Consequently, the GPCR-mediated increased [Ca2+]i in acinar cells promotes saliva secretion, and this saliva moves into the oral cavity through the ducts. In this review, we seek to elucidate the potential of GPCRs, the inositol 1,4,5-trisphosphate receptor (IP3R), store-operated Ca2+ entry (SOCE), and AQP5, which are essential for salivation, as cellular targets in the etiology of xerostomia.Xerostomia, the subjective feeling of a dry mouth associated with dysfunction of the salivary glands, is mainly caused by radiation and chemotherapy, various systemic and autoimmune diseases, and drugs. As saliva plays numerous essential roles in oral and systemic health, xerostomia significantly reduces quality of life, but its prevalence is increasing. Salivation mainly depends on parasympathetic and sympathetic nerves, and the salivary glands responsible for this secretion move fluid unidirectionally through structural features such as the polarity of acinar cells. Saliva secretion is initiated by the binding of released neurotransmitters from nerves to specific G-protein-coupled receptors (GPCRs) on acinar cells. This signal induces two intracellular calcium (Ca Saliva is essential for maintaining a healthy oral environment and overall health. The salivary glands regulate salivation according to the surrounding environment and circumstances, and various factors can affect the volume and composition of saliva. Xerostomia (dry mouth), defined as a subjective feeling of oral dryness , is a teSaliva performs a variety of functions essential for oral and systemic health. These include moisturizing and lubricating the mouth; enabling gustatory and olfactory sensation; protecting the teeth and oropharyngeal mucosa; facilitating speech articulation; allowing mastication, swallowing, and digestion; and maintaining a balanced microbiome ,17,18,19Most of the secretion from the PAR glands occurs in response to stimuli, while the SM and SL glands are responsible for the majority of unstimulated saliva production . These gThe secretion of saliva from the three main salivary glands into the oral cavity occurs through their respective ducts. Stensen’s duct pierces the buccinator and connects the PAR gland to the buccal mucosa adjacent to the maxillary second molar ,26. WharTo understand the mechanism of salivation, it is important to understand the structure of the cell level constituting the salivary gland. The salivary glands are composed of various epithelial cells, including acinar cells, which produce saliva; ductal cells, which transport saliva to the oral cavity; and myoepithelial cells, which facilitate the secretion of saliva .Acini are formed by clusters of several pyramidal secretory cells, acinar cells, and are identified by the expression of markers, such as the water channel aquaporin 5 (AQP5) and the transcription factor muscle, intestine, and stomach expression 1 (Mist1) ,28,29,303−) and potassium (K+) and reabsorbing sodium (Na+) and chloride (Cl−), making the saliva hypotonic. They compose most of the duct systems in the major salivary glands i) stimulated by neurotransmitter–GPCR binding in acinar cells is a major trigger for salivation i in SM gland cells showed little difference in M1 KO mice compared with control mice but was greatly reduced in M3 KO mice and completely lost in double-KO mice i, and the histamine H1 receptor was expressed i, and stimulation of ion transport pathways. When there is an increase in [Ca2+]i in the acinar cells, ion channel activity is regulated in various domains of the cells, the AQP5 channels are translocated to the apical membrane, and water secretion occurs.The salivary gland cells regulate their secretions through neurotransmitter-generated Ca2+ release from the ER and Ca2+ influx via the plasma membrane) are required to increase [Ca2+]i in the salivary glands and maintain the saliva secretion state (2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). The initial increase in [Ca2+]i after external stimulation of acinar cells is triggered by the release of Ca2+ from the ER via the binding of cytosolic IP3 and IP3 receptors (IP3Rs) at the ER membrane. In exocrine gland cells, IP3R2 and IP3R3 are concentrated in the apical pole of the cell [2+]i increases in the apical region, spreads to the basal pole, and activates various ion channels and transporters to coordinate fluid secretion i into a sustained increase essential for long-term salivation [2+ depletion of the ER initiates the activation of SOCE [2+ sensor, which causes a conformational change when the concentration of Ca2+ in the ER is lowered and forms SOCE with Orai channels or transient receptor potential canonical (TRPC) channels expressed in the plasma membrane of acinar cells [2+ to enter the cell. Orai1 is the best characterized among the members of the Orai channel family, and it generates a highly Ca2+-sensitive, inwardly rectifying Ca2+ current when activated by STIM1 [2+-permeable nonselective cation channels, and all members are activated in response to PIP2 hydrolysis stimulated by neurotransmitters [2+ current and a significant loss of fluid secretion [2+ pool and GPCR signaling [The IPlar Ca2+ . Subsequlivation  , a family of transmembrane channel proteins, are responsible for water transcellular permeability in most living organisms . In mammmembrane ,93,94,95membrane , throughmembrane ,97. The membrane . Sympathmembrane ,98,99. AThe majority of the 550,000 patients who undergo radiation treatment for head and neck cancer annually and more than 4 million patients with Sjögren’s syndrome worldwide suffer from salivary gland dysfunction . More thDrugs are the most frequent cause of dry mouth  and are The likelihood of xerostomia increases with the total number of medications taken, regardless of whether the individual medications cause dry mouth. There are many other types of receptors for endogenous substances in the salivary glands that can be causes of drug-induced dry mouth, but anticholinergic actions contribute significantly. Synergistic effects of drug combinations contribute to xerostomia; in addition, although saliva flow does not necessarily decrease with age, older people are more likely than younger people to develop xerostomia due to the increased prevalence of chronic conditions requiring pharmacological treatment ,113. DruSystemic diseases that affect the salivary glands can cause salivary dysfunction, resulting in xerostomia  Table 2Table 2. Radiation therapy is an important treatment for patients with head and neck cancer. However, the salivary glands are often inadvertently irradiated and damaged because they are within the irradiated area . CumulatSalivary gland tumors are uncommon, representing less than approximately 5% of all cancers of the head and neck . SurgeryBoth unstimulated and stimulated salivation decrease with age in humans . Histolo1 and M3 mAChRs. Head and neck radiation-treated patients with established hyposalivation respond minimally to systemic sialagogues [Systemic sialagogues mimic the neural signals that stimulate saliva production in the epithelium. Anterior sialagogues mentioned by the FDA and the National Institute for Health and Care Excellence include pilocarpine and cevimeline ,110, whilagogues . In addilagogues ,10,11.Saliva substitutes or artificial saliva are often prescribed to temporarily relieve xerostomia. They generally contain a thickening agent and have protective properties but poor antibacterial and antifungal properties . Most saMouth rinses, mouthwashes, and toothpaste can provide short-term relief from dry mouth and keep the patient’s mouth, teeth, and gums healthy. Such mouth rinses can greatly increase saliva volume and improve pH buffering . However2+ signaling. Therefore, it is expected that regulators controlling the exocrine intracellular Ca2+ mechanism contribute directly to salivation. Key components involved in intracellular Ca2+ signaling include GPCR, IP3R, SOCE, and AQP5, all of which can be targets of medication or diseases. Conversely, acinar cell-specific GPCR agonists, such as mAChR and P2Y2R agonists, are potential therapeutic candidates that can regulate intracellular Ca2+ signaling at specific cellular units.Normal salivation is important for oral and overall health and wellbeing. Xerostomia is clearly a problem faced by an increasing proportion of the population. Accordingly, elucidating the mechanisms leading to the loss of salivary secretion and those involved in functional rescue should be a major focus of salivary research in the future. Acinar cells are polarized and are responsible for the unidirectional movement of fluid. Cellular heterogeneity among gland types derived from RNA-seq data suggests that various cells are coordinated within the salivary gland. Further elucidating this diversity will require a detailed study of the components of each cell unit. Importantly, saliva secretion is precisely regulated by GPCR-mediated intracellular Ca


Multiciliated cell loss is a hallmark of airway epithelial remodeling in chronic inflammatory airway diseases including cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease. It disrupts mucociliary clearance, which fuels disease progression. Effective clearance requires an optimal proportion of multiciliated and secretory cells. This is controlled by Notch signaling such that between two adjacent cells the one that activates Notch becomes a secretory cell and the one that avoids Notch activation becomes a multiciliated cell. Consequently, blocking Notch by a small molecule inhibitor of the γ-secretase enzyme that cleaves the Notch receptor for signal activation directs differentiation toward the multiciliated lineage. Thus, γ-secretase inhibitor (GSI) treatment may alleviate multiciliated cell loss in lung disease. Here, we demonstrate the therapeutic restoration of multiciliated cells by the GSI LY450139 (semagacestat). LY450139 increased multiciliated cell numbers in a dose-dependent manner in healthy primary human nasal epithelial cells (HNECs) during differentiation and in mature cultures, but not when applied during early epithelialization of progenitors. LY450139 did not impact stem cell proliferation. Basal and apical administration were equally effective. In healthy adult mice, LY450139 increased multiciliated cell numbers without detectible toxicity. LY450139 also increased multiciliated cells and decreased excess mucus secretory cells in CF HNECs and IL-13 remodeled healthy HNECs. LY450139 normalized multiciliated cell numbers in CF HNECs without interfering with the activity of CFTR modulator compounds. In summary, we demonstrate that GSI administration is a promising therapeutic to restore multiciliated cells and potentially improve epithelial function in a wide range of chronic lung diseases.NEW & NOTEWORTHY Our findings show that low-dose, short-term topical or systemic γ-secretase inhibitor treatment may lead to restoration of multiciliated cells without toxicity and potentially improve epithelial function in a wide range of chronic lung diseases. The airway epithelium is a patchwork of luminal multiciliated and secretory cells and an underlying layer of basal stem cells. It safeguards against inhaled pathogens, allergens, toxins, and debris by acting as a physical barrier, participating in immune surveillance and response, and via the mucociliary clearance of contaminants by the directional motility of cilia. In case of injury, the healthy epithelium can promptly and completely regenerate. In chronic inflammatory airway diseases including cystic fibrosis (CF), asthma, chronic obstructive pulmonary disease (COPD), and chronic rhinosinusitis (CRS), cycles of infection, injury, and inflammation lead to structural and functional epithelial changes termed remodeling –4. RemodDamaged and dysfunctional cilia and multiciliated cell loss are readily detectible both histologically and functionally in remodeled airways , 5. CiliThe Notch pathway is critical to maintain the proper proportion of multiciliated and secretory cells , 15. NotEvents linking Notch ligand presentation and downstream motile ciliogenesis in nascent multiciliated cells are only partially understood. However, Notch inhibition increases, while Notch activation blocks, multiciliated cell formation both in in vivo and primary culture models , 23, 24.Multiple GSIs have been developed to therapeutically block Notch signaling to treat a variety of cancers, graft versus host disease, endometriosis, and to restore auditory hair cells , 25, 26.Here, we demonstrate that LY450139 (semagacestat) , a GSI tpassage 0 (P0) or P1 basal stem cells was carried out as we previously described (https://doi.org/10.6084/m9.figshare.22304557).Primary human nasal epithelial cells were obtained by brush biopsy of the inferior turbinates with written informed consent from subjects recruited via the Cystic Fibrosis Center at Stanford University (Human Subjects Protocol No. 42710). Primary air-liquid interface (ALI) culture of nasal epithelial cells from escribed , 29. Briescribed , all froescribed . CF HNECFor wholemount immunofluorescence, HNECs were fixed in −20°C methanol or 4% paraformaldehyde for 10 min as previously described . TransweALI + 21 days HNECs were washed with warm PBS to remove mucus, then the Transwell filter was gently cut out of the plastic support. The filter was carefully folded along the center such that the folded edge exposed the mucosal surface for the evaluation of beating cilia. The folded filter was placed on a microscope slide inside a 100 µm mask, 100 µL of warm medium was added to maintain humidity, and a coverslip was immediately placed over the filter to seal the preparation. The slide was then placed on a heated stage at 37°C and imaged with a digital video microscope fitted with a Keyence VW-9000 series high-speed camera (Keyence). Images were captured at ×2,000 and 500 fps on multiple areas per filter. Average CBF (Hz) was calculated from kymographs generated from the high-speed videos. Ciliary length was measured from still images of the recording where the cilia were orthogonal to the apical surface.Foxj1/EGFP mice have been previously described  administration. In the first experiment, 0.1 mg/kg, or 1 mg/kg LY450139, 10 mg/kg DAPT or saline vehicle was administered twice daily for three consecutive days, then the mice were euthanized on day 7. In the second experiment, 1 mg/kg LY450139 or saline vehicle was administered once daily for five consecutive days per week for 3 wk, then the mice were euthanized on day 30. Body weight was monitored as a surrogate measure of gastrointestinal toxicity. See Supplemental Fig. S3A for more information. For both studies, the lungs were cryoembedded en bloc in OCT compound (Thermo Fisher). Cryosections were formalin-fixed, then labeled with anti-GFP and acetylated α-Tubulin antibodies and stained with DAPI to mark nuclei. Images were acquired using a Leica SP8 confocal microscope and analyzed by three independent blinded investigators as follows. In airways of approximately uniform size viewed in cross section, cells were first identified by DAPI signal, which provided the total luminal cells per airway metric. Cells were then scored either as multiciliated cells (GFP positive) or nonciliated cells (GFP negative). Anti-acetylated α-Tubulin signal was used as an additional identifying feature for multiciliated cells. Data are presented as multiciliated cells per total luminal cells. See Supplemental Fig. S3B for example image. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Stanford University School of Medicine in accordance with established guidelines for animal care.escribed , 33. 10–sc) measurements using standard methods (inh-172 (20 µM) to block CFTR current. For each agonist, signals were monitored until a plateau in current was noted before adding the next agonist. The delta-Isc in response to CFTRinh-172 was used as the chief readout for CFTR-mediated chloride transport.CF HNECs were differentiated from ALI + 0 to 21 days in the presence or absence of 125 nM LY450139 with and without 3 µM elexacaftor and 3 µM tezacaftor (Selleckchem). Transwells were mounted in an Ussing chamber for electrophysiological short circuit current (I methods . SolutioA). The use of homemade culture medium is important, as cultures grown in commercially available Pneumacult medium often contain 85%–95% multiciliated cells at the luminal surface . HNECs were treated with a low and high dose  of each compound during the entire 21 days of ALI differentiation (ALI + 0 to 21 days). The fraction of mature multiciliated cells was quantitated using wholemount anti-acetylated α-Tubulin antibody (marks ciliary axonemes) labeling. We found that, like DAPT, all GSIs we tested were able to induce the formation of extra multiciliated cells . DMSO control treatment had no effect on multiciliated cell number .The γ-secretase inhibitor (GSI) DAPT, is known to block Notch signaling and increase the proportion of multiciliated cells in an in vitro reconstituted respiratory epithelium , 24. AltC), overall morphology , or the number of cilia per cell (not shown). GSI treatment slightly increased ciliary beat frequency, although it remained within the range reported for human sinonasal cilia (C). As expected, both DAPT and LY450139 treatment led to a drastic reduction in cleaved NOTCH1 confirming that they act as inhibitors of Notch signaling in HNECs .Here, we further characterize LY450139, as of the four candidates, it is the most extensively studied GSI in both in vitro and in vivo models as well as clinical trials . We deteal cilia  , followed by differentiation induced, in part, by lifting to ALI . Thus, HTo test if active differentiation is required or if treatment of already differentiated epithelium is sufficient to gain more multiciliated cells, we carried out GSI treatment in mature HNECs. To ensure that the cultures are fully differentiated, we grew cultures to ALI + 30 days. We then treated cells with LY450139 or DAPT for 1 or 2 wk. We found that both GSIs and treatment times led to the robust induction of multiciliated cell formation C and D. Whereas ALI-cultured HNECs are exposed to drugs from the culture medium on their basal sides, we asked if GSI treatment applied to the apical surface is sufficient to induce multiciliated cell formation. This required the long-term culture of HNECs undergoing ALI differentiation under submerged conditions, which has been shown to suppress differentiation , 24. Indday 7 . To quantitate multiciliated cells, treatments were carried out in Foxj1/EGFP transgenic mice that express cytoplasmic GFP under the multiciliated cell-specific Foxj1 promoter (B). We found that 1 mg/kg LY450139 or 10 mg/kg DAPT lead to a modest, but statistically significant increase in the number of multiciliated cells, whereas 0.1 mg/kg LY450139 showed a trend toward more multiciliated cells that did not reach statistical significance . In homemade medium, we also showed that DAPT treatment during differentiation increases the multiciliated cell proportion in CF cultures to that of healthy HNECs (A). As observed in healthy HNECs, GSI-induced multiciliated cells in CF HNECs are structurally normal, and GSI treatment in both differentiating and mature CF cultures can mitigate multiciliated cell loss .We previously showed that CF HNECs cultured from unpassaged or early passage sinonasal basal cells in homemade medium model the reduced multiciliated cell numbers focally exhibited by donor tissue  , which are thought to serve as hotspots for CFTR activity in the airway epithelium (Approximately 90% of people with CF (pwCF) in the United States carry at least one mutant CFTR allele that is compatible with highly effective modulator therapy (HEMT), a triple combination of the small molecules VX-445, VX-661, and VX-770 (elexacaftor/tezacaftor/ivacaftor or E/T/I) . The majithelium . Thus, aThis study serves as proof of principle for the therapeutic targeting of multiciliated cell loss in chronic airway diseases by pharmaceutical grade γ-secretase inhibitors (GSIs) that block the Notch signaling pathway. We previously demonstrated that DAPT, a GSI tool compound increases multiciliated cell numbers in healthy, CF and CRS primary airway epithelial cell cultures . Here weThe primary goal of GSI treatment in chronic airway diseases would be to restore functional multiciliated cells to optimal numbers to improve mucociliary clearance, a vital host defense mechanism. We show that GSI treatment can increase multiciliated cell numbers after only a few days of treatment. LY450139 was able to drive multiciliated cell formation in differentiating as well as mature, homeostatic airway epithelial cultures, suggesting that GSI treatment can restore multiciliated cell numbers in both regenerating and intact portions of the airway epithelium. Importantly, GSIs increased multiciliated cells numbers in a dose-dependent manner, which suggests it may be possible to control the number of extra multiciliated cells that are generated. This is critical, as mucociliary clearance requires an optimal balance of multiciliated and secretory cells, and too many multiciliated cells could be detrimental to host defense. We show that LY450139 treatment during airway stem cell proliferation had no observable effect on subsequent differentiation, which suggests that it does not perturb the stem cell pool.GSIs induce multiciliated cell formation by impinging on cellular differentiation programs, so it is likely to be a disease agnostic treatment for a wide range of chronic inflammatory diseases. In COPD and asthma, where aberrant Notch activation drives mucous cell hyperplasia , 21, GSIWe previously showed that restoring multiciliated cells in CF primary cultures by GSI treatment led to improved barrier capacity as measured by transepithelial electrical resistance (TEER) . Notch i- transport correction induced by E/T/I. We speculate that E/T/I may even lead to greater improvement of CF lung and sinus function in a structurally and functionally normalized epithelium resulting from adjunctive treatment with a GSI.Despite the mitigation of remodeling phenotypes, chronic lung diseases will certainly still require other treatments such as antibiotics, anti-inflammatories, and/or CFTR modulators  in the case of CF. Thus, we propose GSIs as an adjuvant and not a stand-alone therapy. The majority of pwCF receive E/T/I as part of their standard of care treatment regimen . We showDue to their widespread and vital functions, therapeutic targeting of developmental signaling pathways such as Notch naturally carry the risk of on-target toxicity. Gastrointestinal toxicity of GSIs observed in clinical trials is thought to be related to Notch-dependent stem cell maintenance and differentiation in the gastrointestinal epithelium . PreclinGSIs represent only one therapeutic option to block Notch signaling. Anti-NOTCH function-blocking antibodies are currently in clinical trials for solid tumors . A combiFinally, it is important to note that the molecular mechanism through which GSI treatment increases multiciliated cell numbers remains unclear. Mechanisms downstream of Notch ligand presentation are poorly understood in any system, although there is evidence that ligand-receptor complexes are internalized by the signal-sending cell after receptor cleavage, then are degraded . InternaData will be made available upon reasonable request.10.6084/m9.figshare.22304557https://doi.org/10.6084/m9.figshare.22304557.Supplemental Tables S1 and S2 and Supplemental Figs. S1–S4: This work was mentored and financially supported by Stanford’s SPARK Translational Research Program. This project/publication was supported by the Stanford Maternal and Child Health Research Institute through Stanford’s SPARK Translational Research Program , by an AP Giannini Postdoctoral Foundation Fellowship (to E.K.V.), a Cystic Fibrosis Foundation Research Grant , and by the Ross Mossier CF Research Laboratory gift fund (to C.E.M.).1) funding from Stanford Maternal and Child Health Research Institute through Stanford’s SPARK Translational Research Program and an AP Gianni Foundation Postdoctoral Fellowship and 2) US Patent PCT/US2021046742. Z.M.S. discloses the following related relationships/activities/interests: 1) funding from a Cystic Fibrosis Foundation Research Grant and 2) consulting fees from Vertex Pharmaceuticals and AbbVie Inc. J.D.A. discloses the following related relationships/activities/interests: 1) funding from Stanford Maternal and Child Health Research Institute through Stanford’s SPARK Translational Research Program and 2) US Patent PCT/US2021046742. C.E.M. discloses the following related relationships/activities/interests: 1) funding from Stanford Maternal and Child Health Research Institute through Stanford’s SPARK Translational Research Program and Ross Mosier CF Research Laboratories Gift Fund, 2) consulting fees from Vertex Pharmaceuticals, 3) US Patent PCT/US2021046742, and 4) founder shares in Alentar BioSciences Inc. K.K., L.S.R.-H., J.M.S., N.S.J., and R.A.C. have no related relationships/activities/interests to disclose. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.E.K.V. discloses the following related relationships/activities/interests: E.K.V., J.D.A., and C.E.M. conceived and designed research; E.K.V., K.K., L.S.R.-H., J.M.S., Z.M.S., N.S.J., R.A.C., J.D.A., and C.E.M. performed experiments; E.K.V., K.K., Z.M.S., N.S.J., R.A.C., J.D.A., and C.E.M. analyzed data; E.K.V., J.D.A., and C.E.M. interpreted results of experiments; E.K.V. prepared figures; E.K.V. drafted manuscript; E.K.V., Z.M.S., R.A.C., J.D.A., and C.E.M. edited and revised manuscript; E.K.V., K.K., L.S.R.-H., J.M.S., Z.M.S., N.S.J., R.A.C., J.D.A., and C.E.M. approved final version of manuscript.


Despite recent advances, prognosis of acute myeloid leukemia (AML) remains unsatisfactory due to poor response to therapy or relapse. Among causes of resistance, over-expression of multidrug resistance (MDR) proteins represents a pivotal mechanism. ABCG2 is an efflux transporter responsible for inducing MDR in leukemic cells; through its ability to extrude many antineoplastic drugs, it leads to AML resistance and/or relapse, even if conflicting data have been reported to date. Moreover, ABCG2 may be co-expressed with other MDR-related proteins and is finely regulated by epigenetic mechanisms. Here, we review the main issues regarding ABCG2 activity and regulation in the AML clinical scenario, focusing on its expression and the role of polymorphisms, as well as on the potential ways to inhibit its function to counteract drug resistance to, eventually, improve outcomes in AML patients. Acute myeloid leukemia (AML) is an aggressive heterogenous disease arising from the accumulation and clonal expansion of somatic-driven mutations in CD34+/CD38- hematopoietic progenitors, which demonstrate increased proliferation, survival, and impaired maturation capacity ,2. It isDespite this, the occurrence of intrinsic or acquired drug resistance still results in AML being an aggressive disease and a major challenge for clinicians. Clinical multidrug resistance (MDR) is a multi-factorial phenomenon depending on host variations, drug–drug interaction, deregulation of cell death mechanisms, failure of DNA damage response and repair, epigenetic alterations, intratumor heterogeneity, and microenvironment alteration. MDR protects leukemia cell from immune surveillance and from the alteration in intracellular drug concentrations due to overexpression of membrane drug transporter proteins . AcquireThe general architecture of ABC proteins consists of two cytoplasmic nucleotide-binding domains (NBDs), which bind and hydrolyze ATP, and of two sets of hydrophobic transmembrane domains (TMDs), which transport substrates. ABC genes encode either a full transporter or a half transporter with a single TMD domain and a single NBD. Half transporters must dimerize as either homo- or heterodimers to form an active protein . WhereasThe present paper will focus on the role of one specific MDR protein, ABCG2, in AML, summarizing the current knowledge on the impact of its overexpression on disease outcome and on the possibility to counteract its action to improve the efficacy of anti-leukemic therapies.The ABCG subfamily includes five ABC half transporters: ABCG1, ABCG2, ABCG4, ABCG5, and ABCG8.ABCG1 is coded on chromosome 21q.22.3 and contributes to cholesterol transport and to cellular cholesterol homeostasis ..106].p = 0.024) [In drug-selected cell lines such as S1-M1-80 and MCF7/AdVp3000, unique mutations in amino acid position 482 makes cells highly resistant to mitoxantrone and doxorubicin. The replacement of Arg with Gly or Thr at position 482 increases rhodamine and anthracycline efflux compared to the wild-type counterpart . In cont= 0.024) . Many AB= 0.024) . The pre= 0.024) . StudiesAmong the factors mediating the acquisition of the MDR phenotype, the role of intercellular communications has recently emerged. In this setting, extracellular vesicles (EVs) play a pivotal role . EVs areDifferent approaches to overcome ABCG2-mediated MDR have been proposed. Despite some positive results obtained in preventing ABCB1 binding ,130, theAspergillus fumigatus, extensively used in experimental settings to test drug sensitivity in ABCG2 overexpressing cells [So far, the most used method remains an inhibition of the efflux function, re-sensitizing resistant cells to conventional anticancer drugs. The first identified functional inhibitor of ABCG2 was fumitremorgin C (FTC), a mycotoxin produced by ng cells . Since tng cells . Some ofng cells .It must be underlined that many of the molecules with inhibitory activity work only at high concentrations, often not attainable in clinical use. Among novel molecules, Ko143 (an FTC derivative), chromone derivatives, and a recently discovered indenoindol derivative are the most promising agents, having an IC50 in the nanomolar range . Many ABIt must be underlined that, in leukemic cells, ABCG2 expression is significantly lower compared to cell lines and that an efficient inhibition could be obtained even at lower concentrations. Moreover, moving from the original molecule, modifications done according to critical requisites for ABCG2 inhibition could increase the potency of ABCG2 inhibitors while maintaining the bound affinity, to avoid off-target toxicities. Many inhibitors, supposedly selective for ABCG2, actually act as dual inhibitors, either anti-ABCG2/ABCB1 ,144,145 Other new anticancer drugs have recently demonstrated an ABCG2 inhibitory effect in vitro. Sorf et al., demonstrated that ribociclib, a C4C/6 inhibitor approved for the treatment of locally advanced/metastatic breast cancer, inhibits ABCB1- and ABCG2-mediated daunorubicin and mitoxantrone efflux in AML cell lines at IC50 ranging between 1,4–3 μM, suggesting that combination therapy can revert MDR, especially in CD34+/FLT3-WT cases . In vitrDespite the discovery of several molecules with an inhibitory effect, the interest in the clinical development of effective ABCG2 inhibitors has been deprioritized, likely due to the disappointing results attained in ABCB1 inhibition, and at present no clinical trials including ABCG2 inhibitors are ongoing. This is in contrast with the evident contribution of ABC transporters in chemotherapy failure, at least in AML. In this setting, ABCG2 overexpression not only accounts for an increased relapse risk and poor survival after conventional therapy, but also identifies a subset of patients at higher risk of relapse after allogeneic transplantation, which is still recognized as the only “curative” option for high-risk disease . It is pKukal et al., provided an exhaustive review of the network of signaling pathways involved in ABCG2 regulation . This woWith this basis, new chemotherapy protocols could be designed by combining conventional drugs and ABCG2 expression modulators, with the attempt to reverse stem cell properties in overexpressing AML blasts.Great progress has been made in the past decades in deciphering ABCG2 structure, substrate transport, expression, and regulation as well as its impact on AML outcome. It is time to translate this knowledge into the clinics. New approaches should be developed to revert the negative effect of ABCG2 overexpression, including strategies addressed to down-modulate ABCG2 membrane expression, not only impair its efflux activity. The right inhibitor dose, the more appropriate administration timing (during induction therapy or after), the right chemotherapy association , and the management of off-target toxicity and of the frequent co-expression of two or more ABC proteins remain open questions. Moreover, standardized methods for a precise “quantification” of ABCG2 expression should be established, based on protein rather than on mRNA measurement, due to the heavy post-transcriptional regulation of the protein.


They are proved to be the mixture of normal CA2 pyramidal neurons, CA3-like neurons in CA2-CA3 mixed border, some CA2 interneurons and rarely few CA1-like neurons, which are probably the ones projecting to the revealed CA2 downstream targets, VMH, STHY and PMV in WT mice injecting this AAV/M1-Cre virus but not in Amigo2-Cre mice. Though it is still challenging to get a pure CA2 tracking and manipulation system, this tool provides a new, more flexible and extended strategy for in-depth CA2 functional study in the future.Mice hippocampus contains three prominent subregions, CA1, CA3 and DG and is well regarded as an essential multiple task processor for learning, memory and cognition based on tremendous studies on these three subregions. The narrow region sandwiched between CA1 and CA3 called CA2 has been neglected for a long time. But it raises great attentions recently since this region manifests the indispensable role in social memory. Its unique physical position connecting CA1 and CA3 suggests the potential novel functions besides social memory regulation. But the CA2 is too small to be accurately targeted. A flexible AAV tool capable of accurately and efficiently targeting this region is highly demanded. To fill this gap, we generate an AAV expressing Cre driven by the mini Map3k15 promoter, AAV/M1-Cre, which can be easily utilized to help tracing and manipulating CA2 pyramidal neurons. However, M1-Cre labeled a small percentage of M1The online version contains supplementary material available at 10.1186/s13041-023-01038-6. As one of the most studied brain regions, hippocampus is well known for its crucial roles in learning, memory and recognition due to the decades of unstopped study enthusiasm in this field. The best-known function of hippocampus is to act as the key regulator of episodic memory and spatial navigation , 2. ThesHowever, hippocampus contains another region, a narrow region sandwiched between CA1 and CA3, called CA2. CA2 has several distinguished features comparing to CA1 and CA3. The pyramidal neurons in CA2 have simple spines as CA1 pyramidal neurons but they have larger soma body size than CA1. While the pyramidal neurons in CA3 have thorny excrescences (complex spines) on their dendrites and similar soma body size as CA2 pyramidal neurons –10. CA2 The function of CA2 keeps mysterious for a long time. Until recently a bunch of studies indicate that this region actually plays unique roles in social memory formation –15. And To realize accurate targeting to CA2 regions, Hitti and Kohara et al. constructed two knock-in mouse lines respectively, Amigo2-Cre and Map3k15-Cre, specifically labeling CA2 pyramidal cells , 18. But6–8 week-old male C57BL/6 mice were kept group-housed in 4 per cage. Inbred mouse strain Tg (Amigo2-cre) 1Sieg (Amigo2-Cre) was acquired from the Jackson Laboratory and inbred in the Animal Core Facility of Nanjing Medical University  for experiments. The mice were housed under standard laboratory conditions with access to food and water ad libitum, stable temperature (22 ± 1 °C), and 12-h light–dark cycle (lights on at 07:00). All animal care and experimental procedures were followed by the Animal Experimental Ethical Guide of Southeast University and Animal Core Facility of Nanjing Medical University.The promoter of specific CA2 expression gene was identified based on Allen Brain Atlas and Mouse Genome Informatics. The sequences of mitogen-activated protein kinase kinase kinase 15  and regulator of G protein signaling 14  were downloaded from the NCBI. Map3k15 and RGS14 promoter were amplified using mouse DNA as a template. Activity of promoters was detected by OIBO company .And the following primers are used for the amplifications: 5′-gctggaactagggaaggatttc-3′ (Map3k15-2.3 kb promoter-F) & 5’-atcgtagaaggcatcgagcac-3’ (Map3k15-2.3kb promoter-R);5′-ggaggaagtcagggagggaggaaa-3′ (Map3k15-0.5 k promoter-F3) & 5’-gcggggctggcggcttcgaa-3’ (Map3k15-0.5k promoter-R3);5′-tgctccctatctctctgctttctg-3′ (RGS14-1.4 k promoter-F) & 5’-tctgccacaggaagtgtctcta-3’ (RGS14-1.4k promoter-R).The specific CA2 promoter construction plasmids  were packaged into Adeno-associated viruses (AAV)-M1-CRE (Map3k15-0.5 kb), AAV-M2-CRE (Map3k15-2.3 kb), and AAV-RGS14-CRE (RGS14-1.4 kb) by OBIO . AAV-CMV-EGFP (AOV022-1 AAV2/9), AAV-hSyn-DIO-mcherry (H4828 AAV2/9 CK1214 AAV2/9) were purchased from OBIO . AAV-Ef1a-DIO-EGFP , AAV-hSyn-CRE , AAV-Ef1a-DIO-hM4Di-mcherry (PT-0043 AAV2/9) were purchased from VTA . Clozapine-N-oxide  was purchased from APEXbio .12 vg/ml) and AAV-hSyn-CRE (1.09 × 1012 vg/ml) were injected with equal amounts of viral particles by controlling the injection volume, and the volume ratio of AAV-CRE and AAV-DIO was ensured to be 2:3. A syringe pump  was used to inject 100 nl of AAV into the dorsal CA2 region bilaterally at 10 nl/min. The needle was gradually withdrawn 20 min after injection. To prevent hypothermia after anesthesia, the mice were positioned on a heating pad through the procedure until fully awake. The injected mice were monitored daily for recovery status, body weight and infection.P30 mice were anesthetized with isoflurane (2–5%)  and had their scalp immobilized in a stereotaxic apparatus (RWD). After a precise craniotomy, the AAV was loaded in a glass pipette pulled by a heater, which was slowly targeted for injection into the dorsal hippocampus CA2 . AAV-M1-CRE , which consists of two parts, the center (29.7 cm diameter in the center of the box) and the periphery. Mice were allowed to move freely for 10 min in the box. The movement of mice was recorded and analyzed with a webcam and Ethovision XT software .stranger1-Timeempty (stage 1), and social difference score = Timestranger2-Timestranger1 (stage 2) were used to define the social novelty and social memory of the subject mice.The three-chamber behavior apparatus (60 cm long × 40 cm wide × 22 cm high) containing three compartments with equal size, which were separated by two walls with small doors (5 cm wide × 8 cm high). 20 min after intraperitoneal injection of CNO or vehicles, mice were placed in the device and allowed to explore freely for 10 min. The left and right chambers were placed with empty inverted uncapped cages before the experiment. Stranger mice  were placed in the inverted cages in the chamber where the subject mice were not habitually explored. During the social novelty stage, the time that the test mice spent sniffing the novel mice and the empty cage in the other chamber was compared. After 10 min, the test mice were isolated in the middle chamber for 10 min, and at the same time a second novel stranger mouse  was carefully put into the inverted cage that was originally empty in the previous stage. The subject mice were released from the middle chamber to explore the two compartments, and social preference was determined by comparing the time the mice took to sniff S2 and S1 in the last 10 min. Before introducing new subjects into the apparatus, the chambers and inverted cages were thoroughly cleaned with 25% ethanol. Behavior was monitored and scored for social novelty and memory which were represented by the anogenital and nose-to-nose sniffing time of the subject mice. Sniffing time was calculated by a trained observer, who was unaware of genotype and treatment of the mouse. The formulas, social difference score = Time2 bullets, animals were perfused intracardially with ice-cold PBS followed by 4% ice-cold PFA/PBS. Mouse Brains were fixed in 4% PFA overnight and then transferred into 30% sucrose for dehydration. After being embedded in Tissue-Tek® O.C.T., the brains were snap-frozen in liquid nitrogen and stored at − 80 °C. The brains were sectioned into the slices with the thickness of 30 µm using a Leica cryostat .After appropriate anesthesia with the COBrain serial sections were attached to glass slides coated with poly-L-lysine , and then washed for 15 min with PBS. Heated-antigen retrieval was performed for staining. In general, water-bathed sections were incubated at 96 °C for 5 min in 50 mM sodium citrate pH 8.5 . After being washed from the incubator for 5 min, the sections were permeabilized in 0.3% Triton X-100 in PBS (PBT) for 30 min. Subsequently, the sections were blocked at room temperature for 2 h with 10% Fetal Bovine serum (FBS) in 0.3% PBT (10% FBST) before overnight incubation with primary antibodies at 4 °C diluted with 10% FBST (1:500). The next day, the sections were washed 7 times for 35 min in 0.3% PBT and stained with secondary antibody (1:500) in 10% FBST for 1 h, followed by 5 times washing in 0.3% PBT for another 25 min. After the sections were stained with DAPI , the slices were mounted. Images were captured under a Zeiss confocal microscope LSM 700 or LSM 900 . The whole coronal brain image was taken by Palm Microbeam (Zeiss).+ cell number in RGS14 stained region was defined as total CA2 cells (Cell sum) and is normalized as 100%. The formulas for Fig. For RGS14, Calretintin, Calbindin, GAD67, Parvalbumin, NeuN, mcherry, CaMKII-α, PCP4 and GFP staining, the brain sections were incubated with the primary antibodies  and secondary antibodies . For CA2 spine imaging, RGS14 and CaMKII-α were costained on 1.5%PFA fixed brain slices with the thickness of 150 μm prepared by a vibratome. Sections were incubated with 5% FBS in 0.1% PBT for 1 h, then incubated with the primary antibodies against RGS14 (1:500) and CaMKII-α (1:500) in 3% FBS (0.05% PBT) overnight, and visualized using secondary antibody Alexa 555 (1:200) and Alexa 647 (1:200). The cells were counted on the complete coronal hippocampus images which were acquired through stitching the 10 × confocal microscopic diagrams together. RGS14ImageJ software  and ZEN  were used to count cells and measure soma size.Mice were perfused with 1.5% PFA, and the brains were postfixed by the same solution overnight. Then, 150 µm-thick sections were prepared by a vibratome and labeled by Dil-labeled Au particles using Gene gun. After 24 h at room temperature proceed with incubation with antibody to RGS14 (1:500) in 10% FBS for 16 h, and visualized using secondary antibody to mouse Alexa 647.Ordinary one-way ANOVA, post-hoc test Bonferroni’s test, and two-tailed unpaired Student’s test was used to assess statistical significance as described in the figure legends. GraphPad Prism 7  was used for data analysis. Photoshop CS6  was used to merge confocal images to fully present the hippocampus. All the statistical data in the graphs are presented as the mean ± SEM. p < 0.05 was considered significant. All p values were shown in the figures and n represented the number of animals.https://portal.brain-map.org), we decided to choose comparatively more restricted CA2 markers, RGS14 and Map3k15, for further promoter activity and specificity test. We wonder which gene and which promoter region are the best choice for CA2 highly preferential viral plasmid construction. Therefore, several vectors containing different promoter fragments from rgs14 and map3k15 with various lengths were constructed and tested. Using the luciferase assays for the promoter activity tests, we screened out a fragment from map3k15 with the length of around 500 bp, which displays the highest luciferase activity  in CA2  or hSyn driven EGFP expressed cells   mossy fibers from dorsal dentate gyrus  for M1-Cre treatment   mixture performed as before in dorsal CA2  layer also show the larger cell body size than CA1 pyramidal neurons in Py layers as previously reported [+RGS14− neurons from stratum oriens (Or) layer are also quantified and their body size is close to the body size of PV and CB interneurons but not CR interneurons in Py layer  layer of CA2 . But some of them are M1+RGS14− neurons. They can be detected in different area of CA2. Half of them are normal CA2 pyramidal neurons. But half of them are CA3-like neurons (grey) within CA2–CA3 mixed border (Z4 area) or CA2b region tightly close to the border (Z4–Z3 border). The case to detect CA1-like M1+RGS14− neurons in CA1–CA2 border is very rare comparing to the possibility to detect CA3-like M1+RGS14− neurons in CA2–CA3 border.We summarize what we found about CA2 M1The mixed usage of two viruses usually has a lower site accuracy than the single viral usage. It is consistent with our results in Fig. To test if M1 driven system can be utilized for anterograde neural tracing, AAV9/M1-Cre & AAV9/EF1α-DIO-mcherry or AAV9/M1-Cre & AAV9/EF1α-DIO-EGFP were injected and fluorescent signals were traced Fig. A. We sucLast we wonder if AAV9/M1-Cre virus suitable to be applied into neural activity regulation through chemogenetic manipulation in CA2 region. To test chemogenetic efficiency, AAV9/M1-Cre & AAV9/EF1α-DIO-hM4Di-mcherry were co-injected into CA2 Fig. A and sucOverall, we successfully developed a CA2 highly preferential Cre viral system to label and manipulate neural activity in CA2 region. It can efficiently drive CA2 pyramidal neurons as what Amigo2-Cre transgenic mice does. Meanwhile, it even displayed an extraordinary CA2 targeting accuracy comparing to most of current available targeting systems for CA2 and it is much more flexible to be utilized into different mice to realize the CA2 specific manipulation and gene deletion.+RGS14+ and M1+RGS14− pyramidal neurons in Py layers of CA2. M1+RGS14+ neurons occupy around 80% of total M1+ cells and they are all typical CA2 pyramidal neurons. For those M1+RGS14− neurons in Py layers of CA2, their cell body size and CaMKII presence still announce their typical CA2 pyramidal neuron identity. Further studies on spine morphology indicate that the M1+RGS14− CA2 like pyramidal neurons in CA2-CA3 border actually are the mixtures of normal CA2 neurons (with simple spines) and CA3-like neurons (with complex spines). Half of M1+RGS14− neurons are the normal CA2 pyramidal neurons, which occupy around 10% of total M1+ neurons in CA2. Some of M1+RGS14− cells  does localize in Or or SLu layer of CA2. They are probably GAD67+ interneurons with other novel markers need to be further explored. Meanwhile CA2 neurons displayed diverse morphology of pyramidal neuron cell bodies not only in M1+RGS14− neurons in CA2 Py layers, but also in M1+RGS14+ or regular CA2 neurons in Py layer sometimes. This finding transform our former impression on pyramidal neurons (more homogenic in morphology and function comparing to the interneurons) in hippocampus. But it is consistent with the previous reports that CA2 does have multiple pyramidal neuron types [Moreover, our mini-Map3k15 driving Cre system enriched pyramidal neuron types able to be manipulated in CA2. Using this system, we highly preferentially labeled CA2 neuronal cells containing both M1on types , 23.+ neurons in CA2 Py layers cannot colocalize with Amigo2. That is, this portion of M1+RGS14− pyramidal neurons could not be labeled by Amigo2-Cre mice. And it won’t be surprised if this group of pyramidal neurons have different axonal projections with M1+RGS14+ or Amigo2+RGS14+ pyramidal neurons in CA2. And this also could explain why we could trace CA2 projecting axons to VMH, STHY and PMV using our M1-Cre system whereas this result could not be recapitulated in Amigo2-Cre mice. Similar phenomena were observed in other Cell-type specific Cre models [One more thing needs to be noticed. Given the 97% overlapping between RGS14 with Amigo2 as previously reported, ~ 10% of total M1e models , 25. Thee models . But in e models , 27.To further increase the accuracy and usage of the CA2 specific viruses, the following strategies should be considered in the future. First, a more restricted AAV serotypes such as AAV2 or AAV5 is worth testing. Second, the viral tools for different purposes in CA2 region should be developed. An AAV/M1-Cre-EGFP (mcherry) for neural labeling/tracing and an AAV/M1-Cre-hM4Di (or hM3Dq) for neural activity regulation should be generated separately so that one viral usage can be applied instead of using mixed viruses. Third, it is uncertain if AAV/M1-Cre or Amigo2-Cre can accurately label ventral CA2. But the in-situ hybridization (ISH) data for Amigo2, RGS14 and Map3k15 in Allen Brain Atlas display the expression discrepancy of these genes in anterior and posterior part of the hippocampus which means they might not be able to correctly label the posterior CA2 as well as the anterior CA2. Otherwise, CA2 interneurons definitely need to be labeled and manipulated separately to study their own functions in the future. It is necessary to be mentioned that M1-Cre virus is obviously hard to label most of interneurons. GAD67/Map3k15 colocalization events observed in Fig. Additional file 1: Figure S1. for Figure 3 Quantitative analysis for soma size in different types of hippocampus neurons, and M1/PCP4 and M1/RGS14 co-staining comparisons. A Quantitative column chart displaying the soma size of cells in hippocampus. For measurements, images were examined using image J by delineating the soma fluorescence edge. The surface areas of CA1 pyramidal neurons, CA3 PN and CA2-EGFP+ were quantified by measuring AAV9/M1-EGFP-expressing neurons in IHC images. The surfaced areas of PV, CB and CR were quantified by measuring antibodies stained in IHC images. All means were calculated from no less than 5 brain slices of each biological sample. Data are presented as mean ± SEM. B Scatter chart displaying the proportions of M1+PCP4- and M1+RGS14- neurons in the total Map3k15-EGFP neurons. The proportions of PCP4-failed expressed and RGS14-failed expressed neurons in the EGFP were similar. C Pie charts displaying the proportions of M1+PCP4- and M1+PCP4+ in Map3k15-EGFP, and the proportions of M1+RGS14- and M1+RGS14+ in M1-EGFP. D Pie charts displaying the proportions of M1+PCP4- and M1+RGS14- neurons located in CA2 Or and Py. The proportions of M1+PCP4- in Py and Or are the number of PCP4-in Py or Or divided by total PCP4-failedexpressed neurons number. Figure S2. for Figure 5 Description of M1-EGFP+ neurons outside CA2 region, and expression pattern of the AAV9/hSyn-DIO-mcherry and M1-CRE co-injection system. A1-A3 Images of M1-EGFP co-stained with RGS14. Box in A1 displays RGS14-EGFP+ neurons considered as the neuron outside RGS14 stained region. Dash-line boxes in A2 and A3 display virus leakage in the case of small probability. D1-D4, E1-E4 Coronal serial sections of dCA2 from two different mice infected with AAV9/hSyn-DIO-mcherry and M1-CREfrom head to tail. Scale bars: 200 μm, 500 μm.


Xenopus oocytes are encompassed by a layer of follicular cells that contribute to oocyte growth and meiosis in relation to oocyte maturation. However, the effects of the interaction between follicular cells and the oocyte surface on meiotic processes are unclear. Here, we investigated Xenopus follicular cell function using oocyte signaling and heterologous-expressing capabilities. We found that oocytes deprotected from their surrounding layer of follicular cells and expressing the epidermal growth factor (EGF) receptor (EGFR) and the Grb7 adaptor undergo accelerated prophase I to metaphase II meiosis progression upon stimulation by EGF. This unusual maturation unravels atypical spindle formation but is rescued by inhibiting integrin β1 or Grb7 binding to the EGFR. In addition, we determined that oocytes surrounded by their follicular cells expressing EGFR–Grb7 exhibit normal meiotic resumption. These oocytes are protected from abnormal meiotic spindle formation through the recruitment of O-GlcNAcylated Grb7, and OGT (O-GlcNAc transferase), the enzyme responsible for O-GlcNAcylation processes, in the integrin β1–EGFR complex. Folliculated oocytes can be forced to adopt an abnormal phenotype and exclusive Grb7 Y338 and Y188 phosphorylation instead of O-GlcNAcylation under integrin activation. Furthermore, an O-GlcNAcylation increase (by inhibition of O-GlcNAcase), the glycosidase that removes O-GlcNAc moieties, or decrease (by inhibition of OGT) amplifies oocyte spindle defects when follicular cells are absent highlighting a control of the meiotic spindle by the OGT–O-GlcNAcase duo. In summary, our study provides further insight into the role of the follicular cell layer in oocyte meiosis progression. Xenopus amphibians, oocytes are covered with a single layer of flat stellate follicle cells, also named follicular cells with extended macrovilli that contact the oocyte surface  and MAPK–Erk2 pathways  and the subsequent O-GlcNAcylation of Grb7 avoiding its phosphorylation by the FAK–integrin signaling axis in response to EGFR activation. This novel unraveled mechanism allows a correct maturation and spindle anchorage to the plasma membrane. In addition, OGT and O-GlcNAcase (OGA) correct regulation is also necessary for meiotic maturation.In an attempt to understand both the function of Xenopus oocytes were unsheathed from their surrounding follicular cells and injected with RNA encoding the EGFR before nuclei were labeled with Hoechst 33258 (A). EGF stimulation of the receptor results in tyrosine autophosphorylation in both cases (B). One hour before stimulation with EGF, the adaptor protein Grb7 was microinjected at various concentrations. The occurrence of the maturation, or germinal vesicle breakdown (GVBD), is a faithful witness of prophase I to metaphase II transition. It is characterized by the appearance of a white spot at the animal-pigmented pole and is easily scored in oocytes with or without their surrounding follicular cells (C). Without EGF stimulation, neither EGFR expression with or without Grb7 microinjection nor Grb7 alone induces a significant GVBD , in oocytes with or without their surrounding follicular cells as previously described (D and E). The half-maximal time attesting for a maturation (GVBD50) is significantly shorter for unsheathed oocytes expressing EGFR and microinjected with 20 ng or 100 ng of Grb7 or not (respectively 5 h 40 min ± 17 min and 5 h 35 min ± 16 min) compared with oocytes expressing EGFR without Grb7 (7 h 10 min ± 1 7 min) (F). GVBD50 obtained for folliculated oocytes (FCs) had similar timing whatever experimental conditions were applied.st 33258 A. EGF stth cases B. One hoar cells C. Withouescribed , 34, 35.escribed , D and E1 7 min) F. GVBD50A). EGFR immunoprecipitations reveal that the discrepancy between DE and FC EGR/Grb7-expressing oocytes is not linked to the receptor phosphorylation state. No difference in the EGFR tyrosine phosphorylation is detected between DE and FC oocytes with 20 ng of Grb7 (B). Unstimulated EGFR displays no tyrosine phosphorylation. In addition, Grb7 is immunoprecipitated in the EGFR signaling complex in DE and FC oocytes. Integrin β1 and FAK are only linked to the EGFR complex in DE oocytes (B). OGT is only present in the EGFR complex from FC oocytes (B). When the same samples are submitted to glutathione-S-transferase (GST)-Grb7 pull down, DE oocytes display a tyrosine-phosphorylated form of Grb7, whereas this phosphorylation is not detected in FC oocytes for which Grb7 is conversely O-GlcNAcylated (B). To ascertain the specificity of the interactions, immunoglobulin G (IgG) controls for immunoprecipitation are shown in O-GlcNAc protein enrichment on succinylated wheat germ agglutinin (sWGA)-agarose beads (C). Control realized with 0.5 M free GlcNAc as competitor shows no Grb7 detection (C). In addition, integrin coprecipitates with EGFR and Grb7 in deprotected DE oocytes but not in protected FC oocytes (D).We further investigate the possibility of a crosstalk between integrin and EGFR relaying the activated signaling pathways in defolliculated oocytes (DEs). Integrin β1 and FAK are detected in DE, FC, and in isolated follicular cells A. EGFR i of Grb7 B. Unstim oocytes B. OGT is oocytes B. When tAcylated B. To ascse beads  followedse beads C. Controetection C. In add oocytes D.Figure E and F) impede the tyrosine phosphorylation of Grb7 in the EGFR signaling complex. Y188 when used alone only reduces the Grb7 phosphorylation state (F) showing tyrosine phosphorylation of Grb7 occurs on both sites Y188 and Y338 and requires FAK. In these conditions, no O-GlcNAcylation is detected on Grb7. With FC oocytes, competition experiments performed with Y338, Y338 added to Y188, or FAK antibodies could not abolish Grb7 O-GlcNAcylation (G). Control peptides formed by mismatched sequences flanking the tyrosine residue could not modify Grb7 tyrosine phosphorylation or its O-GlcNAcylation (H).To gain better insight into the differences observed in the recruitment of integrin β1 at the level of the EGFR complex and into the discrepancies of post-translational modifications of Grb7, competition experiments were led by using peptides mimicking the major FAK-phosphorylated site in Grb7 and with FAK antibodies. In DE oocytes, both Y188 and Y338 peptides and FAK antibodies , E and Fon state F showingcylation G. Controcylation H.50 (A and B). SH2 domains of Grb7 also reduce the percentage of GVBD obtained compared with controls (A). Into the EGFR complex of oocytes incubated with echistatin, EGFR exhibits no changes in its tyrosine phosphorylation status. Grb7 is recruited with OGT in the EGFR complex, whereas integrin β1 is not (C). Moreover, with the integrin inhibitor, Grb7 O-GlcNAcylation is restored, whereas its tyrosine phosphorylation is lost (D). Competition experiments performed with the SH2 domains of Grb7 lead to the dissociation of full-length Grb7 and integrin β1 from the EGFR complex, whereas, in return, OGT is recruited into the complex (D). Reverse experiments were performed in FC oocytes expressing EGFR, microinjected with Grb7, and incubated with an integrin activator . In these conditions, GVBD50 are significantly lower and similar to those obtained for DE oocytes (A and B). Early GVBD50 is associated with Grb7 and integrin β1 binding and a loss of OGT binding in the EGFR complex. In parallel, we observed that immunoprecipitated Grb7 is phosphorylated on tyrosine and its O-GlcNAcylation abrogated (C).Incubation with the integrin inhibitor echistatin (Sigma–Aldrich), a member of the disintegrin family, at 500 nM or microinjection of Grb7-competing SH2 domains to avoid Grb7 recruitment to the EGFR complex (60 ng) in DE oocytes significantly restores a classical GVBD50 , A and Bcontrols A. Into t1 is not C. Moreov is lost D. Compet complex D. Revers oocytes , A and Bbrogated C.Figure In DE oocytes expressing EGFR–Grb7, the meiotic maturation results in abnormal meiotic spindles that were not anchored to the plasma membrane , 46 Fig. Normal O-GlcNAcylation and phosphorylation, experiments are performed with inhibitors targeting OGA or OGT, respectively, Thiamet G . At 100 μM, Thiamet G induces a significant decrease in GVBD50 similar to a value observed with 15 ng of Grb7 in DE but not in FC (A and C). In addition, abnormal spindle formation is scored in DE oocytes but not in FC oocytes (A and B). About 100 μM of Thiamet G is further used as the lowest dose capable to reduce GVBD50 and to increase the formation of abnormal meiotic spindle in DE oocytes (C and 7A). The value of Gbr7 (15 ng) is chosen as the lowest value capable of significantly reducing the GVBD50 of the EGFR–EGF signaling from DE oocytes (C) and increasing abnormal meiotic spindle formation (A). The concomitant addition of Grb7 and Thiamet G lowers GVBD50 significantly compared with Grb7 or Thiamet G treatment alone and increases significantly the formation of abnormal spindles in DE oocytes (C and 7A), whereas no effects are observed in FC oocytes (D and 7B). Immunoprecipitations and sWGA pulldown to reveal this effect is associated with the recruitment of phosphorylated Grb7 without OGT in the EGFR complex in DE oocytes (C and D). Grb7 phosphorylation is present in DE oocytes after Thiamet G treatment (C and D). When the phosphorylation on Grb7 is prevented in DE oocytes (C), with the integrin inhibitor echistatin (500 nM), GVBD50 and the number of abnormal spindles are similar to those observed for Thiamet G alone (C and 7A). However, in those conditions in DE oocytes, Grb7 is not O-GlcNAcylated (C and D). FC oocytes display no significative decrease in GVBD percentage, GVBD50, or normal spindle formation under Thiamet G addition (B and D and 7B). Moreover, in FC oocytes treated with Thiamet G, Grb7 is O-GlcNAcylated in immunoprecipitated and sWGA pull-down experiments, and OGT is present in the EGFR complex (C and D).To understand the interplay between Grb7 ficantly , B and Dot in FC , A and C oocytes , A and B oocytes C and 7A. oocytes C and incormation A. The co oocytes C and 7A, oocytes D and 7B. oocytes , C and Dreatment , C and D oocytes C, with t G alone C and 7A.Acylated , C and D complex , C and DA and B). A lower concentration of OSMI-4 (15 μM) reduces the percentage of GVBD obtained in DE (to 50% compared with the control 83%) and FC oocytes (50% compared with the control 90%) (A and B). The concentration of OSMI-4 (15 μM) is further used in the experiments as the lowest value capable to induce an effect. The combined action of Grb7 and OSMI-4 (in DE oocytes) decreases the GVBD (to 27%) compared with OSMI-4 alone (50%), whereas in FC oocytes, the GVBD percentage is unaffected  (A and B). GVBD50 in DE and FC oocytes are not accelerated except for Grb7 in DE oocytes and Grb7–OSMI-4–integrin activator in FC oocytes (C and D). DE oocytes treated with Grb7–OSMI-4 never reached GVBD50 (C). In DE oocytes submitted to Grb7, OSMI-4, or Grb7/OSMI-4 and in FC oocytes treated with OSMI-4 or Grb7–OSMI-4, the number of normal spindles is significantly lower (A and B). In addition, in DE oocytes microinjected with Grb7 or Grb7–OSMI-4, Grb7 is phosphorylated, but not O-GlcNAcylated, and OGT is not present in the EGFR complex independently of OSMI-4 treatment (C and D). In FC oocytes, Grb7 is O-GlcNAcylated in the control but not after OSMI-4 or after integrin activator treatments (C and D). Moreover, Grb7 O-GlcNAcylation and OGT recruitment in the EGFR complex are lost after OSMI-4 and integrin activator treatments (C and D). FC oocytes treated with Grb7–OSMI-4–integrin activator that display an accelerated GVBD50 also show a phosphorylated Grb7 (D).OSMI-4 effect was determined on the GVBD induced by EGFR–EGF in DE and FC oocytes expressing Grb7 (15 ng) after its microinjection. A concentration of OSMI-4 (30 μM) blocks the GVBD in DE and FC oocytes , A and Brol 90%) , A and Band 57%) , A and B oocytes , C and Dd GVBD50 C. In DE ly lower , A and Breatment , C and Deatments , C and Deatments , C and Dted Grb7 D.Xenopus oocyte exert multiple roles during oogenesis and could be involved in the regulation of oocyte signaling during meiosis  was described in an O-GlcNAcylated EGFR complex . After anesthesia, ovarian lobes were surgically removed and stored in ND96 medium  at 14 °C until required.All animal experiments were performed according to the rules of the European Union Directive for laboratory animal experimentation 2010/63/EU. The protocol of this study was approved by the local institution . Mature To remove the layer of follicular cells, stage VI oocytes were defolliculated by partial ovarian digestion with collagenase A treatment for 30 min (1 mg/ml) followed by manual microdissection under a stereomicroscope. For oocytes that remained surrounded by their follicular cells, the outer layer was locally pinched opened manually in the equatorial region using a watchmaker's forceps (Moria) to reveal a tiny area of the plasma membrane to allow further microinjection. Sixty nanograms of mRNA of the human EGFR  were micThe human EGFR pOBER vecto was linearized with NotI, and capped cRNA was transcribed using SP6 RNA polymerase  , 33. Grb50) was determined to enable the comparison between the maturation kinetics of 10 to 20 oocytes from three different females in each experiment. In the case of the phenotypic absence of the white spot, oocytes were heat fixed for 15 min at 100 °C and bisected along the animal–vegetative axis to ascertain the presence of the germinal vesicle. Statistical significances (mean ± SD) were determined by two-way ANOVA followed by Dunnett’s multiple comparison tests .Oocytes displaying a white spot, a rise of the germinal vesicle at the animal pole reflecting meiotic progression from prophase I to metaphase II, were individually scored 12 h after EGF stimulation , 86. The2, 1 mg/ml bovine serum albumin, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 μg/ml soybean trypsin inhibitor, 10 μg/ml benzamidine, 1 mM PMSF, 1 mM sodium vanadate, with a ratio of 10 oocytes/100 μl of buffer. After a 12,000g centrifugation for 10 min at 4 °C, the supernatant was denatured in 2× Laemmli buffer  at 96 °C for 3 min. Proteins were separated by 4 to 20% SDS-PAGE gels  for 1 h at 200 V in denaturing buffer  and transferred onto nitrocellulose membrane (Amersham Hybond) by wet transfer  for 1 h at 100 V. Membranes were blocked with 5% (m/v) low fat dry milk or bovine serum albumin in Tris-buffered saline (TBS) added with 0.05% (v/v) Tween and incubated overnight at 4 °C with specific primary antibodies: rabbit polyclonal antibodies were raised against integrin β1 , OGT ; mouse monoclonal antibodies were raised against EGFR , FAK , GST , phosphotyrosine , O-GlcNAc , and goat polyclonal antibody was raised against β-actin . After three washes of 10 min each in TBS–Tween, nitrocellulose membranes were incubated for 1 h with the appropriate horseradish peroxidase–labeled secondary antibodies: anti-rabbit or antimouse antibodies  or antigoat antibodies . After three washes in TBS–Tween for 10 min each, the signals were revealed with a chemiluminescent assay  on hyperfilms (Amersham hyperfilm MP). Follicular cells were removed manually from 30 oocytes and used for Western blot after homogenization in PYT buffer using a grinding pestle (1.5 ml tube). After centrifugation at 12,000g for 10 min, the concentration of the supernatants was evaluated using the Bradford assay (Bio-Rad) at 595 nm . Three independent experiments were performed on three different females.Oocytes were lysed in the following PYT homogenization buffer: 50 mM Hepes (pH 7.4), 500 mM NaCl, 0.05% (m/v) SDS, 0.5% (v/v) Triton X-100, 5 mM MgClg centrifugation for 10 min at 4 °C, pellets were resuspended and homogenized using Eppendorf micropestles (SA). After a 12,000g centrifugation for 10 min at 4 °C, lysates were precleared at 4 °C for 1 h with Protein A Sepharose  under gentle rocking. After brief centrifugation, supernatants were incubated at 4 °C for 1 h with antibodies raised against either EGFR (1/100), GST (1/100), mouse IgG (1/100), or rabbit IgG (1/100) under rotation and followed by incubation with Protein A Sepharose (20 μl of 50% bead slurry) for 1 h at 4 °C under rotation. After three washes with PYT buffer without detergent, the pellets were collected by brief centrifugation, resuspended in 2× Laemmli buffer, and heated at 96 °C for 3 min before SDS-PAGE and Western blots were performed.Oocyte lysates were obtained using PYT buffer as described in the Western blot section. After a 12,000g for 10 min at 4 °C, lysates were incubated with 50 μl of sWGA agarose beads (Vector Laboratories) for 2 h at 4 °C or with sWGA beads preincubated with 0.5 M free GlcNAc (TCI) for 1 h, to control the specificity of the reaction. sWGA-bound proteins were washed four times and eluted from the beads in 2× Laemmli buffer before SDS-PAGE was performed.Twenty oocytes were lysed in 200 μl of radioimmunoprecipitation assay buffer  as described . After aNikon A1R (TISBIO).Abnormal spindles were detected as described previously , 50. Oocp < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, and ∗∗∗∗p < 0.001).Statistical significances (mean ± SD) were determined by two-way ANOVA followed by Dunnett’s multiple comparison tests (∗All data are contained within the article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


DOME receptor gene in Penaeus monodon (PmDOME) and examines the effects of PmDOME and PmSTAT silencing on immune-related gene expressions in shrimp hemocytes during white spot syndrome virus (WSSV) infection. PmDOME and PmSTAT were up-regulated in shrimp hemocytes upon WSSV infection. Suppression of PmDOME and PmSTAT showed significant impacts on the expression levels of ProPO2 (melanization), Vago5 (interferon-like protein) and several antimicrobial peptides, including ALFPm3, Penaeidin3, CrustinPm1 and CrustinPm7. Silencing of PmDOME and PmSTAT reduced WSSV copy numbers and delayed the cumulative mortality caused by WSSV. We postulated that suppression of the JAK/STAT signaling pathway may activate the proPO, IFN-like antiviral cytokine and AMP production, resulting in a delay of WSSV-related mortality.Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway plays an important role in antiviral immunity. This research reports the full-length  Penaeus monodon or black tiger shrimp is one of the most invaluable shrimp in the aquaculture industry contributing to economic prosperity. However, over the past years, the amount of shrimp export has reduced, mainly due to viral disease outbreaks. White spot syndrome virus (WSSV) is a major virus pathogen causing white spot syndrome and accounted for almost 100% mortality within a few weeks after infection1. So far, there is no proven method for 100% preventing WSSV outbreaks. When foreign particles or pattern pathogen-associated molecular patterns (PAMPs) attach to pattern recognition proteins (PRPs) or receptors (PRRs) in shrimp, the innate immune system is activated through intracellular signaling cascades. The humoral immune responses include the melanin synthesis by the prophenoloxidase (proPO) system, blood clotting system and the generation of circulating antimicrobial peptides (AMPs)2. The cellular responses, on the other hand, cause apoptosis, phagocytosis, nodule formation and encapsulation3.4. The JAK/STAT pathway plays a role in haematopoiesis, immune function, cell growth, differentiation and development5 as well as innate immune and adaptive immune systems6. In mammalian, the main components of the JAK/STAT pathway are wide and diverse range of extracellular ligands , transmembrane receptors , four JAK members  and seven STAT proteins 7. On the contrary, the JAK/STAT pathway of invertebrates is simpler than vertebrates with only a few components necessary for the signal transduction8. In Drosophila, the JAK/STAT pathway consists of three unpaired ligands 9, a receptor (Domeless or DOME)10, a JAK (Hopscotch or HOP)11 and a STAT (STAT92E)12. The binding of ligand to the Domeless receptor activates the receptor associated JAK tyrosine kinase Hopscotch, leading to phosphorylation of the receptor/JAK complex, which subsequently phosphorylates STAT92E dimer. Once the activated STAT92E dimers are translocated into the nucleus, they can bind to consensus DNA recognition motifs in the promoters, resulting in a transcription activation13. Several genes including TotA, TotM and virus induced RNA-1 (vir-1) contain STAT-binding sites in their promoter and are activated by the JAK/STAT pathway. Moreover, multiple regulatory proteins have been reported in Drosophila such as protein inhibitor of activated STAT (PIAS), protein tyrosine phosphatases (PTPs) and the suppressor of cytokine signaling (SOCS), the gene expression of which is activated by STAT and thus creates feedback inhibition of the pathway14.The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway was first identified in mammalian systems and shown to transduce the various cytokines and growth factor signalsLitopenaeus vannamei, LvDOMELESS enhanced the wsv069 promoter activity by acting on the STAT-binding motif and knocking down the LvDOMELESS, resulting in lower cumulative mortality of shrimp and less WSSV copies15. Meanwhile, LvJAK silencing caused a higher mortality rate and increased the WSSV viral load16. It was reported that shrimp STAT was hijacked by WSSV to enhance the viral immediate early gene expression17.In Pacific white shrimp Penaeus monodon (PmDOME) and investigate the role of the JAK/STAT pathway during WSSV infection. RNA interference techniques and mortality study were employed and the transcription levels of genes in the Toll, Immune Deficiency (Imd), JAK/STAT, interferon regulatory factor (IRF)-Vago, proPO and AMPs were examined under the silencing of PmDOME or PmSTAT during WSSV challenge. This work might lead to a better understanding of the characteristic of PmDOME and the relationship between the JAK/STAT pathway and the immune-related genes in response to WSSV infection in the black tiger shrimp.In this work, we report the full length of Domeless receptor in the black tiger shrimp PmDOME (2038 bp) was retrieved from the P. monodon EST database. The complete full length of PmDOME was obtained by PCR amplification using specific primers (Supplementary Table PmDOME is 5102 bp including 314 bp of the 5′-untranslated region (5′-UTR), 597 bp of 3′-UTR containing a polyA signal sequence (ATTAAA) and 4191 bp of the open reading frame (ORF) that encoded a putative protein of 1396-amino acid residues  is 5.81. The ORF of PmDOME shared high sequence identity with Pacific whiteleg shrimp LvDOMELESS 15 and DOME from Marsupenaeus japonicus, MjDOME 18. Meanwhile, PmDOME exhibited 21.92% sequence identity with Drosophila melanogaster domeless  and 28.57% sequence identity with human cytokine receptor (CAA41231.1).The partial nucleotide sequence related to PmDOME was clustered into the group of invertebrates and closely related to those from L. vannamei and M. japonicus  phylogenetic tree based on the deduced amino acid sequence. The bootstrap sampling was reiterated 1000 times and demonstrated that cus Fig. A. NetNGlPmDOME possessed 24 residues of a signal peptide, a cytokine binding motif, four fibronectin type III (FNIII) domains and a transmembrane domain. Figure PmDOME and others from seven closely related species. PmDOME shows four FNIII domains, similar to Pacific whiteleg shrimp LvDOMELESS and Chinese mitten crab EsDOME. In contrast, kuruma shrimp MjDOME displayed five FNIII domains, similar to redclaw crayfish Cherax quadricarinatus DOME (CqDOME), DmDOME and cytokine receptor from mud crab Scylla paramamosain (SpCR). Previously, DmDOME was shown most similarities to the interleukin-6 (IL-6) receptor family10. In vertebrates, IL-6 receptors belong to the type I cytokine receptor family, identified by the cytokine binding homology region (CHR). CHR possesses four conserved cysteine residues arranged in a CX-(9–10)-CXWX-(26–32)-CX-(10–15)-C sequence, forming two disulfide bonds at the N-terminal domain and a WSXWS motif at the C-terminal domain19. In this research, the amino acid sequences of PmDOME and cytokine receptors from 11 species were analyzed using the Clustal Omega tool. Multiple sequence alignment showed that PmDOME contained four cysteine residues at the N-terminal domain and an incomplete WSXWS motif at the C-terminus, similar to other invertebrate cytokine receptors. Meanwhile, the vertebrate cytokine receptors, HsCR, MmCR and OmCR showed four conserved cysteine residues, along with a full WSXWS motif -CXWX-(26–32)-CX-(10–15)-C sequence, phenylalanine is present in this particular sequence of CcCR and DmDOME. Based on sequence analysis, PmDOME shared several characteristics with the vertebrate cytokine class I receptors and invertebrate DOME receptors, suggesting that PmDOME is a signal-transducing receptor, involved in the JAK/STAT pathway.From protein motif prediction, PmDOME, PmJAK and PmSTAT transcripts in various tissues of healthy shrimp were determined by quantitative RT-PCR using the elongation factor-1α gene (EF-1α) as an internal control. PmDOME was expressed at the highest level in lymphoid, followed by eyestalk, hemocyte and gill, respectively, while the lowest expression level of PmDOME was found in hepatopancreas  technique was used to knock down PmDOME and PmSTAT expression levels. PmDOME mRNA levels in control and GFP dsRNA treated groups were up-regulated around 2.5- and 2-fold at 6 hpi and 24 hpi, respectively, in comparison to the unchallenged group. Similarly, PmSTAT transcription levels in control and GFP dsRNA injected shrimp increased by twofold at 6 h and 24 h after WSSV challenge. Although WSSV infection seemed to stimulate PmDOME and PmSTAT expression, the transcript levels of these genes remained low in the PmDOME and PmSTAT knockdown shrimp upon WSSV infection.As shown in Fig. PmDOME and PmSTAT knockdown shrimp were determined by qRT-PCR genes. These included genes in the signaling pathways , melanization process (ProPO2), IFN-like gene (PmVago5) and AMPs . As shown in Fig. ProPO2, Penaeidin3 and CrustinPm7 expressions significantly increased by 17, 28 and 14-fold at 6 h and 12, 16 and 2.5-fold at 24 h after WSSV challenge, in comparison with unchallenged shrimp. This suggested that ProPO2, Penaeidin3 and CrustinPm7 play an important role in WSSV defense. Other genes including PmSTAT, PmDOME, PmSOCS2, Vago5 and ALFPm3 were also up-regulated (> twofold) at 6 and 24 hpi. It is worth mentioning that PmDorsal and PmIMD expression levels, representing the Toll and Imd pathway, respectively, were insignificantly changed at 6 and 24 hpi. In addition, CrustinPm1 was up-regulated by 2.5-fold at 6 hpi but its transcript level was not substantially different at 24 hpi, compared with that of the control group.To investigate the role of the JAK/STAT signaling pathway during WSSV infection, the mRNA levels of immune-related genes in PmDOME-deprived shrimp also showed down regulation of PmSTAT and PmSOCS2 at 6 and 24 hpi , Vago5 (interferon-like protein) and AMP gene expressions.Consistent with CS2 Fig. E,F. SuppPmDOME and PmSTAT silenced shrimp were quantified by the detection of conserved VP28 gene using qRT-PCR, in comparison with those in WSSV infected control and GFP treated group. Clearly, WSSV copy numbers in PmDOME and PmSTAT silenced shrimp were significantly lower than that in control and GFP treated shrimp   Fig. A. Althou21. From tissue distribution, both PmDOME and PmSTAT transcript levels were high in the hemocyte  at 6 and 24 hpi, while the mRNA levels of Dorsal and PmIMD, representing the Toll and IMD pathway, in unchallenged and WSSV-infected shrimp showed slight differences. In addition, the gene expressions of ProPO2, Vago5, ALFPm3, Penaeidin3 and CrustinPm7 were up-regulated at 6 and 24 hpi in WSSV-infected shrimp, compared with unchallenged shrimp  and a late gene envelope protein, VP2826. Suppression of CqDOME also decreased CqSTAT phosphorylation, which is required for activation of the IE1 transcript17.Surprisingly, knockdown of either 27. For example, in red swamp crayfish Procambarus clarkia, injection with the recombinant proPO2 could significantly decrease 65% mortality of WSSV-infected crayfish and reduced the amount of WSSV copy number in hepatopancreas and gill28. In addition, the silencing of the proPO gene in freshwater prawn Macrobrachium rosenbergi makes them susceptible to WSSV29. It was reported that the proPO gene was highly up-regulated after LPS stimulation, while the lack of LvSTAT down-regulated the proPO expression. It was speculated that the JAK/STAT pathway activated the proPO system by controlling the differentiation of hemocytes to promote the humoral immune response30. In this research, PmDOME and PmSTAT silencing significantly enhanced the expression levels of proPO2 by 30–60-fold during WSSV infection  regulatory factor (IRF)-like gene identified in L. vannamei could be activated by WSSV and bind the 5′-untranslated region of L. vannamei Vago4 gene31. This suggested that IRF-Vago-JAK/STAT might exist in invertebrates, similar to the vertebrates IRF-IFN-JAK/STAT. Suppression of LvVago 4/5 led to an increase in shrimp mortality by WSSV. Obviously, this result is consistent with our research, showing that Vago5 expression was elevated when the JAK/STAT signaling pathway was disrupted  of average 3 g body weight were obtained from a shrimp farm in Chachoengsao Province, Thailand. Shrimp were acclimated in recirculating water tank system filled with air-pumped seawater with a salinity of 20 ppt at the temperature of 28 ± 4 °C. They were fed with a commercial diet twice a day for at least 1 week before the experiments. This study was conducted under the ethical principles and guidelines according to the animal use protocol 1923021 approved by Chulalongkorn University Animal Care and Use Committee (CU-ACUC). The biosafety guidelines were reviewed and approved by the Institutional Biosafety Committee of Chulalongkorn University (SC-CU-IBC-004-2018).Healthy black tiger shrimp  in the expressed sequence tag (EST) database of P. monodon (http://pmonodon.biotec.or.th)3. The full length of PmDOME gene was amplified from the hemocyte cDNA of P. monodon using the 5′ upstream region sequence (UPDOME-F1 primer) and 3′ downstream region sequence (DOME-R primer). The PCR conditions were as followed; (1) 96 °C for 5 min, (2) 25 cycles of 96 °C for 20 s, (3) 56 °C for 30 s, (4) 68 °C for 5 min, (5) a final extension at 68 °C for 7 min. The PmDOME PCR product was ligated into pGEX 6P-3 vector using an In-Fusion cloning kit (Takara) with the following conditions; 37 °C for 15 min, 50 °C for 15 min and 4 °C for 5 min; and then transformed into Escherichia coli TOP10 (Invitrogen). The recombinant plasmid named pGEX 6P-3-PmDOME was verified by sequencing. The full-length PmDOME gene was deposited in the NCBI GenBank (GenBank accession No. MW187497).The ORF of PmDOME was determined by ExPASy-Translate tool (https://web.expasy.org/translate/) and the protein motif was analyzed by SMART (http://smart.embl-heidelberg.de/) and GenomeNet (https://www.genome.jp/tools/motif/). The polyadenylation site in PmDOME sequences was predicted by Poly(A) Signal Miner (http://dnafsminer.bic.nus.edu.sg/PolyA.html). The predicted molecular weight of PmDOME was calculated by Compute pI/MW (https://web.expasy.org/compute_pi). Clustal Omega program was used to analyze the multiple sequence alignments . The phylogenetic analysis was performed using MEGA 11 software to construct a neighbor-joining phylogenetic tree based on the deduced amino acid sequences. The bootstrap sampling was reiterated 1000 times. The N- or O-glycosylation sites of PmDOME was predicted by NetNGlyc-1.041 or NetOGlyc-4.042 and by GlycoEP server43.The amino acid sequence of 44.WSSV was prepared from the gill tissue of WSSV-infected moribund shrimp using ultracentrifugation and membrane filtration as described in Jaturontakul et al.PmDOME dsRNA, PmSTAT dsRNA and GFP dsRNA were synthesized using T7 RiboMAX™ Express Large Scale RNA Production System (Promega). Two sets of specific primers each for PmDOME, PmSTAT and GFP genes were designed as shown in Supplementary Table P. monodon was used as a template to amplify PmDOME dsRNA and PmSTAT dsRNA, while the recombinant GFP plasmid was used as a template to amplify GFP dsRNA. The two PCR products each of PmDOME, PmSTAT and GFP genes were separately amplified by the specific primer pairs using the following conditions; 94 °C for 2 min, followed by 35 cycles of 98 °C for 10 s, 58 °C for 30 s and 68 °C for 30 s and a final extension at 68 °C for 7 min. Subsequently, the dsRNA of PmDOME, PmSTAT and GFP was produced in vitro using T7 RiboMAX™ Express Large Scale RNA Production System (Promega). The quality of synthesized dsRNAs was examined by 2% agarose gel electrophoresis and the concentration was determined by measuring absorbance at 260 nm and stored at − 80 °C for further use.Tissue samples from healthy and pathogen-infected shrimp were collected and homogenized in FARB buffer . Total RNA was extracted by following the manufacturer’s protocol. Five hundred nanograms of total RNA were converted to the cDNA by RevertAid First Strand cDNA Synthesis Kit . The cDNA was kept at − 20 °C for further use.PmDOME, PmJAK and PmSTAT gene expression levels in each tissue were identified by quantitative RT-PCR using 2 µL of tenfold diluted cDNA template and specific primers shown in Supplementary Table EF-1α) gene was used as an internal control. The expression level in the intestine was used as a control and set as 1.0.Shrimp tissues including hemocyte, eyestalk, epipodite, gill, heart, lymphoid, intestine and hepatopancreas were collected from 9 healthy shrimp and total RNA was extracted by Tissue Total RNA mini kit (Favorgen), followed by cDNA synthesis using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher). ® Universal qPCR Master Mix (NEB) in the following conditions: one cycle at 95 °C for 1 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 30 s, using specific primers as shown in Supplementary Table EF-1α) was used as an internal control. Melt curve analysis was performed at the end of the PCR thermal cycle to determine the specificity of amplification. The relative expressions of PmDOME and PmSTAT were calculated using a comparative CT method with the 2−ΔΔCT45. The data were shown as means ± standard deviations (SD). Statistical analysis was performed using one-way ANOVA followed by Duncan’s new multiple range test. The data were considered for statistical differences with the significance at p < 0.05.The quantitative RT-PCR (qRT-PCR) was carried out using an equal amount of cDNAs in the CFX96 Touch™ Real-Time PCR System and the Luna, shrimp with an average body weight of 3 g were injected with different dosages of either PmDOME dsRNA or PmSTAT dsRNA through intramuscular injection. A single injection of PmDOME dsRNA contained either 5 or 10 µg dsRNA per 1 g shrimp. Meanwhile, either a set of 10 µg dsRNA, followed by 10 µg dsRNA or a set of 10 µg dsRNA, followed by 5 µg dsRNA per 1 g shrimp was used in a double injection of PmSTAT dsRNA with 24 h interval between each injection. The dsRNAs were dissolved in 1X-PBS, pH 7.4 and the experiments were carried out in triplicate. The control group was injected with 1 × PBS (pH 7.4). Three shrimp hemolymphs in each group were collected at 24 h post injection. The hemolymph was collected using a sterile 1-mL syringe with 500 μL of anticoagulant, pH 7.0 . The hemolymph-anticoagulant mixture was then centrifuged at 800×g for 15 min at 4 °C to separate the hemocytes from the plasma.To verify the silencing efficiency of dsRNA in vivoPmDOME and PmSTAT silencing on immune-related genes upon WSSV infection, shrimp with an average body weight of 3 g were divided into ten groups with three shrimps per group. The experiment was carried out in triplicate. Shrimp in groups 1 and 2 were injected with 1 × PBS (pH 7.4), while shrimp in groups 3 and 4 were injected with 5 µg of PmDOME dsRNA per 1 g of shrimp; and shrimp in groups 5 and 6 received 5 µg of GFP dsRNA per 1 g of shrimp as a control of PmDOME silenced group. Shrimp in groups 7 and 8 were double injected with 10 µg of PmSTAT dsRNA per 1 g of shrimp and shrimp in groups 9 and 10 were double injected with 10 µg of GFP dsRNA per 1 g of shrimp as a control of  the PmSTAT silenced group. After 24 h post dsRNA injection, shrimp in the even-numbered groups were infected with ~ 6 × 106 viral copies of WSSV. The hemolymph in each group was collected at different time points, 6 and 24 h post-WSSV infection, before the total RNA was extracted and converted to cDNA. The expression profiles of immune-related genes including PmSOCS2, Dorsal, PmIMD, Vago5, ProPO2, ALFPm3, Penaeidin3, CrustinPm1 and CrustinPm7 were determined by qRT-PCR using gene specific primers in Supplementary Table To examine the effects of PmDOME and PmSTAT suppression on WSSV replication, the WSSV copy number in shrimp was determined. The shrimp’s gill samples were collected in parallel with the WSSV challenge experiment. Shrimp genomic DNA was extracted using FavorPrep Tissue Genomic DNA Extraction Mini Kit  and quantified by NanoDrop™ 2000c Spectrophotometer (Thermo Scientific). The extracted sample was used as a DNA template for viral copy number analysis. qRT-PCR was performed in triplicate using Luna® Universal qPCR Master Mix (NEB) with 2 μL genomic DNA (15 ng/μL) and VP28 primers  calculated by the logrank test.Shrimps were separated into six groups and each contained 10 shrimps per group. The assay was conducted in triplicate. Shrimp in groups 1 and 2 were injected with 1 × PBS (pH 7.4), while those in groups 3 and 4 were either single injected with 5 µg of Supplementary Information.


Cholinergic urticaria is triggered by an increased body temperature after exercise, passive warming, or emotional stresses. The anesthetic management used for two patients with an anaphylaxis history after cholinergic urticaria is described.Patient 1: A 34-year-old female was scheduled for a right-side thyroidectomy. At 27 years old, she experienced post-exercise anaphylaxis and repeated episodes of hives following exercise, sunbathing, mental stresses, and the consumption of spicy foods. Patient 2: A 35-year-old female was scheduled for a laparoscopic ovarian cystectomy. At 19 years old, she experienced anaphylaxis after a long bath and then hives after hot showers, bicycle riding, and long walks. For both patients, intraoperative passive warming was not performed to prevent excessive warming, and prophylactic antiemetics and multimodal analgesia were used to minimize their perioperative stresses.Careful anesthetic management is necessary to prevent anaphylaxis during anesthesia in a patient with a history of cholinergic urticaria. Cholinergic urticaria, which is characterized by pruritic pinpoint-sized wheals, is triggered by an increased body temperature with sweating after exercise, high environmental temperature, or mental stresses , and it Cholinergic urticaria is thought to develop following an immediate-type allergy to autologous sweat antigens or following IgE which increases in response to antigens or certain elements contained in sweat . AcetylcIn a patient with cholinergic urticaria, anesthesiologists are required to manage patients without inducing anaphylaxis during surgery. I present the anesthetic management for two patients with a history of anaphylaxis following cholinergic urticaria.3, and the portion of eosinophils was 10%.A 34-year-old female  was scheduled for a right-side thyroidectomy. She had a history of allergic rhinitis. At 27 years old, anaphylaxis  after exercise in a bathroom had occurred. She was then diagnosed as having cholinergic urticaria based on her history of repeated hives following exercise, sunbathing, mental stresses, and the consumption of spicy foods. Her daily medications were loratadine 20 mg and bilastine 20 mg, and she sometimes used prednisolone 5 mg or d-chlorpheniramine maleate 4 mg as a rescue. Her preoperative white blood cell count was 4900/mmThe patient’s daily medications were taken on the operative day, and her preoperative axillary body temperature was 36.7 ℃. General anesthesia was induced with 90 mg propofol, 0.25 μg/kg/min remifentanil, 200 μg fentanyl, and 40 mg rocuronium. General anesthesia was maintained with 5.0 mg/kg/h propofol and 0.2–0.3 μg/kg/min remifentanil. For antiemesis, 6.6 mg dexamethasone and 4 mg ondansetron were given, and 350 μg fentanyl, 800 mg acetaminophen, and 50 mg flurbiprofen were administered for multimodal analgesia.The patient’s intraoperative body temperature was continuously measured by a rectal thermometer, and it ranged from 37.2° to 37.4 ℃. The surgical data were as follows: surgical duration, 95 min; anesthesia time, 131 min; blood loss, 21 g; urine output, a small amount, total infusion volume, 900 mL. The postoperative axillary body temperature measured periodically during the patient’s hospitalization ranged from 36.5° to 36.9 ℃, and perioperative cholinergic urticaria was not observed.3, and the portion of eosinophils was 4.8%.A 35-year-old female  was scheduled for a laparoscopic ovarian cystectomy. She had a history of atopic dermatitis and cold-induced urticaria. At 19 years old, anaphylaxis  after long-time bathing had occurred. She was then diagnosed with cholinergic urticaria based on her history of repeated hives following hot showers, bicycle riding, and long walks. Her daily medications were levocetirizine 5 mg, cimetidine 400 mg, d-chlorpheniramine maleate 6 mg, and suplatast tosilate 200 mg. Her preoperative white blood cell count was 5000/mmThe patient’s daily medications were taken on the operative day, and her preoperative axillary body temperature was 36.5 ℃. General anesthesia was induced with 7.0 μg/mL propofol , 0.25 μg/kg/min remifentanil, 100 μg fentanyl, and 35 mg rocuronium. General anesthesia was maintained with 2.7–4.0 μg/mL propofol (TCI) and 0.15–0.25 μg/kg/min remifentanil. For antiemesis, 4 mg ondansetron was given. The administration of 300 μg fentanyl and 1000 mg acetaminophen and rectus sheath block were done for multimodal analgesia.The patient’s intraoperative body temperature was continuously measured by a nasopharyngeal thermometer, and it ranged from 36.1° to 36.5 ℃. The surgical data were as follows: surgical duration, 164 min; anesthesia time, 234 min; blood loss, a slight amount; urine output, 500 mL, total infusion volume, 1000 mL. The postoperative axillary body temperature measured periodically during the patient’s hospitalization ranged from 36.2° to 37.3 ℃, and perioperative cholinergic urticaria was not observed.Careful body-temperature management is quite important during anesthesia in a patient with cholinergic urticaria. In general, anesthetics-induced thermoregulatory impairment can induce a patient’s hypothermia during a patient’s surgery , and an As shown by patient 2’s case, cholinergic urticaria is sometimes associated with cold-induced urticaria, and in some individuals with cholinergic urticaria, hives can be induced by colder living conditions with temperature differences , 9. AnesA passive warming test is performed when an individual is suspected of having cholinergic urticaria . Details2 via cyclooxygenase-2 expression, which can induce hyperthermia [Surgical stress produces various cytokines that increase the synthesis of prostaglandin Erthermia . Thereforthermia . In the Each of the two present patients had no history of bronchial asthma, and their preoperative pulmonary function tests showed normal findings. However, the frequency of bronchial hyper-responsiveness in patients with cholinergic urticaria was noted to be 43% even when the patients had no history of bronchial asthma . The patIn patients with cholinergic urticaria, careful anesthetic planning including body-temperature management is necessary to achieve successful anesthetic management.