N = 36, males, between 15 and 25 years of age), we found that a large proportion stopped interacting with the program over time . Our results also indicated that students performed significantly more health-related activities when assigned to the social regret lottery, as opposed to the individual variant. This result was supported by interview responses from active participants: They mainly participated to compete against their peers, and not so much for the prizes. Hence, from this study, we obtained initial evidence on the impact of social and competitive aspects in lottery-based incentives to stimulate engagement levels in lower SES students with an mHealth app.In general, individuals with lower socioeconomic status (SES) are less physically active and adhere to poorer diets than higher SES individuals. To promote healthier lifestyles in lower SES populations, we hosted a digital health promotion program among male vocational students at a school in The Netherlands. In a pilot study, we evaluated whether this target audience could be engaged with an mHealth app using lottery-based incentives that trigger feelings of anticipated regret. Especially, we studied the social and interpersonal aspects of regret lotteries in a within-subject experimental design. In this design, subjects either participated in a social variant , or an individual variant . Additionally, we studied the impact of different payout schedules in a between-subject experimental design. In this design, participants were assigned to either a short-term, low-value payout schedule, or a long-term, high-value payout schedule. From a population of 72 male students, only half voluntarily participated in our 10-week program. From interviews, we learned that the main reason for neglecting the program was not related to the lottery-based incentives, nor to the prizes that were awarded. Instead, non-enrolled subjects did not join the program, because their peers were not joining. Paradoxically, it was suggested that students withheld their active participation until a larger portion of the sample was actively participating. From the subjects that enrolled in the program ( Over the past decade, public health literature has clearly demonstrated a relationship between socioeconomic status  and unhA challenge of lifestyle intervention design is to select the appropriate strategies to stimulate end user engagement with the intervention , 12. GamThis study aims to extend the body of literature on the impact of regret lotteries on engagement levels with an mHealth app in lower SES populations. In this pilot study, we explored the impact on student engagement levels of either social, or individual regret lotteries, by controlling whether lottery outcomes were publicly announced , or not . Because anticipated regret was proposed to be larger if also visible to (significant) others , we hypoParticipants were recruited among first-year and second-year , vocational students at a vocational school  in The Netherlands in April 2020. The entire population comprised 72 students, distributed over 6 different school classes. The study was advertised as a health promotion program that students joined voluntarily. The written consent of students was collected digitally upon registration for the program. All  procedures were approved by the ethics committee of Eindhoven University of Technology .For this study, we have developed a custom web interface using the mHealth app GameBus , 22, to The overall goal of the intervention from the students' perspective was to obtain as many virtual points as possible by performing the suggested health-related activities. In particular, it was set by the school's management to focus on: (1) increasing physical activity, (2) improving dietary intake, and (3) fostering sustainable relationships among students . From tThe entire program lasted 10 weeks, and was divided into five program “waves,” each with a duration 2 weeks. Every wave, a subset of activities were suggested, see To explore different aspects of meaningful (regret) lottery design in a health promotion context, we have designed an experiment with four study arms. In a within-subject experimental design, we have evaluated the impact of a social regret lottery-based incentive, compared to the impact of an individual lottery-based incentive. Subjects in both the first and third study arm  were assigned to the individual lottery-based incentive in the first wave . Subjects in both the second and fourth study arm  were assigned to the social regret lottery-based incentive in the first wave . Additionally, in a between-subject experimental design, we have evaluated the impact of a short-term, low-value payout schedule, compared to the impact of a long-term, high-value payout schedule. Subjects in SA1 and SA2 were assigned to the short-term, low-value payout schedule, whereas subjects in SA3 and SA4 were assigned to the long-term, high-value payout schedule. Lastly, participants were allocated to the different study arms using a randomized block approach , resulting in a study design that was theoretically balanced with 14–20 students per study arm. The entire study design is detailed in all participants, although non-eligible “winners” were not awarded, but instead they were publicly informed on their forgone prize. Also, participants could review each other's position on the leaderboard, and eligibility to win prizes at any time.The social lottery-based incentive was visualized as a leaderboard, that displayed the total number of points a participant had obtained in a given wave, also see The individual lottery-based incentive was visualized as a digital slot machine, also see The school's management had provided several tangible rewards that were likely attractive for the students. The school provided 54 tangible rewards. Hence, only roughly 11 tangible rewards were available per wave, which had to be distributed over a sample that could theoretically contain up to 72 students. To increase participant's overall chances to win a prize, several virtual badges were introduced. Note that these badges did not have any monetary value. The prizes  were distributed throughout the program in a between-subject experimental design, to explore the impact of varying payout schedules in terms of timing and monetary value.Several prizes were distributed in a short-term, low-value payout schedule. The payout schedule was short-term as prizes were distributed regularly . The payout schedule was low-value because the prizes were of lower monetary value , see non-enrolled users. Students that enrolled in the program, but did not engage in the suggested activities were labeled passive users. Finally, subjects that enrolled in the program and did register at least one health-related activity were labeled active users.In mHealth research, engagement is most commonly captured via measures of app usage , 27. UsiA post-test survey was used to collect demographic data  from the entire target population. The survey was distributed at the end of the program to the entire target population. Students were allowed to complete the post-test survey during classroom hours. Additionally, at the end of the program, two semi-structured, focus group interviews were conducted to further elaborate on participants motivation to either participate, or not to participate. The first focus group panel consisted of three randomly selected participants that did not register any activities into the mHealth app, whereas the second focus group panel consisted of three randomly selected participants that were among the participants with the highest number of activity registrations. The interviews were conducted online, via videoconferencing software, and were supervised by two authors . The main focus of the interviews has been to qualitatively derive: (1) the students' preferences for either the social variant, or individual variant, of the lottery-based incentives, and (2) the students' perceptions of attractiveness of the tangible rewards. Additionally, within the panel of non-enrolled participants, we have also focused at deriving the barriers that withheld them from participating in the program. Finally, within the panel of active participants, we have also aimed to derive the students' perceptions of the impact of the program on their overall health status and engagement in health behaviors.p-value with p>0.05 is deleted (p ≤ 0.05). We have not tested whether significant second-order interaction effects existed amongst these variables, as sample sizes in individual clusters would then have been too low for a thorough analysis of second-order interaction effects. In all models we did allow for random intercepts between individual participants. Subsequently, we have used Pearson's Chi-Squared test to confirm our final models do not suffer from overdispersion. Overdispersion of a Poisson model occurs when the variance value of the underlying data is greater than the mean value of that data, suggesting that the data is not likely to be modeled well using a Poisson model , exploratory analyses were performed using mean plots to detect potential differences between study arms, over time. Subsequently, several generalized  hierarchical linear models were estimated for these two outcome variables, using time , and the treatment variables  as predictors. To obtain the final models for both outcome variables, a backward elimination selection procedure was employed . Backwar deleted . This meon model . Lastly,In total, 38 unique participants joined the study. Amongst the participants were 36 students, and 2 teachers . The students were assigned to the different study arms using a randomized block approach: 6 students were assigned to SA1, 17 to SA2, 2 to SA3, and 11 to SA4. Of the 36 students enrolled, 10 students performed at least one activity; the others have only been checking the app. Passive engagement was measured as the number of days participants checked the app within a given wave. p = 0.01). The model was validated not to be harmed from overdispersion. Still, the model may have underfitted zeros, slightly . Finally, although payout schedules were not reported to have a significant impact on passive levels of engagement, From a backward elimination selection procedure we have obtained a final model for predicting the number of days participants checked the app with just one significant predictor. Particularly, we found that the number of days participants checked the app significantly decreased over time . Additionally, it was found that the number of activities participants performed decreased when engaged with the individual lottery-based incentive , as opposed to the social lottery-based incentive. The model was validated not to be harmed from overdispersion and not to be underfitting zeros . Finally, again, I only would have participated in the program when more people would have joined.” Additionally, a participant mentioned: “I would have liked to engage in a competition with my peers, but when I checked the leaderboard, I did not see too many active participants, which in turn did not spark my interest to participate.” Another reason for not engaging with the program was that interviewees found the suggested activities and prizes insufficiently attractive: “Although the suggested activities and prizes looked like they could have been attractive for some, they did not stimulate me to engage with the program.” Nevertheless, the interviewees even speculated that prizes with a higher monetary value would also not have triggered them to actively participate: rather would they have engaged in a program with a higher overall uptake. Lastly, interviewees were hesitant to participate because of  invasions of privacy: “I did not like the fact that I was required to upload photos and videos of myself engaging in the suggested activities, as I felt my privacy could thereby potentially be invaded.”As the main reason for not engaging with the program, participants within the panel of non-enrolled participants designated the overall low uptake with the program: “did not participate to win prizes“: their main incentive was “to engage in a competition with other participants.” One focus group interviewee mentioned that he “wanted to be better than the others.” He added: “Especially, I wanted to beat the ones with a comparable number of virtual points as myself: even when we could not see from each other the number of points we had collected [because we were engaged in the individual lottery-based incentive], we kept each other posted on the number of points we had collected.” “ Whenever I was falling behind, I would make sure to perform more activities to catch up.” The drive to compete against others made participants hesitant to exchange their virtual points for a spin of the slot machine in the individual lottery-based incentive. One subject mentioned that he did not want to spend his points at the digital slot machine, because he was “afraid to loose his points.” He mentioned: “I was told that an other participant had spent all his points without winning anything, so I rather kept my points.” Lastly, participants were unsure whether the program empowered them to change their lifestyle: “I cannot say that the [mHealth] app changed my behavior, because I already engaged in physical activities regularly before the intervention.” However, he mentioned: ‘The [mHealth] app made engaging in healthy lifestyle behaviors more fun, especially when we could compete against each others by collecting points.” Participants agreed that the implementation of (push) notifications could further increase the impact of the intervention.Participants within the panel of active participants agreed that they “In this pilot study we evaluated engagement levels of vocational students with a health promotion program that employed lottery-based incentives. Half of the target population of 72 male students voluntarily engaged with the program and were exposed to the lottery-based incentives. The other half of the population had never engaged with the program. From focus group interviews, we learned that the main reason for non-enrolled subjects to neglect the program was not related to the lottery-based incentives, nor the prizes that were awarded. Instead, non-enrolled subjects did not join the program since their peers were not joining the program. In other words, we found that the program would have been more engaging if more students had actively participated initially. This seems to be a paradox of participation: subjects withhold their active participation until a larger portion of the sample is actively participating. However, if everyone is waiting for their peers to participate, no one will participate. This paradox may potentially be resolved by introducing interdependencies among participants , such thFrom our analyses of subjects that did actually enroll in the program, we found that a large proportion of participants stopped interacting with the program over time . A reason may be that students tried to minimize potential feelings of regret by ignoring the study . AnotherFinally, from our exploratory statistical analyses of user engagement levels it was suggested that the long-term, high-value payout schedule fostered student engagement levels, especially in the last wave of the program. Although we could not statistically validate this observation , this observation may indicate that the impact of a long-term, high-value payout schedule is largest when the deadline approaches . Hence, in line with the proposition of Zeelenberg , a shortN = 72) that only included male subjects. Moreover, only 36 out of 72 students enrolled in the study and, through our randomized block design, subjects were unevenly distributed to study arms . Although measures were taken to attract as many students as possible to join the program , as well as to balance sample sizes over study arms , students participated in the program voluntarily. As a result, the overall sample size and sample sizes over study arms could not be controlled.This study was subject to several limitations. First, this study evaluated the impact of our intervention on a particular target group  within a specific context . Based on the current pilot study, findings cannot be generalized yet as results were derived from a small target population  had expressed a desire for all participating students to have the opportunity to win a tangible reward.Third, additional data measures could have helped to better interpret the results. In particular, we have not measured the levels of regret participants actually perceived from the different lottery-based incentives. Similarly, no data was recorded on the number of times teachers brought up the program in their virtual classrooms, nor the degree(s) of students attending to these classrooms, which could have helped to explain adoption rates. Besides, the study could have benefited from an exploration of social relationships between students in order to be able to assess which students were befriended, which students were most popular and which teachers were beloved. Potentially, this analysis could have helped to target the most influential subjects, and motivated them to actively participate. Presumably, these subjects could have then triggered others to actively participate .Fourth, the experimental setup was vulnerable for fraudulent usage. In theory, participants could upload photos and videos of activities that were not performed by themselves, or that they had already used to claim points. The research team has validated incoming activities on a day by day basis. When any form of fraud was detected in an activity registration, all the points and possible prizes that were obtained from that activity were withdrawn. Participants who committed fraud were also alerted by a pop-up that their user account could be suspended, whenever they continued cheating [i.e., see  for detaFinally, since this study was executed in times of the COVID-19 pandemic, students were not actually physically present at school. Instead, students were educated via videoconferencing. Hence, also our intervention, including participant recruitment by teachers, was hosted entirely online. This may have harmed the effectiveness of the participant recruitment strategy.To counter the paradox of participation that we identified, and to persist with momentum throughout the program, follow-up studies may evaluate the impact of creating interdependencies between participants, as well as using bot accounts to artificially boost the number of active participants. Additionally, follow-up studies could explore combinations of different payout schedules to find the optimal configuration for sustaining user engagement over a longer period of time. Lastly, follow-up studies could benefit from recording participants' levels of perceived regret from different lottery-based incentives. These measurements provide insight into the degree of anticipated regret vocational students perceive from different implementations of lottery-based incentives.We found that lottery-based incentives that trigger feelings of anticipated regret can potentially be employed in lifestyle interventions to promote engagement with mHealth apps among lower SES vocational students. However, besides these lottery-based incentives, practitioners may want to employ other methods to initially attract a larger portion of the population to enroll in the program. Sole deployment of lottery-based incentives seems insufficient to attract an entire target population. But, paradoxically, a higher uptake may increase the actual impact of the lottery-based incentive itself. Additionally, the impact of lottery-based incentives may be fostered by including social and interpersonal  aspects. Particularly, we found that an incentive with social and competitive elements was more engaging than an incentive solely at the individual level. Finally, we have found initial evidence of payout schedules increase engagement, particularly when their deadline approaches. Hence, different payout schedules with alternating deadlines may potentially be employed to foster engagement at different phases throughout a lifestyle intervention. Still this observation has to be studied in more depth.10.6084/m9.figshare.14386754, ' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.RN and PV: conceptualization, methodology, software, and validation. RN and JH: formal analysis, investigation, resources, and writing—original draft preparation. RN: data curation and visualization. RN, PV, PL, AK, PB, and MS: writing—review and editing and supervision. RN, PV, and JH: project administration. PV, PB, AK, PB, and MS: funding acquisition. All authors have read and agreed to the published version of the manuscript.This work was part of the research program Gamification for Overweight Prevention and Active Lifestyle (443001101), which was partly financed by the Netherlands Organization for Health Research and Development (ZonMw).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A family-centered care (FCC) parent participation program that ensures an infant is not separated from parents against their will was developed for the caring of their small or sick newborn at a neonatal intensive care unit (NICU) in Delhi, India. Healthcare provider sensitization training directed at psychosocial and tangible support and an audio-visual training tool for parent-attendants were developed that included: 1) handwashing, infection prevention, protocol for entry; 2) developmentally supportive care, breastfeeding, expression of breastmilk and assisted feeding; 3) kangaroo mother care; and 4) preparation for discharge and care at home. The study aimed to examine the feasibility and acceptability of the FCC model in a NICU in India.A prospective cohort design collected quantitative data on each parent-attendant/infant dyad at enrollment, during the NICU stay, and at discharge. Feasibility of the FCC program was measured by assessing the participation of parent-attendants and healthcare providers, and whether training components were implemented as intended. Acceptability was measured by the proportion of parent-attendants who participated in the trainings and their ability to accurately complete program activities.Of 395 NICU admissions during the study period, eligible participants included 333 parent-attendant/infant dyads, 24 doctors, and 21 nurses. Of the 1242 planned parent-attendant training sessions, 939 (75.6%) were held, indicating that program fidelity was high, and the majority of trainings were implemented as intended. While 50% of parent-attendants completed all 4 FCC training sessions, 95% completed sessions 1 and 2; 60% of the total participating parent-attendants completed session 3, and 75% completed session 4. Compliance rates were over 96% for 5 of 10 FCC parent-attendant activities, and 60 to 78% for the remaining 5 activities.FCC was feasible to implement in this setting and was acceptable to participating parent-attendants and healthcare providers. Parents participated in trainings conducted by NICU providers and engaged in essential care to their infants in the NICU. A standard care approach and behavior norms for healthcare providers directed psychosocial and tangible support to parent-attendants so that a child is not separated from his or her parents against their will while receiving advanced care in the NICU. The first few weeks of an infant’s life are an important time for the formation of healthy attachment between newborn and parents. When an infant is born small or sick, however, separation from the parents during hospitalization can disrupt this bonding process . Modern The family-centered care (FCC) approach was adapted from the Human Neonatal Care Initiative in the 1990s , and exiFCC has been implemented in several high-income countries  includinIn 2008, family engagement was introduced in the NICU at Ram Manohar Lohia (RML) Hospital, a busy tertiary hospital in New Delhi, to aid the nursing team in their duties and overcome severe workforce constraints that limit quality of care. This was the first implementation of family engagement in the NICU in India and the first time such a model has been tested in South Asia. Parents were trained on handwashing, entry protocol, breastfeeding, assisted feeding and skin-to-skin contact for eligible infants, developmentally supportive care, as well as recognition of danger signs and preparation for post-discharge care at home. In 2010–2012 a randomized controlled trial of the approach documented improved breastfeeding rates and no increase in nosocomial infections or adverse events [Following the encouraging results of the trial, an FCC health education program was developed for the parent caring for a small or sick newborn in the NICU, linked to NICU care provider sensitization to effectively communicate with and support parents. This intervention is a paradigm shift in NICU care that transforms parent-attendant roles from mere passive observers to active willful participants with the care team through skills building and demonstrated competence. The program includes parent-attendant training on 1) handwashing skills; importance of infection prevention; protocol for entry to nursery; 2) developmentally supportive care ; 3) kangaroo mother care; and 4) preparation for discharge and care at home. The resulting collaboration between the families and the healthcare providers creates a mutually respectful partnership that fosters communication between the two stakeholders and develops trusting relationships. In 2014, RML Hospital collaborated with the Norway India Partnership Initiative to pilot test the model in five district-level special newborn care units. Based on the study’s results, the Government of India issued a national policy to scale up FCC in all 700 district special newborn care units , 30. In We conducted this study in the 14-bed NICU at RML Hospital in New Delhi, India. It is a referral neonatal unit in a teaching hospital which provides tertiary neonatal care including assisted ventilation and major surgery for sick newborns. NICU care at the study facility, as well as other facilities in the public health system, is absolutely free of cost. However there are private sector facilities in India that provide care at a cost levied to the patient and his/her family. During each shift, the unit is staffed by up to three nurses and two resident doctors (one senior and one junior). The NICU admits an average of 30 infants per month and has 2 sub-divisions: the intensive care area and a step-down care area for care to infants graduating from intensive care. A dedicated breastmilk expression and storage room with a refrigerator has been created in the unit. Kangaroo mother care (KMC) is provided by the bedside. All eligible infants admitted to the unit during the study period were recruited for participation in the study. Daily trainings for parent-attendants were provided in a designated area situated adjacent to the clinical area with seating for 12–15 persons and an LED television set.This feasibility and acceptability study was conducted using a prospective cohort design. The study protocol and data collection materials were developed by the study team at RML Hospital. We collected quantitative data on all study participants at enrollment, during the NICU stay, and at discharge using structured participant files.We examined the feasibility and acceptability of implementing an FCC model using seven measures. Feasibility measures addressed whether interventions were implemented as intended, such as sensitizing sessions for all healthcare providers, training all parent-attendants, and frequency of training sessions. Acceptability measures focused on whether the intervention was acceptable to parent-attendants and healthcare providers as measured by whether healthcare providers conducted parent-attendant training sessions and whether parents were able to complete all activities as intended. Feasibility and acceptability measures are described in Table The study met international ethics requirements and was approved by the RML Hospital New Delhi Ethics Committee, RML Hospital, New Delhi, India, and Quorum Review IRB, Global Alliance to Prevent Prematurity and Stillbirth (GAPPS), Lynnwood, WA, USA (No. 33218). Verbal consent was approved by the Ethics Committee and was obtained from all parent-attendants and healthcare providers participating in the study prior to data collection. We obtained verbal consent in their preferred language to avoid any potential perceived intimidation by requesting a signature, and informed them that their participation would be voluntary and there would be no professional or personal consequences nor benefits of participation. Mothers were given the option to read or hear their consent form according to their literacy level. To avoid possible coercion, no financial incentives were provided.Parent-attendant/infant dyads were eligible to participate in the study if the infant was hemodynamically stable (not on inotropes), not on ventilation (invasive or noninvasive), and the parent-attendant was willing and available to provide care to the infant. Exclusion criteria included the following: the infant was not hemodynamically stable, the infant was on ventilation, and the parent-attendant was unwilling or unavailable to provide care to the infant.Of all 395 NICU admissions that occurred during the study period, 1 parent-attendant refused to participate, and 2 infants did not have an available parent-attendant and were therefore not eligible. Another 59 infants were found not to be eligible due to clinical exclusion criteria during their admission. Parents and family members of all eligible infants admitted to the NICU from June 2016 through July 2017 were included in this study.Data were collected on three groups of participants: 1) parent-attendants, 2) infants, and 3) healthcare providers (doctors and nurses) working in the unit. For each infant admitted to the unit, we identified a primary parent-attendant , who would be available and willing to care for the infant during the study. Some infants were cared for by more than one parent-attendant; however, for the purpose of this analysis we considered data about the primary parent-attendant only. A total of 392 parent-attendant dyads/infant participated in the study. All healthcare providers assigned to the NICU during the study period were included.Data were collected from enrollment through discharge for each parent-attendant/infant dyad. Compliance of the parent-attendant with FCC program activities was directly observed during June and July 2016. All data collected from parent-attendant/infant dyads were recorded by a study nurse and entered into the study database. Data collected include demographics, parent-attendant training session attendance, parent-attendant activities, unit staff sensitization sessions attendance, and staff participation in conducting training sessions for parents. All data were de-identified and analyzed in Excel. Descriptive analyses were completed to summarize study sample characteristics for parent-attendants, healthcare providers, and newborns. All continuous variables were summarized with means, percentages, and ranges. Infants with missing data were excluded from the analysis. However, those who died or left the unit against medical advice, but had complete data recorded, were included in the analysis. Data on healthcare providers were obtained on a quarterly basis from the routine NICU records that did not record personal identifiers.The FCC intervention focuses on developing a participatory collaboration between healthcare providers and parent-attendants in the care of a sick newborn in the NICU. The FCC concept has been implemented in diverse ways, with a common focus on the infant, parent, and healthcare provider triad as a unit of care . For theThe healthcare provider component provided sensitization to the FCC approach and training of the healthcare providers as to how to train and support the parent-attendants in a consistent and standardized manner. Healthcare provider buy-in is essential to this model of care, and therefore sensitization sessions were carried out for healthcare providers at the beginning of the study period and every three months during the duration of the study. This accommodates the system of rotational postings from the pediatric department to the neonatal unit. Provider sensitization/training sessions were conducted by the principal investigator and included an in-person training session on the topics covered in the parent-attendant trainings and a discussion session. In addition to providing medical care to newborns, healthcare providers led skill-building training sessions for parent-attendants that focused on psychosocial and tangible support, and provided ongoing support, supervision, and continuous communication with parent-attendants in the NICU. Mothers were provided three meals daily at no cost. Financial support to families was provided through a hospital social worker in times of economic hardship. The hospital ensured that families did not have to incur any out of pocket expenditure for medical care that their baby received.The parent-attendant training sessions were made available daily and the parent-attendants were granted autonomy to decide to participate or not, and could participate in sessions as often as they liked. A comprehensive audio-visual training tool with four sequential modules was developed with multidisciplinary technical input from a neonatologist, community medicine specialist, psychologist, nurse, and Hindi language expert. The topics included in each session are presented in Table During the first two months of the study, nurses formally observed a subset of parent-attendants as they performed essential newborn care activities to assess uptake of the training topics and evaluate whether parents were able to correctly perform all activities. The nurse in charge of the observation randomly selected five parent-attendants for observation daily, observed all parent-attendant activities, and recorded compliance with proper protocol for all activities performed. During the remainder of the study period, nurses recorded whether each participating parent-attendant completed an activity during each day of admission but did not assess accuracy of the performed skills.Fidelity of the training sessions for the parent-attendants and the sensitization sessions for the healthcare providers were assured through direct supervision from the senior nursing office in the NICU. There were no punitive measures or direct incentives provided for the healthcare providers to deliver the training sessions.Of 395 NICU admissions during the study period, eligible participants included 333 parent-attendant/infant dyads, 24 doctors, and 21 nurses. Most parent-attendants were mothers (68%), between the ages of 20–35 (87.6%), and either illiterate or had completed secondary school (59.2%). Twenty percent of parent-attendants were fathers, and 12% were other family members. The average length of stay in the NICU was 15.8 days and the average age at admission was 8.9 days. Of all infants admitted to the NICU during the study period, only 35.5% were female and only 38% were exclusively breastfed  were held, indicating that program fidelity was high, and most trainings were implemented as intended  to 84% (36/44). The percentage of healthcare providers receiving FCC training increased in the second half of the study, with more than 83% of staff trained in 2017. Provider training data were collected during discrete time points, therefore multiple provider training contacts are possible.The FCC program was acceptable to both parent-attendants and healthcare providers. Parent-attendant acceptability was measured by the percentage of parents who participated in the program trainings and the parent-attendants’ ability to accurately complete program activities. Of the 395 parent-attendant/infant dyads admitted to the NICU, 392 (99.2%) agreed to participate in the FCC program and had at least 1 parent-attendant available. One parent refused participation and two infants did not have an available parent-attendant and were therefore not eligible.Parents and family members were willing to participate in trainings and provide essential care to infants in the NICU. Training session 1 was conducted during study enrollment, and therefore all participating parent-attendants completed training session 1. While only 50% of parent-attendants (167/333) completed all 4 FCC training sessions, 95% (316/333) completed sessions 1 and 2, required for all participants; 75% (249/333) completed session 4 which covered preparation for discharge and care at home; and 60% of the total participating parent-attendants (200/333) completed session 3 which focused on KMC. Most parent-attendants were able to participate in the care of their infants in the NICU by completing the specified FCC activities daily throughout the study period.Observed compliance rates were over 96% for 5 of 10 FCC parent-attendant activities, and 60 to 78% for the remaining 5 activities. Fig.  The newbAcceptability of the FCC intervention to healthcare providers was measured by the percentage of all participating healthcare providers who participated in conducting parent-attendant training sessions after being sensitized to the program. Provider data were collected on a quarterly basis. The percentage of healthcare providers conducting parent-attendant trainings increased steadily during the study period, with more than 50% of healthcare providers participating in training parent-attendants during 2017 Fig. .Fig. 4NOf the 4 million newborn deaths each year, 99% are in low- and middle-income countries . In IndiThe family visiting policy in the NICU was favorable at the study facility. The hospital policy at the time of the study allowed either the mother or father to be present in the NICU without time restrictions, but was limited to one person at a time to avoid overcrowding. In the uncommon instance when the mother or father were not available, an alternate attendant from the family was allowed. Siblings were not allowed.The hospital philosophy and strategic priorities relating to FCC improved gradually as evidence was generated that an FCC approach when practiced in the NICU does no harm and is beneficial to newborns. The hospital leadership has acknowledged that the FCC approach to NICU care improves newborn outcomes, increases parent satisfaction, and is consistent with ethical principles for the provision of medical care.Healthcare provider sensitization and buy-in were key to driving implementation and participation in the FCC intervention. Through iterative learning during implementation we learnt that training only parent-attendants was not sufficient. Parents were already onboard, waiting to be involved in their own baby’s care in NICU. The bigger challenge was overcoming the conventional mindset of healthcare providers that assume a position of commanding authority in the traditional provider-centered care model. Psychosocial and tangible support and education offered to participating family members depends strongly on engaged healthcare providers , 36. HeaRecent studies of FCC implementation have found that this approach results in increased provider satisfaction and job performance, although the immediate impact of the intervention has been found to present challenges for nurses and healthcare providers, such as increased time away from patient care, difficulty in coordinating communication, and increased time spent on supporting and communicating with parents , 37–39. Most research on the implementation of FCC models has focused on the experience of and benefits to parents and family members participating in the intervention . In the Early adoption and sustained demonstration of desired activities is an indication of acceptability of the intervention and impact on behaviors. The overall compliance rates for the FCC activities were over 90%. Low compliance rates for particular FCC activities suggest either greater challenges for parents and family members or lower acceptability of these activities. Compliance with handwashing protocols was lower during the second month of observation than at the beginning of the study period, suggesting that this element may be less acceptable to parents and may make sustained implementation of this intervention challenging. Although not the primary outcome of this study, the decrease in compliance with certain activities may indicate parent-attendant fatigue, provider fatigue, or a particularly challenging activity for parents.Given the required strong focus on handwashing and infection prevention for newborns in hospital settings, the relatively lower compliance with handwashing is an important finding of this evaluation. While the benefits of infection prevention including hand hygiene are significant and demonstrable , achieviEfforts to sustain the fidelity of the FCC intervention were focused on the healthcare providers. The sensitization sessions and training directed to provide psychosocial and tangible support to parent-attendants led by experienced healthcare providers were scheduled regularly as new staff rotate into the NICU. A roster of trained healthcare providers was maintained and these providers were routinely scheduled for the daily parent-attendant trainings. The senior nursing officer in the NICU oversaw all FCC sensitization and training activities with support from the NICU medical director.During the onset of the COVID-19 pandemic in March 2020, practice of FCC in the NICU had a setback. Parent-attendant entry to the NICU was restricted until symptom screening and testing became available. Implementation of FCC has been maintained with appropriate COVID-19 transmission prevention measures in place. There remains an indispensable need for FCC, including the infection prevention training component.This model of FCC is currently being scaled in India at sites across the country, which will lead to additional learning about the implementation process and the feasibility and acceptability of this approach within different healthcare settings in India and other contexts. Initial findings show that to date, 85 districts have implemented FCC, reaching over 13,000 mothers and family members. Eighty-six percent of newborns below 2000 g received KMC and exclusive breastfeeding; 75% continued to benefit from KMC at home; and post-discharge mortality was reduced from 7 to 3% . The preThe generalizability of the findings to other Indian and international contexts has limitations. This intervention was implemented in an urban tertiary level NICU at a publicly supported hospital in New Delhi, and therefore these results may not be representative of all neonatal care facilities in India, especially in rural settings, or at private fee-for-service hospitals. Based on a 2011 review of NICU function, use, and staffing in eight Indian states , the NICThis study used direct observation of parent-attendant compliance with key activities, which was conducted only during the first 2 months of implementation; therefore, this may reflect a wash-out period effect as parent-attendants and healthcare providers became sensitized to the intervention and more familiar with program activities and concepts. In addition, while this study allowed for infants to be cared for by multiple parents or family members, complete data were collected only for the primary parent-attendant. This may have resulted in continuity issues in the care of the infant, and the data used in the present analysis may therefore not be fully representative of all caregivers who participated in the study. Finally, this evaluation was limited to quantitative data and analyses on acceptability and feasibility; a qualitative assessment of this model is published separately . TherefoFCC was feasible to implement in the NICU setting and the program was acceptable to participating parent-attendants and healthcare providers. Parents participated in trainings conducted by NICU providers and engaged in essential care to their infants on a day-to-day basis in the NICU. The FCC program establishes a standard care approach and behavior norms for healthcare providers that direct psychosocial and tangible support to parent-attendants so that a child is not separated from his or her parents against their will while receiving advanced care in the NICU. Expanding this intervention with monitoring across a range of NICUs, including in more rural settings, will be key to inform scaling of the FCC model in India and other LMICs.


The RNA-binding protein Quaking (QKI) is a post-transcriptional regulator of genes encoding polarity proteins in muscle stem cells. Loss of QKI in MuSCs results in reduced myogenic progenitors and a striking muscle regeneration defect. Muscle stem cells (MuSCs) have the ability to carry out the specialized function of cell polarization, which is required for the production of one repopulating cell and one myogenic progenitor cell with muscle regeneration capabilities. The mechanisms which regulate proteins involved in establishing MuSC polarity such as Dmd and Itga7 are currently not well understood. Herein, we define the RNA-binding protein Quaking (QKI) as a major regulator alternative splicing of key MuSC polarity factors including Dmd, Itga7, Mark2, and Numb. We generate a conditional QKI knockout mouse, and for the first time it is shown in vivo that deficiency of QKI in MuSCs results in reduced asymmetric cell divisions, leading to a loss of the myogenic progenitor cell population and striking muscle regeneration defects. Transcriptomic analysis of QKI-deficient MuSCs identifies QKI as a regulator of the splicing events which give rise to the mutually exclusive Itga7-X1 and -X2 isoforms. We observe increased X1 expression in QKI-deficient MuSCs and recapitulate this splicing event using antisense oligonucleotide directed against a quaking binding site within the Itga7 mRNA. Interestingly, recreating this single splicing event is detrimental to the polarization of Itga7 and Dmd proteins, and leads to a drastic reduction of the myogenic progenitor population, highlighting the significance of QKI-mediated alternative splicing of Itga7 in maintaining MuSC polarity. Altogether, these findings define a novel role for QKI as a post-transcriptional regulator of MuSC polarity. Muscle regeneration is a complex process which begins with the activation of tissue-resident quiescent muscle stem cells (MuSCs) . After mThe QKI RBP belongs to the hnRNP K homology (KH)–type family of RBPs . QKI is Itga7, Dmd, Mark2, and Numb. We show that QKI-deficient MuSCs were unable to polarize the location of Itga7 and Dmd proteins within the cell before division. Interestingly, using a 2′-O-methyl antisense oligonucleotide (ASO) to mask the QRE within Itga7 intron 4 near the 3′ splice site of exon 5 was sufficient to induce loss of polarized localization of Dmd and Itga7 proteins, as observed in QKI-deficient MuSCs. These findings define QKI as a regulator of Itga7 AS, which is required for MuSC polarity and the production of myogenic progenitors after MuSC activation.The role of the QKI proteins and AS, in general, in MuSC physiology is not well understood. In the present study, we report that deletion of QKI in mouse MuSCs leads to reduced asymmetric MuSC divisions, causing a deficit in myogenic progenitors and severe muscle regeneration defects after injury. Transcriptomic analysis identified aberrant splicing of known regulators of MuSC asymmetric division, including To define the role of QKI-5–mediated AS during the early stages of activation when MuSCs exit quiescence and begin to proliferate, we confirmed its expression in myofiber-associated MuSCs. The first MuSC division on an ex vivo cultured myofiber occurs at ∼40 h after isolation and by 72 h the MuSCs have proliferated extensively . To moniWe next interrogated QKI-5 expression throughout myogenesis of primary MuSCs. Primary MuSCs were purified from C57BL/6 mice and differentiated in vitro with reduced serum media for 24, 48, and 72 h. Total cell lysates were immunoblotted with antibodies against QKI-5 and the terminal differentiation marker myosin heavy chain to confirm that MuSCs were differentiated. QKI-5 protein was increased immediately after 24 h of differentiation and persisted through 72 h . Togethe2lox/2lox mice with Pax7CreERT2/+ mice  for 5 d were performed on QKI-ctrl and QKI-cKO mice, and myofibers were isolated and cultured ex vivo for 72 h revealing QKI ablation in MuSCs by immunostaining with a pan-QKI antibody (+ MuSCs per QKI-cKO myofibers (8.8 ± 4.0) compared with QKI-ctrl (39.4 ± 8.5)  . These f2 ± 0.07 in QKI-cKO versus 3.4 mm2 ± 0.06 in QKI-ctrl  or muscle fiber architecture (We reasoned that the reduced number of myogenic progenitors in QKI-cKO mice might influence muscle regeneration after cardiotoxin (CTX) injury of the tibialis anterior (TA) hindlimb muscle. Therefore, we injected CTX into the TA hindlimbs of QKI-ctrl and QKI-cKO mice . 3 wk afQKI-ctrl , and disQKI-ctrl . Notablyitecture , consistitecture . TogetheQKI exon 2 was confirmed with RT-qPCR  . CRE-med RT-qPCR . Differe RT-qPCR  and AS a RT-qPCR . A PCA p RT-qPCR  QKI exon  RT-qPCR . Among ttive PCR . The deptive PCR . We thentive PCR . These fTable S1 Differentially expressed genes in QKI-cKO MuSCs. Table S2 Skipped exons in QKI-cKO MuSCs. Primer sequences for RT–PCR and qPCR.Table S3 Differential AS events identified in QKI-cKO MuSCs were further categorized into skipped exon , multiple skipped exon , retained intron , alternative 3′ splice site , and alternative 5′ splice site  . All sigDmd (exon 78 exclusion), Mark2 (exon 15 inclusion), and Numb (exon 9 inclusion) (Dmd exon 78 is known to occur in patients with myotonic dystrophy (DM1), resulting in expression of the embryonic form of dystrophin and leading to defects in mobility and muscle architecture  . Notablyitecture .Itga7) in QKI-cKO  of muscle  in MuSCs, whereas the QKI-cKO MuSCs expressed equivalent X1 (exon 5) and X2 isoforms  by fixing the myofibers after precisely 36 h of culture. Itga7 has been shown to interact with dystrophin-associated glycoprotein complex, and thus is known to co-localize with Dmd in the MuSC . The perThe polarized Dmd protein in the MuSC is inherited by only one of the daughter cells following asymmetric division, whereas during symmetric divisions, Dmd is inherited by both daughter cells . To deteDmd, Mark2, Numb, and Itga7 for the QRE sequence. We found that only exon 5 of Itga7 had a neighbouring QRE sequence (ACUAAY), which was located 14 nucleotides upstream of the 3′ splice site in intron 4 (Itga7 intron 4 which contains the QRE sequence (Itga7-QRE), and a biotinylated RNA harbouring a mutated QRE sequence (Itga7-mutQRE)  . The RNA-mutQRE) . TherefoItga7 isoforms could reproduce the polarity and myogenic progenitor defects we observed in QKI-cKO MuSCs. To re-create this defect, we opted for 2′-O-methyl ASOs targeting the Itga7 intron 4 QRE sequence (ASO-Q1). We also identified a QRE sequence near the 3′ end of Itga7 intron 6 and used this ASO as control (ASO-Q2) and given its distal location from exons 5 and 6, we expected this would not affect the splicing of exon 5 or 6. Wild type primary MuSCs were cultured in vitro and transfected with ASO-Q1 and ASO-Q2 either alone or in combination, or mock-transfected with water as a negative control. The cells were collected and RNA was isolated 2 d after transfection.We next investigated whether the balance of X1/X2 Gapdh, P = 0.0083) . Expression of the X2 isoform (exon 6) was unaffected by treatment with ASO-Q1, and was slightly elevated by treatment with ASO-Q2   or exon 6 (X2) compared with an unspliced region of Itga7 (exon 23) among the ASO-treated MuSCs. Interestingly, we recapitulated the increased expression of the X1 isoform with transfection of ASO-Q1  . Exon 23 0.0083) . Unexpec= 0.026) . Semi-quof Itga7 . The ASOS events . These r compare .Itga7-X1 containing the extracellular linker region could reproduce the polarity defects observed in QKI-cKO MuSCs. Myofibers were isolated from C57BL/6 wild type mice and transfected with ASO-Q1 or mock transfected with water as a negative control (CTRL). Transfection was performed 4 and 16 h after addition of fiber growth medium (fGM), and myofibers were fixed 36 h after the addition of fGM. Co-immunostaining of Itga7 and Dmd revealed a significant reduction of polarization of both proteins in ASO-Q1 transfected myofibers (28.4% ± 1.8%) compared with CTRL cultures (83.0% ± 4.3%) (We next examined whether the ASO-Q1–mediated increased expression of  ± 4.3%) .+ myogenic progenitors. Myofibers were transfected twice with ASO-Q1 or water CTRL at 4 and 16 h after adding fGM and fixed after 72 h in culture. We observed a striking reduction in Myog+ myogenic progenitors per myofiber in ASO-Q1 treated myofibers (10.4 ± 2.8) versus CTRL cultures (52.3 ± 7.6), and a reduction in the total number of MuSCs per myofiber (159.3 ± 15.7 in CTRL versus 35.1 ± 7.4 in ASO-Q1) (Itga7-X1 isoform by targeting the intron 4 QRE re-creates the MuSC polarity and myogenic progenitor defects observed in QKI-cKO MuSCs.ASO-Q1 treatment of myofibers was then extended to 72 h of culture to assess the production of Myog ASO-Q1) . These dItga7 and Dmd undergo defective AS in QKI-deficient primary MuSCs. QKI-deficient MuSCs exhibited a loss of cell polarity and loss of the myogenic progenitor population. Treatment of wild type MuSCs with ASOs directed against the QRE in Itga7 intron 4 led to exon 5 inclusion and expression of the Itga7-X1 isoform known to express extracellular linker domain and interfere with polarization of both Dmd and Itga7. Our findings identify QKI-5 as a critical AS regulator of polarity in MuSCs.In the present study, we define a role for QKI-5 and Itga7 X1/X2 isoforms in MuSC polarity. We report that major polarity-determining factors including The loss of cell polarity in QKI-deficient MuSCs suggests that one possible mechanism through which the pool of myogenic progenitors is depleted is through a defect in asymmetric cell division. Asymmetric cell division is essential for diversification of cell types during development and specification of stem cells in adults . In DuchItga7-X1 isoform in QKI-deficient MuSCs and subsequent loss of MuSC polarity provides evidence that maintaining a high Itga7-X2/Itga7-X1 isoform ratio is a requirement for MuSC polarity.The MuSC surface marker Itga7 has been shown to affect the generation of myogenic progenitor cells and muscle differentiation . The expItga7-X1 (exon 5) and Itga7-X2 (exon 6) isoforms are expressed in a mutually exclusive manner and are regulated during development. The X1 isoform is observed in embryogenesis during the development of skeletal muscle, whereas the X2 isoform in found in adult skeletal muscle  results in exon 5 inclusion. One possible mechanism through which ASO-Q1 increases exon 5 inclusion is through binding and masking a splicing silencer element (SSE). The use of ASOs to restore exon expression through this mechanism has been demonstrated previously. For example, an ASO-walking technique was used to identify an SSE in the IKBKAP pre-mRNA responsible for the pathological skipping of exon 20 in familial dysautonomia  (DMPK1) RNA  . Our finpotonia) . For exaPK1) RNA . Subsequyofibers . Develope models , 2009. Gmyopathy .Itga7 mediated by a QRE-targeted ASO, or in QKI-deficient MuSCs, led to cell polarity defects thus identifying a role for Itga7 isoforms in establishing cell polarity.In summary, we report QKI-5 as a major regulator of splicing in skeletal MuSCs and as being essential for maintaining the splicing isoform pattern that drives MuSC polarity and production of myogenic progenitors. QKI-deficient mice had severe muscle regeneration defects arising from a lack of these progenitor cells. AS of QKI gene (QKI2lox/2lox), and were generated previously (2lox/2lox (QKI-ctrl) mice were crossed with C57BL/6 mice expressing CreERT2 recombinase under the Pax7 promoter  to generate QKI-cKO mice with 4-hydroxytamoxifen-induced QKI deficiency specifically in MuSCs. To induce Cre recombinase, 4-hydroxytamoxifen  was dissolved in corn oil  to 1 mg/ml, and 100 μl was injected intraperitoneally in 6- to 8-wk-old mice once daily for five consecutive days. Experiments were conducted 10 d following the final injection of 4-hydroxytamoxifen. Sex- and age-matched mixed populations of males and females were used for each genotype for all experiments. All mouse husbandry and experiments were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) of McGill University. All animal procedures conducted at McGill were approved by the Animal Welfare Committee of McGill University (protocol #3506).C57BL/6  were the wild type mice used for MuSC FACS purification and myofiber isolation where indicated. QKI-cKO mice are maintained in a C57BL/6 background with two loxP sites flanking exon two of the eviously . QKI2lox2. Whole muscle was then triturated with a plastic disposable Bohr pipette to dissociate individual fibers from the whole muscle as described previously  in DMEM for 30 min at 37°C and 5% COeviously . To mimiCultured myofibers were fixed in 2% PFA at the indicated time points and washed twice with 1× PBS. Fixed myofibers were then permeabilized with 0.2% Triton X-100, 0.125 M glycine in 1× PBS for 15 min at RT. Blocking followed for 1 h at RT with 2% BSA, 5% horse serum and 0.1% Triton X-100. Primary antibodies were diluted in blocking buffer to detect Pax7 , MyoD , Myogenin , Dystrophin , QKI-5 . After 16-h incubation at 4°C, fibers were washed three times with 1× PBS for 10 min. Secondary antibody (AlexaFluor anti-mouse or anti-rabbit 488 or 568 nm), or AlexaFluor-647–conjugated primary antibody against Itga7  was used at a dilution of 1:400 in blocking buffer for 1 h in the dark at RT. Myofibers were washed three times for 10 min with 1× PBS. Finally, the myofibers were transferred to a microscope slide outlined using an ImmEdge hydrophobic barrier pen  and mounted with ProLong Gold Antifade Mountant with DAPI . Fiber-associated MuSCs were then visualized on a Zeiss Axio Imager M1 microscope (Carl Zeiss) or LSM800 Airyscan confocal microscope and resulting images were analyzed using Zeiss’ ZEN Digital imaging suite software.g and resuspended in 1% BSA/PBS. Cells were stained for 15 min RT with Itga7-AlexaFluor-647  for positive selection, and PE-CD45 (Invitrogen), PE-CD11b  for negative selection. Hoescht was used to gate living cells. Cells were washed once with 1% BSA/PBS and pelleted before final resuspension and one last filter through a 40 μM cell strainer. ITGA7+/CD45−/CDCD11b−/Sca1−/Hoechst+ Cells were sorted into full myoblast growth media (GM) using the FACSAriaIII cell sorter (BD Biosciences).Skeletal muscle tissue was isolated from the abdominal and diaphragm muscles of wild type C57BL/6, QKI-ctrl, and/or QKI-cKO mice and MuSCs were isolated as previously described using FACS . Briefly2. The medium was replenished every 2 d. To differentiate MuSCs into myotubes, MuSCs were grown to 90% confluency in GM, washed twice with 1× PBS, and switched to differentiation medium . C2C12 myoblasts were grown in DMEM, 10% FBS, and 1% Penicillin/Streptomycin at 37°C and 5% CO2. To induce differentiation, C2C12 myoblasts were grown to 90% confluency and cultured in DMEM, 1% FBS, and 1% Penicillin/Streptomycin at 37°C and 5% CO2.Purified MuSCs were seeded onto collagen-coated plates and expanded in GM  at 37°C and 5% COThe right TA muscles of QKI-ctrl or QKI-cKO mice were injected once with 50 μl of 10 μM CTX  to induce muscle injury. After 3 wk, mice were euthanized, and the injured and contralateral control TA were dissected using standard dissection techniques. Dissected TA muscles were fixed in 0.5% PFA for 2 h at 4°C, and subsequently equilibrated in 20% sucrose in 1× PBS overnight at 4°C. The following day, TA muscles were rapidly frozen in liquid nitrogen-cooled isopentane and embedded in OCT compound . Frozen TA muscles were cut into 10 μM sections using a Leitz cryostat directly onto Fisher Superfrost Plus microscope slides (12-550-150) for immunofluorescence staining. Resulting tissue sections were permeabilized for 12 min with 0.2% Triton X-100, 0.125 M glycine in PBS at RT. Blocking followed with M.O.M blocking reagent  for 1 h at RT. Primary antibodies were diluted in blocking reagent to detect Pax7  and laminin . After 16-h incubation at 4°C, sections were washed three times with 1× PBS for 10 min. Secondary antibody (AlexaFluor anti-mouse or anti-rabbit 488 or 568 nm) was used at a dilution of 1:400 in blocking reagent for 45 min in the dark at RT. Sections were then washed three times for 10 min with 1× PBS. Tissue sections were mounted with ProLong Gold Antifade Mountant with DAPI  and covered with a coverslip. Visualization was performed on a Zeiss Axio Imager M1 microscope (Carl Zeiss), and resulting images were analyzed using Zeiss’ ZEN Digital imaging suite software.Primary MuSCs were purified from 6- to 8-wk-old QKI-ctrl or QKI-cKO mice  using FACS gating of ITGA7+/CD45−/CDCD11b−/Sca1−/Hoechst+ cells. Purified cells were expanded ex vivo in myoblast growth medium for 72 h, pelleted, and bulk RNA was isolated using the PicoPure RNA extraction kit according to the manufacturer’s protocol . RNA quality was assessed using an Agilent Tapestation 4200. RNA sequencing libraries were generated with TruSeq stranded mRNA Sample Prep Kit with TruSeq Unique Dual Indexes (Ilumina). The resulting libraries were multiplexed and sequenced with 100 bp paired-end reads on the Illumina NovaSeq platform. Samples were subsequently demultiplexed with bcl2fastq v2.20 Conversion Software from Illumina. Illumina adaptor sequences were then removed using Trimmomatic v0.39 software. Trimmed sequences were subsequently aligned to the mm10/GRCm38 genome using STAR v2.7. Mapped reads were then processed using the Cufflinks software suite. FPKMs and fragment counts were scaled using the geometric means of fragment counts across all libraries. Significant changes in transcript expression were classified as having a log fold change greater than 2, base means larger than 20, and FDR less than 0.05. Expression plots were generated using CummeRbund. AS events in QKI-cKO MuSCs were quantified using rMATS v4.0.2 and Gencode vM23 gene annotations using untrimmed paired-end reads. Events with an FDR of less than 0.05 were accepted as significant. Pathway enrichment analysis of DEGs and AS events was performed using Enrichr.TRIzol (Invitrogen) was used to isolate RNA from cultured primary MuSCs per the manufacturer’s instructions. 1 μg of total RNA was used for cDNA synthesis using M-MLV reverse transcriptase , followed by qPCR analysis with targeted primers using PowerUp SYBR Mastermix  run on QuantStudio7 real-time PCR system (Thermo Fisher Scientific). mRNA was quantified with the ΔΔCt method, normalizing to Gapdh and/or Hprt and 18s mRNA levels as indicated. Splicing assays were performed using cDNA isolated as described above and specific primers which flanked the indicated exon for PCR. Resulting PCR products were separated on a TBE-based polyacrylamide gel and DNA was stained with ethidium bromide. All PCR reactions were repeated in triplicate. A complete list of RT-PCR and qPCR primers is provided in Table S3.Proteins from total cell lysate  were separated by SDS–PAGE and transferred onto nitrocellulose membranes using the Trans-Blot turbo transfer system (Bio-Rad). Membranes were stained with Ponceau Red to confirm equal loading, and then blotted with the primary antibodies against myosin heavy chain , QKI-5  overnight at 4°C. After three washes in TBST, membranes were incubated with HRP-conjugated secondary antibodies (Sigma-Aldrich) for 45 min and visualized on X-ray films with Western Lightning Plus ECL (PerkinElmer).FACS-isolated primary MuSCs were transfected with siRNA designed to target QKI (5′-GGACUUACAGCCAAACAAC-3′) and luciferase as a negative control (5′-CGUACGCGGAAUACUUGA-3′). siRNA transfections were carried out using Lipofectamine RNAiMAX reagent  per manufacturer’s instructions. Cells were harvested 48 h after transfection for total RNA isolation. 2′-O-methyl ASOs were designed to target the exon 5 upstream QRE (ASO-Q1: 5′-GUCCUGUUAGUAUUCAAGGUGG-3′), and the downstream QRE (ASO-Q2: 5′-CGAUUACUGUGAGUGAUUAUCCAAC-3′) to induce exon 5 inclusion. For cultured primary MuSCs, 50 nM of each ASO or water control were transfected using the Lipofectamine 3000 transfection reagent  according to the manufacturer’s instructions. Cells were harvested 48 h after transfection for total RNA isolation. For myofibers, a first transfection was performed with 50 nM ASOs 4 h after addition of fGM. A second transfection was performed 12 h after the initial transfection. Myofibers were subsequently fixed 36 or 72 h after addition of culture medium as indicated.Streptavidin Mag Sepharose magnetic beads  were incubated with 1 µg of biotinylated RNA (IDT) for 30 min at 4°C with end-over-end mixing.C2C12 myoblast lysate  was then added to the RNA-Streptavidin mixture and incubated at 4°C with end-over-end mixing for 1 h. The beads were then washed three times with lysis buffer containing increasing salt concentrations . Protein samples were separated with SDS–PAGE and immunoblotted for QKI-5.GSE193899.The RNA-seq data from this publication have been deposited to Gene Expression Omnibus under the accession number


Over 40 million informal caregivers provide care to adults aged 50 or older with a chronic illness. In addition to the negative health and well-being impact, caregivers experience financial difficulties including lost income, retirement benefits, future earnings, and unanticipated out-of-pocket costs. Few evidence-informed programs exist to assist caregivers in understanding and managing these financial tasks. This poster presents preliminary acceptability data from 71 caregivers who completed the newly developed Managing Money: A Caregiver’s Guide to Finances program. The 60-minute program was delivered online by trained community educators and addressed caregiving costs/impacts; future planning; initiating conversations; avoiding financial abuse/fraud; and identifying needs. Participants  were 87.32% female with 77.46% self-identified as White, 9.86%, as Black, and 8.45% as Hispanic/Latino. Using a Likert scale (1=strongly disagree to 5=strongly agree), participants indicated the program: 1) identified financial challenges ; 2) provided information for managing money ; 3) content was easy to read/understand ; 4) program length was appropriate ; and 5) activities were helpful . Participants rated the program as very important  and would highly recommend . Results indicated the program was well-received by participants and highly acceptable. Discussion will highlight key program features designed to promote acceptability along with the importance of measuring acceptability for large-scale implementation along with next research steps.


Measles-negative sera (n = 187) were collected from individuals presenting with other fever and rash illnesses. A total of 7 ELISA kits  and one CLIA method (DiaSorin LIAISON XL) were evaluated. The ELISA kits included two IgM capture methods and five indirect methods. Calculated sensitivities and specificities ranged from 75.0% to 98.1% and 86.6% to 99.5%, respectively. The parvovirus B19 IgM positive sera were noted to cause false-positive results, particularly for the ELISA kits from Serion and NovaLisa; specificities for this subset of samples ranged from 51.4% to 100.0%. The capture IgM ELISA methods provided the best combination of sensitivity and specificity.The World Health Organization (WHO) has targeted measles for global eradication through mass immunization. For effective monitoring of eradication targets, high-quality surveillance is needed. The detection of IgM antibodies, specific to the measles virus, with the use of commercial enzyme-linked immunosorbent assays (ELISA or EIA) is broadly used within the WHO global measles and rubella laboratory network for laboratory confirmation, and in particular, ELISA kits manufactured by Siemens (Enzygnost kits) have been primarily used. Spurred by the discontinuation of these kits, this study aims to report on the clinical sensitivity and specificity of comparable commercial ELISA kits and one automated chemiluminescent immunoassay (CLIA) method. A panel of 239 serum samples was assembled that included sera from confirmed measles cases ( Measles, once typically a childhood illness characterized by high fever and rash, is caused by infection with the measles virus (MeV) and can be prevented with an effective vaccine that has been available since the 1960s , 3. Howe4–The World Health Organization (WHO) has targeted measles for global eradication through mass immunization . For eff10–n = 43). For 6 specimens, an additional third serum was added, due to insufficient volume after the initial top-up. An additional specimen, sourced from a company that supplies sera with known acute measles infection status to proficiency panel providers was included in the confirmed measles panel .The study was conducted retrospectively and employed anonymized residual sera that had been received either at the National Microbiology Laboratory (NML) or the Alberta Public Health Laboratory (ProvLab) for serological testing (convenience sampling). Sera from 68 laboratory-confirmed cases that met the national case definition for measles by local public health authorities and collected during measles outbreaks were sent to the NML in 2013 for confirmatory measles serology. All sera were reported by the referring laboratory to be anti-measles IgM positive or equivocal. The residual volume of all specimens was insufficient for use on the DiaSorin LIAISON XL method, which requires at least 170 μl; thus, the sera were pooled to create a total of 49 specimens with volumes of 170 to 190 μl . The poon = 4), dengue , human herpesvirus 6 (HHV-6) , parvovirus B19 (n = 35), and Zika virus (n = 3). Thirty-seven sera from clinical cases with fever and rash, as recorded on the test requisition, and referred for HHV-6 serology with HHV-6 IgM-negative/IgG-positive results were included. The sera for which collection dates were known were collected between 2001 and 2015. An additional 36 sera, available as part of the inventory of the NML’s rubella serology proficiency panel program, were included. These were archival sera sourced from a variety of suppliers, including commercial rubella IgM-positive controls.A total of 153 residual clinical sera that were confirmed to be IgM positive for other fever- and rash-causing viruses were included in the panel . Agents n = 239) was assembled, randomized, and blinded at the NML. The panel was frozen and sent to the Alberta provincial laboratory for testing on the automated platform DiaSorin LIAISON XL. Upon completion, the panel was refrozen and returned to the NML for all plate-based ELISAs.The final panel , IBL, Microimmune, NovaLisa, Serion, and Enzygnost  or positive (“presumptive positive”). np + z2 – 1 – z √ [z2 – 2 – (1/n) + 4p(nq + 1)]}/2(n + z2) and U [upper limit] = {2np + z2 + 1 + z √ [z2 + 2 – (1/n) + 4p(nq - 1)]}/2(n + z2), where z = 1.96, p is the sensitivity or specificity, and q = 1 – p. If p = 1, then U = 1, since specificity and sensitivity cannot be >100%.) . Confidence intervals were calculated using the score method  .https://www.graphpad.com/quickcalcs/kappa1/).An online calculator was used to calculate the kappa measure of agreement and 95% confidence intervals . The scale of the resulting output values was not comparable across methods, particularly for the Serion activity calculator method, which generated values in the thousands, while most other methods had values of <10. Therefore, the data for each method were normalized to 100 to facilitate their comparison. In Microsoft Excel 2016, the grouped measles and non-measles output values were sorted from highest to lowest value for each method. The highest value for each method was arbitrarily set to 100. To determine the normalized value for the remaining values, each value was divided by the highest value for the method and then multiplied by 100. The normalized values were used to generate the box plots.A panel of 239 sera was assembled that included 50 pooled specimens from confirmed cases of measles and 189 sera that were IgM positive for a number of other viruses  or presented with fever and rash of unknown etiology (the “non-measles” panel) . These vSeveral characteristics, such as choice of antigen, completeness of the kit components, length of time needed for the test, incubation temperatures, and user-friendliness, of the seven commercial microplate-based ELISA kits that were included in the study were compared . Two kitThe measles sera panel, consisting of 52 specimens , was useThe results of testing with the non-measles sera panel, which consisted of 7 subsets of panels for a total of 187 specimens , was usen = 150 sera) or were collected from individuals reported as having fever and rash (n = 37) (All sera in the non-measles sera panel were either IgM positive for other agents that can present with fever and rash symptoms ((n = 37) . To asse(n = 37) . Few falThe predetermined classification of the sera as either true measles positive (using the measles case definition) or true measles negative (80% of the sera were confirmed for a fever rash illness of another etiology)  was usedAll methods generated test results as continuous variables  which were then compared to cutoff values to classify the numerical value to a qualitative result. These output values by sample set (measles and non-measles) were compared for each test kit to assess the spread between positive and negative sample sets . The resPrevious extensive evaluations of commercial measles IgM EIA kits resulted in the broad adoption of the Siemens (previously Behring) Enzygnost kit within the WHO global measles and rubella laboratory network –13. More–17–11–17–n = 15 to 17). Of the panel of 35 parvovirus-positive sera, 20 (57.1%) resulted in a borderline/equivocal or false-positive result with at least one method, and most of these (n = 13) were reactive  with at least two methods. Nine sera had false-positive anti-measles IgM results with 2 or 3 methods. This same panel was used to evaluate 8 anti-rubella IgM kits (manuscript in preparation), and many of the same sera that were cross-reactive with the anti-measles IgM kits were also cross-reactive with anti-rubella IgM kits. Of the 20 sera that had a borderline/equivocal or false-positive result with at least one anti-measles IgM method, 17 also had a borderline/equivocal or false-positive result with at least one anti-rubella IgM method. This suggests false-positive results are due to a general cross-reactivity and are not specific to measles IgM kits.The presence of parvovirus B19-specific IgM was previously demonstrated to be a source of cross-reactivity for anti-measles IgM detection methods , 22–24 a22–In elimination settings, surveillance systems operate with a focus on sensitivity which, for some tests, can come with a cost of false-positive results, a cause for concern. Furthermore, in these settings, the positive predictive value of IgM serology is greatly diminished. Thus, methods with excellent accuracy are needed. For this evaluation, a high bar was set by taking an “always wrong” approach to classify the equivocal results. However, in practice, to meet enhanced surveillance needs, a suspected measles case with an IgM equivocal result would not be discarded but would instead be reflexed to additional investigation, such as reverse transcriptase PCR (RT-PCR), follow up IgM, or IgG testing. In this scenario, an equivocal IgM result could be considered a presumptive or suspected positive. Using such an approach to classify the equivocal results for the methods in this evaluation improved the clinical accuracy of the best-performing methods as well as the two kits from Euroimmun. The lowest-performing kits (NovaLisa and Serion) remained unchanged with accuracies hovering around 90%.This study had some limitations. Because residual, anonymized sera were used, information regarding the individual case, such as vaccination history and rash onset date, were unavailable for the measles sera. It is expected that some of the measles cases were confirmed by RT-PCR, but that information was also unavailable. Overall vaccination coverage rates are high in Canada , and mos26–An additional limitation was that only one lot of each test method was included in the study. While reproducibility was assessed using the results of the external control, it was not evaluated for multiple lots and thus may not be representative of the overall reproducibility of the methods. For the Euroimmun NP kit, however, an additional lot (E181121BK) was evaluated using the entire panel and demonstrated poor concordance with the lot included in this study (data not shown). However, it was noted that the results from one of the three test plates (lot E181121BK) were likely invalid , and so we are unable to determine if the poor concordance was due to variability between lots.Detection of measles IgM antibodies remains an important, high-throughput, robust method of providing laboratory confirmation of measles cases, particularly in settings of high disease prevalence. In settings where measles elimination has been achieved, or nearly so, the positive predictive value of IgM serology is greatly diminished, and additional information (including laboratory tests such as RT-PCR) is required for the confirmation of measles cases , 24, 33.