Repair of DNA double-strand breaks (DSBs) is crucial for genome integrity. A conserved response to DSBs is an increase in chromatin mobility that can be local, at the site of the DSB, or global, at undamaged regions of the genome. Here, we address the function of global chromatin mobility during homologous recombination (HR) of a single, targeted, controlled DSB. We set up a system that tracks HR in vivo over time and show that two types of DSB-induced global chromatin mobility are involved in HR, depending on the position of the DSB. Close to the centromere, a DSB induces global mobility that depends solely on H2A(X) phosphorylation and accelerates repair kinetics, but is not essential. In contrast, the global mobility induced by a DSB away from the centromere becomes essential for HR repair and is triggered by homology search through a mechanism that depends on H2A(X) phosphorylation, checkpoint progression, and Rad51. Our data demonstrate that global mobility is governed by chromosomal conformation and differentially coordinates repair by HR. Double-strand breaks (DSBs) are harmful lesions that constantly challenge the eukaryotic genome. DSBs can lead to deleterious genetic alterations such as modifications of the DNA at the DSB site (insertions/deletions) or global chromosomal rearrangements (translocations). To faithfully maintain genome stability and survive DSBs, cells display a DNA damage response (DDR) involving complex networks that detect, signal, and repair DSBs. Two of the most conserved factors contributing to DSB signaling are the checkpoint kinases Tel1 and Mec1  that catalyze the phosphorylation of histone H2A in yeast and of H2AX in mammalian cells, forming γ-H2A(X) . γ-H2AX. This wiDSBs can be accurately repaired by homologous recombination (HR). This mode of repair, predominant in S phase, uses the sister chromatid as template . When hotrans-contacts between centromeres, and telomeres are bound to the nuclear periphery in a position that depends on the size of the chromosome arms (The non-random organization of chromosomes in the nucleus affects numerous nuclear processes including homology search. In the configuration of budding yeast chromosomes (known as the Rabl configuration), centromeres are clustered close to the yeast microtubule organizing center (spindle pole body), as evidenced by abundant ome arms . It was ome arms . Recombiome arms . Accordiome arms . Likewisome arms .The search for distantly located homologous regions challenges recombination, likely requiring additional sophisticated mechanisms, such as chromatin mobility. Originally observed in yeast, increased chromatin mobility, both at the damaged locus  and elsewhere in the undamaged genome , is a conserved response to DSBs , but not on the Rad9-dependent checkpoint nor on the Rad51 nucleofilament. Therefore, this ‘proximal’ mobility has little influence on the overall HR efficiency, but rather accelerates repair kinetics. In contrast, a subtelomeric DSB induces a mobility that we call ‘distal’. This distal mobility only occurs if a homologous sequence is present and if it is near the centromere. We demonstrate that the donor homologous sequence allows γ-H2A(X) spreading that is the requisite for chromatin motion. This distal mobility shares the characteristics of homology search mobility. It is indeed dependent on Rad9 and the Rad51 nucleofilament, and is necessary for HR. Our data unambiguously demonstrate the role of damage-induced global mobility due to non-random chromosome organization and unify conflicting data on its role in HR repair.mCherry synthesis, using its coding sequence as both recipient and donor sequences. The recipient and donor mCherry sequences cannot be translated into functional fluorescent proteins in the absence of HR. The mCherry recipient sequence is located on a chromosome. It is under the control of the TEF constitutive promoter and contains an I-SceI cut site (cs), followed by a stop codon that results in an mCherry truncation. The donor sequence, is on a centromeric plasmid, hereafter referred to as dCen. It contains an mCherry sequence lacking the promoter and terminator sequences, and shares 539 and 172 bp of homology upstream and downstream of the I-SceI cs of the recipient sequence , and in a subtelomeric position, 10 kb from the right telomere (S strain)  . All str strain) . These dSceI endonuclease on a plasmid under the control of the GAL1 galactose-inducible promoter generates single DSB (+DSB), whereas the empty plasmid not expressing I-SceI was used as a control (-DSB). I-SceI expression, measured by RT-qPCR showed a 6.7±0.85-fold increase after 1 hr and a fivefold plateau at longer times  of V-Vis upon induction by I-SceI in the different C, L, and S strains, in the presence of the plasmids containing or not the donor sequence. For each condition, we tracked the locus by time-lapse microscopy at 100 ms time intervals in 500–1000 cells, as in SceI were grown in galactose for 6 hr. In the absence of I-SceI induction, the V-Vis locus exhibited a subdiffusive behavior, with MSDs exhibiting a power law (MSD ∼ Dtα), where α was ∼0.6. The value of this anomalous exponent below 1 was previously shown at these short and long time scales for many loci scattered along the genome . Altogether these results show that in the absence of homology, the spatial position of the DSB plays a key role in induction of global mobility. A pericentromeric DSB specifically triggers a global mobility that reflects chromatin changes at short times and greater exploration of nuclear space at long times  and ∆rad9 strains and assessed cell survival and cell cycle progression in the ∆rad9 mutant  signal on the actin gene was used as a reference control. In the absence of DSB, low-intensity γ-H2A(X) ChIP signals were detected in both the C and S strains  increased significantly in cis at 50 and 100 kb from the I-SceI CS by approximately 15- and 10-fold in the C and S strains, respectively  spreads around the break and across pericentromeric regions if the DSB is close to the centromere.Phosphorylation of H2A occurs upstream of Rad9 binding after DNA damage. It is necessary and sufficient to induce global mobility when multiple damage occur and it spreads through the centromeres after a pericentromeric DSB . H2A pho strains . ChIP γ-ectively . In the is locus . In the  plasmid . TogetheSceI cleavage efficiency in these mutated strains exhibited similar levels to the WT after 6 hr of I-SceI expression, as shown by qPCR  in mobility by measuring global dynamics in phospho-deficient H2A-S129A and phospho-mimetic H2A-S129E mutants, in the presence or absence of dCen. I- by qPCR . Upon I-ckground . Yet, thckground . In the We next analyzed global mobility upon DSB induction in WT and H2A-S129 mutant strains. In the H2A-S129A mutant, the increase in global chromatin mobility was similarly impaired in the C and S strains harboring or not the donor sequence dCen . In contThe kinetics of HR repair in H2A-S129 mutants was next assessed using the THRIV system . After aRAD51 in the C and S strains, verified that cleavage by I-SceI was as effective in both strains  or RAD51 deletions on mobility, indicating that at least under these conditions, γ-H2A(X) spreading allowed by centromeres clustering is sufficient to enhance chromatin mobility.We observed a first type of global mobility, that we propose to name ‘proximal mobility’, when a DSB is induced near the centromeres. This proximal mobility correlates with and depends solely on γ-H2A(X) that can spread from the site of damage to other pericentromeric regions. This was shown by ChIP in a strain where the break is induced near the centromere (C strain), in agreement with That Rad9 is dispensable for proximal mobility is intriguing and contradicts previous evidence showing a checkpoint requirement for increased global mobility after random damage . This ditrans-propagation of γ-H2A(X) in the pericentromeric region occur? γ-H2A(X) is primarily induced by the H2A kinase Mec1 . In this case, the increased mobility of V-vis is only induced if a donor close to the centromere, either on a plasmid or on the chromosome, is present. This donor-dependent distal mobility requires a more sophisticated mechanism than simple γ-H2A(X) propagation, since it involves checkpoint progression and homology search and is thus triggered in a Rad9- and Rad51-dependent manner.trans contacts . Likewise, Rad51 also collaborates to maintain this local mobility, preparing itself to initiate sampling within the nucleus with an elongated filament. In agreement, ChIP of γ-H2A(X), Rad51, and H2A-S129 mutants show that γ-H2A(X) signal propagation and homology search are apparently directly related . The faccontacts . The disSchizosaccharomyces pombe appears to be important for HR repair  and sealed using VaLaP . For growth curves, overnight cultures were diluted in YPD to a starting density of OD600=0.5. Cell cultures were incubated at 30°C and OD was measured every 45 min. For drop assays, overnight cultures were diluted to bring cells to the exponential phase. These cultures were diluted to a starting density of OD600=1.0 and serial 1:5 dilutions were plated on selective medium (SC-Ade-His) containing raffinose or galactose (2%), and incubated at 30°C for 48 or 72 hr.Yeast cells were grown in rich medium  or synthetic complete (SC) medium lacking that appropriate amino acid at 30°C. Synthetic medium containing 2% raffinose and lacking appropriate amino acids was used to grow the cells overnight prior the induction of I-SceI cutting site at the desired position, a KANMX cassette was integrated into intergenic loci of chromosome IV at positions 453880+453959 (C strain); 853996+854075 (L strain); 1511840+1511919 (S strain). The KANMX cassette was then replaced by HR using CRISPR/Cas9 (pEF526) with the amplified donor sequence mCherry* containing cutting site of I-SceI (5’-ttacgctagggataacagggtaatatagcg-3’), from pCM189-mCherry*-I-SceI (cs), with oligos oEA310/311. The dCen (pEF573) plasmid was obtained by PCR amplifying mCherry sequence with (oEA307/308) oligonucleotides and cloned into pRS412 (ADE2 centromeric plasmid) digested with BamHI and EcoRI. pB07 plasmid containing I-SceI under the control of a GAL1-10 promoter in a HIS3 plasmid was obtained from B Pardo.Strains C, L, and S were constructed by first integrating 256 TetO sequences at the MAK10 gene (YEL053C) as in HTA1 (oEA325/326) and HTA2 (oEA327/328) inserted into the plasmid pJH2971 (CRISPR/Cas9-KANMX-Addgene plasmid#100955) were used to generate a DNA break, afterward repaired with a donor oligonucleotide of 80 nt carrying the desired mutation. All integrations were confirmed by PCR and mutations checked by sequencing. The primers and RNA guides used are indicated in The H2A-S129A and H2A-S129E mutants were constructed as described in RAD9 (YDR217C) and RAD51 (YER095W) genes in the different strains were carried out by integration of the amplified KANMX cassette derived from the Saccharomyces Genome Deletion Project collection.Deletions of For drop assays, overnight cultures were diluted to a starting density of OD600=1.0 and serial 1:5 or 1:10 dilutions were plated on selective medium (SC-Ade-His) containing raffinose or galactose (2%), and incubated at 30°C for 36 hr.2 at a pixel size of 108 nm. We acquired 3 min films consecutively with an exposure time of 100 ms. The complete imaging system including camera, piezo, LEDs (SpectraX) was controlled by the NIS element software (version 4.60).Live cell imaging was done using a wide-field microscopy system featuring a Nikon Ti-E body equipped with the Perfect Focus System and a ×60 oil immersion objective with a numerical aperture of 1.4 . We used an Andor Neo sCMOS camera, which features a large field of view of 276×233 μmParticle tracking and MSD analyses were performed as described in 8 cells were centrifuged for 1 min at 13,000 rpm and 300 mg of glass micro beads, 200 μL of lysis solution  and 200 μL of phenol-chloroform-isoamyl alcohol were added to the cell pellet. The mixture was vortexed for 10 min at 4°C and centrifuged for 10 min at 13,000 rpm; 140 μL of the aqueous phase were transferred into new tubes containing 500 μL of cold 100% ethanol. The mixture was centrifuged for 10 min at 13,000 rpm at 4°C, the pellet washed with 500 μL of 70% ethanol, dried at room temperature, resuspended in 40 μL of TE containing 20 μg/mL of RNAse and incubated at 42°C for 30 min. The DNA was quantified by SimpliNano from Biochrom.Cells were grown to exponential phase in raffinose selective medium and induced by addition of galactose 2% final concentration. 1–2·10For RT-qPCR, RNA was extracted with the Machery Nagel kit, quantified by Nanoview and absence of RNA degradation checked by gel electrophoresis migration. Maxima First Strand was used for RT reaction on 1.5 µg RNA.The qPCR was performed on LightCycler 480 (Roche) on a 96-well plate under the following conditions: pre-incubation (95°C – 5 min – 4.4 °C/min), amplification , melting curve  and cooling (40°C – 30 s - 2.2 °C/min.).The primers oEA312/313  were use9 cells were cross-linked for 10 min with 1% formaldehyde (Sigma F8775) at room temperature on a shaking device. Fixation was quenched by addition of 0.25 M glycine (Sigma G8898) for 5 min under agitation. Cells were washed two times with cold TBS1X (4°C). Dry pellets were frozen and stored at –20°C. Cell pellets were resuspended in lysis buffer  supplemented with 1 mM PMSF, phosphatase inhibitor, and anti-protease  and lysed three times by beads shaking. The lysate  volume was sonicated with a Q500 sonicator . Twenty μL of input material were saved for qPCR. Approximately 180 μL (13 OD) of the input were incubated with 0.5% BSA, 1 μL of DNA carrier (10 mg/mL) and 2 μL of anti-γ-H2A(X) (Abcam 15083) on a rotating wheel overnight at 4°C. The day after, 30 μL of protein G Sepharose beads were washed three times and resuspended in 90 μL final of lysis buffer, which were added to the overnight culture during 2 hr on a rotating wheel at 4°C. Beads were then collected and washed on ice with cold solutions: twice with Lysis buffer , twice with Lysis buffer added with 360 mM NaCl, twice with Washing buffer  and once with TE . Antibodies were uncoupled from beads with 150 μL of Elution buffer  for 20 min at 65°C. Eluates were incubated with 120 μL of TE containing 0.1% SDS at 65°C for 6 hr to de-crosslink. Then 130 μL of TE containing 60 μg RNase A  were added and the samples were incubated for 2 hr at 37°C. Proteins were digested by addition of 20 μL of proteinase K  at 20 mg/mL to the samples and incubation for 2 hr at 37°C. DNA purification was completed with the QIAquick PCR Purification Kit (n28104). Finally, qPCRs were performed in a LightCycler480 (Roche). IP/input ratios were calculated and qPCR results were normalized on ChIP-qPCR Act1 for γ-H2A(X). For each strain, three independent experiments were performed with the corresponding controls.This protocol is adapted from 6 cells were analyzed with a CANTO II flow cytometer (BD Biosciences). Aggregates and dead cells were gated out, and percentages of cells with 1C and 2C DNA content were calculated using FLOWJO software.For cell cycle, cells were grown to mid-log-phase in liquid cultures, and treated or not with Zeocin at 250 mg/μL during 6 hr at 30°C. After incubation, samples were fixed with 70% ethanol and kept at 4°C for 48 hr. Cells were then resuspended in 50 mM sodium citrate (pH 7) containing RnaseA at 0.2 mg/mL final concentration. After incubation at 37°C for 1 hr, Sytox Green was added to a final concentration of 1 mM. A total of 107 cells were removed and centrifuged. All the following steps were done in the dark. The pellet was washed once with cold PBS at 4°C. After removal of the supernatant, the pellet was resuspended in 500 μL of cold PBS and 500 μL of cold PBS with formaldehyde  were added and gently mixed. The cells were incubated for 15 min at 4°C. The cells were then washed three times with sodium citrate (50 mM) and prepared at a concentration of 1.106 cells/mL for the cytometry carried out immediately after (in order to limit the loss of fluorescence). Following steps were performed as above.For the kinetics of fluorescence red cells, 1–2·10 This study is of relevance to the field of DNA repair. It uses a cleverly designed new recombination assay in yeast to address the impact of DNA break position on global genome mobility. A centromere–proximal DNA double–strand break (DSB) induces an H2A(X) phosphorylation–dependent global mobility that accelerates but is not essential for DSB repair, while a centromere–distal DSB triggers global mobility that is essential for repair and which depends on H2A(X) phosphorylation, Rad9 and Rad51. Together, these data support a model where global genome mobility promotes homologous recombination repair, particularly for centromere–distal DSBs, and help settle some recent controversy in the field. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kevin Struhl as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Aurèle Piazza (Reviewer #3).Thank you for submitting your article "Global chromatin mobility induced by a double–strand break is dictated by chromosomal conformation and defines the outcome of homologous recombination" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:eLife. They also raise some important points, most of which can be easily addressed by text changes, although some additional controls seem to be required. We highlight what we consider essential revisions here:The reviewers agree that experiments are well–conducted and support the claims and that the manuscript is suitable for publication in 1. A common concern is the lack of ability to distinguish between damaged (80%) and undamaged (20%) cells for MSD experiments. Addressing this point requires additional experiments, and a possible approach to address this point is proposed by reviewer #3 .2. The discussion should directly focus on the proteins tested (see comments from reviewer #1). It should also highlight the limitations of the system used (caveats of a plasmid–based system).3. Presentation of statistics, sample sizes, etc should be easy to identify within each figure (see comments from reviewer #1).4. Reviewer #2 raises an important point that even after most of the break is likely repaired, there is still elevated mobility. While establishing mobility at even later time points might be outside the scope of this manuscript, this is a point worth discussing.5. In the absence of Rad51, mobility still occurs in the Cen–proximal cut strain. It is important to show HR efficiency in this strain, as pointed out by reviewer #2.6. Reviewer #3 points out interesting observations ("painting model") that the authors might find helpful to integrate in the model/discussion. There are also some issues pointed out by this reviewer about consistency across experiments, which we think should be mostly addressable with text changes, and by acknowledging the different sensitivities of distinct assays. Additionally, how the HR defects of checkpoint mutants might affect7. A discussion of the data and model fit with previous work  should be provided.Reviewer #1 (Recommendations for the authors):General feedback:– Although this study focuses on global mobility effects, it is reasonable to think that the global mobility effects reported here occur in parallel to DSB site–targeted mobility. The introduction and especially the discussion would benefit from explaining how the authors envision the global mobility processes reported here intersecting with the DSB–targeted mobility that is well documented in the literature.– In the model and discussion, highlighting Mec1 as opposed to H2A, Rad9, Rad51 is puzzling. An area for improvement for the manuscript would be to improve the discussion and model to focus on factors directly tested in the study.– Another area for improvement centers around the presentation of error bars, sample sizes, and statistics in a manner that does not necessitate that the reader cross–reference the figure, main text, figure legend, and supplementary tables to determine exactly what was done in a given data panel. Especially complete and clarify the information on sample sizes, replicates, and statistics in the figure legends.– The discussion should include a section on the limitations of the systems used and especially the caveats associated with the use of plasmid–based donor sequences in most experiments.Presentation and clarity:– Figure 2B and other MSD plots: error bars needed to interpret the MSD data accurately are absent; also, the lines in some of the MSD plots are so faded that this reviewer struggled to distinguish some of them. Please consider making the lines thicker or changing their colors.– Figure 4A and 4B: p values are only shown for one of the four graphs when it seems that large and likely statistically significant changes were observed in the other three graphs. Please clarify.– Callout to Figure 4E, S, grey bars near the bottom of page 9 of the text: should this refer to Figure 4D instead of 4E?– Page 12, 2nd paragraph, 2nd line: "end of the IVR chromosome," should be "VR"?– Page 3, paragraph 2: "that allows simultaneously tracking".– Overall, the paper would benefit from detailed English language editing.Reviewer #2 (Recommendations for the authors):It would be valuable to show in the supplemental Figure 1 the frequency of red cells at all of the timepoints with the empty vector (no repair template) both with and without DSB induction to show the false positive rate for red forming cells. In other words, what is the background level of red–forming cells that result from the reversion of the stop codon both before and after DSB induction?Please discuss why the experiments shown in Figure 1 were not performed in the strain harboring the tetO repeats that were used for visualization and MSD analysis. The authors did a great job of classifying repair dynamics using their THRIV system, but then add another component to it (V–Vis) before performing the crucial MSD experiments. Is it because the GFP signal would interfere with the FACS analysis? Maybe the authors can at least do the spot assay as quality control?It is not clear why 6 hours was chosen as the time to use for DSB induction. Is it due to the delay observed between the I–SceI enzyme expression and an effective cutting? If so, please state the rationale. Do 3 hours of DSB induction change the results observed in Figure 2?The results shown in Figure 3 are a highlight of the paper. The authors do a great job utilizing the strength of their system to examine how mobility changes at different stages of the repair process (beginning vs end). However, it is surprising that even after 12 hours of DSB induction and clear evidence of completed repair (red cell), there is still elevated levels of mobility when compared to –DSB control. If repair is complete, why is there still elevated mobility? What are the results of mobility at 24 hours after DSB induction ? How long does it take for the mobility response to completely return to baseline after HR is successful? Please discuss these points.For Figure 5, were the HR kinetics examined in the rad51 mutant using the FACS assay? Does HR repair occur normally in the C strain when rad51 is deleted given that the global mobility response is shown to still occur in that strain?Reviewer #3 (Recommendations for the authors):– Experiments in Figure 2C rule out that increased mobility of undamaged sites is due to a centromere detachment from the SPB, and instead suggests a spreading mechanism. Authors should spell it out clearly in the text.– Lay out more clearly the "painting" model.– Figure 3A–B: red cells have repaired, and are not in the same cell cycle phase (see figure 1D FACS and picture of the red cells), which is known to affect mobility (Cheblah Gasser 2020).– MSDs without DSB are different in Figure 2B and in Figure 2C (0.05 vs 0.03 at 10 seconds). Why? The same thing with C–DSB and C+DSB, values are different in Figure 2B and 2C.– Indicate at which time post–DSB induction the MSD is monitored on the figure panel.– Inconsistent spot assays across figures for the same condition, or similar conditions . For instance, C strain WT somewhat grows on +Gal in Figure 1E and S4B, but not in S3B. The S+dCen strain WT +DSB is dead in Figure 1E but shows excellent recovery in S3B and S4B. There are other inconsistencies within Figure S4B, with C and S strains without DSB showing different growth despite having the same genotype. These spot assays should be redone in parallel, with the same dilution and let grow the same amount of time. The methods indicate that the plating is made from a saturating culture. It is standard practice to plate exponentially growing cells, which may also improve data reproducibility.– Figure S2c: colors are switched in one panel.eLife. Your revised article has been evaluated by Kevin Struhl (Senior Editor) and a Reviewing Editor, in consultation with the reviewers.Thank you for resubmitting your work entitled "Global chromatin mobility induced by a double–strand break is dictated by chromosomal conformation and defines the outcome of homologous recombination" for further consideration by The manuscript has been significantly improved but there are some remaining issues that need to be addressed, as outlined below:1. The addition of text comparing the genetic requirements of local vs. global mobility has improved the paper. Yet, how the authors envision DSB site–focused molecular mechanisms intersecting, or not, with the local vs. global phenomena reported here remains somewhat unclear. Such molecular mechanisms in yeast include the release of centromeres from the SPB , repair protein phase separation , microtubule/motor–dependent DSB mobility , and DSB/global chromatin changes or condensation . How may such molecular mechanisms intersect with the proximal vs. distal homology systems used in this study? Adding a paragraph to discuss the potential intersection of such mechanisms, or lack thereof, to the discussion would further enrich the discussion/paper.2. Limitations related to the common failure of MSD to detect directed mobility and to over–represent random mobility should be added to the limitations section. How this limitation may affect the interpretation of the MSD curves and conclusions in this study should be mentioned.3. We encourage the authors to include the scatter plot showing the MSD for the 'damaged–only', the 'repaired–only', or the 20%–80% mixed population in the supplementary figures, and include a brief discussion about it in the text. Essential revisions:The reviewers agree that experiments are well–conducted and support the claims and that the manuscript is suitable for publication in eLife. They also raise some important points, most of which can be easily addressed by text changes, although some additional controls seem to be required. We highlight what we consider essential revisions here:1. A common concern is the lack of ability to distinguish between damaged (80%) and undamaged (20%) cells for MSD experiments. Addressing this point requires additional experiments, and a possible approach to address this point is proposed by reviewer #3 .The referees raise the important question of the heterogeneity of a population, a central challenge in biology. Here we wish to distinguish between undamaged and damaged cells. Even if a selection of the damaged cells had been made, this would not solve entirely the inherent cell to cell variation: at a given time, it is possible that a cell, although damaged, moves little and conversely that a cell moves more, even if not damaged. The question of heterogeneity is therefore important and the subject of intense research that goes beyond the framework of our work . However, in order to start to clarify if a bias could exist when considering a mixed population (20% undamaged and 80% damaged), we analyzed MSDs, using a scatter plot. We considered two population of cells where the damage is the best controlled, i.e. (i) the red population which we know has been repaired and, importantly, has lost the cut site and will be not cut again (undamaged - only population) and (ii) the white population, blocked in G2/M, because it is damaged and not repaired (damaged - only population). These two populations show very significant differences in their median MSDs. We artificially mixed the MSDs values obtained from these two populations at a rate of 20% of undamaged - only cells and 80% of damaged - only cells. We observed that the mean MSDs of the damaged - only and undamaged - only cells were significantly different. Yet, the mean MSD of damaged - only cells was not statistically different from the mean MSD from the 20% - 80% mixed cell population. Thus, the conclusions based on the average MSDs of all cells remain consistent.2. The discussion should directly focus on the proteins tested (see comments from reviewer #1). It should also highlight the limitations of the system used (caveats of a plasmid–based system).We have rewritten parts of the discussion to better reflect the proteins tested. In particular, our experiments allow drawing a parallel between the local mobility and the distal mobility because both require the Rad51 filament and the control of Rad9.We have added a paragraph called Systems Limitations which include this particular aspect; although the THRIV system was validated also with a chromosomal donor, the plasmid/chromosome contexts are different, and may in particular impact the time or residency of the nucleoprotein filament, as suggested by Reviewer 3.3. Presentation of statistics, sample sizes, etc should be easy to identify within each figure (see comments from reviewer #1).We have now added these data in the legend of each figure.4. Reviewer #2 raises an important point that even after most of the break is likely repaired, there is still elevated mobility. While establishing mobility at even later time points might be outside the scope of this manuscript, this is a point worth discussing.It is noteworthy that at 12h, red cells that have lost the cleavage site for I - SceI and thus can no longer be cut by the enzyme, still show a higher mobility than the undamaged control. The most probable hypothesis is that the H2A phosphorylation has not yet returned to normal at this time point. There are several mechanisms to loose H2A phosphorylation that include exchange of the phosphorylated histone and its possible degradation or dephosphorylation of H2A. Kinetics of H2A dephosphorylation by ChIP and/or measurement of chromatin dynamics in backgrounds mutated for H2A dephosphorylation, should be performed to confirm this hypothesis, experiments that are beyond the scope of this manuscript. However, it is known that while H2A dephosphorylation around the DSB is quite fast (~1h), H2A dephosphorylation at 20kb from the DSB requires more time (~5h) and foci of γH2A are still detectable at ~6h after damage . On the other hand, kinetics of dephosphorylation in trans are not known. Interestingly, this study demonstrated that H2A dephosphorylation might not be complete even at the end of repair, which is consistent with the maintenance of γH2A on chromatin, including in red cells. These red cells could therefore, still exhibit increased mobility.5. In the absence of Rad51, mobility still occurs in the Cen–proximal cut strain. It is important to show HR efficiency in this strain, as pointed out by reviewer #2.In the C strain, when the DSB is induced close to the centromere, cells deleted for Rad51 do not grow, even in the presence of the donor, although an increase in mobility is detected. Poor growth is a sign of ineffective repair that impedes FACS analyses for calculation of HR efficiency. We propose that mobility promotes recombination, but cannot supplant such essential recombination proteins as Rad51 in the recombination reaction.6. Reviewer #3 points out interesting observations ("painting model") that the authors might find helpful to integrate in the model/discussion. There are also some issues pointed out by this reviewer about consistency across experiments, which we think should be mostly addressable with text changes, and by acknowledging the different sensitivities of distinct assays. Additionally, how the HR defects of checkpoint mutants might affectThe paintbrush model was very useful in explaining the distal mobility that indeed is linked to local mobility genetic requirements. It is also helpful to think of a different model than the paintbrush model when pericentromeric damage occurs. To stay in the terms of painting technique, this latter model would be similar to the pouring technique, when oil paint is deposited on water and spreads in a multidirectional manner. It is likely that Mec1 or Tel1 are the factors responsible for this spreading pattern. We therefore propose to maintain the notion of two distinct types of mobilities. Without going into pictorial techniques in the text, we have attempted to clarify these two models in the manuscript.We have corrected the experimental inconsistencies noted by referee 3, mainly due to different incubation times.7. A discussion of the data and model fit with previous work  should be provided.We have added a new paragraph in the discussion to better account for references to previous studies by Rothstein and Gasser labs.Reviewer #1 (Recommendations for the authors):General feedback:– Although this study focuses on global mobility effects, it is reasonable to think that the global mobility effects reported here occur in parallel to DSB site–targeted mobility. The introduction and especially the discussion would benefit from explaining how the authors envision the global mobility processes reported here intersecting with the DSB–targeted mobility that is well documented in the literature.We thank the reviewer for this constructive criticism, because indeed, the parameters that govern the distal mobility that we document are the same as those of the local mobility, highlighting the parallel mechanisms for both mobilities and therefore deserve to be better explained and discussed. This is now done.– In the model and discussion, highlighting Mec1 as opposed to H2A, Rad9, Rad51 is puzzling. An area for improvement for the manuscript would be to improve the discussion and model to focus on factors directly tested in the study.The mechanism of mobility around the centromere, related to the propagation of H2A phosphorylation is intriguing and could be explained by the mode of diffusion of Mec1 or Tel1. As pointed out by the reviewer, the involvement of the Rad51 nucleoprotein filament and of Rad9 in the global mobility induced when the damage is far from the centromere, according to a mechanism similar to that of the local mobility, deserves to be better highlighted with a more precise discussion of these two proteins. We have therefore added this aspect to our discussion.– Another area for improvement centers around the presentation of error bars, sample sizes, and statistics in a manner that does not necessitate that the reader cross–reference the figure, main text, figure legend, and supplementary tables to determine exactly what was done in a given data panel. Especially complete and clarify the information on sample sizes, replicates, and statistics in the figure legends.We have completed as much as possible the legends with the data of sample sizes, replicates and statistics in order to simplify the reading.– The discussion should include a section on the limitations of the systems used and especially the caveats associated with the use of plasmid–based donor sequences in most experiments.We have added a paragraph called Limitations of the systems used.As stated in the general comment, although the THRIV system was validated also with a chromosomal donor, the plasmid/chromosome contexts are different, and may in particular impact the time or residency of the nucleoprotein filament, as suggested by Reviewer 3.Presentation and clarity:– Figure 2B and other MSD plots: error bars needed to interpret the MSD data accurately are absent; also, the lines in some of the MSD plots are so faded that this reviewer struggled to distinguish some of them. Please consider making the lines thicker or changing their colors.As the reviewer noticed, the MSDs curves were not very distinct and the standard deviations were not really visible; we have made the lines thicker and the sd’s more visible– Figure 4A and 4B: p values are only shown for one of the four graphs when it seems that large and likely statistically significant changes were observed in the other three graphs. Please clarify.Pvalues are now added when possible.– Callout to Figure 4E, S, grey bars near the bottom of page 9 of the text: should this refer to Figure 4D instead of 4E?Indeed, the error is now corrected.nd paragraph, 2nd line: “end of the IVR chromosome,” should be “VR”?– Page 12, 2It is the labelled and tracked locus which is located on the right arm of chromosome V. The I – SceI cutting sites discussed in this paragraph are located on the right arm of chromosome IV.– Page 3, paragraph 2: “that allows simultaneously tracking”.Replaced by « that allows simultaneously to monitor…”.– Overall, the paper would benefit from detailed English language editing.We had the text proofread and corrected by a native English speaker.Reviewer #2 (Recommendations for the authors):It would be valuable to show in the supplemental Figure 1 the frequency of red cells at all of the timepoints with the empty vector (no repair template) both with and without DSB induction to show the false positive rate for red forming cells. In other words, what is the background level of red–forming cells that result from the reversion of the stop codon both before and after DSB induction?If we have not indicated in Figure 1F the % of red colonies in the absence of donor it is because it remains constant and low over time around 0.55% ± 0.004 and 1.02% ± 0.85 for the C and S strains, respectively. We have added in Figure Supp.1C quantifications and examples of cells after 24h in the C and S strains in the absence of donor to illustrate our point.Please discuss why the experiments shown in Figure 1 were not performed in the strain harboring the tetO repeats that were used for visualization and MSD analysis. The authors did a great job of classifying repair dynamics using their THRIV system, but then add another component to it (V–Vis) before performing the crucial MSD experiments. Is it because the GFP signal would interfere with the FACS analysis? Maybe the authors can at least do the spot assay as quality control?We apologize if our description of the strains was not sufficiently explicit. These are the same strains in Figure 1 and Figure 2. We had split the description into 2 parts for clarity, but we now state from Figure 1 that these strains contain the cutting sites for I - SceI, the donor sequence and the visualization system.It is not clear why 6 hours was chosen as the time to use for DSB induction. Is it due to the delay observed between the I–SceI enzyme expression and an effective cutting? If so, please state the rationale. Do 3 hours of DSB induction change the results observed in Figure 2?We chose 6h, because it is the induction time for which we have the best compromise, on the number of damaged cells and the beginning of the appearance of red cells. At 3h, as the referee pointed out, the enzyme is expressed but the cutting is not yet effective. The rationale of the long - time is now discussed in the “limitations of the system” paragraph.The results shown in Figure 3 are a highlight of the paper. The authors do a great job utilizing the strength of their system to examine how mobility changes at different stages of the repair process (beginning vs end). However, it is surprising that even after 12 hours of DSB induction and clear evidence of completed repair (red cell), there is still elevated levels of mobility when compared to –DSB control. If repair is complete, why is there still elevated mobility? What are the results of mobility at 24 hours after DSB induction ? How long does it take for the mobility response to completely return to baseline after HR is successful? Please discuss these points.We thank the referee for the appreciation of this difficult experiment, which was indeed very instructive. Concerning the dynamics of the red cells at 12h, the most probable hypothesis is that the H2A phosphorylation has not yet returned to normal at this time point. There are several mechanisms to loose H2A phosphorylation that include exchange of the phosphorylated histone and its possible degradation or dephosphorylation of H2A. Kinetics of H2A dephosphorylation by ChIP and/or measurement of chromatin dynamics in backgrounds mutated for H2A dephosphorylation, should be performed to confirm this hypothesis, experiments that are beyond the scope of this manuscript. However, it is known that while H2A dephosphorylation around the DSB is quite fast (~1h), H2A dephosphorylation at 20kb from the DSB requires more time (~5h) and foci of γH2A are still detectable at ~6h after damage . On the other hand, kinetics of dephosphorylation in trans are not known Interestingly, this study demonstrated that H2A dephosphorylation may not be complete even at the end of repair, which is consistent with the maintenance of γH2A on chromatin, including in red cells. These red cells could therefore, still exhibit increased mobility.For Figure 5, were the HR kinetics examined in the rad51 mutant using the FACS assay? Does HR repair occur normally in the C strain when rad51 is deleted given that the global mobility response is shown to still occur in that strain?As stated in the general comment, in the C strain, when the DSB is induced close to the centromere, cells deleted for Rad51 grow poorly, even in the presence of the donor, although an increase in mobility is detected. Poor growth is a sign of ineffective repair that impedes FACS analyses for calculation of HR efficiency. We propose that mobility promotes recombination, but cannot supplant such essential recombination proteins as Rad51 in the recombination reaction.Reviewer #3 (Recommendations for the authors):– Experiments in Figure 2C rule out that increased mobility of undamaged sites is due to a centromere detachment from the SPB, and instead suggests a spreading mechanism. Authors should spell it out clearly in the text.In our opinion, it is still difficult at this stage to estimate whether or not pericentromeric DSB causes microtubule detachment from kinetochores. It remains possible that the spreading of γH2A weakens this attachment, but only super - resolution microscopy experiments could perhaps resolve this point.– Lay out more clearly the "painting" model.The model related to the formation of the Rad51 filament and the probable residence time of this filament with the donor as well as the propagation pattern of γH2A are now better highlighted, in the text and mentioned in the legend of figure 6.– Figure 3A–B: red cells have repaired, and are not in the same cell cycle phase (see figure 1D FACS and picture of the red cells), which is known to affect mobility (Cheblah Gasser 2020).The distinction between white cells, blocked in G2/M, and cycling red cells effectively highlights the fact that the increased dynamics may be related to a cycling defect. However, it is possible to observe an increase in dynamics in a Rad9 mutant, where the cycle is no longer blocked upon damage , suggesting that G2/M block is not sufficient to explain an increase in dynamics.– MSDs without DSB are different in Figure 2B and in Figure 2C (0.05 vs 0.03 at 10 seconds). Why? The same thing with C–DSB and C+DSB, values are different in Figure 2B and 2C.The differences between MSDs experiments are a good example of biological heterogeneity. This is the reason why in each experiment the pictures are rigorously taken in the control strains without DSB and compared to the strains having undergone the DSB.– Indicate at which time post–DSB induction the MSD is monitored on the figure panel.In the legend, 6h.– Inconsistent spot assays across figures for the same condition, or similar conditions . For instance, C strain WT somewhat grows on +Gal in Figure 1E and S4B, but not in S3B. The S+dCen strain WT +DSB is dead in Figure 1E but shows excellent recovery in S3B and S4B. There are other inconsistencies within Figure S4B, with C and S strains without DSB showing different growth despite having the same genotype. These spot assays should be redone in parallel, with the same dilution and let grow the same amount of time. The methods indicate that the plating is made from a saturating culture. It is standard practice to plate exponentially growing cells, which may also improve data reproducibility.The spot assay was done on cells in exponential phase, we have corrected this error in the methods. We thank the referee for his critical eye, the differences were due to the growth times and the dilution between the different figures  and to the different contrast of that picture. The reason for a longer time is related to the slow growth of the SA mutant. Times and dilutions are now indicated in the legend. All strains grow similarly between experiments.– Figure S2c: colors are switched in one panel.CorrectedReferencesAltschuler SJ, Wu LF. 2010. Cellular Heterogeneity: Do Differences Make a Difference? Cell 141:559–563. doi:10.1016/j.cell.2010.04.033Cheblal A, Challa K, Seeber A, Shimada K, Yoshida H, Ferreira HC, Amitai A, Gasser SM. 2020. DNA Damage - Induced Nucleosome Depletion Enhances Homology Search Independently of Local Break Movement. Mol Cell 80:311 - 326.e4. doi:10.1016/j.molcel.2020.09.002Garcia Fernandez F, Lemos B, Khalil Y, Batrin R, Haber JE, Fabre E. 2021. Modified chromosome structure caused by phosphomimetic H2A modulates the DNA damage response by increasing chromatin mobility. J Cell Sci jcs.258500. doi:10.1242/jcs.258500Hauer MH, Seeber A, Singh V, Thierry R, Sack R, Amitai A, Kryzhanovska M, Eglinger J, Holcman D, Owen - Hughes T, Gasser SM. 2017. Histone degradation in response to DNA damage enhances chromatin dynamics and recombination rates. Nat Struct Mol Biol. doi:10.1038/nsmb.3347Herbert S, Brion A, Arbona J - M, Lelek M, Veillet A, Lelandais B, Parmar J, Fernández FG, Almayrac E, Khalil Y, Birgy E, Fabre E, Zimmer C. 2017. Chromatin stiffening underlies enhanced locus mobility after DNA damage in budding yeast. EMBO J 36. doi:10.15252/embj.201695842Keogh MC, Kim JA, Downey M, Fillingham J, Chowdhury D, Harrison JC, Onishi M, Datta N, Galicia S, Emili A, Lieberman J, Shen X, Buratowski S, Haber JE, Durocher D, Greenblatt JF, Krogan NJ. 2006. A phosphatase complex that dephosphorylates γH2AX regulates DNA damage checkpoint recovery. Nature 439:497–501. doi:10.1038/nature04384Smith MJ, Bryant EE, Rothstein R. 2018. Increased chromosomal mobility after DNA damage is controlled by interactions between the recombination machinery and the checkpoint. Genes Dev 32:1242–1251. doi:10.1101/gad.317966.118The manuscript has been significantly improved but there are some remaining issues that need to be addressed, as outlined below:1. The addition of text comparing the genetic requirements of local vs. global mobility has improved the paper. Yet, how the authors envision DSB site–focused molecular mechanisms intersecting, or not, with the local vs. global phenomena reported here remains somewhat unclear. Such molecular mechanisms in yeast include the release of centromeres from the SPB , repair protein phase separation , microtubule/motor–dependent DSB mobility , and DSB/global chromatin changes or condensation . How may such molecular mechanisms intersect with the proximal vs. distal homology systems used in this study? Adding a paragraph to discuss the potential intersection of such mechanisms, or lack thereof, to the discussion would further enrich the discussion/paper.Our work highlights two types of global  mobilities. As proposed, we have incremented the discussion by a new paragraph.Indeed, discussion of the mechanistic similarities between local and global  mobilities highlights the strength of existing mechanisms to initiate mobility and facilitate repair. Moreover, the possible involvement of cytoskeletal factors in these mobilities broadens the discussion and opens an interesting field of future research.2. Limitations related to the common failure of MSD to detect directed mobility and to over–represent random mobility should be added to the limitations section. How this limitation may affect the interpretation of the MSD curves and conclusions in this study should be mentioned.We have added this limitation in the corresponding section.3. We encourage the authors to include the scatter plot showing the MSD for the 'damaged–only', the 'repaired–only', or the 20%–80% mixed population in the supplementary figures, and include a brief discussion about it in the text.We have added this text p7 lines 192 - 202, included the corresponding figure in Supp Figure 3A along with its legend.


The redeployment of mentors and restrictions on in-person face-to-face mentoring meetings during the COVID-19 pandemic has compromised mentoring efforts in Palliative Medicine (PM). Seeking to address these gaps, we evaluate the notion of a combined novice, peer-, near-peer and e-mentoring (CNEP) and interprofessional team-based mentoring (IPT) program.A Systematic Evidence Based Approach (SEBA) guided systematic scoping review was carried out to study accounts of CNEP and IPT from articles published between 1st January 2000 and 28th February 2021. To enhance trustworthiness, concurrent thematic and content analysis of articles identified from structured database search using terms relating to interprofessional, virtual and peer or near-peer mentoring in medical education were employed to bring together the key elements within included articles.Fifteen thousand one hundred twenty one abstracts were reviewed, 557 full text articles were evaluated, and 92 articles were included. Four themes and categories were identified and combined using the SEBA’s Jigsaw and Funnelling Process to reveal 4 domains - characteristics, mentoring stages, assessment methods, and host organizations. These domains suggest that CNEP’s structured virtual and near-peer mentoring process complement IPT’s accessible and non-hierarchical approach under the oversight of the host organizations to create a robust mentoring program.This systematic scoping review forwards an evidence-based framework to guide a CNEP-IPT program. At the same time, more research into the training and assessment methods of mentors, near peers and mentees, the dynamics of mentoring interactions and the longitudinal support of the mentoring relationships and programs should be carried out.The online version contains supplementary material available at 10.1186/s12909-022-03409-4. Built uization” , novice ization” –11 incluization” . These lization”  and raisization”  of mentoization” .Although supplementing novice mentoring with peer  and elecIt is with this impetus to address the prevailing threats to novice mentoring  and a geIn the absence of mentoring data in PM , 31–33, To enhance the transparency and reproducibility, Krishna’s Systematic Evidence Based Approach –50  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 (henceforth the expert team), allows for an accountable and reproducible approach to the search and review of data..SEBA’s use of the principles of interpretivist analysis , 54–56 e1The expert team was consulted at each stage of the SEBA process.i.Determining title and background of reviewThe expert team, stakeholders and the research team collaborated to determine the overall goals of the systematic scoping review and the population, context and concept to be evaluated.ii.Identifying the research questionThis systematic scoping review in SEBA confines its review of CNEP and IPT amongst physicians and nurses, medical social workers, physiotherapists and/or occupational therapists .ii.Identwhat is known about CNEP and IPT?” The secondary research questions were “what are the features of CNEP and IPT?” and “is CNEP-IPT suitable for the PM setting?”iii.Inclusion criteriaGuided by the population, concept and context (PCC), the teams also determined the primary research question to be “All grey literature, peer reviewed articles, narrative reviews, systematic, scoping and systematic scoping reviews published between 1st January 2000 to 28th February 2021 were included in the PCC and a PICOS format was adopted to guide the research processes , 58. Seeiv.Searchinginformal dynamic advisory relationships within a group of individuals who are similar in experience, education level, and seniority” [synchronous (live video or instant messaging) (60) and asynchronous ” communication [senior, near-peer and/or peer mentors who are medical, nursing, medical social work, physiotherapy and occupational therapy healthcare professionals or students supporting junior healthcare professionals and students in advancing their professional, clinical, personal and academic development” [To broaden the search, ensure a structured approach and reduce omission of critical papers, three separate search strategies were formulated to look for articles about CNEP, IPT and e-mentoring respectively. These search terms were developed based on the following definitions. Near-peer and peer mentoring is defined as “niority” . E-mentonication . IPT refssaging)  and asynSearches on seven bibliographic databases  and five grey literature databases  were carried out and included articles from 1st January 2000 to 28th February 2021. Articles published before year 2000 were not included given evidence that they often failed to clearly delineate distinct mentoring approaches such as leadership, patient, family, adolescent, group, peer, near-peer, novice and e-mentoring , 62, andA manual search of related areas of interest, and an expanded search of the references of the included articles were also carried out. This revealed six additional articles of interest.v.Extracting and chartingThe three separate PubMed Search Strategies may be found in Additonal file Using the abstract screening tool, members of the research team independently reviewed the titles and abstracts found from each database to finalise the list of titles and summaries to be reviewed. Sambunjak, Straus and Marusic’s  approachTo enhance validity of the analysis, the Split Approach  was empldefined from the raw data without any predetermined classification” [Negotiated consensual validation” was used to determine a final list of themes [Concurrently, three members of the second team independently analysed the included articles using Braun and Clarke’s  approachication” . In phasf themes .identifying and operationalizing a priori coding categories” [Enhancing Mentoring in Palliative Care: An Evidence Based Mentoring Framework”, to guide the coding of the articles in the second stage. Any data not captured by these codes were assigned a new code [deductive category application, coding categories were reviewed and revised as required [A third team of three researchers employed Hsieh and Shannon’s  approachegories” –76. The egories”  study tinew code . In keepPrinciple of pragmatism: in ensuring that the focus of the review remains upon the research question,Principle of pluralism: in ensuring that all themes are included in the review,Principle of historicity: in ensuring that the process is reproducible by including the review descriptions of the unfolding narrative,Principle of contestation: in ensuring that all ‘conflicting data’ is considered,Principle of reflexivity: in ensuring that throughout the review, reviewers continually reflect individually and as a team on the emerging findings, andPrinciple of peer review: in ensuring that emerging findings are peer reviewed through use of the split review, peer reviewed data, and that that the stakeholders agree with the data and interpretation.The Jigsaw Perspective brings together the themes and categories identified in the Split Approach to provide a more holistic perspective of the available data. This process is overseen by the expert team and guided by six principles –79:PrincReciprocal translation determines if the themes and categories can be used interchangeably.The process of creating themes/categories is derived from Phases 4 to 6 of France et al.’s , 81‘s ad1) Themes identified through Braun and Clarke’s approach to thematic analysis:Characteristics of CNEP and IPTMentoring StakeholdersStages of CNEP and IPTAssessment methods and criteria2) Categories identified through Hsieh and Shannon’s approach to directed content analysis:Mentoring NatureMentoring StakeholdersMentoring RelationshipsMentoring ApproachesMentoring AssessmentsCharacteristicsMentoring stagesRoles of the host organizationAssessmentsHere the combination of the themes/categories provides triangulation , improveThe themes/categories identified through the Jigsaw Process were reviewed and compared with the tabulated summaries in Additonal file Adapting Phase 5 of France et al’s , 81 apprA total of 15,121 abstracts were reviewed, 557 full text articles were evaluated, and 92 articles were included. See Fig .A summary of the background, theoretical approach and methods, population characteristics, main empirical findings and insights drawn are highlighted in Additonal file CNEP and IPT provide timely, well-resourced, and high-quality –87 reseaThe data garnered suggest that the similarities between CNEP and IPT are consistent with the critical aspects of novice mentoring and have likely evolved from novice mentoring roots, serving to emphasize the likelihood that they may be used to support novice mentoring relationships in the COVID-19 era and beyond when access to senior physician mentors is expected to remain limited.CNEP pivots on in-person face-to-face mentoring , 116–119Near-peer mentors provide mentees with an alternative source of professional, personal, research and clinical support –115, 128IPT is reliant on each team member possessing effective interprofessional communication and teamwork skills , 90, 130From these findings, it is also evident that while CNEP and IPT possess unique characteristics, these elements are able to build upon each other to enhance the effectiveness of a mentoring program and a holistic mentoring environment.CNEP and IPT both exhibit the presence of mentoring stages first described in novice mentoring, reaffirming the notion that they could be used to support novice mentoring programs. These stages include the pre-mentoring stage, mentoring process and the post-mentoring stage.The pre-mentoring stage includes recruitment of mentors and mentees , 134, evThe host organization also determines the matching process , 138. ToHowever, given the venture towards inter-professional mentoring, important considerations in the matching process to best suit the dynamic, complex and multi-level mentoring relationships within CNEP-IPT still remain unclear.The mentoring process begins once a mentee and mentor agree to a match and commence on a mentoring relationship with each other –143. At CNEP programs may employ video conferencing in the initial face-to-face meeting , 144.The post-mentoring stage involves assessments of the mentoring process , 124, th.The host organizations of CNEP and IPT programs play crucial roles in overseeing and running the mentoring programs. The roles are described in Table Evidently, the role of the host organization is integral in ensuring effective support of complex mentoring relationships within CNEP-IPT and in circumnavigating ethical concerns regarding the misappropriation of mentee’s work, disregard for the needs of mentees, and even bullying .Assessment of CNEP and IPT mentoring programs revolve around the mentee’s perspectives and experiences , 125, buThe success of IPT mentoring programs is evaluated based on impact on mentor and mentee welfare, effectiveness and efficiency of the program, project outcomes, research output and improvements in patient care , 159. ThHolistic assessment approaches are especially important in a CNEP-IPT program given the multi-level nature of mentoring and also the large number of stakeholders involved. Longitudinal assessments are also crucial in the continual improvement and development of this novel mentoring approach.To evaluate the impact of grey literature and opinion, perspectives, editorial, letters and non-data based articles (henceforth non-data driven group) drawn from bibliographic databases upon the systematic scoping review, evidence-based data from bibliographic databases (henceforth evidence-based publications) were separated from the non-data driven group and both groups were thematically analysed separately. The themes from both groups were compared and found to be similar, suggesting that the non-data driven publications are unlikely to steer the systematic scoping review away from evidenced data.The systematic scoping review produced from consolidating the themes, categories and tabulated summaries was guided by the Best Evidence Medical Education (BEME) Collaboration guide  and the Recent research provides promising accounts of programs adopting a similar CNEP-IPT concept, which employ “systems of mentors” comprising “senior colleagues, teachers, peers, as well as junior colleagues and students” , 60, 89,In answering its primary and secondary research questions, this review suggests that CNEP and IPT mentoring programs share similarities in their practice, structure, stages, mentoring goals, codes of conduct, assessment processes and mentoring environments that ought to allow a melding of these approaches and the creation of a combined CNEP-IPT mentoring program , 159. ThIt is also clear from our findings that a combined CNEP-IPT mentoring program would need to be part of a formal mentoring program, designed, supported, and overseen by the host organization –100. ThiThe notion of a synergistic relationship between these mentoring approaches also requires further study given implications upon mentoring dynamics in the presence of multiple stakeholders. Mentoring relationships in CNEP-IPT may be more challenging compared to traditional ones given inherent hierarchical boundaries that exist both in terms of disparities in seniority and qualifications, and also across healthcare disciplines. Yet it is also worth noting that with time and the promotion of inter-professional collaboration and teamwork among stakeholders, CNEP-IPT may help to break down these historical barriers. This gives rise to considerations on how mentors, near peers and mentees can be best matched for the task. As a result, this review suggests that a combined CNEP-IPT mentoring program ought to be designed, supported, overseen and assessed by the host organization and be part of a formal program. Indeed the host organization plays a key role in the effective running of the proposed CNEP-IPT mentoring program with the roles and responsibilities of the host organization set out in Table Despite efforts to enhance the reproducibility and transparency of the systematic scoping review, gaps in the methodology and analysis persist. While we have conducted three separate search strategies using a two-tiered approach of both independent searching of selected databases by our expert team and an expanded search of reference lists of publications and manual searches, important papers may still have been missed. Similarly, while use of the ‘Split Approach’ and tabulated summaries in SEBA allowed for triangulation and ensured a holistic perspective was constructed from different and diverse perspectives, inherent biases amongst the reviewers would still impact the analysis of the data and construction of themes. Moreover, SEBA is not evidenced as yet and is time-consuming, raising questions as to its viability and concerns of the need for careful balancing between the promised benefits and the sustainability of continued involvement of the expert teams who were involved in all stages of SEBA.The use of thematic analysis to review the impact of non-evidence-based data improves transparency in the synthesis of the discussion, however the inclusion of these data may still bias results and provide opinion-based views with a ‘veneer of respectability’ despite a lack of evidence to support them. This raises the question as to whether non-evidence-based data should be accorded the same weight as published literature.In addressing its research questions, this systematic scoping review in SEBA offers a glimpse into the future of mentoring in PM but also raises a word of caution. While synergy between CNEP and IPT built on a common ancestry rooted in novice mentoring is evident, there are many aspects of the process that require further study. However, evidence of the desire to continue promoting mentoring in these difficult circumstances underscores its reputation and role within PM. We look forward to engaging in this developing field as advances in the understanding of dynamics, support and oversight within these relationships begin to take shape and help mould a structured approach to this form of mentoring within PM and beyond.Additional file 1. Search Strategies for PubMed.Additional file 2. Summary of Included Articles.


Interstitial deletion in the long arm of chromosome 9 [del(9q)] is a fairly common cytogenetic finding associated with acute myeloid leukemia (AML), seen in approximately 2–5% of AML patients. However, the genomic features of the deletion remain largely unknown. Using chromosome analysis, single nucleotide polymorphism microarray, and next-generation sequencing, we characterized del(9q)s and other genomic alterations in 9 AML patients. We found several distinct features of the del(9q)s. The proximal breakpoints of the deletions are clustered within a 2.5-Mb region  enriched with segmental duplications, which may represent a “hotspot” for genomic rearrangements. However, the distal breakpoints of the deletions vary significantly. In addition, the overall deleted region could be divided into a 14.4-Mb proximal constitutional region  and a 24.0-Mb distal oncogenic region . We further identified a 6.8-Mb common overlapped deletion region (CODR) in the distal region . This CODR carries multiple genes that are reportedly involved in cancer pathogenesis. The prognostic value of the del(9q) in AML apparently depends on additional genomic alterations in the patients. Interstitial deletion in the long arm of chromosome 9 [del(9q)] is a recurrent finding in acute myeloid leukemia (AML). The deletion has been reported to occur predominantly in myeloid disorders, frequently in AMLs with t translocation. It can occur solely or with other abnormalities as a primary or secondary event. However, the pathogenic role of del(9q) is unclear despite numerous studies of the deletion . In addition, the genomic features of the deletion remain mostly undefined, hindering further understanding of the pathogenic significance of the deletion. Integrated genomic analyses, including chromosome analysis, single-nucleotide polymorphism microarray (SNP array), and next-generation sequencing (NGS), make it possible to delineate genomic abnormalities in detail. Using these technologies, we characterized del(9q)s along with other genomic alterations in 9 patients with AML. Our studies revealed distinct genomic features of the deletions, which have not been described previously. These features include clustered proximal and variable distal breakpoints, proximal constitutional and distal oncogenic regions, and a common overlapped deletion region (CODR) that carries known cancer-related genes. In addition, we also detected various additional genomic alterations in these patients, which may affect the prognostic value of the del(9q).Nine patients with AML and del(9q) from clinical cases tested at the University of California San Francisco (UCSF) Cytogenetics Laboratory between 2012 and 2021 were selected for this study  following the manufacturer's instruction.ANKRD26, ASXL1, ASXL2, BCOR, BCORL1, BRAF, CALR, CBL, CBLB, CEBPA, CSF3R, CUX1, DDX41, DNMT1, DNMT3A, ELANE, ETNK1, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HNRNPK, IDH1, IDH2, IL7R, JAK1, JAK2, JAK3, KDM6A, KIT, KMT2A, KRAS, LUC7L2, MPL, NOTCH1, NPM1, NRAS, NSD1, PHF6, PIGA, PRPF40B, PRPF8, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SH2B3, SMC1A, SMC3, SRSF2, STAG2, STAT3, STAT5B, SUZ12, TET2, TP53, U2AF1, U2AF2, WT1, and ZRSR2.AML NGS gene panel studies were clinically requested for all 9 patients, and the tests were performed in UCSF Clinical Cancer Genomic Laboratory, ARUP Laboratories or Foundation Medicine. The gene panels included Interstitial 9q deletions were detected by chromosome analysis in all 9 AML patients. The deletions were confirmed and their sizes and breakpoints were defined by SNP array Table . All delGADD45G, WNK2, MIRLET7A1, MIRLET7D, and MIRLET7F1 . Loss of function or reduction of expression of these 5 genes have been reported to be associated with cancer pathogenesis (see Discussion).By reviewing literature and our internal constitutional microarray database, we found multiple constitutional interstitial del(9q)s that were located within an approximately 14.4-Mb region between 9q21.11 and 9q21.32  . These constitutional deletions completely overlap with the proximal region of the AML-associated del(9q)s in this study s. The common phenotypic features associated with this deletion region include mental retardation with speech delay, epilepsy, autistic behavior, and moderate facial dysmorphology . However, the AML patients apparently did not show such constitutional phenotypic abnormalities. This could be explained by the fact that the AML-associated deletions would only occur in hematopoietic cells, and other cells/tissues of the AML patients were unaffected. Thus, these patients would not show the constitutional phenotypic abnormalities.GADD45G encodes a DNA damage inducible protein in p53 signaling pathway associated with cell cycle arrest. Defects in the GADD45G pathway appear to be involved in initiation and progression of malignancies . WNK2 has been identified as a tumor suppressor in hepatocellular carcinoma, glioma, and pancreatic ductal adenocarcinoma . MIRLET7A1, MIRLET7D, and MIRLET7F1 are microRNAs that were reported to negatively regulate HRAS, KRAS, and NRAS oncogenes. Reduced expression of these microRNAs was associated with adverse prognosis independent of disease stage in lung cancer . Further investigation of these and other genes involved in del(9q)s may lead to a better understanding of the pathogenesis and progression of AML with del(9q)s.It is hypothesized that haploinsufficiency of critical gene(s) within del(9q)s plays a role in AML pathogenesis. Herold et al. [2017] demonstrated a significant impact of haploinsufficiency of del(9q), but were unable to detect pathogenic variants in the genes within the del(9q) region by exome sequencing. Nonetheless, efforts have been made to search for a critical region within 9q deletions, which is responsible for the pathogenesis of AML. Sweetser et al. [2005] identified a 2.4-Mb minimal common deletion region in 43 AML cases with del(9q), but they did not identify recurrent pathogenic variants in all 13 genes within/adjacent to this region either. A similar region was also reported by Naarmann-de Vries et al. [2019]. Our study suggests that the overall region involved in the del(9q)s can be divided into a proximal constitutional region and a distal oncogenic region. The latter contains a 6.8-Mb CODR that is approximately 3.0 Mb distal to the previously reported minimal common deletion region . The CODR carries 5 reported cancer-related genes. Among these genes, PML-RARA; t, RUNX1-RUNX1T1, and biallelic pathogenic variants of CEBPA. Favorable prognoses were observed in these patients who stayed in complete remission until the last available follow-up  seen in AML is believed to be an early event in leukemogenesis and associated with an intermediate risk . However, additional genomic alterations frequently occur with del(9q)s, as seen in this study .The authors have no conflicts of interest to declare.The authors have no funding sources to declare.Zhongxia Qi: study design, data analysis and interpretation, drafting of the manuscript. Kwun Wah Wen: study design, data analysis, critical revision of the manuscript for important intellectual content. Anita Ki: data acquisition and analysis. Sonam Prakash and Scott Kogan: study design, critical revision of the manuscript for important intellectual content. Jingwei Yu: study design, supervision, drafting and editing the manuscript.The data that support the findings of this study are available from the corresponding author upon reasonable request.


In neurons, Ca2+ buffering is particularly important as it helps to shape Ca2+ signals and to regulate numerous Ca2+-dependent functions including neuronal excitability, synaptic transmission, gene expression, and neuronal toxicity. Over the past decade, identification of the mitochondrial Ca2+ uniporter (MCU) and other molecular components of mitochondrial Ca2+ transport has provided insight into the roles that mitochondrial Ca2+ regulation plays in neuronal function in health and disease. In this review, we discuss the many roles of mitochondrial Ca2+ uptake and release mechanisms in normal neuronal function and highlight new insights into the Ca2+-dependent mechanisms that drive mitochondrial dysfunction in neurologic diseases including epilepsy, Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. We also consider how targeting Ca2+ uptake and release mechanisms could facilitate the development of novel therapeutic strategies for neurological diseases.Mitochondria are essential for proper cellular function through their critical roles in ATP synthesis, reactive oxygen species production, calcium (Ca Mitochondria are colloquially referred to as the powerhouse of the cell because of their critical role in the production of ATP through oxidative phosphorylation (OXPHOS) . The pro2−) produced through OXPHOS are converted to H2O2 by enzymes such as superoxide dismutase and subsequently broken down into harmless byproducts . The OMM is permeable to molecules smaller than 5 kDa, including Ca2+. This permeability is ascribed to voltage-dependent anion channels (VDACs) also known as mitochondrial porins that are highly expressed in the OMM   . Recent chondria . The mitf ∼2–2.5 . Earlier10–20 μM . Such a nditions  2 and CaHPO4  is the main pathway mediating Ca2+ efflux from mitochondria in neurons  as a pore-forming subunit of the greater uniporter complex , which are auxiliary subunits that regulate Ca2+-dependent gating of the channel; the mitochondrial Ca2+ uniporter regulator 1 (MCUR1), and an essential MCU regulator (EMRE), the protein critical for the assembly of MCU complex  and Glu243 (E) within the carboxylate rings at the mouth of the channel, which form a so-called DXXE motif that is critical for Caectivity . Notably+ uptake . Previouough MCU .in vivo remains a critical task in the field of mitochondrial biology. Although many in vitro studies  suggested that mitochondrial Ca2+ transport is critical to neuronal function, studies in MCU knockout (KO) mice revealed that MCU deletion had surprisingly limited effects in vivo . It is ubiquitously expressed throughout the body including the brain  the MCU complex forms a dimer with a second MCU complex and in this arrangement the MICU2 subunits interact directly. This also results in MICU1 moving away from the pore of the MCU complex, allowing for Ca2+ uptake  and H+/Ca2+ (mtHCX) exchangers mediate Ca2+ extrusion from mitochondria , a product of gene Slc8b1, represents an essential component of the mitochondrial Na+/Ca2+ exchanger , and possibly other organelles  What are the mechanisms of cellular trafficking of NCLX? 2) What are the functions of NCLX on the plasma membrane, ER and possibly other organelles? 3) Are there additional yet to be identified molecules that mediate Na+/Ca2+ exchange across the mitochondrial membranes?Despite the appreciation of NCLX as a mediator of Caganelles . There i for Na+  and the n by Na+ . In addiectively . These f2+ uptake into the mitochondria is critical for normal cellular function, the entry of excessive amount of Ca2+ into the mitochondria can result in Ca2+ overload and lead to activation of the mitochondrial permeability transition pore (mPTP); this phenomenon has been associated with many different disease states, and thus the mPTP is considered a promising target for therapeutic intervention  on the outer membrane and adenine nucleotide translocators (ANT) on the inner mitochondrial membrane . Althougthe mPTP . Using bsynthase . Subsequsynthase . Followiexposure , and hadexposure . When c-exposure . The delexposure . In HAP1gkrekate . Deletiogkrekate . Howevergkrekate . In suppgkrekate . It is pgkrekate . Howevergkrekate . These f0F1 ATPase plays a critical role in its formation, but also suggest that certain subunits of the ATPase can be compensated for by other mechanisms. The combination of the results for and against the notion that F0F1 ATPase is the mPTP suggest that cell-type specific differences exist and that the mPTP can form via several mechanisms    and spaser (PiC) . Howeverer (PiC) . The key2+ signaling plays an especially important role in neurons because it regulates many aspects of neurotransmitter release and synaptic function  of synaptic vesicles.Cafunction   at neuro-muscular and central synapses  following the initial [Ca2+]i rise to the micromolar levels in response to intense stimulation   , are the2+ transport in synaptic transmission. Inhibiting MCU in the Drosophila mushroom body neurons caused impairment of synaptic function and olfactory memory  channels and Ca2+ signaling  and release of cytochrome c and other proapoptotic factors  is a debilitating neurodegenerative disorder and the most common form of dementia in the elderly. It is characterized by progressive impairment of memory and cognitive functions and may lead to a completely vegetative state and early death . AD curr2+ deregulation is thought to play critical roles during various phases of AD and can manifest in many forms, including chronic elevation of the cytosolic and mitochondrial Ca2+ concentrations, enhanced Ca2+ release from intracellular Ca2+ stores, spontaneous Ca2+ spikes, and enhanced activity of glutamate NMDA receptors  and the formation of neurofibrillary tangles from accumulated tau protein . Accordieceptors    and presenilin 2 (PS2). Mutated PS1 and PS2 modulate IP3R activity and increase Ca2+ release  . Interesligomers . ER Ca2+ release . PS1 mut release , which c with AD . The stontration . Injectitrations . In additoxicity . Glutamatoxicity . Howevertoxicity .2+ dysregulation in AD and other neurodegenerative disorders ultimately converge on mitochondrial Ca2+ overload, a crucial event that triggers mPTP opening, release of proapoptotic factors, ROS generation, and cell death  is a progressive neurodegenerative disorder that is defined by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) . Before thmicity . This unl toxins .2+-overload as a key target in the treatment of PD  function and reduces ER Ca2+ uptake  . Experim+ uptake . The redoverload   is a fatal neurodegenerative disorder that results in progressive loss of motor neurons throughout the brain and spinal cord, leading to death typically within 2.5–3 years after its initial diagnosis . Althougvalbumin . Consistproteins . Furtherlial ALS . Increastoxicity .2+-binding proteins, motor neurons have been shown to contain glutamate AMPA receptors that are more permeable to Ca2+, thereby adding to their susceptibility to excitotoxicity , it was determined that the neurons had enhanced Ca2+ release following stimulation and both mutations also led to upregulation of Ca2+ permeable AMPA and NMDA receptors , protected against kainic acid-induced excitotoxity in vitro, and this appeared to involve a reduction in MCU expression  . The mecthe cell . Many otthe cell . The impn in ALS , studies limited . One of  limited . The apppression . Althoug2+ buffering is essential for normal neuronal function and dysfunctional Ca2+ buffering and subsequent Ca2+ overload is common to many neurological diseases. Thanks to the recent identification of the components of the MCU complex, targeting the mitochondrial Ca2+ uptake and release machinery has become possible and has helped to refine the roles of these processes in neuronal function, as well as enable investigation of the effectiveness of targeting these proteins in disease prevention. Despite progress, many major questions remain unanswered in defining the roles of specific components of mitochondrial Ca2+ uptake and release systems in neurodegenerative disorders, epilepsy, stroke and other neurological conditions. Recent advancements in genetic targeting of all the major components of mitochondrial Ca2+ transport have provided important tools for addressing these questions. Thus, an important task for future research would be to systematically examine the impact of deletion or overexpression of MCU, MCUb, MICU3 and other components of mitochondrial Ca2+ transport in animal models of AD, PD, ALS, epilepsy, and other neurological conditions.Mitochondrial Ca2+ signaling and mitochondria Ca2+ transport play crucial roles in these cells in the context of many neurological diseases (2+ transport in neurological diseases by genetically targeting these molecules in neurons, glia, and immune cells.Even though this review focused primarily on neurons, it is important to point out that glial cells, such as astrocytes and microglia, also play crucial roles in epilepsy and neurodegenerative diseases as highlighted in many excellent reviews on this topic . Equallydiseases . Thus, ain vitro assays, its poor plasma membrane permeability and low stability in aqueous solutions limit its applicability for in vivo research (in vivo in mice subjected to hypoxic/ischemic brain injury (2+, and benzethonium, which inhibits this process (2+ overload is a common neurotoxic mechanism implicated AD, PD, ALS, and epilepsy (2+ uptake in human disease is that only a few drugs and small-molecule inhibitors of the uptake and release mechanisms are currently available. Thus, there is a critical need to screen for such agents, and to develop small-molecule inhibitors that could potentially be effective in treating neurologic diseases in which Ca2+ overload is a key problem.Another important line of future research would be development of potent and selective pharmacological tools that target MCU. Although a commonly used MCU inhibitor Ru360 is both potent and selective in research . A recenresearch . The latresearch . Notablyn injury . Currentn injury , but it n injury . Mitoxann injury . Interesn injury . Given i process . Since mepilepsy , it will


Acute myeloid leukemia (AML) survivors face unique challenges affecting long-term outcomes and quality of life. There is scant literature on the long-term impact of AML treatment in physical and mental health, disease recurrence, and financial burden in survivors.Fatigue, mental health concerns, infections, sexual dysfunction, and increase cancer recurrence occur after AML treatment. Chronic graft-versus-host disease (GVHD) and infections are common concerns in AML after hematopoietic stem cell transplantation (HCT). Survivorship guidelines encompass symptoms and complications but fail to provide an individualized care plan for AML survivors. Studies in patient-reported outcomes (PROs) and health-related quality of life (HRQoL) are sparse.Here we discuss the most common aspects pertaining to AML survivorship, late complications, care delivery, prevention of disease recurrence, and potential areas for implementation. Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by immature myeloid cell proliferation, accumulation, and infiltration of the bone marrow, blood, and other tissues. It is one of the most common types of adult leukemia with 20,500 estimated new cases in 2022 in the United States (US) . The incClinicians are responsible for surveillance testing for recurrent or subsequent primary cancers, disease prevention, management of comorbid conditions, and monitoring for physical and psychological effects of cancer treatment. Although there is a significant need for information regarding AML long-term survival, quality of life, and comorbid conditions, there are limited studies and literature available to help aid with the navigation of survivorship.We summarize the current state of AML survivorship, care delivery for AML survivors, caregiver and social support, and opportunities for improvement in research, practice, and education in AML.Fatigue is a common symptom in AML survivors and is a strong predictor of worse physical, emotional functioning, and decreased survival . In the Infertility and sexual dysfunction are prevalent long-term effects in AML survivors, and clinical counseling and education should be provided prior to receiving treatment. In a self-reported fertility study of the AML survivors who wanted children after treatment, 46% of women and 40% of men felt that they had not been fully informed about fertility-related issues . Many feA cross-sectional study of sexual health and infertility, post-allo-HCT patients reported a decrease interest in sex, sexual activity, pleasure from sex, and ability to have sex . AnotherThe NCCN and ASCO have established recommendations for fertility preservation in cancer patients. Sperm, oocyte, embryo, and ovarian tissue preservation are strategies supported by survivorship guidelines , 21. RelStreptococcus pneumoniae, Neisseria meningitidis, Hemophilus influenzae, Aspergillus, Candida, Pneumocystis jiroveci, cytomegalovirus, and varicella zoster virus are common causes of late infections in AML survivors [Acute myeloid leukemia survivors are predisposed to bacterial and fungal infections due to the duration of prolonged neutropenia, which can often lead to long-term antibiotic exposure, multi-drug resistant organisms, and the alteration of the microbiome . Organisurvivors . Due to With the emergence of the COVID-19 global pandemic, AML survivors are at a higher risk of morbidity and mortality due to their overall immunocompromised state . AML surAdvances in AML have improved survival outcomes but have not fully prevented disease relapse . The mosThe methodologies for MRD include multiparameter flow cytometry (MFC), real-time quantitative PCR (qPCR), and or next-generation sequencing (NGS) . MRD is Patient-reported outcomes (PROs) are an important aspect of survivorship. AML survivors report chemotherapy and stem cell transplant side effects, along with functional status and quality of life . PROs arSince clinical trials are limited to a selective number of patients, real-world data studies in PROs are needed to demonstrate the true impact of AML interventions in the community .AML survivors after intensive treatment or allo-HCT experience psychological and cognitive effects negatively affecting QoL and clinical outcomes . DepressFewer comorbidities, higher family income, working or being in school, and having a solid support system in place are associated with a higher emotional well-being .Cognitive dysfunction has been evaluated in a few studies in older adults with AML. The estimated prevalence on cognitive dysfunction in hematologic cancer survivors was 17.2% in working memory, 35.2% in executive function, and impairment in working memory was associated with worsening survival . AML cheThe NCCN survivorship guidelines have implemented screenings for evaluation of mental health, as well as pharmacological and non-pharmacological interventions . The appThe cost of treatment for adult AML patients is an estimated $1.5 billion and $500 million respectively . The cosPatients who experience cancer-related financial problems often reported a poor quality of life that affects their physical health, mental health, and social life . One mulAlong with financial burden, employment can also be distressing for patients. Among 1-year AML survivors, the percentage of full-time employment improves over time with very few limitations in productivity •. ApproxLate complications and chronic health conditions can appear early after allo-HCT with clinical consequences on long-term outcomes. These complications are not unique to AML patients and can be consequences of pre- and posttransplant comorbidities, chemotherapy and conditioning regimens, psychosocial, chronic GVHD, second malignances, bone disease, and infectious complications leading to decreased survival , 73. StuThe American Cancer Society, American Society of Clinical Oncology, and the National Comprehensive Cancer Network (NCCN) have survivorship guidelines not specific for AML. . AlthougThe ideal guidelines should incorporate high level of evidence recommendations extensive to different clinical and socioeconomic scenarios, considering the patient’s perspective.OncoLife, developed by Livestrong and the University of Pennsylvania, constitutes a valuable tool for individualized care plans in AML. OncoLife provides customized information and recommendations for patients at any phase of their survivorship journey. Providers enter information regarding a patient’s disease state, chemotherapy and allo-HSCT, side effects, and complications, and an individualized care plan is created that can be printed out for both patient and provider . The infClinicians play a key role by guiding AML patients through treatment, disease monitoring, and long-term survivorship . Very feEffective AML survivorship requires the partnership between the cancer institution and the primary care physician (PCP). However, many PCPs are unfamiliar with Cancer Survivorship guidelines and long-term chemotherapy and transplant complications. A survey study demonstrated barriers in the interactions between the referral center and the PCP; PCPs rarely to never received a treatment summary, a third rarely to never received timely updates, and about a third had difficulty transferring patient care responsibilities . Other lIn general, PCPs felt comfortable providing general medical care and general cancer screening for survivors, however, felt that the primary oncologist would be better suited to provide care regarding medical concerns related to the cancer treatment and screening for cancer reoccurrence or secondary malignancies . As PCPsFamily caregivers play a pivotal role in providing supportive care to a cancer survivor, which aids the survivor in coping and adjustment; however, it often comes at a considerable cost to the caregivers such as psychiatric comorbidity, compromising their physiological functioning . As cancFatigue, infertility, and sexual dysfunction are one of the most common symptoms in AML survivors. Biomarkers of fatigue and correlation with HRQoL instruments, as well as therapeutic interventions, are needed. Further studies need to be done to determine the impact of non-pharmacological interventions such as psychosocial intervention, exercise, and complementary medicine versus novel pharmacological approaches .MRD constitutes a valuable prognostic tool in AML, but its role as surrogate endpoint is yet to be determined. Its use as therapy decision-making in real-world require the standardization of MRD methodologies and newer technologies for better prediction of AML recurrence.AML clinical trials are incorporating PROs and HRQol as endpoints, timing for assessments and validation across different studies are needed. Table Establishing a network of survivorship services facilitate an individualized survivorship care plan with detailed information regarding a patient’s cancer history and treatment, define surveillance goals and schedules, and health priorities to ensure potential health problems do not get missed. Communication between oncologists and PCPs should remain decentralized and open, to allow for effective follow-up care for the AML survivors.Clinical counseling and education should be greatly emphasized prior to starting any treatment. As anxiety and depression are linked to worse clinical outcomes, making sure AML patients undergo mental health screening, and get offered mental health and social support throughout treatment and into survival is crucial to improve PROs. The impact of the stress and depression in caregivers is not known.The costs of AML treatment cause financial toxicity in survivors. Novel treatment options, along with hospitalizations and allo-HCT, will continue increasing costs. Shifting towards outpatient AML management, along with safer and effective options, will reduce financial burden and improve survival outcomes.AML survivors pose unique challenges and needs. There is a strong need for disease-oriented survivorship guidelines with individualized SCPs. As new therapeutic options arise, survivorship aspects must be considered since initial treatment response, using PROM and HRQoL instruments at different timepoints.In addition to academic and regional efforts to provide adequate resources for survivorships and caregivers, public health policies in cancer survivorship must direct efforts in implementing personalized information, assessment and referrals, and dissemination and implementing methods and interventions for survivors and caregivers .AML is an aggressive hematologic malignancy requiring intensive treatment measures that profoundly affects survivors HRQOL. As the number of AML survivors continues to grow, there is a need to improve survivorship care to improve survivors’ overall health, wellness, and QoL. PCPs and oncologists need to communicate adequately about a patient’s survivorship care plan, which should include surveillance testing for recurrent or subsequent primary cancers, disease prevention, management of comorbid conditions, and monitoring for physical and psychological effects of cancer treatment. Survivors and caregivers need to have resources available to them in how to navigate back into a new normalcy of life, including care for medical complications, coping mechanisms for mental health problems, resources for financial burden, and social reintegration.