SceI expression to individual DSBs, or DSB-clusters comprising known numbers of DSBs with defined DNA-ends. We find that, similarly to high-LET IR, DSB-clusters of increasing complexity, i.e. increasing numbers of DSBs, with compatible or incompatible ends, compromise classical non-homologous end-joining, favor DNA end-resection and promote resection-dependent DSB-processing. Analysis of RAD51 foci shows increased engagement of error-free homologous recombination on DSB-clusters. Multicolor fluorescence in situ hybridization analysis shows that complex DSB-clusters markedly increase the incidence of structural chromosomal abnormalities (SCAs). Since RAD51-knockdown further increases SCAs-incidence, we conclude that homologous recombination suppresses SCAs-formation. Strikingly, CtIP-depletion inhibits SCAs-formation, suggesting that it relies on alternative end-joining or single-strand annealing. Indeed, ablation of RAD52 causes a marked reduction in SCAs, as does also inhibition of PARP1. We conclude that increased DSB-cluster formation that accompanies LET-increases, enhances IR-effectiveness by promoting DNA end-resection, which suppresses c-NHEJ and enhances utilization of alt-EJ or SSA. Although increased resection also favors HR, on balance, error-prone processing dominates, causing the generally observed increased toxicity of high-LET radiation. These findings offer new mechanistic insights into high-LET IR-toxicity and have translational potential in the clinical setting that may be harnessed by combining high-LET IR with inhibitors of PARP1 or RAD52.Heavy-ion radiotherapy utilizing high linear energy transfer (high-LET) ionizing radiation (IR) is a promising cancer treatment modality owing to advantageous physical properties of energy deposition and associated toxicity over X-rays. Therapies utilizing high-LET radiation will benefit from a better understanding of the molecular mechanisms underpinning their increased biological efficacy. Towards this goal, we investigate here the biological consequences of well-defined clusters of DNA double-strand breaks (DSBs), a form of DNA damage, which on theoretical counts, has often been considered central to the enhanced toxicity of high-LET IR. We test clonal cell lines harboring in their genomes constructs with appropriately engineered I-SceI recognition sites that convert upon I- In addition, the increased LET at the Bragg peak induces complex DNA damage that further increases their efficacy.Radiotherapy is an intricate and essential component of present-day cancer therapy and uses either photons in the form of X-rays, or accelerator-produced charged particles – mainly protons and heavy ions (HI) . The ratThe key DNA lesion induced by IR and responsible for the effectiveness of radiotherapy is the DSB . DSBs arIn mammalian cells, four mechanistically distinct DSB-repair pathways have evolved to mitigate the consequences of DSB induction. Homologous recombination (HR) is the only error-free repair pathway that restores both DNA integrity and sequence at the break site, but its engagement is limited to DNA post-replication stages. Classical non-homologous end-joining (c-NHEJ) rapidly processes DSBs throughout the cell-cycle, but frequently causes mutations  and SCAsAlternative end-joining  is thought to engage when HR or c-NHEJ are inactive or fail – hence often termed backup end-joining . Alt-EJ HR, alt-EJ and SSA are classified as DNA end-resection resection (henceforth simply resection) -dependent DSB-repair pathways, because they share this initial processing step . During The divergent properties and fidelities of the four DSB-repair pathways indicate that they cannot be considered as equivalent alternatives of DSB-repair, and suggest that they may actually also serve to accommodate necessities generated from different sources, including the increased complexity of high-LET IR-induced DSBs . Indeed,However, increased engagement of an error-free DSB-repair pathway in the form of HR is difficult to reconcile with the dramatic increases in radiosensitivity and SCAs-formation that accompany increases in LET . TherefoAmong the levels of DSB-complexity that have been considered hitherto , DSB-cluWe show that increased DSB-clustering suppresses c-NHEJ, promotes CtIP-dependent resection and favors HR, alt-EJ and SSA. However, our results show that on balance, despite the relative increase in the utilization of HR, DSB-clusters cause increased cell killing and increased SCAs-formation through SSA and alt-EJ. The similarities in the processing characteristics between high-LET-induced “complex” DSBs throughout the genome, and of well-defined complex DSBs in form of DSB-clusters, at fixed location in the genome, support the hypothesis that DSB-clusters underpin the biological effects of high-LET IR .2 in air in the presence of 400 μg/ml G418 (Capricorn Scientific). Inhibitors were administered either immediately after transfection or 4 h later. 6-hydroxy-DL-DOPA (RAD52i) (Sigma-Aldrich) was used at a concentration of 20 μM. DNA-PKcs inhibitor NU7441 (DNA-PKcsi) (Tocris Bioscience) was applied at a concentration of 5 μM.Chinese hamster ovary (CHO) clones  carryingISceI plasmid. In some experiments, as indicated, the expression plasmid pISceI-T2A-Trex2-IRES-BFP was utilized  and EdU positive (EdU+) cells were analyzed in distinct cell cycle compartments after staining following standard protocols  blocking buffer (LI-COR) for 1 h at room temperature, followed by overnight incubation with primary antibody (diluted in Intercept™ T20 (TBS) antibody diluent) at 4°C. Membranes were washed three times with TBS-T  and incubated for 1 h with secondary antibody. The following primary antibodies were used: anti-CtIP (D-4) mouse monoclonal (Santa Cruz Biotechnology), anti-RAD51 rabbit polyclonal (Merck Millipore) and anti-GAPDH mouse monoclonal antibody (Merck Millipore). The secondary antibodies were: goat anti-mouse IgG conjugated with IRDye680 or goat anti-rabbit IgG conjugated IRDye800 (LI-COR Biosciences). The proteins on the membranes were visualized by scanning using Odyssey infrared imaging scanner (LI-COR Biosciences).Protein extracts were prepared and run on SDS-PAGE using standard protocols. Proteins were transferred onto nitrocellulose membranes, which were incubated in Intercept6 cells transfected with pCMV-3xNLS-ISceI plasmid were split in three dishes and plated for 24, 30 and 48 h, respectively. To accumulate cells at metaphase, colcemid (Biochrom AG) was added for 2–3 h at a concentration of 0.1 μg/ml. Metaphase spreads were prepared using standard procedures. mFISH was performed using 12XCHamster Multicolor FISH Probe for Chinese Hamster Chromosomes (MetaSystems Probes) according to manufacturer’s protocol. An automated imaging system (MetaSystems) was used to obtain high quality images of metaphase chromosomes, as previously described  following manufacturer’s protocol and program U-23. Briefly, the following oligonucleotides were used for depletion of CtIP: 5′-GUGCAAGGUUUACAAAUAA-3′; 5′-CAAAGUCCCUGCCAAACAA-3′; 5′-AGAAUACUCUCCAGGAAGA-3′ (Eurogentec). Similarly, for downregulation of RAD52, again a cocktail of three specific RNAs was utilized, following the same transfection procedure: 5′-UGAGAUGUUUGGUUACAAU-3′; 5′-ACUGCAUUCUGGACAAAGA-3′; 5′-CCCUGAAGACAACCUUGAA-3′. For efficient RAD51 ablation the following three specific siRNA sequences were used: 5′-GUGCCAAUGAUGUGAAGAA-3′; 5′-GGGAAUUAGUGAAGCCAAA-3′; 5′-GGCGUUCAGAAAUCAUACA-3′. The following negative control RNA (ncRNA) sequence was used: 5′-UUCUCCGAACGUGUCACGUdTdT-3′. ncRNA is used for mock-transfection.For depletion of CtIP, RAD52 and RAD51, a pool of three siRNAs, specific for each protein, as previously described  ). For the one-way analysis of variations test (ANOVA) a network applet was utilized (https://statpages.info/anova1sm.html). The applet also calculates the Tukey HSD (“Honestly Significant Difference”) post-hoc test, to indicate the significance between different groups. Unless otherwise stated, data shown represent means and standard deviations (±SD) from at least three independent biological determinations. The detailed data of the statistical analysis is included in the corresponding Supplementary information .xlsx files.The statistical analysis was carried out by the online version of the MedCalc software , to reduce complications from background signals generated in S-phase cells  . The numA recent study shows that inhibition of alt-EJ using PARP1 inhibitors abrogates SCAs-formation, and that this effect is stronger for DSB-clusters . Here weTo assess the contribution of SSA to SCAs-formation under these conditions, we depleted RAD52, 24 h before transfection for I-SceI expression. Similar to CtIP depletion, RAD52 knockdown has no impact on SCAs in cells sustaining individual DSBs and has no statistically significant effect on cells sustaining DSB pairs with compatible ends . HoweverSceI induced DSBs  shows only relatively small changes with increasing DSB-complexity in cells analyzed in G1-phase, it causes pronounced delays in cells analyzed in G2-phase. Furthermore, the observation that processing of complex DSBs in G2-phase is resistant to DNA-PKcs inhibitors, not only explains this delay, but also suggests that DSB-complexity is a strong suppressor of c-NHEJ (The results summarized above show that increased DSB-complexity, as defined by I-f c-NHEJ .2-phase cells with reference to DSB-processing that also distinguishes them from G1 -phase cells, is that resection and HR are fully active. We therefore infer that resection and HR are intimately contributing to the observed distinct outcomes in G2-phase cells. Indeed, we show that HR engagement increases with increasing DSB-complexity (A key characteristic of Gmplexity . This reNotably, despite increased engagement of HR on complex DSBs, more than 75% of them are still processed by other repair pathways. Since c-NHEJ fails to engage on complex DSBs, it follows that the remaining 75% are processed by alt-EJ or SSA. Since these pathways are inherently error-prone, the results allow us to resolve the conundrum as to why and how increased engagement of HR in high-LET irradiated cells fails to make them radioresistant. Evidently, the majority of complex DSBs are repaired under these conditions by highly error-prone repair pathways leading to the enhanced cell killing and genomic instability observed. The choice among these pathways may be regulated by the degree of resection, an aspect that is presently under investigation . These aSince error-prone DSB-processing causes SCAs, we analyzed the mechanisms underpinning their formation as a function of DSB-complexity. Indeed, the increases in DSB-complexity modelled here , lead toNotably, suppression of HR increases SCAs-formation and sensitizes cells to complex DSBs, as expected from an error-free repair pathway promoting genomic stability and cell survival . AblatioKnockdown of RAD52, a key component of SSA, strongly suppresses SCAs-formation  confirmiSceI, as their removal by TREX strongly suppresses SCAs-formation. It is equally interesting that DNA-ends apparently blunted by TREX, can be processed by DNA-PKcs even when highly complex, as inhibition of DNA-PKcs dramatically increases SCAs-formation. The result of this inhibition further indicates that blunted DNA-ends, actually good substrates of DNA-PKcs (It is intriguing that SCAs-formation utilizes the 4 bp TTAT 3′-overhangs generated by I-DNA-PKcs , can be 2-phase has important parallels to the effects of high-LET IR as compared to low-LET IR. Indeed, the enhanced resection initiated at complex DSBs predicts increased engagement of ATR as it is observed (2-checkpoint (1-phase, where resection is normally suppressed (The important role of resection in the processing of modelled complex DSBs in Gobserved . There aeckpoint . The higeckpoint . Interesppressed .The expansion of particle therapy using protons or HI offers new opportunities for improving cancer care , but req


NRAS, MCL1, EVI1, GATA2, WT1, and PAK1. And the ecDNA (chr11: 58668376-58826008) occurred in five out of 17 AML evolution-related cells, which was associated with the high expression of the GLYATL1 gene and the high expressed GLYATL1 was a poor prognostic factor. In conclusion, the eccDNA profiles of normal hematopoiesis and AML evolution were depicted and the recurrent eccDNAs we revealed might be utilized in the treatment of AML as biomarkers.Elevated extrachromosomal circular DNA (eccDNA) has been reported to accelerate tumor pathogenesis. Although the eccDNA profiles of other tumors have been established, the landscape of the eccDNA of acute myeloid leukemia (AML) has not been revealed. Our study first depicted the eccDNA profile of normal hematopoiesis and AML evolution by exploiting the ATAC-seq and RNA-seq data from nine healthy donors and 12 AML patients, which contained a total of 137 cell samples and 96 RNA-seq samples . We found the number of eccDNAs generally increased with the evolution of normal hematopoiesis and AML. The ecDNAs and ring chromosomes were found to reappear both in normal hematopoiesis and AML cells. Furthermore, we compared the eccDNAs of AML with normal cells. There were almost 300 AML-specific genes, including the known oncogenes  MYC or MLL amplification, complex karyotype, monosomal karyotype, TP53 deletion, and TP53 mutations [Extrachromosomal circular DNA (eccDNA) was currently reported to generate in the process of DNA damage and the corresponding DNA repair , 2. Accoutations , 14. Thoutations . This prThis study analyzed the ATAC-seq data from all cells in normal hematopoietic and leukemia evolution to reveal the eccDNA landscape of normal hematopoiesis and AML.P = 0.039, Fig. P = 0.025, Fig. P < 0.05, Fig. The eccDNAs of 13 cell types of normal hematopoiesis evolution were shown in Fig. To reveal the landscape of microDNAs in different normal blood cells, we proved the distribution of microDNAs of the stem, myeloid, and lymphoid cells by chromosome ideogram plot Fig. . By inteWe also explored the landscape of ecDNAs and ring chromosomes and their functions. Of particular interest in these results was that the distribution frequency of ecDNA and ring chromosome was high in chromosome 2 and chromosome 12, especially in myeloid and lymphoid cells. Figure P = 0.004, Fig. We continued to discover the landscape of eccDNAs in AML evolution. The average number of microDNAs (or tandem gene duplications) and ecDNAs gradually increased as primitive cells differentiate into the terminal cells except for LSC. And the number of ring chromosomes didn’t change much in four cell types Fig. . OncoploWe next dug the landscape of microDNA across HSC, pHSC, LSC, and blast in AML evolution. The distribution of microDNA across pHSC, LSC, and blast was shown in Fig. The landscape of ecDNAs and ring chromosomes across pHSC, LSC, and blast of AML evolution was also investigated. The distribution of ecDNAs and ring chromosomes across pHSC, LSC, and blast was shown in Fig. NRAS (1p13.2), MCL1 (1q21.2), EVI1 (3q26.2), GATA2 (3q21.3), WT1 (11p13), and PAK1 (11q14.1) were amplified in the eccDNAs of AML evolution compared with normal hematopoietic cells. The frequency of gene distribution of AML and normal cells displayed that REL was the most recurrent gene in eccDNAs of AML and normal cells. AML cells containing the eccDNA chr11: 58668376-58826008 highly expressed the GLYATL1 gene, while the frequency of occurrence in normal cells was very low , occurred in five AML evolution-related cells. GLYATL1 only highly expressed in AML cells and AML patients with the high expression of GLYATL1 had a shorter OS. GLYATL1 was also reported to overexpress in primary prostate cancer [TP53 deletion, and TP53 mutations. These studies also reported DMs in myeloid neoplasms commonly harbored MYC, KMT2A, or MLL gene amplification [In AML evolution, the eccDNAs also increased with the evolution from HSC to blast, except for LSC. A recent study revealed that AML patients with double minutes  presented an extremely poor prognosis . Approxin of AML –26. Besie cancer . The pree cancer . Therefofication , 14. WheNRAS, MCL1, EVI1, GATA2, WT1, PAK1, and GLYATL1) in the eccDNAs of AML evolution might contribute to AML progression. Moreover, we speculated that the high expression of AML-specific oncogenes in eccDNA might be associated with some inferior prognostic effects to promote AML progression. GLYATL1 might be a prognostic biomarker in AML. However, our algorithm cannot distinguish between extrachromosomal circles and chromosomal segmental tandem duplications unless the circles are experimentally purified prior to library preparation to remove the linear DNA, and most tandem duplications tend to be short segments. So we refer to these eccDNA as microDNA or tandem duplications. It’s necessary to provide more evidence and information for further research.In conclusion, the eccDNAs generally increased with the evolution of normal hematopoiesis and AML. There were some recurrent eccDNAs both in normal hematopoiesis and AML cells, especially ecDNAs and ring chromosomes. Whether it’s the intra-group comparison in AML cells or the comparison between AML and normal hematopoietic groups, we found that AML blast-specific genes and AML-specific genes were much more than in other groups. Combined with the previous studies, the accumulation of eccDNAs and the oncogenes (+ T cell (CD4), CD8+ T cell (CD8), B cell (B), and natural killer (NK) cell. The LMPP, CD4, CD8, B, and NK cells belong to lymphoid cells. The CMP, GMP, MEP, Mono, and Ery cells are part of myeloid cells. The remaining three cell types of AML evolution are preleukemic HSC (pHSC), leukemia stem cell (LSC), and leukemia blast cell (blast). These samples were exploited to ATAC-seq. And the paired expression data of 96 samples from RNA-seq was enrolled in our study. All original ATAC-seq and RNA-seq data were available under GEO accession GSE74912. Above AML samples were from the article of Corces et al. and obtained from patients at Stanford Medical Center with informed consent and agreement from institutional review board (IRB)-approved protocols  [https://cancergenome.nih.gov).In this study, ATAC-seq data from cells isolated from 9 healthy human donors  and 12 patients with AML . A total of 137 cell samples contained 16 blood cell types of the normal hematopoietic and AML hierarchies. Thirteen cell types were normal hematopoietic cells, including hematopoietic stem cell (HSC), multipotent progenitor (MPP), lymphoid-primed multipotent progenitor (LMPP), common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP), megakaryocyte-erythroid progenitor (MEP), monocyte (Mono), erythroid (Ery), common lymphoid progenitor (CLP), CD4nd 6453) . Other 1g RCF for 5 min at 4 °C and removed all supernatant. Then added 50 ul transposase mixture   in the cells and mixed well. Transposition reactions were incubated at 37 °C for 30 min with agitation at 300 rpm, afterwards purified DNA and prepared the library. More detailed steps of the protocol can be found in this article [All cell samples were separated by flow cytometry analysis and cell sorting (FACS). ATAC-seq data in our study was derived from the fast-ATAC sequencing, which is an optimized protocol for blood cells and requires just 5000 cells. Five thousand cells were pelleted by centrifugation at 500× article . ATAC-se article , with thhttps://github.com/pk7zuva/Circle_finder and https://github.com/pk7zuva/Circle_finder/blob/master/circle_finder-pipeline-bwa-mem-samblaster.sh). Obtained the eccDNAs were annotated by annovar with refGene and cytoBand (https://annovar.openbioinformatics.org/en/latest/). Oncogene and tumor suppression genes were annotated according to oncokb (https://www.oncokb.org/).All ATAC-seq data were processed as previously described . Using bhttp://metascape.org/).The eccDNAs were dived into mircroDNAs and ecDNAs according to length. Most statistical analyses in this study were performed and visualized by the R Bioconductor package, Maftools package . Oncoplohttp://gepia.cancer-pku.cn/), the data from The Cancer Genome Atlas . The endpoint was overall survival (OS). OS was defined as the time from study enrollment to death or last follow-up. The P value with statistical significance was 0.05 for the two-tailed test and the confidence interval (CI) was 95%.The survival analysis was performed by Kaplan–Meier method in GEPIA (Supplementary figuresSupplementary table 1Supplementary table 2Supplementary table 3


An increasing number of genetic and epigenetic abnormalities have been shown to display prognostic value in acute myeloid leukemia (AML) . Risk stTraditionally, multicolour flow cytometry (MFC) and real time PCR based techniques targeting recurrent specific molecular alterations were the most widely used tools for MRD assessment in AML. More recently, next-generation sequencing (NGS) has been introduced. Despite good sensitivity of those methods, there are still some pitfalls in MRD evaluation in AML, for example, mutations associated with nonmalignant clonal hematopoiesis or immunophenotypic abnormalities due to regenerating bone marrow can determine a “background noise” that may result in false positive tests , 9.Liu et al. evaluated a combination of PCR and MFC in a large cohort of AML patients treated with conventional 3 + 7 induction, demonstrating that the integration of the two techniques depends on the specific AML subtype, whereas the post -consolidation time point provided the most significant prognostic information. It is possible however that if more intensive induction therapies are adopted, the optimal timepoint for MRD assessment may differ , according to PCR-based MRD levels after HSCT. They showed that whereas administration of IFNa appears to be the best strategy for patients with low levels of RUNX/RUNXT1 transcript, for patients with higher burden of disease, donor lymphocyte infusions (DLI) represent the most efficient immunological intervention.Differently from acute lymphoblastic leukemia, where almost all therapeutic protocols are MRD driven, in AML, despite the relevant prognostic role of MRD, only a small minority of protocols incorporate MRD information for therapeutic decisions . FurtherGeorgi et al. reported how donor/recipient chimerism assessment may effectively trigger immune intervention in most AML patients.In patients lacking a specific transcript, aspecific markers could be used with the same aim. In this view, Kamel et al.Another issue of MRD assessment in AML is represented by the genetic heterogeneity of leukemic cells, both within an individual patient and between different patients . Even ifBernardi et al. provide an updated and comprehensive review of currently available data, comparing different MRD assessment methods and timepoints. In most studies, MRD clearance in patients treated with HMA + Venetoclax seems indeed to have different kinetics than in intensively treated patients, so that further studies are warranted.Finally, in the last year with the introduction of the combination of hypomethylating agents and novel drugs such as the BCL-2 inhibitor Venetoclax, the treatment of elderly AML patients has dramatically changed –24. HoweIn conclusion, despite significant advances in the standardization of MRD assessment in AML, there are still only few examples of MRD-driven treatment, mostly in AML with specific genetic lesions and in the post-transplant setting.A better understanding of the biology of the disease and the widespread introduction and integration of more sensitive and specific techniques will probably increase the clinical value of MRD information, leading to more MRD-directed clinical trials in the next future.FG: conceptualization. CV: draft writing. FG, CC and AI: draft revision. All Authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


UNRS measures BU severity in the past 24 hours from 0 (no urgency) to 10 (worst possible urgency). Psychometric evaluation of the UNRS showed Clinically Meaningful Improvement (CMI) is This analysis evaluated the proportions of patients in LUCENT studies achieving BU CMI and BU remission.The modified intent-to-treat (mITT) population (patients receiving ≥1 dose of miri or placebo (PBO); N= 1281) was randomized at induction study baseline in a 3:1 ratio to IV doses of 300mg miri or PBO every 4 weeks (Q4W) during induction . Patients achieving Clinical Response, measured by Modified Mayo Score (MMS), to miri during induction were re-randomized at W0 of the maintenance study in a 2:1 ratio to subcutaneous (SC) 200mg miri or PBO Q4W through W40 (52 weeks of treatment). Patients recorded their UNRS score daily in an e-diary. Mean weekly UNRS scores were calculated from diary data if ≥4 days of data were available. Rates of BU CMI and BU remission in the miri v PBO groups were compared at W12 (induction) in the mITT population with a baseline UNRS score ≥3, and W52 (maintenance) among miri clinical responders at W12 with a baseline UNRS score ≥3. Cochran-Mantel-Haenszel tests with non-responder imputation for missing values were used for all treatment comparisons.Patient population: mean age 43 years, 60% male, disease duration 7 years; 63.0% left-sided colitis; 36.3% pancolitis; 46.7% moderate disease (MMS 4-6); 53.2% severe disease (MMS 7-9). Significantly higher proportions of miri versus PBO patients achieved BU CMI (48.7% v 32.2%) and BU remission (22.1% v 12.3%) at W12  in the induction study. Similarly, at W40 of maintenance, significantly greater proportion of miri patients achieved BU CMI (65.2% v 41.9%) and BU remission (42.9% v 25.0%) compared to PBO among miri induction responders .Miri had a highly significant and clinically meaningful benefit on reducing bowel urgency, one of the most disruptive UC symptoms. The Urgency Numeric Rating Scale usefully quantified the baseline level and change in bowel urgency after treatment across a spectrum of severity.OtherEli Lilly and CompanyNone Declared


The oncometabolite 2-hydroxyglutarate (2-HG) plays a key role in differentiation blockade and metabolic reprogramming of cancer cells. Approximatively 20–30% of acute myeloid leukemia (AML) cases carry mutations in the isocitrate dehydrogenase (IDH) enzymes, leading to a reduction in the Krebs cycle intermediate α-ketoglutarate (α-KG) to 2-HG. Relapse and chemoresistance of AML blasts following initial good response to standard therapy account for the very poor outcome of this pathology, which represents a great challenge for hematologists. The decrease of 2-HG levels through pharmacological inhibition of mutated IDH enzymes induces the differentiation of AML blasts and sensitizes leukemic cells to several anticancer drugs. In this review, we provide an overview of the main genetic mutations in AML, with a focus on IDH mutants and the role of 2-HG in AML pathogenesis. Moreover, we discuss the impact of high levels of 2-HG on the response of AML cells to antileukemic therapies and recent evidence for highly efficient combinations of mutant IDH inhibitors with other drugs for the management of relapsed/refractory (R/R) AML. This maAML is a highly complex disease characterized by a plethora of cytogenetic alterations and genetic mutations, which are used to classify AML. The therapeutic approaches are also established on the classification of patients in fit, unfit, and frail. This stratification is based on several features, including age, performance status , and comorbidities . PatientMoreover, despite a good response to first-line treatment (more than 70% of cases), most AML patients relapse within 6–9 months after the consolidation phase. Notably, the treatment options for relapsed/refractory (R/R) patients are limited and new strategies are urgently needed. In this scenario, the development of targeted therapies has led to remarkable successes in some genetic subtypes of AML, leading to improved survival and quality of life , making In this review, we focused on the mutations of IDH1 and IDH2, coding for the cytosolic and the mitochondrial NADP-dependent isocitrate dehydrogenases, respectively . Wild-tyIn this context, patients with IDH mutations could benefit from pharmacological inhibition of IDH enzymes through specific small molecules. In parallel, pharmacological approaches that target altered molecular pathways were also developed. Indeed, both fit and unfit patients with IDH mutations appear more sensitive to the treatment with venetoclax + azacitidine than the IDH1/2 wild type, achieving higher and more durable response rates and more prolonged median OS .p < 0.001) [Venetoclax is a BCL-2 inhibitor, recently approved by the Food and Drug Administration (FDA) in combination with HMA to treat de novo AML patients unfit for intensive chemotherapy, where BCL-2 anti-apoptotic protein is usually upregulated and associated with poor prognosis and resistance to chemotherapy . Clinica< 0.001) . A phase< 0.001) .TP53 and BAX genes are key elements of resistance to venetoclax, explaining venetoclax insensitivity in samples from AML patients who have low expression of these two genes [However, the response to venetoclax-based treatments appears limited in time  and the wo genes .These observations suggest that it is of primary importance to study the effect of genetic mutations in AML development and pharmacological response, as well as to understand the molecular mechanism of drug resistance to prevent and/or counteract it.R132, IDH2R140, or IDH2R172), do not lead to an inactive form of IDH1/2 but allow them to catalyze a new reaction [+ as a cofactor, with the consequent implementation of the NADPH cellular pool [+ and isocitrate [As described above, the accumulation of the oncometabolite 2-HG in AML cells is mainly associated with the mutations of IDH1/2 and their acquired neomorphic activity. Indeed, these mutations, which affect the arginine residues in the catalytic domain  by depleting the main source of electrons used to regenerate the antioxidant systems [The wild-type IDH1/2, together with other enzymes , maintain the NADPH pool and redox homeostasis in non-malignant cells . On the  systems ,46. More systems .Elevated levels of 2-HG are also associated with the reprogrammed metabolism of many tumor types, such as glioma, glioblastoma, and AML ,48, and The oncogenic effects associated with IDH mutations and high levels of 2-HG were demonstrated in human and murine models. Different studies showed that the expression of human mutants of IDH2 synergistically acts with mutated FLT3 and NRAS to promote leukemogenesis by accumulating undifferentiated cells of the myeloid lineage in the hematopoietic compartment of mice ,60. HoweMoreover, identifying high levels of 2-HG in AML patients’ sera suggests that it could also be eligible as a biomarker for diagnosis, prognosis, and as a monitoring tool in AML. Results from different studies reported that the presence of IDH mutations positively correlates with elevated serum 2-HG levels ,66 and tHigh levels of circulating 2-HG are also associated with tumor burden  as well R140 and DNMT3A mutations have a significantly worse prognosis than patients with coexisting NPM1 mutation. In contrast, the association of IDH1R132 and DNMT3A mutations does not affect prognosis. In a recent meta-analysis, IDH1 mutations were associated with a lower chance of CR, worse OS, and EFS, especially in patients with normal karyotype. In contrast, IDH2 mutations were associated with favorable outcomes regardless of the type of mutation [The generation of 2-HG through the neomorphic activity of mutated IDH enzymes leads to the consumption of NADPH, altering the redox homeostasis of AML cells harboring these mutations. Thus, in principle, AML cells with mutant IDH should be more sensitive to pharmacological strategies aimed at increasing the levels of ROS. It is worth noting that the standard chemotherapeutic regimen used to treat AML induction therapy comprises well-known ROS inducers: cytarabine  and an amutation . Anothermutation . These oContrarily, other studies demonstrated an increased sensitivity of cancer cells with IDH mutations to several drugs, including alkylating agents , inhibitChen and colleagues reported that 2-HG released from IDH-mutated AML cells induces the activation of NF-κB pathway in bone marrow stromal cells, thus creating a supportive niche for leukemic cells . The autThe inconclusive results on prognostic impact of IDH mutations are probably due to differences in population, type of study, and influence of additional genomic aberrations. Large, multi-centric clinical studies are needed to drive a conclusion about the role of 2-HG in the response of AML to chemotherapy. This notion could aid clinicians in stratifying patients that will benefit from induction therapy and patients that, instead, need alternative regimens, such as IDH-targeted therapies.Experiments performed in primary human AML cells demonstrated that mutations in IDH1 or IDH2 genes induce dependence of AML cells on the antiapoptotic protein BCL-2 and, thus, higher sensitivity to venetoclax , a speci50) of venetoclax in AML cells, with greater effects observed in cells expressing IDH1R132H mutation, compared to those with wild-type IDH1 [+ hematopoietic stem and progenitor cells , which are also highly resistant to venetoclax treatment ex vivo, suggesting a wide in vivo therapeutic index [The role of 2-HG in AML response to venetoclax was also assessed both ex vivo and in xenograft models. The introduction of octyl-(R)-2-HG, a cell-permeable precursor of 2-HG, significantly reduces the half-maximum inhibitory concentration  and enasidenib (AG-221) are the only drugs approved by the FDA for the treatment of IDH1- and IDH2-mutated R/R AML, when intensive treatment is precluded . HoweverIDH1 gene, thus decreasing 2-HG levels and inducing blast differentiation. Ivosidenib efficiently killed IDH1R132 mutant cancer cell lines at low nanomolar IC50 in vitro, drastically and rapidly reduced 2-HG levels in a mouse xenograft model derived from HT-1080 fibrosarcoma cell line and led to the differentiation of IDH1-mutated AML cells from patients ex vivo [Ivosidenib is the first inhibitor of mutant IDH1 that achieved an anticancer efficacy in clinical trials . It inhi ex vivo . As sing ex vivo . Ivoside ex vivo . Moreove ex vivo . Recentl ex vivo  in a froR140Q-expressing erythroleukemia cell line, inhibited growth factor-independent proliferation, and reversed hypermethylation of histone H3. Moreover, this compound reduced 2-HG levels, induced differentiation of primary IDH2-mutated AML blasts ex vivo and improved the survival of mice bearing an aggressive xenograft established from an AML patient harboring IDH2R140Q mutation [Enasidenib reduced 2-HG levels in a IDH2mutation . Stein amutation . Among tmutation . Interesmutation . The commutation , an advamutation –has a symutation . Moreovemutation . Based omutation . The ratmutation . Interesmutation .Jasra and colleagues recently reported the case of a patient affected by IDH2-mutated AML refractory to induction therapy, who achieved CR upon combined treatment of enasidenib and azacitidine. At relapse, the patient received also venetoclax in combination with the other two drugs, achieving clearance of peripheral blasts . In a phNPM1, DNMT3A, ASXL1, and receptor tyrosine kinase (RTK) genes) was correlated with decreased ORR and CR [Besides ivosidenib and enasidenib, several other IDH inhibitors were developed and tested in preclinical and clinical studies. BAY1436032 is an allosteric inhibitor targeting all IDH1 mutations, which inhibits cell proliferation and self-renewal of leukemia stem cells . The comR and CR . In combR and CR . AnotherR and CR .When using IDH inhibitors in the clinic, two main drawbacks should be taken into account: (i) severe adverse effects exist; (ii) several mechanisms are responsible for the resistance of AML cells to these drugs. A serious adverse effect induced by IDH inhibitors is differentiation syndrome (DS), a potentially lethal complication also induced by ATRA in APL . DS is cNRAS, KRAS, PTPN11, and FLT3, are associated with primary resistance to ivosidenib despite the reduction in 2-HG levels [IDH1 gene and canonical mutations of IDH2, which can co-occur in ivosidenib-resistant AML [IDH2 mutations but reported seven different patterns of clonal evolution, including mutations of IDH1, cytokine receptor signaling genes, hematopoietic transcription factors, components of the spliceosome, and chromosome 7 monosomy, which restored the differentiation blockade in AML cells [Both primary and secondary mechanisms of resistance to IDH inhibitors have been described. Co-occurring mutations in RTK pathway genes, such as G levels . MoreoveG levels . Secondatant AML . Quek anML cells . As isofML cells , combineML cells . InteresML cells .From an epidemiological point of view, AML represents a global health issue. In fact, among leukemias, it has the highest lethality rate worldwide . MoreoveThe identification of specific genetic alterations could broaden the therapeutic scenario, as most mutations are associated to different prognostic outcomes. Indeed, the FLT3-ITD mutations predict a worse prognosis for patients through the activation of several oncogenic pathways  . On the The most interesting mutations reported to have a great impact on AML affect the NADP-dependent isocitrate dehydrogenases IDH1 and IDH2. These mutated enzymes acquire the ability to convert α-KG to 2-HG, a oncometabolite that accumulates in AML cells , inducesAlthough all these observations support the role of 2-HG as a tumor-promoting molecule, its role in the response of AML blasts to therapies is less clear. Unlike other IDH-mutated tumors such as gliomas, AML cells with high levels of 2-HG were reported to be either more resistant to induction therapy or more sensitive to several other therapies like PARP inhibitors  or venetGiven its pathogenic role in AML, 2-HG could be exploited as a molecular target to counteract tumor relapse in IDH mutant cells. Indeed, circulating 2-HG was found to be associated with tumor burden , poor OSIn conclusion, 2-HG has a great potential both as a prognostic marker in IDH-mutated AML and as a biomarker of clinical outcome to therapies in AML patients. Moreover, the potent antileukemic effect achieved through the pharmacological reduction in its levels indicates that 2-HG is a therapeutic target in IDH-mutated AML. Further studies should be carried out to dissect in detail the complex effects of 2-HG on leukemic cells and on healthy bone marrow cells, and its role in the response of AML blasts to therapies, thus improving the management of R/R AML with mutated IDH enzymes.


Immunotherapy is changing the therapeutic landscape of many hematologic diseases. Nevertheless, in acute myeloid leukemia (AML) the anti CD33 antibody-drug conjugate gemtuzumab ozogamicin is the only approved drug. In this review, we aimed at reporting biological mechanisms and their clinical impact of vaccines in AML. The principal vaccination strategies have been analyzed and commented, highlighting advantages in terms of toxicity and possibility to apply in elderly patients. Nevertheless, the clinical results of this strategy in AML are still far from satisfactory. It is necessary to evaluate the best scenario for this approach, whether in a therapeutic, prophylactic, or preemptive setting, considering the poorer results in active or high-burden disease. Finally, we underlined the necessity in AML of further research to optimize immunotherapy-based strategies, among which vaccines might represent relevant actors to contribute to long-term disease control.Immunotherapy is changing the therapeutic landscape of many hematologic diseases, with immune checkpoint inhibitors, bispecific antibodies, and CAR-T therapies being its greatest expression. Unfortunately, immunotherapy in acute myeloid leukemia (AML) has given less brilliant results up to now, and the only approved drug is the antiCD33 antibody-drug conjugate gemtuzumab ozogamicin. A promising field of research in AML therapy relies on anti-leukemic vaccination to induce remission or prevent disease relapse. In this review, we analyze recent evidence on AML vaccines and their biological mechanisms. The principal proteins that have been exploited for vaccination strategies and have reached clinical experimental phases are Wilm’s tumor 1, proteinase 3, and RHAMM. the majority of data deals with WT1-base vaccines, given also the high expression and mutation rates of WT1 in AML cells. Stimulators of immune responses such as TLR7 agonist and interleukin-2 have also proven anti-leukemic activity both in vivo and in vitro. Lastly, cellular vaccines mainly based on autologous or allogeneic off-the-shelf dendritic cell-based vaccines showed positive results in terms of T-cell response and safety, also in elderly patients. Compared to other immunotherapeutic strategies, anti-AML vaccines have the advantage of being a less toxic and a more manageable approach, applicable also to elderly patients with poorer performance status, and may be used in combination with currently available therapies. As for the best scenario in which to use vaccination, whether in a therapeutic, prophylactic, or preemptive setting, further studies are needed, but available evidence points to poorer results in the presence of active or high-burden disease. Given the poor prognosis of relapsed/refractory or high-risk AML, further research is urgently needed to better understand the biological pathways that sustain its pathogenesis. In this setting, research on novel frontiers of immunotherapy-based agents, among which vaccines represent important actors, is warranted to develop new and efficacious strategies to obtain long-term disease control by immune patrolling. Acute myeloid leukemia (AML) is a clonal disorder of hemopoietic cells characterized by the uncontrolled proliferation of neoplastic blast cells conditioning concomitant bone marrow failure . As a coAmong several mechanisms that could be exploited to reduce relapse risk, the immune control of leukemic myeloid proliferation is the base of different therapeutic strategies. In this field, the forerunner approaches are represented by hemopoietic stem cell transplantation (HSCT) and donor lymphocyte infusion (DLI), as efficient graft versus leukemia (GvL) shows that immunological activity can provide a long-lasting disease control. Although effective, HSCT is not applicable to every patient for many possible reasons . Consequently, further immunological strategies are being explored: monoclonal antibodies , bispecific antibodies (BiTE), chimeric antigen-receptor (CAR) T cells, and, lastly, vaccination. In this review, we discuss the state of the art of vaccination strategies aimed to enhance anti-leukemia immunity and consequential clinical outcomes in AML. Vaccines in AML could be macroscopically categorized into two broad groups based on the type of constructs: peptide vaccines and dendritic cell (DC)-based vaccines . In bothThe main leukemic antigens investigated are Wilms’ tumor 1 (WT1), proteinase 3 (PR3), the receptor for hyaluronic acid-mediated motility (RHAMM), and mucin 1 protein (MUC1), all able to elicit a specific cytotoxic T response .c-myc and bcl-2. Mutations of WT1 may lead to the dysregulation of cell growth and differentiation, playing a role in leukemogenesis . Ov. Ov47]. Antibody-based therapies are directed to cell surface antigens which limit their applicability with respect to other neoplasms, such as lymphoid ones, due to the difficulty in identifying robust cell surface targets expressed selectively on neoplastic cells but not on their normal counterpart. To date, as for cell-targeting antibodies, only two toxin-conjugate antibodies are approved for AML therapy, namely gemtuzumab ozogamicin (Mylotarg) for AML and tagraxofusp for plasmacytoid dendritic cell neoplasms. Both agents have been proven to significantly increase event-free survival when added to chemotherapy  or as aThe most-tested immune checkpoint inhibitors in AML are anti-PD-1 antibodies. However, responses are variable and often transient, as PD1-L is not constitutively expressed on all neoplastic myeloid cells . PD1-L eBispecific antibodies are engineered drugs recognizing two different antigens in order to recruit an immune cell (mainly T cell or NK cell) and link it to the neoplastic cell. Antibodies currently under study are directed against different key antigens, mainly CD33, CD123, and CLEC12A/CLL-1 ,54. EffeCAR-T cells are engineered autologous lymphocytes with a polypeptide chain made by the combination of the extracellular antigen-binding site of an antibody and the intracellular CD3ζ chain linked to the signal cascade of the T cell receptor, thus bypassing the need for an HLA-based immune response mechanism, as HLA downregulation is a common way of resistance for leukemic cells. Given the good results of CAR-T cell therapy in lymphoid neoplasms ,57, therIf, in the context of active disease, other immunotherapies show greater efficacy, more has to be studied in different clinical settings, such as relapse prevention and MRD positivity, in which vaccines may provide a maintenance strategy at low risk of toxicities. Moreover, an interesting open question can be made on the use of vaccines in pre-leukemic states  ), in whiAs for other immunotherapies, with the exception of bispecific antibodies and CAR-T, vaccination therapy shares the feature of being an easily combinable agent with standard approved therapy in order to increase response rates and response duration. Nevertheless, the possibility that vaccination may induce tolerance in the effector T cell deserves further investigation, and the association in sequence with immune therapies that rely on these cells as bispecific antibodies and checkpoint inhibitors should be carefully evaluated. Overall, AML remains a proliferating tumor with a high tendency for immune escape, leading to the possible failure of these immunotherapies.Also, differently from CAR-T cell therapy, peptide vaccines can be readily available drugs that can be rapidly administered to patients, while CAR-T cells require weeks for production, during which AML patients can commonly face complications or die from rapidly progressive disease.Lastly, to a different extent, all cited immunotherapies bear a significant economic cost which can constitute a limit in the case of the wide application of these therapies or for their application in developing countries.Nowadays, relapsed/refractory or high-risk AML is still a poor-prognosis disease, with unsatisfying outcomes. New therapeutic approaches are needed to reach deeper and long-lasting disease control and prevent relapse. Immunotherapy offers a good strategy for this purpose and the immune control of leukemic cells can be achieved with different mechanisms and agents. Vaccine-based strategies have been explored in clinical trials, several still ongoing . Surely,


Because of its central importance in physiology, the activity of this transporter is tightly controlled via direct interactions with tissue-specific regulatory micropeptides that tune SERCA function to match changing physiological conditions. In the heart, the micropeptide phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) stimulates SERCA. These competing interactions determine cardiac performance by modulating the amplitude of Ca2+ signals that drive the contraction/relaxation cycle. We hypothesized that the functions of these peptides may relate to their reciprocal preferences for SERCA binding; SERCA binds PLB more avidly at low cytoplasmic [Ca2+] but binds DWORF better when [Ca2+] is high. In the present study, we demonstrated this opposing Ca2+ sensitivity is due to preferential binding of DWORF and PLB to different intermediate states that SERCA samples during the Ca2+ transport cycle. We show PLB binds best to the SERCA E1-ATP state, which prevails at low [Ca2+]. In contrast, DWORF binds most avidly to E1P and E2P states that are more populated when Ca2+ is elevated. Moreover, FRET microscopy revealed dynamic shifts in SERCA–micropeptide binding equilibria during cellular Ca2+ elevations. A computational model showed that DWORF exaggerates changes in PLB–SERCA binding during the cardiac cycle. These results suggest a mechanistic basis for inhibitory versus stimulatory micropeptide function, as well as a new role for DWORF as a modulator of dynamic oscillations of PLB–SERCA regulatory interactions.The ATP-dependent ion pump sarco/endoplasmic reticulum Ca The rate of Ca2+ transport by SERCA2a determines how quickly the heart muscle relaxes during the diastolic phase of the cardiac cycle as the ventricle is filling with blood. SERCA2a function also sets the amplitude of SR Ca2+ release, which determines the heart’s contractile strength during the systolic phase when blood is being ejected from the heart. Pathological decreases in SERCA expression, function, and regulation are associated with heart failure (The type 2a sarco/endoplasmic reticulum (ER) Ca failure , 2, focu failure , 4. So f failure . Therefo2+, decreasing Ca2+ uptake , a single-pass transmembrane micropeptide , blue. P+ uptake . This indent PKA , 8, provto SERCA , 12, 13.ERCA . PLB pentamers represent an inactive, reserve pool  , shown ited Ca2+ . This immyocytes . Howeverpeptides , 30, 31.2+, whereas the DWORF–SERCA interaction is slightly more stable with elevated [Ca2+] , SERCA predominantly resides in the ATP-bound state, E1-ATP , waiting for Ca2+ to bind. During intracellular Ca2+ elevations, SERCA samples all the intermediate states of the Ca2+ transport cycle, but these states are not all equally populated during systole. Rather, there is relative accumulation in the autophosphorylated E1P/E2P intermediates that precede rate-limiting steps in its enzymatic cycle . A hyperbolic fit to the data yielded the maximal FRET efficiency at high protein concentration or FRETmax (representing the intrinsic FRET of the bound PLB–SERCA complex) and the apparent dissociation constant (KD), the protein concentration that yields half-maximal FRET efficiency. The KD value is inversely related to the affinity of the PLB–SERCA complex. Figure 2B shows the conditions that yielded the greatest difference in relative KD for the PLB regulatory complex. PLB bound to the E1-ATP state of SERCA is obtained with a solution containing 3 mM ATP and low Ca2+ and represents the prevailing conformation in resting (diastolic) conditions. Addition of thapsigargin (TG) resulted in a significant right-shift of the binding curve (p = 8 × 10−3) -labeled SERCA2a (FRET donor) and YFP-labeled PLB (FRET acceptor) and quantified the interaction of these proteins using acceptor sensitization FRET microscopy. Protein expression levels in membrane fractions prepared from HEK cells were comparable to protein concentrations in membranes prepared from human heart tissues . To contFig. 2 × 10−3) , indicat × 10−3)  and othe × 10−3) .C and D and 2+ is low, the muscle is relaxed, and the heart is filling with blood. The affinity of PLB for this state was not altered over a range of pH from 6 to 8 . We noted slightly lower KD values for E1PCa and E2P states. The population of SERCA pumps increasingly accumulates in these states during the systolic phase of the cardiac cycle when cytoplasmic Ca2+ is elevated and the heart is contracting to eject blood. DWORF may have a preference for those conformations, though the difference was not statistically significant by one-way ANOVA . In some cells, these Ca2+ elevations coincided with small decreases in PLB–SERCA FRET, shown in F and G as gray data overlaid with a smoothed trendline . Indeed, we were previously unable to detect a PLB–SERCA FRET change over a single Ca2+ transient in electrically paced cardiac myocytes, only observing a modest decrease in average FRET over a period of repeated Ca2+ transients during rapid pacing (2+ elevations were more prolonged (increasing time for quantification), expression of endogenous SERCA/PLB was very low (reducing competing non-FRET interactions), and the cells are noncontractile (eliminating confounding cell motions). These advantages enabled detection of small shifts in the binding equilibria in the present study.Taken together, these data indicate that PLB and DWORF prefer to bind different SERCA conformations corresponding to different enzymatic intermediate states of the transport cycle. To determine how this differential Caeviously . HEK-293eriments , C and Ed pacing . However2+ elevations. These FRET changes were even smaller than those observed for PLB–SERCA binding (H), consistent with DWORF’s relatively flat affinity profile across SERCA enzymatic states (D). The DWORF–SERCA FRET change was easier to appreciate in experiments where a low affinity Ca2+ indicator localized to the ER lumen (R-CEPIA1er) was used instead of a cytoplasmic Ca2+ indicator dye (I), perhaps because ER Ca2+ buffering by the indicator resulted in larger amplitude Ca2+ release events. Interestingly, the direction of the DWORF–SERCA FRET change was the opposite of that observed for PLB–SERCA; we observed modestly increased FRET during Ca2+ transients (H and I), consistent with enhanced DWORF–SERCA binding affinity at elevated cytoplasmic Ca2+ (E) ..2+ eleva2+ signaling determines SERCA–micropeptide binding dynamics, we quantified the rates of change of FRET and [Ca2+]. Specifically, exponential decay fitting of these changes revealed characteristic time constants (τ) for each process. Analysis of the Ca2+ release process (A) showed that DWORF–SERCA binding occurred with a time course that was similar to the rate of rise of Ca2+  (B). In contrast, PLB–SERCA unbinding was slower (τ = 0.74 ± 0.14 s), lagging behind the Ca2+ upstroke (p = 4 × 10−8) (C). In addition, we evaluated how Ca2+-dependent changes in PLB–SERCA binding affects the PLB monomer–pentamer equilibrium, measuring intrapentameric FRET between Cer-PLB and YFP-PLB. Interestingly, PLB–PLB FRET increased rapidly during Ca2+ elevations (2+ release (p = 2 × 10−4) (D). This delay was similar to that of PLB–SERCA unbinding (E), suggesting that PLB–SERCA unbinding may be rate limiting for subsequent PLB oligomerization during Ca2+ elevations. The kinetics of SERCA–micropeptide binding dynamics during Ca2+ release are summarized in To examine how the kinetics of cellular Ca process A showed ctively) B. In con × 10−8) C. In addevations . The inv × 10−4) D. This dnbinding E, sugges2+ declined to baseline during the Ca2+-reuptake phase of the Ca2+ transient (F). DWORF dissociated rapidly from SERCA, as measured by the decrease in the DWORF–SERCA FRET ratio back to baseline (G). This unbinding process occurred with a τ of 1.2 ± 0.4 s, similar to the Ca2+ transient relaxation time. However, PLB–SERCA reassociation displayed a significant lag compared to the Ca2+ transient relaxation, with FRET continuing to increase back toward maximum for several seconds after Ca2+ had already stabilized at a basal level (H). PLB–SERCA reassociation occurred with a τ of 4.9 ± 0.6 s, much slower than the Ca2+ relaxation time .Next, we examined changes in SERCA–micropeptide binding as Caransient F. DWORF baseline G. This ual level H. PLB–SEI) that closely matched the slow PLB–SERCA reassociation (4.9 ± 0.6 s) . The data suggest PLB pentamer dissociation is rate limiting for PLB rebinding SERCA after Ca2+ elevations. This interpretation was further supported by the observation that destabilization of the PLB pentamer by mutagenesis (PLB-AFA) (p = 8 × 10−3), such that the FRET change no longer lagged behind the Ca2+ transient decay   accelerant decay J and S9.p = 2 × 10−6)  J and S10A. The relative population of these two ensemble states is dependent on the relative concentration of Ca2+, as quantified by confocal microscopy experiments. Monomeric PLB (PLB1) can bind either the Ca-free or Ca-bound ensemble of SERCA (E). The relative affinity of PLB for SERCA was constrained to be twofold higher for Ca-free versus Ca-bound ensembles, as determined experimentally from FRET-binding curves (C) (1) is also in equilibrium with the pentameric population (PLB5). The relative affinity of DWORF for SERCA was constrained to increase by 25% between Ca-free and Ca-bound ensembles, consistent with FRET measurements (D) . M. MA. Theurements D (18). Tents (D) .Figure 42+ (measured simultaneously). Figure 4B provides a best-fit model prediction of the FRET compared to a representative experimental measurement. The model captured the rapid reduction of FRET with the rise of the Ca2+ transient. The decrease in PLB–SERCA is followed by a slower restoration of the FRET as Ca2+ returns to baseline. The similarity of the predicted and experimentally measured FRET data suggests that the model appropriately represents key aspects of PLB–SERCA binding dynamics.The computational model provided good descriptions of data from physical experiments showing time-dependent changes in PLB–SERCA FRET and cytoplasmic CaC, purple) during a Ca2+ elevation , consistent with the “subunit model” . However, a fraction of the population of PLB–SERCA dissociated during a Ca2+ transient . This fraction comprised ∼30% of PLB–SERCA complexes under these simulation conditions. This shift impacted the PLB monomer/pentamer equilibrium . Expectedly, there was very little observed change in the small population of PLB monomers, as the dynamic fraction was quickly incorporated into pentamers. These reciprocal shifts are the cause of the transient decrease in SERCA–PLB FRET and increase in PLB–PLB FRET observed by confocal microscopy (C and D).Using these fit parameter values, we simulated the time-dependent changes in PLB–SERCA and PLB–PLB binding dynamics that we observed in HEK cells. Importantly, we observed that a majority of the PLB–SERCA regulatory complexes remained intact C, purplet model” . Thus, Pcroscopy , C and D2+ transients observed in the heart, pacing at a range of frequencies from 60 to 180 beats per minute, to investigate regulatory dynamics under conditions of rest, moderate exercise, and intense exercise. Increasing pacing frequency progressively decreased PLB–SERCA binding  and increased accumulation of PLB in pentamers . The systole–diastole difference in PLB–SERCA binding was small. This oscillation became even smaller with increasing pacing frequency , as the shifts in binding equilibria began to lag behind the rapid Ca2+ changes. These results suggest that the exchange dynamics of PLB may impact adaptive SERCA regulation between resting and exercising heart rates.Next, we simulated the shorter, faster Ca2+ was increased by 50% , decreased PLB–SERCA oscillation amplitude (G), and blunted the responsiveness of these parameters to increasing heart rates (E–G). This suggests frequency-dependent changes in PLB inhibition may be reduced in heart failure.To assess how PLB–SERCA binding dynamics may be altered in disease, we modified the model conditions to reflect changes observed in heart failure. Specifically, the SERCA population was reduced by 40%  and diasilibrium E, decreamplitude G, and blrt rates , E–G. ThA). Increasing the amount of DWORF relative to PLB decreased PLB–SERCA binding  and increased PLB oligomerization . DWORF competes potently with PLB even at low stoichiometry because oligomerization of PLB reduces the effective concentration of the active monomeric species. Interestingly, the Ca2+-dependent increase in DWORF affinity for SERCA exaggerated the oscillations in PLB–SERCA binding during pacing . That is, during Ca2+ elevations DWORF (which binds SERCA better at high Ca2+) increasingly displaced PLB (which binds SERCA better at low Ca2+).Introduction of DWORF–SERCA interactions to the model yielded the expected effect of competition of DWORF and PLB for SERCA binding A. Increa2+) , there was decreased formation of the PLB–SERCA complex (E), and therefore, the effect of DWORF competition was blunted (I). Moreover, since changes in PLB–SERCA binding between systole and diastole were smaller in heart failure, the ability of DWORF to exaggerate this change through calcium-dependent competition was also decreased (I). These results suggest that the impact of DWORF on PLB–SERCA binding dynamics may be reduced in heart failure.Under conditions representing heart failure , and this is the underlying cause of the reciprocal Ca2+-dependent affinities of these micropeptides (E) (C), which is the predominant state at resting Ca2+ . In contrast, DWORF shows a much flatter SERCA-binding profile, with more avid binding to E1P and E2P states . Much of the SERCA population accumulates in these states when cytoplasmic Ca2+ is elevated. This accumulation occurs because of rate-limiting, slow steps in the SERCA enzymatic cycle . A and F show how differential binding of micropeptides leads to exchange of a dynamic fraction of PLB and DWORF from the binding site on SERCA during intracellular Ca2+ elevations. In high Ca2+ (A), DWORF increasingly outcompetes PLB, displacing a fraction of monomeric PLB (“PLB1”), which is increasingly incorporated into PLB pentamers (“PLB5”). Conversely, in low Ca2+ (F), DWORF binding is decreased, and PLB monomers increasingly bind to SERCA because the pump accumulates in the E1-ATP state that PLB prefers. This recovery process is rate limited by the slow dissociation of monomeric PLB from pentamers (I).The principal finding of the present study is that PLB and DWORF preferentially bind to different conformations of SERCA , C and Dpeptides E 18). T. TC and ides (E) C, which ic cycle , 35, whiigh Ca2+ A, DWORF low Ca2+ F, DWORF entamers I.2+ dependence is the key determinant of whether a micropeptide is inhibitory or stimulatory for SERCA function. In particular, micropeptide–SERCA binding affinity may be taken as an index of the relative energetics of the regulatory complex, much as melting temperature may be quantified as a proxy for the stability of a protein–protein complex  is expected to stabilize that complex and favor the population of that state of SERCA. The functional consequence of stabilizing this state is to slow the subsequent Ca2+-binding step in the enzymatic cycle. This may account for the principal inhibitory effects of PLB, reducing the apparent Ca2+ affinity of the pump and slowing pump turnover  , SERCA activity is already low because Ca2+ is low. SERCA is further inhibited in diastole by an increase in the fraction of pumps that bind PLB (F and G). Then, during systole (cardiac contraction), Ca2+ is high, supporting high SERCA activity. This high SERCA activity is further enhanced by increased DWORF binding (H and I), which increasingly displaces PLB and directly stimulates SERCA maximal activity . The dose dependence of these effects revealed in the computational model may provide guidance for future DWORF-based gene therapy approaches , a noteworthy finding since this is significantly slower than the cardiac cycle. Even at a resting heart rate, a 1 s interval between beats would not be sufficient time for the equilibrium to fully relax between systole and diastole. Therefore, Ca2+ signals must also integrate over a longer timescale, such as between rest and exercise. This is visualized in our computational model, which showed increasing accumulation of PLB in pentamers at faster heart rates at the expense of the population of PLB–SERCA (D). Such “kinetic trapping” of PLB in pentamers at faster heart rates is further increased by phosphorylation of PLB by PKA or CaMKII, which stabilizes the pentamer complex . This may explain why the Bowditch effect is blunted in patients with heart failure  D, a noteLB–SERCA D. Such “ complex , 21, 22. KO mice , 45, 46. KO mice , E–G. Th failure .2+ transients. The present data clarify the role of the PLB pentamer as a phosphorylation-tunable kinetic trap that limits the rate of SERCA rebinding by a dynamic fraction of PLB. Future studies will test the functional predictions derived from the present results: DWORF enhances transitions through E1P, E2P states in the SERCA enzymatic cycle; DWORF increases the size of the fraction of PLB that binds/unbinds SERCA with each Ca2+ elevation, exaggerating the SERCA inhibition/stimulation cycle; DWORF and PLB act in concert to increase the cooperativity of Ca2+ transport. Overall, the data suggest that dynamic competition of PLB and DWORF is an important determinant of cardiac function. More generally, the results may provide insight into the interplay of other tissue-specific micropeptide regulators of ion transporters.Dynamic changes in the competition of regulatory micropeptides on seconds and minutes timescales represent an important aspect of the responsiveness of SERCA regulation to Cavia a five amino acid linker to the N terminus of the micropeptide or SERCA  and SERCA constructs consisted of mCerulean3, enhanced YFP, or TagRFP fused or SERCA , 27, 48.or SERCA , 50 and function , 51, 52.function . The datversus 750:1 lipid:protein ratio quantified for native SR (AAV 293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) cell culture medium supplemented with 10% fetal bovine serum (ThermoScientific). Following culture, the cells were transiently transfected using either MBS mammalian transfection kit  or Lipofectamine 3000 transfection kit (Invitrogen) as per instructions provided with the respective kits. Twenty-four hours post-transfection, the cells were trypsinized (ThermoScientific) and replated onto poly-D-lysine–coated glass bottom chambers and allowed to settle down for 1 h before imaging. We have previously estimated that this approach achieves ER protein expression levels on the order of 2000:1 or 4000:1 lipids:protein ratio , versus ative SR , 54. ThuHuman left ventricular tissue was provided by Loyola Cardiovascular Research Institute Biorepository. The sample collection was approved by Loyola University Review Board (IRB number 210940821918) and written informed consent was obtained for collection of heart tissue according to Declaration of Helsinki.2HPO4, 2.5 mM KH2PO4, 2 mM EDTA, and protease inhibitors. Samples were mechanically homogenized with an Omni International GLH-01 homogenizer and rotated for 1 h at 4 °C. Homogenates were centrifuged at 10,000g for 20 min at 4 °C. Supernatants were collected and centrifuged at 48,000g for 1 h at 4 °C. The pellet containing membrane fractions was resuspended in 100 μl of a solution containing 1 M sucrose and 50 mM KCl.Membrane protein enriched fractions were prepared from human heart tissue as previously described . Brieflyg for 10 min at 4 °C. Cell pellets were resuspended in homogenizing solution and mechanically homogenized. Cell homogenates were centrifuged at 4000g for 20 min at 4 °C. Supernatants were collected and centrifuged at 55,000g for 30 min at 4 °C. The pellet containing membrane fractions was resuspended in 100 μl of a solution containing 1 M sucrose and 50 mM KCl.Membrane protein enriched fractions were prepared from transfected HEK cells as previously described . Fourty-Sample total protein concentrations were determined using a Pierce bicinchoninic acid protein assay kit. Thirty micrograms of sample were diluted in 4× Laemmli sample buffer with β-mercaptoethanol at 1:1 ratio, denatured at 90 °C for 5 min, run on a 4 to 15% polyacrylamide gradient gel, and transferred to a polyvinylidene difluoride membrane. The membrane was stained with Revert total protein stain (LI-COR Biosciences) for 5 min to obtain total protein in each lane and then blocked for 1 h at room temperature in Intercept blocking buffer (LI-COR Biosciences) diluted at a 1:1 ratio in PBS with Tween 20 (PBS-T). Blots were incubated overnight at 4 °C with primary antibody diluted 1:1000 in PBS-T: mouse anti-SERCA2 , mouse anti-PLB , or rabbit anti-DWORF  a gift . The blo2 and were at least 40% circular, were automatically scored for Cer, YFP, and FRET fluorescence intensity with a rolling background subtraction using a plugin in Fiji (imagej.net/software/fiji/). FRET efficiency was calculated according to G represents FRET intensity corrected for the bleedthrough of the channels. The parameters a and d are bleedthrough constants calculated as a and d were 0.185 and 0.405, respectively. FRET efficiency for each scored cell was plotted as a function of expressed protein concentration, as determined from the fluorescence intensity of the YFP channel (max [YFP]/(KD+[YFP]), where FRETmax is the maximal FRET efficiency at high protein concentration (representing the intrinsic FRET of the bound complex), [YFP] is inferred from YFP fluorescence emission in each cell, and KD is the apparent dissociation constant .Acceptor sensitization FRET was quantified as previously described . Briefly channel , 28. FRE2, 2 mM EGTA, and 10 mM imidazole, pH 7.0. The base solution was used to characterize SERCA in a ligand-free state, Eapo. The following ligands were used to prepare specific solutions corresponding to SERCA biochemical state: 100 μM TG for E2(TG); 3 mM ATP for E1-ATP; 2.1 mM CaCl2 for E1(Ca2) with free [Ca2+]i = 100 μM  for E1(Ca2-AMPPCP); 2.1 mM CaCl2, 500 μM ADP, 50 μM AlCl3, and 3 mM KF for E1(Ca2–ADP–AlF4−); 0.1 mM orthovanadate for E2(Vi); and 50 μM AlCl3 and 3 mM KF for E2(AlF4−). Concentrations of AMPPCP nucleotide analog and ADP nucleotide were reduced from previously published conditions (3 mM) used to stabilize SERCA in microsomal fractions (KD between buffer conditions using a one-way ANOVA with Tukey’s post hoc test (significance = p < 0.05).To control the conformational poise of SERCA, cells were permeabilized with 0.05 mg/ml saponin in bath solutions, appropriate for stabilization of the transporter in various conformations. Solutions for ligand stabilization of enzymatic intermediate states were prepared by addition of corresponding substrates to a calcium-free base solution, which includes 100 mM KCl, 5 mM MgCl= 100 μM ; 2.1 mM ractions  in order2+, high ATP conditions , a buffer containing 100 mM KCl, 5 mM MgCl2, 2 mM EGTA, 10 mM imidazole, and 3 mM ATP was prepared. The pH was then adjusted to the following concentrations: pH = 6.0, 6.5, 7.0, 7.5, and 8.0. For these experiments, a TagRFP-PLB acceptor construct (pKa = 3.8) was used in place of the YFP-PLB acceptor because YFP fluorescence is sensitive to pH changes in this range (pKa = 6.9)  .2+], solutions were prepared containing potassium aspartate 120 mM, KCl 15 mM, KH2PO4 5 mM, MgCl2 0.75 mM, dextran 2%, ATP 5 mM, Hepes 20 mM, and EGTA 2 mM, pH 7.2. The elevated [Ca2+] buffer was prepared with CaCl2 1.7 mM for a free [Ca2+]i = 2 μM (KD between high and low Ca2+ conditions using a Student’s t test (significance = p < 0.05).For experiments with low and elevated intracellular . FRET ratio data was smoothed using a Savitzky–Golay binomial filter with a 4.08 s averaging window. Changes in FRET ratio and X-Rhod fluorescence associated with Ca2+ uptake were fit to the single-exponential decay function, 2+ release were fit using the single-exponential decay function, x1 is the time in seconds of Ca2+ release, and y1 is the baseline fluorescence prior to x1, defined by the linear function p < 0.05).FRET ratio was determined by dividing the acceptor fluorescence by the donor fluorescence and plotted as a function of time with X-Rhod to indicate concurrent changes in [CaA (kon/koff) was constrained to be twofold higher for Ca-free versus Ca-bound ensembles, as determined experimentally from FRET-binding curves. Likewise, the relative affinity of DWORF for SERCA (kon/koff) was constrained to increase by 25% between Ca-free and Ca-bound ensembles, consistent with FRET measurements. The ordinary differential equation system was numerically solved using the scipy (v1.5.0) SOLV_IVP function, using the experimentally measured intracellular Ca2+ transient as an input to the model. The resulting numerically estimated PLB–SERCA population was compared against the experimentally reported FRET data by assuming that the PLB–SERCA population was proportional to the FRET efficiency. Mean-squared error between the predicted PLB–SERCA population and the FRET data was computed to assess the fit. Fitting was optimized using a genetic algorithm we developed previously (2+ transients with amplitudes and frequencies in place of those measured in HEK cells for fitting purposes. A parameter sensitivity analysis is provided in We implemented systems of ordinary differential equations according to the schematic provided in Figure 4A . This kieviously  that redhttps://github.com/huskeypm/pkh-lab-analyses.All code written in support of this publication are publicly available at This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Career success of women toxicologists requires intentional strategies designed to encourage and support their professional and personal growth. Key among these are mentoring approaches which should be initiated early in their academic careers and continue as their careers progress. While undergraduate and graduate students as well as postdoctoral fellows, women engaged in all STEM fields benefit from one-on-one mentoring experiences offered by both their peers, near-peers and faculty. Here, they not only receive encouragement and lessons on “how to be a good mentee”, but also gain scientific and life skills. Networking opportunities and career planning advice are also important benefits. As woman scientists progress in their careers, they continue to benefit from one-on-one mentoring and structured career development programs adapted to meet their changing needs ultimately culminating in leadership coaching as they reach the pinnacles of their careers. While mentoring success is best facilitated by structured programs that match mentees with mentors and offer training, support and programming, the availability of these programs to women toxicologists is limited. Opportunities for women to participate in structured mentoring programs should be enhanced by institutions, funding agencies and scientific societies as a component of accelerated diversity and inclusion efforts. The success of the next generation of women toxicologists requires an assessment of their future needs and challenges to allow for a correspondingly restructuring of the scientific community to maximize their training and support. The impetus for change arises from the fact that our academic and work environments were founded by men and traditionally best accommodate their needs and work styles . As a coMentoring harbors both relationship and developmental aspects that typically address both career and psychosocial needs . TraditiMentoring often occurs within formal/organized or informal/ad hoc structures . TraditiWhat are aspects of a quality mentoring relationship/a good mentoring match? Efforts to understand the factors that contribute to quality mentoring have often focused on how well the mentor and mentee “mesh” with respect to their personalities, perspectives and expectations. It is important to understand the mentoring relationship as a collaboration wherein both the mentees and mentors engage as “active learners” . PersonaIn addition to mentoring, other forms of support that can play key roles in advancing women toxicologists are coaching and sponsorship. Coaching pertains to support in learning a specific skill and typically involving practical work performed over a short period of time followed by feedback that specifically addresses the performance of the individual . While tAs women progress in their careers, their needs, expectations and benefits with regards to mentorship will undergo corresponding changes. Quality mentoring relationships that appropriately adapt to these changes will benefit not only the mentee, but also the mentor and the scientific community. Mentors receive a number of benefits from the mentoring relationship that include increased productivity, increase in engagement in the workplace and scientific community and a heightened of belonging and professional value. The scientific community benefits from the increased scholarship, creativity and vibrancy. The following sections will focus primarily on mentoring during different stages of women’s academic careers, but many of these concepts can be generalized to other sectors of the workforce.It is well established that undergraduate students who are involved in a mentoring relationship experience better retention in their programs of study, are more satisfied with their choice of academic program and more likely to attain measures of success  . Undergrallies, ambassadors and master-teachers  of their mentees and take a supportive approach towards their working relationship. As ambassadors, faculty introduce their students to their scientific discipline, instill a sense of professional identity and familiarize their students with the types of activities their future careers will entail. As master-teachers, faculty allow students to work relatively independently to demonstrate their research abilities and become expert researchers. Early-career mentees are advised to engage with multiple mentors who are able to provide perspectives from a variety of backgrounds and experiences as well as expertise in specific skills . Thus, our expectations that the status of women toxicologists will continue to improve remain high.The future of women toxicologists is strong as indicated by the relatively high interest of women in obtaining doctoral degrees in toxicology . InteresWhile our understanding of mentoring and how it may be best used to promote the development and growth of individuals at a range of career stages and from diverse backgrounds is growing, the mentoring literature is still dominated by four disciplines; academic medicine, industrial and organizational psychology, education, nursing and psychology . EffectiA few aspects that contribute to unique challenges experienced by women toxicologists should receive greater scrutiny by our professional societies. Early in their academic careers, women toxicologists may face exceptional difficulties in identifying women mentors due to the absence of representation of toxicology within the curriculum of undergraduate-serving communities. In addition, because of the highly interdisciplinary nature of toxicology which spans the full gamut of scientific fields ranging from Earth sciences, biomedical sciences and engineering to social and clinical sciences, potential women mentors are often housed in a variety of academic units making them difficult for a young woman STEM major to locate. Finally, because of the underrepresentation of women at the senior level of faculty ranks within biomedical disciplines of research-intensive universities  the poolWhile women toxicologists have made significant strides with respect to improving the quality and advancement of their careers, significant challenges persist. Mentoring can, in part, address some of these challenges. However, to be effective for enhancing the status of women toxicologists, the needs and potential benefits must be adapted to each woman’s career stage. In many cases, mentoring should be coupled with additional interventions that include coaching and sponsorship and must be valued within the workplace. Scientific communities, funding agencies and academic institutions must work coordinately to provide mentoring opportunities and structures that promote the career advancement of women toxicologists.


Drosophila heart’s ability to adapt to mechanical force. We find Piezo is a sarcoplasmic reticulum (SR)-resident channel and is part of a mechanism that regulates Ca2+ handling in cardiomyocytes in response to mechanical stress. Our data support a simple model in which Drosophila Piezo transduces mechanical force such as stretch into a Ca2+ signal, originating from the SR, that modulates cardiomyocyte contraction. We show that Piezo mutant hearts fail to buffer mechanical stress, have altered Ca2+ handling, become prone to arrhythmias and undergo pathological remodelling.Throughout its lifetime the heart is buffeted continuously by dynamic mechanical forces resulting from contraction of the heart muscle itself and fluctuations in haemodynamic load and pressure. These forces are in flux on a beat-by-beat basis, resulting from changes in posture, physical activity or emotional state, and over longer timescales due to altered physiology (e.g. pregnancy) or as a consequence of ageing or disease (e.g. hypertension). It has been known for over a century of the heart’s ability to sense differences in haemodynamic load and adjust contractile force accordingly , 307–317; Patterson and Starling, J. Physiol., 1914, 48 (5), 357–79; Starling, The law of the heart , 1918). These adaptive behaviours are important for cardiovascular homeostasis, but the mechanism(s) underpinning them are incompletely understood. Here we present evidence that the mechanically-activated ion channel, Piezo, is an important component of the  In most animals over a certain size a heart is necessary to provide the propulsive force for the transport and distribution of materials around the body. The work required of the heart varies in response to bodily demands brought about by changes in activity, emotion, physiology, or by alterations associated with disease. Fluctuations in pressure and haemodynamic load, operating over different timescales require that the heart is able to adapt its performance.2+) within cardiomyocytes, any change to the Ca2+cycle will affect heart performance. Upon cell membrane depolarization, extracellular Ca2+ enters the cardiomyocyte via sarcolemmal L-type voltage-gated Ca2+ channels, activating further Ca2+ release from the sarcoplasmic reticulum (SR) through the ryanodine receptor type 2 (RyR) in a process known as calcium induced calcium release . There is strong evidence that EC coupling is widely conserved in animal hearts, including invertebrate hearts such as Drosophila , and maintained throughout the life of the larva and adult   . Piezo iot ostia .in vivo and the first evidence to show Piezo localises to the SR membrane in cardiomyocytes.To determine Piezo subcellular localisation we used UAS-Piezo:GFP  and a PiPiezoKO hearts function well under normal conditions. End diastolic diameter (EDD), end systolic diameter (ESD), length of the cardiac cycle and beats per minute are not significantly different in PiezoKO hearts when compared to controls , revealing the phenotype to be one of acute response to hypotonic stress rather than one affecting general tissue viability. As the majority of control hearts are able to sustain heart function under conditions of hypotonic stress, whereas the majority of PiezoKO hearts are not, it suggests the existence of an adaptive mechanism to modulate heart function in the face of mechanical stress, and that this mechanism is dependent on Piezo.As mechanically-activated ion channels are suggested to contribute to cardiac adaptive behaviours, such as the Frank-Starling law, we decided to investigate whether Piezo is involved in adapting to fluctuations in mechanical force. To do this we first exposed the hearts to hypotonic stress to induce osmotic swelling, thereby stretching cell membranes and structures anchored to them , and coynchrony . In contc arrest . PiezoPiezoKO but not reaching statistical significance, 35.9% ± 3.5% versus 30.6% ± 2.8% for control and PiezoKO hearts respectively; p = 0.32, n = 12, PiezoKO hearts , whereas control hearts are more contracted under hypotonic conditions relative to the EDD in isotonic conditions   in the conical chamber  . In summith EDTA  and combPiezoKO hearts  or decrease (−64 KPa) in atmospheric pressure for a 10-min period and recording the number/duration of abnormal heart pauses  in 1 minute. . Raisin hearts . A simil hearts .Together, these data provide compelling evidence that Piezo is part of a mechanism that modulates cardiac contractility to buffer the heart against mechanical stress.2+ spark rate in rat ventricular cardiomyocytes  to modulate contractility in response to stretch. This hypothesis is in line with the increase in cytoplasmic Ca2+ in mammalian cardiomyocytes that occurs in response to mechanical stretch, including hypotonic stretch (2+ handling is involved in the Piezo-dependent response to stretch in Drosophila cardiomyocytes we compared systolic Ca2+ transients before (i.e. in isotonic solution) and during hypotonic stress in control and PiezoKO hearts. In control hearts there is a large and significant increase in the peak amplitude of the Ca2+ transient in response to hypotonic stress. The systolic calcium transient amplitude is 67 ± 10%  larger than the pre-hypotonic (isotonic) value. In contrast, there is no change in the level of the Ca2+ transient in response to hypotonic stress in PiezoKO cardiomyocytes. The systolic calcium transient amplitude is 3 ± 4%  smaller compared to the pre-hypotonic (isotonic) value (2+ transients for control (upper) and PiezoKO (lower) cardiomyocytes before and during hypotonic stress. During the change in solution the cardiac cycle pauses. When the cycle resumes, the Ca2+ transients in control cardiomyocytes (now stretched due to hypotonic stress) are significantly higher than they were in isotonic solution. This elevation in the Ca2+ transient is abolished in PiezoKO cardiomyocytes (2+ content ([Ca2+]SR) in PiezoKO cardiomyocytes, as [Ca2+]SR in PiezoKO is significantly higher than control cardiomyocytes  value . Figure myocytes . This ismyocytes . Rather,Piezo affect cardiac structure we compared heart morphology in control and PiezoKO animals by measuring heart length and luminal cross-sectional area  or assuosensors . Indeed,osensors , consistPiezoKO hearts , which is known to block a variety of mechanosensitive ion channels, strongly depressed heart rate in Drosophila  genetic background prior to conducting experiments. w1118 was used as control for all experiments. The Piezo RNAi line used was P{KK101815}VIE-260B. For analysing Piezo expression and localisation the following lines were used: P{Piezo-Gal4.1.0}IIA, P{UAS-Piezo.GFP}IIA-2, P{UAS-Piezo.GFP}IIIA  or Tin-Gal4, P{tinC-Gal4. Δ4} to P{UAS-GsMTx4-FL, P{UAS-GsMTx4-AP or P{UAS-GsMTx4-SR . Experiments on hypertensive flies were carried out at 10 days to allow the haemolymph pressure to build in the body cavity . In some preparations fluorescently labelled Wheat Germ Agglutinin  was used as a counter-stain; in such cases WGA was incubated with live tissue for 20 min at room temperature, followed by fixation in 4% formaldehyde. Heart preparations were mounted in Vectasheild (Vector Laboratories) and imaged on a Nikon A1R FLIM confocal microscope. Maximum intensity projection images were generated using Fiji. In situ hybridisation was carried out using the hybridisation chain reaction v3.0 (HCR v3.0) technique using established protocols (Immunohistochemistry on heart preparations were performed using established protocols . Hearts rotocols  with a p2 and 2 mM CaCl2 (referred to as HBSS below). Briefly, high frame rate (200 fps) movies recording movement of the heart wall were captured on a Zeiss Axioplan microscope with x40 water dipping objective connected to a Prime 95B sCMOS camera (Photometrics) running from Micro-Manager 1.4 software. M-mode kymographs were generated by excising a single pixel horizontal slice from each frame  and aligned horizontally using the “make montage” function in Fiji to provide an edge trace displaying heart wall movement in the y-axis and time along the x-axis.Acquisition and analysis of heart function were based on established protocols from a preparation of semi-intact 3-day old adult flies . Heart pTin-Gal4 was used to drive the genetically encoded Ca2+ sensor UAS-GCaMP6F . The GC2O, whilst maintaining concentration of CaCl2 (2 mM) and MgCl2 (4 mM). Isotonic solution (i.e. HBSS) = 311 mOsm, hypotonic solution = 172 mOsm. For experiments in 2+ transient after hypotonic stretch.Hypotonic solution was made by diluting HBSS 50:50 in dH2 cell culture flask) in which the heart preparation was placed/viewed, connected to either 1) a syringe and one-way valve, to decrease air pressure in the chamber or 2) a bulb pump to increase air pressure in the chamber. Both devices were fitted with a pressure gauge to monitor pressure levels  was added to HBSS. The genetically encoded GsMTx4 strains are described above; these were crossed to either 2+ transients were imaged as described above. Imaging was carried out continuously whilst changing solutions. Heart preparations were imaged for ∼10 s in HBSS to record normal systolic Ca2+ transients, the solution was then replaced rapidly with HBSS +20 mM caffeine to record caffeine-induced Ca2+ transients. Caffeine induces Ca2+ release from the SR, resulting in a caffeine-induced Ca2+ transient larger than the normal systolic Ca2+ transient, which decayed back to resting levels within 10–20 s (2+ transient was estimated by subtracting the peak from the resting level. The obtained value was taken as relative estimate of the SR Ca2+ content between genotypes.Ca 10–20 s . The ampImage analysis was conducted in Fiji and data analysis in Prism. Details of the statistical tests used are provided in figure legends.


Introduction: Safranal, which endows saffron its unique aroma, causes vasodilatation and has a hypotensive effect in animal studies, but the mechanisms of these effects are unknown. In this study, we investigated the mechanisms of safranal vasodilation. Methods: Isolated rat endothelium-intact or -denuded aortic rings were precontracted with phenylephrine and then relaxed with safranal. To further assess the involvement of nitric oxide, prostaglandins, guanylate cyclase, and phospholipase A2 in safranal-induced vasodilation, aortic rings were preincubated with L-NAME, indomethacin, methylene blue, or quinacrine, respectively, then precontracted with phenylephrine, and safranal concentration–response curves were established. To explore the effects of safranal on Ca2+ influx, phenylephrine and CaCl2 concentration–response curves were established in the presence of safranal. Furthermore, the effect of safranal on aortic rings in the presence of ouabain, a Na+-K+ ATPase inhibitor, was studied to explore the contribution of Na+/Ca2+ exchanger to this vasodilation. Results: Safranal caused vasodilation in endothelium-intact and endothelium-denuded aortic rings. The vasodilation was not eliminated by pretreatment with L-NAME, indomethacin, methylene blue, or quinacrine, indicating the lack of a role for NO/cGMP. Safranal significantly inhibited the maximum contractions induced by phenylephrine, or by CaCl2 in Ca2+-free depolarizing buffer. Safranal also relaxed contractions induced by ouabain, but pretreatment with safranal totally abolished the development of ouabain contractions. Discussion/Conclusion: Inhibition of Na+-K+ ATPase by ouabain leads to the accumulation of Na+ intracellularly, forcing the Na+/Ca2+ exchanger to work in reverse mode, thus causing a contraction. Inhibition of the development of this contraction by preincubation with safranal indicates that safranal inhibited the Na+/Ca2+ exchanger. We conclude that safranal vasodilation is mediated by the inhibition of calcium influx from extracellular space through L-type Ca2+ channels and by the inhibition of the Na+/Ca2+ exchanger. Crocus sativus L., commonly known as saffron, is a member of the Iridaceae family. The perennial stemless herb has been used as a spice and as a medicinal plant in Mediterranean countries since ancient times. The major components of saffron include crocin, picrocrocin, and safranal i i . For exar stores . In the safranal D. 2+ metabolism. Therefore, experiments were performed to evaluate the potential antagonistic effect of safranal on phenylephrine- and Ca2+-induced contractions. Increasing the cytosolic Ca2+ level in vascular smooth muscle can be attributed to the activation of voltage-dependent channels, receptor-operated channels, store-operated channels, as well as the Na+/Ca2+ exchanger operating in the reverse mode [−3 M) significantly (p < 0.004) inhibited the contractions induced by the selective adrenoceptor agonist phenylephrine  significantly (p < 0.01) suppressed the contractile responses induced by CaCl2 in calcium-free depolarizing solution  dose-dependently inhibited contractions induced by 60 mM KCl  inhibition of the function of the Na+/ Ca2+ exchanger, which mobilizes Ca2+ into the cell in exchange for Na+ exiting the cell; (iii) efflux of K+ ions out of the cell through Ca2+-activated potassium channels, leading to a relaxation; and (iv) activation of SR Ca2+-ATPase or plasma membrane Ca2+-ATPase. The first possibility gains support from the experiments in which safranal inhibited Phe-induced, Ca2+-induced, and K+-induced contractions. The second possibility gains support from the experiments that showed that incubation of aortic rings with safranal followed by challenge with ouabain prevented these preparations from developing any significant contractions . The fourth possibility cannot be ruled out based on the present experiments, and this is one of the limitations of this study. Another limitation is that inhibition of the Na-K pump by ouabain could raise extracellular K+, as found by other investigators in hippocampal neurons [+ channels. The concentration of ouabain used in the present experiments may not completely inhibit all the Na-K pump population to cause the accumulation of high K+ to cause that effect, since only a 10x increase in ouabain induced a phasic contraction that decayed in about 3 min, and then was followed by a modest relaxation  most of the available Na+/Ca2+ exchanger inhibitors hitherto are lacking selectivity since they interfere with other ionic currents such as L-type Ca2+ current and K+ (IKir) currents. Only recently, some more specific Na+/Ca2+ exchanger inhibitors were reported, and they are in an experimental stage [+/Ca2+ exchanger inhibitor under some pathological conditions where the exchanger, operating in the reverse mode, induces calcium overload in the vasculature or the heart, and hence may increase vasoconstriction or cardiac contractility. (3) Safranal consumption  could interfere with the action of endogenous ouabain function, which has been implicated in hypertension [The findings of this study suggest that safranal inhibited the function of the Naal stage ,44. (2) rtension . (4) Safrtension ,45.2+ channels, but also involved inhibition of the function of the Na+/ Ca2+ exchanger. In light of the lack of specific inhibitors for the Na+/Ca2+ exchanger, the use of safranal under certain pathological conditions, especially when the Na+/Ca2+ exchanger works in the reverse mode, could be assessed.The data presented here show that safranal relaxed rat thoracic aorta by an endothelium-independent mechanism. This vasorelaxation was not affected by the inhibition of nitric oxide synthase, cyclooxygenase, guanylate cyclase, or phospholipase A2. In contrast, the vasorelaxant effect of safranal involved blockade of extracellular calcium influx through voltage-gated Ca