This study evaluated participant satisfaction with “Couplelinks,” an online psychological intervention designed for younger couples coping with breast cancer. The program included six experiential learning exercises , psychoeducational information, and support from a personal mental health professional.The primary objectives were to examine participants’ perceptions of: the online intervention’s structure and content; the value of including a professional facilitator; and benefits and drawbacks of the program.N = 53) approximately 1–2 weeks following the intervention which occurred in the context of a randomized controlled trial. Descriptive statistics were used to summarize satisfaction ratings and generalized linear models with fixed effect for gender were used to test for differences in male-female outcomes. A thematic analysis was undertaken in order to understand, organize and summarize the qualitative textual feedback.A treatment satisfaction questionnaire comprised of Likert indices and open-ended questions pertaining to treatment satisfaction was completed by 26 patients and 27 male partners (SD = 0.54) with patient satisfaction ratings being higher than that of male partners’ (p = 0.01). The majority of participants considered the facilitator’s role to be necessary 4.6 (SD = 0.60), and found the program to be convenient 4.1 (SD = 0.81) despite some participants struggling to keep up with the modules. Subjective data revealed participants valued the convenience and flexibility of the online intervention and appreciated the program’s involvement of both partners. Participants also reported that including a professional facilitator humanized the intervention, served as motivation to progress through the program, facilitated insight into their relationship, and was reassuring. Experiential gains noted by participants included that the program: helped couples to open channels of communication; prompted them to designate quality time for one another; evoked feelings of unity and togetherness; and inspired new insight in the relationship.Participants reported an overall satisfaction rating of 4.3 out of 5 (Such feedback supports the feasibility and acceptability of the Couplelinks program while offering directions for improvement of online couple-based interventions in cancer. Women diagnosed with breast cancer (BC) during their childbearing years tend to face challenges that make accessing traditional psychosocial supports particularly burdensome because they are often juggling myriad family and employment-related responsibilities alongside invasive and taxing treatments . The quaAlthough it is the woman who is directly affected by the life-threatening diagnosis and burden of treatment, the challenges and psychological distress associated with the illness also extend to her partner. Male partners of patients with BC tend to experience reduced psychological wellbeing, lower quality of life, and lower sexual engagement compared to pre-diagnosis . They arBC also generates challenges that are psychologically distressing to both partners concurrently. For example, due to the more common need for gonadotoxic chemotherapy when treating younger BC patients, potential infertility is a distressing fear for many young couples who have not started or completed their families . Sexual Dyadic coping ability is associated with improved couple adjustment to BC and reductions in the individual psychological distress of each partner . CouplesThe development and evaluation of online interventions as a flexible and accommodative alternative to traditional, in-person therapy in cancer care has been a burgeoning area of research. Further support for the feasibility of online modalities in cancer care comes from a review by OurRelationship.com. The participating couples in this large-scale study reported significant improvements in relationship satisfaction (Cohen’s d = 0.69), relationship quality (d = 0.57), and relationship confidence (d = 0.47), and individual functioning 12 months post-treatment  and partners (d = 0.34), and between-group effect sizes for patients on communication around affect (d = -0.35) and problem-solving (d = -0.50) with lower scores signifying improvement.In a recent review of online interventions for couples affected by cancer, d = 0.24) and perceived ability to cope as a couple with BC (d = 0.23), but effects were not maintained at 3-month follow-up and no effect was seen on overall relationship adjustment or satisfaction  administered approximately 1–2 weeks after program completion. The primary research objectives were to understand participant perceptions of: (1) the online intervention’s structure and content; (2) the professional facilitation component; and (3) the ways in which the program did and/or did not benefit them.The study upon which this analysis was based received ethics approval by the following institutions: Sunnybrook Health Sciences Centre and York University in Ontario (ID# 300-209); QEII Health Sciences Centre in Nova Scotia (ID# 2010-357); the British Columbia Cancer Agency (ID# H10-00300); and Cancer Care Manitoba (ID# 2013-017).The current study is based on an analysis of feedback collected from 53 participants representing 30 couples who completed the Couplelinks program as part of an RCT (n = 39) and waitlist control (n = 36) groups. Participants were informed of their randomization outcome after baseline measures had been completed. Seven couples dropped out of the treatment arm . One additional couple was omitted from the analysis as they neglected to read the facilitator’s asynchronously delivered feedback, and thus were considered to not have experienced an integral component of the treatment.Couples were actively recruited for the RCT by health care providers at collaborating institutions in Ontario, Nova Scotia, Manitoba, and British Columbia. In addition, flyers were displayed in hospitals and cancer agencies, announcements were made during hospital meetings and in BC support groups, and links to the informational webpage of Couplelinks were posted on social media and on other online BC resources. The recruitment and treatment period spanned 5 years from 2010 to 2015. A total of 75 couples were randomized to treatment . The TSQ guides participants to rate their degree of satisfaction with the program overall, the program’s convenience, and the quality of the professional facilitation on a five-point Likert scale. To obtain written, more in-depth understanding of the program’s perceived value, limitations and benefits, open-ended questions were included such as, “What did you like best about the program?” and “What did you like least about the program?” In the final sections of the TSQ, participants used both Likert scales and open-ended questions to provide specific feedback on the value of the psychoeducational articles and videos. For the last item, there is a space to provide “any additional comments” regarding the overall program (see Descriptive statistics such as means and standard deviations (SD) were used to summarize the quantitative data obtained from the Likert scales on the TSQ. Generalized linear models with a random intercept for couples to adjust for the within couple correlation using a variance component correlation structure were developed. A fixed effect for gender was entered into the model to test for differences in mean outcome scores. Effect sizes for the difference in outcome scores  were calculated as model estimated value divided by the pooled standard deviation. The data analysis was conducted using SAS/STAT software version 15.2 and the SAS System for Windows version 9.4.For the data obtained from the open-ended questions in the TSQ, n = 3 women and n = 4 men) representing 30 couples in total).1 The 26 participating women were an average age of 38.9 years old (SD = 5.48). There were no age restrictions for the 27 males, although they were on average 40.8 years old (SD = 6.35). Eighty-one percent of the couples were married, 15% were cohabitating, and 4% were living apart. On average, couples had been together for 13.8 years (SD = 7.46). At the time of participation, most women were receiving follow-up care (54%), although a sizable portion were undergoing active treatment (31%), or had just finished active treatment (8%). Only two women (8%) were recently diagnosed. Forty-six percent of women had Stage I BC, 19% had Stage II, and 35% had Stage III. The majority of participants (83%) were White and most had completed university or college (86%).Demographics of the participants and couples are summarized in SD = 0.54) on average, and all but two  indicated that that they would recommend the program to a friend in similar circumstances. In terms of the professional facilitation component, the vast majority of participants agreed or strongly agreed that the facilitator role was necessary , that the level of interaction with the facilitator was sufficient , and that the facilitator’s feedback was important . Participants found the program was generally convenient to use . Male and female participants differed only on the overall satisfaction variable with females’ satisfaction ratings being significantly higher  or “the role playing” (M16) involved in some of the modules. One participant commented, “I found something useful in all the exercises” (M4) and another wrote, “All aspects were very valuable.” (F23) However, not every couple was able to derive maximal benefit from the modules or see their value. The most illustrative example of individual factors shaping couples’ evaluations was the dichotomous responses regarding the enjoyment of the “Facing Cancer as a Unified Front” module. In this module, each couple was tasked with using their creativity to build a graphic representation of their illness on the webpage. While a number of participants wrote explicitly about the value of this module, couples who did not perceive themselves as “artistic” wrote that it was challenging and unenjoyable. As one female participant wrote, “The exercise where we did the drawing was outside my comfort zone—I do not like that sort of thing.” (F16)Each couple was expected to complete the program’s standard curriculum consisting of six Dyadic Learning Modules (DLMs), while reviewing the psychoeducational articles and videos was left to each participant’s discretion. The majority of the participants’ comments revealed favorable evaluations of the DLMs such as couples repeatedly reporting on the value of the “the variety of activities,” In terms of the written psychoeducational materials, slightly more than half of participants reported to have read these, and of those that read the articles, the majority indicated they valued them. Over half of participants also watched the psychoeducational videos and the majority of these individuals found them to contain valuable information. As one participant wrote:(F17)The videos provided additional guided support with the various phases of the program. No one really understands how you feel, the fears you have or how cancer has affected your relationship until they’ve been through it. I found that I could relate to many aspects of the… videos. However, one psychoeducational video was met with constructive feedback by one couple. Reflecting on the value of the videos, one female conveyed how she could not relate to the couple in one video clip:(F22)They [couple in video] seem to have faced an early stage cancer, since they say they were always clear that they would be okay after the treatment. I could not relate with that case because our main concern was always whether I would survive or not. Treatment side effects were always a minor thing compared to the fear of dying and [names of couple in the video] were apparently in a different situation, where the worst part of the journey is associated with the treatment, side effects. dyad, instead of just the single partner alone: “It was the first “cancer” activity that actively involved both of us.” (29F) Positive responses regarding the inclusion of the male partner in the program suggest that couples perceive psychological support in cancer care to be more readily available to the identified patient, the woman with BC. As one participant stated: “By participating in a program that involved us as protagonists, it highlighted how important our relationship is and how much care and attention we need to give in order to feel happy with each other.” (F20)The Couplelinks program importantly provides male partners with the opportunity to receive psychosocial support, addressing each couple as a , feeling “pressure”  or “pressed for time to complete by the deadline” (F30) (n = 13) or that the timeframe was “totally doable” (F15), “appropriate” (M28), and “reasonable” (M19) (n = 8). One participant wrote, “It was very difficult to make the time to complete the modules within the allocated time” (26F) which contrasted with another participant who wrote, “I was given more than enough time to complete each module.” (12F)Couples were asked to complete the Couplelinks program within approximately 8 weeks by reviewing the psychoeducational materials, and completing the experiential dyadic exercises on weekly basis. Feedback was split among participants regarding the time allotted for completion. Participants reported either needing “more time” (M8) to fit the weekly exercises into their schedules (n = 14), while other participants reported that the program’s flexibility allowed for it to be easily incorporated into their week (n = 4). As well, while most participants wrote about the benefits of the program’s convenience and of doing the intervention at their “own pace,” (F10) some wrote about how it was perhaps a little too flexible (n = 5). For these participants, the lack of structure was a challenge and made it harder to complete the weekly exercises. As one female participant explained:The Couplelinks program was designed and structured to function as an asynchronously delivered, self-managed intervention. The ability to decide when to complete the modules was viewed as a benefit for some couples, and a drawback for others. Some participants wrote about the challenge of “finding the time” (16F)It was a little too convenient and I found that despite our ability to do it when we had time, that almost gave us an excuse when we were too busy. For me personally, I need something a bit more structured, and we could have done better if my husband and I scheduled the time between us and kept to the schedule. (16F) continued:Although feedback regarding the Couplelinks program’s design and structure was mostly split between participants, there were also contradictions observed sometimes within a single participant. The same female participant The flexibility. I loved that and hated it too. I’m sort of like a child that needs to be reminded from time to time, and I really appreciated the support from the moderators. Everyone has been very kind and I felt like they were really on my (our) side.n = 22). Participants appreciated the freedom to complete the program and exercises at home on one’s “own terms.” (9M) Accessing the intervention online, and at their own pace, was perceived as an advantage over scheduled appointments or “driving into the city.” (6F) As one participant put it, “We never felt pressured, and found being able to go online at our own leisure was very convenient.” (10M) For young couples with busy schedules, juggling early careers or childcare, the flexibility also made psychological intervention more feasible, when it otherwise might not have been possible: “The phone call check in’s were scheduled for a time that was best for [husband’s name] and I, which meant evenings due to his work schedule.” (11F)The primary benefit identified regarding the virtual nature of the intervention was its accommodative nature (n = 9). Some participants claimed they were simply a “face-to-face kind of person” (5F) and preferred to communicate with others through in-person contact. One man wrote: “[I did not like] that it was almost entirely online. But my own bias is for more face-to-face (or on the phone).” (M29) As well, disclosing personal information through an online modality instead of face-to-face was evidently disconcerting for certain participants. One participant felt “awkward” (5F) talking on the phone to the professional facilitator. Another participant felt “raw” and “expos[ed]” (16F) revealing personal information through the online modality without the immediate feedback or validation received in in-person counseling.While many participants stated that they benefited from the convenient and accommodative nature of the online program, the most commonly reported limitation was the absence of in-person contact “Even though having such a program in an online mode has several advantages, especially for introverted people who may feel a bit uncomfortable and for whom it would be difficult to open and share in front of a third party, it is key to have someone following the process.” (18F) and ensured a smooth progression through the generic aspects of the program.The vast majority of the participants “strongly agreed” or “agreed” that the professional facilitator’s role was necessary and that their feedback was important to their successful utilization of the program. The facilitator allowed participants to feel they “weren’t alone doing some online program” (M16) (n = 19). The facilitator importantly pushed the couples to keep pace and meet their deadlines, as one participant wrote, “if we were left on our own without someone there pushing us a little bit, we may not have completed the course.” (5M) Participants appreciated having an “encouraging, instructive” (19M) facilitator to help clarify instructions, ensure the exercises were done correctly, who offered guidance when they were “experiencing difficulties” (24M) and ensured they “got the most out of each activity.” (24F)Another primary benefit identified regarding the professional facilitator was that he or she served as a motivator and helped couples “stay on track” (11F) helped couples make headway in the program (n = 16). Many participants appreciated the facilitators’ skillful reflections on their progress through each module considering these to be a “major benefit” (17F) of the program. The personalized feedback and support “put a voice and thoughts” (1M) to problems, helped couples “reflect on things,” (5F) identify areas for improvement, and afforded them new directions to consider. According to one participant, the facilitator helped “to reframe, interpret, elaborate, redirect, [and] refer.”(21M)The facilitator’s “invaluable feedback” n = 8). One participant wrote, “I often wondered if my responses to some of the questions were clearly understood, [and] his feedback helped reassure me.” (4F) Another participant wrote, “The feedback was such a great validation to know that we were on the right track and doing well.” (11F)The feedback also importantly served as a source of affirmation and validation, giving couples the confidence to proceed through the program  opening channels of communication; (b) carving out time for each other; (c) gaining insight into their relationship; (d) evoking a sense of togetherness, while a few participants felt; (e) “there wasn’t anything shockingly new” in reference to possible gains from the program.n = 19). The DLMs and experiential exercises facilitated “open” (4F) (n = 10) discussions between partners, “without requiring someone to initiate a “we need to talk” situation” (19M) which could seem more ominous. The exercises encouraged, and enabled, couples to “share their experiences and emotions openly” (7M) and presented an “excuse” (4F) or opportunity for participants to be “more open, and straightforward” (22F) with their partners. The Couplelinks program guided couples through “difficult dialogue” (23F) and for one couple, opened “doors that were closed many years ago” (22M), evoking important conversations that they might not otherwise have had. As one participant commented: it was “helpful to have something to force us to communicate.” (23F)Many participants reported that the intervention facilitated a sense of openness within their relationship (n = 5). For example, one female participant shared a concrete way in which she and her partner’s communication ameliorated: “I learned there were hidden feelings deep inside of me, even though I was being positive and optimistic. It’s okay to feel down and express how I feel to my husband” (27F). The program helped couples “enhance” (11F) their communication skills, and acquire “better” (26F) or “new ways to communicate,” (6M), and “got [them] to talk in a different way.” (5F) One patient summed this up by saying that, “the program provided several tools that we can use in the future in talking about our feelings [and] concerns.” (19F)Some couples reported a distinct change and improvement in their method of communication as a result of the program (n = 10), even “as awkward as the communication might be.” (21F) In one participant’s written feedback, she reflected, “It’s important for us to keep talking to each other and making time for each other” (18F), and another participant wrote that the program helped him realize that, “Taking time to listen and talk is key to keeping a healthy marriage.” (1M)The Couplelinks program helped couples recognize the value of bringing effective and open communication into their relationship ((19F) put it. One participant commented: “It [was] a way to sort of nurture our relationship after a period of not paying attention to our relationship at all… next to no intimacy, no dates -you know- for a long period of time.” (19F) Many wrote about enjoying “dedicated time” (29M) with their partner (n = 6), and as one participant articulated, “Having a project that was just about the two of us. No kids involved, no work involved… just the two of us.” (21F) One participant expressed the gratitude she felt to have the opportunity to spend quality time alone with her partner as “there are also issues of body image and sexuality associated with BC.” (26F)Participation in the program provided couples with a set time to spend together each week, “like a date,” as one woman n = 11). Many couples appreciated that Couplelinks granted them the opportunity to “sit down and breathe” (25M) or take pause “even in busy times” (28F) to explore or think about their relationship (n = 11). As a female participant wrote, “[It was a] good reason to make time to talk about us.” (F18)Partaking in the program also allowed for couples to set specific time aside for focusing on their relationship “I most liked an opportunity for [name of husband] and I to focus on our sexual relationship. It’s something that had been an issue for us before the program and then cancer treatment only made it that much more difficult. The exercise on reconnecting physically was very helpful for us and helped give us a framework to kick-start the physical side of relationship again.” (16F) (n = 15). The DLMs the couples completed together, as a team, allowed participants to remember that they are “coping with BC as a couple” (13M) vs. as individuals. By facilitating the “opportunity to connect,” (28M) Couplelinks allowed couples to “deepen” (F11) their relationships, bringing partners closer together (n = 7). As one participant wrote: “… [Couplelinks] made us be together, closer to each other in many levels.” (22F)Participating in the Couplelinks program helped couples recognize, as one participant put it, that they are “in this together” (M6) As one participant wrote about her husband: “The program allowed me to see him in a new light, it made him more human to me.” (16F) This change of perspective also allowed for the discovery of new realizations about the nature of participants’ relationships. For example, one woman recognized, “My husband and I need to have projects and things to do together, alone without our sons.” (22F)Couples made reference to how completing the program’s exercises and modules enabled them to view their relationship from a “new and different perspective.” n = 18). A female participant wrote: “The program has “showed me” to slow down and be more responsive/considerate and patient with him,” (16F) while her husband learned: “[The program] helped me realize that I have a lot of work to do to make our relationship work. I need to express myself more and I need to be more attentive/aware of what my spouse is going through.” (16M) Another female participant came to identify, “We need to pay more attention to connecting physically and taking an emotional chance with each other in order to do that.” (6F)This enhanced relationship awareness also allowed participants to identify areas in their relationship in need of improvement ((9M) Another participant wrote, “I learned that [wife’s name] and I have a very strong relationship and that I’m proud of how hard we work at it.” (15M)While gaining new insight allowed some participants to identify areas in their relationship in need of improvement, others found affirmation of their mutual bond by, for example, seeing “how strong (our) relationship is.” (29M).” The strategies learned were “useful and practical” (25F) and, as one participant wrote, “identified ‘problems/topics’ and helped you develop techniques to address them.” (17F) Some participants made specific reference to acquiring a framework with which to think about and discuss the relationship as one participant noted, “One concept we still talk about is “Turning Toward and Away” from one another.” (5M)Another type of learning gained by the couples was reportedly a “great set of tools and strategies to help for managing relationship ‘stuff’ 13F While most participants wrote about the many tools, strategies, and insights learned, some participants reported that they did not get “a lot out of it” (15F) (n = 5). As one participant put it, “Except for the physical exercise, the rest of the components didn’t really share anything new for me.” (21F)“There really wasn’t anything shockingly new.” The most common reason reported among couples that expressed a lack of novel learning from the program was due to a perception of an already strong relationship. For example, a female participant wrote:(21M)I, personally, didn’t find most of [the DLMs] helpful, but that, I think, is reflective of the fact that we already think about and talk about the things that were prompted in the modules, so there wasn’t anything new, really. (21F)Some couples who felt they were in particularly high-functioning relationships perceived to have not benefited from the stock components of the program as much as other couples. The same female participant cited above wrote, “My husband and I found that it mostly just reinforced that we have a very good and supportive relationship. But I can see how it would be very valuable for couples who were struggling.” The current study examined participant feedback on an online psychological intervention designed to help young couples cope with BC through the use of dyadic coping strategies, psychoeducation, and weekly experiential learning exercises. Participants unanimously saw the benefit in the convenience and flexibility of the program, and appreciated the intervention’s involvement of both partners. Including a professional facilitator in the online intervention was well received among participants as they humanized the intervention, served as motivation to progress through the program, facilitated insight into their relationship, and offered reassurance. The analysis revealed numerous experiential gains that participants felt they took away from the program. Couplelinks helped to: (1) open channels of communication, (2) designate quality time for couples to spend together each week, (3) evoke feelings of unity and togetherness, and (4) inspire new insight in the relationship.Among young couples affected by BC specifically, the cost effectiveness and flexibility of self-managed interventions provide further value over face-to-face therapy sessions . The Counot targeted by Couplelinks with its relationship enhancement, strengths-based (rather than conflict resolution) focus.It is interesting to consider overall favorable treatment satisfaction findings relative to the RCT outcomes which demonstrated short term improvement in positive dyadic coping but no between group differences in marital adjustment or relationship satisfaction—and given that participants in the current sample represented 30 of the 31 couples randomized to the treatment arm of the RCT . SpecifiMoreover, the Dyadic Coping Inventory, a measure that was evidently more sensitive to the changes provoked by Couplelinks, showed these changes were not maintained at follow-up . In thisOurRelationship.com  and progress through the program.While the couples’ subjective accounts regarding the structure and content of the program were mixed , feedback on their experience receiving the professional facilitator’s individualized support and guidance was consistently viewed as an asset and integral to their satisfaction and successful completion of the program. Past research on self-managed interventions demonstrates the benefits of incorporating a coach or therapist in the program e.g., , a findiship.com . An earlship.com  illustra program . The pre(9F)) While this can suggest that the Couplelinks program may be less effective for couples who embark on the program with a higher level or quality of relationship functioning, other couples were both well-adjusted and able to benefit from the program’s content. Indeed, a primary goal of the Couplelinks program was relationship enhancement, and many functional couples did in fact experience novel learning. This disparity may be due to the fact that the subset of couples who perceived themselves to be well-adjusted and did not report achieving insight through the program viewed the stock curriculum as an affirmation of their relationship strengths ). It may have been that these couples already perceived themselves to possess the relationship skills taught in the DLM, or that they were unable to incorporate new learning into a relationship they already saw as strong. This finding highlights both the limitations of interventions with pre-determined content designed for mass administration, as well as the added value of professional facilitation which allows for additional tailoring and personalization to couples whose feelings of affirmation prevent them from acquiring new skills and learning.A notable weakness of the Couplelinks curriculum is illustrated by the subset of couples that reported that they did not “learn anything shockingly new.” A commonly reported reason for this was that they felt their relationship was already strong and functional  did not include all 62 participants from the RCT treatment arm (n = 31 couples). Thus there is the possibility that the present findings were biased toward more favorable impressions of the intervention. Having said that, the fact that there was representation from 30 of the 31 RCT couples in the present sample, with more males than females participating , lends confidence to the validity of our findings.One limitation of the current analysis is the absence of couple-derived feedback in which partners are addressed together and able to build from one another’s reflections about the program in a co-constructed fashion. Given the intervention is focused on developing “we-ness,” dyadic as well as individual-based evaluations of the program would have been appropriate and likely would have added to the understanding achieved through individual evaluations. An additional consideration is that the sample ; QEII Health Sciences Centre in Nova Scotia (ID# 2010-357); British Columbia Cancer Agency (ID# H10-00300); and Cancer Care Manitoba (ID# 2013-017). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.KF: conceptualization, formal analysis, funding acquisition, investigation, methodology, project administration, resources, supervision, visualization, writing original draft, and review and editing. AT: methodology, data curation, formal analysis, writing original draft, and review and editing. SA: conceptualization, data curation, formal analysis, investigation, methodology, project administration, supervision, visualization, and review and editing. SG: conceptualization, data curation, formal analysis, funding acquisition, methodology, supervision, visualization, writing original draft, and review and editing. EW: conceptualization, funding acquisition, investigation, methodology, resources, and writing–review and editing. DM and JS: conceptualization, funding acquisition, investigation, methodology, resources, and writing-review. WC: conceptualization, investigation, methodology, and writing-review. AP: data curation, investigation, methodology, and project administration. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Early identification of individuals at risk of sudden cardiac death (SCD) remains a major challenge. The ECG is a simple, common test, with potential for large‐scale application. We developed and tested the predictive value of a novel index quantifying T‐wave morphologic variations with respect to a normal reference (TMV), which only requires one beat and a single‐lead ECG.P=0.009). In ARTEMIS, 34 (1.8%) individuals reached the primary end point. Patients with TMV ≥5 had an HR for SCD of 2.86  with respect to those with TMV <5, independently from QRS duration, corrected QT interval, and left ventricular ejection fraction. TMV was not significantly associated with death from a cause other than SCD.We obtained reference T‐wave morphologies from 23 962 participants in the UK Biobank study. With Cox models, we determined the association between TMV and life‐threatening ventricular arrhythmia in an independent data set from UK Biobank study without a history of cardiovascular events  and SCD in patients with coronary artery disease from ARTEMIS . In UK Biobank study, 220 (0.4%) individuals developed life‐threatening ventricular arrhythmias. TMV was significantly associated with life‐threatening ventricular arrhythmias (hazard ratio [HR] of 1.13 per SD increase ; TMV identifies individuals at life‐threatening ventricular arrhythmia and SCD risk using a single‐beat single‐lead ECG, enabling inexpensive, quick, and safe risk assessment in large populations. CCSCanadian Cardiovascular SocietyLTVAlife‐threatening ventricular arrhythmiaMACEmajor adverse cardiovascular eventnon‐SCDdeath from a cause other than SCDQTccorrected QTSCDsudden cardiac deathTMRT‐wave morphologic restitutionTMVT‐wave morphologic variations with respect to a normal referenceTpeT‐peak–to–T‐endA novel index, T‐wave morphologic variations with respect to a normal reference, quantifies abnormal T‐wave morphologic variations from a single beat on a single‐lead ECG.T‐wave morphologic variations with respect to a normal reference is the only ECG marker associated with life‐threatening ventricular arrhythmias in individuals without cardiovascular disease, and it is strongly associated with sudden cardiac death in patients with coronary artery disease independently from QT interval and left ventricular ejection fraction.T‐wave morphologic variations with respect to a normal reference is not associated with death from a cause other than sudden cardiac death.This is the first study evaluating T‐wave morphologic variations with respect to a normal reference; this novel index has the potential for predicting sudden cardiac death when measured from wearables in large‐scale screening.Sudden cardiac death (SCD) is a leading cause of mortality, responsible for approximately half of all cardiovascular deaths.Life‐threatening ventricular arrhythmias (LTVAs) are an important cause of morbidity and SCD in almost all forms of heart disease,At a population level, deviations of traditional T‐wave indexes, like the QTc interval or the Tpe interval, from standard thresholds measured from resting ECGs indicate increased cardiovascular risk.Anonymized data and materials have been returned to UK Biobank and can be accessed per request.Sex‐, heart rate–, and lead‐specific normal T‐wave morphologic references were calculated from standard 10‐second, 12‐lead ECG recordings at rest in a population of 23 962 middle‐aged men and women without a history of cardiovascular events from the UK Biobank  from the ARTEMIS study.The primary end point was SCD or resuscitation from sudden cardiac arrest, whichever occurred first. The definition for SCD was a witnessed death within 1 hour of the onset of symptoms. For unwitnessed deaths, the definition was last being seen alive and stable 24 hours before discovery. The secondary end points were cardiac death , non‐SCD, and all‐cause mortality. Follow‐up was 5 years.γ×tr is the optimal warping function relating the average T‐wave from each participant to its corresponding sex and RR normal T‐wave morphologic reference , with an additional weighted variable that has recently proved to be more robust against noise.In the low‐risk cohort (UK Biobank), we calculated TMV by comparing the average T‐wave from each participant with his/her corresponding sex and RR normal T‐wave morphologic reference in lead I from the reference cohort Figure . In shorWe, then, followed the same procedure to derive TMV in the moderate‐risk cohort (ARTEMIS) from lead I (to ease comparisons across cohorts) and from lead V4 . The derivation of TMV and its association with events in ARTEMIS were performed in a blinded manner.In UK Biobank, the QT and Tpe intervals were measured as the intervals between the QRS onset and the T‐wave end, and between the T‐wave peak and the T‐wave end, respectively, from the averaged heartbeat at rest. Then, we corrected the QT interval using Bazett formula.The 2‐tailed Mann‐Whitney and Fisher exact tests were used for univariable comparison of quantitative and categorical data, respectively. The concordance index (C‐index) was calculated to estimate the performance of TMV in both UK Biobank and ARTEMIS. We estimated the optimal cutoff values for TMV in both low‐ and moderate‐risk cohorts based on the highest sum of specificity and sensitivity above median values with at least 20% sensitivity, as in previous studies.P<0.05 was considered statistically significant. Statistical analyses were performed using R version 4.0.2.Univariable and multivariable Cox regression analyses were performed to determine the predictive value of the risk markers. Models were adjusted by risk factors shown in Table The derived heart rate– and lead‐specific reference T‐wave morphologies for women and men are shown in Figures The low‐risk population consisted of 23 954 men, aged 40 to 73 years (median [interquartile range] of 58 [13] years) after exclusions. The demographic characteristics of this population are shown in Table P<0.001) and had higher body mass index (P=0.019), systolic blood pressure (P<0.001), diastolic blood pressure (P=0.018), glycated hemoglobin (P=0.005), triglycerides (P=0.010), creatinine (P<0.001), and TMV (P=0.003). In addition, they had lower high‐density lipoprotein and albumin/creatinine ratio (P<0.001). Finally, there were significantly more men  showed a positive predictive value of 0.6%, a negative predictive value of 99.6%, sensitivity of 44.6%, and specificity of 66.5%. In univariable Cox analysis, participants with TMV ≥0.0983 had a hazard ratio (HR) of 1.57  compared with participants with TMV <0.0983 , independently of age, male sex, diabetes, body mass index, systolic blood pressure, smoking status, glycated hemoglobin, glucose, low‐density lipoprotein, high‐density lipoprotein, creatinine, albumin, T‐wave inversions, and QTc interval Tables  and S6. The ARTEMIS population consisted of 1835 individuals  after exclusions. The demographic characteristics of this population are shown in Table P<0.001 for all), left ventricular mass index, TMV in lead I (P<0.01 for both), age, total cholesterol, low‐density lipoprotein, QRS duration, and TMV in lead V4  were significantly higher in the SCD group than in the SCD‐free group (Table P<0.001), creatinine clearance, and RR interval (P<0.05 for both) were significantly lower in the SCD group than in the SCD‐free group. Finally, there were significantly more individuals being given insulin (P<0.001), with a history of revascularization, with a Canadian Cardiovascular Society (CCS) grading of angina pectoris ≥2, with T‐wave inversions , or with type 2 diabetes in the SCD group compared with the SCD‐free group , positive predictive value was 3.3%, negative predictive value was 99.1%, sensitivity was 70.6%, and specificity was 61.2%. Individuals in the TMV lead I ≥2.4 group had 3.76‐fold risk  of dying of SCD than those in the TMV lead I <2.4 group (P<0.001), CCS class ≥2 (P=0.029), T‐wave inversions (P=0.014), and TMV >5 in lead V4 (P=0.004) remained significantly associated with SCD in the model (Table P=0.032) when included in the model. The C‐index values of each model were 0.767 and 0.762, respectively .Several T‐wave morphologic indexes have been previously proposed in the literature, like morphology complexity score,In the low‐risk cohort from UK Biobank, well‐established predictors of risk, like resting heart rate, QRS duration, QTc interval, T‐wave inversions, or Tpe interval, did not remain significantly associated with LTVAs, unlike TMV Table . This coIn moderate‐risk patients from ARTEMIS, TMV ≥5 in lead V4 or TMV ≥2.4 in lead I was more strongly associated with SCD than known SCD risk factors, like reduced left ventricular ejection fraction or the QTc interval Table . These fPrevious studies have shown that the T‐wave morphology reflects dispersion of ventricular repolarization,TMV, instead, has been developed on the basis of the hypothesis that it reflects dispersion of ventricular repolarization at rest. In particular, we hypothesized that by comparing an average T‐wave with the corresponding sex‐, heart rate–, and lead‐specific reference T‐wave morphology, any variation attributable to sex, heart rate, or lead would be removed, and the remaining variability would be mainly attributable to dispersion of ventricular repolarization. The benefit of TMV over TMR is that, similarly as the QT or Tpe interval, it can be derived from a single‐lead, 10‐second ECG recording.Recent electrophysiological publications have studied the mechanisms underlying the T‐wave and specific indexes,Several prediction scores integrating ECG risk markers have been proposed,A risk marker with strong potential for clinical translation would require an adequate specificity.Our study has several strengths, including significant sample size in UK Biobank, rigorous adjudication of modes of death in ARTEMIS, robust and automated algorithms to derive the ECG markers, and testing in 2 different cohorts, a low‐ and a moderate‐SCD risk population. In addition, the derivation of TMV and its association with events in ARTEMIS were performed in a blinded manner. However, there are also limitations. Continuous TMV was independently predictive in UK Biobank but not in ARTEMIS, where only the dichotomized TMV was predictive in the multivariable models. The median (interquartile range) of TMV in lead V4 was 4.1 (5) in victims of SCD and 2.5 (2.2) in the SCD‐free group, as shown in Table In conclusion, TMV, an ECG index quantifying T‐wave morphologic variations with respect to a normal reference from a single beat from a single‐lead ECG, is significantly associated with LTVAs in a large low‐risk population, and is a stronger SCD predictor than traditional risk factors in a moderate‐risk population when dichotomized. We demonstrate the potential clinical translation of TMV for risk stratification in large‐scale screening studies.Dr Ramírez acknowledges the “María Zambrano” fellowship support from the European Union–NextGenerationEU and the support from the Marie Sklodowska‐Curie grant 786833. We also wish to acknowledge support by the Medical Research Council grant MR/N025083/1. Dr Lambiase is supported by University College London/University College London Hospital Biomedicine National Institute of Health Research (NIHR). Drs Tinker and Munroe acknowledge the NIHR Cardiovascular Biomedical Research Centre at Barts and Queen Mary University of London, UK. Dr Junttila acknowledges funding from Academy of Finland, Sigrid Juselius Foundation, and Finnish Foundation for Cardiovascular Research.None.Data S1Tables S1–S13Figures S1–S2References Click here for additional data file.


Drosophila imaginal discs. We find that compensatory proliferation is associated with a unique cell cycle profile, which is characterized by short G1 and G2 phases and, surprisingly, by acceleration of the S-phase. S-phase acceleration can be induced by two distinct signaling signatures, aligning with inflammatory and non-inflammatory tissue damage. Specifically, non-autonomous activation of JAK/STAT and Myc in response to inflammatory damage, or local activation of Ras/ERK and Hippo/Yki in response to elevated cell death, promote accelerated nucleotide incorporation during S-phase. This previously unappreciated convergence of different damaging insults on the same regenerative cell cycle program reconciles previous conflicting observations on proliferative signaling in different tissue regeneration and tumor models.Regeneration relies on cell proliferation to restore damaged tissues. Multiple signaling pathways activated by local or paracrine cues have been identified to promote regenerative proliferation. How different types of tissue damage may activate distinct signaling pathways and how these differences converge on regenerative proliferation is less well defined. To better understand how tissue damage and proliferative signals are integrated during regeneration, we investigate models of compensatory proliferation in  Drosophila imaginal discs. We demonstrate that inflammatory and non-inflammatory damage activate distinct proliferative signaling pathways, which accelerate cell cycle progression via reducing gap phase length. Despite the risk of inducing replicative stress and compromising genome integrity, we find that nucleotide incorporation during DNA replication is strongly and safely accelerated, thereby also reducing S-phases length. Our work thus provides an unprecedented perspective on the convergence of different damage and signaling environments on the same regenerative cell cycle program.Regeneration requires high levels of cell proliferation to restore the physiological function a damaged tissue. This can either be achieved by allowing more cells to enter the cell cycle, or by accelerating cell cycle progression. In addition, the spatio-temporal dynamics of proliferation need to account for differences in the types and degree of damage. How different tissue damage environments, proliferative signals and cell cycle controls are integrated to drive tissue regeneration is little understood. We address this open question in regenerating  Tissue regeneration in many systems relies on the induction of cell proliferation in stem cells  or otherEpithelia are tissues with high regenerative capacity. Epithelia cover the surfaces of many organs and execute one core function: to act as barrier between internal and external environments . This esMany tissues that undergo regenerative proliferation increase the number of proliferating cells. For example, quiescent stem cells which reenter the cell cycle can support tissue repair , 14. YetDrosophila imaginal discs [To better understand how the type of tissue damage, proliferative signals and cell cycle controls are integrated during regeneration, we chose to investigate compensatory proliferation in al discs –35. In ial discs , 34, 36 al discs , 37–39. al discs , but theal discs  invited head involution defective (hid) or eiger (egr) under the control of rn(rotund)-GAL4 and a temperature-sensitive GAL80ts for 24 h during third instar stages . Importantly, EdU incorporation rates in early stages were comparable to those in late developmental stages, and lower than those observed in ng discs . Similarng discs . Thus, thid- and egr-expressing domain. As accelerated DNA replication clearly defines the compensatory cell cycle, we used EdU incorporation to faithfully track proliferative domains in both systems. We first mapped activity of the most central stress coordinator JNK, which was previously shown to modulate the cell cycle cell-autonomously [hid-expressing discs displayed mildly elevated TRE>RFP activity in the pouch where cells undergo compensatory proliferation  [hid-expressing discs did not alter EdU incorporation dynamics   in wild dynamics . Combinehid-expressing disc. We focused on pathways known to promote proliferation during tissue regeneration, specifically the growth-promoting and pro-survival pathways Hippo/Yki, Ras/ERK, JAK/STAT and Myc, predicting that the regulation of these pathways would positively correlate with high EdU intensity in hid- or egr-expressing discs.To understand which signaling pathways may then be required to produce a compensatory cell cycle profile, we closely analyzed the signaling environment in hid-expressing pouch, but not in normally cycling cells in the disc periphery  . Strikinery Figs . Similarscs Figs . Utiliziing Figs . Similaring Figs . We concegr-expressing discs, we analyzed the same reporter panel for changes in the domain of proliferating cells. Strikingly, in contrast to hid-expressing discs, Yki was not enriched in nuclei of proliferating cells but instead localized to the nuclei of cell cycle arrested, high JNK-signaling cells in egr-expressing discs, as reported before  in hid-expressing cells . To analyze the growth of clones in egr or hid, experiments were carried out as described in [rn-GAL4, tub-GAL80ts(rnts) and carrying the desired UAS-transgenes were staged by a 6 h egg collection and raised at 18°C at the density of 50 larvae/vial. Overexpression of transgenes was induced by shifting the temperature to 30°C for 24 h at day 6 or 7 after egg deposition (AED), as indicated. Larvae were subsequently dissected for analysis (recovery time point R0) or allowed to recover at 18°C for the indicated time. All images represent R0, unless noted otherwise. Control genotypes were either rnts control crosses, or the sibling larvae  [To induce expression of ribed in , 35, 42 ubGAL80) . At leasWing discs from third instar larvae were dissected and fixed for 15 min at room temperature (RT) in 4% paraformaldehyde in PBS. Washing steps were performed in PBS containing 0.1% TritonX-100 (PBT). Discs were then incubated with primary antibodies in PBT, gently mixing overnight at 4°C. The following antibodies were used: rabbit anti-cleaved Dcp-1 , mouse anti-β-Galactosidase , chicken anti-GFP , rabbit anti-GFP , rabbit anti-H2Av-pS137 , mouse anti-H3-pS10 , rat anti-HA , mouse anti-MMP1 , mouse anti-RFP , rat anti-RFP . Tissues were counterstained with DAPI  or Phalloidin-Alexa Fluor 488/647  or Phalloidin-conjugated TRITC  during incubation with cross-absorbed secondary antibodies coupled to Alexa Fluorophores (Invitrogen or Abcam) at room temperature for 2 h. Tissues were mounted using SlowFade Gold Antifade . Whenever possible, experimental and control discs were processed in the same vial and mounted on the same slides to ensure absolute comparability in staining conditions between different genotypes. Genotypes were distinguished on the slide by deliberately co-expressed fluorescence markers . Of note, the signals of the following fluorescent reporters were further amplified by anti-GFP or anti-mCherry antibody staining: miniCiC-mCherry, Yki-GFP. Images were acquired using the Leica TCS SP8 Microscope (DFG Project 414136422), using sample-matched confocal settings.EdU incorporation was performed after crude dissection and detected using the Click-iT Plus EdU Alexa Fluor 647 Imaging Kit  prior to primary antibody incubation. Briefly, larval cuticles were inverted in Schneider’s medium and incubated with EdU  at RT for 15 minutes. Cuticles were then fixed in 4% PFA/PBS for 15 minutes, washed for 30 minutes in PBT 0.5%. EdU-Click-iT labeling was performed according to manufacturer’s guidelines. Tissues were washed in PBT 0.1%, after which additional immunostainings, sample processing and imaging were carried out as described above.3BO3 pH 8.5. After three washes in PBT 0.5%, discs were incubated with mouse anti-BrdU  in PBTN, gently mixing overnight at 4°C. Tissues were washed in PBT 0.5%, then counterstained with DAPI  and secondary antibody coupled to Alexa Fluorophores (Invitrogen) at room temperature for 2 h. Tissues were washed again in PBT 0.5% and PBS before mounting. Mounting and imaging were performed as described above.Larval cuticles were inverted in Schneider’s medium and incubated with BrdU  at RT for 15 minutes. Cuticles were then fixed in 4% PFA/PBS for 20 minutes and washed in 0.5% PBT. Samples were then incubated in HCl at 2N concentration for 45 minutes and subsequently washed twice for 2 min in 0.1M Nahid-expression using the GAL4/UAS/GAL80ts system, larvae were transferred to fly food containing EdU . Larvae were left feeding for 2 h or 18 h. Only larvae still roaming in the food were chosen to dissect after the feeding. After dissection, larvae were fixed in 4% PFA/PBS for 15 minutes, and EdU was detected using the Click-iT Plus EdU Alexa Fluor 647 Imaging Kit  as described above.After Cell cycle analysis of wing imaginal discs by flow cytometry was performed as described . Wing imWhere possible, control and experimental samples were fixed, processed and mounted together to ensure comparable staining and imaging conditions. Positive results were verified with a minimum of n = 3 replicates. Images were processed, analyzed and quantified using tools in Fiji (ImageJ v2.0.0)   and EdU areas (EdU). Masks were then expanded with the functions "fill holes" and "dilate". Pixel fluorescence intensities for all channels in these masks were obtained using the ‘Save XY Coordinates’ function, either on the whole tissue or in a ROI selected with the ‘Freehand Selection’ tool in Fiji. The percentage area of replicating DNA in the image was calculated as the ratio of the number of EdU-positive pixels in the DAPI mask over the number of pixels in the DAPI mask. This measure approximates the percentage of cells undergoing DNA replication, circumventing technical difficulties of segmenting individual nuclei in the tightly packed pseudostratified wing epithelium. The average intensity of EdU was calculated by averaging pixel intensities within the EdU mask only. If a stack of 3 section was used, only the EdU percentage was calculated.The proportion of cell-cycle phases  was calculated in Fiji/ImageJ using FUCCI-expressing imaginal discs. A DAPI mask was generated to analyze nuclear levels of FUCCI.NLS markers, by following the same steps described above. After intensity-based thresholding based on subtracting disc-specific non-nuclear background, the fluorescence intensities for each nuclear pixel coordinate was exported using the ‘save XY coordinates’ function. The pixel population was then divided into 4 cell-cycle phases: Ubi-GFP.E2f.1-230 positive area (G1), Ubi-mRFP1.NLS.CycB.1-266/CyO positive area (Late S), Ubi-GFP.E2f.1-230 and Ubi-mRFP1.NLS.CycB.1-266/CyO double positive area (G2), Ubi-GFP.E2f.1-230 and Ubi-mRFP1.NLS.CycB.1-266/CyO double negative area (S).hid-expressing discs, the proliferative ROI was created by free-hand selection of the rn-GAL4 defined pouch area. For egr-expressing discs, the proliferative ROI was created by (1) free-hand selection of the JNK-signaling area in the TRE-RFP channel and then (2) subtracting it from a free-hand selection of the pouch area using characteristic folding patterns as guides. This approach created a ROI of the proliferative domain and excluded the JNK-signaling area with G2-arrested cells and inflammatory signaling. Average intensity was measured for the DAPI channel and the γH2A channel in the ROI. All γH2A intensities were normalized by the DAPI intensity.A single confocal section was chosen which maximally captured the nuclear DAPI area of the disc proper. For S1 Fig(A-D) Control wing disc  and wing disc after 8 h of hid-expression in the pouch domain  were assessed for DNA replication activity by EdU incorporation. Discs were stained with DAPI to visualize nuclei and were assessed for DNA replication activity by EdU incorporation. Pyknotic nuclei in (C) confirm onset of hid-induced cell death with the wing disc pouch domain where hid is expressed under the control of rn-GAL4 (cyan dotted line). S-phase-specific incorporation of the nucleotide analogue EdU into replicating DNA is already elevated in the pouch after 8 h of hid-expression (D).  Nota of control wing disc and (E) wing disc after 18 h of hid-expression in the pouch domain and at 6 h into the recovery period (F). Discs express two ‘flip-out’ construct to generate labelled clones, either controlling expression of GFP (green) or of Lac-Z (red). As both constructs are induced independently, clones either express GFP (green), LacZ (red) or both (yellow). (G) Quantification of number of cells per clone in the notum domain, from control or hid-expressing discs. Mean and 95% confidence interval (CI) are shown. Welch’s test was performed to test for statistical significance. . (H) Peripodium of wild type wing was stained with DAPI to visualize nuclei (H), expresses the FUCCI reporter system, ubi-GFP-E2f11-230 (green in overlay) and ubi-mRFP-NLS-CycB1-266 (red in overlay) . Discs were assessed for DNA replication activity by EdU incorporation . (H”“) Composite view of H,H’,H”. (H”“‘) Composite view of H’,H”,H”‘. Euchromatin correlates with lower DAPI staining and is replicated early (magenta arrow). Satellite repeats (heterochromatin) correlate with bright DAPI staining and replicate late (purple arrow). (I) Wild type wing disc expressing the FUCCI reporter system, ubi-GFP-E2f11-230  and ubi-mRFP-NLS-CycB1-266  and stained with DAPI to visualize nuclei. An extremely apical section through the wing pouch visualizes mitotic cells. Mitosis occurs exclusively on the apical surface in imaginal discs. Magnified view in I’ and I” is indicated by blue frame in I. DAPI staining visualizes progression through M-phase: metaphase plates (white arrows) and two separate nuclei (blue arrows). (J) Schematic representation of cell cycle phase identification using the FUCCI reporters validated by EdU incorporation assays and mitotic markers. (K) Flow cytometry analysis of DNA content in undamaged control wing discs (grey) and in wing disc after 24 h of hid-expression (red). The pouch of the wing disc was labeled by rn-GAL4-driven expression of UAS-GFP and thus cells outside the rn-GAL4 domain can be distinguished by the lack of GFP expression. GFP-negative events were plotted. The cell cycle of cells outside of the control and hid-expressing domains in the pouch is not different. (L) Flow cytometry analysis of DNA content in undamaged control wing discs (grey) and in wing disc after 24 h of hid-expression , as shown in hid-expressing discs can be detected. This suggests that hid-expressing discs do not experience a specific increase in events that may represent endoreplicating nuclei. (M-Q) Control wing disc  and wing disc after 24 h of ectopic MMP1 (N), MMP2 (M) and cora-RNAi (Q) expression in the pouch domain. Discs were stained with DAPI to visualize nuclei and were assessed for DNA replication activity by BrDU incorporation or EdU incorporation. Cora-RNAi expressing discs were stained for Cora to assess knock-down efficiency. Maximum projection of the apical domain is shown . Maximum projections of multiple confocal sections are shown in ; Single confocal sections are shown in . Maximum projection of the apical domain is shown . Scale bars: 50 μm.(PDF)Click here for additional data file.S2 Fig(A) Wing disc after 24 h of egr-expression (E) in the pouch domain. Discs also express the JNK-reporter TRE-RFP . The disc was stained with DAPI to visualize nuclei , for the mitotic marker phospho-His3 to visualize M-phase cells  and was assessed for DNA replication activity by EdU incorporation (grey). Compare number of phospho-His3 positive events to the number of EdU labelled nuclei to estimate relatively low frequency of M-phase cells in discs. (B-E) Nota of control wing disc  and and wing disc after 24 h of hid-expression (C) and after 24 h of egr-expression (E) in the pouch domain. Discs were stained with DAPI to visualize nuclei and were assessed for DNA replication activity by EdU incorporation (B’-E’). Cyan star in (E) marks small domain of frequent transdetermination as described in M. I. Worley, L. A. Alexander and I. K. Hariharan, CtBP impedes JNK- and Upd/STAT-driven cell fate misspecifications in regenerating Drosophila imaginal discs, Elife 2018 Vol. 7. Cells in this patch undergo compensatory-like proliferation as part of the transdetermination program and therefore incorporate more EdU. Scale bars: 50 μm.(PDF)Click here for additional data file.S3 Fig(A-E) Wing discs at different developmental stages (A-D), and a wing disc after 24 h of hid-expression (E). Discs were stained with DAPI to visualize nuclei and were assessed for DNA replication activity to visualize S-phase cells by EdU incorporation. Please compare (A-D) to (E). (F-I) Wing disc expressing the FUCCI reporter system, ubi-GFP-E2f11-230 (green in overlay) and ubi-mRFP-NLS-CycB1-266 (red in overlay) at different developmental stages . Discs were stained with DAPI to visualize nuclei. (J) Quantification of cell cycle phase distribution using the FUCCI profile at day 5 and day 8 after egg lay. Phases were defined as described in experimental procedures. A Welch’s test was performed to test for statistical significance between day 5 and day 8 wing discs: G1 (p = 0.0269 *), S (p = 0.3828 ns), Late S (p = 0.9363 ns), G2 (p = 0.0092 **). n = 5 disc for each day. (K-M) Control wing disc (K) and wing disc after 24 h of hid-expression in the pouch domain . Discs were stained with DAPI to visualize nuclei. Discs were assessed for JNK activity by TRE-RFP reporter activity . Discs were assessed for DNA replication activity by EdU incorporation (M).  Control wing disc (N) and wing disc after 12 h of puc-RNAi-expression in the pouch domain (O). Discs were stained with DAPI to visualize nuclei. Basal section of the disc from puc. (P) Wing disc after 24 h of bskDN-expression in the engrailed domain using en-GAL4 . Discs were stained with DAPI to visualize nuclei (P). Discs were assessed for DNA replication activity by EdU incorporation .  Control wing disc (Q) and wing disc after 24 h of co-expressing hid and bskDN in the pouch domain (R). Discs were stained with DAPI to visualize nuclei . Discs were assessed for DNA replication activity by EdU incorporation . Graphs display mean and 95% confidence interval (CI). Single confocal sections are shown. Scale bars: 50 μm. Dotted lines (red) outline stereotypic folds in the wing discs.(PDF)Click here for additional data file.S4 Fig(A-D) Control wing disc , wing disc after 24 h of hid-expression in the pouch domain . Discs either express Yorkie-GFP  (green) or the ERK reporter miniCic-mCherry  (green). Discs were stained with DAPI to visualize nuclei and were assessed for DNA replication activity by EdU incorporation . Magnified view of the pouch domain shown.  Control wing disc (E) and wing disc after 24 h of hid-expression in the pouch domain (F). Discs express the JAK/STAT reporter 10xStat92E>dGFP (green) and DNA replication activity was assessed by EdU incorporation (magenta). Magnified view of the pouch domain shown . Same disc as in (PDF)Click here for additional data file.S5 Fig(A-D) Control wing disc , wing disc after 24 h of egr-expression in the pouch domain . Discs either express Yorkie-GFP  (green) or the ERK reporter miniCic-mCherry  (green). Discs were stained with DAPI to visualize nuclei (A”‘-D”‘) and were assessed for DNA replication activity by EdU incorporation (A’-D’ or magenta). Magnified view of the pouch domain shown.  Control wing disc (E) and wing disc after 24 h of egr-expression in the pouch domain (F). Discs express the JAK/STAT reporter 10xStat92E>dGFP (green) and DNA replication activity was assessed by EdU incorporation (magenta). Magnified view of the pouch domain . Please note that these discs are the same as shown in Figs (G-P) Control wing disc , or wing disc after 24 h of expression in the pouch domain and then analyzed 24 h into the recovery period, after egr-expression  or hid-expression (P) was stopped. Discs either express Yorkie-GFP , the ERK reporter miniCic-mCherry , the JAK/STAT reporter 10xStat92E>dGFP  or an endogenously tagged Myc-GFP construct (M-P). Images with increased brightness show the presence of Myc-GFP in the regenerative domain . We suggest that the Myc-expressing cells in the anterior pouch domain of control disc are killed by egr-expression and a new expression pattern of Myc is set up de novo by tissue damage signals, which is maintained throughout the regenerative period. The interspersed apoptosis and the lack of a JNK-driven wound response program in hid-expressing disc maintains the original myc-expression pattern in the anterior pouch. Discs were stained with DAPI to visualize nuclei. Maximum projections of multiple confocal sections are shown in ; single sections are shown in (A-D). Scale bars: 50 μm. Scale bars: 50 μm.(PDF)Click here for additional data file.S6 Fig A wing disc expressing the act-GAL4 ‘flip-out’ system controlling the mosaic expression of GFP  and UAS-Warts-RNAi (A), or UAS-Hippo-RNAi (B). Discs were stained with DAPI to visualize nuclei  and were assessed for DNA replication activity by EdU incorporation .  Control wing disc (C), and a wing disc heterozygous for ykiB5 (D). Discs were stained with DAPI to visualize nuclei  and were assessed for DNA replication activity by EdU incorporation . (E-H) Control wing disc (E), and a control wing disc after 24 h of Egfr.DN-expression in the pouch domain (F). A control wing disc after 24 h of hid-expression (G) and a wing disc after 24 h of hid- and Egfr.DN-co-expression in the pouch domain (H). Discs were stained with DAPI to visualize nuclei and were assessed for DNA replication activity by EdU incorporation. Egfr.DN does not change EdU incorporation dynamics in wild type and hid-expressing discs. (I-L) Control wing disc (I) and a control wing disc heterozygous for Ras1 (J). A control wing disc after 24 h of hid-expression (K) and a wing disc after 24 h of hid-expression and heterozygous for Ras1 (L). Discs were stained with DAPI to visualize nuclei and were assessed for DNA replication activity by EdU incorporation. Heterozygosity for Ras1 does not change EdU incorporation dynamics in wild type and hid-expressing discs. (M) Quantification of the percentage of DAPI areas that were positive for incorporated EdU in hid-expressing discs and hid-expressing discs heterozygous for Ras1. This serves as a proxy for the number of nuclei undergoing DNA replication. Mean and 95% CI are shown. Welch’s test was performed to test for statistical significance. . (N) Quantification of incorporated EdU, measured as the mean EdU intensity in the EdU area within the pouch of hid-expressing discs and hid-expressing discs heterozygous for Ras1. This serves as a proxy for the dynamics of nucleotide incorporation. A Welch’s test was performed to test for statistical significance. . Single sections are shown in . Maximum projections of multiple confocal sections are shown in . Scale bars: 50 μm.(PDF)Click here for additional data file.S7 Fig Control wing disc after 24 h of egr-expression (A) and a wing disc heterozygous for the Stat92E85C3 null allele after 24 h of egr-expression in the pouch domain (B). Discs were stained with DAPI to visualize nuclei and were assessed for DNA replication activity by EdU incorporation.(PDF)Click here for additional data file.S8 Fig Control wing disc (A), wing disc after 24 h of UAS-InR-DA expression in the pouch domain (B). Discs were stained with DAPI to visualize nuclei and were assessed for DNA replication activity by EdU incorporation. (C) Quantification of the percentage of DAPI areas that were positive for incorporated EdU in control wing discs or UAS-InR-DA expressing wing discs. This serves as a proxy for the number of nuclei undergoing DNA replication . (C’) Quantification of incorporated EdU, measured as mean EdU intensity in the EdU area within the pouch. A Welch’s test was performed to test for statistical significance. . Single sections are shown in . Scale bars: 50 μm.(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


While there is an expanding body of literature on Point-of-Care Ultrasound (POCUS) pedagogy, administrative elements that are necessary for the widespread adoption of POCUS in the clinical environment have received little attention. In this short communication, we seek to address this gap by sharing our institutional experience with POCUS program development and implementation. The five pillars of our program, selected to tackle local barriers to POCUS uptake, are education, workflow, patient safety, research, and sustainability. Our program logic model outlines the inputs, activities, and outputs of our program. Finally, key indicators for the monitoring of program implementation efforts are presented. Though designed for our local context, this approach may readily be adapted toward other clinical environments. We encourage others leading the integration of POCUS at their centers to adopt this approach not only to achieve sustainable change but also to ensure that quality safeguards are in place. Though Point-of-Care Ultrasound (POCUS) program leaders are generally well versed in POCUS education, they may lack the tools to support the broader implementation of POCUS in the clinical environment. While there is an expanding body of literature on POCUS pedagogy –3, adminThe first step to any change initiative is to gain an understanding of the operational environment . A thoroOur program stakeholders include senior management, divisional leadership, content experts, non-clinical partners , and end users. Stakeholder engagement was achieved using different mediums including informal interviews, divisional meetings, and online surveys.The Ottawa Hospital has established programs in Emergency Medicine Ultrasonography (EMUS) and Critical Care Ultrasonography (CCUS). In addition to offering a wealth of experience in program development, these programs have a mature POCUS infrastructure, including hospital-based archiving, that can readily be expanded to other departments. Our environmental survey also showed that there is strong leadership support both at the senior management and divisional level for the implementation of POCUS in General Internal Medicine (GIM).In addition to these enablers, we identified barriers to the broader uptake of POCUS in our division. Similar to barriers that have previously been described , lack ofOnce we had developed a good understanding of our local barriers and enablers, we set out to establish our mission, values, and vision Table  12]. Th. Th12]. Our next step was to identify strategies that would address each barrier Table . This exOnce we had identified the key elements of our program, we set out to plan our specific deliverables . We presFinally, we planned for monitoring of our implementation efforts. We selected indicators that could feasibly be collected, would adequately signal change, and would be actionable Table  18–20].–20.18–20In this paper, we have—through sharing our institutional experience—sought to address a gap in the literature regarding POCUS implementation in the clinical environment. A strength of our program is its focus on quality and patient safety. Our program is designed specifically for our local context but may readily be adapted toward other clinical environments. As such, we encourage others leading the integration of POCUS at their centers to adopt this approach not only to achieve sustainable change but also to ensure that appropriate quality safeguards are in place.