria contacts are increased in AD models, promoting Ca2+ transfer to mitochondria  is the major risk factor for sporadic AD and it can increase ER–mitochondria contacts . ApoE4-eradic AD ; Table 12+ buffering and ATP production, as measured using the mitochondria-targeted GECI and the ATP sensor, although detailed mechanisms are not known. Interestingly, inhibition of IGF-1R reverses altered synaptic release in an APP/PS1 mutant model  levels are found in AD patient and mouse model brain samples . IGF-1R nt model .2+ regulation is especially important for dopaminergic neurons because of their steady and autonomous pacemaker function , PTEN-induced kinase 1 (PINK1), and parkin . Ca2+ refunction .2+ by enhancing ER–mitochondria interaction  stretch of the huntingtin protein .2+ buffering have been observed after applying Calcium Green-5N to isolated mitochondria from HD mouse brain to monitor extramitochondrial Ca2+   is a neurodegenerative disease characterized by progressive muscle paralysis resulting from the degeneration of upper and lower motor neurons. ALS exhibits multiple pathogenic features, including oxidative stress, mitochondrial dysfunction, and protein dysfunction of 43-kDa transactivating response region binding protein (TDP-43) and cytoplasmic Cu1 (SOD1) .2+ in the motor neurons of ALS mutant transgenic mice (SOD1G93A)  N2a cells also show significantly reduced mitochondrial Ca2+ uptake and elevated cytosolic Ca2+  . However neurons . Decreasll death . Other Alic Ca2+ .G93A-transgenic mice show upregulated MCU and MICU1 expression at the end stage of the disease (P115–140)  . However115–140) .2+ homeostasis in HEK293T cells , VAPB, and expanded hexanucleotide repeats in intron 1 of the encoding chromosome 9 open reading frame 72 (C9ORF72). The OMM protein PTPIP51 is a known binding partner of the ER protein VAPB. VAPB–PTPIP51 interaction in mouse motor neurons is disrupted by overexpression of ALS mutant or wild-type TDP-43 and FUS, also leading to disruption of Ca3T cells , 2016. Apression  (Figure pression .2+ regulatory defects. These alterations have been revealed using various experimental methods, including electrophysiological recording and live imaging. However, large part of in-vitro studies for neurodegenerative diseases have performed using cell lines and patient-derived fibroblasts rather than neurons. In addition, Ca2+ signals were triggered by various chemicals, and most conditions are not neurophysiological (2+ uptake capacity and regulatory components can be different (2+ sensors for specific organelles will provide more precise neuron type-specific data (In summary, brain aging and neurodegenerative diseases involve mitochondria- and ER-mitochondria contact-related Caological . Dependiifferent . Therefofic data .2+ regulatory molecular mechanisms have recently been revealed, with some studies showing that the composition of the MCU complex can change during disease progress and differ between mutant types (Finally, mitochondrial Cant types . Thus, rHJ, SYK, FC, and S-KK wrote the manuscript and created the figures and table. S-KK and YC provided guidance and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The intracellular free Ca2+ concentration was measured in confluent monolayers of SH-SY5Y cells and A7R5 cells with the Ca2+-sensitive fluorescent indicator Fluo-4 NW. Only four compounds showed calcium channel blocking activity in SH-SY5Y and A7R5 cells as well as in the aortic ring model. Among them, compound 3 was the most active calcium channel antagonist, which had 3 times higher activity on carbachol-activated SH-SY5Y cells than amlodipine. Two of the compounds were inactive. Compound 4 had 9 times higher calcium agonist activity than the classic DHP calcium agonist Bay K8644. The intracellular mechanism for the action of compound 4 using inhibitor analysis was elucidated. Nicotinic as well as muscarinic receptors were not involved. Sarcoplasmic reticulum (ER) Ca2+ (SERCA) stores were not affected. Ryanodine receptors (RyRs), another class of intracellular Ca2+ releasing channels, participated in the agonist response evoked by compound 4. The electrooxidation data suggest that the studied compounds could serve as antioxidants in OS.A set of six new 4-pyridinio-1,4-dihydropyridine  compounds has been synthesized. The calcium channel modulating activity of these compounds was evaluated in an aorta vascular smooth muscle cell line (A7R5), in an isolated rat aortic ring model, and in human neuroblastoma cell lines (SH-SY5Y). The antagonistic effect of these 1,4-DHP was tested by modulating the impact of carbachol-dependent mobilization of intracellular Ca The dihydropyridines (DHPs), especially 1,4-DHP, are a class of polyfunctional (pleiotropic) redox-active organic compounds.1,4-DHP is an analogue of 1,4-dihydronicotinamide and model compounds of redox-coenzymes NAD(P)H, which participates in redox reactions and can act as deactivators (quenchers) of reactive oxygen species (ROS) and reactive nitrogen species (RNS) i was measured in confluent monolayers of SH-SY5Y or A7R5 cells with the Ca2+-sensitive fluorescent indicator Fluo-4 NW according to the instruction   is shown in , 22.μM. The application of carbachol (100 nM) to Fura-4 NW loaded SH-SY5Y cells stimulated a classic “biphasic” response. The well-known 3rd generation calcium channel inhibitor amlodipine, which is also claimed to act as antioxidant and free radical scavenger i were measured from the fluorescence emitted at 516 nm due to alternate excitation at 494 nm using the microplate reader Tecan Infinite M1000 (Austria). Intracellular Ca2+ concentration was calculated from a standard curve with known amounts of free Ca2+ using the standard Ca2+-EGTA buffering system .Changes in i signal due to the compound 4 was decreased by 50% compared to that in the presence of extracellular Ca2+. This was confirmed by an increase of intracellular Ca2+ ion concentration when Ca2+ ions were added at 1 mM concentration to the Ca2+-free buffer after stimulation with compound 4. If the experiments were performed in a medium lacking Ca2+ ions, the level of the released Ca2+ ions decreased by 50%. A subsequent addition of Ca2+ ions at 1 mM concentration for 870 sec caused a peak of Ca2+ channel activation , normally a voltage-dependent calcium channel (VOC), as well as close structurally situated ryanodine receptors (RyRs). Besides, receptor-operated calcium channels (ROC) may regulate calcium ion influx and efflux.Main two Ca2+ ion influx and calcium ions channel types induced by compound 4, we studied the effect of known antagonists of Ca2+ channels: (1) the classical L-type calcium channel inhibitors—DHP derivatives nifedipine, nicardipine, and amlodipine; (2) atropine, a muscarinic receptor antagonist; (3) mecamylamine, a nonselective and noncompetitive nicotinic receptors blocker.To identify the source of CaμM of amlodipine, nifedipine, or nicardipine inhibited the [Ca2+]i influx mediated by compound 4 by 50%, 42%, or 33%, respectively. Incomplete inhibition indicated that the L-type Ca2+ channels were only partially responsible for [Ca2+]i level changes in this cell line  the structurally different classic L-type Ca2+ channel inhibitors—DHP derivatives nifedipine and amlodipine .The effect of amlodipine on the stimulation of calcium ion entry by compound 4 was different as shown in the literature ; where aμM) had no effect on Ca2+ ion channel activity in the presence of compound 4; thus, nicotinic acetylcholine receptors were not involved in the response.Mecamylamine (at concentrations from 4 to 100 μM) had no effect on Ca2+ ion channel activity in the presence of compound 4; therefore, this compound did not affect muscarinic receptors.Atropine (at concentrations from 4 to 100 2+- (SERCA-) ATPase (membrane transport protein ubiquitously found in the endoplasmic reticulum (ER) of all eukaryotic cells) in the mobilization of Ca2+ ions ([Ca2+]i) from sarco-endoplasmic reticulum in SH-SY5Y neuroblastoma cells treated with compound 4, we measured the increase of Ca2+ ion ([Ca2+]i) concentration for cells placed in medium free of Ca2+ ions. As shown in μM) of SERCA, which depletes Ca2+ stores in ER, had no influence on Ca2+ ion increase induced by compound 4. This indicated that mobilization of intracellular Ca2+ stores in SH-SY5Y neuroblastoma cells induced by compound 4 proceeded without involvement of SERCA.To explore the role of sarco-/endoplasmic reticulum CaHere, the main thesis—results in both cases (with and without TG)—are practically the same.2+]i increase in neuroblastoma cells induced by compound 4. Pretreatment of cells with the cell-permeant IP3R inhibitor 2-APB for 15 min had no effect on the Ca2+ channel response induced by compound 4.We also investigated the possible involvement of Gi/o/Gq/11 G protein-phospholipase C-IP3 receptor pathway in the i increases is expressed more effectively than that of carbachol alone.Carbachol is used and serves as the control (reference) compound—it alone leads to increase of fluorescence . The effμM concentration of compound 4 has substantially higher effect. After reaching the peak level, the oscillatory mode of Ca2+ ion response is remarkable, about one oscillation in 200 s.In the studies on the A7R5 aorta cell line, compound 4 showed a dose-dependent agonist activity, evoking a two-phase response see . It is iThe calcium channel antagonist activity of compound 4 was evaluated in potassium chloride precontracted denuded rat aortic rings. Denuded aortic rings were selected for this purpose to exclude any direct effects of the tested compound on the endothelium, taking into account that some authors have shown a release of vasodilator substances from the endothelium in the presence of 1,4-DHP derivatives .Amlodipine, a well-known calcium channel inhibitor, was used as a positive control. The obtained results are summarized in Compound 4 did not cause contractions of aorta rings; thus, there was no agonist effect in this model.2+ V 1.2 channel subtype.In the case of the aorta model, the studied DHP derivative 4 most likely showed antagonism against the Ca2+ channel antagonist and agonist effects on mainly SH-SY5Y neuroblastoma cells were revealed using fluorescence counting data in a group of six compounds that were derived from 4-pyridinio-1,4-dihydropyridines.Both CaThe manifestation and expression of calcium antagonist/agonist activity depends on the substituent structure of studied 1,4-DHP compounds see .2+ antagonist properties of various degrees. One derivative (compound 4) could be characterized as Ca2+ channel agonist and antagonist.The obtained data revealed that two of the mentioned derivatives (compounds 2 and 5) lacked any activity, while the remaining four derivatives showed Ca2+ ion channel antagonist or agonist activity was observed ; it even is superior comparing to amlodipine (approximately 3 times). Activity is diminished as a result of p-benzyloxy substituent in the phenyl group and insertion of methyl radical in the active methylene group of the phenacyl ; still, activity is similar to that of amlodipine. Antagonist activity is significantly diminished in the case of p-methoxy or p-nitro group at phenacyl moiety (compounds 1 and 4), and it is absent in case of aliphatic N-ethoxycarbonylethyl moiety (compound 2) or transition to isomeric 4-1,4-DHP-pyridinio compound 5. We could observe Ca2+ ion channel agonist activity only in the case of compound 4, so the 4-benzyloxy group and methyl radical in the active methylene groups are beneficial.As shown  and ment2+]i evoked by compound 4 showed a biphasic effect that was comparable to that of the well-known Ca2+ channel agonist Bay K8644. Therefore, by stimulation of the cells using compound 4, a rise of [Ca2+]i occurred not only from the intracellular stores of Ca2+ but also due to influx of Ca2+ into the cell from the extracellular medium. This was confirmed by the 50% decrease of the response in the absence of extracellular calcium ions in the incubation medium.The rise of [Ca2+]i due to compound 4 was inhibited by two different antagonists of the dihydropyridine receptors (DHPRs)—nifedipine that binds to specific sites on the DHPRs, known as dihydropyridine sites  similar to the action of Вау K8644.The fact that the rise of i evoked by compound 4 suggested that this compound had no effect on the muscarinic and nicotinic receptors.The lack of the effect from atropine and mecamylamine on the rise of [Ca2+- (SERCA-) ATPase by thapsigargin had no effect on the rise of [Ca2+]i evoked by compound 4. This confirmed that compound 4 has no effect on the SERCA activity. These observations agree with the conclusions about Ca2+ modulator activity by DHP derivatives published by Copello et al. i was not only due to release from the intracellular stores of calcium but also due to influx of Ca2+ ions into the cell from the extracellular medium.Concerning the mechanism of calcium ion channel activation by compound 4, it was revealed that the ryanodine receptors had the dominant role. Similar to the stimulation of cells using compound 4, the rise of [CaCompound 4 did not cause the contractions of aortic rings; thus, there was no agonist effect in the case of this model. The performed structure-activity study enables better understanding of the interactions between 1,4-dihydropyridines and calcium channel. In addition, the studied compounds being DHP derivatives could serve as electron-donating entities for the prevention of oxidative stress.A new therapeutic strategy, so called the multitarget small molecule (MTSM) approach, is based on the design of drugs able to bind simultaneously at diverse enzymatic systems or receptors involved in pathology . 1,4-DHPCompound 4 has calcium channel agonist and antagonist properties on the same cell line SH-SY5Y.The obtained data, especially the just mentioned peculiar property of compound 4, could be a basis for further studies to obtain similar compounds, to investigate structure-activity relationships of calcium transport modulation (directly or indirectly ), as wel