The advantages of targeted therapy have motivated many efforts to find distinguishing features between the molecular cell surface landscapes of diseased and normal cells. Typically, the features have been proteins, lipids or carbohydrates, but other approaches are emerging. In this discussion, we examine the use of cell surface acidity as a feature that can be exploited by using pH-sensitive peptide folding to target agents to diseased cell surfaces or cytoplasms. Targeting therapeutic agents to diseased tissues can significantly enhance their efficacy by reducing side effects. Successful molecular targeting approaches have usually been based on distinguishing the cells in a diseased tissue from those in a healthy tissue by exploiting surface features. The advantages of targeted therapy have motivated searches for distinguishing features between the molecular cell surface landscapes of diseased and normal cells. In this discussion we emphasize an alternative for targeting: cell surface acidity.Traditional approaches for targeting diagnostic and therapeutic molecules to the site of disease are based on recognition of overexpressed receptors or specific antigens localized at the surface or within cellular membranes . HoweverA very different set of targeting approaches might be based on the recognition of differences in other properties of the cellular membranes in normal and diseased tissues. It has been shown that cancer cells are mechanically softer than normal cells, and that the membranes of cancer cells have increased fluidity . FurtherIn addition to the targeting of biomarkers or membrane properties, cells within diseased tissues have pronounced surface acidity resulting from their metabolism. Such acidity can be specifically targeted by pH-triggered membrane-associated peptide folding, which is the subject of this review.in vivo, such as the translocon, which assist in placing transmembrane sequences across the bilayer , arthritis, stroke, ischemia, and others . In the The cytoplasmic production of acidity is deleterious, so cells have mechanisms to regulate their cytoplasmic pH by exporting the acidity to the extracellular environment. pH regulation is carried out by transmembrane proteins that pump protons from the cytoplasm across the plasma membrane to the extracellular space or to the lumen of various organelles . The flupH (Low) Insertion Peptides (pHLIPs) utilize pH-triggered membrane insertion and folding to target acidic diseased tissues. The insertion can be used for the selective delivery of therapeutic and imaging agents . pHLIPs When the extracellular pH is low, key Asp/Glu residues are protonated, and the overall hydrophobicity (LogP) of the peptide is enhanced. Because the dielectric environment affects the protonation and deprotonation rates, the pKas of the carboxyl groups are raised in the environment of the membrane surface , usefullWhile there may be some agreement concerning the overall concepts discussed above, there is an area in which we believe there is an important conceptual misunderstanding found in the literature in the interpretation of protonation events, polypeptide partitioning and folding in a membrane and the targeting and intracellular delivery within a biological system. We assert:•At each pH, an ensemble of all pHLIP states exists. At high and low pHs the predominant, but still not unique, states are the peptide membrane-adsorbed, and membrane-inserted states, respectively, each of which has a variety of dynamic excursions but is approximately described by the two states . At inte•– groups is dependent on the electrostatic potential, which varies dynamically with the ion concentrations, membrane composition and peptide insertion depth, as well as the presence of other groups dictated by the local dynamics of the peptide. Therefore, the pKa of any individual group is constantly changing in both directions while the average of the pHLIP ensemble of states propagates more deeply into the membrane  fluorescent dye widely used for circulatory imaging . ICG-pHLSince the ICG-pHLIP resides in the blood for hours, imaging applications for angiography may also be useful. ICG-pHLIP interacts with blood proteins and remains circulating in the blood, allowing excellent visualization of blood vessels for a period of time of 1–2 h as compared to minutes for ICG alone. Blood flow imaging has wide ranging applications in a variety of surgical procedures , and hasA different, promising imaging agent is QC1-pHLIP , where QAnimal studies have also demonstrated the potential utility of pHLIP linked to paramagnetic and superparamagnetic iron oxide nanoparticles for MRI, chitosan capped mesoporous silica coated gold nanorods and gold nanostars for computed tomography, photoacoustic imaging, and photothermal therapy .Importantly, in addition to the extracellular delivery of imaging agents, a variety of other cargoes, such as immuno-stimulating molecules including proteins (cytokines), carbohydrates, peptides (like HA-peptide), or small molecule antigens  can be specifically targeted to the surfaces of tumor cells to induce biological responses. Specific antigens to activate either endogenous or exogeneous antibodies could be positioned on tumor cell surfaces by pHLIP, bypassing the need to identify molecular biomarkers for targeting. Endogenous antibodies could be developed against the targeted antigen by immunization. Or, exogenous antibodies or antibody drug conjugates (ADCs) could be administrated to target specific antigens delivered to tumor cells by pHLIP.The pHLIP technology can also be used for the targeted intracellular delivery of molecules by connecting them to the inserting end of the peptide by linkers cleavable in cytoplasm, such as S-S links . If the Membrane-associated folding facilitates the cooperative translocation (flipping) of cargoes across the membrane bilayer directly into the cytoplasm, bypassing endocytotic uptake . This pain vivo targeting of miRNAs, long non-cording RNAs, and mRNAs (Peptide nucleic acids (PNAs) belong to such a class of useful, cell-impermeable polar molecules, which can activate immune responses and regulate cell functions by targeting coding and non-coding RNAs and DNA . PNA wasnd mRNAs . An espend mRNAs . Thus, tnd mRNAs .Targeted pHLIP-mediated delivery of moderately hydrophobic small molecule drugs has proven to be successful as well. Among the drugs that have been delivered are potent inhibitors of tubulin, such as monomethyl auristatin E, poly (ADP-ribose) polymerase inhibitors (PARPi’s) including rucaparib and talazoparib, and other molecules . A notabOther classes of potent therapeutic small molecules, such as the corticosteroids widely used in the treatment of severe inflammations and infections, might be targeted to restrict their action to the site of disease. Corticosteroids are very effective drugs that possess immunosuppressive properties. Dexamethasone is an example of a potent steroid with important clinical utility, but systemic administration and associated systemic immunosuppression are associated with devastating side effects, limiting the dose and duration of its uses . These lTo summarize, pHLIP peptides bind to the surface of tumor cells or cells in inflamed tissues, where the acidity is the most pronounced, followed by folding and insertion as helices across the membrane. Using the insertion of pHLIP, cargoes can be located at an acidic cell surface if the cargo is attached to the non-inserting end (extracellular delivery) and/or the cargo can be directly delivered and released into its cytoplasm if the cargo is attached to the membrane-inserting end via a bond that is unstable in the cytoplasm (intracellular delivery). The following advantages are associated with pHLIP targeted delivery:•Cell-surface acidity and pHLIP targeting is not subject to clonal selection.•pHLIP targeting overcomes the problem of antigen or other marker heterogeneity found within the tumor and between tumors.•pHLIP tumor targeting can reach about 20% of the ID/g.•pHLIP binding to a cell membrane is much less saturable than antigen binding, so larger amounts of cargo can be delivered.•pHLIP can provide additional protection and increased stability of a drug in the blood by interaction with the ∼ 4 kDa pHLIP unstructured polymer.•pHLIP alters the pharmacokinetics and biodistribution of drugs.•pHLIP reduces off targeting and toxicity, especially in bone marrow, and can target highly potent molecules to tumors to enhance their therapeutic index.•pHLIP directly flips cargo into the cytoplasm bypassing endosomal trapping.•pHLIP’s cargo can be polar and large, reducing escape from the targeted cell.pHLIP technology is now taking its first steps into human imaging clinical trials, which potentially will open an opportunity for imaging of acidic diseased tissues, improvement of surgical resections of tumors, and visualization of blood flow. The initiation of imaging trials should be closely followed by the first trials of therapeutic interventions, such as the targeted delivery of cytotoxic and immune-stimulating or immune-suppressive molecules.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.YR, OA, and DE are founders and shareholders of pHLIP, Inc. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Considering the pivotal role of Wnt/β-catenin signaling in AML development and persistence, the current study addresses in AML, the prognostic value of Wnt/β-catenin signaling molecules and the anti-leukemic value of Wnt/β-catenin inhibition. In silico analysis of RNAseq data from AML patients and flow cytometric analysis of primary AML samples revealed that higher levels of Wnt/β-catenin pathway is a poor prognostic marker. Next, we found that pharmacological interference, through small molecule inhibitors of Wnt and/or GSK-3 signaling reduces AML cell survival by sensitizing the leukemia cells to chemotherapeutic agents both in vitro and in vivo. Overall, our findings suggested that Wnt-inhibitory therapy could overcome the prognostic significance of patient risk stratification, standing as a therapeutic response for all subgroups of AML.Wnt/β-catenin signaling has been reported in Acute Myeloid leukemia, but little is known about its significance as a prognostic biomarker and drug target. In this study, we first evaluated the correlation between expression levels of Wnt molecules and clinical outcome. Then, we studied—in vitro and in vivo—the anti-leukemic value of combinatorial treatment between Wnt inhibitors and classic anti-leukemia drugs. Higher levels of β-catenin, Ser675-phospho-β-catenin and GSK-3α  were found in AML cells from intermediate or poor risk patients; nevertheless, patients presenting high activity of Wnt/β-catenin displayed shorter progression-free survival (PFS) according to univariate analysis. In vitro, many pharmacological inhibitors of Wnt signalling, i.e., LRP6 (Niclosamide), GSK-3 , and TCF/LEF (PNU-74654) but not Porcupine (IWP-2), significantly reduced proliferation and improved the drug sensitivity of AML cells cultured alone or in the presence of bone marrow stromal cells. In vivo, PNU-74654, Niclosamide and LiCl administration significantly reduced the bone marrow leukemic burden acting synergistically with Ara-C, thus improving mouse survival. Overall, our study demonstrates the antileukemic role of Wnt/β-catenin inhibition that may represent a potential new therapeutics strategy in AML. Acute myeloid leukemia (AML) is the most common acute leukemia in adults; relapse after chemotherapy still represents a critical problem in most patients, with only a 35–40% five-year overall survival rate for non-promyelocytic AML ,2. NeverWnt/β-catenin signaling is a developmental pathway mostly involved in tissue patterning during embryonic development, through its ability to modulate proliferation, differentiation and motility of embryonic and adult stem cells . The patIn AML, a number of studies support the contribution of the canonical Wnt signaling in the maintenance of leukemia stem cells (LSCs) . Indeed,In this work, we demonstrate that pharmacological interference, through small molecules inhibitors of Wnt and/or GSK-3 signaling reduces AML cell survival by sensitizing the leukemia cells to chemotherapeutic agents both in vitro and in vivo. Consequently, combination treatments with small molecules inhibitors of Wnt/GSK3 axis and chemotherapy may represent a novel therapeutic strategy for a better chance of AML cell eradication.® AQueous One Solution Cell Proliferation Assay  was from Promega . Cytarabine (Ara-C) and Idarubicin (Ida) were provided by the Pharmacy Unit of the University Hospital of Verona.The antibodies used for blast cell identification by Flow Cytometry  were: anti-CD45-VioBlue, anti-CD45-APC-Vio770, anti-CD34-PerCP and anti-CD117-APC all from Miltenyi Biotech . For Western blot analysis, anti-β-catenin, anti- Ser675-phospho-β-catenin, anti-Ser33/37/Thr41-phospho-β-catenin, anti-non-phopho-β-catenin, anti-GSK-3β, anti-Ser9-phospho-GSK-3β, anti-GSK-3α, anti-Ser21-phospho-GSK-3α anti-Histone H3 antibodies and Alexa 488-conjugated secondary antibodies were from Cell Signaling ; anti-GAPDH, and HRP-conjugated secondary antibodies against mouse or rabbit were from Sigma-Aldrich . Wnt modulators used for proliferation and vitality assays, i.e., Wnt-3a, PNU-74654, Niclosamide, IWP-2, Lithium Chloride (LiCl), and AR-A014418, were all purchased from Sigma-Aldrich. For the analysis of cell death, Propidium iodide (PI) and FITC-conjugated Annexin V were from Miltenyi Biotechnology. CellTiter 96+ using MACS CD34 Microbead Kit (MiltenyiBiotec). AML patients characteristics have been described elsewhere [n = 12) and AML patients  after informed consent as previously described [All cell samples were collected from AML patients and healthy donors after written informed consent, as approved by the Ethics Committee of Azienda Ospedaliera Universitaria Integrata Verona . In detail, AML blast cells were obtained from bone marrow or peripheral blood samples of patients with AML at diagnosis (>80% of leukemia cells). Samples with less than 80% of blast cells were enriched with CD34lsewhere  and summescribed ,18. Humaescribed . Cell liImmunoblotting were performed as previously described . BrieflyThe IC50 for each drug were obtained by analyzing treated cells with the colorimetric One Solution Cell Proliferation Assay (MTS), as previously described ,20. Cellp ≤ 0.05) using a one-sample t-test power calculation. With a commitment to providing refinement, reduction and replacement (3Rs), application of factorial design to reduce the minimum group size was applied to obtain a minimum group size of 5–8 mice. Xenograft mouse model were generated in NOD/Shi-scid/IL-2Rγnull  mice as previously described [6) were injected into the tail vein of totally irradiated , 8–12-week-old male mice. At day 9 post-injection, mice  received for 5 days intraperitoneal daily injection of Ara-C (25 mg/Kg) or its vehicle . In case of combined treatment, Ara-C or DMSO were associated with each Wnt/GSK-3 inhibitor for the first two days, followed by three days of Ara-C or DMSO. Mice were sacrificed after 2 weeks following the cell line injection, and bone marrow leukemic burden was evaluated as percentage of human hCD45+ cells. No blinding was done. Wnt inhibitors used for mouse experiments were PNU-74654 (0.5 mg/kg), Niclosamide (10 mg/Kg) and LiCl (25 mg/Kg).Animal care was performed in accordance with institution guidelines as approved by the Italian ministry of health. Mice were purchased from Taconic . Animal experiments were carried in pathogen-free conditions at the animal facility of the Interdepartmental Centre of Experimental Research Service of the University of Verona. Parameters used for sample size are power of 80%, a signal/noise ratio of 2 and a significance level of 5%  and cell death .To study the capability of hBM-MSCs to support AML cell survival, AML cells were cultured in suspension or on a confluent monolayer of hBM-MSCs with complete RPMI 1640 in 96-well plates: 10AML cell lines or AML blast cells identified as CD45+, CD34+, CD38- cells by flow cytometry, were fixed and permeabilized for 30 min at 4 °C. Permeabilized cells were probed with primary antibodies or their specific isotype for 1 h. Subsequently cells were washed and labelled for 30 min with Alexa 488-conjugated secondary antibodies. Protein expression was then analyzed through flow cytometry and expressed as relative median of fluorescence intensity (rMFI), defined as the ratio of the specific antibody fluorescence over the specific isotype fluorescence.To monitor Wnt/β-catenin transcriptional activity, we transfected THP1 with reporter plasmids encoding for an inducible TCF responsive GFP reporter  as previously described  GFP signp < 0.05. This strategy was applied for the gene of the WNT signaling pathways .RNA-seq data from 173 AML patients were obtained as part of The Cancer Genome Atlas project (TCGA). Gene expression data in Z-score format were downloaded from the cbioportal R package cgdsr for the TCGA-LAML. Normalized data were used for Z-score calculation. For each mRNA, a sample showing a Z-score higher or lower than the average Z-score for the whole population was considered as expressing highly or lowly, respectively, the corresponding gene, Z > +/− 1.96; Statistical analysis was performed using GraphPad Prism software . Mann–Whitney and Kruskal–Wallis were used to compare two groups or more than two groups, respectively. All tests were one-sided. Pearson Chi-square analysis was used to test association among variables. Survival curves were calculated by the Kaplan–Meier Method.We first evaluated in three AML cell lines, HL-60, THP1 and U937, the basal expression and activation of the Wnt molecules, including total β-catenin, pan-phosphorylated β-catenin(Ser33–37/Thr41), Ser675-phospho-β-catenin, active non-phospho-β-catenin, GSK-3β  and GSK-3α . These proteins were expressed in all the three cell lines . WesternAXIN, APC, CTNNB1 (BETA-CATENIN), GSK-3A and GSK-3B  project on AML . We obsed GSK-3B . We thend GSK-3B A. Howeved GSK-3B B. Pearsod GSK-3B A. A posid GSK-3B B. The Eud GSK-3B . All then = 12) and AML-hBM-MSCs  samples expressed Wnt components, suggesting a possible paracrine signal between hBM-MSCs and leukemic cells. The presence of active forms of β-catenin, including non-phospho-β-catenin and Ser675-phospho-β-catenin [Considering the importance of bone marrow microenvironment in AML onset and recurrence, ex-vivo co-culture of leukemic cells on bone marrow stromal monolayer represents a good tool for evaluating drug sensitivity ,24. Bone-catenin , revealeWe assessed whether Wnt inhibitors could increase AML cell sensitivity to drugs normally used for AML therapy, such as Idarubicin or Ara-C. AML cells were cultured alone or in the presence of hBM-MSCs for 48 h, with or without Wnt modulators and chemotherapeutic agents. Cells were harvested, stained with annexin and analyzed for apoptosis. Idarubicin (0.5 µM) or Ara-C (10 µM) significantly induced the apoptosis of both AML cell lines and AML primary cells, while co-culture with hBM-MSCs led to a significant rescue effect . The addTo investigate which microenvironmental prosurvival protein network was affected by Wnt pharmacological inhibition, we first analyzed how the Wnt-GSK axis was modulated in AML cell lines treated with above mentioned inhibitors, observing that the pattern of modulation was cell line-dependent, probably reflecting differential modulation of the pathway according to molecular features of the AML cell/samples . BrieflyTo assess whether he anticancer effect of Wnt and GSK-3 inhibitors could be operational in vivo, we generated a xenograft model of AML by injecting the U937 cell line in the tail vein of NOG mice, as previously described . Two weeTargeted treatments using small molecule inhibitors are extensively studied as promising strategies to eradicate drug resistance in cancer and in AML . In thisThe rationale for using pharmacological inhibitors of Wnt signaling as anti-leukemic agents is the evidence of aberrant activation of Wnt pathway in AML samples ,29. ThroWnt/β-catenin signaling is a multiple step cascade. The importance of each step for cancer development is disease- and cell context-dependent ,41. The Our data show that small molecule inhibitors of Wnt or GSK-3 substantially enhance the antitumor effect of Ara-c and Idarubicin in vitro and reduce AML cell survival advantage, thus lowering the leukemic burden in the bone marrow of AML mouse models. The mechanisms involved in Ara-C or Idarubicin-induced cell death include the modulation of many pathways, such as mTor/Akt, Erk, NF-KB, stat3, Bax/Bak etc ,43,44,45FLT3 [The main molecular events related to Wnt pathway activation in AML are both chromosomal translocations and/or mutations of FLT3 . EnhanceFLT3 . HoweverFLT3 ,46. We oFLT3 ,48,49. SFLT3 . Our co-FLT3 . Thus, WOverall, our study demonstrates that because of their capabilities to interfere with AML cell growth and chemoresistance, small molecule inhibitors of Wnt and GSK-3 signaling may represent potential candidates for drug development in AML.


Drug repurposing is a method of drug discovery that consists of finding a new therapeutic context for an old drug. Compound identification arises from screening of large libraries of active compounds, through interrogating databases of cell line gene expression response upon treatment or by merging several types of information concerning disease–drug relationships. Although, there is a general consensus on the potential and advantages of this drug discovery modality, at the practical level to-date no non-anti-cancer repurposed compounds have been introduced into standard acute myeloid leukaemia (AML) management, albeit that preclinical validation yielded several candidates. The review presents the state-of-the-art drug repurposing approach in AML and poses the question of what has to be done in order to take a full advantage of it, both at the stage of screening design and later when progressing from the preclinical to the clinical phases of drug development. We argue that improvements are needed to model and read-out systems as well as to screening technologies, but also to more funding and trust in drug repurposing strategies. Drug repurposing, or its variant-drug repositioning, is an attractive drug discovery approach whereby the exploitation of an already-approved or investigational drug for a new therapeutic indication is attempted ,2,3,4. STo date, most of the successfully repurposed drugs were identified serendipitously through the analysis of their side effects. However, drug repurposing requires systematic, high-throughput and rational methods . Novel mThe most important advantage of repurposing is the reduction of time and costs necessary to deliver compounds to patient’s bedside. The development of a new drug can take 10–17 years at an estimated cost of 2 or 3 billion dollars from research to marketing approval. The reported timeframe for repurposing, instead, equals approximately six years with a clear positive impact on costs . RepurpoThere are hurdles and limitations specific to re-tasking projects. The drug to be repurposed often acts through a new mechanism of action, on new molecular targets or both , therefoThe review presents the state-of-the-art drug repurposing approach in acute myeloid leukaemia (AML), analyses development times and poses the question of how to exploit this approach, both at the stage of screening design and later when progressing from the preclinical to the clinical phases of drug discovery.AML is a heterogeneous group of clonal haematopoietic malignancies underlain by a failure to differentiate and over-proliferation in the stem cell/progenitor compartment producing non-functional blasts . AML’s hDrug repurposing can be divided into ”soft repurposing” (otherwise known as repositioning) and ”hard repurposing” . The forDrug repositioning seems intuitive and meets less resistance than hard repurposing. Soft repurposing in AML concerns clofarabine, cladribine, actinomycin D, azacytidine, melphalan, hydroxyurea and arsenic trioxide.NPM1 mutation with actinomycin D, an antibiotic approved for the treatment of rare cancers, led to a complete remission (CR) lasting 14 months. Future studies are necessary to assess the efficiency of actinomycin D in different AML subtypes [FLT3 mutation seemed to be more sensitive to glucocorticoids than cytarabine-responsive cells [RUNX1-ETO fusion formation, and strong synergy when combined with chemotherapeutics [Clofarabine, a purine nucleoside antimetabolite structurally related to cladribine, is a case of soft repurposing as it was initially approved by the FDA for the treatment of children with relapsed or refractory acute lymphoblastic leukaemia (ALL) . In the subtypes . Azacytisubtypes . Melphalsubtypes . Successsubtypes . Hydroxysubtypes . Arsenicsubtypes ,39,40. Psubtypes . Two recsubtypes ,43. Cytave cells . AdditioInterestingly, salinomycin treatment of primary mouse leukaemia cells resulted in loss of leukaemia repopulation ability following transplantation . FinallyThe chief example of hard repurposing in the field of haematology is thalidomide, a compound developed to alleviate morning sickness during pregnancy, withdrawn from the market due to its teratogenic properties and now approved for the treatment of multiple myeloma . In AML,Metformin, chloroquine and hydroxychloroquine have been initially repurposed in solid tumours. Statins , valproic acid (VPA) anti-convulsion medicine, sertraline and tranylcypromine derivative antidepressants, pioglitazone metabolic regulator, ribavirin and AMD3100 antiviral agents were found to specifically inhibit molecular processes important for the growth of leukaemia. There is also epidemiological evidence of lower cancer incidence in patients under chronical medication in the case of some of the agents.AML1-ETO, MN1, MLL-AF9, HOX9A-Meis1) or a leukaemic niche-like system. One study screened the LOPAC compounds against four primary AML samples and peripheral blood mononuclear cells from a healthy donor. Neoplastic and normal human pluripotent stem cells were used in a screen to identify differentiating agents within a custom library of 590 compounds.In vitro and in silico screening projects pinpointed 19 compounds to be repurposed in AML. Nine large-scale and two small-scale chemical screens have been reported . The larThree groups approached drug repurposing in AML from the in silico angle taking advantage of the Connectivity Map  or 2D structure similarity analysis; the most recent of the three paired a cmap (Built 2.0) query to select compounds that kill LSCs whilst sparing normal haematopoietic counterparts to balance between healthy and leukaemic stem cells with an in vitro secondary screen. Not all studies reported the number (or percentage) of hits identified and only three published their shortlists. Hits comprised 1.6 to 11.5% of agents present in the library analysed. Clearly, this datum depends on experimental system and cut-offs applied during analysis. All projects stated that known anti-cancer and anti-leukaemia drugs made it onto the hit shortlist validating the specific approaches and model systems. The 25 hits listed by Erikson et al were iodoacetamide, NSC95397, brefeldin A, tetraethylthiuram disulphide, ouabain, idarubicin, ammonium pyrrolidinedithiocarbamate, mitoxantrone, quinacrine dihydrochloride, CGP-74514A, parthenolide, 2--ethyltrimethylammonium iodide, emetine dihydrochloride hydrate, 2-chloroadenosine, L-703,606 oxalate, oligomycin A, VER-3323, podophyllotoxin, Bay 11-7085, colchicine, U-73122, cantharidin, diphenyleneiodonium chloride, wortmannin and spermidine trihydrochloride .All hits require careful validation in order to become promising candidates. Thirty-two compounds including antimicrobials (16), neuropsychiatric (6), metabolic (6) and antiarrhythmic (4) agents underwent preclinical validation in AML confirming the potential benefit for AML treatment .There are six anthelmintic compounds with repurposing potential. Albendazole and mebendazole were identified through a chemical screen of selected compounds ,64. BothAntiprotozoal  compounds turned out to be a rich source of promising candidates. Screening over a thousand compounds from the LOPAC library (Sigma Aldrich) led to the identification of quinacrine . It showed low toxicity in normal mononuclear cells and synergistic effect with cytarabine or azacytidine, in both in vitro and in vivo studies, resulting in a decrease of total number of circulating blast cells and increase in mice survival . There iThe list of antimicrobials with repurposing potential also includes tigecycline, an on-patent antibacterial agent, used for the treatment of intra-abdominal or skin infections that was found in a chemical screen of ~300 compounds. Similarly to clioquinol and ivermectin, tigecycline selectively killed leukaemia cells and decreased tumour growth in a xenograft mouse model by blocking mitochondrial protein synthesis . RepurpoAnti-proliferative potential of psychotropic agents was known for over 20 years . Six canFive agents that target cell metabolism are candidates for AML treatment. Amongst oral antidiabetics, application for metformin was found in oncology. Meta-analysis of 561,836 patients from 18 studies showed that metformin use was associated with a 27% reduction in any cancer risk . MetformAmiodarone, digitoxin, ouabain and proscillaridin A are medications for heart conditions and are under evaluation as anti-leukaemia agents. Amiodarone is a potassium channel blocker approved to treat life-threatening ventricular arrhythmias. In vitro studies showed that the combination of amiodarone with ABT-263, an inhibitor of anti-apoptotic BCL2-like protein, increased apoptosis in human leukaemia cell lines . DigitoxIn summary, of 32 compounds considered for repurposing in AML, 27 were successfully validated in the preclinical setting aimed at leukaemia elimination. A different aspect of AML treatment was addressed by another repurposing study. Anti-AML chemotherapy affects rapidly cycling normal multipotent and lineage-committed progenitors causing anaemia, neutropoenia and thrombocytopaenia. Quizartinib, an FLT3 inhibitor frequently mutated in AML, induces the quiescence of normal murine multipotent progenitors conferring a significant protection to mice treated with chemotherapy without affecting the elimination of FLT3-independent tumours by cytotoxic drugs .clinicaltrials.gov database, the main source of information for this section of the review .Five antimicrobials entered into the clinical trial phase. The tolerance and biological activity of oral clioquinol were evaluated in 11 patients with relapsed or refractory haematological malignancy is reported (NCT00963495). Although safe up to a dose of 1600 mg twice-daily, no useful clinical responses were observed, probably, due to low intracellular concentration of the drug resulting in a minimal proteasome inhibition . UniversThe safety of tigecycline was evaluated via an open-label phase I clinical trial in 27 patients (median age 70) with relapsed and refractory AML comprising seven patients with poor risk cytogenetics (NCT01332786). Analogously to clioquinol, the trial demonstrated a favourable safety profile of tigecycline at doses of 300 mg/day and lack of response to therapy leading to the closure of the trial. The inefficacy of tigecycline may be due to its short half-life .clinicaltrials.gov; data accessed: 14 March 2020). One more ongoing trial will assess the efficacy of combining decitabine, a drug used in patients with MDS, with rapamycin or ribavirin in high risk AML patients (NCT02109744); a recruiting status is reported (data accessed: 14 March 2020).Ribavirin has undergone four clinical trials: two completed, one with unknown status and one still recruiting. The first phase II clinical trial (NCT00559091) to assess ribavirin’s efficacy against AML M4 and M5, which enrolled 13 patients, started in 2007, three years after the emergence of preclinical evidence for ribavirin activity against AML . CRs andclinicaltrials.gov. The first phase I/II clinical trial (NCT00512252) to evaluate the safety and efficacy of plerixafor in combination with mitoxantrone, etoposide and cytarabine (MEC), started in 2007 and demonstrated that plerixafor was safe at a dose of 0.24 mg/kg/day; 21/56 patients (median age 52) achieved CR and complete remission with incomplete blood count recovery (CRi) with a median OS of 8.2 months and relapse-free survival (RFS) of 9.0 months. Plerixafor mobilised leukemic blasts into circulation abrogating protective signalling from the niche [Plerixafor in combination with different agents underwent nine clinical trials for the treatment of AML patients as reported at he niche . The samhe niche . The thihe niche . In the he niche . Finallyhe niche .clinicaltrials.gov database, of which nine are completed. VPA has been tested in combination with decitabine, ATRA, theophylline, cytarabine and 5-azacytidine. In 2001, the first in vitro evidence of anticancer activity of VPA emerged [Twelve clinical trials of VPA are registered in the  emerged . The fir emerged . Later t emerged . Two yea emerged , prompte emerged . By cont emerged . The com emerged . A rando emerged . No clin emerged . The res emerged . Currentclinicaltrials.gov, data accessed: 14 March 2020).A trial to assess the safety of thioridazine in combination with cytarabine in relapsed or refractory AML or patients in advanced-stage MDS (NCT02096289) recruited 13 patients (median age >65 years). Leukaemic burden decreased in 8/11 patients during initial thioridazine treatment and continued to decline when cytarabine was added. Blasts increased when thioridazine was stopped, suggesting that chronic administration is required for their suppression; however, thioridazine intake is limited by its cardiotoxicity and neurological events. Thus, modifications are needed to improve tolerability . CurrentMetformin’s efficacy against AML cells was established in 2010 by Scotland and colleagues . Five yeSeveral trials were carried out to assess the potential of statins in AML treatment. Ten years since the first in vitro evidence of pravastatin utility against AML, Kornblau et al hypothesised that the pravastatin added to standard therapy may block adaptive cholesterol responses in AML blasts improving therapeutic efficacy and put this theory to a phase I trial. The combination was safe and efficient: 11/15 newly diagnosed AML patients including those with poor cytogenetics and 9/22 salvage patients achieved CR or CRp . The SWOA trial of digoxin (and not digitoxin) combined with decitabine for the treatment of AML and MDS was started in 2017 and then terminated due to slow accrual (NCT03113071). The remaining candidate compounds never entered into clinical trials.None of the candidate compounds have so-far obtained FDA or EMA approval and, therefore, have not been introduced into AML treatment . The cliThe main issue encountered was the lack of efficacy stalling the agents in phase I and II clinical trials. Clioquinol, tigecycline, ribavirin, VPA in some combinations elicited no, little or transient responses. Ribavirin as monotherapy showed an initial short-term response; it worked better in combination with cytarabine, but the latter altered its absorption diminishing the activity ,127. DivToxicity of the repurposed drugs is an aspect not to be underrated. The combination of VPA and decitabine is associated with marked and unacceptable VPA-dependent encephalopathy. Pravastatin together with idarubicin and cytarabine resulted in multi-organ failure. Additionally, pravastatin with etoposide, mitoxantrone and cyclosporine and thioridazine combined with cytarabine had adverse effects ,143.Last but not least, slow accrual led to a premature closure of six clinical trials , metformin, lovastatin and digoxin); maybe extra funding is necessary for trial success.When reviewing drug repurposing in cancer, one must not forget to mention the Repurposing Drugs in Oncology (ReDO) project. It draws attention to the new potential oncological uses of well-known and well-defined non-cancer drugs through an accurate analysis of the literature on the premise that most drugs act on more than one target within a cell. The ReDO project identified 310 non-cancer agents with “hard” repurposing potential supported by preclinical and clinical data. Most of them (85%) were off-patent compounds and at least 67% had data on patients such as case reports, observational studies or clinical trials supporting their anticancer effects (ReDo database accessed: 7 March 2020) ,145,146.Dogma 1: Drug repurposing saves timeIs the drug repurposing strategy delivering new therapeutic options in AML or is it not? Every scientific publication concerning the re-tasking approach lists a number of statements that have become dogmas, but are they incontrovertibly true?Dogma 2: Phase I clinical trials can be skipped.It took a median of 4.8 (0–13) years from the initial report of compounds’ validation as potential anti-AML agents to their entry into a clinical trial  with tigEight out of fifteen initial clinical trials were phase I trials, as agents were administered at higher doses than for their original therapeutic indication, to assess the safety of combinations even at lower doses than standard dosing or because of a novel route of administration .New safety evaluation as primary trial objective was needed to establish dose-limiting toxicity (DLT) and the maximum tolerated dose (MTD) of oral clioquinol that is normally used topically.Doses different to the original are often required to target new molecular pathways of repurposed agents and their safety has to be re-ascertained. In AML, hydroxychloroquine was given for 21 days at the dose of 600–1400 mg/day against a total of 2000 mg divided over 48 h in acute malaria or 400 mg per week in malaria prevention. Tigecycline required re-testing because doses up to seven times higher than the standard dose of 50 mg/day were given in 3-week cycles compared to its 14-day cycle as antibiotic. The doses of sertraline and pioglitazone were slightly higher than standard . Pravastatin and lovastatin, instead, were used at significantly higher doses (up to 1680 mg/day) than when used in hypercholesterolaemia and similar therapeutic indications (up to 80 mg/day).Dogma 3: Repurposed drugs are safe as their toxicity profile is knownPhase I clinical trials were performed for the combinations involving hydroxychloroquine, plerixafor, thioridazine, sertraline, metformin, pravastatin and atorvastatin. The trial to assess the use of metformin to treat AML specifically sought to determine DLT and MTD of metformin in combination with cytarabine as a primary objective in patients with relapsed/refractory AML; metformin was administrated at its standard dose for the treatment of diabetes type II (standard dosage taken from drugs.com database).Three out of fifteen (20%) trials were stopped because of toxicity.Does the promise hold? Hardly. A very recent review on drug repurposing in AML asked if it were a futile pursuit and responded by saying that even a failed attempt furthers our knowledge of AML biology .What needs to be done to increase the chances of success? One can think of improvements at the level of screening and preclinical validation to identify more robust candidates. Thus, we need better tools to screen. Numerosity of compounds in libraries has brought about the demand for increased throughput leading to the miniaturisation of screening formats and the introduction of droplet-based-, continuous-flow-, digital- and paper-microfluidics . ThThe limited scale and number of studies suggests that money is a real problem when repurposing off-patent compounds. More small clinical trials should be conducted as they are less expensive and quicker. Not-for-profit organization such as universities, hospitals, foundations, research institutes and governments could fund these trials without a contribution from pharmaceutical industry. Recently, the Orphan Drug Act, incentivised rules have been proposed to overcome all these issues for manufacturers licensing generic drugs ,178. AMLIt transpires from the analysis conducted that there may be a kind of distrust towards the candidates to be repurposed and, as a consequence, the trials fail to accrue sufficient numbers. In one case, the move of the principal investigator was a reason for the trial’s closure (NCT00583102). The wariness of new therapeutic options emerging from repurposing may be caused by the full awareness of the clinicians involved that AML is a serious neoplastic disease requiring an aggressive and effective treatment and, therefore, hardly anybody is prepared to try something “new”, but not entirely new. The disease progresses too fast to afford the luxury of calmly testing a potential candidate and switching back to something else should the drug fail to work. The haematologists probably do not want to renounce the “certainties” of standard therapy and exchange them for the unknown efficiency of novel options. Clearly, de novo drug discovery gives no guarantees of success either, but studies are sponsored by the big pharma and are somehow more accepted by clinicians and patients alike.Taken together, it is hard to be enthusiastic about drug repurposing in AML. It identified a host of candidates for the treatment of AML through diverse wet and in silico approaches from antimicrobial, neuropsychiatric, metabolic and cardiac medication categories. Although most candidates belonged to antimicrobials, all but one was unsuccessful in clinical setting. The results obtained for plerixafor with decitabine or plus fludarabine, idarubicin, cytarabine and G-CSF, VPA with 5-azacytidine and pioglitazone with low-dose 5-azacytidine or with standard treatment are the most promising, but the bottom line is that no “old” drug has so far been effectively repurposed and none is close to being approved. The development times have been long and clinical trials were troubled by lack of efficacy, toxicity and slow accrual.Traditionally, anti-AML treatment was based on a relatively small number of molecules with anti-proliferative potential used in all diagnoses. The complexity of molecular pathways deregulated or highjacked by leukaemia and the heterogeneity of disease phenotypes call for specific agents that will target vulnerabilities of individual diseases. Compound libraries are a source of infinite therapeutic options that should be continuously explored although there is no easy way to go about it.


In mammals, there are three unique NCX encoding genes—NCX1, NCX2, and NCX3, that comprise the SLC8A family, and mRNA from all three exchangers is expressed in hippocampal pyramidal cells. Furthermore, mutant ncx2-/- and ncx3-/- mice have each been shown to exhibit altered long-term potentiation (LTP) in the hippocampal CA1 region due to delayed Ca2+ clearance after depolarization that alters synaptic transmission. In addition to the role of NCX at the synapse of hippocampal subfields required for LTP, the three NCX isoforms have also been shown to localize to the dendrite of hippocampal pyramidal cells. In the case of NCX1, it has been shown to localize throughout the basal and apical dendrite of CA1 neurons where it helps compartmentalize Ca2+ between dendritic shafts and spines. Given the role for NCX and calcium in synaptic plasticity, the capacity of NCX splice-forms to influence backpropagating action potentials has clear consequences for the induction of spike-timing dependent synaptic plasticity (STDP). To explore this, we examined the effect of NCX localization, density, and allosteric activation on forward and back propagating signals and, next employed a STDP paradigm to monitor the effect of NCX on plasticity using back propagating action potentials paired with EPSPs. From our simulation studies we identified a role for the sodium calcium exchange current in normalizing STDP, and demonstrate that NCX is required at the postsynaptic site for this response. We also screened other mechanisms in our model and identified a role for the Ca2+ activated K+ current at the postsynapse in producing STDP responses. Together, our data reveal opposing roles for the Na+/Ca2+ exchanger current and the Ca2+ activated K+ current in setting STDP.Sodium Calcium exchanger (NCX) proteins utilize the electrochemical gradient of Na NCX can extrude Ca2+ at a rate far higher than the plasma membrane Ca2+ ATPase pump (kcat 30–250 s−1) o and [Ca2+]o respectively, F is Faraday’s constant, T is temperature (in Kelvin), and R is the molar gas constant. Intracellular calcium and sodium concentrations were 50nM and 10mM respectively. Extracellular calcium and sodium concentrations are 2mM and 140mM respectively. See Weber et al. , and [Ca]i is the intracellular calcium concentration. The Ca2+-dependent functions are described  and pandas (ver. 0.23.4). Plots were generated using matplotlib (ver. 2.2.3), and heatmaps were rendered using PySpike (ver. 0.6). Spike Sync [spike_sync_matrix function from PySpike [SciPy stats (ver. 1.3.0) was used to test array like samples where p = 0.05. The Tukey HSD test from the StatsModels Python module (ver. 0.9.0) was used to compare all pairwise comparisons with TukeyHSD confidence intervals at α = 0.05.Data were processed using ike Sync ,21 heatm PySpike , which cdifflib module); mean firing rate  for each cell was: cell 1 = 15 s-1; cell 2 = 19.3 s-1; cell 3 = 15.2 s-1; cell 4 = 15.9 s-1; cell 5 = 14.6 s-1; cell 6 = 14.4 s-1; and cell 7 = 15.07 s-1. Taken together, we show that our model can simulate realistic activation patterns in response to gamma frequency stimulation.Using a morphologically realistic model from a CA1 pyramidal cell  we simul+/Ca2+ exchange current in dendritic compartments, during forward signaling, we programmatically divided the dendrite into oblique, tuft, and primary regions  ncx oblique–Na+/Ca2+ exchange current model localized to the oblique dendrite; 3) ncx primary–Na+/Ca2+ exchange current model localized to the primary dendrite; and 4) ncx tuft–Na+/Ca2+ exchange current localized to the dendrite tuft. From these data we observed no significant difference in spike-width based on NCX localization using both Mann Whitney U (α = 0.05) and Tukey HSD (FWER = 0.05) tests  tests . Further5) tests  and math5) tests .+/Ca2+ exchange current on back propagating action potentials in CA1, we simulated a depolarizing current to the soma and recorded the resulting voltage change throughout the apical dendrite tree  on peak voltage in the oblique region . Next, we performed a similar set of experiments but this time instead of recording the absolute peak amplitude, we recorded the depolarization time (ms) for each compartment within the oblique , and implemented a similar linear model for Na+/Ca2+ exchange current density also (termed linear). Visual depictions of these linear models are shown in To study the effect of the Naite tree . A repree region , primarye region , and tufe region . From th oblique , primary oblique , and tuf oblique  when ther et al. . The mod+/Ca2+ exchange current may contribute to this potentiation. For this analysis we compared the potentiation response across four different scenarios: 1) when NCX was removed (ncx-/-); 2) when NCX localized only to the primary dendrite (ncxprim); 3) when NCX localized only to the oblique localization (ncxobq); and 4) when NCX localized only to the tuft dendrite (ncxtuft). From these simulations we observed a significantly increased potentiation response when the Na+/Ca2+ exchange current was removed as compared with wildtype (-/- versus wt: p< 0.005). A similar increase in potentiation was observed when NCX was localized to the apical tuft dendrite and to a lesser extent when NCX was localized to the apical oblique dendrite regions . Interestingly, when NCX was localized to the primary dendrite region, which is also the site of synaptic input, the potentiation was reduced to near normal levels (prim versus wt: p > 0.05). Inward sodium current during back propagating action potentials triggered reverse mode NCX activity ; 2) Na+/Ca2+ exchange current linear density model in the primary dendrite (ncxdensity(LI)); 3) Na+/Ca2+ exchange current linear allostery model in the primary dendrite ); and 4) Na+/Ca2+ exchange current linear density and linear allosteric models in the primary dendrite ). No significant differences were observed in the potentiation responses across these four scenarios, suggesting that, at least in our simulations, the density and allostery does not scale with STDP responses  significantly reduced the potentiation response (-/- versus wt: p < 0.05). By inserting the Ca2+ sensitive K+ current into the primary dendrite region, normal potentiation was restored . Inserting the Ca2+ sensitive K+ current into the oblique or tuft dendrite regions did not increase potentiation to the same level as when inserted into the primary dendrite region . This suggests that similar to the Na+/Ca2+ exchange current, the Ca2+ sensitive K+ current likely functions primarily at the postsynaptic site to influence STDP responses. To explore the relationship between the Na+/Ca2+ exchange current and Ca2+ sensitive K+ current during STDP, we examine the double mechanism knockout  which removed both the Na+/Ca2+ exchange current and the Ca2+ sensitive K+ current from the model. In this case we observed reduced potentiation as compared to similar to ncx-/- alone . We also examined K+ currents (IK+) during bAPs and found that the inward INa+ during bAPs led to greater activation of Ca2+ sensitive K+ current channels from reverse mode NCX that increases outward IK+. Removing the Na+/Ca2+ exchange mechanism thereby not only reduces inward ICa2+ but also abrogates the Ca2+ sensitive K+ current by reducing outward IK+: without the ncx-/- mechanism, the outward IK+ was reduced by an average of 23.3% for peak 1 and 37.5% for peak 2 as compared with wildtype. Taken together, our results reveal that the Ca2+ sensitive K+ current is required at the postsynaptic site to promote STDP while the Na+/Ca2+ exchange current is required at the postsynaptic site to normalize STDP responses, which suggest that together they create a negative feedback mechanism to deliver an appropriate set-point for STDP responses.To gain more mechanistic insight into how the Na2+ accumulation during synaptic signaling in the postsynaptic cell is critical in determining synaptic plasticity [2+ channels that result in near global Ca2+ elevations in spines and dendrites where NCX and plasma membrane Ca2+ pumps represent the major clearance mechanisms [2+ homeostasis, it denotes a unique substrate for tuning Ca2+ levels, and in turn synaptic plasticity. Previous work has shown that forward mode NCX functions at the postsynaptic side to promote Ca2+ clearance during depolarization at synaptic terminals [2+ levels from EPSP events; manipulating NCX localization allowed us to uncover a mechanism whereby deletion of NCX results in elevated potentiation during pairing of bAPs with EPSPs. This suggest that NCX functions to set an appropriate set-point for STDP, and that defects in this normalization mechanism results in altered STDP. Our simulation data also demonstrated a role for the Ca2+-activated K+ channel current in promoting potentiation during pairing of bAPs with EPSPs. Previous data from both cortical and hippocampal pyramidal neurons has shown that bAPs can activate small conductance Ca2+-activated K+ channels (SK channels), and thereby function to gate STDP during bAP pairing with EPSP [2+-activated K+ channels may function in opposing ways in postsynaptic cells to adjust STDP during bAP pairing with EPSP. While limitations do exist within our simulation models, our findings are suggestive of an integrative mechanism by NCX and small conductance Ca2+-activated K+ channels to modulate synaptic plasticity.The timescale of Caasticity –25. In Cchanisms . Given terminals . Furtherith EPSP . A novelS1 Fig+/Ca2+ exchange current was localized to the oblique (A), tuft (B), or primary dendrites (C) while modeling linear and constant models of NCX allostery , and linear and constant models of NCX density (linear and const).Depolarization times were recorded during back propagation when the Na(TIF)Click here for additional data file.S2 Fig+/Ca2+ exchange currents were examined during bAPs paired with EPSPs to examine the direction of Ca2+(A) and Na+(B) ion exchange.Na(TIF)Click here for additional data file.


SOX9 and EGR2 was decreased in AML-MSCs as compared with ND-MSCs. Increasing expression of SOX9 decreased adipogenic potential of AML-MSCs and decreased their ability to support AML progenitor cells. These findings suggest that AML-MSCs possess adipogenic potential which may enhance support of leukemia progenitor cells.Bone marrow mesenchymal stromal cells (MSCs) constitute one of the important components of the hematopoietic microenvironmental niche. In vivo studies have shown that depletion of marrow MSCs resulted in reduction of hematopoietic stem cell content, and there is in vitro evidence that marrow MSCs are able to support leukemia progenitor cell proliferation and survival and provide resistance to cytotoxic therapies. How MSCs from leukemia marrow differ from normal counterparts and how they are influenced by the presence of leukemia stem and progenitor cells are still incompletely understood. In this work, we compared normal donor (ND) and acute myelogenous leukemia (AML) derived MSCs and found that AML-MSCs had increased adipogenic potential with improved ability to support survival of leukemia progenitor cells. To identify underlying changes, RNA-Seq analysis was performed. Gene ontology and pathway analysis revealed adipogenesis to be among the set of altered biological pathways dysregulated in AML-MSCs as compared with ND-MSCs. Expression of both  Acute myelogenous leukemia (AML) is characterized by proliferation and/or accumulation of abnormal clone(s) of cells which lack differentiation potential. Concurrently, production of normal hematopoietic cells is curtailed, often resulting in clinically significant cytopenias. There were an estimated 21,380 new cases of AML diagnosed in the United States in 2017 . CompletThe marrow microenvironment includes a complex network of extracellular matrix proteins, soluble growth factors, cytokines, and distinct but possibly overlapping cellular niches, including the endosteal (or osteoblastic) niche, and the vascular niche . While a+, CD45−, and Ter119− cells [In human marrow, the definition of distinct niches and hierarchy of mesenchymal stromal cells (MSCs) is less well-characterized than in murine models. In murine models, MSCs have been classified as CXCL12-abundant reticular cells, stem cell factor expressing cells, nestin-expressing cells, and PDGFR-α, Sca-19− cells . In leuk9− cells –17. SeveThere is experimental evidence in murine models that changes in microenvironmental cells can result in myeloproliferative disease through Notch activation , in myelThere are now several reports of MSC changes in human hematologic malignancies such as MDS , 24, acuIn this work we sought to further compare ND-MSCs and AML-MSCs by phenotype and transcriptome analysis in order to assess effect on outgrowth of leukemia blasts and progenitors. Our findings, using primary marrow from AML patients, show an adipogenic predisposition in MSCs isolated from established AML and suggest that these changes may impact leukemia cell survival in the marrow niche.Please refer to Supplementary Information for more detailed “Materials and methods” descriptions.n = 10; Supplementary Table n = 1; Supplementary Table MSCs were cultured from marrow of 11 AML patients, 8 previously untreated  with STAR-2.5.2b. The list of significant genes was used for pathway analysis using Ingenuity Pathway Analysis software .SOX9 lentiviral activation particles were utilized following manufacturer’s instructions  to upregulate expression of the SOX9 gene. This is a synergistic activation mediator (SAM) transcription activation system. In negative controls, lentiviral activation particles contain SAM activation elements which are deactivated via Cas9 nuclease fused to the transactivation domain and a nonspecific 20 nucleotide guide RNA. The SAM complex does not recognize or bind any specific sequence within the genomic DNA and does not activate transcription of any specific gene.t test was used to test differences between groups. Statistical significance was considered at p < 0.05 utilizing prism .All data were collected from a minimum of three independent experiments. Data were expressed as the mean ± standard error of means from the indicated number of experiments. Comparisons among groups were conducted using one-way analysis of variance. Student’s p > 0.05, n = 5).ND-MSCs had similar morphology as compared with age-matched AML-MSCs as presented in Fig. 5/cm2 in tissue culture flasks for both ND and AML specimens. Early passaged AML-MSCs lagged behind in their growth rate as defined by days between passage to confluence as compared with ND-MSCs  but this difference was not observed in later passaged cells  cells were plated at a density of 5 × 10p = 0.0002, n = 5) per 1 million LDBM cells plated. Representative images of CFU-F from ND and AML-LDBM are shown in the lower panel of Fig. The CFU-F assay was performed using LDBM from ND and AML samples in order to evaluate their fibroblastic clonogenic potential. The CFU-F outgrowth was significantly decreased in AML-LDBM as compared with that from age-matched normal marrow donors Fig. . In the p = 0.35, n = 4).After two rounds of exposure to osteogenic induction medium, no difference was noted between ND- and AML-derived MSCs. This was measured by Alizarin red S staining Fig.  and by aAfter two rounds of induction in adipogenic medium at passage 3, AML-MSCs exhibited more oil Red O stained cells as compared with ND-MSCs from age-matched controls also at passage 3 Fig. . The perq < 0.05). Lists of up- and downregulated genes are shown in Supplementary Tables In order to assess differences in gene expression between ND-MSCs and AML-MSCs, an unbiased RNA-Seq analysis was performed on three ND-MSC and three previously untreated AML-MSC specimens, all from subjects >50 years of age and analyzed at passage 3 in culture. This identified 88 genes significantly differentially expressed between the two groups (SOX9 (SRY-related high mobility group-Box gene 9) and EGR2 β and CEBPσ, we focused whether its overexpression in AML-MSCs could affect lipid production during induction as measured by oil Red O staining. The SOX9 gene was overexpressed using lentiviral activation particles. Oil Red O quantification showed that control AML-MSCs induced to adipogenesis had a higher percentage of oil Red O staining than did those of AML-MSCs induced to adipogenesis after SOX9 activation  via CEBP-β/σ . Downregegulated . Herein,Some groups have found increased osteogenic potential in AML-MSCs whereas others have found increased adipogenic potential as noted here. AML-MSCs have been found to have increased expression of FABP4 in comparison with normal controls and delayed osteogenic potential . They haIn contrast, Battula et al.  found thIn this work where we directly compared ND-MSCs with AML-MSCs, genes involved in pre adipogenesis were altered and FABP4 expression was increased providing evidence for adipogenic propensity of AML-MSCs. As noted previously, MSC heterogeneity across AML subtypes is minimal , 33, 40.Adipogenic niches are involved in solid tumor marrow colonization and growth , 52. Bon+ CD38− selected cells. Genes associated with quiescence and survival of stem/progenitor cells were increased such as ANGP51, SPP1, and SDF-1α [In our study, we found that AML-MSCs supported normal light density marrow survival and CFU-GM and BFU-E outgrowth as well as did ND-MSCs. Whether normal progenitors are differentially supported by AML-MSCs vs. ND-MSCs remains controversial. Chandran et al.  found thd SDF-1α . Others d SDF-1α . Other gd SDF-1α . LikewisThe studies reported upon here have several limitations. They are limited by the short co-culture duration allowed by ex vivo studies with primary AML blasts which do not survive well in culture without growth factor or stromal support. Also, ex vivo MSC cultivation could select subsets which may not be representative of all those found in marrow. Furthermore, MSCs are grown from marrow aspirates which may not extract such cells from all locations equally. While MSCs from human and murine marrow can be prospectively isolated and sorted, these cells are quite limited in number (estimated at 0.001–0.01% of marrow nucleated cells ), and whSOX9 gene was shown here to affect adipogenic potential and resultant support of leukemia progenitor cells. Enhanced marrow MSC adipogenic potential could lead to a pro-tumoral microenvironment and its modulation may therefore represent a novel means to inhibit AML.Our studies have corroborated differences in human marrow-derived MSCs between normal and leukemia subjects and have shown differential expression of genes involved in adipogenesis. Adipocytes support leukemia survival by regulating their metabolic energy, and disruption of fatty acid oxidation in marrow adipocytes may be a therapeutic strategy . ModulatRevised Supplementary Information Clean


Obesity confers enhanced risk for multiple diseases including cancer. The DNA methylation alterations in obesity-related genes have been implicated in several human solid tumors. However, the underlying role and clinical implication of DNA methylation of obesity-related genes in acute myeloid leukemia (AML) has yet to be elucidated.LEP expression was correlated with prognosis among obesity-related genes from the databases of The Cancer Genome Atlas. In the validation stage, we verified that LEP hypermethylation was a frequent event in AML by both targeted bisulfite sequencing and real-time quantitative methylation-specific PCR. Moreover, LEP hypermethylation, correlated with reduced LEP expression, was found to be associated with higher bone marrow blasts, lower platelets, and lower complete remission (CR) rate in AML. Importantly, survival analysis showed that LEP hypermethylation was significantly associated with shorter overall survival (OS) in AML. Moreover, multivariate analysis disclosed that LEP hypermethylation was an independent risk factor affecting CR and OS among non-M3 AML. By clinical and bioinformatics analysis, LEP may be also regulated by miR-517a/b expression in AML.In the discovery stage, we identified that DNA methylation-associated LEP methylation is associated with LEP inactivation, and acts as an independent prognostic predictor in AML.Our findings indicated that the obesity-related gene  Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by clonal proliferation of the hematopoietic progenitor cells . ClinicaLEP, LEPR, NPY, ADIPOQ, FTO, MC4R, PCSK1, and POMC are implicated and have a direct role in obesity [Obesity has been verified as an independent health risk and is significantly correlated with the development of metabolic disorders, including hyperlipidemia, type 2 diabetes mellitus, hypertension, stroke, and cardiovascular disease. Furthermore, strong evidences have proved the links between body mass index (BMI) and various cancers including the most forms of tumor-based cancer and hematological malignancies –6. A num obesity .To the best of our knowledge, epigenetic alterations have been suggested as a molecular mechanism mediating gene expression, and also described as a potential early cancer-related biomarker with strategies for diagnostic, prognosis or cancer screening procedures being developed , 14. To http://bio-bigdata.hrbmu.edu.cn/diseasemeth/analyze.html) was applied to compare the methylation difference between these AML patients with controls.The first cohort of 200 AML patients from The Cancer Genome Atlas (TCGA) databases included in this study was used in the discovery stage for the identification of prognostic methylation-related genes . Among tA second cohort of 25 healthy donors and 111 de novo AML patients treated at the Affiliated People’s Hospital of Jiangsu University were also enrolled, and used in the validation stage for targeted bisulfite sequencing. In addition, expanded samples of the sequencing cohort, including 172 AML patients (161 de novo AML and 11 MDS-derived AML) and 46 healthy donors, were used in the validation stage for real-time quantitative methylation-specific PCR (qMSP). AML patients were diagnosed and classified according to the 2016 World Health Organization (WHO) criteria . TreatmeLEP were shown in Additional file The target gene methylation was detected by Targeted bisulfite sequencing—MethylTarget, which was performed in Genesky Biotechnologies Inc.  as our previous investigations , 23. TheLEP mRNA expression was performed by real-time quantitative PCR (qPCR) using AceQ qPCR SYBR Green Master Mix . The reference gene ABL1 mRNA, examined by 2 × SYBR Green PCR Mix , was detected to calculate the abundance of LEP mRNA expression. The qPCR primers used for LEP expression detection were listed in Additional file LEP mRNA expression was calculated using 2−∆∆CT method.Reverse transcription was carried out as our previous studies , 21. TheLEP methylation level was evaluated by qMSP with primers shown in Additional file ALU methylation level was also detected. Relative LEP methylation level was calculated using 2−∆∆CT method.Genomic DNA was bisulfite converted as our previous reports , 22. TheLEP was identified by the venn analysis (http://bioinformatics.psb.ugent.be/webtools/Venn/) of three websites miRDB (http://mirdb.org/miRDB/), miRWalk  and TargetScan (http://www.targetscan.org/vert_72/) [https://www.r-project.org).Differential expression analysis for RNA/miRNA sequencing data was calculated using the raw read counts with the R/Bioconductor package “edgeR”, all analyses were controlled for the false discovery rate (FDR) by the Benjamini–Hochberg procedure . The miRert_72/) , 26. Allχ2/Fisher’s exact test were used for the comparison of continuous and categorical variables, respectively. Correlation analysis between LEP methylation and methylation/expression was performed by Spearman test. The Receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) were used to evaluate LEP methylation level in distinguishing AML from controls. Complete remission (CR) was evaluated after 1–2 course of chemotherapy. Overall survival (OS) and leukemia free survival (LFS) were defined as previous report [LEP methylation on OS and LFS was analyzed by Kaplan–Meier analysis and Cox regression analysis (univariate and multivariate). A two-sided P less than 0.05 was seen as statistically significant.SPSS 20.0 and GraphPad Prism 5.0 were conducted to perform statistical analyses. Mann–Whitney’s U/Kruskal–Wallis followed by Dunn’s post hoc test and Pearson’s LEP, LEPR, NPY, ADIPOQ, FTO, MC4R, PCSK1, and POMC in AML. Prognostic value of these genes was analyzed in two groups divided by the median expression level of each gene respectively. In total AML and cytogenetically normal AML (CN-AML) patients, Kaplan–Meier analysis showed that only LEP expression was positively associated with OS , LEPR , FTO , PCSK1 , and POMC  genes .In order to explore whether mer Fig. c, and thLEP promoter methylation may be severed as an underlying biological marker for distinguishing AML from controls with an AUC of 0.803  subtypes . Moreover, there were no significant associations between LEP hypermethylation and gene mutations besides N/R-RAS mutations with a trend  of AML patients from TCGA datasets. A total of 83 differentially expressed miRNAs (included 71 positively correlated and 12 negatively correlated) (FDR < 0.05, P < 0.05, |log2 FC|> 1; Fig. miR-10a was identified to be significantly associated with AML with NPM1 mutation [miR-582, miR-517, miR-511, miR-508, miR-518c, miR-520g, and miR-187 were less investigated. Moreover, LEP was identified as a direct target of 69 miRNAs by bioinformatics prediction (Fig. miR-517a/b was shared in both clinical data and bioinformatics prediction, suggesting that LEP may be also regulated by miR-517a/b expression in AML (Fig. Due to a very weak correlation of mutation , whereasion Fig. . Of thesAML Fig. c.Fig. 4MLEP, NPY, and LEPR was involved in tumorigenesis of renal cell carcinoma, and LEPR methylation was associated with prognosis, and predicted renal cell carcinoma recurrence [LEP methylation, negatively associated with LEP expression, was independently associated clinical outcome in AML.Obesity confers enhanced risk for multiple diseases including cancer, and is increasingly recognized as a growing cause of preventable cancer risk –6. The Dcurrence , 28. HerLEP remains controversial in AML. Functional studies have showed that leptin presented an oncogenic role in AML biology by affecting cell proliferation and angiogenesis [LEP mRNA level in BMMNCs but not in serum of AML patients, and were found to be significantly decreased. The decreased expression of LEP may be caused by LEP promoter methylation in AML cells. In accordance with the previous study, we also observed LEP hypermethylation was associated with higher percentage of BM blasts and lower platelets. These results suggested DNA methylation-mediated leptin inactivation was a frequent event in AML cells. The reduction of autocrine of leptin in leukemia cells may negatively feedback regulates the increase of paracrine of leptin from adipose tissues into cancer microenvironment to promote leukemogenesis. Accordingly, further functional studies in vivo and in vitro are required to confirm our hypothesis.The expression pattern and direct role of ogenesis –31. In cogenesis , 33, twoogenesis , 35. In LEP expression in AML. In this study, we identified that LEP expression may be also regulated by miR-517a/b expression. Although few investigations revealed the miR-517a/b expression pattern in AML, a number of studies have reported the oncogenic role of miR-517a/b in diverse human solid tumors [LEP expression in AML biology. Obviously, additional studies are required to confirm the direct links of LEP with miR-517a/b by luciferase assay, and the direct role of miR-517a/b in AML needs further functional studies.Besides the DNA methylation, miRNAs expression was also identified to be associated with d tumors –38. ThesLEP methylation is associated with LEP inactivation, and acts as an independent prognostic predictor in AML.Our findings indicated that the obesity-related gene Additional file 1. Primers used for MethylTarget sequencing, qPCR and qMSP.Additional file 2. The impact of obesity-related genes expression on leukemia-free survival among AML patients from TCGA databases. Additional file 3. Different expressed microRNAs between lower and higher LEP expression groups.Additional file 4. Venn results of microRNAs targeting LEP.


These parameters are potential predictors of successful outcomes for men undergoing artificial reproduction techniques (ARTs), but methods currently available for their determination pose various technical challenges and limitations. Here, we developed a novel strategy employing time-lapse flow cytometry (TLFC) to determine capacitation-related membrane potential (Em) and pHi changes, and progesterone-induced [Ca2+]i increases. Our results show that TLFC is a robust method to measure absolute Em and pHi values and to qualitatively evaluate [Ca2+]i changes. To support the usefulness of our methodology, we used sperm from two types of normozoospermic donors: known paternity (subjects with self-reported paternity) and no-known paternity (subjects without self-reported paternity and no known fertility problems). We found relevant differences between them. The incidences of membrane hyperpolarization, pHi alkalinization, and increased [Ca2+]i were consistently high among known paternity samples , while they varied widely among no-known paternity samples . Our results indicate that TLFC is a powerful tool to analyze key physiological parameters of human sperm, which pending clinical validation, could potentially be employed as fertility predictors.Plasma membrane (PM) hyperpolarization, increased intracellular pH (pH Sexual reproduction involves the successful fusion of the female (egg or oocyte) and male (sperm) gametes, a process called fertilization. Human fertility-related issues are a growing public health problem worldwide . The caui) [2+]i) upon exposure to progesterone  i changes, some reports suggest that sperm cells from men with fertility issues present a decreased response to Pg i changes. We compared all three cellular responses in sperm cells incubated under conditions that either support or do not support in vitro capacitation. We also found promising preliminary results when comparing the incidence of the three capacitation parameters in sperm samples from normozoospermic donors with self-reported paternity (known paternity) versus donors with no self-reported paternity and no known fertility issues (no-known paternity). Altogether, our results lead us to propose TLFC as a robust method for the analysis of physiological parameters in human sperm cells, with the potential for use in male fertility evaluations and ART selection.Taking advantage of this instrument’s unique feature, we developed a novel method, which we have named time-lapse flow cytometry (TLFC). Our main findings suggest that TLFC is suitable for measurements of both membrane potential , abbreviated to Disc), a cationic carbocyanine Em-sensitive probe. This fluorescent dye partitions into the sperm PM according to its Em—when the PM becomes hyperpolarized, Disc will accumulate in the PM due to its cationic nature, while a depolarization favors the dye’s efflux from the cell, resulting in decreased fluorescence i during the protocol for Em measurements. Interestingly, in most of the sperm samples tested, Fluo3 fluorescence rapidly dropped after PM hyperpolarization with valinomycin, reaching a minimum after 60 s, followed by a recovery phase  from each value fluorescence (F) and dividing by F0  was added to the sperm suspension, triggering a fluctuation in [Ca2+]i as previously reported i signaling is fundamental for sperm capacitation, we evaluated the Pg-evoked Ca2+ influx using TLFC. In all samples, we were able to observe the expected increase in [Ca2+]i upon Pg stimulation, consisting of a fast and transient [Ca2+]i increase followed by a sustained plateau [2+]i increase is lost due to recording interruption [2+]i changes in sperm; as mentioned earlier, some of the disadvantages they present  can be overcome by the use of TLFC. In this work, we decided to compare the [Ca2+]i response upon Pg stimulation in sperm cells incubated either under NC or CAP conditions, since previous studies have shown that the [Ca2+]i response increases by at least onefold upon capacitation [2+]i increase in NC versus CAP conditions. An increased response to Pg under CAP conditions was only observed for 61% of all samples analyzed . Previous reports suggest that the differences in responses after a Pg stimulus depend on the basal [Ca2+]i of the sperm cells [2+]i after a Pg stimulus [2+]i responses are relevant to provide predictive values of fertility outcomes. Our results indicate that TLFC is completely suitable to analyze these responses, providing key advantages over other commonly employed techniques.Considering that i changes upon Em manipulation. After valinomycin-evoked Em hyperpolarization, a transient decrease in [Ca2+]i was observed in most cases, and this was followed by a recovery phase that remained either below or similar to resting conditions. Then, a KCl-induced depolarization evoked a Ca2+ influx. These results are consistent with previous reports of voltage-dependent changes in [Ca2+]i [2+]i changes and the sperm’s Pg-induced acrosome exocytosis could be performed using the appropriate combination of fluorescent labels  [Another benefit of TLFC is the possibility of combining different dyes for the simultaneous evaluation of more than one physiological parameter. We co-stained sperm cells with Fluo3 and Disc in order to monitor [Ca [Ca2+]i ,58,59 th [Ca2+]i ,24,60. Actively) . This evctively) ,62.i has been recognized as a hallmark of mammalian sperm capacitation i responses to Pg stimulation, and cytoplasmic alkalinization are physiological parameters that may convey information of the fertilizing potential of sperm samples. Further investigation is required to establish putative normal response ranges for these parameters, as well as to understand the clinical significance whenever a given sample fails to display a response within such ranges. Even though the BD Accuri C6 cytometer is not an instrument commonly found in fertility clinics at this time, we propose that it is a suitable and cost-effective tool to perform sperm TLFC recordings aimed at guiding ART selection. The instrument retrofitting required for this application is reversible and straightforward, involving the use of readily available and inexpensive materials. One of the disadvantages of using this equipment, however, is that the filter/illumination configuration is fixed and thus the dye choices are limited. Nevertheless, the evaluation of the three capacitation-associated parameters described here, and potentially additional ones, could be useful as additional pieces of evidence when assessing the fertilizing potential of a given sperm sample. Moreover, since our method evaluates several parameters related to molecular processes involved in sperm function, it offers the potential of providing new and valuable information about the specific mechanisms that may be altered in sperm samples from infertile men. As such, we believe that our methodology may open up the door to a novel and more specific way of diagnosing and treating male infertility.The results provided in this work support the notion that 3(5)), and 4--4′-methyl-2,2′(ethylenedioxy) dianiline-N,N,N′,N′-tetra acetic acid tetrakis (acetoxymethyl) ester (Fluo3-AM) from ThermoFisher Scientific; ionomycin from Alomone; and all others from Sigma-Aldrich. Fluorescent dye and test compound stock solutions were prepared in Dimethyl sulfoxide (DMSO), except for those of KCl, KOH, NH4Cl, and MnCl2, which were prepared in distilled water.Chemicals were obtained from the indicated sources: 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), 3,3′dipropylthiadicarbocyanine iodide  i in cells in suspension after sequential additions of test compounds i changes. At 120 s after fluorescence recording began, 4 µM Pg was added. To obtain the maximum fluorescence intensity, we added 10 µM ionomycin after recording for 300 s, followed by addition of 5 mM MnCl2 at 540 s to quench Fluo3 fluorescence and thus obtain a minimum fluorescence value.i fluorescence, and pHi values were first separated into the 2 donor classes—known paternity and no-known paternity. In each of these groups, paired comparisons were performed between NC and CAP conditions by a paired t-test or a Wilcoxson signed-rank test, depending on whether or not the data passed the Shapiro-Wilk normality test, respectively. After calculating the ΔEm (EmCAP − EmNC), ΔFPeak (FPeakCAP − FPeakNC), and ΔpHi (pHiCAP − pHiNC), we performed a one-sample t-test comparison against theoretical mean = 0 for each donor class—known paternity and no-known paternity . A probability value (p) < 0.05 was considered as a statistically significant difference. The final versions of the figures were made in Inkscape 0.91 (Inkscape.org).Flow cytometry experimental data from the BD Accuri C6 Plus software were exported in Flow Cytometry Standard (FCS) format and initially analyzed with FlowJo Software version 10.1 (BD). Then, fluorescence and time data were exported into a comma-separated values (CSV) format and analyzed in RStudio version 1.3.1093 . Fluores


Rabbits are one of the most used experimental animals for investigating the mechanisms of human cardiovascular disease and lipid metabolism because they are phylogenetically closer to human than rodents (mice and rats). Cholesterol-fed wild-type rabbits were first used to study human atherosclerosis more than 100 years ago and are still playing an important role in cardiovascular research. Furthermore, transgenic rabbits generated by pronuclear microinjection provided another means to investigate many gene functions associated with human disease. Because of the lack of both rabbit embryonic stem cells and the genome information, for a long time, it has been a dream for scientists to obtain knockout rabbits generated by homologous recombination-based genomic manipulation as in mice. This obstacle has greatly hampered using genetically modified rabbits to disclose the molecular mechanisms of many human diseases. The advent of genome editing technologies has dramatically extended the applications of experimental animals including rabbits. In this review, we will update genetically modified rabbits, including transgenic, knock-out, and knock-in rabbits during the past decades regarding their use in cardiovascular research and point out the perspectives in future. Rabbits were first used for disclosing the pathogenesis of human atherosclerosis a century ago. In 1908, a Russian physician, Alexander I. Ignatowski (1875–1955) fed rabbits with a diet supplemented with animal proteins  and found that these rabbits developed pronounced aortic atherosclerosis.Later, a Russian experimental pathologist, Nikolai N. Anichkov (or Anitschkow) (1885–1964) further demonstrated that it was dietary cholesterol rather than proteins that play the critical role in the pathogenesis of atherosclerosis in rabbits and proposed a causal role of cholesterol in the development of atherosclerosis  rabbits was almost concurrently reported by German  cells and the genome information, it has been considered impossible to create KO rabbits by homologous recombination-based genomic modification as to generate KO mice. Unavailability of KO rabbits also constitutes another obstacle that hampers researchers to study loss-of-functions of genes in rabbits. We strived to use somatic cell nuclear transfer technique to generate KO rabbits after Chesne et al. reported the first cloned rabbit about 17 years ago  and was first reported by Brown et al. 50 years ago . FH is an autosomal dominant genetic disorder characterized by elevated plasma LDL levels due to LDL receptor dysfunctions . Since CETP is genetically absent in rodents (mice and rats) and pigs, rabbits are considered the best model for investigation of CETP functions because rabbits have high levels of CETP in the plasma as humans. Taking this advantage, Zhang et al. created CETP KO rabbits and found that CETP KO rabbits showed higher plasma levels of HDL-cholesterol  is a glycoprotein that transfers plasma lipids between HDLs and apoB-containing particles therefore plays an important role in lipoprotein metabolism. However, it is not known whether inhibition of CETP activity can prevent cardiovascular disease because four CETP inhibitors  failed to prove their efficacy in terms of reduction of cardiovascular risk by clinical trials(ApoAII is the second major apolipoproteins in HDLs. However, its physiological functions are largely unknown compared with apoAI. Interestingly, WT rabbits are genetically deficient in apoAII so their HDLs only contain apoAI. This unique feature makes WT rabbits as a “natural” apoAII KO model. We first made Tg rabbits expressing human apoAII gene and found that hepatic expression of human apoAII inhibits cholesterol diet-induced atherosclerosis (Wang et al., Although genetically modified rabbits are an important experimental model in cardiovascular research, they should not be simply used as a substitute of mice and rats, as discussed above. Because rabbits are more expensive, require larger space, and need more time to breed compared with mice, the generation of genetically modified rabbits should be carefully planned to solve those specific problems that cannot be well-examined in other experimental animal models, such as the development of lipid-lowering drugs (Niimi et al., JF, YW, and YC wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mentoring provides mentees and mentors with holistic support and research opportunities. Yet, the quality of this support has been called into question amidst suggestions that mentoring is prone to bullying and professional lapses. These concerns jeopardise mentoring’s role in medical schools and demand closer scrutiny.To better understand prevailing concerns, a novel approach to systematic scoping reviews (SSR) s is proposed to map prevailing ethical issues in mentoring in an accountable and reproducible manner. Ten members of the research team carried out systematic and independent searches of PubMed, Embase, ERIC, ScienceDirect, Scopus, OpenGrey and Mednar databases. The individual researchers employed ‘negotiated consensual validation’ to determine the final list of articles to be analysed. The reviewers worked in three independent teams. One team summarised the included articles. The other teams employed independent thematic and content analysis respectively. The findings of the three approaches were compared. The themes from non-evidence based and grey literature were also compared with themes from research driven data.Four thousand six titles were reviewed and 51 full text articles were included. Findings from thematic and content analyses were similar and reflected the tabulated summaries. The themes/categories identified were ethical concerns, predisposing factors and possible solutions at the mentor and mentee, mentoring relationship and/or host organisation level. Ethical concerns were found to stem from issues such as power differentials and lack of motivation whilst predisposing factors comprised of the mentor’s lack of experience and personality conflicts. Possible solutions include better program oversight and the fostering of an effective mentoring environment.This structured SSR found that ethical issues in mentoring occur as a result of inconducive mentoring environments. As such, further studies and systematic reviews of mentoring structures, cultures and remediation must follow so as to guide host organisations in their endeavour to improve mentoring in medical schools. Mentoring’s success in nurturing “dynamic, context dependent, goal sensitive, mutually beneficial relationships between an experienced clinician and medical students that is focused upon advancing the development of the mentee” has seen its role in medical school education grow . Yet, meWith mentoring playing a key role in training medical students and with many ethical issues poorly described , 23, a sAn SSR is employed given its wide scope . HoweverThe SEBA process comprises of the following stages: 1) Systematic Approach, 2) Split Approach, 3) Jigsaw Perspective, 4) Reiterative Process and 5) Discussion. This is outlined in Fig. Ensuring a systematic approach to the SSR in SEBA synthesis, the research team consulted the expert team to determine the overall goals of the SSR and the population, context and particular mentoring approach to be evaluated.Building on findings of two recent systematic scoping reviews , 23 of eGuided by the PCC  elements of the inclusion criteria , the priThe inclusion and exclusion criteria are elaborated on below Table .Table 1The focus was confined to ethical issues in mentoring in undergraduate and postgraduate medical schools.Ten members of the research team carried out independent searches of five bibliographic databases  and two grey literature databases (OpenGrey and Mednar) between 5th March 2020 to 7th March 2020 for articles published between 1st January 2000 to 31st December 2019. The PubMed search strategy may be found in Additional file Ten members of the research team independently reviewed all identified article titles and abstracts, created individual lists of titles to be included and discussed them online where ‘negotiated consensual validation’  was emplThree teams comprising of three researchers simultaneously reviewed the 51 included full text articles. The first team independently summarised and tabulated the articles in keeping with Wong and Greenhalgh’s RAMESES publication standards: meta-narrative reviews  and PopaConcurrently, each member of the second team analysed the 51 included articles using Braun and Clarke’s approach to thematic analysis  whilst tThe themes identified were ethical concerns and solutions at the level of the mentee, mentor, mentoring relationship and host organisation.Deductive category application  was emplThere was general consensus within the research team and expert team that the findings reflected the positions of the included articles. The combined findings of the Split Approach are presented in Tables The jigsaw perspective brings data from complementary pieces of the mentoring process such as aspects of the mentoring relationship together to create a cohesive picture of ethical issues in mentoring.Whilst there was consensus on the content and arrangement of the themes/categories identified, the expert team raised concerns that data from grey literature, which is neither quality assessed nor necessarily evidence-based, could ‘slant’ the direction of the narrative synthesis. As a result of these concerns, the research team thematically analysed the data from grey literature and non-research-based pieces drawn from the five bibliographic databases. These included letters, opinion and perspective pieces, commentaries and editorials.Comparisons of themes from grey literature and non-research-based pieces with peer-reviewed evidence-based data revealed that the former two additionally focused on predisposing factors to ethical issues involving mentees, mentors and their mentoring relationships Table .Table 4It comes as no surprise that ethical concerns surrounding mentoring issues in medical schools remain poorly understood and categorised. In curating and analysing the data, this SSR in SEBA found great diversity in what constitutes ethical issues in mentoring. This ranges from ineffective mentoring support and the misappropriation of the mentee’s work , 50–52 tThere appears to be a connection between relatively mild or innocuous issues and severe breaches of professional, ethical and clinical codes of conduct. Without timely and effective interventions, innocuous ethical issues may lead to more serious violations in mentoring practice. Poor access to mentoring support  may exacUpon closer analysis of the data, predisposing factors to breaches in ethical mentoring practice may be categorised at the level of the (i) individual mentee and mentor, (ii) mentoring relationship as well as the (iii) host organisation.At the mentee and mentor level, ethical concerns stem from poor mentee and mentor recruitment and training , 62, 73,At the matching stage, a lack of choice in mentors and poor consideration of mentee preferences for the mentor’s gender, cultural background, experience, interests, personality and working style may have a detrimental effect on the suitability of the mentoring approach and the quality of their interactions , 76–78. At the level of the host organisation, the primary source of ethical issues in mentoring revolves around poor structuring of the mentoring program which includes the lack of clearly established goals, timelines, project outcomes, mentoring approaches, roles, responsibilities, codes of conduct and expectations upon mentee, mentor and the host organisation at recruitment, training and matching processes. These issues, if insufficiently addressed or supported by the institution, will lead to mentoring failures.On deeper consideration of the findings, it could be surmised that the solutions proffered ultimately pivot on the provision of a conducive mentoring environment. To this end, Hee et al. note that an effective mentoring environment comprises of both an efficacious mentoring structure and mentoring culture . The menWith the lack of in-depth discussion in prevailing literature on pertinent characteristics of unethical mentoring practice, it is clear that there are no ‘magic bullets’. However, comparing the findings of this SSR in SEBA with existing reviews on mentoring in medicine and surgery , 23, it Efforts to enhance the reproducibility and transparency of the SSR in SEBA unfortunately appear limited. Whilst the databases used were identified by the expert team, critical papers may still have been omitted despite concerted efforts to employ independent selection processes. Similarly, whilst use of the Split Approach allowed for triangulation and greater transparency of the SSR in SEBA, inherent human biases may have impacted the data analyses. Although use of thematic analysis to review the impact of grey literature also improved the transparency of the discussion, the inclusion of themes derived from grey literature may have skewed the results by providing opinion-based views with a ‘veneer of respectability’ despite a lack of evidence to support them. This raises the question as to whether grey literature should be accorded the same weight as published literature.In answering its primary and secondary research questions, this SSR in SEBA provides a comprehensive, accountable, reproducible and evidence-based perspective of ethical issues in mentoring in undergraduate and postgraduate medical schools. This SSR in SEBA also identifies the pivotal role of the host organisation in identifying and addressing these concerns.Further studies on mentoring structures and mentoring cultures are necessary to further guide efforts on how a more gracious and altruistic mentoring environment could be facilitated. Better prevention and remediation of ethical breaches and violations would also certainly help to reinstate mentoring’s role in medical schools. It is only when these concerns are addressed head-on that all stakeholders will be able to truly reap the benefits of mentoring.Additional file 1. Search Strategy, which shows Search Strategy employed for PubMed.Additional file 2. Summary of Included Articles, which shows Summary of Included Articles.Additional file 3. PRISMA Checklist.doc, PRISMA Checklist, which shows the PRISMA checklist for this review.