Post-traumatic OA (PTOA) is often triggered by injurious, high-impact loading events which result in rapid, excessive chondrocyte cell death and a phenotypic shift in residual cells toward a more catabolic state. As such, the identification of a disease-modifying OA drug (DMOAD) that can protect chondrocytes from death following impact injury, and thereby prevent cartilage degradation and progression to PTOA, would offer a novel intervention. We have previously shown that urocortin-1 (Ucn) is an essential endogenous pro-survival factor that protects chondrocytes from OA-associated pro-apoptotic stimuli. Here, using a drop tower PTOA-induction model, we demonstrate the extent of Ucn’s chondroprotective role in cartilage explants exposed to excessive impact load. Using pathway-specific agonists and antagonists, we show that Ucn acts to block load-induced intracellular calcium accumulation through blockade of the non-selective cation channel Piezo1 rather than TRPV4. This protective effect is mediated primarily through the Ucn receptor CRF-R1 rather than CRF-R2. Crucially, we demonstrate that the chondroprotective effect of Ucn is maintained whether it is applied pre-impact or post-impact, highlighting the potential of Ucn as a novel DMOAD for the prevention of injurious impact overload-induced PTOA. Injurious, high-impact loading events to weight-bearing joints, such as those occurring in the knee during accidents or sporting injuries, often cause cartilage damage, as well as high levels of chondrocyte cell death and a phenotypic shift in the remaining cells towards a more catabolic state ,2,3,4. TAs chondrocytes play an important role in the homeostasis of articular cartilage, their survival is crucial for the maintenance of tissue integrity. However, within the avascular and aneural environment of the cartilage, the paucity of nutrients means that replication of chondrocytes is extremely limited, and hence their loss is permanent. The lack of macrophages within cartilage also means dead cells and their remnants remain within the tissue, inducing a further degradative cascade ,11. As s2+ conductance [2+ overload and chondrocyte cell death [We have previously demonstrated that the short endogenous peptide urocortin-1 (Ucn) and its relevant G-protein coupled receptor (corticotrophin releasing factor receptor 1 (CRF-R1)) are expressed in chondrocytes, where Ucn can function as both an essential endogenous pro-survival factor and protect from OA-associated pro-apoptotic stimuli ,13. We hductance ,15. If lll death ,17. HaviPreviously used models have replicated the force and impact experienced by chondrocytes within the joint. These have included 3D models to study OA ,19, wherTo extend our previous in vitro studies, we have developed an ex vivo model of PTOA, whereby porcine cartilage explants are subjected to various impact events administered from a drop tower. This has allowed us to investigate the extent of Ucn’s chondroprotective role in response to excessive impact load and, by use of Piezo1-modifying agents, enabled us to confirm a role for Piezo1 in this chondroprotective effect.To confidently use the custom drop tower for downstream applications, it was first necessary to optimise the energy of impact. Explants were impacted from increasing heights at 1 cm intervals from 1 cm to 7 cm and visualised with CMFDA (green) and PI (red) staining at 72 h after impact under a macro-confocal microscope a. IncreaHaving defined an appropriate height of impact, the effect of Ucn on impacted explants was investigated. Ucn-treated explants were impacted 30 min after the addition of Ucn and cultured for an additional 72 h before visualisation on a confocal microscope a. ImpactTo provide a more representative model of PTOA in terms of a potential therapy, it was also necessary to investigate the effect of Ucn administration post-impact. Explants were impacted and incubated for 30 min in normal 1% FCS media, before the addition of 0.1 µM Ucn to the media, and imaged at 72 h post-impact d. ImpactBecause Ucn has a similar affinity to both CRF-R1 and CRF-R2, and as both receptors are expressed by chondrocytes, it was important to determine which receptor was responsible for its chondroprotective effect. CRF is a specific agonist for CRF-R1, whereas Ucn2 and Ucn3 both interact more selectively with CRF-R2 . TherefoTo further assess whether this chondroprotection was a direct ligand interaction with CRF-R1 specifically, selective antagonists to the receptors were employed. These were used to treat explants pre-impact to block any potential rescue effect of Ucn c. ExplanPrevious work on chondrocyte cell lines and primary human chondrocytes has identified a link between increased intracellular calcium levels post-impact and cell death ,25,26,273+ to compare the effect of a non-selective cation channel blocker c). Both Gd3+ and post-impact Ucn treatment were able to significantly reduce the number of Fluo-4 AM-stained cells at 1 h post-impact when compared to explants impacted alone  and Piezo1 and 2. In the absence of specific blockers for the Piezo channels, the chondroprotective effect of the spider venom GsMTx4 (a non-selective cationic mechanosensitive ion channel blocker effective against Piezo1 but not TRPV4) ,29 was cTo confirm that the increase in cell death observed in HC-067047-treated explants was due to a lack of chondroprotection and not a cytotoxic effect of the drug , dose response tests for 12.5–75 µM HC-067047 was conducted on explants in the absence of impact c. No sigThe previous calcium influx experiments demonstrated a role of Ucn in the modulation of a non-selective cation channel post-impact in order to reduce the excessive calcium influx observed in impacted explants alone. Given that the stimulus to chondrocyte damage in this case was mechanical trauma, the protective effect of the mechanosensitive ion channel blocker GsMTx4 was used alongside Ucn to investigate whether they had the same protective capabilities a. ExplanTo study the role of Piezo1 in more detail in this system, explants were treated with the Piezo1-specific activator Yoda1 in the absence of impact c. Yoda1-Injurious impact to cartilage results in the initiation of cell death pathways in chondrocytes and the accumulation of dead chondrocytes and their remnants promotes matrix catabolism ,11. A deAs such, there is a need for a chondroprotective agent that can be administered within a short time frame after injury in order to promote chondrocyte health and prevent progression to a catabolic, diseased tissue environment. We have previously demonstrated that the small, 40 amino acid peptide Ucn is an essential chondrocyte pro-survival factor  and in tEk = mgh, where Ek = kinetic energy, m = mass of falling object, g = Earth’s gravitational acceleration and h = height of impact. Using a drop height of 5 cm and a 500 g weight resulted in an impact energy of 0.245 J. This is similar to that used elsewhere in the literature to induce OA-like changes in articular cartilage explants [A simple drop tower model of impact was employed, similar to that widely used in previous studies ,21,22,31explants ,31,32 anexplants ,34.Porcine samples from young, healthy pigs were utilised rather than human tissue due to logistical difficulties in obtaining healthy human cartilage. Porcine cartilage is widely used in the field of OA research as it shares many characteristics with human cartilage in terms of structure, thickness and the anatomy of the joint , and the3+. Further exploration with mechanosensitive ion channel blockers highlighted Piezo1 as the potential channel at work in this system, as Piezo1-specific agonist Yoda1 was able to produce similar levels of cell death which could further be rescued by Ucn. Ucn is able to bind both CRF-R1 and R2, with a slight preference for CRF-R2 [The calcium influx observed was reduced by pre- and even post-impact addition of Ucn, and with the use of non-selective cation channel blocker Gdr CRF-R2 . The CRFAntagonists of CRF-R1 (CP) and CRF-R2 (Ast2B) were also used pre-impact to identify the receptor at work in this system. Both CP and Ast2B-treated explants exhibited a significant increase in levels of cell death compared to non-treated explants. However, Ast2B-treated explants showed significantly fewer dead cells that CP-treated explants, suggesting that blocking CRF-R2 did not elicit as much damage as CRF-R1. The significant levels of cell death in Ast-2B-treated explants could suggest a role of Ucn binding to CRF-R2 in some cases to elicit chondroprotection. It is possible, therefore, that some compensation occurs between CRF-R1 and CRF-R2 when the explant is experiencing extreme stress in the form of impact. However, the results shown with Ucn2 suggest that Ucn binding CRF-R2 is not sufficient to significantly reduce levels of cell death.3+. Chondrocytes express a range of channels to regulate calcium in stress and non-stressful situations, including ligand-gated [3+ results indicate a non-selective ion channel, with mechanosensitive ion channels being the most likely candidates. We thus utilised GsMTx4, a potent blocker of cationic mechanosensitive ion channels [Having established the interaction between Ucn and its receptor, the influence of calcium was investigated using real-time microscopy immediately after impact. Both pre- and post-impact, Ucn was able to significantly reduce calcium influx at a 1 h time point, as was treatment with Gdnd-gated ,38, voltnd-gated ,40 and ond-gated ,42. The channels , along wTRPV4 and Piezo1 are well-discussed in chondrocyte literature  and we hOur findings, particularly that Ucn can elicit its chondroprotective effects even after impact has occurred, highlight a potential clinical use for Ucn in the prevention of PTOA through delivery shortly after joint injury. However, as Ucn is expressed in many other tissues, systemic administration may elicit effects in other tissues. This is particularly pertinent with regard to the effects of Ucn on the cardiovascular system, where it is known to reduce blood pressure due to its properties as a vasodilator ,46. To aTaken together, this study demonstrates that Ucn is a chondroprotective agent in the context of injurious mechanical overload. Its ability to provide chondrocyte cytoprotection post-trauma highlights its potential as a novel DMOAD to maintain cartilage health and prevent progression from injury to PTOA.v/v) foetal calf serum (FCS) , supplemented with 1000 units/mL penicillin and 1 mg/mL streptomycin for the first 24 h, dropping to 100 units/mL and 100 µg/mL, respectively, after this time. Explants were maintained at 37 °C and 5% CO2, with media changes every 48 h.Female 8-week-old suckling pigs were sacrificed by an abattoir according to local guidelines no more than 3 h before cartilage collection. Lateral and medial patellofemoral condyles were collected from both knees and washed in PBS, and 6 mm diameter biopsy punches were taken using a Uni-Core system punch . Punch biopsies that showed any superficial damage were discarded. Thickness of the explants varied due to the anatomical location, with explants demonstrating a thickness no greater than 6 mm retained. Explants were cultured in 24-well plates (one explant/well) containing Dulbecco’s Modified Eagle Medium  with 10% (Cartilage explants were subjected to impact from a custom-made drop tower device capable of applying a 500 g weight under sterile conditions (adapted from ). Prior 2 in media containing the relevant recombinant peptides or modifying agents in 1% FCS media for 30 min prior to impact. Post-impact treated explants were placed into fresh media with the relevant recombinant peptides or modifying agents added 30 min following impact. All explants were incubated for a further 72 h with no further media changes prior to assay.Pre-impact treated explants were incubated at 37 °C and 5% CO3+)  was used for calcium experiments at 500 µM concentration.In order to test the role of Ucn in the prevention of chondrocyte cell death, explants were incubated with 0.1 µM human Ucn (Ucn1)  or Ucn2 . To investigate the role of Ucn receptors, CRF , CP-154526 (CP)  and Astressin 2B (Ast2B)  were used at 0.1 µM. For ion channel identification, Yoda1  and GsMTx4  were also used at 0.1 μM concentration. HC-067047  was used either at 50 µM concentration, or in a concentration from 12.5 µM to 75 µM. Gadolinium  and incubated at 37 °C for 30 min. PI stain  0.5 µM was added and incubated for 1 min before visualisation to allow for penetration of the dye without compromising the number of live cells present in the explant.Macro-confocal images were obtained with the Leica Macro-confocal TCS LSI at 1× magnification. Images were captured in a format of 1024 × 1024 pixels with a scanning speed of 600 Hz and z-stack steps of 10 µm. CMFDA was visualised using a 488 nm laser line; PI was visualised under sequential scanning using the 532 nm laser line.All other confocal images were captured using an upright Leica SP8 multi-photon microscope. Explants were visualised using a 10× objective, with the superficial zone oriented towards the objective in a PBS-lined 30 mm plate. A 1024 × 1024 pixel image size was collected for each explant under a scanning speed of 700 Hz with a step size of 2.5 µm. CMFDA and PI were visualised simultaneously with a laser emission bandwidth of 484–555 nm. The gain of the 488 ATOF laser was adjusted to 10.0 as compensation between the two dyes. To avoid operator bias and to ensure the whole explant was represented, a mark-and-find protocol was used to define 5 fields of view for analysis over the explant, which was kept consistent for each explant during one experiment. Total z-stack range varied between images due to the natural curvature of the explant.Macro-confocal images were analysed using ImageJ software. Images were converted to 8-bit and area and integrated density measurements were collected for each CMFDA and PI image, excluding the edge to eliminate any possibility of edge effect. The ratio of CMFDA:PI integrated density was presented as a green:red reading.All other confocal images were analysed using the Spots function of Imaris v9.64. Cell diameter was determined by taking an average of 10 cells from a single snapshot and kept consistent throughout the analysis of each experiment. Thresholds of fluorescence intensity were adjusted based on one representative image per experiment and kept consistent throughout the analyses. The number of spots observed in the green and red channels were interpreted as the total number of cells seen in the image, with one spot equivalent to one cell.Lactate dehydrogenase (LDH) assays were conducted using the Pierce™ LDH Cytotoxicity Assay Kit  according to the manufacturer’s instructions. At the 72 h time point, 50 µL media from each well was plated in duplicate into wells of a 96-well plate along with a sample lysed with the kit lysis buffer as a positive control of LDH release. Explants were weighed and final LDH readings were adjusted based on weight to compensate for differences in the height of the explant.2, 7 mM glucose, 2.6 mM CaCl2, in dH2O at pH 7.4) containing 1 μM Fluo-4 AM and 5 μM Hoechst-333285 and placed under normal culture conditions in the dark. Post-impact Ucn- and Gd3+-treated explants were administered 0.1 μM Ucn or 500 μM Gd3+ 30 min post-impact. At 1 h post-impact, all explants were visualised using a Leica SP8 confocal microscope.Explants were impacted as described and returned to normal culture conditions in 1% FCS media for 10 min. All explants were then placed into buffer (137 mM NaCl, 6 mM KCl, 10 mM HEPES buffer, 1 mM MgClData from each experiment were collected into Microsoft Excel spreadsheets and collated on GraphPad Prism8. Data were tested for normality using a D’Agostino and Pearson normality test, then analysed using a one-way ANOVA with Tukey’s multiple comparisons test if normally distributed or Kruskal–Wallis with Dunn’s multiple comparisons test if not. Graphs in


However, we found that HeLa cells overexpressing the Orai1 channel displayed enhanced Ca2+ entry and a limited ER depletion in response to the combination of ATP and thapsigargin (TG) in the presence of external Ca2+. As these effects require the combination of an agonist and TG, we decided to study whether the phosphorylation of Orai1 S27/S30 residues had any role using two different mutants: Orai1-S27/30A  and Orai1-S27/30D . Both O1-wt and O1-AA supported enhanced Ca2+ entry, but this was not the case with O1-E106A (dead-pore mutant), O1-DD, and O1-AA-E106A, while O1-wt, O1-E106A, and O1-DD inhibited the ATP and TG-induced reduction of ER [Ca2+], suggesting that the phosphorylation of O1 S27/30 interferes with the IP3R activity. O1-wt and O1-DD displayed an increased interaction with IP3R in response to ATP and TG; however, the O1-AA channel decreased this interaction. The expression of mCherry-O1-AA increased the frequency of ATP-induced sinusoidal [Ca2+]i oscillations, while mCherry-O1-wt and mCherry-O1-DD decreased this frequency. These data suggest that the combination of ATP and TG stimulates Ca2+ entry, and the phosphorylation of Orai1 S27/30 residues by PKC reduces IP3R-mediated Ca2+ release.The overexpression of the Orai1 channel inhibits SOCE when using the Ca Some of them are the voltage-gated Ca2+ channels ER) with the activation of Orai channels at the plasma membrane ER reduction results in Ca2+ unbinding from the EF-hand and the aggregation of this protein, forming the puncta [2+ concentration ([Ca2+]i), remarkably when the SERCA pump is inhibited i due to IP3 action activates PKC i response to the addition of either TG (1 μM) or ATP (10 μM) in the presence of 1.8 mM external [Ca2+]  was slower  with an averaged frequency of 4.4 oscillations in the 3-min evaluation period  as seen with the mock cells . Moreover, the amplitude of the two types of [Ca2+]i responses was enhanced by the expression of the O1-AA channel compared with those expressing the O1-DD channel , and similarly to O1-wt, O1-DD changed the [Ca2+]i oscillation to the baseline type. A single [Ca2+]i oscillation was the most frequent event observed for those cells overexpressing the O1-DD channel  to avoid SERCA pumps diverting Ca2+ entering the cytoplasm to the ER. We detected simultaneous changes in [Ca2+]ER and [Ca2+]i using the combination of Mag-Fluo-4 and Fura-2; the former readily enters the ER [2+]ER reduction, as was the case with Mag-Fluo-4. Since only Mag-fluo-4 can be used together with Fura-2, we decided to use this combination for the rest of the experiments. ATP and TG combined in the mock-transfected cells resulted in a substantial [Ca2+]ER depletion, as expected i response in cells overexpressing only functional Orai1 channels and in the presence of 1.8 mM CaCl2. Such an effect did not require the overexpression of STIM1, a requisite for Orai1 to increase SOCE [2+]i response. This effect required a functional channel because of the absence of external [Ca2+] or overexpressing Orai1-E106A, a dominant-negative mutant of the Orai1 channel, resulting in a slower [Ca2+]i response. This effect of the O1-E106A channel has two possible explanations; the inhibition of endogenous O1 channels or the inhibition of IP3R-mediated Ca2+ release. As the O1-AA-E106A mutant channel did not inhibit the agonist-induced [Ca2+]i response, and O1-AA mutant had a decreased interaction with IP3R in response to the agonists, thus the data with O1-E106A support the idea that the O1 channel inhibits agonist-induced ER Ca2+ release. Furthermore, the expression of all three O1 channels resulted in a higher frequency of transient [Ca2+]i response, particularly for the O1-AA channel. However, both O1-wt and O1-DD resulted in baseline [Ca2+]i oscillating cells. These data suggest that the phosphorylated O1 channel stabilizes the inactivated form of IP3R, resulting in a decreased reduction in the luminal [Ca2+]ER, and this scenario might explain the effect of the agonist on the enhanced interaction between O1 and IP3R seen with PLA.Many reports show that the Orai1 channel overexpression produces a potent inhibition of SOCE ,52, a siase SOCE . The ove2+ entry is seen with the overexpression of functional Orai1 channels involved in the phosphorylation of different amino acids beyond S27 and S30. The Orai1 mutant lacking the PKC-mediated phosphorylation sites, Ser27 and Ser30, displayed an enhanced Ca2+ entry, as seen with the O1-wt. This result agrees with previous publications where the O1-AA mutant supports SOCE equally well i response [3R sensitivity to its agonist or a reduction in the luminal [Ca2+]ER i and the signal was used to normalize the changes in the Mag-Fluo-4 fluorescence. The Fura-2 ratio (340/380) was transformed into [Ca2+]i using Grynkewicz’s equation [2+]i = Kdβ(R − Rmin)/(Rmax − R), and the FeliX32 analysis software (PTI), where Rmax is the fluorescence ratio after the addition of digitonin (200 μM) and the Rmin is the fluorescence ratio after the addition of EGTA (5 mM). This calibration was carried out at the end and for all of the experiments. These ratios were adjusted for viscosity using the correction factor β = 0.75 [d for Fura-2 of 200 nM. The background fluorescence was obtained using 12 mM MnCl2; these values were subtracted from 340 and 380 signals before calculating the ratios. The fluorescence signal reported by Mag-Fluo-4 was normalized to the basal time course (F0) employing a nonlinear fitting algorithm, and it was expressed as a fraction (∆F/F0).After transfection, HeLa cell monolayers were washed twice with PBS, followed by trypsinization for 1.5 min. The cell suspension was placed in DMEM supplemented serum and microfuged at 400× nditions ,46,47,56nditions . Brieflyβ = 0.75  and a KdWe introduced two points mutations (S27D/S30D) in the plasmid p3XFlag-CMV-10-Orai1-S27A/S30A—A kind gift by Dr. Stefan Feske —and the The constructions for mCherry-Orai1-S27A/S30A and mCherry-Orai1-S27D/S30D were carried out using p3XFlag-CMV-7.1-mCherry-Orai1. In this case, we replaced the Orai1-wt cDNA with those for the mutations (either S27A/S30A or S27D/S30D) employing the unique BspEI (codifying region) and BamHI (polylinker) sites. Sanger’s DNA sequencing was used to verify the mutations and for correct ligation for all the new constructions.CFP-Orai1, p3XFlag-CMV-7.1-mCherry-Orai1, and STIM1-CFP were kind gifts from Luis Vaca .2, 5 mM glucose, 25 mM Hepes and 1.8 mM CaCl2, pH 7.4 adjusted with NaOH) and kept for 10 min at 37 °C followed by ATP (10 μM) plus TG (1 μM) stimulation for 3 min. Immediately after stimulation, the cells were lysed with a mix of 2× NP-40 lysis buffer, EDTA-free proteases inhibitors, and 2 mM Na₃VO₄ on an ice bed. The samples were sonicated and centrifuged (1.6 × 104× g for 30 min at 4 °C). The protein pellets were quantified with the BCA protein assay kit and immunoprecipitated with 1 μg of either anti-Orai1 or anti-Flag antibodies to distinguish the Orai1 mutants that have a Flag tag at the amino-terminal domain and 30 μL of protein A agarose by overnight incubation at 4 °C in an oscillating rotor. The microfuged beads  were washed twice with cold PBS, and the proteins were denatured with Laemmli’s sample buffer supplemented with 5% DTT and 5% β-mercaptoethanol at 70 °C for 10 min followed by placing them on ice for 10 min. The supernatant of microfuged beads  was separated in SDS-PAGE (10% acrylamide gel) followed by blotting to a nitrocellulose membrane in semi-humid conditions (0.8 mA/cm2 of blotting paper). Membranes were blocked by incubating them with 10% (w/v) BSA diluted in TBST  solution for 1 h at room temperature. Next, they were incubated with primary antibody diluted (1:500) in 10% BSA overnight at 4 °C, followed by 6× washes with TBST and then incubated for 1 h with horseradish peroxidase-conjugated goat anti-mouse IgG antibody , followed by 6× washes and incubated with SuperSignal West Dura chemiluminescent substrate for 5 min.Immunoprecipitation and Western blot were carried out as previously described ,59. Brie3R signal/Orai1 signal.Bands were quantified with a C-DiGit Chemiluminescent Western Blot Scanner . The data shown are the ratio of IP3R2, diluted 1:50 in a Duolink antibody solution and incubated for 60 min at 37 °C in a humidified chamber. Next, the coverslip was washed twice with Wash Buffer A (5 min at room temperature) and incubated with PLA probes (PLUS and MINUS PLA probes) for 60 min at 37 °C in the humidified chamber. The sample was washed twice with Wash Buffer A (5 min at room temperature) and incubated with ligase diluted in 1× Duolink ligation buffer at 1:40 for 60 min at 37 °C, followed by 2× washes with Wash Buffer A (5 min at room temperature). The amplification reaction was carried out with diluted polymerase (1:80 with 1× amplification buffer) in the dark at 37 °C for 100 min. Next, the sample was washed with Wash Buffer B for 10 min, and one additional wash was done with 0.01× Wash Buffer B for 1 min at room temperature. The excess of Wash Buffer B was removed and followed by Duolink in situ Mounting Medium with DAPI. The samples were analyzed in an epifluorescence inverted microscope  with an image acquisition and analysis system for video microscopy . The PLA signal was excited at 594 nm and collected at 624 nm wavelengths, in addition to DAPI signals and brightfield images of cells.Proximity Ligation Assay was adapted from the manufacturer’s instructions, as previously described , with miPreviously, HeLa cells were grown on 21 mm × 21 mm coverslips, and once they reached 50% confluency, were transfected with 2 μg of DNA of either an empty vector (mock), mCherry-Orai1 wt, mCherry-Orai1-S27D/S30D, or mCherry-Orai1-S27A/S30A for 24 h with Lipofectamine 2000, as described above. HeLa cells were fixed with 2 mL per well of 4% (PBS) cold paraformaldehyde for 10 min at room temperature, follow by two PBS washes and permeabilization with 2 mL per well of 0.5% (PBS) Triton X-100 for 10 min at room temperature. The cells were washed three times with PBS and incubated with DAPI solution  for 15 min. HeLa cell imaging was carried out with a ZEISS LSM700 confocal microscope using 63× objective (NA 1.4). The fluorescence signal from the mCherry protein was excited with the 555 nm laser line at one Airy unit (45 μm) and collected at 580 nm. The image dimensions were 1024 × 1024 pixels, 40 nm pixel size, and 12-bit resolution; these images were then deconvolved  and their intensity profiles were obtained to visualize the Orai1 channel distribution for different mutants. All the data were analyzed with ImageJ software and were scaled in arbitrary units of fluorescence.2+. Images were acquired with one track, two channels, ex/em at 488/510 nm for Fluo-4 and 555/580 nm for mCherry signals. Image sizes were 512 × 512 pixels with a pixel size of 200 nm and 8 bit resolution. A cytoplasmic mask was used to calculate ∆(F − Fmin)/(F0 − Fmin) at a 5-s resolution time during the 4 min recording time. The Fmin was the fluorescence signal in the absence of a cell. The average fluorescence for 1 min before the addition of 10 μM ATP was considered Fo.Confocal calcium imaging. HeLa cells grown and transfected as indicated above were loaded with 1 μM Fluo-4/AM at room temperature for 30 min in the dark. After this time, the cells were washed once with a recording saline solution with Cat-tests with one-tail or one-way ANOVA with Dunnett’s post hoc test (GraphPad Prism 5.0). The difference was statistically significant if the null hypothesis had a * p < 0.05.The data shown are the mean ± SEM, with n being the number of independent experiments carried out for each condition. Depending on the experimental design, data were analyzed with either unpaired


Although Jaw1 is known to interact with inositol 1,4,5-triphosphate receptor (ITPRs), Ca2+ channels on the endoplasmic reticulum, the function of Jaw1 in the Ca2+ dynamics with physiological stimulation remains unclear. In this study, using inducible Jaw1-expressing HEK293 cells, we showed that Jaw1 increases Ca2+ influx by GPCR stimulation via changing the Ca2+ influx oscillation pattern. Furthermore, we showed that Jaw1 increases the Ca2+ release activity of all ITPR subtypes in a subtly different manner. It is well known that the Ca2+ influx oscillation pattern varies from cell type to cell type, therefore these findings provide an insight into the relationship between the heterogeneous Ca2+ dynamics and the specific ITPR and Jaw1 expression patterns.Ca Intracellular Ca2+ is stored in the endoplasmic reticulum (ER), and cell surface receptor-mediated extracellular stimuli trigger Ca2+ influx into the cytoplasm to initiate a physiological response4.Intracellular Ca2+ channels on the ER membrane and cell surface has been described in stimuli-dependent Ca2+ dynamics regulation. Inositol 1,4,5-triphosphate receptors (ITPRs), Ca2+ channels on the ER membrane, release Ca2+ from the ER into the cytoplasm in an IP3-dependent manner when the IP3 is produced upon G protein-coupled receptor (GPCR)-related stimulation6. The Ca2+ efflux from the ER subsequently leads to Ca2+ depletion in the ER, triggering the activation of stromal-interaction molecule (STIM), a Ca2+ sensor protein on the ER8. The activated STIM interacts with Orai, a plasma membrane Ca2+ channel, resulting in Ca2+ influx from the extracellular source into the cytoplasm, called store-operated calcium entry (SOCE). The cytoplasmic Ca2+ is retrieved by sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), a Ca2+ pump on the ER, resulting in Ca2+ replenishment in the ER9. This Ca2+ dynamics series is repeated and observed as a cytosolic Ca2+ concentration ([Ca2+]c) oscillation. Interestingly, recent studies revealed that the oscillation, as well as the cytoplasmic Ca2+ influx level, is important for Ca2+-dependent protein activation11.To date, the involvement of several basic Ca15. We previously reported that Jaw1 is localized on the ER and the outer nuclear membrane, and it is associated with nuclear shape maintenance16. Furthermore, Jaw1 was recently reported to interact with all ITPR subtypes via its coiled-coil domain, and the deletion of Jaw1 in mouse embryonic fibroblast (MEF) reduces Ca2+ influx into the cytoplasm upon ionomycin stimulation, a reagent that increases cell membrane ion permeability13. However, whether and how Jaw1 is involved in the calcium dynamics under physiological conditions, such as GPCR stimulation-related aspects, remains unclear. Furthermore, physiological differences have been reported among ITPR1–3, three ITPR isoforms, such as IP3 affinity, Ca2+ release activity, and in vivo expression patterns20, but it remains elusive whether Jaw1 affects the ITPR activities subtype-specifically.Jaw1, also known as lymphoid restricted membrane protein (LRMP), is a type II integral membrane protein with specific expression in immune cells, taste cells, pancreatic acinar cells, and small intestinal tuft cells2+ influx upon GPCR stimulation, leading to changes in the Ca2+ influx pattern. Furthermore, our data indicated that Jaw1 interacts with all ITPR subtypes, increasing their Ca2+ release activity with subtle differences among each subtype. These results contribute to our understanding of the physiological significance of the heterogeneous Ca2+ dynamics among cell types and the involvement of Jaw1, as an ITPR regulator, in the uniqueness of Ca2+ dynamics in tissues or cells.In this study, we showed that Jaw1 increases cytoplasmic Ca2+ dynamics upon physiological stimulation, we created HEK293 Flp-In T-REx Jaw1 inducible-expressed cells with doxycycline (Dox) treatment-inducible Jaw1 expression. As previously reported, endogenous Jaw1 is expressed in HEK293 cells at a low level21. First, we generated HEK293 Flp-In T-REx Jaw1 KO cells (Jaw1 KO cells) by CRISPR/Cas9 , we prepared cells expressing Jaw1 at medium and high levels  among the cell lines  suggest that the Jaw1–ITPR interaction on the ER increases the Caa2+ Fig. A. The tolls Fig. B,C, indiype Fig. D. Ca2+ iity Fig. E,F. Howells Fig. E,G, indi24. To confirm whether Jaw1 interacts with all ITPR subtypes, we first prepared Jaw1 KO, Jaw1 IE, and Δcoil IE cell lysates and performed co-immunoprecipitation. The results showed that all ITPR subtypes co-immunoprecipitated with Jaw1, but none with Δcoil, suggesting that Jaw1 interacts with all subtypes via its coiled-coil domain , expressing exclusively a single subtype of each ITPR , a Jaw1 homolog, is also known to interact with ITPRs but represses their activity unlike Jaw12+ influx activates the Ca2+ adaptor proteins and enzymes in the cytoplasm, which transmit various signals4. Importantly, recent studies revealed that the oscillation, as well as the cytoplasmic Ca2+ influx level, is important for downstream protein activation30, suggesting that the oscillation-mediated duration of high [Ca2+]c above a certain threshold could be a key factor to induce the physiological responses. Indeed, several cellular events, such as secretion and gene expression, reportedly take a few minutes to initiate a response after the stimulation31. Of interest, as shown in Figs. 2+ imaging in Jaw1 IE cells demonstrated that each kinetic curve is more oscillated than that of Jaw1 KO cells. Particularly, the curve for steady reduction type showed a trend of being highly oscillated, although each ratio was depending on the concentration of ligand as well as the expression of Jaw1. Thus, more frequent Ca2+ peaks brought by the expression of Jaw1 increase the residential time of Ca2+ in the cytosol at single cell level. These data provide us a new hypothesis that the augmentative effect of Jaw1 on the sustained Ca2+ influx contributes to downstream response activation in Jaw1 expression-specific cells. To verify this concept, the Ca2+ dynamics in taste or tuft cells, expressing Jaw1 abundantly under physiological conditions, should be investigated in future studies.The cytoplasmic Ca2+ influx analysis focusing on each ITPR subtype, Jaw1 expression changed the cytoplasmic Ca2+ influx oscillation pattern in R1 and R2 SE cells, but not in R3 SE cells. The ability of ITPR3 to induce continuous cytoplasmic Ca2+ influx is reportedly the lowest among the three subtypes32, which might result in the smaller effect of Jaw1 on the residential time in the R3 SE cells. On the other hand, the Jaw1 expression had more augmentative effects on the maximum amplitude in R3 SE cells. Interestingly, the amino acid sequence homology among the subtypes is approximately 65–70%33, and their interactors are different depending on the subtypes34. These factors might also contribute to the above-described phenotypic differences among the subtypes, although further analyses would be required to clarify these aspects. Intriguingly, the ITPR subtype expression patterns were tissue- and cell-dependently heterogenous. Jaw1 and ITPR2 are expressed in small intestinal tuft cells, chemosensory cells that detect parasitic infection, Jaw1 and ITPR3 in tongue epithelial taste cells that perceive taste, and Jaw1 and ITPR2/3 in pancreatic acinar cells that secrete digestive enzymes. The non-uniform Jaw1 effect on the Ca2+ dynamics in each cell type and Jaw1 depletion in MEF reportedly reduced the Ca2+ influx while it increased that in pancreatic acinar cells15. This heterogeneous Jaw1 effect on each ITPR subtype might explain the non-uniform effect of the Jaw1 depletion on Ca2+ dynamics. It would be also important to investigate the biological significance of how the ITPR subtype-specific Ca2+ influx, as well as ITPR and Jaw1 expression patterns, are involved in the physiological responses in each tissue or cell in vivo.In the Cal-glutamine , 100 U/mL penicillin, and 100 µg/mL streptomycin . Cells were grown in 5% CO2 at 37 °C.Each HEK293 Flp-In T-REx cell line  was cultured in DMEM supplemented with 10% fetal bovine serum , 5.84 mg/mL Fect™A plus . After incubation for 48 h, the cells were subjected to selection by treatment with 2 µg/mL puromycin . The cells were then single-colonized by the limiting dilution cloning method in a 96-well plate and expanded. For the verification of the mutated sequence in each cell line by Sanger sequencing, genomic DNA was purified from each cell line using QIAamp DNA Micro Kit , and the amplified PCR products containing the target region were submitted to the DNA sequence contract service (Genewiz). Two clones for each cell line  from the cells with mutated sequences were randomly selected and used for following assay to remove the bias behind genetic background between clones. The numbers with cell name are original experimental clone number. Of note, there was no differences in the results between two clones. For the HEK293 Flp-In T-REx Jaw1 IE and Δcoil cell generation, HEK293 Flp-In T-REx Jaw1 KO #17 cells were transfected with pcDNA5 FRT/TO Hu SJaw1 or pcDNA5 FRT/TO Hu SJaw1 Δcoil using ScreenFect™A plus. After incubation for 48 h, the cells were selected by treatment with 100 µg/mL Hygromycin B  and expanded.For the generation of HEK293 Flp-In T-REx Jaw1 KO cells and ITPRs SE cells using the CRISPR/Cas9 system, HEK293 Flp-In T-REx cells were transfected with pSpCas9-2A-Puro Jaw1 or ITPRs sgRNA using the ScreenRabbit polyclonal anti-human Jaw1 antibody was raised against truncated Jaw1 encoding amino acids 1–440 (Operon Biotech). The sequence of human Jaw1 was referred from NCBI (NP_00191055). The serum was subjected to affinity chromatography using N-Hydroxysuccunumidyl-Sepharose 4 Fast Flow  coupled with the N-terminal region of Jaw1 encoding amino acids1–144, resulting in purified anti-Jaw1 N-ter antibody.35) was digested with BamHI and XhoI, and the fragment was subcloned into a pcDNA5 FRT/TO vector digested with the same enzymes using a commercial DNA ligation kit . For the production of pcDNA5 FRT/TO Hu SJaw1 Δcoil, pcDNA3.1 (+) Hu LJaw1 (previously described35) was first performed by inverse PCR with primer set 1 resulting in pcDNA3.1 (+) Hu LJaw1 Δcoil. pcDNA3.1 (+) Hu LJaw1 Δcoil was then digested with BamHI and XhoI, and the fragment was subcloned into a pcDNA5 FRT/TO vector (digested with the same enzymes) using the aforementioned DNA ligation kit, resulting in pcDNA5 FRT/TO Hu LJaw1 Δcoil. Furthermore, pcDNA5 FRT/TO Hu LJaw1 Δcoil was digested with HpaI and XhoI, and the fragment was subcloned into pcDNA5 FRT/TO Hu SJaw1 digested with the same enzymes to swap the N-terminal region of Hu LJaw1 to that of Hu SJaw1.For the production of pcDNA5 FRT/TO Hu SJaw1, pcDNA3.1 (+) Hu SJaw1 -2A-Puro (PX459) vector  digested with g for 30 min. The supernatants were added to SDS-PAGE sample buffer and heated at 95 °C for 5 min. The samples were loaded onto a poly acrylamide gel, electrophoresed, and transferred onto polyvinylidene difluoride membranes . The membranes were blocked with tris-buffered saline containing 0.1% Tween20 (TBS-T) and 3% skim milk  for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: rabbit anti-Jaw1 N-ter , rabbit anti-ITPR1 , mouse anti-ITPR2 , mouse anti-ITPR3 , mouse anti-α-Tubulin , or mouse anti-GAPDH . The membranes were washed with TBS-T three times and incubated for 3 h with the following secondary antibodies: anti-Mouse IgG, HRP-Linked Whole Ab Sheep  or anti-Rabbit IgG, HRP-Linked Whole Ab Donkey . The membranes were washed with TBS-T three times and reacted with Immuno-star Zeta  or SuperSignal™ West Atto Ultimate Sensitivity Substrate . The bands were detected using an LAS4000 imaging system. The membranes were cut prior to hybridization with antibodies as shown in Supplementary Fig. Each cell line was seeded onto six-well plates  and incubated overnight. The cells were then treated for an additional 24 h with each Dox concentration to induce Jaw1 expression. After that, the cells were harvested by gentle pipetting, washed once with PBS, and lysed with RIPA buffer  containing a protease inhibitor cocktail . The samples were sonicated for 10 min on ice and centrifuged at 12,000 × 16). After the incubation for 1 h, the cells were washed with PBS three times and incubated for 1 h with Hoechst 33342 along with the following secondary antibodies: goat anti-rat IgG conjugated with Alexa Fluor 488  and goat anti-rabbit IgG conjugated with Alexa Fluor 568 . The cells were then washed with PBS three times and observed using fluorescence microscopy .Each cell line was seeded onto 96-well plates  and incubated overnight. The cells were treated with each Dox concentration to induce Jaw1 expression. After incubation for 24 h, the cells were fixed with 4% PFA in PBS buffer for 15 min. The cells were then washed with PBS once and permeabilized with 0.2% Triton-X  in PBS for 10 min. Next, the cells were washed with PBS three times and blocked with 2% bovine serum albumin in PBS for 1 h. The cells were then incubated with the following primary antibodies: rabbit anti-Jaw1 N-ter  and rat anti-Jaw1 coil  containing 0.5% NP-40 and 1 µL of a protease inhibitor cocktail . The lysates were sonicated for 10 min on ice and centrifuged at 12,000 × g for 30 min at 4 °C. The lysates were diluted with lysis buffer into 0.1% NP-40, then incubated with anti-Jaw1 antibody-conjugated beads for 1 h at 4 °C. The conjugated beads were prepared by mixing the anti-Jaw1 N-ter antibody and Protein A Mag Sepharose  for 1 h at 4 °C. The beads reacted with the cell lysate were then collected by a magnetic stand and washed with lysis buffer three times. For elution, the beads were mixed with SDS-PAGE buffer and heated at 95 °C for 5 min. On the magnetic stand, the supernatants were collected and subjected to western blotting.Each cell line was cultured on a six-well plate and incubated overnight. The cells were treated with 200 ng/mL Dox to induce Jaw1 expression. After incubation for 24 h, the cells were collected by gentle pipetting and centrifuged at 500 × jaw1 and gapdh gene detection, primer sets 6 and 7 were used, respectively.Each cell line was cultured on a six-well plate and incubated overnight. The cells were treated with 200 ng/mL Dox to induce Jaw1 expression. After incubation for 24 h, total RNA was isolated using RNAiso Plus . Total RNA was then reverse transcribed using PrimeScript RT Master Mix , and subsequent PCR was performed using TB Green Premix EX Taq II  on Thermal Cycler Dice Real Time System II MRX . For 2+ condition, the cells were incubated for 24 h, washed with PBS once, and incubated with recording buffer  containing 2 µM Fluo-4 AM  or 2 µM Rhod-2 AM  for 30 min. The cells were then washed with PBS once and incubated with HBSS containing 1.25 mM probenecid for 30 min. For the well scanning calcium assay using a plate reader, Varioskan LUX (Thermo Fisher Scientific) was used. The fluorescence values were recorded every second, and ATP  or carbachol  solution was added at 10 s. The data was measured from three independent wells. For the calcium imaging, we used a fluorescent microscope  equipped with a 20X fluor objective. The fluorescent images were captured every 2 s, and ATP solution was added at 10 s. The data was measured from four independent wells. For the calcium assay under Ca2+ free conditions, the cells were incubated with Ca2+ free recording buffer containing Fluo-4 AM for 30 min, washed with PBS once and incubated with Ca2+ free recording buffer. For the well scanning calcium assay for SOCE activity quantification, a plate reader was used and 2 µM thapsigargin solution was added at 10 s and recording buffer with 2 mM CaCl2 at 480 s. For the calcium imaging under Ca2+ free conditions, fluorescence microscopy was used and the 100 µM ATP solution without Ca2+ was added at 10 s.Each cell line was seeded onto 96-well black wall plates  and incubated overnight. The cells were treated with each Dox concentration to induce Jaw1 expression. For the calcium assay with extracellular Ca0 and the relative fluorescent intensity at each time point was calculated as F/F0. The calculated data was then graphed in GraphPad Prism7.The fluorescence intensity at 0 s was defined as FFor the calcium imaging, fluorescent images of the Fluo-4-loaded cells upon ATP stimulation were analyzed and quantified by Fiji. The calcium imaging was performed in the four independent wells and data from 50 cells in each image was used for the quantification. We randomly chose 50 cells from each image, and the center of each cell (8 × 8 pixel) was selected as a representative area. The fluorescent intensity in the chosen area was measured over time for 5 min.2+ influx types as follows: (1) The first group (single type) included the cells with amplitudes only once for 5 min. (2) The second group (oscillation type) included the cells with more than two oscillations. The oscillation was defined as the relative Fluo-4 intensity decreasing below 125% with a peak that rose again during 5 min. (3) The third group (steady reduction type) included the cells that maintained the relative Fluo-4 intensity higher than 125% of the initial values for 5 min. (4) The fourth group (no response type) included the cells maintaining the amplitude less than 300% of the initial value for 5 min. To calculate the ratio, we used 50 cells for each of the four measurements .Relative Fluo-4 intensity of each cell was plotted for 5 min using GraphPad Prism 7. The relative Fluo-4 intensity curves were then transferred to layouts and manually classified into four Cat-test was used to compare two sample groups. To compare more than two groups, one-way ANOVA followed by Tukey–Kramer’s t-test was used. *, **, ***, and **** indicate statistically significant P values of p < 0.05, < 0.01, < 0.001, and < 0.0001, respectively. n.s.: non-significant.All statistical tests were calculated using GraphPad Prism 7, and the data are represented as the mean ± S.D. The sample numbers are included in each figure legend. Unpaired, nonparametric Student’s Supplementary Information 1.Supplementary Information 2.


Microbial growth leads to a decrease in apical glucose levels. T1Rs detect this change and liberate bitter taste receptor signaling, initiating an innate immune response to both kill and expel pathogens through releasing antimicrobial peptides and increasing nitric oxide production and ciliary beat frequency. However, chronic inflammation due to disease, smoking, or viral infections causes a remodeling of the epithelial airway. The resulting squamous metaplasia causes a loss of multi-ciliated cells and solitary chemosensory cells, replaced by basal epithelial cells. To understand how T1R function is altered during disease, we used basal epithelial cells as a model to study the function of T1R3 on Ca2+ signaling dynamics. We found that both T1R1 and T1R3 detect amino acids and signal via cAMP, increasing the responsiveness of the cells to Ca2+ signaling stimuli. Either knocking down T1R1/3 or treating wild-type cells with MEM amino acids caused a reduction in ER Ca2+ content through a non-cAMP signaled pathway. Treatment with amino acids led to a reduction in downstream denatonium-induced Ca2+-signaled caspase activity. Thus, amino acids may be used to reduce unwanted apoptosis signaling in treatments containing bitter compounds.T1Rs are expressed in solitary chemosensory cells of the upper airway where they detect apical glucose levels and repress bitter taste receptor Ca N-Acyl homoserine lactones [2+) signaling cascade leading to an increase in nitric oxide (NO) production while also increasing ciliary beat frequency to hasten mucociliary clearance, thus providing a means to both kill and remove ensnared pathogens [Upper airway epithelial cells initiate an innate immune response through taste receptor signaling pathways in retaliation against inhaled pathogens. Bitter taste receptors T2Rs) localized to the motile cilia of multi-ciliated airway epithelial cells  detect “Rs localilactones . While mathogens ,5.3, and Gγ13, which signal for intracellular Ca2+ elevations [Pathogens feed on apical glucose in the airway surface liquid. Solitary chemosensory cells (SCCs) are a rare epithelial cell type that contain both T2Rs and sweet taste receptors (T1Rs) ,6,7,8. Ievations ,15,16. Tevations . This pa2+) signaling through detection of apical glucose [2+-dependent release of anti-microbial peptides from neighboring multi-ciliated cells into the apical lumen of the airway to combat infections. Thus, in a typical, heathy epithelial layer, T1Rs and T2Rs function as sentinels of the upper airway.In airway SCC’s, T1R2 and 3 are expressed  and hypo glucose . Once pa glucose . T2Rs thHowever, chronic inflammation, caused by diseases such as chronic rhinosinusitis (CRS) , COPD 2, smoking2+ stores. Thus, umami receptor T1R1/3 may play a pivotal role in reducing apoptosis activated by bitter drugs in infected airway cells.While bitter compounds increase NO production, we have also shown that T2R activation induces apoptosis in squamous and/or basal airway cells . MoreoveFor a detailed list of reagents used in this work, please refer to g lysates were estimated by Bradford DC Assay  then 60 µg protein/lane was loaded into a 4-12% Bis-Tris gel. After electrophoresis, the resulting gel was transferred to nitrocellulose and the membrane was blocked in 5% milk in Tris-Tween  for at least 1 h. The primary antibody was diluted 1:1000 in Tris-Tween containing 5% bovine serum albumin and incubated for at least 2 h. The secondary antibody goat anti-rabbit IgG-horseradish peroxidase was diluted 1:1000 in Tris-Tween containing 5% milk. After extensive washing, blots were incubated in Clarity ECL  as per manufacturers’ protocol, and resulting immunoblots were visualized using a ChemiDoc MP Imaging System . Final images were processed using Image Lab Software .For endogenous T1R1 and T1R3 immunoblot visualization, cell pellets were resuspended in RIPA Buffer  and lysed via sonicating water bath. Protein concentrations of post 800× 2+ measurements, cells were loaded with either 5 µM of Fluo-8 AM or Fura-2 AM for 1 h in HEPES-buffered Hank’s Balanced Salt Solution in the dark at room temperature. For intracellular or nuclear cAMP, biosensors were transfected 48 h prior to measurements. Fura-2 AM images were taken using Fura-2 filters  while all other images were taken utilizing FITC or TRITC filter sets (49002-ET or 49004-ET Chroma) on an Olympus IX-83 microscope (20× 0.75 NA objective) with fluorescence xenon lamp and X-Cite 120 LED boost lamp , excitation and emission filter wheels , Orca Flash 4.0 sCMOS camera  and MetaFluor software .For intracellular CaPrimary human sinonasal cells were isolated from residual surgical material acquired in accordance with the University of Pennsylvania guidelines for use of residual clinical material along with the U.S. Department of Health and Human Services code of federal regulation (Title 45 CFR 46.116) and the Declaration of Helsinki. Informed consent from each patient was obtained in addition to institutional review board approval (#800614). Patients were >18 years old and either receiving surgery for sinonasal disease or other procedures.2 for 2 h in PneumaCult-Ex Plus medium  with 100 U/mL penicillin and 100 µg/mL streptomycin to remove non-epithelial cells. After 2 h, unattached cells were then plated into a tissue culture dish and propagated in PneumaCult-Ex Plus medium. Basal cell identity was confirmed by positive staining for basal cell markers CD49f and p63  and transfected with either 10 nM of non-targeting, TAS1R1.6, or TAS1R3.2 RNAi duplex (Integrated DNA Technologies) for 48 h using lipofectamine 3000  as per manufacturer’s protocol. Knockdown was validated using qPCR as described below.Cultured cells were resuspended in TRIzol  and either stored at −70 °C or immediately used. RNA was purified via Direct-zol RNA kit  subjected to RT-PCR using High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). Resulting cDNA was then utilized for qPCR using Taqman Q-PCR probes in QuantStudio 5 Real-Time PCR System (ThermoFisher Scientific). Data was analyzed using Microsoft Excel and graphed in GraphPad PRISM v8.t-tests was used. ANOVA was utilized for more than two comparisons where Tukey–Kramer’s post-test was used for comparisons of all samples, Dunnett’s post-test was used to compare multiple samples to one control, and Bonferonni’s post-test was used for selective pair comparisons. In all figures, (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001, “n.s.” . All data represent the mean ± SEM of a minimum of 3 experiments.For comparisons of only two data sets TAS1R1, TAS1R2, and TAS1R3, the genes encoding T1R1, T1R2, and T1R3, respectively, were expressed in both the Beas-2B and primary cells in both basal and differentiated cultures  and 1× non-essential amino acids (NEAAs)  , we did ignaling a. Additie Fura-2 b.2+ elevation that was completely inhibited by a 1-hour pretreatment with 20 mM of umami antagonist lactisole (2+ elevations were from 1× MEM AA’s (~92%) while 1× NEAAs had very little contribution (~6%). While this observation is interesting, it remains to be determined if this is physiologically relevant. We first wanted to study the function of T1R1/3 under normal physiological pH, thus we continued experiments with buffers with pH corrected to 7.4 for the rest of this study.Fortunately, we found that the addition of both 1× MEM AA and 1× NEAA’s to HBSS containing 20 mM HEPES decreased the pH to 6.5, and at this pH, we found that both 1× MEM AAs and 1× NEAAs induced an intracellular Caactisole . The add2+ signaling was unaffected by 1× MEM amino acids at pH 7.4, we also evaluated cAMP signaling. Utilizing genetic encoded fluorescent biosensor Flamindo2 to measure intracellular cAMP levels [Though CaP levels , we obseP levels , we alsoTAS1R3 expression in Beas-2Bs  and ryanodine (RYR) receptors [2+ content.Elevations in cAMP alters many downstream signaling pathways, canonically through protein kinase A (PKA) or exchange protein directly activated by cAMP (EPAC). Using Beas-2B cells expressing PKA biosensor AKAR4 ,35 whicheceptors ,38,39,402+ content, we utilized the SERCA pump inhibitor thapsigargin. Without the constant uptake of Ca2+ into the ER, cells treated with thapsigargin will release their ER Ca2+ content into the cytosol where it can be measured using the intracellular Ca2+ binding dye Fluo-8. The Ca2+ will then dissipate into the extracellular space where it will be chelated by EGTA in the surrounding 0-Ca2+ HBSS buffer. This allows us to observe the release of total ER Ca2+ content without re-uptake of Ca2+ across the plasma membrane or ER membranes. The addition of 1× MEM AAs caused a 35% decrease in ER Ca2+ content  then treated with 15 mM denatonium. To our surprise, 1× MEM AA treatment caused a 30% increase in denatonium-induced Ca2+ release based on the population average  also reduced denatonium-stimulated Ca2+ release by ~50% per responsive cell, demonstrating that MEM AA treatment modifies intracellular Ca2+ release rather than Ca2+ influx  ER Ca2+ content decreased, (2) cultures released less calcium per cell in response to denatonium, (3) and the number of cells responding to denatonium increased. Together, from the perspective of ER Ca2+ content, these data suggest that MEM AAs might be disrupting the T1R1/3 activity. If T1R1/3 is providing a baseline regulation of ER Ca2+ content, one explanation is that something in the MEM amino acids may be acting as an inverse agonist. Additional research would be necessary to determine that this decrease in ER Ca2+ content is solely due to amino acid-T1R1/3 interaction and not the action of another receptor. Furthermore, additional, more detailed pharmacological studies, beyond the scope of this study here, would be needed to test inverse agonism of the MEM-amino acid mixture.Here, we show that and T1R1 and T1R3 are expressed in basal airway epithelial cells. There, they likely function to detect components of MEM AAs and signal via cAMP elevation. While knocking down T1R1 or T1R3 leads to a reduction in ER Ca2+ content, forskolin treatment did not. Given these observations, it seems unlikely that cAMP alone is responsible for the reduction of ER Ca2+. However, either knocking down T1R1/3, treating with 1× MEM AA, isoproterenol, or forskolin all led to an increased sensitization of cells to agonists that signal via Ca2+ elevations. Thus, we hypothesize that while cAMP elevations may increase the number of cells that respond to agonists that signal via intracellular Ca2+ pathways, the reduction of ER Ca2+ content may occur through a separate, distinct pathway. Moreover, as there are many transcription factors that are sensitive to cAMP levels, the data showing changes in nuclear cAMP may also suggest a possible role for T1R1/3 in amino acid-induced transcriptional changes, which could be investigated in future studies.While isoproterenol treatment also led to a decrease in ER Ca2+ signaling [T1R3 has been previously characterized to detect some L-amino acids via heterologous expression studies ,42. One ignaling . A separThe physiological agonist (s) activating T1R1/3 in basal airway cells also needs to be determined in order to identify whether this is an immune sensor  or a sensor of host metabolic or nutritional state. An important caveat to our study is that we do not yet know the identity of the endogenous agonists. Future work is needed. However, MEM AAs have been used as a T1R1/3 agonist in other studies’ cells ,32, and 2+ release in response to 1× MEM AA treatment at physiological pH. However, when added to a standard HBSS formulation containing 20 mM HEPES without any pH corrections, 1× MEM AA and 1× NEAA reduced the pH from 7.5 to 6.5. We observed Ca2+ elevations in response to amino acids when the HBSS was at pH 6.5. At this lowered pH, the majority of the Ca2+ response was due to the 1× MEM AAs. This Ca2+ elevation was also inhibited by pretreatment with 20 mM T1R3 antagonist lactisole, which did not alter the final pH of the solution. While this suggests a function of T1R1/3 during conditions where pH is lowered, the physiological relevance is not yet determined. Additionally, while we have not observed Ca2+ elevations in response to amino acids at a physiological pH; this could also be due to variations in G protein subunits bound to T1R1/3 in our cell models used here. The promiscuity of GPCRs for G protein subunits has been well documented [We did not observe an increase in intracellular Cacumented ,44,45,462+ signaling pathway in pancreatic b cells [2+ signaling pathways reported here is likely independent of mTOR [2+ signaling like MEM-AAs. While ERK1/2 and mTORC1 may still be activated in our models, this is a distinct, non-canonical pathway of umami receptor signaling. Parallel effects of ERK1/2 and/or mTORC1 activation will be investigated in future studies.Through detecting amino acids, T1R1/3 was shown to activate ERK1/2 and mTORC1 through a Ca b cells ,32 and i b cells . We show of mTOR ,32 as ra2+ content led to a reduction in intracellular Ca2+ elevations signaled by denatonium. Thus, amino acids may play a protective role in reducing apoptosis via the umami T1R1/3 receptor. When we previously measured NO production from bitter compounds such as denatonium, flufenamic acid [2+-signaled NO response even in the presence of 1× MEM AAs, our current work suggests that the addition of 1× MEM AAs to a topical T2R agonist therapeutic may help to diminish apoptotic signaling pathways without preventing stimulation of NO production. Thus, while we do not know the identity of the physiological agonist (s) activating T1R1/3 in airway basal cells, our data do reveal the potential therapeutic utility of 1× MEM AAs activation of T1R1/3 to protect airway epithelial cells against pro-apoptotic insults such as bitter agonists.Ultimately, this reduction in ER Camic acid , or diphmic acid , we util2+ content. Furthermore, the addition of 1× MEM AAs reduced ER Ca2+ content and greatly impaired apoptosis signaling via denatonium treatment. Therefore, amino acids or other umami receptor agonists may be useful to employ in therapeutics containing bitter compounds to repress unwanted induction of apoptosis.Our findings demonstrate a previously undescribed function for T1R1/3 in airway epithelial cells as a receptor for amino acids that signal through cAMP and as a regulator of ER Ca


Refugee and migrant women experience personal, cultural and structural challenges as they adapt to new lives in host countries. Peer mentoring programs are used to facilitate resettlement, build empowerment and improve job-readiness for refugee and migrant women; however, the effectiveness of these programs is not well understood. A systematic search of five databases, plus grey literature from January 2005 to December 2020, was undertaken, resulting in 12 articles. A narrative synthesis using thematic analysis identified the key components and outcomes of effective programs. Most mentoring programs were co-designed with community-based service providers, using participatory approaches to ensure cultural acceptability. Communication and sharing were facilitated using workshops and individual in-person or telephone mentoring. The training and support of mentors was critical. However, differences in expectations between mentors and mentees at times resulted in attrition. Qualitative evaluation revealed enhanced social support, greater empowerment and confidence for the women. There was improved access to the social determinants of health such as education, but limited success in obtaining employment. Mentoring programs can enhance refugee and migrant women’s wellbeing and social connectedness in resettlement contexts. However, it is unclear whether these benefits can be sustained over the longer term. Future programs should be rigorously evaluated through qualitative and quantitative analyses to generate conclusive evidence for best practice. There is a high and growing number of displaced people globally due to civil conflicts, war and climate impacts. This includes those who are internally displaced, and refugees and asylum seekers who are forced to leave their homelands and seek shelter in other countries due to civil conflicts, persecution and poor employment opportunities. More than 100 million people are reported to have been forcibly displaced as of June 2022, the vast majority of whom were hosted in neighboring countries, often in temporary facilities [One durable solution available to people experiencing displacement is resettlement through the United Nations High Commissioner for Refugees (UNHCR), usually in high-income countries, which often results in permanent residency or citizenship in the new country . BetweenIn addition to migrants fleeing persecution, many skilled migrants make the conscious decision every year to move to other countries to take up employment opportunities and reunite with family. Combined, global migration in 2020 was 281 million with the increasing feminization of migration occurring as the number of women migrants increase ,8. In thMigration is recognized as being a significant stressor, especially for refugees who experience substantial displacement, emotional and physical trauma, and the associated cultural dissonance of resettlement experiences, before and after migration to the host country ,11. RefuPost-migration stressors such as concerns about housing, particularly shared housing, difficult interactions with government and community service providers, and limited help from governments and charities have been found to be significantly correlated with general mental health problems amongst refugees ,18. InseResettlement policies fail to recognize the unique challenges faced by female refugees, with interventions focusing instead on males’ access to employment and financial independence . More reFor example, in Australia, female refugees experience higher rates of mental health issues and psychological distress than men ,23. In aPeer mentoring is usually undertaken by two or more people who are peers, with one acting as a mentor. Mentoring demonstrates a belief in the value of the individual and expresses a commitment to ongoing development, capacity building and enhancing agency . Peer mePeer mentoring programs with marginalized populations integrate the principles of social justice, access and equity . In the Two main questions underpinned the review (1) “What are the impacts of community-based peer mentoring programs on the personal and employment outcomes for refugee and migrant women in resettlement countries?”, and (2) “What factors need to be considered in the design of a community-based participatory peer mentoring program to engage refugee and migrant women in resettlement countries?“.Specific objectives were to: (i) describe peer mentoring programs that have been offered with refugee and migrant women in resettlement countries; (ii) identify social and wellbeing outcomes experienced by refugee women participants of peer mentoring programs; and (iii) identify key components of effective mentoring programs.This systematic review was undertaken using standard methods according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) . NarratiLiterature was sourced from the following electronic databases: ProQuest, Scopus, PubMed, Google Scholar and Wiley Online Library and included studies from 2005–2020. Reference lists of studies that matched the eligibility criteria were manually searched to identify further possible studies. Boolean operators and truncating of the distinct keywords in each search strategy combined with AND, OR and NOT were used to combine terms with each strategy and * was used for truncation where required.The target population under review were CALD refugee and migrant  women greater than 18 years of age;The interventions and programs of interest were ‘peer mentor’/‘peer-led’/‘peer to peer mentoring programs’ targeted at CALD refugee and migrant women in resettlement countries with search terms related to the types of peer mentoring programs, ‘community based’, ‘participatory’, ‘structured’, ‘coaching’;The outcomes were ‘resettlement’, ‘migrant support’, ‘peer mentoring process’, ‘integration’, ‘social inclusion’, ‘community support’.The search terms used for:Contextual and semantic text differences between studies required flexibility in the search process because certain words and phrases implicitly became substitutes for our original criteria keywords. For example, whereas ‘peer(s)’ may not initially surface in a keyword search, words such as, ‘friends’, ‘community’, and ‘support’ can tend to suggest some similarity in models of engagement with and among migrants and refugees.Qualitative, quantitative and mixed-methods studies which presented the outcome/evaluation of peer mentoring/peer-led programs targeted at refugee and migrant CALD women, studies written in English, were considered in the review. Review articles, discussion papers, opinion papers, dissertations and theses, books, personal blogs, commentaries, articles from web pages, editorials, and articles written in languages other than English, were excluded. Despite an initial focus on peer mentoring programs for women only, studies that evaluated mixed gender refugee mentoring programs have been included in our evaluation if specific outcomes for women were reported. Studies were excluded if the mentoring programs were only for male participants, or where specific findings for female participants were not distinguishable. Mentoring programs for skilled migrants, typically located in the organizational context, were excluded as skilled migrants face different challenges to lesser-skilled refugees and migrants. A number of articles identified in the initial search provided overviews of general refugee/migrant support programs offered by community organizations to facilitate resettlement, some of which included aspects of peer support. However, if the programs did not include a specific mentoring program the articles were excluded.The initial search process identified 152 potential articles. Removal of duplicates resulted in the retention of 133 studies for abstract review to determine relevance and eligibility. Review of abstracts and titles led to the removal of a further 103. A total of 30 studies were retained for full review after 103 records were excluded. After full review, 18 were excluded as they did not meet the inclusion criteria. The Critical Appraisal Skills Program (CASP) checklists were used to assess each article . Due to The review objectives guided the data extraction process. The main characteristics that were extracted from the included articles were the country where the mentoring program was delivered, descriptions of mentees and mentors, key components of the mentoring program, methods of evaluation and outcomes . The larn = 4, 33%), Spain ; the US , Canada  and Sweden . Participant numbers in the included studies ranged from 6–172 [Of the 12 included studies , all focom 6–172 ,35. Partom 6–172 , Sudanesom 6–172 ,48,49, Bom 6–172 , Hmong, om 6–172 ,50, Syriom 6–172 , Bhutaneom 6–172  or of noom 6–172 ,53.n = 4), social connectedness and social capital (n = 7) and employment (n = 1).Five peer mentoring programs were delivered specifically to women and six were delivered to mixed gender groups. The remaining study focused on the mentors’ experience but provided information on key components of the program. As much as possible, only the outcomes for the female cohort of the program have been included in the review. All of the peer mentoring interventions were designed to improve refugee isolation and depleted social networks  and to improve host countries’ responses to the needs of resettled refugees ,50. SpecDescriptions of each of the mentoring programs, along with the main findings of the quantitative and qualitative evaluations, are outlined in There were a number of common approaches to the development and composition of the peer mentoring programs. These are outlined below.In keeping with a participatory approach, the peer mentoring programs were commonly developed in collaboration with participants, via pre-intervention interviews or consultation ,49. ThisCommunity organizations were commonly the source of participant recruitment. Users of the programs and services offered by the community organizations were approached and invited to participate. When participants were recruited from the general community, without the support of a community organization, researchers had difficulty recruiting sufficient participants ,53. In tIn keeping with the principles of participatory research, the content of the programs evolved over time. Both mentees and mentors were consulted at various points to ensure the programs were meeting specific needs and content was added in response to ongoing feedback ,53.In accordance with the core principles of mentoring, the mentoring programs emphasized communication and sharing. Mutual exchange of knowledge was a key feature, either between mentors and mentees ,51,52,53There was a mixture of formats utilized by the mentoring programs, but were generally a combination of group workshops, and individual mentoring either face to face or by telephone. A total of 3 programs used workshops/group sharing only ,51,53,54The workshop/group sharing format promoted socialization and allowed for the sharing of knowledge and the development of social capital between group members ,48,50, pThe individual mentoring format provided mentors with the flexibility to attend to individual participant needs ,50. ThisTelephone mentoring was offered in several studies ,48,49. TAll mentoring programs took place in the community with workshops and group sessions being conducted at community centers, and individual home visits in some cases ,52. It wThere were conflicting findings on the optimum duration of the mentoring programs. Two mentoring programs lasted 8 weeks  and 10 wConsiderable focus was dedicated to the recruitment, training and support of mentors, and these were highlighted as essential components of the mentoring programs ,50,52,53The mentor training consisted mostly of responsibilities and roles, facilitation skills, strategies for assisting mentees with additional needs, the role of community services, and particular discussion themes ,53. The Where the content of training programs was outlined, it contained a combination of communication skills, workshop facilitation skills, refugees’ emotional and practical needs, mentor responsibilities and understanding the migration experience ,53. For Consideration was given to the matching process where mentor/mentee dyads were formed. Dyads were matched according to ethnicity and gender , age, chThere were a number of cultural considerations highlighted in the studies. It was considered important to have a culturally appropriate approach, particularly in the discussion of sensitive personal issues. Western-style counselling approaches with an individual focus were not considered appropriate . LearninThe importance of the mentees’ families and communities accepting their participation in the mentoring program were highlighted, especially if this involved the mentor visiting the family home ,52. The Cultural issues sometimes resulted in attrition from the mentoring programs making it difficult for mentees to realize the full benefit ,52. FactThematic analysis of the qualitative findings of the evaluations of the mentoring programs was undertaken to determine key outcomes . InitialQualitative evaluations found that the mentoring programs resulted in improved access to social support and connection, including feeling less isolated and learning there are others in the same position ,50,51,52Attrition was noted in several studies, indicating that social connections were not always an outcome ,53. AuthTwo of the studies measured wellbeing outcomes quantitatively and found a significant positive impact on quality of life, distress, and post-traumatic growth ,53. The Empowerment was a noted outcome in several studies ,50,53,54Quantitative measures found increased scores post-mentoring in English proficiency, citizenship knowledge and satisfaction with resources . These aThere was little discussion of employment as an outcome for refugee women in the studies reviewed. Quantitative analysis of employment outcomes in Sweden for refugee participants in a mentoring program specific to enhancing employment noted no real impact for female refugee participants . The autThis review sought to identify common core components of mentoring programs for refugee and migrant women in resettlement countries, and assess their effectiveness in improving wellbeing, social connection, and access to the social determinants of health such as employment. Results indicate that mentoring programs are effective in enhancing social connection and promoting wellbeing, but that these results are not necessarily sustained over time. Only one study measured the longer-term impact of participation in a mentoring program, and further research is needed to evaluate longitudinal impacts.This review highlights that being able to participate in mutually supportive relationships is beneficial for refugee women. This may be with a mentor from the same language and/or culture, or with a locally born mentor, and benefits and disadvantages have been outlined for both approaches. There is value in providing opportunities for refugee women to share their stories with a mentor or group that practices empathic listening, prioritizes social connections and validates refugee women’s experiences ,53.Particular benefits were noted for using mentors with lived experience of migration and preferably forced migration . SupportBuilding trusting relationships between organizations, communities and individuals takes time. In particular, refugee women are often a ‘seldom-heard’ group  and shorThis review was constrained by the scarcity of relevant research outputs. Despite published refugee and migrant research spanning decades, it is apparent that peer mentoring of refugee women, and particularly, migrant women has not been well-examined. Furthermore, there are methodological limitations in the existing evaluations of mentoring programs. Very few studies are able to identify causality between specific components of programs and outcomes . A clue There is little evidence that peer mentoring programs enhance employment outcomes for female refugees. This group may have gender-related limitations on labor force participation, such as family and caring responsibilities, language barriers, and limited work experience and training. Mentoring programs may be unable to address these . ProgramMany of the studies in this review concluded that outcomes for female refugee and migrant populations can be improved through better understanding of women’s values, perspectives, and expectations ,49,50. TSuccessful outcomes have also been achieved in health care research by building on women’s resilience and engaging with community networks to promote wellbeing ,58,67.Topic- and age-specific workshops may overcome confidentiality concerns for participants and enable facilitators to keep discussion to topics directly relevant to participants at different life stages. For example, workshops with a focus on employment or education issues . UtiliziThere is scope for further research on this topic that could include: establishing the most effective content material for mentor training programs; and systematic and rigorous investigation of the effects of mentoring programs on participants’ wellbeing, self-reliance and social connectedness, especially over the longer term, using empirical methods suited to collectivist cultures’ definitions of wellbeing ,68. FurtThis much needed and timely review provides valuable insights into the key components, challenges and contributing factors to successful outcomes in peer mentoring programs for refugee women. Mentoring programs can enhance female refugees’ wellbeing, build networks, improve interpersonal communication and social connectedness. However, the review highlights that for benefits to be sustained over the longer term, adequate support and continual funding is critical. The body of work in this area is currently limited and additional research is necessary to trial and rigorously evaluate other interventions to generate conclusive evidence for best practice.