P < .001) and presented with leukopenia more frequently . There was no difference between patients with and without hypocellular AML in terms of the presence of antecedent hematologic disorders . FLT3-ITD and NPM1 mutations were less common in hypocellular than non-hypocellular AML . No differences were seen between the hypocellular and non-hypocellular AML groups in the complete remission rate  or early death rate . The OS and EFS did not differ between the hypocellular and non-hypocellular AML groups . Hypocellular AML is more frequently observed in older-aged patients and have fewer FLT3-ITD and NPM1 mutation, but the clinical outcomes of hypocellular AML do not differ from those of non-hypocellular AML.The hypocellular variant of acute myeloid leukemia (AML) is defined as bone marrow cellularity of <20% in a biopsy specimen at presentation. We performed a retrospective analysis of the clinical features and survival outcomes of hypocellular AML in a Korean population. We reviewed the medical records of all patients diagnosed with AML at nine hospitals participating in the Korean AML registry from 2006 to 2012. Overall survival (OS) and event-free survival (EFS) rates were calculated from the time of diagnosis until death or an event, respectively. In total, 2110 patients were enrolled and 102 (4.8%) were identified as having hypocellular AML. Patients with hypocellular AML were older than those with non-hypocellular AML (median age: 59 vs 49 years;  Patients with hypocellular AML were older than those with non-hypocellular AML, and presented with cytopenias more frequently. Al-kali et al also reported that the treatment outcomes and prognosis of hypocellular AML did not differ from those of non-hypocellular AML. However, most studies have been based on data from a small number of patients or a single institute. Therefore, we used Korean AML/MDS Working Party registry data to conduct a nationwide population study of the clinical features, molecular features, and prognosis of hypocellular AML.A recently published study conducted by Al-kali et al at MD Anderson Cancer Center reported that ∼10% of AML patients had hypocellular AML, defined as bone marrow cellularity of <20% in a biopsy specimen at presentation.22.1FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations and nucleophosmin-1 (NPM1) mutations were assessed using genomic DNA extracted from bone marrow aspirates with polymerase chain reaction assays.We conducted a retrospective study of all patients newly diagnosed with AML at nine hospitals participating in the Korean AML registry between 2006 and 2012. The data cut-off point was October 2014. For all patients, core biopsies and bone marrow aspirate smears were completed at the time of presentation. Bone marrow aspirate smears were assessed using Wright-Giemsa stain followed by cytochemical analysis for myeloperoxidase and non-specific esterase. Cytogenetic analysis was performed using G-banding, with at least 20 metaphases counted. Hypocellular AML is defined as AML with a bone marrow cellularity of <20%. We classified the patients according to French-American-British (FAB) and cytogenetic and molecular risk status stratified by National Comprehensive Cancer Network (NCCN) guidelines.Treatment response was assessed according to criteria proposed by the 2003 International Working Group. For instance, complete remission was defined as the presence of <5% blasts in bone marrow with recovery of the peripheral blood count. Information on antecedent hematologic disorders and prior chemotherapy or radiotherapy was collected.The study protocol was approved by the Institutional Review Board of Chungnam National University Hospital . The requirement for informed consent was waived because of the retrospective nature of the analysis.2.29/L for 3 days or a neutrophil count ≥1.0 × 109/L for 1 days after the start of induction chemotherapy. Platelet recovery was defined as a platelet count ≥20 × 109/L without a transfusion.The primary endpoints were clinical features, including differences in antecedent hematologic disease, cytopenias, cytogenetics, molecular genetics, complete remission rate, and early mortality. The secondary endpoints were survival outcomes, such as overall survival (OS) and event-free survival (EFS), and neutrophil and platelet recovery time after induction chemotherapy. Early mortality was defined as death from any cause within 2 months after diagnosis. The OS was calculated from the time of diagnosis until death, and EFS from the time of complete response to relapse or death from any cause. Neutrophil recovery was defined as a neutrophil count ≥0.5 × 102.3t test and the chi-squared test. Logistic regression was used to examine correlations. Overall and EFS were assessed using the Kaplan–Meier method. Survival rates were compared using the log-rank test. A P-value < .05 was considered significant. All statistical analyses were performed using SPSS software .Categorical variables were compared using Student's 33.1P < .001). The hypocellular AML group had a median bone marrow cellularity of 15% (range: 5–20%). The gender ratio and rate of antecedent hematologic disease did not differ by group. The median white blood cell count was 1690/uL  in the hypocellular AML group, which was significantly lower than that in the non-hypocellular AML group (P < .001). The most common subtype of AML according to the FAB classification was M2. This subtype accounted for 38.2% and 36.0% of hypocellular and non-hypocellular AML cases, respectively. However, the unclassifiable subtype was statistically significantly higher in the hypocellular AML group than in the non-hypocellular AML group (P < .001).A total of 2110 patients were enrolled from nine hospitals participating in the Korean AML registry (based on WHO criteria) between January 2006 and December 2012. Of these patients, 102 (4.8%) had a bone marrow cellularity of <20% and were diagnosed with hypocellular AML. The characteristics of patients with hypocellular AML and non-hypocellular AML are presented in Table 3.2P = .104). Rates of chromosomal abnormalities that are frequently observed in MDS, such as deletion 5 or 5q and deletion 7 or 7q, were also similar . Rates of translocation t and of chromosome 16 inversion or translocation t were also similar in both groups (t(8:21): 8.8% of hypocellular AML patients and 8.7% of non-hypocellular AML patients, P = .956; inv(16) or t(16:16): 1.0% of hypocellular AML patients and 3.8% of non-hypocellular AML patients, P = .136). A total of 1778 patients (64.3%) were tested for the FLT3-ITD mutation, and 883 patients (41.8%) were tested for the NPM1 mutation. None of the hypocellular AML patients tested had the NPM1 mutation, compared to 79 of the 830 non-hypocellular patients tested (9.5%) (P = .019). The FLT3-ITD mutation was found in 1 of the 84 hypocellular AML patients tested (1.2%) and in 242 of the 1694 non-hypocellular AML patients tested (14.3%) (P < .001).Risk status based on validated cytogenetics according to NCCN guidelines did not differ between the two groups Table . Interme3.3P = .112). Idarubicin plus cytarabine or enocitabine was most common initial induction regimen in both groups, followed by daunorubicin plus cytarabine or enocitabine (Table P = .002).In total, 64 of 102 patients in the hypocellular AML group 63.7%) and 1480 of 2008 patients in the non-hypocellular AML group (73.7%) were treated with standard induction chemotherapy. This difference was not statistically significant (.7% and 1P = .139). The early mortality rate was 14.7% in the hypocellular AML group, which was similar to the rate in the non-hypocellular AML group . The median platelet recovery time also was similar, at 22.5 days (range: 15–57 days) in the hypocellular AML group and 22.0 days (range: 8–58 days) in the non-hypocellular AML group (P = .787). However, hypocellular AML patients had lower median cellularity after induction chemotherapy  than non-hypocellular AML patients   showed no difference in the median neutrophil recovery time between the hypocellular AML group  and the non-hypocellular AML group   and profound cytopenias. Also, Hu et al found that hypocellular AML was associated with lower white blood cell counts and older age . In both studies, hypocellular AML was also more frequently associated with antecedent hematologic disorders. This trend was not replicated in our study, which is thought to be the result of the limited amount of past medical history data in registry-based studies. To compensate for this limitation, we analyzed chromosomal abnormalities frequently observed in MDS. Rates of chromosomal abnormalities  did not differ between the hypocellular and non-hypocellular AML groups. Al-kali et al reported similar findings in the chromosomal abnormalities.In this study, hypocellular AML was observed in older patients and associated with more severe leukopenia. This is consistent with the findings of previous studies.FLT3-ITD mutation is found in about 20% to 30% of de novo AML patients.,13 In hypocellular AML, we found a markedly lower rate of FLT3-ITD. This result is consistent with the studies by Al-kali et al and Hu et al. The FLT3-ITD mutation is a well-known adverse prognostic factor in AML and the patients with the FLT3-ITD mutation had increased white blood cell counts, because it suppresses apoptosis and proliferates the dysregulated cells.–16 Despite the low frequency of the FLT3-ITD mutation in hypocellular AML patients, the prognosis is similar to that of non-hypocellular AML patients. It is thought that the poor prognosis caused by FLT3-ITD mutation was offset in non-hypocellular AML patients, because those were relatively younger and had undergone more allogeneic hematopoietic stem cell transplants than hypocellular AML patients. The NPM1 mutation was also positively correlated with high white blood cell counts in our AML patients, and it frequently found in conjunction with the FLT3-ITD mutation.,18 The NPM1 mutation is present in ∼30% of AML cases, but it is less common in hypocellular AML than in non-hypocellular AML. NPM1 mutation is favorable prognostic factor in AML patients in the absence of FLT3-ITD mutation.,20 However, hypocellular AML patients had significantly lower rate of both NPM1 mutation and FLT3-ITD mutation in this study. Therefore, it is thought that the prognostic effect of the NPM1 mutation was weakened in hypocellular AML patients.The ,22 Chen et al, for instance, used single-cell targeted sequencing of leukemic stem cells to demonstrate a nonlinear pattern of clonal evolution from MDS to AML. In our study, non-hypocellular AML patients had a higher rate of allogeneic hematopoietic stem cell transplantation compared to the hypocellular AML patients, but survival outcomes were similar in both groups. This is probably because non-hypocellular AML tended to occur at a younger age than hypocellular AML, but genetic mutations associated with poor prognosis, such as FLT3-ITD, were more frequently observed in non-hypocellular AML.Hypocellular AML was expected to be associated with a higher mortality rate or a slower rate of bone marrow recovery after induction chemotherapy, due to its profound cytopenias, but early mortality (death before 8 weeks) and bone marrow recovery time did not differ between the hypocellular and non-hypocellular AML groups. Interestingly, the hypocellular AML cases retained low cellularity after BM recovery of induction chemotherapy, which data has not been previously reported in other articles. This suggests that patients with hypocellular AML are unique in having low BM cellularity regardless of leukemia. The development and increasingly widespread use of molecular genetic technologies, such as next-generation sequencing and single-cell RNA sequencing, is expected to shed further light on this issue.FLT3-ITD and NPM1 mutations. However, the clinical outcomes did not differ between hypocellular and non-hypocellular AML.In conclusion, we found that hypocellular AML was more frequently observed in older patients, who were less likely to exhibit We would like to thank all centers of the Korean Society of Hematology AML/MDS Working Party for their contributions to this analysis.Conceptualization: Ik-Chan Song, Deog-Yeon Jo, Hee-Je Kim.Data curation: Ik-Chan Song, Deog-Yeon Jo, Hyeoung-Joon Kim, Hee-Je Kim.Formal analysis: Ik-Chan Song, Yoo-Hong Min, Hee-Je Kim.Funding acquisition: Ik-Chan Song.Investigation: Ik-Chan Song, Dae Sik Hong, Won-Sik Lee, Ho-Jin Shin, Je-Hwan Lee, Jinny Park, Hee-Je Kim.Methodology: Ik-Chan Song, Hee-Je Kim.Project administration: Ik-Chan Song, Hee-Je Kim.Supervision: Hee-Je Kim.Writing – original draft: Ik-Chan Song, Deog-Yeon Jo.Writing – review & editing: Ik-Chan Song, Je-Hwan Lee, Hee-Je Kim.


Myeloid sarcomas (MS) are defined as rare extramedullary masses composed of immature myeloid cells. MS mostly develops in patients with acute myeloid leukemia (AML), and involves primarily the skin, soft tissues, bones, and lymph nodes. Pleura and pericardium involvement of MS are extremely uncommon. Polyserositis is also a very rare extramedullary presentation of acute myeloid leukemia (AML).A 30-year-old woman with a complaint of right neck mass combined with coughing for 2 months as well as fever and systemic edema for the last 10 days, was admitted to our center on July 11, 2019. Initial positron emission tomography (PET) scan indicated systemic lymphadenopathy, bilateral pleural effusion, and pericardial effusion.The initial pathological diagnosis of lymph nodes was MS. Subsequent bone marrow analysis confirmed AML.Conventional IA induction regimen followed by high-dose cytarabine (HiDAC) regimen.Complete absorption of pericardial and pleural effusion after the first cycle of IA induction chemotherapy.Polyserositis can be an extramedullary presentation of AML. Patients with polyserositis should undergo routine flow cytometric analysis. For AML with extamedullary infiltration, systemic chemotherapy should be administered in all confirmed cases. Most commonly, MS involves the skin, soft tissues, bones, and lymph nodes. Polyserositis as a result of leukemic infiltration, on the other hand, is an extremely rare extramedullary presentation of AML with only one case documented to date. In this report, we present a case of acute myelogenous leukemia with myeloid sarcoma confirmed in the lymph nodes. Notably, AML also manifests as polyserositis.Myeloid sarcoma (MS) is a rare form of localized malignancy that is caused by extramedullary proliferation and infiltration of myeloid primitive cells and immature myeloid cells.218F-FDG in the spine, pelvis, and femur was detected. Additionally, systemic lymphadenopathy with a maximum standard uptake value (SUVmax) of 6.6, thickening region of the right pleura with SUVmax of 4.3, and bilateral pleural effusion and pericardial effusion were also evident.A 30-year-old woman with a complaint of right neck mass combined with coughing for 2 months as well as fever and systemic edema for the last 10 days, was admitted to our center on July 11, 2019. The patient was diagnosed with a systemic infection. Prior to admission, the patient suffered from acute pericardial tamponade that required emergency pericardiocentesis. Otherwise, she was previously healthy. Her initial chest CT is shown below in Figure 9/L, with blasts accounting for 83%. Tests on bone marrow aspirate indicated a myeloblast proportion of 79.5%. Flow cytometric analysis revealed that the bone marrow blasts were positive for CD7, CD34, CD33, CD38, and HLA-DR, and negative for CD117, CD3, CD20, CD10, CD19, CD56, CD14, CD64, CD11b, CD13, GlyA, CD7, CD61, cMPO, cCD3, cCD79a, cCD22, and cTDT, confirming a diagnosis of AML , antifungals (amphotericin B and cancidas), and hemostasis drugs  were administered. Eventually, she refused further treatments and died 69 days after the initiation of induction chemotherapy.According to National Comprehensive Cancer Network guidelines, the patient subsequently received a conventional 3 + 7 induction regimen: 125 mg/mon Figs.  and 7. B3–7 According to a recent study, approximately 55% of MS cases also expressed CD7. CD7-postive AML exhibited high blast cell counts and poor outcomes.–10 Additionally, it has been reported that leukemic pleural effusion could also be an initial manifestation of CD7-positive AML.MS as extramedullary infiltration of immature myeloid cells is a very rare condition. It mostly develops in patients with AML. Previous reports have suggested no significant difference between the prognosis of isolated MS and MS with concomitant AML. However, leukemic pleural effusion is rare, and no more than 20 cases have been reported.,11,13–26 The average age of patients reported in the literature was 53 years , of whom 14 were male and 4 were female. One of these cases was CD7 positive AML. However, the exact incidence rate of leukemic pleural effusion is difficult to estimate. Awasthi et al found that 18% of 898 pleural fluid samples were malignant, yet none of them were AML. Cakir et al reviewed 4684 pleural fluid specimens in Turkey and found only one case of AML. Although the prognostic implication of leukemic pleural effusion in AML is undetermined, most cases have a poor prognosis. Nevertheless, both induction chemotherapy and stem cell transplantation were proven efficacious against this particular condition.,17As far as pleural effusion is concerned, it can occasionally occur during the clinical course of any hematological malignancies, especially in Hodgkin and non-Hodgkin lymphomas. a condition for which only 4 documented cases can be found.–33 The average age of these 4 patients was 42 years , of whom 3 were male and 1 was female. 2 of 4 patients died prior to any possible treatment. Therefore, the prognostic value of leukemic pericardial effusion needs further investigation. Nevertheless, early recognition may improve clinical outcomes.In contrast, pericardial effusion can occur in 21% of patients with AML at initial diagnosis, but only 0.5% manifests as pericardial tamponade,,35 Treatments for MS include systemic chemotherapy, surgical resection, and local radiotherapy. Anti-AML induction remission therapy is sufficient and effective for MS.,35 Our patient received IA and HiDAC induction therapy according to AML protocols, which resulted in complete resolution of pericardial and pleural effusion. However, complete remission (CR) was not achieved in the bone marrow, indicating different biological characteristics from extramedullary infiltration. Since the patient refused further examination, a definite validation regarding the potential difference was no longer possible.In addition to pleural and pericardial effusion, our case of MS also involved lymph nodes. MS can be the initial manifestation of AML, particularly for M5 (monocytic) and M4 (myelomonocytic) subtypes.In conclusion, although polyserositis as a primary clinical manifestation of AML is extremely rare, patients with polyserositis should still undergo routine flow cytometric analysis in order to avoid delay in diagnosis. For AML with extramedullary infiltration, systemic chemotherapy should be administered in all confirmed cases.Data curation: Yubo Pi, Beining Wang.Supervision: Lihong Wang, Hanyun Ren.Writing – original draft: Yubo Pi.Writing – review & editing: Lihong Wang, Hanyun Ren.


An elevation in [Ca2+]cyt levels is one of the earliest responses in plant cells after exposure to a range of environmental stimuli. Advances in understanding the role of [Ca2+]cyt in plant development has been facilitated by the use of genetically-encoded reporters such as GCaMP. Most of these studies have relied on promoters such as Cauliflower Mosaic Virus (35S) and Ubiquitin10 (UBQ10) to drive expression of GCaMP in all cell/tissue types. Plant organs such as roots consist of various cell types that likely exhibit unique [Ca2+]cyt responses to exogenous and endogenous signals. However, few studies have addressed this question. Here, we introduce a set of Arabidopsis thaliana lines expressing GCaMP3 in five root cell types including the columella, endodermis, cortex, epidermis, and trichoblasts. We found similarities and differences in the [Ca2+]cyt signature among these root cell types when exposed to adenosine tri-phosphate (ATP), glutamate, aluminum, and salt, which are known to trigger [Ca2+]cyt increases in root cells. These cell type-targeted GCaMP3 lines provide a new resource that should enable more in depth studies that address how a particular environmental stimulus is linked to specific root developmental pathways via [Ca2+]cyt.Cytoplasmic calcium ([Ca Because Ca2+ acts as a stress signal, it is one of the first responses plants have to environmental stimuli. This fast response is achieved through an influx of extracellular Ca2+ and an efflux of stored Ca2+ from the vacuole, endoplasmic reticulum (ER), and mitochondria that is achieved via a concentration gradient between the intracellular Ca2+ stores and the cytoplasm ..2+-bindiM domain .2+ sensors in plants are driven by constitutive promoters such as Cauliflower Mosaic Virus 35S (35S) and Ubiquitin 10 (UBQ10) [2+ sensors enabled the functional study of [Ca2+]cyt changes across entire plant organs [2+]cyt signature. While [Ca2+]cyt changes typically observed in the entire plant organ likely originate from more than one cell type, we lack tools to monitor these changes within specific tissues or cells cyt changes in roots treated with chemicals previously shown to induce [Ca2+]cyt increases in plants.To date, expression of CGaMP and other genetically-encoded Ca (UBQ10) ,13. Altht organs , it is lor cells . To addrromoters . We demo2+]cyt sensors that are available to the scientific community are driven by constitutive promoters. A. thaliana lines expressing the UBQ10:GCaMP3 construct, which we generated and used previously to study [Ca2+]cyt oscillations in root hairs   solution without ATP to roots. We found that applying MS solution triggered a rise in [Ca2+]cyt that was delayed and lower in amplitude compared to the [Ca2+]cyt change caused by ATP  . In mamm2+]cyt . While p [2+]cyt ,26,27. C decline A 5,25].,25.2+]cyd by ATP A. This oresponse ,20,27,282+]cyt signatures. For example, the PIN2:GCaMP3 lines, which expressed GCaMP3 in the epidermis and cortex , NaCl treatment resulted in cell-specific [Ca2+]cyt transients in the early elongation zone [UBQ10:GCaMP3 exhibited late-onset [Ca2+]cyt transients  in cells of the elongation zone  using freshly prepared MS plates for planting seeds and (2) pretreating roots two times with MS solution prior to application of the actual chemical solution. These modifications in our treatment protocols were tested using ATP which typically generated peaks with the highest amplitude.As shown in the preceding sections, the 0.5X MS control solution induced [Cad cortex . Althoug2+]cyt transients triggered by the MS solution were significantly dampened when fresh plates were used for the experiments . Under these modified growth and pretreatment conditions, the ATP-induced [Ca2+]cyt increases were lower in amplitude when compared to the [Ca2+]cyt increases shown in 2+]cyt transients, these [Ca2+]cyt responses were better separated from the [Ca2+]cyt responses induced by MS solution, particularly in lines expressing UBQ10:GCaMP3 and those in which GCaMP3 was expressed in root surface cell types and the cortex , and analyzed the relationship between chemicals using Analysis of Variance (ANOVA) with Tukey Post-Hoc test (3+ showed similar responses (except ATHB8:GCaMP3 to Al3+) while adapted ATP:MS showed significantly slower reaction times to both ATP and glutamate  [2+]cyt transients when compared to UBQ10:GCaMP3 lines , is required for root growth recovery after encountering high salinity conditions cyt transients in the cortex revealed by the PEP:GCaMP3-expressing lines could be a mechanism to maintain root growth integrity. The cell type GCaMP3 lines described here should be useful in testing this hypothesis.The line that was most similar to . (2010)  . The [Ca2+]cyt transients observed after MS treatment were reminiscent of touch-induced [Ca2+]cyt responses [2+]cyt signaling [2+]cyt imaging involved planting seed of the reporter lines on a thin layer of agarose to secure the root while at the same time allowing for the root cells to be accessible to the various chemical treatments  (http://neb.com) and cloned into a modified pCAMBIA1390 vector cyt-dependent GCaMP3 fluorescence, images of growing roots were acquired every 1 s for 10 to 15 min using the Leica SP8-X confocal microscope. Images were captured at a scanning speed of 600 MHz and pixel resolution of 512 × 300. Baseline GCaMP3 fluorescence of the roots was first acquired for 1 min after placing seedlings on the stage of the microscope. Twenty µL of the treatment solution was then added on top of the agarose medium where the root was growing using an adjustable volume pipette while imaging continued. From the collected images, the average fluorescence intensity was acquired by marking a rectangular region of interest corresponding to the specific cell type using the rectangular selection tool of the SPX-8 LAS software. GCaMP3 fluorescence (F) values were normalized using the formula To measure [Caonstruct . Another


MLL gene rearrangements (MLL-r). The histone methyltransferase DOT1L is involved in supporting the proliferation of MLL-r cells, for which a target inhibitor, Pinometostat, has been evaluated in a clinical trial recruiting pediatric MLL-r leukemic patients. However, modest clinical effects have been observed. Recent studies have reported that additional leukemia subtypes lacking MLL-r are sensitive to DOT1L inhibition. Here, we report that targeting DOT1L with Pinometostat sensitizes pediatric AML cells to further treatment with the multi-kinase inhibitor Sorafenib, irrespectively of MLL-r. DOT1L pharmacologic inhibition induces AML cell differentiation and modulates the expression of genes with relevant roles in cancer development. Such modifications in the transcriptional program increase the apoptosis and growth suppression of both AML cell lines and primary pediatric AML cells with diverse genotypes. Through ChIP-seq analysis, we identified the genes regulated by DOT1L irrespective of MLL-r, including the Sorafenib target BRAF, providing mechanistic insights into the drug combination activity. Our results highlight a novel therapeutic strategy for pediatric AML patients.Pediatric acute myeloid leukemia (AML) is an aggressive malignancy with poor prognosis for which there are few effective targeted approaches, despite the numerous genetic alterations, including  FLT3) mutated and mixed lineage leukemia rearranged (MLL-r). Therefore, at present, improved therapies for pediatric AML remains an unmet need.Acute myeloid leukemia (AML) is a genetically heterogeneous group of myeloid malignancies that account for approximately 20% of pediatric leukemias . CurrentMLL-r leukemias. DOT1L is the only known methyltransferase catalyzing non-processive mono-, di-, and trimethylation of lysine 79 on histone H3 (H3K79), and the presence of H3K79me2 strongly correlates with active gene transcription [MLL-r leukemias, DOT1L is recruited on ectopic target sites, and its mislocation promotes the aberrant overexpression of developmentally important genes—for instance, HOXA cluster genes and MEIS1 [MLL rearrangements [Recent studies have elucidated the role of the histone methyltransferase disrupter of telomeric silencing 1-like (DOT1L) in cription , and a nnd MEIS1 ,6,7. Accngements ,12,13,14S-adenosyl-L-methionine (SAM). Due to the encouraging results obtained in preclinical studies [MLL-r leukemias, showing, however, modest clinical efficacy [MLL-PTD, IDH1/2-, NPM1-, and DNMT3A-mutated subtypes, were found to be sensitive to DOT1L inhibition [MLL-r. Several in vitro studies also demonstrated a combination benefit of DOT1L inhibitors in conjunction with chemotherapeutic agents or chromatin modifying drugs [Pinometostat is a small molecule that blocks DOT1L activity by competing with the methyl-donor  studies , Pinometefficacy . Moreovehibition ,19,20,21ng drugs ,23.In light of the above, we hypothesized that DOT1L inhibition could sensitize pediatric AML cells to further treatment because of the impact on the regulation of genes involved in hematopoietic cell maintenance. In particular, we sought to investigate whether DOT1L inhibition with Pinometostat could sensitize AML cells, irrespectively of MLL fusions, to Sorafenib, a multi-kinase inhibitor with activity against FLT3, members of the RAF/MEK/ERK signaling pathway, VEGFR2, PDGFR, and KIT .MLL-r. We also tried to determine the mechanisms underlying Pinometostat sensitization of AML cells. Through ChIP-seq analysis, we identified the DOT1L targets shared between MLL-r and non-MLL-r AML cells that play relevant roles in cancer biology, including RAF1 and BRAF, which encode for RAF/MEK/ERK signaling members inhibited by Sorafenib, and we validated the notion that strong BRAF down-modulation is a mechanism by which Pinometostat enhances Sorafenib efficacy. Collectively, our findings describe a promising approach to improve outcomes for pediatric AML patients.Our study demonstrates that combined treatment dramatically impacts AML cell proliferation and survival, despite the poor response to Pinometostat as a single agent and irrespective of MLL-r in a dose- and time-dependent manner. As expected, MV4-11, MOLM-13, and NOMO-1 cell lines, carrying MLL-AF4 or MLL-AF9 fusions, showed a dramatic decrease in cell number, with IC50 values below 1 µM following 8 days of treatment. In contrast, proliferation of U-937 or HL-60 cells, both lacking MLL-r, was unaffected by Pinometostat. Surprisingly, DOT1L inhibition had no effect on MLL-AF9-positive THP-1 cells, whereas it significantly decreased proliferation of the non-MLL-r OCI-AML3 cell line. These results demonstrate that the impact of DOT1L inhibition on cell proliferation is not strictly dependent on MLL fusions and suggest that additional factors could affect Pinometostat sensitivity. First, we tested the efficiency of 1 µM Pinometostat in inhibiting DOT1L methyltransferase activity, confirming that such a concentration, chosen so as not to exceed those used in vivo , caused 0/G1 phase cells was detected  of gene expression profiles revealed a main cluster comprising most of the AML cell lines and including both -r cells A, thus i-r cells . A p val-r cells B. To sea-r cells C. In parMLL-r, we hypothesized that treating both MLL-r and non-MLL-r AML cells with Pinometostat would induce sensitization to further treatment with the multi-kinase inhibitor Sorafenib.Due to the strong impact of DOT1L inhibition on proliferative signaling and the induction of myeloid differentiation irrespective of 50 of 6 µM after 48 h treatment  or MLL fusions . Although the favorable interaction between Pinometostat and Sorafenib was not seen in all the primary samples - which is not surprising because of the high heterogeneity of AML- it should be noted that combined treatment was particularly effective in inhibiting the proliferation of non-MLL-r primary samples. In addition, analysis of Annexin-V positive cells mirrored this trend, demonstrating the co-treatment’s ability to increase apoptosis in comparison to both single agent treatments  were collected with informed consent according to institutional guidelines. This study was conducted according to the principals of the Declaration of Helsinki and was approved by the Independent Ethics Committee of the University Hospital of Bologna “S. Orsola-Malpighi” . Primary AML samples were isolated using Ficoll-Paque  and were co-cultured on a layer of human stromal HS-5 cells in DMEM medium supplemented with 20% FBS, penicillin–streptomycin, and human cytokines  [WSU-AML cell line was kindly provided by St. Jude Children’s Research Hospital, Memphis, TN, USA. All other AML cell lines were obtained from DSMZ and were cultured in RPMI-1640 , IMDM (MV4-11), or MEM-Alpha  media  supplemented with 10%–20% Fetal Bovine Serum (FBS) (Thermo Fisher Scientific Inc.), 100 U/ml penicillin, and 100 μg/ml streptomycin  at 37 °C, in a humidified atmosphere of 5% COdon, UK) . Human APinometostat, Sorafenib, Vemurafenib, and AZ628 were purchased from Selleck Chemicals , and GW5074 was purchased from Tocris . All the drugs were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) to obtain a 10 mM stock solution. AML cell lines and primary AML cells were cultured for up to 28 days or for 48 h in the presence of Pinometostat or Sorafenib, respectively, or DMSO (control cells). Overall, the final DMSO concentration during all incubations was kept below 0.1%, in order to avoid effects on the differentiation potential of AML cells.5 and 106 cells/mL, respectively. Growth media and Pinometostat were replaced every 4 days, and, on days of media replacement, leukemic cells were collected, counted, and analyzed as required. For cell viability analysis, the drugs were added at increasing concentrations . For drug-combination experiments with Pinometostat and Sorafenib, both AML cell lines and primary AML samples were first treated with 1 µM Pinometostat for 4 or 8 days, and then 1 µM Sorafenib was added for a further 24 or 48 h . For all other analyses, Pinometostat was employed at 1 µM. For drug-combination experiments with Sorafenib, Vemurafenib, GW5074, and AZ628, AML cell lines were plated at a density of 0.2 × 106 cells/mL and treated with single or simultaneously combined agents at increasing concentrations (from 1 nM to 10 µM) for 24 h. For long-term culture, AML cells and primary samples were plated at a density of 10t-test or one-way ANOVA with GraphPad Prism Software . A p value of <0.05 was considered statistically significant.Data are presented as mean values ± SD and were statistically analyzed using a two-tailed unpaired Additional materials and methods are described in the MLL-r, and they provide a comprehensive picture of the cellular functions likely regulated by DOT1L methyltransferase activity. Importantly, our preliminary data on the favorable interaction between Pinometostat and Sorafenib provide the rationale to investigate this drug combination as a novel and more effective approach to treat pediatric AML patients. Although further in vivo analyses are required, our results are of particular interest in light of the limited therapeutic strategies for pediatric AML patients. In summary, our data show that DOT1L inhibition has drastic repercussions for the gene expression program in AML cells irrespectively of


There is a pressing need for improved sensors, but improving them is challenging because testing them involves a low throughput, labor-intensive processes. Our goal was to create synthetic, excitable cells that can be activated with brief pulses of blue light and serve as a medium throughput platform for screening the next generation of sensors. In this live cell system, blue light activates an adenylyl cyclase enzyme (bPAC) that increases intracellular cAMP . In turn, the cAMP opens a cAMP-gated ion channel. This produces slow, whole-cell Ca2+ transients and voltage changes. To increase the speed of these transients, we add the inwardly rectifying potassium channel Kir2.1, the bacterial voltage-gated sodium channel NAVROSD, and Connexin-43. The result is a highly reproducible, medium-throughput, live cell system that can be used to screen voltage and Ca2+ sensors.Genetically encoded fluorescent biosensors are powerful tools for studying complex signaling in the nervous system, and now both Ca B) The △F is the difference between the baseline fluorescence and the maximum fluorescence of each peak. The mean and S.E.M of the Ca2+ transients in [Kir2.1]:[NavD] optimized Kuhl-CKNa cells C) Blue line indicates point of 20s blue light stimulus. The time of each fluorescence peak was recorded and is indicated by a black dot. D) The total number of Ca2+ transients per cell (ROI) for experimental [Kir2.1]:[NavD] optimized Kuhl-CKNa cells. E) Representative duration of elevated R-GECO1 fluorescence over time per Ca2+ transient. Black dot indicates the Ca2+ transients FWHM for each peak. F) Black dot indicates the time between Ca2+ transient events per cell. Red bars indicate mean and ± S.E.M (n = 150 cells per condition).A) The total peaks for each Ca(TIF)Click here for additional data file.S5 Fig2+ transient within a trial was recorded, and the experiment with [1e8 Kir2.1]:[1e8 NavD] VG/μL had the greatest recorded peaks. B) The △F is the difference between the baseline fluorescence and the maximum fluorescence of each peak. The mean and S.E.M of the Ca2+ transients in [Kir2.1]:[NavD] optimized Kuhl-CKNaCx cells C) Blue line indicates point of 20s blue light stimulus. The time of each fluorescence peak was recorded and is indicated by a black dot. D) The total number of Ca2+ transients per cell (ROI) for experimental [Kir2.1]:[NavD] optimized Kuhl-CKNaCx. E) Representative duration of elevated R-GECO1 fluorescence over time per Ca2+ transient. Black dot indicates the Ca2+ transients FWHM for each peak. F) Black dot indicates the time between Ca2+ transient events per cell. Red bars indicate mean and ± S.E.M (n = 150 cells per condition).A) The total peaks for each Ca(TIF)Click here for additional data file.S6 Fig2+ transient within a trial was recorded, and the experiment with [Cx 1e5] VG/μL had the greatest recorded peaks. B) The △F is the difference between the baseline fluorescence and the maximum fluorescence of each peak. The mean and S.E.M of the Ca2+ transients in Cx-43 optimized Kuhl-CKNaCx cells. C) Blue line indicates point of 20s blue light stimulus. The time of each fluorescence peak was recorded and is indicated by a black dot. D) The number of Ca2+ transients per cell (ROI) for Cx-43 optimized Kuhl-CKNaCx cells. E) Representative duration of elevated R-GECO1 fluorescence over time per Ca2+ transient. Black dot indicates the Ca2+ transients FWHM for each peak. F) Black dot indicates the time between Ca2+ transient events per cell. Red bars indicate mean and ± S.E.M (n = 150 cells per condition).Corresponding (TIF)Click here for additional data file.S7 Fig2+ transient within a trial was recorded, and the experiment with [CNG 1e3] VG/μL had the greatest recorded peaks. B) The △F is the difference between the baseline fluorescence and the maximum fluorescence of each peak. The mean and S.E.M of the Ca2+ transients in CNG optimized Kuhl-CKNaCx cells C) Blue line indicates point of 20s blue light stimulus. The time of each fluorescence peak was recorded and is indicated by a black dot. The wells without CNG and Cx were very interesting because it appears that without blue light there is a D) The total number of Ca2+ transients per cell (ROI) for experimental CNG optimized Kuhl-CKNaCx cells. E) Representative duration of elevated R-GECO1 fluorescence over time per Ca2+ transient. Black dot indicates the Ca2+ transients FWHM for each peak. F) Black dot indicates the time between Ca2+ transient events per cell. Red bars indicate mean and ± S.E.M (n = 150 cells per condition).Corresponding (TIF)Click here for additional data file.S8 Fig2+ transient within a trial (T = 300s) was recorded. The experiment with [HCN2 1e2] VG/μL had the greatest recorded peaks. B) The △F is the difference between the baseline fluorescence and the maximum fluorescence of each peak. The mean and S.E.M of the Ca2+ transients in HCN2 optimized Kuhl-HKNaCx cells C) Blue line indicates point of 20s blue light stimulus. The time of each fluorescence peak was recorded and is indicated by a black dot. D) The total number of Ca2+ transients per cell (ROI) for experimental HCN2 optimized Kuhl-HKNaCx. E) Representative duration of elevated R-GECO1 fluorescence over time per Ca2+ transient. Black dot indicates the Ca2+ transients FWHM for each peak. F) Black dot indicates the time between Ca2+ transient events per cell. Red bars indicate mean and ± S.E.M (n = 150 cells per condition).Corresponding (TIF)Click here for additional data file.S9 Fig2+ transient within a trial (T = 25m) was recorded. B) The △F is the difference between the baseline fluorescence and the maximum fluorescence of each peak. The mean and S.E.M of the Ca2+ transients in optimized Kuhl-CKNaCx cells C) Blue line indicates point of 20s blue light stimulus. The time of each fluorescence peak was recorded and is indicated by a black dot. D) The total number of Ca2+ transients per cell (ROI) for optimized Kuhl-CKNaCx cells. Representative duration of elevated R-GECO1 fluorescence over time per Ca2+ transient. Black dot indicates the Ca2+ transients FWHM for each peak. F) Black dot indicates the time between Ca2+ transient events per cell. Red bars indicate mean and ± S.E.M (n = 150 cells per condition).Corresponding (TIF)Click here for additional data file.S10 Fig2+ transient within a trial (T = 25m) was recorded. B) The △F is the difference between the baseline fluorescence and the maximum fluorescence of each peak. The mean and S.E.M of the Ca2+ transients in optimized Kuhl-CKNaCx cells C) Blue line indicates point of 20s blue light stimulus. The time of each fluorescence peak was recorded and is indicated by a black dot. D) The total number of Ca2+ transients per cell (ROI) for optimized Kuhl-CKNaCx. Representative duration of elevated R-GECO1 fluorescence over time per Ca2+ transient. Black dot indicates the Ca2+ transients FWHM for each peak. F) Black dot indicates the time between Ca2+ transient events per cell. Red bars indicate mean and ± S.E.M (n = 150 cells per condition).Corresponding (TIF)Click here for additional data file.S11 Fig2+ transient frequency (n = 150 cells per condition).A) Activity was optimized by varying the viral concentration of CNG. Differences were analyzed using Dunnett’s multiple comparison test on the Carequency  compared(TIF)Click here for additional data file.S1 TableThe table shows the experimental set up, the final viral concentration used, and their corresponding figures.(TIF)Click here for additional data file.S2 TableThe table shows all test run using Dunnetts where stated in the manuscript.(TIF)Click here for additional data file.S1 MovieThe authors do not know what these come from.(AVI)Click here for additional data file.S2 Movie2+ bursting rosette pattern following a stimulus, while HCN2 seems to have a spreading wave-like pattern.Rose like patterns. The behavior of HCN2 was visually different from CNG. While both wells have random activity, there is a brief somewhat coordinated activity after a blue light stimulus  Figs. S2(AVI)Click here for additional data file.S3 MovieWave-like spreading pattern.(AVI)Click here for additional data file.S4 MovieCells imaged for 15 minutes with robust, repeatable activity.(AVI)Click here for additional data file.S5 MovieAn image created by the OptoSplit II is shown where the green and red emission from both sensors are split and imaged on two different portions of the EMCCD camera.(AVI)Click here for additional data file.S6 MovieActivity of Kuhl-CKNaCx cells with the GEVI ArcLight.(AVI)Click here for additional data file.S7 MovieActivity of Kuhl-CKNaCx cells with the GEVI ArcLight from the same well.(AVI)Click here for additional data file.S8 MovieActivity of Kuhl-CKNaCx cells with the GEVI ArcLight from the same well.(MOV)Click here for additional data file.


V3.2 (T‐type Ca2+ channel encoded by the α‐3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca2+ influx to trigger Ca2+ sparks and large‐conductance Ca2+‐activated K+ channel feedback in vascular smooth muscle. We hypothesize that this mechanism of Ca2+ spark generation is affected by age. Using smooth muscle cells (VSMCs) from mouse mesenteric arteries, we found that both Cav3.2 channel inhibition by Ni2+ (50 µM) and caveolae disruption by methyl‐ß‐cyclodextrin or genetic abolition of Eps15 homology domain‐containing protein (EHD2) inhibited Ca2+ sparks in cells from young (4 months) but not old (12 months) mice. In accordance, expression of Cav3.2 channel was higher in mesenteric arteries from young than old mice. Similar effects were observed for caveolae density. Using SMAKO Cav1.2−/− mice, caffeine (RyR activator) and thapsigargin (Ca2+ transport ATPase inhibitor), we found that sufficient SR Ca2+ load is a prerequisite for the CaV3.2‐RyR axis to generate Ca2+ sparks. We identified a fraction of Ca2+ sparks in aged VSMCs, which is sensitive to the TRP channel blocker Gd3+ (100 µM), but insensitive to CaV1.2 and CaV3.2 channel blockade. Our data demonstrate that the VSMC CaV3.2‐RyR axis is down‐regulated by aging. This defective CaV3.2‐RyR coupling is counterbalanced by a Gd3+ sensitive Ca2+ pathway providing compensatory Ca2+ influx for triggering Ca2+ sparks in aged VSMCs.Caveolae position Ca Cav3.2‐RyR axis in smooth muscle cell is down‐regulated by age‐related ultrastructural alterations of caveolae and reduced Cav3.2 expression. This defective Cav3.2‐RyR coupling is counterbalanced by a Gd3+ sensitive Ca2+ pathway providing compensatory Ca2+ influx for triggering Ca2+ sparks in aged VSMCs. Although genetic caveolin‐1 deletion leads to a complete lack of caveolae from the VSMC plasma membrane, data interpretation is limited because Cav1 deletion may affect SR Ca2+ load and is known to increase the density of BKCa channels in VSMCs  to cholesterol‐rich membrane microdomains  receptor activation with phenylephrine in mesenteric arteries of mice  CaV1.2−/− mice and a novel Eps15 homology domain‐containing protein (EHD2) knockout mouse model, which leads to destabilization of caveolae at the plasma membrane  and thereby reduces SR [Ca2+] load ‐induced peak fluorescence was measured to monitor maximal RyR Ca2+ release from SR stores. Our data showed that thapsigargin decreased concentration‐dependently caffeine‐induced cytosolic [Ca2+] peaks  VSMCs  VSMCs compared to Cav1.2+/+ control cells. As illustrated in Figure 2+] peaks were larger in Cav1.2+/+ cells compared to SMAKO Cav1.2−/− VSMCs, consistent with the idea that L‐type CaV1.2 channels are critical for SR Ca2+ load and peak [Ca2+] release. We compared the role of Ca2+ uptake into SR in these cells. 15 min after the first caffeine pulse, subsequent application of caffeine induced a strong [Ca2+] peak in Cav1.2+/+ control compared to Cav1.2−/− (SMAKO) cells  in VSMCs  proper function of the T‐type CaV3.2‐RyR axis requires sufficient high [Ca2+]SR load.Thapsigargin inhibits the SR Cas Figure a–d. The s Figure ,c . In cons Figure ,c. Theses Figure . We alsos Figure i–k. Thess Figure e–h. STOCs Figure f, while s Figure e,h. Toge2.3V3.2‐RyR axis in old VSMCs could result from alterations in the ultrastructure of caveolae, where Cav3.2 channels reside to drive RyR‐mediated Ca2+ sparks , a cholesterol‐depleting drug, which is known to disturb caveolae and inhibit a significant fraction of Ca2+ sparks in VSMCs  did not further reduce Ca2+ sparks in methyl‐β‐cyclodextrin treated VSMCs neither from young nor old mice. Next, we evaluated the ultrastructure of caveolae in young versus old VSMCs. Although caveolae were present in cells of both groups, the density of caveolae was reduced in old VSMCs compared to young VSMCs  mouse model. Since EHD2 localizes to the caveolar neck region of all caveolae, genetic abolition of EHD2 increases ubiquitously detachment of caveolae from the plasma membrane  VSMCs compared to control cells. Furthermore, Ca2+ spark frequency and the percentage of cells firing Ca2+ sparks were diminished in VSMCs from EHD2 del/del mice  had no effects on Ca2+ sparks in young VSMCs  cation channels providing sufficient Ca2+ influx to generate Ca2+ sparks.We noticed that there was a fraction of Cay Figure . Surpriss Figure . In conts Figure l,m. Toge2.5V3.2‐RyR relationship to regulate arterial tone, we performed video microscopic measurements on isolated arteries. In young wild‐type mesenteric arteries, the Cav3.2 blocker Ni2+ 50 µM increased myogenic tone from 9.2% ± 1.2% to 13.04% ± 0.8% at 60 mmHg, from 11.6% ± 1.2% to 19.7% ± 0.5% at 80 mmHg, and from 17.7% ± 2% to 27.8% ± 1.3% at 100 mmHg  and old (60.7% ± 2.1%) pressurized arteries  and EHD2 genetic knockout mice. Our studies demonstrate that caveolar Cav3.2 channels‐RyR axis is impaired in aged VSMCs. We observed age‐related ultrastructural alterations of caveolae, which together with decreased Cav3.2 expression, may underlie incomplete caveolae‐Cav3.2‐RyR coupling for extracellular Ca2+ influx to trigger Ca2+ sparks and BKCa feedback in aged vascular smooth muscle.In this study, we analyzed the effects of aging on the Ca3.1v1.2 channels provide the predominant Ca2+ pathway for Ca2+ spark generation in VSMCs  can activate RyRs from the SR luminal side of the receptor to produce Ca2+ sparks , a well‐known activator RyRs, to induce SR calcium release. Caffeine evoked smaller Ca2+ transients through SR Ca2+ release in SMAKO Cav1.2−/− VSMCs, in which T‐type Cav3.2 channels play a minor role in providing Ca2+ influx to induce Ca2+ sparks. These findings support the view that Ca2+ influx through L‐type Cav1.2 channels, but not T‐type Cav3.2 channels, represents the main source for luminal SR calcium load  abolished all Ca2+ sparks in young VSCMs  and found that gadolinium had no effects on these Ca2+ sparks. Although gadolinium has been identified as nonspecific blocker  has been upregulated to compensate for loss of T‐type CaV3.2 channels driving Ca2+ sparks in aged VSMCs  has been described previously  was generated by homologous recombination in R1 embryonic stem cells  into a Cav1.2 knockout allele (L1) specifically in SMCs  and corresponding control mice  (12–14 weeks each) were i.p. injected with tamoxifen (30 µg/g body weight/day) for five consecutive days and sacrificed within 2–4 days after the injections. EHD2 del/del or EHD2 del/+ littermates (as control) mice (20–35 weeks each) were used as previously described  and the animal welfare officers of the MDC . There are no ethical concerns.In this study, young (12–14 weeks) versus old (48–56 weeks) male mice were used. The generation and usage of mice deficient in the smooth muscle Ca4.22‐5% CO2) physiological salt solution (PSS) of the following composition (mM): 119 NaCl, 4.7 KCl, 1.2 KH2PO4, 25 NaHCO3, 1.2 MgSO4, 1.6 CaCl2, and 11.1 glucose. The arteries were cleaned, cut into pieces, and placed into a Ca2+‐free Hank's solution (mM): 55 NaCl, 80 sodium glutamate, 5.6 KCl, 2 MgCl2, 1 mg/ml bovine serum albumin , 10 glucose, and 10 HEPES (pH 7.4 with NaOH) containing 0.5 mg/ml papain (Sigma) and 1.0 mg/ml DTT for 37 min at 37°C. The segments were then placed in Hank's solution containing 1 mg/ml collagenase  and 0.1 mM CaCl2 for 17 min at 37°C. Following several washes in Ca2+‐free Hank's solution (containing 1 mg/ml BSA), single cells were dispersed from artery segments by gentle triturating. Cells were then stored in the same solution at 4°C.Arterial VSMCs from mesenteric arteries were isolated as previously described  and pluronic acid  for 60 min at room temperature in Ca2+‐free Hanks’ solution . Images were recorded using a Nipkow disc‐based UltraView LCI confocal scanner  linked to a fast digital camera . The confocal system was mounted on an inverted Nikon Eclipse Ti microscope with a x40 oil‐immersion objective . Images were obtained by illumination with an argon laser at 488 nm and recording all emitted light above 515 nm. Ca2+ spark analyses were performed off‐line using the UltraView Imaging Suite software (Perkin Elmer). The entire area of each image was analyzed to detect Ca2+ sparks. Ca2+ sparks were defined as local fractional fluorescence increase (F/F0) above the noise level of 1.5. The frequency was calculated as the number of detected sparks divided by the total scan time. Caffeine‐induced peak was measured as previously described , images were obtained following a single pulse of 10 mM caffeine. Maximal amplitude of caffeine‐induced peak fluorescence was normalized by the initial fluorescence value (0F/F) and considered as an index of total SR Ca2+ load.Ca4.42, and 0.05 EGTA (pH 7.2). The patch pipette solution was supplemented with 200 µg/ml Amphotericin B, dissolved in dimethyl sulfoxide (DMSO), to measure K+ currents in the whole‐cell perforated‐patch mode. The external bath solution contained (in mM): 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES (pH 7.4); holding potential was −60 mV. Whole‐cell currents were recorded using an Axopatch 200B amplifier (Axon Instruments/Molecular Devices) or an EPC 7 amplifier (List) at room temperature. Data were digitized at 5 kHz, using a Digidata 1440A digitizer  and pClamp software versions 10.1 and 10.2. STOC analysis was performed off‐line using IGOR Pro (WaveMetrics) and Microsoft Excel software (Microsoft Corporation). A STOC was identified as a signal with at least three times the BKCa single‐channel current amplitude  were averaged for each group.Quantitative assessment of caveolae was carried out as previously described . Vessels were cleaned of perivascular fat, and all tissues were immediately placed on dry ice and kept at −80°C until use. Samples were homogenized in RIPA buffer  containing protease inhibitors (Sigma‐Aldrich). Tubes containing homogenates were freeze‐thawed three times at −80°C and 37°C, respectively, and then centrifuged at 11,200 g for 20 min at 4°C. After determining protein concentration, samples prepared in Laemmli buffer  were boiled for 2 min, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) on 7% polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. Membranes were blocked in 5% nonfat dry milk in phosphate‐buffered saline (PBS) containing 0.1% Tween 20 and then incubated overnight at 4°C with primary anti‐CaV3.2 antibody . After washing, membranes were incubated with anti‐mouse IgG‐peroxidase‐linked secondary antibody  for 1 hr at room temperature. Blots were washed and incubated in enhanced chemiluminescence reagents , after which bands were detected using a ChemiDoc XRS+ Imaging System (Bio‐Rad). An anti‐Actin antibody  was used as a loading control, and Precision Plus Protein Prestained Standard (Bio‐Rad) was used as a molecular weight marker.Mesenteric arteries were isolated from mice and placed into cold physiological saline solution (PSS) previously oxygenated for 30 min , and afterward, vessels were dissected, and tissue pieces were further fixed for 4 hr in 4% PFA, transferred to 15% sucrose  for 4 hr and incubated in 30% sucrose overnight. After embedding in TissueTek (Sakura), the tissue is frozen at −80°C and 8‐µm sections were obtained in a Leica cryostat at −30°C. For immunostainings, the cryostat sections were incubated with blocking buffer (1% donkey serum/1% Triton X‐100/PBS), the first antibody was applied overnight at 4°C, and after washing with PBS/1% Tween, the secondary antibody and DAPI were applied for 2 hr. Afterward, the sections were embedded in ImmoMount (Thermo Scientific #9990402). The stained sections were analyzed with Zeiss LSM700 microscope provided with Zeiss 40x objective, and images were analyzed by ImageJ/Fij. Antibodies: anti‐beta‐actin‐mouse (Sigma #A2228), anti‐Cav3.2‐rabbit (Alomone Labs #ACC‐025), anti‐mouse‐Alexa488 (Invitrogen #R37114), anti‐rabbit‐Cy3 (Dianova #711‐165–152), and DAPI (Sigma #D9542).4.82 −5% CO2; pH, 7.4; 37°C) containing (mM): 119 NaCl, 4.7 KCl, 25 NaHCO3, 1.2 KH2PO4, 1.6 CaCl2, 1.2 MgSO4, and 11.1 glucose. The intravascular pressure was incrementally elevated from 20 to 100 mmHg using a pressure servo control system (Living System Instrumentation), and the inner diameter of the vessel was measured (Nikon Diaphot). The recording system was connected to a personal computer for data acquisition and analysis (HaSoTec). Arteries were equilibrated at 15 mmHg for 60 min and contractile responsiveness assessed by applying 60 mM KCl before starting experiments.Vessel myography was performed as previously described . Thapsigargin was purchased from Alomone Laboratories. All salts and other drugs were obtained from Sigma‐Aldrich or Merck. In cases where DMSO was used as a solvent, the maximal DMSO concentration after application did not exceed 0.5%  or Mann–Whitney U test. p‐values < .05 were considered statistically significant; “n” represents the number of cells.Data are presented as means ± None declared.G.F., M.K., Y.C., D.T, C.M., S.K., C.Z., S.Z., and Y.X. were responsible for data collection, analysis, and interpretation. M.K. and M.G. were responsible for the conception and design of the experiments. G.F. and M.G. drafted the manuscript. All authors were responsible for interpretation of the data, contributed to the drafting, and revised the manuscript critically for important intellectual content. All authors have approved the final version of the manuscript and agreed to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.Fig S1Click here for additional data file.


DA released from receptor (type I) cells during chemostimulation is predominantly inhibitory, acting via pre- and post-synaptic dopamine D2 receptors (D2R) on type I cells and afferent  terminals respectively. By contrast, co-released ATP is excitatory at postsynaptic P2X2/3R, though paracrine P2Y2R activation of neighboring glial-like type II cells may boost further ATP release. Here, we tested the hypothesis that DA may also inhibit type II cell function. When applied alone, DA (10 μM) had negligible effects on basal [Ca2+]i in isolated rat type II cells. However, DA strongly inhibited [Ca2+]i elevations (Δ[Ca2+]i) evoked by the P2Y2R agonist UTP (100 μM), an effect opposed by the D2/3R antagonist, sulpiride (1–10 μM). As expected, acute hypercapnia , or high K+ (30 mM) caused Δ[Ca2+]i in type I cells. However, these stimuli sometimes triggered a secondary, delayed Δ[Ca2+]i in nearby type II cells, attributable to crosstalk involving ATP-P2Y2R interactions. Interestingly sulpiride, or DA store-depletion using reserpine, potentiated both the frequency and magnitude of the secondary Δ[Ca2+]i in type II cells. In functional CB-petrosal neuron cocultures, sulpiride potentiated hypercapnia-induced Δ[Ca2+]i in type I cells, type II cells, and petrosal neurons. Moreover, stimulation of type II cells with UTP could directly evoke Δ[Ca2+]i in nearby petrosal neurons. Thus, dopaminergic inhibition of purinergic signalling in type II cells may help control the integrated sensory output of the CB during hypercapnia.Dopamine (DA) is a well-studied neurochemical in the mammalian carotid body (CB), a chemosensory organ involved in O Though perfusion of the same culture with 10 μM dopamine (DA) alone had no effect on basal [Ca2+]i in either cell type, the UTP-evoked Δ[Ca2+]i in the type II cell was markedly inhibited by DA  inhibited the UTP-evoked integrated Δ[Ca2+]i (mean inhibition by ~70%) as well as the duration of the intracellular Ca2+ signal . Also, when present alone, sulpiride had no effect on the basal intracellular Ca2+ levels in type II cells at the concentrations used, suggesting it did not cause a non-specific elevation in intracellular Ca2+ transients in The inhibitory effects of DA at the CB chemosensory complex have been attributed largely to the presence of both pre- and post-synaptic D2 receptors (D2R) ,10,15,172+-dependent activation of ATP-permeable pannexin I channels i in a type I cell that was part of a cell cluster; note that the neighboring type II cell (identified by the presence of a UTP-induced Ca2+ transient) was unresponsive to hypercapnia. However, when the hypercapnic stimulus was re-applied to the same cells, but in the presence of sulpiride (10 μM), there was a potentiation of the type I cell Ca2+ response and the unveiling of a delayed Δ[Ca2+]i in the type II cell . We attribute the potentiation of the type I cell Ca2+ response by sulpiride to the prevention or removal of autocrine-paracrine negative feedback inhibition mediated by released DA acting on D2R on type I cells i in a type I, but not type II, cell when applied alone. By contrast, when the high K+ stimulus was re-applied in conjunction with sulpiride to the same cells there was an unveiling of a delayed, robust Ca2+ response in the type II cell. This effect was reversible after washout of sulpiride .To obtain further support for the posit that DA inhibits ATP-mediated crosstalk between type I and type II cells we used a high K I cells ,17,22,30ulpiride D. Summar2 ~15–20 mmHg), though under our experimental conditions the intracellular Ca2+ responses in type I cells tended to be smaller and were detectable at a lower frequency than for high K+ or hypercapnic stimuli [2+]i in both type I and neighboring type II cells in the presence of sulpiride. However, the responses were poorly reversible after washout of the drug (data not shown). Taken together, the combined data from these studies suggest that during chemoexcitation the paracrine excitatory effect of ATP-P2Y2R signalling on Ca2+ transients in type II cells is blunted by the concurrent paracrine action of co-released DA acting via sulpiride-sensitive DA receptors.We also tested the effects of hypoxia , suggesting that purinergic signalling contributed significantly to crosstalk in these conditions, as previously demonstrated for control cultures i) were routinely obtained from type I and type II cells, and in some cases from cocultured petrosal neurons as well. The procedures were similar to those described in detail in previous studies from this laboratory i according to the Grynkiewicz equation [2+ were identical to those described previously [2+ transient during stimulus application was >20 nM above baseline or preceding ‘control’ intracellular Ca2+ levelFura-2 measurements of intracellular Caboratory ,21,27. Iequation ,45. The 3 (24), NaCl (115), glucose (5), KCl (5), CaCl2 (2) and MgCl2 (1) at pH ~7.4, maintained by bubbling a 5% CO2-95% air mixture. Isohydric hypercapnia  was generated by doubling the concentration of NaHCO3 to 48 mM (maintaining osmolarity by reducing NaCl to 91 mM) and aerating with 10% CO2–90% air (pH ∼7.4). Hypoxia was generated by bubbling standard perfusion solution (as stated above) with 5% CO2–90% N2 (pH ∼7.4). High extracellular K+ solution (30 mM KCl) was made by equimolar substitution of NaCl for KCl. UTP (94370), dopamine hydrochloride (H8502), (±)-sulpiride (S8010), and reserpine (R0875) were purchased from Sigma Aldrich . Drugs were perfused over the cells for 1 min before, 1 min during, and 1 min after the stimulus was applied.The perfusion solution  was maintained at 35–37 °C and was composed of (in mM): NaHCOt test was used where noted. Results were considered statistically significant at p < 0.05.For each experiment, dissociated carotid bodies (CBs) from one litter of 10–12 pups were divided and plated into four equal fractions at similar densities. For data analysis, the indicated (n) values in the text refer to the number of litters used for each experiment. Typically, 3–20 cells were sampled per dish. Statistical analysis was carried out using either one way or repeated measures ANOVA with Tukey’s post hoc tests (for non-parametric data). Student’s 2+ that is required for the activation of ATP-permeable pannexin-1 channels in type II cells [This study uncovered a previously unrecognized pathway by which dopaminergic inhibition helps control the CB chemoafferent discharge. This pathway involves the glial-like type II cells, previously proposed to contribute to CB excitation by boosting synaptic levels of the excitatory neurotransmitter ATP via the mechanism of ‘ATP-induced ATP’ release . Our curII cells . By releII cells ,21,35.


The captured information is represented graphically as a signed directed graph. Each signaling relationship is associated to an effect (up/down-regulation) and to the mechanism  causing the up/down-regulation of the target entity. Since its first release, SIGNOR has undergone a significant content increase and the number of annotated causal interactions have almost doubled. SIGNOR 2.0 now stores almost 23 000 manually-annotated causal relationships between proteins and other biologically relevant entities: chemicals, phenotypes, complexes, etc. We describe here significant changes in curation policy and a new confidence score, which is assigned to each interaction. We have also improved the compliance to the FAIR data principles by providing (i) SIGNOR stable identifiers, (ii) programmatic access through REST APIs, (iii) bioschemas and (iv) downloadable data in standard-compliant formats, such as PSI-MI CausalTAB and GMT. The data are freely accessible and downloadable at  In SIGNOR 2.0, each causal interaction is represented as a source entity that affects  a target entity and is annotated with the mechanism underlying the regulation . Causal interactions are visualized as a dynamic and customizable graph where nodes are the entities and edges represent the causal relationships between them  curation. However, dedicated efforts were made to increase the coverage of domains of interest in our group. For instance, a specific literature search aimed at extending the coverage of relationships that modulate the activity of Transcription Factors (TFs) and the relationships between TFs and their validated target genes. To date, 571 TFs (35% of the TFs defined in the list of Lambert group)  have at To support a group effort aimed at linking ‘cancer genes’ to cancer Hallmark phenotypes , we haveA dedicated curation effort was also devoted to post-translational modifications, focusing on phosphorylation and dephosphorylation modifications. Only modifications with a known effect (activation/inactivation) on the target protein were considered. As a consequence, the number of causal phosphorylation reactions have increased from 4900 to 9088 and the dephosphorylation reactions from 250 to 1072. 381 kinases and 119 phosphatases are now integrated in the SIGNOR causal network.www.guidetopharmacology.org) resource that contains expert-curated interactions between targets and ligands  extracted from pharmacology and drug discovery journals .We have also annotated the large G protein-coupled receptors (GPCRs) interaction dataset recently published by Inoue et al. , coverinSIGNOR 2.0 is mainly a non-organized collection of causal relationships. User feedback, however, has ascertained that the organization of the annotated relationships in pathways has some value for users. Since pathways are only useful mental abstractions, they cannot have a unique definition and different experts are likely to associate different relationships to the same pathway. Our criterion in defining the size of a subgraph representing a pathway is the ease in the interpretation of the corresponding graph layout. As a consequence, the pathways annotated in SIGNOR 2.0 are limited to a maximum of approximately 30 nodes and 50 edges. SIGNOR 2.0, in its latest release, offers the annotation of 49 curated pathways. This pathway list is available in the SIGNOR 2.0 home page through a drop-down menu that allows pathway selection for graphical representation. Moreover, the pathways stored in SIGNOR 2.0 can be viewed in the NDEx resource  and visuOver the past few years, the signaling community has worked within the HUPO-PSI and GREEKC frameworks , to devehttps://www.ebi.ac.uk/ols/ontologies/mi/terms?iri=http%3A%2F%2Fpurl.obolibrary.org%2Fobo%2FMI_2233). For example, all the terms annotated as ‘activates/inactivates’ have been converted into ‘up-regulates/down-regulates’ or into more granular child terms (e.g. up-regulates activity or down-regulates quantity by destabilization).In addition, the signaling community has agreed on establishing guidelines dubbed ‘minimum information about a molecular interaction causal statement (MI2CAST)’ that recommends ontologies, CVs and other related details to represent causal interactions in a standardized and reproducible way. We have updated our internal control vocabulary in compliance with the new standards. All terms have been mapped to the PSI-MI Controlled Vocabulary terms under the root term ‘causal interaction’ .SIGNOR 2.0 also contains data on fusion proteins, proteins generated by translocation events involving critical regions of proto-oncogenes. The rearrangements generate chimeric proteins directly promoting abnormal cell proliferation or differentiation blocks. Fusion proteins actively contributing to carcinogenesis, as for example the fusion proteins AML1-ETO and PML-RARα in Leukemia . Fusion Mus musculus and Rattus norvegicus. Mapping was obtained by extracting orthology data from the InParanoid resource  by causal interactions. In the search box, the user has now the possibility to insert one, two or more entity names or identifiers and, depending on the number of inserted entities, to get access to different functionalities. If only one entity is entered in the field, the search returns the ‘entity page’ listing all the relationships pertaining to the query entity. If the entity names are more than one, by clicking the ‘all’ check-box button, one obtains a graph showing all the relationships in which the query entities are involved, when the ‘connect’ check-box is selected only the interactions connecting the query entities will be displayed. An extension of this latter functionality permits to draw graphs connecting the query proteins via additional ‘bridge’ proteins (‘include first neighbors’ check-box).Finally, a new algorithm was implemented to identify the shortest directional path between any two nodes in the causal graph. The algorithm can be launched when the query field contains two entities. In this case the ‘shortest path’ check box can be selected to launch an algorithm that finds the shortest directional path (and those that are one step longer) between the two entities. The results of such a query are shown both in a graph and a table view Figure , where tThe data in SIGNOR 2.0 can be freely downloaded. In the download page, the user can choose to download various subsets of the data, i.e phosphorylation data, specific interactions based on relation ID, transcriptional relations and pathway-specific relations in various formats, such as SBML and GMT. The entire dataset of binary causal interactions is offered both as a stable quarterly release and as a ‘live’, up-to-date file containing all the recently curate interactions. Files are offered in a tab-delimited format as well as a CausalTab. SIGNOR 2.0 entity data, and detailed information about CV, is available for download as well.To facilitate programmatic access to SIGNOR 2.0, data can also be retrieved via a REST API that is based on simple URL queries using selected parameters, as shown in In order to join the effort toward increasing ‘searchability’ and ‘findability’ of biological data, the Schema.org markup has been included in the main SIGNOR 2.0 page code. This includes descriptive information about the database and its contents, plus information about data formats and retrieval.The new SIGNOR 2.0 score combines four features, each taken as evidence of the support for the functional relevance of the considered causal relationship. The features can be divided into two categories; the first one is based on the annotation within SIGNOR 2.0, whereas the second one employs external resources.For the first two features, we count (i) the number of annotated articles (PMIDs) reporting works that support the interaction in the annotation in SIGNOR 2.0 and (ii) the number of occurrences of the specific relationship in a pathway that is already annotated in SIGNOR 2.0.The remaining two features are independent from SIGNOR 2.0 annotations and consider the support that is possible to extract from two of the major curated pathway databases: Reactome  and UniPsource–target relationship we count how many times the target entity is mentioned in the UniProtKB entity page of the source.For the ‘Reactome feature’ we count the number of occurrences of the specific interaction pair in the ‘Human Protein Interactions’ file that can be downloaded from the Reactome website. As for the UniProtKB feature, we surmise that if entity X (protein or phenotype) is mentioned in the UniprotKB page of protein Y, the two entities are more likely to be functionally correlated. Thus, for a given n’  that we associate to a ‘partial score’ Y according to the following formula in order to normalize each value in the 0–1 range.Each feature returns a figure ‘C was empirically set to 1.5 for all the features with the exception of the number of supporting PMIDs that was set to 2.2, since this feature has much lower values than the other ones. The final combined score (r) is obtained by averaging the four partial scores.The constant The score ranges between 0 and 1. However the score can never be 0 since all SIGNOR relations are supported by at least one reference. The combined score distribution is depicted as a bar diagram .Signaling information can be captured by a variety of different models each with a different level of description granularity and coverage potential. Some established databases, such as KEGG and REACTOME use reaction-based models, which capture some mechanistic details and—kinetic constants available—the dynamics of networks containing up to a few dozen entities. Models based on networks of logic relationships, such as SIGNOR, on the other hand, have a higher potential to describe larger systems at the expenses of mechanistic details. Users have the possibility to choose a specific resource in the signaling resources panorama, according to their needs or interest.The number of stored logic relationships has almost doubled.Focused curation on relationships involving membrane receptors, proteins mutated in cancers and pharmacologically relevant proteins.Clearer definition of curation rules, controlled vocabularies and standards for data exchange.REST APIs for programmatic access.New graph analysis tools to explore functional connections between any pair of network entities.Here we have presented SIGNOR 2.0, which is a much improved version of the database published in 2016 . The moset al. under submission to the NAR database issue) and muscle regeneration  on any other node of the network can be estimated by looking the graph paths connecting the two query nodes. The result of such a procedure is not unambiguous as the different paths that are retrieved may predict opposite effects on the target protein or phenotype. There are a variety of reasons for this apparently contradictory response. For instance, not all proteins are equally expressed in the different systems and a different proteome context may favor one or the other outcome. Although the newly developed reliability score may help in limiting the analysis to paths covering more trustworthy relationships, it is left to the ‘expert’ users to filter out the paths that, based on their experience, are unlikely to be biologically relevant or to test them experimentally.SIGNOR 2.0, with its growth and improvements, is now a more mature resource that offers valuable tools to design experiments and interpret the results.Importantly, in the new version of SIGNOR 2.0, we followed FAIR data principles in order to increase data interoperability. SIGNOR 2.0 now adopts stable identifiers, offers REST APIs for programmatic access, implements bioschemas and allows data download in standard-compliant format.https://signor.uniroma2.it/downloads.php.SIGNOR 2.0 interaction data are available and freely downloadable at: https://signor.uniroma2.it/APIs.php.Furthermore, programmatic access is available through the APIs section at https://signor.uniroma2.it/.All described database features are accessible via the main SIGNOR 2.0 website: gkz949_Supplemental_FileClick here for additional data file.


Arrhythmogenic Ca2+ alternans in cardiac myocytes has been observed in experiments under abnormal mitochondrial depolarization. However, complex signaling pathways and Ca2+ cycling between mitochondria and cytosol make it difficult in experiments to reveal the underlying mechanisms of Ca2+ alternans under abnormal mitochondrial depolarization. In this study, we use a newly developed spatiotemporal ventricular myocyte computer model that integrates mitochondrial Ca2+ cycling and complex signaling pathways to investigate the mechanisms of Ca2+ alternans during mitochondrial depolarization. We find that elevation of ROS in response to mitochondrial depolarization plays a critical role in promoting Ca2+ alternans. Further examination reveals that the redox effect of ROS on ryanodine receptors and sarco/endoplasmic reticulum Ca2+-ATPase synergistically promote alternans. Upregulation of mitochondrial Ca2+ uniporter promotes Ca2+ alternans via Ca2+-dependent mitochondrial permeability transition pore opening. Due to their relatively slow kinetics, oxidized Ca2+/calmodulin-dependent protein kinase II activation and ATP do not play significant roles acutely in the genesis of Ca2+ alternans after mitochondrial depolarization, but their roles can be significant in the long term, mainly through their effects on sarco/endoplasmic reticulum Ca2+-ATPase activity. In conclusion, mitochondrial depolarization promotes Ca2+ alternans acutely via the redox effect of ROS and chronically by ATP reduction. It suppresses Ca2+ alternans chronically through oxidized Ca2+/calmodulin-dependent protein kinase II activation.Mitochondria are vital organelles inside the cell and contribute to intracellular calcium . However, the underlying mechanisms behind these experimental observations remain unclear. Here, we focus on revealing the underlying mechanisms of cellular Ca2+ alternans in the context of mitochondrial depolarization. Using our newly developed action potential model that incorporates a 3D network of Ca2+ release unit with a 3D network of mitochondria, we dissect out individual roles of mitochondrial Ca2+ and complex signaling pathways  in the genesis of cellular Ca2+ alternans. The key finding is that mitochondrial depolarization promotes Ca2+ alternans acutely via the redox effect of ROS and chronically by ATP reduction, and oxidized Ca2+/calmodulin-dependent protein kinase II activation may suppress Ca2+ alternans chronically. We believe that this study reveals insightful information for understanding the mechanisms of the genesis of Ca2+ alternans in the context of mitochondrial depolarization and provides valuable simulation results to guide future experiments.Mitochondrial dysfunction has been implicated in life-threatening cardiac arrhythmias. Experimentalists have shown that mitochondrial depolarization could cause period-doubling behavior in cytosolic Ca Time traces of voltage and Ca for control and mitochondrial depolarization cases at PCL = 500 ms. B. Time traces of voltage and Ca for mitochondrial depolarization case at PCL = 300 ms. Note that we plot two consecutive beats on top of each other to better observe the alternans.(TIF)Click here for additional data file.S3 Fig2+ transients vs. the whole-cell averaged open probability of mPTP. The open probability of mPTP was measured at the end of each simulation of Bifurcation diagram of peak values of Ca(TIF)Click here for additional data file.S4 FigA. Time traces of whole-cell averaged cytosolic Ca2+ transient and mitochondrial Ca2+ for αMCU = 1 (red) and 0.5 (black). αmPTP = 60. B. Same as A, but mitochondrial NCX was at control (red) and 50% reduction (black). αmPTP = 60, PCL = 500 ms.(TIF)Click here for additional data file.S5 FigA. Time traces of the whole-cell average cytosolic Ca2+, mitochondrial Ca2+, where the mitochondrial Ca2+ was clamped to 0.4 (black), 0.5 (red), and 0.6 (blue) μM at t = 25 sec. B. Same as A, but the cytosolic Ca2+ was clamped to 0.2 (black), 0.5 (red), and 1 (blue) μM at t = 25 sec.(TIF)Click here for additional data file.S6 Fig2+ alternans amplitude on αmPTP and 2+ transient peaks in a simulation of 30 sec. PCL = 500 ms.Dependence of Ca(TIF)Click here for additional data file.S7 Fig0 and C1 are the two closed states. O represents the open state.Three-state mPTP model. C(TIF)Click here for additional data file.


Monarda fistulosa L. essential oils. To address this issue, we isolated essential oils from the flowers and leaves of M. fistulosa and analyzed their chemical composition. We also analyzed the pharmacological effects of M. fistulosa essential oils on transient receptor potential (TRP) channel activity, as these channels are known targets of various essential oil constituents. Flower (MEOFl) and leaf (MEOLv) essential oils were comprised mainly of monoterpenes (43.1% and 21.1%) and oxygenated monoterpenes (54.8% and 77.7%), respectively, with a high abundance of monoterpene hydrocarbons, including p-cymene, γ-terpinene, α-terpinene, and α-thujene. Major oxygenated monoterpenes of MEOFl and MEOLv included carvacrol and thymol. Both MEOFl and MEOLv stimulated a transient increase in intracellular free Ca2+ concentration ([Ca2+]i) in TRPA1 but not in TRPV1 or TRPV4-transfected cells, with MEOLv being much more effective than MEOFl. Furthermore, the pure monoterpenes carvacrol, thymol, and β-myrcene activated TRPA1 but not the TRPV1 or TRPV4 channels, suggesting that these compounds represented the TRPA1-activating components of M. fistulosa essential oils. The transient increase in [Ca2+]i induced by MEOFl/MEOLv, carvacrol, β-myrcene, and thymol in TRPA1-transfected cells was blocked by a selective TRPA1 antagonist, HC-030031. Although carvacrol and thymol have been reported previously to activate the TRPA1 channels, this is the first report to show that β-myrcene is also a TRPA1 channel agonist. Finally, molecular modeling studies showed a substantial similarity between the docking poses of carvacrol, thymol, and β-myrcene in the binding site of human TRPA1. Thus, our results provide a cellular and molecular basis to explain at least part of the therapeutic properties of these essential oils, laying the foundation for prospective pharmacological studies involving TRP ion channels. Little is known about the pharmacological activity of  Monarda L. (Lamiaceae) includes 20 species native to the United States . ,11. Monap-cymene . In contl (5.5%) . The culreported ,44,45,46i in HEK 293 cells overexpressing TRPA1. HEK rTRPA1 cells were treated with increasing concentrations of MEOFl and MEOLv, with Hanks’ balanced-salt solution (HBSS) washes included between each dose, as indicated in M. fistulosa essential oils dose-dependently induced transient [Ca2+]i influx. Transient changes between baseline to peak [Ca2+]i induced by 20 and 25 µg/mL of MEOFl (0.095 ± 0.008 and 0.105 ± 0.007) and MEOLv (0.145 ± 0.007 and 0.150 ± 0.006) had similar magnitudes; hence, we chose 20 µg/mL as the working concentration for further experiments, since it was the concentration where the agonist effect plateaued. Analysis of the AITC (TRPA1 agonist)-induced increase in [Ca2+]i confirmed the functional expression of TRPA1 channels in these cells. Summarized data showing the quantified transient increases in [Ca2+]i induced by increasing concentrations of the essential oils are shown in Fl and MEOLv, respectively.Essential oils and their components have been reported previously to modulate TRP channel activity ,53,54,55Monarda essential oils elicited a transient increase in [Ca2+]i exclusively through TRPA1 channels, we evaluated the specific TRPA1 antagonist HC-030031 [2+]i elicited via MEOFl and MEOLv were eliminated in HEK rTRPA1 cells when cells were pretreated with HC-030031 (20 µM) for 20 min, and reversible inhibition was confirmed by a subsequent transient increase in [Ca2+]i elicited by the essential oils following washout of HC-030031 from the perfusion chamber with the control buffer , we performed real-time calcium imaging in HEK 293 cells stably overexpressing either hTRPV1 or hTRPV4 channels. Representative traces show that 20 µg/mL of MEOFl and MEOLv failed to induce any increase in [Ca2+]i in HEK hTRPV1 and HEK hTRPV4 cells  and GSK-1016790A (TRPV4 agonist) were evaluated to confirm the functional expressions of TRPV1 and TRPV4, respectively ,58. The ed cells .M. fistulosa essential oils on TRPA1 channels, parental HEK 293 cells were subjected to real-time calcium flux analysis in the presence of MEOFl and MEOLv, and representative traces illustrating the effects of TRP channel agonists and M. fistulosa essential oils on [Ca2+]i in parental HEK 293 cells are shown in 2+]i in parental HEK 293 cells. Similarly, no changes in [Ca2+]i were observed when parental HEK 293 cells were treated with either MEOFl or MEOLv , had no effect on the nontransfected/parental HEK 293 cell viability. Indeed, receptor-effector coupling of these cells after essential oil treatment was robust upon treatment with ATP, which induced a pronounced increase in [Ca2+]i and demonstrated that the cells were still viable and functional after essential oil or monoterpene treatment.Although we did not evaluate the cytotoxicity of the Monarda essential oils activated TRPA1 channels, we next investigated the TRPA1-activating effects of their constituent monoterpenes to identify the active compounds. Sixteen commercially available compounds, including six major compounds , were evaluated for agonist effects on [Ca2+]i in HEK rTRPA1 cells; however, only carvacrol, thymol, and β-myrcene exhibited significant agonist activity 2C= fragment of β-myrcene, occupy the same area of space in the vicinity of the hydrophobic Ile811, Tyr812, and Tyr842 side chains. Hydroxyl groups of carvacrol and thymol are located close to each other in their superimposed docking poses and are directed to the OH and carboxyl groups of Tyr812 and Glu808, respectively. As for the inactive p-cymene, there was a noticeable difference in location of the molecule in the binding site when compared with the active compounds. There was a displacement of the p-cymene aromatic ring relative to the docking poses of the active aromatic compounds (carvacrol and thymol) and with respect to the location of the β-myrcene hydrocarbon chain.We performed molecular docking studies on carvacrol, thymol, β-myrcene, and try 3J9P ) as the try 3J9P  using thM. fistulosa represent a rich source of bioactive monoterpenes with a broad range of therapeutic potential, as summarized in Monarda is represented by 20 native species, with M. fistulosa (wild bergamot) essential oils being used medicinally to treat cardiovascular defects, wounds, colds and flu, gastrointestinal disorders, insomnia, and infant convulsions i measurements, all cell types were grown as monolayers until 70–80% confluency. Cells were incubated at 37 °C for 30 min with 5-µM Fura 2-AM in complete medium, followed by two gentle washes. De-esterification was allowed for a further 30 min, and then, the monolayers were mounted on the stage of an Olympus IX-81 inverted fluorescence microscope . Cells were superfused continuously with HBSS  at a flow rate of 2 mL/min. Cells were exposed to compounds under investigation by switching from the control medium (HBSS with Ca2+) to the compound-containing medium (working concentrations of treatment prepared in HBSS with Ca2+) for 30s, unless noted otherwise. Control buffer and treatments were always kept in separate chambers to prevent any potential interaction between the compounds. Relative [Ca2+]i was allowed to return to baseline after a response before exposing the cells to any successive treatment. Cells were pretreated with the TRPA1 antagonist HC-030031 (20 µM) for 20 min to block TRPA1 activation with essential oils. ATP (25 µM) was routinely utilized to confirm receptor-effector coupling in control parental HEK 293 cells. The [Ca2+]i measurements were recorded in individual cells using a fluorescence imaging system  equipped with a multi-wavelength DeltaRAM monochromator  and a QuantEM 512SC electron multiplying charge-coupled device camera , as previously described [2+]i data were acquired using an alternating excitation wavelength protocol  and emission wavelength of 510 nm. Background fluorescence was automatically corrected for the experiments using Easy Ratio Pro software. The signals obtained at 340 and 380 nm were calculated as the 340/380 ratio and used as an index to measure relative changes in [Ca2+]i from the baseline [For real-time [Caescribed . Images baseline  and exprThe structure of TRPA1 determined experimentally by electron cryo-microscopy  was downp-cymene were downloaded from the PubChem database  and imported into MVD. Docking was performed with a rigid receptor and flexible ligands. For each compound, 300 docking runs were performed. For the docking poses obtained, additional geometry optimizations were carried out using the “Energy Minimization” and “Optimize HBonds” options of the MVD program.Three-dimensional conformers of carvacrol, thymol, β-myrcene, and t-test, one-way ANOVA, and two-way ANOVA, as appropriate. For all statistical analyses, GraphPad Prism 8.0 software was used . p-values are as stated in the figure legends.The data are expressed as mean ± SEM. Statistical comparisons were made with an unpaired