Web-based sexual health resources are typically evaluated in terms of their efficacy. Information is lacking about how sexual health promotion websites are perceived and used. It is essential to understand website use to address challenges with adherence and attrition to Web-based health interventions. An existing theoretical framework for examining loyalty to electronic health  interventions has been not yet been applied in the context of sexual health promotion nor has the association between e-loyalty and intended intervention efficacy outcomes been investigated.The objectives of this study were to investigate users’ loyalty toward a sexual health website , measure user perceptions of the website, and measure the association between e-loyalty and perceived knowledge increase and intent to change behavior.Over 4 months, website users  participated in an open, online, cross-sectional survey about their user experiences that measured e-loyalty, user perceptions, and intended website efficacy outcomes. Relationships between user perceptions and e-loyalty were investigated using structural equation modeling (SEM). Associations between e-loyalty and website efficacy outcomes were tested using Spearman rank correlation.P<.001), their intent to have safer sex (ρ(171)=.24, P=.01), and their intent to get tested for sexually transmitted infections (ρ(171)=.37, P<.001). The SEM showed that trustworthiness, overall experience, active trust, and effectiveness were directly related to e-loyalty. Finding the website “easy to understand” was significantly related to active trust .A total of 173 participants completed user perception questions and were included in the analysis. E-loyalty was high for both clients and providers and was significantly correlated with clients’ perceived knowledge increase (ρ(171)=.30, E-loyalty may be related to the efficacy of the selected website in improving one’s sexual health and was significantly associated with all three intended knowledge and behavioral outcomes. To increase e-loyalty, trustworthiness and active trust are important user perceptions to deliberately engender. Our findings indicate that understanding a website contributes to active trust, thereby highlighting the importance of considering eHealth literacy in designing health promotion websites. Our study confirms the relevance of e-loyalty as an outcome for evaluating the antecedents of the use and efficacy of online public health interventions across disciplines by adapting and validating an existing e-loyalty framework to the field of sexual health promotion. Our findings suggest that e-loyalty is positively associated with measures of website efficacy, including increased knowledge and intent to change behavior. Longitudinal research with larger samples could further investigate the relationships between e-loyalty, website understandability, and outcomes of online health interventions to determine how the manipulation of website characteristics may impact user perceptions and e-loyalty. Web-based resources for sexual health promotion, including the resources on prevention of sexually transmitted infections (STIs) and human immunodeficiency virus (HIV), have been accessible for over 15 years, by virtue of the advantage of the Internet to deliver personal or tailored sexual health information to large audiences ,3. Althouse of health interventions, which seeks to understand why individuals in the general public choose to engage with and remain loyal to a particular website  of providers had college-level education or higher vs 69.4% [75/108] of clients).Females had higher mean scores than males and other genders for e-loyalty  lead us to hypothesize that e-loyalty contributes to the efficacy of this website in improving sexual health. A meta-analysis of Web-based health interventions found conflicting results regarding the relationship between intervention adherence  and behavioral outcomes but concluded they are likely to be related . More loOur SEM provides further insights into associations between user perceptions and e-loyalty for health promotion websites, confirming previous findings and revealing new relationships.Trustworthiness had the strongest direct effect on e-loyalty in this model, and its effect was also partially mediated by active trust. This finding is in line with prior social marketing research that showed active trust partially mediating the effect of website credibility on behavioral intent . In our the delivery of clear information [There is a large body of literature recognizing the importance of trust in the use of Web-based health information -23. Trusormation ,25. Howeormation . The onlSSR also has other features known to be associated with trustworthiness among specific populations. When searching for sexual health information online, youth have been shown to assess credibility largely upon a website’s domain name—dot com, dot gov, or dot org—or its association with government ,27,28. AIn our model, the effect of trustworthiness on e-loyalty was also partially mediated by active trust. Active trust is characterized by a user’s confidence in acting upon information presented on a website, the only user perception in the e-loyalty model that speaks to action. As the ultimate client-oriented objective of an interactive Web-based resource such as SSR is to provide information that contributes to behavior change , active trust is arguably the most important user perception to engender along the pathway to e-loyalty.Increasingly, people are seeking health information on the Internet before going to health care providers, and youth are more likely than older adults both to go online first and to trust online health content . As the Our hypothesis that “understanding” may be related to e-loyalty was supported by the data. The SEM indicates that respondents who reported better understanding of content were more likely to have active trust; in other words, understanding was an antecedent to active trust along the pathway to e-loyalty.These findings suggest an overlap between the theoretical frameworks of e-loyalty and eHealth literacy. As much as understanding is a component of eHealth literacy, we acknowledge that understanding content is only one small piece; eHealth literacy comprises much more than having the reading skills to understand information at an appropriate reading level ,32. UndeLinks between understanding, active trust, and e-loyalty are not yet well developed in the literature, but our findings are congruent with prior research, including a study showing strong support that understanding impacts trust beliefs and intention to use health information websites . A studyThe main limitation of this study was the small sample size. Our challenges with attrition, despite offering an incentive, are reflective of the fundamental challenges of open online surveys. The use of a lottery-style incentive was selected based on cost and feasibility, informed by a literature search in 2013 -38, and We also recognize the potential for bias in an open, online survey in which participants could respond more than once. In 37 (7%) instances, responses originated from the same IP address as other responses. We retained these responses in our sample, as we were unable to differentiate between duplicate responses from the same individual and different individuals accessing the survey from a shared computer or an institutional network, which may be likely among survey respondents .Another limitation is evident in the selection process and the differences between respondents who completed and did not complete the e-loyalty section. As our participant recruitment method was related to e-loyalty , and as completers of the e-loyalty section were more likely to be returning SSR users and health care providers, our findings may be biased toward visitors with higher e-loyalty. This is also evident in that returning visitors reported significantly higher e-loyalty scores, leading us to consider whether self-reported, cross-sectional surveys are an appropriate way to capture e-loyalty. Nevertheless, user perceptions from 173 unique respondents supported an SEM with adequate fit and statistically significant relationships between variables that provides useful insight into relationships between user perceptions, including the novel user perception, “understanding,” as well as knowledge and behavioral outcomes.Finally, as a cross-sectional survey, we are not able to draw any conclusions regarding causation or determine whether the intended behavioral outcomes  did, in fact, occur. Future research that includes a longitudinal component  to validate our findings is needed.This exploratory study contributes to a growing field of literature on the applications of an e-loyalty framework to Web-based public health interventions. With the addition of a novel user perception to a theoretical framework of e-loyalty, we have shown that users’ understanding of the content affects their confidence in acting upon information presented, which in turn affects their e-loyalty to the site. Furthermore, we found positive associations between e-loyalty and measures of intervention efficacy . We propose that e-loyalty and related user perceptions, including trustworthiness and understanding, are constructs relevant to consider in addition to efficacy measures in studies that evaluate Web-based sexual health interventions.There is a need to further investigate how sexual health websites can deliberately engender understanding, trust, and active trust to develop e-loyalty among their users, for example, by manipulating website characteristics to change user perceptions. We echo the call to action to bring sexual health interventions to their full potential on the Internet by continuing to explore ways to increase their use and impact . As the


This study focuses on the impact of a program aimed at reducing heat-related mortality among older adults residing in central Rome by counteracting social isolation. The mortality of citizens over the age of 75 living in three Urban Areas (UAs) located in central Rome is compared with that of the residents of four adjacent UAs during the summer of 2015. The data, broken down by UA, were provided by the Statistical Office of the Municipality of Rome, which gathers them on a routine basis. During the summer of 2015, 167 deaths were recorded in those UAs in which the Long Live the Elderly (LLE) program was active and 169 in those in which it was not, implying cumulative mortality rates of 25‰  and 29‰ , respectively. Relative to the summer of 2014, the increase of deaths during the summer of 2015 was greater in UAs in which the LLE program had not been implemented (+97.3% vs. +48.8%). In conclusion, the paper shows the impact of a community-based active monitoring program, focused on strengthening individual relationship networks and the social capital of the community, on mortality in those over 75 during heat waves.  Heat-related mortality is a growing public health concern worldwide because of the increased number of older adults highly susceptible to the effects of heat. In fact, heat-related mortality amongst those over 75 is becoming a major issue because of the high prevalence of frail individuals, who show a higher risk of death as a consequence of psycho-physical impairment and/or lack of socio-economic resources ,2,3. In Several factors prevent an accurate assessment of the reduction in the impact of heat waves following the implementation of heat-related mortality prevention programs; in practice, most prevention programs are aimed at the general population of a city (or even a country), so that most studies are based on historical research rather than on a comparison of those sections of a population that are included or not included in prevention activities ,17. MoreThe Long Live the Elderly (LLE) program has been running for 13 years in some Urban Areas (UAs) of central Rome . It was The study design is a quasi-experimental retrospective cohort study. The impact of the intervention program has been assessed by comparing the death rate between two populations living in the first administrative urban district of Rome , Table 1Tappmax), which is a discomfort index based on air dew point temperature, calculated using the following formula [The definition of heat wave made by the Heat Health Watch Warning Systems (HHWWS), and implemented in Italy at the national level by the national prevention program of heat-related risk effects on population health, results from a combination of two factors: the air-mass model  with the maximum apparent Temperature  to contact all those over 75 for the purpose of offering them a periodic assessment of their social and health needs, health promotion campaigns , assistance in handling bureaucratic matters or seeking formal or informal care and to provide details of the office, active from 8 a.m.–5 p.m., Monday–Friday, to contact if necessary; (b) to strengthen the community network around sick and/or socially-isolated individuals by involving people living or working near them in volunteer care actions; and (c) to increase the awareness of the community about the needs of older adults.The LLE program promotes a proactive approach to reach the whole targeted population, so as to avoid some individuals being neglected because of a lack of awareness of their care needs. Based on a list provided by the municipality, all those over 75 receive a letter and then a phone call to obtain their consent to be part of the program; the percentage of refusal is lower than 2%. If the client accepts, then a multidimensional evaluation of their care needs is performed, and the service begins. According to the risk of a negative event, as assessed by the multidimensional evaluation, an individual care plan is drafted , and the client is included on the list for periodical phone calls: the higher the risk of negative events, the more frequently the person will be called, with a maximum frequency of once every two weeks, unless specific actions need to be taken. The activities of the program are intensified when a heat wave occurs: all those over 75 assisted by the program are traced by phone, and if necessary, the staff intervene with a home visit, bringing food and/or medicines as necessary, or involving the client’s network of relationships. It must be taken into account that a part of the population took holidays out of the city and that some of the declared residents were not in fact living at their official home addresses. Throughout the year, the operators act as a liaison between the older adults assisted by the program and the community, in order to increase the social capital of both. Healthy 75 year olds, however, are also contacted at least three times a year and during climatic emergencies, in order to monitor their situation.This analysis is focused on the death rate during the summer of 2015 and is based on data collected through standard procedures and periodically published by the Statistical Office of the Municipality of Rome . However, a special dataset for this study was provided to the authors by the Statistical Office of the Municipality see the : publishConceptual issues are raised by the variation of death rates where services have been implemented to take care of segments of the population, like those over 75, with a higher risk of death . In factAge-specific death rates of those over 75 and the percentage variations of age-specific death rates for summer 2015 have been compared with those of summer 2014 in order to assess the impact on mortality of the greater number of heat alerts in 2015. A list of variables see  has beenThe two populations were made up of 6483 cases and 5724 controls, comparable for age and gender .From June–September 2015, 4720 older adults were contacted by the LLE program, by phone; 2963 received more than one phone call  and 502  more than four phone calls. The discrepancy between the official residents and the citizens contacted by the program is mainly due to a real residence different from the official one and to the absence of the citizen due to spending holidays outside the city. The authors assume that this discrepancy should not affect cases and controls differently. The LLE program operators carried out 660 interventions other than phone calls for 143 older adults (3.0%): most of these interventions were “visiting the patients when admitted to the hospital”  and “bringing food/medicine” at home . Other frequent interventions were “contact relatives/friends” (88 cases) and “search for a paid assistant” (73 cases): these interventions all together represent about 54% of the total number during the summer of 2015.During the summer of 2015, 167 deaths were recorded in the UAs in which the LLE program was active, and 169 in those in which it was not, implying cumulative mortality rates of 25‰  and 29‰ , respectively. The increase of deaths during the summer of 2015, compared to the summer of 2014, was more prominent in UAs in which the LLE program had not been implemented . Interestingly, the mean property tax valuation is associated with the overall mortality from May 2014–December 2016 , but is not associated with either the 2015 or the summer of 2015 mortality  on populations of older adults. It was noted that the impact of cold snaps lasts up to three weeks after the event ends ,40, whilThe results of this study should be treated with caution, because of its limitations: first of all, the data reported here are based on the assisted people’s administrative residence, which does not correspond to the place where they live. In fact, it is estimated that, in Rome, at least 10% of the people living in UAs are not resident, while the same percentage are resident, but living in a different place. Since mortality data are based on the official home address, some inaccuracies might occur, although the authors assume that they would affect all the UAs equally. A second limitation could be the lack of individual information on factors that can affect mortality, such as the presence of air conditioning in the house or the vacation time spent outside Rome during the summer of 2015. These conditions, which are likely to be enjoyed by the more affluent people among those under observation, can be taken into consideration by including in the model a proxy of the socio-economic status like the mean property tax valuation in a given UA. However, this variable did not affect the 2015 mortality, even if it showed a strong correlation with the overall 2014–2016 mortality. This could be an effect of the interventions, based on an assessment of multidimensional frailty, including socio-economic conditions, and focused on frail people, including those lacking socio-economic resources. The program probably reduces inequalities, providing support to those most in need, based on psycho-physical impairment and/or a lack of socio-economic resources. We do not know the immediate causes of deaths, even for those that occurred during heat waves, which are usually not so different from those under normal circumstances (mainly cardiovascular and/or respiratory diseases), so we cannot establish a direct relationship between heat waves and deaths that occurred during the summer. Again, the authors do not think this limitation should have different effects between different UAs.In conclusion, the paper shows the impact of a community-based active monitoring program focused on strengthening individual relationship networks and the social capital of the community on the mortality of those over 75 during heat waves. The mitigation of mortality during the 2015 heat wave summer compared to non-heat wave summers is likely to be the outcome of the increase of resilience resulting from the implementation of the LLE program throughout the year and especially during climatic crises. Investments in social services associated with assessments of frailty carried out routinely in order to draft individual care plans could be a good strategy for improving people’s health and quality of life, at least during heat wave episodes.


Carvedilol is used in the management of hypertension, ischemic heart disease, heart failure and most recently, portal hypertension. It has been associated with improved outcomes regarding variceal bleeding, hepatic decompensation and death when compared to propranolol and endoscopic band ligation. The main cause of portal hypertension is cirrhosis and therefore carvedilol is increasingly used in these patients. Due to its extensive hepatic metabolism, carvedilol is contraindicated in severe hepatic impairment. However, there are no dosage adjustments in the manufacturer’s labelling for mild to moderate hepatic impairment.We present a case of cardiogenic shock that occurred after carvedilol 25 mg orally was administered to a patient with cirrhosis. As there was no overdose, the diagnosis was based on clinical recognition of the toxidrome. The patient was successfully treated with glucagon 5 mg bolus followed by infusion.Patients with cirrhosis represent a special at-risk group for beta blocker toxicity. The typical threshold for carvedilol toxicity in overdose is 50 mg but in patients with cirrhosis this is not applicable. Nurses and physicians need to recognize the toxidrome early. Hospitals where carvedilol is used in patients with cirrhosis should have glucagon in formulary at doses to treat toxicity (bolus and infusion). Finally, dose adjustment and slow uptitration of carvedilol in cirrhosis is recommended. Carvedilol is used in the management of hypertension, ischemic heart disease, heart failure and most recently, portal hypertension. Beta blockers decrease portal hypertension and are the mainstay of pharmacologic prophylaxis for gastroesophageal varices. The original beta blockers studied were nadolol and propranolol, but carvedilol recently emerged as an alternative . There iA 56-year-old man with liver cirrhosis (secondary to hepatitis C) presented with hematemesis. The patient did not have diabetes, ischemic heart disease or other comorbidities. He did not regularly see a primary care physician and did not take any medications prior to admission. Laboratory investigation and imaging confirmed cirrhosis, with an alanine aminotransferase (ALT) of 101 IU/L, aspartate aminotransferase (AST) of 51 IU/L, albumin of 2.9 g/dL, total bilirubin of 1.4 mg/dL and international normalized ratio (INR) of 1.4. On imaging there was slight ascites and he did not have encephalopathy on examination, with Child-Pugh score of 7. An esophagogastroduodenoscopy confirmed bleeding esophageal varices, and band ligation was performed. Post intervention, the patient had an uneventful course and 7 days after presentation carvedilol 12.5 mg twice daily orally was started. Echocardiography on admission showed cardiac function was normal with a preserved ejection fraction. The other medications being administered were rifaximin and pantoprazole, and a final dose of ceftriaxone (1 g/day for 7 days) given only as prophylaxis. The first dose of carvedilol was administered after lunch and no reaction was noted acutely. At night the second dose was given. From this point on, the heart rate and blood pressure are charted in Fig. The following morning, 8 h after administration, the patient was hypotensive with a blood pressure of 80/45 mmHg, pulse of 51 beats/min and had a respiratory rate of 18 breaths/min. The patient was fully alert and oriented and expressed his wish to not have placement of any central venous access. A total of three liters intravenous crystalloid fluid boluses were administered but there was only a transient rise in blood pressure  and hypoglycemic (serum glucose 40 g/dL) but the rest of the physical exam was unchanged. To note, the patient was not diabetic and had not received any insulin or other hypoglycemic medications. Laboratory investigations showed a stable hemoglobin and no signs of infection. Passive rewarming was started and intravenous dextrose and albumin given. Despite these measures repeat blood pressure was 69/42 and the patient began to become delirious. An electrocardiogram showed sinus bradycardia. A bolus of 5 mg intravenous glucagon was given. Within 5 min of administration the heart rate had increased to 65/min and blood pressure began to increase. This was followed by a glucagon infusion in 5% dextrose at 1 mg/h. As shown in Fig. 1-andrenoreceptor antagonist activity, respectively. Its vasodilating effect is theorized to contribute to its potency in reducing mortality and morbidity in the settings of ischemic heart disease and portal hypertension [Pharmacologically, carvedilol is a unique beta blocker. The formulation is a racemic mixture that forms S(−) and R(+) enantiomers which enable the drug to possess both non-selective β-adrenoreceptor antagonist and also αrtension . Comparertension .As a therapeutic class the beta blockers have a good safety profile. Carvedilol has been widely used for decades, and at a dose of 25 mg daily, postmarketing surveillance has shown that it is generally well tolerated . To the Due to its extensive hepatic metabolism, carvedilol is contraindicated in severe hepatic impairment. However, there are no dosage adjustments in the manufacturer’s labelling for mild to moderate hepatic impairment. The pharmacokinetics of carvedilol are greatly altered in cirrhosis at both hepatic and systemic levels. Hepatic blood flow and drug extraction are reduced, leading to impaired metabolism and elimination of the drug. To add to this, carvedilol is a highly protein bound drug and hypoalbuminemia in cirrhosis affects the unbound serum concentrations.Despite its widespread use in these patients, there is limited data on pharmacokinetics outside of healthy volunteers or patients with heart failure. Rasool et al. performed physiologically based pharmacokinetic modelling to simulate use in cirrhotic patients. They suggested that in these patients, to maintain drug exposure equivalent to 25 mg carvedilol in healthy individuals, the administered doses should be reduced to 12.5 mg, 6.25 mg, and 3.125 mg stratified by Child-Pugh class A, B, and C. Additionally, in liver cirrhosis the unbound systemic concentration of carvedilol increases much more in comparison to that of total systemic concentration of carvedilol .Carvedilol decreases portal pressure after acute and long-term administration. Some authors have suggested that the clinical benefit of carvedilol, as reflected by reduction in HVPG, is directly proportional to dosage. In the studies examined Table , investiDespite its widespread use, from our review of PubMed, ToxLine and International Pharmaceutical Abstracts, there are only two published case reports (English) of carvedilol toxicity and both of these were in the setting of overdose , 18. TheCarvedilol is a highly lipophilic drug and mental status should be monitored closely. In carvedilol toxicity, the signs of hypoglycemia are masked and a high index of suspicion is needed. With normal mental status and a stable airway, the focus of management should be on hemodynamic stabilization. We successfully used crystalloids and albumin followed by glucagon as a high dose intravenous bolus and infusion. Atropine has been recommended by some as first line treatment but has poor results in severe beta blocker toxicity . In carvThe treatment of beta blocker toxicity continues to be an area of active investigation. In beta blocker poisoning with bradycardia and hypotension, high-dose glucagon is considered the first-line antidote . GlucagoThere are no studies on glucagon use in humans and current therapy is guided by animal studies and case reports. It would be unethical to undertake a randomized clinical trial investigating treatment of beta blocker toxicity. In the five studies of animal models of beta-blocker overdose in systematic review by Bailey, glucagon increased the heart rate (at least transiently) but appeared to have no effect on mean arterial pressure . In thisBeta blocker toxicity is usually the result of overdose. Patients with cirrhosis represent a special at-risk group that can have toxicity at standard doses. Healthcare providers need to recognize the toxidrome early. In this case reversal was achieved with intravenous glucagon as evidenced by raised cardiac output and blood pressure. Hospitals where carvedilol is used in patients with cirrhosis should have glucagon in formulary at doses to treat toxicity (bolus and infusion).


Poverty is a key determinant of health that leads to poor health outcomes. Although most healthcare providers will work with patients experiencing poverty, surveys among healthcare students have reported a curriculum gap in this area. This study aims to introduce and evaluate a novel, student-run interprofessional inner city health educational program that combines both practical and didactic educational components.Students participating in the program answered pre- and post-program surveys. Wilcoxon signed-rank tests and descriptive thematic analysis were used for quantitative and qualitative data, respectively.A total of 28 out of 35 participants responded (response rate: 80%). Student knowledge about issues facing underserved populations and resources for underserved populations significantly increased after program participation. Student comfort working with underserved populations also significantly increased after program participation. Valued program elements included workshops, shadowing, and a focus on marginalized populations.Interprofessional inner city health educational programs are beneficial for students to learn about poverty intervention and resources, and may represent a strategy to address a gap in the healthcare professional curriculum. Socioeconomic status (SES) is a key determinant of health. Recent data have shown that one in seven or 4.8 million Canadians live in poverty.11We believe that the majority of physicians and healthcare professionals will work with patients experiencing poverty and homelessness, regardless of their practice location or specialty and thus, healthcare students need to understand the impact of poverty on health status, know how to intervene to treat poverty, and directly train with vulnerable populations. However, surveys among healthcare students indicate a perceived curriculum gap in addressing poverty and providing resources for low income patients.17In response to feedback from health profession students at the University of Toronto indicating the need for more directed training when working with marginalized populations, two medical students (TH and KAC) developed an interprofessional inner city health experience program. The goals of the program were to: 1) enable students to gain knowledge on issues relevant to inner city populations; 2) practice their skills, such as interviewing patients and creating management plans, while shadowing at an interprofessional clinic serving primarily inner city populations; 3) develop an understanding of how to intervene and treat poverty and learn about available resources; and 4) learn about the role of other healthcare providers in poverty intervention. The program had several components: 1) interactive workshops from guest speakers with lived experience and health practitioners, which focused on topics such as addictions, refugee health, non-medical interventions for poverty, and advocacy, and included interprofessional small group discussions; 2) clinical shadowing at the student-run IMAGINE clinic  and other inner city clinics; and 3) a facilitated reflection session held in interprofessional small groups. A focus of the program was on interprofessional education: this was achieved through a variety of means such as facilitating case studies, workshop discussions, and reflection in interprofessional small groups as well as shadowing at an interprofessional clinic. At the clinic, a pair of students from different professions conducted the initial interview and examination with patients and brought this information to the larger interprofessional group of preceptors and students. A discussion was held with input from each profession to determine an effective management plan for each patient, which enabled students to learn about the scope of practice for each profession.This study aimed to evaluate this student-run inner city health educational program among interprofessional healthcare students in order to improve the program for future students and add to the literature informing interprofessional training in inner city health care.This study was approved by the institutional ethics review board of the University of Toronto . Written informed consent was obtained from all students participating in the program. Participants were current students in medicine, social work, nursing, physician’s assistant, occupational therapy, physical therapy, speech-language pathology, pharmacy, and medical radiation sciences.Participants were asked to complete pre- and post-program surveys. Each survey involved a quantitative Likert-scale portion  and an open-ended response portion. The pre-program survey asked students about what skills and experiences they hoped to develop and their understanding of other health professions’ roles in treating poverty. The post-program survey asked about skill competencies developed during the program , what students had learned about interprofessionalism, valuable aspects of the program, and program improvements.All data were de-identified prior to analysis. Analysis of quantitative data was completed using Wilcoxon signed-rank tests with significance assigned at p < 0.05. Qualitative survey data were analyzed iteratively using descriptive thematic coding conducted by two separate reviewers using a consensus approach to resolve discrepencies.18The overall cohort consisted of 35 participants , of whom 28 consented to survey completion . Participant average age was 24 ± 2.6 years. Disciplines represented were medicine (36%), nursing (14%), pharmacy (18%), social work (18%), and other professions (14%). About 52% of participants were in the first year of their programs, and 48% were in their second year. The proportion of students in their first year by discipline were: 42% (medicine), 100% (nursing), 40% (pharmacy), 40% , and 67% (other professions).Overall, the program was very well received by participants. Students, asked to reflect on their personal reasons for joining this program, reported wishing to learn about and help underserved, marginalized populations and participate in an interprofessional team approach to providing healthcare to underserved populations. Four themes emerged from students’ written reflections:Many participants desired to learn about and help underserved and marginalized populations. Some had previous experience working with inner city populations, but most were neophytes. When asked why they joined this program, one participant answered: “to learn more about the less privileged communities and issues related to their access to healthcare.” Another was “interested in healthcare geared towards marginalized populations.”Several participants described not getting enough exposure to inner city health within their respective disciplines. One noted: “This is a great learning opportunity I don't think I will be able to gain solely from my schooling.”Interprofessionalism emerged as a major theme in participants’ reasons for joining the program. One participant described the program as “a unique opportunity to longitudinally work with students across several disciplines.” Another signed up “to witness and participate in an interdisciplinary team approach to providing healthcare to an underserved and often misunderstood population.”Shadowing emerged as one of the most valuable program features. One participant wrote: “Shadowing a family physician who treated youth at a youth shelter. This was a valuable experience because I noticed many of the youth came in for surface issues such as a cold, but had many underlying mental health issues that couldn’t be addressed because there was no time.” Participants noted that shadowing would not have been as beneficial without the first day of workshops to orient them. As one participant noted: “I think one without the other really would have made the program incomplete.”Participants valued the educational aspect of the workshops. One student specifically highlighted the value of “hearing about the actual practical steps I could take to help future patients, for example, with income security.” Other valuable program elements included the “poverty tool”Similar to other community service learning programs,Given the significant number of people living in poverty and their unique health and social needs, it is important that medical and other health profession schools promote social responsibility by offering educational programs highlighting poverty intervention resources and enabling students to train directly with marginalized populations.Study limitations include a relatively small sample size, as this was the first program iteration. However, our data suggest that students found that training with other professions in a program focused on inner city health was beneficial for professional development. This interprofessional inner city health educational program appears to be a worthwhile strategy to address a gap in the health profession curriculum. Areas for future research include long-term outcomes from program participation such as impact on career choice.


Their expression is tightly regulated during brain development with nuclear QKI-5 being the most abundant during embryogenesis followed by QKI-6 and QKI-7 that peak during myelination. Previously, we generated a mouse conditional qkI allele where exon 2 is excised using Olig2-Cre resulting in QKI-deficient oligodendrocytes (OLs). These mice have dysmyelination and die at the third post-natal week. Herein, we performed a transcriptomic analysis of P14 mouse brains of QKI-proficient  and QKI-deficient  OLs. QKI deficiency results in major global changes of gene expression and RNA processing with >1,800 differentially expressed genes with the top categories being axon ensheathment and myelination. Specific downregulated genes included major myelin proteins, suggesting that the QKI proteins are key regulators of RNA metabolism in OLs. We also identify 810 alternatively spliced genes including known QKI targets, MBP and Nfasc. Interestingly, we observe in QKIFL/FL;Olig2-Cre a switch in exon 2-deficient qkI mRNAs favoring the expression of the qkI-5 rather than the qkI-6 and qkI-7. These findings define QKI as regulators of alternative splicing in OLs including self-splicing.The  The deletion of these QKI isoforms in neural progenitors, muscles and monocytes causes severe differentiation defects4. A mouse conditional null allele of qkI has been generated by deleting exon 2 encoding part of the KH domain2. The deletion of the QKI isoforms in OLs using Olig2-Cre  leads to severe CNS hypomyelination with tremors by post-natal day 10 (P10) and death at the third post-natal week. Ablation of qkI in adult mice using PLP-CreERT results in hindlimb paralysis, thoracic kyphosis and immobility by the third to fourth week post-4-hydroxytamoxifen (OHT) administration2. This work defines the QKI isoforms as major regulators of OL differentiation and maintenance. QKI also plays a pivotal role in monocytes, as its deletion using RNA interference blocked their differentiation into macrophages4. Moreover, a qkI haploinsufficient patient5 also exhibits monocyte to macrophage differentiation defects4. Indeed, in humans, the essential role of QKI is evidenced by a marked intolerance to loss-of-function (LoF) genetic variants: an analysis of protein-coding genetic variation in >60,000 human exomes6 shows that LoF variants are observed 14 times less frequently than expected by chance, with an associated probability of LoF intolerance of 0.96.The quaking  encoding QKI-5, -6, and -7 that differ in their C-terminal 30 amino acids7. QKI-5 contains a nuclear localization signal in its unique C-terminal sequence and is exclusively nuclear8. Thus QKI-5 participates in nuclear roles such as in the regulation of alternative splicing. QKI-6 is distributed throughout the cell and forms heterodimers with QKI-5 and may perform both nuclear and cytoplasmic functions9. QKI-7 is predominantly cytoplasmic and both QKI-6 and QKI-7 are thought to function in mRNA export, mRNA stability and protein synthesis15. During embryonic brain development, QKI-5 isoform is the most abundant and its expression declines after the first two post-natal weeks, while the QKI-6 and QKI-7 isoforms are expressed later in embryogenesis and peak during myelination at the third post-natal week16. What regulates the qkI gene and its alternative splicing is unknown.The 1–20)-UAAY17. Transcriptome-wide CLIP (crosslinking and immunoprecipitation) demonstrate that the QKI isoforms in cellulo bind the QREs and also showed that QKI associates predominantly with intronic sequences implying that alternative splicing regulation is one of its main nuclear functions18. The QKI isoforms were shown to regulate pre-mRNA splicing of myelin components19, and later this function of QKI was shown not to be restricted to brain, as QKI-5 also fulfills this role in vascular smooth muscle cells, monocytes and skeletal muscle20.The QKI isoforms all share identical KH domains flanked by QUA1 and QUA2 sequences that mediate sequence-specific recognition of an RNA element termed the QKI response element (QRE) with the following sequence ACUAAY- and performed RNA-sequencing at an average of 133 million reads/sample 24. Genome-wide loss of QKI resulted in widespread AS changes, with 1,438 ASEs detected in 810 genes  events  over the distribution of the ACUAA motif frequency in a random set of control sequences  within a defined subset of genes allowed us to identify 31 QKI-regulated ASEs in oligodendrocytesnts Figs  and S2. nts Figs . In partces Figs  and S2b.ces Figs , again rces Figs . In addiQKIFL/FL;Olig2-Cre, such as for exon 7 of Bcas1, exon 18 of Sema6a and exon 9 of Capzb . We have also quantified the exon inclusion/exclusion levels normalized over a constitutive exon of qkI (in exon 1) shown on the right of each semi-quantitative PCR  and 7b (qkI-6) and the increase in the inclusion of exon 7c of qkI-5 were confirmed by RT-qPCR  and 7b (qkI-6) as well as their 3′-UTRs. To confirm this, we transfected plasmids expressing green fluorescent protein (GFP) alone or GFP-QKI-5, -QKI-6, -QKI-7, mutated versions of QKI-5 that either cannot dimerize (GFP-QKI-5:E148G), or a version that cannot bind RNA (GFP-QKI-5:V157E) in HEK293T cells and assessed the inclusion of exon 7b of human QKI-7 by RT-PCR. Note, in humans the qkI-7 exon is 7b and not 7a as in mice  only when GFP-QKI-5 was transfected, but not when GFP-QKI-6, GFP-QKI-7 or the mutated versions of QKI-5 were transfected  inclusion levels normalized over a constitutive exon of qkI (exon 1), and observed a significant increase in exon 7b inclusion when QKI-5 was transfected , axon ensheathment, and myelination to be the most downregulated. Surprisingly, we identified qkI as the top alternatively spliced event by DEXseq and we show that QKI-5 regulates the alternative splicing of the qkI-6 and qkI-7 isoforms. Inspection of the qkI pre-mRNA reveals the presence of many QREs distributed across the intronic regions of these pre-mRNAs. Our findings define the QKI isoforms as master regulators of RNA metabolism in oligodendrocytes and show that QKI-5 isoform autoregulates alternative splicing of its own gene.The presence of a QKI response element in >1400 mRNAs suggested that the QKI isoforms regulate many genesqkI-5 mRNA is highly expressed during embryogenesis and maintained until approximately P14 at which point its expression declines thereafter. In contrast, the mRNA expression of qkI-6 and qkI-7 begins embryonically and peaks at P14 and is maintained into adulthood25. Thus our observation in P14 wild type mice  that qkI-6 and qkI-7 are the main mRNA isoforms observed is expected. What is unexpected is that qkI-5 mRNA is the major isoform observed at P14 in the brains of QKIFL/FL;Olig2-Cre mice. These findings suggest that the complete absence of the QKI proteins favors the splicing of the qkI-5 isoform. It also suggests that the partial qkI promoter deletion observed in the quaking viable mice7 may decrease the transcriptional output of the qkI gene such that the levels of qkI-5 are insufficient to drive the subsequent pre-mRNA splicing of qkI-6 and qkI-7 and their expression. Therefore, it is the qkI isoform imbalance that blocks cellular differentiation and these findings support the tumor suppressor function of the QKI proteins26, as their deletion or isoform imbalance favors cell proliferation. Furthermore, the haploinsufficient expression of the human qkI gene5 is enough to block cellular differentiation, as observed with its monocyte to macrophage differentiation defect4.Northern blot analysis shows that the qkI haploinsufficient patient exhibits intellectual disabilities, hypotonia, seizures, brain anomalies and specific dysmorphic features5. She carries a balanced reciprocal translocation  , where a break point in one of her qkI alleles causes reduced expression of QKI mRNA and protein4. RNA-seq analysis of her monocytes and macrophages compared to her control sibling revealed 1,513 alternative splice events4. These spliced events were indeed enriched for QREs next to these regulated exons4, suggesting these events are mainly direct targets of the QKI isoforms. Low QKI levels is a prognostic marker for poor survival for patients with NSCLC26. The human lung/bronchus epithelial cell line, BEAS-2B, was depleted of the QKI isoforms using shRNAs26. RNA-seq was performed and 451 ASE were identified of which 81 were validated by RT-qPCR26. Recently, transcriptomic profiling and alternative splicing analysis was performed in wild type versus qkI-deficient neural stem cells either in the presence or absence of Pten and Trp53 identifying 388 and 215 genes that are differentially regulated and alternatively spliced, respectively27.We next compared our mouse alternative splice analysis with two other RNA-seq analyses that were performed in human cells. The et al.26, de Bruin et al.4 and Shingu et al.27, we have 96, 93, and 46 common gene names, respectively. If we compare all ASEs from all 4 gene sets, we can assemble a common signature of 4 genes in QKI-depleted cells . Interestingly, this list of 4 gene names spans mouse, human, oligodendrocytes, neural stem cells, monocyte/macrophages and lung epithelial cells. The other alternative splice targets are, therefore, more tissue- or cell-specific. Other genes commonly identified in three out of four gene sets include HP1BP3, MAP3K7, SBF1, SPAG9 and VLDLR.Our RNA-seq analysis identified a total of 1438 ASEs Tables  to S5 wiIn sum, we define the QKI RNA binding proteins as master regulators of RNA metabolism driving oligodendrocyte differentiation. Our RNA-seq analysis defines QKI as regulators of alternative splicing and herein we define many spliced targets including self-regulation.QKIFL/FL;Olig2-Cre and QKIFL/FL;- mice were generated as described2. Exon 2 of the qkI gene is flanked with loxP sites and the recombination with Cre results in a null. Total RNA was extracted from brains of QKIFL/FL;- and QKIFL/FL;Olig2-Cre P14 mice (n = 3 for each genotype) with appropriate amounts of TRIzol® reagent according to the manufacturer’s instructions (Invitrogen). 30 µg of RNA were DNase I (Promega) treated for 30 mins at 37 °C.All mouse procedures were performed in accordance with McGill University guidelines, which are set by the Canadian Council on Animal Care. Experimental protocols involving animal use were approved by the McGill University Animal Care Committee. QKIFL/FL;- and QKIFL/FL;Olig2-Cre brains  were purified using polyA selection and converted into cDNA by Illumina TruSeq and sequenced on a HiSeq 2000 (Illumina) with paired ends 100 bp in length  at an average of 133 million reads/sample. Illumina sequencing adapters were removed from the reads, and all reads were required to have a length of at least 32 bp. Sequencing runs were processed with Illumina CASAVA software. Trimmomatic v0.3228 was used to trim reads, including removal of low-quality bases at the end of reads (phred33 < 30), clipping of the first three bases and clipping of Illumina adaptor sequences using the palindrome mode. We performed quality trimming with a sliding window, cutting once the average quality of a window of four bases fell below 30. We discarded reads shorter than 32 base pairs after trimming. Trimmed reads were aligned to the reference genome mm 10 using STAR v2.3.0e29. Quality control was performed using metrics obtained with FASTQC v0.11.2, SAMtools30, BEDtools31 and custom scripts. This showed a good proportion of high quality reads and mapping rates higher than 90% in all samples. Bigwig tracks were produced with custom scripts, using BEDtools and UCSC tools. Data were visualized using the Integrative Genomics Viewer (IGV)32.Total RNA from 33 and normalized using DESeq234. Global gene expression changes were assessed by unsupervised hierarchical clustering of samples and PCA, as previously described35. Differential expression analysis between QKIFL/FL;- and QKIFL/FL;Olig2-Cre mice was performed using DESeq234 for all UCSC known genes. Genes were considered differentially expressed if they had an adjusted p-value < 0.05 and a base mean >100. Expressed genes were defined as all genes with the DESeq2 base mean higher than the first expression quartile.Expression levels were estimated by featureCounts v1.5.0 using exonic reads24. Since rMATS requires reads of equal lengths, we performed the analysis on untrimmed reads after verifying that the adaptor content was low in the FASTQC reports. We ran rMATS with standard parameters and analyzed both outputs: AS events identified using reads mapping to splice junctions and reads on target (JC + ROT), and AS events identified using reads mapping to splice junctions only (JC). The list of putative AS events was obtained by including all events with an adjusted p-value (FDR) <0.05 in either JC + ROT or JC. We also computed differential exon usage with DEXSeq v1.12.223. To generate counts compatible with DEXSeq, we used featureCounts to count reads mapping to all annotated exons in Ensembl GRCm38.84 and converted them to the standard DEXSeq input format using scripts available from Vivek Bhardwaj . Differential exon usage analysis between WT and QKI KO mice was performed using standard DEXSeq parameters. Exons with a minimum length of 15 nucleotides and with an adjusted p-value > 0.05 were considered statistically significant.We used rMATS v3.2.2 to identify alternative splicing (AS) eventsTo obtain a reduced set of stringent, robust ASE candidates, we combined predictions from DEXSeq and rMATS, since the underlying methods and the type of information  used by each approach are quite dissimilar. Significant events called both by rMATS  and by DEXSeq  were intersected based on genomic location. For this, we first merged the two rMATS outputs (JC + ROT and JC) so that each AS event was represented by one entry. We then used the BEDTools intersect function to identify the genomic regions of overlap between the results from rMATS and DEXSeq, keeping the information from rMATS Table . Since a36. The lists of differentially expressed genes and genes containing AS events were compared to a background list of expressed genes, consisting of all expressed genes in the complete dataset . For differentially expressed genes, upregulated and downregulated genes with a base mean higher than 100 and an absolute fold change greater than 2 were used for the analysis. For AS, we used the genes in which stringent AS events  were observed.GO term enrichment analysis was performed GOrillaQKIFL/FL;Olig2-Cre mice and SEs with higher exclusion in QKIFL/FL;Olig2-Cre mice (QKI activated). To determine the position of the core motif relative to the exon, we used the FIMO tool37 (MEME suite) and searched for exact matches to ACUAA.We searched for the motif ACUAA in windows of 200 nucleotides neighbouring each side of the identified SE events corresponding to “cassette” or “mixed” exons . To verify enrichment of this motif in SEs and compute empirical p-values, we generated a set of control exons  that met the following criteria: “cassette” or “mixed” exons, good coverage at the exon and gene level , not alternatively spliced  and with a minimum length of 15 nucleotides. To compute empirical p-values, we performed 1,000 iterations, where in each iteration we randomly selected a number of control exons equal to the number of observed AS events, and computed the frequency of occurrence of the ACUAA motif within neighbouring windows of 200nt in these controls exons. Motif analyses were performed successively for all SEs, SEs with higher inclusion (QKI repressed) in qkI alternative splicing was in exon 6 and is the following: 5′-CCT CAC CCA ACT GCT GCA ATA G-3′. The reverse primer to detect exon 7 of QKI-7 inclusion is: 5′-CAT GAC TGG CAT TTC AAT CC-3′. Human constitutive exon: forward, 5′-GTC GGG GAA ATG GAA ACG AAG-3′ and reverse, 5′-GGT TGA AGA TCC CGC AGA AG-3′. Human GAPDH forward: 5′-CCG CAT CTT CTT TTG CGT CG-3′, Human GAPDH reverse: 5′-TGG GTG TCG CTG TTG AAG TC-3′.HEK293T cells were plated in 6-well plates and transfected with 2 µg of the indicated GFP expression vectors encoding QKI-5, QKI-6, QKI-7, QKI5:E148G and QKI5:V157E. The common forward primer for RT-PCR detection of qkI isoforms they were normalized to a constant exon region.1 µg of RNA was reverse transcribed using oligo(dT) primer and M-MLV reverse transcriptase according to the manufacturer’s protocol (Promega). cDNAs were then amplified by PCR using the following conditions: 95 °C for 30 seconds, 60 °C for 30 seconds, 72 °C for 1 minute and repeated for 30 cycles. The PCR products were then separated by agarose gel electrophoresis. For real-time PCR, primers were designed and efficiency tested according to the MIQE guidelines. Real-time PCR was performed in triplicates with a 1:4 dilution of cDNA using SyBR Green PCR Mastermix  on 7500Fast Real-Time PCR System . All quantification data were normalized to GAPDH using the ΔΔCt method. All quantification data were normalized to GAPDH using the ΔΔCt method or for the Mouse 1: 5′-TCC TTG AGT ACC CTA TTG AAC CC-3′; Mouse 2: 5′-GGG CTG AAA TAT CAG GCA TGA C-3′; Mouse 3: 5′-TTA GCC TTT CGT TGG GAA AGC-3′; Mouse 4: 5′-GGT CTG CGG TCA CAA TCC TTT G-3′; Mouse 5: 5′-TAG GTT AGT TGC CGG TGG C-3′; Mouse 6: 5′-GGC CTT CTT ACC GTT CG-3′;. Mouse constitutive exon : forward, 5′-GTC GGG GAA ATG GAA ACG AAG-3′ and reverse, 5′-GGT TGA AGA TCC CGC AGA AG-3′. Mouse GAPDH forward 5′-CCC AGC TTA GGT TCA TCA GG-3′. Mouse GAPDH reverse: 5′-CAA TAC GGC CAA ATC CGT TC-3′. Mouse Bcas1 forward: 5′-GAC ACT GGT TTC ACC TAA CAA G-3′. Mouse Bcas1 reverse: 5′-GTA GCC TTT GTC TTC CCT C-3′. Mouse Bacs1 constitutive forward: 5′-CCC TCT GAT GGC GTT TCT CA-3′. Mouse Bcas1 constitutive reverse: 5′-CCA CGG AGT TTG ACG TTT GG-3′. Mouse Sema6a forward: 5′-GAG ACT GTC ACA ATT CCT TCG-3′. Mouse Sema6a reverse: 5′-GAA CAA GCT GGT CGT TGC-3′. Mouse Sema6a constitutive forward: 5′-CCA AAC ATG CCA ACA TCG C-3′. Mouse Sema6a constitutive reverse: 5′-TGA CAG TCC TGA GGG TAG GG-3′. Mouse Capzb forward: 5′-CTG GTG GAG GAC ATG GAA AAC-3′. Mouse Capzb reverse: 5′-CTG CTG CTT TCT CTT CAA GG-3′. Mouse Capzb constitutive forward: 5′-GTG GTG GAA GTG CAG GAG-3′. Mouse Capzb constitutive reverse: 5′-AGC CGG ATT TGT TGG TTT GC-3′.Supplementary informationSupplementary tables


EHMT1) gene causes Kleefstra syndrome, which is associated with the typical triad of distinct facial appearance, (childhood) hypotonia, and intellectual disability. A high degree of psychopathology is associated with this syndrome. A few parents with a mosaic EHMT1 mutation have been detected upon testing after a child was diagnosed with a germline EHMT1 defect. At first glance, carriers of a mosaic EHMT1 mutation appeared to function normally. However, recent studies have shown that de novo, postzygotic mutations in important developmental genes significantly contribute to autism spectrum disorder (ASD). Therefore, we hypothesized that EHMT1 mosaicism could cause neuropsychiatric defects. To investigate this, we performed a detailed investigation of cognitive neuropsychiatric parameters in parents identified with EHMT1 mosaicism.Genetic mosaicism is only detected occasionally when there are no obvious health or developmental issues. Most cases concern healthy parents in whom mosaicism is identified upon targeted testing of a genetic defect that was initially detected in their children. A germline genetic defect affecting the euchromatin histone methyltransferase 1 , the mini Psychiatric Assessment Schedules for Adults with Developmental Disabilities (mini PAS-ADD), the Vineland Adaptive Behavior Scales (VABS), and the Cambridge Neuropsychological Test Automated Battery (CANTAB). These measures were compared with our previously reported data from Kleefstra syndrome patients with confirmed (germline) EHMT1 defects.Three adults  with a genetically confirmed diagnosis of All three subjects achieved maximum total scores on the VABS, indicative of adequate (adaptive) functioning. In all, scores above cutoff were found on the ADOS for ASD and on the mini PAS-ADD for major depressive disorder (lifetime). Finally, results on the CANTAB showed impaired cognitive flexibility in all subjects.EHMT1 mosaicism seem to have increased vulnerability for developing severe psychopathology, especially ASD and mood disorders. Although at first glance they appear to be well-adapted in their daily functioning, they may experience significant psychiatric symptoms and show reduced cognitive flexibility in comparison to the general population.Individuals with  Almost all cases of KS are due to de novo microdeletions or intragenic loss-of-function mutations in this gene ,(RP13_467E5-)[45/50].Subject 3: After the detection of a 9q34.3 deletion in the son (arr [hg 19] 9q34.3(140350924 140551108)× 1), the array analysis on DNA from subject 3 suggested the presence of a mosaic deletion in 9q34.3. Subsequent FISH analyses on blood lymphocytes showed an abnormal signal pattern in 29 of 50 cells analyzed, suggesting 60% mosaicism: ish del(9)(q34.3q34.3)(RP11-48C7-)[29/50].The genetic, psychopathological, and neurocognitive characteristics are presented in Table The developmental age of all three mosaic carriers was 12 years or older. This maximum score on the clinical interview VABS is in line with the developmental age of the general population. Additionally, all subjects fulfilled the diagnostic criteria for ASD and MDD either currently and/or in the past. In contrast to the ADOS, the mini PAS-ADD did not formally determine ASD as the total subscale scores were just below the cutoff score. Notwithstanding, ASD items scored in the mini PAS-ADD were similar to the clinical observations of the ADOS. During the clinical observation, all participants scored on limited eye contact and minimum use of emotional gestures, when testing was focused on the communication. For social interaction, all three showed impaired social reciprocity, a limited range of facial expressed emotions, and an inadequate description of their role in social relations.In addition to estimating the degree of psychopathology, we used the CANTAB to measure several cognitive functions of our subjects. The percentages represent the subjects’ performance compared to the normative group with the same sex and age. Subject 1 had low scores on the PRM and IED. Subject 2 had overall low scores on the tests. Subject 3 experienced problems with the IED. Detailed results are presented in Table EHMT1 mosaicism on psychopathology in three adult subjects. The three seemingly unaffected parents included in this study had been referred to the clinical genetics department only because of the diagnostic trajectory of each parent’s respective child. The discovery of EHMT1 mosaicism in these subjects was unexpected [EHMT1 mutation had a variable occurrence in the mosaic individuals. The results are indicative of a vulnerability to develop severe psychiatric disorders that carriers of an EHMT1 mosaic mutation possess. This correlates with the high prevalence of psychopathology, which has been reported for individuals with the full KS phenotype due to a germline EHMT1 mutation [In this report, we describe the consequences of expected –5. The dexpected . Despitemutation , 8, 21. mutation . These dEHMT1 mosaicism and consequent neuropsychiatric assessment is significant. Optimal treatment of the psychopathology may improve daily functioning and positively influence the well-being of the individuals treated and their families, including the child with KS, as well as their participation in society. Based on our findings, personalized psychiatric evaluation and treatment should be considered for subjects diagnosed with EHMT1 mosaicism. The diagnostic procedure should include a special attention to ASD and mood disorders. Furthermore, these cases illustrate that presence of mosaic deletions could potentially be missed, since the genetic defects were only discovered during the diagnostic phase of the children with the full KS phenotype. None of the subjects had been referred to a medical specialist for their own psychiatric problems prior to our studies. These subjects tend to have a more subtle presentation, but this can nevertheless cause impaired  functioning. Therefore, our study also demonstrates that genetic analysis should be considered in patients with a psychiatric disorder and other characteristics of KS without the classic appearance.Psychopathology has a direct impact on adaptive functioning. The adaptive behavior measured with the VABS implies a developmental age above 12 years, consistent with adult functioning, for all of our subjects. Nevertheless, all three of them struggle to fulfill their role in society, most likely as a direct consequence of their neurodevelopmental problems. They became socially isolated and experienced difficulties in finding the right type of education and employment that would suit their strengths and weaknesses. Therefore, the clinical relevance for diagnosing these subjects with EHMT1 mosaicism and each had different levels of mosaic expression. Interestingly, in recent studies, it was shown that rare postzygotic mutations in important developmental genes contribute significantly to ASD [EHMT1 are in line with these reports. EHMT1 is an important developmental gene that is extremely intolerant to loss-of-function mutations (http://exac.broadinstitute.org) [EHMT1 mosaicism, although the extremely low prevalence of such mosaic defects hampers the careful collection of symptoms in larger cohorts. For future research, we recommend to include more specific measures on IQ, ASD, and mood disorders in subjects with an EHMT1 mosaicism. These measures should be in addition to a (semi)structured clinical observation as people with neurodevelopmental disorders, like ASD, are more likely to have deviating scores on questionnaires [Despite the similarities in psychopathology between our subjects, the consequences of mosaicism in general are widespread and unique for each individual . The phey to ASD , 13. Ourute.org) . Furtheronnaires , 28. ThuEHMT1 mosaicism seem vulnerable to developing psychiatric disorders, especially ASD and mood disorders. However, since the behavioral outcome of mosaicism is unpredictable, these results should be interpreted with caution. Based on our findings in this small but unique cohort, we recommend screening subjects with an EHMT1 mosaicism for psychopathology. In our experience, multidisciplinary care, combining expertise in psychiatry, clinical genetics, and clinical neuropsychology, is of additional value in these complex cases where a broad perspective is required. Ideally, a careful diagnostic assessment of psychopathology, cognition, and physical health should be performed to provide an integrative diagnosis. Deficits in daily functioning and behavioral problems may well be a result of an underlying genetic or otherwise somatic cause, which is often not clearly present. Clinical considerations should therefore include the possibility of a genetic mosaicism in parents who have one or more children with a neurodevelopmental disorder of genetic origin and a typical combination of complaints, as illustrated in this study.Subjects with


Many payment for ecosystem services (PES) programs, such as the Slope Land Conversion Program (SLCP), are passive and require full participation by impacted households. In contrast, this study considers the alternative of “active and incomplete” participation in PES programs, in which participants are not obliged to contract their own land, and have the right to select into the program or not. This type of program has been popular over the last decade in China; however, there have been few studies on the characteristics of willingness to participate and implementation. As such, this paper uses the Choice Experiment (CE) method to explore ways for inducing effective program participation, by analyzing the effects of different regime attributes. The case study used to analyze participation utility was the Jing-Ji Afforestation Program for Ecological and Water Protection (JAPEWP), a typical active-participation forestry PES program, and a key source of water near Beijing in the Miyun Reservoir Catchment (MRC). Analyzing rural household survey data indicated that the program faces a variety of challenges, including long-term maintenance, implementation performance, cost-effectiveness, and monitoring approaches. There are also challenges with one-size-fits-all payment strategies, due to ineffective program participation or imperfect implementation regimes. In response, this study proposes several policies, including providing secure and complete land tenure to the participants, creating more local off-farm employment opportunities, designing performance-based monitoring systems that are integrated with financial incentives, applying differentiated payment strategies, providing capacity building to support forestation activities, and establishing a comprehensive implementation regime that would address these challenges. These policy conclusions provide valuable lessons for other active-participation PES programs as well. Over Willingness to participate is an inherent part of a PES program, and creates opportunities and the space for stakeholders to negotiate agendas, policies, programs, roles and partnerships –15. FurtInternationally, many case studies have described rural household’s willingness to participate in PES programs , 23–31. China leads global PES efforts ; most ChOver the last decade, many studies of SLCP have discussed the program’s impacts on rural households. These impacts include off-farm labor allocation , 37, graThe second type of PES in China is referred to as “incomplete and active participation.” With this type, rural households have the right to choose to participate or not participate in the programs. An example is the Sand Control Program in the Beijing-Tianjin Sand Source Regions –49. MoreAs an international mega-city with a population exceeding 20 million, Beijing is suffering from an unprecedented water crisis –52. SincThis study uses the Choice Experiment (CE) method to explore ways to induce effective program participation, by analyzing the effects of various program attributes on participation utility. The goal was to address a variety of challenges, including long-term maintenance, implementation performance, cost-effectiveness and monitoring approaches. This research was needed due to ineffective program participation and imperfection implementation regimes in the JAPEWP program. This program is a typical active-participation type of forestry PES program, in the key source water area of Beijing, the Miyun Reservoir Catchment (MRC). The study provides valuable and unique policy insights into designing active and effective programs, as well as implementing efficient program regime policies for typical emerging active-participation PES programs.2 and a maximum water storage capacity of 4.375 billion m3 and is the largest reservoir in the North China. It receives water mainly from the Chao and Bai Rivers [3 per year for urban water use; this accounts for 25% of total water used [The Miyun Reservoir, built in the 1960s, is in northeast Beijing, approximately 100 kilometers away from the city center. The reservoir originally had a surface area of 188 kmi Rivers –57. The ter used –59.2, 80% of which is located in the separate Hebei Province. This cross-jurisdictional characteristic brings about a series of challenges in terms of development interests, for both upstream and downstream stakeholders [The MRC, primarily the upper watershed area, has significant strategic importance for the water security of Beijing . Howevereholders .Initiated in 2009, JAPEWP has invested a total of US$143.2 million to build an ecologically important 66,666 ha water protection forest within the Miyun and Guanting Reservoir Catchment of Hebei Province . The proFirst, young and capable labor forces are moving into urban areas to work as migrants , 46, lea2 of watershed area, covering two primary MRC water sources, the Chao river and Bai river [This study involved a rural household survey in the MRC area of Hebei Province. Before that, however, the research team completed a pre-survey in Fengning, Luanping, and Chicheng counties in October 2012. Among these, Fengning county occupied more than 4,400 kmai river . As suchWithin Fengning county, based on pre-survey findings and reviews of local statistical yearbooks, Tanghe, Heishanzui, and Humaying Townships were selected as experimental sites. Of these, Tanghe Township is in the Bai river sub-catchment; Heishanzui and Humaying Township are located in the Chao river sub-catchments. In terms of local livelihoods, Tanghe Township is more of a traditional agriculture township compared to Heishanzui and Humaying Townships, where there are emerging mining industries . As such, the rural households in Heishanzui and Humaying Townships have more diversified livelihood sources and depend less on agriculture than the Tanghe Township. Together, these three townships effectively represent MRC from the perspectives of geological scope and livelihood diversification.In our study, rural household’s willingness to participate (participation or no participation), which represents their preferences for attributes of the program, was treated as a discrete variable. This allowed the application of the CE method, using discrete econometric models to analyze the determinants of willingness to participate. CE is a Stated Preference Method rather than a Revealed Preference Method; as such, it relies on Lancastrian consumer theory  and randSince the 1990s, CE has been applied to study public willingness to participate in agricultural and forest management –76, biodThis case assumed that rural households maximize utility in a given choice set . The choHere, we assume that ε follows a Type I extreme value distribution (Gumbel distribution), with an independent and identical distribution across alternatives and individuals . As suchDue to the non-linear nature of the MNL model, the raw coefficients do not reflect the effects of per unit attribute changes on the choice probability. Given this, the Marginal Effect (ME) is applied to measure the relative importance of each attribute affecting choice . Based on/MEp, where MEn and MEp are the marginal effects of non-monetary and monetary attributes. Based on this expression, the total net willingness to accept (TWTA) at different utility levels can be estimated by: TWTA = -(V0-V1)/βp. In this expression, βp is the estimation coefficient of price or monetary attribute. TWTA estimates the minimum bid that a rural household would be willing to accept to sign up for a program that yields utility V1, as compared to the value obtained from remaining with status quo V0 [MWTA is estimated using the expression MWTA = -MEs quo V0 .CLENGTH), freedom to leave (RELEASE), afforestation survival rate (SRATE), financial penalty (PENALTY), inspection method (INSPECTION), and cash subsidy (CSUBSIDY).During the October 2012 pre-survey, we developed a rough shortlist of choice attributes on the JAPEWP program policy. These were informed by consulting with focus groups, such as local authorities and rural households. A test using the draft choice sets consisting of these attributes was then tested in one village. After that, minor revisions were made to improve the efficiency of choice sets, in accordance with respondent feedback. Finally, the key factors assumed to most impact rural household’s willingness to participate in the JAPEWP program were listed into the final choice sets . These i2×23×5 total potential policy options. In our study, a choice set includes 4 options: alternative policies A, B, and C, and option D, which is no participation. These facilitate respondent choice behavior [As behavior . The expbehavior . To achibehavior . This restratified sampling method ensured the sample representativeness with respect to wealth status and participant willingness, and minimized the sample bias to the largest extent possible.The field survey was jointly conducted by the Beijing Forestry Society (BFS) and Beijing Forestry University (BJFU) in August 2013. At the beginning of the field survey, with assistance from local township staffs, all villages within each selected township were grouped into rich, medium and poor groups according to their wealth status. Then, the survey team randomly sampled a number of villages from each group. Similarly, under the help of local village heads, a number of rural households were also selected with the above sampling approach in each sampling village . The strDuring the survey, the enumerators, led by local village heads, visited the sampled households and used the structural questionnaire interview to solicit respondent decisions on different choice sets. Improving on previous experience that the presence of village heads during the formal interview may impact respondent’s decisions and generate unnecessary choice bias. In order to ensure the validity and accuracy and the responses elicited, we thus requested that the village heads initially introduced the enumerators and respondents to know each other and would not be involved the following interview.During the in-person interview phase, the enumerator first introduced the survey goals and background about the JAPEWP policy by reading through the following narrative wording on the first page of questionnaire for the respondent.As the sole source of surface water, the Miyun Reservoir is important for supplying domestic water to Beijing city. The MRC area has a particularly strategic importance for providing water for the reservoir. The water resource status, however, is becoming increasingly impacted as the inflow from the MRC has decreased annually. Thus, the Beijing Municipal Government recently has made great PES efforts to restore the ecosystem services in the MRC area.The JAPEWP program funded by the Beijing Municipal Government intends to restore ecosystem services in the MRC area and to guarantee the water security for Beijing via forestation approaches. The program participation is voluntary for the local rural household, that is, you can choose participate in or not participate in the program while there is no need to contract your land with the program like the SLCP program. However, once one decides to sign up to the program, the participant would be asked to obey the following program regulations. Firstly, the survival rate of the plantation should meet the qualification requirements proposed by the program. Secondly, the planting would receive inspection for the survival rate after afforestation, and might receive an economic penalty if the survival rate is insufficient. Finally, on the basis of a qualified performance the participant would receive some amount of cash subsidy each year. In addition, the program would not only have other requirements for the participant, such as contract length, release and penalty options but also provide relevant capacity building and technical services.In order to assist the decision maker to formulate more efficient program policy, the organizer hopes to make sense of the real willingness to participate and the preferences to the implementation regime via the survey. Please make your choices and indicate the information related to you and your family following my instructions. Any of your responses and suggestions would be highly appreciated.Then, the enumerators explained the meaning, level, and range of six attributes . After aThe socio-demographic characteristics of rural households affect choice utility . As such, In contrast, the higher off-farm income in the Humaying township suggests that local mining resource development may yield a more developed off-farm employment market. This form of well-established off-farm employment market could help households reallocate labor forces, and avoid over-supplying labor for traditional agriculture . In summThe MNL model was applied to analyze the effects of each attribute on choice utility. CLENGTH and INSPECTION. Increasing increments on both RELEASE and CSUBSIDY variable increase the associated utility level provided by the choice. The presence of either SRATE or PENALTY attribute decreases the utility. Every township showed similar estimation outcomes.Overall, the outcomes of the Chi-square test indicate that the estimated results fit the empirical model well. Most estimated coefficients are statistically significant at the 1% level, except for the variables In this study, ASC reflects the utility derived from choosing to participate in the program, keeping all other attributes at status quo levels. In fact, the participant’s willingness to participate might be an interaction between local welfare status and the program attributes . The sigRELEASE and CSUBSIDY attributes are significantly positively correlated with participation utility. The highest incremental utility occurs when participants are free to leave the contract. In other words, the ability for a participant to be freely released from the JAPEWP program contract has the largest positive effect on the willingness to participate.Both On one hand, because the subsidy level is relatively low compared to the other income sources , the subThere is a big difference between active and passive SLCP participation; participating farmers are unlikely to exit as long as the payments are being delivered . This maSRATE and PENALTY attributes negatively impact participation utility. A higher survival rate entails more labor input, as forestation is a labor intensive work in general among which planting, tending and fire patrolling all cost significant labor. Meanwhile, the relevant forestry knowledge and experience of the worker would be another pre-requisite because a skilled forestry laborer can provide better management [SRATE reflects a lack of a sufficient and qualified local labor force due to higher opportunity costs. In addition, the stay-at-home laborers, who lack physical strength and relevant forestry knowledge, may worry about the possible economic losses that would occur if they experience a disqualifying survival rate. That is why the PENALTY attribute also has a negative effect on the rural household’s willingness to participate. Therefore, along with increasing and diversifying income sources, more local off-farm employment opportunities should be created to attract more qualified labor forces home [As anticipated, the nagement . As noteces home , 39, 41.PENALTY above SRATE (-0.874 VS. -0.632). As mentioned above, Tanghe Township depends more on traditional agriculture, whereas Humaying Township depends more on off-farm employment. Heishanzui Township falls somewhere in between. Households that are more vested and experienced in agriculture, with less exposure to off-farm labor markets, manage planted trees better [There are a few differences in the importance of specific variables between the townships. Tanghe Township households value s better . As suchCLENGTH nor INSPECTION attributes significantly affect participation utility. Findings related to the contract length attribute were consistent with the “RELEASE” attribute discussed above. Because participants have stronger interests in having the freedom to be released from a contract, it is not unexpected for them to prefer a shorter-term contract, or to be insensitive to contract length at all. The insignificance of the INSPECTION attribute for all sample units indicates that ongoing inspection methods (regular or irregular inspection) do not provide sufficient guarantees to support program implementation. Therefore, more innovative policy methods and assessment mechanisms are needed.Overall, neither the To better understand the heterogeneity of household preference, we also assessed several other household-specific variables. These variables do not vary across household choices; as such, they drop out of the probability model. They can, however, be included in the model by assessing their interactions with the attributes that do vary across household choices . IncludiThen, we added these household-specific variables interacted with each of the choice attributes into the MNL model estimation. Therefore, the following potential measures should be taken into consideration to release the constraints on market and institution. Firstly, lowering dependence on traditional agriculture, while attracting more qualified labors to return home by creating new local income sources associated with JAPEWP program implementation, should be considered. Secondly, providing a set of complete and secure forest land tenure for the participants via forest land tenure reform would promote the willingness to participate and even further incent their land quality investment to some extent . Lastly,Ceteris paribus, an increase of 200 Yuan, results in a 0.5% increase in the likelihood of participation. These results suggest that depending on the importance of the cash subsidy is not a cost-effective way to motivate willingness to participate in the active type of program.The ME of the cash subsidy attribute suggests that increasing the cash subsidy by one Yuan increases the likelihood of program participation by 0.003% . CeterisTurning to another continuous variable, a 1% increase in the required survival rate results in an 18% decline in the probability of participation. With the dummy variables, allowing participants the freedom to leave the program contract increases the likelihood of participation by 14%. As such, this variable should be considered an effective policy tool to induce willingness to participate. In contrast, the presence of the “financial penalty” attribute decreases the likelihood of participation by 15%.The results associated with these attributes have important implications for policy designs, if the goal is to induce more household’s willingness to participate in the JAPEWP program. In addition, ME fluctuations among the three townships generally follow the above analysis of raw coefficients.Using the ME estimate outcomes, Figs For example, the probability density function shows that to achieve an enrollment probability of 0.5, a subsidy of no less than 5250 Yuan/year/ha should be paid to farmers, if all other attributes remain at status quo levels. However, once the “freedom to leave” and “financial penalty” interventions are introduced, the cash subsidy fluctuates to 750 and 9750 Yuan/ha/year, respectively. This indicates that different policy interventions could save significant cost for the program.ceteris paribus (Scenario A and C), the enrollment probability is 0.297 and 0.289, respectively. From this, it appears that technical training may help alleviate the pressure of survival rate.For the other two continuous variables, the “contract length” and “required survival rate” attributes, we designed three different policy scenarios to simulate enrollment probability; other attributes were kept at the status quo level. Scenario A required a 1-year contract and 75% survival rate; Scenario B assumed a 1-year contract, but required a 100% survival rate; Scenario C required a 10-year contract and 75% survival rate. 0) to the most desirable attribute level (V1) , the total net willingness to accept (TWTA) will drop to just 65 Yuan/ha/year. This further verifies the finding above that cost-saving goal can be achieved by reforms and additional supporting services.When considering specific attributes, the participants are willing to accept an income loss of 4293 Yuan/ha/year if they are free to leave the contract. The other three attributes are all associated with a participant need for additional compensation for each unit of improvement. Based on the equation introduced in the Methods section, if the program is implemented in a way in that adjusts the utility level from the status quo . This suggests this township may have the greatest willingness to sign up for the program. Increasing local off-farm employment may increase participation rates at a given subsidy level , 39, 41.Along with the dramatic development of PES programs overall in China, there are an increasing number of active-participation PES programs. In these programs, participants are not obligated to provide their own land to the program, and have the right to select into program participation or not. As such, it is projected that these programs can play more important roles in preventing environment degradation and alleviating poverty. However, with insufficient willingness to participate or ineffective implementation, this kind of program also faces challenges associated with long-term maintenance, implementation performance, and cost-effectiveness. Within this context, this study used the JAPEWP program as a case study to explore the characteristics of rural household preferences; the goal was to induce more active and effective program participation and establish an efficient implementation regime.Program participation is currently voluntary at the household level. However, the program is unable to sustain long-term willingness to participate, due to the lower subsidy levels and higher labor opportunity costs. This study proposes that participant household income may be increased by diversifying income sources. In particular, the survey found that forestland tenure reform has not been completely undertaken in the area , 91–92. Today, an increasing number of young and knowledgeable laborers have moved out of rural areas to work as migrants in cities. The survey also indicated that the majority of stay-at-home laborers are often old and ailing, and have less related knowledge and experience. The shortage of young and qualified laborers generates risks for program implementation and benefits . To encourage migrant labors to return home, more local off-farm employment opportunities would need to be created , 93–94. The insignificant effect of the inspection method attribute on willingness to participate is in stark contrast to the significant impact of the financial penalty attribute. This suggests that the financial penalty is likely to ensure program implementation to some extent, but, overuse may decrease future rural household’s willingness to participate. This finding also indicates that the ongoing monitoring systems previously used in similar set-aside programs (e.g. SLCP) may not efficiently ensure program implementation . A scienHousehold willingness to participate in the program varies across townships with different socio-economic characteristics. However, programs still use a one-size-fits-all payment strategy, regardless of the interests of local rural households, and regardless of project location. Failing to spatially differentiate and target more effectively may result in losses in efficiency , 27, 43.In conclusion, the JAPEWP program subsidy is no less than another similar PES program in the United States , which had an average subsidy level of 705 Yuan/ha/year in 2016 . As suchS1 FileAppendix A. Demographic data of rural household in the Miyun Reservoir Catchment area, China.(XLSX)Click here for additional data file.S2 FileAppendix B. Choice Experiment data and household-specific variables of rural household in the Miyun Reservoir Catchment area, China.(XLSX)Click here for additional data file.