ADCYs were detected using the 3D Genome Browser, after which the functions of these FANGs were predicted using Metascape tools. Cell viability and apoptosis were assessed using CCK-8 and Annexin V-FITC/PI kits. Expression levels of ADCYs were higher in AML cells lines and in bone marrow-derived mononuclear cells from AML patients than in control cells, and were predictive of a poor prognosis. A total of 58 ADCY FANGs were identified from the topologically associating domains on the basis of the Hi-C data. Functional analysis of these FANGs revealed abnormal activation of the MAPK signaling pathway. Drug sensitivity tests showed that fasudil plus trametinib or sapanisertib had a synergistic effect suppressing AML cell viability and increasing apoptosis. These findings suggest that dysregulation of ADCY expression leads to altered signaling in the MAPK pathway in AML and that the ADCY expression profile may be predictive of prognosis in AML patients.We explored the roles of adenylyl cyclases (ADCYs) in acute myeloid leukemia (AML). Expression ADCYs in AML and their effect on prognosis was analyzed using data from Oncomine, GEPIA and cBioPortal databases. Frequently altered neighbor genes (FANGs) of  Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy , 2. Thre+2/calmodulin [+2- independent and are stimulated by G proteins [Adenylyl cyclases (ADCYs) have been attracting increased attention in recent years . These elmodulin . Group 2proteins . Group 3proteins . In addiproteins .Aberrant expression of these isoforms can lead to changes in receptor-mediated activation of ADCYs, as well as alterations in the downstream signaling pathways . HoweverIn the present study, we used bioinformatics analysis with online public data to explore possible functions of ADCYs and their unique prognostic value in AML. We also examined the regulation of ADCY-related pathways as potential targets for therapeutic intervention.ADCYs were highly expressed in cases of kidney tumor and leukemia. In Stegmaier’s dataset, a 3.002-fold increase in ADCY1 expression was detected in AML as compared to control tissues. Similar increases in ADCY1 expression in AML were also seen in Haferlach’s dataset (4.766-fold), Valk’a Leukemia Statistics (1.503-fold), and Andersson’s Leukemia Statistics (1.298-fold) (ADCY2 expression (1.034-fold) was also detected in AML patients vs. control samples. Consistent with that finding, Valk et al.’s data showed that ADCY2 was upregulated 1.823-fold in AML, while Stegmaier et al. showed it to be upregulated 1.8331-fold. In addition, ADCY3 expression was upregulated 1.312-fold in AML vs. control, and ADCY5 and ADCY6 expression was upregulated 1.025-fold and 1.105-fold, respectively. In Haferlach’s dataset, ADCY9 expression increased 1.292-fold in AML, while Valk Leukemia Statistics showed a 1.137-fold increase  . Elevateincrease .ADCYs between AML and matched control data from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases were compared on the basis of the Gene Expression Profiling Interactive Analysis (GEPIA) dataset (http://gepia.cancer-pku.cn/). These results showed that the expression of genes for all 9 ADCY isoforms was higher in AML samples than in control samples . Using Kaplan-Meier analysis, we found that increased mRNA expression of ADCY2 (P = 0.03994), ADCY3 (P = 0.01924), ADCY4 (P = 0.02211), and ADCY7 (P = 0.01772) were significantly associated with poor overall survival (OS) in AML patients  and mutation (MUT) groups . We deteADCY alterations, gene relevance, and the interaction networks using the cBioPortal database for AML . ADCY alterations were detected in 32.52% of 163 AML patients . This enabled us to simultaneously examine the gene regulatory events and the 3D genomic organization. Topologically associating domains (TADs) of ADCYs were identified through analysis of Hi-C data from THP1 cells, which enables us to predict the potential target genes . The neighborhood protein network, where proteins were densely connected, was identified using the MCODE algorithm.We then constructed a network for et genes . The top dataset . The resth ADCYs . In addihttps://david.ncifcrf.gov/summary.jsp) was used to predict potential functional pathways of ADCYs as well as their associated genes. Biological processes analysis showed that the cAMP-mediated signaling (GO: 0019933), the cAMP biosynthetic process (GO: 0006171), and the adenylate cyclase – activating/inhibiting G-protein coupled receptor signaling pathway (GO: 0007193 and 0007189) were significantly regulated by ADCYs in AML  (s in AML . Using ms in AML . In addiL (ADCYs . Most ofhttps://www.proteomicsdb.org/). We found these genes were enriched in six important pathways related to AML leukemogenesis: endocrine resistance pathway, purine metabolism pathway, the Calcium signal transduction pathway, the MAPK signaling pathway, the cGMP-PKG signal transduction pathway, and the Rap1 signal transduction pathway , trametinib (MEK inhibitor) and sapanisertib (mTOR inhibitor) with Kasumi-1, MOLM13, OCI-AML3 and OCI-AML2 cells, we found that all cells were sensitive to 50 μM fasudil, with about 50-60% of cells remaining viable after treatment for 48 h. Kasumi-1 cells were the most sensitive to trametinib (10 nM), with 50% of cells viable after 48 h , and MOLGiven the ability of fasudil, trametinib and sapanisertib to reduce the viability in AML cell lines, we next sought to validate the effects of these inhibitors using mononuclear cells isolated from the bone marrow of primary AML patients. Consistent with the results from cell lines, patient cells treated with fasudil and trametinib or sapanisertib were less viable than cells treated with a single inhibitor. The synergistic efficiency of fasudil plus trametinib was greater than that of fasudil plus sapanisertib . ConsistDysregulation of ADCY expression has been reported in various solid cancers , 15, 17,Functional analysis suggested that cAMP may affect extracellular signaling in several tumors . In the Smoking is thought to be a major risk factor for AML in older adults and childhood leukemia , 22. We Our findings suggest dysregulation of ADCY expression leads to altered signaling in the MAPK pathway in AML, and that the ADCY expression profile may be predictive of prognosis in AML patients.Between 2019 and 2020, a total of 5 AML patients newly diagnosed at the Sun Yat-sen University Cancer Center were enrolled in this study. All participants provided written informed consent in accordance with the regulations of the Institutional Review Boards of the Hospitals in agreement with the Declaration of Helsinki.Inhibitors fasudil (S1573), trametinib (S2673) and sapanisertib (S2811) were obtained from Selleck Chemicals LLC .5 cells/well. To determine the cytotoxicity of the inhibitors tested, cells were separately incubated with appropriate concentrations of each inhibitor. After 48 h, 10 μL of reagent from a Cell Counting Kit-8  were added to each well. The samples were then incubated for an additional 4 to 6 h, at 37°C and the absorbance at 450 nm was measured using a spectrophotometer. An Annexin V FITC/PI staining kit  was used to detect apoptotic cells induced by the inhibitors.AML cell lines  were obtained from Da-Wei Wang  and Da-Jun Yang’s lab (Sun Yat-sen University Cancer Center) and cultured in RPMI-1640  supplemented with 10% fetal bovine serum . Cells were seeded into 96-well plates at a density of 10https://www.oncomine.org/re-source/login.html) was utilized to obtain ADCY gene expression data from cancer and control samples. ADCY expression was compared between clinical cancer specimens and paired normal controls using Student’s t test.The Oncomine website  is an open-access website that contains over 225 tumor genomics datasets. ADCY alterations in the LAML samples were analyzed using this website. Genetic information, including mutations, gene splicing and copy number variations (CNVs), was also found in this dataset. The neighboring genes were excluded unless the frequencies were > 20%.The cBioPortal  were used to calculate the combination indexes according to the average fraction of viable cells in the cytotoxicity assays , 27. TheSupplementary Table 1Supplementary Table 2


Here, we employ time-resolved two-photon excitation fluorescence imaging in acute hippocampal slices and in cortex in vivo to find that resting [Ca2+] predicts the scale (amplitude) and the maximum (peak) of astroglial Ca2+ elevations. We bidirectionally manipulate resting [Ca2+] by uncaging intracellular Ca2+ or Ca2+ buffers and use ratiometric imaging of a genetically encoded Ca2+ indicator to establish that alterations in resting [Ca2+] change co-directionally the peak level and anti-directionally the amplitude of local Ca2+ transients. This relationship holds for spontaneous and for induced (for instance by locomotion) Ca2+ signals. Our findings uncover a basic generic rule of Ca2+ signal formation in astrocytes, thus also associating the resting Ca2+ level with the physiological “excitability” state of astroglia.Astroglia regulate neurovascular coupling while engaging in signal exchange with neurons. The underlying cellular machinery is thought to rely on astrocytic Ca  •2+ signals in brain astroglia are controlled by the local basal [Ca2+]Ca•2+] is associated with higher peak level but lower amplitudeA high basal [Ca•2+ signals in vitro and in vivoThis rule applies to Ca•2+ affects peak and amplitude of Ca2+ signals accordinglyChanging basal Ca 2+] increases the maximum level but decreases the amplitude of local Ca2+ signals. This fundamental relationship holds over a wide range of preparations, ranging from hippocampal astrocytes in acute slices to locomotion-associated Ca2+ signals in the awake animal.King et al. show that, in brain astroglia, a high resting [Ca A multitude of signaling pathways enable astroglia to convert neuronal activity into cytosolic Ca2+ increases, which often involve Ca2+ stores  via inositol 1,4,5-trisphosphate receptors (IP3Rs) is determined by the receptor opening probability, which has a bell-shaped dependence on cytosolic [Ca2+]  of organic Ca2+ indicators. We combined these measurements with photolytic release of Ca2+ and Ca2+ buffers inside individual astrocytes to establish a causal relationship between astroglial resting [Ca2+] and astroglial [Ca2+] transients.Thus, our hypothesis was that resting Ca2+ dependence of the fluorescence lifetime of dyes from the Oregon Green BAPTA family was used to accurately measure [Ca2+] in astroglia in situ and in vivo by performing FLIM (The Caing FLIM . We chos2+ (1 and C2), in which the numbers of detected photons were counted before their ratio was taken  were calibrated for free Ca2+ A. For annstrated . It was ted  via a whole-cell patch pipette into astrocytes. After an equilibration period of 10–20 min, astroglial Ca2+ transients were evoked by pressure application of the mGluR agonist DHPG from a pipette placed immediately adjacent to the astrocyte territory while collecting OGB2 fluorescence , and the amplitude (concentration increase with respect to the baseline/resting level) to capture dependencies across a recorded astrocyte . Where detected, progressive decreases in Ca2+ transient amplitudes were not due to saturation of OGB2, because the maximum peak [Ca2+] observed in a cell, averaged across the entire population, was 191.2 ± 38.2 nM. This is equivalent to photon count ratios about halfway between their minimum and maximum . We also tested whether variations of the resting [Ca2+] could influence the kinetics of Ca2+ transient, which was however not the case . This result suggested that the baseline [Ca2+] is not primarily determined by local astrocyte morphology. In contrast, we found negative correlations between the local VF and peak [Ca2+]  and between VF and the amplitude . The latter observation indicates that, independently of baseline [Ca2+], the amplitude and peak [Ca2+] of mGluR-dependent transients are higher when the local VF is low, i.e., in the small astrocyte branches.We next asked whether this finding can be explained by local astrocyte morphology. Because the fine structural details cannot be resolved by diffraction-limited two-photon excitation microscopy , we used2+] display transients with a higher peak [Ca2+], but lower amplitudes, within individual cells. Higher baseline [Ca2+] could decrease Ca2+ entry into the astrocyte cytosol by reducing Ca2+ influx either from extracellular space or from Ca2+ stores. Theory dictates that 50 nM changes in basal [Ca2+] lead to only <10% changes in its extracellular driving force  was added to the intracellular solution: UV photolysis of diazo-2 drastically increases its affinity for Ca2+  and CaCl2 (3 mM) to the intracellular solution and using UV illumination to release Ca2+  and occurred at an average frequency of 1.40 ± 0.159 events per minute . Note that these are free Ca2+ concentration estimates in the presence of 200 μM added exogenous buffer (OGB2) and that the free cytosolic Ca2+ concentration may rise much further in its absence.Although DHPG pressure application reliably evoking timed astroglial Ca imaging , withoute n = 7; A. On ave2+ transients are often quantified by normalizing the fluorescence intensity of the Ca2+ indicator at the peak to its pre-event baseline value (FP/F0). Assuming that the fluorescence of the Ca2+-free indicator is negligible , FP/F0 can be derived from the law of mass action to be [Ca2+]P × (KD + [Ca2+]0)/([KD + [Ca2+]P] × [Ca2+]0), where KD is the indicator’s dissociation constant and [Ca2+]0 (baseline) and [Ca2+]P (peak) correspond to FP and F0. Using a KD of 350 nM for Fluo-4, our measurements correspond to FP/F0 values of 1.86 ± 0.118 , which is well within the range of published data, both in vitro and in vivo  to monitor intracellular [Ca2+] and its transient changes in an unbiased and consistent way throughout a cell.Therefore, we used a mouse line that conditionally expresses the Capression . After cpression  and tamo2+ transients recorded from acute slices  via a nearby pipette onto GCaMP5g/tdTomato-expressing astrocytes in acute slices . Again, the resting Ca2+ level correlated negatively with the amplitude and positively with the peak of Ca2+ transients , which is in line with previous studies (2+] varied considerably between sub-compartments (average CV ∼0.4).In this study, we set out to establish how the variability of the local resting [Ca studies . Within 2+ transients depends on the local resting [Ca2+] using FLIM of OGB2-loaded astroglia. Here, we used activation of metabotropic glutamate receptors (DHPG), a robust method to induce astroglial Ca2+ signaling in the hippocampus at various stages of development , although the peak of Ca2+ signals was at 118 ± 52.2 nM (mean ± SD). Within this concentration range, a rise of the resting [Ca2+] will increase the open probability of IP3Rs, irrespective of the IP3 concentration and IP3R subtype . This study did not generate new unique reagents.Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Christian Henneberger , and kept in the slicing solution at 34°C for 15 minutes before being stored at room temperature (21-23°C) in an extracellular solution containing (in mM) NaCl 126, KCl 2.5, MgSO4 1.3, NaH2PO4 1.25, NaHCO3 26, CaCl2 2, and glucose 10. All solutions were continuously bubbled with 95% O2/ 5% CO2. Experiments were performed at ∼34°C.Acute hippocampal slices from rats and mice were prepared as previously described . Briefly2+] measurement, Oregon Green 488 BAPTA-2  was loaded into individual astrocytes as previously described (2/ 5% CO2). In these experiments, neuronal activity was inhibited by adding the sodium channel blocker TTX  to the extracellular solution. Putative astrocytes were identified using DIC optics and patched (Multiclamp 700B) with an intracellular solution containing (in mM) KCH3O3S 135, HEPES 10, di-Tris-Phosphocreatine 10, MgCl2 4, Na2-ATP 4, Na-GTP 0.4 , OGB2 0.2, and Alexa Fluor 594 0.1 . Cells were held in current clamp or voltage clamp at their resting potential. Astroglial cells were identified by their low resting membrane potential (< −80 mV), low input resistance (< 10 MΩ), symmetric, ‘passive’ responses to current injections  and their characteristic morphology. Recordings were rejected if the access resistance was initially above 20 MΩ or changed more than 30% during the recording. After dye equilibration for 10-20 minutes, Ca2+ imaging experiments started. After acquisition of a baseline period, the mGluR agonist DHPG was pressure-applied through a patch pipette placed immediately adjacent to the astrocyte territory. DHPG (300 μM) was dissolved in extracellular solution also containing Alexa Fluor 647 (3 μM) to visualize the ejection of DHPG into the tissue.For astroglial  was estimated using WebMax Chelator (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/webmaxc/webmaxcS.htm) taking into account the concentration of Ca2+, Ca2+-buffers, ATP and Mg2+. The relationship between [Ca2+] and the photon count ratio and amplitude-weighted average decay constant could be approximated by a Hill function (2+]. The OGB2 decay time constants (τ1-3) differed slightly between intracellular measurements and calibrations  and in time windows of 600 ms  was automatically calculated.For experimental frame scanning TCSPC data, detected photons were pooled across automatically set regions of interest . UV uncaging was performed using a computer-controlled UV-LED  with the output power set to 2.5 mW and activated with 10 pulses of 50 ms delivered at 10 Hz. The UV-stimulus was delivered via a light guide  submerged in the extracellular solution and positioned just above the hippocampal slice in the vicinity of the investigated astrocytes.In a subset of experiments, FLIM  transient peaks and amplitudes on the local basal [Ca2+] within individual cells, a measure of correlation is needed. Since the exact mathematical relationship between these parameters is not known a priori and may also differ between cells, a non-parametric approach was required. We used Spearman’s rank correlation coefficient (R) to extract a single and unbiased measure that can be used across different cells, techniques and preparations. In this analysis, Spearman’s R is the biological relevant statistical unit . It is a dimensionless variable reporting the degree of x-y correlation. In our case, it captures how well the relationship between the basal [Ca2+] and the peak/amplitude of Ca2+ transients can be described using a monotonic function . See 2+] and enabled us to compare different cells irrespective of the precise values, which can vary considerably from one cell to another. The overall average dependence of peak and amplitude on resting [Ca2+] across a set of cells was then calculated as the mean ± s.e.m. and displayed together with individual data points in boxplots (see above and To establish the dependence of astrocytic Ca+ transie2+ concentrations, fluorescence intensities are color-coded using ImageJ’s color lookup tables (red hot and green fire blue, see Estimates for the calculations presented in The datasets and custom code supporting the current study have not been deposited in a public repository but are available upon reasonable request from the corresponding author.


Acute myeloid leukemia (AML) with myelodysplasia-related changes (AML-MRC) is a distinct biologic subtype of AML that represents 25–34% of all AML diagnoses and associates with especially inferior outcomes compared to non-MRC AML. Typically, patients with AML-MRC experience low remission rates following intensive chemotherapy and a median overall survival of merely 9–12 months. In light of these discouraging outcomes, it has become evident that more effective therapies are needed for patients with AML-MRC. Liposomal daunorubicin–cytarabine (CPX-351) was approved in 2017 for adults with newly diagnosed AML-MRC and those with therapy-related AML (t-AML), and remains the only therapy specifically approved for this patient population. Other studies have also demonstrated the efficacy of the hypomethylating agent (HMA) azacitidine as upfront therapy for AML-MRC patients, which, to date, is the most common treatment employed for patients unable to tolerate the more intensive CPX-351. HMAs and venetoclax combinations have also been evaluated, but additional studies utilizing these agents in this specific subgroup are needed before conclusions regarding their role in the therapeutic armamentarium of AML-MRC patients can be reached. Currently, many studies are ongoing in attempts to further improve outcomes in this historically ill-fated patient group. Acute myeloid leukemia (AML) is a disease of the myeloid lineage of blood cells that results in a block in differentiation of myeloid cells and uninhibited growth of leukemic blasts that constrain the growth of normal blood cells  ClinicalAML with myelodysplasia-related changes (AML-MRC) is one such subtype of AML that is estimated to represent a sizeable 25–34% of all AML cases and is more commonly seen in older AML patients, with a median age of 73 years . FurtherNPM1 or biallelic CEBPA mutations, even in the presence of bone marrow findings demonstrating multilineage dysplasia [NPM1 mutation or biallelic CEBPA mutation [NPM1 and biallelic CEBPA mutations [AML-MRC was first introduced in the 2008 World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia and expanded on the prior category of AML with multilineage dysplasia . The 200ysplasia . This chmutation ,8,9. As utations ,11.RUNX1, TP53, SETBP1, epigenetic regulators, and splicing factors. Furthermore, the molecular MRC-like pattern was identified in over 10% of patients not classified as MRC per WHO criteria but who experienced a similarly poor overall survival (OS), suggesting the definition of AML-MRC may need expanded to include this molecularly-determined subset as well.With the introduction of next generation sequencing (NGS), our understanding of AML-MRC is again evolving with the discovery of the emerging role that common molecular mutations play in AML-MRC and their impact on prognosis. An analysis by Baer and colleagues sought to classify AML-MRC on the basis of molecular aberrations and were able to detect 96–99% of patients with AML-MRC per WHO definition criteria . MutatioTET2 or RUNX1) followed by a second “hit” in a gene impacting cellular proliferation and survival  eventually result in leukemic transformation from antecedent MDS [The precise mechanism through which MDS progresses to AML remains unclear. However, several factors have been implicated in this phenomenon . Epigenedent MDS ,17.TET2, U2AF1, and TP53) then accumulate additional mutations (such as in NOTCH2 and KMT2C), resulting in subclones that can develop into both MDS blasts and pre-AML/AML stem cells. However, pre-MDS/MDS stem cells require other additional mutations in order to develop into AML blasts . Chen et al. also show that stem cells harbor a higher complexity of subclonal mutations than blasts cells, and the size of different clones varies from MDS blasts to AML blasts. Therefore, dominant and passenger mutations cannot be determined only on the basis of the size of the clone.Recently, newer platforms such as targeted deep sequencing and single-cell sequencing have led to an updated non-linear theory of MDS to AML progression. A compelling study by Chen and colleagues proposes that MDS does not directly evolve into AML, but rather parallel clonal evolutionary changes occur in which pre-MDS stem cells and/or MDS stem cells concurrently develop into two separate cell populations: an MDS population and a separate population of pre-AML/AML-stem cells . The latIn current practice, targeted NGS studies of the blood or bone marrow blasts is performed in order to determine disease mutations in AML but lacks specificity for being able to differentiate between AML and MDS blast populations. Further work is needed to determine how new models of MDS leukemic transformation will be translated into the clinic.As compared to non-MRC AML, AML-MRC has, overall, been associated with inferior outcomes such as lower remission rates, ranging between 30–50%, and shorter overall survival ,19,20,21One of the main challenges of treating patients with AML-MRC is that, compared to non-MRC AML which typically has a flatter incidence of occurrence, AML-MRC tends to occur in an older patient population ,22,28. TAnother challenge in tailoring treatment for patients with AML-MRC is the high likelihood that many of these patients will have had prior treatment with cytotoxic agents preceding their leukemia diagnosis. Although such patients are technically considered to have therapy-related AML (t-AML), there is significant overlap between t-AML and AML-MRC . A recenp = 0.05), 3-year event free survival , and relapse-free survival once achieving a CR  [p = 0.12), patients with AML-MRC demonstrated a worse CR rate after a second induction course  [AML-MRC is reported to occur in approximately 21% of pediatric AML patients (per 2008 WHO criteria), and tends to decline in frequency with increasing age. In fact, 37.5% of AML patients ≤2 years old were found to have MDS-related cytogenetic abnormalities compared to 20.9% and 18.5% of AML patients 3–14 years old and 15–21 years old, respectively ,35. In a = 0.06) .Furtherm < 0.01) .Interest < 0.01) The treatment of AML, until recently, had remained largely unchanged for decades, and consisted primarily of intensive chemotherapy with a cytarabine and anthracycline-based backbone (“7 + 3”) followed by allogenic stem cell transplant  for curative intent. Although the overall CR rate for the 7 + 3 regimen is reported to be 60–80%, patients with AML-MRC are reported to have lower CR rates with this regimen, typically between 30–50% ,23,32. Gp = 0.016)) and median overall survival (OS) (9.56 months vs. 5.95 months (hazard ratio (HR) 0.69)) compared to those who received 7 + 3. Importantly, survival post alloHSCT was also significantly improved in CPX-351 patients , indicating that the depth of remission achieved in these patients was perhaps superior to the quality of CRs achieved in 7 + 3 patients.CPX-351 is a liposomal encapsulation of cytarabine and daunorubicin in a fixed 5:1 molar ratio, being approved by the Food and Drug Administration (FDA) in August 2017 for adults with newly diagnosed AML-MRC and t-AML. To date, it is the only therapy specifically approved for this AML subgroup ,36. The An exploratory subgroup analysis of this trial in the 246 enrolled AML-MRC patients showed that a higher number of patients with AML-MRC obtained a CR/CRi (CR with incomplete hematologic recovery) with CPX-351 than with standard 7 + 3 therapy  . This ulA recent retrospective study of 30 younger patients <60 years old with secondary AML, previous cytotoxic therapy, or AML-MRC induced with CPX-351 showed an ORR of 46.7%, CR rate of 17.2%, and CRi rate of 10.3% with an OS of 7 months (0.5–12.4 months) . There iThe toxicity profile of CPX-351 is comparable to that of 7 + 3 overall, with febrile neutropenia being the most common adverse event . Howeverp = 0.036).For many older unfit patients with AML-MRC, the HMAs remain a cornerstone therapy in the management of their disease. In the pivotal phase 3, multicenter, randomized trial AZA-AML-001, azacitidine was compared to several conventional care regimens (CCR), which included 7 + 3 induction chemotherapy, low-dose cytarabine (LDAC), or only supportive care in 488 older patients with newly diagnosed AML . AzacitiA central review of bone marrow samples from the AZA-AML-001 study revealed that many more patients carried the diagnosis of AML-MRC than in the original analysis; therefore, azacitidine was further explored in this expanded population of 262 patients . Again, This study also attempted to determine whether the subtype of AML-MRC or the cytogenetic risk group affected outcomes . Patientp = 0.11). Unfortunately, dedicated analyses pertaining to AML-MRC patients specifically was not performed for this trial, but it did analyze patients with secondary AML separately. In these 171 patients with secondary AML, decitabine failed to show a significant improvement in median OS (p = 0.6357), and on multivariate analysis there was no significant difference in OS between de novo and secondary AML (p = 0.35) [p = 0.016) [Decitabine was initially studied in 485 older patients with newly diagnosed AML in a randomized, multicenter, phase 3 trial comparing decitabine (5 consecutive days every 4 weeks) to treatment choice  . In cont = 0.35) ,40. Inte= 0.016) .A separate phase 2 trial investigated 10 day cycles of decitabine administered every 4 weeks in patients aged ≥60 . Again ap  <  0.00001; HR for decitabine 0.86, 95% CI: 0.73–1.02, p = 0.08) [Although HMAs are commonly employed in the treatment of AML-MRC patients, a number of practical considerations remain. At present, in the absence of head-to-head comparisons, it is not known whether choice of HMA selection matters. However, thus far, combined analysis of the five published phase 3 randomized control trials of HMAs supports an overall survival advantage with azacitidine compared with decitabine  . AdditioVenetoclax has recently been approved for newly-diagnosed AML patients ≥75 years, or those unable to tolerate intensive chemotherapy, in combination with either an HMA or low dose cytarabine (LDAC) . The appVenetoclax was investigated in combination with HMA therapy in a phase1b open-label, international dose-escalation/expansion study of 145 patients ≥65 years . PatientWhen studied in combination with LDAC in 82 AML patients ≥60 years, including those previously treated with HMAs for MDS, the CR/CRi rate was 26% and 28%, respectively, with a median OS of 10.1 months (95% CI: 5.7–14.2) . UnfortuIn a separate study, venetoclax monotherapy was also investigated in seven patients with secondary AML (five of which had prior MDS) who relapsed after HMA therapy . Of the The most common and most important side effects venetoclax are myelosuppression, gastrointestinal toxicities, and tumor lysis syndrome. Additionally, venetoclax is a substrate of CYP3A4, and thus dose adjustments must be made when given along with strong or moderate CYP3A inhibitors or P-glycoprotein inhibitors, specifically when antifungal prophylaxis is indicated.p = 0.7). A second study by Lee et al. of 138 patients showed similar results with no significant difference in 3-year OS (41% vs. 57%), CIR (35% vs. 21%), and NRM (24% vs. 22%) between patients with and without AML-MRC following alloHSCT [p = 0.40) after alloHSCT. Interestingly, MDS-related cytogenetics was shown to be an independent prognostic factor following alloHSCT , but multilineage dysplasia or history of MDS was not significant. Therefore, patients with AML-MRC who are able to undergo alloHSCT should be encouraged to receive this therapy.AlloHSCT is advised for all patients with AML-MRC and remains the only known potentially curative therapy in this patient population . Indeed,alloHSCT . Again, At present, multiple clinical trials for AML-MRC patients are in progress in an effort to improve responses and outcomes . As CPX-AML-MRC remains a common and highly aggressive biologic subtype of AML and is associated with poor long-term outcomes. Recent studies have focused on the genetic mechanisms that underlie disease progression in patients with antecedent myelodysplasia as well as towards identification of more tailored therapies for AML-MRC patients. Currently, only CPX-351 is approved for this indication, however, newer treatments for AML have shown promise for AML-MRC patients as well. Future studies directed towards halting leukemic progression in MDS patients are urgently needed, as well as more effective and well-tolerated treatments for those who present with AML-MRC at the time of initial diagnosis.


NPM1 mutations and their consequences, the underlying leukemia pathogenesis is still unclear. Recent studies that focused on dysregulated gene expression in AML with mutated NPM1 have shed more light into these mechanisms. In this article, we review the current evidence on normal functions of NPM1 and aberrant functioning in AML, and highlight investigational strategies targeting these mutations.Nucleophosmin (NPM1) is a ubiquitously expressed nucleolar protein involved in ribosome biogenesis, the maintenance of genomic integrity and the regulation of the ARF-p53 tumor-suppressor pathway among multiple other functions. Mutations in the corresponding gene cause a cytoplasmic dislocation of the NPM1 protein. These mutations are unique to acute myeloid leukemia (AML), a disease characterized by clonal expansion, impaired differentiation and the proliferation of myeloid cells in the bone marrow. Despite our improved understanding of  NPM1) is one of the most commonly detected genomic alterations in AML. It is found in 20–30% of newly diagnosed AML and in 50% of those with a normal karyotype [NPM1 lead to leukemia have been described. However, despite an increased understanding of these mechanisms, there has not been to date any targeted therapy for NPM1-mutated AML. In this review, we summarize the current knowledge on the normal and aberrant functions of NPM1, highlight mechanisms of leukemogenesis and discuss rational therapeutic strategies.Acute myeloid leukemia (AML) is a clinically and genomically heterogeneous malignancy despite having a relatively low number of genomic alterations compared to other cancers ,2. Mutataryotype . NPM1 isNPM1 is a ubiquitous and abundant protein that resides in the nucleoli under normal physiologic conditions but continuously shuttles between the nucleus and the cytoplasm. It is composed of 294 amino acids with a molecular weight of 37 kDa. It is divided into three structural and functional domains: N terminus, central and C terminus . The N tThe functions and localization of NPM1 are regulated by post-translational modifications ,16. NPM1NPM1 mRNA has been shown to inhibit the processing of pre-rRNA [The nucleolus—where NPM1 resides in physiologic states—is the site of the active transcription of the ribosomal ribonucleic acid (rRNA) and ribosome assembly . Ribosompre-rRNA . Thus, NNPM1 knockout mice have uncontrolled centrosome duplication, which leads to supernumerary chromosomes and aneuploidy [NPM1 maintains genomic stability by binding to centrosomes and restricting their duplication to once per cell cycle . The traeuploidy ,32. Finaeuploidy .NPM1 is involved in the regulation of proliferation and growth suppression pathways. NPM1 overexpression correlates with uncontrolled cell growth ,34. NPM1NPM1 is a transcriptional target of MYC [t of MYC ,40. MYC t of MYC ,41.NPM1 gene is highly conserved between humans, rodents, chicken and fish. Human NPM1 is located on chromosome 5q35 and is composed of 12 exons with sizes ranging from 58 to 358 base pairs (bp) [NPM1 gene has 11 exons encoding 294 amino acids. There are two other isoforms in addition to the wild-type NPM1 gene (NPM1.2 and NPM1.3); however, less is known about their expression and function. The inactivation of NPM1 in the germ line is embryonically lethal. Npm1−/− mutant mice have aberrant organogenesis and die between embryonic day E11.5 and E16.5 because of severe anemia caused by defects in primitive hematopoiesis [The irs (bp) . The regopoiesis . Other dopoiesis .NPM1 locus is involved in translocations causing hematologic malignancies such as acute promyelocytic leukemia where t leads to NPM1–RARα, anaplastic large cell lymphoma where t leads to NPM1-ALK, and myeloid neoplasms where t leads to NPM1-MLF1 [NPM1 in AML. Subsequently, Falini and colleagues discovered that NPM1 mutations are relatively common in AML [NPM1-mutated gene (NPM1c), and there are no NPM1 mutations with NPM1 remaining in the nucleolus. NPM1 mutations are exclusively heterozygous, which implies that NPM1c is able to form a dimer with wild-type NPM1, recruit it to the cytoplasm and perturb its normal function [NPM1 is frequently overexpressed in multiple malignancies including gastric, colon, ovarian, bladder and prostate cancers, as reviewed by Grisendi and colleagues ,44,45,46PM1-MLF1 ,48,49. In in AML . These mn in AML ,53,54,55function .NPM1c through the addition of misprimed nucleotides causing these frameshift mutations [FLT3-ITD), which frequently co-occur with NPM1 mutations in AML [FLT3-ITD mutations occur twice as often in AML with mutated NPM1 compared to AML with the wild-type NPM1 gene [NPM1 mutations alone are not sufficient to cause AML and require cooperating mutations in other genes  also co-occur with NPM1c in 60% of AML cases [DNMT3A mutations occur in hematopoietic stem cells and lead to a clonal expansion of these cells, a process known as clonal hematopoiesis (CH), which is an age-related process that predisposes to leukemia [NPM1c mutations are only detected in overt myeloid malignancies and not in clonal hematopoiesis [DNMT3A likely develop first in hematopoietic stem cells (or other mutations causing CH), followed by NPM1c second in a CMP, and FLT3 mutations (or RAS) third  has been implicated in the development of utations . The norutations . Interess in AML . FLT3-ITPM1 gene ,54,55,59er genes . A knockopoeisis . Howeveropoeisis ,61. Mutaleukemia ,64,65,66opoiesis ,68. Thesopoiesis ,69. UsinS) third  [1,54,70NPM1c possibly offer the best clues on the mechanisms underlying their neoplastic effect: (1) NPM1c mutations are only detected in myeloid malignancies and not in any other cancer despite the ubiquitous expression of NPM1 in cells from various tissues; (2) All NPM1c mutations lead to a translocation of NPM1 from the nucleus to the cytoplasm; (3) All NPM1c mutations in AML are heterozygous; therefore, they likely act as a dominant negative mutant leading to the retention of wild-type NPM1 in the cytoplasm; (4) NPM1c alone without co-operating mutations is not sufficient for leukemogenesis. Therefore, it is not clear based on current knowledge whether NPM1c mutations drive leukemia predominantly through loss of function, gain of function or both.Given all the various important functions of NPM1 in maintaining homeostasis, any disruption of these functions could perturb numerous mechanisms and lead to malignancy . HoweverNPM1 is a central mechanism contributing to leukemogenesis. Npm1+/− mice have a significant degree of genomic instability that increased their susceptibility to oncogenic transformation [NPM1c are most common in normal karyotype AML, indicating no evidence of genomic instability at least at the cytogenetic analysis level. It is possible that NPM1c cells retain their capacity to control centrosome duplication in those cases. Additional chromosomal abnormalities are detected in 15% of AML with an NPM1 mutation. In a recent analysis, the presence of a complex karyotype in AML with an NPM1-mutated gene, with negative or low burden FLT-ITD, was associated with lower response rates and shorter survival despite earlier reports showing no impact on prognosis [NPM1 regulates centrosome duplication, and NPM1 loss affects genomic stability. However, it is unclear whether the disruption of this function following mutations in ormation . On the rognosis ,72,73.The tumor suppressor Arf residing in the nucleolus is stabilized by NPM1. Arf is delocalized to the cytoplasm by NPM1c, which in turn exerts a dominant negative effect on wild-type NPM1, and it limits the inhibitory effect of NPM1 on this protein’s turnover, thus decreasing the half-life of Arf . HoweverNPM1 stabilizes both TP53 and MDM2, the TP53 negative regulator, hence modulating cellular growth suppression through p53-dependent and independent mechanisms. Loss of NPM1 could affect levels of TP53, leading to oncogenic transformation. However, mouse cells and embryos that are Npm1 null have stabilized p53, indicating that NPM1 is not critical for p53 induction ,31.NPM1c binds to cleaved caspase-6 and 8, which are the active forms of these proteins, thereby exerting a gain of function by inhibiting their proteolytic activity . This afNPM1 gene compared to other subtypes [NPM1.Cells with NPM1c have decreased the degradation of Myc by acting on the F-box protein Fbw7gamma, a component of the E3 ligase complex involved in the ubiquitination and proteasome degradation of this oncogene . Myc prosubtypes . This peNPM1c is associated with the upregulation of the homeobox cluster or HOX genes, which are genes that are important for hematopoietic development. Specifically, members of this distinctive stem cell signature include the genes HOXA, HOXB and their DNA-binding cofactor MEIS1 [NPM1c had not been clear until recently. To assess whether the presence of NPM1c, its cytoplasmic location and the overexpression of HOX genes are just an association or a series of events related to the mutation, Brunetti and colleagues employed a series of experiments modulating these factors genetically or pharmacologically [HOX genes are also upregulated in leukemias with a rearrangement of the Mixed Lineage Leukemia (MLL) gene  [MLL-rearranged leukemia, and the dependence of MLL rearrangements on their DNA-binding cofactor menin to exert an oncogenic effect, recent studies have shed more light into how NPM1c induces the aberrant expression of HOX genes [NPM1 gene was found to be dependent on menin and on MEIS1 [MLL complex and HOX genes in causing this leukemia, the mechanism by which NPM1c interacts with these epigenetic regulators is still unknown.AML with or MEIS1 ,60,61,81ogically . They for KMT2A) . MLL1 isOX genes ,84. Usinon MEIS1 ,86. ThouNPM1 gene is a distinct subtype according the 2016 World Health Organization (WHO) classification, due to its specific mutational profile, immunophenotype, clinical behavior and mutual exclusiveness to other recurring genomic alterations [NPM1c mutations are associated with the female sex, even though AML is more common in men, in those with a higher median age (age 39–47 years), and in those with higher bone marrow blast, white blood cell and platelet counts compared to other subtypes of AML [NPM1c AML is most commonly classified under the M4 (Acute myelomonocytic leukemia) or M5 (Acute monocytic leukemia) subtypes of the French–American–British (FAB) classification [NPM1-mutated AML typically have a distinctive fish-mouth appearance. It has a relatively better prognosis with higher rates of remission and longer survival compared to other subtypes of AML [FLT3-ITD mutations confer a worse prognosis, especially when their allelic burden is high [FLT3-ITD is most pronounced when present in combination with mutations in NPM1 and DNMT3A [NPM1 and FLT3-ITD mutations, especially when the FLT3-ITD allelic burden is high [AML with the mutated erations . NPM1c ms of AML ,59,88,89fication . The blas of AML ,55,88,90 is high . Further is high ,93.NPM1c mutations are persistent throughout the course of AML and disappear with remission. This finding highlights their clinical significance and use in the monitoring of minimal or measurable residual disease (MRD) following treatment. MRD provides powerful prognostic information and is increasingly incorporated in the routine management of AML. Detectable MRD is consistently associated with an increased risk of relapse and worse long-term outcomes [NPM1c transcripts, Ivey and colleagues established the importance of NPM1c as a prognostic MRD marker in AML [outcomes . Using ar in AML . These mr in AML .NPM1c has resulted in multiple innovative and promising therapeutic strategies. The current treatment of AML with the NPM1-mutated gene consists of combinations of chemotherapeutic agents with the backbone of an anthracycline and cytarabine [NPM1c AML, where the FLT3 mutational burden is low, leading to prolonged survival compared to other subtypes [NPM1c, and the current treatment can be associated with a significant morbidity, especially in older patients. Therefore, novel therapeutic strategies can further improve outcomes in this entity, especially when specifically targeting NPM1c—a leukemia-initiating event  was identified as an effective addition to combination strategies [NPM1c, resulting in higher response rates in this entity compared to other mutational profiles [NPM1c AML (among six patients with refractory NPM1c AML) [The combination of all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO), a curative treatment for acute promyelocytic leukemia, leads to the degradation of NPM1c, inducing apoptosis and cell differentiation in ed cells ,98. Giverategies ,100. Bclrategies ,102. Theprofiles . DactinoM1c AML) .HOX genes and an anti-leukemia effect [The cytoplasmic location of NPM1c is a key event in leukemia development. The translocation of NPM1 to the cytoplasm depends on the interaction with the CRM1 export protein, also known as Exportin 1 or XPO1. Selinexor is an orally bioavailable selective XPO1 inhibitor approved for the treatment of advanced multiple myeloma and is under investigation for the treatment of lymphoma and AML. Brunetti and colleagues targeted XPO1 with selinexor in an NPM1c model, which led to the relocalization of NPM1 to the nucleus, inhibition of the a effect . Howevera effect . The lowa effect .HOX genes in NPM1c pathogenesis and the interaction with the MLL chromatin complex, recent studies have leveraged the use of small molecule inhibitors of the MLL co-factor menin to target NPM1-mutated AML [Npm1 single mutant and Npm1/Dnmt3a double mutant mouse model in addition to patient-derived xenograft models, Uckelmann and colleagues tested the effect of VTP-50469, a small molecule inhibitor of menin [MEIS1 expression and led to the significant differentiation of Npm1c leukemic cells in vitro, with a potent anti-leukemic activity on NPM1c in pre-leukemia and in overt leukemia. Interestingly, the expression levels of HOX genes were not affected by this drug despite the dramatic anti-tumor effect, indicating maybe that MEIS1 could be the shared vulnerability for AML with NPM1c. Similarly, Klossowski and colleagues used the menin inhibitor MI-3454 in a mouse model of NPM1-mutated AML and in patient-derived xenograft models, and showed remarkable anti-leukemic activity, this time, however, downregulating HOX genes and MEIS1 [HOX genes and MEIS1 versus MEIS1 alone would add to efficacy or affect tolerability.Given the growing knowledge on the central role of ated AML ,107,108.of menin . They dend MEIS1 . Both ofNPM1c mutations produce the same alternative reading frame in the C-terminus domain. Therefore, this can be considered a shared neoantigen with the NPM1-mutated gene in AML, which can be targeted with immunotherapy. Neoantigens arise from tumor-specific mutations and are limited to malignant cells [NPM1-mutated AML [nt cells ,111,112.ated AML .Other investigational strategies to target NPM1c, including proteasomal degradation or inhibiting NPM1 oligomerization, are summarized In NPM1 mutations which lead to a unique leukemia has expanded. The most important next steps are to translate these discoveries in the clinical setting and improve therapy for patients with this illness. NPM1 mutations can be used as a robust biomarker indicating MRD. Therefore, future studies should focus on how to incorporate this biomarker into clinical decision making. The obvious consequence of NPM1 persistence following standard treatment would be the use of allogeneic stem cell transplants to eradicate MRD. However, all targeted therapies under development for NPM1-mutated AML should leverage this disease, indicating characteristics for the monitoring of therapy. There are likely multiple mechanisms leading to leukemia development triggered by NPM1c, but the critical question is which mechanism would be considered a shared therapeutic vulnerability. Ultimately, similar to what is seen with targeted therapies in multiple cancers, a resistance caused by clonal heterogeneity or escape could emerge; therefore, safe combination strategies will be key for achieving cures for all AML patients with a mutated NPM1 gene.NPM1 is involved in critical mechanisms that maintain cellular homeostasis. Our knowledge on the aberrant function of


Results showed that the level of cytoplasmic Ca2+ increased and SR Ca2+ decreased in skeletal muscle fiber during late torpor (LT) and inter-bout arousal (IBA), but both returned to summer active levels when the animals aroused from and re-entered into torpor , suggesting that intracellular Ca2+ is dynamic during hibernation. The protein expression of ryanodine receptor1 (RyR1) increased in the LT, IBA, and ET groups, whereas the co-localization of calsequestrin1 (CSQ1) and RyR1 in GAS muscle decreased in the LT and ET groups, which may increase the possibility of RyR1 channel-mediated Ca2+ release. Furthermore, calcium pump  protein expression increased in the LT, IBA, and ET groups, and the signaling pathway-related factors of SERCA activity  all increased, suggesting that these factors may be involved in the up-regulation of SERCA1 activity in different groups. The increased protein expression of Ca2+-binding proteins CSQ1 and calmodulin (CaM) indicated that intracellular free Ca2+-binding ability also increased in the LT, IBA, ET, and POST groups. In brief, changes in cytoplasmic and SR Ca2+ concentrations, SR RyR1 and SERCA1 protein expression levels, and major RyR1 and SERCA1 signaling pathway-related factors were unexpectedly active in the torpor stage when metabolic functions were highly inhibited.To explore the possible mechanism of the sarcoplasmic reticulum (SR) in the maintenance of cytoplasmic calcium (Ca We hypon = 8 for each batch). The first batch was used for cytoplasmic and SR Ca2+ level measurement. The second batch was used for molecular biology experiments, including protein extraction, western blot analysis, and protein co-localization analysis. At the end of surgical intervention, the animals were sacrificed by an overdose injection of sodium pentobarbital.All animal procedures and care and handling protocols were approved by the Committee on the Ethics of Animal Experiments of Northwest University (Permit Number: SYXK 2010-004). Daurian ground squirrels were caught within the Weinan region in Shaanxi Province, China. After transfer to our laboratory, the ground squirrels were individually housed in 50 cm × 50 cm × 20 cm cages. Animals were weight-matched and divided into six groups : (1) summer active (SA): samples were collected in mid-June from ground squirrels with a body surface temperature (Tb) range of 36–38°C; (2) pre-hibernation (PRE): samples were collected in mid-September from ground squirrels with a Tb range of 36–38°C; (3) late torpor (LT): samples were collected 2 months after first entering torpor from ground squirrels with a Tb range of 5–8°C for more than 5 days (d); (4) inter-bout arousal (IBA): samples were collected 2 months after first entering torpor from ground squirrels with a Tb range of 34–37°C for less than 12 h (h); (5) early torpor (ET): samples were collected 2 months after first entering torpor and after animals entered a new torpor bout with a Tb range of 5–8°C for less than 24 h; (6) post-hibernation (POST): samples were collected in March of the following year from ground squirrels aroused from torpor with a Tb range of 36–38°C for more than 2-day. The body surface and environmental temperatures are shown in 2 and 5% CO2 gas mixture to ensure the muscle fibers were completely digested, after which the solution was removed with PBS and the muscles were agitated gently and repeatedly with pipettes , then rinsed with 20 mL of phosphate-buffered saline , acutely dissociated with 3 mL of enzymatic digestion solution consisting of 0.35% collagenase I and 0.17% neutral protease , and finally incubated at 37°C on an orbital shaker for 2 h. The enzymatic digestion solution was saturated with 95% Opipettes . The dis2+ binding, to determine cytoplasmic free Ca2+. The experimental method is the same as that in our previous report  , which demonstrates increased fluorescence upon Cas report . Briefly2+, was used to indicate SR free Ca2+, as described previously (2+ mentioned above.Magnesium-Fluo-4-acetoxymethylester (mag-Fluo-4/AM) , which exhibits an increase in fluorescence upon binding to Caeviously . BrieflyTM BCA Protein Quantification kit . The supernatants were mixed with 1 × SDS loading buffer  at a 1:4 v/v ratio, followed by boiling and then storage at −20°C for further analysis.A Nuclear/Cytosol Fractionation Kit was used to extract cytoplasmic protein . As described previously , solubleThe muscle protein extracts were then separated via SDS-PAGE . After electrophoresis, the proteins were electrically transferred to polyvinylidene fluoride (PVDF) membranes (0.45-μm pore size) using a Bio-Rad semi-dry transfer apparatus. The blotted membranes were blocked with 1% BSA in Tris-buffered saline  and incubated with primary antibodies in TBS containing 0.1% BSA at 4°C overnight. The primary antibodies used for western blot analysis are listed in We cut 10-μm thick frozen muscle cross-sections from the mid-belly of each muscle at −20°C with a cryostat , followed by storage at −80°C for further staining . Immunofpost hoc test was used for multiple comparisons among groups. Differences were considered significant at P < 0.01 because the Daurian ground squirrels were wild captured, and thus individual differences were larger. Data in the tables are expressed as means ± standard deviation (means ± SD); data in the figures are expressed as individual sample values, and median, average, upper and lower quartiles, upper and lower edges, and extreme outliers are reported. All statistical analyses were conducted using SPSS 19.0.The normality of data and homogeneity of variance were tested by Levene tests. Single factor analysis of variance (one-way ANOVA) was used to determine group differences. The Tukey P < 0.01). Thus, only slight skeletal muscle atrophy occurred in the different measured stages in hibernating ground squirrels . Overall, cytoplasmic Ca2+ was elevated to varying degrees in the different measured stages in hibernating ground squirrels , but partially recovered when the animals aroused from and re-entered into torpor.As shown in 2+ levels were observed in the three different skeletal muscles. Compared with that in the SA group, SR Ca2+ levels in the SOL, EDL, and GAS muscles decreased by 20–80% in the PRE, LT, and IBA groups. Compared with that in the LT group, SR Ca2+ increased by 136–401% in the ET and POST groups. In addition, SR Ca2+ was 42–472% higher in the ET group relative to the IBA group (P < 0.01). Overall, SR Ca2+ decreased during torpor and recovered when animals aroused from and re-entered into torpor, opposite to the pattern observed for cytoplasmic Ca2+.As shown in P < 0.01)  .P < 0.01)  .P < 0.01)  compared with that in the SA group . Similar < 0.01) .P < 0.01). The co-localization of RyR1 and FKBP12 was similarly measured and found to be unchanged among the six groups in all three skeletal muscles.As shown in P < 0.01) (The reticulate subcellular distributions of SLN and SERCA1 fluorescently labeled proteins are shown in  < 0.01) .2+ concentration and a reduction in SR Ca2+ concentration were observed in the different skeletal muscles of the LT group compared to that of the SA group. This is similar to that found in non-hibernators under the disuse state   . However2+ release may have increased. The co-localization levels of DHPR and RyR1 in the three skeletal muscles decreased in the LT and ET groups compared to that in the SA and PRE groups. The structural combination of these two proteins is the basis for excitation-contraction coupling during skeletal muscle depolarization  during torpor  were increased, which may reduce the inhibition effect of PLB on SERCA (Mesocricetus auratus) and dormice (Dryomys nitedula) participates in the regulation of SERCA activity . In addition to several Ca2+-handling-related proteins, cytoplasmic Ca2+ homeostasis is also affected by other Ca2+ stores (such as mitochondria) or Ca2+-binding proteins . However, as the remaining skeletal muscle samples could not satisfy the needs of these experiments, the effects of these factors on cytoplasmic Ca2+ homeostasis during hibernation will be explored in future work.In this study, the protein expression levels of RyR and SERCA and their co-localization with regulatory factors only reflected the potential biological functions of the CaAll datasets presented in this study are included in the article/Ethics approval and consent to participate all animal procedures and care and handling protocols were approved by the Committee on the Ethics of Animal Experiments of the Northwest University (Permit Number: SYXK 2010-004).Y-FG, ZW, and H-PW conceived and designed the experiments. ZW, X-FM, JZ, HC, XP, and S-HX performed the experiments. ZW and X-FM analyzed the data. ZW, JZ, and Y-FG wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In vitro inoculation of chicken embryo kidney cells with recombinant and wild-type viruses demonstrated that the accessory protein 5b is involved in the delayed activation of the interferon response of the host after IBV infection. Embryo mortality after the inoculation of 8-day-old embryonated chicken eggs with recombinant and wild-type viruses showed that rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b had an attenuated phenotype in ovo, with reduced titres at 6 h p.i. and 12 h p.i. for all viruses, while growing to the same titre as wild-type rIBV at 48 h p.i. When administered to 1-day-old chickens, rIBV-Δ3a, rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b showed reduced ciliostasis in comparison to the wild-type viruses. In conclusion, individual deletion of accessory genes in IBV H52 resulted in mutant viruses with an attenuated phenotype.Avian coronavirus infectious bronchitis virus (IBV) infects domestic fowl, resulting in respiratory disease and causing serious losses in unprotected birds. Its control is mainly achieved by using live attenuated vaccines. Here we explored the possibilities for rationally attenuating IBV to improve our knowledge regarding the function of IBV accessory proteins and for the development of next-generation vaccines with the recently established reverse genetic system for IBV H52 based on targeted RNA recombination and selection of recombinant viruses in embryonated eggs. To this aim, we selectively removed accessory genes 3a, 3b, 5a and 5b individually, and rescued the resulting recombinant (r) rIBV-Δ3a, rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b.  Infectious bronchitis virus (IBV) is primarily a respiratory pathogen of domestic fowl and is the cause of economic losses worldwide. It enters the host through the respiratory tract, causing the destruction of the epithelium with consequent respiratory distress and inclination towards the development of secondary bacterial infections . DependiGammacoronavirus, family Coronaviridae, order Nidovirales. It is an enveloped virus containing a positive-sense RNA genome that is 27.6 kb in length, coding for at least 10 open reading frames (ORFs) characterized by the following organization: 5′UTR-1a-1ab-S-3a-3b-E-M-5a-5b-N-3′UTR. The first two-thirds of the genome comprises the replicase gene, which is expressed through 2 polyproteins, pp1a and pp1ab, which are cleaved by 2 types of virus-encoded proteinases, resulting in 15 non-structural proteins. In addition, the genome encodes four structural proteins, spike glycoprotein (S), small membrane protein (E), integral membrane protein (M) and nucleocapsid protein (N), and a set of accessory proteins [IBV is a member of the genus Coronaviridae, although differences in number, location along the genome and amino acid composition have been observed between viruses belonging to this family [in vitro [in vivo [in vivo [The presence of accessory proteins is a common feature of the members of the family s family . With thin vitro . Further[in vivo . Until r[in vivo . In our [in vivo . The rIB[in vivo , showed [in vivo . Howeverin vitro, in ovo and in vivo. The data gathered provide useful information regarding the significance of IBV’s accessory proteins, and increase the options towards the development of a new generation of rationally attenuated live vaccines.Here, we selectively deleted individual accessory genes of IBV H52 in order to assess how the absence of a single protein influences the phenotype of the virus in vitro-transcribed donor RNA obtained from p-IBV-Δ3a, p-IBV-Δ3b, p-IBV-Δ5a and p-IBV-Δ 5b, respectively. At 7 days post-inoculation embryonic death was observed in all the groups. The embryos showed stunting and curling typical for IBV infection. Replication of rIBV-Δ3a, rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b  were inoculated with LR7 cells that had been infected with murine IBV (mIBV) and transfected with The genetic identity of the viruses was evaluated by RT-PCR targeting the genomic regions where the mutations were introduced. Sequencing of amplicons obtained with primer set D  confirmein ovo growth kinetics of the recombinant viruses were assessed by inoculating 8-day-old ECEs with 102 EID50 of working stock per egg and determining the relative viral load in the AF of five eggs per virus at 6, 12, 24, 36 and 48 h post-infection (p.i.) by RT-qPCR. At the time points 6 and 12 h p.i. the viral loads observed for all viruses in which one of the accessory genes was deleted were slightly lower than for IBV H52 BI and rIBV-wt , as indicated by luciferase activity, in comparison not only to the wild-type viruses, but also to the other deletion mutants and IBV Beaudette. The luciferase activity measurements following IBV-Δ5b infection were also the highest of those for the recombinant viruses at the two following time points. A significant difference in the luciferase activity between rIBV-Δ3a or rIBV-Δ3b and the wild-type viruses was only observed at 30 h p.i., while rIBV-Δ5a showed similar luciferase activity to the wild-type viruses at each time point.The type I interferon response was evaluated for each virus at 6, 12, 18, 24 and 30 h after the inoculation of chicken embryonic kidney cells (CEKs). The supernatants were collected from CEKs at the different time points and used to inoculate cell culture wells containing CEC-32 cells, a reporter cell line expressing luciferase under the control of an IFN-responsive chicken Mx promoter. A one-way analysis of variance (ANOVA) with Tukey’s honestly significant difference (HSD) post hoc test was used to assess whether the differences between the deletion mutants and the wild-type viruses were significant. The results are reported in in vivo by inoculation in 1-day-old SPF chickens with a dose of 103 EID50 per animal of IBV H52 BI, rIBV-wt, rIBV-Δ3a, rIBV-Δ3b, rIBV-Δ5a, or rIBV-Δ5b. As a readout for attenuation, the ciliary activity of 10 tracheal sections per bird at 7 days post-inoculation was evaluated. All rIBV deletion mutants showed reduced ciliostasis compared to wild-type H52 and recombinant H52, with rIBV-Δ5a showing the most attenuated phenotype (P<0.05) in ciliary activity between the deletion mutants and rIBV-wt in infected chickens (P<0.05).The phenotype of the deletion mutants was assessed henotype . Statistchickens . There wIn the present study, the recently developed RGS (based on targeted RNA recombination and selection in embryonated chicken eggs) of IBV H52 BI was used either to remove the accessory genes encoding the 3a, 3b and 5a proteins or to inactivate 5b protein expression. We performed a comprehensive analysis that showed for the first time that the four accessory proteins of IBV all contribute to the pathogenicity of the virus.To this aim, the single-deletion mutants were inoculated in 8-day-old embryonated chicken eggs, in which they showed a slightly delayed but otherwise similar growth kinetics compared to those of the wild-type viruses, hence confirming that IBV accessory proteins are not essential for viral replication in embryonated chicken eggs . DiffereCoronaviridae are known to be involved in the mechanisms to escape the type I IFN response [in vitro approach in which we infected CEK cells with rIBVs and used the collected inactivated supernatants from these to perform the type I IFN luciferase bioassay. rIBV-Δ5b showed an earlier onset of type I IFN production in comparison with both the wild-type viruses and the other single-deletion mutants, as indicated by an increase in the luciferase activity. This finding is in accordance with a previous study, which identified protein 5b as being responsible for the shut-off of the host translation machinery for the cell culture-adapted IBV Beaudette [Ifnβ transcription [Subsequently we investigated whether IBV accessory proteins are involved in evading the immune response of the host. Viral replication in a host relies on the ability of the virus to delay or counteract the type I IFN response  and acceresponse . As the response , we initresponse . We conseaudette . As a coeaudette , while pcription . Taken ain vitro corresponded with an attenuated phenotype in vivo. We infected 1-day-old SPF chickens with the single mutants and the wild-type viruses and evaluated the ciliary activity at 7 d p.i. All rIBV single mutants showed an attenuated phenotype in comparison to the wild-type viruses. Of all the recombinant viruses tested, rIBV-Δ5a showed the most attenuated phenotype in vivo. To the best of our knowledge, this is the first report demonstrating the involvement of protein 5a in the pathogenicity of IBV. The data gathered in this study suggest that protein 5a is not a type I IFN antagonist and that it is dispensable for replication in ovo. Similarly, the deletion of gene 6 from SARS-CoV does not affect the replication in vitro, but results in lower titres and lower morbidity and mortality in vivo [in vivo and that its deletion results in lower viral replication rates in chickens and consequently in an attenuated phenotype.Subsequently, we assessed whether the inability of the deletion mutants to delay or counteract the type I IFN response  in vivo . We can in vivo. Furthermore, we demonstrated that the reverse genetics system for IBV based on targeted RNA recombination allows the rational modification of IBV strains, and that this system could not only be used effectively for studies on gene functions, but also for the generation of rationally attenuated viruses, as the rescued recombinant viruses proved to be less virulent than the wild-type viruses.The data presented in this study show that each individual IBV accessory protein is accountable for the pathogenicity of the virus, despite being involved in different pathways, and that the deletion of one of these genes is sufficient to cause an attenuated phenotype l-glutamine (Lonza), 10 % foetal calf serum  and 0.05 mg ml−1 gentamicin (Gibco Invitrogen) at 37.0 °C and 5 % CO2. CEK cells were obtained by aseptically removing the kidneys from 17- or 18-day-old chicken SPF white leghorn egg embryos . A cell suspension was obtained by trypsinization and the resulting CEK cells were seeded in 24-well plates at 1.5*105 CEK cell/well in Alpha MEM (Gibco Invitrogen) supplemented with 10 % FCS (BioWhittaker), 4 mM l-glutamine (Lonza) and 0.1 % penicillin/streptomycin (Gibco Invitrogen). The CEC-32 quail reporter cell line expressing luciferase under the control of the chicken Mx promoter [2. Specific pathogen-free (SPF) white leghorn eggs  were incubated at 37.5 °C and 45–65 % relative humidity. At day 8 of incubation the embryonated chicken eggs (ECE) were inoculated with virus into the allantoic cavity and candled daily. ECEs with dead embryos were transferred to 4 °C for 16–24 h, and then the AF and CAMs were collected. The virus titre in ovo was expressed as the 50 % embryonic infectious dose (EID50) ml−1, as determined at day 7 p.i. according to Reed and Muench [50 were pooled after incubation for 24 h.Murine LR7 cells  were culd Muench . For preIBV strain H52 BI , recombinant IBV wild-type (rIBV-wt), IBV strain Beaudette  and murinized (m)IBV (strain #1B3-IIA) were propagated as described previously .Immunohistochemistry (IHC) was performed on CAMs collected from ECEs using monoclonal antibody (MAb) Ch/IBV 26.1 directed against the IBV S2 protein  .RNA from the AFs of ECEs was isolated using the QIAamp viral RNA Mini kit  according to the manufacturer’s protocol. To assess the relative viral load in the AFs, a semi-quantitative one-step RT-qPCR was performed using a previously described protocol . For seqThe donor plasmid was designed as previously described . The conMssI-linearized plasmids p-IBV-Δ3a, p-IBV-Δ3b, p-IBV-Δ5a and p-IBV-Δ5b using the mMessage mMachine T7 kit (Ambion by Thermo Fisher Scientific). The in vitro-transcribed RNA was transfected by electroporation into LR7 cells previously infected with mIBV, allowing the introduction of the IBV spike ectodomain into mIBV genome by targeted RNA recombination, as previously described [−1–10−5) and replication was assessed by IHC and RT-qPCR. After sequence analysis of the modified regions, recombinant viruses underwent two further rounds of end-point dilution in 8-day-old ECEs, and virus stocks were grown and titrated. The entire 3′ end (from nt 18 612 onwards) of passage 4 of all rIBV delta variants was sequenced using the oligo sets reported in As described previously , capped escribed . IBV rec50 ml−1 [Comparison of the growth kinetics of rIBV-Δ3a, rIBV-Δ3b, rIBV-Δ5a, and rIBV-Δ5b against those of H52 BI and rIBV-wt was performed by RT-qPCR on viral RNA extracted from the AF of inoculated 8-day-old ECEs. Previously a standard curve was obtained using a 10-fold dilution series of IBV H52 BI with known EIDCEK cells obtained from kidneys of 16-day-old ECEs were seeded in 24-well plates and inoculated at an m.o.i. of 10 with the following viruses: IBV Beaudette, IBV H52 BI, rIBV-wt, rIBV-Δ3a, rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b. Supernatant from infected cells was collected at 6-h intervals starting at 6 h p.i. Cells to which PBS of the same volume as the AF was added served as negative controls.Mx promoter [5 cells/well and incubated with the supernatant of CEK-infected cells for 6 h. Luciferase activity was measured using a luciferase assay kit (Promega) following the manufacturer’s instructions. A standard curve was created by culturing CEC-32 with a twofold dilution series of a known concentration of recombinant IFNα (6 U ml−1). A one-way ANOVA with Tukey's HSD post hoc test was performed to analyse whether the luciferase activities were statistically different between the groups. A P value <0.05 was considered statistically significant.The chicken type I interferon (chIFN) response was assessed using a bioassay based on the CEC-32 quail reporter cell line expressing luciferase under the control of the chicken promoter . First, 3 EID50 in 0.1 ml of IBV H52 BI (n=15), rIBV-wt (n=15), rIBV-Δ3a (n=15), rIBV-Δ3b (n=15), rIBV-Δ5a (n=15) and rIBV-Δ5b (n=15), or not inoculated , respectively. At 7 days post-infection the animals were humanely killed for sampling of the trachea and subsequent evaluation of the ciliary activity. Briefly, for each trachea ten 1 mm transverse sections were collected  and examined under low-power microscopy. The ciliostasis of each tracheal section was scored on a scale from 0 (100 % ciliary activity) to 4 (no ciliary activity), and the average ciliostasis score was calculated for each group of animals [P value <0.05 was considered statistically significantOne hundred layer-type chickens were hatched from SPF white leghorn eggs at BIAHO from eggs obtained from Valo Biomedia, Osterholz-Scharmbeck, Germany. At 1 day of age the birds were divided into seven groups and kept in separate isolators under controlled housing conditions. Animals were inoculated via eye-drop with 10 animals . Eventua


Growing concerns over ethical issues in mentoring in medicine and surgery have hindered efforts to reinitiate mentoring for Palliative Care (PC) physicians following the easing of COVID-19 restrictions. Ranging from the misappropriation of mentee’s work to bullying, ethical issues in mentoring are attributed to poor understanding and structuring of mentoring programs, underlining the need for a consistent approach to mentoring practices.Given diverse practices across different settings and the employ of various methodologies, a novel approach to narrative reviews (NR)s is proposed to summarize, interpret, and critique prevailing data on novice mentoring. To overcome prevailing concerns surrounding the reproducibility and transparency of narrative reviews, the Systematic Evidenced Based Approach (SEBA) adopts a structured approach to searching and summarizing the included articles and employed concurrent content and thematic analysis that was overseen by a team of experts.A total of 18 915 abstracts were reviewed, 62 full text articles evaluated and 41 articles included. Ten themes/categories were ascertained identified including Nature; Stakeholders; Relationship; Approach; Environment; Benefits; Barriers; Assessments; Theories and Definitions.By compiling and scrutinizing prevailing practice it is possible to appreciate the notion of the mentoring ecosystem which sees each mentee, mentor, and host organization brings with them their own microenvironment that contains their respective goals, abilities, and contextual considerations. Built around competency based mentoring stages, it is possible to advance a flexible yet consistent novice mentoring framework. At the heart of concerns amongst administrators, program designers, and curriculum advisor as well as some mentors and mentees are growing concerns over reports of ethical issues in mentoring.To address this lacuna and better inform the structuring of mentoring programs, review of novice mentoring data is required. However such an endeavor must acknowledge the fact that prevailing data involves a variety of methodologies and practices and does not often consider mentoring’s evolving nature that requires a longitudinal perspective of mentoring nor its context specific nature that limits comparisons of mentoring data across different research, clinical, academic, practice and healthcare settings, and mentoring goals. Scrutiny of novice mentoring is also limited by poorly defined terms and the mistaken intermixing of mentoring and educational approaches such as peer, near-peer, group, leadership, patient, family, and e-mentoring as well as advising, sponsoring, role modeling, tutoring, coaching, supervising, and networking which have their own distinct approaches and roles in medical education. Such conflation of terms and practices also compromises structured search processes which relies on clearly defined terms to guide the search process. In addition many review fail to consider the socially constructed aspect of mentoring nor the need to include longitudinal mentoring experiences and perspectives of mentees, mentors, and other stakeholders that are often captured in gray literature. These considerations and the need for a multidimensional socioculturally informed perspective of mentoring from the viewpoint of mentees, mentors, the host organization, and other stakeholders also underline the need for a constructivist approach and the use of a relativist lens.1518 we will seek to study prevailing novice mentoring data in IM and extrapolate these findings to PC.In the face of data suggesting that mentoring can be extrapolated from accounts of novice mentoring in Internal Medicine (IM),19 and the hidden systems that drive the mentoring process2028 across different research traditions.27 An NR is also better equipped to contend with data from gray literature and forward a better understanding of why21 and how mentoring’s socioculturally informed processes evolve and affect stakeholders and their mentoring relationships.252937With use of systematic and scoping reviews limited by poorly defined mentoring terms that will compromise structured searches, a narrative review (NR) is proposed given its ability to delve into the underlying38 whilst its relativist lens allows for the collation of historically, socioculturally, ideologically, and contextually influenced views, experiences and accounts of mentees, mentors, and host organizations (henceforth stakeholders).To address concerns that NRs lack transparency, accountability, and structure in the synthesis of narratives, we adopt Krishna’s novel methodology called Systematic Evidenced Based Approach (SEBA). Built upon a constructivist perspective, a SEBA guided NR (henceforth NR in SEBA) is able to map complex topics from multiple angles3941 This enhances the accountability of the search process.Guided through each stage of the synthesis of an NR in SEBA by an expert team comprised of medical librarians from the Yong Loo Lin School of Medicine (YLLSoM) at the National University of Singapore and the National Cancer Centre Singapore (NCCS), and local educational experts and clinicians at the NCCS, the Palliative Care Institute Liverpool, YLLSoM, and Duke-NUS Medical School, and guided by the principles of interpretivist analysis, the research team immerse and chart their review, and analysis of the qualitative data. Team discussions and expert guidance ensured that the data was pieced together in a meaningful, transparent, and reproducible manner.Outlined in what is known of novice mentoring in Internal Medicine (henceforth IM)?” In order to inform the design, structure, and oversight of novice mentoring programs, the secondary research questions were determined to be “what are the key characteristics of novice mentoring in IM?” and “what processes contribute to their success?”Ensuring a systematic approach to the synthesis of NRs in SEBA, the expert and research team established the overall goals of the NR and the population, context, and concept to be evaluated. The research question was determined to be “43 This is outlined in A Population Intervention Comparison Outcome Study Design (PICOS) format was adopted to guide the research process.Supplemental Appendix 1.Ten members of the research team carried out independent searches of 7 bibliographic databases  and gray literature databases  between 17 December 2019 and 17 January 2020. Focus on mentoring in IM rather than PC was in acknowledgement of a dearth of mentoring data in PC and evidence that extrapolation of data from IM and PC is possible. The PubMed Search Terms and Strategy may be found in 44 approach to “negotiated consensual validation” was used to achieve consensus on the final list of titles to be reviewed. The research team then independently reviewed each full-text article from this final list, created individual lists for inclusion, held team discussions online and arrived at a consensus on the final list of full-text articles to be included in the NR in SEBA. To circumnavigate the limitations caused by poorly delineated mentoring terms, the research teams reviewed the references of the included articles and snowballing their findings to reduce the chance of omitting relevant articles.Using an abstract screening tool, each member of the research team independently reviewed the titles and abstracts found in each database and drew up a list of titles to be reviewed. Comparing these individual lists via online meetings, Sambunjak et al’s45 approach to thematic analysis whilst the second team employed Hsieh and Shannon’s46 approach to directed content analysis. Used to enhance the trustworthiness of the results, concurrent thematic and content analysis is the hallmark of the Split Approach The third team summarized and tabulated the included articles, keeping with recommendations drawn from Wong et al’s38 RAMESES publication standards: meta-narrative reviews and Popay et al’s47 “Guidance on the conduct of narrative synthesis in systematic reviews.” The tabulated summaries serve to ensure that key aspects of the included articles are not lost. They are presented in Supplemental Appendix 2.Dividing into 3 teams, the researchers simultaneously reviewed the included full-text articles. The first team adopted Braun and Clarke’s4850 These codes were then organized into themes deemed to best represent the whole data set.51 Each member independently reviewed and refined their themes with negotiated consensual validation used to establish a final list of themes.52A reiterative step-by-step thematic analysis was carried and this saw codes constructed from the “surface” meaning of the articles.53Directed content analysis was employed to enhance validity of the themes identified and addressed the relative failure of Braun and Clarke’s approach in resolving contradictory data.54 reviewers drew codes from Krishna et al’s17 “Mentoring stages: A study of undergraduate mentoring in palliative medicine in Singapore,” the first clinically evidenced account of novice mentoring in Palliative Medicine. Deductive category application was employed to identify any relevant data not captured by existing codes and subsequently assigned a new code.55 Each member independently reviewed and refined their codes with negotiated consensual validation used to establish a final “code book.”Identifying and operationalizing a priori coding categories,The jigsaw perspective brings together complementary data and is especially important in ensuring that critical aspects are not lost in the “funneling” process that follows. This “funneling” process saw the themes and categories identified in the independent analyses compared and combined to present a holistic perspective of novice mentoring thus facilitating its effective analysis and interpretation.As part of the reiterative process, the findings were discussed with the expert team and concerns were raised over the influence of gray literature on the results as they were neither peer reviewed nor clearly evidence based. This saw the research team differentiating gray literature such as correspondence, letters, editorials, and perspective pieces extracted from academic databases from data driven and research based peer reviewed articles. Both were thematically analyzed independently and the themes derived from the gray literature were found to be in agreement with themes from the peer-reviewed literature.In total, there were 10 themes/categories revealed through Braun and Clarke’s and Hsieh and Shannon’s approach. As extensive overlaps were observed, they will be presented and discussed together.Supplemental Appendix 3. The following themes/categories pertaining to novice mentoring was discerned: (1) Definitions, (2) Nature, (3) Stakeholders, (4) Relationship, (5) Approach, (6) Environment, (7) Benefits, (8) Barriers, (9) Assessments, and (10) Theories.A total of 18 915 abstracts were reviewed, 62 full-text articles evaluated and 41 articles included. This is outlined in the PRISMA Flowchart in 56 definition which dominates prevailing practice characterizes novice mentoring as “an evolving relationship between an experienced clinician and junior clinicians and/or students that is focused upon creating personalised and enduring mutually beneficial mentoring relationships.”Wesley et al’sHowever 5 other considerations were highlighted:(a) the host organization’s role in the mentoring relationship.(b) the host organization’s role in nurturing the mentoring environment through the establishment of clear codes of conduct and provision of support for consistent, longitudinal and holistic assessments, matching processes, and training programs.(c) the presence of mentoring stages in a structured mentoring process.(d) the need to balance between a consistent mentoring approach, compliance with codes of practice, and flexibility so as to accommodate for evolving needs, goals, and expectations of stakeholders.(e) novice mentoring’s evolving, goal-sensitive, context-specific, mentee-, mentor-, organizational- and relational-dependent nature and thus implication on research and support.As briefly outlined in the final consideration above, novice mentoring possesses critical characteristics further elaborated in 58 These mentee- and mentor-specific traits are delineated in Supplemental Appendices 4 and 5 but may be broadly categorized into personal and professional characteristics.By virtue of their intrinsic involvement, stakeholders also naturally influence the mentoring process and their outcomes. This underscores the gravity of the matching process in ensuring that would-be mentees and mentors possess desirable traits that complement one another and the overall goals of the program.17 Indeed, the host organization, oft forgotten as a pivotal stakeholder, plays a crucial role in overseeing, supporting and assessing various facets of the mentoring program—including mentor training, mentee briefing, the matching process, mentoring relationship, mentoring approach, and mentoring environment.60In addition, their ability to fulfil their respective roles and responsibilities foregrounds the importance of longitudinal training, assessment and support.58 argue that mentoring relationships are shaped by shared values and beliefs between mentees and mentors, their success hinged on the presence of quality and reciprocal interactions between all stakeholders, including the host organization.58 The mentoring relationship has notable bearings on the professional identity formation of mentees and the motivations of mentors in seeking out avenues to groom them. Indeed the success of the overall mentoring program in realizing its mentoring goals is said to pivot upon the nurturing of lasting and personalized mentoring relationships.60Qiao Ting Low et al60Most mentoring programs adopt either a formal or informal mentoring approach. Formal mentoring offers a more rigorous structure to the matching process and provides clearer specifications with regards to goals, learning objectives, roles and responsibilities, codes of conduct, standards of practice, and the type and duration of interactions expected.revolves around the idea of apprenticeship in medicine”57 and sees more ad-hoc influence by learners, tutors, and the host organization.60 Whilst it proffers a more collegial environment which facilitates open communication beneficial toward the development of stronger ties between mentees and mentors, the lack of protected time and poor oversight, transparency, and support from the host threaten the viability of informal mentoring processes.Conversely, informal mentoring “Indeed regardless of which mentoring approach is used, for its full efficacy it should be sensitive to the personal, academic, research, professional, social, and emotional considerations of individual stakeholders involved. This is paramount as mentee and mentor knowledge, experience, preferences, needs, and limitations affect their availability and commitment toward developing the mentoring relationship. Whilst acceptable practice parameters must be adhered to, the importance of a flexible mentoring approach is underpinned here.60 suggest that the mentoring environment has 2 interwoven features, the mentoring structure and mentoring culture.Hee et althe framework that shapes the learning approach.” It includes logistical considerations such as the provision of protected time; the location, frequency, and duration of mentoring, tutorial, and feedback sessions; and the presence of confidential avenues for raising concerns directly to the host organization. The mentoring structure lays the foundation for the provision of streamlined professional and personal support to mentees and mentors alike.The mentoring structure is defined as “the norms, values, beliefs, practices and support moulding the socioemotional environment in which learning occurs.” These include permissible topics and sanctioned behaviors during the mentoring process. It is informed by the stakeholders, the formal mentoring structure and the informal curriculum. The latter referring to opportunistic and idiosyncratic instruction-giving, including the transmission of values and beliefs that underlie prevailing actions and practices of mentors and the host.The mentoring culture on the other hand refers to “The diverse benefits of mentoring to mentees are highlighted in 60Although not commonly addressed, benefits to mentors may be personal and or professional. Personal benefits include the chance to share their knowledge and experience leading to satisfaction, joy, fulfilment, and pride when witnessing the success of their mentees. It also reportedly encourages professional growth, improved job performance, accelerated research productivity and promotions, and provides opportunities to forge new liaisons with collaborators.16 These hinder the fostering of fruitful mentee-mentor relationships.Barriers to effective mentoring include a lack of protected time, availability of mentors and difficulties in balancing disparate needs, goals, and expectations of the stakeholders involved.60 No validated tool to assess mentoring experiences or their outcomes as presently available.Traditionally under the purview of the host organization, assessments and evaluations of the mentoring process remain poorly studied.61 there are no prevailing theories that comprehensively capture all its components.Despite recent efforts to forward a theory of mentoring,62 and the STORIES  statement. In addressing its primary and secondary research questions, this NR in SEBA builds on the 10 themes/categories identified to map and discuss prevailing data on novice mentoring.The narrative produced was guided by the Best Evidence Medical Education (BEME) Collaboration guideyet maintain a consistent mentoring approach that exists within the confines of codes, standards of practice, and program expectations.With the new findings, novice mentoring may be characterized as the process of creating personalized, enduring, and mutually beneficial mentoring relationships between stakeholders. Its success necessitates the guidance of a mentoring structure that is able to balance demands for flexibility in accommodating for evolving mentoring needs, goals, circumstances of stakeholders, and the mentoring environment To support novice mentoring’s dynamic, entwined, evolving, adaptable, context-specific, goal-sensitive, mentee-, mentor-, host organization-, mentoring environment-, mentoring approach-, and mentoring relationship-dependent nature, the host organization must oversee, assess, and support the mentees, mentors, matching process, mentoring relationship, mentoring approach, and mentoring environment.This definition will help to focus the course and content of this NR in SEBA.6388 yet flexibility697389103 within the mentoring program. By spearheading the program, the host organization is deeply involved in formalizing the mentoring approach and its structure. It establishes mentee and mentor roles, responsibilities, and expectations;104 practice standards and codes of conduct;105 and the milestones of each mentoring stage.96 Albeit under the aegis of the mentoring program as a whole, guidelines established by the host steer mentees and mentors as they set their specific goals,107 objectives,106 and timelines.Recent reviews suggest that the host organization plays a critical role in ensuring consistency88 is enhanced by flexible accommodations to their particular setting, goals, needs, and capabilities. Understanding the host organization’s role in the mentoring process and in operationalizing the mentoring structure it has adopted underlines the influence of the mentoring culture and introduces the notion of mentoring dynamics.Whilst a consistent and transparent program imbues confidence in the mentoring relationship, the mentee’s and mentor’s sense of autonomy, connectivity, and advocacy of the program108 which enables the host to instil and influence mentoring and education philosophies as well as goals and values of the program.109 The mentoring culture consequences recruitment and retention of stakeholders110112 and informs stakeholders of characteristics desired of them.Mentoring dynamics refer to the quality of interactions between stakeholders. It is critical to the development of mentoring relationships is influenced by particularly influenced by the mentoring cultureThe notion that each stakeholder influences the mentoring dynamic raises the idea of a mentoring ecosystem with each stakeholder bringing with them individual “micro-environment.” For mentees and mentors, their micro-environment consists of internal and external factors. Internal factors account for individual characteristics, availabilities, abilities, motivations, and goals. These are impacted by external factors such as particular sociocultural, curricular, personal, academic, clinical, professional, ethical, and research factors; prevailing geopolitical, care and educational financing as well as healthcare and educational systems. Changes in these factors affect their micro-environment and influences their ability to productively participate in the mentoring process.The host organization’s microenvironment is more complex. Despite nurturing the program’s mentoring environment, the host organization’s micro-environment does not on its own shape it. Instead the host organization’s “micro-environment” which is informed by internal and external factors including the mentoring structure, the nature and dynamics of interactions between stakeholders and the informal, formal and hidden curricula shape the program’s own mentoring environment.The mentee’s and mentor’s micro-environment are influenced by the program’s mentoring environment as early as the recruitment stage where participation is contemplated. At the matching stage where mentees are introduced to potential mentors, their micro-environment begin to intermingle. Within this “meso-environment,” elements within the mentee’s micro-environment affect the mentor’s ability to function within the mentoring program and vice versa.The mentoring relationship begins with the mentee’s and mentor’s formal agreement to enter into a mentoring relationship with each other under the aegis of the mentoring program. With this, the mentee’s and mentor’s meso-environment fuse with the host’s microenvironment as well as the program’s mentoring environment to form the macro-environment.This macro-environment sees the mentor and mentee influenced by wider factors affecting the mentoring program and host organization. The macro-environment will change as the stakeholders’ micro-environment interact with one another and as the mentoring relationship moves through the mentoring stages. Thus the evolution of the mentoring micro-, meso-, and macro-environments is directed and heavily influenced by the overarching mentoring structure and its constituents. This gives rise to the idea of a mentoring ecosystem which comprises of these dynamic elements and interactions. How micro-, meso-, and macro-environments form under the aegis of the mentoring structure also uncovers the novel role of competency based mentoring stages.17 posit clear mentoring stages in the form of recruitment; aligning of expectations; the matching process; pre-mentoring meetings and training processes; and the subsequent mentoring relationship. For more targeted support and efficacious mentoring outcomes, it was suggested that each stage contain specific competencies to be met by the mentees in order for the mentoring relationship to advance. Not only do these competency based stages shed light as to how micro-, meso-, and macro-environments interact and respond to rigorous mentoring structures but it also further explains the importance of balancing between such need for consistency and demands for flexibility.The more traditional view of mentoring relationships is that it begins with and is sustained by a “fit for purpose” match that brings mentees and mentors with complementary interests and goals together. A newer perspective however sees the development of mentoring relationships occur in stages. To create a stable, effective and nurturing environment, Krishna et alIn order to conceptualize these dynamic elements and interactions within the mentoring ecosystem, 17 (As PC programs wrangle with concerns about structural issues in mentoring programs that may predispose ethical issues and abuse, we forward a mentoring framework built around the mentoring stages posited by Krishna et al17 . Advanci38 In addition although use of Levac et al’s113 adaptation of Arksey and O’Malley’s114 methodological framework to conduct a systematic scoping review rather than “traditional” scoping reviews115 was used to enhance transparency and reproducibility of the NR and use of broad search terms, the presence of papers drawn from a common stable raises concerns about inherent biases and the omission of critical papers. These factors stifle efforts to deepen understanding of a complex process116 despite use of the Split Approach and involvement of expert teams. However, the expert team concurred on the narrative presented and believed that it would be useful for various parties interested in designing, assessing, supporting, and expanding novice mentoring programs.Whilst NRs attempt to build on linked papers, the lack of data on novice mentoring and the common root from which they stem from hinders efforts to create a “coherent paradigm.”Conceiving mentoring as a flexible process within structured mentoring stages helps explain the process of balance and underlines the ability of the mentoring structure to personalize mentoring processes and contend with evolving mentoring dynamics across different stages without compromising mentoring standards and breaching codes of practice. Perhaps more importantly having the competencies and the standards of practice of the specific stage agreed upon by the stakeholders makes for more timely, personalized, appropriate, specific assessments of the mentoring dynamics, relationship, and progress. Moving forward, future studies must also consider the impact of PC’s multidisciplinary practice and the impact of multiple stakeholders in the mentoring process and enhance the structuring of the mentoring process accordingly. Assessments of mentoring processes and mentor training must be the focus of coming studies if mentoring in PC is to regain its place in PC education.Click here for additional data file.Supplemental material, Appendices_xyz46891a36bb924 for Enhancing Mentoring in Palliative Care: An Evidence Based Mentoring Framework by Lalit Kumar Radha Krishna, Lorraine Hui En Tan, Yun Ting Ong, Kuang Teck Tay, Jia Min Hee, Min Chiam, Elisha Wan Ying Chia, Krish Sheri, Xiu Hui Tan, Yao Hao Teo, Cheryl Shumin Kow, Stephen Mason and Ying Pin Toh in Journal of Medical Education and Curricular Development