PARP and other DDR-related genes in acute leukemia, selective vulnerabilities have been reported in several disease subgroups, along with a “BRCAness phenotype.” AML carrying the RUNX1-RUNX1T1 or PML-RARA fusion genes or mutations in signaling genes (FLT3-ITD in combination with TET2 or TET2 and DNMT3A deficiency), cohesin complex members (STAG2), TP53 and BCOR as co-occurring lesions, IDH1/2 and ALL cases expressing the TCF3-HLF chimera or TET1 was highly sensitive to PARPi in preclinical studies. These data, along with the warning coming from the observation of cases of therapy-related myeloid malignancies among patients receiving PARPi for solid tumors treatment, indicate that PARPi represents a promising strategy in a personalized medicine setting. The characterization of the clonal and subclonal genetic background and of the DDR functionality is crucial to select acute leukemia patients that will likely benefit of PARPi-based therapeutic regimens.The members of the Poly(ADP‐ribose) polymerase (PARP) superfamily are involved in several biological processes and, in particular, in the DNA damage response (DDR). The most studied members, PARP1, PARP2 and PARP3, act as sensors of DNA damages, in order to activate different intracellular repair pathways, including single-strand repair, homologous recombination, conventional and alternative non-homologous end joining. This review recapitulates the functional role of PARPs in the DDR pathways, also in relationship with the cell cycle phases, which drives our knowledge of the mechanisms of action of PARP inhibitors (PARPi), encompassing inhibition of single-strand breaks and base excision repair, PARP trapping and sensitization to antileukemia immune responses. Several studies have demonstrated a preclinical activity of the current available PARPi, olaparib, rucaparib, niraparib, veliparib and talazoparib, as single agent and/or in combination with cytotoxic, hypomethylating or targeted drugs in acute leukemia, thus encouraging the development of clinical trials. We here summarize the most recent preclinical and clinical findings and discuss the synthetic lethal interactions of PARPi in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Despite the low frequency of genomic alterations of  The majylation) , (2) theylation) .Table 1BThis review aims to discuss the potentials of PARP inhibitors (PARPi)-based therapeutic strategies in acute leukemia. To this aim, we first summarize the role of PARP proteins in the DDR and the mechanisms of action of PARPi, which helps understanding their preclinical and clinical successes and failures in acute leukemias. We then focus on synthetic lethal interactions of PARPi in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), which opens a promising therapeutic window for specific disease subgroups.PARP1, PARP2 and PARP3 are the most studied enzymes for their involvement in the DDR. In mammalian cells, they act as DDR sensors in response to different types of DNA damages. In particular, PARP1 responds to single-strand breaks (SSBs), DNA cross-links, stalled replication forks and double-strand breaks (DSBs) . PARP2 sBase modifications can arise by endogenous sources or errors during DNA replication and both PARP1 and PARP2 enzymes play a central role in their repair throughout base excision repair/single-strand break repair process (BER/SSBR) . PARP1 PIn eukaryotic cells, DSBs are repaired through HR, cNHEJ or aNHEJ repair pathways, according to the cell cycle phase . PARP1 pcNHEJ is the chosen mechanism of DDR during interphase (G0/G1 in particular) . PARP1 PPARP1 has a crucial role in the regulation of aNHEJ repair, that is mostly active in S and G2 phases of cell cycle , 47  or deletions , while they accounted for 1.4% of pediatric AML cases . In adults, PARP2 was found mutated in one case and amplified in another one in the TCGA-LAML cohort . PARP3 was deleted in two cases of pediatric AML and ALL  and mutated in one patient from the Beat AML cohort .According to public genomic data available on acute leukemias on the cBio portal   and below 1.0% in the Beat AML study . Deletions, missense and truncating mutations, that likely induce loss of function phenotypes, are the most recurrent alterations in BRCA1 and BRCA2 genes (Table RAD51 (135C) and its paralog XRCC3 (241M) has been associated with an increased risk of de novo and t-AML [Mre11A and ATM on chromosome 11 has been described in t-AML patients and leads to alterations in both NHEJ and HR as Mre11A is an early factor in these two pathways [FANCA gene were found in a small percentage of AML patients [FANCC point mutation was identified [ATM, PRKDC, ATR, RPA1, DSS1, NBN, RAD51, RAD54, CHEK1, CHEK2, ERCC1, POLB, FEN1 and CDK12 have shown synthetic lethality in combination with PARPi [Mutations, copy number alterations and/or polymorphisms of other DDR genes have been described, that may result in the deregulation of the HR pathway activity. Mutations in the nd t-AML . Deletiopathways . Alteratpathways . Heterozpatients  and havepatients . In T-ALentified . Genomicth PARPi  and are th PARPi .PALB2, a gene of the FA pathway that interacts with BRCA1 and BRCA2 in the DNA damage repair [. Patients with PALB2-mutated solid tumors have been reported to be responsive to PARPi [PALB2 [. In addition, another phase II trial is enrolling patients with advanced HER2-negative breast cancer and other solid tumors with mutations in HR genes (excluding BRCA1/2) for the evaluation of the treatment with talazoparib (NCT02401347). Therefore, PALB2 deletion is a potential marker of HR defects, that may also suggest a potential vulnerability to PARP inhibition in AML.Moreover, we have recently reported that about 5% of AML cases carry heterozygous copy number loss of e repair . Patientto PARPi –83, and i [PALB2 . In addiAs already mentioned, SSBs arise frequently in proliferating cells and they are repaired through PARP-dependent DNA repair mechanisms . Efficient SSB repair is essential for the survival of proliferating cells Fig. A. All thAs an additional mechanism of action, PARPi can trap PARP1 and PARP2 enzymes on DNA  Fig. 4CC4C. It h+ T and CD8+ T cells [+ T cells functions as a costimulatory signal. Paczulla and colleagues showed that PARP1 represses NKG2D ligands expression in leukemia stem cells, thus contributing to their selective escape from immune surveillance by NK cells [In addition to a direct activity on the DDR pathway, PARPi exert an immunomodulatory function, as demonstrated in multiple cancer types. Numerous lines of evidence now suggest that the DDR plays an important role in driving sensitivity and response to immune checkpoint blockade . First,  T cells . In para T cells . BindingNK cells . AccordiNK cells . These dThis section summarizes the main preclinical data available regarding the efficacy of different PARPi in acute leukemias.RUNX1-RUNX1T1 rearranged AML cell line, KASUMI1; erythroleukemia cell line, KG1a) and xenograft models (mice transplanted with KG1a cells), both in monotherapy and when used in combination, by increasing apoptosis and reducing cell proliferation [KMT2A-AFF1 rearranged AML cell line, MV4-11; FLT3-ITD AML cell line, PL21) by disrupting BER, which was required for repairing decitabine-induced DNA lesions through recruitment of XRCC1 at DNMT1 foci and repair of trapped DNMT1 [KMT2A-MLLT3 rearranged AML cell line, MOLM13; NPM1/DNMT3A double mut AML cell line, OCI-AML3) and ALL  cell lines, resulting in DNA damage accumulation and cell death by apoptosis, as also confirmed in an AML murine model [Olaparib  is an orally bioavailable and well-tolerated PARPi. Olaparib binds four members of the PARP super family, PARP1-PARP4  and is tferation . Moreoveed DNMT1 . Impairmne model . The comDNMT3A mut OCI-AML2 and T-ALL RPMI-8402 cells) and in vivo in AML (mice transplanted with KMT2A-MLLT3 AML primary cells) and ALL (mice transplanted with pre-T-ALL primary cells) showing elevated efficacy [Rucaparib (Rubraca/AG-0146991) is an orally bioavailable PARPi, with cancer cytotoxicity levels comparable to olaparib . A recenefficacy . Upon ruefficacy .FLT3-ITD cells MOLM-14) with activation of the ATM pathway, increased production of ROS, decreased mitochondrial membrane potential, and induction of apoptosis. The efficacy of the triple combination was confirmed also on primary leukemic cells  in which the combination induced DNA damages (H2AX marker) and triggered apoptosis (cleaved PARP1 and Caspase 9) [Niraparib (MK-4827) is an orally bioavailable PARP1/2 inhibitor, also showing cancer cytotoxicity levels comparable to olaparib . Few stuspase 9) . The mecVeliparib showed modest cytotoxic activity in ALL and AML cell lines in single agent, obtaining a significant reduction in the cell viability only at concentration 5- to 33-fold higher than plasma concentrations achieved in either animals or humans –115. How+ AML, MDS and CMML samples [FLT3-ITD and complex karyotype leukemic cells [RUNX1-RUNX1T1 rearranged AML cell line, KASUMI1) and in the xenograft model (MV4-11 cells). The authors demonstrated that the combination induced cell apoptosis and cell cycle arrest in G2/M phase and promoted DNA damage [Talazoparib is nowadays the most potent PARP inhibitor due to its ability to induce strong PARP trapping . The num samples . The str samples , which iic cells . Strong A damage . MoreoveA damage .BRCA1/2 genes [BRCA1/2 mutations in acute leukemia, expression changes have been reported. Scardocci and colleagues showed that the median expression of BRCA1 mRNA in AML samples is lower compared to normal bone marrow [BRCA1 downregulation was mediated by promoter hypermethylation and was associated with chromosomal aberrations or therapy-related AML. Of note, PARP1 overexpression and BRCA1 proficiency are predictive of resistance to ex vivo olaparib treatment in AML blasts, whereas formation of γH2AX foci is a marker of sensitivity [PARPi can act through synthetic lethality, whereby genetic DNA repair defects are enhanced by drug-induced defects in a compensatory pathway . Today, /2 genes , 123. Ine marrow . BRCA1 dsitivity .BRCA1/2 germline-mutated cases an impaired functionality of the HR pathway, despite carrying functional BRCA1 and BRCA2 [PALB2 is a potential marker of HR defects, that may also suggest a potential vulnerability to PARP inhibition [During the last decades, it has become clear that oxidative stress and genomic instability, due to mutations in DNA repair genes but also to replication stress, sensitize cancer cells to PARP inhibition –129. In nd BRCA2 . The “BRhibition .This section reports data on sensitivity and resistance biomarkers to PARP inhibitors, which are summarized in Table + umbilical cord blood cells expressing RUNX1-RUNX1T1, genes involved in the FA pathway , together with genes in the ATM and ATR and the BER pathways were downregulated, compared with control cells [RUNX1-RUNX1T1 chimera displayed a compromised functionality of the HR pathway, as proved by the reduced levels of RAD51, ATM, BRCA1 and BRCA2 [RUNX1-RUNX1T1-driven leukemia is characterized by a mutator phenotype, which implies a predisposition to the acquisition of mutations, both spontaneously and under the pressure of genotoxic agents [Several AML-related molecular alterations, including fusion proteins and mutations, were demonstrated to induce a selective vulnerability to PARPi, through deregulation of the DDR pathway Fig. A and B. ol cells . Consistnd BRCA2 . Both muc agents . This obPML-RARA fusion gene [KMT2A rearrangement induced a modest sensitivity to PARPi. Two independent studies investigated the effect of PARPi inhibition on KMT2A-MLLT3 rearranged leukemia encoded by the t translocation [KMT2A-MLLT3 chimera to doxorubicin and cytarabine [KMT2A-MLLT3 leukemia seems to be strictly dependent on resolution of DSBs originated from oxidative stress [KMT2A-MLLT3-transformed cells led to the upregulation of DDR target genes such as RAD51 and BRCA2 and enhanced resistance to PARP inhibition, that could be reversed by HOXA9 suppression [.The pressure exerted by antineoplastic agents as combination therapy may cause an accumulation of DSBs until a point of no return [KMT2A-MLLT3 rearranged patients should not be candidate to PARPi in single agent, as their benefit will likely be modest. In addition, treatment with PARPi was synthetic lethal in proliferating and quiescent AML and ALL cells deficient for DNA-PK–mediated NHEJ [BCR-ABL1-driven leukemias, due to tyrosine kinase-mediated downregulation of BRCA1 and DNA-PKcs protein expression. Taken together, these findings are relevant to the design of personalized therapeutic approaches aided to the eradication of quiescent leukemia stem cells, that are spared by chemotherapy regimens.High sensitivity to PARPi was also reported in human and murine models expressing the ion gene . The spelocation , 133. Boe stress , causinge stress . Indeed,pression .The prested NHEJ . These cFLT3-ITD expressing cells, the increased activity of the HR pathway was mediated by the overexpression of RAD51 [FLT3-ITD mutation, where the treatment with the FLT3 inhibitor AC220 induced the BRCAness phenotype in FLT3-ITD-mutated cells, with downregulation of HR and NHEJ proteins including BRCA1, BRCA2, PALB2, RAD51, and LIG4 and enhanced sensitivity to talazoparib and olaparib [FLT3-ITD quiescent and proliferating leukemia stem cells, as well as leukemic progenitors. A recent study showed that the co-occurrence of TET2 or DNMT3A mutations with FLT3-ITD had opposite effect on the response to PARPi. In particular, the co-occurrence of FLT3-ITD and TET2 mutations exacerbated the sensitivity of Lin−CD34+ primary AML cells to olaparib. On the contrary, cells expressing FLT3-ITD alteration alone or in combination with DNMT3A mutations were resistant to the treatment with the inhibitor [FLT3-ITD, JAK2V617F, MPLW515L, NRASG12D) showed that Tet2-deficient cells were sensitive to PARPi, while Dnmt3a-deficient cells were not. The combination of talazoparib and quizartinib exerted a strong in vivo inhibitory effect against FLT3-ITDITD;Tet2−/− leukemia, while it was neither effective nor enhanced the effect of the combination in FLT3-ITDITD;Dnmt3a−/− cells. The sensitivity was associated to an increase in lethal DSBs linked to accelerated forks progression and correlated with a reduced expression of BRCA1 and LIG4 proteins in FLT3-ITD;Tet2−/− and FLT3-ITD;Tet2−/−;Dnmt3a−/− compared with FLT3-ITD and FLT3-ITD;Dnmt3a−/− cells. On the contrary, PARP1 was downregulated in FLT3-ITD;Dnmt3a−/− cells, consistently with reduced HR/cNHEJ and aNHEJ activity, respectively. Finally, regarding FLT3-ITD AML, Le et al. described the protecting role of the TGF-β1-TGFβR kinase-SMAD3 pathway in the presence of PARPi in the bone marrow. The use of TGFβR inhibitors in combination with FLT3i in FLT3-ITD;Tet2−/− mice enhanced the effect of the combination and prolonged the survival of treated mice [Besides chimeric proteins, activating mutations and/or genomic alterations in the signaling pathway, that affect 59% of AML cases , were alof RAD51 , 151 andof RAD51 . These dolaparib . The comnhibitor . In lineted mice .KIT-mutated AML expressing the RUNX1-RUNX1T1 fusion gene [JAK2V617F, where the pharmacological treatment caused G2/M arrest associated with activation of the ATR-CHK1 pathway [Pten, which is targeted by deletions in about 3.0% and 1.0% of pediatric and adult AML, respectively [The same synthetic lethality effect linked to protein kinase inhibition was also described in ion gene . The inh pathway . Regardiectively , causes ectively  which isSTAG2 mutations, a gene encoding for a subunit of the cohesin complex, which regulates sister chromatids during cell division. About 6% of myeloid neoplasm is characterized by the presence of loss-of-function mutations in STAG2, representing the most altered gene (51% of cases) among the cohesin family members [STAG2 mutations promoted high levels of DNA damage and sensitivity to PARP inhibition [STAG2-mutated cells were preferentially dependent on members of the base excision repair (PARP1), homologous recombination , mismatch repair machinery  and DNA replication , in order to avoid a massive accumulation of genomic instability. The loss of STAG2 was associated with an increase in stalled replication fork and, therefore, sensitivity to PARPi, that was confirmed in vitro and in vivo. Notably, U937 and K562 cells bearing inactivating mutations in SMC1 or RAD21, other two members of the cohesin complex, responded to PARPi to a similar extent of STAG2-mutated cells [STAG2-mutant cells and DNA damage repair genes was common to other cancer types carrying STAG2 mutation [A synthetic lethal effect of PARPi was recorded in presence of  members . In parthibition  in AML. ed cells . The synmutation .TP53 mutations, thus opening a novel potential therapeutic window for a subgroup of high-risk patients [Trp53/Bcor-mutant tumors (carrying wildtype Dnmt3a and Tet2) were highly sensitive to talazoparib and veliparib, independently of BRCA1/2 status.Sensitivity to PARPi was also reported in the presence of patients . In acutIDH1/2-mutated enzymes impaired the HR pathway. Indeed HEL cells expressing mutant IDH1/2 exhibited levels of DSBs repair defects comparable to those detected in BRCA1/2-mutated cell lines and were sensitive to olaparib [IDH1R132-mutated AML [IDH1/2-mutated AML, while IDH1/2 inhibitors antagonized with PARPi and daunorubicin [Finally, it has been demonstrated that 2-hydroxyglutarate, the oncometabolite produced by olaparib  and talaolaparib . Furtherated AML . In detaated AML , 156. Moorubicin .TET1 gene [TET1 expressing T-ALL cells are highly sensitive to olaparib  gene, that encodes for a G2/M cell cycle checkpoint regulator, is downregulated by the TCF3-HLF fusion protein resulting in the attenuation of HR activity and in the upregulation of the anti-apoptotic factor BCL2, which suppresses HR activity by interfering with BRCA1 [Recent studies revealed that PARP enzymes are highly expressed in T-ALL patients and regulate the expression and post-transcriptional modification of the ET1 gene , 158. Foand NHEJ , Piao anth BRCA1 .Overall, in the setting of acute leukemia, only preliminary clinical data on PARPi exist. Ongoing or finished clinical trials are summarized in Table Talazoparib was investigated in adult patients with advanced hematological malignancies, including AML, in a single-agent Phase 1 study (NCT01399840). Thirty-three participants were enrolled: Twenty-one of them were affected by AML and four by myelodysplastic syndromes (MDS). For AML, dose-limiting toxicity (DLT) for single-agent talazoparib was demonstrated at a dose of 200 mg/day; no responses were seen in AML, and a hematological improvement was reported in one patient with MDS. Stable disease was reported in 12 of 24 evaluable patients with AML or MDS .+ cells, as marker of DNA damage accumulation [+ leukemia cells was again confirmed by the increase in H2AX phosphorylation. Moreover, FANCD2 monoubiquitination was a positive prognostic factor with the combination proposed in the trial.Veliparib has been evaluated in two combinatorial regimens in AML. The combination therapy with temozolomide was tested on 48 patients (NCT01139970). DLT level was reached at 200 mg of veliparib, and the phase 2 trial explored the efficacy of the combination between veliparib 150 mg and temozolomide. Complete remission (CR) was documented in 8/48 patients (16.6%). Of note, responders exhibited a veliparib-induced increase in γH2AX in CD34mulation . In an umulation . DNA damOf note, in the investigational clinical trials with PARPi, blood count was ever used as an exclusion criterion, for the intrinsic nature of AML and MDS. PARPi have the class effect of lowering platelets production, being PARP an essential enzyme in the physiology of platelet formation . HoweverTET2 and TET2/DNMT3A-mutant AML for their high sensitivity to DNA damage [IDH1/2 mutant (NCT03953898) and in cohesin mutant (NCT03974217) AML. The preclinical evidence reported in IDH1/2 [Seminal results, that will drive future development, are expected from the combination of PARPi with decitabine (NCT02878785), particularly in the sub-cohort of A damage , and fron IDH1/2 , 156 or n IDH1/2 , 155, asDespite the clinical efficacy of PARPi in oncology, the scientific community is constantly evaluating the risk/benefit ratio of the inhibition DDR pathways for cancer patients. Indeed, the main raised concern is related to the potential genotoxic effect of PARPi in healthy normal cells. The development of secondary myeloid neoplasm (t-MNs) following the use of PARPi in breast, ovarian and pancreatic cancers highlighted the potential toxicity of PARP1 inhibition in healthy hematopoietic precursors , 164. InOlaparib (2014), Rucaparib (2016), Niraparib (2017) and Talazoparib (2018) are currently FDA approved, each with specific indications, for the treatment of advance ovarian, fallopian tube, peritoneal and Her-negative metastatic breast cancer.The available data on acute leukemia suggest potential windows for successful PARPi treatment in disease subgroups, defined on the basis of molecular markers and/or DDR activity, and/or in combination with genotoxic or targeted agents or immunotherapy, with a particular attention to the disease stage .p = 0.026), with a median latency between first PARPi exposure and disease development of 17.8 months (8.4–29.2) [TP53, PPM1D and CHEK2, that are involved in clonal hematopoiesis, occur with increased frequency in cancer patients that were exposed to treatment, and in particular to platinum or topoisomerase II inhibitors or radiation therapy [However, we have to take into account the evidence that in solid tumors treatment with PARPi is associated with a significantly higher risk of therapy-related MDS and AML   . Accordi.4–29.2) . Recent  therapy , indicat


Isolated coronary arteries and cardiomyocytes were assessed for vascular reactivity and cellular Ca2+ transport using a multiwire myography system and a cell-contraction-ion detection system, respectively. Allicin administration improved cardiac function and myocardial pathology, reduced myocardial enzyme levels, and increased H2S and H2S synthetase levels. Allicin administration resulted in concentration-dependent effects on coronary artery dilation, which were mediated by receptor-dependent Ca2+ channels, ATP-sensitive K+ channels, and sarcoplasmic reticulum (SR) Ca2+ release induced by the ryanodine receptor. Allicin administration improved Ca2+ homeostasis in cardiomyocytes by increasing cardiomyocyte contraction, Ca2+ transient amplitude, myofilament sensitivity, and SR Ca2+ content. Allicin also enhanced Ca2+ uptake via SR Ca2+-ATPase and Ca2+ removal via the Na+/Ca2+ exchanger, and it reduced SR Ca2+ leakage. Notably, the protective effects of allicin were partially attenuated by blockade of H2S production with PAG. Our findings provide novel evidence that allicin-induced production of H2S mediates coronary artery dilation and regulation of Ca2+ homeostasis in AMI. Our study presents a novel mechanistic insight into the anti-AMI effects of allicin and highlights the therapeutic potential of this compound.Acute myocardial infarction (AMI) is a condition with high morbidity and mortality, for which effective treatments are lacking. Allicin has been reported to exert therapeutic effects on AMI, but the underlying mechanisms of its action have not been fully elucidated. To investigate this, a rat model of AMI was generated by ligating the left anterior descending branch of the coronary artery. DL-propargylglycine (PAG), a specific hydrogen sulfide (H Acute myocardial infarction (AMI) is a condition with high morbidity and mortality . Despite2+ homeostasis, which is regulated by Ca2+-induced excitation-contraction coupling, is a critical determinant of cardiac contractile function. In fact, Ca2+ dyshomeostasis may lead to impaired systolic-diastolic function of cardiomyocytes after AMI  plays a crucial role in cardiovascular homeostasis. In the human body, H2S production is predominantly catalyzed by cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST). To date, most scholars have proposed that CSE is the primary H2S-producing enzyme in the cardiovascular system  was purchased from Tianjin Tanabe Pharmaceutical Co., Ltd. . Potassium chloride (KCl), 5-hydroxytryptamine (5-HT), endothelin-1 (ET-1), tetraethylamine , 4-aminopyridine , barium chloride  and glibenclamide  were purchased from Sigma-Aldrich . All other reagents were of analytical purity.Allicin was provided by Xinjiang Ailexin Pharmaceutical Co., Ltd. . DL-propargylglycine  was purchased from Shanghai Yuanye Bio-Technology . Diltiazem  rats (weighing 200–220 g) were utilized in the present study.n = 14–16 per group) by a random number table: sham, model, diltiazem (8.1 mg/kg), allicin (14 mg/kg), allicin (7 mg/kg), and allicin (14 mg/kg) + PAG (32 mg/kg) groups (The AMI model was generated via left anterior descending coronary artery (LAD) ligation in SD rats. Briefly, rats were anesthetized with 1% pentobarbital sodium. After left thoracotomy, the heart was exteriorized, and the LAD was ligated approximately 2 mm below the left atrium with a 6–0 silk suture. AMI was confirmed by elevation of the ST segment on an electrocardiogram and bulging of the relevant segment of the left ventricle (LV). In the sham group, the suture was removed without tying, and no infarction was generated. After establishment of the AMI model, rats were divided into six groups  groups . All groCardiac function was assessed using a Vevo 3100 echocardiography system . Rats were anesthetized with 1.5–2% isoflurane via continuous inhalation and warmed on a heated pad (37°C). Ultrasound transmission gel was applied to the chest, and echocardiography (M-mode and B-mode imaging) was performed. The LV internal diameter and thickness of the anterior wall at end-diastole  and end-systole , as well as LV fractional shortening (FS), ejection fraction (EF), and stroke volume (SV) were measured in each rat in a blinded manner. All values were averaged using three to five cardiac cycles per rat. Rat hearts were harvested after 1% pentobarbital sodium overdose via intraperitoneal injection and sliced into five sections of 1-mm thickness across the left ventricular long axis under the ligature. To identify the infarction area, heart slices were incubated with nitro-blue tetrazolium chloride (Sigma-Aldrich) for 3 min at 22 ± 2°C. Infarction areas were measured using Image-Pro Plus software  and presented as a percentage of infarct area to ventricular area or total area.Before the rats were sacrificed, blood samples were collected from the abdominal aorta. Serum was incubated at 22 ± 2°C for 30 min and centrifuged at 975.87 ×g for 10 min. The supernatant was collected for determination of serum cardiac troponin I (cTnI), lactate dehydrogenase (LDH), and CSE levels. Levels of serum cTnI, LDH, and CSE were separately quantified with commercially available cTnI , LDH , and CSE ELISA kits ; antibody and chromogenic agent were added according to the manufacturer’s instructions. Absorbance was measured at 450 nm, detected by a microplate tester. Levels of LDH, cTnI, and CSE were calculated according to the standard curve .The border zone of myocardial infarction tissues were fixed with 4% (v/v) paraformaldehyde and incubated with dimethylbenzene for 30 min before serum blocking for 60 min. Specimens were incubated with CSE antibody  for 24 h at 4°C prior to incubation with goat anti-rabbit IgG (H + L) fluorescein isothiocyanate-conjugated polyclonal antibody  in the dark at 37°C for 60 min. After washing specimens in phosphate buffer solution, nuclei were stained with 4′, 6-diamidino-2-phenylindole (Sigma-Aldrich). Images were obtained using an upright fluorescence microscope  under ×400 magnification.2S in serum and the border zone of myocardial infarction tissue were measured using methylene blue spectrophotometry at 665 nm according to manufacturer’s instructions .Levels of HHearts were harvested, weighed, washed in phosphate buffer solution, fixed in 4% paraformaldehyde overnight, and embedded in paraffin. Each paraffin-embedded heart was cut into 4-µm thick sections through the infarction area and stained with hematoxylin and eosin (H&E) for morphological observation. Specimens were stained with Masson’s trichrome stain to evaluate collagen volume. Sections were imaged using a stereomicroscope .2, 1.5; NaH2PO4, 1.2; MgCl2, 1.2; NaHCO3, 15; Glucose, 5.6; pH 7.4]. RCARs were prepared and performed as previously described  of SD rats were then isolated and placed in a cold Krebs buffer [composition (mM): NaCl, 119; KCl, 4.6; CaClescribed .–2 M) until a plateau of contraction was reached. The rings were then divided equally into two groups (8 rings each): PAG or control. RCARs were incubated with PAG (10–2 M) or the same volume of saline, respectively, for 5 min. Both groups were treated with allicin (10−5–10–4.2 M) and cumulative concentration-response curves were obtained.RCARs were contracted with KCl (6 × 102+ channels (VDCCs), whereas U46619, 5-HT, and ET-1 mediate the opening of receptor-dependent Ca2+ channels (RDCCs). In this study, RCARs were divided into three groups (8 rings each): allicin, allicin + PAG, and control groups. Rings were incubated with allicin (10–4.8 M), allicin (10–4.8 M) + PAG (10–2 M), or the equivalent volume of saline, respectively, for 5 min. Cumulative concentration-response curves for KCl (10–1.54–10–1.42 M), U46619 (10−8–10–5 M), 5-HT (10−8–10–4 M), and ET-1 (10−9–10–6 M) were obtained.KCl mediates the opening of voltage-dependent Ca2+-sensitive potassium channels (KCa), voltage-dependent potassium channels (Kv), inwardly rectifying potassium channels (Kir), and ATP-sensitive potassium channels (KATP) to allicin-induced vasodilation, the corresponding inhibitors, TEA (10–3 M), 4-AP (10–3 M), BaCl2 (10–5 M) and Glib (10–5 M) were applied. RCARs were divided into two groups (8 rings each): control and inhibitor groups. The rings were incubated with the four respective inhibitors for each channel or the same volume of saline for 5 min. Cumulative concentration-response curves for allicin (10−5–10–4.2 M) were obtained.To investigate the contribution of Ca−5–10–4.2 M) were obtained as described above.If the maximum vasodilatory effect in the inhibitor group was lower than that in the control group, subsequent experiments were conducted, as follows. The rings were divided into three groups (8 rings each): inhibitor, PAG, and PAG + inhibitor groups. Cumulative concentration-response curves for allicin (102+ Channel-induced Contraction” section). Contraction-response curves for caffeine (3 × 10–2 M) were obtained.The grouping and intervention for RCARs were performed as described above , and placed in a cell chamber. Myocytes were stimulated to contract at a pacing frequency of 1 Hz with 4 ms of electrical stimulation. Myocytes were exposed to 340 or 380 nm excitation wavelengths, and the emitted fluorescent signal was detected at 510 nm. Sarcomere length and fluorescence intensity (a proxy of Ca2+ concentration) were synchronously recorded with a cell contraction-ion detection system . Contractility parameters including amplitude, peak time, systolic half-time of decay (T50), diastolic T50, and myofilament sensitivity were measured. Ca2+ transient parameters including amplitude, maximum ascending and descending velocity, and Ca2+ decline time constant were also recorded.Isolated cardiomyocytes were loaded with 2 μM Fura-2 AM (Sigma-Aldrich) in the dark for 30 min at 22 ± 2°C. Cells were washed, resuspended twice in Tyrode’s solution .SR Ca release . Short p2+ release and assess the contribution of the Na+/Ca2+ exchanger (NCX) and slow transport systems . The contribution of the slow transport system to Ca2+ removal is only 1% and is often overlooked  uptake and NCX Ca2+ efflux with superfusion of Tyrode’s solution. Based on these factors, the time of SERCA-mediated Ca2+ removal was calculated (Tau).Rapid and continuous application of 10 mM caffeine was employed to induce SR Caerlooked . With co2+ leakage levels were assessed by perfusing myocytes with 1 mM tetracaine and reperfusing in 10 mM Na-, Ca-free Tyrode’s solution (containing Li+ and EGTA instead of Na+ and Ca2+). Levels of SR Ca2+ leakage were calculated based on the difference in cytosolic Ca2+ concentration before and after tetracaine perfusion.Ca50) and maximum relaxation (Rmax) or contraction (Emax) were determined using concentration negative logarithm-effect curves. The differences in vasomotor responses to allicin, as well as in the levels of KCl, 5-HT, U46619, and ET-1 were compared by a two-way analysis of variance (ANOVA), with a post hoc Bonferroni test for group comparison. Statistical significance was determined through a one-way ANOVA with Dunnett’s test for multiple-group comparisons in other experiments. *p < 0.05, **p < 0.01 vs. sham group, △p < 0.05, △△p < 0.01 vs. AMI model group, #p < 0.05, ##p < 0.01 vs. allicin 14 mg/kg group in all figures. All data are presented as the mean ± S.E.M.The SPSS statistical software  was used for statistical analysis. Vasodilation and vasocontraction are expressed as the percentage of precontraction amplitude. The negative logarithms of the concentration that produced the half-maximal effect  and total heart area (ITT) were significantly lower in the sham group  than in the model group . ITV and ITT were significantly reduced by treatment with 14 mg/kg of allicin  and 8.1 mg/kg of diltiazem . However, in the allicin 14 mg/kg + PAG group, ITV and ITT were significantly higher  than in the allicin 14 mg/kg group, but significantly lower than in the model group . Hence, cTnI and LDH levels were significantly higher in the model group than in the sham group , indicating that the model successfully mimicked AMI conditions. Levels of cTnI and LDH were significantly lower in the diltiazem 8.1 mg/kg group , allicin 14 mg/kg group , allicin 7 mg/kg group , and allicin 14 mg/kg + PAG group  than in the model group. cTnI and LDH levels were significantly higher in the allicin 14 mg/kg + PAG group than in the allicin 14 mg/kg group. These data suggest that treatment with diltiazem or allicin significantly alleviated the changes in cardiac function induced by AMI, and PAG partially reversed this effect .H&E and Masson staining revealed that in the sham group, the structure of cardiomyocytes remained intact, the transverse and fiber striations of the myocardium were clear, and cells were arranged regularly. No degeneration, necrosis, hemorrhage, inflammatory cell infiltration, or collagen deposition was observed in the sham group. In the model group, the arrangement of myocardial fibers was disordered, swollen, and disjointed; the septum of the fiber bundles was widened; transverse lines of cells were absent, striations were disordered, normal cell structure was disrupted; and extensive cardiomyocyte necrosis, interstitial vascular hyperplasia, hyperemia, edema, inflammatory cell infiltration, and collagen deposition were observed. These features are typical of myocardial infarction. In the allicin 14 mg/kg and diltiazem 8.1 mg/kg groups, the arrangement of cardiomyocytes was slightly disordered, myocardial fibers were neatly arranged, the interstitium was broadened, the septum of the fiber bundles was not significantly widened, no cell necrosis was observed, and minimal cardiomyocyte edema and collagen deposition were noted. Hence, the degree of pathological damage observed in the allicin 7 mg/kg and allicin 14 mg/kg + PAG treatment groups was intermediate compared with that noted in the model and allicin 14 mg/kg groups .2S in serum and the border zone of the myocardial infarction tissues were significantly lower in the model group  than in the sham group  , allicin 14 mg/kg , allicin 7 mg/kg , and allicin 14 mg/kg + PAG groups  than in the model group, suggesting that these treatments alleviated myocardial infarction symptoms. High levels of allicin were more effective than lower levels, as H2S serum and tissue levels were significantly lower in the allicin 7 mg/kg and allicin 14 mg/kg + PAG groups than in the allicin 14 mg/kg group. Finally, the levels of H2S in the serum and myocardial tissue were significantly higher in the allicin 14 mg/kg group than in the model group.The levels of the Hctively) . On the 2S production.Immunofluorescence analysis revealed that the average optical density (AOD) of CSE in myocardial tissue was significantly lower in the model group (0.039 ± 0.014) than in the sham group (0.141 ± 0.028). CSE showed a significantly higher level in the diltiazem 8.1 mg/kg (0.091 ± 0.009), allicin 14 mg/kg (0.101 ± 0.008), allicin 7 mg/kg (0.077 ± 0.010), and allicin 14 mg/kg + PAG groups (0.073 ± 0.003) than in the model group. On the other hand, CSE levels tended to be lower in the allicin 7 mg/kg and allicin 14 mg/kg + PAG groups than in the allicin 14 mg/kg group, but this trend did not reach statistical significance . Similar50 between the two groups.Allicin-induced dilation in rat coronary arteries precontracted with KCl in a dose-dependent manner. The maximum vasodilation reached 85.11 ± 2.11% of the pre-contraction amplitude. After the application of PAG, the maximum relaxation induced by allicin was 49.37 ± 6.94%, which was significantly lower than that in the control group . The inhmax and pEC50 of KCl-induced concentration-contraction curves were observed after administration of allicin or allicin + PAG . Following administration of allicin + PAG, the Emax values of the concentration-contraction curves induced by 5-HT, U46619, and ET-1 were 133.91 ± 8.65%, 124.09 ± 6.64%, and 144.02 ± 5.22%. These values were significantly lower than those of the control group but were significantly higher than those of the allicin group , whereas the Rmax of allicin was significantly lower in the Glib group (46.98 ± 3.86%) than in the control group. No significant differences were observed in the Rmax of allicin among the PAG, PAG + Glib, and Glib groups. Altogether, these data suggest that the response to allicin is mediated by receptor-dependent, rather than voltage-dependent, Ca2+ channels (The Rchannels .–2 M) induced a transient and rapid contraction of rat coronary arteries in Ca2+-free solution. The contraction amplitude was 40.70 ± 10.09% of the contraction induced by KCl (6 × 10–2 M). After administration of allicin, the contraction amplitude induced by caffeine was 28.45 ± 5.42%, which was significantly lower than that in the control group. Caffeine-induced contraction amplitude was significantly higher in the allicin + PAG group (40.28 ± 6.05%) than in the allicin group (Caffeine (3 × 10in group .50 (0.056 ± 0.017 s), and diastolic T50 (0.128 ± 0.044 s) were significantly longer in the model group than in the sham group . Contraction amplitude in the allicin 14 mg/kg (6.76 ± 2.43%) and allicin 14 mg/kg + PAG groups (4.65 ± 1.53%) was higher, whereas peak time, systolic T50, and diastolic T50 in the allicin 14 mg/kg group  was significantly lower than that in the model group. The addition of PAG significantly reduced the contraction amplitude in the allicin 14 mg/kg + PAG group (4.65 ± 1.53%) compared with the allicin 14 mg/kg group. Similarly, peak time, systolic T50, and diastolic T50 were significantly longer in the allicin 14 mg/kg + PAG group  than in the allicin 14 mg/kg group, indicating that PAG significantly alleviated the effects of allicin.2+ transients in the model group  compared with the sham group . The Ca2+ decline time constant in the model group (0.35 ± 0.08 s) was significantly longer than that in the sham group (0.23 ± 0.07 s) as well. Ca2+ transient amplitude in the allicin 14 mg/kg group (0.15 ± 0.03 RU) and maximum ascending and descending velocities of Ca2+ transients in the allicin 14 mg/kg  and allicin 14 mg/kg + PAG groups  were significantly higher than in the model group. The Ca2+ decline time constants in the allicin 14 mg/kg (0.25 ± 0.10 s) and allicin 14 mg/kg + PAG groups (0.28 ± 0.08 s) were significantly shorter than in the model group. Compared with the allicin 14 mg/kg group, the allicin 14 mg/kg + PAG group exhibited significantly lower Ca2+ transient amplitude and maximum ascending and descending velocities of Ca2+ transients, as well as significantly longer Ca2+ decline time constant  was significantly lower than in the sham group (3.86 ± 1.76). By contrast, myofilament sensitivity in the allicin 14 mg/kg and allicin 14 mg/kg + PAG groups was significantly higher than that in the model group. Myofilament sensitivity was significantly lower in the allicin 14 mg/kg + PAG group than in the allicin 14 mg/kg group  than in the sham group . Compared with the model group, the TauNCX and TauSERCA of the allicin 14 mg/kg group  and allicin 14 mg/kg + PAG group  were both significantly decreased. No significant differences were observed in TauNCX and TauSERCA between the allicin 14 mg/kg and allicin 14 mg/kg + PAG groups  than in the sham group (1.51 ± 0.37 RU). On the other hand, the allicin 14 mg/kg (1.43 ± 0.18 RU) and allicin 14 mg/kg + PAG (1.17 ± 0.13 RU) treatments induced significantly higher SR Ca2+ content when compared with the model group. SR Ca2+ content was significantly lower in the allicin 14 mg/kg + PAG group than in the allicin 14 mg/kg group.The SR Ca2+ leakage was significantly higher in the model group (0.097 ± 0.033 RU) than in the sham group (0.052 ± 0.040 RU), whereas allicin 14 mg/kg (0.056 ± 0.05 RU) and allicin 14 mg/kg + PAG (0.073 ± 0.044 RU) administration significantly decreased leakage compared with the model group. Ca2+ leakage was significantly higher in the allicin 14 mg/kg + PAG group than in the allicin 14 mg/kg group , and ET-1, by binding to their corresponding G protein-coupled receptors, are the main activators of RDCCs, whereas VDCCs are predominantly activated by cations, including K+. We demonstrated that allicin significantly inhibited the contractions induced by 5-HT, U46619, and ET-1 in rat coronary arteries, but it did not alter contractions induced by KCl, suggesting that RDCCs, rather than VDCCs, mediated the extracellular Ca2+ influx that contributed to vasodilation. Receptors mediating SR Ca2+ release include the inositol triphosphate and ryanodine receptors (RyRs), of which, the latter mediates more than 90% of SR Ca2+ release. In order to study the effects of allicin on RyR-mediated calcium release, we used caffeine in Ca2+-free solution, which increased the intracellular Ca2+ concentration by completely activating the RyRs in the SR (2+ release mediated by RyRs.In AMI, coronary microvascular dysfunction results in the insufficient supply of blood and oxygen and may affect myocardial function at rest and during stress . Given tlar tone , we focum the SR . Ca2+ inn the SR . Our fin+ channels, which leads to cell membrane hyperpolarization by promoting intracellular K+ outflow, is a way to decrease intracellular Ca2+ concentration and induce vasodilation. To date, four types of K+ channels with different activation mechanisms have been identified: these are the KV, KCa, Kir, and KATP channels (2+ influx. Further, they increase intracellular Ca2+ efflux by stimulating the NCX, thus reducing intracellular Ca2+ and causing vasodilation. To investigate the involvement of K+ channels in allicin-induced vasodilation, we applied 4-AP, TEA, BaCl2, and Glib separately on coronary arteries to observe their effects on allicin-induced vasodilation. Only Glib application significantly inhibited allicin-induced vasodilation, suggesting that the vasodilatory effects of allicin on coronary arteries were at least partly attributed to KATP channel opening but did not involve other K+ channels.The opening of Kchannels . These c2+-mediated excitation-contraction coupling is a requirement for correct cardiomyocyte contraction and relaxation (2+ enters the cytoplasm through L-type Ca2+ channels, which triggers the activation of the RyRs in the SR and leads to extensive Ca2+ release from the SR. Intracellular Ca2+ binds to troponin to cause cell contraction (2+ can be rapidly recaptured by SERCA on the SR and transported out of cells through the NCX on the cell membrane, which causes cardiomyocytes to enter a diastolic state. Ca2+ transients reflect the rapid dynamic changes in cytoplasmic Ca2+ during cardiomyocyte contraction and relaxation, and the changes in velocity and amplitude represent changes in myocardial contractility. In addition, the sensitivity of cardiomyocyte myofilaments to Ca2+ affects the contractile function of cardiomyocytes. Under pathological conditions, there is a decrease in the amplitude and velocity of Ca2+ transients of cardiomyocytes, SR Ca2+ content, and myofilament sensitivity to Ca2+. This leads to a decrease in cardiomyocyte contraction and relaxation function (D.M. 2+ transients, and myofilament sensitivity of cardiomyocytes. Allicin also decreased the peak time, systolic T50, diastolic T50, and elimination constant of Ca2+ transient time. Collectively, these findings indicated that allicin significantly improved the contraction and relaxation function of myocardial cells.Cardiomyocyte dysfunction is a direct consequence of AMI and leads to a decrease in cardiomyocyte vitality, which eventually results in heart failure and even death. Calaxation . During traction . Intraceion D.M. . In this2+ is not reabsorbed by SERCA or expelled by the NCX in a timely manner at the end of cardiomyocyte contraction. Rather, intracellular Ca2+ accumulates in the cytoplasm, which hinders the separation of thin and thick myofilaments and leads to myocardial diastolic dysfunction. On the other hand, under physiological conditions, the elimination of Ca2+ transients is predominantly mediated by SERCA recapture, Ca2+ efflux by the NCX, and slow transport systems during end-diastole. In this regard, the contribution of slow transport systems is less than 1% (2+ content and Ca2+ efflux induced by the NCX, we continuously perfused coronary arteries with caffeine in a Ca2+-free environment, which completely opened the RyRs. This enabled all Ca2+ in the SR to be completely released and temporarily offset SERCA-induced Ca2+ reabsorption. Therefore, cytoplasmic Ca2+ concentration reflected the SR Ca2+ content, and the elimination of Ca2+ transients was considered to be predominantly accomplished by the NCX. We demonstrated that allicin significantly enhanced SERCA recapture and increased Ca2+ extrusion by the NCX, and increased the SR Ca2+ content. Furthermore, RyR dysfunction is known to cause Ca2+ leakage during diastole (2+ overload, SR Ca2+ content decrease, and eventually, abnormal cell contraction (2+ leakage, suggesting its ability to regulate Ca2+ homeostasis.In pathological states, intracellular Ca than 1% . In ordediastole , which ltraction . Our dat2S functions as a gasotransmitter, similar to nitric oxide and carbon monoxide. H2S exerts various cardiovascular effects, including vasodilation, blood pressure reduction, and myocardium protection. Endogenous H2S, a potent vasodilator, is synthesized via the metabolic breakdown of L-cysteine by CSE, CBS, and 3-MST (2S production in organs that express all three enzymes (2S. Furthermore, our previous research demonstrated that allicin reduced blood pressure by promoting H2S production. Here, we expand upon our previous findings by demonstrating that the anti-AMI effects of allicin are at least partially underpinned by the production of H2S mediated by CSE. In this regard, PAG, a CSE inhibitor, significantly attenuated, but did not completely abrogate, the positive effects that allicin exerts on AMI injury, coronary artery vasodilation, and calcium transport regulation in cardiomyocytes. In the present study, allicin increased the levels of H2S and CSE with or without PAG, but PAG reversed this effect, indicating that PAG partially impedes the allicin-induced production of H2S and CSE. Therefore, we speculate that allicin directly induces H2S production both directly and indirectly by increasing the levels of CSE. Several studies have shown that the mechanisms underlying H2S-induced vasorelaxation include the opening of KATP channels, closing of VDCC, and decreased concentration of intracellular Ca2+ (2S-donor, improved diabetic cardiomyopathy by regulating the Ca2+-handling system in the SR (2+ and K+ currents are strictly superimposable to those exhibited by H2S. These findings suggest that H2S is involved in the anti-AMI effects of allicin, but other mechanisms are involved. Unexpectedly, our results showed that PAG weakened the effect of allicin on CSE production, causing a decrease in CSE levels. However, we are puzzled as to why PAG, an inhibitor of CSE activity, also reduces CSE level. After preliminary literature research, we found that quite a few studies reported that PAG could decrease CSE level (Hnd 3-MST . Found pnd 3-MST , CSE con enzymes . In addi enzymes . Allicinlar Ca2+ . It has n the SR . ConsistSE level . The imp2S. The results showed that the CSE inhibitor significantly weakened the vasodilatory effects of allicin on isolated rat coronary arteries, indicating that CSE is indeed involved in allicin-induced vasodilation by mediating H2S production. However, a previous study on swine coronary arteries suggested that CBS was the most important enzyme for the production of H2S under hypoxic conditions, and that the vasodilatory contribution from CSE and 3-MST becomes apparent only upon inhibition of CBS (2S-producing enzymes in coronary arteries is still debatable. In this study, we only evaluated the role of CSE, but not that of CBS or 3-MST; hence, this study cannot fully elucidate the role of each enzyme in the rat coronary artery. Our results showed that the CSE inhibitor inhibited allicin-induced vasodilation in coronary arteries by 42.4%, indicating that a considerable amount of CSE exists in rat coronary arteries, and that its role in inducing H2S production cannot be neglected, as previously suggested in some studies (2S-producing enzyme in coronary arteries, four reasons could explain such discrepancies. First, the expression and activity of CSE, CBS, and 3-MST may vary across different species, and under different physiological or pathological conditions. Second, the levels of enzyme expression may not be proportional to the levels of activity, and indeed an inverse relationship may be present where lower expression results in higher activity. Third, enzyme activity may vary under different experimental conditions and methods, and lastly, the predominance of one H2S-producing enzyme over the others may differ with or without drug intervention. Therefore, further in-depth studies are needed to better understand the dominant role of CSE, CBS, and 3-MST in coronary arteries.An additional point worth noting is that most scholars believe that the CSE enzyme is mainly expressed in the cardiovascular system . Therefon of CBS . Anothern of CBS . Therefo studies . Regardi2+ homeostasis via H2S production. Allicin induced the vasodilation of coronary arteries via favoring H2S production by inhibiting the opening of RDCC, promoting the opening of KATP, and decreasing the Ca2+ release induced by the RyRs. Allicin regulated Ca2+ homeostasis by increasing cardiomyocyte contraction, Ca2+ transient amplitude, myofilament sensitivity, and SR Ca2+ content, and reducing SR Ca2+ leakage via H2S production. Moreover, allicin enhanced the Ca2+ uptake induced by SERCA and Ca2+ removal induced by the NCX, in which H2S is not involved. (Our study demonstrates that allicin may exert cardioprotective effects in a rat model of AMI injury by inducing coronary artery vasodilation and maintaining Canvolved.  This new


The Bcl2 inhibitor venetoclax is a breakthrough therapy in acute myeloid leukemia (AML). We show that venetoclax induces caspase-dependent degradation of AMPK, a central regulator of cellular energy metabolism, with implications in the anti-Leukemic activity of venetoclax in AML.The treatment of acute myeloid leukemia (AML) remains a challenge especially among the elderly. The Bcl-2 inhibitor venetoclax recently showed significant survival benefits in AML patients when combined to low-dose cytarabine or azacitidine. Bcl-2 inhibition initiate mitochondrial apoptosis, but also respiration and cellular ATP production in AML. AMP-Activated Protein Kinase (AMPK) is a central energy sensor activated by increased AMP:ATP ratio to restore the cellular energy balance. Unexpectedly, we observed that venetoclax inhibited AMPK activity through caspase-dependent degradation of AMPK subunits in AML cells. On the other hand, genetic models of AMPK invalidation and re-expression suggested that AMPK participated to the early stages of apoptotic response through a negative regulation of multi-domain anti-apoptotic effectors such as Mcl-1 or Bcl-xL. Together our results suggested a new link between AMPK and Bcl-2-dependent mitochondrial apoptosis that participated to the anti-leukemic activity of venetoclax in AML. Acute myeloid leukemia results from the proliferation of bone marrow immature myeloid progenitor cells. AML is uniformly treated by intensive chemotherapy in young patients, and with low-dose chemotherapy or hypomethylating agents in older and frail patients . UnfortuBcl-2 is an anti-apoptotic protein localized to the outer membrane of mitochondria, sequestrating pro-apoptotic effectors such as BAK or BAX and thus preventing the formation of mitochondrial pore for the release of cytochrome c and subsequent apoptosis cascade triggering . In addiWhile venetoclax inhibits mitochondrial respiration in AML, the consequences of Bcl-2 inhibition on the master intracellular energy sensor AMP activated protein kinase (AMPK) are largely unknown. Heterotrimeric AMPK complexes bind adenosine nucleotides through their γ-subunit, with AMP strongly enhancing AMPK activity, attested by increased AMPKα phosphorylation at Threonine 172 (T172), mediated by liver kinase B1 (LKB1) and calcium/calmoduline dependent protein kinase kinase 1 (CAMKK1) dependent on energy and calcium levels, respectively . InhibitIn AML cells sensitive to apoptosis induction by venetoclax, we observed a drop in intracellular ATP content, but unexpectedly, a rapid decrease of AMPK activity occurred in these cells. In vitro kinase assays excluded that venetoclax directly inhibited AMPK, while instead AMPK degradation was due to the on-target activity of venetoclax through caspase activation in apoptotic cells. Finally, AMPK contributed to the anti-leukemic activity of venetoclax, as AMPK depletion protected venetoclax-treated AML cells from early apoptosis, which correlated with an increased amount of multi-domain anti-apoptotic effectors Mcl-1 and BclxL. Our findings suggested a role for AMPK at the early steps of apoptosis induction by venetoclax in AML.Bcl-2 not only constitutively represses the formation of mitochondrial pores and caspase activation, but also participates to mitochondrial respiration and cellular energy homeostasis . We incuAs ATP depletion generally favors AMPK activation by increasing the AMP:ATP ratio, we investigated AMPK activity in AML cells incubated with venetoclax during short-term periods. Surprisingly, AMPK phosphorylation at Thr172 was moderately inhibited in MOLM-14, and more strongly in OCI-AML2 and HL-60 cells, respectively, correlating with a proportional decrease of AMPKα amount, while both phospho-AMPK and AMPKα remained unaffected in the THP-1 cell line C. MoreovWe investigated whether venetoclax could directly inhibit AMPK activity in vitro. We first observed that the phosphorylation of recombinant AMPK increased in the presence of ATP, suggesting that AMPK autophosphorylation occurred in vitro A. MoreovAs inhibition of AMPK phosphorylation induced by venetoclax correlated to a decreased AMPK protein detection, we performed cycloheximide chase assays  to measuCaspase 3 is the downstream effector of mitochondrial apoptosis activated after Bcl-2 inhibition and subsequent cytochrome c release . From thKO cells were deficient for AMPK activity as attested by the absence of AMPKα protein expression and of ACC, ULK-1 and AMPKα phosphorylation . Notably, addition of doxycycline had no impact on the viability of AML cells incubated with venetoclax over a short-term period (data not shown), in contrast to observations made using the same combination during a longer time , heated at 90 °C for 5 min, and resolved by SDS-polyacrylamide gels electrophoresis, transferred to nitrocellulose membranes, and probed with primary antibodies. Protein signals were revealed by chemoluminescence  at 37 °C for 30 min without or with 100 µM ATP . Enzymatic reactions were then stopped by 20 mM EDTA, then solubilized in Leammli sample buffer (1X), and submitted to immunoblotting.Cells were washed in annexin V binding buffer, incubated with Phycoerythrin-coupled annexin V (BD biosciences) and 1/100 4’, 6-diamidino-2-phénylindole (DAPI), as reported. Flow cytometry assays were analyzed using the Flowjo software .n = value. All experiments were repeated at least three time separately. Differences between the mean values of two experimental groups were analyzed using the two-tailed Student’s t test or a Mann-Whitney test in case of nonparametric data. In comparisons involving more than two groups, we used analysis of variance (ANOVA) and Tukey’s multiple comparison tests. Statistical analyses were performed using Prism software 8.1.1 .The number of independent replicates of each experiment is indicated in the figures legends as We showed that Bcl2 inhibition by venetoclax induced caspase-dependent AMPK degradation in AML. Moreover, AMPK could participate to early apoptosis induced by venetoclax through the regulation of apoptotic effectors in AML. These results add a new layer in our understanding of the therapeutic activity of venetoclax in AML.


After a long time of relative disinterest in skeletal muscle SOCE, this mechanism is now recognized as an essential contributor to muscle physiology, as highlighted by the muscle pathologies that are associated with mutations in the SOCE molecules STIM1 and Orai1. This review mainly focuses on the peculiar aspects of skeletal muscle SOCE that differentiate it from its counterpart found in non-excitable cells. This includes questions about SOCE localization and the movement of respective proteins in the highly organized skeletal muscle fibers, as well as the diversity of expressed STIM isoforms and their differential expression between muscle fiber types. The emerging evidence of a phasic SOCE, which is activated during EC coupling, and its physiological implication is described as well. The specific issues related to the use of SOCE modulators in skeletal muscles are discussed. This review highlights the complexity of SOCE activation and its regulation in skeletal muscle, with an emphasis on the most recent findings and the aim to reach a current picture of this mesmerizing phenomenon.Current knowledge on store-operated Ca Twice  (rev in ). Fast a (rev in ). 2+ elevation and, eventually, skeletal muscle contraction. It is initiated by the release of acetylcholine from the motor neuron nerve, which binds to its cognate ionotropic receptors at the neuromuscular junction (NMJ). The resulting cation influx depolarizes the plasma membrane (PM), activates Nav1.4 voltage-gated sodium channels, and triggers action potentials (APs) that propagate along the PM and into the t-tubules of skeletal muscle fibers. Within the t-tubules, the AP is sensed by L-type Cav1.1 channels, which are also called dihydropyridine receptors (DHPRs). Through physical interaction, the change in DHPR conformation is transmitted to the RyR1 Ca2+ channel, which is located in the membrane of the jSR. The opening of RyR1 leads to an explosive and simultaneous release of Ca2+ from the SR into the cytoplasm across sarcomeres, initiating muscle contraction. After stimulation, Ca2+ clearance from the cytosol is accomplished by the SERCA pumps, which mediate the re-uptake of Ca2+ into the SR store. Due to the very brief muscle AP and the intrinsic biophysical properties of Cav1.1, there is virtually no Ca2+ flux through these channels during membrane depolarization  to open and permit muscle contraction. The non-requirement of Ca2+ entry for EC coupling in part explains the delayed interest for SOCE in skeletal muscle.Excitation–contraction (EC) coupling depicts the succession of events leading to cytosolic Ca (rev in ). Hence,2+ to enter the cells in response to a decrease in the ER/SR Ca2+ content. This peculiar mechanism was originally described in 1986 in the pioneering work of J. Putney on salivary glands, where this mechanism was called capacitive Ca2+ entry at that time cyto transients t-sys and derive a measure of SOCE during SR Ca2+ release [2+ release was induced by lowering the free cytosolic magnesium concentration ([Mg2+]cyto), which removed the Mg2+-dependent inhibition of the RyR1 and caused a cell-wide Ca2+-release [2+] or [Mg2+] will always refer to the respective free ion concentrations. Under these conditions, SOCE was activated within one second upon exposure to low [Mg2+]cyto. This was the first demonstration of fundamental differences in skeletal muscle SOCE because it was at odds with the classical view of SOCE activation as observed in, e.g., immune cells: STIM oligomerization, recruitment of PM and ER contact sites, puncta formation, and activation of Orai channels. SOCE, as observed in these early experiments, activated way too fast to allow for such a complicated mechanism of activation. A refinement of the technique led to further insights. Thus, Edwards et al. demonstrated that SOCE in mouse EDL muscle even occurs on a millisecond timescale cyto. Besides a cell-wide Ca2+ release, low [Mg2+]cyto commonly induces the emergence of propagating Ca2+ waves across the preparation, a condition under which SR Ca2+ buffers are constantly depleted and refilled, and SOCE can be studied. A low-affinity Ca2+-sensitive dye loaded into the t-system of a skinned fiber allowed for the continuous monitoring of SOCE, which reported the respective activation of SOCE only 27 ms after SR Ca2+ release had occurred. This fast kinetics indicated a steep relationship between changes in [Ca2+]SR and activation of SOCE and suggested a physical pre-coupling between STIM1 and Orai1 within the triad region to account for the observed fast kinetics [2+]cyto cyto reached during an AP (see below). Moreover, it enabled calculating the amount of Ca2+ released during every muscle twitch and thereby allowed for determining the dependence of pSOCE on SR Ca2+ release cyto from 1 to 3 mM, a well-known condition that inhibits RyR activity, largely suppressed SR Ca2+ release, in agreement with previous findings cyto did not affect t-system excitability cyto that was increased from an approximately physiological level of 67 nM to 200 nM and 1.3 µM led to an increase in [Ca2+]SR and a subsequent reduction in pSOCE [2+]cytoper se, as preloading the fiber at an increased [Ca2+]cyto of 200 nM and then returning it to the original [Ca2+]cyto of 67 nM immediately before the pSOCE assessment  resulted in marked suppression of pSOCE at the beginning of the recording, again in agreement with an increased distance to the threshold of SOCE activation.What were the main arguments for actually defining this Caon an AP , which i of EGTA . Second,nd pSOCE . Moreovefindings ,113,114,findings . Of noteis study , increasin pSOCE , propose2+ levels were barely depleted SR was reported at about 0.5 mM in intact FDB fibers [tibialis anterior (TA) muscle [2+]cyto set to 67 nM, free [Ca2+]SR was determined to be 0.7 mM SR is between 0.5–0.7 mM, STIM1 luminal Ca2+-binding sites must be mostly occupied; bulk [Ca2+]SR does not drop significantly during an AP [2+]SR is maintained by the strong buffering of CASQ, which is the main Ca2+-buffer in the SR [2+ to be released despite maintaining [Ca2+]SR at a steady level. This is accomplished by the high buffering capacity of CASQ, which is further increased upon polymerization [2+]SR is high, CASQ forms long polymeric tendrils that are anchored to the RyR1 via triadin and junctin SR SR during EC coupling have been limited to global averages of bulk [Ca2+]SR SR and hence react with much less sensitivity. Their role is likely not an immediate one, but may involve recruitment of additional Ca2+ entry pathways upon heavy demand or functional impairment. Under resting conditions, free SR . These S leakage ,126,127 [Ca2+]SR ,46,112. to JP-45  in mousese sites ,38,41 in2+-channels other than Orai1, e.g., from the TRP family of ion channels, could mediate pSOCE, although some TRP channels have been neglected as mediators of cSOCE. For example, cSOCE was shown to be unaffected by the knockdown of TRPC3 cyto, in particular within the triadic cleft, reaches levels equaling that within the t-system during a twitch such that pSOCE operated near the reversal potential of Orai with a strongly reduced driving force . The hih muscle ,143, pSOh muscle , indicatm e.g., ,145), an, an2+ lo2+ concentrations at a high micromolar [Ca2+]cyto, as is the case during a twitch. Irrespective of the biophysical mechanism behind the phenomenon, the data suggests that pSOCE, in cooperation with the t-system Ca2+-uptake proteins Na+/Ca2+ exchanger (NCX) and/or plasma membrane Ca2+ ATPase (PMCA), equilibrates Ca2+ gradients across the t-system membrane in a bidirectional manner, leading to an influx of Ca2+ if [Ca2+]cyto transients are lower and an efflux of Ca2+ if [Ca2+]cyto transients are higher than “normal.” These considerations suggest that pSOCE regulates fiber Ca2+ on a twitch-to-twitch basis by correcting changes in SR Ca2+ release. Thus, rather than refilling depleted SR Ca2+ stores, the role of pSOCE is probably better described as maintaining SR Ca2+ levels. One could also see pSOCE as a mechanism that prevents excessive build-up of Ca2+ within the t-system when [Ca2+]cyto levels peak during a muscle twitch. By “clamping” the t-system Ca2+ levels to the levels found in the extracellular space, the function of pSOCE would therefore avoid significant diffusional loss of Ca2+ from the t-system into the external environment of the fiber [Another explanation for the decline in pSOCE amplitude with low cytosolic buffering could be provided by the reported inward rectification of the current–voltage relationship of Orai1 . While the fiber ,111.2+ store depletion and might involve additional membrane structures [As mentioned previously, it remains to be confirmed whether pSOCE depends on the very same protein machinery as cSOCE, i.e., STIM and Orai proteins, but it is also possible that their physiological function and localization are different. Based on what is known today, one can postulate (at least) two pools of SOCE to exist in skeletal muscle cells: one endogenous pool that can be activated rapidly and most likely relies on preformed clusters within the triads (see above); the other one, an inducible pool, is recruited only slowly after strong SR Caructures ,85, poteructures ,146, andructures . It is pSince even before the molecular mechanisms of SOCE were elucidated, drugs from the group of imidazoles , di3+ and La3+) were among the first modulators ever to target Orai1. They bind Orai1 and its isoforms Orai2 and Orai3 with high potency by blocking the pore [3+ and Gd3+ were widely used in skeletal muscle as standard SOCE inhibitors at micromolar concentrations –200 µM; , their u–200 µM; ,38,159).channels ,162. IndE; e.g., ,164,165)., [v1.1 . 2+ influx blocker [2+ [ATP channels [2+ pumps in human endothelial cells [50 of ~10 µM [2+ entry into skeletal myotubes and muscle fibers mediated by SOCE, ECCE, or APACC with similar potency.SKF96365 was first described as a receptor-mediated Ca blocker  and only blocker . Studiescker [2+ ,166,168,cker [2+ , and KATchannels . Moreoveal cells  and NCX f ~10 µM . To blocf ~10 µM ,59. Withf ~10 µM ; it also2+ fluxes and ICRAC in a variety of tissues and cell types, including skeletal muscle [3R antagonist [CRAC at low concentrations (1–10 µM), while higher concentrations (20–50 µM) inhibit ICRAC [2-Aminoethyldiphenyl borate (2-APB) was widely used to modulate Cal muscle ,174,175.tagonist ,177,178,tagonist ,180,181.tagonist ,183, as tagonist ,168. Intit ICRAC ,184. Sucit ICRAC ,182,184.CRAC inhibitor [2+ handling was provided [2+ handling in rat skinned EDL fibers at multiple levels. It inhibited SOCE at low concentrations (1–5 µM) but, interestingly, also impaired RyR1 activity at higher concentrations. Thus, it blocked AP-induced SR Ca2+ release and RyR leak at >10 µM. The impairment of RyR1 occurred in an indirect manner, probably by affecting the DHPR [3,5-bis(trifluorometyl)pyrazole (BTP) compounds were characterized for the first time as nuclear factor of activated T-cells (NFAT) activators and cytokine production inhibitors of T cells ,151,152.nhibitor ,186,187.nhibitor  and to bnhibitor . Despitenhibitor ,190,191 provided . The autthe DHPR , which ithe DHPR ).CRAC inhibitors. GSK-7975A was shown to inhibit Orai1-mediated currents with an IC50 of ~4 µM in a heterologous expression system, presumably by altering the Orai1 pore geometry [2+ entry upon store depletion in myoblasts expressing TAM-associated Orai1 mutants, and thus suggested it as a potential candidate drug for TAM treatment [2+ channels at 10 µM [GSK-7975A and relatives  belong to the class of pyrazole derivatives developed by GlaxoSmithKline ,194,195 geometry . A recenreatment . Neverthat 10 µM . The mecat 10 µM . To the 2+ and Na+ channels [Synta66 is structurally closely related to BTP2 and inhibits Orai1 activity at micromolar concentrations via a pore-blocking mechanism . It has channels , and othchannels . Howeverchannels . Togethe50 of 2 µM. Ca2+ influx through SOCE measured in rat skinned EDL fibers upon exposure to caffeine and low Mg2+ showed a comparable increase by about 20–30% at 10 µM IA65. IA65 exhibited some selectivity, enhancing Orai1 activity but only marginal affecting Orai2 and Orai3 [Recent attempts have led to the synthesis of novel SOCE inhibitors, of which CIC-37 seems the most promising candidate , but alsnd Orai3 ,202. It 2+ influx pathways across the t-tubule membrane are difficult to discriminate with current pharmacology; (ii) the importance of several proteins to the function of SOCE, be it direct  or indirect , greatly increase the probability for off-target effects; (iii) the RyR plays a pivotal role in SOCE activation [v1.1 and Nav1.4, which are known to be affected by current SOCE pharmacology. What can be learned from the existing data? While drug specificity is always of concern when applying pharmacology, it seems that this particularly applies to SOCE: (i) the different Cativation ,59,89,97tivation ). In partivation . Regardi2+ entry induced by STIM1L. A reduction in ER-PM contacts could lead to less Ca2+ accumulation in the proximity of the Orai1 channel to reduce CDI and hence augment SOCE. Another explanation is linked to different channels that are activated by STIM1L. Electrophysiological recordings in HEK cells expressing STIM1 or STIM1L showed that STIM1L is indeed more prone to activating TRPC1 compared to STIM1 [2+ entry than STIM1 and interacts more strongly with TRPC (in that case TRPC3 and TRPC6) than STIM1. Interestingly, in differentiated human myotubes, it was shown that STIM1L indeed physically interacts with TRPC1 and TRPC4 [2+ release [2+ .2+ level was reduced both in myoblasts and myotubes, in line with several reports showing the role of STIM2 in the maintenance of resting Ca2+ levels in different cell types [2+ content was higher and, consequently, KCl- and caffeine-induced Ca2+ release was more pronounced in the case of STIM2 downregulation. The cytosolic part of STIM2 was identified as the binding region to SERCA1a [2+ release was reduced in STIM2-downregulated cells and repeated KCl stimulations rapidly led to SR Ca2+ store depletion [2+ level elevation that was observed under this condition [2+ level was, however, not seen in CD4+ T cells [2+ entry during muscle formation, considering that STIM1L expression also increases during muscle differentiation and promotes SOCE [2+ homeostasis during muscle formation, but also in mature fibers. We are now only at the beginning of understanding the complex regulation of SOCE that is linked to the expression of STIM proteins, and future studies should bring exciting new findings to better understand the interplay between the four STIM isoforms that are expressed in skeletal muscle.The other member of the STIM family, namely, STIM2, is expressed in muscle as well and plays a role in differentiation. The downregulation of STIM2 in human skeletal muscle led to an impaired myoblast differentiation, a decreased expression of myogenin and myocyte enhancer factor-2 (MEF2), and a reduction in SOCE . In addill types . Interesll types . Considell types ), one call types . Besidesll types . Accordi SERCA1a . In contepletion . The conepletion ,214. Intepletion ,215. Thetes SOCE . It is wtes SOCE . Hence, 2+ release upon an AP has a higher amplitude and shorter duration than in slow-twitch fibers [Based on the different properties regarding the speed of contraction, metabolism, and fatigue resistance, different types of muscle fibers can be distinguished in skeletal muscle. According to their contraction velocity, two main types are defined, namely, type I and type II, which are also called slow- and fast-twitch fibers, respectively . The difh fibers . The fash fibers  and a moh fibers . Skeleta2+ than that of EDL muscles. Hence, in soleus muscles, the force of contraction decreased more strongly in the presence of BTP2 or the absence of extracellular Ca2+ [The contribution of SOCE toward maintaining contractile force during repetitive muscle activity in the different fiber types is not clearly defined for the time being. Ex vivo experiments showed that the contractile force of soleus muscles, upon stimulation at high frequencies, is more dependent on extracellular Calar Ca2+ , showing2+ entry during EC coupling [A critical role of SOCE in slow muscle was demonstrated recently in muscle-specific Orai1 knock-out mice . The redcoupling . It shoucoupling  and rev coupling ). Why sl2+. In mice, TAs are found in old animals almost exclusively in type IIB fibers [2+ entry for a stronger contraction and are sensitive to SOCE inhibitors [2+ overload. Slow fibers, with a higher mitochondrial density and a preferential oxidative metabolism, might be better protected against Ca2+ overload, thus not requiring TAs formation, but this remains hypothetical. As reported in a recent comprehensive review on CEUs, the structural rearrangement that occurs upon strenuous exercise was so far only studied/observed in fast muscle [2+ handling between the two fiber types, it would not be surprising that SOCE is also divergent in terms of localization within the fiber and regarding respective activation/regulatory mechanisms.Another interesting point is related to the formation of tubular aggregates (TAs), which are abnormal structures of the SR that strongly express STIM1 and Orai1 . In humaB fibers , which aB fibers ). IntereB fibers . In thishibitors , which whibitors . Indeed,hibitors . The dechibitors . From tht muscle . It woulFinally, what do we know about the expression of the SOCE molecules in the different fiber types? Not much is known besides a study that reported a higher expression level of STIM1L in rat soleus muscle compared to rat EDL muscle . The lev2+ flux. The picture of SOCE that emerges is a multifaceted one, envisaging SOCE as both static and dynamic in nature, where SOCE is not confined to the triad alone but acts presumably across multiple sites within a single fiber, where it likely serves different physiological functions. Skeletal muscle is not a homogenous tissue but comprises different types of fibers that have an extremely broad range of activity. The physiological function(s) and regulation(s) of SOCE in this context are so far only poorly understood. Several basic questions remain open, including the localization, mobility, and interaction of the involved SOCE molecules, as well as their differential expression among the different fiber types. Current methods for studying SOCE in muscle, some of which are specific to this tissue, such as the skinned fiber preparation and the force/fatigue measurements, have highlighted the peculiar and fascinating nature of this small but important Ca


OBJECTIVES/GOALS: The goal of this project was to develop and evaluate a pilot mentor training program for clinical research professionals. This project presents an evidence- and theory-based mentoring program that has been developed, implemented, and evaluated for this group of translational research professions. METHODS/STUDY POPULATION: The curriculum for the program was designed for aspiring mentors and aligned with the topics of existing Entering Mentoring curriculum for translational workforce . Eleven experienced CRPs participated in the pilot training program. The training was delivered in two-hour meetings over eight weeks. Qualitative e-mail interviews and a validated mentoring competency assessment  and mentor role assessment  tool were used for process and outcome evaluation. Cases studies specific to the CRPs work environment were developed and used to facilitate discussions throughout the training. RESULTS/ANTICIPATED RESULTS: Pre- and post-training scores for mentoring competency assessment were compared across six sub-indexes. Paired t-tests showed a significant difference for the maintaining effective communication competency, p = 0.0202. Comparisons of individual items also showed positive changes in the promoting professional development competency, p = 0.0161). Qualitative assessment revealed that most mentor trainees recognized a distinction between a mentor and a supervisor or on-the-job-trainer. Furthermore, most have been informal mentors without a formal role assignment, the need for ongoing mentoring, and potential of mentoring networks. DISCUSSION/SIGNIFICANCE OF IMPACT: CRPs is a diverse group of research support professionals who may hold the roles of research study coordinators, research nurses, regulatory and compliance specialists. Tailored mentoring can provide essential infrastructure for ongoing professional development and support talent retention.


Rhododendron (Ericaceae) extracts contain flavonoids, chromones, terpenoids, steroids, and essential oils and are used in traditional ethnobotanical medicine. However, little is known about the immunomodulatory activity of essential oils isolated from these plants. Thus, we isolated essential oils from the flowers and leaves of R. albiflorum  and analyzed their chemical composition and innate immunomodulatory activity. Compositional analysis of flower (REOFl) versus leaf (REOLv) essential oils revealed significant differences. REOFl was comprised mainly of monoterpenes (92%), whereas sesquiterpenes were found in relatively low amounts. In contrast, REOLv was primarily composed of sesquiterpenes (90.9%), with a small number of monoterpenes. REOLv and its primary sesquiterpenes  induced intracellular Ca2+ mobilization in human neutrophils, C20 microglial cells, and HL60 cells transfected with N-formyl peptide receptor 1 (FPR1) or FPR2. On the other hand, pretreatment with these essential oils or component compounds inhibited agonist-induced Ca2+ mobilization and chemotaxis in human neutrophils and agonist-induced Ca2+ mobilization in microglial cells and FPR-transfected HL60 cells, indicating that the direct effect of these compounds on [Ca2+]i desensitized the cells to subsequent agonist activation. Reverse pharmacophore mapping suggested several potential kinase targets for these compounds; however, these targets were not supported by kinase binding assays. Our results provide a cellular and molecular basis to explain at least part of the beneficial immunotherapeutic properties of the R. albiflorum essential oils and suggest that essential oils from leaves of this plant may be effective in modulating some innate immune responses, possibly by inhibition of neutrophil migration. Rhododendron belongs to the Ericaceae family of plants and includes more than 1000 identified species i) in human neutrophils were measured with a FlexStation 3 scanning fluorometer (Molecular Devices), as previously described i. After a 10 min incubation at 37 °C, the fluorescence baseline was recorded again, and 10 µM fMLF was added to evaluate inhibitory effects on agonist-induced [Ca2+]i. Responses were normalized to the response induced by 10 µM fMLF.For analysis of Ca2+ influx experiments, curve fitting (at least five or six points) and calculation of median effective concentration values (EC50 or IC50) were performed by nonlinear regression analysis of the dose–response curves generated using Prism 7 .For all Ca+ containing 2% (v/v) heat-inactivated fetal bovine serum (2 × 106 cells/mL), and chemotaxis was analyzed in 96-well ChemoTx chemotaxis chambers , as described previously i, which is a key component of phagocyte activation [Lv treatment increased [Ca2+]i, with EC50 values of 18.6 µg/mL and 22.8 µg/mL in neutrophils and C20 microglial cells, respectively  showed that these compounds also activated neutrophil Ca2+ influx, with the most potent being viridiflorol -β-ocimene, γ-terpinene, p-cymene, terpinolene, linalool, and terpinen-4-ol, were all found previously to have no activating effects on neutrophil Ca2+ mobilization [Fl is also inactive.In contrast to REO [Ca2+]i . Similarll types . Previoulization ,31. Thus2+ influx, specific receptors, or other unidentified molecular targets by agonists can result in the desensitization and subsequent downregulation of neutrophil responses [2+]i and inhibit cell migration i  also inhibited agonist-induced Ca2+ mobilization and thus desensitized human neutrophils to further agonist activation [We next evaluated the effects of individual constituent compounds on agonist-induced Ca2+]i see  desensittivation .Fl (8.9%), on agonist-induced neutrophil or HL60-FPR1/FPR2 Ca2+ influx showed that it had no effect -β-ocimene, γ-terpinene, p-cymene, terpinolene, linalool, and terpinen-4-ol) showed that they had no inhibitory effect on agonist-induced neutrophil Ca2+ mobilization [Fl does not desensitize neutrophil agonist-induced activation.Evaluation of the effects of β-phellandrene, one of the principal monoterpenes in REOo effect , which io effect . In addilization ,31,32. TLv for 10 min dose-dependently inhibited fMLF-induced human neutrophil chemotaxis, with an IC50 of 3.3 µg/mL  and test compounds at various concentrations (up to 25 µM) in HL60 cells during 30 min and 2 h incubation periods. These incubation periods are comparable to the times used to measure Ca2+ mobilization (up to 30 min) and cell migration (up to 1.5 h). As shown in Lv, REOFl, viridiflorol, spathulenol, curzerene, and germacrone had minimal effects on cell viability during a 30 min incubation, verifying the absence of cytotoxicity during the Ca2+ influx assay period  [2+-activated Cl− currents and K+ channel activity [The sesquiterpene compounds identified in REOaw edema . This coaw edema . Curzereaw edema  and SKMEaw edema . Spathulaw edema , as wellaw edema . Spathuler 72 h) . Finallyer 72 h) . Germacrer 72 h)  and has er 72 h) ,65,66,67er 72 h) . It has er 72 h) . Finallyactivity .Lv) was previously reported [Salvia hydrangea [Aloysia gratissima [Eremophila mitchellii [Merremia dissecta [Elytropappus rhinocerotis [Annona squamosal [Artemisia annua [Cleome spinose [Despite the various biological activities reported for these compounds, little is known about their specific cellular targets. Thus, we performed reverse-pharmacophore mapping on the molecular structures of viridiflorol, curzerene, spathulenol, germacrene B, and germacrone to identify potential biological targets. Note that pharmacophore mapping of bicyclogermacrene  [2+ mobilization [MAPKAPK2, JNK1/3, CD11a, and PIM1 represent potential targets that could contribute to the direct inhibitory effects of REO (CD11a) . In neut (CD11a) . Imperatlization . In addilization . Likewislization .Based on the possibility that MAPKAPK2, JNK3, and PIM1 could interfere with phagocyte migration, we evaluated the binding affinity of pure viridiflorol, curzerene, spathulenol, and germacrone toward these three kinases but did not observe any binding activity. Nevertheless, we still cannot exclude a role for integrin α-L (CD11a) as a target for viridiflorol and spathulenol in human neutrophils.We calculated the most important physico-chemical parameters for these sesquiterpenes using SwissADME  Table 7Table 7 aR. albiflorum essential oils and, more specifically, individual component compounds on innate immune cells in vitro. Since we observed potentially beneficial immunomodulatory effects and low cytotoxicity, the next step would be to evaluate the selected compounds in vivo, and these studies are being considered. Indeed, previous studies have shown that in vivo treatment with sesquiterpenes can be beneficial for various clinical problems. For example, sesquiterpenes are currently under clinical evaluation for cancer treatment . Likiewed in  and Alzhiewed in ,94. Furtiewed in . Thus, tR. albiflorum contain a high amount of sesquiterpenes (up to 91%), and these essential oils can induce human neutrophil and microglial cell Ca2+ influx, which desensitizes these cells to subsequent agonist-induced functional responses. Moreover, the major constituents of R. albiflorum leaf essential oils  exhibited the same effects, inhibiting agonist-induced Ca2+ mobilization and chemotaxis in human neutrophils and agonist-induced Ca2+ mobilization in microglial cells and FPR-transfected HL60 cells. Thus, our data provide a molecular basis to explain at least part of the beneficial therapeutic effects of R. albiflorum essential oils and component compounds and suggest that inhibition of innate immune cells by component compounds of this essential oil might have anti-inflammatory effects. Future studies are now in progress to evaluate the potential of Rhododendron essential oils as therapeutic remedies for various disorders with immune and/or inflammatory mechanisms, including Alzheimer’s disease, as well as to determine the molecular targets of the active compounds.Essential oils isolated from the leaves of