The present study aims to investigate the effect of the 4-F  group-based program on physical, clinical, and biological outcomes in young patients suffering from severe psychiatric disorders.A pilot study with a naturalistic design was conducted to investigate the effect of a group-based intervention on young patients.M = 13.65, SD = ±1.93) compared to T1 ,  and the general increase in mental well-being from baseline to the end of the program according to the type of psychopathology . A slight modification in eating behavior, with a tendency towards a decrease in TFEQ hunger levels, was also noticed. The ANCOVA showed no difference in outcomes between the groups based on diagnosis.A descriptive analysis revealed that out of the 61 outpatients initially enrolled in the program, with a mean age of 26.9 years old , 71% were overweight or obese. Paired T-tests for the difference between T0 and T1 were used to evaluate the evolution of the outcomes. The 24 patients who completed the full program showed no significant decrease in weight or body composition. Despite the limitations, the main findings of this study were the significant improvement in muscular endurance and coordination (from T0 (Despite its limitations and the small sample size, this pilot study provides valuable insights, demonstrating the feasibility of the program and its positive impact on physical well-being and improved mental health in young patients with psychiatric disorders, sedentary behavior, and unhealthy lifestyles. These encouraging results warrant further research in controlled, larger population samples to deepen our understanding of the potential effects of such interventions. People with moderate to severe mental illness have higher rates of somatic comorbidities and premature mortality than the general population , 2. FactNew evidence suggests that balanced dietary habits, physical activity (PA), and other lifestyle modifications may modulate brain function and promote changes in adaptive neural plasticity . In an i2 max and a 9 bpm decrease in resting heart rate)  .Furthermore, several clinical studies have investigated PA programs and suggested that exercise can relieve both depressive and anxiety disorders. An association between depression, sedentary behavior, and lack of PA has previously been reported . Based oFinally, a group-based program should improve medium-to-long-term adherence to healthy habits, resulting in better compliance with appropriate psychotropic treatment, better quality of life, prevention of weight gain, and metabolic and cardiovascular outcomes .It is challenging in clinical psychiatric practice to focus on improved physical and mental health conditions; however, we believe that it is crucial to investigate the effects of a multidisciplinary, group-based program aimed at improving the physical, nutritional, and mental well-being of young patients suffering from psychiatric disorders. Based on the current knowledge of the positive effects of regular PA on anxiety, depression, and psychotic symptoms, the 4-F program was established in the psychiatric department (PD) of the University Hospitals of Geneva, focusing on physical activity, education on healthy eating, and improved body perception and emotion regulation , 31.In addition, we recently conducted a scientometric analysis of PA, mental health, and well-being to evaluate key themes and trends over the past decades. Our results strengthen and expand the central role of physical activity in public health and call for an optimization of the implementation of physical activity in health policies .We can therefore assume that such an intervention can prevent weight gain and improve mental and physical health in young patients aged 18–35 years with severe psychiatric disorders. On this basis, we conducted a pilot study with a naturalistic design to investigate the effect of a group-based intervention.The aim of this pilot study was to investigate the feasibility of the 4-F program  and to assess the effect of this group-based intervention on physical, clinical, and biological outcomes over time in a cohort of 24 young outpatients with moderate-to-severe psychiatric disorders.The 4-F program was set up in June 2017 in the psychiatric department (PD) of the University Hospitals of Geneva to optimize the measures and strategies needed to prevent and avoid weight gain and metabolic disorders in young patients with psychiatric disorders. This departmental program is naturalistically and systematically focused on PA, a healthy diet, and improved body perception.This pilot study was conducted over three evaluation periods of the program: Visit 1 at the beginning and Visit 2 at the end of the program (at baseline and after 8 weeks), and one follow-up  12 weeks after the inclusion date . ClinicaFrom August 2018 to October 2020, we recruited outpatients in an ambulatory mental health center in Geneva, patients with moderate-to-severe psychiatric disorders [MINI International Neuropsychiatric Interview ], both mThe monitoring was centered and conducted in a naturalistic and systematic way by qualified professionals consisting of senior psychiatrists, psychologists, PA coaches , dieticians, and registered nurses. This collaboration involved the physicians of these patients.A special study procedure was set up in addition to the usual 4-F program procedure . In addi2), umbilical perimeter, VO2 max , and body composition  were recorded using a Body stat impedance meter at baseline and T1 with the same conditions prior to testing. An ECG was obtained for all patients in the study upon inclusion.The vital signs, height, weight , BMI  to evaluate the patient's physical activity, body weight, and clinical condition [CGI ].As part of the 4-F program, patients participated in two sessions of moderate exercise intervention for 60 min each per week. One of the sessions (“Fit”) consisted of a progressive warm-up and moderate-intensity aerobic exercise in the form of circuit training. The other session (“Fun”) consisted of a warm-up followed by a chosen sport  at moderate-to-sustained intensity . Patients were assessed with the Eurofit physical fitness test battery   to evaluA therapeutic session (“Feel”) with interdisciplinary co-facilitators (a psychologist and a dietician) provided a therapeutic space for talking: participants shared their experiences with weight gain and their body perception and exchanged information about their therapeutic program. The facilitators had the dual objective of motivational interviewing and nutritional listening on the one hand and psychological support  on the other.The dietician and the psychologist organized bimonthly sessions (“Food”) to share educational counseling on nutrition knowledge and behavioral goals according to well-defined topics. The bimonthly sessions helped the participants share their experiences with eating habits, weight gain, and healthy lifestyles.Descriptive analysis of the data was expressed as mean (±SD), percentage, or number of patients. Paired T-tests for the difference between T0 and T1 (8 weeks after the beginning of the program) were used to evaluate the evolution of anthropometric , psychometric , and nutritional (TEFQ) outcomes.post hoc test was performed as a follow-up to examine the between-group effect.In addition, a general linear model of covariance (ANCOVA) was first performed to examine between-group differences in anthropometric, psychometric, and nutritional outcomes in the three groups of patients . The ANCOVA allowed for controlling for gender, and a pairwise comparison with a Bonferroni-adjusted p ≤ 0.05. Statistical and demographic analyses were performed with the Statistical Package for Social Sciences (SPSS) software version 25.0. .Global statistical significance was set at The study was conducted in accordance with the protocol and the principles of the current version of the Declaration of Helsinki and the guidelines approved by the local ethics committee of Geneva .From August 2018 to October 2020, 61 patients with a mean age of 26.9 years old  were included in the 4-F program. Among these patients, 73% were overweight or obese , with a MetS prevalence of 39.3%, and 57.4% suffered from anxiety and mood disorders, while 26.2% suffered from psychotic disorders. Most of the patients received at least one psychotropic treatment  .Due to the COVID-19 outbreak, the lockdown periods, and the usual dropout rate (20%–50% in outpatients) , 41, almN = 8), the COVID-19 outbreak and national quarantine period (N = 8), worsening of psychiatric status and hospitalization (N = 8), and a few patients declining to start the program (N = 6). For the patients enrolled before the COVID-19 outbreak (January 2020) and the consequent lockdown measures, we decided to adopt the 4-F program via online videoconference format , and 11 completed at least 50% of the training sessions (mean participation = 57.4%). Reasons for dropping out were lack of motivation and attrition (e format . MoreoveN = 24) are shown in The sociodemographic and clinical characteristics of this cohort of patients . Among them, 93.8% were single, 56.3% were Swiss or European, 43.8% lived alone, and 79.2% were overweight or obese , with a 37.5% prevalence of MetS, 62.5% suffering from anxiety and mood disorders, and 33.3% from psychotic disorders. Most of the subjects were receiving at least one psychotropic treatment  .F (1.17) = .818, p = .66], waist circumference (WC) , and waist-to-hip ratio .Concerning anthropometric outcomes, univariate tests indicated a significant difference between men and women at baseline and T1. We found no difference in the above variables over time while controlling for gender. No significant differences were observed from baseline to T1 for fat-free mass (kg) . No significant differences were observed for the other two subscales of the EUROFIT test: flexibility , nor whole body balance   from T0 (p = .93] .t(26) = −.228, p = .821]; .At the post-intervention point, there was no difference in weight, BMI, or body composition compared to baseline. In particular, we observed no change in fat mass or fat-free mass (lean mass) at T1 compared to baseline . No significant difference was observed for the other two subscales of the questionnaire, such as dietary restriction  and disinhibition   = .−.899, p = .38], total cholesterol (Tot-Cs) , LDL cholesterol (LDL-Cs) , HDL cholesterol (HDL-Cs) , HDL cholesterol , fasting blood glucose , serum leptin levels , and serum ghrelin levels   to T1 , . A significant difference was observed for the general well-being (WEMWBS)  from T0  to T1 , with patients with anxiety, depression, and psychotic disorders increasing their general well-being from T0  to T1  (t(3) = −4.12 p = .026; t(4) = −2.91 p = .044).Concerning those patients with anxiety or mood disorders, MADRS scores decreased slightly at 8 weeks from baseline T0  = 4.25, p < .05]. In particular, patients with anxiety-mood disorder symptoms increased their general mental well-being (WEMWBS) from T0  to T1  . Concerning patients with psychosis, no significant difference was found for the PANSS total score  and a tendency for a decrease in abdominal circumference was observed at T1 ,  compared to T0 . No difference in abdominal circumference (AC) was observed at T1 in patients with mood disorders  or in those with anxiety disorders .There was a significant effect of the program on mental well-being according to the type of psychopathology , was observed in addition to a slight (not significant) change in glucose from T0  to T1 , . No significant difference in plasma leptin levels or blood glucose levels was observed in patients with anxiety-depression symptoms   compared to T1  and to T2, after 4 weeks after the end of the program , p = 0.86 and p = 0.36, respectively . A slight change in eating behavior was observed over time, with a tendency towards a decrease in the TFEQ hunger scores.Patients who completed the 8-week program improved muscular endurance and coordination, with no change in flexibility or whole-body balance. In line with previous reports , the EurIn the 4-F program, a deliberate choice was made regarding the dosage of exercise. An 8-week group-based intervention was implemented following the recommendations of the World Health Organization (WHO) and the American College of Sports Medicine (ACSM) . The proDespite some discrepancies in methodological design, these interventions had a real impact on the physical and mental health of patients suffering from severe psychiatric disorders.It appears crucial to address their motivation for non-adherence with behavioral and motivational counseling .An improvement in general mental well-being from baseline to post-intervention and a slight positive change in depression scores were observed in the 8-week program, while no difference in positive or negative scores on the PANSS was noted. This is in line with a previous 12-week program consisting of moderate-to-intense aerobic PA in which the authors observed enhanced cardiorespiratory fitness, well-being, and reduced psychiatric symptoms . In a loThis is consistent with a randomized control study that reported better weight management in the intervention group  in drug-naïve patients with first-episode psychosis in the first three months following the introduction of atypical antipsychotic drugs compared to a control group .Not surprisingly, in the final sample of this study, 79.2% of the subjects were overweight or obese, and the prevalence of MetS was 37.5%. In the general population, this constellation of risk factors has been associated with the development of cardiovascular disease . This reInterestingly, for this cohort, no significant decrease in weight, BMI, WC, or body composition was observed. This may be due to the short duration of the program, the low participation rate, and the characteristics of the population studied (most of them were overweight or obese with multiple diagnoses).In contrast, in an 18-month controlled study, the active group with a weight control program that included an educational activity significantly reduced their body weight and WC . In factHowever, in this study, only the subgroup of patients suffering from psychosis had a slight decrease in the abdominal circumference (AC) after 8 weeks of the program. These results were confirmed in a 14-week aerobic interval training program with 25 individuals with a first psychotic episode. The authors observed the effectiveness of aerobic training in reducing weight and waist circumference while also improving maximal oxygen uptake .This suggests that AIT may induce a faster reduction in WC than traditional exercise training in a psychiatric population, as has been demonstrated in the general population . However2 max was observed at the end of the program, whereas other authors reported a significant increase in VO2 max in a 14-week aerobic interval training (AIT) program  over time, as in a previous controlled study of an exercise and dietetic program conducted in young patients with first-episode psychosis . Since tInterestingly, a slight decrease in the TFEQ hunger score was observed from baseline to post-intervention at T1, in line with previous works, which reported that the TFEQ disinhibition and hunger scores increased according to body mass index . PA may Finally, the effects of the 4-F program on clinical global impression (gravity and improvement) did not differ after 4 weeks of intervention, in line with a previous study showing that improvements on the Calgary Depression Scale (CDS) in both yoga and aerobic exercise groups at 3 months of intervention remained stable at 18 months of follow-up in both intervention groups .The results of this pilot study demonstrate the feasibility of the 4-F program in assessing 8 weeks of adapted physical activity levels and improvements in muscular endurance and coordination in young patients with moderate-to-severe psychiatric disorders. Additionally, it highlights a notable increase in the overall mental well-being of these patients.However, there are some limitations to this study. First, the hypotheses could not be tested due to a lack of adequate power and sample size calculations.Second, the study was non-randomized and non-controlled due to the absence of an active control group. Nevertheless, the effect of a specific 8-week program on mental and physical outcomes was evaluated in an outpatient setting with a 4-week follow-up.Third, the longitudinal analyses were conducted on a small sample size of participants; therefore, the results and conclusions should be regarded as preliminary and do not allow for definitive conclusions to be drawn. The dropout rate reached the upper limit of the range reported in previous findings [20 to 50% in outpatients ], and thFinally, the Eurofit test as a speed of limb movement by plate tapping is expected to have a large learning effect when performed for a second time, so these preliminary results should be interpreted with caution , 43. DesThe feasibility of this program will help us implement and develop a structured research plan, considering a larger cohort of patients with a control group, to strengthen our methodology and obtain strong results.Despite the acknowledged limitations, these preliminary findings suggest that the implementation of an 8-week group-based program may be feasible and may lead to potential increases in muscular endurance and coordination, along with improvements in the general mental well-being of patients compared to baseline. This pilot study has the potential to guide the refinement of procedures to foster better patient involvement and adherence to the program, thus potentially reducing dropout rates. It also suggests the benefits of a multidisciplinary approach that focuses on both physical and mental health.Additionally, the outcomes of this study provide a rationale for initiating subsequent longitudinal follow-up research in a larger and more controlled cohort of patients. Such extended investigations could significantly advance the understanding of these interventions, which have already shown promise in improving the quality of life and reducing morbidity and mortality in individuals dealing with severe mental illnesses.


Industry and higher education sectors devote considerable, but independent resources to deliver postgraduate nursing education. This leads to duplication, uncertainty among students, and critical gaps in nursing education. Establishing and sustaining meaningful partnerships between invested university and industry stakeholders can strengthen workforce capability and improve patient care.To evaluate the feasibility and effectiveness of using a University-Industry Integration Framework to develop a postgraduate nursing education program. Prospective mixed methods cohort study (STROBE). A co-design approach, using an established University-Industry Integration Framework, leveraged expert stakeholder partnerships to contextualise knowledge and service need for developing a postgraduate education program for cancer care nurses.All participants (n = 46) were 100% satisfied with the online resources, support, and communication processes applied. Qualitative data generated three major analytical interpretations , highlighting the experiences and connections and how the partnership evolved. Program participants (n = 15) undertook a six-week cancer education program with eight responding to the survey with overwhelming satisfaction (100%), increasing their knowledge and skills. While barriers were evident, three quarters (n = 6) indicated these were addressed and enabled progress in the program. However, 63% (n = 5) were not satisfied with the program workload.University and industry partners can apply the University-Industry Integration Framework and deliver a successful postgraduate education program for cancer care services. Within a co-design partnership it is possible to develop strategies and processes to overcome barriers and deliver a program for mutual benefit. The culmination of this successful education program has enhanced collaborations between partners and likely will sustain the offering of future co-design endeavours.The online version contains supplementary material available at 10.1186/s12912-023-01419-1. Health systems depend on the specialised knowledge of nursing staff and high-functioning, team-based approaches to deliver person-centred care. Postgraduate professional education should facilitate an educated health workforce and research-literate nurses who can implement evidence-based practice within scope of regulatory frameworks and organisational requirements –3. EvideResearch suggests that post-registration nurses benefit from a judicious blend of practical and theoretical learning experiences . Such exUniversity and industry sectors invest significant but separate financial and human resources to deliver postgraduate nursing educational programs . This edThe University-Industry Integration Framework  recommenThe aim of this study was to evaluate the feasibility and effectiveness of the University-Industry Integration Framework  when appThe research questions are:Is it feasible for university and industry partners to use the University-Industry Integration Framework  to co-deWhat strategies and processes enabled and/or hindered university and industry stakeholders to collaborate, develop, implement, participate and evaluate the educational program?What were the Educational Program Partnership Group’s and program participants’ experiences of a co-designed postgraduate educational program?This prospective cohort study employed mixed methods to evaluate a co-design approach and framework used to develop, evaluate and deliver a postgraduate nurse education program. This approach drew on Sanders and Stappers  conceptuThis research was conducted over six months across two large metropolitan health services in Queensland, Australia and in the School of Nursing of a large metropolitan university.The purposeful sample of program participants were registered nurses (RNs) who were beginning practitioners working in cancer care services at the participating sites. As this program aimed to support beginning cancer nurses to transition safely into the workplace, RNs who possessed a postgraduate qualification in cancer were excluded. Industry partners advised that new staff would need at least three months experience to meaningfully engage with the program.Project Team were a group of stakeholders from each organisation responsible for conceptualising and developing the agreed educational program; An Advisory Committee, who provided high-level strategic direction and input on program deliverables; a Working Group of industry managers, nurse educators, university lecturers, academic leaders and researchers who provided expert advice to co-design, develop, implement and evaluate the program. Directors of Nursing from each industry site who authorised the nurses’ program participation.The Educational Program Partnership Group comprised key personnel from each site. The The postgraduate educational program was designed to advance knowledge and professional skills among participating nurses and provide opportunities for them to demonstrate autonomy and judgement as an introduction to the specialty of cancer nursing. The program was delivered over six weeks via modules located in a Blackboard Learning Management System. The modules taught Population Health, Cancer Biology, Classisfication, and Diagnosis. Learning was self-directed and self-paced, requiring program participants to invest approximately eight to 10 h per week. Modules were comprised of: learning objectives; core theoretical content, with reference to literature; and a range of interactive blended learning activities. Three synchronous webinars were delivered to support program participants’ online learning and were facilitated by industry nurse educators and university lecturers. Program participants were encouraged to complete the two assessment activities, a case study, and a brief oral presentation of the case.Identification of potential program participants occurred over a two-month period through line managers and nurse educators who were able to identify all new staff who met the inclusion criteria. The Senior Research Assistant conducted information sessions and provided the participant information sheet and consent forms. Written consent was also obtained from the Educational Program Partnership Group. Completed consent forms certified participation in the study and subsequent provision of evaluation specific to each Educational Program Partnership Group . The decision to participate was voluntary and participants could withdraw at any time without consequence. All data was confidential and deidentified at data collection. Recruitment, engagement, follow up and data collection occurred over a six-month period (1 January 2021–31 June 2021).Data included transcribed audio recordings of semi-structured interviews, reflective notes, action meeting notes and surveys conducted between February and June 2021.Semi-structured interviews with the Project Team and Directors of Nursing explored the co-design and collaborative process of achieving mutual understanding in developing and delivering the education program. The Senior Research Assistant conducted interviews in a private room (face-to-face) or online. Recorded audio and data were anonymised prior to transcription. Interview questions included, How did you reach common goals? What are your views on sustainability? and What makes a successful education program?To support reflection in action and reflection on action , programTo gain an appreciation of engagement and interaction, action notes were collated from the Project Team, Advisory Committee, and the Working Group stakeholders’ meetings. These notes assembled information related to tasks, actions, structures, and processes, which enabled or hindered collaboration and integration. These notes were de-identified and confidential.Surveys were used for the program participants, Advisory Committee, Working Group and Director of Nursing stakeholders to gain an understanding of the level of satisfaction with involvement and perceived relevance of the final education program. Surveys were developed by the research team and consisted of eight core questions with a five-point Likert scale for responses ranging from very satisfied, satisfied, neutral, dissatisfied and very dissatisfied. Respondents were also able to add comments to an open-ended section.Qualitative analysis followed a critical and iterative review of data generated from the semi-structured interviews, reflective notes and action meeting notes . Each daSurvey data was entered into IBM SPSS Statistics (version 27) . DescripData integration was guided by the research questions, with the quantitative findings synthesised according to the themes generated from the qualitative data. The quantitative and qualitative data combined subsequently informed the synthesised narrative of results in the discussion and conclusions. Trustworthiness was adhered to in this study based on Sandelowski’s  argumentA total of 15 program participants (RNs) consented to undertake the six-week cancer educational program. All 15 participants accessed the online educational platform at least once during the program. Two participants did not engage in the program after week three. Thirteen (86.7%) participants presented a case study online for peer and educator feedback during the program. One participant, citing family reasons, withdrew immediately prior to the final summative assessment. Three other participants chose not to submit the final assessment. At the program’s completion, nine participants submitted and passed the summative assessment.Of the 15 participants who took part in the cancer educational program, eight (53%) responded to the survey. See Tables The Educational Program Partnership Group consisted of 31 members, of which 25 completed the survey, providing a response rate of 80%. Results indicated that the majority  were satisfied that mutual understanding was realised within the group and that involvement in the program contributed to enhanced collaboration between university and industry partners. Respondents were strongly satisfied with their involvement in the project  and contribution to building staff capability . Satisfaction with the educational program and supportive resources was reported by 18 (72%) participants. Respondents were less certain about the ability of the educational program to be replicated in the future with 11 (44%) not satisfied that this would be possible. Likewise, 10 (40%) respondents were not satisfied that barriers that arose during the project were sufficiently addressed to progress the program. A total of seven (28%) respondents were not satisfied with the communication processes used during the project. Of these, four (57%) were from the Participant Industry Manager or were Nurse Educators; two (28%) were from the Working Group; and one (14%) was from the Advisory Committee. See Table A sub-set of questions were asked of the Participant Industry Managers, Nurse Educators and.Lecturers (n = 10) with a 70% (n = 7) response rate. The majority  were not satisfied with their engagement with participants, the level of engagement by program participants , or their ability to manage the workload associated with the project .The findings from all qualitative data in this study were synthesised,  and generated three major analytical interpretations, with a focus on the partnership process that evolved between university and industry during the study. The interpretations overlap demonstrating interconnectedness and complexity, as the partnering relationships changed.We can’t keep doing what we are doing … it is counterproductive. (Project Team Interview 3)The process of establishing mutual understanding commenced through the natural alignments and familiarity of three stakeholders with existing relationships. Building on these relationships, the new Project Team expanded to five members who explored a shared concern for contemporary graduate capabilities and what nursing practice entails. Identifying this enabled the group to consider the different perspectives of how education could be developed to suit the desired outcomes of university and industry stakeholders. They recognised the current context of duplication was unproductive, unsustainable and did not meet student needs.Setting a platform of collegiality was crucial for these beginning conversations, as the Project Team focused on new ways of working together established on trust, authenticity, and openness to ideas. The ability to speak up, as well as the acceptance of respectful, robust discussion and appreciation of previous knowledge and expertise developed through the co-design process. An intentional flattened hierarchy enabled the Project Team to collaborate as “equal partners with equal responsibility” (Project Team Interview 3). Members became active and accountable by working with their strengths. Despite each stakeholder operating on different sites, “nothing was done in isolation” (Project Team Interview 5). Their sharing of ideas was enabled by a back-and-forth approach as they investigated the problem and explored possible solutions—an approach which continued throughout the research. A shared vision of a quality learning package to support student learning and safe practice was established on their willingness to participate in this cooperative process.Right from the word go … it wasn’t ‘we can’t do this’, it was ‘we need to do this, how can we do this’. (Nursing Director Interview 1)A critical dimension of reciprocity was the cooperative movement within and beyond the Project Team. The dimension and spirit of reciprocity was to underpin how this team, from their shared vision, interacted with the other groups within the project. Working across three sites was considered ‘next generation’ due to the extensive development of the Program beyond stakeholder consultation which required clear communication and commitment flowing from all involved.A cohesive dialogue formed from the Project Team’s vision was joined by each group as they became accountable for their roles. While natural alignments existed within these groups, the Project Team facilitated a communication conduit so that timely iterative changes could be made.… so I think there was really good synergy between all three to try and get this product moving … so having a really engaged team from my perspective was a really strong enabler to get that done. (Nursing Director Interview 2)This approach enabled groups and individual members to engage meaningfully with the work.Reciprocity kept the co-design and implementation process moving forward as there was a “genuine belief that working together gives insight” (Project Team Interview 3). It was a novel and innovative approach—the ‘next generation’ of building on network connections, ideas and learning together. This collaborative approach even in the design phase was shifting the conversation from previously siloed education packages across three institutions to partnering education and evolving the curricula so that nursing education could be relevant to industry, universities and RNs.… it is not that we don’t want to, but these are the boundaries within which we are able to work and recognising that we have different structures and processes for the different organisations that must be maintained. (Project Team Interview 1)The realisation of this deeper process raised awareness of boundaries and competing interests between university and industry. Expressing these boundaries required frank and honest conversations within the Project Team. These conversations occurred as team members recognised the complexity of the different structures within their institutions. Yet, while there was a readiness to address boundaries, for some members these disclaimers were “more revealing than anticipated” (Project Team Interview 1). The different interests and pressures from each institution meant that members were not free to make decisions early in the project.This brought an initial uncertainty as the team took time to reformulate their ideas, actively listen to each other, and then begin to recognise that they were using different terms to describe the same idea.Everyone stepped back from their vested interest and looked at what could be the best outcome, so that we were getting a better product and better engagement. (Project Team Interview 3)The Project Team continued this process of negotiating towards clarity with the other groups involved in the project. By giving opportunity for expression, members of the wider groups were able to explore the socio-political landscape and find a mutual space to collaborate rather than compete. This was vital as some protective elements were apparent in sharing knowledge and skills across the three institutions.Significantly, as the work evolved, collaboration made use of each group’s strengths and resources. For industry, learning could occur close to care provision and intentionally focus on the student experience, while the access to a learning designer and lecturer from university created a professional learning package that utilised a multi-modal approach.This nicely evolved. They worked exceptionally well to really focus on the student experience, to really focus on what was needed … everyone managed to step up because everyone could see the value in it and wanted to try things in a different way … I was really impressed with the standard. (Project Team Interview 4)Flexible adaptations aligns with considering the right people for the right roles within the project. These deliberations required time, wisdom, and prudence as the Project Team wanted a whole and fulsome approach. Project Team members worked closely across all other teams, providing direction when needed and making space for other team members to flourish. This was specifically evident in the co-design of the education package in the Working Group. Within this group, education experts from each institution worked together to create an engaging quality postgraduate education product. While curriculum expertise was available from the Project Team, they did not impose on the Working Group. The same principles of trust, respect, communication, and connection to the vision harnessed the collective, expert knowledge of this group.My role was limited to releasing staff to attend their requirements. (Industry Manager Interview 2)Flexible adaptations was also expressed as groups considered future directions, sustainability and replication following implementation. For industry, the Project Team recognised the need for greater enablement at the ward level to support sustainability. Some Industry Managers felt disconnected through limited communication, which affected their engagement. They were also concerned that the program impacted the integration of new staff to the ward.I think we need to think about different ways of being more flexible in how we do industry/academic engagement in ward time. (Project Team Interview 5)It was apparent that better lead times for clinical areas would support roster requirements and facilitate offline time for students, which hospital wards could manage. This would also enable webinar scheduling and assessment presentations to align with ward programs.At the university level, flexible enrolment options that match hospital intakes and nursing work also need to be considered. However, this would require a shift in the way university education is currently organised, since rigid enrolment processes do not currently meet the needs of students or industry.It is nice when you can align your learning and theoretical components to the space where it happens in the workplace. (Nursing Director Interview 1)The Project Team intentionally sought to provide an environment for co-design where the blending of different expertise could transpire. The project’s inclusion of two hospitals was considered the ‘next generation’ of developing postgraduate education differently. This approach enabled working with practitioners and professionals who could bring authentic value to the design and implementation. The integration of their resources generated a substantive learning package from the new collegial relationships across the three institutions. What emerged was the development of a credible and highly valued product that met a “need in developing new to practice cancer nurses” (Nurse Educator Interview). Importantly, authentic learning facilitated the progression of sound theoretical grounding to the bedside.I think it is organisations’ or industry’s responsibility and accountability to make sure that we set nurses up to achieve what they need to achieve with that academic flavour. (Nursing Director Interview 1)While this work raised awareness of how industry can partner in the postgraduate education space, there were concerns regarding workloads for the Nurse Educators heavily involved in implementation and assessment. Conjoint positions, review and rationing of current work practices and sharing the financial burden could be future considerations. However, the industry partners understood the significance of their contribution to cancer nursing education in this co-operative approach.We need to be investing in our staff. We need to be investing in their education. We need to be investing in their career progression and further education and we all have an obligation in doing so. That is why I see this as being critical. The evidence tells us that a highly qualified educated workforce provides better outcomes. (Nursing Director Interview 2)Likewise, this approach meets the need of investing in safe patient care and nursing professionalism.The modules are great for the novice nurse to gain a better understanding of patient and diagnosis. (Program Participant Interview 1)Program participants came to the course with an expectation of building their understanding and nursing knowledge of cancer care in order to practice safely. They anticipated a specific focus on grading, staging, pathophysiology and treatment to foster their workplace learning as a novice cancer nurse.… gave me a better understanding of the patient experience, how things work at the doctor’s end and allowed me to start thinking about how this might impact the patient at different stages, understanding better treatment options in relation to their specific disease and stage. (Program Participant Interview 4)The partnering approach provided a holistic learning package where program participants were supported to critique their knowledge, skills and practice and work towards assessment close to their daily work. It became a unique learning experience, with knowledge being consolidated through regular feedback sessions and onward opportunities for assistance and clarification. Access to specialist cancer nurses facilitated a sound understanding of the patient experience including diagnosis, testing and treatments.…when my patient comes to have cancer treatment, I will look at the diagnosis and find out what stage they are. Then I will find out what type of diagnostic tests they needed to be diagnosed. Then link this to why the doctor has chosen the exact treatment for this specific patient. (Program Participant Interview 3)Interactive modules and assessment situated in the students’ context guided their education to generate experiences for further research. The case study presentations created spaces for the students to hear from each other’s experiences. Importantly, students linked their growing knowledge base to understanding the patient’s context. This supported practice confidence, interprofessional relationships and the ability to provide safe person-centred care.This partnership heralds the future of early nursing career development, linking industry and academia; and has the capacity to strengthen nursing professionalism. (Working Group action meeting note reflection)For many involved, the partnership across the three institutions was considered a watershed. The university-industry-partnered education program accomplished an innovative approach to design and delivered an authentic postgraduate education program in cancer care services.Students also gained a relevant understanding of cancer nursing to support their care provision as novice practitioners. Notably, the sharing of new knowledge, resources and the new relationships established will be invaluable to future collaborations.Developing nursing education programs in isolation prevents optimal creation of quality sustainable postgraduate education for the future nursing workforce. This co-creation requires a fundamental rethink of how industry and university partnerships work to deliver products that support student learning and enable safe, educated clinicians. Despite their mutual interest, there is a dearth of literature that explores how these two sectors can partner to deliver postgraduate education. Yet, recognising and working with the different perspectives from each group can facilitate a shared understanding and innovative solutions . Co-creaadd-on value of capacity-building which occurred as members worked closely across the two sectors. The Nurse Educators gained a deeper understanding of formal assessment requirements for the Australian Qualifications Framework as they worked with the university partner. University partners also raised collective awareness for what volumes of learning students and industry valued. Students created their own community of practice through the blended learning and close workplace learning approaches [The partners in this study recognised that existing postgraduate education processes are not beneficial or meeting nurses’ postgraduate education needs. As these partnerships take courage to appreciate and work with the nuances of each group, there is capacity to skilfully adapt to achieve an alignment of learning for all and enhance workplace safety. Building communication between the groups is essential for members to work with their strengths . Our resproaches . This waThe Framework enabled the partners to adapt to the complexity of the healthcare environment. Shared ownership within the partnership supported each Project Team member to use innovative ways of keeping their wider participants/groups connected and informed as the research progressed. As highlighted in the work of Gustafsson et al., , ProjectThe University-Industry Integration Framework provides a unique guide for academic, industry and student connections in postgraduate education. While there are upfront development costs in co-designing a Program, it was designed to support industry to deliver education for beginning practitioners to be safe at the bedside. Once the Program is established, the ongoing revision and update is anticipated to occur only every three years. The ongoing profession and university benefit lies in the uptake of life-long learning and subsequent enrolment in postgraduation nursing courses. This research was supported with project funding, education providers may create opportunities through established industry relationships or new partnerships. A guide for University-Industry Integration (2021) was developed and is available from the Queensland Government website . Future This research provides an exemplar for health services to initiate an effective partnership to improve education for novice nurses. High quality, relevant education in postgraduate nursing specialties cannot be effectively delivered by either universities or industry in isolation, this research provides implications for delivery of future sustainable education. The value of the framework guiding this project allow for adaptation to meet the needs of the partnership. That is, the industry partner can amend to join with any university provider, and the university may also deliver a co-design approach with private industry partners.The commitment and investment of established partnerships clearly enhanced this research, though this may not be the case for others embarking on similar ventures as trust and an understanding of ‘ways of working’ need to be established upfront. The timing of the program’s delivery was not flexible, and this created challenges for stakeholders. Short notice was impacted by advanced rostering of participants to clinical duties and limited preparation time and delivering the program early in the year meant that most of the work was undertaken over December and January. These are known to be busy times for health industries employing new graduates and for universities preparing for semester one courses in Australia.Due to the small sample of program participants, results need to be viewed with caution and may not be generalisable for developing future postgraduate education programs. As purposeful sampling was used, we acknowledge this raises risk of bias and therefore reduces generalisability to all nurses. A further limitation is this research took place at two large metropolitan health services and a metropolitan university that were located in close proximity. This allowed some face-to-face work, which may have enhanced trust development. This research did not examine the costs associated with developing and delivering nursing education in partnership, and this is likely to be a determining factor in the replication and sustainability of this initiative. The study was undertaken through a global pandemic, so collaborations and deliverables were second to pandemic health service workloads and priorities.University and industry partners can apply the University-Industry Integration Framework  to achieEmbarking on the postgraduate cancer care education program was not without the hindrances of workloads, timing, staff availability, completing legal contractual agreements in a timely way, and recruiting from unique health service areas. Further research needs to be undertaken to evaluate the benefits, financial and job satisfaction to stakeholders. Overall, stakeholders were highly satisfied with co-designing a postgraduate education program for cancer care nurses, leading to enhanced collaboration between university and industry partners and increased knowledge and skills in cancer care program participants.Below is the link to the electronic supplementary material.Supplementary Material 1


Uchida & Yamasaki  have developed a universal prevention program to enhance understanding and regulating others’ emotions for elementary and junior high school students. In recent years, affect and emotions are popular research topics in the domains of psychology and brain science. Most research has thus far focused on the effects of explicit affect on health and adjustment. However, an increasing number of studies have started to examine the effects of implicit affect on psychological outcomes. Although the program was developed for enhancing coping of explicit emotions for health and adjustment, the effectiveness needs to be examined also in terms of implicit affect.The purpose of this study was to examine the effectiveness of this program in terms of implicit and explicit affect.Participants were 6th-grade children in a public elementary school in Japan. The final sample was 61 children (32 boys and 29 girls). Participants completed a battery of two questionnaires just before (Time 1) and just after (Time 2) the intervention program. The questionnaires were the Implicit Positive and Negative Affect Test for Children (IPANAT-C) for assessing implicit positive and negative affect (IPA and INA) and the Japanese version of the Positive and Negative Affect Schedule for Children (PANAS-C) for measuring explicit positive and negative affect (EPA and ENA). The universal intervention program that was one of the programs we developed for children’s health and adjustment was implemented over four regular classes targeting all children in their homeroom classes.Fs  = 6.82 and 4.54, p < .05, respectively), revealing in the post hoc tests that they significantly increased at the post-intervention period. In contrast, regarding ENA and INA, there was no significant main effect of periods. Moreover, regarding EPA, there was a significant main effect of sex. No significant period x sex interactions were found in any affect.Data were analyzed by 2 (pre-intervention and post-intervention periods) x 2 (boys and girls) analyses of variance (ANOVA) with the hoc tests using Holm corrections. First, regarding the EPA and IPA, there was a significant main effect of periods (These results revealed that the current program was effective in enhancing explicit and implicit positive affect. On the other hand, no significant effects were found in enhancing explicit and implicit negative affect. The necessity of future research that will examine the sustainability of the effectiveness of the program is discussed, along with several limitations.None Declared


Work in embryonic stem cells has shown that the splicing factor Quaking (QKI) regulates alternative splicing during cardiomyocyte differentiation. However, the relevance and function of QKI in adult cardiomyocytes remains unknown. In this study, we aim to identify the We generated mice with conditional deletion of QKI in cardiomyocytes by the Cre-Lox system. Mice with cardiomyocyte-specific deletion of QKI died during the foetal period (E14.5), without obvious anatomical abnormalities of the heart. Adult mice with tamoxifen-inducible QKI deletion rapidly developed heart failure associated with severe disruption of sarcomeres, already 7 days after knocking out QKI. RNA sequencing revealed that QKI regulates the alternative splicing of more than 1000 genes, including sarcomere and cytoskeletal components, calcium-handling genes, and (post-)transcriptional regulators. Many of these splicing changes corresponded to the loss of muscle-specific isoforms in the heart. Forced overexpression of QKI in cultured neonatal rat ventricular myocytes directed these splicing events in the opposite direction and enhanced contractility of cardiomyocytes.Altogether, our findings show that QKI is an important regulator of the muscle-specific alternative splicing program that builds the contractile apparatus of cardiomyocytes.  RBM20 is the most studied splicing factor in the heart, which has been shown to regulate networks of splicing events, particularly in sarcomere and calcium-handling genes.3–5 Mutations in RBM20 are a frequent cause of familial dilated cardiomyopathy (DCM) which highlights the functional impact of aberrant splicing in the heart.7 Loss-of-function studies in mice also uncovered roles for other muscle restricted-splicing factors, namely RBM24 and RBFOX1 in the heart,9 but the list of RNA-binding proteins (RBPs) controlling alternative splicing in the heart is far from complete.Alternative splicing, the process by which exons from one mRNA can be spliced in different arrangements, is a crucial mechanism to expand protein diversity. It is now established that more than 95% of all human genes undergo alternative splicing to produce protein isoforms that are functionally or structurally specialized for the cell type in which they are expressed.10 QKI has three main isoforms, i.e. QKI-5, QKI-6, and QKI-7, that differ in the C-terminal region. QKI-5 is the predominant isoform in the heart and is the only one with a nuclear localization signal, which allows it to act as a splicing factor by binding nuclear pre-mRNAs.11 QKI-6 and QKI-7 are cytosolic proteins, and they are mainly involved in binding mRNAs outside of the nucleus to control their stability.12 The role of QKI in the nervous system has been well studied, in fact, the name ‘quaking’ stems from the phenotype of the ‘quaking viable’ mouse model that has reduced levels of myelin in its nervous system, leading to a shaking behaviour, with frequent seizures.13–15 In the heart, the function of QKI is not fully understood. Its activity in cardiomyocytes has been associated with a better outcome after ischaemia/reperfusion (I/R)17 and after doxorubicin toxicity.18 Systemic loss of QKI in mice (using the betaGeoQki mouse line) resulted in embryonic lethality around Day E9.5–E10.5, which was attributed to the failure of blood circulation in the yolk sac, in which vascular remodelling was impaired.19 Other abnormalities that were observed at this embryonic stage were open neural tubes, incomplete embryonic turning, and pericardial effusion, while the hearts seemed normal.19 In a subsequent study using the same betaGeoQki mouse line, several splicing changes were detected in the hearts of the KO embryos.20 Work in human embryonic stem cell-derived cardiomyocytes (hESC-CM) indicated that QKI is required for the proper splicing of genes involved in myofibrillogenesis, such as ACTN2, NEBL, and TTN.20 Despite this first evidence for splicing regulation during differentiation of cardiac progenitors to early cardiomyocytes and in the specification of cardiac mesoderm,21 the functional relevance of QKI-mediated splicing in the embryonic and adult heart remains unknown.The splicing factor Quaking (QKI) is a member of the Signal Transduction and Activator RNA (STAR) family of RBPs, which is ubiquitously expressed with highest levels in brain and heart.In this study, we investigated the function of QKI in the mouse heart by selectively deleting QKI in cardiomyocytes. To circumvent embryonic lethality due to impaired yolk sac development and to be able to ascertain a cardiomyocyte-autonomous role for QKI, we utilized the Cre-LoxP system to conditionally delete QKI from cardiomyocytes. Mice with cardiac-specific deletion of QKI died during the early foetal stages (E14.5), without obvious structural abnormalities of the heart. In the adult myocardium, loss of QKI (using the tamoxifen-inducible MerCreMer) induced dilation of the ventricles and a rapid decline in cardiac function, which was associated with severe disruption of sarcomere organization. RNA sequencing revealed splicing changes in more than a thousand genes in the adult QKI KO hearts, a large subset of which represents muscle-specific isoforms. While deletion of QKI in adult myocardium induced a rapid heart failure phenotype, we also show that forced overexpression of QKI in neonatal cardiomyocytes is able to enhance sarcomere contractility. Altogether, we are the first to show that QKI is a crucial splicing factor in the adult heart to maintain a muscle-specific splicing program.2.2.1Animal studies were approved by the Institutional Animal Care and Use Committee of the University of Amsterdam and carried out in compliance with the guidelines of this institution and the Directive 2010/63/EU of the European Parliament. Animal husbandry was performed by the Animal Research Institute AMC.Qki were generated by crossing the previously generated Qki-floxed mice13 with the conditional Myh6-Cre (Jackson Laboratory stock #011038) and with the tamoxifen-inducible Myh6-MerCreMer (Jackson Laboratory stock #005657)22 in C57BL/6N background. In all experiments, mice had a single copy of the Cre or the MerCreMer (MCM) recombinase allele. Males and females were included in the experiments.Cardiomyocyte-specific knock-out mice of MerCreMer recombinase activity, a tamoxifen solution  was injected intraperitoneally for 4 consecutive days  in 12–17-week-old mice .For inducing Echocardiography and electrocardiogram (ECG) recordings were performed as indicated in the 2.2Table S1.For adult mice, total RNA was extracted from the left ventricle by using TRI reagent  following the manufacturer’s instructions. For E12.5 embryos, RNA from both ventricles was extracted using the kit ReliaPrep RNA Miniprep Systems . RNA quality was determined using the Agilent RNA 6000 Nano Kit and the Agilent 2100 Bioanalyser. All samples had an RNA Integrity score ≥8. cDNA synthesis and (q)RT–PCRs were performed as described in the Table S2.Western blotting and histology were performed as depicted in the 2.3RNA from left ventricle samples of five adult ciQKI WT and five adult ciQKI KO mice, and four cQKI WT and four cQKI KO E12.5 ventricles was used for paired-end RNA sequencing. Details of library preparation, RNA sequencing, and data analysis are found in the 2.423 (Bioconductor release 3.13). Transcripts Per Million (TPM) for each gene was also calculated. Genes with TPM value ≥0.5, an absolute log2 fold change cut-off ≥1.0, and adjusted P-value cut-off ≤ 0.05 were deemed significantly differentially expressed.Differential gene expression analysis was performed using the R Bioconductor package, DESeq224 (Bioconductor release 3.13). Only genes with a TPM value ≥ 0.5 were considered. Exons with an absolute log2 fold change cut-off ≥ 1.0, and adjusted P-value cut-off ≤ 0.05 were deemed significantly differentially spliced.Differential exon usage analysis was performed using the R Bioconductor package, DEXSeq2.5Isolation, transduction, culture and contraction measurements of neonatal rat ventricular myocytes (NRVMs) are depicted in the 2.6P < 0.05 was considered statistically significant.Data were analysed using GraphPad Prism 9 . Data are presented as mean ± standard deviation unless differently stated. Results were analysed with appropriate statistical tests, as indicated in the respective figure legends. A value of 3.3.1Qki-floxed mice13 with mice carrying the Myh6-Cre transgene,25 in which the onset of Cre expression is as early as embryonic stage E7.5.26 In this conditional knockout allele, exon 2 of Qki is flanked by loxP sites. Exon 2 encodes part of the KH RNA-binding domain, common to all QKI isoforms, and its deletion generates a frameshift, resulting in a null allele13 . We refer to the homozygous fl/flQki; TgMyh6-Cre as conditional QKI knock-out mice (cQKI KO), fl/wtQKI; TgMyh6-Cre as conditional QKI heterozygous mice (cQKI HET), and wt/wtQki; TgMyh6-Cre as conditional QKI wild-type (cQKI WT) mice.To investigate the function of the splicing factor QKI in the heart, we generated a cardiomyocyte-specific QKI knockout mouse model by crossing Table S3). To pinpoint the timepoint of embryonic death, we collected embryos at different stages and found cQKI KO embryos at expected Mendelian ratios until Day E14.5 (Table S4). From that timepoint onwards, all cQKI KO embryos were dead or reabsorbed. At E14.5, cQKI KO embryos displayed pericardial effusion and signs of necrosis . No differences in embryo and heart size were detected between the three genotypes at E10.5 and E12.5 . Furthermore, Hematoxylin & Eosin (H&E) stainings did not reveal obvious defects in cardiac anatomy at Day E10.5, E12.5, or E14.5 , suggesting the QKI cKO hearts were functionally compromised, not anatomically.cQKI HET mice were born at the expected Mendelian ratios and were viable, but cQKI KO mice were not found at birth, indicating that QKI expression in cardiomyocytes is essential during embryonic development . Of note, QKI is also expressed at lower levels in other cell types of the heart , which may contribute to the background expression of QKI in the knockout hearts. Next, we validated the knock-out approach by immunostainings for QKI and the cardiomyocyte marker cardiac troponin I (cTNI). In WT embryos, we could detect QKI protein in cardiomyocytes of the four chambers of the developing heart, however, QKI expression was clearly reduced in the QKI cKO embryos at Days E12.5 and E14.5 . At E14.5, residual QKI expression was observed particularly in cells of the compact myocardium and the middle axis of the heart. We do not have a clear explanation for the pattern of remaining cells, but it could be explained by cardiomyocytes not expressing the Cre recombinase or by other cell types in that area expressing QKI.To assess the deletion efficiency by Myh6-Cre, we performed qRT–PCR on RNA isolated from E12.5 ventricles using primers spanning exons 2 and 3 of 27 Overall, this indicates that QKI regulates key processes in cardiomyocytes during development.In conclusion, the heart-specific cQKI KO mice were unable to progress beyond embryonic day E14.5. This is substantially later than the lethality caused by whole-body ablation of QKI, which occurs around E9.5, and which has been attributed to abnormalities in vascular remodelling of the yolk sac.3.2Qki-floxed mice with mice carrying the Myh6-mER-Cre-mER transgene (TgMyh6-MCM).22 To induce the Cre-loxP recombination, we injected tamoxifen intraperitoneally in 12- to 17-week-old WT , ciQKI HET , and ciQKI KO  mice for 4 consecutive days . We noted that within 5 days after the last tamoxifen injection a subset of ciQKI KO mice started to become inactive, while the tamoxifen-injected WT and ciQKI HET mice appeared healthy. As low physical activity can be indicative of heart failure in the ciQKI KO group, we terminated the experiment 7 days after the last tamoxifen injection. qRT–PCR on heart tissue revealed that Qki mRNA levels were reduced by 80% in the ciQKI KO and by 40% in the ciQKI HET hearts, 7 days after the last tamoxifen injection . QKI protein levels were reduced by 80% in ciQKI KO mice, as shown by western blotting .To overcome embryonic lethality and to be able to study QKI function in the adult heart, we created a cardiac-specific tamoxifen-inducible QKI (ciQKI) knockout mouse line by crossing the Figure and F). Echocardiography confirmed a heart failure phenotype with severe systolic dysfunction in ciQKI KO mice. Specifically, ejection fraction (EF) was reduced by ∼45% in ciQKI KO mice compared with the cQKI WT, and this was associated with a ∼14% increase in left ventricular (LV) internal diameter during systole . No differences in heart weight and ECG parameters were found in ciQKI KO mice .Inspection of global cardiac morphology and histological analysis on H&E-stained sections, 7 days after the last tamoxifen injection, showed enlargement and thinning of the ventricles  and evaluated them by confocal microscopy. This showed, besides a nearly complete loss of QKI protein, that the organization of the myofibrils was disrupted in the ciQKI KO hearts. Immunohistochemistry for alpha-actinin  and titin  yielded a comparable picture of disorganized myofilaments . Of note, in the ciQKI KO hearts, we observed variation in the extent of filaments disruption, as some regions were more affected than others. No disrupted filaments were observed in the WT or ciQKI HET hearts. Ultrastructural analysis of the adult myocardium using transmission electron microscopy showed these defects in greater detail . Whereas WT hearts exhibited intact sarcomeres with clearly defined Z-discs and well-organized myofibrils, ciQKI KO hearts displayed degenerated sarcomeres, and this was associated with glycogen deposits around sarcomeres .Next, we performed double immunocytochemistry for QKI and desmin (an intermediate filament connecting sarcomeric Z-discs to the cytoskeleton) on adult ciQKI WT, ciQKI HET, and ciQKI KO hearts  and in E12.5 embryonic hearts (four WT and four cQKI KO). To be able to analyse gene expression and splice isoforms, cDNA libraries were subjected to ultra-deep sequencing, with around 250 million paired-end reads per sample.Figure , Online data 1). Among the up-regulated genes, there were markers of cardiac stress, such as Acta1, Casq2, Ccn2, Myh7, Nppb, and Rcan1. Among the down-regulated genes, there was Actn2, a previously characterized target of QKI in hESC-CM.20 Western blotting was performed to confirm the down-regulation of ACTN2 and the up-regulation of CAMK2D protein in the ciQKI KO hearts . To assess alternative splicing changes in the adult hearts, we detected differential exon usage with DEXSeq24 and found 1023 differentially spliced genes in ciQKI KO hearts. Specifically, 927 exon bins were up-regulated and 1089 exon bins were down-regulated in the ciQKI KO hearts . Among the mis-spliced genes, there were multiple genes directly involved in cardiac contraction, such as Atp2a2, Cacna1c, Cacna2d2, Sorbs2, Vcl, Tpm1, or Ttn.We first performed differential gene expression analysis on the adult hearts, and we found 409 genes significantly up-regulated and 444 down-regulated in the ciQKI KO hearts . Principal component analysis on exon usage and gene expression in the embryonic and adult hearts shows that splicing changes are more distinct than gene expression changes at both timepoints . Although there was a smaller number of differentially spliced transcripts in the E12.5 cQKI KO embryos compared with the adult ciQKI KO hearts (148 in embryos vs. 1023 in adults), a substantial proportion of these mis-spliced genes (68%) did overlap with the ones observed in the adult QKI KO hearts . A scatterplot depicting the correlation between differentially spliced exons in embryo and adult hearts also reveals that the fold changes are larger in the adult heart . Pathway analysis of the 1023 mis-spliced genes in the adult ciQKI KO heart showed enrichment for gene ontology ‘biological process’ terms related to muscle biology, such as ‘sarcomere organization’ and ‘striated muscle cell development’ .RNA-seq in the ventricles of E12.5 cQKI WT and cQKI KO mice also yielded differences in gene expression and splicing , regulatory kinases (Camk2d and Akap9), calcium channels (Cacnb1 and Ryr2), and transcriptional and splicing regulators . Interestingly, ciQKI HET hearts did not show intermediate splicing changes, except for a subtle difference in Ttn and Ryr2 splicing. Sashimi plots were generated to visualize splicing changes at the transcript level . Without QKI, exon 4 of the Z-disc component Ldb3 (/ZASP/Cypher) is partially skipped and exons 5–7 are aberrantly included; exons 25 and 26 of the Z-disc component nebullete (Nebl) are skipped; exons coding for Z-repeats of titin (Ttn) are skipped (exons 11–13);28 and the terminal exons of alpha-tropomyosin (Tpm1) are alternatively spliced.29 In conclusion, QKI depletion in the embryonic and adult heart leads to massive splicing changes in genes crucial for muscle biology.To validate the splicing changes observed by RNA-seq, we performed RT–PCR in adult heart tissue  for a panel of genes involved in muscle biology. We could confirm robust splicing changes for all genes tested. As shown in 3.430 We compared the presence of motifs surrounding the differentially spliced exons in adult ciQKI KO hearts with motifs surrounding an equal number of exons not differentially spliced. We limited our analysis to the proximal upstream and downstream regions, encompassing 200 nt in the flanking introns and 10 nt within the exon. As anticipated, the binding motif of QKI (ACUAAY) was the most enriched sequence in the regions flanking the mis-spliced exons . Interestingly, one of the other enriched sequences was the binding motif for RBFOX1. Like QKI, RBFOX1 is a splicing regulator with enriched expression in brain and striated muscle. As multiple splicing targets are shared between RBFOX1 and QKI ,8 it is conceivable that they cooperate in splicing networks.To examine the presence and location of putative QKI-binding sites within the transcript, we performed an unbiased enrichment analysis of RBP motifs using ATtRACT, a database for RBPs and associated motifs.31 Also for QKI it has been reported that the location of ACUAAY motifs within the transcript determines whether QKI acts as a repressor or as an activator of exon inclusion.32–35 Therefore, we quantified and compared the presence of QKI-binding sites in the proximal upstream and downstream regions of QKI-activated (exons skipped in the ciQKI KO heart), QKI-repressed (exons included in the ciQKI KO heart), and QKI-independent exons  . We observed an increase in the number of motifs upstream of the QKI-repressed exons and an increase in the number of motifs downstream of QKI-activated exons, further illustrating how the positioning of QKI relative to the exon determines whether the exon is included or excluded in the final transcript. Examples of QKI-activated exons with downstream ACUAAY motifs (exon 4 of Akap9 and exon 101 of Dst) are shown in Figure . Mutually exclusive exons 3a/3b of Slc25a3 and 10a/10b of Rbfox2 were found to contain the motif upstream of the QKI-repressed exon and downstream of the QKI-activated exon.Splicing factors can have opposing effects on exon usage, depending on whether they bind to their motifs up- or downstream of the exon.3.532 and we wondered whether this is also the case for QKI-regulated isoforms. First, we checked whether the QKI-dependent splicing events that we observed occurred exclusively in the heart or were present in other tissues as well. To do so, we performed RT–PCR on a tissue panel of adult mice, consisting of ‘non-muscle’ tissues , tissues with a high content of smooth muscle cells , and striated muscle tissues . To be able to interrogate isoform expression across multiple tissues, we selected only those QKI targets that are expressed in a ubiquitous manner. As can be appreciated from Figure , the picture emerges that QKI depletion prevents the expression of muscle-specific isoforms. For instance, the isoform of Slc25a3 containing exon 2A is highly enriched in striated muscle, but in the ciQKI KO hearts, this isoform is completely lost. The same holds true for the muscle isoforms of Tmed2, Akap9, and Nebl. Other QKI-dependent splicing events, like Ank3 and Cacnb1, are not restricted to striated muscle types but are expressed in smooth muscle tissues as well. Interestingly, loss of QKI in the heart often results in the expression of isoforms that are normally present in the cerebral cortex , which is remarkable since QKI is also highly expressed in the nervous system .Splice isoforms are often produced in cell-type and/or developmental-specific manner,35 tissues with relatively high levels of QKI. Interestingly, Qki mRNA levels increase in the heart throughout development, whereas in other tissues  Qki mRNA levels seem to decrease over time .36 We used our RNA-seq data to investigate whether loss of QKI in the adult heart would lead to the expression of more embryonic splice isoforms. As such, we compared QKI-dependent splicing events that we identified in the adult hearts with the isoforms present in wild-type E12.5 hearts. As shown in the heatmap in Figure , there were 55 genes that switched to an embryonic isoform in the absence of QKI. Four examples of QKI-dependent genes that reverted to an embryonic isoform are illustrated in the bedgraphs of Figure . Overall, our findings illustrate that QKI controls the expression of numerous muscle- and developmental-specific splice isoforms in the heart. This puts QKI at the centre of co-ordinated splicing networks that regulates cardiac tissue identity and cardiac development.Alternative splicing is a fundamental mechanism for organ development, especially in the brain and heart,3.6Figure ), and this was associated with an increase in cell size . QKI expression was increased approximately by 2.5-fold at the protein level  and RT–PCRs showed a shift in splicing opposite to the one observed in the ciQKI KO hearts . This shift towards more muscle-specific isoforms consisted of increased skipping of exon 16 in Ank3, higher levels of the isoform δ-C of Camk2d, more inclusion of exon 4 and skipping of exons 5–7 in Ldb3, and increased skipping of exon 2a in Myocd.Given our observation that QKI regulates a muscle-specific alternative splicing program that shapes the contractile function of cardiomyocytes, we wondered whether overexpression of QKI in NRVMs (i) would be sufficient to push the expression of adult QKI-dependent splicing isoforms and (ii) would affect contractility of cardiomyocytes. We limited our studies to QKI-5, since it is the predominant isoform in the heart and the only one with a nuclear localization signal. We made use of a bicistronic lentiviral vector to simultaneously express QKI-5 and GFP after transduction. As a control, we overexpressed GFP alone. Five days after transduction, NRVMs presented QKI protein overexpression in the nucleus . Representative cell contraction transients of GFP- and QKI-5-transduced NRVMs are depicted in Figure . Interestingly, QKI-5 overexpression increased the peak height of the contraction transients, and the departure and return velocities (i.e. the maximum velocities reached on the contraction and relaxation phase of the transient) . In summary, our in vitro experiments indicate that QKI-5 overexpression is sufficient to increase muscle-specific splicing and enhance the contractile properties of neonatal cardiomyocytes.To test whether the splicing changes induced by QKI-5 overexpression affected the contractility of the NRVMs, we made use of CytoCypher, a high-throughput microscopic system able to measure cardiomyocyte contraction by calculating pixel displacement over time. We performed the measurements on Day 4 after transduction, before the increase in cell size occurs , cytoskeletal genes , calcium-handling genes , and (post-)transcriptional regulators . Forced overexpression of QKI-5 in cultured neonatal cardiomyocytes directed these splicing events in the opposite direction, which led to increased contractility of cardiomyocytes.In this study, we investigated the function of QKI in the heart. We demonstrate that QKI is required in cardiomyocytes to maintain normal cardiac function during development and in the adult heart. Most strikingly, loss of QKI in adult cardiomyocytes rapidly induces heart failure, with LV dilation and severe disruption of sarcomeres, and this occurs already 7 days after deleting QKI from the heart. We show that QKI regulates the integrity and structural composition of sarcomeres, at least in part, by controlling alternative splicing of hundreds of protein-coding genes. Analysis of splicing events in ciQKI KO hearts revealed defective splicing in numerous sarcomere genes  pathway.20 Within Ttn, exons 11–13 are skipped in the absence of QKI. These exons are located in the Z-band region of TTN and alternative splicing in this region generates a variable number of Z-repeats, presumably changing the binding sites for the scaffold protein ACTN2, and thus influencing the mechanical stability of the sarcomere.38 In Tpm1, we observed splicing changes in the terminal exons that define TPM1 affinity for actin. Specifically, in the absence of QKI, we observed loss of striated muscle-specific exons 9a and 9b, in favour of exons 9c and 9d. Exons 9a and 9b encode for specific amino acid residues that are critical for the interaction of troponin with tropomyosin on the thin filaments.39 For CaMKIIδ, we observed a switch from the δ-C to the δ-A isoform, resulting from the inclusion of exons 15 and 16. The δ-C isoform is located in the cytoplasm, where it phosphorylates RyR2 and phospholamban, while the δ-A isoform is associated with the intercalated disc and T-tubules, where Ca2+ channels and activated CaMKIIδ are concentrated.41 The increased δA levels may lead to Ca2+-handling disturbances in QKI KO cardiomyocytes, but this warrants further investigation. The transcription factor MEF2C is a crucial regulator of myogenesis by controlling the expression of genes that encode a network of structural proteins. Here, we show that QKI is required for the expression of the MEF2C isoform containing the mutually exclusive α2 exon. The α2 isoform has potent myogenic activity, whereas the isoform containing α1 does not.42 It is conceivable that mis-splicing of MEF2C in the QKI KO hearts contributes to the sarcomere phenotype by reducing the transcription of muscle genes.Because QKI regulates the splicing of such a large number of developmental- and disease-relevant genes, it is currently not possible to pinpoint how each defectively spliced gene contributes to the QKI KO phenotype we observe. Nevertheless, the functional relevance of a number of these splice isoforms has been reported previously. For instance, skipping of exon 8 in 20 Genes involved in contractile function were found to be alternatively spliced in the absence of QKI, including ACTN2, NEBL, ABLIM1, PDLIM5, and TTN. Consistent with this work, Fagg et al.21 compared alternative splicing events in three cell lineages differentiated from hESCs , and showed that QKI plays a critical role in the specification of cardiac mesoderm and the formation of cardiomyocytes. One of the studied splicing targets in that study was Bridging Integrator1/Amphiphysin 2 (BIN1), a protein known for its role in cardiac development and myofibrillogenesis.44 In our QKI mouse models, we did not find altered splicing or gene expression of Bin1, neither in the adult QKI KO hearts, nor in the embryonic QKI KO hearts. This may relate to the timing of QKI deletion using the Myh6 promoter, which starts to become active from E7.5 onwards, which is after cardiac mesoderm formation. In conclusion, these human stem cell studies uncovered a role for alternative splicing in early lineage-specific gene expression and implicate QKI as a cardiac mesoderm-enriched RBP required for the differentiation of cardiomyocytes. A role for QKI in myofibril formation is, however, not limited to cardiomyocytes, as recent studies also provided evidence that QKI controls the formation of myofibrils of skeletal muscle in zebrafish45 and regulates a splicing program during smooth muscle development.46 In addition, QKI deficiency in muscle stem cells using a conditional QKI mouse model in combination with the Pax7-Cre driver was shown to result in loss of the myogenic progenitor cell population and muscle regeneration defects.47Two recent studies in human stem cells already uncovered a critical role for QKI in cardiomyocyte differentiation. Chen et al. employed CRISPR/Cas9 gene editing to delete QKI from human embryonic stem cells (hESC). By differentiating these stem cells into cardiomyocytes, they revealed that QKI is essential for the formation of myofibrils during cardiomyocyte differentiation.48 Maintenance of sarcomeres is a complex process, in which new proteins are constantly incorporated into sarcomeres to replace old ones without compromising mechanical function. The half-lives of sarcomere proteins in the heart are estimated in the order of days to weeks.48 Our finding that sarcomeres in the adult heart degenerate already 1 week after QKI depletion suggests that sarcomere maintenance is compromised by the incorporation of aberrant protein isoforms. This is particularly relevant, as there is evidence that QKI expression is lost during multiple forms of heart disease. This was shown in a mouse model of doxorubicin-mediated cardiotoxicity,18 in a rat model of ischaemia/reperfusion,16 in the diabetic mouse heart,49 and in human heart failure.18 Accordingly, reduced levels of QKI may very well contribute to the disease mechanism of sarcomere dysfunction and decreased contractility in heart failure. In this light, it has been shown that more than 1000 transcripts express aberrant isoforms in ischaemic and dilated cardiomyopathies, indicating that alternative splicing is a highly regulated process and an important layer of gene regulation during pathological remodelling of the heart.51 It will be very interesting to see whether therapeutic up-regulation of QKI using gene therapy in acquired heart disease will be able to shift splicing towards more cardioprotective isoforms and improve cardiac remodelling and function. This is strengthened by our observation that QKI overexpression is sufficient to increase contraction and relaxation velocities of NRVMs. Moreover, Gupta et al. recently provided evidence that therapeutic overexpression of QKI is beneficial in a mouse model of doxorubicin-mediated heart failure.18 They showed that adeno-associated virus serotype 9 (AAV9)-mediated overexpression of QKI attenuates apoptosis and atrophy of cardiomyocytes in this model. Alternative splicing of sarcomere genes was not investigated in this study, instead certain circRNA molecules were identified as mediators in the observed anti-apoptotic effect. A role for QKI in cardiomyocyte apoptosis was also reported in a study by Guo et al., who showed that QKI inhibits ischaemia/reperfusion-induced cardiomyocyte apoptosis by regulating Foxo1 mRNA stability.16 Notably, we did not find altered expression or splicing of Foxo1 mRNA in our QKI KO datasets.Once assembled, cardiac sarcomeres require constant maintenance as the continuous contractions of the heart are accompanied by mechanical stress, which in turn predisposes proteins to misfolding and aggregation.3 RBM24,9 SRSF10,52 ASF/SF2,41 and RBFOX1,8 were shown to regulate alternative splicing of a large number of transcripts in the developing and/or adult heart. By comparing their splicing targets, the picture emerges that these splicing factors regulate a distinct, but overlapping set of transcripts. For instance, the switch in CaMKIIδ, from the δ-C to the δ-A isoform that we identified in QKI KO hearts, has also been found in RBM20 and ASF/SF2 KO mice.41 The inclusion of the mutually exclusive exons 3a/3b of the mitochondrial phosphate carrier Slc25a3 is regulated by QKI and RBM24.9 Splicing of TTN is regulated by RBM20 and by QKI; however, these factors control splicing of different exons. Whereas RBM20 is responsible for the inclusion of I-band exons, a region of TTN that is crucial for the elastic property of sarcomeres, QKI regulates the inclusion of Z-band exons, a region that is thought to be important for the mechanical stability of the sarcomere.37 We found that one of the most enriched sequences in the flanking introns of QKI-regulated exons is the binding motif for RBFOX1, a splicing regulator that is also enriched in brain and striated muscle. As multiple splicing targets are shared between RBFOX1 and QKI , it is conceivable that QKI and RBFOX1 engage in a complex regulatory network of alternative splicing.8 The full extent of how QKI cooperates and competes with other splice factors is yet to be determined. Finally, our observation that QKI also regulates alternative splicing of Rbfox1 itself adds another layer of complexity and warrants further exploration.It has become evident that alternative splicing occurs in co-ordinated networks, rather than in isolated events. Several splicing factors, including RBM20,20 and in the specification of cardiac mesoderm.21 Future studies are needed to investigate whether QKI dysregulation contributes to the mechanism of contractile dysfunction in heart disease. Specifically, the fact that QKI is down-regulated in human DCM,18 and our observation that forced overexpression of QKI enhances cardiomyocyte contractility, may open avenues for therapeutic strategies to adapt cardiac isoform expression and improve cardiac function in heart failure patients.In conclusion, we are the first to show that QKI acts as a major regulator of striated muscle identity in the cardiomyocytes of the adult heart, by directing the expression of muscle-specific isoforms of numerous pre-mRNAs. This extends previous studies showing a role for QKI in cardiomyocyte differentiation during the transition from cardiac progenitors to early cardiomyocytesP.M.A. and E.E.C. contributed to the experimental design, writing, and editing of the manuscript. P.M.A., S.A., E.N.S., I.v.d.M., and E.E.C. contributed to the acquisition of data, analysis, and interpretation. L.C.O., A.C.E., S.R., D.W.D.K., and V.M.C. provided experimental resources. S.R., D.W.D.K., V.M.C., Y.M.P., and E.E.C. revised the intellectual content of the manuscript. Y.M.P. and E.E.C. supervised the project. All authors read and approved the manuscript.cvad007_Supplementary_DataClick here for additional data file.


Generations Uniting through Sport (GUS) stimulates mutual understanding between youngsters and elderly people with sports activities. The project aims to promote intergenerational relations and increased physical activity by setting up an innovative and sustainable program. The concept for the GUS program is to identify, recruit, train and support a cohort of experienced practitioners. These professionals and volunteers will facilitate local-scale collaboration projects between youngsters and elderly people to increase their participation in physical activity and sport. The program will bring together young and old beneficiaries, through trained practitioners. The aim of the current research was to generate a state of the art overview of available evidence and practices that facilitate the development and implementation of intergenerational programs.The literature search consists of two parts. One part focuses on identifying scientific literature on working principles within intergenerational programs. The search will be performed in Google Scholar, Pubmed and PROSPERO and is restricted to literature from the year 2000 onwards and languages used within the consortium. Main search terms are ‘adolescent’, ‘elderly’, ‘intergenerational’, ‘activity’/’program’ and synonyms of these terms.The second part of the search is performed within the project consortium and focuses on good practices from national and local settings. Searches will be performed in grey literature and through personal networks in France, Spain, Bulgaria, United Kingdom and Slovenia.Literature analysis focuses on activities, target groups, involved stakeholders, behavioral change models, barriers and outcomes/impact.The literature search results in a set of principles for programs and activities tailored towards generations uniting through sport/movement in Europe. More specific, it synthesizes behavioral change models incorporating methodologies that motivate youngsters and elderly people to exercise together and reduces some of the motivational and structural barriers to intergenerational programs.The final results and conclusions will be presented at the conference.