Ca2+ homeostasis preservation is essential for maintaining skeletal muscle structure and function. Store-operated Ca2+ entry (SOCE), a Ca2+-entry process activated by depletion of intracellular stores contributing to the regulation of various function in many cell types, is pivotal to ensure a proper Ca2+ homeostasis in muscle fibers. It is coordinated by STIM1, the main Ca2+ sensor located in the sarcoplasmic reticulum, and ORAI1 protein, a Ca2+-permeable channel located on transverse tubules. It is commonly accepted that Ca2+ entry via SOCE has the crucial role in short- and long-term muscle function, regulating and adapting many cellular processes including muscle contractility, postnatal development, myofiber phenotype and plasticity. Lack or mutations of STIM1 and/or Orai1 and the consequent SOCE alteration have been associated with serious consequences for muscle function. Importantly, evidence suggests that SOCE alteration can trigger a change of intracellular Ca2+ signaling in skeletal muscle, participating in the pathogenesis of different progressive muscle diseases such as tubular aggregate myopathy, muscular dystrophy, cachexia, and sarcopenia. This review provides a brief overview of the molecular mechanisms underlying STIM1/Orai1-dependent SOCE in skeletal muscle, focusing on how SOCE alteration could contribute to skeletal muscle wasting disorders and on how SOCE components could represent pharmacological targets with high therapeutic potential.Intracellular Ca Cellular Ca2+ homeostasis is maintained through the precise and coordinated function of Ca2+ transport molecules, Ca2+ buffer/binding proteins such as calsequestrin or calreticulin, and several calcium channels. These include the plasma membrane calcium ATPases (PMCAs) that actively pump Ca2+ out of the cell [2+-release-activated-Ca2+ (CRAC) channel located in the plasma membrane (PM) and activated by the endoplasmic/sarcoplasmic reticulum (ER/SR)-Ca2+ release; and the sarco-/endoplasmic reticular calcium ATPase (SERCA) located in the ER/SR that transport Ca2+ back into the ER/SR [2+ entry (ECCE), and store-operated Ca2+ entry (SOCE). EC coupling is the process mediated by mechanical coupling between the dihydropyridine receptor (DHPR) in the transverse tubule membrane, specialized invaginations of the sarcolemma, and the ryanodine receptor type 1 (RYR1) ion channel located in the ER/SR membrane. In this process, an action potential in the transverse tubule and the voltage-dependent conformational change of DHPR trigger the release of Ca2+ from the sarcoplasmic reticulum to drive muscle contraction [2+ entry pathway mediated by the DHPR, RYR1, and by a yet to be identified Ca2+ entry channel with properties corresponding to those of store-operated Ca2+ channels. It is triggered by sustained or repetitive depolarization and contributes to muscle contractility [2+-entry process activated by depletion of intracellular stores that contributes to the regulation of various functions in many cell types. It is mediated by the interaction between stromal-interacting molecule-1 (STIM1), the Ca2+ sensor of ER/SR [2+ signaling in skeletal muscle, thus causing or contributing to the pathogenesis of various skeletal muscle disorders. Therefore, therapies focused on restoring SOCE mechanism and targeting SOCE-associated proteins are promising for the treatment of SOCE-related skeletal muscle disorders. The present review aims to provide a brief overview of the molecular mechanisms underlying STIM1/Orai1-dependent SOCE in skeletal muscle, focusing on how SOCE alteration may contribute to muscle diseases.In skeletal muscle fibers, intracellular Captations . Ca2+ hoopathies ,4, muscuopathies ,6,7, cacopathies  and age-opathies ,11,12,13the cell ; the Ca2he ER/SR . In skeltraction . ECCE isactility ,18,19. Sof ER/SR , and Oraof ER/SR . Aberran2+ channels regulated by Ca2+ content in intracellular deposits. Due to their strong functional connections to ER/SR and their small but selective conductance for Ca2+, they have preferential access to Ca2+ response pathways and provide Ca2+ to refill the ER/SR after Ca2+ is released and pumped through the plasma membrane [2+ concentration within the ER/SR provide a signal for SOCCs activation at the sarcolemmal membrane, which play an important role in maintaining Ca2+ homeostasis in physiology, as well as in determining calcium homeostasis dysregulation in pathological condition. The critical components of SOCCs responsible for the SOCE mechanism are: the stromal interaction molecule-1 (STIM1) protein located in ER/SR [Store-operated-calcium channels (SOCCs) are plasma membrane Camembrane . Changesin ER/SR ,24, and in ER/SR ,25,26.2+ sensors for SOCE. STIM1 knockdown and mutagenesis studies strongly contributed to clarify the Ca2+ sensor property associated with these proteins [2+ sensing domain is located at the N-terminus region of STIM1, facing the ER/SR luminal side, and consists of a canonical EF-hand (cEFh), a non-canonical EF-hand (ncEFh), and sterile-motif (SAM) domains. SAM is followed by the transmembrane (TM) domain. Although Ca2+ binds only to the cEF-domain, the stability of the whole EF-hand-SAM domain is important for its Ca2+ sensing role [2+ levels in the ER/SR [C-terminus region, placed in the cytoplasmic side of ER/SR. These binding sites include: three conserved cytosolic coiled-coil (CC) domains , a proline/serine-rich domain and, at the very end of the C-terminus, a lysine-rich domain, which participates in Orai1-independent plasma membrane targeting of STIM1 [C-terminus region includes the C-terminal inhibitory domain (CTID), which interacts with the Ca2+ entry regulatory protein SARAF in the resting state and is responsible for the regulation of the slow Ca2+ inactivation dependent on Orai1 [2+. For example, several studies have shown that STIMATE (STIM-activating enhancer), an ER/SR transmembrane protein encoded by the TMEM110 gene, interacts directly with STIM1, favoring the conformational change of STIM1 and contributing to maintaining the correct structure of the ER/SR-PM junctions [2+ entry in skeletal muscle compared with other cell types [Stromal interaction molecule (STIM) proteins are single-pass transmembrane proteins located in the ER/SR, where they act as ER/SR Caproteins ,28. In mproteins . The Ca2ing role ,31. Furthe ER/SR ,32. The of STIM1 ,34. The of STIM1 . Furtherof STIM1 . The CADof STIM1 . Furtheron Orai1   between their key domains , but work differently as Ca of SOCE . Althougto STIM1 . Successtivation . A subsetivation . In additivation . Furthertivation .2+ release-activated Ca2+ channel (CRAC channel) [2+-selective ion pores. Three Orai isoforms  encoded by homologous genes and two versions of Orai1, Orai1α and Orai1β, arising from alternative translation initiation [CRAC current in human T cells, suggested the link between Orai1, in both Orai1α and β isoforms, and CRAC channel function [2+-selective pore [N- and C-regions facing the cytoplasm that mediate the interaction with STIM1, STIM2, and other regulatory proteins [2+ pore is formed by six TM1 domains surrounded by TM2-TM3, which provide stability to the structure [C-terminus. The glutamate at position 106, situated at the extracellular end of TM1, provides the binding site for Ca2+ ions inside the channel and confers the high Ca2+ selectivity to the CRAC channel [N-terminal (ETON) region is present. This region contributes to the interaction between the N-terminus of Orai1 and STIM1 [C-terminus [N-terminus and promotes a permissive conformation of Orai channel [C-terminus in the Orai1 complex, that are subsequently transmitted to the pore region [2+ entry through Orai1 via a SOCE mechanism [2+ permeation, are different from those of Orai1, suggesting that these proteins are little involved in SOCE [Orai proteins have been identified as key components of the Cachannel) ,51 and achannel) ,53. Partitiation , were iditiation . The prefunction ,56,57,58ive pore . Each Orproteins   regulation necessary for channel activity and desensitization, a calmodulin/inositol 1,4,5-trisphosphate receptor-binding (CIRB) site and a Ca2+ binding EF-hand [2+ homeostasis during sustained contractile muscle activity and is considered the best candidate to conduct Ca2+ influx during SOCE. TRPC1, which acts as a SOCE channel, participates in myoblasts migration and fusion via calpain activation during the terminal differentiation process [2+ entry was important to produce normal-sized myotubes. Conversely, overexpression of STIM1 with TRPC4 or TRPC1 increased SOCE, accelerated myoblast fusion, and produced hypertrophic myotubes [2+ entry through TRPC1 has been shown to play a role in myoblast differentiation and in muscle regeneration by modulating the PI3K/Akt/mTOR/p70S6K pathway. In fact, in myoblasts derived from −/−Trpc1 mice, the phosphorylation of both Akt and p70S6K proteins, as well as the activation of PI3K, were inhibited. This evidence suggests that Ca2+ entry through TRPC1 plays a role in the activation of this pathway [2+ transients during repetitive membrane depolarizations. This process, in turn, promotes myogenesis and maintains a fast repetitive Ca2+ release in human skeletal myotubes [CRAC [soc. However, this observation does not rule out the TRPC channels’ involvement in SOCE. Experimental studies have shown that STIM1 interacts with and directly activates TRPC1 channels not only by binding through its SOAR region with TRPC1, but also through electrostatic interactions between highly positively charged lysines located in the STIM1 lysine-rich domain and the TRPC1 aspartate residues [2+ entry triggers the recruitment of TRPC1 into the PM where it is then activated by STIM1 [N- and C- terminals of TRPC3 or TRPC6. Actually, it is not clear if they are able to act as SOCE channels without Orai1 interaction [In skeletal muscle, the non-selective transient receptor potential channels (TRPCs) have also been proposed as emerging SOCE channels . Indeed,ectivity . TRP chaectivity ,74. TRPCytoplasm . The cyt EF-hand  . In the second case, depletion of the ER/SR store essentially occurs following the stimulation of plasma membrane G protein-coupled receptors (GPCRs) through the activation of phospholipase C (PLC) subtypes and the related release of inositol 1,4,5-triphosphate (IP3); IP3 binds to the IP3R on the ER/SR membrane, triggering Ca2+ release from the ER/SR lumen into the cytosol [2+ independently of receptors and IP3 [2+ store depletion, a secondary influx of extracellular Ca2+ through CRAC channels in the plasma membrane results in a more sustained increase in cytosolic Ca2+ levels. Different SOCE models in skeletal muscle have been proposed, including the interaction between STIM1 and Orai1 [SOCE, also known as capacitive calcium entry (CCE), consists in a Caeostasis . The con+ influx . Success+ influx . General cytosol . Experim and IP3 ,89,90. Hnd Orai1 , STIM1 and Orai1 , and STInd Orai1 . Among t2+ from the ER/SR leads to Ca2+ dissociation from the luminal STIM1 N-terminal EF-hand domains. This determines the beginning of dramatic conformational changes in STIM1 that lead to the formation of an unfolded STIM1 protein and more closely associated luminal EF-hand domains. Unfolding and activation of STIM1 upon Ca2+ dissociation causes SARAF dissociation [C-terminus and the PM phospholipids [N- and C- termini of Orai1 through SOAR region and activate Orai1 channels for Ca2+ entry from the extracellular environment [2+ entry through Orai1 channels since it is required for Orai1 recruitment into STIM1 puncta and for STIM1-Orai1 interaction [2+-dependent inactivation [In the resting state, STIM1 is a dimer associated with its regulator SOCE-associated regulatory factor (SARAF), a single-pass transmembrane protein . The proociation  and STIMociation  by assocociation  and by tociation . Successholipids , resultiholipids . At the holipids . After pironment ,93,95,96ironment  and bioceraction . Furthertivation .2+ influx [2+ influx during store depletion. Accordingly, in skeletal muscle, SOCE occurs in less than a second, i.e., significantly faster than in other types of cells where it can require up to several seconds [Another hypothesis on the SOCE mechanism postulates that, in skeletal muscle, STIM1 and Orai1 pre-localize under resting conditions within the triad junction, a specialized macrostructure composed of a parallel transverse tubule and two opposing ER/SR membranes. They remain inactive until ER/SR depletion triggers conformational changes in STIM1 and direct activation of Orai1-mediated Ca+ influx ; this al seconds .C-terminal fragments (in a 1:1 STIM1:Orai1 stoichiometry) [C-terminal tail, leaving the remaining STIM1 subunit free to cross-link with a different Orai1 channel  [The precise stoichiometry of the STIM1-Orai1 complex that forms the functional core of the CRAC channel still needs clarification and it has long been a subject of debate . Severaliometry) ,101,102.iometry) . More reiometry) .2+ by ER/SR SERCA protein or following the export of cytosolic Ca2+ to the extracellular area by PMCAs [2+ rebinds to the STIM1 EF-hand, STIM1 dissociates from Orai1, and STIM1 and Orai1 revert to their diffuse distributions [SOCE terminates following the reuptake of Caby PMCAs . Upon stibutions .2+ stores to maintain the appropriate environment within the ER/SR for protein folding/processing, vesicle trafficking, and cholesterol metabolism [2+ entry via SOCE has the crucial role in short-term and long-term muscle function.Generally, the SOCE mechanism has traditionally been known for serving as the primary route to rapidly replenish depleted intracellular Catabolism . In skel2+ refilling during repolarization cycles, to complement Ca2+ recycling to the ER/SR by the SERCA and support ER/SR Ca2+ release [2+ ions from the extracellular environment via STIM1/ORAI1-mediated SOCE represents a mechanism that allows the ER/SR Ca2+ refilling to maintain Ca2+ release during periods of high-frequency repetitive stimulation.In regard to short-term function, i.e., muscle contractility, the faster SOCE mechanism is required for ER/SR Ca release . Further2+-dependent signals such as the nuclear factor of activated T-cells (NFAT), mitogen-activated protein (MAP) kinase and ERK1/2 [Importantly, SOCE has also been proposed to contribute to key myogenic events important for long-term skeletal muscle functions, such as myoblast fusion/differentiation and muscle development ,109. Thid ERK1/2 . Interesd ERK1/2 ,71,110. d ERK1/2 . It has d ERK1/2 .2+ loss across the transverse tubule system during EC coupling [The SOCE mechanism in adult skeletal muscle also reduces fatigue during periods of prolonged stimulation ,111,112,coupling .2+-mediated processes. This leads to different skeletal muscle disorders including muscular hypotonia and myopathies associated to STIM1/ORAI1 mutations [According to this key role in a plethora of muscle determinants and functions, abnormal SOCE is detrimental for skeletal muscle and results in loss of fine control of Cautations ,4, muscuutations ,7, cacheutations  and sarcutations ,11,12,13As detailed above, proper functioning of SOCE is important for maintaining healthy skeletal muscle processes. Involvement of SOCE in genetic skeletal muscle diseases has been proposed when a missense mutation (R91W) in the first transmembrane domain of Orai1 was found in patients suffering from severe combined immunodeficiency (SCID) and presenting myopathy, hypotonia and respiratory muscle weakness . SuccessTo date, thirteen different STIM1 and Orai1 LoF gene mutations have been described , all of them resulting in a marked reduction of SOCE function . LoF R912+ entry [In contrast, GoF mutations in STIM1 and/or Orai1 induce the production of a protein that is constitutively active and results in SOCE over-activation and excessive extracellular Ca2+ entry ,119. In 2+ entry ,119,120.2+ entry ,122. Mus2+ entry ,124. Som2+ entry , a rare 2+ entry  and prom2+ entry .2+ level, leading to an augmented concentration of intracellular Ca2+ [2+ selectivity and its sensitivity to external pH and to STIM1-mediated gating [To date, fourteen different STIM1 GoF mutations are known in TAM/STRMK patients, including specifically twelve mutations in the EF-domain  and two mutations in luminal coiled-coil domains  ,126,127.lar Ca2+ . Regardilar Ca2+ ,123,128 2+ concentration and the augmented SOCE characterizing STIM1 mutant muscle cells matched with a reduced cell multinucleation and with a distinct morphology and geometry of the mitochondrial network indicating a defect in the late differentiation phase [Only a few STIM1 and Orai1 mutations have been functionally characterized in native skeletal muscle cells, most of them having been studied in heterologous expression systems. This represents an overt limitation both for the limited reliability of the cellular model and for the translation of drug efficacy in humans. TAM animal models exist and broadly recapitulate the clinical signs of human disorders but, unfortunately, only partially replicate muscle symptoms . Particuon phase . These f2+ homeostasis, finally increasing intracellular Ca2+ levels [2+ due to extracellular Ca2+ entry approximately twice that of control muscle fibers [2+ influx triggers the activation of calpains, phospholipase A2 and Ca2+-activated kinases, such as PKC, and may act in a reinforcing loop with the mitochondrial dysfunction and the production of reactive oxygen species (ROS) [2+ level [2+ overload is yet under investigation. At first, Boittin and colleagues hypothesized that products of Ca2+-independent PLA2, such as lysophosphatidylcholine, are able to activate the SOCE process through a Ca2+-independent pathway without changing the threshold for SR Ca2+ [2+ influx to the dystrophic phenotype of mdx mice. Indeed, as a contributing cause of higher Ca2+ entry in mdx dystrophic muscle fibers, higher SOCE is reported through Orai1 upregulation or Stim1 overexpression [2+ entry in dystrophic myotubes. Indeed, studies on muscle-specific transgenic mice with a TRPC3 overexpression showed that Ca2+ influx across this TRP channel isoform contributes to the dystrophic muscle phenotype [2+ [Muscular dystrophies are a group of inherited skeletal muscle diseases that affect both children and adults and mainly involve muscle tissues causing progressive muscle degeneration and contractile function reduction with severe pain, disability and death . To date+ levels . Particue fibers ,137,138.e fibers . A key re fibers . The inces (ROS) . Then, ces (ROS) ,142,143.2+ level . Althoug SR Ca2+ . Successpression . Importapression . In addihenotype . Furthertype [2+ . In skel2+ homeostasis and the SOCE mechanism that contribute to impaired muscle functions, poor quality of life and disease progression.Several pathological conditions are characterized by loss and/or impairment of muscle and muscle wasting. When muscle wasting is present, it is always related to greater morbidity and reduced survival in chronic disease states, favoring the onset of negative outcomes and death . The maj2+ overload observed in cachectic skeletal muscle, probably due to SOCE-independent mechanisms, is associated with a reduced response to the application of depolarizing solution or caffeine, as well as with a reduced SOCE in terms of functional activity and gene expression. Particularly, a down-regulation of STIM1, ORAI1, RyR1 and Dhpr muscle gene expression was observed in cachectic animals with respect to controls [2+ overload and SOCE alteration observed in cachectic muscle can exert deleterious effects that lead to muscle damage. This is due to the activation of Ca2+-activated proteases  and the disruption of the integrity of the sarcolemma, all events contributing to the loss of strength muscle [Cachexia is defined as a debilitating wasting that manifests in several types of cancer and, at the same time, represents a serious and dose-limiting consequence of cancer chemotherapy . Cachectcontrols . Considecontrols . Ca2+ ovh muscle .2+ ions available to activate the contractile filaments, associated with a decrease in DHPR expression and consequent uncoupling between DHPR and RYR1 proteins [Aging is a multifactorial biological process characterized by a progressive decline of the main physiological functions that gradually leads to dysfunctions of various tissues including skeletal muscle . Normal proteins . Moreoveproteins . Skeletaproteins . This oxproteins . At the proteins ,159. In proteins . Furtherproteins .2+ homeostasis alteration has been recently proposed for fast-twitch muscle fibers of elderly mice. In particular, it has been demonstrated that dysfunctional accumulation of proteins forming TAs, which include also STIM1 and Orai1, together with a concomitant SOCE alteration, were associated with a reduced ability to restore internal deposits of Ca2+ from the extracellular environment in aged skeletal muscle [Similar to TAM, a correlation between TAs formation and Cal muscle . All theDespite several studies performed over the last years, the exact role of SOCE in sarcopenia remains controversial. For example, Edwards and colleagues demonstrated that SOCE remains unaffected in the skeletal muscle of aged mice despite an approximate 40% decline in STIM1 protein expression not accompanied by any alteration of Orai1 expression .2+ homeostasis and SOCE mechanism can be compromised in skeletal muscle pathological conditions involving proteins and/or intracellular organelles not directly related to SOCE, such as Ca2+ buffer proteins and/or mitochondria [2+ buffer proteins levels, such as calsequestrin or sarcalumenin, seems to be correlated to an altered SOCE [−/−) mice, showed that the absence of sarcalumenin enhanced muscle SOCE mechanism ameliorating muscle fatigue resistance. The parallel increase in muscle MG29 expression suggested the occurrence of a compensatory change in Ca2+ regulatory proteins that affect SOCE when sarcalumenin is reduced or absent [−/−) mice, showed that the absence of calsequestrin induced an increase of muscle SOCE mechanism with an increase of STIM1, Orai1, and SERCA expression associated with a high density of Ca2+ entry units (CEUs) [2+ entry channels during Ca2+ entry and capture large amounts of Ca2+ entry [Accumulating evidence has demonstrated that intracellular Cachondria ,164,165.red SOCE ,164. Zhar absent . Similars (CEUs) ,167. In 2+ entry . This evAs knowledge about the role of SOCE in skeletal muscle diseases accumulates, there has been a growing interest in developing molecules targeting SOCE and identifying therapies that can be used for specific treatments. Indeed, several studies recently aimed to develop SOCE modulators to reduce SOCE activation following the pathological skeletal muscle GoF mutations mentioned above. For example, Rahaman and colleagues used in silico screening to identify FDA-approved drugs able to suppress the SOCE mechanism. Particularly, leflunomide and teriflunomide, FDA-approved drugs for the treatment of rheumatoid/psoriatic arthritis and multiple sclerosis, respectively, were able to inhibit SOCE at therapeutically-achievable concentrations; furthermore, lansoprazole, tolvaptan and roflumilast resulted in even more selective molecules to suppress the SOCE mechanism . Recentl2+ are in progress. The spider venom toxin AT-300/GsMTx4, a peptide that blocks the mechanosensitive Ca2+ channels, for example, prevented the rise of intracellular resting Ca2+ with modest benefits in mdx mice [2+ signal alteration in DMD, and focused studies in this field are highly desirable.Regarding dystrophies, and DMD in particular, at present there are no effective treatments and the glucocorticoids which act as anti-inflammatory agents are often used to stop progressive muscle damage ,174. Premdx mice . Another2+ homeostasis alteration and SOCE decrease in skeletal muscle of cachectic rats [2+ homeostasis observed in the cachectic muscles suggesting that SOCE could be considered a potential target for cachexia therapy. Likewise, sarcopenia cannot be completely reversed by conventional nutritional support and/or increased physical activity, and SOCE could be considered a potential biomarker and target for therapeutical interventions for prevention or for counteracting sarcopenia. To achieve this goal, additional focused studies are still needed. In this context, the evaluation of senolytics and senostatics drugs, molecules considered to be revolutionizing in the field of aging research [Finally, regarding skeletal muscle wasting disorders, knowledge of the real role of the SOCE mechanism, in particular during cachexia and aged-sarcopenia, is a fundamental requirement for finding a potential therapy. Nutrition is a key factor for the therapy of these conditions because both the quality and quantity of nutrients are pivotal for improving muscle anabolism, reducing catabolism, and lightening the prognosis . Howevertic rats . Interestic rats . A directic rats . All theresearch , on the 2+ influx mechanisms operated by store depletion (for example STIM1 coupling to TRPC or Orai1/TRPC channels) and of a series of SOCE regulators (for example SARAF). The importance of a proper SOCE in skeletal muscle is evidenced by the observation that mutations in STIM1 and/or Orai1 genes or defects in STIM1/Orai1-mediated SOCE cause or contribute both directly and indirectly to the pathogenesis of various skeletal muscle disorders, including myopathies, dystrophies, cachexia, and age-related sarcopenia (The identification of STIM and Orai1 as the key molecules mediating SOCE had essential implications for skeletal muscle biology. Importantly, in recent years, several studies have helped to understand the basic molecular mechanisms of SOCE and have revealed the presence of other possible Carcopenia . Thus, t


Sarcoptes-induced oxidative discrepancy in rabbits. The goal of this study was to investigate if turmeric extract can alleviate side effects in Ivermectin-treated rabbits while simultaneously improving their performance and antioxidant status. The results indicated that turmeric extract was utilized with varied efficacy against mites, and also helped rabbits recover faster and improved Ivermectin’s miticidal efficacy by improving performance and compromised immunity. Turmeric extract has strong antioxidant properties, suggesting that it could be used as an adjunctive remedy to reduce the side effects of Ivermectin while treating clinical rabbit sarcoptic mange. Furthermore, no adverse effects were observed in turmeric extract adjunctively supplemented rabbits, and the provided dose regimen of these supplements was found to be safe.Sarcoptic mange is a common rabbit disease that can be spread directly from sick to healthy rabbits. Infection in rabbits causes intense pruritus, head shaking, and scabby lesions on the inner side of the pinnae and on the external ear canal, as well as diminished growth performance and an increased rate of oxidative stress. Ivermectin has been shown to be an effective treatment for sarcoptic mange in rabbits; however, it has side effects on their performance. Botanicals such as turmeric extract have a remarkable antioxidant potential contributing to the deterrence of Sarcoptes-infested rabbits aged 60 days were randomly allocated into three groups, with 21 rabbits in each group, to receive either no TE or TE supplementation (1 or 2 mg/kg diet) for 30 days after being subcutaneously injected with Ivermectin (IVM) 1% w/v at a dose of 0.2 mg/kg body weight twice a week. Another 21 healthy rabbits were used as the control. Treatment with IVM + 1 and 2 mg TE improved body weight (BW), body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) in infested rabbits (p < 0.05). The IVM alone treatment had the highest mortality rate compared with the other treatments. Rabbits treated with IVM + 1 and 2 mg TE demonstrated progressive recovery manifested by improved nutrient digestibility and nitrogen balance. On day 7, the serum total protein, albumin, and albumin/globulin (A/G) ratio were significantly lower in the treated groups compared with the control group; also, the lowest values were observed in rabbits treated only with Ivermectin, followed by IVM + 1 and 2 mg TE. Treated rabbits had higher thiobarbituric acid reactive substance (TBAR) levels, but lower total antioxidant capacity (TAC), superoxidase dismutase (SOD), and glutathion peroxidase (GSH-Px) levels compared with the control group. On day 30 post-treatment, the rabbits in the IVM + 1 and 2 mg TE treatment groups showed progressive recovery manifested by improved biochemichal parameters, as well as a remarkable improvements in the oxidant/antioxidant balance towards normalcy (p < 0.05), and became comparable to that of the control compared with IVM alone treatment. In conclusion, turmeric extract improved rabbits’ performance toward normalcy, and has remarkable antioxidant properties and can be used in conjunction with a miticide to treat sarcoptic mange in rabbits.In this experiment, the protective effect of turmeric extract (TE) on side effects of Ivermectin-treated rabbits, while improving their performance, blood characteristics, and antioxidant status, was investigated. Sixty-three clinically  Sarcoptes scabiei, is a highly contagious, non-seasonal, pruritic skin disorder in rabbits [Sarcoptic mange, caused by the mite  rabbits , and has rabbits . In addi rabbits ,4, it is rabbits . In rabb rabbits . Because rabbits . El-Ashr rabbits  reportedsarcoptic scabies, combined with declining therapeutic efficacy, necessitates the development of innovative control strategies in the form of adjuvant alternative therapies. Ivermectin (IVM) has been shown to be an effective treatment for sarcoptic mange in rabbits . UnfortuCurcuma longa) is a perennial herb that belongs to the ginger family, Zingaberaceae, and is used extensively in Ayurvedic medicine in India. Turmeric contains three different compounds, curcumin, demethoxycurcumin, and methoxycurcumin, all of which are known as curcuminoids [In this context, antioxidants from natural sources, especially medicinal plant extracts like turmeric extract, have gained much attention. Turmeric , Alexandria, Egypt.Curcuma longa) rhizome mash was weighed and mixed in 1 L of distilled water and was allowed to boil for 20 min. The mixture was allowed to stand for 24 h, then the supernatant was removed and introduced in a Buchi rotavapor at 60 °C under a vacuum for sample drying, where 350 mg of aqueous extract was obtained. The obtained concentration of curcumin was 4.5 mg/100 mg of aqueous extract of Curcuma longa.About 500 g of macerated turmeric  for 30 days after being subcutaneously injected with a commercial formulation of Ivermectin 1% w/v at a dose of 0.2 mg/kg body weight twice a week. All rabbits were kept under observation in well-ventilated buildings, and were individually housed in galvanised metal wire cages and maintained under equal managerial, hygienic, and environmental conditions in a room with an ambient temperature of 23 ± 2 °C, humidity 55–65%, and a photoperiod of 16 h light/8 h dark. All cages were equipped with feeding hoppers and drinking nipples, and feed and fresh water were available ad libitum during the experiment. Rabbits were fed a commercial pelleted diet to meet their requirements, according to NRC [Sixty-three Sarcoptes (n = 63) were not given any ectoparasiticides or steroid anti-inflammatory medications for 15 days before being examined clinically and dermatologically. Rabbit’s feces were examined for any infection with helminth parasite eggs following the sedimentation technique [In this experiment, male rabbits infected with echnique , which r® SPT-ITH, Fisher Scientific, Pittsburgh, PA, USA) and were manually transferred to glass slides containing 2−3 drops of Hoyer’s solution (Mounting fluid). The samples were then covered with glass slide covers and kept in an incubator for 48 h prior to examination. The morphological characteristics of the samples were then examined under a magnification microscope  at 40× magnification. Mite identification was performed using the previously described dichotomous key [The mites were examined under a stereoscopic microscope , and at the end of the experiment at 90 days of age to determine the final body weight (FBW). Feed intake (FI) was recorded daily during the experiment. The body weight gain (BWG) and feed conversion ratio (FCR) were calculated, and the mortality rate was recorded daily and the percentage was recorded for each group at the end of the experiment.2O solution to avoid any bacterial contamination. Nitrogen intake (NI), nitrogen excreted in urine (NU), and nitrogen excreted in the feces (NF) were estimated by the amounts of feed ingested and the excreted nitrogen of the urine and feces. Retained nitrogen (RN) was calculated according to the following equation:A digestion and nitrogen balance trial was conducted on 7 rabbits chosen at random from each treatment during the final week of the experiment, according to Perez et al. . Rabbitsg for 20 min at 20 °C. Serum was separated and kept at −20 °C for biochemical analyses to determine total protein, albumin, cholesterol, triglyceride, high density lipoprotein (HDL), low density lipoprotein (LDL), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity, while globulin was calculated as . Blood biochemicals were determined calorimetrically using standard kits supplied by  based on the procedure outlined by the manufacturer.On day 7 and 30 of the experiment, five ml of blood samples were obtained from the ear vein of 7 rabbits in each treatment, in clean centrifuge tubes and immediately centrifuged at 3000× The antioxidant enzymes, such as glutathione peroxidase (GPx), superoxide dismutase (SOD), total antioxidant capacity (TAC), and thiobarbituric acid-reactive substances (TBARS) as an index of lipid peroxidation in blood were also determined. The antioxidant indices of blood samples were determined according to the manufacturers’ instructions using commercial assay kits .ij is the measured value, μ is the overall mean effect, Di is the ith treatment effect, and εij is the random error associated with the jth rabbits assigned to the ith treatment. While, data of serum biochemical parameters and serum antioxidants status were analyzed as a randomized complete block design using PROC MIXED of SAS. The obtained data of rabbits were subjected to factorial analysis of variance (2 × 2) according to the following model:i is the effect of the ith level of factor treatment, βj is the effect of the jth level of factor time, and εijk is the random error associated with the kth replicate in cell . Differences among means were considered to be significant at the p < 0.05 level, as determined using Duncan’s multiple range test.Data were statistically analyzed using the General Linear Model procedure of the Statistical Analysis System . Data obSarcoptes scabiei var. cuniculi   . Observas, 1758) a, legs Is, 1758) b, as wels, 1758) c.p < 0.05) improvement in growth performance parameters (p < 0.05), followed by IVM + 1 mg of TE. The improvement in growth performance parameters in rabbits treated with IVM + 1 mg TE was comparable to that of the control group, with the exception that the BWG in the IVM + 1 mg TE group was higher than in the control group, while the rabbits treated with IVM + 2 mg TE had better growth performance than the other treatments. The group treated with IVM alone had the highest mortality rate followed by the IVM + 1 mg of TE group, while the lowest mortality rate occurred in the IVM + 2 mg of TE group that was similar to that of the control.Both doses of TE inclusion used in the study were effective against the negative effect of IVM alone on rabbit performance during sarcoptic mange treatment, and showed a significant (rameters . When cop < 0.05) when injectable IVM was used alone. There were no significant differences in nutrient digestibility between the control group and IVM + 2 mg TE group. Rabbits treated with IVM + 1 and 2 mg of TE demonstrated progressive recovery manifested by improved NI, DN, RN, DN/NI, RN/NI, and RN/DN, which was verified by comparison with the control group on day 30   . The totp < 0.05) TBARS levels, but lower TAC, SOD, and GSH-Px levels when compared with the control (p < 0.05). The increased TBARS of these groups was significantly (p < 0.05) lowered in comparison with the value obtained on day 7, and this level became comparable to those in the control rabbits by day 30 post-treatment. The levels of TAC, SOD, and GSH-Px were significantly (p < 0.05) higher in infested rabbits treated with IVM + 1 and 2 mg of TE when compared with the values obtained on day 7, and were found to be comparable to that of the control rabbits. However, the rabbits treated with IVM alone could not achieve comparable values to those of the control rabbits by day 30 post-treatment.On day 7, infested rabbits treated with either IVM alone or IVM + 1 and 2 mg of TE had significantly higher  amelioration in the aforementioned serum biochemistry towards normalcy more rapidly compared with the rabbits treated with IVM alone. Many factors can affect albumin circulation in the blood, including chronic liver disease, which can lower albumin levels in the blood and parasitism by ecto-or endo-parasites, which can cause inflammation and necrosis in the animals’ livers, resulting in a decrease in albumin production . CertainSarcoptes-infested rabbits in the IVM alone group were found to be in a state of severe oxidative stress; this result is consistent with previous scientific reports that have related altered antioxidant systems to states of oxidative stress in animals with parasitic skin infestations, such as sarcoptic mange [Psoroptes cuniculi. By day 30 post-treatment, TE may have a complementary impact with IVM injection as an antioxidant defense system of Sarcoptes-infested rabbits, significantly improving the healing effect of IVM and avoiding IVM’s unfavorable effects on rabbit performance and physiological function. TE indirectly enhances the miticidal effect of IVM due to a variety of factors, including immunomodulatory and antioxidant properties, which help the animal’s body fight off Sarcoptes mites.ic mange . Amelioric mange  reportedSarcoptes-induced oxidative discrepancy in rabbits. It can also help rabbits recover faster and improve Ivermectin’s miticidal efficacy by improving performance and compromised immunity. Furthermore, no adverse effects were observed in turmeric extract adjunctively supplemented rabbits, and the provided dose regimen of these supplements was found to be safe. The findings recommend turmeric extract as an adjunctive remedy along with the miticide (Ivermectin) to treat clinical rabbit’s sarcoptic mange. Large-scale research is also required to confirm these assumptions.Turmeric extract has a remarkable antioxidant potential contributing to the deterrence of


We want to understand how much of the Ca2+ signaling from Gi-coupled receptors is due to G protein promiscuity and how much is due to transactivation and crosstalk with other receptors. We propose a possible mechanism underlying the apparent switching between different G protein signaling pathways. We show that chemokine-mediated Ca2+ flux in HEK293T cells expressing CCR5 can be primed and enhanced by ATP pretreatment. In addition, agonist-dependent lysosomal exocytosis results in the release of ATP to the extracellular milieu, which amplifies cellular signaling networks. ATP is quickly degraded via ADP and AMP to adenosine. ATP, ADP and adenosine activate different cell surface purinergic receptors. Endogenous Gq-coupled purinergic P2Y receptors amplify Ca2+ signaling and allow for Gi- and Gq-coupled receptor signaling pathways to converge. Associated secretory release of GPCR ligands, such as chemokines, opioids, and monoamines, should also lead to concomitant release of ATP with a synergistic effect on Ca2+ signaling. Our results suggest that crosstalk between ATP-activated purinergic receptors and other Gi-coupled GPCRs is an important cooperative mechanism to amplify the intracellular Ca2+ signaling response.Many G protein-coupled receptors (GPCRs) signal through more than one subtype of heterotrimeric G proteins. For example, the C–C chemokine receptor type 5 (CCR5), which serves as a co-receptor to facilitate cellular entry of human immunodeficiency virus 1 (HIV-1), normally signals through the heterotrimeric G protein, Gi. However, CCR5 also exhibits G protein signaling bias and certain chemokine analogs can cause a switch to Gq pathways to induce CaThe online version of this article (10.1007/s10571-020-01002-1) contains supplementary material, which is available to authorized users. G protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors that mediate many important physiological processes. They are activated by ligand binding on the extracellular side and then couple preferentially to a unique Gα protein sub-family on the intracellular side to activate specific downstream signaling pathways. The Gα subunit in complex with the Gβγ subunits make up the heterotrimeric G proteins. There are four main families of Gα proteins: Gi/o, Gq/11, Gs, and G12/13, which we refer to as Gi, Gq, Gs, and G12. Once activated by the GPCR, the G protein dissociates into the Gα and Gβγ subunits, and both of these subunits can go on to mediate downstream effector activity by activating or inhibiting enzymes or channels. Some GPCRs do not couple to a unique Gα protein and have the ability to signal through more than one Gα protein class  is one such example of a GPCR that displays G protein-subtype signaling bias. The human immunodeficiency virus 1 (HIV-1) uses CCR5 as a co-receptor to infect immune cells /Ca2+ system via P2 purinergic receptors  or its breakdown product, adenosine (P1 A-type receptors). The P2Y family of purinergic receptors are GPCRs that couple to specific G proteins . Carbachol was from Abcam . Triton X-100 was from Polysciences . RANTES was from Peprotech  and RANTES analog, PSC-RANTES was a gift from Oliver Hartley (Université de Genève). HEK293T cells were from American Type Culture Collection (ATCC) . Dulbecco’s Modified Eagle’s Medium GlutaMAX (DMEM), FluoroBrite DMEM, Dulbecco’s phosphate-buffered saline without Ca2+ and Mg2+ (DPBS), Hanks' Balanced Salt solution (HBSS), and HEPES buffer were from Fisher Scientific . Lipofectamine 2000 and trypsin–EDTA  were from ThermoFisher Scientific . Fetal bovine serum (FBS) was from Gemini Bio-Products . Clear and clear-bottom black 384-well microplates were from Greiner . FLIPR Calcium 6 Assay and Flexstation II 384 Plate Reader were from Molecular Devices . 384-well transfer tips for the Flexstation were from Axygen .NF157, MRS2500, suramin, AR-C 118925XX, CGS15943, pertussis toxin (PTX), ATP, and ATPγS were from Tocris Bioscience . YM-254890 (YM) was from Wako Pure Chemical Industries . Bovine serum albumin (BSA) fraction V fatty acid-free was from Roche . Poly-2. Cells were transiently transfected with a synthetic vector encoding human CCR5 cDNA in pcDNA3.1(+) with a C-terminal 1D4 epitope tag (TETSQVAPA). Transfections were done directly ‘in-plate’ in 384-well microplates using Lipofectamine 2000 according to manufacturer’s instructions with some modifications. Briefly, 30 ng per well of CCR5-1D4 plasmid DNA was mixed in DMEM GlutaMAX (no FBS). In a separate mixture, the total Lipofectamine 2000 (2.5 µL per µg DNA) was mixed in DMEM GlutaMAX (no FBS) and incubated for 5 min. The Lipofectamine 2000/DMEM mixture was mixed with the DNA/DMEM and incubated for 20 min. Cells were then trypsinized, re-suspended in DMEM supplemented with 20% FBS, and counted. Cells were mixed with the DNA/Lipofectamine 2000/DMEM mixture, and directly plated onto 0.01% poly-d-lysine coated, black, clear-bottom, tissue culture treated 384-well plates at a density of 30,000 cells per well in 20 µL total volume.HEK293T cells were maintained in DMEM GlutaMAX supplemented with 10% FBS (passage numbers 5 to 16) at 37 °C under 5% CO2 for 24 h. On the day of the experiment, FLIPR Calcium 6 dye was diluted to a 4 × concentration in Hanks' Balanced Salt solution supplemented with 20 mM HEPES, pH 7.4 (HBSS-H) with 0.4% BSA. Then, 10 µL of the 4 × FLIPR Calcium 6 dye was added to each of the wells. Further, 10 µL of YM at a final concentration of 1 µM in HBSS-H with 0.4% BSA was added to the appropriate wells. The plate was placed back into the incubator at 37 °C under 5% CO2 for 1.5 h. In the meantime, the ligand plate was prepared in a clear 384-well microwell plate, with 5 × concentration of each ligand in HBSS-H with 0.4% BSA. The FlexStation II 384 Plate Reader (Molecular Devices) will take out 10 µL from these wells and inject into the appropriate wells in the assay plate with the cells. The ligands used were PSC-RANTES , RANTES , ATP , carbachol , ATPγS  as well as HBSS-H + 0.4% BSA (buffer only). After 1.5 h incubation, 10 µL of purinergic receptor inhibitors, NF157, MRS2500, suramin, and AR-C 118925XX at a final concentration of 100 µM in HBSS-H with 0.4% BSA were added to the appropriate wells. Note that 10 µL of apyrase at a final concentration of 0.2 U/well in HBSS-H with 0.4% BSA were added to appropriate wells as well. For single injection experiments, additional inhibitors PTX  and CGS15943  were also incubated.HEK293T cells were transfected as described above and incubated at 37 °C under 5% COt2, raw injection signal) minus mean signal between 0 and 20 s , and for injection 2 as the mean signal between 150 and 250 s  minus mean signal between 130 and 150 s   or 250 s (double injection experiments) time course at 2.5 s intervals. For the single injection experiments, 10 µL of the agonists PSC-RANTES, RANTES, ATP, carbachol, ATPγS or buffer were added to the cells at a pipette height of 35 µL at 17 secs. Similarly, for the double injection experiments, at 17 s, the first injection added 10 µL of ATP or buffer to the cells at a pipette height of 20 µL with two trituration steps of 20 µL each. At 150 s, the second injection added 10 µL of agonists to the cells at a pipette height of 20 µL with two trituration steps of 20 µL each. It is important to note that as we have two compound transfer steps, half of the plate was read at a time to account for the number of 384-well transfer tips per rack. This was also to ensure the cells were not incubating in the FLIPR Calcium 6 dye for over 2 h as this may lead to cytotoxicity. Relative fluorescence units (RFU) are calculated for injection 1 as the mean signal between 20 and 120 s (2+ indicator composed of a circularly permuted green fluorescent protein (cpGFP), calmodulin and the M13 domain of the myosin light chain kinase , was used for these transfections as the phenol red can interfere with GFP fluorescence signals. Cells were incubated at 37 °C under 5% CO2 for 24 h. On the day of the experiment, the media from half of the wells were aspirated carefully using the 8-port manifold. To those wells, 30 µL of HBSS-H with 0.4% BSA was added and to the other half 10 µL of HBSS-H with 0.4% BSA was added. Further, 10 µL of YM at a final concentration of 1 µM in HBSS-H with 0.4% BSA was added to the appropriate wells. The plate was placed back into the incubator at 37 °C under 5% CO2 for 1.5 h. In the meantime, the ligand plate was prepared as described above. After 1.5 h incubation, the purinergic receptor inhibitors were added to the appropriate wells. For single injection experiments, CGS15943 was added at three concentrations  for 30 min.GCaMP6s is a genetically encoded CaPrior to measurement, the plate was incubated at 37 °C for an additional 30 min in a pre-warmed FlexStation II 384 Plate Reader. Fluorescence readings were collected at 485 nm excitation and 535 nm emission using the same settings to stimulate the cells and collect the data as outlined above. The same analysis as above was conducted to obtain the corrected injection values.2 for 24 h in FluoroBrite DMEM, as described above. On the day of the experiment, the steps described above were followed to prepare the assay plate as well as the ligand plate. In the ligand plate, the CCR5 ligands were prepared along with 0.1% Triton X-100 . The FlexStation added 10 µL of ligand to the cells at 17 s (first injection) and then 10 µL of Triton X-100 at 150 s (second injection). Similar analyses were conducted to obtain corrected mean RFU values. Injection 1 is calculated as the mean signal between 20 and 120 s  minus mean signal between 0 and 20 s , as above. However, for injection 2 the mean signal between 0 and 20 s  is subtracted from the mean signal between 150 and 250 s  as we wish to calculate the total increase in fluorescence to quantify total cell count in each well.HEK293T cells transfected with 20 ng per well of CCR5-1D4 plasmid DNA and 10 ng per well of GCaMP6s plasmid DNA were incubated at 37 °C under 5% COData analysis was performed using SoftMax Pro Software version 5, Microsoft Excel, and GraphPad Prism version 8.2+ levels induced by stimulation by a second ligand. HEK293T cells were transiently transfected with CCR5 and intracellular Ca2+ levels were detected by FLIPR Calcium 6 assay dye, a permeable dye sensitive to Ca2+. The cells were first subject to an injection of either 10 µM of ATP or buffer at time 17 s. Then, 130 s after this first injection, cells were subject to a second injection of either PSC-RANTES , RANTES , ATP , carbachol , ATPγS  or buffer only, and the increase in intracellular Ca2+ levels were monitored. The concentrations of these agonists cause maximal Ca2+ flux. In particular, the EC50 values for PSC-RANTES (25 nM) and RANTES (32 nM) were previously determined by fitting a dose–response curve for CCR5-mediated Ca2+ flux stimulated by these two agonists  minus mean signal between 0 and 20 s , t2 −  t1. Similarly, injection 2 was calculated as the mean signal between 150 and 250 s (t4) minus mean signal between 130 and 150 s (t3), t4 − t3. The corrected mean RFU values for the first injection are plotted in Fig. S1. As expected, the buffer injection (black bars) did not give a significant increase in mean RFU. The ATP injection induces Ca2+ flux from the CCR5-expressing cells (gray bars), which is decreased by differing amounts upon incubation with the purinergic receptor inhibitors.From the raw data described in Fig. t4 − t3), comparing cells pre-stimulated with 10 µM ATP with those that were not  and is caused by injection artifacts, as explained above. However, we noticed that after ATP pre-injection, a buffer injection stimulated a significant increase in Ca2+ flux (7170 RFU) that is inhibited by incubation with purinergic receptor inhibitors  was subtracted from the respective second injections of all other ligands incubated with the same inhibitor. This was done for both buffer and ATP pre-injection signals and the results are shown in Fig. S2. The major trends for each ligand are reproducible compared with those pre-correction in Fig. We then plotted the mean RFU for the second ligand injections . Apyrase, AR-C 118925XX, MRS2500, and NF157 all decrease ATP-induced Ca2+ increase to about half of the Ca2+ increase of the untreated cells, to about 8000 RFU. Interestingly, suramin, the non-selective P2 purinergic inhibitor, is an exception and caused an ATP-induced Ca2+ increase of 12,600 RFU. ATP pre-stimulation does not increase the Ca2+ flux induced by a second ATP injection  to 11,200 RFU (ATP pre-stimulation). This is due to desensitization of the receptors and reveals that repeated application of the same ligand will reduce the Ca2+ response. Accordingly, the effects of some of the purinergic receptor inhibitors were not as significant as compared to the buffer pre-stimulation case. The Ca2+ flux was significantly reduced for cells incubated with NF157 and MRS2500, with mean RFU levels lower than those cells that received a buffer pre-stimulation.In the absence of any inhibitors (buffer incubation) and buffer pre-injection, ATP induces an increase in intracellular Ca2+ levels of 9900 RFU in the absence of inhibitors and without ATP pre-stimulation  to 15,600 RFU (ATP pre-stimulation) .RANTES, the native CCR5 ligand, induces an increase in intracellular Ca2+ levels of 17,200 RFU for both buffer and ATP pre-stimulated cases , ATPγS induces an increase in intracellular Ca2+ flux can be monitored in the same way  − mean RFU(Buffer)]. This effectively shows the increase or decrease in Ca2+ flux caused by ATP pre-stimulation. For each ligand, the mean RFU(ATP) − mean RFU(Buffer) was compared with that of the control buffer incubation case (gray bar) and assessed for significance using a Dunnett’s multiple comparison test  − mean RFU(Buffer) for the buffer control case  when cells are incubated with all inhibitors except for AR-C 118925XX, the P2Y2 receptor blocker.As mentioned before, a buffer injection should not stimulate an increase in Caase Fig. a. This p2+ flux from the second ATP injection. Apyrase, which hydrolyses ATP to ADP and AMP, serves as a good control, in which the mean RFU(ATP) − mean RFU(Buffer) becomes slightly positive and is significantly different from the buffer incubated cells (p < 0.1). The ATP from the pre-stimulation is digested by the apyrase and the receptors are not desensitized for the second injection. All other purinergic receptor inhibitors show a slightly less negative difference, as the effects of the ATP pre-stimulation are dampened, and receptor desensitization is not as prominent. It seems that the pre-stimulation of ATP does not affect the Ca2+ flux of cells stimulated by ATPγS, with most mean RFU(ATP) − mean RFU(Buffer) values hovering around zero  − mean RFU(Buffer).For ATP stimulation, the mean RFU(ATP) − mean RFU(Buffer) is negative for the buffer incubation control Fig. b. This iero Fig. e. This s2+ flux for CCR5-expressing cells stimulated with PSC-RANTES, and the mean RFU(ATP) − mean RFU(Buffer) is positive for the buffer incubation control . Perhaps incubation with NF157 does not allow a substantial number of purinergic receptors to be stimulated by ATP in order to amplify CCR5-Ca2+ flux. If most of the Gq activation is through P2Y11 activation, incubation with the other inhibitors, which specifically inhibit other P2Y receptors, should show a smaller effect. This is reflected in our data. These data support the hypothesis that the enhancement in Ca2+ flux seen with ATP pre-stimulation is due to CCR5 cross-talking to purinergic receptors, particularly P2Y11.An ATP pre-stimulation moderately increases Carol Fig. c. This p2+ flux and the largest difference in the mean RFU(ATP) − mean RFU(Buffer) for the buffer control  upon incubation with all inhibitors, indicating that RANTES stimulation of CCR5-expressing cells is more sensitive to the purinergic receptor crosstalk. The activation of all purinergic receptors by ATP pre-stimulation is able to amplify the Ca2+ flux, as specifically inhibiting any one decreases the amplification. Similar to what was seen for PSC-RANTES, pre-incubation with NF157 essentially abolishes the effect of the ATP pre-stimulation, suggesting that P2Y11 is strongly involved in the crosstalk and Ca2+ flux amplification.The ATP pre-stimulation has the largest effect on CCR5-expressing cells stimulated with RANTES, leading to a great increase in Carol Fig. d. This l2+ flux, is specifically affecting CCR5 and no other endogenous GPCRs.Lastly, ATP pre-stimulation has a slight increasing effect on CCR5-expressing cells stimulated with carbachol, leading to a positive mean RFU(ATP) − mean RFU(Buffer) for the buffer control Fig. e. Howeve2+ flux, confirming that our most significant observations are not altered upon this correction. Here, as the data are being manipulated twice, the error accumulates and reduces the statistical significances when comparing to the buffer control data in a Dunnett’s multiple comparisons test. This may be why the significant reduction in the ATP-dependent enhancement of PSC-RANTES-stimulated Ca2+ flux by the purinergic receptor inhibitors was lost, although overall reduction trends look reproducible.These observations were also seen when the data corrected for second buffer injection, as outlined above, were used to calculate the mean RFU(ATP) − mean RFU(Buffer) . Here, the purinergic receptor inhibitors all show a significant reduction of ATP-dependent enhancement of RANTES-stimulated Ca2+ flux  to be activated by ATP, and so the reduction should not be as drastic. Are the adenosine receptors really playing a critical role in cross-talking with CCR5 to enhance PSC-RANTES- and RANTES-induced Ca2+ flux?Interestingly, incubating cells with PTX, which inhibits Gi protein activation, completely abolishes RANTES-mediated CCR5-Calux Fig. c, d. On 2+ flux measurement. The FLIPR Calcium 6 dye uses “masking technology”, in which the Ca2+ dye is added to the cells alongside a loading buffer, which contains chemicals that reduce background fluorescence originating from residual extracellular Ca2+ indicator, media, and other components. Loading buffers may be hypertonic, which creates undefinable and uncontrolled conditions within the wells from dead or lysed cells due to this change is osmotic potential , the Ca2+-binding protein calmodulin (CaM), and CaM-interacting M13 peptide with a chromophore within the cpGFP β barrel. Upon CaM binding to Ca2+, there are Ca2+-dependent conformational changes causing increased brightness of the chromophore which can be conveniently monitored at the same wavelengths as those of the FLIPR Calcium 6 dye  which contributes adenosine to the wells.As we have shown that the GCaMP6s system works well in our hands to reproduce the effects seen from the purinergic receptor inhibitors on CCR5-mediated Ca2+ flux signals upon ligand injection. To account for this, we quantified the cell loss by once again, using the double injection feature of the FlexStation. We injected 10 µL of 0.1% Triton X-100  after the cells were stimulated by their respective ligands. This lyses the cells and releases GCaMP6s, which causes a measurable increase in GFP fluorescence as they come into contact with free extracellular Ca2+. In this way, the corrected mean RFU signals from the second injections quantify the total cell count in the washed or non-washed wells . The wash step reduced the fluorescence from 6400 RFU (non-washed) to 5800 RFU (washed) indicating about a 9% cell loss.We were concerned that aspiration and replacement of the media in a 384-well may lead to cell loss, which may affect the Ca2+ flux of cells incubated with the inhibitors, as compared to those that were not washed  are not affected by media exchange. This confirms our hypothesis that PSC-RANTES- and RANTES-stimulated CCR5 are cross-talking predominantly with P2Y receptors and not with the adenosine receptors. The large reduction from before may have been a combined effect of the FLIPR Calcium 6 loading buffer and accumulation of ATP, FBS and adenosine in the assay wells. We suspect that there may also be a non-specific effect on CCR5 at the highest concentrations of CGS15943, as the PSC-RANTES- and RANTES-stimulated Ca2+ fluxes are both reduced drastically when wells are not washed , and unsurprisingly, caused the most significant loss in ATP pre-injection mediated Ca2+ flux amplification.PSC-RANTES may not be as sensitive to this enhancement because it couples CCR5 to Gq effectively. There is still an amplification from the activated P2Y receptors, but the pre-injection of ATP does not have as pronounced of an effect. Cells incubated with AR-C 118925XX, MRS2500, and suramin do not show a significant decrease in the Ca2-adrenergic receptors (β2AR) transactivated Gq-coupled purinergic receptors and increased intracellular Ca2+ upon activation of β2AR by agonist isoproterenol (ISO)  and potency (increase in EC50) of ISO-promoted Ca2+ increase. This means that the extracellular mediator, ATP, released upon β2AR activation, is depleted and no longer able to transactivate P2Y11. Although we did not test any inhibitors of the P2X receptors, we similarly found that the highly expressed P2Y11 was predominantly responsible for the increase in intracellular Ca2+ and incubation with both NF157 and apyrase abolished the increase in intracellular Ca2+. This example of purinergic receptor crosstalk with a GPCR is slightly different from our example, however, as β2AR-mediated release of ATP stimulates P2Y11 to invoke a Ca2+ flux when there otherwise is none. In our example, chemokine-stimulated CCR5 induces Ca2+ flux, but there is an enhancement when ATP first stimulates P2Y11. Together with our results, these findings demonstrate that both Gs- and Gi-coupled GPCRs can crosstalk with Gq-coupled purinergic receptors activated by ATP. We speculate that this crosstalk could be the underlying mechanism of the reported off-target effects of YM and FR900359 on Gs and Gi signaling (Gao and Jacobson A recent study showed that the Gs-coupled β2+ channels to release Ca2+ stored in the smooth endoplasmic reticulum (sER)  pump Ca2+ into the sER from the cytosol to maintain the high concentration of Ca2+ inside. Once the cytosolic concentrations of Ca2+ increase, there is a cascade of intracellular activity. However, an important point is that Ca2+ is also required for the release of ATP to the extracellular side. N-ethylmaleimide-sensitive factor (NSF) is a AAA ATPase involved in fusion of the ATP-rich lysosome to the cell membrane, and the exocytotic ATP release is Ca2+ dependent  from secretory vesicles, which are full of ATP in the same way lysosomes are, and so their release leads to a concurrent liberation of ATP  or reuptake (monoamines). Thus, synergistic signaling of the two components will dramatically sharpen the precision of the local signal, which is essential for chemotaxis and also for neurotransmission.There is also a second mechanism of purinergic amplification. The second mechanism is from secretory release of GPCR ligands  and sequestering free βγ subunits  by acting as an inexpensive intermediary agonist to boost intracellular Ca2+ signaling via crosstalk with purinergic receptors.More broadly, the binding of Gαq to a PLC-β isoform (possibly PLC-β3) is thought to increase its affinity for the βγ subunits derived from activated δ-opioid receptors in NG108-15 cells, due to a relief of steric hindrance  from secretory vesicles. Both methods of ATP release allow for crosstalk between ATP-activated purinergic receptors and Gi-coupled GPCRs to amplify the intracellular Ca2+ signaling response.In conclusion, we have found that CaElectronic supplementary material 1 (DOCX 3166 kb)Below is the link to the electronic supplementary material.


Despite advances in the understanding of disease pathobiology, treatment for relapsed or refractory acute myeloid leukemia (R/R AML) remains challenging. The prognosis of R/R AML remains extremely poor despite chemotherapy and bone marrow transplants. Discoveries on recurrent and novel genetic mutations, such as FLT3-ITD and IDH1/IDH2, critical signaling pathways, and unique molecular markers expressed on the surface of leukemic cells have been under investigation for the management of R/R AML. Other than monoclonal antibodies, diabodies, and triabodies are new targeted therapies developed in recent years and will be the new direction of immunotherapy. Targeted agents combined intensive regimens can be viable options for salvage therapy and as bridges to allogeneic transplant. Future directions will focus on novel, efficient and targeted combinations, low-toxicity maintenance, and individualized precision strategies. Here, we review the major recent advances of targeted therapies in the treatment of R/R AML. Acute myeloid leukemia (AML) is a clinically and biologically heterogeneous disease, characterized by clonal proliferation of myeloid precursors. Acquired somatic mutations accumulated in hematopoietic stem and progenitor cells are the main pathogenesis of AML . ClinicaThe identification of unique molecular markers expressed on the surface of leukemic cells and discoveries on recurrent and novel genetic mutations are pivotal for the discovery of novel targeted therapies against R/R AML. Traditional chemotherapy acts not only on AML blasts but also on normal cells and produces toxicity. Targeted agents mainly aim at genetic or molecular lesions specific to, or enrich in, AML cells. This difference will make targeted therapy be more effective and less toxic than conventional chemotherapy. In recent years, the development and application of whole-genome sequencing have given us a macroscopic understanding of the AML gene mutation spectrum . Based oFMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations in patients with acute myeloid leukemia (AML) are associated with early relapse and poor prognosis. FLT3-ITD mutation can lead to constitutive autophosphorylation of FLT3 and activation of its downstream effectors including RAS/RAF/MEK, MAPK/ERK, PI3K/AKT, and JAK/STAT signal pathways, result in uncontrolled cell proliferation, survival, and differentiation of AML, while FLT3-ITD inhibitors can inhibit these downstream pathways through specific FLT3 inhibition . The firp = 0.002 and 83% vs. 50% of OR, p = 0.001), so as the 1-year OS  and the 1-year PFS . Subgroup analysis showed that the CR and OR rates in sorafenib+ chemotherapy+donor lymphocyte infusion (DLI) were higher than that in monochemotherapy , and they were similar to that in sorafenib+ chemotherapy and chemotherapy +DLI  [Sorafenib. Sorafenib is a multi-targeted small molecule inhibitor of RAF kinase, VEGFR-2, c-KIT, and FLT3, with activity of down-regulation of the MAPK pathway, Mcl-1 (Myeloid cell leukemia-1), and growth inhibition of AML cells with FLT3-ITD mutations -20. Sora> 0.008) . The com> 0.008) .FLT3 mutations. In this phase III trial, 3277 patients were enrolled to determine whether the addition of midostaurin to standard chemotherapy would prolong overall survival in untreated AML with FLT3 mutations. The mOS was significantly longer in the midostaurin group (74.7 months) than in the placebo group , as was event-free survival (8.2 vs. 3.0 months). The 4-year OS rate was 51.4% and 44.3%, respectively [FLT3-mut AML patients, several clinical trials have been conducted , and there was no difference in overall survival between the two arms [Lestaurtinib. Lestaurtinib demonstrated no surprising results in R/R AML. A phase I/II open-label trial of lestaurtinib single-agent therapy in R/R or poor-risk AML showed an ORR of 29% . The restwo arms .Quizartinib. Quizartinib was approved by the Ministry of Health, Labor and Welfare (MHLW) of Japan for R/R AML with FLT3 mutation. Compared with the first-generation TKIs, quizartinib has a stronger selectivity for FLT3. Monotherapy of quizartinib demonstrated a great efficacy in R/R AML, with a CR rate of 40-50%, and OS of 5-8 months . A phaseFLT3-mutated were enrolled to compare the efficacy and survival of gilteritinib and chemotherapy. This study showed that gilteritinib resulted in significantly longer survival and higher percentages of patients with remission than salvage chemotherapy with OS 9.3 months vs. 5.6 months and median event-free survival 2.8 months vs. 0.7 months, respectively. The ORR was 34.0% in the gilteritinib group and 15.3% in the chemotherapy group, and CRR was 21.1% and 10.5%, respectively. The most common AEs of grade 3 or higher in the gilteritinib group were febrile neutropenia (45.9%), anemia (40.7%), and thrombocytopenia (22.8%), but all these AEs occurred less frequently in gilteritinib group than in chemotherapy group [Gilteritinib. Gilteritinib is a small molecule dual inhibitor of FLT3/AXL . Phases py group ,50. The py group . In the py group . AccordiPexidartinib. Pexidartinib is a selective small-molecule kinase inhibitor of CSF1R, KIT, and FLT3-ITD. A phase I/II study of pexidartinib monotherapy in R/R AML demonstrated an ORR of 21% and CRR of 11%, which was lower than the ~40% to 50% CRc rates observed with quizartinib  or gilteIsocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) mutations are recurrent mutations in AML. Somatic point mutations in IDH1/2 lead to the excessive secretion of D-2-hydroxyglutarate (D-2HG). D-2HG plays an important role in the development of both hematological and solid tumors. It mainly promotes the occurrence of tumors by interfering with cellular metabolism and epigenetic regulation, thus leading to the expansion and differentiation of hematopoietic stem cells ,56. In amIDH1 AML, single agent treated with ivosidenib achieved a CR/CRh rate of 42.4% and mOS of 12.6 months [Ivosidenib. Ivosidenib (AG-120) is an oral, targeted, small-molecule inhibitor of mutant IDH1. It can restore normal differentiation and results in clinical responses in a subset of patients with mIDH1 R/R AML. The outgrowth of RTK pathway mutations and 2-HG-restoring mutations contributed to acquire resistance . In a pr6 months .Enasidenib. Enasidenib (AG-221) is an oral small-molecule IDH2 inhibitor that is approved by FDA in 2017 for treatment of adult patients with mutant-IDH2 R/R AML at an initial dose of 100 mg once daily. The phase I/II study of enasidenib induced overall responses in 40.3% of patients with R/R AML, with 19.3% of patients achieving CR. The mOS was 9.3 months, and 19.7 months in those with CR. Hyperbilirubinemia and IDH-differentiation syndrome (IDH-DS) were the most prominent toxicities . In a dop < 0 .001) and the 6-month projected OS was 80% versus 12% [Venetoclax. Venetoclax (VEN) is highly selective, oral small-molecule B cell leukemia/lymphoma-2 (BCL2) inhibitor. In patients with relapsed and refractory (R/R) AML, VEN had a modest single-agent activity (19% CR/CRi) . In contrsus 12% . Anotherrsus 12% . A retrorsus 12% . The oversus 12% . In a phrsus 12% . In a rersus 12% . Thereforsus 12% . The objDrug–drug interactions with moderate to strong CYP3A4 inhibitors, which are “azole” antifungals, are an important factor to considered for venetoclax plus HMA clinical trials, and these therapies were not permitted in most patients ,72. CYP3p = 0.0004). The most common AEs were cytopenias and gastrointestinal events (mostly grade 1-2) in glasdegib plus LDAC arm [Glasdegib is the hedgehog pathway inhibitor. Glasdegib in combination with low-dose cytarabine (LDAC) was approved by FDA for treatment of newly diagnosed AML in adults who are ineligible for intensive chemotherapy in November 2018 . For AMLLDAC arm . TavaresLDAC arm . SimilarLDAC arm . FurtherADC is a novel therapy that combines a monoclonal antibody with targeting specificity and a small molecule with high toxicity. CD33 antigen is a pleasant target for R/R AML. CD33 is expressed on more than 90% of AML patients, while expressed not on pluripotent hematopoietic stem cells, thus avoiding permanent inhibition of the hematopoietic system. Gemtuzumab ozogamicin (GO) is an ADC composed of an anti-CD33 monoclonal antibody covalently linked to the DNA-cleaving cytotoxic agent calicheamicin. The phase III study showed that monotherapy of GO can prolong the OS and recurrence-free survival of newly-treated or R/R AML patients who cannot tolerate standard chemotherapy . In R/R Not only ADCs but also bifunctional antibodies are under investigation. The results of a phase I clinical trial showed that the CR/CRi rate of CD123 × CD3 bifunctional antibody (Flotetuzumab) in patients with relapsed AML after chemotherapy was 31%, but no treatment response was observed in patients with refractory AML ,86. The The demethylating drugs decitabine or azacitidine have shown encouraging effects in the treatment of R/R AML, especially in elderly patients who are unsuitable for intensive chemotherapy or transplantation. Due to the different conditions of the enrolled patients, the ORR of HMA combined therapy is about 30%-60% ,25-27,36Targeted agents provide new options for the treatment of R/R/AML and obtain a certain remission rate. However, the median duration time of CR is short, and the prolongation of patient OS is not ideal. The results of targeted agents for R/R AML/MDS are listed in More than one driver mutation participates in the pathogenesis of AML. The genomic and epigenomic landscapes of AML reported 13 coding mutations in genes, with an average of 5 of these are recurrently mutated in AML . Previouin vitro and in vivo. This indicates that CAR T-cell immunotherapy in combination with small molecule inhibitor can be used to exert anti-leukemia efficacy [Tumor immunotherapy has been proven to have continuous immune surveillance. Studies of targeted therapy combined with immunotherapy are ongoing. Preclinical study showed that FLT3-inhibitor can upregulate the surface expression of FLT3 specifically on FLT3-ITD+ AML cells and enhance the recognition of FLT3-CAR T-cells efficacy . Preclinefficacy .In summary, the emergency of therapeutic strategies targeting mutated genes, cell surface markers, cell signal transduction pathways, and immune responses making R/R AML treatments gradually diversified and achieved well therapeutic effects. More individualized and precise treatment should be involved for the treatment of R/R AML in the future.


Airway smooth muscle (ASM) is a key resident cell type, responding to hyperoxia with increased contractility and remodeling , making the mechanisms underlying hyperoxia effects on ASM significant. Recognizing that fetal lungs experience a higher extracellular Ca2+ ([Ca2+]o) environment, we previously reported that the calcium sensing receptor (CaSR) is expressed and functional in human fetal ASM (fASM). In this study, using fASM cells from 18 to 22 week human fetal lungs, we tested the hypothesis that CaSR contributes to hyperoxia effects on developing ASM. Moderate hyperoxia (50% O2) increased fASM CaSR expression. Fluorescence [Ca2+]i imaging showed hyperoxia increased [Ca2+]i responses to histamine that was more sensitive to altered [Ca2+]o, and promoted IP3 induced intracellular Ca2+ release and store-operated Ca2+ entry: effects blunted by the calcilytic NPS2143. Hyperoxia did not significantly increase mitochondrial calcium which was regulated by CaSR irrespective of oxygen levels. Separately, fASM cell proliferation and ECM deposition (collagens but not fibronectin) showed sensitivity to [Ca2+]o that was enhanced by hyperoxia, but blunted by NPS2143. Effects of hyperoxia involved p42/44 ERK via CaSR and HIF1α. These results demonstrate functional CaSR in developing ASM that contributes to hyperoxia-induced contractility and remodeling that may be relevant to perinatal airway disease.Supplemental O However, prolonged hyperoxia is also recognized as contributing to pediatric airway diseases such as wheezing and asthma that persist beyond the neonatal period , cell proliferation, and extracellular matrix (ECM) deposition  enhances [Ca2+]i) regulation in human fetal ASM (fASM) from lungs of 18–22 week fetuses, a developmental period of rapid bronchial growth and proximate to neonatal survival in the ICU. However, compared to adult, the mechanisms of [Ca2+]i regulation and of O2 effects are less-studied. We previously characterized the suitability of isolated fASM cells in exploring regulation of [Ca2+]i  level of ~1.7 mM compared to adult (~1.2 mM) . The CaSR is well-recognized for its role in the parathyroid, kidney and bone toward regulation of body calcium levels  or 1 μM NPS2143   with perfusion used to alter [Ca2+]o or add agonists as necessary. Baseline, peak, and amplitude of [Ca2+]i responses were recorded and analyzed.Previously described techniques for fura-2 based fASM i responses to 10 μM histamine (p < 0.05). Here, cells in normoxia showed increasing [Ca2+]i responses with higher [Ca2+]o levels. This [Ca2+]o sensitivity was substantially increased in 50% O2-exposed cells. The contribution of CaSR to this increased sensitivity to [Ca2+]o was shown by the significant inhibitory effect of NPS2143 (p < 0.05).We previously showed that [Cao groups . However2+]ivia endoplasmic reticulum (ER) Ca2+ release involving IP3 receptors . In cells exposed to 50% O2 that showed higher [Ca2+]i responses, Xestospongin C had greater inhibitory effects (p < 0.05). In the presence of the CaSR blocker NPS2143, Xestospongin C effects were reduced  induced by sarcoplasmic reticulum Ca2+ depletion  is used to assess [Ca2+]i reflecting SOCE. fASM cells exposed to 50% O2 showed significantly higher SOCE (p < 0.05) which was substantially blunted by NPS2143 (p < 0.05).Given their proliferative tendency, fASM cells are more reliant on plasma membrane Ca2 with/without the CaSR inhibitor NPS2143. Interestingly, in rhod-2 loaded fASM cells, 50% O2 did not significantly influence mitochondrial Ca2+ responses to histamine . These data using MTS assay were corroborated by Western blots for PCNA  but interestingly not of fibronectin. Hyperoxia effects on these ECM proteins showed some sensitivity to [Ca2+]o but was not consistent between the collagens or to specific [Ca2+]o levels. Nonetheless, NPS2143 significantly blunted hyperoxia effects on these ECM proteins (p < 0.05).Exposure of fASM to 50% O2 increased p42/44 MAP kinase phosphorylation (p < 0.05) but did not alter p38 phosphorylation. NP2143 blunted hyperoxia effects on p42/44 MAP kinase phosphorylation (p < 0.05).Exposure to 50% O2 compared to 21% O2, consistent with being a hypoxia mimetic . Additionally, NPS2143 did not substantially alter nuclear-cytosolic HIF1α levels in the CoCl2 condition  on [Ca2+]i responses to bronchoconstrictor agonist, cellular proliferation and deposition of ECM in the context of airway hyperreactivity and remodeling. Data showing functional CaSR in fASM is consistent with our previous reports   channels in pulmonary artery smooth muscle . For immunofluorescence, proliferation, ECM or signaling studies, this was easily accomplished since cells were fixed or otherwise processed immediately following O2 exposure. For [Ca2+]i imaging, the fura-2 loading was also done in 50% O2 for this group, and the imaging protocols were relatively short-duration and thus it was assumed that any reversal of hyperoxia effects were minimal.Neonatal hyperoxia, i.e., O2 effects in specific cell types and the types of changes are dependent on extent of exposure structure/function, where with higher levels of O2 promote bronchopulmonary dysplasia  and increased ECM and thus more fibrotic airways  effects on [Ca2+]i of developing ASM to the CaSR. Our data first show that 50% O2 enhances fASM CaSR expression. Thus, even if there were no additional effects of O2, in vivo the increased CaSR would allow for greater responsiveness to the higher [Ca2+]o of the developing lung and thus enhance [Ca2+]i or other CaSR effects. We further show that hyperoxia increases CaSR sensitivity in increasing the amplitude of [Ca2+]i response to agonist (but not baseline Ca2+), which is suppressed by the negative allosteric modulator NPS2143. In this regard, our data show that hyperoxia enhances intracellular Ca2+ release via IP3 receptor channels (inhibited by Xestospongin C) consistent with a known role for the PLC/IP3 pathway in CaSR effects. Therefore, we expected NPS2143 to further Xestospongin C effects, but instead noted alleviation. While the reasons for this effect are not clear, it is possible that the presence of both Xestospongin C and NPS2143 results in compensatory responses of ryanodine receptor or calcium influx activation following histamine stimulation. More nuanced exploration in zero extracellular calcium  are necessary to determine such pathways that may or may not involve CaSR.The results of the present study now link moderate hyperoxia  although our data do not necessarily indicate a role for CaSR in that NPS2143 did not reverse these effects. However, NPS2143 increased HIF1α in hyperoxia only (effects of NPS2143 on HIF1 α nuclear-cytoplasmic ratio were not exhibited in normoxia) and NPS2143 did not reduce HIF1α activation in CoCl2, suggesting some linkage between these pathways that remain to be explored further and could be relevant at least to hypoxia-induced changes in the airway, perhaps even during normal development.While the focus of this study was the effect of hyperoxia on ASM, it is interesting to note that hypoxia in vivo efficacy of CaSR modulation in alleviating oxygen effects in the developing lung.Overall, the present study points to potentially important mechanisms by which CaSR inhibition could be used to blunt oxygen effects, particularly in the context of contractility and remodeling that is unlikely to be responsive to current therapies such as bronchodilators or even corticosteroids. Accordingly, future studies need to explore the The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Mayo Clinic IRB. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.AR and JR equally contributed to develop concepts, perform initial experiments, analyze data, and write manuscript drafts. CB, BP, MS, BR, and LN contributed to additional concepts, experiments, and analyses. CP, RM, PM, and YP collaboratively developed concepts and design, interpreted data, and helped with various drafts. YP prepared the final/submitted version that was reviewed and approved by all authors. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.