Delirium is a frequent complication of hospital admission among older people. Multicomponent interventions which can reduce incident delirium by ≈one-third are recommended by the National Institute of Health and Care Excellence. Currently, a standardised delirium prevention system of care suitable for adoption in the UK National Health Service does not exist. The Prevention of Delirium (POD) system of care is a theory informed, multicomponent intervention and systematic implementation process which includes a role for hospital volunteers. We report POD implementation and delivery processes in NHS hospital wards, as part of a feasibility study.A comparative case study design and participatory, multi-method evaluation was performed with sequential six month preparatory and six month delivery stages. Six wards in five hospitals in Northern England were recruited. Methods included: facilitated workshops; observation of POD preparatory activities; qualitative interviews with staff; collection of ward organisational and patient profiles; and structured observation of staff workload.POD implementation and delivery was fully accomplished in four wards. On these wards, implementation strategies informed by Normalization Process Theory operated synergistically and cumulatively. An interactive staff training programme on delirium and practices that might prevent it among those at risk, facilitated purposeful POD engagement. Observation of practice juxtaposed to action on delirium preventive interventions created tension for change, legitimating new ways of organising work around it. Establishing systems, processes and documentation to make POD workable in the ward setting, enhanced staff ownership. ‘Negotiated experimentation’ to involve staff in creating, appraising and modifying systems and practices, helped integrate the POD care system in ward routines. Activating these change mechanisms required a particular form of leadership: pro-active ‘steer’, and senior ward ‘facilitator’ to extend ‘reach’ to the staff group. Organisational discontinuity (i.e. ward re-location and re-modelling) disrupted and extended POD implementation; staff shortages adversely affected staff capacity to invest in POD. Findings resulted in the development of ‘site readiness’ criteria without which implementation of this complex intervention was unlikely to occur.POD implementation and delivery is feasible in NHS wards, but a necessary context for success is ‘site readiness.’ Delirium is a frequent complication of hospital admission in older people . It is aIn the first research phase we developed a novel delirium prevention intervention and multifaceted implementation strategy, the Prevention of Delirium system of care (POD) that would be sustainable in NHS hospital wards . Our staPOD targets people at risk of delirium . It comprises actions summarised in protocols addressing ten modifiable risk factors associated with the development of delirium among vulnerable patients. Action is directed at changing ward practice with patients to optimise hydration and nutrition, reduce environmental threats, increase orientation to time and place, improve communication, support and encourage mobility, and effect better pain, infection and medication management. Intervention content is based on individual assessed need, and its form is flexible, for example, ‘stimulating activities’ could include music, games or reminiscence work, delivered individually or in groups. Since these practices are pertinent to optimising patient experience and recovery, systematic engagement of wards in delirium prevention has potential to enhance patient care.coherence, cognitive participation, collective action and reflexive monitoring . This included: observation of preparatory implementation activities ; informant conversations with implementation team members individually and together over time; collection of relevant preexisting documents  and new materials developed and used during delivery. Researchers observed ward practices  to capture ‘usual care’ pertinent to delirium and how it changed with POD. Qualitative interviews with ward staff and volunteers were carried out toward the end of delivery to explore perceptions of POD implementation, delivery and impact [total: 28]. The research team maintained a diary /events log to provide a contemporaneous account of unfolding processes and action in time: communication with teams , problems encountered, solutions arrived at, and relevant contextual features . We organised a one-day central workshop to share learning, build on what was similar and unique in different ward contexts and elicit participants’ perceptions of implementation.A ‘dependency-acuity-quality’ method was adopted at the start of implementation planning and during delivery to gauge its impact on ward staff workload , 21. ResInterviews and workshop proceedings were audio-recorded, transcribed fully, entered onto a database (NVivo 9) for initial coding, sorting and linking. Observations and conversations were captured in contemporaneous jottings, then written up as expanded accounts . QualitaQuantitative data were analysed using appropriate parametric and non-parametric statistical methods to give summary descriptions and investigate comparisons. Staff workload data analysis included investigation of the relationship between dependency, activity and other variables , 21.The study was approved by an NHS Research Ethics Committee (10/H1302/66). Before commencing fieldwork, meetings were organised with staff on each participating ward and information leaflets distributed; researchers were also available at designated times to answer questions about the study. Posters about the study were displayed on ward noticeboards and in staff rooms. Participation was voluntary and individuals could withdraw at any time.We describe features of organisational contexts within which POD was inserted and examine POD implementation and delivery. We consider whether the theory of change underpinning POD is supported by the findings, critically review feasibility and implications for a future randomised controlled trial to determine clinical outcomes.Five NHS hospitals in Yorkshire, UK, varying in size and socio-economic context were initially recruited Table .Table 1Profile of participating wards: [see Table : Of six wards recruited, ward 5 in Hospital D withdrew prior to commencing preparatory work, citing staffing difficulties. Among the remaining five study wards, three were care of older people and two were surgical orthopaedic. Bed occupancy was high: varying between 89 and 100%. Occupancy rates over 85% have been associated with regular bed shortages, periodic bed crises and increased numbers of hospital-acquired infections [fections ; and clifections .Table 2Several wards were understaffed, being most pronounced on care of older people wards, resulting in routine use of bank and agency staff to cover shifts. Based on UK Royal College of Nursing recommendations , ward 4 Patient profile [see Table : On all wards, among those ≥65 years, patients in advanced older age dominated. Patient dependency was high, particularly on care of older people wards (around 90% were dependent on staff for most of their care needs). Lower dependency on trauma orthopaedic wards (around two-thirds had high-dependency needs) reflected a wider age profile on these wards.Although change is ubiquitous in large, complex organisations like the NHS, there were additional sources of turbulence during the research. Most study wards moved physical location or changed their ward model immediately prior to or during POD implementation, as a consequence of local reorganisations.Identifying individuals to lead POD implementation involved discussions with hospital middle managers, practice development leads, clinicians, ward and voluntary services managers. Interest in participation was multiple and varied. Clinicians were keen to increase delirium awareness among nursing and care staff; senior nurses with specialist training in delirium viewed it as opportunity to improve care quality; senior ward staffs’ interest was sparked by volunteer involvement to enhance patients’ hospital experience; and voluntary services staff were enthusiastic about expanding volunteering. Melding these multiple interests toward a common purpose presented an implementation challenge.Negotiation centred on identifying at minimum a senior ward team member to facilitate the programme’s reach to ward staff; a voluntary services manager to act as the conduit to volunteer participation; and a middle manager/clinician with positional authority to mobilise human and material resources. Preparatory work in the first site suggested that although the contribution of these stakeholders was necessary to make implementation happen, it was insufficient to drive it; a pro-active site steer was essential. Among ward managers, the exigencies of day-to-day care delivery and managing patient flow constrained their capacity to enact the role; senior clinicians perceived their involvement as maintaining a supportive presence than being active participants; and voluntary services managers viewed their remit as task specific. In hospitals A and E and wards 1, 2 and 6, a matron/deputy matron came forward during early meetings to drive change. Clinical interest in delirium and positional authority shaped their emergence. Capacity for relationship building through vertical and horizontal networking between ward and directorate levels created a facilitative environment, and activated the change process. Whereas the research team initiated meetings, organised workshops and introductory events, it was the backstage work of these ‘drivers’ in negotiating with, and engaging relevant others that proved decisive in pursuing implementation as a collaborative enterprise. They co-ordinated the work of team members in developing, testing and refining systems and paperwork for assessing risk, developing delirium care plans and volunteer roles, and modifying exemplar tools for incorporation into ward/hospital systems. Dedicated time negotiated within their work space made enactment of the driver role feasible.This form of pro-active leadership proved difficult to achieve on other wards. Attempted in ward 3, it was not carried through. A working group had been established of relevant stakeholders led by a clinical manager, but it was primarily a discussion forum. It was assumed that the ward manager would take charge of implementation. Six months into the preparatory phase, little progress had been made: burdened by nursing staff vacancies, the ward manager engaged intermittently in the group. In ward 4, negotiations about participation were protracted – in a stop/re-start dynamic with periods of resurgent communication between. Staffing difficulties were cited as contributory factors. As a consequence, the research team introduced a modification: financial support to second a staff nurse for the equivalent of one day a week over six months to assist with practical POD preparatory tasks, intended to support but not substitute for, directorate and senior ward staff. Threaded through accounts of POD preparatory work are factors implicated in how this investment worked in particular local contexts and not in others.Implementation planning proceeded as intended in wards 1, 2, 3, and 6, albeit timescales were protracted in wards 3 and 6; it was pursued in ward 4 with some success in some aspects .The conduct of observations/audits by implementation teams varied. In wards 1 and 2, they were carried out by different professionals , their respective disciplinary lenses and seniority viewed as enhancing ownership. They were completed by the seconded staff nurse on wards 3 and 4, and on ward 6 by nurses at different levels of seniority and varied experience with older people. Nevertheless, the function observations performed was similar: creating tension for change through ‘seeing’ how practices militated against aspects of quality patient care pertinent to POD delivery:It’s been a massive learning curve for me … just in seeing things… there were some patients who didn’t eat because they were waiting so long that the meal was cold when it got to them and they didn’t want it …We were also so busy with the red tray system that patients who might need help but who didn’t have a red tray … could get missed.Staff nurse, Ward 4.Field note, Ward 2.The staff member reported that during her observation patients were sat up in bed for dinner and they could reach drinks; tea was poured before the meal arrived and was cold when dinner was served. The ensuing discussion referred to a decision by middle managers to deploy domestic staff in making drinks: because of protected meal times they had to stop cleaning. The meeting concluded that the ward manager would ask domestic staff to help give out meals (something they were not currently doing) and provide a hot drink after the meal.Observations highlighted unintended consequences of decisions taken to optimise efficiencies in deploying staff. The following was recorded by the researcher in a field note during a feedback meeting after staff had completed their observations.Some changes considered by implementation teams as pertinent to delirium prevention, impacted on patient care generally. For example, the pace of work early morning was observed as an extended period of frenetic activity. Ward and visiting staff were described as simultaneously launching into bays and rooms: healthcare assistants to wash and dress patients; nurses to do clinical observations; domestic staff to serve breakfast; doctors to do ward rounds. They then swept out and moved on, leaving patients alone, tired and dozing. Discussed and introduced was an approach to ‘slowing the pace’ by extending the time during which morning work routines were completed, allowing staff time to spend with patients.Creating tension for change among implementation teams was necessary but insufficient. Contingent on extending POD’s ‘reach’ was that the wider staff team also ‘saw’ the need for change. On wards 1, 2, 3 and 6, via routine and special meetings, ward managers facilitated discussion and reflection on audit findings with their staff. On ward 4, barriers at several levels operated to constrain team engagement: senior staff were preoccupied with managing patient flow with prolonged staffing difficulties, and ‘reach’ was compromised by heavy reliance on bank and agency workers and staff discontinuity.Nursing, therapy and care staff on all wards reported minimal knowledge of delirium and its adverse consequences for patients and caregivers, similar to studies in acute settings elsewhere –31. It wOn wards 1, 2, 3 and 6, formal training was provided to all staff; the mode of delivery modified to maximise participation. For example, small groups of 3/4 staff with the matron in huddles over several sessions, each of 20 min duration (ward 6); multiple, short 10–15 min ‘breakfast events, led by the seconded nurse until all material was covered with all staff (ward 3); jointly delivered multiple group sessions with a mental health practitioner and senior nurse (wards 1 and 2). Training conveyed delirium knowledge with reflection on staff experience and current practice through the lens of delirium prevention, to engage staff in thinking differently about it.We had the meeting to feedback audit observations to staff; but I linked training on delirium with observations and with examples of patients on the floor… We talked about … a patient to whom this was currently happening… and we’d then link the key step on the training programme with the person…Linking knowledge about delirium to experience and practice on the ward…made it very easy to engage staff …in how they could adapt what they do every day to prevent delirium or pick it up early.Matron, Hospital E.A comparison of wards 3 and 4, each with a seconded nurse to steer implementation and lacking pro-active support at directorate level, illustrates the significance of local contingencies in accomplishing planning tasks. The ward 3 nurse had informal support of a senior clinician with authority to negotiate through, and secure resolution of competing priorities that threatened implementation planning. This contrasted with the limited influence the ward 4 nurse could exert on colleagues as a consequence of discontinuity within the staff group and senior ward staff’s lack of capacity to provide support. Absence of training, moreover, meant that delirium prevention was neither understood nor accorded value. Only changes within the nurse’s direct sphere of influence occurred, which included supporting volunteers. Given demand pressures, preventive interventions subject of formal surveillance, accountability and sanctions  were prioritised.Preparatory work comprised development of systems, processes and associated documentation to identify at-risk patients, establish how staff would act on interventions, communicate with each other, with families and volunteers, and review patients’ progress.we’ve got to see the use of it and what a difference it makes and ideally it’s not going to add to the workload already’. They prioritised trying out different ways of capturing, recording and reviewing how changes could be adapted to fit local systems for optimal workability. ‘Negotiated experimentation’  cultivated staff engagement with system construction, which in turn helped to embed new practices.All four full implementer wards incorporated a delirium risk tool into ward admission assessments, conducted by nurses. A care plan, pertaining to identified risks was completed for each patient. POD leads acknowledged that staff participation in new systems and processes required that ‘Participating hospitals had a long tradition of volunteering, the size of their volunteer base varying between 300  and 700 . Voluntary services had established systems for recruiting, training and supporting volunteers who performed a diversity of roles in different locations, similar to the national picture : ‘meetinPOD volunteers were not specifically recruited to the programme initially: they were drawn from among those who had applied to become volunteers and had expressed interest in working with older people. Motivations were diverse: students wishing to pursue a career as a health professional; working age young people and adults to enhance their employability; retired people seeking something meaningful to do; and those who wished to support the hospital for the care they or a relative had received.Involving volunteers in POD encompassed reciprocal agreement between ward staff and voluntary services on work roles, competencies and training to enact them; establishing methods for communicating information about which patients to work with, in relation to what tasks; creating mutually supportive working arrangements, and engendering a sense of common purpose.Volunteer specific training was aimed at reinforcing POD as a collaboration between staff and volunteers. This conception, however, was not carried through in all sites. POD specific training was pursued on wards 1 and 2 only. On these wards, additional to addressing hospital procedural rules, typically on infection control, confidentiality and patient safety, training articulated the purpose and content of the POD system of care, placed emphasis on staff and volunteers working together on a common objective, and contextualised reciprocal volunteer/staff communication and staff support within that joint endeavour.On wards 3, 4 and 6, standard hospital volunteer training was supplemented with an information leaflet about POD, although ward 6 later altered its approach. On wards 4 and 6, training was limited to hospital procedural rules. On ward 3, it was more expansive: task specific training depending on role, communication skills and dementia awareness. Their rationale for not deviating from standard training was pragmatic: they nether had the resources to do things differently nor did they see the need for it. Their existing model facilitated substitutability of volunteers across the diversity of roles performed in a hospital context: if the person did not ‘fit’ in one service environment, s/he might ‘fit’ in another. Given the investment of voluntary services staff in recruiting and training volunteers, substitutability was viewed as important.Even if the volunteer doesn’t feel they are in the right environment…they can fit into a different type of voluntary work. It doesn’t mean we lose them.Voluntary service manager, Hospital C.They follow a task description that … we use... And I didn’t want to detract them from that, I wanted them to carry that on.Voluntary services manager, Hospital B.On these wards, voluntary services established from the outset the tasks volunteers would perform and for which training prepared them. Although not dissimilar to POD role descriptions, they were conveyed as incidental to, rather than central to delirium prevention.On wards 1 and 2, volunteer training was followed by informal introductory meetings with ward staff in a social setting. On ward 4, voluntary services organised a similar gathering to introduce volunteers to staff and to familiarise them with the ward environment. On wards 3 and 6, usual practice pertained: a voluntary services staff member accompanied the volunteer the first occasion s/he went on to the ward.At conclusion of the preparatory phase, all five wards had recruited varied numbers of volunteers: 18 for wards 1 and 2, nine and ten respectively for wards 3 and 4 and five for ward 6, where recruitment was ongoing. Core features of the role were: providing emotional and social support, engaging in stimulating activities, ensuring patients were drinking enough and providing practical assistance as needed. On wards 1 and 2, therapists developed guidance for volunteers to assist patients with what they termed ‘social mobility’, accompanying those able to walk independently but lacking confidence to do so.It was intended that volunteers would work one 2/3 h session weekly. Assigning volunteers to patients and tasks was to occur via the patient care plan, and a symbol on the patient board identifying delirium. Volunteers were to complete the care plan at each visit; wards also maintained a book for them to raise issues/make comments.POD was fully delivered in wards 1, 2, 3 and 6 over six months. Delivery involved some adaptation of the systems and processes established during the preparatory phase as new challenges unfolded requiring resolution.Slowly you started noticing at handovers …that staff… wouldn’t say Mrs X was confused; the word ‘confused’ went, and people talked about whether she may have a delirium…it became a clinical thing, not something to be dismissed…delirium wasn’t just about the person walking up and down…they were picking up on people who were quiet. They started associating a patient’s sudden change in behaviour with possible delirium and doing something about it.Senior nurse, Ward 6.On these four POD wards, enhanced knowledge of delirium had seeped into work practices prior to delivery through knowledge sharing and practice reflection. Increasing use of the term ‘delirium’ instead of ‘confusion’ was symbolic of new understanding with consequences for action.just seeing a ‘problem’ patient’. Whereas nursing and care staff might have previously reacted impatiently to a patient “behaving badly,” they were more likely to spend time with the patient, seeking to ease their confusion and distress.Staff suggested that delirium knowledge facilitated empathic connection with the experience of patients as opposed to ‘It was a means to pull staff through the nursing process: you’ve assessed someone’s risk, now it’s about planning work relating to it, evaluating what you’ve done and what’s happened as a result… the difference between putting a tick beside hygiene care and not thinking what it means.Matron, Hospital E.The new systems and paperwork aligned with delirium knowledge projected a collective sense of thinking and acting on delirium prevention. Since most patients on care of older people wards and older patients on trauma orthopaedic wards met criteria for delirium risk, the risk tool’s primary function was to reflexively produce an individual patient care plan specifying action to be taken on each risk factor identified:Work relating to delirium care plans served to extend action in respect of other aspects of routine practice. For example, patients at risk of malnutrition were identified through the Malnutrition Universal Screening Tool (MUST) on ward admission, triggering a dietician assessment. For nurses doing the admission assessment, identifying a patient at risk of poor nutrition in the delirium care plan meant that action on nutrition encompassed those who might require encouragement or assistance to eat.Our ‘Health Cares’ are coming to us nurses and saying, ‘look this patient has only taken sips of fluid, we need to work on this together’… we were always notoriously bad at filling in fluid balance charts …but now hydration, glasses have become more part of the general routine…there’s a purpose to it.Staff nurse, Ward 3.Care plans were reviewed regularly by senior ward nurses. Compliance was fostered through review of action during daily ‘handovers’ with nursing and care staff. Changes in behaviour patterns were noted and implications for further action considered. The division of labour on delirium prevention highlighted the contribution of health care assistants because of their direct patient knowledge. This in turn enhanced confidence in their ability to note, make sense of, and draw attention of qualified staff to subtle changes in patients that required concerted action.Attaching meaning actions by locating them in context of delirium prevention was important in reinforcing practice change.discharges, equipment, home visits’, OT students worked therapeutically with cognitively impaired patients at risk of delirium. Transforming the day room into a therapeutic space, OTs used reminiscence materials, games, music, and conversation to connect patients with their pre-hospital lives and relationships. They involved relatives directly in eliciting knowledge about patients’ interests and preferences, encouraging them to bring in favourite music, videos and books. The organisational context in part facilitated this focus. As an orthopaedic trauma ward, dedicated therapists with a visible ongoing presence were the norm, contrasting with care of older people wards where therapists were not physically located there, but attending to work with individual patients and for multidisciplinary meetings.Direct ward engagement in POD delivery did not extend to therapists, except for ward 6, where a senior therapist was in the implementation team. It was planned that cognitive stimulation for patients with dementia, at particularly high risk of developing delirium, would be prioritised. This was enacted through deploying student occupational therapists (OTs) in a different way. Instead of focusing on what had become their core activity: ‘On ward 4, although improvements in some aspects of care had occurred as a consequence of preparatory work , systematic engagement of the ward team in delirium prevention did not happen, reinforcing the significance of implementation work in creating a facilitative change environment, embracing the wider staff team.Preparatory work had generated considerable enthusiasm for volunteers among staff who had invested time in creating a welcoming and supportive environment, to ensure the smooth integration of volunteers onto the ward. In practice effecting integration was more complicated.The first weeks of delivery were especially vulnerable to volunteer attrition. Between a fifth and a third of volunteers never started or left after a few sessions either for personal reasons  or because they found working with older people too difficult. Personal experience in caring for older people could be a hindrance or a spur to sustaining volunteering: the former where it unearthed painful emotions; the latter when it generated confidence in care tasks and empathic connection with the patient as person.Going onto a ward where there are quite a lot of really ill people …I’d done loads of preparation for it and I still found it daunting so, yeah. But I think once you’ve done it once or twice you kind of know what it’s like and you know what to do…and staff were welcoming… helpful and reassuring and showed me what to do …until they knew I’d got the hang of it.Student volunteer 01, Ward 1.Even those who persisted, found it intimidating in the beginning.It was difficult …with a patient with dementia I didn’t know how to handle the situation…more often because of their distress…I learned how to get into a conversation… it can seem disjointed…but you learn to listen to the flow and relate to that…there is a massive sense of fulfilment.Student volunteer 03, Ward 6.Unfamiliarity of the environment and in engaging with older patients, particularly those who were frail or had a cognitive impairment, provoked anxiety which took knowledge and experience to overcome.It’s taking me a long time to get to know the staff because they’re busy, they turnover… I can ask them but if they’ve got jobs to do … It’s difficult isn’t it …I have to be a little bit careful that I’m not treading on the toes of the healthcare assistants and I’m conscious of that and … occasionally I feel by being eager I can be seen to chase their tails when they’re feeling very busy and because I’m only in for a short time – about 6 h – I can afford to be walking around and available anytime I’m on the ward but it’s hardly sustainable for the staff to do that …but if I see someone in a pickle I want to go and help...Retired volunteer 04, Ward 4.Volunteers with expertise in caring for older people either by personal experience or previous employment, found that negotiating the boundary between ‘being helpful’ and not undermining staff, was tricky:Nevertheless, there were multiple examples of how volunteers developed creative and person-centred approaches to communicate with frail and cognitively impaired older people not as patients but as individuals with a past, present and fears and hopes for a future. They supported at-risk patients in eating meals together in a social space that also involved assisting them to move beyond the bed/bay. In several wards, volunteers worked closely with individual staff: on ward 2, the creation of a communal dining space for patients was initiated jointly with the nutrition assistant, who had assumed day-to-day responsibility for supporting volunteers. On ward 6, volunteers worked alongside OTs in group and individual reminiscence and social activities. Among volunteers in wards 1, 2 and 6, there was a strong sense of involvement in a joint programme with staff. In ward 4 by contrast, high level commitment of several retired volunteers was animated by their personal values of affording dignity in older age.The number of hours contributed weekly to each ward by volunteers was modest , although this is likely an underestimate . This did not reflect lack of individual commitment: some contributed up to eight hours a week. On ward 3, where few volunteers took part, two completed ≈56 of the 81 h recorded during delivery; at the other end of the spectrum one volunteer did not return after the first shift, and the second left after completing two shifts.Except for ward 6, voluntary services did not pursue recruitment after POD preparatory implementation. Wards tried out different methods to support and retain volunteers with mixed success: ‘buddying’ a new volunteer with one who was experienced; organising feedback meetings to identify need for support. In absence of ongoing recruitment and with attrition on these wards, under a third of the original cohort remained to end of delivery, of whom most were planning to continue.On ward 6, from an inauspicious start (only two of five volunteers started and one left shortly after to take up employment), volunteer numbers slowly built up by adopting novel recruitment methods and establishing bespoke training and support structures. A direct approach to a local 6th form college, led to recruitment of first one, then three further students interested in a health career. Each new volunteer was taken under the wing of a senior staff member and spent the first couple of weeks shadowing ward staff and an ‘experienced’ volunteer until confident of managing independently. Reciprocal peer support was fostered; encouraging a ‘link’ experienced volunteer to assume responsibility for it, made it happen. Recruitment and training were re-worked toward the end of delivery for a new volunteer intake: one-to-one interview with a senior nurse; then a dedicated, half-day POD training event with ward senior staff and older person’s lead. Within six months of the delivery phase ending, a further eight student volunteers, additional to the four who continued beyond the study end, were working on the ward. Persistence of staff in investing in new methods for recruiting, training and supporting volunteers was underpinned by commitment to sustaining POD and extending it to other wards.Structured ward observations demonstrated only small changes in nursing input in direct patient care  in NHS hospital wards, an exemplar of a complex intervention . AlthougThe preparatory work of POD implementation was fully accomplished in four of five participating wards, and subsequently delivered over six months in those wards. The implementation strategy components worked synergistically, interactively and broadly as intended to effect the hypothesised mechanisms of change theorised in NPT, albeit in specific conditions. Thus, observations of practice pertinent to each of the preventive interventions using the audit tool served the function of enabling staff to ‘see’ how and in what ways intervention delivery was similar to and different from existing practice. Creating tension for practice change only occurred in circumstances where what was observed and the implications for action were conveyed and reviewed within the wider staff team. Developing understanding of delirium through education and training was more than conveying knowledge of its presentation and risk factors. It required engagement of staff in thinking differently about it. Knowledge aligned with interpreting experience against a new mental model facilitated organisational learning and forging networks of practice, as elsewhere . Re-visiA major study learning point was that activating the mechanisms of action to implement change required a particular form of leadership to drive it: individuals with formal authority from their organisational position to enlist stakeholders in the work; skills in networking, negotiating and engaging relevant others; and headspace to enact the role. Alternatively, the introduction of an experienced staff nurse with limited dedicated time could organise and accomplish planning tasks in the absence of such a steer in specific conditions: assistance of a senior professional or middle manager to unblock barriers arising from competing hospital priorities; and support from senior ward staff to facilitate engagement of the wider staff group. In ward 4, where the resource of a dedicated nurse to pursue change had marginal ward-level impact, the absence of local leadership and staffing difficulties created an environment in which staff had neither the support nor the headspace to pursue preparatory POD implementation.Subsequently, delivering POD shifted responsibility to senior ward staff as the primary drivers. The transition from implementation planning to delivery was viewed in hindsight as requiring focused work: to ensure that the systemic, relational and practice dimensions of delirium prevention were being pursued and that the systems and processes in place to embed POD in organisational routines were operating to secure intended benefit. This highlights ongoing appraisal work as critical to facilitating and maintaining cultural and practice change , 36. MorIn this study, three broad contextual features were critical in the work of preparing for, and delivering POD: structure, organisation and timing. Structural features of ward contexts, especially staffing difficulties, impeded preparatory implementation and POD delivery, even in the presence of interest and enthusiasm among senior staff to make it happen. Thus, ward 4 without a basic level of staffing (and staff continuity) was unable to engage with the work involved. Adverse organisational factors, such as ward re-organisation, change in location and discontinuity among senior ward staff risked subverting POD implementation and delivery (ward 6). Here, although preparatory POD implementation work was extended in time, the configuration of human resources mobilised in this site cut through organisational constraints: senior management investment to procure dedicated time of a matron to steer implementation; support throughout from the directorate lead for older people and a new senior ward team who embraced the intervention as an opportunity to enhance practice. ‘Timing’ was another contextual factor interfacing with POD which could either facilitate or subvert implementation and delivery. The proposed closure of ward 1, raised in the first weeks of delivery, preoccupied staff and adversely affected morale at a critical point in the change process. Although closure was postponed, structural and organisational factors (bed closures and staff moves) resulted in POD gradually fading away. It is possible that if the proposed changes had occurred later on when POD was more integrated into ward routines, the move to the new ward might have temporarily disrupted POD but not effaced it.The mechanisms of change to implement and deliver POD demonstrated potential to change ward practice with positive impact on staff, staff/patient interaction and practices pertaining to delirium prevention. This was achieved with modest resources to support preparatory implementation work and transition to delivery .Our model of delirium prevention included a role for hospital volunteers to enhance ward practice in delirium prevention, particularly regarding those tasks that appear difficult for staff to undertake consistently . Although volunteer involvement was highly valued by staff, recruitment and retaining volunteers was more difficult than anticipated. Wards varied in the numbers recruited at outset and volunteer attrition was high, with specific points of vulnerability: between recruitment and starting; shortly after starting; then a slower, less predictable drift. Different factors appeared to operate at each point: where general interest collided with the reality of the commitment involved; where engagement with older people in an acute care context was found to be too difficult; and changing circumstances . For some, their motivation to become volunteers could be both a barrier to persistence and a source of sustainability. In three of the four full delivery wards, between a quarter and a third of those indicating interest in volunteering persisted to the six months end, and most intended to continue. The time contribution of individual volunteers was enormously varied. This was the case in wards in which implementation teams had invested heavily in processes and action to establish a supportive environment for volunteers and to involve them conjointly with staff in delivering POD; and in wards in which an integrated approach, for various reasons, did not happen. Different methods introduced to support volunteers had positive effects in sustaining involvement in some contexts but not in others. The pattern of attrition was not dissimilar to that in a recent study of mealtime assistance .There is considerable descriptive knowledge about the motivations and characteristics of volunteers and the benefits they derive from, and contribute to individuals and organisations, through volunteering, from North American , 43 and Our study primarily addressed the work of preparing implementation and enacting delivery through the lens of those involved. Although we interviewed volunteers about their experience with the programme, this only included those who persisted to end of delivery. Similarly, interviews with ward staff over time would have provided insight into the processes whereby practice change occurred from their different perspectives. Nevertheless, strengths of the study included informal observation of preparatory implementation activities and work practices over time, structured observation of work and workload at different time points and completion of documentation pertaining to new systems and processes.We conclude that POD is feasible to implement and deliver in an NHS ward context in specific conditions which we have encapsulated as ‘readiness factors’:A named, individual ‘driver’ at senior level whose professional authority and vertical networks legitimate the work of POD implementation in face of competing priorities. A ward-based ‘facilitator’, typically the ward manager, to provide support and encouragement to legitimate staff time devoted to POD and extend its reach to the wider staff team. A voluntary services manager to recruit and support volunteers and facilitate their introduction to the ward and POD.Dedicated time of around one day per week for around four months to support the day-to-day tasks of implementation and transitioning to delivery.Implementation of what is an augmented system of care requires capacity and resources to deliver at least a basic standard of care. Staffing difficulties including reliance on bank or agency staff means that there is not the headspace for staff to pursue cultural and practice change.These criteria do not imply knowledge, interest, or prior work on delirium prevention. Rather, they relate to the presence of contingent factors that are necessary to allow selection of wards which are realistically going to be able to implement this complex intervention.Our findings resulted in a re-assessment of the role of volunteers within delirium prevention in the feasibility trial. The model of delirium prevention we had adopted and described here included a prominent role for hospital volunteers. As most wards were unable to recruit or sustain the number of volunteers needed to have a major impact on delivery; the revised model adopted a flexible approach to inclusion of volunteers.Additional file 1. Staff Workload.


Catch-up growth after insults to growing organs is paramount to achieving robust body proportions. In fly larvae, injury to individual tissues is followed by local and systemic compensatory mechanisms that allow the damaged tissue to regain normal proportions with other tissues. In vertebrates, local catch-up growth has been described after transient reduction of bone growth, but the underlying cellular responses are controversial. We developed an approach to study catch-up growth in foetal mice in which mosaic expression of the cell cycle suppressor p21 is induced in the cartilage cells (chondrocytes) that drive long-bone elongation. By specifically targeting p21 expression to left hindlimb chondrocytes, the right limb serves as an internal control. Unexpectedly, left–right limb symmetry remained normal, revealing deployment of compensatory mechanisms. Above a certain threshold of insult, an orchestrated response was triggered involving local enhancement of bone growth and systemic growth reduction that ensured that body proportions were maintained. The local response entailed hyperproliferation of spared left limb chondrocytes that was associated with reduced chondrocyte density. The systemic effect involved impaired placental function and IGF signalling, revealing bone–placenta communication. Therefore, vertebrates, like invertebrates, can mount coordinated local and systemic responses to developmental insults that ensure that normal body proportions are maintained. The coordination of organ growth is necessary to attain correct individual organ sizes and body proportions. While extensive studies in insects have revealed that both intra-organ and inter-organ communication mechanisms are involved in regulating organ growth, vertebrate studies have lagged behind. Here, we developed a new mouse model to examine cellular mechanisms underlying growth regulation after a developmental insult. The cell cycle suppressor p21 was expressed in the cartilage that drives growth of the long bones, targeting the left limb exclusively and leaving the right limb as an internal control. By triggering the insult during the last gestational week, we found that left–right limb symmetry was maintained due to the following 2 compensatory mechanisms: (1) hyperproliferation of the spared cells within the targeted cartilage, which indicates that these cells respond to a signal coming from the arrested cells, and (2) a growth reduction in the rest of the body, an effect that correlates with changes in the levels of placental insulin-like growth factor (IGF) signalling and that can be rescued by boosting placental efficiency. These results reveal that the response to developmental insults is quite evolutionarily conserved across species as well as open new avenues of future research for the development of therapies to treat growth disorders. Drosophila larvae, injured imaginal discs can undergo compensatory proliferation  and andIgs7Tpromoter  Fig 1B)Igs7TRE-Lelements . Consequembryonic day (E) 12.5 until birth (ePit-Col-p21 model), analysis at E14.5–E17.5 revealed the expected cartilage-exclusive expression of tdT, mainly in the right skeletal elements, and p21 expression preferentially in the left limb cartilage, albeit in a mosaic fashion. For example, 36%–67% versus 0.8%–23% of chondrocytes were found to be p21+ in left versus right proximal tibia . As we previously observed with the Cre transgene [p21 expression was more widespread in the left hindlimb than in the left forelimb . Therefore, we focused our initial analysis on the hindlimb. As expected, proliferation was inhibited in p21+ proximal tibia chondrocytes at E15.5 and E17.5 . Although a potential consequence of p21 misexpression in proliferative zone (PZ) chondrocytes could have been their premature differentiation, we did not find precocious expression of chondrocyte maturation markers hypertrophic chondrocytes (which do not express the transgene) appeared slightly fainter in the distal femur and proximal tibia cartilage, and RNA sequencing (RNA-seq) analysis of the combined proliferative and pre-hypertrophic zones revealed that their normalized counts were diminished in the left versus right cartilage . At E17.5, the left bones were 0.2–0.3 mm (approximately 10%) shorter in animals misexpressing p21 , indicating that blocking chondrocyte proliferation resulted in decreased bone growth. However, the effect was milder than expected, given that between one-third and two-thirds of chondrocytes were being arrested. This result suggested that compensatory mechanisms that minimized the impact of the p21 insult had been activated in the left limbs. Indeed, at E15.5 or E17.5, no major changes in the length of the proliferative or hypertrophic zones of the growth plate were found  showed no obvious difference in their left/right length ratio compared to ePit-p21 control littermates. The one exception was a transient small reduction in the size of the left radius compared to the right . These results suggested that the right  bones in ePit-Col-p21 mice responded to the impact of p21 expression in the left limbs via a systemic effect or inter-organ communication, such that their growth was reduced similarly to that of the left bones.We next took advantage of our unilateral approach that provides an experimental and a control limb within an animal and performed left–right intra-individual comparisons to determine the degree of asymmetry. Unexpectedly, most ePit-Col-p21 mice could be due to both bone-intrinsic cellular mechanisms and the aforementioned apparent extrinsic regulation. We first tested for any organ-intrinsic responses and started by examining proliferation of the spared chondrocytes in the hindlimbs. Indeed, the left/right ratio of 5-ethynyl-2’-deoxyuridine (EdU) incorporation by p21− chondrocytes was higher in experimental animals as compared with controls at E17.5 and P0 but not E15.5 , while tending to be slightly reduced, was not significantly different, indicating that the compensatory proliferation phenomenon is quite effective , and this depletion was not due to inactivation of rtTA activity .The compensatory response observed in the limbs of ot E15.5 , revealirol mice , the hypffective . MoreoveePit-Col-p21 tibiae (together in the same well) for 2 d with Dox, in the absence of surrounding mesenchyme (n = 3), whereas EdU incorporation was not detected in the inner core of the proximal tibia , probably because of insufficient nutrient diffusion. We therefore focused our analysis on the distal epiphysis. Similar to our findings in vivo, EdU incorporation in p21− chondrocytes was significantly higher in the left as compared to the right cultured cartilages (n = 4) or separately (n = 6) from the left bones.As a means to examine whether compensatory proliferation was only dependent on local cell–cell interactions, we cultured left and right E15.5 senchyme . We founsenchyme , n = 3, rtilages , suggestrtilages  and foun+ chondrocytes in the growing cartilage influences the extent of compensatory proliferation. Given that forelimb bones show a lower proportion of p21+ chondrocytes than hindlimb bones within each embryo analysed , we first tested whether compensatory proliferation was triggered in the proximal humerus. We found that although there was a trend towards increased proliferation in left p21− chondrocytes, the difference was not significant (n = 3), suggesting that compensatory proliferation requires a minimum insult threshold to be triggered. Because intrinsic differences between forelimb and hindlimb bones might exist in regards to the compensatory proliferation response, we also tested the threshold hypothesis using only hindlimb bones. In order to induce p21 expression in fewer chondrocytes than in ePit-Col-p21 mice, we made use of a newly generated Col2a1-tTA line   and Pit-tTA-p21 tibiae, at E17.5 (or E15.5 plus 2 d ex vivo). Segmental linear regression analysis revealed that the extent of EdU incorporation by p21− chondrocytes did not correlate with the proportion of p21+ neighbours when this proportion was below 35%, but beyond this threshold, there was a linear correlation between both parameters (n = 26 bones). These results suggest that compensatory proliferation is due to a signal produced in proportion to the number of arrested chondrocytes, that the signal needs to reach a certain threshold to be effective, and that it remains active until at least P0 despite the dilution of p21+ chondrocytes.We next addressed whether the proportion of p21nificant ; n = 3, line see  in place1 model) . tTA exp21 model . To furtePit-Col-p21 mice that could contribute to the growth compensation and potentially correlate with the number of insulted chondrocytes. Because an alteration in cell density can influence organ size, we tested whether cell density was changed in the PZ of ePit-Col-p21 mice. That was indeed the case, and we found a temporal association between the occurrence of compensatory proliferation in the ePit-Col-p21 model  and statistically significant reduction of cell density in the left PZ as compared to the right (ePit-p21 mice (n = 12). Moreover, in line with the threshold hypothesis, we found that, at E17.5, there was a certain value of cell density below which EdU incorporation sharply increased in p21− chondrocytes (n = 20 bones).We next asked whether additional cellular changes occur in the left limbs of he right . Notablyp21 mice , n = 12.ePit-Col-p21 embryos as compared to ePit-p21 littermates (n = 7 for ePit-p21 and n = 13 for ePit-Col-p21 embryos). We found a very good correlation between both types of measurements (n = 80 bones) and therefore used flat micrographs for all measurements in the study. We first tested whether the growth reduction affected the whole body. We found that, in addition to a decrease in right bone length, body weight of E17.5 and P0 ePit-Col-p21 mice—but not E15.5 or E16.5 embryos—was approximately 10% lower than in ePit-p21 littermates  showed enrichment of several pathways related to stress and immune responses in the left cartilage  between left and right cartilage  of single at E17.5 . Indeed,artilage . In partartilage  and veriartilage  or in siartilage . Relaxinentified ,27 alarm.5 or P0 , suggestePit-Col-p21 embryos (n = 19) as compared to ePit-p21 controls (n = 17), such that the placenta/body weight ratio was increased . Increased expression of IGF2 by the placenta has been seen as part of a placental stress response triggered by prenatal insults such as alcohol exposure, which is also associated with placental functional impairment, increased placental/body weight ratio, and foetal growth restriction [Igf2r , which encodes a decoy receptor that can decrease local IGF2 availability [We reasoned that the most likely foetal organ to control systemic growth by responding to a circulating alarm signal is the placenta because in rodents it produces higher IGF levels than any other organ  and is cncreased . This retriction ,31. Therlability . Furtherlability .ePit-Col-p21 embryos was due to impaired IGF signalling within the placenta, we injected pregnant dams  that does not cross the placental barrier, can only bind IGF2R (and not IGF1R), and thus was shown to increase placental efficiency [ePit-Col-p21 embryos were compared between litters that were either treated or not treated with Leu27-IGF2 to determine the degree to which body/organ size was rescued, and they were also compared with ePit-p21 embryos within treated litters to determine whether Leu27-IGF2 differentially affected experimental and control embryo growth. Boosting placental function led to the following results:As a means to test whether the systemic growth reduction in ficiency . Body anePit-Col-p21 embryos was significantly increased compared to untreated experimental embryos , and within treated litters, femur length was not significantly different between ePit-Col-p21 and ePit-p21 littermates (n = 6), demonstrating preferential rescue of the mutant embryos , and within-litter comparison revealed that body weight was not only rescued but also slightly increased beyond that of ePit-p21 littermates (n = 6), suggesting that ePit-Col-p21 embryos are especially sensitized to the systemic growth factors produced by the placenta . Taken together, these results suggest that, in the presence of local growth defects, a systemic growth reduction is necessary to maintain limb–body proportions. Unexpectedly, rescue of the systemic effect did not result in left–right asymmetry in ePit-Col-p21 embryos . As a consequence, the amount of cartilage scaffold being laid down is not reduced, and thus endochondral ossification can proceed at an almost normal rate. This proposed response fits with stereological studies that showed that, in growth plates with low proliferative rates, it is mainly the production of extracellular matrix that contributes to bone growth . In thiseduction .+ chondrocytes—we posit that density plays a permissive rather than an instructive role and that stress signals emanating from p21+ chondrocytes are needed as well. These stress signals could be diffusible molecules or cell–cell interactions, and they could act either in short range, affecting only cells in their proximity, or in long range, via a self-propagated travelling wave [When the insult is extensive enough for cell density to drop below a certain threshold, compensatory proliferation is triggered in spared chondrocytes, to an extent proportional to the number of affected chondrocytes. Because we found that the extent of compensatory proliferation does not linearly correlate with cell density—but it does with the proportion of p21ing wave  and lead+ chondrocytes have been replaced by p21− cells and the threshold cell density has recovered, compensatory proliferation stops, such that overgrowth is not generated.Once p21Our results reveal a mild but consistent systemic growth reduction (approximately 10%) in response to a local insult in the cartilage. We propose that the stress response generated in the left cartilage is somehow communicated to the placenta, which in turn systemically reduces growth . We sugg+ chondrocytes, p21− chondrocytes, the perichondrium, or cells at the cartilage–bone interface—are released into circulation, and, when they surpass a threshold, impact on placental efficiency .ePit-Col-p21 organs are equally reduced or whether the musculoskeletal system  is primarily affected. Resolution of the latter question is currently difficult because the embryos are too small for individual organs to be weighed reliably. Volumetric analyses using mesoscopic techniques such as optical projection tomography [With regards to potential growth correction treatments, it would be important to determine whether all mography  on embryePit-Col-p21 concepti through maternal Leu27-IGF2 treatment, long-bone growth is not enhanced to the same extent as body weight, resulting in a reduction in the ratio of bone length to body weight  according to MSKCC institutional guidelines.ePit-Col-p21 or Pit-tTA-p21) to the average measurement for its control littermates (ePit-p21 or Pit-p21). This is important because the absolute measurements vary significantly between litters, in part because they differ in exact developmental stage, number of embryos, and age of the mother [http://powerandsamplesize.com/Calculators), based on the average SD observed in pilot experiments, to achieve an effect size of 0.03 in the left/right bone length ratio, 0.5 in the left/right ratio of EdU incorporation, or 10% in normalized systemic measurements, with a power of 0.8 and a 95% CI. In ePit-Col-p21 and one from the eCol-tdT populations) were abnormally small, possibly dead, and were excluded from the analysis. For comparison of qualitative expression, a minimum of 2 specimens per stage and 5 across several stages were used. The investigator measuring bone length was blinded to the treatment/genotype of the specimens. No blinding was done for other measurements. No randomization was used for animal processing.To correct for interlitter variability when studying the effect of p21 misexpression on systemic growth, we normalized each measurement from an experimental animal , by 1-way ANOVA (1 variable and ≥3 conditions), or by 2-way ANOVA (2 variables and 2 or more conditions) following a matched (paired) design when possible (indicated when not). When left and right measurements were compared within experimental animals only, paired 2-tailed t test was used. For all ANOVA, alpha = 0.05. All relevant parameters for the statistical tests can be found in When data were available for control and experimental, a normalized measurement (left/right ratio or percentage of average control mice) was calculated for both. Between different time points, the normalized measurements were compared by multiple unpaired Igs7TRE-LtSL-p21 mouse line, the NruI-STOP-loxP-tdTomato-SnaBI fragment in the Ai62(TITL-tdT) Flp-in replacement vector [NruI-tdTomato-STOP-loxP-MluI-HpaI-SnaBI cassette, to generate an empty DRAGON vector. A PCR-amplified Kozak-Cdkn1a cassette was subsequently cloned into the MluI and SnaBI sites to generate the DRAGON-p21 vector. This vector was then used for recombinase-mediated cassette exchange into Igs7-targeted G4 ES cells [Col2a1-tTA line, a Kozak-tTA fragment was PCR-amplified from plasmid pEnt L1L3 tTA-3  and cloned into a vector containing the regulatory region of mouse Col2a1 obtained from plasmid p3000i3020Col2a1 [Col2a1-tTA allele. The progeny of one of those (founder number 92) expressed the tTA faithfully in the highest percentage of chondrocytes and was bred with Pitx2-Cre animals to generate breeders for the experiments.To generate the t vector  was replES cells . Two suc20Col2a1 . BackbonCol2a1-tTA mice were maintained on an outbred Swiss Webster background and genotyped using primers Col2a1-F (CCAGGGTTTCCTTGATGATG) and tTA-R (GCTACTTGATGCTCCTGATCCTCC) and a standard PCR program with 55°C annealing temperature. The Pitx2-Cre [Col2a1-rtTA [Ai9 (R26CAGGS-LSL-tdTomato) [Ai62 (Igs7TRE-LSL-tdTomato) [Igs7TRE-LtSL-p21 animals were genotyped like Ai62 mice. Pitx2-Cre/Cre; Col2a1-(r)tTA/+ females and males homozygous for the conditional misexpression allele were crossed to generate experimental and control animals. Noon of the day of vaginal plug detection was considered E0.5. The equivalent of E19.5 is referred to as P0.itx2-Cre  (kind gi2a1-rtTA  (kind gidTomato) , and Ai6dTomato)  mouse liDox hyclate (Sigma) was added to the drinking water at a final concentration of 1 mg/ml, with 1% sucrose to increase palatability.2) in 24-well plates with serum-free DMEM (Gibco) containing 0.2% Bovine Serum Albumin (BSA), 0.5 mM L-glutamine, 40 U/ml penicillin/streptomycin (Gibco), 0.05 mg/ml ascorbic acid (Sigma), and 1 mM betaglycerophosphate (Sigma). The medium additionally contained 1 ng/μl Dox to maintain transgene expression. After 2 d, the bones were incubated with 10 μM EdU for 90 min, then fixed in PFA and processed for histological analysis.A previously described protocol  was sligNote that after 2 d, we consistently observed growth of 19% to 23% in control limbs as compared to the original length. This is less than in vivo (approximately 87% growth between E15.5 and E17.5), and the main difference seemed to be at the level of the proximal cartilage, which does not proliferate, likely due to insufficient diffusion of nutrients because it is larger than the distal cartilage. We therefore focused our analysis on the distal cartilage, which at these stages is expected to contribute one-third of total growth , i.e., a27-IGF2  was prepared at 500 ng/μl in sterile 0.01 N HCl solution and kept at 4°C in between injections. From E15.25 to E17.25, the pregnant dam was subcutaneously injected every 8 h, for a total dose of 1 μg/g of body weight per day.Human LeuStaining of cartilage and bone was performed as described . Bone leWhole femora and tibiae were scanned using a Scanco μCT 35  system. Six-μm voxel size, 45 KVp, 0.36-degree rotation step (180-degree angular range), and a 400-ms exposure per view were used for the scans, which were performed in air. Scanco μCT software  was used for 3D reconstruction and viewing of images. After 3D reconstruction, ‘Distance 3D’ tool was used for measuring the length of the ossified region. Three measurements were taken and the average derived for each bone. The observer was blinded to the genotype of the mouse.Mouse embryos were euthanized by hypothermia in cold PBS. Mouse pups were euthanized by decapitation after hypothermia-induced analgesia. Knees (or isolated full tibiae and femora) were dissected out, skinned, and fixed by immersion in 4% paraformaldehyde  in PBS for 2 d at 4°C. After several washes with PBS, the tissue was then cryoprotected, first by brief incubation with a solution of 15% sucrose and then 30% sucrose in PBS for at least 4 h at 4°C, and then embedded in Cryomatrix (Thermo) using dry-ice-cold isopentane (Sigma). The knees were oriented sagittally and facing each other, with the tibiae on the bottom of the block . Serial 7-micron sections were collected with a Leica Cryostat on Superfrost slides, allowed to dry for at least 30 min, and stored at −80°C until used. For all histological techniques, frozen slides were allowed to reach room temperature in a closed box, and Cryomatrix was washed away for 15 min with warm PBS (37°C).Sections were incubated in citrate buffer  for 15 min at 90°C, allowed to cool down, washed with PBSTx (PBS containing 0.1% Triton X-100), blocked with 5% BSA in PBSTx for 30 min at room temperature, and incubated with the primary antibody over night at 4°C (see list of antibodies below). After PBSTx washes, incubation with Alexa647- and/or Alexa555-conjugated secondary antibodies  was performed for 1 h at room temperature. After PBSTx washes, the slides were mounted with Fluoro-Gel (Electron Microscopy Sciences). For TUNEL staining, endogenous biotin was blocked after antigen retrieval using the Avidin/Biotin blocking kit (Vector #SP-2001), and TdT enzyme and Biotin-16-dUTP (Sigma #3333566001 and #11093070910) were subsequently used following manufacturer instructions. Biotin-tagged DNA nicks were revealed with Alexa488- or Alexa647-conjugated streptavidin  during the incubation with the secondary antibody.Arf , and p16-INK4A .Antibodies  included tdT , p21 , p19Col2a1, Col10a1, and Ihh (provided by Dr. Licia Selleri), the templates for most riboprobes were generated by PCR from embryonic cDNA, using primers containing the SP6 or T7 RNA polymerase promoters. Sequence of the primers is available upon request. After purification of the PCR product (Qiagen PCR purification kit), DIG-labelled probes were transcribed following manufacturer instructions (Roche), treated with DNAase for 30 min, and purified by LiCl-mediated precipitation in alcoholic solvent. Probes were kept at −80°C in 50% formamide (Fluka). For immunohistochemistry after in situ hybridisation, sections were incubated in citrate buffer  for 15 min at 90°C, allowed to cool down, washed with PBSTx, and incubated with 1% H2O2 in PBSTx for 1 h to block endogenous peroxidases. After BSA blocking and primary antibody incubation, endogenous biotin was blocked using Avidin/Biotin Blocking kit (Vector #SP-2001), and then the slides were incubated with a biotinylated secondary antibody. A brown precipitate was obtained using a peroxidase-coupled streptavidin-biotin complex  and DAB substrate (Vector #SK-4100), following manufacturer instructions.The protocol described in  was follFor in vivo experiments, sagittal sections of the knees were imaged, focusing on the region between both menisci and analysing at least 2, and typically 4, sections per limb. For cultured distal tibiae, frontal sections were used because they allow for better identification of the different epiphyseal regions. The transition between round (resting) and flat (columnar) nuclei, roughly describing an arch between the upper point of the wedges formed by the groove of Ranvier, was chosen as the start of the PZ, while the transition towards bigger, more spaced nuclei (pre-hypertrophic) was chosen as the end of the PZ. The point where pericellular matrix is sharply reduced around enlarging chondrocytes was considered as the start of the HZ, while the end of the HZ was marked as the distal end of the last intact chondrocyte. Bright-field and fluorescence images were taken on a Zeiss inverted microscope (Observer.Z1) using Axiovision software (Zeiss). Mosaic pictures were automatically reconstructed from individual 10× (bright-field) or 20× (fluorescence) tiles.Five mg/ml EdU in PBS was injected  1.5 h before euthanizing the mice. EdU was detected using the Click-iT Alexa488 Imaging Kit  once the immunohistochemistry and/or TUNEL staining were finished on the same slides. The fraction of nuclei that were positive for EdU, p21, or tdT in the PZ of the cartilage was determined using ImageJ or CellProfiler, followed by manual curation.2 using the resolution and scale information. The DAPI channel was segmented and quantified using Cell Profiler. Cell density was calculated as the number of chondrocytes per area unit.The PZ was cropped from 20×-imaged sections of left and right experimental and control proximal tibial cartilage, stained for DAPI, p21, and EdU. The area of the region of interest (PZ) was measured in pixels using the Histogram tool in Adobe Photoshop and converted into mmePit-Col-p21 proximal tibial cartilage. tdT+ chondrocytes were segmented and counted, and their individual area was measured using Cell Profiler.The PZ was cropped from 20×-imaged sections of left and right Pit-Col-p21 embryos was dissected in cold PBS, the condyles and hypertrophic zones removed using a microknife, and the perichondrium removed by a combination of collagenase type II treatment  and mechanical dissection. Left and right cartilage fragments from each embryo  were kept in separated tubes and flash-frozen in liquid nitrogen. RNA was extracted using Trizol (Invitrogen) and a mechanical tissue disruptor.The distal left or right femoral and proximal tibial cartilage from E17.5 p-value ≤ 0.05. NMF library tools were used to generate heatmaps. Enrichment analysis was performed using DAVID [High-quality RNA was deep sequenced (≥50 million paired-end reads) by the New York Genome Center. Aligned reads were analysed using DESeq2 tool in R. A paired design was used, such that left and right comparison was performed for each specimen, which minimized the effect of sequencing batch and interspecimen variability. DEGs were obtained using a threshold of adjusted ng DAVID  and WebGng DAVID .−ΔCT method, with Gapdh (for cartilage) or Tbp (for placenta) as a reference.cDNA was synthesized from purified RNA using iScript reverse transcriptase (RT) as described by the manufacturer (Bio-Rad). Each target was amplified in triplicate to obtain an average per sample, using SYBR Green (Applied Biosystems) on a StepOnePlus real-time PCR system (Applied Biosystems). Primer sequences are shown in S1 DataePit-Col-p21 growth plates.RNA-seq data from left and right  Left (L) and right (R) proliferative and pre-hypertrophic zones from 3 different E17.5 ePit-Col-p21 embryos were dissected and sequenced. Growth plates from femur and tibia were pooled. Normalized counts are shown. The original numbering (#386–388) was changed to 1–3.(TXT)Click here for additional data file.S2 DataePit-Col-p21 cartilage at E17.5.List of DEGs between left and right  The DESeq2 tool (adjusted p ≤ 0.05) was used to obtain the list.(TXT)Click here for additional data file.S3 DataIndividual values of the measurements presented throughout the study.(XLSX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S1 FigPitx2-Cre females were crossed with Ai9 males to characterize the specificity of Cre-mediated labelling. Seven-μm sections from left and right hindlimbs are shown at 2 different stages: E12.5 (A–D) and E18.5 (E–F’), n = 4 for each stage. Boxed regions in panel E and panel F are shown in E’,  Dynamics of tdT and CDKN1A (p21) activation in ePit-Col-p21 embryos, 1 d  and 2 d  after Dox administration to the pregnant female. Boxed regions in panel G and H are shown in G’ and H’. Note that activation of the transgene starts to be detectable 1 d post Dox administration, but it is not complete until 2 d post Dox. Asterisks indicate autofluorescent cells. Of note, the Pitx2-Cre allele is consistently left-predominant only when inherited from the female. (I–J”) Same as above, but E17.5 elbow sections are shown. (K) Intra-individual comparison of the proportion of p21+ nuclei in the left proximal humerus versus left proximal tibia PZ (n = 3). See also p-Value for 2-tailed paired t test is shown. Cre, recombinase from P1 bacteriophage; Dox, doxycycline; E, embryonic day; PZ, proliferative zone; tdT, tdTomato.(A–F’) (TIF)Click here for additional data file.S2 FigCdkn1c, Col10a1, and Ihh is not ectopically triggered by p21 misexpression , but their expression is qualitatively and quantitatively diminished in the left cartilage , nor to ectopic cell death at E15.5 or E17.5 . (F) Hematoxylin–eosin staining of E15.5 femora and E17.5 proximal tibiae from ePit-Col-p21 embryos. (G) Comparison of the length of the left and right proliferative and hypertrophic zones (PZ and HZ) of the femora from ePit-Col-p21 (n = 4) and ePit-p21 embryos (n = 3) at E15.5 . (H) Left/right ratios of EdU+ incorporation in the PZ of ePit-p21 and ePit-Col-p21 embryos at E15.5 (n = 4 and n = 3), E17.5 (n = 5 and n = 5), and P0 (n = 4 and n = 8). Comparison by 2-way ANOVA for Genotype and Stage . p-Values for Sidak’s post hoc test are shown in the graphs. (I) Cell size of tdT+  chondrocytes was characterized for ePit-Col-p21 embryos at E17.5 . For panel G–I, see (A–C) The expression of chondrocyte maturation markers (TIF)Click here for additional data file.S3 Fign = 3). (B) Right tibiae show the same extent of proliferation regardless of whether they are cultured together (n = 4) or separated (n = 6) from the contralateral tibia. See also (A) After 2 d of culture, the distal tibial cartilage does not show signs of senescence, as shown by lack of p16 immunostaining ((TIF)Click here for additional data file.S4 FigCol2a1-tTA allele. See ref. [Col2a1 regulatory region used. In the absence of Dox, the tTA is activated around E12.5  [+ chondrocytes in the PZ of left proximal tibia of Pit-tTA-p21 embryos unexposed to Dox, at E15.5, E17.5, and P0 . Comparison by 1-way ANOVA (p = 0.0368), followed by Tukey’s post hoc tests (shown). (B) Left/Right ratio of EdU incorporation in PZ chondrocytes of Pit-tTA and Pit-tTA-p21 mice at E15.5 (n = 3 each), E17.5 (n = 4 each), and P0 (n = 3 each). Comparison by 2-way ANOVA for Genotype and Stage . (C) Percentage of p21+ or p21− chondrocytes that have EdU+ nuclei in the PZ in the left and right tibias of E17.5 ePit-p21 (Control) and ePit-Col-p21 (Exp) embryos. p21− cells from Control and Exp mice were compared by 2-way ANOVA with Side and Genotype as variables . n as in panel B. (D) Length of P0 Pit-p21 (n = 6–10 depending on the bone) and Pit-tTA-p21 (n = 3–7) right bones, normalized to the average value of control littermates. Comparisons were done by 2-way ANOVA with Genotype and Bone identity as variables; p-values are shown. (E) Weight of pooled E17.5 and E18.5 Pit-p21 (n = 9) and Pit-tTA-p21 (n = 11) mice, normalized to the average value of control littermates and compared by unpaired 2-tailed Mann-Whitney test. (F) Left/right length ratio for femur and tibia from newborn Pit-p21 (n = 10) and Pit-tTA-p21 (n = 3–8) mice. Comparisons by 2-way ANOVA with Genotype and Bone identity as variables; p-values are shown. (G) Cell density in left and right PZ of E17.5 Pit-p21 (n = 4) and Pit-tTA-p21 (n = 4) mice. Comparisons were done by 2-way ANOVA with Genotype and Side as variables; p-values are shown below the graph . For panel A–G, see also (A) Left: schematic of the new See ref.  for deta allele) . Right: (TIF)Click here for additional data file.S5 Fign = 7 ePit-p21, n = 13 ePit-Col-p21). (C) Correlation analysis between the μCT measurements and the measurements done on micrographs of the same bones (n = 80). Spearman’s correlation coefficient (and 95% CI) is shown. (D) Left panel: weight of E15.5 and E16.5 ePit-p21 (n = 10 and n = 5) and ePit-Col-p21 (n = 11 and n = 6) embryos, normalized to the average control littermate and compared by 2-tailed unpaired Mann-Whitney test. Right panel: comparison of right bone length at P0. n = 4 ePit-p21 and n = 4 ePit-Col-p21 pups. Comparison by 2-way ANOVA with Bone and Genotype as variables. p-Values for Sidak’s post hoc test are shown on the graph. (E) Analysis of tdT expression in E17.5 Pitx2-Cre/+; Col2a1-rtTA/+; Igs7TRE-LSL-tdT/+ embryos  does not reveal spurious activation outside the left cartilage templates (n = 2). The embryos were bisected sagittally to facilitate sectioning. For panel B–D, see (A–B) Representative 3D reconstruction (panel A) and normalized measurements (panel B) of E17.5 bones scanned by μCT ((TIF)Click here for additional data file.S6 Figp ≤ 0.05. (E) Normalized counts for Cdkn1a (p21) and Rln1  are shown for each sample. Note that Rln1 is virtually absent from control and experimental cartilage. See also Cdkn1a. The 4 examples shown are involved in cellular stress response [(A) Schematic of the experimental approach. After dissection and perichondrium removal, left and right cartilage elements were deprived of condyles and hypertrophic zone and were flash frozen. Left and right samples from each embryo were kept separated, and RNA was extracted for deep sequencing. (B) Unsupervised hierarchical clustering of 6 samples (left and right cartilage from 3 embryos). Note that each sample is closest to its contralateral one. (C–D) MA plot (panel C) and clustered heatmap (panel D) of the 285 DEGs (red dots in panel C) obtained by a paired DESeq2 design with adjusted response –52. For (TIF)Click here for additional data file.S7 Fig(A–B) Two alternative mechanisms that could underlie compensatory proliferation in response to a stress signal, classified based on whether they work at the whole growth plate level  or just by proximity to the stress signal (panel B). Coloured outlines identify chondrocytes producing the stress molecule. Note that in panel A, the self-propagating signal could be the same as the original stress molecule. t1–t3 refer to subsequent times of the travelling wave. (C) Potential relay of the stress signal into circulation, which in turn impacts on placental function, causing a systemic reduction in growth (stunting).(TIF)Click here for additional data file.


CDKN1A gene. It is a major p53 target gene involved in cell cycle progression that has been extensively evaluated. To date, p21 has been reported to regulate various cell functions, both dependent and independent of p53. Besides regulating the cell cycle, p21 also modulates apoptosis, induces senescence, and maintains cellular quiescence in response to various stimuli. p21 transcription is induced in response to stresses, including those from oxidative and chemotherapeutic treatment. A recent study has shown that in response to metabolic stresses such as nutrient and energy depletion, p21 expression is induced to regulate various cell functions. Despite the biological significance, the mechanism of p21 regulation in cancer adaptation to metabolic stress is underexplored and thus represents an exciting field. This review focuses on the recent development of p21 regulation in response to metabolic stress and its impact in inducing cell cycle arrest and death in cancer cells.Cancer cells possess metabolic properties that are different from benign cells. These unique characteristics have become attractive targets that are being actively investigated for cancer therapy. p21cip1/waf1, also known as Cyclin-Dependent Kinase inhibitor 1A, is encoded by the  Eukaryotic cells utilize complicated mechanisms to regulate cell division and quiescence in response to both internal and external stimuli. These cells rapidly divide under certain conditions such as embryonic development and wound healing. Conversely, they stop proliferating in response to adverse conditions and enter cell cycle arrest and post-development quiescence. Hence, the cells develop a sophisticated braking system required for survival. Deregulation of this mechanism can result in loss of genome integrity and cancer development. p21 (Cip1/WAF1) was first identified as a Cyclin-Dependent Kinase (CDK) regulator that inhibits the retinoblastoma gene (Rb) phosphorylation and G1/S cell cycle progression ,4,5. p21p21 transcription can be regulated by either a p53-dependent or -independent manner. These regulations were described in the early years of p21 studies, where p21 induction in response to radiation-induced DNA damage was characterized as p53-dependent . In contDuring normal tissue development and serum-stimulated growth in vitro, p21 is primarily regulated in a p53-independent manner by a number of transcription factors . Under vEpigenetic processes can also regulate p21 transcriptional activation. They can be induced through the p53-independent pathway by chromatin remodeling following acetylation of histones H3 and H4 in the p21 promoter region ,38,39,40p21 is also subjected to post-translational modification. Most notably, it is regulated by serine-threonine kinases phosphorylation at Ser-130, Ser-146, Thr-57 and Thr-145. These phosphorylation events impact both the function and stability of p21. For example, Thr-145 phosphorylation by AKT kinase disrupts p21 binding to PCNA and enhances its cytoplasmic localization. This, in turn, promotes cell survival and transformation in breast cancers ,45,46,47Inhibition of cell cycle progression is one of the main cellular functions of p21. It mainly occurs by inhibiting cyclins A/CDK2, E/CDK2, and D1/CDK4 activities, thereby preventing the phosphorylation of Rb protein . The abip21 has been demonstrated to regulate apoptosis in a paradoxical manner, depending on the cellular context ,54,55. Ip21 is also a major regulator of cellular senescence, a complex program involving multiple signaling pathways. p21 interacts with p16INK4A tumor suppressor to inhibit cyclin-dependent kinases, suppressing retinoblastoma protein phosphorylation and the expression of cell proliferation-associated genes . SimilarA hallmark of stem cells is their ability for self-renewal. Most stem cell populations are in the quiescent state until they are stimulated. p21 has been reported to maintain cellular quiescence ,67 and pApart from its well-established roles, p21 may also directly or indirectly influence cancer metastasis. However, the association between p21 expression and metastatic ability is inconsistent in different cancers. In some cancers, p21 levels are inversely related to the metastatic phenotype, where a higher p21 expression predicts a favorable disease outcome ,73,74,75Both p21 inactivation and activation have been implicated in cancer therapy resistance. p21 inactivation is associated with paclitaxel and 5-Fluorouracil (5FU) resistance in noncancerous breast epithelial and colon cancer cells, respectively ,81. In cMetabolism is a cornerstone for various cellular functions. It is so important that Major metabolic enzymes and regulatory pathways are conserved from unicellular organisms to mammals. Metabolic stress can be triggered by either nutrient unavailability or malfunction of important proteins involved in the metabolic machinery. Adaption to metabolic stress is essential for cell proliferation and survival. p21 is involved in cellular responses to various stressors ,87,88. Rp21 plays an important role in cell response to nutrient deficiency. Studies have shown that p21 is transcriptionally upregulated in mice under short-term fasting ,90. A maCellular nutrient uptake can stimulate intracellular nutrient-sensing mechanisms. This, in turn, activates anabolic pathways aiming at increasing biosynthesis. Apart from nutrient availability, the stimulation of growth factor signaling also accelerates anabolism. Nutrient deprivation, particularly glucose, is sensed by the AMP-activated kinase (AMPK), a key regulator of the catabolic-anabolic balance. AMPK is an allosteric enzyme that is directly regulated by the AMP to ATP ratio, which is a readout for cellular energy status ,96. GiveWhile it is not the focus of the current review, it is important to note that AMPK can also sense DNA damage stress and induce p21 transcription in both p53-dependent and-independent manners ,100,101.Amino acids are important building blocks and signaling intermediators. Their deficiency can alter many cellular metabolic functions that, in turn, regulate cell cycle progression and survival through complex regulations, including those involving p21 ,107,108.In cancer, p21 is generally perceived as a tumor-suppressor protein. This hypothesis is supported by the observations that p21-null mice spontaneously developed tumors . AdditioCompared with their benign counterparts, cancer cells have distinct metabolic characteristics that present therapeutic opportunities. One of the most recognized signatures of cancer metabolism is the Warburg effect, or aerobic glycolysis, which describes the preference of cancer cells using glycolysis over oxidative phosphorylation to metabolize glucose and derivatives for energy production. As a result of incomplete oxidation, most cancer cells consume glucose to generate more metabolic intermediates and lactic acid than benign cells ,117. MulDespite their dependence on glycolysis in the metabolism, mitochondria respiration in cancer cells is important for generating energy and nucleic acid synthesis . As a maRAS mutation driven-oncogenesis accounts for one-third of human cancers. The relevance of the RAS mutation becomes even more apparent if the aberrancies in relevant pathways such as EGFR  mutations and MAPK (Mitogen-Activated Protein Kinases) signaling components are also taken into consideration ,136,137.To better understand the mechanisms underlying p21 elevation in response to ICMT inhibition and its role in cancer cell proliferation and survival, we evaluated a panel of cancer cells from diverse tissue origins. Cancer cells that are sensitive to ICMT inhibition show a constellation of changes related to metabolism that include the reduction of mitochondria respiration, the induction of autophagy, cell cycle arrest and apoptosis ,146,147.To better understand how p21 regulation leads to cell adaptation following metabolic distress, it is necessary to briefly discuss the cellular process of autophagy—a fundamental eukaryotic cell adaptation in response to a nutrient-depleted state. The double membranous structure of autophagosomes forms as a self-digestive organelle in the lysosomal pathway to degrade damaged/aged organelles and protein aggregates; this maintains homeostasis in cells under normal conditions . AutophaIn evaluating the impacts of ICMT inhibition in cancer cells, we found that p21 is induced only in the sensitive cell lines upon ICMT suppression. Marked reduction in mitochondrial respiration and consistent depletion of cellular “fuel molecules”, such as ATP and other NTPs, were also noted following exposures to the ICMT inhibitor. The mitochondria targeting effect has been shown to be responsible for most of the anti-proliferative effects . The pheAs discussed earlier, p21 regulates cell cycle progression and apoptosis. Therefore, it makes physiological sense that p21 would be involved in sensing and transmitting the signal of metabolic stress. Indeed, when we further evaluated the role of p21 in response to ICMT inhibition, we found that p21 is an upstream regulator of cell cycle progression, autophagy and apoptosis, through the transcriptional regulation of downstream effectors that include BNIP3 (BCL2 Interacting Protein 3), LC3 (Microtubule-associated protein 1A/1B light chain 3B) and ULK1 (Unc-51 Like Autophagy Activating Kinase 1). Both ULK1 and LC3 are well-known proteins that promote the initiation and extension of autophagosomes. BNIP3 is a mitochondrial member of proapoptotic BCL2 family proteins containing a motif similar to the BH3 domain, which is a regulator of both autophagy and apoptosis ,165,166.In summary, we have discussed the evidence that supports p21 as a key regulator and signaling node in mediating cancer cells’ response to nutrition and fuel depletion. Overall, the evidence supports that p21 functions to integrate catabolism activities, cell cycle progression, and ultimately programmed cell death B. While The field of p21 has grown tremendously in the last three decades. Since its discovery in the 1980s as a Cyclin-Dependent Kinase inhibitor, p21 has attracted significant attention from researchers in many fields, as evident from the thousands of research publications on this protein. To date, p21 is known not only as a cell cycle regulator, but also as a key player in various other cellular functions including senescence, apoptosis, and self-renewal. p21 has also been long regarded as a stress response protein. In this regard, p21 can function as a coordinator for DNA damage repair pathways. Adding to its portfolio, p21 has recently been reported to act as a central regulator in cell adaptation to metabolic stress, particularly energy depletion in response to either starvation or treatment with mitochondria respiration inhibitors. Although this aspect has only been recently investigated with relatively few publications, the impact of these findings in understanding basic cell biology and the development of potential new therapy is apparent. For example, although recent findings demonstrate that stress-induced levels of p21 primarily leads to cell cycle arrest and cell death, the exact outcomes of p21 activation are very much dependent on the type, intensity and duration of particular stress, as well as the cell type upon which the stress acts . AnotherThis review hopefully highlights (i) the significance of emerging p21 research as a promising therapeutic approach for biologists and oncologists, and (ii) the necessity to continue the effort in understanding p21’s function as a coordinator of metabolic stress responses. As mentioned above, because of its dichotomy function, it is a challenging task to fully understand how p21 regulates various biological and cellular functions, particularly those associated with the cancer metabolism and its potential therapeutic implementation. Concerted explorations to understand p21 in adaptation to stress, metabolic and other stimuli, are essential for its translational application in cancer therapy.


Research investigating female gaming has begun to emerge despite gaming being traditionally more popular with males. Research in the 21st century has drawn attention to the role of women in culture, society, and technology, and female gaming is one of the growing phenomena not to have been researched in depth. The aim of the present paper was to review female gaming  and identify any associated psychopathological symptomatology. The review adapted the Sample, Phenomenon of Interest, Design, Evaluation, Research (SPIDER) model in conducting a narrative literature review. A search of three scientific electronic databases yielded 49 papers for further evaluation. From a methodological perspective, studies had to fulfill the following criteria to be included: i) published between the years 2000 and 2018; ii) assessed female gaming or the female position within gaming culture, iii) contained quantitative, qualitative, or mixed methods approaches to produce empirical data or discuss theoretical implications through reviews, iv) be retrievable as a full-text peer-reviewed journal paper, and v) published in English, German, Polish, Spanish, Italian, Portuguese, or French. Four categories emerged from the papers: i) the benefits of female gaming, ii) why women might play video games less than men, iii) perceptions and realities of female characters within video games, and iv) women’s position in gaming culture. The main findings showed playing video games has benefits for women in terms of enhancing cognitive, social, and physical abilities. However, they are less encouraged to play video games due to negative expectations based on gender and/or experiences during game play. Video games are associated with stereotypical male characteristics, such as being overly aggressive, and frequently contain sexualized content. Female gamers appear to require coping strategies to handle online harassment. Females look for different things in video games, which are not often included in game designs thereby limiting their abilities. For instance, female avatar representation—which is exaggerated and hypersexualized—can prompt social comparisons and lead to feelings of decreased self-esteem, depression, and other impacts on well-being. Overall, there are still obstacles for women playing video games even though they comprise half of the gaming population. Over the past two decades, the number of female video game players has increased, and females today make up half of the gaming population according to both the Entertainment Software Association (ESA)  and the Despite the growing female gaming population, almost all research on gaming addiction is focused on male gamers. Currently, there is no agreement on the prevalence of gaming addiction due to its conceptualization and methodological problems within the research that has been conducted to date. This is because most research i) comprises surveys using non-representative samples ; ii) uses different scales to assess problematic gaming . Thus, it is not possible to draw conclusions about the extent to which sample demographics across the studies in this review reflect female participants only, the population within a particular geographic region, or across the nation. The findings only reflect those based on gaming research in Western culture.Additionally, the specific scientific databases selected and the inclusion criteria used to conduct this review may have excluded some sources, especially from other disciplines outside of psychology and medicine, although https://www.heforshe.org/en) started by the United Nations is an excellent case in point addressing gender equality, whereby both women and men are encouraged to commit to standing in “solidarity with women to create a bold, visible and united force for a gender equal world.” In the context of women’s position in gaming culture, this contemporary feminist movement helps support the view of women as being just as capable and skilful gamers as men and require just as much respect and recognition from gaming culture, which they are part of. It may influence beliefs that women are inferior within gaming and encourage more females to play video games. It may also open up communication in such a way that harassment is reduced, and designers consider video games with women as their audience more so than they have done previously. According to the ESA , AW (Method and Results), and DK (Discussion). Review and editing were done by OL-F, DK, AW, and MG.via multi-platforms.”The present study was supported by the Psychology Department QR Funding at Nottingham Trent University, through a Kickstarter grant (2018) awarded to OL-F to develop studies on “Female gaming: A cross-cultural study of pathological and non-pathological gaming The authors declare that the research was conducted in the absence of any commercial of financial relationships that could be construed as a potential conflict of interest.