Coxiella burnetii that have cases reported in humans and animals almost everywhere. The aim of this study was to describe the seasonality of Coxiella burnetii in the wild rabbit (Oryctolagus cuniculus) and the tick Hyalomma lusitanicum in a meso-Mediterranean ecosystem. (2) Methods: two populations of wild rabbits that differ in whether or not they share habitat with ungulates, mainly red deer (Cervus elaphus) were sampled for a year to collect ticks, blood and vaginal or anal swabs. Presence of C. burnetii DNA in swabs and the tick H. lusitanicum was determined by PCR and serum antibodies by ELISA. (3) Results: C. burnetii DNA was detected in 47.2% of 583 rabbits, in 65.5% of sera, and in more than half of the H. lusitanicum. There were small variations according to sex and age of the rabbits but significant according to the habitat (4) Conclusions: The results indicate that C. burnetii circulates freely between wild rabbits and H. lusitanicum and the sylvatic cycle in meso-Mediterranean environments relies in the presence of wild rabbits and H. lusitanicum above all if sharing habitat with red deer.(1) Background: Q fever is a worldwide zoonosis caused by  Coxiella burnetii . Except in New Zealand, the infection has been widespread throughout the world, where we find endemic or epidemic areas [Q fever is a worldwide zoonosis first described in Australia  caused bic areas . It is aCoxiella burnetii is an obligate intracellular Gram-negative bacterium with two infectious variants, a small cell variant as spore-like form with environmental resistance and a large cell variant that has greater metabolic activity [C. burnetii into the environment occurs mainly during parturition by birth products [C. burnetii have been reported: ingestion (mainly drinking raw milk), person-to-person transmission, via trans placental, intradermal inoculation, blood transfusion or sexual transmission, although the aerosol route is the primary mode of infection [activity ; it can activity . Besidesactivity . The infactivity ,8. Sheddproducts ,10, but nfection . Little nfection , in spitnfection ,13,14,15Coxiella [C. burnetii in Dermacentor marginatus Sulzer, Rhiphicephalus sanguineus Latreille, Rhiphicephalus pusillus Gil Collado and Hyalomma lusitanicum Koch [C. burnetii in nature, as a bridge between wild and domestic animal hosts [Dermacentor andersoni Stiles, Haemaphysalis bispinosa Neumann, Haemaphysalis humerosa Warburton & Nuttall, Hyalomma aegyptium L, Hyalomma asiaticum Schulze & Schlottke, Ixodes holocyclus Neumann and R. sanguineus Latreille) [In addition to all these sources of transmission, ticks are able to spread the pathogen and to be a source of infection . It has Coxiella , and thaCoxiella ,17. The cum Koch . In consal hosts ,6,19. ItC. burnetii and wild rabbits in wild meso-Mediterranean environments [Hyalomma lusitanicum ticks from nymph to adults has been recently reported [C. burnetii remains unknown. Furthermore, in meso-Mediterranean ecosystems the contact between C. burnetii, H. lusitanicum and susceptible wild animals could occur due to coincidence of the main lambing seasons of wild rabbits and that of red deer. The highest infestation of both host species by immature and/or adults stages of H. lusitanicum and the maximum activity of host-seeking adults of this tick specie [Coxiella burnetii in the wild rabbit and the tick Hyalomma lusitanicum in two populations of wild rabbits in a meso-Mediterranean ecosystem that differ in whether or not they share habitat with ungulates, mainly red deer.Some studies suggested the close relationship between ronments ,13,20,21k specie ,23,24. T(1) = 3.69; p ≤ 0.055). Regarding the age, most captures were adults (63.5%) and sub adults (24.9%) and just a few young rabbits (11.3%) were sampled, being the relative proportion of captures from each group of age within sex similar in the overall population (χ2(2) = 2.46; p ≤ 0.292). Similar sex distribution was observed in both populations although the percentage of adults was higher in the rabbits area (R-A) (74.1%) than in rabbits and ungulates area (RU-A) population (55.2%).Taking into account the overall rabbit population, females were more frequently captured than males (57.3% vs. 42.7%), proportions being marginally non-significant , the relative proportion of each species is showed in Rhipicephalus pusillus was present practically all year round  ((3) = 1435.36; p ≤ 0.0001) were found for each of the two major tick species  ≤ 93 for R. pusillus and H. lusitanicum), indicating a strong association with area.Four tick species were collected: ar round , increasin RU-A) . When coHyalomma lusitanicum which is the species of interest in this study was collected in rabbits from June to November in the RU-A (n = 1270) whilst in R-A only 15 specimens were found (13 in August and 1 in June and September).On external inspection of the animals, we found that 96.8% of the animals were healthy and only 3.2% showed some characteristic sign of myxomatosis  in February, July, August and December. Signs of other diseases were not observed.Coxiella burnetii DNA was detected in 47.2% of 583 swabs analysed, with slightly higher prevalence in females than in males (49.1% and 44.7%) and it was similar at all ages (42.5% to 45.7%), but young females excreted more bacteria than sub-adult and adult females  being differences not significant, χ2(2) = 2.23; p ≤ 0.328)  = 8.46; p ≤ 0.006) being higher in the RU-A than in R-A indicating a strong association with the habitat.≤ 0.328) . When C.C. burnetii antibodies were detected in 65.5% of sera from wild rabbits tested (n = 602) and it was similar in females and males (65.2% and 65.7%) but varied with age. Thus, seroprevalences in adults and sub-adults were significantly higher than in younger individuals  = 112.51; p ≤ 0.0001), irrespective of sex  = 12.71; p ≤ 0.0004) in rabbits collected in the RU-A than in those collected in the R-A.Anti-e of sex . SeropreC. burnetii DNA in more than half of the H. lusitanicum ticks but there were small variations according to sex and age of the rabbits where ticks were collected  than from females (51.9%) and from adults (55.3%) than young and sub adults rabbits (50.0% each). The number of H. lusitanicum ticks processed in each area of capture was quite different (7 in R-A and 171 in RU-A) so it is difficult to make a direct comparison. Nevertheless C. burnetii detection in both situations was over 40% of the sampled H. lusitanicum (42.9% in R-A and 55.0% in RU-A).It appears that, overall, it was possible to detect the presence of ollected . So, DNAThere were apparently no major differences between males and females in the seasonal pattern of antibody or DNA excretion . The dynC. burnetii DNA excretion and subsequent antibody production . This previous situation is in agreement with  who repoRhipicephalus pusillus and Hyalomma lusitanicum are in agreement with the studies of other authors [Haemaphysalis hispanica and Ixodes ventalloi being also typical of wild rabbits do not usually appear with such intensity. González et al. [H. lusitanicum is the predominant species in wild rabbits in this ecosystem above all in spring and summer and R. pusillus is present all the year in lower amounts. In present study R. pusillus—all stages—was the predominant specie and was found along all the year whilst H. lusitanicum—immature stages—was mainly found in summer. The differences were due to the effect of trapping location which strongly influenced tick populations by changing host abundance [H. lusitanicum adults as revised by Valcárcel et al. [H. lusitanicum.Due to the methodological design we could not establish a direct comparison with other studies on the number of ticks per rabbit because we only sampled those ticks attached to the ears of the rabbits thus we underestimated the total tick index. Nevertheless, the abundance of  authors ,23,24,36z et al.  indicatebundance . So, ticl et al. . In contThe inhalation of birth products is the main route of infection of Q fever; in consequence, it is presumed that ticks play a minor role in Q fever transmission in the domestic cycle but they may play a role as the natural reservoir in the sylvatic cycle  where trC. burnetii transstadially and it was thought that most of them could do it transovarially [C. burnetii in several tick species, the consideration of tick abundance as a risk factor for Q fever is controversial. Some studies found no significant correlations due to the low global prevalence of C. burnetii in ticks (<5%) [C. burnetii DNA was detected in more than 50% of H. lusitanicum, a similar percentage to that reported by González et al. [It was reported that most soft and hard ticks are able to transmit varially . Howeverks (<5%)  and otheks (<5%) ,42,43. Tz et al. , suggestz et al. ,44 and mz et al. .Rhipicephalus, we focused on H. lusitanicum because of its known role in the transmission of C. burnetii and the high presence of this bacterium in this tick species [C. burnetii from H. lusitanicum collected from both infected and uninfected wild rabbits confirming previous results in this species [Hyalomma lusitanicum to transmit C. burnetii. However, there are field evidences in both studies of González et al. about the adaptation of the bacteria to the pass from one stage to the next of naturally infected H. lusitanicum and that at least transstadial transmission is happening in meso-Mediterranean environments. In addition, DNA can also be detected in 33% of the batches of offspring from engorged females, suggesting transovarial transmission; unfortunately, in both studies the viability of the bacteria was not tested.Some ticks can carry a pathogen without being able to transmit it . Althoug species ,22. In f species ,22 as al species . The absC. burnetii in wild rabbit has also been found with higher prevalence where host density is greater as it happens in our study [C. burnetii DNA, which was slightly higher in adult females than in adult males due to the high concentration of bacteria in birth products despite the fact that it can also be excreted in faeces, urine, and milk of infected animals [C. burnetii prevalence in wild rabbits.The presence of ur study ,21,39. S animals . Our resOther studies suggested the essential role of wild rabbits in the maintenance of the bacteria in wildlife environments ,22. We aC. burnetii infection is primarily due to the Th1 response and IFN production while antibodies are dispensable [C. burnetii antibodies, however the regular decline of antibodies after DNA depletion may indicate that they are short-lived.DNA detection was similar at all ages; however, seral prevalence increased with age, as younger rabbits had less opportunity to become infected than sub-adults and adults. The DNA presence in vaginal swaps of young females was higher than that of sub-adult and adult females, but their seroprevalence was low, probably because at the time of sampling they were freshly infected and did not have time to develop antibodies. Therefore, it is reasonable to conclude that the first infection occurs early and, consequently, as the immune response develops, bacterial shedding decreases. Control of the first pensable . ConsequC. burnetii infection, reflecting a failure of the Th1 response, or it may be due to weak reinfections. We cannot rule out persistent infections even though we have not found any evidence of altered health status and it is supposed to occur in both areas. However, it is possible the reinfection through bacteria excreted by red deer or transmission by H. lusitanicum ticks.It would be expected that DNA excretion and antibody production would be similar in both areas throughout the year, but this only happened during the first nine months of the study. In the beginning there should be a finishing active infection in which immune responses cause DNA excretion to decrease until May, rabbits should not be re-infected in these first months because there was a posterior antibody reduction. Later, a new infection coinciding with the main spring breeding season occurred with an increase in DNA and subsequent antibodies. In summer and autumn, rabbits spend more time out of the burrow and there is little or no reproduction, so the re-infection chance among rabbits is low in R-A, which explains the reduced DNA and antibodies in September that remained in low levels since then. However, from September to January antibody production remained at high levels in rabbits from RU-A despite a similar decrease in DNA prevalence. In this point it is needed to note that the number of captures at the end of the study was very low and results should be carefully considered. The differences could be due to the existence of an antigenic stimuli in RU-A sufficient to stimulate the immune response by producing antibodies. This antigenic stimulus could have two origins, the progression of persistent C. burnetii can survive long periods in soil due as resistant spore-like forms [Wild rabbits and red deer share habitat in the RU-A so it is possible that wild rabbits are infected by a spore-like form bacteria excreted during red deer calving in spring or in September-October during the mating call of red deer because ke forms  and theyke forms . This poCoxiella burnetii is multiplicity primarily inside gut epithelial cells of other tick species remaining viable inside the tick’s body between 200, in Hyalomma aegyptium [Ornithodoros turicata [3 to 108 viable bacteria for up to 635 days [C. burnetii in H. lusitanicum but transmission through saliva has been recently reported in artificial feeding [H. lusitanicum faeces but do not rule out the possibility. If confirmed, it would be a serious source of infection of rabbits as ear damage from fighting rabbits is very common . The site covers an area of 13,000 ha at a mean altitude of 669 m above sea level; annual average rainfall is 650 mm and temperatures range from −4 to 43 °C. This reserve harbours a wild fauna and flora representative of the meso-Mediterranean bioclimatic environment ).For the sampling we selected two sampling areas which were separated 20 years ago by a hunting fence. Both areas had similar flora and fauna, and there are available feeders and drinkers near to the burrows, which are checked daily by the farm staff for proper functioning. The first one is an area of 540 ha where wild rabbits and ungulates share habitat (RU-A: rabbits and ungulates area) whilst in the other area, 340 ha, there is no presence of ungulates (R-A: no ungulates area or rabbits area)  except for certain areas, “visors” which remained open for the rabbits to familiarise themselves with and pass through on a daily basis. The netting was not laid in a linear arrangement, but formed “shelter pockets” every 20–30 m. In the early morning of the day of the capture, when the rabbits are in the sowing area, the “visors” are lowered and the netting is firmly fixed to the ground. When the rabbits try to return to their burrows, they find the path closed and take refuge in the shelter pockets and are all collected by hand by the farm’s qualified personnel, who place them in authorised transport cages.After capture wild rabbits were quickly transported to the laboratory located in the same reserve. Immediately after identification and data and sample collection, animals were transported again to the same sampling point where they were captured. Captures, management and sample collection was performed with the authorization of the Animal Ethic Committee of the Regional Government (PROEX 14-2018 “Monitorización del estado sanitario del conejo de campo”) to preserve animal welfare in accordance with the general conditions for the housing and care of animals set out in Article 6 of Royal Decree 53/2013 of 1 February, which establishes the basic rules applicable to the protection of animals used for experimental and other scientific purposes, including teaching.®), sexed and identified by placing metal ear tags (Hauptner®). The age of the animals was established based on live weight and assigned to one of the three groups of age as follows: young <800 g, subadult = 800–1100 g and adult = > 1100 g) [Each rabbit was handled individually in the laboratory. Firstly, they were weighed with a portable scale (Cobos precision model CR-20 1100 g) . Subsequ® tubes with 70% alcohol until identification by specific keys [To maintain rabbit welfare by reducing handling time, only those ticks attached to ears were picked up with fine tweezers and were individually stored in labelled Eppendorffic keys ,55,56,57A 0.5 mL sample of blood was collected from marginal ear vein and preserved in tubes without anticoagulant. Vaginal or anal samples from females and males were respectively removed with sterile swabs. Ticks and swaps were stored at −20 °C prior DNA extraction and blood samples were stored at 4 °C prior ELISA testing.H. lusitanicum were individually processed and placed into a well of a 96-well collection plate . Each vial included 3 stainless steel beads (3 mm)  and 200 μL miliQ water. Plates were sealed and shaken in a TissueLyser machine  at 30 Hz (2 cycles of 6 min) for disruption and homogenization. After this, every sample (150 µL) were mixed with a solution containing 20 µL of proteinase K  and 30 µL of ATL buffer ; then the mix was kept at 56 °C overnight stirring at 900 rpm.Ticks were 70%-ethanol-disinfected and then adult ticks were cut with scissors to assist DNA extraction. Specimens of Vaginal and anal swabs were also individually put into a well of the collection plate with a solution containing 20 µL of proteinase K and 400 µL of ATL buffer, then were kept at 56 °C overnight stirring at 900 rpm.DNA was extracted using BS96 DNA Blood Kit  following the protocols for tissues (ticks) or swabs respectively in a BioSprint 96 workstation . More detailed descriptions can be seen in González et al. ,21.C. burnetii positive control kindly supplied by Institute of Health Carlos III (strain Nine Mile phase II). We evaluated a sample to be positive until a threshold cycle (CT) value 40. All controls were negative and C. burnetii positive controls presented their corresponding positive results and did not show any cross-reaction.Cross-contamination during DNA extraction was excluded by running in parallel negative controls ; one negative control every 20 samples was included per plate. We used a Coxiella burnetii using the commercial kit LSIVetTMRuminant Q Fever-Serum/Milk  according to the manufacturer’s instructions. Basically, “samples and controls are spread on the plate coated with C. burnetii antigen, where specific anti-C burnetii antibodies bind to the antigen. After washing, a peroxidase-labelled G-protein conjugate (HRP) is added which binds to the antibodies previously attached to the plate. The unbound conjugate is removed by washing, followed by the addition of a chromogenic substrate. Oxidation of the substrate by the HRP conjugate produces a blue colour. After stopping the reaction, the colour turns yellow and the results are then read using an ELISA plate reader. The intensity of the yellow colour present in positive samples is proportional to the amount of specific antibody in the sample.Blood samples were centrifuged to obtain serum for subsequent serological testing by performing an indirect ELISA assay for The S/P (Sample/Positive) ratio is calculated for each sample by calculating the mean OD  of the positive control (ODm PC), and that of the negative control (ODm NC)”.n = 284) needed to know the starting situation and to get the chance of recapturing them along the year. In the following twelve months (February to January) a total of 727 wild rabbits were captured then a total of 1011 captures were performed along the thirteen months of the study. A total of 149 wild rabbits were recaptured two or more times . As the aim of the study was to obtain a global overview and a seasonal description of the prevalence of C. burnetii in rabbits and its percentage in H. lusitanicum ticks, recaptured rabbits were only considered once (the first capture) in the data processing.In the first month (January) we performed a strong effort to assure an enough number of rabbits  but no large differences between wild rabbit sex with apparently more DNA excretion by rabbits during breeding season and that bacteria harboured by H. lusitanicum is enough to maintain and transmit the bacteria. Further studies are needed to determine the viability of the bacterium in the different stages and faeces of H. lusitanicum, as well as the role of other tick species and micromammals in its maintenance in wildlife and possible connections with the domestic cycle.This study confirms that


The COVID-19 pandemic disrupted almost all sectors of academic training and research, but the impact on human immunodeficiency virus (HIV) research mentoring has yet to be documented. We present the perspectives of diverse, experienced mentors in a range of HIV research disciplines on the impact of COVID-19 on mentoring the next generation of HIV researchers.In November to December, 2020, we used an online data collection platform to cross-sectionally query previously-trained HIV mentors on the challenges related to mentoring during the pandemic, surprising/positive aspects of mentoring in that context, and recommendations for other mentors. Data were coded and analyzed following a thematic analysis approach.Respondents (180 of 225 mentors invited [80% response]) reported challenges related to relationship building/maintenance, disruptions in mentees’ training and research progress, and mentee and mentor distress, with particular concerns regarding mentees who are parents or from underrepresented minority backgrounds. Positive/surprising aspects included logistical ease of remote mentoring, the relationship-edifying result of the shared pandemic experience, mentee resilience and gratitude, and increased enjoyment of mentoring. Recommendations included practical tips, encouragement for patience and persistence, and prioritizing supporting mentees’ and one's own mental well-being.Findings revealed gaps in HIV mentors’ competencies, including the effective use of remote mentoring tools, how to work with mentees in times of distress, and the prioritization of mentor well-being. Mentors are in a unique position to identify and potentially address factors that may lead to mentees leaving their fields, especially parents and those from underrepresented backgrounds. We discuss implications beyond the COVID-19 pandemic. The mounting barriers to successful retention of ESIs in academic medicine, including high student loan burdens, decreased availability of tenure-track positions, and declining funding for research, have intensified this need. In early 2020, the SARS-CoV-2 pandemic introduced an unprecedented and prolonged disruption to almost every sector of the globe. In academia, the pandemic  has the potential to derail the career trajectories and mentoring of ESIs across several disciplines, particularly those within the diverse pipeline of investigators addressing critical issues in the fight against human immunodeficiency virus (HIV).The importance of effective mentoring to early-stage investigators (ESIs) in academic research has been well documented.,6 Moreover, the priority of the National Institutes of Health (NIH) and other funding agencies to diversify the pool of investigators from underrepresented minority backgrounds is particularly at risk, as the communities from which these investigators come are often disproportionately impacted by COVID-19 related morbidity and mortality. In addition, HIV researchers who formerly focused on HIV vaccines, novel therapeutics, cure strategies, social determinants of health, and behavioral prevention efforts have been pulled into important COVID-19 clinical and research endeavors. While the resulting clinical and research workforce of HIV experts pivoting their attention to COVID-19 is impressive, this redirection may result in a diminishing bandwidth to provide mentoring to ESIs focused on the still paramount challenges facing HIV prevention and treatment research.Among COVID-19 threats are setbacks and derailments to progress made toward ending the global HIV epidemic.–11 By design, the Mentoring the Mentors Program concentrates on US-based HIV investigators, as the program includes a focus on NIH and other funding opportunities available primarily to US investigators. The goal of this study was to examine challenges expressed by these researchers of mentoring during the COVID-19 pandemic; positive or surprising aspects of mentoring presented by the pandemic; and recommendations made to other mentors for effective mentoring during this and future crises. To accomplish our goal, we leveraged a planned long-term evaluation of the Mentoring the Mentors Program among prior participants who had completed the training in which we collected qualitative data on experiences with mentoring during the pandemic.The purpose of this paper is to explore the perspectives of experienced HIV research mentors across the United States regarding the impact of the COVID-19 pandemic on their mentoring of ESIs who are building research careers in HIV. For almost 10 years, the University of California, San Francisco Center for AIDS Research Mentoring the Mentors Program has conducted 2-day intensive mentor training workshops to systematically build mentoring competency among domestic HIV researchers with an emphasis on mentoring diverse mentees in HIV science. To date, our program has trained over 226 mentors from across the country, and the evaluation results have been described previously.22.1The current investigation used a qualitative phenomenological research approach.2.2–11 we assessed respondents’ perceptions of the impact of COVID-19 on their mentoring practices, along with suggestions for addressing challenges associated with mentoring during the pandemic.As part of the long-term, cross-sectional evaluation of mentors who participated in 1 of 7 two-day intensive HIV-related mentor training workshops between 2012 and 2020,In November of 2020, a total of 226 unique participants who had completed at least one of our annual workshops were invited by email to complete a web-based survey via Qualtrics  that included descriptive program evaluation questions, background and demographic questions, and 3 open-ended questions related to mentoring during COVID-19. Following initial invitations, we sent reminders weekly to those who had not responded and those who had started, but not completed, the survey. As part of the invitation, mentors were told that those who completed the survey would receive a US$10 gift code for an online retailer and entered into a raffle for an additional 5 US$100 gift codes, which we randomly selected and distributed following closeout of the survey.The primary objective of the survey was to solicit data on the long-term impact of the program. However, we took the opportunity to include additional content related to mentoring during the SARS-CoV-2 pandemic by eliciting answers to the following open-ended questions:1.What are the challenges, worries, or concerns you face while mentoring early-career investigators/trainees during COVID-19?2.Are there any positive or surprising aspects to mentoring during COVID-19? Please explain.3.What suggestions would you have for other mentors who are struggling with mentoring during COVID-19?These questions were generated through discussions among the authorship team based on their experiences and concerns while mentoring during the early phases of the COVID-19 pandemic.2.3The current investigation was conducted as part of a program evaluation, and thus ethics committee approval was not required.2.4 First, the authors independently read the first 20 responses to each of the 3 questions and separately generated themes and an initial coding scheme. They then discussed and refined the preliminary codes until a set of initial codes were generated. Then they separately coded a set of 40 survey responses, which were discussed and reconciled, until revisions to the codes were finalized. All responses were then divided up for final coding. The survey also assessed demographic and background data for each respondent, which are reported as provided by respondents in order to characterize the sample. Missing data were minimal across qualitative and quantitative domains.Survey respondents provided open-text responses to each of these questions. These responses remained blinded to respondent identity, demographics, and other characteristics and were entered into qualitative coding and analysis software  and then coded by the first and senior authors using a thematic analytic approach.33.1Of 226 mentors invited, 180 (80%) completed the survey. Of those who did not complete, 2 (<1%) responded that they were now retired or were no longer in a mentoring role; 32 (14%) never responded to multiple invitations; 3 (1%) opened but did not complete the survey; 9 (4%) opted out of completing the survey (an option offered through the survey platform that indicates they received the invitation but chose not to participate); and 1 (<1%) had a non-functioning email address and we were unable to locate more current contact information.3.2Respondents’ primary work locations demonstrate geographic diversity throughout the US see Fig. . Table 1Approximately two thirds reported currently being in a leadership role related to mentoring or training, such as directing training programs, leading mentoring or developmental cores for research centers, or having positions in academic affairs. The mean number of current primary and secondary mentees for this group of mentors were 4.6 and 5.0, respectively. Mentors reported working with mentees from the following groups: 98% with mentees who are female; 96% with mentees from racial or ethnic minority backgrounds; 79% with mentees from socioeconomically disadvantaged backgrounds; 46% with mentees who identify as gender minorities; 83% with mentees who identify as sexual minorities; 27% with mentees with disabilities; and 74% with mentees who are the first in their immediate families to attend college.3.3The qualitative data analysis was structured around 3 domains of the survey: challenges, positive/surprising aspects, and suggestions for other mentors.3.3.1Respondents reported a range of challenges, concerns, and worries about mentoring during the COVID crisis. These included difficulties with establishing or maintaining inter-personal relationships with mentees, concern over mentees’ and their own well-being, awareness of disruptions in their mentees’ progress, and uncertainty about the future  either did not provide a response  or explicitly stated that there were no positive elements . However, the two-thirds who did provide responses pointed to the logistical ease of remote mentoring and the related increased capacity for mentoring across geographic distance; the deepening of relationships resulting from the shared pandemic experience; an increased enjoyment of mentoring; and heightened awareness of their mentees’ gratitude and resilience  and increased workloads were noted.3.3.2.3Many commented on how the pandemic has imposed a dramatic shared experience between the mentor, the mentee, and others in their lives. Some described this experience as strengthening their connections with their mentees.3.3.2.4Some noted that the shared experience of the pandemic increased their sense of connection with their mentees, as the use of video meetings from home provided glimpses into each other's personal lives that helped to humanize each other and bring them closer together in the mentoring relationship.3.3.2.5Several respondents commented that the stresses of the COVID pandemic demonstrated the resilience of their mentees in times of turmoil, which heightened their respect for them and increased their optimism about their mentees’ futures. Similarly, some noted that mentees seemed highly appreciative and grateful for the mentor's efforts, even if they were sometimes just brief check-in emails, calls, or text messages.3.3.2.6Some, when commenting on the above positive aspects of the current circumstances, stated that they had an enhanced sense of enjoyment in their mentoring related to providing what they saw as much-needed support during a critical time that was highly appreciated.3.3.3When asked for suggestions for other mentors who are struggling with mentoring during COVID-19, respondents offered a range of strategies Table .3.3.3.1Several respondents suggested that consistency and persistence were critical to effective mentoring during the pandemic. This included establishing and adhering to weekly schedules for each mentee, even if there are no pressing deadlines and even if it appeared that progress had been minimal.3.3.3.2In addition to the need for consistent, regular mentoring meetings, many mentors suggested additional and frequent check-ins between meetings to take the place of hallway conversations or other impromptu interactions that would be occurring if mentors and mentees were working on site together. Some suggested drop-in Zoom office hours or being available at specific times by phone or text if questions arose between formal mentoring meetings. Some suggested walking, outdoor, masked, in-person meetings to augment the less intimate Zoom meetings.3.3.3.3Some respondents recommended that others learn how to use Zoom and other remote tools more effectively, including camera placement, use of screen sharing, annotation, and open chat features on team software applications, and recognizing the need for more breaks during remote compared to in-person meetings.3.3.3.4A common theme across respondents was the importance of taking the time to check in with mentees on how they were doing mentally and physically. This took the form of brief check-ins at the beginning of each mentoring session, acknowledging and normalizing the unprecedented stress of current circumstances, and providing space for mentees to see the mentor's own struggles with the pandemic.3.3.3.5Several respondents indicated the importance of slowing down and allowing more time for tasks than prior to the pandemic and of validating the forces that have caused delays in mentees’ progress. This was especially true for mentees who were struggling psychologically and those who had increased childcare responsibilities.3.3.3.6While recognizing the need to slow down, several mentors noted that slowing the pace should not be confused with pausing or giving up on mentees. Rather, several stated the importance of persistence and “hanging in there,” and remaining a stable and supportive presence in mentees’ lives.3.3.3.7Consistent with the prior theme of mentor well-being, several respondents noted the importance of mentor self-care during times of stress, with the implication that if mentors did not take care of themselves, they would not be an effective resource nor source of support for mentees.3.3.3.8“I think we should be gathering and sharing wisdom and tips. And yet no one I know has more time for gathering and sharing wisdom.”Finally, some suggested that mentors reach out to other mentors to share strategies for mentoring during stressful times. As 1 respondent said: 4This survey reports qualitative findings from 180 mentors across the U.S. who had previously attended a designated Mentoring the Mentors training workshop at University of California, San Francisco for HIV researchers, and showed how the pandemic has affected mentoring, offering both challenges and solutions. Among the key findings are the need for additional supports and tools for mentors working in times of disruption. Academic programs should help mentors stay consistent with mentoring, even remotely; provide support and offer stories of personal challenges during the pandemic; build mentor capacity to use remote tools more effectively; and validate productivity delays while maintaining persistence. Like most other aspects of life, the COVID-19 pandemic has disrupted mentoring of ESIs working to build careers in academic research, making the need for effective mentoring all the more important.–17 this report is the first to provide data from a large number of geographically diverse and experienced mentors on the impact of COVID-19 on their mentoring practices. It also demonstrates the position from which mentors may witness and intervene on the processes by which the COVID pandemic may differentially impact the careers of women and other caregivers, as well as scientists from underrepresented backgrounds.Although the current survey focused on the specific impact of the COVID-19 pandemic on mentoring, the pandemic occurred alongside other broad societal challenges related to racial injustice and political divisiveness. Indeed, 2020 saw a convergence of pandemic-related academic disruption, social unrest, financial uncertainty, and political turmoil that created a context unlike any other in history. Understanding the collective impact of these forces on mentoring is paramount. While there have been published perspective essays and opinion pieces on the topic,In terms of challenges, first, with the exception of a few respondents who had previously been doing considerable virtual mentoring, moving to full-time remote mentoring felt abrupt and difficult. This illuminated a major gap in mentors’ competencies related to the effective use of remote technologies for mentoring. These competencies relate to technical challenges and to relational challenges for building and maintaining effective and productive mentoring relationships on virtual platforms. Second, the findings from this analysis revealed a notable gap in mentors’ perceived capacity to identify and manage mentee distress during turbulent times. This may be due to discomfort with the topic area, difficulty connecting via remote methods, or most likely both. Third, the confluence in research delays, sudden diversions of effort to childcare or clinical work, and anxiety about the future created an environment in which mentees were not meeting targets, and mentors did not always know how to help. Finally, the data showed that mentors were not always able to prioritize their own self-care during stressful times, a dynamic that can lead to burnout and other negative consequences. The mentors surveyed offered a range of suggestions for addressing these gaps, and the findings provide a rationale for ongoing, targeted mentoring training in these areas.In terms of solutions, a number were offered including consistency with mentorship, persistence while acknowledging reductions in productivity, support, sharing personal stories of hardship during the pandemic, and becoming more versed with virtual tools. We propose that designated training for mentors to react nimbly to barriers to mentee progress, including the use of adaptive Individual Development Plans, may be warranted to better equip mentors in the future. Likewise, the pandemic gave mentors a look inside the home lives of many of their mentees, which allowed a vantage point into family and personal life contexts. Finding ways to better appreciate the non-work challenges that mentees face is non-pandemic times may be beneficial in providing the support and structure that is needed. Findings also support the importance of mentors being aware and connected to local, institutional, and other resources for mentees struggling during such challenging periods. These may include mental health resources, childcare options, and financial support to offset delays in training and research offered by the institution, locality, and professional organizations.There are limitations that should be considered when generalizing the results. The mentors surveyed in this study had self-selected to both previously participate in an intensive in-person mentor training workshop and also agreed to complete the survey when invited. Therefore, the current sample may represent mentors that are particularly dedicated to mentoring ESIs in HIV research. Likewise, those who did not respond may have been particularly affected by the COVID-19 pandemic to the extent that they did not have the time or bandwidth to participate. The online survey format allowed for the efficient text entries used in the present analyses. However, the relatively brief responses may not have fully captured the rich and nuanced underlying themes that might have been better expressed through in-depth interviews with structured probing of relevant content. The pool of mentors surveyed reflected investigators who, at least at the time of their participation in the Mentoring the Mentors Program, were based at US institutions. Therefore, the perspectives of non-US-based mentors were not evaluated. Likewise, by design, the data represent a narrow slice in time during the COVID-19 crisis, which allows for a specific vantage point, but does not reflect the challenges yet to be faced as the pandemic enters subsequent stages, such as the roll-out of vaccines and return to in-person or hybrid mentoring practices. We also did not directly query about specific subgroups of mentees, such as women, parents, and sexual and gender minorities; such a focus may have revealed more nuanced data pertinent to those groups. Finally, although this paper focuses on the perspectives of mentors working primarily in HIV research, the findings ostensibly are not confined to this context. Rather, the results are more general to the challenges of mentoring across a wider range of academic research mentoring settings. In spite of these constraints, we contend that the findings presented from this analysis offer a unique and valuable contribution to the mentoring literature and illuminate strategies and opportunities to offer effective mentoring during times of crisis.In conclusion, findings reported here reveal a range of factors that affected HIV research mentoring resulting from an unprecedented pandemic leading to global disruption. The forced catalysis of remote mentoring, the shared experience of the pandemic, and the simultaneous threats to resources and well-being have clear impacts on mentoring in a way that provides a natural laboratory to examine what works and what does not in HIV research mentoring. While such a set of circumstances is unique and time limited, the perspectives of experienced mentors in this context have the potential to inform future efforts to build mentoring capacity even in the absence of such extraordinary events.MJ conceived of the study and led the data collection, analysis, and writing. MG, JF, JAS, ER, LS, and MS collaborated on the collection of data and drafting of the manuscript. JMS collaborated on the data analysis and interpretation and drafting of the manuscript.Conceptualization: Mallory O. Johnson, Monica Gandhi, Jonathan D. Fuchs, John A. Sauceda, Michael S. Saag, Elise D. Riley, Jae M. Sevelius.Data curation: Mallory O. Johnson, Jonathan D. Fuchs, Jae M. Sevelius.Formal analysis: Mallory O. Johnson, Jae M. Sevelius.Funding acquisition: Mallory O. Johnson, Monica Gandhi, Michael S. Saag.Investigation: Mallory O. Johnson, Lauren Sterling, Elise D. Riley.Methodology: Mallory O. Johnson, Lauren Sterling, John A. Sauceda, Jae M. Sevelius.Project administration: Mallory O. Johnson, Lauren Sterling.Resources: Mallory O. Johnson, Monica Gandhi, Lauren Sterling, Michael S. Saag.Software: Jae M. Sevelius.Writing – original draft: Mallory O. Johnson, Jae M. Sevelius.Writing – review & editing: Mallory O. Johnson, Monica Gandhi, Jonathan D. Fuchs, Lauren Sterling, John A. Sauceda, Michael S. Saag, Elise D. Riley, Jae M. Sevelius.


RBE) of ultrasoft X-rays. We simulated the yields of DSB induction and predicted them in the presence and absence of oxygen, using a Monte Carlo damage simulation (MCDS) software, to calculate the RBE. Monte Carlo excision repair (MCER) simulations were also performed to calculate the repair outcomes . Compared to 60Co γ-rays, the RBE values for ultrasoft X-rays  for DSB induction were respectively 1.3, 1.9, 2.3, and 2.6 under aerobic conditions and 1.3, 2.1, 2.5, and 2.9 under a hypoxic condition (2% O2). The RBE values for enzymatic DSBs were 1.6, 2.1, 2.3, and 2.4, respectively, indicating that the enzymatic DSB yields are comparable to the yields of DSB induction. The synergistic effects of DSB induction and enzymatic DSB formation further facilitate cell killing and the advantage in cancer treatment.This paper estimates the yields of DNA double-strand breaks (DSBs) induced by ultrasoft X-rays and uses the DSB yields and the repair outcomes to evaluate the relative biological effectiveness ( RBE) depends on photon energy. In particular, RBE of ultrasoft X-rays (<20 keV) increases with decreasing energy [RBE is on account of the short traveling distances (~7 nm) of the secondary electrons [The relative biological effectiveness  pathways ,10 and nBE value . In thesBE value .2 [2. Thus, if O2 is present when the DNA damage occurs, the damage is fixed and cannot be reversible repaired by chemical repair by thiols, which is widely regarded as the oxygen fixation hypothesis [2 does not fix the damage directly; rather, it modifies the pathway and final chemical products [Most clustered damage on DNA involves the presence of O2 . When clpothesis . Howeverproducts .In general, ultrasoft X-rays are not used for conventional X-ray therapy (XT) because they penetrate to a shallower depth . However2 on damage yields for cells irradiated by GT and further investigate the RBE of DSB induction and DNA repair for cells irradiated by GT.For cells that are irradiated by GT, the processes of DSB yield are almost exclusively due to the low-energy secondary electrons , and the−3, as used in a previous study [A cylindrical geometry has been used to mimic the typical cell culture experiments ,30,31. Tus study .8 source particle histories, using the 2011 version of PENELOPE. All primary and secondary electrons were followed down to 50 eV [The PENCYL program in the version 2011 penetration and energy loss of positrons and electron (PENELOPE) Monte Carlo code ,33,34 wato 50 eV . PENELOPto 50 eV  that mer1, C2, WCC, and WCR whose definitions and roles were explained previously [1 controls the average angular deflection that is produced by multiple elastic scattering of electrons along the step between hard events and C2 controls the maximum average fractional energy loss in the step. WCC and WCR respectively represent the cut-off energy loss for hard inelastic collisions and for hard Bremsstrahlung emission. The values of parameters in this study were set as C1 = 0.00, C2 = 0.01, WCC = 10 eV, and WCR = 10 eV, as suggested for the simulation of very low energy (0.1–10 keV) electrons in microdosimetry applications [The simulation of electron transport processes in PENELOPE was controlled by the parameters Ceviously . Brieflyications .56Fe [2) [The MCDS is a fast algorithm that calculates the yield of clustered damage within a cell that is irradiated with photons, mono-energetic electrons, protons, or heavy ions, such as 56Fe ,38,39. T56Fe [2) ,38,39.+, 2SSB, DSB+ and DSB++, and total SSB and DSB were defined previously [+), two or more strand breaks on opposite strands that do not constitute DSB (2SSB), DSBs with additional break(s) on a strand within 10 base pairs (DSB+), and more than one DSB within 10 base pairs (DSB++).The MCDS is not a track structure simulation code and its algorithm generates DNA damage data similar to those using computationally-expensive but detailed event-by-event simulations. The types of DNA damage can be grouped into base damage (BD), single strand breaks (SSBs), and DSB. Various complex SSB or DSB, i.e., SSBeviously . BrieflyThe MCER simulation was used to estimate the probabilities of the repair outcomes in the BER and BER/NER pathways for DNA damage in cells that are irradiated with electrons, protons, and helium ions ,42 and h−4, polymerase error for LP BER and for NER = 1.0−6, probability of incorrect insertion opposite a damaged base = 0.75, and probability of incorrect insertion opposite a lost base = 0.75. All four pathways  were simulated. The results show that the NER/LP BER pathway has the lowest probability of correct repair and highest probability for mutation and DSB formation, which is similar to the results for 60Co γ-rays [The following input parameters were used and are the same as those for a previous study : inhibito γ-rays , protonso γ-rays , and helo γ-rays . These r60Co γ-rays were calculated with the dose-weighted formula [E) is the energy fluence of secondary electrons of energy E that are generated in the cell medium from ultrasoft X-rays or 60Co photon interactions. The value for unrestricted LET (stopping power) from the National Institute of Standards and Technology is used for electrons with energy that is greater than 1 keV [LET was calculated by fitting to the data of Emfietzoglou and Nikjoo (2007) [T is the electron energy in eV.The enzymatic DSBs for ultrasoft X-rays and  formula :(1)Yi=∫0an 1 keV . For eneo (2007) , i.e., to that under hypoxic conditions [a and h, respectively denote aerobic and hypoxia conditions.The oxygen enhancement ratio is generally used to quantify the effects of oxygen concentration on DNA damage and cell killing and defined as the ratio of the hypoxic dose to the aerated dose that is required to achieve the same biological outcome . Some stnditions  to quantRBE is originally defined as the ratio of the dose of low LET reference radiation to the dose of any other radiation that is required to achieve an equal biological effect [RBE can also be interpreted as the ratio of the DSB yields of cells irradiated with different radiation sources because DSB induction is linearly proportional to the absorbed dose, D, up to a hundred Gy under aerobic condition (21% O2) and severe hypoxia (0.1% O2) [The l effect . The RBE0.1% O2) ,50, as sU and γ, respectively denote ultrasoft X-rays and γ-rays. The DSB yield for 60Co γ-rays is the reference for all reported RBE values.The subscripts, RBE calculated from the measured DSB induction relative to 60Co γ-rays increased from 1.4 to 2.7 as the photon energy decreased from 4.55 keV to 280 eV [RBE for DSB induction agreed well (within ±5.3%) with the RBE values using the measured DSB induction for photon energies of 280 eV to 1.49 keV  listed in +, and DSBs++ of RBE values estimated based on DSB induction relative to 60Co γ-rays were increasing to the range 1.3–2.9 and 1.4–4.4, respectively (The complexity of the DNA damage was significantly affected by indirect action and oxygen. The yield of DNA damage was simulated at an oxygen concentration of 2%  and loweectively .RBE for DSB induction for cells irradiated with copper L-shell photons (0.96 keV) increased from 2.3 (RBE for cells irradiated with carbon K-shell photons (280 eV) increased from 2.7 to 2.9 (7% increase) as oxygen concentration decreases from 21% to 2%. Under severe hypoxia (0.1% O2), the RBE values for cells irradiated with copper L-shell and carbon K-shell photons further increase to 3.3 and 4.4, respectively. The RBE values for lower energy photons such as carbon K-shell photons (280 eV) under hypoxic conditions were greater, because there was a greater decrease in the yield of DSBs for 60Co γ-rays. The change in DSB yield for different oxygen concentrations is quantified using HRF, considering that carbon K-shell photons (280 eV) had a lower HRF than 60Co γ-rays , for which the HRF for 60Co γ-rays and titanium K-shell X-rays increased as oxygen concentration increased, while that for aluminum K-shell, copper L-shell, carbon K-shell, and 3.31 MeV helium ions (linear energy transfer (LET) =120 keV/μm) [The HRF for DSB induction by all radiation types considered here increased as oxygen concentration decreased, but the HRF for non-DSB clusters varied in different ways.  keV/μm)  decrease60Co γ-rays, titanium K-shell, aluminum K-shell, copper L-shell, and carbon K-shell ultrasoft X-rays, the probability of correct repair, mutation, and DSB formation were calculated (LET = 120 keV/μm) [In terms of DNA repair via the LP BER pathway for cells irradiated by lculated . The pro keV/μm) . The proRBE values (1.3–2.6) for DSB induction was greater than that calculated from DSB conversion . The range of 1.6–2.4; .RBE values were also computed under various oxygen concentrations, using 60Co γ-rays as the reference radiation. In the following sections, we compare our results with experimental results and discuss the effects of oxygen.In this study, the yields of induction and conversion of DSB for cells irradiated by ultrasoft X-rays were calculated. 60Co γ-rays, the measured RBE for DSB induction in cells irradiated by titanium K-shell, aluminum K-shell, copper L-shell, and carbon K-shell X-rays increased from 1.4 to 2.7 [RBE of MCDS-derived DSB yields were comparable (within 7.1%) to the experimentally measured yield of ultrasoft X-rays (4.55 keV to 280 eV) [RBE of 2.7 relative to 60Co γ-rays (8.6 per Gy per Gbp), which was the same value as that experimentally obtained by de Lara et al. (2001) [Relative to 4 to 2.7  as the p 280 eV) ,31. The 2. The RBE for DSB induction increases from 1.3–2.6 under aerobic condition (21% O2) to 1.3–2.9 under moderate hypoxia (2% O2) and further 1.4–4.4 under severe hypoxia (0.1% O2). These results demonstrated that oxygen plays a significant role in DSB induction via indirect action, especially for photon energy above 4.55 keV. The DSB induction for the reference radiation, 60Co γ-rays, reduces 53% as oxygen concentration reduces from 21% to 0.1% while that for carbon K shell (280 eV) reduces 27% only.Data summarized in +, 2SSB, DSBs, DSBs+, and DSBs++, increased  at an oxygen concentration of 21%  was 21.2LET of < 10 keV/μm [LET ~1 keV/μm) was 1.68 [However, the HRF values obtained from a Monte Carlo simulation using track structures, which simulate the effect of oxygen, were about 3.0 for X-rays or particles with an 0 keV/μm . Experimwas 1.68 . The difwas 1.68 .60Co γ-rays or titanium K-shell X-rays, aluminum K-shell, copper L-shell, and carbon K-shell X-rays. The HRF for 60Co γ-rays and titanium K-shell X-rays increased as oxygen concentration increased, which shows that the presence of oxygen increased the number of instances of single-lesion DNA damage due to oxygen fixation [60Co γ-rays was similar, possibly due to the energy of the secondary electrons induced by titanium K-shell X-rays, which is about 4.55 keV. The DNA damage profiles are similar when the electron energy is >2 keV [60Co γ-rays [LET radiation (LET = 120 keV/μm). That may be explained that the sparse DNA damage pattern is present for low-LET radiations (the secondary electrons produced through the irradiation of 60Co γ-rays and titanium K-shell X-rays on cells), while the denser pattern is present for higher-LET radiations. The presence of oxygen intensifies the effects of indirect actions and it turns out that the number of DNA damage with one lesion increases as the oxygen concentration increases (LET radiations (sparse pattern). In terms of complex DNA damage, the HRF values for ultrasoft X-rays such as aluminum K-shell, copper L-shell, and carbon K-shell X-rays were 1.4–2.0, which are comparable to that for helium ions  [60Co γ-rays, was 3.0 [RBE values for Chinese hamster V79 cells, baby hamster kidney (BHK) fibroblasts, and human lymphocyte cells irradiated with carbon K-shell X-rays are 2.4, 2.6, and 2.1, respectively, relative to 60Co γ-rays [RBE for genetic effects could be partly attributed to the highly localized energy deposition of secondary electrons on a micrometer or nanometer scale [The respective adiation . The RBErrations . The RBEickness)  irradiat was 3.0 . If chroer scale ,58. Intrer scale ,60. The RBE values for DSB induction and enzymatic DSB yields. The RBE values for DSB induction and enzymatic DSBs were in the range 1.3–2.6 and 1.6–2.4, respectively, suggesting that the enzymatic DSB yields are comparable to the yields of DSB induction. Under hypoxia conditions, the enzymatic DSB yields may be reduced due to the reduction of non-DSB DNA damage  and severe hypoxia (0.1% O2) increase to the range of 1.3–2.9 and 1.4–4.4, respectively, indicating the advantage of GT for hypoxic tumor cells.The MCDS- and MCER-derived


Loss or rearrangement of genetic information can result from incorrect responses to DNA double strand breaks (DSBs). The cellular responses to DSBs encompass a range of highly coordinated events designed to detect and respond appropriately to the damage, thereby preserving genomic integrity. In analogy with events occurring during viral infection, we appropriate the terms Immediate-Early, Early, and Late to describe the pre-repair responses to DSBs. A distinguishing feature of the Immediate-Early response is that the large protein condensates that form during the Early and Late response and are resolved upon repair, termed foci, are not visible. The Immediate-Early response encompasses initial lesion sensing, involving poly (ADP-ribose) polymerases (PARPs), KU70/80, and MRN, as well as rapid repair by so-called ‘fast-kinetic’ canonical non-homologous end joining (cNHEJ). Initial binding of PARPs and the KU70/80 complex to breaks appears to be mutually exclusive at easily ligatable DSBs that are repaired efficiently by fast-kinetic cNHEJ; a process that is PARP-, ATM-, 53BP1-, Artemis-, and resection-independent. However, at more complex breaks requiring processing, the Immediate-Early response involving PARPs and the ensuing highly dynamic PARylation (polyADP ribosylation) of many substrates may aid recruitment of both KU70/80 and MRN to DSBs. Complex DSBs rely upon the Early response, largely defined by ATM-dependent focal recruitment of many signalling molecules into large condensates, and regulated by complex chromatin dynamics. Finally, the Late response integrates information from cell cycle phase, chromatin context, and type of DSB to determine appropriate pathway choice. Critical to pathway choice is the recruitment of p53 binding protein 1 (53BP1) and breast cancer associated 1 (BRCA1). However, additional factors recruited throughout the DSB response also impact upon pathway choice, although these remain to be fully characterised. The Late response somehow channels DSBs into the appropriate high-fidelity repair pathway, typically either ‘slow-kinetic’ cNHEJ or homologous recombination (HR). Loss of specific components of the DSB repair machinery results in cells utilising remaining factors to effect repair, but often at the cost of increased mutagenesis. Here we discuss the complex regulation of the Immediate-Early, Early, and Late responses to DSBs proceeding repair itself. As an entegrity . Repair,ntegrity . Correctntegrity .Interestingly, endogenous sources of DNA damage due to normal metabolism , rather than exogenous sources , are more important with respect to generating mutations that drive the cancerous phenotype . DNA damSingle strand break (SSB) repair is often considered a ‘specialised’ form of BER, as most SSB repair proteins are also involved in either short-patch or long-patch repair, despite the damage being to the sugar-phosphate backbone instead of the base . In BER,Because SSBs and DSBs are repaired through different mechanisms, these damage pathways are often viewed separately. However, DSBs can be thought of as two closely-spaced SSBs on opposite strands that cannot be repaired by SSB repair , while SThe signal transduction inherent to the Early response to many DSBs is largely carried out by ataxia-telangiectasia mutated (ATM) protein kinase  and an ADP-ribosyl transferases (ART) subdomain. Interestingly, PARP2 and PARP3 lack the N-terminal ZnF1-3 and BRCT domain present in PARP1 but are still able to bind to DNA via the retained WGR domain.PARPs have been extensively studied in the context of SSB repair and BER . There aPARP1/2 are nuclear and depend upon an accessory factor, histone PARylation factor 1 (HFP1), for their activity . Due to . In SSB repair, PARP1 and its associated PARylation events recruit X-ray repair cross-complementing protein 1 (XRCC1) to SSB sites, with XRCC1 functioning as a scaffold for the subsequent binding of SSB repair proteins  . It accuproteins . It is cproteins . A seconproteins . Howeverproteins .An observation favouring the involvement of PARPs in the DSB response is that many DSB response proteins bind PAR through their BRCT and forkhead-associated (FHA) domains . PAR-depin vitro and is recruited to DSBs in vivo via its C2H2 motif. ZNF384 then functions as an adaptor of KU70/80, which promotes the assembly of KU70/80 at DSBs for repair by cNHEJ  and, as PARG hydrolyses the PAR chains, NUDT5 is able to convert the ADP-ribose into ATP, providing energy for downstream processes  . RegulatInterestingly, the state of chromatin condensation plays an important role in activating and maintaining the DSB responses leading to DSB repair. Condensed chromatin is regulated by the binding of HP1 . HP1β isIn heterochromatin after damage, HP1β is phosphorylated (on T51) by casein kinase 2 (CK2), causing its displacement from H3K9me3 . CK2, whin cis or in trans, or if this phosphorylation is necessary for activation of ATM or just a marker of active ATM  and the consequent decondensation of the chromatin around DSBs . In undaTogether, the highly complex regulation of a multitude of histone modifications in the chromatin flanking DSBs contributes to the activation of ATM and its downstream targets, as well as contributing significantly to downstream pathway choice. While initiated during the Early response, the dynamics of chromatin modification are fluid, continuously adjusting to the specific circumstances of each DSB throughout the entire DSB response.Once ATM is activated by TIP60-dependent acetylation, the cell continues with the Early DSB response. The signal transduction pathway initiated by active ATM results in the ubiquitination of histone H2A variants on K13/15 [termed H2A(X)K13/15ub]. The known order of recruitment to chromatin in the vicinity of DSBs is ATM, MDC1, MRN, RNF8, L3MBTL2, and RNF168 . These EOnce activated , the ATMPrior to damage, a proportion of the MDC1 scaffold is already bound to NBS1 via the SDTD domain of NBS1, requiring CK2-dependent phosphorylation of the SDTD motif in MDC1 . Once moRecent data suggests another potential mechanism, loop extrusion, which could facilitate γH2AX propagation . In thisThe phosphorylation of the TQXF motif of MDC1 by ATM provides a binding site for RNF8, an E3 ubiquitin ligase . RNF8 inIn addition to their transduction of the ATM damage signal, RNF8 and RNF168 also have other regulatory roles in the Early DSB response . For exaIt is likely that there are many other undiscovered regulators of ATM recruitment and early phosphorylation events. A further example is the transcription factor SP1, which is phosphorylated by ATM and co-localises with γH2AX and members of the MRN complex, although its mechanism of interaction and regulatory impact have not yet been reported . FinallyEmerging data has demonstrated that two histone modifications are critical for pathway choice between NHEJ and HR . UbiquitPreviously we discussed the regulation of H2A ubiquitination [H2A(X)K13/15ub], which appears to be the critical damage-dependent regulatory event of the Early response. Di-methylation of H4 (H4K20me2) is largely constitutive and widely distributed throughout the genome. Importantly, for BARD1-BRCA1 recruitment, immediately post DNA replication, newly incorporated nucleosomes are transiently unmethylated (H4K20me0), although the existing nucleosomes retain methylation (H4K20me2) . H4K20meIn the context of a DSB, and the associated H2A ubiquitination [H2A(X)K15ub] of the flanking chromatin, the greatest binding affinity of 53BP1 and BARD1 is to H4K20me2 and H4K20me0, respectively . AlthougThe structure of 53BP1 allows it to bind to the bivalent damage-induced H2A(X)15ub and the largely constitutive H4K20me2 , SHLD1, SHLD2, and SHLD3 is recruited to DSBs to block resection . Shieldi53BP1 is clearly important for slow-kinetic cNHEJ and regulation of HR. These roles are supported by its recruitment into chromatin spanning megabases of DNA either side of a DSB, as well as its focal recruitment into large, micron sized, phase-separated condensates . 53BP1 fRegulation of 53BP1 is even more complex than its histone modification-dependent recruitment. 53BP1 is recruited to DSBs that are ultimately repaired by either slow-kinetic cNHEJ or HR. As discussed, its role in slow-kinetic cNHEJ is not just limited to inhibition of resection but it may also have active roles in slow-kinetic cNHEJ , while in HR 53BP1 promotes fidelity by preventing excessive resection . Given sThe regulation of 53BP1 and BRCA1 recruitment to DSBs defines the Late response to DSBs that occur prior to repair by specific pathways, and is clearly complex and not yet fully understood. Emerging data demonstrates that both 53BP1 and BARD1-BRCA1 can bind to related bivalent histone marks, providing a DNA damage histone code. Both factors can bind to the chromatin flanking the same DSB. Details of how BRCA1 outcompetes 53BP1 at some DSBs are emerging. In particular, at one-ended DSBs produced at replication forks specific recruitment of BARD1-BRCA1 drives repair towards HR. Precisely how BRCA1 outcompetes 53BP1 at two-ended DSBs destined for repair via HR is as yet unknown. Furthermore, improved resolution of how 53BP1 and BRCA1 are physically segregated within three-dimensional space proximal to DSBs could provide important insight into pathway choice.Despite being two of the fastest-recruited proteins in the DSB response, KU70/80 and MRN have additional roles in repair choice that occur during the Early and Late response. Earlier we mentioned that proteins in the Immediate-Early damage response do not form visible foci, such as the ionising radiation induced foci (IRIF) that occur during the Early and Late response. While MRN foci have been well characterised, KU70/80 has also been shown to form detectable foci-formation at later timepoints . Focal rImportantly, it appears that while KU70/80 must be retained at DSBs for slow-kinetic cNHEJ, for HR KU70/80 has to be evicted during the Late response. KU70/80 eviction is achieved by a combination of nucleolytic and proteolytic activities. Interestingly, the major nuclease implicated in KU70/80 eviction is MRE11, indicating crosstalk between these two end binding complexes, while CtIP also plays a role . ProteolIt is possible that MRN could also contribute to the proximal ‘melting’ of the broken DNA ends to facilitate the loading of RNA polymerases. The resulting non-coding RNAs (ncRNAs) have been reported to be processed by the RNAses DROSHA and DICER to become the so-called DNA damage response or damage-inducible RNAs (DDRNA or diRNA), reported to regulate the DSB response . AnotherAfter the Late DSB response, largely constituting a delicate balance between 53BP1 and BRCA1 recruitment, the remaining DSBs can be repaired by either slow-kinetic cNHEJ or HR . Howeverde novo DNA synthesis, likely improving fidelity  resection, is additionally termed resection-dependent cNHEJ . This pafidelity . Howeverfidelity . Intrigufidelity .Alt-EJ encompasses vestigial NHEJ repair pathways that do not require KU70/80, XRCC4, or LIG4 . These rin trans homology, and resecting 2–6 kb, while in mammalian cells the minimal in trans homology is unclear, but resection can occur for up to 3.5 kb (In mitotic cells HR has three main subdivisions: GC, BIR, and SSA . GC is to 3.5 kb . In mammo 3.5 kb . This reo 3.5 kb . GC has o 3.5 kb . In SDSAo 3.5 kb . On the o 3.5 kb .BRCA defective breast and ovarian cancers. Furthermore, BRCA defective cancer cells are sensitive to PARP inhibition (It is interesting to note that the involvement of BRCA1 and BRCA2 in HR is an area of intensive research stimulated by the roles of these DSB repair factors in heritable hibition , and thihibition . Howeverhibition , consistBIR is a sub-pathway of HR which uses the invading strand for long-range DNA synthesis without the engagement of a second DSB end . It therin cis homologous repeat sequences which then anneal together with the resulting 3′ flap structures being removed (SSA is not dependent on a sister chromatid for homology and results in deletions . Resecti removed . Studies removed . In yeas removed .trans lesion synthesis, excision repair, strand invasion, and resolution. During S-phase, there are complex repair requirements at single or converging forks, while replication-independent ICL repair can also outside of S-phase (When considering the DDR, there are other pathways that tie into these described repair pathways that have not been discussed in this review, for example, DSBs arising at a replication fork. The kinase ATR can be activated in response to resected DSBs, but is most often activated in response to the elevated levels of ssDNA couated with RPA, that occurs at stalled replication forks. Such structures can be converted into DSBs by nucleolytic attack or fork collapse . Alterna S-phase . ProcessIt is important to note that cNHEJ, both fast- and slow-kinetic, as well as HR appear to be the default pathways in healthy wild-type mammalian cells and they are not usually error prone as they have evolved to operate with high fidelity . The phyHere, we have presented an integrated view of the pre-repair DSB response at its three main stages, Immediate-Early, Early, and Late. Although there is no clear consensus on a precise DSB sensor in the field, the Immediate-Early response consists of the initial DSB sensing and signalling that occurs within seconds of DSB formation. While PARPs have well defined roles in SSB repair, there is emerging data implicating some roles for PARP1, PARP2, and PARP3 upstream of DSB repair. Furthermore, the complex recruitment and interplay between PARP, KU70/80, and MRN contributes to downstream pathway choice. In addition, we support emerging evidence for fast-kinetic cNHEJ responsible for the rapid repair of most DSBs during the Immediate-Early response. The remaining breaks require processing before repair. The activation of ATM and the associated chromatin dynamics constitutes the Early response. This culminates in damage-dependent ubiquitination events permissive for recruitment of Late response proteins, such as the 53BP1 and BRCA1 scaffold proteins.. However, much remains to be discovered about how these responses crosstalk, overlap, and compete.Every step of the pre-repair responses, Immediate-Early, Early, and Late, appears to be important for pathway choice. This requires complex integration of multiple factors to achieve the optimal outcome, which in turn will be specific to the context of each DNA lesion. These factors include the complexity of the DSB itself, chromatin context, cell cycle phase, and availability of the specific repair factors required to achieve the highest fidelity possible. Critical molecular events include the PARP-dependent PARylation response, the recruitment of KU70/80 and/or MRN, dynamic chromatin decondensation and condensation, the activation of ATM, and damage-dependent histone modifications defining a histone code for DSB repair. While there is crosstalk between the Immediate-Early, Early, and Late responses, according to our current understanding it is not until after 53BP1 and BRCA1 recruitment that a cell commits to a specific DSB repair pathwayA key emerging question is apparently simple, yet of deep complexity: for those breaks that are not immediately ligated, at what stage is a DSB committed to a specific repair pathway? Instead of pathway choice occurring downstream of 53BP1 and BRCA1, could it not be more useful to consider pathway choice as a continuous process? It is likely that regulation and crosstalk between the pre-repair pathways allows integration of the many factors required for normal maintenance of genome stability. A related question is whether, if repair fails, can the repair machinery backtrack and attempt to repair the lesion using an alternative high-fidelity approach, before resorting to a more error-prone mechanism. Additionally, the interplay between the PARP-dependent Immediate-Early response and the ATM- and chromatin-dependent Early response has not been fully deciphered. Also, despite its pleiotropic roles throughout the DSB response, how CK2 is activated to specifically regulate so many steps remains enigmatic. It is important to consider that highly error prone mechanisms are unlikely to be physiologically relevant under normal conditions, and are likely to be rare events in normally growing unstressed wild-type cells. Under suboptimal conditions, such as the loss of specific DSB factors that occurs during cancer, or where elevated and non-physiological levels of damage are induce by exogenous agents, repair outcomes become skewed towards mutation. Under such conditions, and if apoptosis is not triggered, repair is likely to proceed using whatever machinery is available. Full understanding of the DSB response remains a challenge for the future. No doubt, these challenges will be met and will expand our evolving understanding of how Immediate-Early, Early, and Late DSB responses are coordinated and integrated to achieve the optimal downstream repair outcomes.


N = 40) compared with healthy volunteers (N = 40). The overall survival time was significantly shorter in patients with higher CD27-AS1 expression than that in patients with lower CD27-AS1 (P < 0.01). Furthermore, downregulation of CD27-AS1 in AML cells suppressed proliferative ability, arrested cell cycle in G0/G1 phase, and induced apoptosis. However, CD27-AS1 overexpression further enhanced the malignant phenotype of AML cells. Additionally, CD27-AS1 was proved to increase PBX3 expression through sponging miR-224-5p. CD27-AS1 knockdown blocked the MAPK signaling through PBX3 silencing and further inhibited the cell growth of AML cells. Taken together, we demonstrate that CD27-AS1 may be a potential prognostic biomarker of AML, and our finding also provides a new insight for non-coding RNA-based therapeutic intervention of AML.Acute myeloid leukemia (AML) is a hematological malignancy with a low cure rate, especially in the elderly. Previous studies have shown that long non-coding RNA (lncRNA) may be an important factor in the pathogenesis of hematological malignancies, including acute myeloid leukemia (AML). However, the biological roles and clinical significances of most lncRNAs in AML are not fully understood. LncRNA CD27 Antisense RNA 1 (CD27-AS1), as a member of lncRNA family, has rare reports on its function. In present study, we found that the expression of CD27-AS1 examined by quantitative real-time PCR was markedly increased in the AML patients ( AML occurs especially in the elderly population2. The cure rate of AML patients under 60 years old is 20–35% higher than that of patients over 60 years old1. Combined with the heterogeneity of AML cytogenetics, the therapeutic methods have become more thoughtful and specific3. Although the development of novel agents and improved treatment strategies have improved the prognosis and survival rate of AML patients, relapse and drug resistance after treatment are still the main problems affecting the cure of AML patients4. Therefore, exploring new diagnostic molecules or therapeutic targets is of great significance for the treatment of AML.Acute myeloid leukemia (AML) is a hematological malignancy derived from abnormally differentiated clonal myeloid cells5. LncRNA CD27 Antisense RNA 1 (CD27-AS1), as a member of lncRNA family, has rare reports on its function. Ma et al. showed that CD27-AS1 expression was significantly upregulated in melanoma, and knockdown of CD27-AS1 suppressed the malignant phenotype of melanoma cells6. However, the expression, function and molecular mechanism of CD27-AS1 in AML are completely unclear. According to the integrated analysis from the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases, we found that the expression of CD27-AS1 in AML patient samples was significantly increased and negatively correlated with the prognosis of AML patients. We hypothesized that CD27-AS1 may exert important roles in the occurrence and development of AML.Long non-coding RNAs (lncRNAs), with a length longer than 200 nucleotides, are involved in the regulation of various physiological and pathological processes, including cancers7. It has been reported that downregulation of miR-224-5p in chronic myeloid leukemia cells was related to cell survival and chemoresistance8. LncRNAs can sponge miRNAs to affect downstream gene expression and function indirectly9. It was predicted that CD27-AS1 may be a potential target of miR-224-5p, and miR-224-5p may also bind to PBX Homeobox 3 (PBX3) from the online bioinformatics tools of Starbase and TargetScan. PBX3 was significantly upregulated in AML samples, and its expression was negatively correlated with AML patient prognosis10. Additionally, PBX3 could activate the mitogen-activated protein kinases (MAPK) signaling pathway to regulate cellular activities in several cancers13. Therefore, those findings suggest a miR-224-5p/PBX3/MAPK signaling pathway may be the downstream of CD27-AS1 to regulate AML progression. In this study, we aimed to investigate the expression and function of CD27-AS1 in AML and study the possible underlying mechanism involved in it.A microRNA (miRNA) mainly regulates gene expression by silencing or degrading its target mRNA molecules to further affect various cellular processes, including tumor progression and metastasisThe bone marrow samples of 40 AML patients  and 40 healthy donors  were obtained between January 2018 and June 2020. These samples were collected from the Affiliated Hospital of Jining Medical University according to the guidelines of the Declaration of Helsinki. All patients and healthy volunteers had signed informed consent before tissue collection. All experiments were approved by the Ethics Committee of the Jining Medical University.6 cells/ml. After centrifugation, the cells were collected and incubated with Anti-Human CD34 PE staining buffer (5 μl) at 4 °C in the dark for 10 min. The cells were collected after centrifugation and resuspended with a staining buffer (500 μl), followed by flow cytometry for CD34+ cell sorting. As previously described14, recombinant human TPO (hTPO)  was used to induce CD34+ cell proliferation.Bone marrow mononuclear cells (BMNCs) were separated from bone marrow samples. The bone marrow samples were diluted with PBS in the same volume, and the diluted bone marrow was spread to the upper part of the separation liquid. After centrifugation, the buffy coat containing the mononuclear cells was collected. For CD34+ cell sorting, the mononuclear cells were then diluted with PBS and stained with trypan blue to count and the cell density was adjusted to 1 × 102.The human AML cell lines HL-60, Kasumi-1, and KG-1 were purchased from Procell . HL-60 and KG-1 cells were incubated in Iscove’s Modified Dulbecco Medium  supplemented with 20% fetal bovine serum  and Kasumi-1 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Procell) supplemented with 20% FBS (HyClone). All the media were placed in an incubator at 37 °C and 5% CO8 TU/ml. AML cells were infected with relevant lentiviral vectors at a multiplicity of infection (MOI) of 10. Culture medium containing the lentivirus plasmids was replaced with fresh IMDM containing 20% FBS 24 h after incubation. Following functional analysis of the cells were carried out 72 h after infection.The recombinant lentivirus plasmid pFUW  containing full length of CD27-AS1 (LV-CD27-AS1) between the BamHI and HpaI sites was constructed. Lentivirus plasmid Tet-pLKO-puro  was used to achieve CD27-AS1 knockdown by inserting specific short hairpin RNAs (shRNAs) of CD27-AS1 into the region between the AgeI and EcoRI restriction sites: LV-CD27-AS1-Sh1 (target sequence: 5′-GCCTGTGTTCTGTCTCTTA-3′) and LV-CD27-AS1-Sh2 (target sequence: 5′-GCAGAAGGAGATCCGATG-3′). Pre-mir-224 (5′-GGGCTTTCAAGTCACTAGTGGTTCCGTTTAGTAGATGATTGTGCATTGTTTCAAAATGGTGCCCTAGTGACTACAAAGCCC-3′) and miR-224-5p sponge (5′-CTAAACGGAACCACTAGTGACTTGAcgatCTAAACGGAACCACTAGTGACTTGAtcacCTAAACGGAACCACTAGTGACTTGAtttttt-3′) were also ligated into the Tet-pLKO-puro vector to attain miR-224-5p overexpression and knockdown, respectively. Lentivirus plasmid pLVX-IRES-puro overexpressing PBX3 (LV-PBX3) was purchased from Fenghbio . The viral titer for all the lentivirus plasmids was 1015 or RT primer. The RT primer (for miR-224-5p) was: 5′-GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACCTAAAC-3′. Acquired cDNA was mixed with specific primers, SYBR Green , and Taq DNA mix (Tiangen) to undergo quantitative real-time PCR reaction using an ExicyclerTM 96 fluorescence meter . Relative expression levels were quantified through the 2−ΔΔCT method. The primers were: has-miR-224-5p forward: TCAAGTCACTAGTGGTTCCGTTTAG, and reverse: GCAGGGTCCGAGGTATTC. U6 forward: GCTTCGGCAGCACATATACT, and reverse: GCAGGGTCCGAGGTATTC. CD27-AS1 forward: TGTGACCTGCTAATGAATG, and reverse: GCTTGGGAGACAGAGTGA. GAPDH forward: GACCTGACCTGCCGTCTAG, and reverse: AGGAGTGGGTGTCGCTGT. The CD27-AS1 expression was expressed as fold-change relative to GAPDH, and U6 served as an internal reference for miR-224-5p expression.Total RNA was extracted using RNA simple Total RNA Kit . Reverse transcription reaction was performed through TIANScript M-MLV Reverse Transcriptase (Tiangen), combined with RNase inhibitor (Tiangen), dNTP, oligo (dT)Total protein samples were extracted by RIPA lysis buffer (Solarbio) supplemented with 1 mM phenylmethylsulfonyl fluoride . Mitochondrial protein extraction kit  was used to extract mitochondrial protein samples from infected AML cells. Protein concentration was quantified by a BCA Protein Assay Kit (Solarbio) according to the manufacturer’s instruction. Certain amount of protein samples  were loaded to undergo SDS-PAGE, which were then transferred to PVDF membranes . Membranes were blocked in 5% non-fat milk powder  or 5% BSA , followed by primary antibody incubation at 4 °C overnight. The primary antibodies were: anti-Ki67 , anti-PCNA , anti-cyclinD1 , anti-cyclinE , anti-CDK2 , anti-CDK4 , anti-p-RB (Ser795) , anti-P21 , anti-P53 , anti-BCL-2 , anti-Bax , anti-cleaved caspase-3 , anti-cleaved caspase-9 , anti-cleaved PARP , anti-cytochrome c , anti-P38 , anti-p-P38 (Thr180/Tyr182) , anti-c-Jun NH 2 -terminal kinase , anti-p-JNK (Thr183/Tyr185) , anti-p-C-raf (Ser338) , anti-p-MEK1/2 (Ser218/Ser222) , anti-extracellular signal-regulated kinase , anti-p-ERK (Thr202/Tyr204) , anti-PBX3 , anti-COX IV , anti-GAPDH . Corresponding secondary antibodies goat anti-rabbit IgG-HRP  and goat anti-mouse IgG-HRP  were used to incubate membranes at 37 °C for 1 h. Enhanced chemiluminescence  reagent was then added to the membranes and densitometric analysis of protein bands was performed using Gel-Pro-Analyzer . The results were expressed as fold-change relative to GAPDH.3 cells (HL-60 or KG-1) were plated on 96-well-plates per well, with 100 μl medium containing relevant lentivirus and/or 10 μM U0126, a MEK1/2 inhibitor , for 72 h. Five replicates were set in each condition. CCK-8 reagent  was then added, followed by further culturing for 2 h at 37 °C in 5% CO2. The value of optical density at 450 nm was assessed using a Biotek ELX800 absorbance microplate reader.A total of 5 × 10Flow cytometry was mainly used to detect cell cycle progression and apoptosis. Cell cycle analysis kit  was purchased to evaluate cell cycle distribution based on propidium (PI) staining, while an Annexin V-FITC apoptosis detection kit  was used to evaluate cell apoptosis through double staining of PI and Annexin V-FITC.2, clone formation in petri dishes was recorded.Cells in each group were harvested and centrifuged to collect cell precipitation. After supernatant removal, complete medium was added to completely resuspend the cells, and the cells were counted. After that, cells were seeded in petri dishes (100 cells each dish) and cultured in the IMDM medium (Procell) containing 30% FBS and 0.9% methylcellulose. After placed in an incubator for 2 weeks at 37 °C with 5% COHoechst staining kit (Beyotime) was used to evaluate cell apoptosis after infection according to the manufacturer’s instruction. Mounted cells were observed under a fluorescence microscope  at 400× magnification.AML cells (HL-60 and KG-1) were used to perform the dual-luciferase reporter assay. Potential binding sequences between CD27-AS1 and miR-224-5p, and miR-224-5p and PBX3 were predicted using Starbase and TargetScan. Cells were co-transfected with the luciferase plasmid pmirGLO  containing predicted wildtype or mutant sequences of binding targets and miR-224-5p mimic (or mimic-NC) through transfection reagent Lipofectamine 2000  according to manufacturer’s instruction. Relative luciferase activities (three replicates for each) were evaluated through the ratio of firefly luciferase to renilla luciferase using Dual-Luciferase Reporter Gene Assay Kit (Keygen Biotech) in accordance with the manufacturer’s protocol.P < 0.05 was regarded to be statistically significant.All data were presented as mean with standard deviation (SD). All experiments were repeated at least three times. GraphPad Prism 8 was used to analyze data in this work, and ordinary one-way ANOVA in combination with Tukey’s multiple comparison test was applied to analyze differences between groups among three or more groups. The expression correlation between CD27-AS1 and miR-224-5p was analyzed via Pearson correlation analysis. N = 40) and healthy volunteers (N = 40) using qRT-PCR. As presented in Fig. According to the integrated analysis from the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases, we found that the expression of CD27-AS1 in AML patients was significantly increased, and negatively correlated with the prognosis of AML patients Fig. . In thisTo study the effects of CD27-AS1 on cell proliferation, the three AML cell lines and normal CD34+ cells were infected with LV-CD27-AS1 and LV-CD27-AS1-Sh1/2. Both infections were efficient Fig. . CCK-8 aAML cells HL-60 and KG-1 with higher expression of CD27-AS1 were used to infect with LV-CD27-AS1-shRNAs, to study the effects of CD27-AS1 on AML cell cycle distribution and colony growth. Results from flow cytometry showed that CD27-AS1 knockdown arrested more cells in the G1 phase, leading to significantly reduced cells in S/G2 phases Fig. . FurtherEffects of CD27-AS1 knockdown on AML cell apoptosis were evaluated subsequently. Both HL-60 and KG-1 cells infected with LV-CD27-AS1-shRNAs showed significantly increased cell apoptosis rate from flow cytometry results Fig. . HoechstTo figure out whether MAPK signaling was involved in the regulation of CD27-AS1 in AML cells, three MAPKs and their phosphorylated forms were checked by western blotting. It was shown that the phosphorylated form of all three MAPKs  was enhanced in CD27-AS1 overexpressed AML cells (HL-60 and KG-1), while decreased in the AML cells with CD27-AS1 knockdown Fig. . Especiahttp://www.oncolnc.org/) analysis showed that the expression of miR-224-5p was positively associated with the overall survival of AML patients  was negatively correlated with CD27-AS1 , and its expression is positively correlated with CD27-AS1 (http://gepia.cancer-pku.cn/). Moreover, Wang et al found that Suppressor of Ty Homologue-6 (SUPT6H) was upregulated in AML and regulated AML cell growth21. SUPT6H is a potential RNA binding protein that that is predicted to CD27-AS1 mRNA (https://rnact.crg.eu/). The evidence above suggests that CD27-AS1 expression may be regulated by transcription factors, such as SP1, or RNA binding proteins, such as SUPT6H.LncRNAs have been found to be dysregulated in various cancer types, and aberrant expression allows them to modulate downstream gene expression and cell homeostasis abnormally, leading to malignant transformation23. Studies have shown that CD27-AS1 was upregulated in melanoma cell lines, and knockdown of it suppressed the growth and migration of melanoma cells6. The expression and effects of CD27-AS1 in melanoma development were similar with that in AML from current study. However, we know little about the specific roles of CD27-AS1 in other cancer types from published articles. Additionally, some other lncRNAs were also identified to participate in the AML progression and prognosis. Studies indicated that lncRNA urothelial carcinoma-associated 1 (UCA1) was upregulated in the AML cell lines and exerted oncogenic functions in AML25. IRAIN (insulin-like growth factor type I receptor-IGF1R antisense intragenic noncoding RNA) was downregulated in AML cells and patient samples, promoting intrachromosomal interaction of insulin-like growth factor receptor (IGF-1R) that can regulate AML cell growth26. Besides, lncRNAs like maternally expressed 3 non-protein-coding gene (MEG3), RUNX1 overlapping RNA (RUNXOR), nuclear paraspeckle assembly transcript 1 (NEAT1) were also dysregulated and exert specific functions in AML. However, their underlying molecular mechanism remains unclear27.The gain-of-function analysis showed that CD27-AS1 overexpression promoted cell proliferation in HL-60 and KG-1 cells. Additionally, CD27-AS1 overexpression also promoted normal CD34+ cell proliferation. Considering a basal expression of CD27-AS1 in normal CD34+ cells, it is not unpredictable that CD27-AS1 may also play a functional role in normal CD34+ cells. So far, CD27-AS1 has only been reported to be aberrantly expressed in several cancers, such as melanoma and cervical cancer28. As members of MAPKs, the ERK signaling is mainly activated by peptide growth factors, while the P38 and JNK signaling pathways are activated by a variety of stimuli, such as endoplasmic reticulum (ER) stress, oxidative stress, and inflammatory cytokines29. In our study, we detected all three MAPK signaling in the AML cells, and found that phosphorylated forms of all three MAPKs were enhanced significantly in the AML cells by CD27-AS1 overexpression, indicating that all three MAPKs were involved in the cell development through regulation of CD27-AS1. Activation of MAPK signaling can activate transcription factors and further increase cell proliferation and protect cells against apoptosis30. For instance, the ERK/MAPK pathway induced the activation of transcription factor AP-1 and further promoted the transcription of cyclin D132.MAPK signaling is known to regulate various aspects of cellular activities, including proliferation, migration, differentiation, survival, and death33. Herein, we demonstrated that CD27-AS1 promoted AML progression through targeting miR-224-5p. Aside from CD27-AS1, miR-224-5p was indeed regulated by multiple lncRNAs, such as LncRNA MALAT134 and NEAT135 in other human diseases. MALAT1 promoted the malignant phenotype of AML cells36, whereas lncRNA NEAT1 inhibited the development of AML37. These above findings indicate the possibility that miR-224-5p is regulated by multiple lncRNAs in AML. Meanwhile, a single lncRNA is considered to sponge multiple miRNAs. Bioinformatics website predicts that CD27-AS1 may target miR-204-5p and miR-628  dimerization could inhibit cell proliferation and suppressed Tribbles Homolog 2 (TRIB2) expression55. TRIB2 is pseudokinase identified as an oncogene in AML56, and its deficiency resulted in impaired activation of MAPKs in AML cells57. In papillary thyroid carcinoma cells, PBX3 increased the expression of Kinase Insert Domain Receptor (KDR), a type III receptor tyrosine kinase58. Inhibition of KDR with pharmaceutical inhibitors60 or neutralizing antibody59 showed anti-AML effects−inhibited cell proliferation and induced cell apoptosis. Interestingly, blockade of KDR abrogated MAPK signaling transduction in AML cells61. The above literatures indicate that PBX3 at least interacts with TRIB2 and KDR to activate MAPK pathway, thereby promoting proliferation of AML cells and inhibiting their apoptosis. Collectively, a novel CD27-AS1/miR-224-5p/PBX3 axis was studied here to regulate AML cell activity through MAPK signaling pathway.The miRNA recognition element on coding genes allows miRNA to directly target them and perform miRNA-mediated regulationCollectively, CD27-AS1 was shown to be significantly upregulated in the AML patient samples and AML cells. Knockdown of CD27-AS1 suppressed cell proliferation and increased cell apoptosis in HL-60 and KG-1 cells. However, forced expression of CD27-AS1 showed the opposite effects. CD27-AS1 increased PBX3 expression through sponging miR-224-5p. CD27-AS1 knockdown or miR-224-5p overexpression blocked the MAPK signaling through PBX3 silencing. Therefore, the results suggested that CD27-AS1 could regulate AML cell progression through a miR-224-5p/PBX3/MAPK signaling pathway, which may provide new insights for noncoding RNA-related therapeutic intervention of AML treatment.Supplementary Figure LegendsSupplementary Figure 1.Supplementary Figure 2.Supplementary Figure 3.Supplementary Figure 4.