There was no discernible impact on receptor agonist-induced contractions of the U&LP after blocking intracellular calcium pathways, suggesting that this tissue is more sensitive to alterations in the availability of extracellular calcium. However, an alternative mechanism of action for GPCR-mediated contraction was identified to be the activation of Rho kinase, such as when Y-27632 significantly reduced the GPCR-mediated contractile activity of the U&LP by approximately 50% . This suggests that contractile responses of the bladder U&LP do not involve a significant release of calcium from intracellular stores, but that Gq/11-coupled receptor activation causes calcium sensitization via Rho kinase. This study highlights a key role for Rho kinase in the urinary bladder, which may provide a novel target in the future pharmaceutical management of bladder contractile disorders.The influence of extracellular and intracellular calcium on smooth muscle contractile activity varies between organs. In response to G protein-coupled receptor (GPCR) stimulation, the urinary bladder detrusor muscle has shown a 70% dependence on extracellular calcium, whereas the urothelium and lamina propria (U&LP) has a 20%–50% dependence. However, as this only accounts for partial contractile activity, the contribution of intracellular calcium and calcium sensitization pathways remains unclear. This study assessed the role of intracellular signaling pathways on GPCR-mediated urinary bladder U&LP contraction. Porcine U&LP responses to activation of the G An elevated concentration of intracellular Ca2+ is essential for the normal contractile activity of the urinary bladder. Levels of Ca2+ in the cytosol are dependent on the influx of extracellular Ca2+ via plasma membrane Ca2+ channels, as well as Ca2+ release from intracellular stores. However, there remains uncertainty about whether this intracellular Ca2+ concentration is due to Ca2+ influx entering the membrane from extracellular sources or the release of Ca2+ from the sarcoplasmic reticulum (SR) -activated contraction of the smooth muscle is known to be dependent on extracellular sources of calcium (Ca2+) , extraceol (DAG) . These stivation . An incrlum (SR) , 10, 11.2+ concentrations (2+-dependent pathway in contraction and has been shown to play an important role in the spontaneous contractile activity of the intact urinary bladder (2+ sensitization events inhibit myosin light chain phosphatase (MLCP) and cause a leftward shift of the Ca2+-force response curve at consistent Ca2+ and MLCK levels. Agonists acting on Gq/11-coupled receptors induce Ca2+ sensitization via the activation of Rho kinase. This occurs when the small monomeric G protein, RhoA, activated from GTP binding, phosphorylates the regulatory subunit of MLCP, which inhibits phosphatase activity and leads to sustained contractions  is a membrane-penetrable inhibitor of IP3-induced Ca2+ release -1-ethyl-1H-imidazopyridin-6-yl]oxy]phenyl]-4-[2-(4-morpholinyl)ethoxy]benzamide)   are seleq/11-coupled receptor systems in the bladder U&LP. This includes identifying whether intracellular mechanisms of action are different between muscarinic, histamine, 5-hydroxytryptamine (5-HT), neurokinin, prostaglandin, and angiotensin receptors. The influence that both Ca2+ release from intracellular sources, as well as Ca2+-independent pathways through the Rho kinase system, plays in urinary bladder U&LP contractions will be assessed.There is increasing evidence to support a prominent role of the U&LP in overall bladder contractile activity , 24. In 3 24.9 mM, d-glucose 11.7 mM, KCl 4.6 mM, MgSO4 2.41 mM, CaCl2 1.9 mM, and KH2PO4 1.18 mM), maintained at 4°C, to the University research facilities and used within 5 h of the organ’s retrieval.Urinary bladders were obtained from Large White-Landrace-Duroc pigs  from the local abattoir. As no animals were bred, harmed, culled, interfered, or interacted with as part of this research project, animal ethics approval was not required for this use of offal . After cAdjacent paired strips (2 cm × 0.5 cm) of isolated U&LP were separated from the underlying detrusor smooth muscle of whole urinary bladders . Strips N-pyridin-6-yl]oxy]phenyl]-4-[2-(4-morpholinyl)ethoxy]benzamide) were obtained from Tocris Bioscience . 5-Hydroxytryptamine (5-HT) was obtained from Toronto Research Chemicals , and Y-27632 hydrochloride (Y-27632) from AdooQ BioScience . Prostaglandin-E2 (PGE2), angiotensin-II (ATII), and ruthenium red were obtained from Cayman Chemicals . All ingredients for the Krebs solution were from Sigma Aldrich . PGE2 was dissolved in 100% ethanol, and CPA and fasudil were dissolved in 100% dimethyl sulfoxide (DMSO). All other pharmaceutical agents were soluble in distilled water. Concentrations selected for the agonists and antagonists were chosen based on their selectivity at each receptor and consistent with concentrations used in previous studies utilizing porcine tissue. In all cases, the receptor agonist concentration used induced submaximal contraction. For this assessment, preliminary studies using concentration-response curves were performed to identify maximal contractions. The agonist concentrations were then chosen as the dose that induced ∼80% of this contraction, allowing for a strong, submaximal contraction of the tissue.Carbamylcholine chloride (carbachol), histamine dihydrochloride (histamine), 2-aminoethyl diphenylborinate (2-APB), and fasudil hydrochloride (fasudil) were obtained from Sigma Aldrich . Neurokinin-A (NKA), cyclopiazonic acid (CPA), and GSK269962 , histamine (100 µM), 5-hydroxytryptamine (100 µM), neurokinin-A (300 nM), prostaglandin-E2 (10 µM), or angiotensin-II (100 nM) was applied to the U&LP tissue strips following a 30-min equilibration period. Baseline tension , frequency , and amplitude (mN) of spontaneous phasic contractions was measured before and after the application of the GPCR agonist with an isometric force transducer  and recorded on a PowerLab system using LabChart v7 software (ADInstruments). Throughout this manuscript baseline change in tension (ΔmN) is related specifically to baseline force.2+-ATPase pump inhibitor, CPA (10 µM), a wash-out method was employed to exhaust the intracellular Ca2+ stores from the SR to avoid reuptake by Ca2+-ATPase. Initially, tissues were washed with warm Krebs containing a DMSO vehicle control  for the control tissues, or CPA (10 µM) for experimental tissues and equilibrated for 30 min. After equilibration, a single dose of carbachol (1 µM) was added and when contraction reached its peak, tissues were washed with either control or CPA Krebs three times to remove any excess agonist. This process was repeated a total of three times for all tissues, and after the third wash, a single dose of a select GPCR agonist was added. During the wash-out procedure, spontaneous phasic activity was disrupted, and as such, only baseline tension (mN) was recorded and analyzed for CPA studies.2-Aminoethyl diphenylborinate (300 µM), ruthenium red (10 µM), fasudil (30 µM), Y-27632 (1 µM), and GSK269962 (1 µM) were applied separately to tissues for a 30-min incubation period before a single dose of GPCR agonist was added. In separate experiments using the intracellular Cat tests when comparing tissues with their paired, direct control tissues. On one occasion (see Spontaneous Phasic Activity of the U&LP in Response to GPCR Agonists), an unpaired Student’s t -test was applied when overall contractile responses between all receptor agonists were assessed. For all statistical analyses, P < 0.05 was considered statistically significant. Throughout this manuscript, n (number of tissues) values quoted are from paired tissue strips, and as such, the number of animals (N) used can be calculated using P values presented used for descriptive analysis, rather than the formal testing of a prespecified statistical null hypothesis. Effect sizes (d (d = 0.2), medium (d = 0.5), and large (d = 0.8).Data were graphed and analyzed using Prism 9.4.1 for Windows . All values were reported as mean change ± standard deviation (SD). Statistical analysis was conducted using paired Student’s two-tailed y nature , 32 withct sizes  were calsizes , frequency of 4.03 ± 0.91 cycles/min (n = 241), and amplitude of 6.29 ± 0.24 mN (n = 241). In all cases, responses to GPCR agonists increased baseline tension (P < 0.001) and the frequency of spontaneous phasic contractions (P < 0.05). A decrease in the amplitude of contractions was observed with all agonists except histamine and NKA  was significantly inhibited by 62% . There was no difference to the change in baseline tension for responses to carbachol, 5-HT, NKA, PGE2, or ATII in the presence of 2-APB  and a decrease in the amplitude of contraction peaks  compared with controls. In addition, ATII (100 nM) in the presence of 2-APB, was observed to decrease the frequency of spontaneous phasic contractions  with no change to amplitude.The responses to six GPCR agonists in the presence of an inhibitor of IP3-induced Caof 2-APB . PGE2  and presence of CPA, indicating an effective depletion of Ca2+ stores. Following the wash-out method, in the presence of CPA , there was no difference between responses to 1 µM carbachol, 100 µM histamine, 100 µM 5-HT, 10 µM PGE2, or 100 nM ATII compared with control contractions . However, tension responses to 300 nM NKA were enhanced in the presence of CPA .After equilibration with CPA (10 µM), a wash-out method was undertaken to deplete SR intracellular Ca2+ signaling, ruthenium red (10 µM), the baseline tension responses to histamine (100 µM) were inhibited by 61% . However, there were no significant differences between the absence and presence of ruthenium red for frequency and amplitude of spontaneous phasic contractions in response to histamine. In all other cases, there were no differences between observed responses in the absence and presence of ruthenium red  to carbachol (1 µM), 5-HT (100 µM), NKA (300 nM), PGE2 (10 µM), or ATII (100 nM).In the presence of the nonspecific inhibitor of intracellular Can = 50, P < 0.001). No change was observed from the addition of a DMSO vehicle control during equilibration.In the presence of the Rho kinase inhibitor, fasudil (30 µM), the baseline tension for all tissues decreased during the 30-min equilibration period by ∼8.63 mN (n = 8) appeared significantly greater than its effect on 5-HT , where fasudil inhibited contraction by 67% for histamine  and 17% for 5-HT .After activation of the six GPCRs with agonists, the baseline tension of the U&LP increased . In the n = 8, P < 0.01) compared with the control tissues. Furthermore, in response to carbachol  and 5-HT , the amplitude of spontaneous phasic contractions was altered in the presence of fasudil compared with in the absence of the Rho kinase inhibitor.The frequency and amplitude of spontaneous phasic contractions produced by the U&LP tissues in response to each of the receptor agonists were investigated in the absence and presence of 30 µM fasudil . No chanTo further explore the involvement of Rho kinase in mediating GPCR-induced contractions, the Rho kinase inhibitor Y-27632 was applied to the tissue before activation by any agonist. Y-27632 (1 µM) significantly inhibited the tonic contractions of the U&LP observed in response to tissue stimulation by all GPCR agonists. This inhibition to the baseline tension of contractions was around 50% .n = 8 for all) were not altered in the presence of Y-27632 (1 µM). In addition, there was no change in the amplitude of spontaneous phasic contractions with Rho kinase inhibition after the addition of 1 µM carbachol, 100 µM histamine, 100 µM 5-HT, 300 nM NKA, and 100 nM ATII . However, the amplitude of spontaneous phasic activity was significantly different after stimulation with PGE2 .The frequency of responses to the GPCR agonists (The Rho kinase inhibitor GSK269962 (1 µM) inhibited baseline tension responses to all GPCR agonists except for 5-HT . The eff2+ influx across the plasma membrane and intracellular Ca2+ release from the sarcoplasmic reticulum contribute to the increase in cytosolic Ca2+ concentration  except those to NKA or PGE2. This is consistent with past findings identifying Y-27632 as the inhibitor with the higher affinity to Rho kinase (2+ channels (Across the three Rho kinase inhibitors, Y-27632 was the most effective and consistent inhibitor of the Cao kinase  and seleo kinase  as well channels . More re2+ and Rho kinase in the mediation of G protein-coupled receptor contractions presents the potential for new and novel targets to be investigated for the pharmacological management of bladder contractile dysfunctions, such as underactive bladder. In elderly people with lower urinary tract symptoms, over 45% exhibit an underactive bladder, presenting this as an increasingly important and clinically relevant syndrome (The role of Casyndrome . Howeversyndrome . In addisyndrome . Howeversyndrome , and wit2+ into the cytoplasm, there was a lack of inhibition on intracellular Ca2+ sources. The inhibitor 2-APB has been considered a reliable blocker of store-operated Ca2+ entry, however, may be an inconsistent inhibitor of IP3-induced Ca2+ release (It should be noted that although three independent drugs were used to block the SR release of Ca release . To acco release , althoug2+-mediated contraction of the U&LP is typically stimulated by both extracellular Ca2+ influx and release from intracellular stores. However, in the urinary bladder U&LP, responses to G protein-coupled receptor stimulation are more sensitive to extracellular Ca2+ for the mediation of contractions, supported by the lack of any discernible impact on receptor agonist-induced contractions of the U&LP to 2-APB, CPA, and ruthenium red. An alternative mechanism of action for GPCR-mediated contraction for the muscarinic, histamine, 5-HT, neurokinin, prostaglandin, and angiotensin receptors was identified to be the activation of Rho kinase. The unique activity of the receptors investigated in this study presents novel directions for future research into the mechanisms underlying bladder contraction, and how these systems may be altered in disease states. The results suggest that Ca2+ regulation is important for activating the contractile machinery of the urinary bladder and highlight an equally important and complementary role for Rho kinase pathways. This presents potential novel targets for the pharmaceutical management of bladder disorders, in particular, overactive and underactive bladder.CaData will be made available upon reasonable request.C. Phelps was supported by an Australian Government Research Training Program Scholarship.No conflicts of interest, financial or otherwise, are declared by the authors.C.P. and C.M. conceived and designed research; C.P. performed experiments; C.P. and C.M. analyzed data; C.P., R.C.-W., and C.M. interpreted results of experiments; C.P. prepared figures; C.P. and C.M. drafted manuscript; C.P., R.C.-W., and C.M. edited and revised manuscript; C.P., R.C.-W., and C.M. approved final version of manuscript.


Chrysomya spp., only 1.5% (43) were recaptured during the 9-day sampling period. The majority of the blowflies (79%) were recaptured 1 km from the release point, while 20.9% were caught about 2-3 km from the release point. One individual blowfly travelled as far as 3 km and before being recaptured, which was the maximum dispersal distance recorded in this study. This result suggests that the estimated locations of the mammals detected from blowfly-derived iDNA is likely to be within 1-2 km radius from the origin of the blowfly sampling location. However, a more accurate estimated distance between the target mammal and the blowfly sampling location requires further investigation due to various factors, such as blowfly species, wind speed and direction that may potentially affect the blowfly dispersal activities. This study contributes further understanding on the development of a blowfly-derived DNA method as a mammalian monitoring tool in the tropical forests.Mammalian DNA extracted from the invertebrates, especially blowfly-derived DNA, has been suggested as a useful tool to complement traditional field methods for terrestrial mammal monitoring. However, the accuracy of the estimated location of the target mammal detected from blowfly-derived DNA is largely dependent on the knowledge of blowflies' dispersal range. Presently, published data on adult blowfly dispersal capabilities remain scarce and mostly limited to temperate and subtropical regions, with no published report on the adult blowfly dispersal range in the Tropics. We seek to determine the blowfly flight range and dispersal activity in a tropical plantation in Malaysia by mark-release-recapture of approximately 3000 wild blowflies by use of rotten fish-baited traps for nine consecutive days. Out of the 3000 marked  Invertebrate-derived DNA (iDNA) has recently been suggested as an alternative to traditional field methods for surveying and monitoring mammalian biodiversity . InverteThe mobility of the iDNA-carrying fly species could impact the spatio-temporal resolution of the iDNA data . ConsideCalliphoridae, Sarcophagidae, Muscidae, Drosophilidae and Tephritidae) by marking techniques, study locations and dispersal ranges are summarised in Table Knowledge of the invertebrate dispersal range is essential for estimating the location of the mammal species relative to the location where the invertebrates were collected . InadequCalliphoridae) might have on the development of a mammal monitoring tool via blowfly-derived DNA, we seek to determine the dispersal activities and flight range of blowflies in the tropical forests by conducting a mark-release-recapture study of Chrysomya spp. in a selected plantation in Malaysia.Considering the implications of the dispersal capabilities of blowflies  by using specimen vials. The blowflies were then provided with sugary solution and maintained at room temperature (27°C-33°C) and relative humidity 70-80%. One day prior to release to the field, the blowflies were marked by orange-coloured fluorescent dust  by mass dusting (Adult blowflies were collected using traps baited with ca. 200 g rotten fish (hereafter referred to as blowfly traps)  within tya genus . The ide dusting . This me dusting . The flu dusting . Most im dusting .Release and recapture of blowfliesOn 27 December 2015, the marked blowflies, approximately 3,000 individuals, were released at 10:00 h, i.e. within the active flight activity of blowflies , in the Identification of trapped fliesCaptured blowflies were examined for the presence of fluorescent powder on their bodies under ultraviolet (UV) light in a dark room. The number of marked blowflies recaptured at different days-after-release (DAR) and distance from the release point were recorded accordingly , followed by 2-DAR , 3-DAR , 4-DAR , 5-DAR  and 6-DAR . The only one blowfly recaptured at 3 km radius from the release point was recaptured at 5-DAR. No blowflies were recaptured after 6-DAR although the trapping lasted for nine days following the initial release  was 1 : 2.4 : 1.8 : 1.6. This showed that more blowflies were heading to the east, followed by south and west directions and the least recaptured were in the north direction of the field site. At 2 km radius, out of the eight marked blowflies, there was no fly recaptured in the north, but only one (12.5%) recaptured in the east. Most of the marked blowflies headed to the south  and southwest . The single blowfly recaptured at 3 km radius from the same release point was also recaptured in the southwest.Chrysomya blowflies was between 1 to 3 km within 6 days after release. Most of the blowflies (79%) were recaptured at 1 km from the release point throughout the sampling period, whereas approximately 21% were recaptured 2-3 km away from the release point. No blowflies were recaptured at a distance of more than 3 km from the release point. This suggests that Chrysomya spp. did not disperse widely, in the range of six days. The daily dispersal distance of < 3 km recorded for Chrysomya spp. is similar to the estimated daily dispersal of 2.20 km and 2.35 km reported for Chrysomyaalbiceps and Chrysomyamarginalis, respectively in the subtropical region of South Africa  throughout the sampling period. This result is similar with the widely-reported low recapture rates in most of the blowfly dispersal studies .File: oo_864901.docxhttps://binary.pensoft.net/file/864901Ping Shin Lee0774FA9F-D634-544C-A517-F1C96F5D765810.3897/BDJ.11.e108438.suppl2Supplementary material 2Supplementary Table 2Data typetableBrief descriptionRecords of daily mean wind speed, maximum wind speed and wind direction during the sampling period.File: oo_866731.xlsxhttps://binary.pensoft.net/file/866731Ping Shin Lee


Dear Editor,The clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR–Cas) system has exhibited powerful abilities to manipulate genomes of animals and plants . Up to nIt has been reported that BhCas12b v4, BvCas12b, and AaCas12b recognize 5′-DTTN-3′ , 5′-ATTN-3′, and 5′-TTN-3′ as PAM sequences, respectively . To compGiven that BhCas12b v4 is the most efficacious Cas12b, we tried to further improve its editing efficiency by introducing a C/G pair into sgRNA  or addinin vivo, we tested it at two target sites with 5′-ATTN-3′ (PAM) in the myostatin (MSTN) gene in rabbit embryos  . These r–/–) and wild-type (WT) rabbits, their body weights were recorded weekly. As shown in –/– rabbits showed significant increase in body weight in comparison with WT rabbits. To further investigate whether the mutations induced by BhCas12b v4 disrupt the expression of MSTN gene in the rabbits, we performed real-time quantitative PCR (RT-qPCR) to analyse MSTN expression in the gluteus maximus of MSTN–/– and WT rabbits using three sets of primers. As shown in –/– rabbits compared with that of WT controls. A typical double-muscled phenotype was also observed in MSTN–/– rabbits at the age of 12 weeks , it is a promising tool for adeno-associated virus-mediated vector delivery Journal of Molecular Cell Biology online. The authors thank Peiran Hu and Nannan Li at the Embryo Engineering Center for critical technical assistance. This study was financially supported by the National Key Research and Development Program of China Stem Cell and Translational Research (2022YFA1105404), the Program for Changjiang Scholars and Innovative Research Team in University (IRT_16R32), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030501 and XDA16030503), and Key Research & Development Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory (2018GZR110104004). Y.J., L.L., and Z.Li conceived and designed the experiments. Y.J., T.W., D.Z., and J.L. performed the experiments. Z.Liu and Y.J. analyzed the data. Y.J., S.C., and T.S. wrote the paper. All authors have read and approved the final manuscript.][mjac076_Supplemental_FileClick here for additional data file.


Despite the critical roles of IP3R-mediated Ca2+ responses, the structural underpinnings of the biphasic Ca2+ dependence that underlies Ca2+ oscillations are incompletely understood. Here, we collect cryo-EM images of an IP3R with Ca2+ concentrations spanning five orders of magnitude. Unbiased image analysis reveals that Ca2+ binding does not explicitly induce conformational changes but rather biases a complex conformational landscape consisting of resting, preactivated, activated, and inhibited states. Using particle counts as a proxy for relative conformational free energy, we demonstrate that Ca2+ binding at a high-affinity site allows IP3Rs to activate by escaping a low-energy resting state through an ensemble of preactivated states. At high Ca2+ concentrations, IP3Rs preferentially enter an inhibited state stabilized by a second, low-affinity Ca2+ binding site. Together, these studies provide a mechanistic basis for the biphasic Ca2+-dependence of IP3R channel activity.Inositol 1,4,5-trisphosphate receptors (IP 3Rs) are intracellular Ca2+ channels that produce Ca2+ oscillations in response to GPCR and RTK activation. Here, Paknejad et al. resolve the conformational landscape of IP3Rs that gives rise to the biphasic dependence on Ca2+ for channel activity.Inositol 1,4,5-trisphosphate receptors (IP Expressed in the endoplasmic reticulum (ER), IP3Rs mediate the flow of Ca2+ from the ER into the cytoplasm and other cellular compartments where Ca2+ contributes to the regulation of cell division1, differentiation2, metabolism3, migration5, and cell death6. Consequently, dysregulation of IP3Rs is associated with numerous pathologies including cancer9, neurological11, cardiac12, and immune13 diseases. IP3R activation requires nanomolar cytosolic Ca2+ and the second messenger IP3, whose production is stimulated by receptor tyrosine kinase and G protein-coupled receptor signaling pathways20. Notably, IP3Rs are inhibited by micromolar cytosolic Ca2+ concentrations, resulting in a biphasic dependence on Ca2+ for channel activity. The recursive nature of IP3R regulation by its permeant ion results in the emergent phenomenon of Ca2+ oscillations in cells. The Ca2+ dependence of both activation and inhibition are further modified by the concentration of IP3 as well as ATP, ER Ca2+ and numerous protein interaction partners23. In this manner, IP3Rs integrate multiple upstream signals to tune the frequency and amplitude of Ca2+ oscillations that encode regulatory information for diverse cellular processes such as mitochondrial oxidative metabolism24, gene expression25, lymphocyte activation26 and neuronal development27.Inositol-1,4,5-trisphosphate receptors  ion channels such as voltage-gated ion channels and TRP channels, as well as a large cytosolic domain (CD) that contains all of the known regulatory ligand-binding sites, including two Ca2+ binding sites, and shares some homology with the Ryanodine Receptor (RyR)35. When both Ca2+ binding sites are occupied, the pore remains closed regardless of IP3 binding status29. In contrast, a recent structure suggests that the pore opens when only one of the Ca2+ binding sites is occupied in the presence of IP333. However, many additional conformations have been resolved whose functional corollaries remain unclear. More broadly, the conformational landscape that enables IP3Rs to pivot from activation to inhibition to generate Ca2+ oscillations remains unknown. Here, we collect electron cryomicroscopic images of human type 3 IP3R (hIP3R3) vitrified in a broad range of Ca2+ concentrations and treat particle abundance as a proxy for the relative free energy of each state to establish high-resolution thermodynamic models of IP3R activation and inhibition, which combined with cellular Ca2+ imaging elucidates the structural basis for IP3R-generated Ca2+ oscillations.Structural snapshots of IP3 and Ca2+ together activate the channel, and high Ca2+ concentrations inhibit the channel, we collected transmission electron cryomicroscopic (cryo-EM) images of purified human type 3 IP3 receptors (hIP3R3) prepared with saturating (200 µM) IP3, saturating (1 mM) ATP, and five concentrations of nominal free Ca2+ spanning a range from 1 nM to 10 µM  ring, armadillo repeat domain 2 (ARM2), the juxtamembrane domain (JD) ring and the pore  and lipid environments (PDB: 7LHE)32, we assigned this conformation as a resting state. Two similar minor states were also present that share the overall conformation of the resting state but differ slightly in the conformation of the TMD with much weaker density for the peripheral S1-S4 domain 33. As the second and third states differ from the activated state only in their closed pores and intact JD rings, we assigned them as a preactivated state and a preactivated+Ca2+ state, respectively. The preactivated state, with its intact BTF ring and retracted ARM2 domain and closed pore, is similar to previously reported structures of IP3Rs in the presence of IP3 37, but the preactivated+Ca2+ state has not been previously described.The second and third major states also have intact BTF and JD rings and a closed pore, but their ARM2 domains adopt the retracted conformation, where ARM2 is rotated towards the central linker domain (CLD) 29. While we were able to resolve structures for these states, we observed significant continuous heterogeneity among these asymmetric classes. Therefore, we combined these particles into an ensemble that we call the resting-to-preactivated transitions for quantification. We also observed classes with asymmetric features in the JD and TMD that otherwise resembled the resting or preactivated states. The pore in these classes has undergone movements that result in either two-fold pseudosymmetric (~C2) or four-fold pseudosymmetric (~C4) dilations compared to the closed states 33, where BTF ring disruption confines IP3-mediated conformational changes to the CD, so we assigned the major state as an isolated inhibited state and the minor state as a higher-order assembly of inhibited states.In the fifth major state, the BTF ring is disrupted, ARM2 is retracted, the JD ring is intact, and the pore is closed Fig. , and 11.2+, activated and inhibited states consistent with an IP3 bound at the BTF2-ARM1 interface and with a Zn2+ and an ATP bound in the JD of all five C4 symmetric major states 42. As observed previously29, IP3 can bind the channel via two modes , IP3 binding results in a contraction of the IP3-binding pocket through movement of a loop (Leu265-Ser278) on BTF2 43. Specificity for adenine bases46 is imparted through the primary amine of the base forming interactions with the backbone carbonyl oxygen of His2558 and thiolate of Cys2538. The triphosphate moiety of ATP extends away from the JD with clear densities corresponding to the α and β phosphates, which are directly coordinated by Lys2152 and Lys2560, respectively  with Cal-520-AM, a fluorogenic calcium-sensitive dye, and then stimulated intracellular IP3 generation by adding carbachol to the bath solution  were added to cells to minimize potential stimulus-dependent effects on the IP3R response in cells53. Consistent with earlier reports52, no detectable changes in cytosolic Ca2+ were observed in IP3R-null cells , we extracted traces from segmented cells, then smoothed and adjusted the baseline to automatically identify peaks. For wild-type hIP3R3 the mean inter-spike interval was 21.7 seconds, which is within the range of times measured for endogenous IP3R-mediated cytosolic Ca2+55.The multimodal regulation of IPtes Fig. . To asseion Fig. 52. Saturnel Fig. . We assense Fig. . For IP3ell Fig. . Finally2+ oscillations of wild-type hIP3R3, we next examined the effects of perturbing the Ca2+-binding sites. We transiently expressed hIP3R3 with mutations to the JD site (Glu1882Gln+Glu1946Gln), the CD site (Glu1125Gln) or both sites (Glu1125Gln+Glu1882Gln+Glu1946Gln) in IP3R-null cells. Robust Ca2+ oscillations were observed in cells expressing the CD mutant , one with ARM1a, and a second with BTF1a  in cells expressing the dARM2 mutant despite both being expressed in a similar fraction of cells  and assessed the effects of its loss on Caons Fig. . Comparells Fig. . Also di2+-bound preactivated+Ca2+ state with the open, Ca2+-bound activated state allows us to observe the conformational changes that enable the pore to open. Although both states share a common ligand-binding profile, large conformational changes can be observed extending from the JD Ca2+ binding site through the JD to the TMD , is connected to the peripheral S1-S4 domain. The pore domain and the domain-swapped S1-S4 domain are connected by a short amphipathic S4-S5 linker helix Fig. . In the 1-Met2191The second segment of the JD, which we call JD-B (Cys2538-Met2608), is directly linked to the cytosolic end of the pore-lining S6 helix. In the activated state, rotation of the JD pulls the S6 helices away from the center of the pore, stabilizing a 13˚ bend and ~30˚ rotation about the helical axis of S6 with Gly2514 being the pivot for both. Together, the tilt and rotation of S6 reposition Phe2513 and Ile2517, which seal the pore in the closed states, out of the ion conduction pathway to create an open pore with a minimum radius of 4 Å Fig.  and 10. 2+ binding may yield uncoupled movements of the two segments that would be a barrier to opening the pore, consistent with the prevailing model for ATP potentiation through sensitizing the channel to Ca2+ activation without affecting maximal open probability or high-Ca2+ inhibition21. Supporting the critical role of a rigid JD domain in channel activation, even a single cysteine-to-serine mutation at the JD Zn2+ binding site results in a complete loss of function without diminishing protein expression or IP3 affinity65.Our structural analyses indicate that both segments of the JD contribute to pore opening, with the rotation of JD-A relaxing the belt around the S6 helices and JD-B pulling the S6 helices apart. At the interface between these segments is an ATP Fig. . The ade3R-specific S1’ and S1” helices in channel gating, we monitored cytosolic Ca2+ responses following carbachol stimulation in cells transduced with hIP3R3 lacking S1’ and S1” (dS1’-S1”)  Fig. . Compare2+ and activated states reveals that the JD also adopts an intermediate conformation. Whereas the JDs are both separated and rotated in the activated state, the JDs in the ~C4 transition are only separated.In the ~C4 subclass, the S4-S5 linkers and the S6 helices of all four protomers are outwardly displaced, creating a partially dilated pore Fig. . However2+ binding in the preactivated+Ca2+ state. Once Ca2+ is bound, the channel can sample the ~C2 transition, where two opposing JDs shift outwards, disrupting the JD ring and loosening the S4-S5 linker belt leading to an outward movement of two of the four S6 helices. Then, the remaining two JDs are displaced away from the pore axis, resulting in a partial dilation of the pore. Finally, in the activated state, the JDs rotate about the helical axis of S6 to stabilize a fully-open pore where the hydrophobic gating residues Phe2513 and Ile2517 are repacked away from the permeation pathway. Notably, we do not observe any conformational changes in the pore helix or selectivity filter between the high-resolution closed and open states, indicating that the positions of Phe2513 and Ile2517 determine the gating state of the pore.Interpolating the ~C2 and ~C4 preactivated TMD transitions into a trajectory that begins with the resting state and ends with the activated state suggests a progression of JD rearrangements that facilitate gating in the pore Fig. . First, 3Rs is thus weaker than the associations described between the pore and the cytosolic gating domains of other 6TM cation channels such as the BK channel (Slo1)67.Subclasses with ~C2 and ~C4 distortions of the pore are also present in the ensemble of resting TMD transitions. In contrast to the preactivated TMD transitions, the JD ring remains intact in these subclasses, suggesting that the pore conformation is not strictly coupled to that of the JD ring  interface through its CLD, one promoter forms what we call the minus (−) interface through its ARM2 domain while the remaining two protomers do not participate in the interaction Fig. . The + i2+, and activated states permit each tetramer to form only a single interaction with an adjacent tetramer while the extended position of ARM2 precludes either interaction from occurring in the resting states. Although there is a substantial entropic cost to these linkers adopting stable conformations, their extensive interactions suggest that the enthalpic gains from their ordering result in an overall reduction of free energy. In the inhibited state the increased flexibility of the CD following BTF ring disruption may offset this entropic penalty.Higher-order assemblies were notably absent from the other states. By examining potential inter-tetramer interfaces, we found that the ability to form both the + and - interactions was unique to the inhibited state Fig. . The con2+-dependence of the assembled inhibited state differs from that of the isolated inhibited state with the assembled inhibited state reaching a plateau at 100 nM Ca2+ while the isolated inhibited state increases in abundance to 10 µM  mutant produced Ca2+ oscillations with a rising-phase slope that is comparable to wild-type channels, the mean inter-spike interval of 12.2 seconds is significantly shorter  or mVenus (cryo-EM)83 followed by a human rhinovirus 3 C protease84 cut-site and then human type 3 IP3R. Plasmids were transformed into DH10Bac cells to generate bacmids29. 100–200 µg of purified bacmid were incubated with 400 µg of 25,000 MW polyethyleneimine  in 1.4 mL water at 55 °C for 30 minutes to sterilize, then added to 50 mL of Sf9 cells at 1×106 cells/mL grown in suspension at 27–30 °C. The Sf9 TNM-FH (Grace’s modified) media was supplemented with 1% penicillin/streptomycin, 0.1% Pluronic F-68 nonionic surfactant (Gibco Cat# 24040), and 4-8% fetal bovine serum to stabilize the virus. Virus titer was amplified to P3 and separated from cell debris by centrifugation. P3 virus was used to infect mammalian HEK293S GnTI- (ATCC CRL-3022) cells at a density of 3×106 cells/mL at a ratio of 50 mL virus for 800 mL cells and simultaneously stimulated with 4.5 mM valproic acid . Pellets were harvested from cells by centrifugation at 48–72 hours after infection and snap frozen.All constructs were N-terminally tagged with 10xHis followed by EGFP , 150 mM sodium chloride (NaCl), 20 mM HEPES pH 7.5, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2.5 µg/mL aprotinin (Sigma Cat# A1153), 2.5 µg/mL leupeptin (Alfa Aesar Cat# J61188), 10 µg/mL pepstatin A (GoldBio Cat# P-020-25), 0.5 mM 4-benzenesulfonyl fluoride hydrochloride , and a few flakes of lyophilized deoxyribonuclease . The resulting cell lysate was centrifuged at 75,000x g for 40 minutes. The supernatant was incubated with sepharose-coupled GFP nanobody affinity purification beads for 4 hours with gentle agitation85. The protein-GFP-nanobody-bead mixture was isolated in a column and washed with 50 mL of gel filtration buffer containing 150 mM NaCl, 50 mM Tris-HCl pH 8.0, 0.02% LMNG, and 2 mM dithiothreitol (DTT). The protein was eluted from the affinity column by cleavage with genetically modified human rhinovirus 3 C protease overnight. Size exclusion chromatography was performed with a Superose 6 Increase column and the resulting protein peak was pooled and concentrated to 20 mg/mL in a 1 mL, 100 kDa MWCO concentrator (Cytiva VivaSpin Cat# 28932258).All surfaces, vessels, and transfer plastics were washed extensively with reverse osmosis water prior to use to minimize contaminating Ca2+ to protein preparations. We opted to produce our own low-Ca2+ blotting paper by treating standard blotting paper (Ted Pella Standard VitroBot Blotting Paper Cat# 47000-100) with an extensive washing protocol. Over several days and multiple buffer exchanges, we treated with approximately 6 L of 100 µM EGTA in reverse osmosis (RO) water, then 6 L of RO water with Ca2+ chelating beads (BIO-RAD Chelex 100 Resin Cat#142-1253), and finally 6 L of RO water alone. The treated paper was then stacked between extensively washed glass plates and subjected to vacuum for 24 hours to remove moisture and resume a flat shape. The treated filter paper is predicted to contain substantially less than 1 mM contaminating Ca2+ (predicted starting condition of blotting paper29) and 100 µM residual EGTA (first wash condition).Cryo-EM sample blotting paper contributes a significant quantity of contaminating Ca2+ concentrations, we engineered a 5X ligand and Ca2+ chelator cocktail. By combining 2 mM each of EDTA (Kd 30 nM), EGTA (Kd 127 nM), BAPTA (Kd 153 nM), HEDTA (Kd 4.8 µM) with 1 mM of ATP (Kd 183 µM), we calculate that our buffer ensures a semi-log-linear relationship between free and total Ca2+ from 1 nM to 300 µM86. The least well-controlled range for free Ca2+ was between 1 nM and 10 nM requiring addition of 864 µM total Ca2+, and the largest was between 10 µM and 100 µM, requiring addition of 2.0 mM total Ca2+. Thus, our total contaminating Ca2+ must be greater than 864 µM to generate a maximum 1-log-fold error in our target free Ca2+ across the entire titratable range, ensuring that we maintain the semi-log-linear relationship between free and total Ca2+ despite contaminating Ca2+. To minimize the impact of widely varying kinetic properties of the chelators, we premixed the 5X solution containing 10 mM of each chelator, 5 mM ATP, 1 mM IP3, and 2.5 mM fluorinated fos-choline-8 (Anatrace Cat# F300F), a detergent that does not interact with hydrocarbons, to protect the protein from the air-water interface. Sensitivity analysis using MaxChelator (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/webmaxc/webmaxcE.htm) revealed that inaccurate pH was the largest contributor to deviations from the predicted free Ca2+, and thus we carefully adjusted all solutions to pH 8, and added an additional 50 mM Tris pH 8.0 to the ligand and Ca2+ chelator cocktail. CaCl2 and MgCl2 were added in varying quantities to generate the desired free Ca2+ concentration and a constant 3 mM free Mg2+ concentration. During grid preparation, 3.2 µL of purified protein was added to the grid and incubated for 30 seconds, after which we added 0.8 µL of the ligand and Ca2+ chelator cocktail directly to the droplet on the grid, immediately blotted with our low-Ca2+ blotting paper for 2 seconds, then plunge-frozen using a ThermoFisher Vitrobot Mark IV. Since the Ca2+ and chelators are premixed, the free Ca2+ is at equilibrium in the master mix, and pipetting error when adding to the protein on the grid will have no effect on free Ca2+. The only deviations due to pipetting error would be [IP3] and [ATP], both of which are above saturating concentrations and so we assume those to be inconsequential for this analysis. Due to the non-equilibrium nature of grid preparation  we acknowledge that while the semi-log-linear relationship established across the titration range is very precise, there is likely inaccuracy in our nominal free Ca2+ concentrations. The final grid conditions have varying free Ca2+, but constant 200 µM IP3, 1 mM ATP, 3 mM free Mg2+, 1.6 mM dithiothreitol (DTT), 2 mM EDTA, 2 mM EGTA, 2 mM BAPTA, 2 mM HEDTA, 50 mM Tris pH 8.0, 120 mM NaCl, 500 µM fluorinated fos-choline-8, and 159 µM LMNG.To further control our sample Ca2+ calibration curves were generated using Fura-2  and Invitrogen Calcium Calibration Buffer Kit (#C3008MP). Briefly, we prepared 50 µL samples for multiple specific free Ca2+ concentrations by mixing buffer A  and buffer B  in the manner prescribed by the manufacturer. Fura-2 fluorescence emission at 510 nm was collected for an excitation scan from 280 to 450 nm in a clear bottom 96-well plate  using Molecular Devices SpectraMax M5e microplate reader at room temperature. A calibration curve for Ca2+ was generated by calculating the fluorescence emission ratios at 340 and 380 nm  with a nominal free Ca2+ concentration of 100-104 nM was added to 40 µL of 62.5 µm Fura-2  diluted in gel filtration buffer ) to achieve a final concentration of 50 µM Fura-2 and 1X Ca2+ chelator cocktail. Samples were excited at 340 nm and 380 nm and fluorescence emissions were collected at 510 nm using a Molecular Devices SpectraMax M5e microplate reader at room temperature. GraphPad Prism 9 was used to estimate free Ca2+ concentrations by interpolating the fluorescent emission ratios at 340 nm and 380 nm excitations  for a total dose of 66 e-/Å2 in automated fashion using SerialEM88. Five datasets were collected during the same session for each Ca2+ concentration on a series of grids that were prepared sequentially resulting in 637 movies at 1 nM, 2150 movies at 10 nM, 6126 movies at 100 nM, 1372 movies at 1 µM, and 3136 movies at 10 µM. A sixth dataset of 4312 movies collected at nominal 100 nM free Ca2+ from a grid prepared later in the sequence was collected as a technical replicate to assess experimental error , resting-like stack with weak TMD density , preactivated-like stack with weak CD density , activated-like stack , preactivated-like stack with weak TMD density , and inhibited-like stack . These stacks were subjected to one final round of classification revealing the five primary C4 symmetric states called resting , preactivated , preactivated+Ca2+ , activated , and inhibited  states and several heterogeneous conformational ensembles. Notably, local conformational changes can still be observed in the five major states by 3DVA at the end of the hierarchical classification due to the large size and overall flexibility of the channel. Our final maps and the corresponding models represent the average of the particles that comprise these states.Each of these six stacks were refined enforcing C4 symmetry to improve signal for reference-based corrections prior to Bayesian Polishing in Relion v3.1.32+ state, 2.9 Å (BTF1/BTF2/ARM1) to 3.3 Å (ARM2) for the activated state, and 2.5 Å (TMD/JD) to 3.4 Å (BTF1/BTF2/RM1) for the inhibited state  a mask containing a single chain from the tetramer (2) the entire cytosolic domain consisting of residues 1-1697 from a single chain 3) BTF1, BTF2, and ARM1 consisting of residues 1-664 from a single chain (4) CLD, ARM3 consisting of residues 665-1100 and 1586-2074 from a single chain (5) ARM2 consisting of residues 1101-1586 from a single chain (6) TMD, JD consisting of residues 2111-2611 from a single chain. The masks generated from these models were dilated by 4 pixels and a cosine soft-edge was applied for 40 pixels, thereby avoiding ringing and mask artifacts that occur when converting hard edges in real-space to reciprocal space. Therefore, this mask retains 100% of the information at ~3 Å away from the model, and 50% of the information at ~25 Å away from the model. CryoSparc v3.3.1 Local Refinement resulted in resolutions ranging from 2.5 Å (TMD/JD) to 3.3 Å (ARM2) for the resting state, 3.6 Å (TMD/JD) to 6.5 Å (ARM2) for the preactivated state, 3.3 Å (BTF1/BTF2/ARM1) to 4.2 Å (ARM2) for the preactivated+Ca BTF1, BT94 guided only by experimental density (no model) and employing a lenient mask that contains all proteinaceous and detergent micelle density, an approach we have used previously97. As part of the procedure, the final maps are sharpened using a half-map derived factor. The resulting density modified and sharpened maps were cropped to a single chain and used for iterative model building using coot98, ISOLDE99 and composite map generation using a 20-residue sliding window cross-correlation (Phenix v1.20.1-4487 Combine Focused Maps)100, which we found to produce artifact-free maps when compared to Chimera’s ‘vop maximum’ command101. For the highest resolution composites  the density-modified local refinements were super-sampled prior to composite generation to aid interpretation of ligands, ions, and waters. Inspection of the resulting composite maps showed that they were free of model-based overfitting, for example density for ions, ligands, and lipids remain intact despite being removed from the input model. The final models were refined against the composite map with Phenix v1.20.1-4487 Real-Space Refinement102.The local refinements were independently subjected to Phenix v1.20.1-4487 Resolve Cryo-EM36. We relaxed our assumptions about symmetry by performing C4 symmetry expansion on each class. For the resting-like ensemble with weak CD density, resting-like ensemble with weak TMD density, preactivated-like ensemble with weak CD density, and the preactivated-like ensemble with weak TMD density, we performed 3DVA with a full channel mask and filter resolution between 5 and 8 Å and clustered each of 3 modes independently into 5 groups. Occasionally, one or two clusters would be populated with very few particles, suggesting that a fewer number of clusters was adequate to represent the underlying heterogeneity. We then refined each class  and assessed the resulting structures, selecting the mode of variability that contained our features of interest. From the resting-like and preactivated-like stacks with weak TMD density, we obtained the ~C2 and ~C4 TMD transition states presented in Fig. The remaining classes represent highly-heterogeneous conformational ensembles that we interrogated via 3D variability analysis (3DVA)To calculate an octameric reconstruction of two adjacent tetramers in a higher-order assembly, the nonsymmetry expanded stack of 1045k inhibited state particles was subjected to CryoSparc v3.3.1 Heterogeneous Refinement seeded with 24 identical references of the inhibited state. Strong density for an adjacent tetramer was present in three of the resulting classes, totaling 246k particles. This classification was used to quantify the particle distributions for clustered versus isolated inhibited states in Fig. In addition to the masks covering the interfaces, we used the same set of local refinements that we used for the five C4 symmetric states to produce a well-resolved composite map of each protomer in the two associated tetramers. For each population, we first produced a consensus tetrameric alignment, and then proceeded to subject each protomer of the tetramer to the six local refinement masks that were used to produce the C4 symmetric inhibited state . Each of these local refinements was then subjected to Phenix Resolve Cryo-EM, and finally to Phenix Combine Focused Maps to produce a tetrameric composite map of each half of the interaction. For the depiction in Fig. 2+ concentrations via unique identifiers in the micrograph names in order to calculate the relative abundance of each state at each condition. Because we collected images from two grids prepared at 100 nM Ca2+, we combined the particles and report the weighted average in Fig. Once all the particles were assigned to a specific state or ensemble in CryoSparc, we determined the number of particles that originated from each of the Ca101. For the depictions of ARM2 density in Fig. 104. For the depictions of the wedge loop density in Supplementary Fig. 106. For the electrostatics calculations in Supplementary Figs. 107 was utilized via PyMol plug-in.For the depictions of the composite maps in Fig. 108. Multiple sequence alignments were performed using the MUSCLE109 algorithm in DNASTAR LaserGene MegAlign Pro 17.3.The MSK High Performance Computing (HPC) resource provides a GPU cluster built for computing large volume data over a range of applications from drug discovery to deep learning and image processing. It contains 120 nodes connected by a 100 Gigabit ethernet backbone. The nodes used for this project each contain Intel Xeon Platinum 2.2 GHz CPUs and 1 TB DDR4 RAM. Each node also contains four A100 GPUs interconnected using NVLink. The cluster runs the CentOS operating system and is supported by a 4 PB high-speed GPFS-based parallel filesystem. A 200 TB NVMe-based Weka ultra-fast tier was used as scratch space. The CPU to GPU communication is established over PCIE 4.0. The project used IBM Spectrum LSF as the orchestrator of shared resources and parallelization is further achieved by MPI over the ethernet network. All cryo-EM software excluding CryoSparc was maintained via HMS SBGrid3R3 constructs detailed in this study was performed using either two concurrent single-primer reactions (PMID: 19566935) or NEBuilder HiFi DNA Assembly kit (NEB Cat# E2621) in Mach1 T1 cells . Oligonucleotides (Supplementary Table https://nebuilder.neb.com/) was utilized for designing oligos for mutagenesis based on NEBuilder HiFi DNA Assembly. All oligos were acquired from Integrated DNA Technologies.Site-directed mutagenesis and cloning for all hIP3R-null cells were obtained through Kerafast52 and cultured to a confluency of ~75-80% on 100 ×20 mm tissue culture treated dishes in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin at 37 °C with 5% CO2. For imaging, cells were then split in a 1:4 ratio and plated on poly-D-lysine coated, 35 mm diameter, optical quality glass-bottom culture dishes  and incubated for ~18-24 hours. At ~60% confluency, cells were transduced with a 200 µl baculovirus followed by incubation at 37 C, 5% CO2 for another 24 hours. All constructs used for Ca2+ imaging in this study were overexpressed in HEK293T IP3R-null cells using the BacMam system110.HEK293T IP2+, Mg2+ free, Hank’s balanced salt solution ] followed by incubation for 1 hour at 37˚C and 5% CO2 in 1.8 ml of imaging buffer containing 3 mM Cal-520-AM  Cal-520-AM-loaded cells were removed from the CO2 incubator and equilibrated at room temperature for 5 minutes prior to IP3 stimulation by the addition of 200 µl of 1 mM carbachol , a Gαq-coupled M3 muscarinic receptor agonist. Carbachol was added at least 10 mm away from the imaging site and allowed to diffuse to a final concentration of 100 µM. Movies of carbachol-induced Ca2+ release in cells were collected at 20x with LD Plan-Neofluar 20X/0.4 Korr M27 objective, for 10 minutes, at 3×3 binning (912×736 pixels post binning), with an exposure time of 250 ms on a Zeiss Axio observer D1 inverted phase-contrast fluorescence microscope equipped with an Axiocam 506 Mono camera (Zeiss). Cal-520-AM imaging was carried out by exciting the sample at 493 nm and monitoring emission at 515 nm using X-Cite Series 120Q illumination system and Zeiss filter set 38 HE.24 hours after baculovirus transduction, cells were gently washed with imaging buffer [20 mM HEPES supplemented Ca2+ imaging movies were processed using ImageJ111, Fiji112 and MathWorks MATLAB 9.12.0.1884302 (R2022a) to extract Cal-520-AM fluorescence traces from individual cells. Movie stacks were background-subtracted with a 200-pixel rolling ball radius in ImageJ. Maximum intensity projection of the stack was used to generate a difference of gaussian image, which was used for edge detection and cell segmentation using MATLAB’s Image Processing Toolbox. Traces were then extracted from segmented cells, smoothed over 41 frames using a Savitzky–Golay filter of polynomial order 2, normalized by Z-score, and baseline adjusted using the linear method of MATLAB’s 1-D data interpolation function with a custom MATLAB script called Baseline Fit113. In the baseline-adjusted traces, the smallest observed Ca2+ oscillation peak value was used to manually threshold and identify other peaks automatically. Detected peaks were then used to calculate inter-spike intervals using MATLAB’s Signal Processing Toolbox. All statistical tests were performed using GraphPad Prism 9. Data reported are from 3 independent biological replicates.Ca113 and normalized between 0 and 1. The first peak of each oscillation/transient was identified and a window of 75 seconds on both sides of the peak was extracted and aligned at the peak position. Mean and 95% confidence intervals were calculated using GraphPad Prism and overlayed on traces from a single biological replicate. A 1 second window on both sides of the mean data point corresponding to half maximal intensity were fit to a straight line and used to calculate the mean rising phase for constructs exhibiting transients/oscillations. Traces with oscillations were sorted based on the maximum number of distinguishable peaks and plotted as a fraction of total oscillatory traces.For analysis of peaks from individual replicates, traces with transients/oscillations were baseline adjusted in MATLAB using Baseline FitFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Supplementary Movie 9Supplementary Movie 10Supplementary Movie 11Reporting SummarySource Data


The study aimed to investigate the relationship between the patients who applied to the emergency department with acute urticarial and meteorological factors and determine the associated weather conditions. The relationship between acute urticaria patients who applied to the emergency department of a training and research hospital in a city with high altitude and continental climate characteristics in the eastern part of Turkey between January 2019 and December 2019 and meteorological data was evaluated retrospectively. The necessary data for the weather were obtained from the data of the Erzurum Meteorology Directorate, and the patient data were obtained from the hospital information management system and patient files. The meteorological data of the patients at the date of admission and the characteristics of the patients at that date were compared. The study identified 691 patients who applied to the emergency department diagnosed with urticaria in 2019. According to the seasons, it was observed that the patients applied most frequently in the summer months . In the univariable regression model, which was created by taking the values of weather events as a reference on the days when the urticaria presentation was not observed (Group I), it was determined that every 1-hour increase in the sunshine hour increased the probability of urticaria by 7.4% (p = 0.018). When the meteorological parameters on the days without urticaria (Group I) and the days with urticaria presentation (Group II) were compared, the sunshine hours were 7.9 (4.0–10.6) hours in Group II and 6.65 (3.3–8.85) hours in Group I (p = 0.001). Regarding relative humidity, higher humidity rates were observed in Group I compared to Group II (p = 0.009). In terms of mean temperature, daily maximum, and minimum temperature, higher temperature rates were detected in Group II (p<0.001). A relationship was determined between urticaria attacks and relative humidity and daily maximum and minimum temperature in patients who applied to the emergency department with acute urticaria. Urticaria is defined as the sudden development of transient hives  and angioedema or both. A wheal is characterized by a circumscribed superficial edema of the skin, mostly surrounded by a bright red erythema and associated with a strong itching or burning sensation .Patients often apply to emergency departments because of the sudden onset of uncomfortable skin lesions and the possibility of developing life-threatening anaphylaxis. Acute urticaria is the most common skin condition leading to emergency room visits in both adults and children , 3. AcutAcute urticaria is defined as the occurrence of papules, angioedema, or both for 6 weeks or less. More than 50% of acute urticaria cases have triggering factors. The most common triggers are infections (40%), drugs (9.2%), and food (0.9%). The most common drugs held responsible are angiotensin-converting enzyme (ACE) inhibitors and nonsteroidal anti-inflammatory drugs (NSAIDs); however, approximately 50% of the triggering factor remains idiopathic . CertainUrticaria is caused by either immunoglobulin E-mediated or non-immunoglobulin E-mediated release of other inflammatory mediators—mainly histamine—from mast cells and basophils .We saw that on some days there was no admission to the emergency department because of acute urticaria, and on some days many patients were admitted. We thought that the weather events that society is exposed to on days with many patient admissions may be effective. In fact, about half of the factors triggering acute urticaria could not be detected. Many studies have shown that some diseases are associated with weather events. For example, low average temperatures, low sunlight, high humidity, and high wind speeds have been found to increase the risk of stroke .With the hypothesis that weather events may cause an increase in allergic reactions, this study aimed primarily to evaluate the effect of weather and climate parameters on the formation of urticaria and to investigate whether meteorological parameters affect the number of patients presenting with urticaria.Patients over the age of 18 who were diagnosed with acute urticaria between January 1, 2019, and December 31, 2019, in the emergency department of a hospital in eastern Turkey, were included in the study. Patients with chronic diseases and a history of drug use related to these diseases were excluded. Prior to the study, local ethics committee approval was obtained (dated 03.01.2022 and numbered 2021/23–296). Patient data were obtained with the hospital information management system and archive files. Demographic data such as age, gender, admission dates, and meteorological factors at that time were used.Diagnoses used for screening acute urticaria according to ICD-10  of L50.0 (Allergic Urticaria), L50.1 (Idiopathic Urticaria), L50.2 (Cold and Hot Urticaria), L50.3 (Dermatographic Urticaria), L50.4 (Vibratory Urticaria), L50.5 (Cholinergic Urticaria), L50.6 (Contact Urticaria), L50.8 (Chronic or Recurrent Urticaria), and L50.9 (Unspecified Urticaria) were used. Meteorological data on days without urticaria applications were defined as Group I, and meteorological data on days with urticaria applications were defined as Group II. The data available in Group II were divided into two further subgroups, according to the number of patients admitted: Group IIA for days with 1–4 patient admissions and Group IIB for days with 5 or more applications. When the total number of patients was evaluated, 1215 patients were diagnosed with acute urticaria. Starting January 1, the data of patients diagnosed with acute urticaria were obtained by scanning the hospital information management system. Patients who presented to the emergency department with acute urticaria and whose trigger factors were known or who were diagnosed with physical urticaria were excluded from the study. We did not have a patient who was diagnosed with urticaria because of physical factors. The excluded factors are shown in 2), pressure , cloudiness (8 Octa), wind speed (meter/second = m/s), relative humidity (%), and temperature (degrees Celsius = °C) were evaluated for use in the study. These parameters were used as the daily average, maximum, and minimum values.The parameters were obtained from the data of the 12th Regional Directorate of Meteorology and used. Among the meteorological parameters, sunshine duration (hours), sunshine intensity  of the independent meteorological variables were calculated with univariate and multiple logistic regression models to predict Group II. A multivariate logistic regression analysis was created by performing stepwise variable selection on those variables with a univariate p-value of <0.25. Multinomial logistic regression analysis was used to identify the effects of each meteorological parameters. All statistical analyses were conducted using SPSS 19.0 for Windows  and R software version 3.6.2. All p-values of less than 0.05 were considered to indicate statistical significance.The total number of patients admitted to our emergency department in 2019 was 239,271. Acute urticaria patients constituted approximately 0.5% of the applications.The seasonal  percentages of total admissions to the emergency department were similar to each other . Percentage of patients with acute urticaria of unknown origin were highest in summer  and lowest in winter  .In our study, 691 (56.8%) patients in whom the triggering factor could not be determined were identified among 1215 patients who applied to the emergency department with the diagnosis of acute urticaria in 2019. Of these, 58% were female, 42% were male, and the median age of the patients was 34.5 (25.0–46.0) years. In 2019, the number of days with admission to the emergency department with the complaint of urticaria was 276 (75.6%). In When the meteorological parameters on Group I days were compared with those on Group II days, the sunshine hours were 7.9 (4.0–10.6) hours in Group II and 6.65 (3.3–8.85) hours in Group I (p = 0.001). In terms of relative humidity, higher humidity rates were observed in Group I compared to Group II (p = 0.009). Regarding mean temperature and daily maximum and minimum temperatures, higher temperature rates were detected in Group II (p<0.001). The comparisons of other meteorological data are presented in In the univariable regression model, which was created by taking the values of the weather events on the days without urticaria (Group I) as a reference, it was determined that every 1-hour increase in the sunshine hours increased the probability of urticaria by 1.074 [(1.012–1.139) p = 0.018]. Furthermore, each 1-degree rise in temperature predicted Group II by 1.056 (1.029–1.083). The power of other meteorological variables to predict Group II was not statistically significant . The mulThe median daily total global solar radiation (GSR) and temperatures of Group I, Group IIA, and Group IIB are summarized in Figs The multinominal logistic regression model created with reference to Group I, estimating Group IIA and Group IIB, was analyzed. In the univariable analysis, the odds ratio for each 1-hour increase in sun exposure to predict Group IIB was 1.148 (1.043–1.264) (p = 0.005). Similarly, OR values of 1.047 (1.020–1.074) for Group IIA and 1.118 (1.070–1.167) for Group IIB were determined for each degree Celsius increase in temperature and were statistically significant . The multivariable regression model revealed that the temperature increase was effective in predicting both Group IIA and Group IIB .In this study, a positive correlation was observed between the increase in temperature, sunshine hours, and low humidity rates and the number of acute urticaria cases admitted to the emergency department. Accordingly, it was determined that the number of patients who applied to the emergency department with acute urticaria in the summer months when the temperature and sunshine duration was high and, on the days when there was low humidity, was high.Zhang J. et al. investigated the relationship between 6-year outpatient visits of urticaria patients in Lanzhou, meteorological data, and air pollution and demonstrated that the daily average temperature increased the outpatient visits of urticaria patients.It was observed that the number of female patients was higher in urticaria admissions. High temperatures have been suggested to increase the risk of urticaria for people aged 0–14 and 15–59 years, while low temperatures increase the incidence of urticaria for people over 60 years of age . Our stuLosappio L et al. detected triggering factors in 42% of patients who presented to the emergency department with acute urticaria, whereas drugs were detected in 20.7%, insect bites in 10.2%, food in 7.4%, and contact urticaria in 3.7%; 58% of the cases did not have a triggering factor . In the In a study performed in Brazil, where meteorological factors were examined in the etiology of acute asthma attacks in allergic asthma—which is similar to the pathophysiology of urticaria—temperature variability of 0–7 days was examined, and each 1°C increase was associated with a 1.0% increase in hospitalizations for asthma  . AnotherA study determined that asthma exacerbations were more frequent when the air humidity increased in patients over 65 years of age. The authors considered that this effect might be because of the increase in air pollutant particles . The preIn a meta-analysis of 16 studies, 31% (5/16) of studies indicated that temperature was the only factor associated with asthma hospitalization or emergency room admissions. In six studies (37%), both temperature and relative humidity were related to hospitalization . In thisIn this study, in which we examined the effect of meteorological factors in patients who applied to the emergency department with acute urticaria and had no suspected etiological factors, we found that sunshine hours, temperature, and humidity rates were effective. There was an increase in the number of applications to the emergency service in the summer months when the sunshine hours were high and on the days when the high temperatures and low humidity rates. When the clinician cannot detect the triggering factor in patients who present to the emergency department with acute urticaria, we recommend that the meteorological factors be evaluated.This study was based on data applied to a third-level hospital in our city and was a single-center study. Only patients who applied to our hospital from the city center were included in our study. Patients admitted from rural areas could not be enrolled.S1 Data(XLSX)Click here for additional data file.


This systematic review identified various bioactive compounds which have the potential to serve as novel drugs or leads against acute myeloid leukemia. Acute myeloid leukemia (AML) is a heterogeneous hematopoietic malignancy that arises from the dysregulation of cell differentiation, proliferation, and cell death. The risk factors associated with the onset of AML include long‐term exposure to radiation and chemicals such as benzene, smoking, genetic disorders, blood disorders, advancement in age, and others. Although novel strategies to manage AML, including a refinement of the conventional chemotherapy regimens, hypomethylating agents, and molecular targeted drugs, have been developed in recent years, resistance and relapse remain the main clinical problems. In this study, three databases, PubMed/MEDLINE, ScienceDirect, and Google Scholar, were systematically searched to identify various bioactive compounds with antileukemic properties. A total of 518 articles were identified, out of which 59 were viewed as eligible for the current report. From the data extracted, over 60 bioactive compounds were identified and divided into five major groups: flavonoids, alkaloids, organosulfur compounds, terpenes, and terpenoids, and other known and emerging bioactive compounds. The mechanism of actions of the analyzed individual bioactive molecules differs remarkably and includes disrupting chromatin structure, upregulating the synthesis of certain DNA repair proteins, inducing cell cycle arrest and apoptosis, and inhibiting/regulating Hsp90 activities, DNA methyltransferase 1, and histone deacetylase 1. From the data extracted, over 60 bioactive compounds were identified and divided into five major groups: flavonoids, alkaloids, organosulfur compounds, terpenes and terpenoids, and other known and emerging bioactive compounds. The mechanisms of actions of the analyzed individual bioactive molecules differ remarkably and include disrupting chromatin structure, inducing cell cycle arrest and apoptosis, inhibiting Hsp90 activities, DNA methyltransferase 1, and histone deacetylase 1, with the consequent hypomethylation of the DNA. In acute leukemia, the cells produced by the bone marrow are abnormal, dysfunctional, and fail to mature, passing into the circulation as immature white blood cells called “blasts.” A percentage of ≥20% blasts in the bone marrow is needed to establish the diagnosis of acute leukemia  leukemia (AML) is characterized by the abnormal proliferation of blasts of myeloid lineage and has emerged as the most common form of leukemia in the adult population, possessing a bimodal distribution in terms of interested age groups. In 2015, AML affected at least 1 million individuals and caused 147,000 deaths worldwide  AND (“acute myeloid leukemia” OR “AML”).2.2To be eligible for inclusion, the selected articles had to meet the following inclusion criteria. To begin, the article must have used generally recognized research models  and reported on the effects of the bioactive compound(s) against AML. Second, the article must have been published in English.2.3We excluded studies that investigated other types of cancers. Duplicate articles from different databases were screened and only one was retained. Two authors evaluated the title and abstract and references of each article.33.1A total of 518 articles were identified through database searching and other sources. Of this number, 487 papers were identified through database searching while 31 additional articles were identified through other sources (hand searching) Figure , namely 3.2Based on the data obtained from this study Table , we idenThe cytotoxicity of the flavagline family on AML cells, which occurs via several mechanisms, has been studied and established ‐expressing AML cells. Another study by Ye et al.  are sulfur‐containing organic compounds Figure  found abOrganosulfur compounds such as diallyl sulfide (DAS), diallyl disulfide (DADS), diallyl trisulfide (DATS), and thiosulfonate, deoxysulfone, and ajoene Figure  exhibit 2O2  are involved in the processes of invasion and metastasis in human malignancies, and the expression levels of MMP‐2 and MMP‐9 have been shown to decrease in MV4‐11/DDP cells treated with high oridonin concentrations. Oridonin may have the capacity to suppress human AML cell invasion and metastasis and is likely to be effective in inhibiting the development of drug‐resistant AML cells. Combination treatment with oridonin and cisplatin has become a useful way of treating human AML cells resistant to cisplatin. Both compounds exerted synergistic antitumor effects, and cisplatin tolerance in human AML cells was essentially reversed. Meanwhile, under these laboratory conditions, opioid toxicity had to be minimized (Zhang, Wang, et al., Besides terrestrial organisms, marine organisms are also valuable sources of anticancer agents such as peptides, alkaloids, polyketides, phloroglucinols, polyphenols, sulfated polysaccharides, sterols, carotenoids, and polysaccharide derivatives obtained from chitin, chitosan, and chitooligosaccharides, etc. (Chakraborty et al., 4Acute myeloid leukemia is one of the most common kinds of leukemia, resulting in significant human mortality in both children and adults. It is a type of hematological malignancy characterized by dysregulation of cell differentiation, proliferation, and death induced by epigenetic and genetic changes in hematopoietic progenitor or stem cells. Despite the development of innovative treatments in recent years, such as modifications in conventional chemotherapies, hypomethylating medicines, and molecular targeted medications, resistance and relapse remain the key clinical concerns of AML care. As a result, there is an urgent need for ongoing research on better alternatives. This study has identified various promising bioactive compounds, many of which pertain to the class of flavonoids and polyphenols. Other important groups are alkaloids, organosulfur compounds and terpenoids. All of the identified compounds showed promising effects on AML cells.The authors declare no conflict of interest.None.


Although PIP2 depletion and PKC activation have been strongly suggested to contribute to muscarinic inhibition of M currents (IM), direct evidence is lacking. We investigated the mechanism involved in muscarinic inhibition of IM with Ca2+ measurement and electrophysiological studies in both neuronal (rat sympathetic neurons) and heterologous (HEK cells expressing KCNQ2/KCNQ3) preparations. We found that muscarinic inhibition of IM was not blocked either by PIP2 or by calphostin C, a PKC inhibitor. We then examined whether muscarinic inhibition of IM uses multiple signaling pathways by blocking both PIP2 depletion and PKC activation. This maneuver, however, did not block muscarinic inhibition of IM. Additionally, muscarinic inhibition of IM was not prevented either by sequestering of G-protein βγ subunits from Gα-transducin or anti-Gβγ antibody or by preventing intracellular trafficking of channel proteins with blebbistatin, a class-II myosin inhibitor. Finally, we re-examined the role of Ca2+ signals in muscarinic inhibition of IM. Ca2+ measurements showed that muscarinic stimulation increased intracellular Ca2+ and was comparable to the Ca2+ mobilizing effect of bradykinin. Accordingly, 20-mM of BAPTA significantly suppressed muscarinic inhibition of IM. In contrast, muscarinic inhibition of IM was completely insensitive to 20-mM EGTA. Taken together, these data suggest a role of Ca2+ signaling in muscarinic modulation of IM. The differential effects of EGTA and BAPTA imply that Ca2+ microdomains or spatially local Ca2+ signals contribute to inhibition of IM.Acetylcholine can excite neurons by suppressing M-type (KCNQ) potassium channels. This effect is mediated by MThe online version contains supplementary material available at 10.1007/s10571-022-01303-7. Kruse and Whitten recently developed a model that describes altering M1R surface density and PI-5-kinase activity regulating the excitability of rat SCG neurons  sympathetic neurons and HEK cells expressing KCNQ channels.IThis study was reviewed and carried out in accordance with the Institutional Animal Care and Use Committee (IACUC) at Sungkyunkwan University School of Medicine (SUSM). SUSM is an Association for Assessment and Accreditation of Laboratory Animal Care International accredited facility and abides by the Institute of Laboratory Animal Resources Guide. The animals were maintained in standard environmental conditions , were given ad libitum access to water and food, and were housed under veterinary supervision at the Laboratory Animal Research Center, SUSM. Sprague–Dawley rats were purchased from Orient Bio Inc. . The authors understand the ethical principles under which The Journal of Physiology operates and the experiments comply with the animal ethics checklist described in Grundy Grundy .SCG neurons were cultured from 3- to 4-week-old male rats as previously described . Filtered signals (1–2 kHz) from a patch-clamp amplifier were fed into an AD/DA converter , digitized at 5 kHz and stored digitally in later analysis. Electrodes were pulled from borosilicate capillaries  using a pipette puller  and positioned precisely with a micromanipulator . The pipettes had a resistance of 2–3 MΩ when filled with pipette solution. Data were not corrected for liquid junction potential (−9 mV). For dialysis, we waited > 5 min before starting the experiment. The perfusion system was a homemade 100-µl perfusion chamber through which solution flowed continuously at 5 ml/min. All recordings were carried out at room temperature (22–24 °C).2PO4, 11.1 glucose, 0.5 MgCl2, 1.8 CaCl2, and pH 7.4 adjusted with NaOH. The pipette solution was as follows (in mM): 126 KMeSO4, 14 KCl, 10 HEPES, and 3 MgCl2 (pH 7.24 adjusted with KOH). 500 nM of TTX  and 0.1 μM of CdCl2 were included in external solution to block Na+ and Ca2+ currents.The normal external solution for HEK293 cell and rat SCG neuron recording was as follows (in mM): 143 NaCl, 5.4 KCl, 5 HEPES, 0.5 NaH2+]I, the concentration of intracellular free Ca2+, was measured by loading cells with 2-μM Fura 2-acetoxymethyl ester (Fura 2-AM)  for 30 min in the recording medium. Changes in Ca2+ were estimated from the ratio of Fura 2 fluorescence (at 500 nm) with alternate excitation at wavelength of 340 and 380 nm, generated by a Lambda DG-4 monochromator (Sutter Instrument Company). Fura 2 ratio data (F340/380), [Ca2+]i, were measured at a frequency of 2 Hz. Intensity of illumination was minimized to reduce bleaching of Fura 2, while maintaining adequate levels of signal. Fura 2 images for digital analysis were generated using a system based on an Olympus IX71 microscope coupled to an image intensifier and CCD camera and were analyzed with MetaFluor software .i, we tested whether Ca2+ mediates muscarinic modulation of IM using 20-mM BAPTA. When the pipette solution contained 20-mM BAPTA, oxo-M minimally affected IM . The oxo-M-induced inhibition of IM was determined as a proportion of oxo-M-sensitive currents to XE991-sensitive currents at + 20 mV as in Fig. n = 19, Fig. n = 7), p < 0.01). We then tested whether the effect of BAPTA on suppressing muscarinic inhibition of IM could be attributed to inhibiting local Ca2+ signals. To do this, we tested the effect of EGTA on muscarinic inhibition of IM. BAPTA is a fast Ca2+ buffer that effectively buffers all types of Ca2+ signals, whereas EGTA is a slow Ca2+ buffer that selectively spares local Ca2+ signals . We confirmed that proportions of IM to total outward currents in the presence of 20-mM EGTA (49.60 ± 3.48% [n = 4]) at + 20 mV) were similar to those in the presence of 20-mM BAPTA (52.40 ± 2.46% [n = 19]).Since muscarinic receptor stimulation elicited an increase of [Ca IM Fig. Aa. Superls Neher . With 202+]i on M currents in SCG neurons. We perfused cells with a bathing solution containing 10-μM ionomycin, a Ca2+ ionophore, and 10-mM CaCl2 to raise [Ca2+]i. We found that as [Ca2+]i increased, the M current amplitude declined in parallel (SI 3). This is consistent with previous data showing the sensitivity of KCNQ2/KCNQ3 channels to [Ca2+]i  can suppress muscarinic inhibition of IM . We referred diffusion coefficient of free Ca2+ ion (223 μm2/s) as reported by Allbritton et al. , or by blocking PKC activation with a specific blocker, calphostin C, or by blocking both PIP2 and PKC pathways. As other potential signaling pathways may have been involved, we examined whether Gβγ subunits of G protein or trafficking of channel proteins were involved in muscarinic inhibition of IM. The results showed that neither seemed to have a critical role.In addition, our results showed that muscarinic inhibition of M channel was not attenuated by blocking PIP2 depletion is not the ‘principal’ contributor to the inhibition of IM by muscarinic signaling, in contrast to the PIP2 depletion hypothesis for muscarinic action. Clearly, KCNQ channels are PIP2 sensitive: they are directly activated by exogenous PIP2 Below is the link to the electronic supplementary material.


Urticaria is a common skin condition encountered across various specialties in medicine, especially in dermatology and allergy/immunology practice. It has a heterogeneous presentation hence it is unsurprising that many skin conditions may be confused with urticaria. Urticaria may present as acute or chronic urticaria, the latter can be further categorised into chronic spontaneous and chronic inducible. In this article, we explore, explain, and summarise various skin lesions that are considered mimickers of urticaria, to promote understanding of each of the conditions highlighted, improve recognition, and reduce misdiagnosis. Urticaria is a common presentation in general practice and a common referral to clinicians including dermatologists, allergists, and immunologists. It is an inflammatory skin disorder characterised by wheals, hives, and often intractable itch, with an estimated lifetime prevalence of 20% globally . A typicAtypical urticarial lesions may appear more indurated, more discoloured, dusky, or bruised in appearance, and last longer. The lesions may heal with an eventual hyperpigmented mark. Instead of pruritic, they are painful. Angioedema is typically absent. Systemic features may coexist, including arthralgia, fever, and malaise.Urticaria can be categorised as acute or chronic. Acute urticaria is recurrent flares of hives lasting less than 6 weeks. It occurs most often in children, in the setting of upper respiratory viral infection in most cases . In otheChronic urticaria is defined as the appearance of hives occurring on most days of the week lasting for more than 6 weeks. A majority of chronic urticaria cases are chronic spontaneous urticaria (CSU), with two distinctive subtypes being recognised: autoallergic CSU (type 1 autoimmunity) with IgE auto-antibody involvement and autoimmune CSU (type IIb autoimmunity) with IgG auto-antibody involvement , 5. ChroThere is, of course, a list of differential diagnoses to be considered for CSU. These include CIndU, Schnitzler syndrome, cryopyrin-associated periodic syndrome, Still's disease, and urticarial vasculitis, with specific questions and aspects of physical examination that could guide further evaluation before a final diagnosis is determined .There are also several skin lesions with morphology that resemble an urticarial lesion, which may pose challenges to even the most experienced clinician. Here, we discuss a list of cutaneous lesions that may be regarded as mimickers of urticaria to improve understanding of a common presenting problem in dermatology or allergy/immunology practice.Mastocytosis is a clonal disorder characterised by abnormal mast cells in various organs, including the skin, bone marrow, liver, and gastrointestinal tract. It is the result of abnormal expansion and focal accumulation of neoplastic mast cells driven by a gain-of-function mutation in KIT, a membrane protein that keeps growth and activation of mast cells in check. The most common mutation described is KIT D816V, though there are other less commonly described mutations. The spectrum of mastocytosis presentation is heterogeneous, ranging from cutaneous to systemic involvement. Cutaneous mastocytosis (CM) is largely benign, whereas systemic mastocytosis often has serious implications, particularly increased risks for anaphylaxis and lymphoproliferation.CM is further categorised into maculopapular cutaneous mastocytosis , diffuse cutaneous mastocytosis, and mastocytoma of the skin. Most cases of CM occur in childhood, and in most cases, lesions usually regress by puberty . In mostUrticaria pigmentosa was first reported in 1,869 in a 2-year-old child who presented with urticaria with a brownish stain . It is bSkin biopsy is characterised by increased mast cell numbers in the papillary dermis and around blood vessels. Immunochemistry staining with mast cell tryptase antibody helps detect mast cell infiltrates. Using CD117 immunostaining, the mast cell distribution pattern and the percentage of mast cells in the inflammatory infiltrates can be determined .Erythema multiforme (EM) is a cutaneous and mucosal hypersensitivity reaction triggered by a stimulus. Infectious aetiology accounts for most cases of EM, with herpes simplex virus (HSV) being the most common cause in adults . MycoplaThe emergence of the novel Coronavirus disease-19 (COVID-19) infection has seen cases of EM being reported as a cutaneous complication, either to the infection itself or to the vaccine , 15. EM EM has an acute presentation. It starts with a painful eruption, often an erythematous papule, that eventually progresses to form a target lesion. Its resemblance to urticaria is related to its lighter oedematous appearance surrounded by a peripheral erythematous border . One of Mycoplasma pneumoniae, and chest x-ray looking for pulmonary infiltrates. A skin biopsy should be performed if in doubt. Characteristic histopathological findings include keratinocyte necrosis, particularly in the centre of the targetoid lesion  test for HSV from a concurrent skin lesion , nasal PCR test for d lesion . Necrosi, and drugs  can all cause UV, though in the majority of cases, the cause is unknown.Urticarial vasculitis (UV) is characterised by long-standing urticarial rashes and histopathologic findings of leukocytoclastic vasculitis . The lesThere are two types of UV, known as normocomplementaemic UV and hypocomplementaemic UV (McDuffie syndrome). Each type has unique pathophysiology, clinical manifestations, and prognosis. UV is considered a complement-mediated disease that is considered a type-3 hypersensitivity reaction. The pathophysiology is a complex interplay between the interaction of antibodies with antigens resulting in complement cascade activation and production of C3a, C5a, and C5b-9 via the classical pathway , 27. SysThe unique role of anti-C1q autoantibodies in hypocomplementaemic UV (HUV) is explained by the activation of complement cascade by autoantibodies targeting the collagen-like region of C1q . The patThe hallmark symptom of an urticarial vasculitis lesion is a painful and burning sensation, in contrast to the wheals of acute or chronic urticaria which are typically pruritic. It is a common mimic of urticaria, however, additional clues to UV include a bruised appearance, longer duration of lesions, often for days (in contrast to acute or chronic urticaria lesions that tend to resolve within a few hours), and healing with residual post-inflammatory ecchymotic hyperpigmented marks.Constitutional symptoms, such as malaise and fever, as well as internal organ involvement, may occur. Arthralgia may be present in approximately 50% of cases . Any joiClinical assessment for UV should always include a systemic review given internal organ involvement, especially in HUV. Physical examination may reveal tell-tale signs of UV, especially from the cutaneous aspect. Thorough investigations including full blood count, inflammatory markers, serum complements, renal function, and liver enzymes are considered basic. Further exploration into a potential autoimmune, infective, or neoplastic aetiology should be undertaken, including antinuclear antibodies, extractable nuclear antigens, anti-double-stranded DNA antibodies, rheumatoid factor, anti-citrullinated protein antibodies, cryoglobulins, anti-neutrophil cytoplasmic antibodies, viral hepatitis serology, anti-streptolysin O titres, full blood picture, lymphocyte immunophenotyping, serum protein electrophoresis, serum free light chain, tumour markers, and urine Bence-Jones protein. Urine analysis looking for the presence of protein, blood, or casts, should be performed. Renal biopsy may be required, especially to confirm or establish the type of glomerulonephritis. In the setting of pulmonary involvement, chest x-ray, chest computed tomography, lung function tests, and blood gas measurement are all useful. With musculoskeletal involvement, a skeletal survey is recommended.Skin biopsy demonstrates features suggestive of leukocytoclastic vasculitis. The key feature is neutrophil predominance, in contrast to non-vasculitic urticarial samples, which may show mostly the presence of eosinophils. A novel histopathological diagnostic scoring system was established recently to distinguish UV from CSU. A combined quantitative assessment of three key features of leukocytoclasia, fibrin deposits, and extravasated erythrocytes convincingly distinguishes UV from CSU in skin histopathology reading . This scErythema marginatum (EM) is a reticular, serpiginous, erythematous lesion that may be mistaken for urticaria. Unlike urticaria, EM is not pruritic and is less widespread. EM was first described by Dinckelacker in 1882. EM is commonly associated with Hereditary Angioedema (HAE), where it may occur alone, or together with fatigue and malaise, as a prodromal symptom experienced by patients with HAE prior to an imminent acute attack of angioedema. In certain parts of the world, including Central and Northern Australia and Southeast Asia where rheumatic fever is endemic, EM is one of the major criteria for this infection caused by Streptococcus.EM may occur on any part of the body, including the trunk and limbs. Given its morphology mimicking urticaria, it is unsurprising that patients with HAE presenting with EM and angioedema potentially may be misdiagnosed as having an allergy. Recent studies have highlighted the rate of misdiagnosis or delayed diagnosis of HAE given misinterpretation of EM as urticaria. Diagnostic delay for 2 years in HAE patients with EM had previously been found .Prodromal symptoms occur hours to days before an acute attack in HAE, with almost two-thirds in a large German population study experiencing the symptoms within 6 h before an attack . The synIn the setting of HAE, EM may occur in HAE with C1 esterase inhibitor (C1-INH) deficiency as well as with normal C1-INH levels. This was observed in a Japanese survey on prodromal symptoms in patients with HAE. The same study reported EM distribution on the forearm as the predominant site, followed by the abdomen, upper arm, and precordium .HAE is an inherited condition with or without C1-INH deficiency. In type 1 HAE, both C1-INH function and antigenic levels are low. In type 2 HAE, C1-INH function is low, whereas C1-INH antigenic level may be normal or elevated. Both types are caused by SERPING1 gene mutations . Known gUrticarial dermatitis (UD) is an intensely pruritic skin eruption that may exist in papules and plaques resembling urticaria. Unlike urticaria, which has a rapid resolution, UD often lasts longer than 24 h. It is recognised that UD may persist for days to even weeks . Common Skin biopsy is helpful when certain features unique to other dermatoses are absent. Overall, non-specific findings are seen on skin biopsy taken from a UD lesion. These include epidermal oedema with perivascular inflammatory (often lymphocytic) infiltrate, with eosinophils present in the papillary and upper reticular dermis . Dermal Bullous pemphigoid (BP) is an autoimmune, heterogeneous, blistering skin disease affecting mostly older adults. It may involve both the skin and mucous membranes. BP is intensely pruritic in the prodromal phase, characterised by non-bullous lesion eruption, often in the form of excoriated, urticarial, or eczematous lesions , 52. As Skin biopsy of lesional tissue and direct immunofluorescent (DIF) staining of the perilesional tissue specimen are key investigations. Key histopathological findings include subepithelial blister formation and eosinophilic spongiosis . Skin biPruritic urticarial papules and plaques of pregnancy (PUPP) is a benign inflammatory disorder that is the most common dermatosis in pregnancy, appearing most commonly in the last weeks of pregnancy. Itchy small papules and plaques start in stretch marks and spare the periumbilical area. The eruption generally spreads to the trunk and limbs but rarely involves the face. There is no mucosal involvement. It resolves rapidly postpartum, leaving no pigmentation or scarring.The cause of the condition is unknown but several factors are considered, including rapid abdominal distension, hormonal changes, and placental and foetal factors. One theory suggests that connective tissue damage within striae is a factor.It is seen in primigravidas primarily and does not typically recur in later pregnancies . The incBiopsy specimens from lesions show non-specific features and direct immunofluorescence is usually negative. Routine laboratory tests are within normal limits.Symptomatic treatment is usually all that is required with topical steroids and antihistamines commonly prescribed. If severe, causing sleep disturbance, a very short course of oral corticosteroids may be considered.Apart from the discomfort, there are no negative consequences; pregnancy and maternal prognosis are not affected .Characteristic features and investigations of each of the conditions are summarised in There are many dermatological conditions that resemble the appearance of urticaria, making diagnosis sometimes challenging. With an understanding of underlying disease associations and pathology, together with a recognition of pathognomonic features, a diagnosis can be established with the aid of relevant investigations.