Introduction: Between 2006 and 2012 the Dutch government funded a nationwide program for reducing the use of seclusion. Although an initial first trend study showed that the reported number of seclusions declined during the program, the objective of a 10% annual decrease was not met. We wished to establish whether the decline had continued after funding ended in 2012.Method: Using quasi Poisson time series modeling, we retrospectively analyzed the nationally reported numbers of seclusion and involuntary medication between 1998 and 2019, i.e., before, during and after the end of the nationwide program, with and without correction for the number of involuntary admissions.Results: With and without correction for the number of involuntary admissions, there were more seclusions in the seven years after the nationwide program than during the nationwide program. Although the reported number of involuntary medications also increased, the rate of increase was slower after the end of the nationwide program than before.Conclusions: Rather than continuing to decrease after the end of the nationwide program, the number of seclusions rose. This may mean that interventions intended to reduce the use of seclusion within this program are not properly sustained in daily clinical care without an ongoing national program. If other interventions in psychiatry fail, in many countries seclusion and restraint are often used as a last resort to manage disruptive and violent behaviors. Though both may prevent injury to the patient, others, or property, they also have negative side effects for patients and staff. These include not only negative feelings like anger, humiliation, anxiety and aggressive feelings, but also injury, disruption in the therapeutic relationship, and posttraumatic stress disorder –3.Due to the relatively high seclusion rates in the Netherlands , 5, the Internationally, there have been few studies of seclusion-reduction programs on such a large scale as an entire state and/or nation.One such example covered the Seclusion and Restraint Reduction Program in Pennsylvania, where, after state-wide policy changes had led to a range of interventions, the state hospital system successfully reduced the use of seclusion and restraint nearly to zero between 1990 and 2000 . Until 2There are several reasons why it is important to determine long term effects of such nationwide programs, for example the considerable investments of money and time and because little to nothing is known about their long-term effects.The aim of this study was therefore to determine whether the nationwide decline in seclusion achieved during the reduction program had continued after funding ended. Even though it is not the primary focus of the program and our study, we also wished to determine whether there had been any changes in the national number of notifications of involuntary medications. We did this to ensure that a potential decrease in the use of seclusion did not lead to a concomitant rise in the number of involuntary medications .Between 2006 and 2012, the Dutch government awarded grants to Dutch psychiatric hospitals that had specific plans for preventing the use of seclusions, and for carrying out any remaining seclusions more humanely. The most important criteria for qualifying for the grant, besides a seclusion reduction intervention, was that a psychiatric hospital had to monitor its results, and it had to match the sum it received .In total, 73 (84%) of the 87 Dutch psychiatric hospitals with a permit for involuntary hospitalizations participated in the national program . We assSeclusion was defined as locking a patient in a room designed for this purpose without opportunities to leave. Involuntary medication was defined as any medication administered  against a patient's will. In the Netherlands, coercive interventions may only be used within an emergency measure (short term) or as part of a specifically elaborated involuntary treatment (long term) , 20. TheUnder the mental health law that applied at the time, the Special Admissions Act , seclusTo model the time series data, we used a quasi Poisson Generalized Linear Model with a log link function and to account for autocorrelation we used the number of seclusions or involuntary medications in the previous year . To captTo correct for changes in the number of involuntary hospitalizations, all models were replicated with the log of the number of involuntary hospitalizations per year as offset. Sensitivity analyses were conducted that limited the time series to 2006–2018. The purpose was to correct for possible registration bias caused by administrative start-up difficulties in the period to 2005, and also to correct for adaptations concerning legislative changes in 2019–2020 preparatory to the implementation of a new Dutch mental health law in 2020.Between 1998 and 2005, the number of notifications of seclusions reported to the DHCI increased by 32.6%, representing an overall linear annual increase of 4.2%. Although the overall number of seclusions decreased  during the nationwide program, the number of seclusions increased by 5.7% in the seven years after the end of the program . The model presented in What was remarkable in With regard to involuntary medications, the number reported to the DHCI between 1998 and 2005 increased by 64.1%, effectively a linear annual increase of 7.3%. The increase of the number of involuntary medications both during and after the nationwide program are comparable to the period before the program. But, in contrary to the numbers of seclusions, the annual number of involuntary medications was almost always greater than that in the previous year. Remarkably, however, there was also a steep decrease in 2018 and 2019.The model  shows noThe sensitivity analyses showed similar results. These analyses excluded the period before and the years 2018 and 2019. This means that these periods had no significant effect on the final estimates.From 1998 until 2019, the number of involuntary hospitalizations increased . Since tThe number of seclusions reported to the DHCI using involuntary hospitalizations as an offset, decreased before, during and after the nationwide program with annual fluctuating percentages ranging between 2.5 and 2.9% per period see . And agaWith regard to involuntary medications, the number reported to the DHCI per involuntary hospitalization increased by only 1.0% before the nationwide program . The annual increase was greater both during the program (5.7%) and after it (3.0%). In the time series model, the number of involuntary medications per involuntary hospitalization was greater after the program, although the slope was less steep see .For the estimates of the final time series models see .Again, sensitivity analyses showed similar results.To our knowledge, few studies have examined national programs for reducing the use of seclusion, and no national scale or statewide study has examined the effects of such a program after it has ended. Ours is therefore the first to examine the longer-term sustainability of continued reductions after such a program. To determine whether a decrease in seclusions led to a concomitant increase in the use of involuntary medication, we also examined the use of involuntary medication. We found that, after funding ended, the number of seclusions and number of involuntary medications both increased.The decrease in the number of seclusions that took place during the nationwide program did not continue after the program ended: instead the numbers rose, an effect that remained even after correction for the increasing number of involuntary hospitalizations. This may mean that the effects of the nationwide program were not sustained in daily clinical care. Although one cannot predict what the number of seclusions would have been without any subsidy or funding, one may question whether the program justified the investment made in it.The effects of the program may have been greater and better sustained if, in order to qualify for the government grant, the individual psychiatric hospitals had been obliged first to use only evidence-based methods to reduce the use of seclusions, and secondly to continue using them in daily clinical care after the end of the program. This would have ensured that the activities transcended the project itself by becoming embedded in normal daily practice.We can only speculate about the lower number of seclusions in 2018 and 2019, which may be a new trend, with a cause that is as yet unknown, or otherwise an effect of ongoing efforts to reduce the use of seclusion and coercive measures. It may also be part of the varying pattern of seclusion numbers since 2012. As there was a similar decrease in the number of involuntary medications, it would be interesting to track these developments over the next few years.During the 20 years under study, the registered number of involuntary medications in the Netherlands has continued to increase.A partial explanation for the increase after 2008 may lie in the effect of certain changes in the Mental Health Act between 2004 and 2008, which were intended to broaden the options for involuntary treatment . For exaWhile it is conceivable that involuntary medication replaced seclusion, the higher number of seclusions in the last period make this unlikely.Finally, we believe that the increased use of involuntary medications in the Netherlands reflects psychiatric health care workers' changing ideas and beliefs about treatment. It suggests a greater focus on using medication, whether voluntary or involuntary, to improve (in)patients' mental health rather than using seclusion to protect bodily integrity, which can be harmed by intramuscular injections . These cAlthough legal and cultural contexts differ between countries, it is interesting to compare our results with those of other national or statewide studies on reducing seclusion, and to see whether and how these results were sustained.Pennsylvania's Seclusion and Restraint Reduction Program very successfully reduced the number of seclusions , 15. As The effect of the Dutch nationwide program we describe may be similar to that of the Finnish national plan for 2009–2015 , which, In the international literature on the sustainability of seclusion reduction programs, we found only one study—in the Netherlands—in which Mann-Poll et al. investigated the long-term effects of this nationwide program in three participating hospitals . AlthougBoumans et al. found that organizational context is very important. Although they found an initial reduction in the use of seclusion after the implementation of an innovation project in a psychiatric hospital, they also found during a later period of organizational turmoil that the staff's work engagement decreased and the use of seclusion increased again. This shows the vulnerability of innovations within an organizational context of continuous changes in mental healthcare .Whitley et al. studied facilitating and barrier factors in the implementation of an innovation project. He found that leadership, organizational culture, training, staff and supervision played meaningful roles in determining the success or failure of its implementation, which was facilitated through strong leadership, an organizational culture that embraced innovation, effective training, and committed staff . These qAnother initiative that might help to support the difficult task of sustaining a change in culture toward ongoing reductions in seclusion was proposed by Colton and Xiong, who developed a questionnaire intended to measure staff perceptions of organizational activities and staff attitudes toward the use of the interventions to reduce the use of seclusion .Due to the retrospective observational design of our study, we cannot say whether the changes we observed in use of the coercive measures resulted directly from the government initiative.The use of seclusion and involuntary medication may have been underreported to the DHCI , especiaAs an extra check, we corrected the registered number of seclusions and involuntary medications for the number of processed involuntary hospitalizations. Although we believe this accurately represents the population at risk, it is not the actual number of involuntary hospitalizations, and comprises requests for short involuntary hospitalizations as well as those for longer ones. In addition, reasons for involuntary hospitalization might differ from reasons for seclusion and involuntary medication.It is also possible that the increase in both the number of reported seclusions and involuntary medications was due to registration bias: in other words, that it was the product of better registration instead of an actual increase in the use of coercive measures. Although this may have been true for the years up until the start of the program, good registration of the number of seclusions and other involuntary measures then became mandatory as a condition for participation. For this reason, a form was developed on which detailed information could be entered on all the coercive measures applied, including seclusion and involuntary medication. This form, named Argus, was implemented nationwide, becoming mandatory from 2012 onwards for reports to the DHCI , and remRather than continuing the decrease after the end of the nationwide program, the number of seclusions rose. As this effect remained even after correction for the increasing number of involuntary hospitalizations, it may mean that interventions intended to reduce the use of seclusion within this program are not properly sustained in daily clinical care without an ongoing national program. To ensure that the effects of future seclusion-reduction programs or other national mental healthcare interventions are sustained after their subsidization ends, we recommend that such subsidies are granted only if these initiatives involve the implementation of evidence-based interventions in normal daily care. As advocated above, these initiatives should be accompanied by the Six Core Strategies for Reducing Seclusion and Restraint Use by Huckshorn, and an ongoing developmental process of implementation of the seclusion-reduction program in psychiatric hospitals.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.FV, AW, EN, HN, and CM conceived and designed the study. FV collected and restructured the data. AW advised closely on the appropriate method of this study and performed the main analyses and critically revised the methods and results sections. FV wrote the first and following drafts, which EN and CM revised critically for important intellectual content. All authors approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Coffee is usually subjected to a roasting process which is responsible for the formation of aroma and flavours but also of some undesirable compounds such as furan and alkyl furans. These compounds are known as process contaminants of the roasting process and exhibit some harmful effects. In order to evaluate the exposure to these compounds in coffee, it is necessary to know the levels of contamination as well as consumption habits. The degree of consumers’ loyalty to specific coffee brands could also be an important driver affecting the level of exposure. This research aimed to evaluate the levels of consumption and the degree of loyalty to coffee brands available in Belgian markets, as well as the factors affecting the choice and the consumption of coffee products and coffee brands. Data were collected in Belgium through an online survey. The results show that for the 1930 participants, 87% reported daily coffee consumption and 13% never or occasionally consumed coffee. The global median coffee consumption was 3 cups per day, and the median for individual daily consumers only was 4 cups per day. The level of consumption of ground coffee was about twice higher than coffee beans, followed by instant coffee and relatively very low consumption of coffee substitutes. In total, 78% of participants reported brand loyalty but to different degrees. Two coffee brands sold in Belgian regions were listed together by more than 20% of the survey participants. The most frequent criteria for selecting a specific coffee brand were taste and price, followed by tradition and habit. The age of coffee consumers and several sociodemographic characteristics have significant effects on coffee consumption. The type of coffee product, the degree of loyalty, and also the type of packaging should be further considered (when available) in the exposure assessment to furan compounds. Coffee is one of the most consumed beverages in the world. Millions of tons of coffee beans are produced annually, making it an important economic source for producing countries . During 10) for neoplastic effects (hepatocellular adenomas and carcinomas) equal to 1.31 mg/(kgbw × day) was derived from a chronic 2-year mouse study : 6; p-value < 0.001). The association between consumption habits and consumer’s age groups was assessed using the correspondence analysis (CA) shown in the Concerning the age of participants, the results of Pearson chi-squared test of independence performed on 2: 6.97; df: 2; p-value: 0.03).According to the chi-squared test performed on 2: 5.39; df: 2; p-value: 0.07).When analysing the data on smoking habits, it can be seen that the percentage of daily coffee drinkers is slightly higher in the group of smokers (93.3%) than in the group of non-smokers (88.1%) . Howeverp-value < 0.05) (data not shown).Regarding the type of activity, the CA highlights that the probability of an occasional consumption habit is greater for ‘students’ than for ‘workers or retired’ .Finally, a significant dependence of coffee consumption with the participant’s institute is shown in n: 1672) is 4 cups per day. Compared with these values, the median of the global coffee consumption obtained by the BNFCS achieved in 2014 was 2 cups per day.The global median of coffee consumption of occasional and daily consumers is 3 cups per day, whereas the median of consumption of daily consumers only (The distribution of daily coffee consumers is presented in a Pareto chart . This fin: 1238). This model displays that type of activity (working/studying status) had no significant effect on coffee consumption with a p-value = 0.74. Therefore, a simplified model was created with only the significant variables (gender and age categories). This model highlights the absence of the interaction between these variables. The medians of consumption for age groups are shown in The multifactorial analysis described in the statistical tests The participants were asked whether they usually consumed a particular type of coffee ). The results reveal that 89% of participants who answered the question consumed a particular type of coffee at home, while this percentage decreased to 55% at work (data not shown).2: 21.75; df: 3; p-value < 0.001).The distribution of consumption of the different types of coffee as a function of the place of consumption was not homogenous and is displayed in Regarding the type of packaging, only the ground coffee was addressed in this study, as it is usually sold in different types of packaging such as bags, capsules, pads, and filters.2: 18.48; df: 4; p-value = 0.001).The distribution of ground consumption based on the choice of packaging is indicated in the n: 1749) consume particular brands at home, while 33% of those who answered the second question (n: 1745) consume particular brands at work.Participants were asked two questions about the consumption of a particular coffee brand at home and at work. According to the results, 63% of those who answered the first question  expressed their loyalty to a specific coffee brand but to different degrees. Indeed, 26% of the respondents were ‘somewhat’ loyal to brand, 20% ‘a little’, 24% ‘a lot’, and 8% ‘fully’ loyal (do not drink any other brand). The remaining 22% were ‘not loyal at all’.The results reveal that 78% of participants who answered the question . The CA performed to evaluate the association between the genders and loyalty shows that the probability of women being in ‘a little’ or ‘a lot’ loyalty categories is higher than in the other loyalty categories. In contrast, the probability of men being in ‘not at all’ or ‘somewhat’ loyalty categories is higher than that in other loyalty categories (data not shown).The contributions of men and women in each loyalty category were different and appear in the contingency table presented below  . The CA shows that the probability of those who consume coffee at home who are in the ‘a lot’ loyalty category is higher than that of those in the other loyalty categories, whereas the probability of those who consume coffee at work who are in ‘not at all’ or ‘a little’ loyalty categories is higher than that of those in the other loyalty categories (data not shown).The participants who consumed particular brands were asked to express their degree of loyalty to the brand consumed according to the place of consumption. The degree of loyalty of participants who answered the question is illustrated in the This section investigates in detail the reasons that may influence the decision for selecting a specific coffee brand. p-value > 0.05).As shown in this figure, the ‘taste’ is the main reason for choosing a brand with the highest median score ‘4’, followed by the ‘price’ with a median of ‘3’. Both, the ‘fair trade aspect’ and ‘tradition & habit’ were in third position with a median equal to ‘2’ and non-significant difference between them . The CA displays that the choices of younger participants (18–24 years old) were more related to ‘caffeine level’ than those of older ones, whereas the choices of older participants were more associated with ‘taste’, ‘packaging’, ‘organic’, and ‘fair trade’ aspects  display a significant association between the age and brand choice . The results of the CA suggest that both men and women select their brands based on ‘taste’, while men are more likely to purchase their brands based on the ‘local’ and ‘fair trade’ aspects than women who select the brands based on the ‘price’ and the ‘organic’ aspects (results not shown).The CA also displays a significant association between the participants’ gender and reasons for brand selection  for a more realistic risk assessment of furan.The global median daily coffee intake obtained in the Belgian sample is 3 cups per day, which is slightly higher to the one obtained by the BNFCS conducted in 2014. The difference could be partly explained by the population sampling specific to this work, where the questions were addressed mainly to workers and employees with a higher education level. In addition, the COVID-19 pandemic may also have influenced the coffee intake in 2020. Indeed, the Belgian purchase of coffee beans increased up to 15% per year during the pandemic ,26. The Although the ‘packaging’ factor did not score highly for brand choice , it coulThe most consumed type of coffee remains ground coffee, followed by coffee beans, instant coffee, and finally, liquid coffee and coffee substitutes. In terms of exposure to furan compounds, the type of packaging should be taken into account when assessing the risk, as the final concentration of these compounds in a cup of coffee depends on the condition of brewing, as mentioned above.In addition, the distribution of coffee consumers according to the type of coffee and packaging  could beIt can also be seen that Belgians choose a particular brand of coffee because they like its taste and the pleasure they get from drinking it . Price iIt is also interesting to note that younger participants select their brands based on the level of caffeine to benefit from its stimulating effect, while older participants choose mainly on the basis of ‘taste’, ‘organic’, and ‘fair trade’ aspects .The degree of loyalty of the studied Belgian sample to specific brands varied from ‘not at all’ to ‘fully loyal’. It can be concluded that the studied sample display slight characteristics of coffee loyalty, with 24% ‘a lot loyal’ and 8% ‘fully loyal’, whereas the remaining participants do not have clear preferences for brands. In term of exposure, the level of exposure to furan compounds in coffee is not directly related to the brand but rather to the conditions of roasting and brewing, as well as to the amount of furans precursors in green coffee, as discussed above. However, the degree of consumer attachment to a particular brand could also determine the systematic exposure to the contaminant, as the green coffee of a particular brand is often provided from the same origin and roasted under similar conditions. Therefore, both the type of coffee and the degree of loyalty (when it is available) should be taken into account in further exposure assessments.The results obtained in this study suggest that coffee consumption is influenced by several factors that depend mainly on the age of the consumer, gender, and place of consumption. Some of these factors such as age groups and gender might be easily introduced in a model to achieve an accurate evaluation of furan exposure. The global median of coffee intake of the Belgian participants in this work is 3 cups per day. This value should be considered when performing a realistic evaluation of the exposure of Belgian coffee consumers to furans in coffee. This intake is slightly higher than that obtained in a previous consumption survey, which might be due to the increased demand for coffee products associated with the current COVID-19 pandemic and crisis. The type of coffee product, the degree of loyalty, and the packaging should be recommended to be taken into account when assessing exposure to furan compounds. The distribution of coffee consumers according to coffee type and packaging could be used to allocate coffee samples in a sampling plan created to construct a furan contamination dataset. Taste, price, tradition, and habits are the main driving factors influencing the selection of a particular coffee brand.


Most formats of currently used community-based health education for cognitive impairment prevention are limited to one-way communication, such as distributing leaflets, pasting posters, or holding a lecture, and they lack comprehensive evaluation. Here we aim to design, test, and evaluate a novel pilot cognitive health education program combined with psychosocial interventions (CHECPI).We designed the CHECPI program and tested it among adults aged 60 and over in an aging-friendly community in 2018. Multidimensional cognitive functions were measured by the Montreal Cognitive Assessment (MoCA) before and three months after the CHECPI program. Quantitative and qualitative analyses were performed based on the RE-AIM  framework to evaluate the program. Wilcoxon signed-rank tests were used to assess changes in multidimensional cognitive functions.n = 22) and younger elderly with an average age of 65.32 years. Effectiveness: 19 participants finished≥6 courses as well as the follow-up survey. Although their MoCA scores did not improve significantly, they had increased their visuospatial ability significantly (with the average score increasing by 0.42). Adoption: the community officers, lecturers, and participants highly recommended the program, but they agreed that the lack of professional instructors may hinder its popularization. Implementation: the program was implemented in full accordance with the pre-program design. Maintenance: three months after the program, 17 participants had maintained at least one of the seven healthy behaviors that were introduced in the program.The CHECPI program was comprised of 12 courses and introduced 5 kinds of psychosocial interventions. Reach: 28 older adults participated in the program, of whom most were female (Younger female elderly were more willing to participate in the program. It enhanced participants’ visuospatial ability, but a sufficient number of professional instructors are crucial for large-scale promotion. China’s population is the largest in the world and is rapidly aging. This presents China with enormous challenges. By the end of 2016, there were 231 million people aged over 60 in China, accounting for 16.7% of the total population. The Chinese elderly population is expected to exceed 300 million in 2025 and reach a peak of 487 million in 2053, when it will account for one-quarter of the global elderly population [Cognitive dysfunction has become one of the most urgent public health problems for aging societies. Currently, among individuals aged over 60, the prevalence of mild cognitive impairment (MCI) is 12%-18% . MeanwhiThe decline of cognitive function can be slowed if the elderly are aware of cognitive impairment-related risk factors and consciously take preventive measures , 10. ForHowever, the format of community-based health education for cognitive impairment prevention, in terms of programs that have actually been implemented, is currently limited to health counseling, distribution of health handbooks, poster publicity, and lectures. Briant  implemenTo date, academic studies have mainly evaluated new cognitive interventions by examining their effectiveness. Although effectiveness is a crucial aspect of an intervention, its application is affected by many other factors, such as accessibility, applicability, and sustainability. There are many programs of proven efficacy that fail when implemented in real-world settings, resulting in wasted resources and unmet needs. A new health promotion or education program should be evaluated on the basis of a broad array of issues so that it can genuinely help decision-makers to make more informed judgments and effective use of scarce resources . The ReaIn this pilot study, we aimed to design, test, and evaluate a novel program of cognitive health education combined with psychosocial interventions (CHECPI). We designed the program by integrating content corresponding both to risk factors and to psychosocial interventions. We then tested it among older adults in an aging-friendly community in Xiamen, China. Afterward, we evaluated it based on the RE-AIM framework. The results of this study informed preliminary estimates of effectiveness and acceptability and feasibility of delivery in real-world settings.The modifiable influencing factors of cognitive decline can be summarized as comorbidities, lifestyles, and psychological factors , 26. TheBefore the CHECPI program was tested, we carried out a publicity program to recruit participants from an aging-friendly community in Xiamen, China. The recruitment notice was sent out through several channels such as a WeChat group, WeChat official account tweets, and posters at the community notice bar. People who were interested in the program participated in the publicity activities voluntarily at the community elderly activity center. The publicity activities included the introduction of the CHECPI program by posters as well as oral presentations, questionnaires, screening of cognitive function, and playing of simple cognitive games. It attracted 91 middle-aged and older adults, of whom 83 were aged over 60 years see . Since tCognitive function was measured by the Montreal Cognitive Assessment (MoCA), which is widely used as a credible preliminary screening tool for mild cognitive impairment . The MoCReach, Effectiveness, Adoption, Implementation, and Maintenance of this pilot program. Referring to original definitions, we interpret the Reach as the absolute number and basic demographic information of those who were attracted by the program publicity activity and persisted in participating in the program. The Effectiveness was measured by the changes in the MoCA scores of the 19 participants who attended at least six courses and finished the 3-month follow-up survey. The score changes were assessed by a Wilcoxon signed-rank test. The Adoption was evaluated by the opinions of relevant participants and personnel gathered by semi-structured qualitative interviews. We interpreted the Implementation by the degree of program completion. The Maintenance was measured by the retention rate of the seven kinds of healthy behaviors that had been introduced in the CHECPI courses.The RE-AIM framework was used to evaluate the The program consisted of 12 courses, which were conducted once a week with application of the five types of psychosocial interventions. Following the example of the existing literature, we included living habits, comorbidity, and mental health as intervention factors. n = 58) and married (n = 72). We then recruited participants on a first-come-first-served basis, resulting in a total of 28 older adults participating in at least one course , with an average attendance of 8.04 courses. As shown in n = 22).The program publicity activity attracted 91 middle-aged and older adults, of whom 83 were aged over 60 years. Among the 83 respondents, the oldest was 77 and the mean age was 65.61. Most of them were female , participants did increase their visuospatial ability . The other five cognitive subdomains did not show statistically significant changes (p>0.05).As shown in The interviews were semi-structured with questions concerning opinions on the best part of the program, obstacles, and advice for long-term implementation of the program. 2 community officers, 5 lecturers/teaching assistants, and 25 participants were interviewed after the CHECPI program. All interviews were conducted face-to-face and lasted 10–15 minutes. At the organizational level, the community officer expressed a high willingness to adopt the program, but the fact that implementation of the program was restricted by the lack of professionals was their main concern. At the individual level, it was difficult for the lecturers to carry out the program over an extended time because of busy work schedules or traffic issues. The older adults believed that the program could improve their health, but occasionally there were time conflicts that forced them to be absent from the courses. Some specific interview contents and feedback are shown in The implementation process of this pilot program is shown in Yes, No, and Unknown). The behaviors included doing finger exercises, ingesting foods that are good for the brain, practicing abdominal breathing, doing Baduanjin exercise , communicating with others frequently, being willing to meet new friends, and participating in group activities. Among the 19 participants, 17 had maintained at least one type of the behaviors described above, and 6 had maintained more than two types. However, some of the participants reported that they had almost forgotten the teaching content and needed a review.In the follow-up survey that was performed three months after the CHECPI program, we asked the participants about their retention of the seven kinds of healthy behaviors introduced in the CHECPI program. This was assessed by the question, “Have you developed the following behaviors according to the content of the CHECPI courses?” Three options were provided  health education interventions help participants to acquire and maintain knowledge, and guide them to put this knowledge into practice. Therefore, the effects would proceed in stages: an increase in knowledge of what to do, behavioral changes, and translation of the behavioral changes into actual performance changes . As a rep<0.05 , 40. HowIn recent years, there have been many programs aimed at improving cognitive function that have mainly focused on cognitive training, aerobic exercise, and health education at home and abroad. Most studies have shown that cognitive training and aerobic exercise can improve cognitive function , 42. HowThe program was carried out strictly according to plan, but there were some emergency situations such as a sudden change of course time due to bad weather. Some participants were hard to reach by WeChat, resulting in absences from those course sessions. In the future, we can establish a group leader to make emergency notifications. Community officers gave a high appraisal of the program and were willing to extend it to other communities. However, it was recognized that the lack of professional health educators may limit the success of the program.We designed the CHECPI program, a combination of psychosocial interventions and cognitive health education, and evaluated it based on the RE-AIM framework. Some limitations should be acknowledged. First, we conducted the pilot program in an aging-friendly community that had available support from the officers and a well-equipped classroom. Whether the program is suitable for communities with inadequate cooperation remains to be studied. Second, due to the absence of a control group, we could not evaluate the effect of the psychosocial interventions. Different psychosocial interventions might be of benefit to the elderly to different extents, which can be studied in the future for the improvement of the program. Third, due to the lack of a long-term follow-up survey, we cannot evaluate the maintenance of the CHECPI program. Fourth, the sample size was small in this pilot study, and thus the results provided an preliminary estimate of effectiveness that should be tested in a larger trial.S1 Dataset(XLSX)Click here for additional data file.


As the need for effective scalable interventions for mental health conditions such as depression, anxiety, and stress has grown, the digital delivery of mindfulness-based stress reduction (MBSR) has gained interest as a promising intervention in this domain.This study aims to evaluate the changes in perceived stress following a 10-week digital MBSR program that combined an app-based digital program with weekly one-on-one remote sessions with a health coach.This study used a retrospective, observational design. A total of 229 participants with moderate-to-high perceived stress scores as assessed by the Perceived Stress Scale (PSS)-10 enrolled in the 10-week Vida Health MBSR program. The program included weekly remote sessions with a certified health coach and digital content based on concepts fundamental to mindfulness practice. The PSS-10 was used to evaluate perceived stress. Of the 229 participants, 131 (57.2%) were considered program completers and provided at least one follow-up PSS-10. A secondary analysis examined the changes in stress scores at 6 months. This analysis was restricted to participants who had been enrolled in the program for at least 6 months (n=121). To account for random and fixed effects, linear mixed effects modeling was used to assess changes in stress scores over time. An intention-to-treat approach was used to evaluate the changes in perceived stress across the entire study cohort, including those who were lost to follow-up. In addition, a reliable change index was computed to evaluate the changes in scores from the baseline.P<.001) at 12 weeks. We observed an average reduction in stress scores of 3.17 points (95% CI −3.93 to −2.44) by program week 6 and 4.86 points (95% CI −5.86 to −3.85) by program week 12. Overall, 83.2% (109/131) of participants showed a reduction in stress scores by week 12, with 40.5% (53/131) of participants showing reliable improvement at 12 weeks and 47.8% (56/131) of participants showing a shift to a lower stress level category . The intention-to-treat analysis revealed a significant, although attenuated, reduction in stress scores at 12 weeks . Participants who completed more lessons had an increased likelihood of moving down at least one stress level category . In assessing medium-term outcomes, among participants who had completed at least 6 months in the program, 48.8% (59/121) of members provided a 6-month assessment. We observed a significant reduction in stress scores at 6 months , with 61% (36/59) of participants showing reliable improvement.The findings revealed a significant positive association between program time and stress reduction (B=−0.365; The findings of this retrospective, observational study suggest that a blended, digital mindfulness-based intervention may support program uptake and meaningful, sustained reduction in stress outcomes. It is without question that the demand for mental health services has substantially increased in recent times, given the health, social, and economic ramifications of the COVID-19 pandemic . EstimatBarriers to seek care that are unique to mental health have been well documented in the literature. Digital mental health interventions (DMHIs) delivered via apps, web-based platforms, or text messaging have shown promise in addressing these concerns. They appear to reduce barriers to access conventional forms of mental health services, including cost, mental health stigma, and accessibility -10. In aMore recently, there has been a growing interest in the clinical utility of mindfulness-based interventions (MBIs) in improving mental health, stress management, and well-being. Mindfulness meditation is the act of purposefully paying attention to the present moment and being aware of mental states and processes with a sense of openheartedness, curiosity, and kindness in a nonjudgmental manner ,15. It hResearch on MBIs and mental health outcomes has spanned two decades, and the general consensus has been that MBIs have a significant effect on improving stress and anxiety . HoweverIn their meta-analytic review of web-based MBIs, Spijkerman et al  observedHealth coaching has recently emerged as a promising behavioral intervention for improving health outcomes and adherence to mobile app platforms ,27. DefiIn summary, although there exists an established body of research evaluating the effectiveness of digital MBIs, there has been less focus on the emerging trend of exclusively remote individualized health coaching combined with digital mindfulness tools. Furthermore, there is considerable variability in the structure of digital mindfulness interventions . In this study, we evaluated the Vida Health MBSR digital intervention for moderate-to-high perceived stress. Vida Health is a commercially available app that offers tailored digital health programs paired with one-on-one coaching with health education specialists or licensed therapists. The Vida Health app is available in all 50 states with program offerings, including MBSR, cognitive behavioral therapy for depression and anxiety, and chronic disease management.The purpose of this study is to evaluate the effect of a mindfulness-based DMHI program delivered via a smartphone on perceived stress. The program pairs individualized remote health coaching, with tailored lessons and tools to introduce and facilitate the practice of mindfulness. Our primary hypothesis was that participants enrolled in the Vida Health MBSR program would show improvements in perceived stress scores at the end of the program. In addition, we predicted that program engagement, as measured by the extent of coach interaction or program content completed, would be positively associated with reductions in perceived stress scores.This study used an observational, retrospective design to evaluate changes in perceived stress among participants who completed Vida Health app-based and coach-supported MBSR program. Individuals from across the United States were invited to use the Vida platform, paid for by their employers. Participants joined the Vida MBSR program between March 2018 and May 2020. The institutional review board (Western IRB) waived the requirement for informed consent because the study was determined to have minimal risk, and the data were fully anonymized before analysis.Stress was assessed using the clinically validated 10-item Perceived Stress Scale (PSS-10) . The PSSThe delivery of the PSS assessment occurred via the Vida app . Participants were recruited between March 2018 and May 2020 from employers that offered the Vida Health MBSR program to employees and spouses as part of their health plan. They were recruited through a combination of email announcements, paper flyers, and onsite events at their employer and were directed to download the Vida Health app from the App Store (iOS version) or Google Play Store (Android version). On downloading the app, participants enrolled in the program by completing a brief set of registration questions that included name, contact information, basic demographics , existing health conditions, and main health goals  accredited program. Coaches performed a video call–based intake in which they determined whether the client was still a good fit for the program. Participants excluded from the study were referred to Vida Health Care Navigators, licensed mental health professionals, who then performed a psychosocial assessment, based on which the participants were referred either internally to the Vida Health Therapy program or external local resources. Conditions that led to exclusion criteria included self-reported moderate-to-severe depressive or anxiety symptoms, reports of suicidality, homicidality or presenting psychosis, active posttraumatic stress disorder, addiction to substances or alcohol, or significant health problems exacerbated by breathing exercises or yoga. Participants scoring in the high perceived stress range (PSS-10 >27) were also offered the option of completing an assessment with a licensed mental health professional to discuss treatment options .Following enrollment, participants received the Vida Health MBSR program, a 10-week digital therapeutic intervention for moderate perceived stress. The intervention was delivered via a mobile app. The evaluated program had three core components: (1) remote personal health coaching with licensed providers, (2) educational lessons and meditation or mindfulness practices, and (3) progress tracking.At the start of the MBSR program, participants were asked to select a health coach with whom they would have 30-minute weekly video call sessions throughout the course of the 10-week intervention. During the video call sessions, the coach and participant routinely discussed progress, set goals, and reviewed strategies for applying learned concepts and skills to the participants’ daily lives. In addition to weekly video calls, participants had unlimited app-based messaging support from their coaches. Digital content, which was based on MBSR techniques and could be audio-, video-, or text-based, was automatically sent to participants’ mobile apps every few days. The materials reviewed core concepts of MBSR, including the mind-body connection, meditations, body and breath awareness, and yoga and mindfulness techniques. Health coaches could also send additional content depending on the participants’ needs. Participants were asked to engage with the lessons and practices as they came, but they had access to all of the content throughout the program. Finally, participants were asked to track their stress levels every 2 weeks via the PSS-10 survey. Examples of how the participants experienced these core program components are shown in The core intervention involved lessons and tools built on concepts fundamental to integrating daily mindfulness-based approaches. These lessons and mindful practices focused on building mindful awareness, establishing healthy relationships with existing stress or stressors, and integrating skills to increase a participant’s autonomy of control over managing and responding to stressors. The lessons and practices were designed for consumption in short intervals, and participants had the option of completing the same lesson or tool multiple times. Were there lessons that were not part of the core?In addition to the core components of the program, participants received access to various app features to support them in reducing their stress. This included graphs detailing the participant’s progress over time and a home screen that used machine learning models to recommend helpful actions for the participant to take or useful information for the participant to know. These models could prompt the participant to read a specific piece of content, send a message to their coach, log their stress, or view their progress in relation to other members using the Vida app. Participants could also generate habits, or actions to take in real life, that the app reminded them to complete. Participants were able to access and engage with all the functionality of the Vida app during their leisure time.t test. As participants were drawn from multiple employers, a Kruskal-Wallis test was performed to assess any potential differences between organizations.All data preparation and analyses were performed using Python version 3.7.9 (Python Software Foundation) and STATA version 16.1 (StataCorp LLC). Baseline was defined as the PSS-10 score completed on the program intake. The week of follow-up assessment completion was computed as the difference in weeks between the survey completion date and the program start date. Baseline differences in stress scores between program completers and noncompleters were assessed using a two-tailed The assessment schedule allowed the participants to complete the PSS-10 survey at any time in a 2-week period. In other words, a participant could complete the survey on the day of receipt or 10 days thereafter. Owing to this inconsistent cadence of completion, only a subset of participants completed the survey in any given week. We used curve fitting, a previously described data imputation technique , which cProgram engagement was operationalized as two variables: the number of coach consultations and the number of core lessons completed during the intervention. The program engagement factors were each scaled. As described in an earlier paper , to adjuTo determine the effect of program time and engagement on changes in perceived stress scores, we used a linear mixed effects model (MLM). Linear MLMs address potential heterogeneity in the data because of possible differences across organizations and provider effectiveness. In this analysis, employer organization and health coaches were entered into the model as random effects. Change in PSS-10 scores from the baseline was entered into the model as the outcome variable. Fixed factors included baseline PSS-10 scores, program time in weeks, gender, number of lessons completed, and number of coach consultations. The reliable change index was computed to estimate the proportion of participants who experienced a reliable improvement in stress outcomes at the end of the intervention and at 3 months post intervention . All anaA total of 229 participants with a baseline PSS score ≥14 (moderate stress or higher) were enrolled in the Vida MBSR program between March 2018 and May 2020. Study enrollment was restricted to participants who completed at least one in-app program lesson or had at least one coach interaction (video consultation or text message to coach) during the study period. The PSS-10 score reported at program intake was used as the baseline PSS-10 score. A schematic of the participant flow is shown in t test that showed a nonsignificant difference in baseline PSS-10 scores between groups (P=.10). However, a two-tailed chi-square test suggested that more program noncompleters scored in the higher perceived stress range (PSS-10 ≥27) than participants in the treatment cohort . This is expected, as participants scoring in the higher severity range were typically referred to external services or Vida Health Therapy rather than remaining in the MBSR program. There were no significant gender (P=.20) or age-based baseline differences between groups (P=.45). As noted earlier, participants were drawn from employer-based organizations. The results of the Kruskal-Wallis test showed no significant differences in baseline PSS-10 scores between organizations (P=.38).To address potential systematic baseline differences between program noncompleters and the treatment cohort, we performed a two-tailed 129=1.93; P=.06). All participants engaged with their coach either via text messages or remote consultations. Of 131 participants, 96 (73.3%) participants had completed at least one consultation with their coach during the intervention period and 130 (99.2%) participants had messaged their coach during the program intervention. A summary of program engagement is presented in The treatment cohort comprised 131 participants. Baseline characteristics are presented in P<.001) such that increased program time was associated with greater perceived stress reduction (P<.001). Higher baseline scores were associated with greater reductions in PSS-10 scores by program week 12. We observed a nonsignificant trend, suggesting that women showed a greater reduction in perceived stress than men .Overall, 83.2% (109/131) of participants experienced a reduction in PSS-10 scores from baseline by program week 12. Of 131 participants, 56 (47.8%) moved down at least one perceived stress level  and 53 (40.5%) had a reliable improvement in perceived stress scores. There was a significant effect of program week on reduction in PSS-10 scores relative to baseline . However, there was a significant effect between core lesson completion and reduction in stress scores by program week 12 . Participants who completed a higher number of core lessons had an increased likelihood of achieving at least one-level reduction in perceived stress scores .The number of coach consultations had no significant effect on the perceived stress scores . By 6 months, participants showed an average reduction of 6.77 points (95% CI 5.45 to 8.09) in perceived stress scores relative to baseline. A reduction of at least one stress level was observed in 57% (34/59) of participants, and 61% (36/59) of the participants showed reliable improvement in stress outcomes relative to baseline. A Hedges g calculation suggested a large effect size .In addition, we examined the three-month postintervention outcomes. A total of 92.4% (121/131) of participants had completed at least 6 months from the start of program enrollment. Of this cohort, 48.8% (59/121) provided a follow-up PSS-10 assessment at month 6. A paired The Vida Health digital MBSR program is an app-based mental health intervention that combines one-on-one weekly remote video sessions with a coach along with tailored digital content based on core concepts of mindfulness practice. The goal of this study is to assess changes in stress outcomes following this MBSR intervention among participants with moderate-to-high perceived stress scores at baseline. The results showed a significant and reliable reduction in perceived stress scores within 12 weeks, which seemed to be maintained at month 6. Higher program engagement, as measured by the completion of core lesson content, was associated with an increased likelihood of a shift to a lower stress-level category . Although our findings suggest that the Vida Health digital MBSR intervention is associated with improvements in perceived stress, the study design  limits our ability to draw causal inferences from these results.P<.001), suggesting that perhaps the coaches serve as a program anchor. In summary, this study observed a significant reduction in perceived stress scores following a 10-week digital MBSR intervention. In addition, the study provides preliminary insight into the role of program content engagement as a possible moderator of this effect. Together, they lend support to the utility and possible efficacy of digital DMHIs that incorporate sound-validated mental health interventions, adding MBSR to the arsenal of options.The results of this study are consistent with previous research ,13,21 thThis study used a retrospective observational design that lacked a comparison group. The possibility of self-selection bias and the lack of randomization limit the generalizability of our findings and the ability to draw causal inferences regarding the effect of the digital MBSR intervention on stress reduction. As noted earlier, 42.7% (98/229) of the enrolled participants failed to provide an assessment after their initial intake. Although we did not observe any significant systematic demographic differences across program nonstarters and those who remained in the program, it is possible that the groups differed on factors not assessed in this study, such as the presence of other comorbid mental health conditions. We did note that more program nonstarters scored in the high stress range than participants who remained in the program. Although participants with high stress scores were eligible to enroll in this study, participants scoring in the high perceived stress range (PSS-10 >27) were offered the services of a Vida Health Care Navigator to assess the suitability of the MBSR program in addressing their mental health concerns. Indeed, as shown in Perceived stress was the key outcome metric assessed in this study. However, MBSRs have been associated with improvements in additional measures of mental health, such as depression, anxiety, mood, and well-being ,22,24. ADMHIs through the integration of mindful awareness lessons, practices, and health coaching can be effective in improving mental health care accessibility, cost-effectiveness, and increasing support services to a larger demographic. Future research should involve equivalence trials comparing DMHIs and in-person behavioral health interventions on MBIs for stress management, further examining the importance of the role of health coaches in DMHIs.The recent growth and accessibility of smartphones has facilitated the continual development and deployment of mobile-based apps, making it practical for individuals to access the DMHI. Mobile phones facilitate the ability for interventions to enter into the daily lives of individuals, allowing unobtrusive monitoring of activities and contexts, and promote the possibility for interventions at opportune moments, that is, when most needed or desired . Mobile


Membrane protein recycling systems are essential for maintenance of the endosome-lysosome system. In yeast, retromer and Snx4 coat complexes are recruited to the endosomal surface, where they recognize cargos. They sort cargo and deform the membrane into recycling tubules that bud from the endosome and target to the Golgi. Here, we reveal that the SNX-BAR protein, Mvp1, mediates an endosomal recycling pathway that is mechanistically distinct from the retromer and Snx4 pathways. Mvp1 deforms the endosomal membrane and sorts cargos containing a specific sorting motif into a membrane tubule. Subsequently, Mvp1 recruits the dynamin-like GTPase Vps1 to catalyze membrane scission and release of the recycling tubule. Similarly, SNX8, the human homolog of Mvp1, which has been also implicated in Alzheimer’s disease, mediates formation of an endosomal recycling tubule. Thus, we present evidence for a novel endosomal retrieval pathway that is conserved from yeast to humans. VPS35 has been associated with Alzheimer’s disease (AD) and Parkinson’s disease  in the endo-lysosome system are essential for the normal assembly and function of the lysosome. The best-characterized recycling cargo is yeast Vps10. Vps10, the first member of the sortilin receptor family, is a transmembrane (TM) protein receptor that sorts carboxypeptidase Y (CPY) into vesicles at the Golgi . After C disease . Retrome disease . Thus, r disease .Sorting nexin (SNX) is an evolutionarily conserved protein family comprising a Phox homology (PX) domain that binds to phosphatidylinositol 3-phosphate (PI3P), allowing its selective interaction with endosome . Some SNVPS1 mutations , it was still localized on the endosome   . Strikin1Δ cells . Vps55-G1Δ cells . These rpep12Δ cells, Mvp1-GFP lost its endosomal localization and was distributed to the cytoplasm  using the LAP  linker  linker , becausefunction . Cells lfunction . Consistfunction , Vps1-GFfunction , C. We afunction . Severalfunction .K42Avps1 mutants. In this mutant, Vps55-mNeonGreen localized to the vacuole membrane  hydrolysis induces conformational changes in the dynamin ring, leading to membrane fission . Subsequmembrane , suggesteonGreen . These rmvp1Δ cells, but Vps1-GFP is still localized on endosomes , Kex2 (serine protease on the Golgi), and Neo1 (P4 type ATPase on the endosome)  . Vps10-Gtssec18 mutants to a non-permissive temperature (37°C), Vps55-GFP lost its endosomal localization and was distributed in the cytoplasm  is cleaved by β-secretase (BACE) at the endosome, and then its fragments (Aβ) are unconventionally secreted . Interesvps35Δ snx4Δ mvp1Δ triple mutants, the retromer and Snx4 complexes may also recruit Vps1 to catalyze the fission of recycling vesicles. Future analysis will be necessary to characterize how Vps1 acts on retromer- and Snx4-mediated recycling tubules. In mammalian cells, dynamin mainly localizes not only on the PM, but also to the endosome  allows cells to maintain organelle identity and function. In this study, we reveal that Mvp1 mediates a novel endosomal recycling pathway. Our findings pave the way toward understanding how cells maintain the composition and function of each organelle. However, several questions remain to be answered. (1) To form a uniform size of recycling vesicles, the activity of Mvp1 and Vps1 should be temporally and spatially regulated, but its regulation mechanism(s) remains unknown. (2) How Mvp1 is uncoated from the recycling vesicles also requires investigation. (3) How is Vps55 recycled, before endosome fusion with the vacuole, is still unclear. (4) Other membrane proteins and lipids delivered through the Mvp1 pathway need to be identified. (5) How do SNX-BAR family proteins contribute to the appropriate distribution of phospholipids? Future studies should address these critical issues.Saccharomyces cerevisiae strains used in this study are listed in Hela cells were kindly provided by Dr. Anthony Bretscher (Cornell University). Cell lines were verified to be free of mycoplasma contamination and the identities were authenticated by short tandem repeat (STR) profiling.2 in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin.Hela cells were maintained at 37°C and 5% COPlasmids used in this study are listed in Fluorescence microscopy was performed using a CSU-X spinning-disk confocal microscopy system (Intelligent Imaging Innovations) or a DeltaVision Elite system .A CSU-X spinning-disk confocal microscopy system is equipped with a DMI 6000B microscope (Leica), a ×100/1.45 numerical aperture (NA) objective, and a QuantEM electron-multiplying charge-coupled device (CCD) camera (Photometrics). Imaging for yeast cells was done at room temperature (RT ) in YNB medium using GFP and mCherry channels with different exposure times according to each protein’s fluorescence intensity. Imaging for mammalian cells was done at 37°C in FluoroBride DMEM media (Thermo Fisher) using GFP, mCherry, and far-red-fluorescent dye channels. Images were analyzed and processed with SlideBook 6.0 software (Intelligent Imaging Innovations).A DeltaVision Elite system is equipped with an Olympus IX-71 inverted microscope, a DV Elite complementary metal-oxide semiconductor camera, a ×100/1.4 NA oil objective, and a DV Light SSI 7 Color illumination system with Live Cell Speed Option with DV Elite filter sets. Imaging was done at RT in YNB medium using GFP and mCherry channels with different exposure times according to each protein’s fluorescence intensity. Image acquisition and deconvolution  were performed using DeltaVision software softWoRx 6.5.2 (Applied Precision).Anti-FLAG-conjugated magnetic beads were prepared according to the manufacturer’s protocol. In brief, N-hydroxysuccinimide ester (NHS) ferrite-glycidyl methacrylate (FG) beads (Tamagawa Seiki) were treated with methanol and then incubated with anti-DYKDDDK antibody (Wako) at 4°C for 30 min. The magnetic beads were mixed with 1.0 M 2-aminoethanol, pH 8.0, at 4°C for 16–20 hr to quench the conjugation reaction, washed three times with the bead wash buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-NaOH [pH 7.2], 50 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), and 10% glycerol), and stored in wash buffer containing 1 mg/ml bovine serum albumin (BSA) .g for 5 min at 4°C, and the resultant supernatants were subjected to high-speed centrifugation at 17,400xg for 10 min. The cleared supernatants were incubated with pre-equilibrated anti-FLAG-conjugated magnetic beads and rotated at 4°C for 1 hr. After the beads were washed with wash buffer containing 0.1% Triton X-100, the bound proteins were eluted by incubating the beads in sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer at 98°C for 5 min.To examine the Mvp1-Mvp1 interaction, cells expressing Mvp1-FLAG and Mvp1-GFP were grown to mid-log phase and washed twice with wash buffer . The cells were lysed in IP buffer , and 1 x protease inhibitor cocktail [Roche]) and lysed by beating with 0.5 mm YZB zirconia beads (Yasui Kikai) for 1 min. IP buffer containing 0.2% Triton X-100 was added to the lysate , and the samples were rotated at 4°C for 10 min. The solubilized lysates were cleared at 500xThe Mvp1-Vps55 interaction was examined similarly as the Mvp1-Mvp1 interaction with some modifications. Cells expressing Vps55-FLAG and Mvp1-GFP were washed twice with phosphate-buffered saline (PBS) wash buffer . Cells were lysed in PBS IP buffer  as above. The lysate was solubilized with 1.0% saponin at 4°C for 60 min. After centrifugation, the cleared supernatant was incubated with pre-equilibrated anti-FLAG-conjugated magnetic beads and rotated at 4°C for 4 hr. After the beads were washed with wash buffer containing 1.0% saponin, the bound proteins were eluted by incubating the beads in elution buffer  at 4°C for 30 min. Eluted samples were mixed with 2x SDS-PAGE sample buffer, then incubated at 42°C for 5 min.The Mvp1-Vps1 interaction was examined similarly as the Mvp1-Mvp1 interaction with some modifications. Cells expressing Mvp1-FLAG and Vps1-GFP were washed twice with high-salt wash buffer . Cells were lysed in high-salt IP buffer  as above. The lysate was solubilized with 1.0% Triton-X 100 at 4°C for 60 min. After centrifugation, the cleared supernatant was incubated with pre-equilibrated anti-FLAG-conjugated magnetic beads and rotated at 4°C for 1 hr. After the beads were washed with wash buffer containing 1.0% Triton X-100, the bound proteins were eluted by incubating the beads in SDS-PAGE sample buffer at 98°C for 5 min.To examine the Mvp1-retromer interaction, cells expressing Vps5-FLAG, Vps17-HA, Vps26-Myc, and Mvp1-GFP or cells expressing Mvp1-FLAG and Vps17-HA were washed twice with PBS IP buffer. Cells were lysed in PBS IP buffer as above. The lysate was solubilized with 0.5% Triton-X 100 for 4°C for 10 min. After centrifugation, the cleared supernatant was incubated with pre-equilibrated anti-FLAG-conjugated magnetic beads and rotated at 4°C for 2 hr. After the beads were washed with wash buffer containing 0.5% Triton X-100, the bound proteins were eluted by incubating the beads in SDS-PAGE sample buffer at 98°C for 5 min.g for 5 min at 4°C, and the resultant supernatants were subjected to high-speed centrifugation at 17,400xg for 10 min. The cleared supernatants were incubated with pre-equilibrated GFP-TRAP_A beads (Chromo Tek) and rotated at 4°C for 1 hr. After the beads were washed with SDS wash buffer , the bound proteins were eluted by incubating the beads in SDS-PAGE sample buffer at 98°C for 5 min.To analyze the ubiquitination status of Vps55, cells expressing Vps55-GFP were washed twice with 400 mM N-ethylmaleimide (NEM). Cells were lysed in urea cracking buffer  and lysed by beating with 0.5 mm YZB zirconia beads (Yasui Kikai) for 1 min. High-salt IP buffer with 20 mM NEM and 0.2% Triton X-100 was added to the lysate, and the samples were rotated at 4°C for 10 min. The solubilized lysates were cleared at 500x25S75E5 buffer . The cells were lysed in H25S75E5 buffer supplemented with 1 x protease inhibitor cocktail (Roche) and lysed by beating with 0.5 mm YZB zirconia beads (Yasui Kikai) for 1 min. The lysates were cleared at 500xg for 5 min at 4°C twice, and the cleared supernatants were incubated with anti-FLAG-conjugated magnetic beads and rotated at 4°C for 1 hr. After the beads were washed with H25S75E5 buffer, the immunoisolated structures were eluted by incubating the beads in SDS-PAGE sample buffer at 98°C for 5 min. For the EM analysis, immunoisolated structures were not eluted, but subjected to negative-staining EM analysis.To immunoisolate Vps55-FLAG- or Vps10-FLAG-containing structures, cells expressing Vps55-FLAG or cells expressing Vps10-FLAG and Vps55-GFP grown at 26°C were incubated at 37°C for 60 min before harvest. Cells were washed twice with HImmunoisolated Vps55-FLAG-containing structures on anti-FLAG-conjugated magnetic beads were applied to the carbon-coated electron microscope grid, stained with 2% ammonium molybdate, and imaged on FEI Morgagni 268 TEM.Vps55-GFP/mNeonGreen, Vps10-GFP, Kex2-GFP, and GFP-Neo1 localization was classified into two categories: punctate structures and vacuole membrane localization. Cells having both punctate structures and vacuole membrane localization were classified in the vacuole membrane localization category. For each experiment, at least 30 cells were classified, and the data from three independent experiments were used for the statistical analysis.Mvp1-GFP and Vps1-GFP localization was classified into two categories: punctate structures and cytoplasmic localization. For each experiment, at least 30 cells were classified, and the data from three independent experiments were used for the statistical analysis.g for 10 min at 4°C, the cells were washed once with 100% acetone and then were lysed in SDS-PAGE sample buffer  SDS, 10% glycerol, 100 mM dithiothreitol (DTT), and bromophenol blue) by beating with 0.5 mm YZB zirconia beads (Yasui Kikai) for 2 min. The lysates were then heated at 98°C for 5 min. After centrifugation at 10,000xg for 1 min at RT, supernatants were analyzed by SDS-PAGE and immunoblotting using anti-GFP and anti-G6PDH antibodies.Cell lysates were prepared as follows. Cells were grown to mid-log phase at 26°C. Aliquots of cells were mixed with trichloroacetic acid at a final concentration of 15%, and the mixtures were incubated for 30 min at 4°C. After centrifugation at 17,400xS. cerevisiae genomic library used for the suppressor screen was prepared as described previously , pelleted, decanted, then resuspended in 5 ml fix buffer , and incubated at RT for 1 hr with gentle agitation. After fixing, the cells were washed twice with 5 ml 0.1 M sodium cacodylate (pH 7.4), then rinsed with 5 ml pre-spheroplast buffer . To prepare for spheroplasting, the cells were re-suspended in 5 ml pre-spheroplast buffer and incubated for 10 min at RT. The cells were then rinsed with 5 ml spheroplast buffer . The cells were then re-suspended in 1 ml spheroplast buffer containing 0.25 mg/ml zymolyase and incubated at RT for 30 min. After spheroplasting, the cells were gently washed twice with 1 ml staining buffer  to remove sorbitol. The cells were embedded in 50 µl of 2% ultra-low-melt agarose and then cut into ∼2 mm3 blocks. The blocks were postfixed/stained in 1 ml osmium staining solution  for 1 hr at RT. The blocks were washed four times with water, then stained with 1 ml 1% uranyl acetate overnight. The blocks were washed four times with water, then dehydrated through a graded series of ethanol: 50%, 75%, 95%, and 2 × 100% for 10 min each (1 ml). The blocks were transitioned to 1:1 propylene oxide:ethanol for 10 min, then for 2 × 5 min in 100% propylene oxide (1 ml). The blocks were embedded in 1:1 propylene oxide:epon resin  and left on a rotator overnight to allow the propylene oxide to evaporate. The blocks were transferred to fresh epon resin and polymerized for 24 hr at 60°C.Cells were grown in YNB medium overnight and 40 ODs of cells were harvested at mid-log growth phase the next morning. To fix, the harvested cells were rinsed in 10 ml sodium cacodylate buffer  by the addition of 250 mM isopropyl β- d-1-thiogalactopyranoside (IPTG) for 16 hr at 16°C and purified using TALON Metal Affinity Resin (Clontech) according to the manufacturer’s protocol. The recombinant proteins were eluted with SUMO protease (Milipore) for Vps1-GFP and Mvp1.His-SUMO-Vps1-GFP and His-SUMO-Mvp1 were expressed in Purified proteins were incubated with anti-GFP magnetic beads at 4°C for 30 min. After the beads were washed three times with PBS with 0.1% TX-100, the bound proteins were eluted by incubating the beads in SDS-PAGE sample buffer at 98°C for 5 min.Transient transfections were carried out using FuGENE (Promega) according to the manufacturer’s instructions, and the experiments were performed 48 hr after transfection.Hela cells seeded on coverslips were fixed in 3.7% formaldehyde/PBS for 10 min at RT, washed with PBS, and permeabilized with 0.2% Triton-X-100/PBS for 5 min, washed, and blocked with 2% FBS/PBS for 10 min at RT. Cells were stained for 1 hr at RT with anti-EEA1 antibody in 2% FBS/PBS. After washing in PBS, cells were stained for 1 hr at RT with Alexa Fluor 647 anti-rabbit antibody in 2% FBS/PBS. Cells were washed with PBS and mounted using Prolong Gold Hardmount and imaged by a fluorescent microscope. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Acceptance summary:This paper will be of broad interest to the vesicle trafficking field, as it implicates an evolutionarily conserved SNX-BAR protein in cargo recognition, carrier formation, and recruitment of a dynamin-like scission factor. An extensive set of experiments reproduces and builds on past work and comes to some new conclusions.Decision letter after peer review:eLife. Your article has been favorably reviewed by 3 peer reviewers, and the evaluation has been overseen by Suzanne Pfeffer as the Senior and Reviewing Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Christian Ungermann (Reviewer #1); Todd R Graham (Reviewer #3).Thank you for submitting your article "A PX-BAR protein Mvp1/SNX8 and a dynamin-like GTPase Vps1 drive endosomal recycling" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. We hope you will consider all the points raised with focus on strengthening the data related to Mvp1 recognition of Vps55 and Vps1 as described below. We hope you will find these comments constructive.Essential revisions:1. Show that Mvp1 binds Vps55 cytosolic sequences in vitro, and that this is blocked by the mutation that disrupts sorting, would provide stronger evidence that Mvp1 recognizes and sorts Vps55. Furthermore, transplanting this signal to a heterologous protein would test if this sequence is sufficient for Mvp1-dependent sorting; inclusion of a control cargo that does not bind would also be helpful.2. Further defining how Mvp1 binds Vps1 would increase the novelty of the study. It is interesting that mutating the Mvp1 dimerization interface didn't block localization to endosomes, but did block sorting. This could mean that Mvp1 oligomerization creates the tubule that recycles Vps55. Alternatively, as suggested by Chi et al., Mvp1 oligomerization could further constrict an existing tubule to promotes the recruitment of Vps1. Can this dimerization mutant recruit Vps1 to endosomes?3. The authors show that overexpression of Vps55 causes its mislocalization to the vacuole in Figure 3G-J. Does overexpression of Mvp1 or the Vps55-interacting protein Vps68 suppress the overexpressed Vps55 missorting to the vacuole? This would address whether or not one of these proteins is the limiting component for sorting Vps55.4. Please revise the text throughout to provide a more complete description of the relevant prior literature as described below.Reviewer #1 (Recommendations for the authors):1. Figure 3,4: The authors nicely dissect the role of Vps1 for the Mvp1 pathway, and Vps1 K42 clearly affects recycling via Mvp1. Is Vps1 required for all three recycling pathways? The authors nicely show that Vps55 is affected in the Vps1 K42A mutant, but should also test the other cargoes (like Atg27).2. In Figure 6, the authors analyze the triple recycling mutant and its deficiency. Is the same Mup1 trafficking defect and duramycin sensitivity observed in vps1 mutants alone or in combination with snx4 or vps35 deletions? This again would address if Vps1 is required for all three pathways or just for the Mvp1 pathway.3. Figure 6: The authors show that the triple mutant of mvp1 snx4 and retromer has a strong sensitivity to heat stress . So far, only Vps55 has been characterized as a cargo, whose function remains unclear. Having a recycling deficient mutant of Vps55, the authors could ask if the Vps55 mutant  combined with snx4 and retromer deletion results in the same phenotype.4. The authors have evidence that Nhx1 is recognized by all three pathways. It would be nice if the authors could speculate on the sorting motif responsible for general vs. specific (e.g. Mvp1 dependent) recycling.Reviewer #2 (Recommendations for the authors):Demonstrating that Mvp1 binds Vps55 cytosolic sequences in vitro, and that this is blocked by the mutation that disrupts sorting, would provide stronger evidence that Mvp1 recognizes and sorts Vps55. Furthermore, transplanting this signal to a heterologous protein would test if this sequence is sufficient for Mvp1-dependent sorting.Further defining how Mvp1 binds Vps1 would increase the novelty of the study. It is interesting that mutating the Mvp1 dimerization interface didn't block localization to endosomes, but did block sorting. This could mean that Mvp1 oligomerization creates the tubule that recycles Vps55. Alternatively, as suggested by Chi et al., Mvp1 oligomerization could further constrict an existing tubule to promotes the recruitment of Vps1. Can this dimerization mutant recruit Vps1 to endosomes?Figure 3B: Why are there asterisks marking two of the residues? This should be explained in the figure legend. These two residues seem to cause a drastic sorting defect. Why are they not included as important in the model shown in F?Reviewer #3 (Recommendations for the authors):1. The authors describe Mvp1/SNX8 as a coat protein involved in Vps55 retrieval. One clear role for coat proteins is in the selection of cargo and it would be great to see the data supporting a role for Mvp1 in recognition of cargo sorting signals strengthened. Figure 3E shows a co-IP experiment between Mvp1 and either WT Vps55 or Vps55-4Ala . The difference in the two co-IPs was rather slight and was not quantified. Quantitation of these results and evidence of reproducibility would improve confidence in this result. It would also be worth discussing if the YXTXXFM motif is similar to other sorting signals recognized by retromer or other sorting nexins.2. Related to the first point – the authors show that overexpression of Vps55 causes its mislocalization to the vacuole in Figure 3G-J. Does overexpression of Mvp1 suppress the overexpressed Vps55 missorting to the vacuole? This would address whether or not Mvp1 is the limiting component for sorting Vps55.3. Figure 4 shows that Vps1 membrane recruitment is lost in a mvp1 vps35 snx4 triple mutant. This is surprising because I would have assumed Vps1 would also be required for scission of vesicles produced by AP-1, AP-3 and GGA from the Golgi. Perhaps the authors could address this point in the Discussion.eLife. Your revised article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by Suzanne Pfeffer as the Senior and Reviewing Editor. For the most part, the reviewers were satisfied with the changes made during revision. However one reviewer felt that additional textual changes would still be required as follows.Thank you for resubmitting your work entitled "A PX-BAR protein Mvp1/SNX8 and a dynamin-like GTPase Vps1 drive endosomal recycling" for further consideration by (1) The authors were asked to test for direct interaction between Mvp1 and Vps55. Experiments were performed but they failed to detect a direct interaction. They were also unable to provide evidence of sufficiency by transplanting this sequence into a naïve reporter. They have provided better support for the co-IP between these proteins by the new Figure 3—figure supplement 1C. However, they should provide a more cautious interpretation of this result as it is possible that the sorting signal identified in Vps55 is not directly binding to Mvp1. I suggest revising line 156 to include something like "…, although it is also possible that the Vps55-Mvp1 interaction is bridged by another protein." Line 399 in the Discussion should also be revised to "…Mvp1-dependent sorting of Vps55 requires its YHTSDFM sorting signal, which does not…".The authors' response was disappointing in that the desired outcome was not obtained but the primary conclusions of the study are still valid. I believe the suggested revisions to the text will be sufficient.(2) I am satisfied with the response to this point regarding the Mvp1-Vps1 interaction.(3) I do not think the response to this point is adequate. The authors now state that overexpression of Vps68 suppresses the mislocalization phenotype caused by Vps55-GFP overexpression. However, the new data included in the manuscript  does not show any difference that I can detect between the vector only and Vps68 OE. No quantitation of this result was provided and it is unclear how the authors came to the conclusion described in the rebuttal. In addition, line 167 of the manuscript states that "Overexpression of Vps68 suppressed degradation of Vps55-GFP ,…". There is no evidence of degradation in the data provided. Was this supposed to have been a western blot? If there is actual data to suggest Vps68 is the limiting component, the authors should include in the Discussion the possibility that the Vps55 sorting signal define is actually a Vps68-interaction motif.Reviewer #2:(4) The authors have improved their treatment of the literature in the revised manuscript and I think their response to this point is adequate. One more citation they should incorporate into the Discussion is PMID: 29437695, which provides evidence for a sorting nexin recruiting dynamin-2.The authors have carried out a considerable number of new experiments in response to the reviewers' comments. In most cases, these experiments have addressed my concerns, and those of the other reviewers. In other instances, I am satisfied that the authors tried hard to carry out the requested experiments even if the results were inconclusive; resolving these issues will require more extensive work that I agree is beyond the scope of this paper. I also appreciate the changes made to the text to recognize others contributions to the field. In conclusion, I am satisfied by this revised version and the authors responses to the previous reviews. Essential revisions:1. Show that Mvp1 binds Vps55 cytosolic sequences in vitro, and that this is blocked by the mutation that disrupts sorting, would provide stronger evidence that Mvp1 recognizes and sorts Vps55. Furthermore, transplanting this signal to a heterologous protein would test if this sequence is sufficient for Mvp1-dependent sorting; inclusion of a control cargo that does not bind would also be helpful.E. coli and performed in vitro pull-down assay 4A-GFP (recycling sequence mutants) are still localized at the endosome in these mutants . These results strengthen our conclusion that the Vps55 sorting signal is required for the association with Mvp1.As suggested by the reviewer, we prepared a recombinant cytoplasmic region of Vps55 and Mvp1 from  mutants . StrikinAs suggested by the reviewers, we transplanted the cytoplasmic region of Vps55, including the recycling signal to Vps10 or Ear1, which are endosomal membrane proteins. However, both of them failed to express. It could be because Vps55 has four transmembrane domains, whereas Vps10 and Ear1 only have one. We tried to generate another chimeric protein using tetra-spanning transmembrane proteins, but we could not obtain a suitable TM protein, which is stably localized at the vacuole membrane. Hence, we were not able to address this point.2. Further defining how Mvp1 binds Vps1 would increase the novelty of the study. It is interesting that mutating the Mvp1 dimerization interface didn't block localization to endosomes, but did block sorting. This could mean that Mvp1 oligomerization creates the tubule that recycles Vps55. Alternatively, as suggested by Chi et al., Mvp1 oligomerization could further constrict an existing tubule to promotes the recruitment of Vps1. Can this dimerization mutant recruit Vps1 to endosomes?Thank you for your constructive comment. As suggested by the reviewers, we examined that the Vps1-GFP localization in Mvp1 dimerization mutants. In these mutants, Vps1 failed to localize at the endosome , suggesting that Mvp1 oligomerization is required for the endosomal localization Vps1.3. The authors show that overexpression of Vps55 causes its mislocalization to the vacuole in Figure 3G-J. Does overexpression of Mvp1 or the Vps55-interacting protein Vps68 suppress the overexpressed Vps55 missorting to the vacuole? This would address whether or not one of these proteins is the limiting component for sorting Vps55.Thank you for pointing this out. As suggested by the reviewers, we overexpressed Vps68, which suppressed the missorting of the overexpressed Vps55 . On the other hand, overexpression of Mvp1 did not affect the localization of Vps55 . These r4. Please revise the text throughout to provide a more complete description of the relevant prior literature as described below.Thank you for your constructive comments. We have carefully revised the manuscript .Reviewer #1 (Recommendations for the authors):1. Figure 3,4: The authors nicely dissect the role of Vps1 for the Mvp1 pathway, and Vps1 K42 clearly affects recycling via Mvp1. Is Vps1 required for all three recycling pathways? The authors nicely show that Vps55 is affected in the Vps1 K42A mutant, but should also test the other cargoes (like Atg27).vps1(K42A) mutants . So far, only Vps55 has been characterized as a cargo, whose function remains unclear. Having a recycling deficient mutant of Vps55, the authors could ask if the Vps55 mutant  combined with snx4 and retromer deletion results in the same phenotype.vps55Δ cells. It was partially defective , we examined Mup1 sorting in efective . Based o4. The authors have evidence that Nhx1 is recognized by all three pathways. It would be nice if the authors could speculate on the sorting motif responsible for general vs. specific (e.g. Mvp1 dependent) recycling.vps35Δ, snx4Δ, or mvp1Δ), but impaired in vps35Δ snx4Δ mvp1Δ cells. Based on the results, we speculate all three SNX-BAR complexes recognize Nhx1’s sorting signal. Identification of this general sorting signal is an essential question in feature study.Thank you for pointing this out. We did not observe any defect of Nhx1 recycling in each mutant (Reviewer #2 (Recommendations for the authors):Demonstrating that Mvp1 binds Vps55 cytosolic sequences in vitro, and that this is blocked by the mutation that disrupts sorting, would provide stronger evidence that Mvp1 recognizes and sorts Vps55. Furthermore, transplanting this signal to a heterologous protein would test if this sequence is sufficient for Mvp1-dependent sorting.Thank you for pointing this out. As mentioned above, we tried in vitro binding assay and constructed chimeric proteins, but it did not work.Further defining how Mvp1 binds Vps1 would increase the novelty of the study. It is interesting that mutating the Mvp1 dimerization interface didn't block localization to endosomes, but did block sorting. This could mean that Mvp1 oligomerization creates the tubule that recycles Vps55. Alternatively, as suggested by Chi et al., Mvp1 oligomerization could further constrict an existing tubule to promotes the recruitment of Vps1. Can this dimerization mutant recruit Vps1 to endosomes?Thank you for pointing this out. As mentioned above, we examined Vps1 localization in Mvp1 dimerization mutants and found that its dimerization is required for Vps1 recruitment .Figure 3B: Why are there asterisks marking two of the residues? This should be explained in the figure legend. These two residues seem to cause a drastic sorting defect. Why are they not included as important in the model shown in F?Sorry for this confusion. These mutants marked with asterisks were more highly expressed than other mutants, which indirectly caused the missorting of Vps55. We carefully revised the Figure legend.Reviewer #3 (Recommendations for the authors):1. The authors describe Mvp1/SNX8 as a coat protein involved in Vps55 retrieval. One clear role for coat proteins is in the selection of cargo and it would be great to see the data supporting a role for Mvp1 in recognition of cargo sorting signals strengthened. Figure 3E shows a co-IP experiment between Mvp1 and either WT Vps55 or Vps55-4Ala . The difference in the two co-IPs was rather slight and was not quantified. Quantitation of these results and evidence of reproducibility would improve confidence in this result.As mentioned above, we performed in vitro binding assay, but it did not work.It would also be worth discussing if the YXTXXFM motif is similar to other sorting signals recognized by retromer or other sorting nexins.Thank you for pointing this out. We revised the manuscript to reflect this 2. Related to the first point – the authors show that overexpression of Vps55 causes its mislocalization to the vacuole in Figure 3G-J. Does overexpression of Mvp1 suppress the overexpressed Vps55 missorting to the vacuole? This would address whether or not Mvp1 is the limiting component for sorting Vps55.Thank you for pointing this out. As suggested by the reviewer, we overexpressed Mvp1, but it did not rescue the Vps55 missorting . However3. Figure 4 shows that Vps1 membrane recruitment is lost in a mvp1 vps35 snx4 triple mutant. This is surprising because I would have assumed Vps1 would also be required for scission of vesicles produced by AP-1, AP-3 and GGA from the Golgi. Perhaps the authors could address this point in the Discussion.vps1Δ cells exhibited the growth defect at 26 degrees , but vps35Δ snx4Δ mvp1Δ cells did not , suggesting that Vps1 may have another function beyond recycling. This is an important question to be clarified by future studies.That is an important point. We did not see the colocalization of Vps1-GFP with the TGN localized Sec7-mCherry , suggesting that Vps1 might only be transiently recruited to the TGN. Strikingly, For the most part, the reviewers were satisfied with the changes made during revision. However one reviewer felt that additional textual changes would still be required as follows.(1) The authors were asked to test for direct interaction between Mvp1 and Vps55. Experiments were performed but they failed to detect a direct interaction. They were also unable to provide evidence of sufficiency by transplanting this sequence into a naïve reporter. They have provided better support for the co-IP between these proteins by the new Figure —figure supplement 1C. However, they should provide a more cautious interpretation of this result as it is possible that the sorting signal identified in Vps55 is not directly binding to Mvp1. I suggest revising line 156 to include something like "……., although it is also possible that the Vps55-Mvp1 interaction is bridged by another protein." Line 399 in the Discussion should also be revised to "…Mvp1-dependent sorting of Vps55 requires its YHTSDFM sorting signal, which does not…".The authors' response was disappointing in that the desired outcome was not obtained but the primary conclusions of the study are still valid. I believe the suggested revisions to the text will be sufficient.As suggested by the reviewer, we have carefully revised the manuscript .(3) I do not think the response to this point is adequate. The authors now state that overexpression of Vps68 suppresses the mislocalization phenotype caused by Vps55-GFP overexpression. However, the new data included in the manuscript  does not show any difference that I can detect between the vector only and Vps68 OE. No quantitation of this result was provided and it is unclear how the authors came to the conclusion described in the rebuttal. In addition, line 167 of the manuscript states that "Overexpression of Vps68 suppressed degradation of Vps55-GFP ,…". There is no evidence of degradation in the data provided. Was this supposed to have been a western blot? If there is actual data to suggest Vps68 is the limiting component, the authors should include in the Discussion the possibility that the Vps55 sorting signal define is actually a Vps68-interaction motif.As suggested by the reviewer, we have added the quantification of “Figure 3—figure supplement 1E” . Also, we have included the possibility suggested by the reviewer in the Discussion (Line 403).(4) The authors have improved their treatment of the literature in the revised manuscript and I think their response to this point is adequate. One more citation they should incorporate into the Discussion is PMID: 29437695, which provides evidence for a sorting nexin recruiting dynamin-2.As suggested by the reviewer, we have incorporated suggested paper in the Discussion (Line 442).