Mentorship is a process in which structured support is provided to new graduates of the profession to facilitate theireffective transitional journey to professional autonomy.To explore and describe the mentoring process as experienced by newly qualified midwives and experienced midwives during thetransition period.Aqualitative approach was used. Five hospitals were selected from Limpopo province. The study was conducted in a maternity unit of each selected hospital. Population comprised of all newly qualified midwives as well as all experienced midwives working at institutions under study. Non-probability, purposive sampling method was used to select twenty-five newly qualified and twenty-five experienced midwives working in maternity wards of selected hospitals. In-depth face-to-face interviews were conducted for data collection.Ineffective mentoring processes were reported, where only a few experienced midwives seemed ready to provide informal and unstructured support to graduates. Experienced midwives recognised their mentoring role however, felt they did not have sufficient knowledge and skills regarding mentoring process. Shortage of staff and increased workload were reported as challenges which negatively affected the mentoring process.Mentoring is an effective process for facilitation of graduates' transition process to become registered autonomous midwifery practitioners. However, they were not effectively mentored; consequently, negatively affecting their development to professional maturity. Transition from being a midwifery student to becoming a registered midwifery practitioner is a stressful process which demands provision of structured support and guidance through mentorship to achieve competence; without which newly qualified midwives are at risk of developing anxiety resulting in reality shockIn a study conducted by Kensington, Campbell, Gray, Dixon, Tumilty, Pairman, Calvert and Lennox, mentors played an important role in helping midwifery graduates regarding setting of goals, debriefing and identification of learning areasAccording to the Ugandan Nurses and Midwives Council (UNMC), mentoring should be provided by a mentor who is defined as a nurse or midwife who has completed an accredited mentorship training by the Council and is skilled, knowledgeable and with a positive attitude to guide students according to their learning needs10Based on literature from the Royal College of Midwives (RCM), lack of support of newly qualified midwives, contributes to attrition ranging from five to ten percent whereby graduates leave the profession during the first year of practiceThe South African National Department of Health (SANDoH) reported that the duration of training for a comprehensive nursing programme, especially in relation to midwifery, is perceived to be too accelerated and does not give student midwives requisite competencies; thus limiting their ability to function within the scope of practice prescribed for registered midwivesThe researchers are lecturers responsible for midwifery students' learning in Limpopo province. During accompaniment of midwifery students at the hospitals, the researchers observed on several occasions that newly qualified midwives placed in maternity units of the hospitals where midwifery students were placed for clinical learningwere acting as the most seniors staff members in charge of a labour ward; taking full accountability for the patients as well as junior staff members including students. This is contrary to the aim of midwifery graduates' compulsory placement during transition period. Failure to provide effective mentoring process to newly qualified midwives result in provision of compromised care to patientsA qualitative approach, with anexploratory, descriptive and contextual design was used to explore and describe mentoring process during transition periodfor newly qualified midwives. One hospital was selected from each of the five districts of Limpopo province, and the study was therefore conducted in five maternity units from the five selected hospitals.Population comprised of two groups: (1). All newly qualified midwives who have undergone a comprehensive nurses' training programme based on R425 of 19 February 1985 as amended, and qualified as nurses  and midwives; as well as (2). All midwives practicing in hospitals selected for the study in Limpopo province.All newly qualified midwives working in maternity during their first year of clinical practice following graduation were included in the study, whereas those working in general wards were excluded. Those who have been practicing for more than a year were also excluded from the study.Experienced midwives who have been working in thematernity unit for at least five years were included in the study. Experienced midwives whose work experience in maternity was less than five years as well as those who were not working in maternity during conduction of the study were excluded.Two groups of participants were sampled using anon-probability, purposive sampling strategy, as follows: (1). Five newly qualified midwives working in amaternity unit during their first year of clinical practice following their graduation, as well as; (2). five experienced midwives who have been working in amaternity unit for at least five years.The sample was made up of twenty-five newly qualified midwives and twenty-five experienced midwifery participants.What are your experiences regarding mentoring of newly qualified midwives during transition period?” Probing was done to facilitate unrestricted expressions and responses from the participants. Observation of participants was done during the sessions. Field notes were documented during interviews and were given meaning through the researcher's reflections. Each interview session lasted for about an hour; and enabled the researcher to explore the mentoring process for newly qualified midwives. Interviews were conducted until data saturation was reached.Individual interviews were conducted as means of data collection; as the researchers aimed at obtaining in-depth information regarding mentoring process as experienced by newly qualified midwives and experienced midwives during transition period. A voice recorder was used to capture participants' responses after obtaining their permission. An open-ended question was posed as: “Criteria for ensuring trustworthiness based on Lincoln and Guba's principles as described in De Vos, Strydom, FoucheandDelport were ensuredQualitative data analysis was done using Tesch's open coding method including the following steps: careful reading of all the transcripts by the researcher to get a sense of whole; compilation of a list of similar topics; grouping of data according to themes and sub- themes and coding and categorization of field notes. Results were contextualised through literature controlThe findings emerged from the interviews conducted with two sampling groups as follows: (1). Twenty-five newly qualified midwives placed in maternity units of selected hospitals.These were graduates practicing during their first year following their completion of training. Five graduates were sampled from each hospital. Females constituted 94.8% whereas males composed 5.2% of the total sample. In terms of ethnic groups, 38.8% were Pedi, 39.1% Tsongas, 8.7% Swati and 13.4% Venda. Of all the participants, 70% have undergone their training at the universities whereas 30% undergone their training at the college of nursing. Newly qualified midwives shared their experiences regarding the mentoring process during the transition process. (2). Twenty-five experienced midwives who have been practicing in maternity units for at least five years.Five experienced midwife participants were sampled from each hospital. All participants were females. In terms of ethnic groups, 22.5% were Pedi, 23,6% Tsongas, 12% Swati and 41,9% Venda. Data from participants were consolidated and linked to each other to form clusters; two themes and four sub-themes emerged as presented in Newly qualified midwives expressed dissatisfaction about the mentoring process, especially because the environment in the midwifery units was non-conducive for learning and support.Serious concerns were raised by newly qualified midwives regarding lack of formal mentoring process, which negatively affected their transition process. Based on the findings, experienced midwives seemed not to be ready to take responsibility for their role as mentors.According to participants, clinical placement during the first year of transition was meant for mentoring; but to their surprise, they were expected to work as if they were experienced. Participants expressed their frustration regarding lack of supervision and mentoring, especially during conduction of labour. They reported that experienced midwives do not allocate time for attending to them they just observe them as they pass. Graduates are referred to their colleagues for assistance if experiencing problems. This was reflected in the following participants' quotes:Some experienced midwives observe you as they pass, if you are doing something right they just keep quiet. If what you are doing is wrong, some will just say ‘do this and this’. Some say ‘ask your colleagues to help you. This is bad because as a new graduate, I need somebody who will take me by my hand and show me the way as my mentor.” “Honestly speaking there is no supervision and mentoring. When a patient is in labour, I progress her and even deliver her alone without any supervision or assistance. It is really unfair.” Newly qualified midwives appreciated a very few experienced midwives who demonstrated a certain degree of willingness to assist; though the help they offered was not formal and structured. The findings revealed that newly qualified midwives were not satisfied about the way support was offered to them during transition period as mentoring process was undermined.“Graduates described midwifery units as being non-conducive for effective transition, as some of the questions they had regarding practice were not positively attended to, instead they were expected to practice as though they were experts in the midwifery field. The environment was considered unsupportive and therefore hindered graduates' growth during transition period.Newly qualified midwives described the environment in maternity unit as nonconductive for learning, as their learning needs were notmet. When graduates asked questions, theywere told that there is no time to attend to their questions as the ward was busy. Graduates were also told that the ward environment was not meant for teaching but practice. ‘All you have to do is to work since you have passed your examinations.’Participants needed somebody with experience to boost their confidence as they provided care to patients; especially during performance of difficult procedures such as resuscitation of a new-born baby. Learning through trial and error was the order of the day, which really put patients' lives at risk.It is not that we were not taught during training....no, we were taught, and we know how to do these procedures. But you need somebody who is experienced to support you as you stand so that you gain confidence to perform procedures as an independent practitioner.” “The situation is not good at all; in some instances, you have to learn through trial and error. I was so scared of resuscitating a newborn baby, until one day in which I had to practice it all by myself. Fortunately, the baby cried whilst I was still struggling with the tubing. We were well prepared academically, but you need to have more time in scary areas such as resuscitation of a new-born baby.” Failure to receive effective support in the midwifery units in which graduates were placed, did not only have a negative impact on their performance, but their confidence level too.“Experienced midwives expressed positive attitudes towards their role as mentors, but raised concerns regarding their level of preparedness on the management of the responsibilities accompanying the mentoring role as well as shortage of staff.It was evident from the findings of the study that participants recognised their role inmentoring and supportinggraduates however, they felt they did not have sufficient knowledge and skills regarding the mentoring process.Experienced midwives expressed a need to be empowered so that they could be up to date with the new developments related to provision of quality midwifery care. They also needed to be trained on how they canprovide mentoring process.My problem with mentoring of the graduates is that, things are always changing in the health system. Maybe you need some refresher courses so that you are up to date.”One participant said: “I did my training a long time ago and a lot has changed since then. For me to be able to support them I need some training on how to do it. Otherwise, I don't think I can.” “Mentoring is important, and I was also mentored when I was a junior member in this profession. But, for you to do it effectively, you need some guidance on how to do it.”Another participant confirmed: “Participants expressed their willingness to take their mentoring role, however lack of readiness and preparedness in terms of knowledge and skills regarding the mentoring process, failed them.Experienced midwives raised issues of shortage of staff and increased workload as factors that hindered their ability to fulfil their mentoring role. The fact that they are being overworked, makes it difficult for them to effectively mentor newly qualified midwives even if they would want to.Experienced midwives reported that they gave priority to provision of patients' care, as they are expected tocare for patients despite staff shortage. To experienced midwives, mentoring and supervision of graduates are time consuming, therefore result in compromised patients' care. The following participants' quotes serve as confirmation: “We are short staffed... you find that most of the time we are few and even if you want to mentor them, you can't because the ward is very busy. Therefore, you give priority to the patients.”Another participant said: “The workload is very high in such a way that there is no time for mentoring. You are on your feet most of the time, the little time you get you are so exhausted, you can't even think well.”Shortage of staff and increased workload were reported as serious challenges that negatively impacted on experienced midwives' role as mentors.The findings of the study revealed that mentoring of newly qualified midwives during their transition period was ineffective. Only a few experienced midwives seemed ready to provide support to newly qualified midwives, even though the support was not based on any formal or structured programme. Lack of commitment to provide mentoring to newly qualified midwives led to frustration and anxiety which negatively affected graduates' confidence. This is consistent with what was reported in a study on ‘clinical practice of midwifery graduates during community service placement, in Limpopo province of South Africa’, SimaneNetshisaulu and Maputle revealed that, graduates felt anxious and frustrated during transition period as they were expected to function all by themselves without mentors or any formal and structured support18. Participants expressed lack of confidence to stand on their own, resulting in shock frustration and angerThe findings also revealed that newly qualified midwives failed to experience sense of belonging as they felt abandoned and ignored during the time they needed support most. This is supported by what was reported by Cummins et al, whereby graduates to whom a mentor was allocated, valued mentoring process and reported positive relationship which developed between them and the more experienced midwives which was very helpful in boosting their confidenceBased on the findings of the study, midwifery units were not conducive for clinical support and learning; as there was no formal mentoring process in place. When graduates raised, questions related to care provision, experienced midwives did not attend to them. Some experienced midwives were not supportive. Newly qualified midwives had to learn through trial and error, which put patients' lives at risk. Therefore, graduates were denied the opportunity to learn, which negatively affected their competence. Hence, effective mentoring is necessary for establishment of a supportive professional environment which enhances competency and professionalization, thus resulting in positive patient care outcomes. This was supported by Tarimo, Moyo, Masenga, Magesa and Mzava who asserted that, effective and good quality learning environment should be created as it serves as a form of support for newly qualified midwives during transition periodAccording to the findings of the study, experienced midwives recognised their role in mentoring and supporting graduates as they believed that mentoring promotes professional and personal development of the graduates, however, they felt they did not have sufficient knowledge and skills regarding the mentoring process. This is in line with the findings of the study conducted by Mubeezi and Gidman, in which mentors valued role modelling and provision of knowledge as key priorities of support, however, perceived that they had gaps in the knowledge and skills necessary for mentoringExperienced midwives reported shortage of staff and increased workload as a burden which hindered their mentoring role. Results also revealed shortage of resources as a challenge associated with the role of mentor. This was supported by Mubeezi and Gidman who reported that increased workload due to large numbers of patients and staff shortages resulted in limited time for mentoringThe student confirmed the challenges of lack of effective mentoring of graduates in the African continent, which seems to be common as supported bywhatwas reported in Nigeria that newly qualified midwiveswereleft with no choice, but just to practice through trial and error, as there was nobody to rely on for support, supervision and mentoring which put patients' lives at riskOn the contrary, in Singapore, the National University Hospital which is a member of the National University Health System; developed a 3-year structured program specifically designed to address the identified needs of new graduates. During the first year, emphasis is given to induction and opportunities to develop clinical competence and confidence through a close working relationship with a registered nurse buddy. In Year 2, graduates commence a one-year structured mentorship program which focuses on workplace relationship issues, and provides a dedicated mentor for coaching in professional development and fostering a sense of professional identity. Year 3 focuses on clinical rotations, professional role consolidation, and preparation for an area of specialty practiceThe findings of the study revealed that, newly qualified midwives were not effectively mentored during transition period. Only a few experienced midwives seemed ready to provide support to newly qualified midwives, though the mentoring they provided was informal and unstructured. Experienced midwives recognised their mentoring role however, felt they did not have sufficient knowledge and skills regarding mentoring process. Shortage of staff and increased workload were reported burdens which negatively affected experienced midwives' role as mentors. Formal and structured mentoring programme for supporting newly qualified midwives should be put in place, otherwise competence and confidence level of graduates are compromised. This in turn negatively affects the quality of care provision to patients.The following are the recommendations made by the researchers:Experienced midwives should be trained to empower them with the necessary knowledge and skills regarding mentoring process. Refresher courses should be implemented to keep experienced midwives up to date with new developments, so that they can be able to address challenges experienced by graduates during care provision. The number of nurses and midwives should be increased to enable them to managethe workload, therefore they will have enough time to attend to the support needs of newly qualified midwives.Thatnursing service managers were supposed to have formed part of the participants as they are responsible for staff establishment, development and training of mentors and provision of equipment necessary for enhancing mentoring process. Future research should be conducted focusing on development of a mentoring programme to facilitate effective transition process for newly qualified midwives.


Our data indicate that this action cannot be explained by an increased agonist-induced IP3 production or an overloaded SR Ca2+ pool in smooth muscle cells from guinea pig urinary bladder. The incubation of these cells with three different PKC inhibitors, such as Go6976, Go6983, and BIM 1, resulted in a higher SR Ca2+ leak revealed by inhibition of the SERCA pump with thapsigargin. This SR Ca2+ leakage was sensitive to protein translocation inhibitors such as emetine and anisomycin. Since this increased SR Ca2+ leak did not result in a depleted SR Ca2+ store, we have inferred there was a compensatory increase in SERCA pump activity, resulting in a higher steady-state. This new steady-state increased the frequency of Spontaneous Transient Outward Currents (STOCs), which reflect the activation of high conductance, Ca2+-sensitive potassium channels in response to RyR-mediated Ca2+ sparks. This increased STOC frequency triggered by PKC inhibition was restored to normal by inhibiting translocon-mediated Ca2+ leak with emetine. These results suggest a critical role of PKC-mediated translocon phosphorylation in regulating SR Ca2+ steady-state, which, in turn, alters SR Ca2+ releasing activity.PKC inhibitors stimulate Ca This action results in a chemical gradient that provides a driving force for Ca2+ discharge upon activation of Ca2+ release channels  and ryanodine receptor (RyR)  . The leaRCA pump . These cgulation . Despitechannels . In othechannels . Translochannels . This ch channel , reducinanslocon . Moreoveanslocon .2+ leak   . Agonisteceptors  or PLCβ eceptors , resulti2+ release in smooth muscle cells, particularly Go6976 because this inhibitor stimulates Ca2+ release while inhibiting the carbachol-induced IP3 response. This apparent paradox was clarified by showing that PKC inhibitors trigger SR Ca2+ leak that seems to be occurring via translocon, and SERCA pumps compensate for this leak. This new steady-state in the luminal SR Ca2+ level facilitates agonist-induced Ca2+ release. Our results suggest that PKC activity might be modulating translocon-mediated SR Ca2+ leak, which modifies SR Ca2+ releasing activity.We have studied the mechanism used by inhibitors of PKC to facilitate agonist-mediated Caad libitum. Precautions were implemented to minimize animal use and reduce pain and distress.All animal care and experimental procedures were performed according to the Mexican Official Norm for the Use and Care of Laboratory Animals (NOM-062-ZOO-1999). The protocol was reviewed and approved by the local Ethics Committee on Animal Experimentation  with 0306–06 and renewed with the reference number 0131–15. Animals were bred and housed in the Cinvestav Animal facility with clean air and controlled light and temperature. Food and water were given Single smooth muscle cells were isolated using the previously described method . The uriSmooth muscle cells were loaded with 2 μM Fura-2/AM in the dark for 1 h at room temperature. Fura-2 loaded smooth muscle cells were placed on the stage of an inverted microscope and dually excited with a PTI DeltaRamV attached to a Nikon Diaphot TMD inverted microscope. Fluorescence excited at 340 and 380 nm were recorded at 510 nm in response to the addition of 20 mM caffeine, 100 μM carbachol, or 10 μM thapsigargin dissolved in recording saline solution and applied using a puffer pipette placed 10 μm away from the cell. These compounds were applied to the cell with a WPI PV830 pico pump .2+]i , single muscle cells were patched in the whole-cell configuration and held at 0 mV. The frequency of STOCs was determined for 1 min. Digitized ion current data was stored to be analyzed offline. The frequency of STOCs was determined using the peak detector tool of the Origin program using a threshold ≥20 pA .3 production, we followed a procedure previously described , EDTA (50 mM), and KOH (1.5 M). The amount of IP3 produced was calculated using the fraction of labeled IP3 released from cerebellar IP3R microsomes with the equation: IP3 = k (1-b)/b, where the k value was obtained for each determination using 75 and 150 pmol of IP3 standards. The b value was obtained with the equation b = (sample-NS)/(Bo-NS), where Bo was one of the standards, and NS was the nonspecific binding signal . The recording saline solution contained (in mM): 137 NaCl, 5 KCl, 4 NaHCO3, 2 CaCl2, 2 MgCl2, 0.42 KH2PO4, 10 glucose, and 10 HEPES, pH 7.4 (NaOH). The pipette internal solution contained (in mM): 80 glutamic acid, 80 KOH, 5 NaCl, 40 KCl, 2 MgCl2, 2 Na2ATP, 0.1 GTP, 20 HEPES, pH 7.2 (KOH). The internal solution also contained 100 μM of Fura-2 acid in experiments where SR and cytosolic calcium were measured simultaneously. The Li solution contained (in mM): 120 NaCl, 10 LiCl, 4 KCl, 2 NaHCO3, 2 CaCl2, 1 MgSO4, 10 glucose, and 10 HEPES, pH 7.4 (NaOH).The dissociation solution contained (in mM): 55 NaCl, 6 KCl, 5 MgClMag-Fluo4/AM, Fura-2/AM, and Fura-2-free acid were purchased from Molecular Probes . Thapsigargin was purchased from RBI. All other chemicals were from Sigma-Aldrich. Fura-2/AM, Mag-Fluo4/AM, Go6976, Go6983, BIM1, and Thapsigargin were dissolved in a DMSO 1000x stock solution and stored before use. Fura-2-free acid and carbachol were dissolved in water, and aliquots were stored separately at -20°C before being used. Daily freshly dissolved caffeine was prepared in the recording saline solution. All experiments were performed at room temperature (24°C).3 [Ca2+]i, luminal SR Ca2+ level, and STOCs are reported as mean ± SEM, where n represents either the number of cells studied or the number of independent experiments to determine IP3 production. Statistical analysis was carried out with Student`s t-test or ANOVA using Dunnett or SNK posthoc analysis. The differences were considered significant when p < 0.05.Data for IP3) and diacylglycerol (DAG). The incubation with carbachol (100 μM) for only 5 s significantly increased more than double the IP3 production . Although the presence of lithium should be inhibiting 1IP phosphatase in our assay conditions  and 200 nM Go6976 for 30 min before the stimulation of IP3 production with carbachol  to 718 ± 67 nM (n = 19), the peak rate of rise (n = 11) to 919 ± 105 nM/s (n = 19) while the rising time (n = 11) to 1.96 ± 0.18 s (n = 19) by the inhibition of PKC with Go6976. Since Go6976 did not promote a larger carbachol-induced IP3 production, we studied the possibility that the incubation with Go6976 could result in an overloaded SR Ca2+ store. The smooth muscle SR is endowed with the IP3 receptor and Ryanodine Receptor (RyR); these receptors share the same SR Ca2+ store in this cell type  to 233 ± 25 nM  in cells incubated with 200 nM Go6976. A saturating concentration of caffeine (20 mM) that activates all RyR at once can be used as an indirect indicator of the size of the SR Ca2+ store. In this case, caffeine-induced [Ca2+]i response did not display any difference between cells incubated with Go6976 (n = 8) and control (n = 8). These data show that inhibition of PKC potentiates both the carbachol- and caffeine-induced [Ca2+]i responses, but it seems unlikely that this was due to an overloaded SR Ca2+ store.Despite the strong reduction in the carbachol-induced IPresponse  went fro of rise  increaseing time  was signell type . The app2+ level, we simultaneously recorded changes in Mag-fluo4 and Fura-2 fluorescence to measure modifications in the luminal SR (2+]i (2+ store does not present an evident Ca2+ leak under our recording conditions (2+]i because this pump no longer buffers the plasma membrane Ca2+ entry (2+ leak for the recorded time (2+ availability to release channels in smooth muscle cells (2+]i response was significantly diminished (2+]i response was 159 ± 15 nM (n = 7). However, the same protocol but in cells incubated with 200 nM Go6976 resulted in a clear reduction in the SR Ca2+ level in response to the inhibition of SERCA pumps with thapsigargin . The third application of carbachol-induced no further reduction of the luminal SR Ca2+ level nor any increase in the [Ca2+]i  was not different  when cells were incubated with 200 nM Go6976. If Go6976 had induced a Ca2+ overloaded SR, a smaller Ca2+ leak should be expected since a less leaky SR would be easier to overload with Ca2+. Nevertheless, Go6976 increased the SR Ca2+ leak.To study the effect of Go6976-induced PKC inhibition on the luminal SR Canditions . Indeed,le cells . Accordi [Ca2+]i . These o2+ leak similar to the one caused by Go6976 /sec), n = 4) was similar to the one obtained with only Go6976. These data imply that the effect of Go6976 was due to the inhibition of PKCs.Go6976 inhibits classic PKCs and PKD . To deteeduction , red bareduction . We decieduction . The slo2+ leak, implying the activation of release channels (IP3Rs or RyRs) or some other type of Ca2+-permeable channel in the SR. 2 mM MgCl2 inside the pipette solution inhibits the RyR-mediated SR Ca2+ leak. Yet, this condition did not interfere with the Go6976-induced SR Ca2+ leak  did not increase the BKCa current, while caffeine activated the BKCa current with the same increase in the [Ca2+]i. The frequency of STOCs increases with a higher RyR activity (n = 5), suggesting that the inhibition of PKC increased the spontaneous activation of RyRs. Moreover, the inhibition of IP3Rs with 5 mg/ml of heparin in the pipette solution did not modify the effect of Go6976 (n = 5). However, the presence of emetine (10 μM) fully inhibited the Go6976-induced increased STOCs frequency (n = 6) without interfering with the STOCs amplitude. Collectively, these data suggest that the inhibition of PKC in smooth muscle cells induces a translocon-mediated SR Ca2+ leak that appears to be compensated by an increase in the SERCA pump activity. This new steady-state seems to facilitate the spontaneous activation of RyRs.Spontaneous transient outward currents (STOCs) are potassium currents carried out by Ca of RyRs . Figure activity . The inc2+ release from the SR in response to carbachol and caffeine in smooth muscle cells isolated from guinea pig urinary bladder. However, we could not find evidence of an overloaded Ca2+ store to explain this effect. Moreover, PKC inhibition reveals a translocon-mediated SR Ca2+ leak without reducing the agonist-induced [Ca2+]i responses. We have inferred that this situation reflects increased compensatory SERCA pump activity. This new steady-state facilitates Ca2+ release by both IP3Rs and RyRs. The latter resulted in a higher STOCs frequency. Overall, these data point to the SR Ca2+ leak role in determining the steady-state luminal SR Ca2+ level, particularly the participation of translocon in defining the SR Ca2+ leakage.We have found that PKC inhibition stimulated Ca2+ level upon inhibition of the SERCA pump. This SR Ca2+ leak was not inhibited by MgCl2 or heparin in the pipette solution, arguing against RyRs or IP3Rs being the SR Ca2+ leak channels responding to the inhibitors of PKC. However, emetine and anisomycin inhibited this SR Ca2+ leak supporting the idea that Sec61 translocon participates in this SR Ca2+ leak.The inhibition of PKC with different inhibitors such as Go6976, Go6983, and BIM1 resulted in a reduction of the luminal SR Ca2+ leak in different cell types , Sec62, and Sec63 proteins, oligosaccharyltransferase complex , TRAM, and the TRAP protein complex  . Puromycll types . Howevera2+ leak . A resul2+ pool depletion. Accordingly, an extensive account of molecular mechanisms reduces translocon Ca2+ leak activity  have preferential access to Ca2+ trapped in luminal proteins (2+ release event. While the leak channels control SERCA pump activity by modifying the free luminal ER/SR [Ca2+], implying much smaller Ca2+ fluxes than when the total Ca2+ capacity of the ER/SR is involved. This separation between Ca2+ release channels and leak channels allows the ER/SR to function as a Ca2+ source without triggering ER stress by Ca2+ depletion or other activities that require an elevated free luminal [Ca2+]ER.The calcium release from internal stores is organized in discrete units , resultior IP3Rs . The ER/behavior . There aproteins , making 2+ leak activity of translocon, which appears to be compensated by a higher SERCA pump activity to avoid SR Ca2+ depletion. This increased steady-state results in accelerated Ca2+ release in response to activation of either IP3Rs or RyRs. These results point to the scenario where the translocon Ca2+ leak modulates the Ca2+-releasing activity of the SR Ca2+ store.Altogether, our results indicate that PKC inhibitors increase the Ca


Work-related musculoskeletal disorders (WRMSDs) are considered nowadays the most serious issue in the Occupational Health and Safety field and industrial exoskeletons appear to be a new approach to addressing this medical burden. A systematic review has been carried out to analyze the real-life data of the application of exoskeletons in work settings considering the subjective responses of workers.The review was registered on PROSPERO. The literature search and its report have been performed following the PRISMA guidelines. A comprehensive literature search was performed in PubMed, EMBASE, Web of Science, and Scopus.Twenty-four original studies were included in the literature review; 42% of the papers retrieved included automobilist industry workers, 17% of the studies evaluated the use of exoskeletons in logistic facilities, and 17% of articles involved healthcare. The remaining six papers recruited farmers, plasterers, wasting collectors, construction workers, and other workmen. All the papers selected tested the use of passive exoskeletons, supporting upper arms or back. Usability, perceived comfort, perceived exertion and fatigue, acceptability and intention to use, occupational safety and health, and job performance and productivity were the main topic analyzed.Exoskeletons are not a fix-all technology, neither for workers nor for job tasks; they tend to show more of their potential in static activities, while in dynamic tasks, they can obstacle regular job performance. Comfort and easiness of use are the key factors influencing the user's experience. More research is needed to determine the most effective and safe ways to implement exoskeleton use in occupational settings.https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=275728, identifier CRD42021275728. The European Agency for Safety and Health at Work (EU-OSHA) has recently reported that around 60% of all workers with a work-related health problem, identify work-related musculoskeletal disorders (WRMSDs) as their most serious issue, representing also one of the most common accidents. According to EU-OSHA 2019 report , 4. WRMSIndustrial exoskeletons can be classified by kinematic structure and type of actuation. Concerning kinematics, rigid-structure devices can be classified as anthropomorphic and nonanthropomorphic devices, whereas soft exosuits do not present any kinematic structure. Moreover, the structure can range from full-body devices to devices that assist only one joint, thus making them potentially tailorable. Actuation types include passive, semi-active, and active systems. The passive exoskeletons do not have actuators or electronic components, such as transducers or controllers, but the force necessary to assist the actions performed by the user is released by exploiting the elasticity of the materials . The advantages offered by this type are in the lightness, economy, and simplicity of redesign, unlike the powered (semi-active and active) ones. Due to those peculiar characteristics, exoskeletons work in tandem with the wearer, lightening the load and increasing the physical capabilities of the wearer, as well as solving potential ergonomic issues of holding and working with tools.Several experimental studies and literature reviews have been conducted to analyze the biomechanical effects of the available exoskeletons on users. Three recent systematic reviews analyzed the effects of industrial exoskeletons on exertion, muscle activity, and partially on user experience –39. TheyOur review aimed at filling the gap in the literature about the worker's experience using the exoskeleton in real working tasks, since studies performed in the lab may have a limited value, not considering the complexity of a work environment as well as the experience of workers, who are the final users of industrial exoskeletons. The purpose of this systematic review is to analyze real-life data on the use of exoskeletons in work settings. In particular, this paper aims to outline a picture of the possible impact, benefits, and criticalities of exoskeletons in the workplace by reviewing and analyzing the subjective responses of workers.This systematic review was registered on the PROSPERO register at number CRD42021275728 on September 30th 2021. The literature search and its report have been performed following the PRISMA guidelines :Working environment or lab setting in which working tasks are performed.Workers of all types.Use of any kind of exoskeleton in an occupational setting (field study/laboratory study reproducing working tasks).When present, the use of no exoskeleton for the performance of the same tasks.User's subjective experience/perception/compliance/usability of the device.A first screening of the results was performed by two independent reviewers (LGL and AM) through titles and abstracts of the reports identified. A further selection was made by analyzing the full text of the articles. The judgment about the inclusion of each paper was performed separately by the investigators; disagreements were solved by the discussion with a third reviewer (AB).Data were manually extracted in a chart jointly developed by the authors, including relevant data from the papers retrieved.The inclusion criteria followed the SPICE scheme mentioned above; we included articles focusing on the application of exoskeletons in the occupational field. We selected papers considering the use of exoskeletons by workers acting in real occupational tasks, both in the field and in the lab. The studies selected took into account the subjective perspectives of workers wearing the exoskeletons regarding comfort, usability, and personal experience.Articles written in languages other than English were excluded. Studies analyzing stereotyped tasks which did not reproduce a real working task, or not considering the subjective experience of the participants were also excluded. We also excluded articles that did not conduct experiments on real workers. Also, review articles were not included in the literature synthesis but were discussed in other paragraphs.The quality assessment was performed using the Mixed Methods Appraisal Tool (MMAT) , 45 whicThe online search retrieved a total of 600 papers. The selection process is shown in At the end of the selection, 24 original studies were included in the literature review. Most (sixteen) of the selected articles have a cross-sectional design, administering validated and not validated questionnaires at the end of an experimental period in which the workers perform their job wearing the exoskeleton. Four studies adopted a mixed-method approach and 1 was purely qualitative; the experiment performed is similar to that of a cross-over study: the workers wear the exoskeleton for a period and then evaluate their experience. Three articles described prospective cohort studies of two working populations studied for several months. Fourteen studies were published in Europe, 8 in North America, 1 in Iran, and 1 in Korea. The overall quality of the studies, evaluated with the MMAT tool, ranges from 20 to 80%: 5 studies scored 80%, 13 scored 60%, and 6 scored 40%. The details of the quality appraisal are included in n = 10) of the selected articles included automobile industry workers, who work in the assembly lines of vehicles; 17% (n = 4) of the studies evaluated the use of exoskeletons in logistic facilities, 17% (n = 4) of the articles involved healthcare workers such as surgery team members and professional caregivers. The remaining 6 papers (25%) recruited farmers, plasterers, wasting collectors, construction workers, and other workmen. 42% (All the papers selected tested the use of passive exoskeletons. Two main types of exoskeletons were worn by the workers: exoskeletons supporting the upper limbs and exoskeletons supporting the back. The types of exoskeletons were chosen according to the job performed. Only one study provided results of usability on lower limbs supporting exoskeleton (Hyundai CEX chair exoskeleton) .ad hoc- not validated - questionnaires and qualitative approaches to investigate the worker's experience wearing the exoskeleton. Usability was measured with the SUS – System Usability Scale  investigated the concept of usability of the exoskeleton worn by workers. The scale used to evaluate usability was heterogenous, the most used was SUS , which was adopted by 4 studies. The scales used by each study are reported in Thirteen studies (54%) addressed the perceived comfort and discomfort of the workers wearing the exoskeletons. There was no homogeneity in the scale used to measure physical comfort or discomfort. The most used scale was a Likert scale measuring discomfort, adopted by four studies. The physical comfort of the device is mainly expressed in the direct interface between the exoskeleton and the body. In logistic workers, participants reported the highest discomfort in the shoulder regions where the rigid frame of the exoskeleton and the user's body interacted, or body parts onto which the exoskeletons were attached. These aspects influenced the score of usability . In the Thermic comfort was recognized as a critical issue for the comfort of the exoskeleton, both for those supporting the back  and thosNine studies (39%) analyzed the exertion and fatigue perceived by workers wearing the device. The most used scale was the RPE scale, adopted by three studies. Overall, as for usability and comfort, it was found great heterogeneity in the methods used to measure exertion and fatigue. In plastering workers, the perceived exertion was reduced using the upper limb supporting exoskeleton, in particular for activities at the ceiling, while during tasks involving various movements passive exoskeletons were found not as effective as during less varied tasks . In the Only three studies addressed the issue of improvement of the participant's health and prevention of injuries, and one study considered the possible harmfulness of the exoskeleton regarding safety in the workplace. In an 18-month experiment, automotive operators reported an equivalent or slightly better perceived safety and slightly better perceived performance. Those who used the ASE were roughly half as likely to make a medical visit that involved an injury to or pain in the upper extremity . In the Eight (33%) studies considered the topic of job performance and productivity. The efficacy of the exoskeleton widely varies according to the task performed. In the automotive setting, tasks involving heavy material handling were reported to be easier to perform with the exoskeleton, while more dynamic tasks which involved various postures were classified as harder to perform, and sometimes the exoskeleton might disturb rather than help. Also, dynamic tasks, which require adjustments in the exoskeleton support, were difficult to manage due to time pressure . Exoskelper se for its characteristics and potential than its use in a real field social aspects come into play, linked to how others may judge an individual who decides to use the device or how the person perceives the decision and the imposition of the exoskeleton (Twelve studies (50%) dealt with the acceptability and the intention to use exoskeletons by workers. Fit and comfort are in general key determinants of the possible adoption of the exoskeleton by workers , 58, 65,skeleton . In a stskeleton , 61. Amoskeleton  and mostskeleton . The aspskeleton . In the skeleton . The exoskeleton . In a geskeleton , the intThis systematic review included 24 studies, in which the exoskeletons were tested on workers performing real occupational tasks, both in the lab setting and directly in the field. Despite the online research being performed without any date restriction, all the studies were published in the last five years, mostly in 2020 and 2021. This testifies to the novelty of the introduction of industrial exoskeletons in real working settings.All the papers included in our review used passive exoskeletons which use the restoring forces of springs, dampers, or other materials to support the human movement. The movements of the user generate the energy stored in a passive exoskeleton, and forces are redistributed to protect specific body regions. The improvement of the performance of the exoskeleton user is not given by additional physical strength (as it would be for active devices) but by the ability provided by passive exoskeletons to maintain exhausting positions over a longer period of time . PassivePrevious studies and reviews have focused on the effects of exertion and reduction in muscle activity provided by industrial exoskeletons, mainly through lab experiments. Only recently, since the beginning of the use of exoskeletons in working settings, user experience has gained relevance in research. Currently, prospective studies analyzing the intervention of using exoskeletons to reduce the incidence of WMDS are still missing. Industrial exoskeletons were born and marketed with the aim of reducing WMSDs, assuming that the reduction in exertion, which is the outcome most analyzed in the current literature, corresponds to a reduction in musculoskeletal disease risk factors. WRMSDs have a complex pathogenesis , and sucThere are several different opinions on how industrial exoskeletons might be considered. They can be defined as Personal Protective Equipment , or as pAs the introduction of exoskeletons is becoming more and more frequent in industrial settings, studies designed to study the potential risks linked to exoskeletons are needed . In our In the field, studies show overall less brilliant results than lab studies, in terms of user satisfaction. The work tasks are far more complex than the experimental tests and exoskeletons are the most useful during static postures, for example, when it is necessary to keep the arms above the shoulders for a long period of time . The differences in the performed tasks, with those in real working environments far more complex than those studied in the lab, is the first great difference between lab and field studies. Recent research has also pointed out how wearing an exoskeleton and performing complex tasks impose greater motor adaptation and neurocognitive efforts, which may almost offset the biomechanical advantages of exoskeletons . IndustrComfort and fit indeed are the main factor influencing the usability and acceptance of the device by workers. Workers may accept the exoskeleton only if the benefits perceived will overcome the possible negative residual drawbacks, like the overall discomfort  . DespiteThis review has several limitations and points of strength. First, the evidence retrieved is subject to the quality of the articles included in the review. The selected papers have a cross-sectional design for the most part, and in some cases adopted a qualitative approach, and this impacts the level of confidence of the evidence. The lack of standards in evaluating the outcomes and the use of not-validated questionnaires makes it hard to compare studies and generalize the findings. Second, as mentioned above, the participants of the studies and the occupational settings involved may not be fully representative of the general working population and working environments in general. Regarding the methodology adopted in this systematic review, although performed according to PRISMA guidelines and with a rigorous search and selection strategy, it is possible relevant studies may have been missed. However, to the best of our knowledge, this is the first review that systematically addressed on-field studies involving real workers wearing exoskeletons and analyzing the depth of their user experience. It identified major points to consider when introducing exoskeletons on the field, such as comfort, job performance, acceptance from workers, task specificity, safety, and health issues.The use of exoskeletons in occupational settings is a relatively new phenomenon with a limited amount of research available on the topic. A systematic review of the literature was conducted in order to identify and evaluate the available evidence on the field use of exoskeletons from an occupational safety and health perspective. Twenty-four scientific articles were identified that met the inclusion criteria for the review. The findings of the review suggest that exoskeletons have the potential to reduce the risk of musculoskeletal injuries in a variety of occupational settings. However, there is a lack of evidence on the long-term safety and efficacy of exoskeleton use in the workplace. Additionally, there are several potential concerns that need to be addressed, such as safety issues and ergonomics, considering that is not clear if the exoskeleton itself can cause the discomfort, or if the discomfort should be due to the lifting tasks that the workers are called to perform. More research is needed to determine the most effective and safe ways to implement exoskeleton use in occupational settings.Our review explored the experience of real workers wearing industrial exoskeletons. The lack of longitudinal studies is the core limitation when analyzing such data and medical data regarding the possible prevention of WMDs using exoskeletons are currently missing. On-field studies, which addressed workers' experience wearing exoskeletons, showed overall less brilliant results than lab experiments, due to the higher complexity of work tasks compared to stereotyped exercises. Exoskeletons are not a fix-all technology, neither for workers nor for job tasks; they tend to show more of their potential in static activities while in dynamic tasks they can obstacle regular job performance. Comfort and easiness of use are the key factors influencing the user's experience.The existing literature on the field use of exoskeletons is mostly anecdotal, consisting of case reports and small case series. There is a need for larger, well-designed studies on the field use of exoskeletons in order to better understand the potential safety and health risks associated with their use.The first draft, conceptualization, and the original idea were provided by AB and LGL. The literature search was performed by LGL and AM. The selection of articles was performed by LGL, AM, and AB. The study was critically revised and reviewed by NM, GA, FC, and LF. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Similarly, the unfolded protein response of the endoplasmic reticulum (UPRER) is responsible for maintaining proteomic stability in the endoplasmic reticulum (ER). Since the mitochondria and endoplasmic reticulum are the primary centers of energy metabolism and protein synthesis in cells, respectively, a synergistic mechanism must exist between UPRmt and UPRER to cooperatively resist stresses such as hyperglycemia in T2D. Increasing evidence suggests that the protein kinase RNA (PKR)-like endoplasmic reticulum kinase (PERK) signaling pathway is likely an important node for coordinating UPRmt and UPRER. The PERK pathway is activated in both UPRmt and UPRER, and its downstream molecules perform important functions. In this review, we discuss the mechanisms of UPRmt, UPRER and their crosstalk in T2D.The mitochondrial unfolded protein response (UPR ER and UPRmt, respectively)  . The endn the ER . Furthern the ER . One succtioning . Recent ctioning . In addictioning . Mitochoctioning . TherefoMitochondria are organelles with double membranes, which contain proteins and RNAs encoded by the nuclear genome and are also capable of encoding proteins and RNAs related to OXPHOS . Mitochomt is a mitochondrial stress response that maintains proteostasis in the mitochondria . In C. elegans, mitochondrial matrix proteins can be hydrolyzed to generate short peptides by CLPP-1 when the processing capability of chaperone proteins cannot afford the aggregation of misfolded and unassembled proteins , has recently been found downstream of mitochondrial dysfunction-driven chromatin reorganization. Histone methyltransferases, MET-2 and LIN-65, are involved in chromatin reorganization, and regardless of which is inhibited, they would reduce the methylation of histone H3K9 and block the induction of UPRmt downstream of CLPP-1. In addition, it was found that MET-2 was necessary for the accumulation of LIN-65 in the nucleus, which could promote the opening of a portion of chromatin, facilitating the binding to open regions and the transcriptional activity of DVE-1  as mRNA, producing XBP1s, which is an active transcription factor. Once XBP1s are encoded and translated, they can enter the nucleus to facilitate the production of chaperones, components of ER-associated degradation (ERAD), and foldases, which participate in protein trafficking, degradation, and folding by driving the activation of UPRER-related genes  increased in PERK- eIF2- ATF4 , and maintaining homeostasis is not only important for the organelle itself, but also for the whole cell. As an essential mechanism to cope with homeostasis disruption, UPRty liver . For ins obesity . The geng OXPHOS . In addiuch mice . Notably studies mt plays a crucial role in T2D, from insulin resistance to beta cell dysfunction. Increased glucose and fatty acid metabolism in cells in hyperglycemia and hyperlipidemia result in increased ROS production and protein aggregation  and pancreatic beta cell dysfunction. Initially, factors such as obesity led to insulin resistance, forcing pancreatic beta cells to exert excessive normal action for more insulin to maintain normal glucose levels. However, prolonged overstimulation results in beta cell dysfunction and death, ultimately leading to hyperglycemia and gradually leading to diabetes. UPRregation . Consequregation . UPRmt rwith T2D . Moreoveght gain .ER coordinates with UPRmt in T2D by activating the PERK pathway. Downstream PERK has been reported to be involved in T2D. The homeodomain transcription factor of pancreas/duodenum homeobox protein 1 (Pdx1) and human diabetes genes partially regulate beta cell survival via direct regulation of the activating transcription factor ATF5. Significantly, the absence of ATF5 decreased the survival rate under stressful conditions. ATF5 is parallel to ATF4 downstream under the control of eIF4E-binding protein 1 (4ebp1), a component of the mammalian target of rapamycin (mTOR) pathway that slows the translation of proteins, based on loss-of-function and chromatin occupancy tests. Consequently, the absence of ATF5 decreased the stress-induced inhibition of almost the whole translation, thereby increasing the vulnerability of cells to stress-induced death . Second, specific CHOP deletion in ß cell can protect aged HFD-fed mice against liver hepatomegaly and steatosis and not influence basal glucose homeostasis. Third, the Ca2+ buffering capacity within ER is reduced and glucose-induced Ca2+ oscillations is regulated in terms of CHOP depletion in cells, causing a result of changes in transcriptional profile of the ER chaperone. Finally, there is decline of pancreatic insulin content and liver triglycerides under the operation of exerting CHOP antisense oligonucleotide conjugated with GLP1. In conclusion, these results suggest a novel therapeutic strategy via targeting CHOP in β cells to mitigate the development of T2D, while regulating insulin secretion and alleviating syndromes of consequent fatty liver disease .As a member of TGF-β superfamily, the growth differentiation factor-15 (GDF-15) cytokine has an impact on the development of several metabolic disorders via plasmatic level regulation, including T2D , cardiovmt is still in its infancy. Despite significant progress in the past few years, the crucial action of UPRmt in metabolism, such as T2D, is gradually being understood. Here, we describe our current understanding of UPRmt and its crosstalk with UPRER, mainly through the PERK signaling pathway. Furthermore, we discuss the functions and molecular mechanisms of UPRmt and coordinated UPRER in the context of T2D. Although our knowledge of the topics in this review has largely improved, many fundamental questions remain unanswered: Do UPRER components other than PERK also affect UPRmt? Does UPRmt also regulate UPRER? Which components of UPRmt respond to insulin resistance development in T2D? Answers to these questions will help identify new candidate interventions for T2D.The study of UPR


Despite advancements in the treatment of hematological malignancies, disease relapse is still a major concern in the management of acute myeloid leukemia (AML). It is believed that chemotherapeutic resistance in leukemic stem cells is the driving factor behind disease relapse. The role of in vitro 3D models in studying the mechanisms of chemotherapeutic drug resistance in leukemic stem cells is not well-researched. Herein, we review the recent advancements in using 3D in vitro models in studying AML. In addition, we describe their role as potential platforms for understanding mechanisms of resistance and relapse in AML and in identifying and testing therapeutic modalities.The complexity of the bone marrow (BM) microenvironment makes studying hematological malignancies in vitro a challenging task. Three-dimensional cell cultures are being actively studied, particularly due to their ability to serve as a bridge of the gap between 2D cultures and animal models. The role of 3D in vitro models in studying the mechanisms of chemotherapeutic resistance and leukemia stem cells (LSCs) in acute myeloid leukemia (AML) is not well-reviewed. We present an overview of 3D cell models used for studying AML, emphasizing the recent advancements in microenvironment modeling, chemotherapy testing, and resistance. Despite advancements in understanding acute myeloid leukemia (AML) pathogenesis and its therapy, most patients with AML continue to have grim long-term outcomes. Standard conventional chemotherapy induces remission in 50% to 75% of adult patients with AML, but unfortunately only 20% to 30% of these patients enjoy long-term leukemia-free survival . In addi6 leukemic blasts), but they are capable of propagating AML in appropriate xenograft models [Biologically, MRD might include different cellular components, including leukemic stem cells (LSCs), which are believed to contribute to disease initiation at the time of relapse . Using pt models . Currentt models . These LTo understand LSCs survival mechanisms, it is crucial to understand how leukemia cells interact with different elements of the 3D bone marrow microenvironment . Such nehttps://pubmed.ncbi.nlm.nih.gov/, accessed on 1 April 2022) to look for articles containing the specific keywords mentioned below, in the period between 2012 and 2022. We also examined the references section of initially reviewed articles for other relevant articles. Keywords used for the retrieval of articles included “three-dimensional”, “in vitro”, “acute myeloid leukemia”, and “drug resistance”.In this minireview, the literature review was performed by using the online search engine PubMed  and demonstrated superior results in terms of chemoresistance to daunorubicin and increased N-cadherin expression when compared to 2D models or suspension conditions , which aTo study the cellular response of AML cells under environmental stress, Velliou et al. used a highly porous PU collagen-coated scaffold to evaluate the cellular proliferation and metabolic profile of AML cells in vitro. Results showed that when the supply of oxygen and glucose to K-652 AML cells is limited, cells still maintain their adaptability and survival mechanisms. In contrast to 2D models, 3D models serve as accurate platforms to predict cellular metabolic mechanisms, particularly under environmental stress . While sOn the other hand, biological scaffolds resemble the tissue of origin, as they contain some constituents of BM ECM. One model utilized decellularized Wharton jelly matrix (DWJM) as a BM ECM mimicking model and found that compared to suspension, their model showed an increase in the markers of leukemic cell quiescence and adhesion-induced chemoresistance compared to suspension . BorellaAlthough 3D in vitro models are continuously advancing, their static nature limits our ability to investigate the dynamic interactions occurring in vivo. Accordingly, bioreactors were introduced to provide a dynamic platform and reproduce normal physiological processes similar to those in the tissue microenvironment. Bioreactors are enclosed complex systems which regulate the occurrence of normal biological processes by connecting their sensors to advanced software. The significance of this technology lies in its role in regulating oxygen and nutrient supply, waste removal, and mimicking mechanical and shear stresses occurring in vivo. Additionally, it can be equipped with fully-humanized vasculature and circulating chemokines. As an example, one model has successfully regulated in vivo physiological processes via providing strict control of oxygen gradient, temperature, pH, and accurate spatiotemporal gradient in solid organ tumors .In a 3D bioreactor model, which compared UCSD-AML1 cell line differentiation between one scaffold-free and two different scaffold-based models (osteoblastic and vascular), results confirmed that in the presence of a scaffold in the model, cells were able to expand and differentiate into their mature type. Additionally, when AML cells were added to the cell culture in the model, the resistance to chemotherapy was more pronounced when compared to 2D models. Additionally, leukemic progenitor/stem cells were retained in the scaffold compared to the mature phenotype, which was released in the supernatant . This reIn addition, we note that when AML cells were cultured with components of the osteoblastic niche, there were discrepancies in terms of gene expression and differentiation compared to when cultured with stromal vascular niche components .On the contrary, metabolic profiling of cultured cells would recapitulate the BM microenvironment more accurately and would provide insight into the metabolic behavior of malignant and non-malignant cells in vitro. Despite creating dynamic conditions within models with the use of rotators, stirring techniques, and pumps, there is no direct control over the movements of cells and scaffolds in the culture system, thus resulting in the disruption of cells or the scaffold architecture through collision. Zippel and Raic et al. proposed a dynamic perfused triculture 3D model that mimics both healthy and leukemic BM niches. Investigators introduced a novel method of inducing dynamicism by applying external magnetic fields to the model . SubsequSeveral models have previously been developed to recapitulate the BM microenvironment. One such model from Chou et al. used a micro-physiologic device to demonstrate hematopoietic stem and progenitor cell function in a 3D model with media flow through a vascular channel to provide physiologically relevant nutrient and waste exchange across an endothelial cell barrier . A similThe development of a functional model capable of mimicking the heterogeneity and dynamism of the BM microenvironment with translational potential is still a major challenge. The complexity of the BM microenvironment makes studying hematological malignancies in vitro a challenging task. For a better understanding of AML behavior in the BM niche, we need to take into consideration multiple factors that influence the BM niche. These factors include highly heterogeneous structure, variability in oxygen gradient, discrepancies in BM stiffness, continuous ECM remodeling, and vascularization. Even though the BM microenvironment is dynamic, there is no stringent evidence to favor the use of dynamic models over their static counterparts. This brings us to an unmet need, which is to conduct further experiments under both static and dynamic conditions.In the context of choosing between scaffolds, Garcia et al. compared phenotypic expression and expansion of AML when cultured in a dynamic 3D stromal vascular model, once with collagen and separately with hydroxyapatite and detected no differences . Thus, tMultiple models debated whether extracting MSCs from the BM is more accurate than from umbilical cord tissue. MSCs from both sources can be used in modeling AML, as both can support the BM microenvironment niche. 3D models that utilized umbilical cord tissue cells for culture reported similar results to BMMSCs in terms of chemoresistance, signaling, and proliferative capacity ,24. On tDespite advancements in cancer biology research, AML relapse is almost inevitable and represents a major hurdle to overcome to improve the quality of care for AML patients. The high relapse rate and drug resistance in AML patients are closely related to the properties of cancer stem cells (CSCs), particularly their quiescent state, complex interplay with the bone marrow (BM) microenvironment, phenotypic plasticity, and their relapse-initiating capacity. Therefore, studying LSCs and their interactions with the BM microenvironment in vitro is an unmet clinical need that could potentially provide invaluable evidence to overcome drug resistance and consequently reduce relapse rates.Currently, the present models have attempted to accurately mimic the BM microenvironment to obtain strong evidence of the relationship and the interaction between AML cells in the BM niche. Nonetheless, few models focused on studying LSCs and their biological behavior in vitro. LSCs are unique, with characteristics markedly distinguishable from mature AML cells, allowing them to induce relapse. Namely, these cells have the intrinsic ability to differentiate and self-renew ,28.In addition, LSCs can avoid being killed by chemotherapeutic agents and radiotherapy by interacting with their microenvironment and surrounding tissues such as adipose tissues ,30. CollTo successfully reduce the burden of relapse and chemoresistance caused by LSCs, it is crucial to initially identify phenotypically distinctive cell surface markers and the molecular mechanisms that govern their survival. In vivo studies have shown that CD123 and CD96 are highly expressed on the membrane of LSCs, whereas they are weakly expressed on the surface of HSCs and are also involved in the development of leukemia ,35. ThusDisease relapse is a major challenge facing clinicians during AML management. Relapsed disease affects almost 10–40% of young patients and a significant percentage of patients 60 years and above . While yConsequently, targeted immunotherapies are continuously emerging as a promising management modality for relapsed AML . HoweverInvestigators have attempted to conduct clinical trials targeted toward inhibiting pathways related to LSCs initiation, cellular pathways, and survival pathways. In terms of drugs targeting CD123 as an LSCs surface receptor, results have shown insufficient efficacy in treating relapsed AML . On the Collectively, the future approach to in vitro modeling of AML should include studying LSCs and their specific properties as they significantly contribute to the resistance to therapy and thus the relapse of AML.The integration of culturing LSCs in a 3D model can potentially take in vitro modeling a step closer to personalized medical plans. By designing 3D in vitro models that closely resemble the BM microenvironment, we can examine these cells and use this information to develop therapeutic plans specific to the phenotype of AML, with fewer side effects and more clinically efficient results. By initially obtaining samples from the patient’s BM and/or peripheral blood and isolating the nuclear cell component, we can subsequently test the isolated cells for their immunophenotype, metabolic patterns, and cellular signaling. This will be followed by culturing patients’ samples in in vitro 3D models that recapitulate BM microenvironmental interactions in order to cultivate and isolate LSCs. During and following culture, these samples could be investigated for specific LSCs cellular signaling pathways, survival mechanisms, and their interaction with the surrounding microenvironment. Identified signaling pathways will serve as potential targets for treatment plans and further provide us with evidence in terms of efficiency and chemoresistance. Furthermore, these newly discovered drugs can also be tested as a mono-therapeutic agent or in conjunction with conventional treatment plans for AML and LSCs cells simultaneously. Collectively, this approach can help us eradicate both AML and LSCs cells .Three-dimensional modeling of the BM microenvironment in AML has strengthened our understanding of the intricacies of leukemic cells and important pathogenic mechanisms related to AML survival and adaptation in vivo. Moreover, they have provided us with invaluable techniques to further optimize the designs of potential in vitro models, particularly approaches related to improving the biomimicry of the actual BM microenvironment. With exclusive evidence highlighting the key role of LSCs in disease relapse, there is a clear need to shift our in vitro models toward better identifying LSCs features contributing to disease relapse. While relapse and chemoresistance have always been major hurdles in the management of AML, the literature is lacking models designed specifically to investigate how LSCs induces relapse and chemoresistance. In this mini-review, we believe that potential approaches should be directed toward uncovering LSCs interactions and designing reliable biomimetic platforms for the testing of future therapeutic modalities.


Contractions and intracellular Ca2+ concentrations were blunted in Notch3−/− vessels. ANG II responses in precapillary muscle arterioles were similar between the WT and Notch3−/− mice, suggesting a focal action of Notch3 in renal vasculature. Abolishing stored Ca2+ with thapsigargin reduced Ca2+ responses in the renal vessels of the two strains, signifying intact intracellular Ca2+ mobilization in Notch3−/−. EGTA (Ca2+ chelating agent), nifedipine (L-type channel-blocker), or mibefradil (T-type channel-blocker) strongly reduced contraction and Ca2+ responses in WT mice but had no effect in Notch3−/− mice, indicating defective Ca2+ entry. Notch3−/− vessels responded normally to KCl-induced depolarization, which activates L-type channels directly. Differential transcriptomic analysis showed a major down-regulation of Cacna1h gene expression, coding for the α1H subunit of the T-type Ca2+ channel, in Notch3−/− vessels. In conclusion, renal resistance vessels from Notch3−/− mice display altered vascular reactivity to ANG II due to deficient Ca2+-entry. Consequently, Notch3 is essential for proper excitation–contraction coupling and vascular-tone regulation in the kidney.Notch3 plays an important role in the differentiation and development of vascular smooth muscle cells. Mice lacking Notch3 show deficient renal autoregulation. The aim of the study was to investigate the mechanisms involved in the Notch3-mediated control of renal vascular response. To this end, renal resistance vessels (afferent arterioles) were isolated from Notch3 Notch is a family of membrane receptors participating in cell growth, differentiation, and apoptosis ,2. Out oIn humans, a mutation in Notch3 causes the disorder cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), which is an inherited small-vessel disease that causes ischemic strokes, mental disorders, and premature death ,8. In a −/− mice displayed a significantly blunted renal vascular response after systemic bolus injections of vasoactive agents, such as norepinephrine, angiotensin II (ANG II), bradykinin, or prostacyclin, consistent with deficient renal autoregulation. Furthermore, chronic infusion of ANG II was associated with increased mortality and severe renal damage in Notch3−/− mice [2+ responses of renal resistance vessels’ freshly isolated afferent arterioles (AAs) from Notch3−/− mice.The importance of renal vascular reactivity in controlling blood pressure led our group to study renal haemodynamics in mice lacking the Notch3 receptor . Compare−/− mice . These d2+ channels in many segments of the renal vascular tree [Despite the expression of Calar tree , we focular tree  and is tlar tree . Furtherlar tree ,14.−/− and wild-type mice were challenged with ANG II. The use of calcium channels or intracellular Ca2+ inhibitors indicated that the absence of Notch3 compromised entry of extracellular Ca2+. The underlying mechanism of this defect was related to a significant downregulation of the α1H subunit belonging to the T-type Ca2+ channel. It thus appears that Notch3 regulates renal vascular tone by controlling the expression and function of specific Ca2+ channels.Therefore, AAs isolated from Notch3−/− AAs  and muscle resistance vessels . As expected, the mean diameter of renal AAs isolated from wild-type mice decreased in a dose-dependent manner, reaching 60% of baseline at 10−7 M ANG II . However, when vessels were challenged with ANG II, contraction and Ca2+i responses were weaker in AAs isolated from Notch3−/− kidneys , contractions were significantly blunted in vessels from wild-type mice. In contrast, the addition of these inhibitors did not affect the Ca2+ responses or contractility of Notch3−/− AAs. Again, the strain difference in the contractility and Ca2+ responses disappeared, suggesting that the reason for the improper response of the Notch3−/− vessels to ANG II was due to dysfunction in extracellular Ca2+ entry.When Ca−/− mice B,F. As arough L- C,G or T-rough L- D,H Ca2+ 2+i mobilization also contributed to blunted contractions, we treated AAs with the Ca2+ ATPase inhibitor thapsigargin, which abolishes intracellular calcium stores (2+ response in both strains (p < 0.001), indicating that intracellular Ca2+ mobilization was equally efficient in the two strains.To examine whether compromised Cam stores A,B. Thap2+-entry in vessels from Notch3−/− mice using KCl-induced depolarization. Depolarization produced identical contractions and Ca2+ responses in both strains  underlying the abnormal renal vascular response in mice lacking Notch3. To this end, we examined ANG II-induced contractility and Ca−/− mice showed no differences in terms of body or kidney weight compared to wild-type littermates. Despite a thinner vessel wall of uneven thickness, as reported before in Notch3−/− [2+ concentrations. When challenged with ANG II, however, it was apparent that contractions and Ca2+ responses to ANG II were much weaker in renal vessels of Notch3−/− mice , suggesting dysfunctional L-type channels. However, the expression of L-type channels did not differ between the vessels of the Notch3−/− and WT . In this case however, we observed a major decrease in the expression of the α1H subunit of the T-type channel (Cacna1h) in Notch3−/− renal vessels , i2+ entry from L-type channels also compromised? One possibility is that the decreased response of Notch3−/− afferent arterioles to ANG II is not due to a dysfunction of Ca2+ entry from its channels but is caused by an abnormality somewhere in the signalling from the ANG II receptor to the Ca2+ channels. This hypothesis is not supported by our previous study testing renal vascular responses in Notch3−/− mice [−/− kidneys to other vasoconstrictors (such as norepinephrine or thromboxane) and to mechanical stress as well . This deficient response is extended to vasodilators, such as prostacyclin or bradykinin as well, showing that the deficient vasoconstriction is not due to a compensatory activation of a vasodilatory signalling.The question, however, remains: Why was Ca−/− mice . In the 2+ entry requires the appropriate functioning of both. The T-type channels are involved in excitability [2+ depend on each other to initiate contraction. In rabbit AAs, L-type Ca2+ entry is reliant on functional T-type Ca2+ channels [2+ channel has no effect on mice lacking the L-type channel [+2 entry in afferent arterioles depend mainly on L-type channels with little contribution from T-type channels [2+ entry and mobilization in afferent arterioles make up a very complex mechanism involving interactions between L- or T- type, transient receptor potential, and K+ and/or Cl− channels as well [2+ responses and contractility.Another hypothesis is that L- and T-type channels are interacting or cross-communicating, and a proper Catability ,19,20,21channels . Convers channel . In line channel . Using Kchannels ,24,25. I as well . It is p2+ entry can be compromised in Notch3−/− renal vessels because the lack of the Notch3 gene is accompanied by defects in vascular smooth muscle cell maturation [−/− afferent arterioles displayed an uneven thickness due to an incomplete maturation of vascular smooth muscle cells [Alternatively, Caturation ,6. Suppoturation . Insteadturation . Based oturation . In agre−/− mice depend mainly on intracellular Ca2+i stores in order to respond to cardiovascular stress and vasoconstrictor stimuli. During control conditions, this appears to be adequate for maintaining vasoregulatory functions. During pathological conditions, however, such as those induced by prolonged exposure to exogenous ANG II [+ influx into the cells, mice lacking Notch3 fail to regulate renal haemodynamics, causing severe renal dysfunction and injury.Whatever the nature of this interaction, our data show that the afferent arterioles of Notch3s ANG II , requiri−/− mice, we found a blunted and likely insufficient increase in RVR compared to wild-type ones after injecting 0.5 ng ANG II into the bloodstream. In contrast, blood pressure, indicating total peripheral resistance, increased equally in the two strains [−/− mouse must have a normal contractile response to ANG II. Consistent with this notion, we found a normal response to ANG II in muscle resistance vessels from Notch3−/−, and we have previously obtained similar data from skin resistance vessels [In our previous study of renal haemodynamics in Notch3 strains . Consequ vessels .−/−, but not wild-type, mice, while the blood pressure responses were similar [1G, was shown to be downregulated several-fold in Notch3−/− cerebral vascular smooth muscle cells . Taken Cacna1h (encoding the T-type Ca2+ channel), several genes affecting vessel contractility were also downregulated in the absence of Notch3. Examples from this list include connexin 43 (gap-junctions), transient receptor potential C6 , integrtraction ), and rhtraction ). Thus, Notch3 gene (Notch3−/−) on a C57Bl6/J background, as described elsewhere [−/− mice, weighing 18–25 g, were bred at the animal facility at the Institute of Biomedicine at the University of Bergen, fed ordinary mouse pellets, and had free access to tap water.Experiments were performed on mice lacking expression of the −/−mice, and we compared them with afferent arterioles from their WT littermates. The analytical description for the isolation of preglomerular vessels, measurements of vessel diameters, and fura-2 intracellular Ca2+ (Ca2+i) responses to ANG II and KCl and chemicals are provided in the Because the objective of the study was to understand the deficient myogenic response in the kidney that we have observed previously , and sinAfferent preglomerular arterioles and precapillary muscle arterioles were isolated using an agarose-infusion/enzyme-treatment technique adapted from Loutzenhiser and Loutzenhiser . In shor−7 M or dose–response curves) or depolarised with KCl (50 mM) to study the Notch3 effects on contractility and Ca2+ transients, (see 2+ ratio using fura-2 340/380 nm emission was recorded at 37° C. AAs from both sexes were used after pilot studies showed no gender differences on contractility. Nifedipine (10−7 M), mibefradil (10−6 M), and thapsigargin (10−7 M) were applied to the vessel bath 15 min before the recordings.Isolated vessels were stimulated with ANG II  and frozen until use. A quantity of 15–20 mg of tissue was used to prepare the total RNA using rNeasy . RNA was quantified using a NanoDrop ND-1000 spectrophotometer . Reverse transcription was performed using the RT core-kit  with 200 ng RNA. Real-time PCR was performed on an ABI-prism 7900-HT sequence-detection system at the Norwegian Microarray Consortium in Bergen using custom-made microfluidic cards: Taqman Low-Density Array . A total of 47 genes known to be involved in the contractile mechanism, Notch- or calcium-signalling, and 18s were selected for RT-PCR . Gene ex2+i responses with EGTA, nifedipine, and mibefradil vs. normal medium were analysed using ANOVA followed by the student–Newman–Keuls post hoc test in SigmaStat 3.1. Diameter and Ca2+i responses in Notch3−/− vs. wild-type and thapsigargin Ca2+i responses vs. normal medium were analysed using student’s t-test in SigmaStat 3.1. Gene expression was analysed using student’s t-test and Bonferroni’s correction. The genes were ordered after significance (p < 0.05 was considered statistically significant. All values are expressed as means ± SEM.Vessel diameter and Caificance , and the2+ entry as a major vasoactive signalling pathway dependent on Notch3 for proper function, which results in a serious defect of the calcium handling in the renal resistance vessels of mice lacking Notch3−/− expression. In AAs lacking Notch3, ANG II stimulation initiated Ca2+ mobilization, but that was not followed by Ca2+ entry. These data explain our previous observation that Notch3−/− kidneys are unable to cope with acute or prolonged ANG II infusion, and they also provide clues for the vascular function of Notch3 in other vascular beds, most notably the brain.The present study is, to our knowledge, the first to identify deficient Ca


To assess the association between clinical history and AML outcomes in the context of allogeneic hematopoietic cell transplantation (HCT), we retrospectively analyzed 739 patients with de novo AML, 125 with antecedent hematologic disorder (AHD)/AML, and 115 with therapy-related AML who received first allografts while in first or second remission. Relative to patients with de novo AML, relapse rates were similar for patients with AHD and therapy-related AML after multivariable adjustment, as were relapse-free survival and overall survival. Non-relapse mortality was, however, higher for AHD AML. These data suggest that the clinical history by itself contains limited prognostic value for adults with AML undergoing allografting, supporting the most recent approach to use this information as a diagnostic qualifier rather than a disease entity.p < 0.001), gender (p < 0.001), disease risk (p = 0.005), HCT-CI score (p < 0.001), blood count recovery (p = 0.003), first vs. second remission (p < 0.001), remission duration (p < 0.001), measurable residual disease , and conditioning intensity (p < 0.001). Relative to patients with de novo AML, relapse rates were similar for patients with AHD  and therapy-related AML  after multivariable adjustment, as were relapse-free survival  and overall survival . Non-relapse mortality was higher for AHD AML . (4) Conclusions: These data suggest that the clinical history by itself contains limited prognostic value for adults with AML undergoing allografting, supporting the most recent approach to use this information as a diagnostic qualifier rather than a disease entity.(1) Background: Secondary acute myeloid leukemia (AML), i.e., AML arising from prior therapy (therapy-related) and/or an antecedent hematologic disorder (AHD) is generally associated with worse outcomes compared to de novo AML. However, recognizing the prognostic importance of genetic characteristics rather than clinical history, secondary AML is now considered a diagnostic qualifier rather than a separate disease entity. (2) Methods: To assess the association between clinical history and AML outcomes in the context of allogeneic hematopoietic cell transplantation (HCT), we retrospectively analyzed 759 patients with de novo AML, 115 with AHD AML, and 105 with therapy-related AML who received first allografts while in first or second remission. (3) Results: At the time of HCT, these three cohorts differed significantly regarding many patient and disease-specific characteristics, including age ( Secondary acute myeloid leukemia (AML), i.e., AML arising after prior exposure to cytotoxic therapy (therapy-related AML) and/or antecedent hematological disorder (AHD), has generally been associated with a worse prognosis compared to de novo AML ,3,4,5,6.Given these mixed data, and the notion that post-HCT outcomes within the subset of patients with secondary AML might differ depending on the details of clinical history  ,22, we eWe included all adults with AML (2016 WHO criteria)  who procSecondary AML was defined as disease following an AHD  or treatment with systemic chemotherapy and/or radiotherapy for a different disorder. All previous cytotoxic regimens were considered for the main analysis, including methotrexate, mercaptopurine, and cyclophosphamide for auto-immune disease. Immunosuppressive treatment using nonchemotherapeutic agents was not considered cytotoxic. Patients developing MDS, MPN, or MDS/MPN between the chemotherapy or radiation treatment for their primary disease and the diagnosis of AML were classified as therapy-related AML unless stated otherwise. In sensitivity analyses, we explored the impact of new classification criteria for therapy-related AML  and AHD (requirement for AHD diagnosis ≥3 months before AML diagnosis) ,11. The The HCT-specific comorbidity index (HCT-CI) and the Treatment-Related Mortality (TRM) score were calculated as previously described ,27. We cp < 0.05. Statistical analyses were performed with R .Categorical variables were reported as numbers (with proportions) and compared using the Chi² test or Fisher’s exact test, as appropriate. Continuous variables were reported as medians (with interquartile range [IQR]) and compared using the Mann and Whitney test. We estimated unadjusted probabilities of relapse-free survival  and overall survival  with the Kaplan–Meier method and compared them with the Log-Rank test. Associations with RFS and OS were assessed using Cox regression models. Probabilities of relapse (with NRM as a competing event) and NRM (death without relapse with relapse as a competing event) were reported using cumulative incidence estimates. Associations with relapse and NRM were assessed using cause-specific regression models . All tesn = 39, 34%), breast cancer , or an auto-immune condition , and 125 patients had an AHD . Due to the high number of therapy-related AML after treatment for breast cancers, there were more females in the therapy-related group  and, as prior cancer is used to calculate the HCT-CI, patients in this group were more likely considered as high-risk according to this score . On the other hand, patients with AHD were less likely to be classified as favorable risk  and they were more likely to have incomplete blood count recovery  and pre-HCT MRD  whereas the time from the last remission to HCT was shorter . Most patients with secondary AML, either therapy-related or AHD, were transplanted in first remission  and they were more likely to receive non-MAC regimens .The characteristics of the 979 patients included in the analysis are summarized in n = 3), and, by analogy, mercaptopurine (n = 1) or cyclophosphamide (n = 1), and 34 patients with AHD diagnosed ≤3 months before AML diagnosis  as de novo AML. As summarized in As the definitions of sAML were recently changed ,11, we ap = 0.099; p = 0.058). This translated into statistically significant lower RFS  and OS  in patients with AHD. Using the updated classification schemes for secondary AML moderately modified these observations, with relapse being statistically significantly higher in patients with AHD , there were 308 relapses, 460 deaths, and 191 NRM events. Relapse was non significantly higher in patients with AHD , relapse , RFS , and OS  whereas therapy-related AML was not (p = 0.001), relapse , RFS , and OS . Similar C-statistic values (indicating similar predictive accuracy) were observed when considering AHD without any time requirement before AML diagnosis or with a three-month time requirement .To study the relationship between AML after AHD and post-HCT outcomes in more detail, we evaluated univariable and multivariable regression models for the endpoints of NRM, relapse, RFS, and OS. In univariable analysis, AML after AHD was associated with NRM  but not relapse , RFS , or OS . In these patients, adverse cytogenetic risk  and positive pre-HCT MRD by MFC  were associated with relapse and positive pre-HCT MRD was associated with RFS  and OS , as was OS , relapse was non-significantly higher  and RFS was non-significantly lower  in younger patients. In contrast to the whole study cohort, non-MAC was not associated with NRM, RFS, or OS.After multivariable adjustment, AML after AHD remained significantly associated with NRM  but not relapse (p = 0.2), RFS (p = 0.8), or OS (p = 0.085). In patients who received MAC, AHD was significantly associated with NRM  but not for relapse  and OS . After multivariable adjustment, AHD remained statistically associated with NRM  but not with relapse , RFS , or OS  in patients receiving MAC. On the other hand, in patients who received non-MAC, AHD was not significantly associated with NRM  but was associated with relapse  whereas OS was similar  in the subset of patients with AHD AML. Similar results were observed when distinguishing RIC from non-myeloablative conditioning (p = 0.018) and showed a borderline association with RFS , but not NRM  or OS .To further explore the relationship between disease status and post-HCT outcomes, we performed subset analyses in which we studied patients who underwent transplants after MAC or non-MAC conditioning separately. These analyses were further motivated by a significant interaction between conditioning intensity (MAC vs. non-MAC) and AHD for NRM , some of the parameters for when exactly an AML should be considered secondary have changed. Specifically, it is now proposed that patients who developed AML after receiving cytotoxic therapy such as methotrexate for auto-immune conditions, or patients for whom the time interval between AHD and AML diagnosis was less than three months, should no longer be considered as secondary AML. Nevertheless, for our main analysis, we chose to consider these patients as secondary AML for comparability with prior studies ,22,40,41Without adjustments, we observed that AML after AHD but not therapy-related AML was associated with post-HCT outcomes in our cohort. This finding is consistent with a recent report on a cohort of patients developing myeloid neoplasms after prior therapy for solid tumors . The finAs in previous reports ,16,17, wOf note, conditioning intensity was not equally associated with post-HCT outcomes in patients with AHD. As in one previous report , but notThe retrospective nature of our study analyzing post-HCT outcomes of patients for whom conditioning intensity was nonrandomly assigned limits our ability to draw definitive conclusions regarding the management of patients with AHD AML. Our general management has been to use MAC whenever it was felt that it could be safely administered based on patient age and comorbidities. Another limitation is the fact that mutational profiles were available only for a small subset of patients.The fact that AML developed post cytotoxic therapy or after AHD provides little prognostic value (except for an increased risk of NRM in patients with AHD) supports the most recent proposal to use this information largely as a diagnostic qualifier rather than a prognostic variable.