CRIP1 as clinically prognostic in t  AML patients. However, the expression and role of CRIP1 in AML are poorly understood. We used the single-cell RNA sequencing and gene expression data from t  AML patients to analyze the immune and regulation networks of CRIP1. Two independent cohorts from GSE37642 and The Cancer Genome Atlas (TCGA) datasets were employed as validation cohorts. In addition, the methylation data from TCGA were used to analyze the methylation effect of the CRIP1 expression. Gene expression profile from t  AML patients showed that the CRIP1-high group exhibited an enrichment of immune-related pathways, including tumor necrosis factor (TNF)α signaling via nuclear factor kappa B (NFκB) pathways. Further studies using CIBERSORT showed that the CRIP1-high group had a significantly higher infiltration of exhausted CD8 T cells and activated mast cells. The CRIP1 expression was validated in the GSE37642-GPL96, GSE37642-GPL570, and TCGA datasets. In addition, with the methylation data, four CpG probes of CRIP1  were identified as negatively associated with the CRIP1 gene expression in AML patients. Our data provide a comprehensive overview of the regulation of CRIP1 expression in AML patients. The evaluation of the TNFα-NFκB signaling pathway as well as the immune heterogeneity might provide new insights for exploring improvements in AML treatment.Acute myeloid leukemia (AML) is a highly heterogeneous hematological malignancy that imposes great challenges in terms of drug resistance and relapse. Previous studies revealed heterogeneous leukemia cells and their relevant gene markers, such as  RUNX1-RUNX1T1  fusion gene, accounts for approximately 7% of adult primary AML (Acute myeloid leukemia (AML) is a highly heterogeneous hematological malignancy characterized by the malignant proliferation of clonal myeloid precursor cells that progress rapidly . t  immune system. Immune dysfunction of AML, including the downregulation of major histocompatibility complex class II genes, is associated with relapse . In addi+CD117dim leukemia cells and their characteristic gene markers  in t  AML patients  belongs to the LIM/double-zinc finger protein family and is abnormally expressed in a variety of tumors, including breast cancer, colorectal tumors, and thyroid cancer  AML patients  AML leading to poor outcomes as well as the clinical significance of CRIP1 in AML remain unclear. This study analyzed the immune infiltration state as well as the epigenetic effect on the CRIP1 expression in AML.Among the markers of the CD34d cancer . CRIP1 moperties . In detasubtypes . Using tpatients . Howeverhttp://www.biosino.org/node/project/detail/OEP000629). Detailed treatment information was provided, as previously described   AML patients were downloaded from our previous studies, which were deposited in the National Omics Data Encyclopedia (escribed . This ster.gov/) . GSE3764RUNX1-RUNX1T1 transcript, Salmon (www.broadinstitute.org/gsea) and the Molecular Signatures Database, as described previously  > 0.5 and an adjusted p value <0.05. The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (https://cn.string-db.org) was then used to predict the protein–protein interaction (PPI) network  (The gene expression data of cancer cell line encyclopedia (CCLE) were extracted from the dataset (le/data)  for analhttp://mexpress.be)  (CRIP1 expression and methylation levels at multiple DNA sites. The CistromeDB Toolkit (http://dbtoolkit.cistrome.org/) (MEXPRESS (ress.be)  and cBioal.org/)  were utime.org/)  was usedhttps://www.cancerrxgene.org/) (RNA-seq and predicted chemotherapeutic response were downloaded from the Genomics of Drug Sensitivity in Cancer (GDSC) database (ne.org/) . The prep value. Statistical analyses were performed using R software .Univariate COX regression analysis and forest analysis were performed using the forestplot package. The Kaplan–Meier method was employed to estimate the probabilities of OS and relapse-free survival, and the log-rank test was used to compare the +CD117dim populations were extracted using the criteria of an average log Fold Change (avg_logFC) >0.5 and an adjusted p value <0.05 (https://cn.string-db.org) to analyze the PPI network  AML patients were used . The sigue <0.05 . We then network .CRIP1 was demonstrated to be the prognostic of t  AML patients using the COX regression model, and it was highly expressed in AML subtypes, including M0–M7  count, and gene mutations. The AML patients with a higher WBC count had a significantly higher CRIP1 expression (p < 0.001), while the gene mutations, including TP53, RUNX1, and FLT3-ITD, had no significant correlation with CRIP1 expression  AML patients, the gene expression profile from previous RNA-seq data of 62 de novo t  AML patients was analyzed  had an enrichment of immune-related pathway, such as IL2-STAT5 signaling, IL6-JAK-STAT3 signaling, and tumor necrosis factor (TNF)α signaling via the nuclear factor kappa B (NFκB) pathways (CRIP1-low group (n = 31), we observed a significant enrichment of DNA repair and E2F target pathway  AML patients.First, to explore the upregulation pathway of analyzed . The patpathways . For the pathway . This hiCRIP1-high group had a significantly higher infiltration of T cells CD8 and Mast cells activated  and CD8 T cells . In contrast, the CRIP1 expression had a negative correlation with resting mast cells  and neutrophils   and TIM-3  and a negative correlation with TCF-1 (HNF1A)   AML patients was analyzed and compared based on the CIBERSORT algorithm . Of the ctivated , whereas resting . Further= 0.036) . The hig= 0.012) .CRIP1 expression in t  AML patients. A previous study had compared the gene mutations of RTK/Ras family, transcription factors, epigenetic modifiers, cohesion complexes, or signaling pathways. No significant differences in gene mutations mentioned above were observed between the CRIP1-high and -low groups  using the single-cell RNA-seq (scRNA-seq) data of t  AML patients, and TNF was identified as the main regulator , RELA , and JUNB , all of whom participated in the TNFα–NFκB pathway  AML pathogenesis, we questioned whether the CRIP1 expression was regulated by the fusion gene. As a result, there was no significant correlation with the coefficient of 0.21 with a p = 0.094  nor RUNX1T1 (ETO) were included as the top putative targets  for the Cistrome  were the targets . InsteadCRIP1 expression was regulated by the TNFα–NFκB pathway, we analyzed the drug-induced gene expression change across different cell lines following the exposure to the NFκB inhibitor bortezomib from GSE116438 to mimic the knockdown of the TNFα–NFκB pathway. For the 55 cell lines tested, when treated with bortezomib for 24 h, the CRIP1 expression was significantly lower (p < 0.0001) (CRIP1 was regulated by the TNFα–NFκB pathway.To test whether the  0.0001) , which fCRIP1 expression in t  AML patients. GSEA showed that the CRIP1-high group has a significant enrichment in histone deacetylase (HDAC) and HDAC7 targets (CRIP1-low group showed enrichment in H3K27ME3 (We then explored the epigenetics’ influence on the  targets . HoweverH3K27ME3 .CRIP1 expression and the methylation level of the CRIP1 promoter in AML (CRIP1 expression with the methylation level from the Human Methylation 27k (HM27) platform of TCGA AML patients was analyzed using the cBioPortal  that were negatively associated with the CRIP1 gene expression in AML patients.Next, we explored the relationship between the r in AML  data usir in AML . In addiioPortal . The DNApression . In addiCRIP1 expression of AML cell lines was analyzed .The IC50 distribution of cytarabine and all-trans retinoic acid (ATRA) was analyzed using data from the GDSC dataset. The tarabine , though CRIP1 expression in APL and non-APL AML patients using the TCGA AML data. Compared with APL patients, non-APL AML patients had a significantly higher CRIP1 expression (p < 0.001) .In addition, we compared the < 0.001) .CRIP1 expression in non-APL patients made us explore the pattern of CRIP1 expression in normal myeloid differentiation. Using data from BloodSpot  AML was first reported by our group  AML cohort (OEP000629) and three other validation cohorts. This activation of E2F targets was consistent with the enrichment result of the CD34+CD117bri population, which showed a lower CRIP1 expression in our previous studies  AML patients. This was further validated in two independent cohorts of AML patients.Previous studies have shown that AML leukemia cells can produce endogenous TNFα, which then activated the downstream NFκB-signaling pathway, thereby resulting in leukemia cell proliferation and drug resistance . The tra pathway . In thisCRIP1 expression was lower, while the monocyte had a rather high level of CRIP1 expression. However, the role of CRIP1 in the myeloid differentiation still needs further exploration.Through further exploration of the expression pattern during myeloid differentiation, we found that at the late promyelocyte stage, the CRIP1-high group had a higher proportion of exhausted CD8 T cells. In fact, the dysfunction of immune cells, especially T cells, was revealed to affect the prognosis of patients with tumors, including nonsmall cell lung cancer, colorectal cancer, and melanoma (Up to date, the treatment of AML still faces challenges of drug resistance and relapse. Multiple studies have shown that immunotherapy is playing an integral role in AML . The sinmelanoma . HoweverCRIP1 expression in AML patients. The evaluation of the TNFα-NFκB signaling pathway as well as the immune heterogeneity might provide new insights for exploring improvements in AML treatment.In conclusion, our data provide a comprehensive overview of the regulation of


MaR2 increased [Ca2+]i and stimulated secretion. MaR2 and MaR1 stimulate conjunctival goblet cell function, especially secretion, by activating different but overlapping GPCR and signaling pathways, and furthermore counter-regulate histamine stimulated increase in [Ca2+]i. Thus, MaR2 and MaR1 play a role in maintaining the ocular surface and tear film homeostasis in health and disease. As MaR2 and MaR1 modulate conjunctival goblet cell function, they each may have potential as novel, but differing, options for the treatment of ocular surface inflammatory diseases including allergic conjunctivitis and dry eye disease. We conclude that in conjunctival GC MaR2 and MaR1, both increase the [Ca2+]i and stimulate secretion to maintain homeostasis by using one set of different, but overlapping, signaling pathways to increase [Ca2+]i and another set to stimulate secretion. MaR2 also resolves ocular allergy.Specialized pro-resolving mediators (SPMs), including Maresins (MaR)-1 and 2, contribute to tear film homeostasis and resolve conjunctival inflammation. We investigated MaR2′s signaling pathways in goblet cells (GC) from rat conjunctiva. Agonist-induced [Ca The ocular surface including the cornea and conjunctiva is covered by a protective tear film. The innermost layer of the tear film is the mucous layer, which consists of electrolytes, water, and mucins produced by conjunctival goblet cells . The mucMaR1 and MaR2 are biosynthesized in macrophages and other tissues and are derived from the endogenous ω-3 fatty acid docosahexaenoic acid (DHA) . MaR1 an2+]i and stimulates glycoprotein secretion. MaR1 increased [Ca2+]i and stimulated glycoprotein secretion by activating PLC and its downstream effectors, IP3, PKC, and by activation of PLD, Ca2+-calmodulin kinase (CaMK) II and extracellular regulated kinase (ERK) 1/2 i) that triggers mucin secretion. Therefore, measuring the [Ca2+]i was used as functional readout in the study presented in this manuscript. We identified the intracellular pathways MaR2 uses by measuring [Ca2+]i and high molecular weight glycoprotein secretion including mucin secretion. We used pharmacologic inhibitors of different signaling pathways followed by addition of MaR2. Furthermore, the effect of MaR2 on histamine was investigated, because the MaRs are thought to play a central role in allergy i, in cultured rat conjunctival goblet cells i rat conjunctival goblet cells in a concentration-dependent manner  for 10−10 M, 93.48 ± 12.66 nM (p = 0.002), for 10−9 M and 189.20 ± 14.54 nM (p = 0.029) for 10−8 M, (n = 3). The highest increase in peak [Ca2+]i was triggered by MaR2 10−8 M; thus, this concentration was used in further experiments. MaR1 at 10−8 M and histamine at 10−5 M also increased [Ca2+]i.Multiple SPMs that stimulate goblet cell secretion, also increase i and stimulate secretion by binding to the receptor in rat conjunctival goblet cells [2+]i and to stimulate glycoconjugate secretion in conjunctival goblet cells [2+]i. The formyl peptide receptors (FPRs) are a family of receptors including three subtypes in mammals; FPR1, FPR2 and FPR3. The first described ligand of the receptor was a formylated peptide from Escherichia coli, which binds with high affinity . The Lip43 (C43) ,22. The 43 (C43) ,23,24,25et cells ,26,27. Met cells . Thus, w−4 M) for 30 min prior to stimulation with MaR2 (10−8 M). MaR1 (10−8 M) and LXA4 (10−9 M) were used as positive controls [2+]i over time. MaR2 significantly increased [Ca2+]i to 184.66 ± 18.00 nM (p = 7× 10−6) while the MaR2 10−8 M treated with BOC2 increased [Ca2+]i to 182.33 ± 37.50 nM . MaR1 added alone significantly increased [Ca2+]i to 223.30 ± 47.88 nM (n = 5). BOC2 significantly decreased the MaR1 response to 66.98 ± 8.95 nM. LXA4-stimulated [Ca2+]i increase was 178.10 ± 24.68 nM (n = 5). In the presence of BOC2 the LXA4 response was significantly reduced to 83.13 ± 29.00. These results show that MaR2 does not activate the ALX/FPR2 receptor to increase [Ca2+]i, but the positive controls MaR1 and LXA4 do.Rat conjunctival goblet cells were incubated with the ALX/FPR2 inhibitor N-BOC-Phe-Leu-Phe-Leu-Phe (BOC2) . The MaR2 response was not significantly inhibited by BOC2, while secretion stimulated by the positive controls MaR1 was decreased and LXA4 was significantly inhibited by the ALX/FPR2 inhibitor. These data indicate that MaR2 does not utilize the ALX/FPR2-receptor to increase [Ca2+]i or stimulate secretion.To explore the role of the ALX/FPR2-receptor in glycoconjugate secretion, goblet cells were incubated with BOC2 for 30 min prior to stimulation with MaR2 and the controls MaR1 and LXAve basal b; n = 4.4 activates the GPCR receptor, BLT1, to cause chemotactic, pro-inflammatory actions [2+]i, rat conjunctival goblet cells were treated with an inhibitor of the BLT1 receptor, U-75302 (10−8–10−6) for 30 min. MaR2 10−8 M significantly increased [Ca2+]i to 240.96 ± 55.34 nM . Preincubation with the BLT1 receptor inhibitor U-75302 caused a MaR2 stimulated [Ca2+]i increase to only 83.06 ± 17.80 (p = 0.021), 113.05 ± 29.34 (p = 0.07) and 62.88 ± 10.55 (p = 0.01) (n = 6) with 10−8 M, 10−7 M and 10−6 M inhibitor, respectively. The positive controls MaR1 at 10−8 M and LTB4 at 10−9 M increased [Ca2+]i that was blocked by U-75302 (n = 6). These results suggest that MaR2, similarly to the positive controls MaR1 and LXA4, is dependent on the BLT1 receptor to increase [Ca2+]i. LTB actions . SPMs in actions ,29. To e = 0.01) c; n = 6  U-75302 c; n = 6.−6 M) for 30 min before addition of MaR2. MaR2 increased secretion 1.8 ± 0.3 -fold above basal (n = 4). MaR2 was not significantly inhibited by U-75302. Secretion stimulated by the positive controls MaR1 and LTB4 was significantly inhibited by U-75302. These data indicate that MaR2 uses the BLT1-receptor to increase [Ca2+]i, but not secretion.To examine the dependency of MaR2- stimulate secretion on the BLT1 receptor, rat conjunctival goblet cells were treated with U-75302  or MaR1 (10−8 M) alone. Then as a control to ensure that each of the MaRs can desensitize themselves, cells were stimulated with MaR2 10−8 M or MaR1 10−8 M followed by MaR2 or MaR1, respectively. Then MarR1 was added before MaR2 to determine if MaR1 desensitized MaR2 and Mar2 before MaR1 to determine if MaR2 desensitized MaR1. MaR2 caused an increase in peak [Ca2+]i to 190.59 ± 21.78 . MaR2 addition before MaR2 caused an increase in peak [Ca2+]i to 93.84 ± 25.20 that was significantly decreased compared to MaR2 alone . Addition of MaR1 before MaR2 caused an increase in peak [Ca2+]i to 112.51 ± 7.02 that was significantly decreased compared to MaR2 alone . Both MaR1 and MaR2 are SPMs derived from the ω-3 fatty acid docosahexaenoic acid (DHA). To determine if the two MaRs desensitize each other and thus would activate the same receptor, we incubated rat conjunctival goblet cells two minutes with MaR2 (10−8 M) caused an increase in [Ca2+]i to 208.58 ± 44.19 . Addition of MaR1 before MaR1 caused an increase to 55.41 ± 8.61 that was significantly decreased compared to MaR1 alone . Addition of MaR2 before MaR1 caused an increase to 188.28 ± 26.07 that was not different from MaR1 alone . MaR1  or its inactive control U-73343 (10−7 M) before stimulating with MaR2 (10−8 M) (n = 4), or the positive controls MaR1 (10−8 M) (n = 4) or Cch 10−4 M (n = 4). 2+]i over time. MaR2 10−8 M caused an increase in peak [Ca2+]i to 196.87 ± 25.62 . Treatment with U-73122 or U-73343 followed by MaR2 caused an increase in peak [Ca2+]i to 99.28 ± 36.77 (p = 0.072), and 118.95 ± 47.13 (p = 0.20), respectively, that were unchanged when compared to MaR2 stimulation. MaR1 and Cch stimulation, in contrast to that of MaR2, is dependent on PLC to increase [Ca2+]i as their action on [Ca2+]i was decreased by U-73122, but not by U-73343 (n = 4). The PLC pathway is activated in a variety of cellular processes, including exocytosis and fluid secretion . This in(10−8 M) a; n = 4,(10−8 M) a; n = 4 h 10−4 M a; n = 4. U-73343 a; n = 4.n = 4). The response was significantly blocked by U73122 to 0.9 ± 0.2 (p = 0.003), but not by the inactive control U73343 (3.6 ± 2.1) (p = 0.44). MaR1- and Cch-stimulated increase in secretion were also significantly blocked by U73122, but not by U73343 (n = 4). We conclude that MaR2 is dependent upon activation of the PLC pathway to stimulate glycoprotein secretion, but MaR2 does not use PLC to increase [Ca2+]i.The action of PLC on MaR2-stimulated increase in glycoconjugate secretion was next investigated. MaR2 increased secretion 1.9 ± 0.1 -fold above basal b; n = 4.y U73343 b; n = 4.3 which binds to its intracellular receptor on the ER causing release of Ca2+ from intracellular calcium stores increasing [Ca2+]i. To determine if MaR2 is dependent of the downstream molecules that activation of the PLC pathway produces, cells were treated with the IP3-receptor inhibitor 2APB (10−5 M) and then stimulated with MaR2 10−8 M. MaR1 (10−8 M) and Cch (10−4 M) were used as positive controls. MaR2 caused an increase in peak [Ca2+]i to 212.28 ± 69.55 . Treatment with 2APB (10−5 M) did not alter the increase in peak [Ca2+]i  compared with the action of MaR2 alone. In contrast treatment with 2-APB blocked the action of MaR1 and Cch on [Ca2+]i (n = 5). We conclude that the action of MaR2 is independent of the action of IP3 on its receptor to increase [Ca2+]i. Activation of the PLC pathway produces IPp=0.016, c; n = 5. [Ca2+]i c; n = 5.n = 6). 2APB significantly decreased MaR2-stimulated response to 1.3 ± 0.2 -fold . The action of the positive control, Cch, on secretion was also significantly inhibited by 2APB (n = 6). We conclude that the action of MaR2 on [Ca2+]i, but not secretion, is independent of the action of IP3 on its receptor to increase [Ca2+]i.The effect 2APB on MaR2-stimulated increase in glycoconjugate secretion was then explored. MaR2-increased secretion was 3.3 ± 0.5 -fold above basal d; n = 6. by 2APB d; n = 6.2+] by other mechanisms than PLC-IP3 pathway, we used the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin. Thapsigargin blocks the uptake of Ca2+ into intracellular stores so that the cytoplasmic [Ca2+]i increases by a passive leak from the ER. If an agonist uses the same intracellular Ca2+ store as thapsigargin, the increase in [Ca2+]i by an agonist added after thapsigargin will be decreased. Conjunctival goblet cells were treated by thapsigargin (10−5 M) for 15 min that releases Ca2+ from the intracellular stores and then stimulated with MaR2 (10−8 M), or the positive controls MaR1 (10−8 M) or Cch (10−4 M). MaR2 increased [Ca2+]i to a peak of 219.10 ± 21.63 nM . Treatment with thapsigargin caused a MaR2 increase in [Ca2+]i to a peak of 85.68 ± 4.02 nM that was significantly decreased compared to MaR2 alone . A similar effect of thapsigargin was detected using MaR1 and Cch. Thus, MaR2 is dependent on a release of Ca2+ from intracellular calcium stores to increase [Ca2+]i.To determine if MaR2 is using intracellular calcium stores to increase [Ca2+]i as neither the active PLC inhibitor nor the inhibitor of IP3 for its receptor on the ER blocked MaR2 increase in [Ca2+]i. MaR2, however, does appear to use PLC to stimulate secretion as the inhibitors tested are in agreement on their actions. The inhibitor of SERCA on the ER does block MaR2 increase in [Ca2+]i, but this could belong to an as yet unidentified pathway. MaR2 does not use PLC to increase [Ca2+ to increase [Ca2+]i i over time. MaR2 with CaCl2 increased [Ca2+]i to a peak of 95.66 ± 28.34 nM , while MaR2 without CaCl2 increased [Ca2+]i to a peak of 64.74 ± 15.25 nM, a non-significant decrease (p = 0.37). A similar finding was detected for MaR1 (10−8 M) (n = 4). In contrast [Ca2+]i stimulated by Cch (10−4 M) was significantly decreased in the absence of extracellular Ca2+ (n = 4). We conclude that MaR2, similarly to MaR1, is independent of influx of extracellular Ca2+ to increase [Ca2+]i, but Cch is not.In conjunctival goblet cells MaR1 is not dependent on extracellular Ca [Ca2+]i . To dete(10−8 M) ; n = 4. lar Ca2+ ; n = 4. 3. PKC can also be activated by other signaling pathways. To determine if MaR2 is dependent on PKC to increase [Ca2+]i we incubated conjunctival goblet cells with the PKC inhibitor RO317549 (10−7 M) for 30 min, then stimulated with MaR2 (10−8 M), or the positive controls MaR1 (10−8 M) and Cch (10−4 M). 2+]i over time. MaR2 increased [Ca2+]i to a peak of 475.49 ± 125.41 nM , while treatment with RO317549 significantly decreased [Ca2+]i to a peak of 76.67 ± 12.84 nM . Similar results were obtained with MaR1 and Cch (n = 3). Thus, MaR2 is dependent on PKC to increase [Ca2+]i, as are MaR1 and Cch.Protein kinase C (PKC) is activated by diacylglycerol (DAG) produced by activation of PLC when PLC also produces IP and Cch a; n = 3.−7 M). MaR2 increased secretion 1.9 ± 0.1 -fold above basal  and MaR2 stimulation was significantly blocked by RO317549 to 0.9 ± 0.2 -fold above basal (p = 0.001). The positive control, MaR1, increased secretion to 2.2 ± 0.8 -fold above basal (p = 0.03) and preincubation with RO317549 significantly decreased the response to 0.8 ± 0.2 -fold above basal (p = 0.03). Both MaR2 and MaR1 are dependent upon PKC to increase [Ca2+]i and stimulate secretion.The effect of RO317549 on MaR2-stimulated increase in glycoconjugate secretion was determined after a 30 min incubation with RO317549 (102+]i we used the PLD-inhibitor 1-butanol (1-but) at 0.3% and the inactive control t-butanol (t-but) at 0.3%. 2+]i over time. MaR2 (10−8 M) caused an increase in [Ca2+]i to a peak of 475.49 ± 125.41 nM . MaR2 (10−8 M) after 1-butanol  increased [Ca2+]i to a peak of 97.89 ± 6.41 nM that was significantly decreased compare to MaR2 alone (p = 0.040). MaR2 (10−8 M) added after t-butanol  increased [Ca2+]i to a peak of 72.40 ± 17.05 nM that was significantly decreased from MaR2 alone (p = 0.033). For the positive controls, MaR1 and carbachol stimulation of peak in [Ca2+]i was decreased by 1-butanol, but not by t-butanol (n = 3). Although the peak in [Ca2+]i was reduced by 1-butanol, we cannot definitively conclude that the action of MaR2 is dependent on PLD, due to inhibition by the inactive control t-butanol. In contrast, MaR1 and Cch stimulation were dependent on the activation of PLD.Activation of Phospholipase D (PLD) is controlled by multiple mechanisms, activates distinct pathways and is important in cellular functioning . To expl-butanol a; n = 3.p = 0.007, n = 3). 1-butanol (p = 0.02), but not t-butanol (p = 0.50), significantly inhibited MaR2-stimulated secretion. The positive controls, MaR1 and Cch stimulated glycoconjugate secretion above basal and 1-butanol, but not t-butanol, significantly decreased the response for each agonist. This indicates that MaR2 utilizes PLD to stimulate glycoconjugate secretion, as do MaR1 and carbachol.To explore the role of PLD in MaR2-stimulated glycoconjugate secretion, rat conjunctival goblet cells were incubated with 1-butanol or t-butanol then stimulated with MaR2. MaR2 increased secretion 3.5 ± 0.7 -fold above basal (2 to increase [Ca2+]i we used the PLA2-inhibitor Aristolochic Acid (AA). 2+]i over time. MaR2 (10−8 M) caused an increase in [Ca2+]i to a peak of 301.53 ± 17.7 nM . When incubated with AA 10−5 M or AA 10−6 M, MaR2 caused an increase in [Ca2+]i to a peak of 108.77 ± 14.83 nM or 129.09 ± 25.71 nM, respectively values that were significantly decreased from MaR2 alone (p < 0.001 for AA 10−5 M and p = 0.001 for AA 10−6 M). The action of Cch on peak increase in [Ca2+]i was blocked by AA at 10−5 M (n= 4). Thus MaR2 activates PLA2 to increase [Ca2+]i.To examine if MaR2 is dependent on PLAt 10−5 M c; n= 4. 2 to stimulate glycoconjugate secretion, conjunctival goblet cells were preincubated with AA. MaR2 increased secretion 1.7 ± 0.2 -fold above basal . When incubated with AA at 10−5 M MaR2 increased secretion to 1.7 ± 0.8 -fold above basal (p = 0.97), not a significantly different value from stimulation with MaR2 alone. The increase in glycoconjugate secretion stimulated by the positive control, Cch was significantly decreased by AA (n = 6). This indicates that MaR2 uses PLA2 to increase [Ca2+]i, but not to stimulate glycoconjugate secretion.To determine if the action of MaR2 is dependent on PLAed by AA d; n = 6.−5 M) for 30 min prior to stimulation with MaR2 (10−8 M), or the positive controls VIP (10−8 M) and MaR1 (10−8 M). 2+]i over time. MaR2 increased [Ca2+]i to a peak of 91.27 ± 17.97 nM . Incubation with H89 increased [Ca2+]i to a peak of 40.66 ± 1.94 nM that was different from MaR2 alone (p = 0.023). The action of MaR1 was not inhibited by H89, but of VIP was blocked (n = 5). This indicates that MaR2 activates PKA to increase [Ca2+]i.When a ligand activates Gαs, adenylyl cyclase (AC) catalyzes ATP to cAMP that in turn stimulates the activity of cAMP dependent protein kinase A (PKA). This is one among a variety of functions of cAMP . To expl blocked a; n = 5.−5 M) 30 min prior to addition of MaR2. MaR2 stimulated secretion to 3.4 ± 0.5 -fold above basal . Incubation with H89 significantly decreased MaR2-stimulated secretion to 1.8 ± 0.3 -fold above basal (p=0.004). Secretion stimulated by the positive control VIP was also significantly inhibited by H89. These data indicate that MaR2, but not MaR1, is dependent on activation of PKA to increase [Ca2+]i and glycoconjugate secretion.To determine the dependency of MaR2 on PKA to stimulate glycoconjugate secretion, conjunctival goblet cells were incubated with H89 (101–H4), causing vasodilatation, and vascular permeability contributing to inflammation [2+]i and stimulates glycoconjugate secretion [2+]i and glycoconjugate secretion [2+]i over time. Histamine caused an increase in [Ca2+]i to a peak of 269.47 ± 28.92 nM . When incubated with MaR2 for 30 min before addition of histamine, the increase in peak [Ca2+]i was attenuated to 116.79 ± 16.80 nM by MaR2 at 10−10 M (p = 0.010), to 150.66 ± 50.78 nM by MaR2 at 10−9 M (p = 0.11) and to 90.73 ± 5.55 nM by MaR2 10−8 M (p=0.0040). The positive control MaR1 at 10−8 M also blocked the histamine stimulated increase in [Ca2+]i. We conclude that MaR2 similarly to MaR1 inhibits the histamine stimulated increase in [Ca2+]i.Histamine has a key role in inflammatory allergic diseases, and is mainly secreted by mast cells and basophils. Histamine uses four receptors  for 30 min prior to stimulation with histamine (10−5 M). Histamine stimulated secretion to 1.75 ± 0.13 above basal . Preincubation with MaR2 decreased the response to 1.09 ± 0.19 above basal (p = 0.029). This indicates that MaR2 inhibits histamine stimulated glycoconjugate secretion.Dartt et al. demonstrated that histamine stimulates an increase in [Caet cells . To inve4 is a chemoattractant involved in inflammation and immune response and activates inflammatory cells [4 binds to the BLT1 receptor and to the ALX/FPR2-receptor [4-stimulated increase in [Ca2+]i, we pre-incubated rat conjunctival goblet cells with MaR2 (10−8 M) or MaR1 (10−8 M) for 30 min, then stimulated with LTB4 (10−9 M). 2+]i over time. LTB4 caused an increase in [Ca2+]i to a peak of 128.71 ± 27.51 nM . Incubation with MaR2 caused a LTB4-stimulated increase in [Ca2+]i to a peak of 89.62 ± 31.53 nM (p=0.39) that was not different from stimulation by LTB4 alone. Incubation with MaR1 significantly decreased the LTB4-stimulated increase in [Ca2+]i to a peak of 47.65 ± 7.65 nM (p=0.030). We conclude that MaR2 does not inhibit LTB4-stimulated increase in [Ca2+]i, while MaR1 does. The leukotriene LTBry cells . LTB4 bireceptor . To dete2+]i that stimulates secretion and blocks overproduction of mucin stimulated by histamine, an allergic mediator. Both of these actions are used by MaR2 to maintain homeostasis in both health and disease  is a parasympathetic neurotransmitter that like MaR2 and RvD2 stimulates PKA [2+]i through a mechanism that is similar to that used by RvD2 and slightly different from that used by MaR2. The difference is that VIP and RvD2 stimulate PLC activity to produce IP3. IP3 then binds with its receptors on the ER to release Ca2+ and cAMP that interacts with the IP3 receptors to increase Ca2+. In contrast, MaR2 does not activate PLC to produce IP3. Thus IP3 receptors are not involved in the action of MaR2. MaR2 activation of PKA would then increase [Ca2+]i by a different mechanism than RvD2. Further studies are warranted to determine the specifics of the MaR2 cAMP-dependent actions and to compare them with those of RvD2 and VIP. Consistent with MaR2 and MaR1, interacting with different receptors or different sites on the same receptor, these SPMs differ in the use of the cAMP/PKA signaling pathway. MaR2, but not MaR1, increases cAMP levels and activates PKA to increase i by release of intracellular Ca2+ stores, confirmed by inhibition of secretion when the [Ca2+]i was decreased by the Ca2+ chelator BATPA/AM and when MaR2 and MaR1 stimulated increase in [Ca2+]i was blocked by the SERCA inhibitor thapsigargin that depletes intracellular Ca2+ stores [2+]i and stimulate secretion in conjunctival goblet cells PLA2, PLD and PLC. Neither MaR2 nor MaR1 activate PLA2 to increase [Ca2+]i but MaR2 uses it to stimulate secretion. Both MaR2 and MaR1 activate PLD to increase [Ca2+]i and stimulate secretion, although the negative control for MaR2 and PLD increase in [Ca2+]i was also inhibitory. MaR2 and MaR1 both activate components of the PLC pathway. MaR2 and MaR1 activate PLC, but only MaR1 uses the downstream molecule IP3R and only MaR2-stimulated secretion is dependent on these components. Both MaR2 and MaR1 activated PKC to increase [Ca2+]i and stimulate secretion. Surprisingly, both MaR2- and MaR1- stimulated increase in [Ca2+]i are independent of extracellular Ca2+. Thus, the PLC pathway has some differences between MaR2 and MaR1 activation, especially in the targets of PLC activation. Whereas MaR1 stimulates the increase in [Ca2+]i and secretion by the well-known PLC pathway that produces IP3 that releases Ca2+ from intracellular stores and produces DAG to activate PKC to stimulate secretion, MaR2 only uses these processes to stimulate secretion. As MaR2 and MaR1 activate the PLD pathway, they could use PLD to activate PKC via an increase in Ca2+. In contrast, MaR2 does not use PLC to increase Ca2+ and activate PKC, while MaR1 does. As there are multiple PKC isoforms in conjunctival goblet cells some of which are Ca2+ -dependent and Ca2+-independent PKC isoforms, MaR2 and MaR1 may be activating different PKC isoforms to stimulate secretion i and secretion decrease. This suggests that MaR2 can block the inflammatory effect of histamine on goblet cells decreasing mucin secretion. Many other SPMs, including LXA4, RvD1, RvE1, and MaR1, similarly counter-regulate the effect of histamine in cultured rat conjunctival goblet cells  i. Thus, MaR2 and MaR1 play a role in maintaining the ocular surface and tear film homeostasis in health and disease. As MaR2 and MaR1 each modulate conjunctival goblet cell function, they each may have potential as novel, but differing, options for treatment of ocular surface inflammatory diseases including allergic conjunctivitis and dry eye disease.We conclude that MaR2 and MaR1 stimulate conjunctival goblet cell function especially secretion, by activating different, but overlapping GPCR and signaling pathways, and furthermore counter-regulate histamine stimulated increase in  was measured with a ratio imaging system  using wavelengths of 340 and 380 nm and an emission wavelength of 505 nm. A minimum of 10 goblet cells was selected and the Ca2+ response was followed for approximately 2 min. MaR2 was either added alone or treated with inhibitors before addition of either MaR2 or the positive controls MaR1, carbachol, or histamine. Cultured rat conjunctival goblet cells were transferred after trypsinization to 35-mm glass bottom dishes and incubated in 37.0 °C overnight. The cells were then incubated in 37.0 °C for 1 h with KRB-HEPES containing 0.5% BSA, 0.5 mM fura2/AM, 250 mM Sulfinpyrazone and 8 mM Pluronic acid F127. [Ca2+]i was calculated by subtracting the average basal [Ca2+]i from the peak [Ca2+]i.The inhibitors H89, BOC2, BLT1, RO 317549, 2APB, U73122, U73343 were added 30 min prior to the agonist. 1-butanol, t-butanol and thapsigargin were added 15 min prior to the agonist. Change in peak [Ca−10–10−8 M) was then added alone or the cells were incubated with an inhibitor for 30 min, and then stimulated with MaR2 (10−10–10−8 M) or carbachol (10−4 M) for 2 h. The amount of goblet cell high molecular weight glycoconjugate secretion was measured using the lectin UEA-1 in an enzyme linked lectin assay (ELLA). Glycoconjugate secretion is shown as -fold increase above basal (which was set to 1).Cultured rat conjunctival goblet cells were trypsinized and transferred to 24 well plates. The cells were serum starved in free RPMI 1640 media containing 0.5% bovine serum album (BSA) for 120 min. MaR2  and GraphPad Prism (version 9.3.1).Data are expressed as mean ± SEM. N indicates cells cultured from different animals. Data were analyzed by either Student’s As MaR2 and MaR1 each modulate conjunctival goblet cell function, they each have potential as novel, but differing, options for treatment of ocular surface inflammatory diseases including allergic conjunctivitis and dry eye disease.


During heart contraction, the mitochondrial Ca2+ concentration and NADH oxidation rate increase synchronously. Without a mechanism to excrete increased intramitochondrial Ca2+, the Ca2+ overload causes excessive oxidative phosphorylation, which produces reactive oxygen species (ROS); this, in turn, induces ventricular arrhythmias. The Na+/Ca2+/Li+ exchanger (NCLX) is responsible for mitochondrial Ca2+ excretion. While NCLX is thought to contribute to mitochondrial homeostasis, chronic activation of NCLX in heart failure conditions, where ATP production has shifted from being fatty acid dependent to being carbohydrate dependent, is expected to result in mitochondrial Ca2+ deprivation. Thus, the evaluation of disease status using mitochondrial function is critical and, at the same time, largely complicated. In order to understand such complex biological phenomena, Dr. Takeuchi and Prof. Matsuoka emphasize the importance of mathematical model analysis using computers. The heart is a necessary organ for sustaining life in mammals, and it is the first organ to function during early development. Once this primary engine begins to operate, it continues to pump blood throughout the body until the individual animal dies. The miraculous homeostasis of the heartbeat, which involves tremendous energy consumption, is naturally synchronized with systemic homeostasis. The minimum unit of energy in the body is called the ATP, which is produced in large quantities in organelles called mitochondria. Recently, it has been shown that the quality maintenance of cardiac mitochondria is achieved by mobilizing the immune system . In otheKs and IKr, more than four bifurcations have been observed. Each bifurcation is an arrhythmia with a different molecular mechanism. In addition, different underlying models naturally lead to different results; in some cases, EAD can be explained by action potential reactivation, while in others, it is accompanied by intracellular Ca2+ release. Eventually, the accumulation of precise experimental data and trial and error in the analysis seem to be indispensable.Dr. Tsumoto and Prof. Kurata reviewed the bifurcation analysis on the cardiac action potentials, a type of mathematical model analysis . In the 2+ release preceding the action potential is reflected in depolarization via a Na+–Ca2+ exchanger (NCX). Surprisingly, Dr. Namekata et al. pharmacologically excluded the need for an NCX in guinea pig heartbeat. Indeed, similar reports have previously been reported in cellular experiments [2+ release preceding the action potential, and the action potential preceding Ca2+ release [On the other hand, to elucidate the mechanism of cardiac function, Dr. Namekata et al. and Dr. Okada et al. employed a strategy of comparing different animal species ,5. To oueriments . Further release . It seem release . They reThe study of heart rhythm in this way has been a passion, and there are a wide variety of experimental and analytical methods. The most straightforward approach is to inhibit specific ion channels in the study of arrhythmias. For example, inhibition of human ether-a-go-go-related gene (hERG) channels is known to produce ventricular arrhythmias, and Dr. Tamura et al. reported that synthetic estrogen has a protective effect against hERG channel inhibition . Dr. ShiBy the way, even though the molecular mechanisms of cardiac excitation and contraction are extremely well regulated, there is one heart region that is innately susceptible to arrhythmias: the pulmonary veins. The pulmonary veins are the source of the most frequent arrhythmia: atrial fibrillation. Bredeloux et al. have provided a review in this Special Issue on the history and latest findings in pulmonary vein research , and OkaFinally, Dr. Omatsu-Kanbe discovered atypical cardiomyocytes (AMCs), which she reviewed in this Special Issue . AMC is


During carcinogenesis, Ca2+ signaling is significantly remodeled due to mutations and/or ectopic expression. Here, we summarize current knowledge on how alterations in Ca2+ signaling contribute to the development of different cancer hallmarks. Emphasis is placed on the structure/function relationship of the well-studied store-operated Ca2+ channel, i.e., Orai1, and the Ca2+-activated K+ channel, i.e., SK3, alongside their individual and joint roles in cancer. This review lays out the current state of knowledge of Ca2+-signaling effectors and proteins as potential targets for the treatment of certain cancer types, with Orai1 and SK3 presented as emerging therapeutic targets.Cell fate is ultimately determined by the precisely coordinated action of the Ca2+-permeating and Ca2+-regulated ion channels in cancer cell development, proliferation, and migration make them promising molecular targets in cancer therapy. In particular, the co-regulation of the Orai1 and SK3 channels has been well-studied in breast and colon cancer cells, where it finally leads to an invasion-metastasis cascade. Nevertheless, many questions remain unanswered, such as which key molecular components determine and regulate their interplay. To provide a solid foundation for a better understanding of this ion channel co-regulation in cancer, we first shed light on the physiological role of Ca2+ and how this ion is linked to carcinogenesis. Then, we highlight the structure/function relationship of Orai1 and SK3, both individually and in concert, their role in the development of different types of cancer, and aspects that are not yet known in this context.Cancer represents a major health burden worldwide. Several molecular targets have been discovered alongside treatments with positive clinical outcomes. However, the reoccurrence of cancer due to therapy resistance remains the primary cause of mortality. Endeavors in pinpointing new markers as molecular targets in cancer therapy are highly desired. The significance of the co-regulation of Ca In this review, we first describe the general role of Ca2+ in tumor development. Since the co-regulation of the Ca2+ channel: Ca2+ release-activated Ca2+ (CRAC) channel, and the Ca2+-regulated channel: Ca2+-activated K+ channel, or SK3, have been extensively reported to play a role in certain cancer types in the last century, we delineate here the current knowledge of the molecular mechanisms of both channels individually and in co-regulation. We highlight critical factors that determine their structure/function relationship, their roles in carcinogenesis identified to date, and outstanding questions in this context.Every year, 18.1 million cases of cancer are diagnosed worldwide, of which 9.5 million lead to death. These numbers are expected to rise 1.6-fold by 2040 ,4,5,6,7.2+ ions are versatile intracellular signals that regulate a plethora of cellular processes including gene transcription, proliferation, and cell migration [2+ concentration on the extracellular side and in intracellular organelles is in the range of mM, and it is 10,000-fold lower in the cytosol [2+ gradient defines the versatility of this signal ion in the life cycle of a cell, to initiate and drive processes such as immune cell activity, neurotransmitter release, or muscle contraction [2+ levels that occur either through Ca2+ release from intracellular stores  or Ca2+ influx across the plasma membrane (PM) from the extracellular space [Caigration ,9. They  cytosol ,12,13. Ttraction ,9,14. Acar space . 2+ signaling is orchestrated by Ca2+-transporting and Ca2+-sensing proteins. Ca2+ level enhancements can be initiated by the stimulation of a membrane receptor ), membrane depolarization , diacylglycerol (DAG)) that activate Ca2+ ion channels in intracellular compartments  and/or the plasma membrane  to allow Ca2+ flux into the cytosol. Cytosolic Ca2+ elevations are sensed by a number of proteins such as Calmodulin (CaM) and downstream targets including CaM kinase (CaMK), calcineurin (CN), or protein kinase C (PKC)  or to the extracellular side (PMCA) [Cellular Cachannels ), or mecchannels ). Ca2+ s C (PKC) A, which e (PMCA) . 2+-signaling components, disrupting the healthy Ca2+ balance [2+ acts at different stages of cancer signaling cascades either directly via Ca2+ signaling proteins  or indirectly via transcription factors  or oncogenic routes. The two major oncogenic pathways [2+ and these oncogenic pathways can affect each other in a bidirectional manner to promote cancer progression. On the one hand, distinct Ca2+ signals can tune oncogene-dependent signaling [2+ ions with Ras regulatory factors, such as CaM [2+ signals, as evidenced by the fact that the oncogene Ras interacts with and activates PLCε to produce IP3, which triggers ER-Ca2+ store-depletion [In cancer cells, genetic and epigenetic alterations can lead to the remodeling of Ca balance ,6,19,20. balance . Ca2+ acpathways  that havpathways . Within pathways . In the pathways . , cAMP-responsive element binding protein (CREB), and AP1  families [2+ also drives their complex formation  at the end of G1 to finally ensure the transition to the S phase. The progression of the G1 phase is further fine-tuned by CDK inhibitor proteins , whose action is controlled directly by Ca2+ via Ca2+-sensing proteins, CaM, CaMKII, and calcineurin (CN) and the tumor suppressor p53 [2+ is also essential for subsequent cell cycle phase transitions  and associated rearrangements of centrosomes are triggered by Ca2+ oscillations acting in concert with CaM and CaMKII [The multitude of Cafamilies . These cssor p53 . Ca2+ isd CaMKII ,27  target Myc to modulate cell cycle progression [2+ route can control Ca2+ signaling. The Wnt-Ca2+ pathway is initialized by Frizzled receptors, which initiate a classical G-protein-coupled signaling cascade that results in the production of IP3 and DAG and is thus directly linked to Ca2+ signaling pathways [A variety of other cancer signaling pathways  and anti-apoptotic  factors at the mitochondria ,32. Dysrproteins ,41,42,43m the ER ,25 , cell migration, invasion, angiogenesis, and intravasation, all of which are controlled by Ca2+ ,25. EMT 2+ . In seve2+ ,51,52,53er types ,32. For er types . Furtherinvasion ,51,54,55-catenin  , followed by actomyosin contraction and disassembly of focal adhesions. The latter is governed by the Ca2+-sensitive protease calpain, which cleaves focal adhesion kinase (FAK), vinculin, talin, and integrins to disconnect the membrane from the cytoskeleton. Conversely, directed actin polymerization and growing focal adhesions located at the leading edge of migrating cells to form membrane extension are stimulated by spatially confined Ca2+ signals. This is modulated by small GTPase, such as Ras and Rac1, Ca2+-dependent factors, including CaMK and proline-rich tyrosine kinase 2 (PYK2), and FAK [2+ levels in tumor cells can further promote FAK disassembly and contribute to the reduction of cell adhesions that promote cell migration. Moreover, the formation of membrane extensions (invadopodia) is driven by the expression of Ca2+-dependent pro-invasive enzymes ) that contribute to the degradation of the extracellular matrix (ECM) [Directional movement of migratory cells is usually driven by highly localized Ca and FAK   ,32,40  and non-voltage Cachannels ,40. Despchannels ,72. Non-channels , receptochannels ,75, SOC channels ,76, acidchannels , and mecchannels . A numbechannels ,87,88,89channels ,91,92. T2+ ion channel, the CRAC channel, in the co-regulation with Ca2+-dependent K+ ion channels in cancer, we focus initially on its currently known structure/function relationship as well as its individual role in cancer. It is composed of the stromal interaction molecule, STIM, containing its Ca2+ sensor embedded in the ER lumen [2+-selective ion channel Orai in the PM [2+ entry mechanisms in most cancer cells [Due to the essential role of the most studied store-operated CaER lumen ,94,95,96n the PM ,101,102.er cells ,109,110 er cells ,109,111.2+ from the ER. Precisely, ligand binding to PM receptors leads to the activation of G-proteins, which initiate the production of phospholipase C to generate IP3 from phosphatidylinositol-biphosphate (PIP2). Subsequently, IP3 couples to the IP3 receptor in the ER membrane, which triggers ER-Ca2+ store depletion [2+ ion channels [CRAC channels, composed of STIM and Orai proteins, are distinguished by their activation mechanism via the release of Caepletion ,116,117.epletion ,118 and channels  in the Pchannels ,121  ,124,128 2+ levels, and the C-terminus relays the activation signal to Orai1 in the PM [In general, the STIM structure consists of a single-pass TM domain with the N-terminus embedded in the ER lumen and its long C-terminal tail exposed to the cytosol. The N-terminus senses fluctuations in Can the PM . The STIn the PM ,137,138 2+-sensing EF-hand domain in a complex with the sterile alpha motif (SAM) region. In the resting state, the EF-SAM domain is folded into a compact structure that is stabilized by hydrophobic interactions [2+ store depletion, Ca2+ ions dissociate, leading to destabilization of the EF-SAM complex, which initiates signal transmission to the C-terminus [Specifically, the luminal side of STIM1 comprises the Caractions . Upon Caterminus ,143,144.2+ store depletion is conveyed from the N- to the C-terminus via the STIM1 TM domain. Two TM domains within a STIM1 dimer are considered to form a crossing angle that alters upon activation [Next, the activation signal of Cativation . In supptivation .2+ store depletion, the STIM1 C-terminal domain undergoes an extensive conformational rearrangement to change from a folded quiescent to an extended state, which leads to the exposure of STIM1 oligomerization and Orai1 coupling sites. The STIM1 C-terminus is composed of three typical protein–protein interaction domains known as coiled-coil regions , the inhibitory [Upon Cahibitory  or CRAC hibitory , the michibitory , and thehibitory ,148  and CC3 of monomers within a dimer is further modulated by intrahelical interactions within two other parts of CC1 (CC1α2 with CC1α3) ,150,151.2+ influx.A minimal portion of the STIM1 C-terminus called CAD (CRAC-activating domain) or SOAR (STIM1-Orai1-activating region) is sufficient for coupling to and activation of Orai channels. They include mainly CC2 and CC3 ,154,155.2+-selective ion pores in the PM. The Orai protein family comprises three human Orai paralogs, namely, Orai1–3  connected by two extracellular and one intracellular loop and flanked by a cytosolic N- and C-terminus ,166,167.Drosophila melanogaster Orai (dOrai) and corresponding gain-of-function (GoF) dOrai  mutants consistently exhibit a hexameric assembly of Orai subunits. Based on the high homology of dOrai to human Orai1 (hOrai1) within the TMs, it is assumed that hOrai1 also forms a hexamer. Of the four TM domains, the first TMs (TM1s) form an inner ring lining the pore in the center of the channel [2+ accumulation region at the extracellular side followed by the selectivity filter (E106), the hydrophobic cavity , and at the cytosolic side, the basic region. Interestingly, TM1 expands helically approximately 20 Å into the cytosol, forming an extension of the Ca2+ ion pore [2+ ion pore, another essential feature of the Orai1 complex represents its periphery, composed of TM4 and the C-terminus [Structural resolutions of the  channel ,178,179. filter E6, the hyion pore ,136,180.ion pore . In additerminus ,170. Theterminus ,170. Theterminus ,170,174,terminus ,182. Theterminus ,182.The general structure of the three Orai isoforms is comparable, yet they have an overall sequence identity of 50–60%, with TM1 being identical among the three Orai proteins, whereas the other TM domains are approximately 81–87% similar C. The cyIn summary, CRAC channel activation represents a multistep activation cascade involving STIM1 unfolding, STIM1 oligomerization, STIM1-Orai1 coupling, and Orai1 activation. Resolving these different intermediate activation states could help researchers to find new targets for selective therapeutic strategies also in cancer. In particular, the isoform-specific differences of Orai channels might be promising in cancer-type-specific drug discovery and therapy development . Neverth2+ ion channels may involve their altered arrangement and interplay in the membrane due to structural rearrangements of the channel, assembly of the channel complex, or channel interaction with regulatory proteins that affect their function [2 and PI4P, cholesterol, and sphingomyelin [Increasing evidence reveals that membrane proteins are localized in microdomains containing especially cholesterol, sphingolipids such as sphingomyelin, and glycosphingolipids . These cfunction . In the function ,189, altfunction . This defunction . The fungomyelin ,196,197.2 modulates STIM1 function via direct binding to its C-terminal end, a lysine-rich region [2 and PIP3 located in cholesterol-rich regions in the PM [2 and PIP3 in cholesterol-rich regions before interacting directly with Orai1 [2 and is therefore essential for modulating Orai1 in membrane domains with different PIP2 content [2 on Orai1 due to its dependence on STIM1. Moreover, the precursor of PIP2, PI4P, regulates CRAC channel function [Specifically, PIPh region ,200,201.n the PM ,204,205.th Orai1 . The N-t content . Howeverfunction ,208,209,Cholesterol affects CRAC channel function via direct interaction with STIM1 and Orai1. Both CRAC channel proteins contain a cholesterol-binding motif, namely, the cholesterol recognition amino acid consensus motif (-L/V-(X)(1-5)-Y-(X)(1-5)-R/K-; X represents from one to five any amino acids before the next conserved residue) ,212,213.2+ store depletion. However, whether this modulatory role occurs due to direct binding or allosterically is still unknown [Sphingomyelin, which is also abundant in cholesterol-rich regions, controls CRAC channel function. Indeed, the application of sphingomyelinase (SMase) D diminished CRAC channel currents without impacting Ca unknown ,216.Additionally, STIM1 and Orai1 undergo S-acylation, the posttranslational tethering of a medium-length fatty acid, to a cysteine—in these cases, C437 in STIM1 and C143 in Orai1. S-acylation of STIM1 controls its puncta formation and maximal activation of CRAC channels . In Orai2, of the membranes, thereby having various effects on the modulation of STIM1/Orai1 coupling and function. Additionally, ER- or PM-associated proteins located in the ER-PM junctions, in particular, septins [Additionally, perturbations to plasma membrane lipids affect other proteins that impact STIM1 and Orai1 expression and function. These include a variety of lipid- or ER-PM transition-dependent accessory proteins at the ER–PM contact sites , as we r septins ,226,227, septins ,229, RAS septins , SARAF [ septins ,231,232, septins ,235,236  septins ,239,240, septins ,242,243.The fact that the CRAC channel complex is not a closed entity but can be modulated by a variety of proteins and lipids in the vicinity of the ER–PM contact sites highlights the multiple regulatory possibilities that may also play a role in the pathogenesis of cancer and could be exploited for therapeutic applications.2+ influx to maintain healthy body functions [2+ influx into cancer cells, where they are linked to tumorigenesis. This is due to either altered expression of CRAC channel proteins [Although CRAC channels are one of the most important pathways for the cellular Caunctions , there iproteins ,245,246,proteins ,250,251 Distinct expression levels of STIM and Orai compared to corresponding healthy tissues have been detected in cells of the breast ,252,253,2+ ion channels, for instance, the canonical transient receptor potential channels, which have been found in breast cancer (TRPC1 and TRPC6) [2+ signaling in dependence with not only STIM1 and Orai1, but also TRPC1 [2+ entry occurs via a molecular complex of TRPC6, STIM1, and Orai1 controlling cell proliferation [2+ entry through Orai1 activation via the accessory protein Secretory Pathway Ca2+-ATPase 2 (SPCA2) has been identified [2+ pump, where it is involved in protein glycosylation, sorting, and processing. However, in this cancer cell line, there is a direct interaction of the N- and C-termini of SPCA2 with Orai1 at the cell surface, which controls EMT [Notably, other molecular components also contribute to STIM- or Orai-dependent cancer progression. Altered expression and function of the molecular components of SOCE include not only CRAC channel components, such as Orai1 and Orai3, but also other Cad TRPC6)  and ovard TRPC6) . Chronicso TRPC1 . Moreoveferation . In the entified . Physiolrols EMT ,278. The2+ entry via the STIM1/Orai1 machinery affects the various cell cycle effectors, either directly via Ca2+-binding proteins or indirectly via transcription factors, Ras/ERK, or PI3K/Akt pathways.In several tumor types, enhanced proliferation correlates with ectopic STIM1/Orai1 expression ,276,277.+ breast cancer cells, SOCE is mediated exclusively by Orai3 and STIM1/STIM2, whereas in ER- breast cancer cells, the canonical CRAC channel consists of STIM1/Orai1, which is sufficient to trigger SOCE [+) breast cancer cells (MCF-7), Ca2+ entry via Orai3 targets the proto-oncogenic transcription factor c-myc via the MAPK  pathway to trigger cell proliferation arrest [+ breast tumorigenesis [In some cancer types, Orai3 and/or STIM2 expression exerts pro-proliferative effects by influencing the cell cycle via various signaling cascades A. In ER+ger SOCE . Specifin arrest , contribigenesis . Indeed,igenesis . In pancigenesis ; however2+ concentration, which controls cell proliferation via Ca2+/CN-dependent activation of the transcription factor NFAT. Interestingly, in mouse models, Orai3 silencing controls the G1/S phase cell cycle by decreasing cyclin D1 expression. Conversely, Orai3 overexpression promoted proliferation [In other cancer types, enhanced Orai3 expression increased Orai1/Orai3 heteromeric formation and thus reduced the number of functional Orai1 channels  Figure A. In proferation . Anotherferation  suggesteferation ,259. In feration . This paferation  providesApoptosis resistance is another cancer hallmark connected to up- or downregulated expression and function of CRAC channel components.2+ crosstalk between ER and mitochondria and/or balance between pro- and anti-apoptotic factors is altered and thus determines apoptosis resistance. Nevertheless, prostate cancer cells benefit from the abrogated SOCE, whereas upregulated SOCE has a pro-survival and pro-migration effect in several other cancer cell types [The androgen-independent stage of prostate cancer, which represents an aggressive phenotype, is manifested by downregulated Orai1 expression, which leads to apoptosis resistance. In accordance, SOCE is abrogated in these androgen-independent prostate cancer cells . Supportll types ,275,276.ll types . Moreovell types . These fCRAC channel components are further of great importance in EMT, migration, and invasion of tumor cells ,278,280.The expression of important markers of EMT and requisite regulators of mesenchymal cell migration, such as vimentin or fibronectin, is downregulated in esophageal  and gastAmong factors that promote cell migration, the expression level of small GTPases Ras or Rac is critical in STIM1- and/or Orai1-dependent cancer types, as reported in breast  and esop2+ influx via Orai1 [2+-dependent Wnt signaling. Specifically, increased Ca2+ levels phosphorylate CaMKII and ß-catenin, thereby switching off Wnt and subsequently inhibiting EMT [The reversal of EMT in a highly aggressive type of breast cancer depends on the enhanced expression of SPCA2, which triggers constitutive Caia Orai1   playing a role in the prostaglandin  synthesis. It is activated by the Ras/ERK pathway ,284,285 and CREB   by which STIM1 and COX-2/PGE2 affect EMT  as well as migration have not been explored in detail.2+ levels can underlie the upregulation of Orai channels; however, the mechanism underlying their upregulation remains unknown. Regarding Orai1, it has been shown to upregulate in breast, colon, and glioma cancer cells due to hypoxia together with TRPC6 [2+ levels due to hypoxia were diminished by the application of SKF-96365, a CRAC channel blocker [Hypoxia-dependent enhancements in Cath TRPC6 . This en blocker ,286,287. blocker   , stomach2+ levels, which might be responsible for the cancer development. Mechanistically, most of these mutations  are located at key checkpoints in the Orai TM domains controlling pore opening. Their manipulation likely initiates a global conformational change that finally induces pore opening [All these cancer-related Orai1 mutants have been reported to lead to constitutive activity independent of STIM1 . This li opening . Positio opening  and cont opening ,294. How2+ influx through Orai1 channels. The mechanistic role of S116N, located in the non-canonical EF hand, is still unknown. Several other cancer-related mutants are located in the STIM1 C-terminus, which is essential in both maintaining the quiescent state and establishing CRAC channel activation. For instance, W350L is located in the CC2 of STIM1, part of the minimal region sufficient to activate Orai1, and G446C/V, T517I, and S521L are located in the flexible portion of the STIM1 C-terminus. Further studies are still required to understand their mechanistic impact.Among the STIM1 mutants identified to occur in cancer, several  are loc2+ ion channels and their interconnectedness with cancer-promoting signaling pathways has opened new possibilities for future therapeutic strategies.The altered expression of CaA variety of CRAC channel drugs are available, which has provided valuable insights into CRAC channel mechanisms, but only a few have reached clinical trials due to low selectivity or undesirable side effects. In the treatment of cancer, SKF-96365, a SOCE/Orai1 blocker, has been shown to impair cell proliferation of GBM cells in vitro  and meta2+ overload and inhibition of Ca2+-dependent survival pathways [Carboxyamidotriazole (CAI) interferes indirectly with SOCE via the PI3K/Akt pathway. Tested as a potential therapeutic, it affects the expression of BCL2 members ,300 in opathways .Drebrin, an actin-reorganizing protein boosting SOCE, is linked to prostate cancer cell invasion, especially at earlier stages of cancer development. There is evidence that BTP2, an Orai1 inhibitor, targets drebrin . Notably2+ levels through Orai3 and caused cell death of AMLC lines [Additionally, store-operated channels in acute myeloid leukemia cells containing Orai3 have been reported to be targeted by tipifarnib, a farnesyltransferase inhibitor, that prevents farnesylation of Ras . This enLC lines .RP4010, a synergistic drug, prevents SOCE activation by inhibiting Orai1. The drug is undergoing clinical trials and might serve as a treatment for pancreatic ductal adenocarcinoma  and esopIn addition to their function as drug targets, CRAC channel proteins can facilitate the circumvention of a therapeutic effect, known as therapy resistance. This is particularly challenging in the case of the chemotherapeutic agents 5-fluorouracil (5-FU) and cisplatin, that induce autophagy and cell death in cancer cells. In hepatocarcinoma tissues, overexpression of Orai1 impairs the effect of 5-FU , making In summary, although several studies have reported the effect of CRAC channel drugs on various cancers, several questions remain to be addressed, such as why cancer cells continue to proliferate and migrate even though the applied CRAC channel drug blocks store-operated currents. Moreover, of the selective CRAC channel drugs currently available, only a few have made it to clinical trials . Thus, t2+, but other channels also sense Ca2+, either by direct binding of Ca2+ or indirectly by Ca2+-binding proteins, such as CaM [2+ channels, Ca2+ often has an inactivating effect, providing a negative feedback mechanism that protects against too much Ca2+ entering the cell [2+, such as Ca2+-activated K+ channels and Ca2+-activated Cl- channels [2+-activated K+ channels and their role in cancer.An important property of many ion channels is not only the transport of Cah as CaM ,311,312.the cell . Other ichannels ,322,323.channels ,330,331.2+-activated K+ channels include the large (BK), intermediate (IK or SK4), and small conductance Ca2+-activated K+ channels  [+ channels, containing six TM regions (S1–S6) with the pore region located between TM5 and TM6 and both the N- and C-termini located at the cytosolic side [Caand SK3) ,333, whelic side   in the C-terminus of each α-subunit. Functional studies of SK channels have demonstrated that the CaM C-lobe is constitutively bound to the SK channel, and the N-lobe is bound upon cytosolic Ca2+ elevations [2+-dependent interaction of the CaM N-lobe is formed with the S4–S5 linker of the SK channel and triggers a conformational change within the channel complex to induce pore opening [45A, which then moves to the cytosolic side. Additionally, S45B moves away from the pore region, leading to further changes in S6, finally causing channel activation [SK ion channels are activated by changes in intracellular Ca+ levels ,315,332. outside . Howeverevations ,312,337. opening . Specifitivation   , glioma SK4 is expressed in different cancer types, including glioma , glioblaThe catalogue of somatic mutations in cancer (COSMIC)  reports 2+-dependent manner. Specifically, an increase in intracellular Ca2+ levels in SK channel-expressing cells leads to their activation and thus to potassium (K+) efflux, which in turn promotes Ca2+ entry via Ca2+ channels in a positive feedback loop. The increase in intracellular Ca2+ levels achieved in this manner controls Ca2+-dependent cancer hallmarks [The development of cancer features triggered by SK channels generally occurs in a Caallmarks ,292,325,2+ entry, as inhibition of SK4 led cells to accumulate in the G1 phase, whereas the number of cells in the cell cycle S phase decreased [2+ entry, cell cycle progression, and the proliferation rate of primary breast tumor cells [Insights into the influence of the SK channel on cell cycle progression have only been gained for SK4. It is able to indirectly control cell cycle progression by driving cellular Caecreased ,368. In or cells   that trigger EMT. Indeed, silencing of SK4 expression abolished the expression of certain EMT markers   Figure .Overall, there is evidence that SK channels are present in cancer cells and can control the development of cancer features. However, knowledge about the influence of SK channels on individual steps in signaling cascades regulating the cell cycle or migration is limited. In this context, identification of the effects on key players modulating, for instance, the focal adhesion dynamics  is required. A detailed understanding in this regard could improve therapeutic options for cancer cells.Drugs targeting SK channels include peptides extracted from scorpions, sea anemone or bee venom, synthetic analogues, and chemically synthesized modulators . However, all of these modulators arose as limited in medical use due to their high toxicity or low efficiency. Among the blockers, particularly those targeting SK4 have been suggested as promising for the treatment of various cancers . SpecifiTo overcome therapy resistance , SK3 and2+ signaling, which is crucial for EMT progression, contributing to therapeutic escape [The alkyl-ether lipids edelfosine and Ohmline have emerged as promising lipid-antimetastatic agents. Both block SK3-dependent cancer cell migration and metastasis, likely due to lipid membrane reorganization, which inhibits SK3 channels . Edelfosc escape . Ohmlinec escape  and Enzac escape . Moreovec escape ,323.Overall, alkyl ether lipids appear to be the most promising and selective drug candidates for the treatment of SK3-dependent cancers.+ channels interact with Ca2+ ion channels to control various cellular functions, including also the fine-tuning of the intracellular Ca2+ concentration. Voltage-gated K+ (Kv) and KCa2+ channels have been classified as Ca2+ amplifiers because of their interplay with Ca2+ channels [+ channels, when activated, induce membrane hyperpolarization either by increased intracellular Ca2+ levels in the case of KCa2+ channels or by membrane depolarization in the case of Kv channels, thereby enhancing the driving force for Ca2+ influx. Several studies indicate that co-regulation of K+  and Ca2+ channels  channels) plays a significant role in cancer cell progression, growth, proliferation, and invasion/migration [V3.2, promoting the proliferation of prostate cancer cells [2+ entry [2+ influx through store-independent (SICE) Orai1 activation. This trio complex triggers collagen I-induced proliferation and survival of breast cancer cells [Ca2+ channels, in terms of structural and functional requirements and their role in cancer, represents that of Orai1 and SK3 [In addition to their individual role, Kchannels ,327,360.igration ,327,360.igration  . This suggests, in agreement with previous findings in cancer cells [2+ levels specifically through Orai1, which together with SK3 triggers the positive feedback mechanisms [2+ entry in SK3-Orai1 expressing cells, and NFAT translocation was only marginally enhanced. This indicates that already, very low local alterations in Ca2+ levels are sufficient to trigger the SK3–Orai1 interplay. Moreover, we found that K+ currents in SK3 and Orai1 co-expressing cells could be reduced to the level of SK3-expressing cells not only when the general Ca2+ channel blocker La3+ was applied, but also the CRAC channel-specific inhibitor GSK-7975A [+ currents compared to the presence of wild-type Orai1, despite their co-localization being maintained  or abolished (1000 µM EGTA) SK3 K+ currents, both in the absence and presence of STIM1  and an intact pore geometry, as demonstrated by a set of LoF-Orai1 mutants. They were unable to boost SK3 channel activity without affecting the co-localization with SK3. However, STIM1 coupling to Orai1 or an intact STIM1 coupling site within Orai1 (L273) is not required for intact Orai1-SK3 interplay, as evidenced by the preserved boost of Orai1-mediated SK3 channel activity in CRISPR/Cas9 STIM1/Orai1 DKO HEK 293 cells. In agreement with this, Orai1 L273D was also able to trigger the amplification of SK3 Kcurrents . It is scurrents . This recurrents ), two pucurrents ,386), a currents ), and a currents ). It rem+ current amplification under physiological conditions, as STIM1 activation removes Orai1 from SK3, and locally increased Ca2+ levels cannot affect SK3 channel activity. Indeed, global Ca2+ level increases also enabled the amplification of SK3 K+ currents in the presence of STIM1 and Orai1 [2+ elevations are sufficient to govern the interplay of SK3 and Orai1 in the absence of STIM1 [Interestingly, co-expression of STIM1 with Orai1 and SK3 showed an inhibitory effect on Knd Orai1   and the Akt pathway promotes the activity of Rac1/Calpain, which amplifies SOCE, and consequently Akt. Simply stated, SOCE triggers activation of the SK3 channel and the Akt pathway, which mediates the activity of Rac1/Calpain, which in turn leads to the amplification of SOCE  are not yet elucidated in detail. For instance, proliferation can be directly regulated by Ca2+ via Ca2+-sensing proteins or influence transcriptional activity to modulate the expression of cell cycle effectors. Alternatively, PI3K/Akt or RAS/ERK pathways might be affected by either enhanced expression levels of Ca2+ ion channels and/or the effect of Ca2+ on certain oncogenes in the PI3K/Akt or RAS/ERK pathways. Therefore, it remains open whether Ca2+ modulates only a specific path or multiple ones to impact cancer progression in a given cancer type. In most recent studies, the proliferative activity of related cancer cell lines is monitored upon siRNA-mediated inhibition of ion channels in vitro. Nevertheless, evidence of tumor behaviors in vivo is lacking. At this point, the question arises whether the effects of ion channels on proliferation are not merely coincidental because they interact closely with each other, or, possibly with different more relevant components of the Ca2+ signaling cascade. Concerning the role of CRAC channel proteins in apoptosis, several have been identified to contribute to pro- or anti-apoptotic mechanisms; however, the detailed effects on certain regulators of cell survival/cell death signaling cascades are not yet fully understood. Key questions include how altered STIM/Orai expression might influence the expression of pro/anti-apoptotic factors, and how mitochondrial/ER Ca2+ levels and their interplay are dysregulated. Moreover, the molecular mechanisms of mutations identified to be critical in cancer are only beginning to be deciphered, and their connections to Ca2+ dysregulated signaling pathways remain open. In particular, for SK channels as well as their interplay with STIM1/Orai1, the detailed links to various cancer signaling pathways are insufficiently described.Cell fate is determined by the finely tuned interplay of signaling cascades involving various cellular components such as CRAC and SK channels. However, their dysregulation/-function can be linked to novel biological functions associated with cancer progression. Mechanistically, dysregulation of CRAC and SK channels can lead to altered activity of a number of effectors of distinct signaling pathways, such as CaM/CaMKII/CN ,273,276,The multifaceted interplay of these ion channels with intracellular signaling pathways is extended by their interaction with each other and with a set of other signaling components, as exemplified by Orai1-SK3 and Orai1-SPCA2 interplay. For such scenarios, the formation of macromolecular complexes appears favorable. For example, SK3 and Orai1 colocalized in cholesterol-rich regions in breast cancer cells , the disAltered expression or mutation of CRAC and SK channels is responsible for poor prognosis in cancer patients. Therefore, these channels could serve as suitable prognostic markers as well as attractive targets for cancer therapy, also in an isoform-specific manner. However, of a large number of CRAC and SK channel drugs available, only Ohmline currently has the potential for future clinical use in SK3/Orai1-driven cancer progression. In addition, the expression of CRAC and SK channels increases resistance to chemotherapeutic agents and promotes cell proliferation, necessitating the need to address alternative targets via potential therapeutic strategies.2+ signal transduction apparatus, CRAC and SK channels play critical roles in the development of certain cancers. Their modulatory role in Ca2+ signaling affects proliferation, apoptosis, and/or migration. Knowledge of these ion channels and their associated cancer features is extensive but insufficient to develop appropriate therapeutic strategies. A more detailed understanding of the mechanisms of ion channels and their connection to cancer signaling pathways will provide new targets for future and more specific therapeutic strategies against cancer.Overall, among the various members of the Ca


Urticaria is a common condition presenting both as acute and chronic disease within primary care. To those without specialist training it is poorly understood from the points of view of diagnosis and management. It causes a considerable disease burden to sufferers with marked impact on quality of life.The recent publication of the EAACI/GA²LEN/EuroGuiDerm/APAAACI Guideline for the Definition, Classification, Diagnosis and Management of Urticaria guideline prompted us to take this excellent resource and re‐configure its findings and recommendations to a non‐specialist audience with particular reference to the needs of the primary care team. The reported prevalence of Chronic Spontaneous Urticaria (CSU) varies from study to study, with a recorded prevalence 0.6% in SpainAge prevalence differs in subtypes, for example, cholinergic urticaria has a peak prevalence between 15 and 30 years of age, CSU around 40 years of age.3PC clinicians recognise that there is a deficiency in their dermatology training at all career levels.Although the GP is considered the first port of call for patients suffering from acute urticaria, there is evidence to suggest that frequently the ED is the initial point of contact, reflecting the urgent desire of patients to attain a diagnosis and treatment in what is essentially a non‐emergency condition. A study from the UK suggested that presentations with urticaria to primary care were more or less static whereas hospital attendances were increasing, perhaps as a consequence of patients using ED as first port of call,A detailed analysis of admissions performed in Spain demonstrated that urticaria accounted for 8.7% of attendances at the ED.4,Many patients with urticaria are inappropriately referred to allergy departments in the belief, by both patient and physician that those patients are suffering from food allergy,2LEN/EuroGuiDerm/APAAACI guideline for urticaria provides authoritative recommendations for the management of urticaria and has implications for primary care.The recently published 2021 update and revision of the EAACI/GA5The purpose of this clinical update on urticaria is to facilitate earlier diagnosis and appropriate treatment, minimise unnecessary investigations and facilitate early identification of those who might benefit from a specialist referral in response to and drawing from this revision. This should, in turn, lead to efficient, patient‐centred, cost‐effective care yielding improved outcomes for patients with urticaria.5.1A collaborative distillation of specialist guidelines by the guideline authors and knowledgeable primary care physicians: The guidelines referred to were contextualised and placed in the context of the non‐specialist to translate that guideline into a useful tool for non‐specialists.6Urticaria is a condition characterised by the development of wheals (hives), angioedema, or both. It is further classified as acute (<6 weeks) or chronic (>6 weeks).Wheals are superficial skin swellings of variable size, usually surrounded by reflex erythema, and they come with an itching or sometimes burning sensation. They are short lived, with the skin returning to its normal appearance, usually within 30 min to 24 h. .Angioedema is the sudden, pronounced erythematous or skin coloured swelling of the lower dermis and subcutis or mucous membranes. It is sometimes painful, rather than itchy, and its resolution is slower than that of wheals (can take up to 72 h). (cf: photographs of angioedema). It is to be noted that the angioedema occurring with urticaria is mast call mediated and does not result in fatalities: It tends to be rapid in onset. Bradykinin induced angioedema  induced angioedema) differs in that there is often a slower evolution with the potential for fatality.Chronic Urticaria (CU) is further divided into two broad categories: CSU, and more rarely, chronic inducible urticaria (CIndU), which has clear precipitating factors .6.1A patient presented with acute urticaria subsequent to a viral infection in the early 2000's. Standard dose antihistamines proved ineffective, however symptoms were controlled with oral corticosteroids (OCS). In view of ongoing symptoms when steroids were withdrawn, the patient was referred to a dermatologist who performed multiple investigations. She was tried on varying combinations of antihistamines to no avail. Ciclosporin was effective but renal impairment became rapidly evident. Other treatments were tried, doxepin, methotrexate, mycophenolate mofetil and psoralen + ultraviolet light A, to no benefit. Some 10 years later she was referred to a dermatologist with a special interest in urticaria. A diagnosis of CSU was confirmed. The patient was weaned off OCS with a rapid increase of Urticaria Activity Score to nearly the maximum. Omalizumab was commenced, with rapid resolution of symptoms. After 6 months, omalizumab was discontinued with a return of symptoms. She now receives omalizumab regularly with a break every 6 months to determine whether spontaneous remission has occurred.This vignette illustrates many of the pitfalls of CSU management: delay in confirming diagnosis, overexposure to oral steroids and inappropriate use of other medications.There is a general perception by non‐specialists that urticaria, whether acute or chronic, is an allergic phenomenon, promoting a cascade of expensive and inappropriate investigations, the results of which may be misleading. Although urticaria is often thought to be due to type 1 hypersensitivity, (immunoglobulinE (IgE) mediated allergy), this is infrequently the case and very rarely without involvement of other body systems  and very rarely the case for chronic urticaria. Urticaria, may be precipitated by upper respiratory tract viral infections or the intake of non‐steroidal anti‐inflammatory drugs (NSAIDs), other medications or even foodstuffs.Allergic acute urticaria must be distinguished from systemic anaphylactic reactions to allergens where wheals and/or angioedema occur together with extracutaneous signs and symptoms Table 28.A retrospective study of anaphylaxis presenting to ED revealed that two‐thirds of anaphylaxis patients (67.1%) presented with wheals/urticaria and 41.3% with angioedema.6.2Acute urticaria, like all urticaria, can present with wheals, angioedema, or both. Most episodes of acute urticaria, independent of symptoms, resolve within 2–3 days. Single wheals usually resolve in 24 h but may recur in a different location, Suggest replace but with however but angioedema may take up to 72 h to resolve.Patients with inducible urticaria often come to their GP after first experiencing wheals in response to a physical trigger: cold, heat, pressure, water UV light etc. To date there is limited data on the natural course of inducible urticarias. Like in CSU, most patients are expected to experience remission after several years of having the disease.6.3Most cases of acute urticaria are acute spontaneous urticaria, rather than acute inducible urticaria. Independent of symptoms, acute urticaria usually resolves within 2–3 days and by week 6 the latest.The natural history of CSU is one of resolution, which when it occurs, occurs rapidly: some 50% will resolve by 6 months after diagnosis, 30% at 3 years 10% at 10 years with some 8% suffering for more than 25 years.6.4CIndU, a subset of CU, lasts for several years in most patients before it shows spontaneous remission.6.5Approximately 40% of patients with CSU will achieve disease control with high dose (4x standard dose) second‐generation oral antihistamines: this further increases up to 70% when using omalizumab with very much smaller incremental benefits gained from addition of other treatments (leukotriene receptor antagonists (LTRAs), ciclosporin etc, which should be administered only by specialists).Inducible urticarias are more difficult to treat than spontaneous despite the fact that patients with some forms of CIndU can avoid precipitating factors to some extent; exposure to cold, heat, UV radiation, pressure for example.6.6Currently, in common with allergyFrom the investigations which are requested there appears to be a belief by both clinicians and patients that urticaria is an allergic phenomenon, with allergy investigations performed in more than half patients seen, along with many other irrelevant investigations including serology for infections or antibody profiling: however, this is as true for specialists as for GPs.,Many patients with CSU are also likely to be undertreated by both dosing and type of medication with many patients receiving first generation antihistamines even though these provide inferior outcomes and greater side effects.In addition, there is a problem with angioedema. We know that patients with hereditary angioedema (HAE) also experience long delays in diagnosis and high rates of misdiagnosis. The most common misdiagnoses are “allergic angioedema” and appendicitis. Allergic angioedema (and HAE) is often suspected by GPs in patients with CSU who present with recurrent angioedema with no wheals.6.76.7.1The presentation of the patient may occur during the event or after the event has resolved Figure . In eith7Time of onset of diseaseShape, size, severity, frequency/duration and distribution of whealsLocation, frequency and duration of angioedemaAssociated symptoms, for example, bone/joint pain, fever, abdominal crampsFamily and personal history regarding wheals and angioedemaInduction by physical triggers or sweat‐including activities/situations (e.g. exercise)Occurrence in relation to daytime, weekends, menstrual cycle, holidays, and foreign travelOccurrence in relation to foods or drugs Occurrence in relation to infections, stressPrevious or current allergies, infections, internal/autoimmune diseases, gastric/intestinal problems or other disordersSocial and occupational history, leisure activitiesPrevious diagnostic procedures/resultsPrevious therapy and response to therapy including dosage and durationFull skin examinationFurthermore, given the anxiety and/or depression which frequently accompanies CU it is important that such co‐morbidities are actively sought in the consultation.8confirming the diagnosis, consider differential diagnosis and medication history and include hypersensitivity reactions to insect bites which may resemble urticaria Ruling out anaphylaxis cf Table  in case43 If anRule out drug allergy .Rule out presentations of other dermatological disorders.The diagnostic approach should include Figure :confirmiIf the clinical diagnosis is confirmed, performing further investigations is the exception not the rule. Patients should not be tested for underlying causes, unless clues from the history point to an allergic reaction.In the absence of systemic symptoms, reassurance is of great importance to the patient or the parents of the patient as the rash and especially angioedema may appear very threatening causing fearfulness of further progression to anaphylaxis. Patients should be told: that generally the causes of acute urticaria are unknown although stress and infection can be contributing factors and, importantly, require no investigations. Specifically, cold induced urticaria is potentially serious and caution about swimming in cold water and eating ice cream should be given before expert review occurs.Referral at this stage should be considered for suspected drug reaction  or anaphylaxis if this is considered a possibility. Elucidating the role of NSAID hypersensitivity is very complex and may require specialist assessment.Other factors which should prompt referral are suspicion of urticarial vasculitis , autoimmune disease, or if the patient has atypical symptoms such as bone pain or is systemically unwell.Referral should also be considered in patients with angioedema in isolation for investigation of bradykinin‐mediated angioedema.9The whole of the skin should be examined to detect whether any lesions are present and whether their appearance is that of urticaria/angioedema. A photographic record retained in the patient's electronic medical record may be useful. Also look for colour of the lesions, skin pain and systemic signs or symptoms. Given the association with thyroid disease it is worthwhile checking for the presence of a goitre.1011Urticaria and angioedema photos used with permission from:https://www.urtikaria.net/de/fuer‐betroffene/galerie/album‐urtikaria.html, who hold patient consent to use their photographs for publication and education. Further photographs are available by clicking in the link above (text in German).1212.1The aim of treatment is complete symptom control achieved through reassurance, education and judicious use of medication.Arguably the single most important factor in management is the giving of explanation of what is happening/has happened and reassurance concerning the long‐term outlook Cf Table , which iReassurance acknowledges that acute urticaria is frightening and can cause transient problems with sleep disturbance and body image but is not dangerous; the cause of acute urticaria is rarely precisely identified: that the majority resolve within 3–5 days: that if it persists there is still no indication to refer until 6 weeks have elapsed, unless symptoms are deteriorating or becoming more severe. That at 6 weeks, limited blood tests will be performed  coupled with increasing the dose of second generation antihistamines up to fourfold standard dose accompanied by monitoring disease activity and control with easy to use tools such as the urticaria activity score (UAS)12.2If the diagnosis reached is acute spontaneous urticaria there is no indication for investigations of any kind. This needs to be communicated transparently to the patient who often seeks investigations to determine what has caused their distressing symptoms and documented in the notes.If the urticaria progresses and becomes chronic spontaneous urticaria it is worthwhile, at this time, performing a full blood count and differential and either CRP, ESR, so that these can be included in the referral letter.12.3Urticaria is a phenomenon largely driven by the release of histamine, thus the logical approach is to treat the disorder with standard dose of long‐acting non‐sedating antihistamine.The patient should be told that apart from antihistamines there is no other effective treatment available in primary care: oral steroids are rarely indicated, due to their side effects: even short courses of oral steroids in otherwise healthy individuals are associated with a bewildering array of adverse events.If patients (or parents) are very distressed, a very short burst of oral steroids could be given for 3–5 days at no higher than 1 mg per Kg bodyweight daily, but it is important that the recipient is fully appraised of the potential risk benefit ratio.Treatment should commence with a standard‐dose second generation antihistamine.The use of first‐generation antihistamines is discouraged in all age groups due to their poor side effect profile.Note: There is no role in primary care for mixing different antihistamines nor for adding H2 blockers  unless recommended in local guidelines which may have been formulated adapted to local availability of medications. Similarly, there is no role for leukotriene receptor antagonists (e.g. montelukast), although there are differences in opinion between Europe and North America.12.4CU occurs when symptoms have continued to occur for 6 weeks Figure .If symptoms are controlled on any dose of a long‐acting non‐sedating antihistamine up to a maximum of four times the standard dose or if the symptoms have spontaneously remitted, no referral is necessary. Note: it may take 1–2 weeks before symptoms come under control. It is important to impress on the patient of taking the medication daily in order to retain remission or symptoms will relapse. However, when remission is achieved, therapy should be withdrawn every 3–6 months to discover whether natural remission has occurred.The corollary of this is that all patients who remain symptomatic and who have been adherent to medication, should be referred for specialist evaluation. CU is a complex disease area, suggesting that the referral should be to a person with extensive knowledge of this disorder in order to achieve the best outcome.13If urticaria is thought to be associated with a drug reaction, food allergy or anaphylaxis, urgent referral should be made to an allergist or dermatologist without undue delay.By definition, acute urticaria is a self‐limiting disorder with a duration of less than 6 weeks which will not benefit from further investigation or referral.Referral should be undertaken if the patient has had symptoms for six or more weeks in spite of an adequate trial of high dose second generation antihistamine therapy (four times standard dose) or if there is any diagnostic uncertainty for example, urticarial vasculitis. The rationale for referral should be explained to and discussed with the patient Table .The specialist may perform further investigations with three aims in mind (1) excluding differential diagnoses, (2) assessment of disease activity, impact and disease control and (3) to identify triggers or if suspected, underlying causes.The primary care physician or ED clinician does not need to understand the complexity of investigations which may be performed by those who are expert in this area. However, they should have a broad appreciation of what will happen next and be able to explain it. (see below). They should also perform a Full Blood Count and ESR or CRP to be included in the referral.13.1The details of the events to date with a clear description of the rash and timings and therapy tried will be detailed in the referral letter along with any photos if possible. This will be accompanied by the results of the simple blood tests performed mentioned above. The patient will attend with photographs taken during events, as well as documentation of disease activity and control by use of the Urticaria Activity Score 7 (UAS7),It is likely that a more detailed history will be taken in the search for any precipitating triggers, facilitating conditions and comorbidities. Based on the clues from the detailed history, additional investigations may be performed, especially in patients with longstanding and severe disease. There are a variety of provocation tests and blood tests which may be performed by the specialist in order to determine if there is a treatable condition that contributes to a patient's urticaria. In many patients, no such precipitating factors are identified. However, even in specialist care there is a clear need to be judicious in the use of investigations.If CIndU, in which urticaria develops in response to clearly defined stimuli such as cold, vibration, radiation, heat, water etc is suspected, provocation testing should precede treatment. Trigger threshold testing is useful, but availability of threshold testing is often limited to specialist centres.Specialists will likely confirm that antihistamines at up to four times the standard are ineffective before progressing to other treatment options. Adherence to antihistamines and the effects on signs and symptoms will be documented, often with the help of the urticaria activity score and/or the angioedema activity score.These tools will also be used to monitor and optimize the therapy with third line treatment options such as omalizumab and/or cyclosporin.14In summary, urticaria is relatively simple to manage if recognised and treatment started in partnership with the patient. Reassurance, shared decision making and simple standard treatments are the bedrock of management. For CU one of the challenges is to get the patient to the specialist in an appropriate time frame in order to confirm the diagnosis and relieve suffering if the patient is unresponsive to treatments easily provided in primary care; a major need is the construction of a simple value based care pathway ending at a facility/clinic which can deal effectively with this disease.There is a clear need to resist the temptation for any unnecessary investigations which, although well intentioned, reveal nothing and may be counterproductive by increasing uncertainty.There is a need to determine at what frequency intervals biologic treatment for CSU should be withdrawn in order to determine whether spontaneous resolution has occurred which will facilitate treatment withdrawal.https://doi.org/10.1111/all.15273.Please note: Figures Dermot Ryan: Conceptualization (Lead); Data curation ; Formal analysis ; Methodology ; Project administration (Lead); Writing – original draft (Lead); Writing – review & editing (Lead). Luciana K. Tanno: Data curation ; Formal analysis ; Writing – original draft ; Writing – review & editing . Elizabeth Angier: Writing – original draft (Supporting); Writing – review & editing (Supporting). Evangeline Clark: Resources (Supporting); Writing – review & editing (Supporting). David Price: Writing – original draft ; Writing – review & editing . Torsten Zuberbier: Conceptualization ; Formal analysis ; Methodology ; Writing – review & editing . Marcus Maurer: Conceptualization ; Formal analysis ; Methodology ; Project administration ; Writing – original draft ; Writing – review & editing .David Price has advisory board membership with Amgen, AstraZeneca, Boehringer Ingelheim, Chiesi, Circassia, Viatris, Mundipharma, Novartis, Regeneron Pharmaceuticals, Sanofi Genzyme, Teva Pharmaceuticals and Thermofisher; consultancy agreements with Amgen, AstraZeneca, Boehringer Ingelheim, Chiesi, GlaxoSmithKline, Viatris, Mundipharma, Novartis, Pfizer, Teva Pharmaceuticals and Theravance; grants and unrestricted funding for investigator‐initiated studies  from AstraZeneca, Boehringer Ingelheim, Chiesi, Circassia, Viatris, Mundipharma, Novartis, Pfizer, Regeneron Pharmaceuticals, Sanofi Genzyme, Teva Pharmaceuticals, Theravance and UK National Health Service; payment for lectures/speaking engagements from AstraZeneca, Boehringer Ingelheim, Chiesi, Cipla, GlaxoSmithKline, Viatris, Mundipharma, Novartis, Pfizer, Regeneron Pharmaceuticals, Sanofi Genzyme and Teva Pharmaceuticals; payment for travel/accommodation/meeting expenses from AstraZeneca, Boehringer Ingelheim, Circassia, Mundipharma, Novartis, Teva Pharmaceuticals and Thermofisher; funding for patient enrolment or completion of research from Novartis; stock/stock options from AKL Research and Development Ltd which produces phytopharmaceuticals; owns 74% of the social enterprise Optimum Patient Care Ltd  and 74% of Observational and Pragmatic Research Institute Pte Ltd (Singapore); 5% shareholding in Timestamp which develops adherence monitoring technology; is peer reviewer for grant committees of the UK Efficacy and Mechanism Evaluation programme, and Health Technology Assessment; and was an expert witness for GlaxoSmithKline. Marcus Maurer declares no COI relevant to this MS. Outside of it, he is or recently was a speaker and/or advisor for and/or has received research funding from Allakos, Alnylam, Amgen, Aquestive, Aralez, Astria, ArgenX, AstraZeneca, BioCryst, Blueprint, Celldex, Centogene, CSL Behring, Dyax, FAES, Genentech, GIInnovation, GSK, Innate Pharma, Kalvista, Kyowa Kirin, Leo Pharma, Lilly, Menarini, Moxie, Novartis, Pfizer, Pharming, Pharvaris, Roche, Sanofi/Regeneron, Shire/Takeda, ThirdHarmonicBio, UCB, and Uriach. Dermot Ryan, Torsten Zuberbier, Elisabeth Angier, Luciana Tanno and Evangéline Clark declare no COI relevant to this MS.