Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are malignant clonal diseases of the hematopoietic system with an unsatisfactory overall prognosis.The main obstacle is the increased resistance of AML and ALL cells to chemotherapy. The development and validation of new therapeutic strategies for acute leukemia require preclinical models that accurately recapitulate the genetic, pathological, and clinical features of acute leukemia. A patient-derived orthotopic xenograft (PDOX) model is established using surgical orthotopic implantation. They closely resemble human tumor progression and microenvironment and are more reliable translational research tools than subcutaneous-transplant models. In this study, we established PDOX models by direct intrafemoral injection of bone marrow and peripheral blood cells from AML and ALL patients, characterized their pathology, cytology, and genetics, and compared the model's characteristics and drug responsiveness with those of the corresponding patients. Leukemia is a common malignancy worldwide. Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are monoclonal neoplastic proliferations of myeloid precursors or lymphoid progenitor cells. Ongoing advances in treatment have significantly improved 5-year survival, but the overall prognosis of acute leukemia is not satisfactory . About 5null (NSG) strains [Preclinical experimental models of acute leukemia are limited because they differ from human disease in genetic heterogeneity, pathological characteristics, and drug responsiveness . Patient strains , 8. In o strains , 10. Act strains . To furtPrkdc  and Il2rg (Common gamma chain receptor) genes were knocked out on NOD/ShiltJGpt background. The genetic background of NOD/ShiltJGpt resulted in natural immunodeficiency, such as complement system and macrophage defects [Prkdc gene leads to the inability of V(D)J recombination to occur, resulting in the inability of T cells and B cells to mature. Il2rg is a common subunit of various interleukin cytokine receptors, and the inactivation of Il2rg leads to the loss of six different cytokine signaling pathways, resulting in NK cell defects [This study investigated the usefulness of PDOX mouse models of AML and ALL for the study of drug sensitivity in NCG mice. NCG mice as a kind of more severe immune-deficient strain being fairly similar to NSG mice was established by CRISPR/Cas9 technology.  defects . Meanwhi defects . Loss of defects . We estaPeripheral blood or bone marrow samples were obtained from AML and ALL patients at the China–Japan Union Hospital of Jilin University. The study was reviewed and approved by the Institutional Ethics Committee, and written informed consent for study enrollment and blood or bone marrow collections was obtained from all patients.Antibodies (Abs) used for flow cytometry, PE mouse antihuman CD33  and APC mouse antihuman CD45  were purchased from BD Biosciences, PerCP/Cy5.5 antihuman CD19  was purchased from Biolegend. Cytarabine (Cat#: BD8499), imatinib (Cat#: BD42606), and ibrutinib (Cat#:BD254580) were purchased from Bide Pharmatech . Epirubicin  was purchased from Shandong New Time Pharmaceutical Co., Ltd., Company . Vincristine (Cat#: MB1298) was purchased from Melone Pharma . Cladribine (Cat#:CSN10004) was purchased from CSNpharma .The immunodeficient NCG (NOD/ShiLtJ-Prkdc em26Cd52 Il2rg em26Cd22) mice used in this study were purchased from GemPharmatech Co., Ltd. Company  and maintained in pathogen-free conditions at Shanghai LIDE Biotechnology Co., Ltd., Company .6 peripheral blood or bone marrow cells of AML or ALL patients after being anesthetized with a 1.25% avertin solution, respectively.6- to 8-week-old NCG mice were injected intrafemorally with 1–2 × 10+CD33+ expression in AML and CD45+CD19+ expression for B-ALL were assayed. A 5–15% dual positivity indicated AML or ALL PDOX engraftment. The procedures of PDOX establishment are shown in Fig. Engraftment of the orthotopic models was monitored by flow cytometry of peripheral blood from inoculated mice. The frequencies of CD45The presence of gene mutations that commonly occur in AML and ALL patients was investigated by next generation sequencing of peripheral blood and bone marrow samples obtained from the AML and B-ALL patients and the xenografts in the PDOX mouse spleen samples. Chromosomal analysis of human AML and ALL cells in mouse spleen samples from the established PDOX models was performed by karyotyping and standard G-banding.Cladribine, cytarabine, imtatinib, or epirubicin in combination with vincristine were used to validate the in vivo efficacy of standard chemotherapies in the AML and ALL PDOX models. Cladribine and imatinib was given daily via gavage at 0.4 mg/dose and 25 mg/kg, respectively. Cytarabine was given on days 1–5 by intraperitoneal injection at 10 mg/kg. Epirubicin was given weekly by intraperitoneal injection at 1 or 2.5 mg/kg. Vincristine was given weekly by intravenous injection at 0.5 mg/kg. The body weight of each animal was recorded every day, and doses were normalized against the individual weight to ensure consistency among groups. Flow cytometry was performed weekly to determine AML/ALL progression and the anti-tumor effectiveness of each treatment.In the validation phase, we identified potential markers and targets in patient samples by next generation sequencing, G-banding, and flow cytometry, and the results are shown in Table +CD33+ cells or CD45+ cells in mouse peripheral blood was monitored weekly by FACS beginning 3–4 weeks after transplantation. To confirm the engraftment of human AML/ALL in the mouse models, P0 indicates the primary passage of patient cell suspension prepared from patient blood or bone marrow samples. When the percentage of positive cells reached 10%, single cell suspensions prepared from the mouse spleen were inoculated intrafemorally into naïve NCG mice (P1), or cryopreserved in liquid nitrogen until inoculation (FP0 + 1). The percentages of leukemia cells at P0, P1, and FP0 + 1 in 12 representative PDOX models are shown in Fig. +CD33+ or CD45+ at P1. Successfully established PDOX required stable morphologic and molecular characteristics for at least two passages. In terms of our success tumor take rates, 10.45% (7/67) of AML patient samples and 40%(8/20) of ALL patient samples successfully initiated PDOX models. In addition, our study indicated that leukemia developed initially from the bone marrow of mice. After 5 weeks of transplantation in LD1-0024-361,280 AML PDOX model, the samples of the peripheral blood (PB) or bone marrow (BM) in 6 mice were collected at the same time for FACS detection. The results showed that the mean percentage of CD45+ CD33+ cells in the peripheral blood (PB) was 13.80% and the mean percentage of CD45+ CD33+ cells in bone marrow was 62.02%.Patient characteristics, diagnosis, and treatment history, are shown in Table + or CD45 + CD33 + cells in mouse peripheral blood were determined by FACS at P0 (black lines), P1 (red lines), and FP0 + 1 (blue lines) of established PDOX models. Individual mice are shown as point values. Group results are shown with bars that give the standard deviation.Cell suspensions prepared from patient samples were injected intrafemorally into NCG mice to generate the PDOX models. The frequency and percentage of CD45 + and CD38+, suggesting that CD19+CD10+CD38+ cell populations might be a useful as a panel to determine the establishment of PDOX in this type of B-ALL. The series of ALL-associated antigens revealed that the immunophenotype of PDOX cells was consistent with the primary specimens for most antigens tested and that they preserved the disease characteristics of the patients that they originated from.FACS analysis of cell surface markers in peripheral blood of patient samples and the PDOX models established using the samples are shown in Table G-banding karyotype analysis showed normal karyotypes and revealed that the karyotypes of PDX cells of LD1-0041-362,073 and LD1-0041-362,021 were similar to that of patient specimens. However, LD1-1041-362,519 showed a different karyotype in the PDX versus patient, the discordance between cytogenetic result of clinical specimen and PDOX on 1q21 and 18q21 may result from clonal selection. Moreover, because there is a complex structural variation on chromosome #9, it is uncertain whether it is a discordance  or different resolution, it would be better to have more clinical data to verify this.  had a mutation on exon 12 of NPM1, a typical hot-spot alteration seen in AML patients without chromosome abnormalities . As showValidation using the same treatments that were administered to patient was performed in five of the established PDOXs. Cytarabine, which is commonly given to AML/ALL, patents had anti-tumor activity in both the AML and the ALL PDOXs Fig. B–E. In t+, CD33+, and/or CD19+) were monitored weekly until the end of the study. Data are means ± standard deviation and p < 0.05 indicates statistical significance vs. control.AML PDOX  and ALL PDOXs  models were used for standard of care validation. Treatment of the model mice began after randomization to the indicated regimen. Cell surface markers (CD45Patient-derived xenografts(PDX) are based on transferring primary tumors directly from the patient into an immunodeficient mouse. There are several advantages with PDXs including retaining the histological characteristics of patient tumors, such as tumor heterogeneity and tumor microenvironment . When coγ-deficient mice has produced triple immunodeficient NCG, NSG, or NOG mice that lack functional T, B, and NK cells and are available from a number of suppliers [Heterogeneity of the malignant cells, with multiple mutant clones and subclones is a hallmark of acute leukemia , 18. Tumuppliers . The useuppliers –29. TripThe implantation site of cancer cells affects the growth characteristics and phenotype of the PDX model . Early PNPM1 gene is the most frequently mutated gene in AML. We found that the NPM1 gene mutation in the PDOX model was consistent with the patient’s bone marrow sample, suggesting the PDOX model has stable genetic profiling.An ideal research model should recapitulate the phenotypic and genetic characteristics of the original tumor in patients. In this study, the pathology of the mouse spleen and the surface markers of the engrafted cells from the PDOX models faithfully resembled the phenotypes of the patient leukemia samples. The karyotypes of PDOX cells were also highly consistent with those of the patient samples. We also compared the genetic profiles of PDOX models, and the clinical samples used for grafting by next generation sequencing. The In conclusion, it is feasible to establish AML and ALL PDOX models using the triple immunodeficient NCG mouse model. The established PDOX models preserve the pathological, phenotypic, and genetic characteristics of the clinical samples, and they also have similar drug sensitivity to the corresponding patients. The PDOX mouse model of acute leukemia provides a clinically relevant platform for testing novel chemotherapy drugs. Ultimately, the PDOX mouse model may be used for developing precision medicine approaches to treat leukemia.


Leukemia or blood cancer has been treated with chemotherapy since the 1970s. The treatment had undergone little change until the last decade when many new molecules were approved. Many different targeted agents that are effective in subgroups of leukemia targeting specialized pathways or in patients with specific mutations are now available. Classification systems have advanced with time from being based on the appearance of leukemia underneath the microscope to now being based on the presence of mutations and other genetic characteristics. We have explored multiple different approaches to fighting leukemia based on modulating and priming the immune system or with the help of cellular therapy and bone marrow transplants. Many newer pathways to targeting cancer are being tested in the laboratory and are being brought to the bedside through clinical trials. This is an exciting decade for researchers and clinicians and many major changes are expected to unravel in the treatment of leukemia. Acute Myeloid leukemia (AML) is a clinically heterogeneous disease with a 5-year overall survival of 32% between 2012 to 2018. The above number severely dwindles with age and adverse risk of disease, presenting opportunities for new drug development and is an area of dire unmet need. Basic science and clinical investigators across the world have been working on many new and old molecule formulations and combination strategies to improve outcomes in this disease. In this review, we discuss select promising novel agents in various stages of clinical development for patients with AML. Acute myeloid leukemia (AML) is an aggressive hematological malignancy that has been at the forefront of investigational agent development. Immature myeloid precursor cells proliferate rapidly, causing varied symptoms due to decreased production of normal hematological cells and increased immature cells. Immature myeloid cells are sticky and increased numbers can cause decreased tissue perfusion. Since the 1970s, conventional chemotherapy followed by post-remission therapy with chemotherapy combinations of cytarabine has been the backbone of treatment for acute myeloid leukemia. A deeper understanding of the molecular landscape of AML from the knowledge gained from routine next-generation sequencing employed in clinical practice and research settings has paved the way for the identification of key genetic changes and molecular mutations that drive leukemia. By selectively targeting aberrant molecular events, these investigational agents offer the potential for enhanced efficacy and reduced toxicity compared to conventional chemotherapy. We have seen a total of 10 new agents approved for acute myeloid leukemia over the last decade and many more agents that are promising in the pipeline. These agents include midostaurin, enasidenib, ivosidenib, olutasidenib, venetoclax, gemtuzumab, glasdegib, gilteritinib, oral 5-azacitidine, and CPX-351.Despite advances in therapy, the overall prognosis for AML remains poor, with high relapse rates and limited treatment options among patients with complex karyotypes and poor risk mutations who are resistant to conventional chemotherapy. Non-intensive induction strategies with comparable remission rates to conventional chemotherapy such as the combination of a BCl-2 inhibitor and a hypomethylating agent  have posSome strategies that have been gaining traction include empowering the body’s own immune system using immune checkpoint inhibitors, cellular therapies such as chimeric antigen receptor T-cell (CAR-T cells), and modulation of the innate immune system through Natural Killer (NK) cells. Differentiation agents that force immature AML cells to mature and small molecule targets identified through reverse engineering which provide a survival advantage to leukemia clones are all also being evaluated. Although decreased risks of cytokine release syndrome and neurotoxicity among frailer patients have been quoted as advantages of NK cell therapy, its inability to overcome the immune-suppressive tumor microenvironment has prevented this strategy from producing durable remissions and efficacy . SimilarKMT2A, previously known as mixed lineage leukemia 1 or the MLL1 gene and has a paradoxical oncogenic role in the proliferation of KMT2A-rearranged leukemias. MLL1 or KMT2A translocations, found in about 5–10% of all adult AML cases, lead to a fusion protein between KMT2A and about 80 different fusion partners, all of which bind to menin at the N- terminus end of KMT2A. This interaction between menin and the KMT2A fusion protein is essential for leukemic transformation by increasing homeobox A9 (HOXA9) gene expression [NPM1) gene mutations are common and occur in 30% of all reported AML cases. NPM1 mutations lead to increased HOX pathway expression and require the interaction between KMT2A and menin for proliferation. Disruption of this interaction leads to differentiation in NPM1mut AML [KMT2A-rearranged and NPM1mut AML cell lines [NPM1 mutated/KMT2A-rearranged AML that showspromising results. Issa et al. reported updated results from the AUGMENT-101 phase 1 trial in 68 patients with heavily pretreated (median 4 lines) R/R AML who received the menin inhibitor SNDX-5613 (now known as revumenib) with dose escalation [Originally described in the context of multiple endocrine neoplasia 1 (MEN1) as a tumor suppressor, it was later found that menin regulates hematopoiesis and myeloid transformation. Chen et al. described the role of menin in normal bone marrow hematopoiesis and the proliferation of MLL-AF9 mutated leukemic cells . Menin ipression . Nucleop1mut AML . These fcalation ,12,13. GTP53-mutated AML. In this phase 1b study, using 75 mg/m2 azacitidine on days 1 to 7 of a 28-day cycle combined with a magrolimab priming dose of 1 mg/kg followed by a ramp up to 30 mg/kg weekly or fortnightly for patients with newly diagnosed TP53-mutated AML, the ORR was 48.6% in 72 patients with p53-mutated AML [TP53-mutant AML. ENHANCE-3 is a phase 3, randomized, double-blind, placebo-controlled study of magrolimab vs. placebo in combination with venetoclax and azacitidine in newly diagnosed AML. Many other CD-47-related molecules are in different stages of clinical development either in combination with chemotherapy, monoclonal antibodies, or immune checkpoint inhibitors.CD47 is a transmembrane protein expressed on a wide array of cells and interacts with its ligand signal regulatory protein alpha (SIRPα) on macrophages to inhibit phagocytosis, now widely known as a “don’t eat me” signal . Work frated AML . The medp = 0.071). OS was in fact lower with volasertib 5.6 months vs. 6.5 months (p = 0.757), likely owing to the increased grade ≥ 4 cytopenia and infections and increased death observed with volasertib. Factors like an extremely long half-life of 5 days and lack of PLK1 selectivity are being offered as possible explanations for its failure. Zeidan et al. reported that the oral, more selective PLK1 inhibitor onvansertib with either low-dose cytarabine 20 mg/m2 on days 1 to 10 or decitabine 20 mg/m2 on days 1 to 5 in R/R AML was well tolerated in their phase 1b study [Numerous signaling pathways work together to form checkpoints during cell division, especially during the transitions between the G1 and S phases and G2 and M phases. Serine-threonine kinases like cyclin-dependent kinase (CDK), Polo-like kinase (PLK), and Aurora kinases are involved in regulating mitosis at different levels . Five di1b study . The pha1b study  and is m1b study  such as 1b study . We beli1b study  could po2 on Days 1, 3, and 5 with azacitidine 75 mg/m2 on Days 1 to 5, 8, and 9 every 28 days. Grade 3 anemia and neutropenia were noted in 30% of patients along with increased liver function tests in 6% of patients with newly diagnosed AML unfit for intensive induction aged more than 60 years [2 as the phase 2 dose for pevonedistat and reported a to-date ORR of 40% [Proteasome inhibitors that target the ubiquitin-proteasome system are used in many malignancies. In multiple myeloma, they prevent the degradation of intracellular proteins. This leads to the accumulation of these proteins that trigger a cascade of events that lead to cell death. Neural cell developmentally downregulated 8 (NEDD8) is a protein homologous to ubiquitin and NEDD-activating enzyme (NAE) leads to neddylation and thereby degradation of certain proteins . However60 years . An enco60 years . Based o60 years , two sepR of 40% . Short e2 IV on days 1 to 5 with or without idasanutlin 300 mg, PO BID daily in 28-day cycles in R/R AML did not meet its primary endpoint OS [TP53 mutations are uncommon in AML accounting for 6–8% of all cases. However, overexpression of MDM2, a negative regulator of the p53 axis  causing point OS . The medpoint OS . Siremadpoint OS . A promipoint OS . The duapoint OS .TP53-mutated AML as a separate entity under “myeloid neoplasms with mutation TP53” to delineate the poor outcomes and highlight the need for better therapies irrespective of blast percent [MET) which has been shown to covalently bind to a mutated p53 protein and restore its wild-type conformation. It also increases its proapoptotic activity via induction of Bax and thereby caspase activation [2 subcutaneous injections daily from days 4 to 10 of a 28-day cycle followed by consolidation with allogenic SCT. The same combination was offered at a lower dose as a maintenance strategy. Common side effects noted were febrile neutropenia (37%) and neurological adverse events (40%). Grade 3 neurological events were reported in only 6% and were all reversible with drug discontinuation and showed no recurrence when the drug was resumed with dose reduction. Efficacy analysis showed an ORR of 33% in AML patients and 62% in MDS patients. Another parallel study from the US reported their combined results with the French GFM group at the ASH meeting in 2021 with no additional safety signals noted [2 once daily from days 1 to 5 of a 28 day cycle in TP53-mutated AML and MDS patients as a post-transplant maintenance regimen and reported that the 1-year RFS of 60% has met their prespecified hypothesis of ≥50%, as historical data correspond to 30% RFS [TP53 mutant MDS failed to meet its primary endpoint CR [p = 0.13). As a result, the company halted further development of this agent.TP53 is a tumor suppressor gene and mutations in TP53 in AML often cause poor outcomes. The international consensus classification (ICC) now has  percent . The Fre percent  of eprentivation . Maslah ls noted . Mishra  30% RFS . A phasepoint CR . In the 2 IV administered on days 1 to 3 followed by cytarabine 2 g/m2 IV on days 6–8 and mitoxantrone IV 40 mg/m2 IV on day 9 versus 7 +3 in 165 patients with newly diagnosed AML led to an increased CR rate of 70% with the TST with FLAM, compared to 46% with 7 + 3 induction (p = 0.003) [Cyclin-dependent kinases are serine-threonine kinases that act as important checkpoints in the cell cycle. Flavopiridol, also called alvocidib, a multi-CDK inhibitor, has activity against CDK4, 7, and 9 and leads to apoptosis of leukemia cells primarily due to its effects on transcriptional regulation due to CDK9 inhibition . Alvocid= 0.003) . However= 0.003) .Advancement in multiparametric flow cytometry and immunohistochemistry has led to the recognition of multiple surface antigens that help distinguish blast cells and leukemia stem cells from other progenitors and mature blood cells. Monoclonal antibodies directed against these antigens are able to activate immune effectors via the complement system, leading to antibody-dependent cellular cytotoxicity which leads to phagocytosis by antigen-presenting cells, resulting in tumor antigens being presented on major histocompatibility complex (MHC) that activate CD8+ve cytotoxic T cells.Vadastuximab was a CD33-directed antibody conjugated to pyrrolobenzodiazepine that, post successful early phase trials, underwent a phase 3, double-blind, placebo-controlled CASCADE trial where vadastuximab was given with a hypomethylating agent (azacitidine or decitabine) in comparison to only HMA in older patients with newly diagnosed AML . However225 (named Actimab-A). This was tested in a phase 1/2 trial in R/R AML given on Day 8 following a salvage regimen of CLAG-M and reported safety data on 21 patients [Bispecific engagers help bring effector cells  using the surface antibody part of their construct directly to the tumor cells recognized by a second antibody. In a phase 1 trial, AMG330 was given as a continuous infusion in 55 patients with R/R AML in a dose-step-up approach with dexamethasone prophylaxis . Cytokinpatients . Dose-li2 on days 1 to 7 and venetoclax 400 mg in 71 patients and had an ORR of 51%. Common side effects reported include febrile neutropenia in 30% of patients, pneumonia in 16%, and grade 3 or higher infusion-related reactions in 3%. APVO436 is a novel bispecific anti-CD123x anti-CD3 ADAPTIR molecule. Results from the expansion phase study in AML and MDS were presented at the ASH 2022 meeting, demonstrating the safety of APVO436 as a single agent and in combination across four different cohorts [A novel Dual Affinity Retargeting (DART) structure was utilized in the making of flotetuzumab, a bispecific antibody with CD3 and CD123 as targets. Uy et al. brought this molecule to clinical practice in their phase 1 trial on R/R AML patients . Safety  cohorts . This wa cohorts .2 on days 1 to 7 of a 28-day cycle [Tumor necrosis factor uses CD70 as its ligand. CD70 is expressed on AML LSC and is not present in normal cells or HSCs. Cusatuzumab is a monoclonal antibody against CD70 with enhanced antibody-dependent cellular cytotoxicity. Based on preclinical studies that showed HMA treatment increased expression of CD70 on LSC, Riether et al. conducted a phase 1 trial combining cusatuzumab with azacitidine 75 mg/may cycle .Mutations in the spliceosome complex are commonly found in myeloid malignancies and account for about 30% of all mutations seen in AML . Given thi cells representative of the bone marrow population was sorted by flow cytometry, the CD3 +ve cells were higher in the relapsed AML vs. new AML vs. HD as 81.1% vs. 78% vs. 60.3% (p = 0.02), but no differences were found in the CD4 +ve or CD8 +ve T cell subsets. They also noticed a significant difference in the immunosuppressive T regulatory cells in relapsed AML vs. new AML vs. HD as 3% vs. 2.1% vs. 1.7% (p = 0.02). Other markers often used as a surrogate for responses to checkpoint inhibitors like PD1-positive CD4 or CD8 T cells were higher in the order relapsed AML > newly diagnosed AML > HD (p < 0.01). They also noted that in the subset of TP53-mutated patients, there was a higher frequency of PD-L1 +ve blasts compared to AML patients with wild-type P53. This finding was further explored in the study conducted by Sallman et al. in 103 MDS or AML patients using their bone marrow mononuclear cells [p < 0.01). Sallman’s group also measured the MYC expression in relation to the PD-L1 positivity and found a positive correlation  and also found lower expression of microRNA MiR-34a in the TP53-mutant group (p < 0.01). This further supports the hypothesis put forth by Williams et al. that P53 regulates PDL1 expression through miR-34a which first influences MYC expression [2 on days 1 to 7 with or without a PD-L1 inhibitor  on day 1 of every 28-day cycle in 129 older newly diagnosed AML patients and demonstrated good tolerability [The widespread success of immunotherapy in multiple solid tumors such as melanoma and renal cell carcinoma led researchers to explore the tumor microenvironment in AML. Based on solid tumor cancer data, they agreed that bone marrow and not peripheral blood would be representative of the tumor microenvironment. Williams et al. first described the immune cell makeup in the bone marrow of AML patients compared to age-controlled healthy donors (HD) . They fopression . The incpression . This lerability . Numericrability . Dual corability  are ongorability . During Immunotherapy with adoptive/engineered T-cells called Chimeric Antigen Receptor (CAR)-T cells has had major breakthroughs in the treatment of Acute lymphoblastic leukemia  and diffAn essential vitamin for human survival, vitamin C has numerous functions in our body. It acts as a cofactor for numerous enzymes, some of which are involved in epigenetic modulation like ten eleven translocase-2 (TET2) . HematopMany giant leaps in drug discovery and development for cancer over the past decade were made, and this has also had a positive influence on AML, with over 10 molecules now FDA-approved for this cancer over this period. Many more promising newer agents are currently in the nascent stages of development and provide new a ray of hope to our patients and newer tools at our disposal. Small molecule inhibitors, antibody-drug conjugates with targeted cytotoxicity, immunotherapy, cell therapy, and advances in stem cell transplants are leading to a paradigm shift in the way we risk stratify and treat acute myeloid leukemia. Survival among older patients has significantly improved with the addition of venetoclax to the backbone of HMA therapy, however, myelosuppression and infectious complications have to be monitored closely.The recognition of preleukemic disease entities, Clonal Hematopoiesis of Indeterminate Potential (CHIP), and Clonal Cytopenia of Uncertain Significance (CCUS), and their inclusion in the WHO classification , will leThere have been several unique challenges faced by researchers who work on this disease. AML is a disease of the older population, and we still do not have a clear system or prognostic tool to help identify individuals who are unable to tolerate traditional chemotherapy and should be considered upfront for other treatment modalities. The unique tumor microenvironment and heterogeneous nature of this disease have posed challenges to the development of chemo-free options including immunotherapy, targeted therapy, and cell therapy. Participation in clinical trials has been shown to improve outcomes, however, the recent pandemic had posed unique challenges to clinical trial accruement and participation . Surviva


Medical education has enjoyed mixed fortunes nurturing professional identity formation (PIF), or how medical students think, feel and act as physicians. New data suggests that structured mentoring programs like the Palliative Medicine Initiative (PMI) may offer a means of developing PIF in a consistent manner. To better understand how a well-established structured research mentoring program shapes PIF, a study of the experiences of PMI mentees is proposed.Acknowledging PIF as a sociocultural construct, a Constructivist approach and Relativist lens were adopted for this study. In the absence of an effective tool, the Ring Theory of Personhood (RToP) and Krishna-Pisupati Model (KPM) model were used to direct this dual Systematic Evidence-Based Approach  study in designing, employing and analysing semi-structured interviews with PMI mentees and mentoring diaries. These served to capture changes in PIF over the course of the PMI’s mentoring stages.Transcripts of the interviews and mentoring diaries were concurrently analysed using content and thematic analysis. Complementary themes and categories identified from the Split Approach were combined using the Jigsaw Approach and subsequently compared with mentoring diaries in the Funnelling Process. The domains created framed the discussion.A total of 12 mentee interviews and 17 mentoring diaries were analysed, revealing two domains—PMI as a Community of Practice (CoP) and Identity Formation. The domains confirmed the centrality of a structured CoP capable of facilitating longitudinal mentoring support and supporting the Socialisation Process along the mentoring trajectory whilst cultivating personalised and enduring mentoring relationships.The provision of a consistent mentoring approach and personalised, longitudinal mentoring support guided along the mentoring trajectory by structured mentoring assessments lay the foundations for more effective mentoring programs. The onus must now be on developing assessment tools, such as a KPM-based tool, to guide support and oversight of mentoring relationships.The online version contains supplementary material available at 10.1186/s12909-023-04748-6. The team comprised of a senior female medical librarian with more than 15 years of research experience from the National University of Singapore’s (NUS) Yong Loo Lin School of Medicine (YLLSoM) and local educational experts and clinicians at Duke-NUS Medical School, the National Cancer Centre Singapore, YLLSoM, and the Palliative Care Institute Liverpool. The five male clinicians were all physicians with more than 15 years of clinical, educational and research experience whilst the male educationalists at Duke-NUS Medical School and the Palliative Care Institute held more than 20 years’ experience in medical and clinical research as trained psychologists. All members of the expert team had successfully published at least 5 studies using the SEBA methodology in peer-reviewed journals.With their primary role of guiding and overseeing the research process, the expert team was not involved in the PMI and thus did not share any contact or prior professional ties with the participants. Concurrently, the research team was tasked with carrying out the research process, data analysis and the writing of the manuscript. Both the expert and research teams were responsible for crafting the research questions.The lead author was a Palliative Care physician and a senior mentor in the PMI with an extensive repertoire of qualitative research experience and peer-reviewed journal publications in medical education and palliative care. As a senior mentor who oversaw the PMI, he was accorded an ‘insider’ role within the study, replete with built-up knowledge and pre-established immersion in the organisation . His in-However, we also recognised the potential pitfalls that came with being an insider. Prior knowledge on the nuances of the PMI could affect and bias how the lead author viewed, interpreted and analysed the data. Moreover, having a senior mentor of the PMI as the potential interviewer of the study might pose a barrier in encouraging clarifications and unconventional perspectives due to a perceived shared knowledge between the mentor interviewer and mentee participant . The leaIn circumventing these roadblocks, we practiced reflexivity by acknowledging and challenging our biases in order to advance trustworthy and honest data. This, for instance, was captured by our recruitment of two trained interviewers who, while part of the PMI, did not share working relationships with the mentees. The absence of a dependent relationship between the interviewers and participants helped mitigate social desirability bias in an ‘even playing field’. Whilst trained on current insights on mentoring and PIF concepts that facilitated effective prompts for robust interviews, the interviewers’ roles as ‘outsiders’ also heightened awareness to unchallenged assumptions otherwise glossed over in shared ‘insider’ relationships .In maintaining reflexivity in data interpretation, the semi-structured interviews were triangulated against anonymised mentoring diaries to enhance data reliability. Furthermore, the lead author consulted members of the expert and research teams comprising of experienced physician-tutors, psychologists, methodologists, and educational scholars to check for any personal biases and attenuate his impact as a senior PMI mentor on the study. The backgrounds of the research and expert teams are detailed in Table To ensure input from all members of the team, synchronous and asynchronous in-person and online meetings and Sandelowski and Barroso ’s approaIn the absence of an effective tool to evaluate PIF, the expert team advised on the design of semi-structured interview questions drawn from recently published reviews and articles on PIF and mentoring that focused on the PMI framework , 20, 38,The combined summaries from Stage 2 guided the design of semi-structured interviews and peer-mentoring diaries around the KPM and RToP , 28. A cRecruited through purposive sampling, eligible participants for this study comprised of all PMI mentees in order to best elucidate and capture lived experiences of mentoring and PIF within a structured program. All PMI mentees were sent email invitations by the interviewers which contained the aims of the study, as well as their rights to privacy, anonymity and to withdraw without prejudice.The semi-structured interviews were conducted over the Zoom video conferencing platform between February and May 2021 by experienced and trained interviewers, AP and CQWL. These 30–45-min online interviews took place in vacant offices that fostered a conducive environment for safe and private in-depth conversations between the interviewers and participants. The two female interviewers did not hold any previous contact with the participants. The participants were informed that both interviewers were experienced in carrying out semi-structured interviews and were employed and trained on current insights on PIF and mentoring for the purposes of this study. Participants consented to having their interviews audio-recorded by a password-encrypted audio-recording device. Using the NVivo 12 Software, all audio recordings were subsequently transcribed verbatim. All transcripts were anonymised.The peer-mentoring diaries were hosted on Google Forms and were completed between March to December 2021. Anonymisation of mentoring diaries for analysis was expediated by independent research team members not involved the PMI or the semi-structured interviews.Ethics approval (reference number: 202010–00084 and 202103–00057) was obtained from the Singhealth Combined Institutional Review Board. Informed written and oral consent was obtained from all participants.Facilitated by three independent teams of researchers, the Split Approach involved the simultaneous thematic and directed content analysis of the data gathered from the semi-structured interviews and mentoring diaries. The concurrent application of thematic and directed content analysis helped minimise biases, streamline interpretations of terminology by different research team members and address the shortcomings of each method of data analysis. The use of content analysis, for instance, accounted for thematic analysis oft-ignored contradictory data –53 and iThe Split Approach also attended to the neglect of Cohen’s Kappa to assess the degree of consensus between researchers coding the same data . In partIn analysing data collated from the semi-structured interviews, the first team of researchers adopted Braun and Clarke ’s approaTo begin, researchers first actively read the anonymised transcripts to discern meaningful patterns in the data –63. Subs“A scoping review of professional identity formation in undergraduate medical education” and Teo et al. [“Assessing professional identity formation (PIF) amongst medical students in Oncology and Palliative Medicine postings: a SEBA guided scoping review”. Dubbed the ‘coding agenda’ [Simultaneously, the second team of researchers utilised Hsieh and Shannon ’s approao et al. ’s review agenda’ , 66, the agenda’ , 67–70 w agenda’ . Consens agenda’ , 71.Notably, Hsieh and Hannon’s approach  to direcRepeating the steps of the two methods detailed above, the third research team carried out thematic and content analysis of the peer-mentoring diaries.Central to the Jigsaw Perspective were Phases 4 to 6 of France et al. 's adaptaThus, the identified themes and categories were envisioned as pieces of a jigsaw puzzle wherein complementary pieces were merged to paint broader themes. Researchers compared themes and subthemes with the categories and subcategories identified. Similarities were verified through comparisons of the codes contained within them. Should they be complementary in nature, the subtheme and subcategory were combined to create a bigger piece of the jigsaw puzzle.The Funneling Process centered on the latter aspects of Noblit et al. 's seven A total of 20 mentees fulfilled the criteria for participation. Six mentees declined to participate in light of preparations for their examinations. Two mentees did not reply in time. A total of 12 mentees thus participated in the semi-structured audio-recorded interviews while 17 mentees contributed to the mentoring diaries. Table The interviews and mentoring diaries were evaluated independently. There were no repeat interviews and the concomitant transcripts were member-checked before anonymisation by the interviewers for analysis by the research team.The two domains identified from Stage 7 were: 1) the PMI as a Community of Practice, and 2) Identity Formation.An initial review of the data indicated that the PMI did display features of a CoP identified in Cruess’ influential paper . KeepingTable Table Table Based on Venkataramana et al. ’s study,“Knowing that our research topic is an important and relevant one and that keeps me going!” (M5)“This experience made me recognise that the research we are doing isn’t just distant and theoretical, but has important implications in clinical practice as well.” (MD5)Here, mentees’ regnant values, beliefs and principles were consistent with those of the projects, thus ensuring continued engagement:“Shared common values… pass it on… and pay it forward… really resonated with me, especially when an established senior clinician still holds these values and is actively seeking ways to pay it forward… That's the role model that I aspire to be… that's why I joined.” (M9)“My SM (senior mentor) shared stories about people in his life and showed us why our research was important. At the point, I was starting to feel a little jaded and that meeting was important to remind me the meaning behind our work and gave me a renewed sense of determination to come up with something that would benefit doctors facing the same issue.” (MD1)“It has shown me how research and its findings can contribute to the current practices within society and that medical students can also play a role in this. I feel like I would be able to make a difference to the medical community and patients if my work is able to get published and perhaps I could be recognised for my work potentially.” (MD17)“Through our mentoring relationship, I learnt to be a lot more open-minded about things in general, not just in medicine. So, you learn to see things beyond what they are. That's another thing. Resilience because it ties in together with open-mindedness because... partly why I started exploring other non-clinical options for career progression is because I struggled a lot during housemanship.” (M1)Shared values, beliefs and principles empowered mentees to act:“I didn't do that well for one of my assessments for school and then, I felt that I didn't know if I was capable of taking up so many, too many things at once. And at first, I felt afraid to speak to my mentor about my academic difficulties. Because I was afraid that maybe I wouldn't get opportunities in the future because I cannot juggle my academics with my extra-curricular stuff.” (M3)“I feel like I learned that it's very important to dedicate a right amount of time to doing the work because we might feel that, okay, I can finish this within a week. But then, I realised that we shouldn't set such a solid time because you might not know that we actually need more time to go more in detail and find out more about the paper and analyse it to a deeper extent.” (M4)In the Individual Ring, participants encountered the need to balance personal goals with their own expectations: “I was very afraid to ask questions and clarify because I was scared that I'll be burdening the team or hampering the effectiveness in the whole efficiency process.” (M3)In the Societal Ring, conflicts between socially-inspired self-expectations and obligations to the team also emerged:Disharmony was reflected in the conflicts that occurred within a single ring of the RToP:“I was neglecting family more… I wouldn't say I'm balancing my time well because I’m putting other things on hold.” (M6)“I guess I feel more inclined to take on more work, especially in the smaller team of three. I felt bad because you can't say “I have to take less work because I've other commitments outside” … Because if I don't do it, someone else has to do it and I'm sure everyone is equally busy as well.” (M8)Between the Individual and Relational RingsBetween the Individual and Societal RingDyssynchrony was evidenced in the conflicts experienced between different rings of the RToP:“Not having a say over the topics you can get to do or how many papers you get to write… because all of the important critical decisions were made at the top and the information is disseminated downwards.” (M6)“I was quite scared of seniors in a sense, I didn't really dare to chat seniors up so much and ask things beyond the curriculum.” (M1)Here, mentees highlighted the tensions between being in a hierarchical program and their desire to partake in the research process more autonomously or discuss matters outside of research:How do mentees develop PIF in a structured mentoring program?”.Drawing upon data in Tables knowledge base, set of beliefs, values, history and experiences” [As a CoP replete with a structured mentoring approach, clear boundaries and competency-based assessments that direct personalised, longitudinal, timely and holistic mentoring support through trained mentors, accessible communication platforms and a curated mentoring environment, the PMI is able to aptly support the Socialisation Process. In turn, the inculcation of the PMI’s shared “riences”  is made riences” –87. NotaThis new theory depicts the PMI as a circle, containing two smaller concentric circles reflecting the graduated progress—first from mentee to peer-mentor and subsequently, from peer-mentor to the mentor’s role at the heart of the PMI. The structure within the CoP is also supplemented by data on the gradual progress from legitimate peripheral participation upon entry into the PMI towards a more central role. This gradual growth is constructed around the PMI’s consistent framework and supported by timely, longitudinal and personalised mentoring support . SuccessUpon further training and achievement of these wider skills, knowledge and competencies, ‘senior’ mentees may enter the second ring within the CoP to become peer-mentors. Finally, following a period of mentored oversight of their progress and successful completion of a number of PMI project, alongside achievements of mentoring, leadership, assessment and supportive skills, the peer-mentor may be invited to be a mentor and take their place at the heart of the CoP. It is the mentor and peer-mentor who influence the culture, structure, and environment of the CoP, as well as the mentoring approach and relationships within the PMI. This can be seen from the results in Tables Depicting the course through the PMI as a gradual spiral-like process, this proposed theory of PIF in the PMI also aptly discusses the Socialisation Process. Here, we present two spiral trajectories. The size of the spiral represents the speed—a slower progression or rapid adaptation—towards the centre of the PMI. Delineated by the ‘closed’ yellow spiral, this first spiral comprises the ideal PIF trajectory wherein the mentee’s progress occurs uninterrupted towards the centre of the PMI. This is especially so if the mentoring program’s values and practices are synchronous with the mentee’s own identity. This is contrasted against the second spiral in Fig. belief system due to developing competencies, experiences and insights. Together, this leads to a more ‘stable’ belief system that is less likely to require significant adaptations which, in turn, requires less significant identity work and thus, births more consistent self-concepts of personhood and identity.Moreover, the second spiral highlights two critical considerations. One, the import of longitudinal support, assessment and oversight by trained peer-mentors and mentors. Two, the attenuation of the magnitude of adaptations to the The changing nature of these two considerations underscores the importance of longitudinal assessments. With stage-specific mentoring outcomes used as proxy for absent PIF tools, much of it would seem to rest on the shoulders of peer-mentors and mentors. Presently, stage-based mentoring outputs are employed to assess PIF. This underlines the importance of effective selection, mentor and peer-mentor training, matching, longitudinal support and protected time to carry out their mentoring duties. This also highlights the potential for mentoring portfolios that include reflective diaries, stage-based mentoring assessments and reviews by mentors, peer-mentors and peers within the project. Concurrently, the need for adaptive, personalised and timely support of mentees is clear as more robust belief systems and more consistent concepts of professional identity develop. This gradual development of a nuanced context-specific identity and inculcation of PMI values may culminate in an invitation to become a peer-mentor whilst the experiences, insights and growing confidence give mentees a greater sense of ‘belonging’ or identification as part of the PMI, echoing Wenger’s notion of ‘modes of identification’ .How do mentees develop PIF in a structured mentoring program?”. Indeed, this Dual-SEBA guided study underlines the importance of assessment driven, longitudinal, adaptive, individualised and timely mentoring umbrella-based support platformed on an enduring and personalised mentoring relationship. It also underlines that other than the structure and environment of the CoP-like features of the PMI and the role of the curated mentoring environment in nurturing PIF, programs like the PMI rely heavily upon its trained mentors, peer-mentors and its host organisation’s support of them.These insights help answer our primary research question, “This qualitative study has utilised interviews to garner greater insight into the experiences of mentees within the PMI. However, there are some limitations. Whilst it has served to highlight the key elements of PIF, this is first time that the SEBA methodology has been employed in a prospective study, thus requiring further study. Second, whilst the SEBA methodology’s use of independent research and external expert teams helps assuage some issues of bias, continued concerns of bias surrounding some authors’ involvement in the PMI cannot be completely attenuated. Lastly, the use of interviews and mentoring diaries functions as snapshots in time or retrospective recounts which may limit the depth of the data collected.A critical feature to be added to this triumvirate of mentoring programs behaving like a CoP supporting the Socialisation Process; the development of enduring and personalised mentoring relationships; and the provision of personalised mentoring umbrella-based support by trained faculty is the need for effective assessments of PIF. As a result, any program in Palliative Medicine and beyond hoping to nurture PIF in a consistent manner requires effective assessment processes built around clear program boundaries; robust and an agreed upon set of expectations and codes of conduct; a well-described mentoring trajectory; aligned expectations; personalised, appropriate and timely application of support akin to the mentoring umbrella; and a nurturing environment. This then will form the focus of our coming research as we look forward to deeper discussions on this increasingly critical area of medical education.Additional file 1.


However, a recent study showed that flecainide may also prevent fibro-adipogenic differentiation by inhibiting store-operated Ca2+ entry (SOCE) and thereby suppressing spontaneous Ca2+ oscillations in C-MSCs isolated from human ACM patients (ACM C-hMSCs). Herein, we briefly survey ACM pathogenesis and therapies and then recapitulate the main molecular mechanisms targeted by flecainide to mitigate arrhythmic events, including Nav1.5 and RyR2. Subsequently, we describe the role of spontaneous Ca2+ oscillations in determining MSC fate. Next, we discuss recent work showing that spontaneous Ca2+ oscillations in ACM C-hMSCs are accelerated to stimulate their fibro-adipogenic differentiation. Finally, we describe the evidence that flecainide suppresses spontaneous Ca2+ oscillations and fibro-adipogenic differentiation in ACM C-hMSCs by inhibiting constitutive SOCE.Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder that may lead patients to sudden cell death through the occurrence of ventricular arrhythmias. ACM is characterised by the progressive substitution of cardiomyocytes with fibrofatty scar tissue that predisposes the heart to life-threatening arrhythmic events. Cardiac mesenchymal stromal cells (C-MSCs) contribute to the ACM by differentiating into fibroblasts and adipocytes, thereby supporting aberrant remodelling of the cardiac structure. Flecainide is an I Ot. OtPKP2)with ACM  , owith ACM  ; hc AAD flecainide ((RS)-N-(piperidin-2-ylmethyl)-2,5-bis benzamide); C17H20F6N2O3) ,els  study . The CAShythmias . Neverthhythmias ,36,37,38hythmias , confirmhythmias . In addihythmias .v1.5 channels represents the most obvious mechanism to explain the anti-arrhythmic effects exerted by flecainide in ACM i) regulate MSC differentiation into a variety of cells belonging to multiple lineages ER) in proximity of discrete sub-compartments of the peripheral ER falls below a threshold concentration because of ER Ca2+ release through InsP3Rs [2+]ER, and Orai, which contributes the Ca2+-selective channel protein on the plasma membrane [2+]ER stimulates STIM proteins to assemble into oligomers that redistribute to peripheral ER-plasma membrane junctions, known as puncta, while extending their cytosolic COOH-termini, which contain the STIM Orai-activating region/CRAC-activating domain (SOAR/CAD), towards the inner leaflet of the plasma membrane  CRAC is t+ levels . Convers content ,71 as we content ,72. In ativation ,74,75. Ove cells ,77,78. Tve cells ,55,56, wM-mMSCs) ,80,81 anM-mMSCs) .2+ oscillations have been described not only in BM-hMSCs ER, also because of SERCA2B up-regulation, in ACM C-hMSCs; (4) the enhanced ER Ca2+ loading, on the one hand, sensitises InsP3Rs to open at a higher frequency; on the other hand, it increases the availability of free intraluminal Ca2+ that can be released during each Ca2+ spike. In this context, it is worth clarifying that, although the SOCE signal driving the spontaneous Ca2+ oscillations in C-hMSCs has been termed constitutive i not only in C-hMSCs but also in many other cell types i, the Ca2+/CaM complex can bind to two neighbouring subunits, thereby enabling CaMKII to autophosphorylate on threonine 287 (Thr287). Autophosphorylation traps Ca2+/CaM and prolongs CaMKII activity even when the [Ca2+]i returns to the baseline. Low-frequency Ca2+ transients are less effective than high-frequency Ca2+ transients to evoke autophosphorylation and Ca2+-independent activity since the Ca2+/CaM complex can dissociate from the holoenzyme between two consecutive Ca2+ spikes i returns to the baseline [2+ overload since it has long been known that CaMKII is able to stimulate SOCE [2+ oscillations and fibro-adipogenic differentiation in ACM C-hMSCs [The mechanism by which flecainide inhibits SOCE requires further investigation. Likewise, the molecular determinants that increase constitutive SOCE may go beyond STIM1 overexpression and include the derangement of other regulatory factors, such as an increase in STIM1 expression or a reduction in Orai2 or Orai3 protein levels. Nevertheless, these preliminary findings strongly hint at flecainide as a pleiotropic drug that owes its efficacy to its ability to target multiple pro-arrhythmic signalling pathways in ACM. In vivo investigations will have to assess whether it does interfere with cardiac fibrofatty remodelling in transgenic mouse models of ACM. It will also be imperative to assess whether flecainide does interfere with CaMKII hyperactivation in ACM C-MSCs and, if so, to untangle the signalling pathways that are recruited by CaMKII to stimulate fibro-adipogenic differentiation. In accordance with this, Maione et al. did not assess whether inhibiting SOCE or InsPbaseline ,148. Thiate SOCE ,150,151.c AAD that proved to be effective in the management of heart rate by targeting NaV1.5, which mediates the fast inward Na+ current, and RyR2, which mediates aberrant SR Ca2+ release. Flecainide is emerging as a promising therapeutic tool to treat ACM, which involves a complex remodelling of the Ca2+-handling machinery in ventricular cardiomyocytes. In addition, studies conducted on C-MSCs isolated from ACM patients and transgenic mouse models of ACM revealed that intracellular Ca2+ dynamics is also dysregulated in the stromal compartment of the heart. SOCE, which represents the main intracellular Ca2+ entry pathway in non-excitable cells, is constitutively activated in ACM C-hMSCs and causes Ca2+ overload in the InsP3-sensitive ER Ca2+ reservoir. This results in an increase in both the amplitude and frequency of spontaneous Ca2+ oscillations that finely tune MSC fate, thereby favouring fibro-adipogenic differentiation in ACM C-hMSCs. Therefore, SOCE up-regulation in C-hMSCs could play a pathogenic role in ACM by stimulating the cardiac structural remodelling that increases the incidence of arrhythmic events. Intriguingly, flecainide, when applied at doses known to inhibit Nav1.2 and RyR2 channels, inhibited constitutive SOCE, thereby suppressing the spontaneous Ca2+ oscillations that occur during fibro-adipogenic differentiation and preventing ACM C-hMSC differentiation into fibroblasts or adipocytes. These pieces of evidence provide the first proof-of-concept that flecainide is also effective against SOCE and indicate that SOCE could be targeted for therapeutic purposes in ACM.Flecainide is a class I


Inositol 1,4,5-trisphosphate receptors (IP3Rs) on the ER, glucose-regulated protein 75 (GRP 75) and voltage-dependent anion channel 1 (VDAC1) on the outer mitochondrial membrane are the canonical component of the Ca2+ transfer unit at ERMCS. These are often reported to form a Ca2+ funnel that fuels the mitochondrial low-affinity Ca2+ uptake system. We assess the available evidence on the IP3R subtype selectivity at the ERMCS and consider if IP3Rs have other roles at the ERMCS beyond providing Ca2+. Growing evidence suggests that all three IP3R subtypes can localise and regulate Ca2+ signalling at ERMCS. Furthermore, IP3Rs may be structurally important for assembly of the ERMCS in addition to their role in providing Ca2+ at these sites. Evidence that various binding partners regulate the assembly and Ca2+ transfer at ERMCS populated by IP3R-GRP75-VDAC1, suggesting that cells have evolved mechanisms that stabilise these junctions forming a Ca2+ microdomain that is required to fuel mitochondrial Ca2+ uptake.Membrane contact sites (MCS) circumvent the topological constraints of functional coupling between different membrane-bound organelles by providing a means of communication and exchange of materials. One of the most characterised contact sites in the cell is that between the endoplasmic reticulum and the mitochondrial (ERMCS) whose function is to couple cellular Ca For cellular homeostasis, intracellular compartments are required to cooperate via functional junctions in order to regulate various processes including Cafficking , glucosefficking , mitochofficking , autophafficking , organelfficking  and varifficking . MCS arefficking . First rfficking . Tetheri2+ to the mitochondria. Specifically, we will highlight the role and regulation of component proteins that drive the stability and delivery of Ca2+ at these junctions.The composition of the ER–mitochondrial MCS (ERMCS) has been thoroughly reviewed previously . The mor2+ signals is one of the most characterised functions of these contact sites. Ca2+ must permeate the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM) to reach the mitochondrial matrix. The OMM has a high Ca2+ permeability. This is possibly due to its abundant expression of voltage-dependent anion-selective channel proteins (VDACs) (of which there are 3 subtypes (VDAC1-3)); although VDAC1 is predominantly implicated in mitochondrial Ca2+ dynamics, it was shown to selectively co-immunoprecipitate with inositol 1,4,5-trisphosphate receptors  was identified as the mediator of Ca2+ transport through the IMM into the mitochondrial matrix ,  is evolutionary important to avoid unregulated Ca2+ overload of the mitochondria for triggering apoptosis . There moverload . Followil matrix . The Ca2. 2018), ). The lopoptosis . The acco the ER . Betweeno the ER . The canchondria . It is iunctions .2+ at ERMCS not only shapes the spatiotemporal properties of global Ca2+ signals but also directly affects mitochondria functions. For example, the activity of three of the important enzymes in the electron transport chain pyruvate dehydrogenase, isocitrate dehydrogenase, oxoglutarate dehydrogenase (OGDH) and α-ketoglutarate dehydrogenase is Ca2+-dependent  and release into the cytosol of apoptotic factors such as cytochrome c, procaspase 9 and apoptosis-inducing factor 9 . The loss of KRAP reduces the number of immobile IP3R clusters in a cell, abrogates Ca2+ puffs and global Ca2+ signals  between the ER and mitochondria and re-expression of IP3Rs including a non-Ca2+ conducting mutant in HEK293 cells rescued the contacts between the ER and mitochondria  is an important regulator of T cell development in the thymus which shares sequence homology  with KRAP  under physiological conditions. However, upon ER stress, such as during ER Ca2+ depletion, sigma-1 dissociates from BiP and translocates to ERMCS, stabilising IP3R3 at these junctions and promoting the prolonged release of Ca2+ from the ER into the mitochondria  ovarian killer (BOK) is an ER-resident pro-apoptotic protein. It has been shown to bind IPstimulus . BOK is stimulus . The loscroscopy . Furthertenuated . Converson assay . The BOKse cells . Altoget3R2  and regulate IP3R-mediated Ca2+ transfer and cellular bioenergetics (3R at these junctions.The translocase of the outer membrane (TOM) family of proteins accounts for a significant proportion of the OMM protein content. TOM proteins, together with the translocase of the inner membrane (TIM), are key regulators of the trafficking of mitochondrial proteins encoded in the nucleus . Unlike ergetics . Of noteergetics . This su3R3–GRP75 interaction in MEFs, suggesting that TG2 is a negative regulator of IP3R-GRP75  is an important mediator of post-translational modifications of proteins and localises to various cellular compartments including the cytosol, mitochondria and nucleus . Mass sp3R-GRP75 . TG2 alsmulation . Althoug reduced . One arg2+ to the mitochondria via IP3Rs in ER stress  . AlthougR stress . Depleti signals . Althoug3R, VDAC and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) during starvation  are serine/threonine kinases mainly located in the mitochondria where they regulate the activity of pyruvate dehydrogenase (PDH) in glycolysis . There ale cells . Overexpat ERMCS . The kinat ERMCS . Inhibit content . Using mchondria . This sul matrix , therefoic cells , but it 2+ exchange have been described in metabolic regulation, neurodegenerative disease models and in cancer. In metabolic regulation, abrogation of the tethers VAPB on the ER membrane and protein tyrosine phosphatase interacting protein 51 (PTPIP51) on the mitochondrial membrane at ERMCS that regulate autophagy attenuates the IP3R3–VDAC1 interaction and subsequent IP3R mediated Ca2+ signals to the mitochondria  recruitment  changes the spatial distribution of IPtoxicity . Furtherunctions . DJ1 regechanism . Actin pruitment .3R-mediated Ca2+ to the mitochondria. In addition, Furthermore, FATE1 has also been suggested to attenuate apoptosis in these adrenocortical carcinoma cells , which encodes a cancer-testis antigen has been shown to localise to MAMs . There ima cells . Upregul3R-GRP75-VDAC complex at ERMCS mediate Ca2+ exchange in various cell types. This poses the question of how ERMCS are regulated? Furthermore, the dogma that the IP3R–GRP75–VDAC tripartite complex forms a funnel through which Ca2+ is exchanged from the ER to the mitochondria, requires closer scrutiny. Firstly, there is evidence that other binding partners can interact with IP3R to form ERMCS where Ca2+ exchange occurs, as is the case for IP3R interacting with the AKAP1 transmembrane domain or TOM70 on the mitochondrial membrane (3R–GRP75–VDAC complex (SECTION 3.3); therefore, TOM70 may well be a coincident reporter of the same IP3R–GRP75–VDAC junctions. Secondly, disruption of other tethering complexes such as between VAPB and PTPIP51 on the OMM, also disrupts Ca2+ uptake into the mitochondria -N,N,N′,N′-tetraacetic acid (EGTA)  . A tight2+ microdomain is maintained by a complex of different tethers and binding proteins working synergistically to promote ERMCS contact sites where IP3R sits to power the low-affinity mitochondrial Ca2+ uptake mechanism. Further work is required to elucidate the interdependence of various tethering complexes on mitochondrial Ca2+ uptake and their role in health and disease. Whilst current technologies are limited in their capabilities, it is also important to assess how the individual tethering complexes at ERMCS change simultaneously in response to metabolic demands, as it will help answer the longstanding questions around the role of the multiple complexes found at these junctions.We suggest that the ER–mitochondrial Ca


Syphilis has resurged in many countries, which has called attention to vaccine development. Based on the immunization-based rabbit model of infection with the Nichols strain, this study explored the protective immune response of a controversial syphilis vaccine candidate, TprK, and found that immunization with full-length rTprK was effective in attenuating lesion development and accelerating lesion resolution, which could reduce the probability of the pathogen spreading to distant tissue sites to prevent the progression of the disease to some extent. Furthermore, the results revealed that immunization with rTprK not only rapidly induced a strong Th1-like cellular response but also elicited a humoral immune response to produce opsonic antibodies to enhance macrophage-mediated opsonophagocytosis. Although complete protection against infection was not achieved, the study provided a comprehensive and in-depth exploration of the immunogenicity of TprK and highlighted the importance of TprK as a promising syphilis vaccine component. Treponema pallidum subsp. pallidum , threatening the health of people all over the world2. Although penicillin is effective for treatment, the incidence of syphilis has dramatically increased in many countries5. This has called attention to the urgent need for a vaccine for global containment strategies.Syphilis is a systemic disease caused by T. pallidum without affecting highly labile immunogenic antigens to achieve complete protection against the homologous strain of T. pallidum, the importance of antigens in T. pallidum as syphilis vaccine candidates has been highlighted6. Among them, the surfaced-exposed proteins have attracted attention, however, research has been greatly hampered by the incredibly slow growth of the pathogen and the paucity of surface-exposed proteins8. With the development of structural modeling of proteins, more information about putative surface-exposed proteins as candidate vaccinogens is being obtained9. Among them, TprK may be a controversial protein not only for the location of the proteins but also for its role in syphilis vaccine12. An early study using syphilitic infection experiments showed that immunization with a fragment (aa 37–348) of TprK attenuated syphilitic lesion development, and the variable domain was a target of opsonic antibody to promote phagocytosis13. However, the result was not reproduced in another laboratory, which used a 100-fold lower challenge dose but failed to observe any alteration of lesion development by immunization with variable domains of TprK, and the anti-TprK antibody did not significantly enhance phagocytosis14. Although subsequent research used the N-terminal portion (aa 37–273) of TprK for immunization, showing that immunization with the N-terminal portion of TprK retarded lesion development15, the protective immune response induced by immunization with TprK requires careful and comprehensive exploration.Indeed, since the Miller study using γ-irradiation to treat freshly isolated tprK gene of the standard Nichols strain, which was revealed to have almost a single tprK sequence17, was used as a template for the expression of recombinant TprK protein (rTprK). By employing immunized-infected rabbit models, we sought to fully investigate the role of TprK in the protective immune response against T. pallidum and revealed the induced cellular and humoral immune response, thus providing important information for understanding the immunogenicity of TprK.Here, the full-length T. pallidum, the challenge sites of each rabbit were monitored. The immunized rabbits first developed small and measurable erythema at 3–5 days postchallenge, and the erythema became more obvious in the next two days, showing clinical evidence of a typical DTH response. For the control rabbits, a delayed and atypical DTH response appeared at 5–7 days postchallenge. Then, with the DTH response of the two groups resolved, the control lesions enlarged rapidly and developed typical chancres, and the mean diameter of lesions between the immunized and control rabbits gradually diverged at 7–15 days postchallenge. At 15 days postchallenge, the mean diameter of lesions in the immunized rabbits was significantly smaller than that in the control rabbits . Subsequently, the lesions of the two groups of rabbits gradually shrank. At 30 days postchallenge, all the lesions in the immunized rabbits were unmeasurable, while the control rabbits still had visible lesions, and the mean diameter of the lesions was 3.95 ± 4.86 mm. Significant differences in the overall diameter development of the lesion between the immunized and control rabbits were observed  at 23 days postchallenge in the immunized rabbits. However, the lesions in the control rabbits were more severe, and 100% (50/50) of lesion sites in the control rabbits progressed to ulceration at 23 days postchallenge. There were significant differences in the proportion of lesions progressing to ulceration between the immunized and control rabbits (P < 0.001). At 30 days postchallenge, all the lesion sites in the immunized rabbits were completely resolved. The control rabbits still had ulceration proportion of 38.0% (19/50) and induration and resolution proportions of 8.0% (4/50) and 54.0% (27/50), respectively , and analysis of T. pallidum RNA yielded similar results (P < 0.001–0.01). Then, the T. pallidum load in the blood of all rabbits was measured every four days, and T. pallidum DNA could be detected in the blood of all rabbits over 30 days. Nevertheless, the overall trend for the T. pallidum burden in the bloodstream was reduced to a greater extent in the rTprK-immunized rabbits (P < 0.001), and the immunized rabbits exhibited less T. pallidum in the bloodstream at every time point  , although the T. pallidum concentration in the two groups was slightly low.To better assess the protective ability of immunization with rTprK, qPCR was performed to determine the 01) Fig. . HoweverIn addition, hematoxylin and eosin (H&E) staining was used to visualize histopathological changes in the tissues at 30 days postchallenge. Examination of the testis, as an easily invaded organ, showed that seminiferous tubules in the control rabbits exhibited atrophy, irregular arrangement, and thickening of the interstation. The degree of inflammatory infiltration in the testis and liver of the rTprK-immunized rabbits was decreased. Notably, abundant immune cell infiltration was observed in the spleen of the immunized rabbits, while the cells in the control rabbits were relatively sparse . None of the recipient rabbits of the immunized groups develop orchitis, although there was no significant difference observed (0% (0/5) vs. 60% (3/5), P > 0.05). Besides, the recipient rabbits of the immunized groups showed less probability to be detected motile T. pallidum in the dark-field microscopy than the recipient rabbits of the control groups (20% (1/5) vs.100% (5/5), P < 0.05) . Their expression was significantly different at 7, 15, 23, and 30 days postchallenge (P < 0.05), especially at 7 days postchallenge, when their secretion was robustly increased  were isolated from the immunized and control rabbits, and rTprK was applied to stimulate PBMCs. Higher proliferation of T cells was found in the cells from the immunized rabbits . For the secretions of the Th2-like cytokines IL-10 and IL-4, there were also no significant differences found between the two groups  were also obtained from the two groups of rabbits, in which the immunized rabbits maintained a steady high anti-rTprK antibody titer ; in contrast, the control rabbits only produced a low level of anti-rTprK antibody titer (lower than 1:6400)  of the two groups was positive, the recipient rabbits of the immunized groups did not develop orchitis and took a longer time to seroconvert, which indicated that immunization with rTprK quickly mobilized the immune reaction and executed the clearance of pathogens at primary sites to prevent the progression of the disease to some extent.Generally, syphilis transmission occurs by contact with infectious lesionsT. pallidum22. In the study, within 7 days postchallenge, a typical DTH response was observed in the rTprK-immunized rabbits. Meanwhile, the levels of the Th1-like cytokines IFN-γ, TNF-α, and IL-12 were significantly increased in the sera of rTprK-immunized rabbits, and at 7 days postchallenge, the secretion of these cytokines peaked. IFN-γ is a marker of the DTH response, and the significantly increased expression of this marker corroborated the occurrence of a typical DTH response in the rTprK-immunized rabbits. There was also some indirect evidence of abundant immune cell infiltration in the spleen of the rTprK-immunized rabbits, suggesting that the cellular immune response in the immunized rabbits was intense. In combination with the significantly reduced number of T. pallidum in lesions of the immunized rabbits, the results indicated that immunized rTprK could rapidly evoke the host to robustly activate the Th1 immune response to limit the number of T. pallidum in lesions, thus altering lesion development23.Based on the examination of histopathology of primary lesions in human syphilis infection as well as rabbit infection models, the production of Th1-skewed cytokines to induce a strong DTH response was critical for limiting the number of T. pallidum due to the presence of opsonic antibodies that facilitate the ingestion and killing of T. pallidum by macrophages25. However, whether TprK is a target of the opsonic antibodies has been a subject of debate between two laboratories14. They both applied rabbit peritoneal macrophages incubated with T. pallidum in the presence of rabbit anti-TprK antisera, which was directed to the purified recombinant variable domain of rTprK, but their findings were opposite, with one demonstrating that the anti-TprK antisera could opsonize T. pallidum (Nichols strain) for phagocytosis and the other demonstrating that the anti-TprK antisera did not promote phagocytosis. The reason is still unclear. Given the current diversity and sophistication of research tools, we applied flow cytometry analysis and indirect immunofluorescence microscopy to analyze the role of anti-rTprK sera. The opsonophagocytosis results showed that macrophage-mediated opsonization of T. pallidum with prechallenge sera from the rTprK-immunized rabbits led to a higher phagocytosis rate. Furthermore, with postchallenge sera from the immunized rabbits, macrophage-mediated opsonization of T. pallidum was significantly strengthened. The findings proved that antibodies against rTprK could enhance opsonophagocytosis of T. pallidum.The humoral response is thought to be important in the clearance of T. pallidum strains with highly variable tprK genes. In addition, it would be better to prolong the observation period, more information could be obtained which might help to understand the battle between T. pallidum and host immune response. And the study could not explain why more T. pallidum was detected in the spleen of the immunized rabbits and whether it was related to abundant immune cell infiltration in the spleen of the immunized rabbits. More work is needed to determine the reason.Although this study exhibited the whole immune response by rTprK immunization, it should be noted that the exploration of Th1-cell responses is still insufficient. More research on the Th1 cell responses induced by TprK needs to be performed. More importantly, the study did not further investigate the epitopes directed to the T-cell response and B-cell response, which would shed light on the development of effective measures to cope with clinical T. pallidum was effective in attenuating lesion progression and reducing the T. pallidum burden. Immunized rTprK not only induced a strong Th1-like cellular response but also produced opsonic antibodies to enhance macrophage-mediated opsonophagocytosis. Although complete protection against infection was not achieved in this study, the study provided a comprehensive and in-depth exploration of the immunogenicity of TprK and highlighted the importance of TprK as a promising syphilis vaccine component. Moreover, the study provides a good foundation for further investigation of the effect of variations in TprK on the immune response.In summary, the immunogenicity of full-length rTprK against This study was approved by the Institutional Ethics Committee of Zhongshan Hospital of Xiamen University, School of Medicine, Xiamen University. All rabbit experimental protocols strictly followed the parameters outlined by the Institutional Animal Care and Use Committee and were approved by the Animal Experimental Ethics Committee of the School of Medicine, Xiamen University.T. pallidum Nichols Seattle strain was kindly provided by Lorenzo Giacani, Ph.D. , and the frozen spirochete was propagated intratesticularly in New Zealand White rabbits and the Nichols strain was harvested at peak orchitis26.The strain was propagated for three rapid passages and used for subsequent challenge experiments.The tprK gene amplification was performed using the following primers: sense 5′- ATGGGTCGCGGCGAATTCATGATTGACCCATCTG- 3′ and antisense 5′- GTGGTGGTGGTGGTGCTCGAGCTACCAAATCAAGCGAC- 3′27. The amplicon was cloned into the pET28a vector  with an N-terminal 6×His tag between the EcoRI and XhoI sites and transformed into the E. coli expression strain BL21(DE3)  according to the manufacturer’s instructions.DNA extraction was directly from the residuals of the frozen spirochete suspension and full-length E. coli cells were pelleted by centrifugation at 6000×g for 10 min, 4 °C and lysed in buffer A . Then cell suspensions were collected by centrifugation at 12,000×g for 20 min 4 °C and resuspended in buffer B . Resulting samples were loaded onto affinity chromatography columns loaded with Ni-nitrilotriacetic acid (Ni-NTA) agarose . The eluted step was performed with 10 mL elution buffer . By SDS-PAGE analysis and Coomassie blue staining, the elute fractions were determined whether contained contaminating proteins. Then the fractions were dialyzed for 48 h at 4 °C with the appropriate molecular weight cut-off against 1× phosphate-buffered saline (PBS) containing gradually decreasing concentrations of elution buffer and concentrated using a spin concentrator with further filtering 15 kDa proteins . After dialysis, soluble proteins were analyzed by western blot using anti-His tag antibody, sera from rabbits infected with T. pallidum Nichols strain. Moreover, the sera from rTprK-immunized rabbits were used to react with Nichols whole cell lysates to further confirm the availability of the purified rTprK. All blots were processed in parallel and derive from the same experiments  test were randomly selected in this study. Five rabbits were immunized with 500 μg of rTprK in an aqueous buffer with TiterMax Gold adjuvant  at a 1:1 ratio. Immunizations were administered subcutaneously (250 μg) and intramuscularly (250 μg) three times at two-week intervals. Five control rabbits administered PBS were used under the same administration conditions. All rabbits were fed antibiotic-free food and water, and were housed at 18–20°C.Outbred Male New Zealand White rabbits  with negative Venereal Disease Research Laboratory (VDRL) test and 7 /mL fresh T. pallidum (Nichols strain) at 10 sites on their shaved backs. Challenge sites were monitored for erythema, induration, and ulceration and measured every two days to assess lesion diameter. At 7, 15, and 30 days postchallenge, the two sets of skin lesions were biopsied (4-mm punch) from three rabbits in each group for T. pallidum DNA and RNA measurement and histological analysis. The remaining two rabbits in each group were only used for the observation. One milliliter of blood was collected every 4 days for measurement of T. pallidum DNA, cytokine secretion, and the serum antibody response and for the opsonophagocytosis assay. At day 30 postchallenge, the challenge sites of rTprK-immunized rabbits had resolved, all rabbits were euthanized with pentobarbital via intravenous injection at 90 mg/kg body weight, and the liver, spleen, testes, and popliteal lymph nodes were removed for T. pallidum DNA measurement and histological analysis. The popliteal lymph nodes were also transferred to the testes of randomly assigned naïve rabbits to observe infectivity29. A positive RIT was considered if seroconversion appeared, and/or orchitis developed, and/or motile T. pallidum was observed under dark-field microscopy in the recipient animals. After a three-month observation, the negative RIT animals were euthanized, and their popliteal lymph nodes were transferred to a new naïve rabbit again , all rTprK-immunized rabbits and control rabbits were anaesthetized with acepromazine via intramuscular injection at 1 to 3 mg/kg body weight and challenged intradermally with 0.1 mL of 1 × 1030. Total RNA from the lesions was isolated using the RNeasy Kit  and was then treated with DNase I . The concentration of DNA/RNA was obtained from spectrophotometric measurements. Reverse transcription of RNA was performed using a high-capacity cDNA reverse transcription kit . The DNA/cDNA samples were quantified by targeting polA through quantitative real-time PCR (qPCR) using a 96-well reaction plate with a LightCycle 480 system . The primers used for targeting polA were as follow: sense 5′- TACGGTGCAAGTGCTCAGAC- 3′ and antisense 5′- CAGGCACATTGTCGGAGGAA- 3′. A standard curve was constructed using 10-fold serial dilutions of linearized plasmid (for polA) DNA to calculate the polA copies. The results were normalized to the total tissue DNA/RNA concentration, expressed as copies per μg of extracted tissue DNA/RNA. The polA in blood was expressed as copy number per mL of blood31.DNA was extracted from the lesions/tissues/blood using a DNeasy Blood and Tissue Kit  according to the protocol recommended by the manufacturer, and careful precautions were implemented to avoid DNA cross-contaminationSamples taken from the lesions, livers, spleens, and testicles were fixed in formalin, mounted, bisected, vertically sectioned at 4 μm and processed for H&E staining to observe the cellular infiltration and structural changes in tissues. Meanwhile, a naïve control animal that was neither immunized nor challenged was also include to analyzed.Blood samples were obtained via ear bleeds from all rTprK-immunized and control rabbits every four days. The expression of Th1-like cytokines  and Th2-like cytokines (IL-10 and IL-4) in the sera was measured using ELISA kits  according to the manufacturer’s instructions.T. pallidum, all rTprK-immunized and control rabbits were bled for analysis of the T-cell response in vitro. PBMCs were isolated from all obtained blood samples. A methyl thiazolyl tetrazolium (MTT) assay was used to detect T-cell proliferation. Briefly, approximately 105 cells in 200 μL of culture medium were plated in triplicate wells of 96-well plates. rTprK (2.5 μg) was added and incubated at 37 °C in a 5% CO2 atmosphere. Concanavalin A  was used as the positive control, and PBS was used to measure background reactivity. After 48 h of incubation, 50 μg MTT was added to each well and incubated for 4 h. Then, 150 μL of dimethyl sulfoxide (DMSO) was added, and the absorbance was measured at 490 nm. In parallel, 48 h after exposure to rTprK, ConA, or PBS, the supernatants of cells were also collected to detect the secretion of Th1-like cytokines  and Th2-like cytokines (IL-10 and IL-4) using ELISA kits as mentioned above.At 10 days after the last rTprK booster, that is, before challenge with 5 PMBCs were plated on ELISpot M96-well plates and incubated with 2.5 μg rTprK for 48 h. ConA (0.5 μg/well) and PBS were used as the positive and negative controls, respectively. Spot-forming cells were counted using a Cellular Technology Limited S6 Universal Analyzer , and data were processed with ImmunoSpot®7.0 software.In addition, specific T-cell responses quantification with IFN-γ was performed using an ELISpot kit  according to the manufacturer’s protocol. Briefly, approximately 1029. In brief, 50 μL of 1 μg/mL rTprK was used as the coating antigen and incubated at 4 °C overnight. A goat anti-rabbit IgG H&L antibody  was used as a secondary antibody at a 1:10,000 dilution, and the absorbance was measured at 450 nm. The cutoff value was 2.1 times the mean value of the negative sample. Individual sera were tested in triplicate wells per serum sample.The antibody titer against rTprK was monitored with an enzyme-linked immunosorbent assay32. Serum samples from the rTprK-immunized and control rabbits at prechallenge (10 days after the last rTprK booster) and postchallenge  were used to assess opsonization capacity. In brief, macrophages were cultured in DMEM medium with 10% serum (heat inactivated at 56 °C for 30 min), and fresh T. pallidum was added at an MOI of 10:1 for 12 h of incubation. Fluorescence-activated cell sorting (FACS) analysis was used to evaluate T. pallidum internalization by macrophages33. Anti-Tp47 mouse monoclonal antibody at a 1:50 dilution was used, which was generated by the Boson Biotech company . And phycoerythrin (PE)-conjugated anti-mouse secondary antibody at a 1:100 dilution  was used. Counts of PE-labeled cells determined by FACS Canto II flow cytometer  represented the percentage of internalized spirochetes. Meanwhile, immunofluorescence microscopy was performed using the anti-Tp47 mouse monoclonal antibody and FITC-conjugated goat anti-mouse IgG . Fluorescence images were captured by a laser scanning confocal microscope 33.Proteose peptone-induced rabbit peritoneal macrophages were isolated from normal rabbits with negative VDRL test and TPPA testt test and repeated-measures ANOVA. Qualitative materials were used in the Chi-square test to identify significant differences between the groups. A two-sided P < 0.05 was considered statistically significant.All statistical analyses were performed using SPSS version 19.0 . Quantitative materials were reported as the mean ± SD. Differences between the two groups were analyzed by Student’s Supplementary Informationreporting-summary


Mentoring is important for a successful career in academic medicine. In online matching processes, profile texts are decisive for the mentor-selection. We aimed to qualitatively characterize mentoring-profile-texts, identify differences in form and content and thus elements that promote selection.hot topics) were labelled on the selected texts and their impact on student evaluation was statistically examined.In a mixed method study first, quality of texts in 150 selected mentoring profiles was evaluated . Second, based on a thematic and content analysis approach of profile texts, categories and subcategories were defined. We compared the presence of the assigned categories between the 25% highest ranked profiles with the 25% lowest ranked ones. Finally, additional predefined categories (hot topics in texts significantly correlated with a positive evaluation.Students rated the quality of texts with a mean of 5.89 ± 1.45. 5 main thematic categories, 21 categories and a total of 74 subcategories were identified. Ten subcategories were significantly associated with high- and four with low-rated profiles. The presence of three or more The introduced classification system helps to understand how mentoring profile texts are composed and which aspects are important for choosing a suited mentor.The online version contains supplementary material available at 10.1186/s12909-023-04804-1. Mentoring can significantly contribute to a successful academic career in medicine –4. IncreThus, finding a suitable mentor has a major influence on a successful mentoring relationship. Although, finding a suitable mentor during formal or informal academic events is said to be the most promising way to initiate a successful mentoring relationship, identifying the desired mentor can be a challenge , 7. TherAlthough potential proteges were able to identify key information on mentoring profiles that matched their expectations and goals for entering a mentoring relationship, the most mentioned reason for choosing a mentor was “likeability” based on the online profile text composed by the mentor . NeverthGoal of this study was to characterize online mentoring profile texts, identify elements that could discriminate against positively evaluated and therefore often selected mentoring profiles. We focus on mentoring dyads between medical students and physicians in different positions.The Ethics Committee of the Medical Faculty of LMU Munich waived approval for the study. A multi-method approach was applied to answer the above-mentioned research objectives. Since 2008, the Medical Faculty of LMU Munich runs a successful, large-scale mentoring program that allows medical students to match a suitable mentor among more than 400 possible candidates . The menIn a first step, we selected 150 online mentoring profiles that fulfilled certain criteria mentioned within the next section  basic demographic data and ii) a profile-text which had to be at least 200 characters long.Eleven medical students, who were not part of the study team, were randomly selected as evaluators: 8 women and 3 men were selected, the majority had a mentor, and all had completed the preclinical section of medical school. These students ranked all 150 mentoring profiles on a Likert scale ranging from 1 to 10  based on the following question: “How do you assess the quality of the profile with regard to the selection of a suitable mentor ?”. One data set was excluded due to an unplausible rating pattern. The mean and standard deviation was calculated for each profile.All profiles were divided into quartiles and the 25% best rated and 25% worst rated profiles with a standard deviation less than 2 (for increased homogeneity among the selected profiles) were included in further analyses Fig. . Thus, 3Two experienced researchers  independently defined categories using a thematic and content analysis approach. We used a modified approach to qualitative content analysis according to Kuckartz  and screhot topics) based on a previous survey [Additionally, we used deductively predefined categories . All three researchers agreed on the anchoring examples. To avoid cognitive biases, calibrate responses and therefore increase reliability, coders were trained by using 20 mentoring-profiles. For the actual analysis each rater labelled the five major thematic categories with corresponding 74 subcategories and the seven main interest topics on every profile using a binary classification, depending on whether a category was present in the text or not.p-value < 0.05 was considered significant, the effect size was calculated using Cramer´s V. Microsoft Excel (Microsoft Corporation. (2018). Microsoft Excel, Redmond, Washington USA) and GraphPad Prism  were used for statistical analysis and creation of the Figures.Inter-rater reliability was measured using Cohen’s kappa. We applied a Chi-squared test to detect differences between “high quality profiles” and “low quality profiles” concerning the presence of the different categories and whether these profiles responded to the seven major interests of the students. A n = 3) with a standard deviation > 2 the upper  and lower  quartiles were further quantitatively assessed.Overall, students rated all 150 mentoring-profiles with 5.89 ± 1.45 (mean ± standard deviation). A linear distribution pattern was detected  information on the mentor's studies and 3) work experience, 4) mentoring  or 5) personal information, e.g., on the mentor's family or hobbies.The main thematic categories include 1) As an example, for the main thematic category "studies", information about the doctoral thesis of the mentor was subsumed, which is divided into five further categories and the three subcategories "experimental research", "clinical research" and "statistical research"  characters, the 38 highest rated profiles 1,385  and the 35 lowest rated profiles 1,143  . Assessment of the 73 extracted mentoring-profiles according to the presence or absence of individual categories by three individual raters reached a Cohen's kappa of 0.8.The most frequently represented subcategories were the statement "working at a university hospital" , "working in patient care"  and "fully formulated text" .p-value < 0.01; Fig. We identified ten subcategories that significantly correlated with a higher student profile ranking . Analysis of the profile texts also reveals that there is a significant association between the number of hot topics addressed and the student rating (Chi-squared test). Naming of at least three hot topics in profile texts correlates with a positive perception by the students   seem to impact students' perception with highest significance in both directions (positive respectively negative). In addition, there is a trend that positively rated profiles contain longer free text than negatively rated profiles. At first glance formal aspects do not seem to be related with likability, and therefore we interpret these findings as an indication that students use time and motivation of the mentor when creating a profile text as a decision criterion. However, the effort involved might give some hints about character traits of the mentor, which according to the Fundamental Principle of Liking (FPL) are again significantly influencing likeability [First, formal categories  as well as mention of personal struggles with studies or exams as well as the mentor's own mentoring experience have a positive influence on the student's evaluation. This factors most likely, give hints to the notion of approachability, openness, and similitude, all factors that contribute to the perception of likability . In linehot topics) emphasize the importance of predefined goals by the students when entering a mentoring relationship [Third, our results  Mentors should be made aware of formal criteria such as the formulation of a continuous text, usage of a salutation or impact of soft factors discussed above when creating a profile. ii) Addressing at least three Finding the right mentor in an academic environment is a challenge for medical students, especially when using online mentoring databases. We have therefore defined categories that positively or negatively influence the likeability of a mentor based on his composed profile text. Formal text criteria and the addressing of known important topics to mentees in a future mentoring relationship have a significant influence. Other influencing factors in students' perception of mentoring profiles need to be examined. Although data on the influence of mentoring profile text quality on the quality of the mentoring relationship are pending, we believe that the here mentioned factors could significantly improve online matching processes.Additional file 1:Supplement Table 1. Characteristics of mentors related to the randomly selected mentoring profiles. Information was collected based on the profile data where available. Supplement Table 2. Full category-system for mentoring profile text analysis including 5 main categories, 21 categories and 74 subcategories. Supplement Table 3. Statistical analysis of results presented in Figure 2 and Figure 3.