Improving practice nurses’ (PN) adherence to smoking cessation counseling guidelines will benefit the quality of smoking cessation care and will potentially lead to higher smoking abstinence rates. However, support programs to aid PNs in improving their guideline uptake and adherence do not exist yet.The aim of this study was to assess the effects of a novel computer-tailored electronic learning (e-learning) program on PNs’ smoking cessation guideline adherence.A Web-based randomized controlled trial (RCT) was conducted in which an intervention group (N=147) with full access to the e-learning program for 6 months was compared with a control group (N=122) without access. Data collection was fully automated at baseline and 6-month follow-up via online questionnaires, assessing PNs’ demographics, work-related factors, potential behavioral predictors based on the I-Change model, and guideline adherence. PNs also completed counseling checklists to retrieve self-reported counseling activities for each consultation with a smoker (N=1175). To assess the program’s effectiveness in improving PNs’ guideline adherence , mixed linear and logistic regression analyses were conducted, thus accommodating for the smokers being nested within PNs. Potential effect moderation by work-related factors and behavioral predictors was also examined.PHolm-Bonferroni =.048), indicating a positive program effect on adherence for PNs with a more than average level of counseling experience. Mixed logistic regression analyses regarding adherence to individual guideline steps revealed a trend toward moderating effects of baseline levels of behavioral predictors and counseling experience. More specifically, for PNs with less favorable scores on behavioral predictors  and high levels of counseling experience, the program significantly increased adherence.After 6 months, 121 PNs in the intervention group  and 103 in the control group  completed the follow-up questionnaire. Mixed linear regression analysis revealed that counseling experience moderated the program’s effect on PNs’ overall guideline adherence , it is important to assess how to support a wider range of HCPs.Netherlands Trial Register NTR4436; http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=4436 (Archived by WebCite at http://www.webcitation.org/6zJQuSRq0) Smoking is the most preventable cause of illness and premature death worldwide ,2. In thAn earlier study investigating PNs’ needs for guideline adherence support found that they were interested in an individually relevant, easy-to-use, and practically applicable program or intervention . MoreoveAdditionally, providing PNs with online access to tailored content  enables them to consult it time-efficiently and whenever and wherever they desire ,18. PrevThe aim of the study described here was to assess the effects of the CT e-learning program on PNs’ smoking cessation guideline adherence in a randomized controlled trial (RCT). We hypothesized that PNs’ guideline adherence would significantly improve as a result of exposure to the CT e-learning program.We conducted an RCT to investigate the effectiveness of the CT e-learning program on PNs’ smoking cessation guideline adherence, compared with no intervention. A full description of the design of the RCT can be found elsewhere . EvaluatThe CT e-learning program was structurally based on previously developed CT programs ,23 and cPNs across the Netherlands were contacted through email, newsletters, and website messages via national organizations for PNs or primary care professionals in general, as well as via a project website  and sociIndividual PNs who were randomly allocated to the intervention group of the trial had access to all e-learning and general modules described above and received a tailored feedback letter based on their answers to the baseline questionnaire; this letter provided individual PNs with a summary of various pieces of tailored advice  and instructions on where to find more elaborate advice in the e-learning modules. PNs in the control group only had access to the general modules. During a 6-month time period , PNs in the intervention and control group were free to visit the modules of the CT e-learning program that were available to them based on their group allocation as many times as they wanted. PNs could directly print content from the modules and save this content on their computer.During the trial, PNs in both the intervention and control group were asked to engage in smoking cessation counseling with their smoking patients when the opportunity arose. All PNs were asked to recruit these smokers to partake in the trial . When smBaseline and 6-month follow-up questionnaires for PNs were informed by the ICM  and wereAfter providing online informed consent, every PN was requested to fill out their first and last name, gender, date of birth, and smoking status . Subsequently, they filled out in how many general practices they worked, how many hours they worked per week, and whether or not they were listed in the Dutch Stop Smoking Quality Register . The final questions concerned the practice in which a PN worked most hours per week; they filled out practice name, experience in smoking cessation counseling in years, the presence of designated smoking cessation consulting hours (yes or no), and whether patients’ smoking status was systematically registered in their patient files (yes or no).Several socio-cognitive factors were assessed as potential predictors of guideline adherence, informed by the ICM : intentiDo you intend to use the STIMEDIC quit smoking guideline?), using the same answering scale .PNs’ intention to use a smoking cessation guideline was assessed by two questions addressing the intention to use (1) Any evidence-based smoking cessation guideline and (2) The most recent Dutch counseling protocol, specifically . As such, PNs scored points for every statement they appropriately identified to correctly reflect the content of the counseling protocol (range 0-18).PNs’ knowledge of evidence-based smoking cessation guidelines was assessed by 18 true-false items concerning the content of the STIMEDIC guideline  and seven items about perceived disadvantages  of using an evidence-based smoking cessation guideline . These items were subsequently combined into separate scales for perceived advantages  and perceived disadvantages .PNs’ attitude was assessed by seven items about perceived advantages  and asking PNs how difficult they would find it to follow a guideline in each of these situations . All items were combined into a self-efficacy scale .PNs’ level of self-efficacy was assessed by ten items describing potentially difficult situations when trying to adhere to an evidence-based smoking cessation guideline , five items about social support , and five items about social norms . All items assessed the potential influence of important others within and outside the general practice  and were subsequently combined into separate scales for social modeling , social support , and social norms . Scores that represented a not applicable answering category were assigned a neutral score.PNs’ perceived social influence was assessed by three items about social modeling , whereas coping plans addressed their aspiration to develop a concrete plan for dealing with potentially difficult situations . Subsequently, sum scores for both action plans (range 0-8) and coping plans (range 0-10) were computed.PNs’ intention to make action plans and coping plans was assessed by eight and ten items, respectively (yes or no). Action plans addressed specific activities for preparing a smoking cessation consultation with a patient  during complete smoking cessation trajectories  of their last ten patients (range 0-10). These data on PNs’ adherence were used to create CT advice for PNs in the intervention group regarding their behavior. In the effect analyses, PNs’ baseline guideline adherence score was used as a covariate. Additionally, during the trial period, guideline adherence was assessed by asking PNs to self-report their adherence to each guideline step  after every consultation with a smoking patient (yes or no) using the counseling checklist . This resulted in a score from 0 (none of the steps were adhered to) to 9  for each individual consultation with a smoker. Checklists of consultations with the same smoker were combined into a single score for guideline adherence, reflecting a PN’s adherence during a complete counseling trajectory of a smoking patient, which was the primary outcome measure in the effect analyses.Questions on guideline adherence concerned the nine evidence-based counseling steps, as described in the STIMEDIC guideline : (1) advWe calculated the required sample size based on the possibility to detect a difference of medium effect size  between intervention and control group PNs . As a result, at least 95 PNs per condition at the end of the trial would be sufficient . Howevert tests and chi-square tests determined significant (P<.05) baseline differences between intervention and control group PNs. Logistic regression was used to determine selective dropout of PNs after baseline, including variables potentially related to PNs’ guideline adherence . On the basis of analyses for baseline differences and selective dropout, statistically significant variables were identified and included as covariates in further analyses.Reliability analyses  were conducted using R version 3.4.0 , and other statistical analyses were conducted using SPSS version 23.0 (IBM Corp). Descriptive analyses were conducted to summarize PNs’ characteristics, whereas independent-samples As smokers were nested within PNs participating in the trial, mixed regression analyses were conducted to assess the effects of exposure to the CT e-learning program on PNs’ smoking cessation guideline adherence. Both PNs’ overall adherence score (range 0-9) was used as outcome measure and their adherence score for each guideline step separately . Therefore, both linear and logistic mixed models were run, including the same covariates. Effect moderators were tested by including interaction effects with PNs’ group allocation  to the regression models tested. On the basis of literature, several work-related factors  and As 211 PNs  completed at least one checklist , it meant that 58 PNs were excluded from effect analyses. For this reason, sensitivity analyses were conducted by replacing missing values on the primary outcome measure (guideline adherence) with scores assuming some dependency between the score being missing and the adherence score itself, either following an optimistic or a pessimistic scenario. In both scenarios, missing data were imputed for these 58 PNs based on the average number of patients counseled during the trial per PN. Furthermore, PNs’ dropout status was taken into account, as some PNs, who did not complete any checklists, also did not fill out the follow-up questionnaire . One might expect PNs who did complete the follow-up questionnaire  to be more motivated and to be more adherent if they would have completed the checklists during the intervention period. This was taken into account when imputing data. In the optimistic imputation scenario, retained PNs were assumed to be adherent in 90% of the consultations with smokers, whereas PN dropouts were assumed to be adherent in only 80% of their consultations. In the pessimistic scenario, a 50% probability of adherence was assumed for retained PNs and a 20% probability of adherence for PN dropouts. The datasets obtained under these two imputation scenarios were analyzed with the mixed regression models, as obtained after backward deletion of nonsignificant (interaction) effects in the analysis of only the complete cases.The baseline sample of PNs  had a me21=10.1; P=.001). Therefore, the presence of designated consulting-hours was included as a covariate in all effect analyses.Baseline characteristics of PNs were comparable between intervention and control group, except for the presence of designated smoking cessation consulting hours, which was significantly more often reported by PNs in the control group  of intervention group and 87.3% (103/118) of control group PNs, respectively . Attritigroup  and a main effect of perceived advantages of guideline use (P=.03). Subgroup analyses showed that for PNs with more than average counseling experience , allocation to the intervention group  resulted in a significantly higher overall adherence compared with the control group . The subgroup analysis for less experienced PNs  revealed no significant intervention effect.group in the presence of a significant interaction. Regarding adherence to step 1 , a significant main effect was found of PNs’ baseline adherence to step 1 (P=.002). Regarding adherence to step 2 , a significant interaction effect of group allocation with perceived advantages of guideline use (P=.001) was identified. Regarding adherence to step 3 , an interaction effect with self-efficacy (P=.02) was found. Regarding adherence to step 4 , a borderline significant interaction effect of group allocation with counseling experience (P=.06) was found, as well as main effects of baseline adherence to step 4 (P=.01) and of perceived advantages of guideline use (P=.03). Regarding adherence to step 5 , significant interaction effects of group allocation with social modeling (P=.009) and with social support were found (P=.046). Regarding adherence to step 6 , a significant main effect was found of perceived advantages of guideline use (P=.045). Regarding adherence to step 7 , significant interaction effects of group allocation with perceived disadvantages (P=.01) and with self-efficacy (P=.001) and a borderline significant interaction effect with counseling experience (P=.05) were found. Furthermore, a significant main effect of social modeling (P=.01) was found. Regarding adherence to step 8 , significant interaction effects of group allocation with counseling experience (P=.01), with social support (P=.001), and with social norms (P=.005) were found. Concerning adherence to step 9 , an interaction with social support (P=.04) was found.Detailed results on subgroup analyses for each significant interaction effect are presented in optimistic imputation scenario, results for overall guideline adherence indicated that only the main effect of perceived advantages remained significant. Regarding step-based adherence, similar results were found for adherence to guideline steps 1, 2, and 8. Concerning adherence to steps 5 and 7 only, the interaction effects with social modeling and self-efficacy could be replicated, and concerning adherence to step 4, only the main effect of perceived advantages remained significant. Main and interaction effects concerning guideline steps 3, 6, and 9 were no longer or only marginally significant after conducting these optimistic sensitivity analyses.After replicating the final mixed regression models following an pessimistic imputation scenario, main and interaction effects for overall adherence were no longer or only marginally significant. Regarding step-based adherence, similar results were found for adherence to guideline steps 1, 6, 8, and 9, whereas regarding step 4, only main effects of baseline adherence to step 4 and perceived advantages remained significant. Main and interaction effects concerning guideline steps 2, 3, 5, and 7 were no longer or only marginally significant after conducting these pessimistic sensitivity analyses.After replicating the final mixed regression models following a The present RCT tested the effectiveness of a novel CT e-learning program for PNs to improve their adherence to evidence-based smoking cessation guidelines in Dutch general practice. Our results suggest that among PNs with more than average smoking cessation counseling experience, access to the CT e-learning program resulted in significantly better guideline adherence. A more detailed inspection of PNs’ guideline adherence revealed comparable results for several specific guideline steps . Additionally, a trend was observed that for PNs reporting less favorable baseline levels for behavioral predictors such as self-efficacy and favorable levels of perceived social support, the e-learning program was effective in improving guideline adherence.Regarding PNs’ counseling experience, subgroup analyses illustrated that more experienced PNs in the intervention group adhered significantly better to the guideline  at follow-up compared with equally experienced PNs in the control group. This finding means that the CT e-learning program successfully promoted guideline adherence among PNs with more counseling experience, which is in line with the fact that a significant positive association between counseling experience and PNs’ baseline guideline adherence was found . ExperieResults regarding PNs’ step-based guideline adherence illustrated that, besides counseling experience, several behavioral predictors could explain better adherence scores of PNs in the intervention group compared with the control group. PNs’ perceived advantages and disadvantages of guideline application, level of self-efficacy, social modeling, social norms, and social support at baseline all moderated the e-learning program’s effect on PNs’ step-based adherence. Although moderation effects could not be replicated precisely for each separate guideline step, a trend was observed that especially a high level of baseline social support and lower baseline levels of the other predictors were related to better effects of the program on adherence. As the content of the e-learning program was designed to especially target such behavioral predictors of guideline adherence , it is lA strength of the present trial is that we enrolled a substantial number of PNs and managed to retain 83.3% (224/269) of them at 6-month follow-up. In comparison, in other studies among nurses, retention rates were considerably lower, eg, studies among hospital nurses that reported 3-month and 6-month follow-up retention rates of 68% and 58% , or 56% t390=−6.73, P<.001). As we observed possible ceiling effects in these adherence scores, smoker-reported data were deemed to be unreliable for being used as outcome measure. An explanation could be that smokers overestimated their PNs’ guideline adherence, as they were recruited for participation in the trial by their PN, who also supported them with smoking cessation. Perhaps they were afraid that providing critical answers to questions about their PNs’ performance could influence the relationship with their PN. Although smokers were informed that data were treated anonymously and were not reported back to PNs, the phenomenon of social desirability is often observed when collecting data from patients in a health care setting [Nevertheless, we also experienced some challenges during the trial. Our intention was to triangulate data on PNs’ guideline adherence from both smokers’ and PNs’ perspectives to compute the primary outcome measure . Unfortu setting . It, hen setting . To exam setting . Yet, moIn light of the issues described, it would be worthwhile to investigate additional methods to gather data on PNs’ guideline adherence. One such method could be qualitative data collection, by conducting content analyses of audio or video recordings of smoker-PN consultations -47. In tFurthermore, the results of the present trial illustrated that the CT e-learning program successfully improved smoking cessation guideline adherence of experienced PNs. Unfortunately, it was also established that usage of the program by PNs in the trial was quite limited . This means that the program’s effectiveness could potentially be increased when implementation of the program by PNs  improves. During posttrial interviews, PNs mentioned that receiving a reminder to visit the program more often could be a potential strategy to stimulate program usage. Furthermore, a process evaluation could inform improving alterations to the program’s tailored content to extend the program’s effectiveness beyond experienced PNs. When such strategies would be combined with program implementation among a larger population of PNs, this could further substantiate the impact of the CT e-learning program on the quality of smoking cessation care in the Netherlands.Providing PNs access to a novel CT e-learning program resulted in significantly better adherence to evidence-based smoking cessation guidelines among Dutch PNs experienced in smoking cessation counseling, compared with similar PNs without access to this program. More favorable improvements on behavioral predictors of guideline adherence among intervention group PNs may explain better adherence scores. To further substantiate the effectiveness of e-learning programs on guideline adherence by HCPs, alternative methods of collecting data on guideline adherence should be explored, and strategies are needed to promote program usage and to also support less experienced PNs to adhere to evidence-based smoking cessation guidelines. This could subsequently inform widespread implementation of the e-learning program among PNs.


Tobacco control and prevention in rural areas are possible as demonstrated by a community-driven tobacco-free village program in India. Success factors included community ownership with supportive program guidance, motivated and committed local leaders, collaboration with grassroots organizations, rewards and sanctions to establish new social norms, and provision of other income-generating options for vendors who sell tobacco. While the program required time and dedicated effort and was not successful in all villages, it holds promise for helping to achieve the goals of tobacco control policies, especially in resource-scarce settings. Tobacco control and prevention in rural areas are possible as demonstrated by a community-driven tobacco-free village program in India. Success factors included community ownership with supportive program guidance, motivated and committed local leaders, collaboration with grassroots organizations, rewards and sanctions to establish new social norms, and provision of other income-generating options for vendors who sell tobacco. While the program required time and dedicated effort and was not successful in all villages, it holds promise for helping to achieve the goals of tobacco control policies, especially in resource-scarce settings. India has 274 million tobacco users and a tobacco use prevalence of 38% in rural areas. Tobacco consumption causes 1 million deaths and costs the health system nearly US$23 billion annually. Tobacco control policies exist but lack proper implementation. In this article, we review the Tobacco-free Village (TfV) program conducted in Maharashtra state in India and describe its process to help villages in rural India achieve “tobacco-free” status . We reviewed program documents and conducted 22 qualitative interviews with program staff and village-level stakeholders. From 2008 to 2014, Salaam Mumbai Foundation implemented the TfV program in 60 villages in Maharashtra state. The program used a number of strategies to help villages become tobacco free, including collaborating with a community-based organization, leveraging existing health workers, conducting a situation analysis, training health workers, engaging stakeholders, developing TfV assessment criteria, mobilizing the community, conducting health education, imposing sanctions, and offering incentives. By 2014, 4 villages had achieved tobacco-free status according to 11 assessment criteria. Successful villages demonstrated strong local leader involvement, ownership of the program, and commitment to the cause by residents. The TfV program faced barriers including poor motivation of health workers, difficulty in changing social norms of tobacco use, and refusal of local vendors to stop tobacco sales due to financial losses. This low-cost, community-driven program holds promise for helping public health practitioners and governments implement and achieve the goals of tobacco control policies, especially in resource-scarce settings. The aim of the Tobacco-free Village (TfV) program, implemented between 2008 and 2014, was to stop the sale and use of tobacco in villages in Chandrapur district, in rural Maharashtra state, in order to reduce tobacco-related morbidities and mortality.We analyzed program documents and conducted 22 qualitative interviews to describe the TfV program process and identify success factors and barriers in implementing a tobacco-free program. The program documents included operational plans, annual reports, meeting minutes, quarterly and monthly visit reports, local village council resolutions, financial budgets, and expenditure reports. We conducted 22 qualitative interviews with purposively identified program managers and coordinators, health workers, and village-level stakeholders.The TfV program was an initiative of the Salaam Mumbai Foundation (SMF), the rural wing of the Salaam Bombay Foundation (SBF), a nonprofit organization. SBF works on life-skills development for tobacco prevention and cessation with children and adolescents from low-income municipal schools in the city of Mumbai (previously Bombay), capital of the state of Maharashtra in western India. Inspired by the success of the urban program, SBF started SMF in 2007 to implement a similar program with children and adolescents in other districts of the state, with a special emphasis on rural areas.sakhis (good friends), who conduct interpersonal counseling and referrals for government health services. SMF strategically implemented the TfV program in collaboration with an established organization like ACF to leverage ACF's grassroots presence in communities and its existing network of sakhis.In 2008, SMF signed a formal collaborative agreement to implement the TfV program with Ambuja Cement Foundation (ACF), the corporate social responsibility division of a large cement manufacturing company in India. ACF works on livelihood programs related to health and agriculture in several districts in Maharashtra, including Chandrapur. ACF implements its health programs in villages via trained female health workers called SMF initiated the TfV program in Chandrapur district in eastern Maharashtra, starting with 10 villages in 2008. The TfV program extended to 60 villages in Chandrapur district by 2014—specifically, 23, 22, and 15 villages in the 3 blocks  of Rajura, Korpana, and Jiwati, respectively—serving a population of nearly 48,882 people in 11,375 households.Between 2008 and 2014, the Tobacco-free Village program extended to 60 villages in Chandrapur district in Maharashtra state.SMF spent 1.74 million Indian Rupees (INR)  to implement the TfV program from 2008 to 2014; 75% was spent on program activities and the remainder on staff salaries in villages. Additional allocations of INR 500,000  on a part-time manager or coordinator at SMF and INR 245,000  on local and interdistrict travel were made over these 7 years. In the fiscal year April 2014 through March 2015, after ACF took over program implementation from SMF, it spent INR 300,000 . Thus, between April 2008 and March 2015, the cost of implementing the program in 60 villages was approximately INR 2.8 million . This amount does not include costs such as electricity, telecommunication, rent, and other organizational expenses.The first step of the TfV program was to conduct a situational analysis of tobacco consumption and socioeconomic factors in program villages in order to provide program planners with a contextual understanding to design appropriate change strategies. The findings of this analysis are summarized below.sakhis used interpersonal communication and wall paintings as their main channels of communication.The program villages had 683 residents, on average, with farming as the most common occupation, followed by work in coal mines and cement factories. Program villages had a total of 24 government schools that served grades 1 to 4 and 36 schools that served grades 1 to 7. Each village had more than 1 shop that sold tobacco. The villages were characterized by poor connectivity to district centers, with a transportation service frequency of 2 state-run buses each day. Few households owned televisions; kharra, which is a mixture of tobacco, chuna, and cardamom , generally has influence over the community. In Thutra village, for example, the sarpanch owned a shop that sold tobacco and initially refused to be a part of the TfV program. After SMF made several visits to discuss the issue with the sarpanch, he agreed to participate and ultimately closed his shop and became a champion for tobacco control in his village as well as neighboring areas.After training the Other influential stakeholders included members of the village council, local police officers, principals and teachers of local schools, and members of self-help groups, community-based organizations, and religious groups. SMF conducted an average of 30 meetings with village-level stakeholders every year to equip them with the information and skills needed to mobilize public support for tobacco control.The TfV program relied on a list of criteria for assessing the progress of villages in becoming tobacco-free. These criteria provided set targets and a road map to implement activities and monitor and evaluate the program in each village. SMF staff developed the criteria by holding meetings with stakeholders to discuss the provisions from the COTPA and then deliberating on which provisions to include and adapt as criteria for assessing tobacco-free villages. The final list included 11 criteria .The Tobacco-free Village program developed a list of 11 assessment criteria based on the Cigarettes and Other Tobacco Products Act of 2003.sakhi, sarpanch, or other villagers assigned to the task used these 11 criteria to monitor the program  to raise awareness about problems associated with tobacco use. Schools organized tobacco-prevention events around commonly celebrated religious festival days, as well as World No Tobacco Day and World Cancer Day, encouraging participation from families. School principals and staff were encouraged to use every possible opportunity to talk to parents about the harms caused by tobacco use. Parents attended anti-tobacco sessions in the schools, conducted by trained self-help groups.In schools, the program trained teachers to conduct classroom sessions on the harmful effects of tobacco use, and students learned how to refuse if an adult asked them to buy tobacco. Competitions—including drawing, essay writing, rallies, and poster campaigns—incentivized students to become involved in tobacco prevention efforts. The program helped schools form student groups called Sakhis conducted home visits, held group meetings, and trained group members to conduct educational sessions in the villages.The TfV program organized community-based activities, such as wall paintings, poster campaigns, street plays, and village rallies, to focus public attention on the tobacco problem. Since few households owned televisions, the program staff organized tobacco-prevention film screenings followed by group discussion every month. The films were entertaining and were considered stress relievers after hard days of work in the fields. More importantly, this medium was persuasive—it got villagers to consider tobacco-related behavior change. Community activities reinforced the interpersonal and school-level efforts for tobacco prevention and behavior change.Tobacco-prevention film screenings were a persuasive medium to encourage villagers to change tobacco-related behavior.sarpanch, village council members, and police officers attended such community-based events and encouraged villagers to quit tobacco use and choose a healthier life.Finally, program staff set up educational booths during village fairs and cultural festivals, where they spoke about the benefits of a life without tobacco. Stakeholders such as the sarpanch from Thutra village and a motivated sakhi from a non-TfV village each received the coveted cash award based on their approaches and efforts in tobacco control. The disbursal of financial awards to someone they knew motivated sakhis and stakeholders to amplify their tobacco control efforts; and helped push the tobacco control agenda further in the selected villages. These incentives also encouraged winners to intensify and sustain their work. While financial rewards were found to be helpful in the initial phase of the TfV program, the program had to include nonmonetary incentives to ensure sustainability, such as appreciating the efforts of sakhis during biannual meetings and public recognition in village meetings and events.The village governing councils in Mangi and Thutra passed resolutions that made the sale of tobacco punishable by law; these sanctions acted as a major facilitator in prohibiting the sale and use of tobacco in order to meet the criteria for becoming tobacco-free villages. Financial incentives in the form of a cash award of INR 100,000  for outstanding work in tobacco control were awarded by SMF's parent organization, Narotam Sekhsaria Foundation. The sakhis served the program on a voluntary basis. After this year, SMF provided 5-year funding to ACF for implementing the TfV program, so ACF would not have to rely entirely on voluntary resources. Sakhis received financial compensation and program staff had higher budgets to conduct comprehensive activities. SMF integrated all TfV program activities into the ACF operational system for seamless implementation in the future. After the 5-year funding period ended, ACF continued to implement the TfV program with internal financing.Until 2011, ACF's network of sakhis, conducted day-to-day monitoring of program activities. At the second level, local school teachers and village-level stakeholders acted as watchdogs by periodically assessing the program status and providing feedback to the program staff. At the third level, senior SMF staff conducted annual observational visits and comprehensive monitoring of all program activities and adherence with the 11 tobacco-free village criteria. Additionally, ACF field teams sent monthly monitoring reports to SMF. Regular meetings also were held between the SMF and ACF program teams, as well as annual review meetings for the sakhis.Program staff and stakeholders conducted 3 levels of monitoring, using the 11 assessment criteria. At the first level, ACF staff, including Addressing the long-standing tradition of tobacco use proved to be the greatest challenge for program staff. It was difficult to persuade villagers to stop using a product they had been consuming for years and that was embedded in their daily routines. It was considered normal for a child, adolescent, adult, or elderly person to consume tobacco. Furthermore, villagers had little or no information about the ill effects of tobacco consumption. In the 4 tobacco-free villages, influential leaders and stakeholders, positive role models, consistent discussions at all levels about the harms of tobacco, and sanctions changed the existing community norms around tobacco use.Persuading villagers to abandon long-standing traditions of tobacco use was the greatest program challenge.sakhis were unable to successfully broach the topic of tobacco among households, as many of the sakhis were tobacco users themselves. As a result, villagers questioned their credibility as health workers counseling on the ill effects of tobacco. ACF addressed this behavioral barrier through an organizational initiative in which they provided sakhis with individual counseling and cessation support in the form of information about nicotine withdrawal symptoms and management techniques, oral health screenings, and reminders by supervisors to maintain positive behaviors. ACF also passed a no-tobacco policy that prohibited all program staff, including sakhis, from using tobacco. Although ACF conducted oral health screenings for villagers in some program villages, regular monitoring, follow-up, and cessation enforcement were not possible administratively.At the start of the TfV program, Sakhis received support to stop using tobacco themselves, which improved their credibility as counselors on the ill effects of tobacco.sarpanch, village council member, or other important stakeholder owned a tobacco shop or used tobacco, stopping the sale and use of tobacco was difficult because people pointed to this leader as justification for their own tobacco use behavior. Even in villages where tobacco was prohibited, it was possible that some people went to neighboring villages to buy tobacco products and consumed it in the privacy of their homes.In villages where the A complete ban on the sale of tobacco products meant that shopkeepers would face losses in income, which made them unwilling to participate. Providing alternative means of income for tobacco vendors was challenging, as the sale of tobacco products was more lucrative than the sale of many other consumer products. Wherever tobacco vendors were reluctant to stop sales after many rounds of discussion, local police intervened. However, in some villages, local police officers refused to intervene because they had a connection with the vendors. In such cases, ACF and SMF program staff lobbied with higher-level officers in the district to intervene and stop tobacco sales.sakhis in tobacco control was time-consuming and took almost a year. Since the sakhis did not receive monetary benefits for their work until 2011, they viewed TfV-related tasks as a burden beyond their regular duties, and they did not want to spend much time on them. Conducting regular follow-up visits to monitor and enforce tobacco-free status of the villages was therefore difficult. In the absence of financial compensation, these programmatic barriers were addressed by providing the sakhis with nonmonetary rewards and recognition during meetings and events. Another barrier was that any change in the team's composition affected program implementation. This challenge was addressed by integrating TfV processes into ACF's system such that any staff member could conduct all program activities seamlessly.Training the Beginning with 10 villages in 2008, the TfV program expanded to 60 villages by 2014. The TfV program declared the 4 villages of Thutra, Khairguda, Mangi, and Loldoha to be tobacco free according to the 11 program criteria. Although 2 other villages—KoolimaduThe Tobacco-free Village program declared 4 villages tobacco free according to its criteria.sarpanch, school principals, teachers, health workers, owners of shops previously selling tobacco, and police officials.Why did some villages meet the TfV criteria while the overwhelming majority did not? We gained insights into the differences between these villages through interviews with village-level stakeholders such as the All the program villages, which were distributed across 3 blocks, had similar socioeconomic profiles, with farming and employment in coal mines and cement factories as the main sources of livelihood. Tobacco-free villages were found to be smaller compared with other villages, in terms of households (126 versus 194 on average) and population (531 versus 835 on average), although there was no significant statistical difference.sarpanch who was responsible. In Khairguda village, which achieved TfV status in 2013, an inspired tobacco seller lit a symbolic bonfire of tobacco products from his shop, which made a persuasive case for others to join the movement. Mangi village also achieved tobacco-free status in 2013 after its local governing council passed a resolution and involved the police in stopping sale of tobacco. In Loldoha village, which became tobacco free in 2014, a committed sakhi worked adeptly through regular group meetings and door-to-door visits to inspire behavior change among villagers.Motivated and committed local leadership stood out as the critical success factor for change in each of the 4 tobacco-free villages. Thutra village became tobacco free in 2012, and it was a motivated Motivated and committed local leadership stood out as the critical success factor for change in each of the 4 tobacco-free villages.Achieving tobacco-free status has to be a process owned and led by the community. It must involve local leaders who are passionate and committed, have influence over people, and are willing to volunteer their time and efforts. Identifying, recruiting, and motivating the right leaders are the most important steps for achieving tobacco-free villages. This has to be followed by strengthening the capacity of leaders and stakeholders, providing supportive supervision of activities, offering feedback, and creating reward and recognition systems. This kind of a community-led model seems useful for tobacco control in resource-scarce settings; however, generating community ownership is an arduous process. For instance, bringing together local leaders and villagers to form a tobacco control committee required constant, consistent guidance and follow-up from program staff. Program planners need to sustain community participation in and ownership of activities, and enable families to champion the cause, rather than let the TfV program be perceived as an external agency's agenda.Many villages that did not achieve tobacco-free status lacked the involvement of local leaders and community stakeholders, especially the village governing council, highlighting the need for strategic approaches to achieve buy-in and ownership from these influential community members. Many of the village governing council members are tobacco users themselves. There is also the issue of political patronage of tobacco vendors. The long-standing normative practice of tobacco use in rural India was another major obstacle to achieving tobacco-free status in the large majority of the villages. However, using a contextually appropriate, multitiered approach of community education and mobilization, advocacy with influential members including government officials, along with village-level sanctions, helped the TfV program to change norms in the 4 tobacco-free villages. Changing norms is difficult; it can take time. Therefore, an effective social and behavior change communication strategy at the state or national level has to be designed and implemented for tobacco control in rural India.In 2016, the Public Health Department of Maharashtra circulated guidelines for setting up district-, block-, and village-level enforcement committees to ensure COTPA implementation.One of the major lessons learned was that collaboration with grassroots organizations is critical for successful implementation and sustainability of the rural tobacco control model. Working with established agencies and leveraging their existing staff, networks, resources, and tools were beneficial from a financial standpoint. However, we also learned that getting buy-in from the staff and frontline workers of the organization is required from the start; in many cases, they become burdened by multiple responsibilities and may not be compensated appropriately. Need-based training of staff for implementing tobacco control measures is also required.Collaborating with a grassroots organization and leveraging its existing staff and resources was key to successful implementation and sustainability.Formative research and needs assessment among the villagers, tobacco users, vendors, and workers of grassroots organizations are useful for examining many factors: sociodemographic characteristics, knowledge, attitudes, and levers of change among various community groups; capabilities of frontline workers with respect to delivery of tobacco control interventions; and ways in which vendors can be informed and involved in the TfV process.Providing alternative income-generating options for vendors who sell tobacco is another critical factor in achieving tobacco-free status in rural areas. Program planners have to include assistance for such stakeholders in the form of financial training or informational support on government schemes for alternative livelihoods. While such measures may convince vendors to switch from selling tobacco to other consumer products, police and legal intervention mechanisms also have to be built into the program. Furthermore, villagers have to be trained in advocacy measures, and provided with timely support and contacts of influential stakeholders who can successfully work with reluctant tobacco vendors who have connections with the local police and political groups.Interviewers with stakeholders revealed that some of the TfV criteria were difficult to achieve, such as criterion 2, which bans the use of tobacco, and criterion 10, which prohibits visitors from bringing tobacco products into the village. Stakeholders agreed that it was possible that people used tobacco within the private confines of their homes, and residents or visitors purchased tobacco from neighboring areas and smuggled it into the village. There was no mechanism to check if residents or visitors carried tobacco in their bags or pockets when they entered the village. However, stakeholders emphasized that tobacco use in the open public areas visibly stopped due to a change in social norms in tobacco-free villages.Providing other income-generating options for tobacco vendors is critical in achieving tobacco-free status in rural areas.Although these 11 TfV criteria were developed through participatory discussions with local stakeholders using the COTPA provisions as a basis, the criteria need to be reexamined with respect to feasibility and accountability. This should occur in the context of a broader dialogue between state and national health ministry officials, international agencies such as WHO, and local village council leaders. Rural tobacco prevention would be well served by meetings that foster dialogue between global, local, and national stakeholders.Despite national tobacco control policies in India, contextual barriers and lack of proper implementation have enabled tobacco use to persist, especially among rural populations. While tobacco control requires time and dedicated efforts, the TfV program demonstrates that rural tobacco consumption can be controlled and prevented through continuous engagement with village-level stakeholders, motivated local leaders and community workers, contextual and tailored activities, and sanctions and incentives that establish new social norms in villages. This low-cost, community-driven program holds promise for helping public health practitioners and governments implement and achieve the goals of tobacco control policies, especially in resource-scarce settings. Future research, especially in the form of a community trial, is required to further examine program effectiveness. By building on the lessons learned from the TfV intervention, nongovernment and government institutions can ensure better implementation of existing tobacco control policies.


These results indicate that multi-Lac-β-CD (DSL5.6) diminished intracellular cholesterol levels in NPC-like HepG2 cells via asialoglycoprotein receptor (ASGPR)-mediated endocytosis.Niemann–Pick type C (NPC) disease, characterized by intracellular accumulation of unesterified cholesterol and other lipids owing to defects in two proteins NPC1 and NPC2, causes neurodegeneration and other fatal neurovisceral symptoms. Currently, treatment of NPC involves the use of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD). HP-β-CD is effective in the treatment of hepatosplenomegaly in NPC disease, albeit at a very high dose. One of the methods to reduce the required dose of HP-β-CD for treatment of NPC is to actively targeting hepatocytes with β-cyclodextrin (β-CD). The aim of the present study was to synthesize a novel multi-lactose-appended β-CD (multi-Lac-β-CD) and to evaluate its cholesterol-lowering effect in U18666A-treated HepG2 (NPC-like HepG2) cells. Further, the study aimed at delivering β-CD to hepatocytes via cholesterol-accumulated HepG2 cells, and indicated that the newly synthesized multi-Lac-β-CD had an average degree of substitution of lactose (DSL) of 5.6. This newly synthesized multi-Lac-β-CD was found to significantly decrease the concentration of intracellular cholesterol with negligible cytotoxicity as compared to HP-β-CD. An increased internalization of TRITC-multi-Lac-β-CD (DSL 5.6) as compared to TRITC-HP-β-CD was observed in NPC-like HepG2 cells. Further, the dissociation constant of peanut lectin with multi-Lac-β-CD (DSL5.6) was found to be extremely low (2.5 × 10 NPC1 and/or NPC2 gene causes the NPC disease. The majority of the mutations occur in the NPC1 gene that produces NPC1 protein responsible for binding [NPC2 gene that encodes the NPC2 protein as a lysosomal protein. Excessive accumulation of cholesterol in endolysosomes in children, results in severe hepatosplenomegaly and neurodegeneration [The Niemann–Pick type C (NPC) disease is a lipid storage disorder with the accumulation of membrane lipids such as cholesterol and multiple sphingolipids especially in lysosomes. The excessive accumulation of these lipids results in a progressive neurologic and visceral dysfunction –3. A mut binding –5. Meanwneration . Hence, NPC1-knockout (Npc1−/−) mice significantly prolonged the survival rate by reducing the cholesterol levels [Cyclodextrins (CDs) are cyclic glucose oligosaccharides used by the pharmaceutical industry to enhance solubility, stability, and bioavailability of drugs. CDs improve the above properties of drugs through complex formation, when formulated as injectable solutions, sprays, eye drops, powders, and tablets –8. In rel levels –11. In aN-acetylglucosamine (GlcNAc) receptor, is highly expressed on the sinusoidal cell surface of hepatocytes. ASGPR is responsible for the binding, internalization, and subsequent clearance of glycoproteins containing terminal galactose or GlcNAc residues from the circulation [The asialoglycoprotein receptor (ASGPR), a hepatic galactose or culation . Hence, culation . In factculation . Furtherculation –16. HoweConsequently, in the present study, we synthesized a novel multi-lactose-appended β-CD (multi-Lac-β-CD) to enhance the targeting ability to ASGPR, and evaluated its cholesterol-lowering effect in NPC-like HepG2 cells.5) to obtain a hepatocyte-specific cholesterol-lowering agent. The schematic representation of the synthesis of multi-Lac-β-CD (5) is shown in per-NH2-β-CD (4) was synthesized by chlorination (per-chloro-β-CD (2)) and azidation (per-azido-β-CD (3)) of primary hydroxy groups of β-CD 1, as reported previously [per-iodo- or per-bromo-β-CD instead of per-chloro-β-CD. However, the iodo-β-CD may be unstable due to the too high reactivity. Therefore, we prepared per-chloro-β-CD with sufficient reactivity. Finally, lactose groups were inserted in per-amino-β-CD by treatment with cyanotrihydroborate in dimethyl sulfoxide (DMSO) at 90 °C for 24 h. The product yield of multi-Lac-β-CD was 25%, and no unreacted compounds were confirmed by thin-layer chromatography (TLC). The MALDI–TOF MS spectrum of multi-Lac-β-CD revealed several peaks at m/z 2804, m/z 3006, m/z 3139, m/z 3296, and m/z 3454, corresponding to tri-, tetra-, penta-, hexa- and hepta-lactose-substituted β-CDs, respectively (1H NMR spectrum indicated that the degree of substitution of lactose (DSL) was 5.6. This value was obtained from the integral values of the anomeric protons of lactose and glucose in β-CD , cell viability was examined after treatment with multi-Lac-β-CD (DSL5.6) by the water-soluble tetrazolium (WST)-1 method . U18666A−8 M, which was comparable with that obtained for the positive control asialofetuin . In contrast, β-CD did not show any response in the frequency of detection unit of QCM, thus indicating a negligible interaction with PNA  with peanut agglutinin (PNA), a galactose-binding lectin, using the quartz crystal microbalance method (QCM) . The diswith PNA . Hence, ®. The fluorescence intensity of TRITC in cells was measured using the software of the BZ-II analyzer  can enter ASGPR-expressing cells, we examined the intracellular distribution of tetramethylrhodamine isothiocyanate (TRITC)-multi-Lac-β-CD (DSL5.6) in NPC-like HepG2 cells (ASGPR positive cells) analyzer . Meanwhianalyzer . In summThe effects of β-CD, HP-β-CD, mono-Lac-β-CD and multi-Lac-β-CD (DSL5.6) on intracellular cholesterol levels were studied in NPC-like HepG2 cells using Filipin III, which can specifically bind to unesterified cholesterol . TreatmeNpc1−/− mice and NPC-diseased human fibroblasts [10HP-β-CD, one of the therapeutic candidates for the treatment of NPC disease, attenuates free cholesterol accumulation in various organs and prolongs the lifespan of roblasts –1019. Toblasts 10–21. Ther–4 M, whereas those of triantennary and tetraantennary compounds with ASGPR were 5 × 10−9 M and 9 × 10−9 M, respectively [−8 M) with PNA, probably a result of its clustering effect in ASGPR recognition. The further elaborate studies to compare the binding affinity of multi-Lac-β-CD (DSL5.6) to PNA with mono-Lac-β-CD are necessary. In addition, to demonstrate the ASGPR-expressing cell-selective binding and cholesterol-lowering effects of multi-Lac-β-CD (DSL5.6), the comparative studies using ASGPR-negative cells are also needed.The galactose density is a crucial factor for regulation of galactose affinity to ASGPR. Stokmaier et al. revealed that the binding affinity of galactose to ASGPR elevated 100–1000 fold from mono- to triantennary galactose structures, probably due to clustering effects . The disectively . Previouectively . ASGPR iectively –16. TherSeveral lines of evidence indicate that the lowering effect of CDs on the accumulation of cholesterol is attributed to endocytosed CD . The CDsIn this study, we developed multi-Lac-β-CD (DSL5.6) to reduce cholesterol levels in NPC-like HepG2 cells. The multi-Lac-β-CD (DSL5.6) was internalized in NPC-like HepG2 cells via ASGPR-mediated endocytosis and was found to diminish the accumulation of cholesterol in the endolysosomes. In conclusion, the findings from the present study may be crucial for the development of an active targeting CD-based therapy for the treatment of hepatosplenomegaly in NPC disease.β-CD was a kind gift from Nihon Shokuhin Kako . Lactose monohydrate was purchased from Wako Pure Chemical Industries . Tetramethylrhodamine isothiocyanate (TRITC) was obtained from Funakoshi . Biotinylated-peanut agglutinin (PNA) and bovine serum albumin (BSA) were purchased from Cosmo Bio  and Boehringer Mannheim K.K. , respectively.per-6-Amino-β-CD (4) was synthesized as reported previously [per-6-amino-β-CD. For this experiment, 20 mL of DMSO containing per-6-amino-β-CD , lactose monohydrate (1.52 g), and sodium cyanotrihydroborate (2.79 g) were mixed and incubated at 70 °C for 24 h. After dialysis by Spectra/pore (MWCO: 1000) in water at room temperature for 48 h, the sample was concentrated in a rotary evaporator and freeze-dried to obtain multi-Lac-β-CD. 1H NMR spectra for multi-Lac-β-CD consisted of protons of both β-CD and lactose. The ratios of peak areas of the anomeric proton of β-CD and protons of the lactose were approximately 5.6. This result indicated that β-CD covalently binds to lactose in a molar ratio of 1:5.6  δ (from TMS), 5.00 , 3.84−3.62 , 3.54−3.43 , 4.56−4.41 (anomeric proton of lactose).eviously . Lactoseof 1:5.6 . A 25% y5 mU/mL), streptomycin (0.1 mg/mL) and supplemented with 10% fetal bovine serum (FBS), in a humidified incubator with 5% CO2. NPC-like HepG2 cells accumulated with high amounts of cholesterol were obtained after 48 h treatment with DMEM containing 1.25 μM U18666A.HepG2 cells (human hepatocellular carcinoma cell line) were grown in Dulbecco's Modified Eagle's Medium , containing penicillin (1 × 10The viability of NPC-like HepG2 cells treated with multi-Lac-β-CD (DSL5.6) was assayed by the WST-1 method. A cell counting kit (CCK) from Wako Pure Chemical Industries  was used for the analysis. The WST-1 method has been described in-depth in our previous publication .Kd) were obtained using Q-UP software equipped in the Single-Q.The molecular interaction of multi-Lac-β-CD (DSL5.6) with PNA, one of galactose-binding lectins, was analyzed with a QCM analyzer . Avidin was used for immobilizing biotinylated-PNA on the sensor cuvette. The biotinylated-PNA was blocked with 1% BSA in PBS (pH 7.4). Two μL of multi-Lac-β-CD (DS5.6)  was added to PBS (pH 7.4). The binding curves and dissociation constants (The intracellular distribution of TRITC-multi-Lac-β-CD (DS5.6) in NPC-like HepG2 cells was assayed by confocal laser scanning microscopy, as reported previously .The intracellular cholesterol levels in multi-Lac-β-CD (DS5.6) treated NPC-like HepG2 cells were detected by a cholesterol cell-based detection assay kit , as reported previously . The flup-value < 0.05 was considered statistically significant.Quantitative data was expressed as the mean ± standard error of the mean (S.E.M.), while the statistical comparisons were made using the Scheffe's test. A


A total of 52 schools participated in the Experimental Implementation Phase of the Project P.A.T.H.S. After completion of the Tier 1 Program, 8,057 students responded to a Subjective Outcome Evaluation Form (Form A) to assess their views of the program, instructors, and perceived effectiveness of the program. Based on the schools' evaluation reports, results of secondary data analyses on four open-ended questions showed that: (a) students felt that they had learned things at the personal, interpersonal, familial, and societal levels; (b) they appreciated the program design, instructors' performance, learning process, and program effectiveness; (c) they generally had positive comments on instructors attitude and teaching process; and (d) they made some suggestions on how the program and its implementation could be improved. The present study, based on qualitative data of subjective outcome evaluation, provides additional support for the effectiveness of the Tier 1 Program of the Project P.A.T.H.S. in Hong Kong.


Tobacco mosaic virus (TMV) causes severe plant diseases worldwide; however, effective antiviral agents for controlling TMV infections are not available. This lack of effective antiviral agents is mainly due to the poor understanding of potential targets associated with TMV infections. During infection, the coat protein (CP), which is delivered by viral particles into susceptible host cells, provides protection for viral RNA. Here, we found that Ningnanmycin (NNM), a commercially used plant antibacterial agent, inhibits the assembly of the CP by directly binding several residues. These interactions cause the disassembly of the CP from discs into monomers, leading to an almost complete loss of pathogenicity. Substitutions in the involved binding residues resulted in mutants that were significantly less sensitive to NNM. Thus, targeting the binding of viral CPs through small molecular agents offers an effective strategy to study the mechanism of NNM. Plant viral diseases cause significant losses in agriculture and low-cost agents capable of effectively controlling such diseases are in great need. Antiviral compounds that target RNA and chemicals that aim at stimulating plant disease resistance have received intensive attention in recent years , 2. Howein vitro through physical regulation  and 10 mmol/L β-mercaptoethanol). The cells were thawed, lysed using a supersonic device, and centrifuged at 13,400 ×g for 30 min at 4°C. After passing through a 0.22-mm syringe filter , the supernatant was loaded onto a Ni2+ Sepharose High-Performance Column , washed with five column volumes of 40 mmol/L imidazole and eluted with 400 mmol/L imidazole. The elute was concentrated using an Amicon Ultra centrifugal filter device (Millipore) with a 10 kDa MW filter and then loaded onto a HiLoad 16/60 Superdex 200 pg column equilibrated with a dialysis solution (10 mM sodium phosphate buffer and 100 mM sodium chloride solution [pH 7.2]). After thrombin digestion of 6×His tags at 4°C overnight, TMV CP was further purified by SEC using a Superdex 200 10/300 GL column  and a buffer containing 10 mM sodium phosphate and 100 mM sodium chloride solution (pH 7.2). Proteins were then concentrated to 8.7 mg/mL for biochemistry experiments using amicon ultra centrifugal filter units (Millipore) with a 10 kDa MW cutoff. The target proteins were briefly stored at 4°C. TMV (common strain) was isolated from infected tobacco leaves (grown in the greenhouse at Guizhou University) and purified [To prepare TMV CP discs, TMV was purified using the method described by Gooding and modified by Shire , 26. TMVpurified , 26. The2+ beads and the tags were cleaved using thrombin. TMV CP was examined for the formation of oligomers with SEC methods. Discs were formed by incubating the oligomers in 10 mM sodium phosphate buffer containing 100 mM sodium chloride (pH 7.2) at 22°C for more than 12 h [The recombinant proteins with 6×His tags were purified using Niβ-lactoglobulin (35 kDa), ribonuclease A (13.7 kDa), cytochrome (13.6 kDa), aprotinin (6.51 kDa) and vitamin B12 (1.36 kDa). The protein was monitored by measuring the absorbance at a wavelength of 280 nm. For TEM [2O. The grid was stained with 20 μL of 2% aqueous solution of tungstophosphoric acid for 90 s as a negative stain. Images were obtained at the Electron Microscope Lab of Zunyi Medical University using a Hitachi H-7650 transmission electron microscope  with 80 kV accelerating voltage. The 17% native PAGE [−1 (ΔH), binding constant in mol−1 (K) and the number of molecules per TMV CP proteins (n) were floating during the fit. The binding free energy, ΔG, and reaction entropy, ΔS, were calculated using the equations, ΔG = −RTlnK  and ΔG = ΔH−TΔS. The Kd was calculated as 1/K. Then, the binding was calculated for MST [N. tabacum cv. K326 were mechanically inoculated with equal volumes of TMV (0.5 mg/mL). After 72 h, 1-cm diameter leaf discs were removed. The leaf discs were floated on solutions of NNM and on buffer  as a negative control. Discs of healthy leaves were floated on buffer as a mock. All of the leaf discs were kept in a culture chamber at 28°C for 48 h, and then, the TMV concentration in the leaf disc was determined. Leaf discs were ground in 5 × protein loading buffer , and then, 5 μL of sample were loaded on a polyacrylamide gel (5% stacking gel and 12% separating gel). After SDS-PAGE, TMV protein bands were transferred at 90 mA for 1 h onto a polyvinylidene fluoride membrane  using an electrotransfer system (Bio-Rad). The membrane was washed in TBST  and blocked with 5% nonfat milk powder in TBST for 1 h at 37°C. The membrane was washed three times, each time for 3 min with TBST, and reacted with a mixture of 1:30,000 alkaline phosphadase-conjugated anti-rabbit IgG  and 1:200 polyclonal antibody of TMV for 2 h at 37°C. After it was washed three times, each time for 3 min with TBST, the membrane was incubated in substrate buffer  with 330 μL/mL nitrotetrazolium blue chloride and 165 μL/mL 5-bromo-4-chloro-3-indolyl phosphate for 3–5 min in the dark until the bands of the CP were clear. For the MD simulation, the X-ray crystal structure of TMV CP was downloaded from (Protein Data Bank) PDB (PDB ID: 4GQH). The initial structure was revised by adding lost residues and hydrogen atoms and checking bonds and bumps. Subsequently, the energy was minimized for 2,000 steps of the steepest descent calculations and 2,000 steps of conjugated gradient calculations by using Sybyl 7.0  and Gaussian03 program at the HF/6-31+G* level [NNM and TMV CP binding was initiated by adding 5 mM anti-TMV compounds to 0.5 mM (8.7 mg/mL) TMV CP discs and incubating for 1 h. Then, 0–10 mM NNM was added to the reactions for 1 h. The oligomer status of TMV was then analyzed by SEC . SEC washrome 13. kDa, apr For TEM , 23, selive PAGE -31 was pive PAGE  were recive PAGE  were per for MST , 35 Monoin B12 1. kDa. TheG* level . The atoG* level . During −1Å−2. Water and counterions were minimized. The backbone atoms of the protein were then fixed with the ligand, side chains, and other atoms that are free to move. Finally, the entire system was fully minimized without any constraint. In each step, energy minimization was first performed using the steepest descent algorithm for 2,000 steps, and subsequently, the conjugated gradient algorithm was used for another 3,000 steps. The MD simulation was performed under periodic boundary conditions using the sander module of the Amber9 program. First, the system was fixed to heat only the water and counterions for 10 ps to make sure the solute was fully solvated. Then, the entire system was gradually heated from 10 K to 300 K using the weak-coupling method and equilibrated for 100 ps with the protein backbone fixed [Based on the docking results, two binding models were selected for MD simulation. Prior to the simulation, the electrostatic potential and partial atomic charges were determined by performing an electrostatic potential fitting according to the Merz–Singh–Kollman scheme with the Gaussian-optimized geometries , 41. Thene fixed . Lastly,ne fixed , 46, whine fixed . The timTo verify the results of the MD simulation, four mutations , S15G, SNicotiana glutinosa (N. glutinosa) were mechanically inoculated with the reconstructed full-length virus or the reconstructed mutated virus (see the TEM methods). The local lesion numbers were recorded 3–4 days after inoculation.Leaves of


Although the importance of evaluating implementation fidelity is acknowledged, little is known about heterogeneity in fidelity over time. This study aims to generate insight into the heterogeneity in implementation fidelity trajectories of a health promotion program in multidisciplinary settings and the relationship with changes in patients’ health behavior.n = ± 70). Higher fidelity scores indicate a more complete implementation of the program’s core components. A hierarchical cluster analysis was conducted on the implementation fidelity scores of 17 organizations at three different time points. Quantitative and qualitative data were used to explore organizational and professional differences between identified trajectories. Regression analyses were conducted to determine differences in patient outcomes.This study used longitudinal data from the nationwide implementation of an evidence-informed physical activity promotion program in Dutch rehabilitation care. Fidelity scores were calculated based on annual surveys filled in by involved professionals (n = 9), ‘moderate and improving fidelity’ (n = 6), and ‘unstable fidelity’ (n = 2). The stable high fidelity organizations were generally smaller, started earlier, and implemented the program in a more structured way compared to moderate and improving fidelity organizations. At the implementation period’s start and end, support from physicians and physiotherapists, professionals’ appreciation, and program compatibility were rated more positively by professionals working in stable high fidelity organizations as compared to the moderate and improving fidelity organizations (p < .05). Qualitative data showed that the stable high fidelity organizations had often an explicit vision and strategy about the implementation of the program. Intriguingly, the trajectories were not associated with patients’ self-reported physical activity outcomes  = − 1032, p = .303).Three trajectories were identified as the following: ‘stable high fidelity’  contains supplementary material, which is available to authorized users. Once a health promotion program  has shown to be effective in changing individual behavior towards a healthier lifestyle, the next step is to implement the program on a larger scale , 2. HoweImplementing core components of a health promotion program in a multidisciplinary healthcare setting, such as rehabilitation care, can be complex due to the involvement of professionals with different specializations and the heterogeneous target population , 11. ConAlthough the importance of evaluating implementation fidelity in health promotion programs is widely acknowledged , 17, notTherefore, the aims of this study were (1) to identify implementation fidelity trajectories of a health promotion program in a multidisciplinary setting, (2) to explore which organizational and professional characteristics are associated with these trajectories, and (3) to test whether changes in patients’ health behavior are different between these trajectories.To gain more insight into the heterogeneity of the implementation fidelity trajectories, we used data from the nationwide implementation of the Rehabilitation, Sports and Exercise (RSE) program. The RSE program is a multicomponent physical activity promotion program in which core components are defined based on results of a previous RCT-study , 20. ThiThis study has theoretical and practical contributions. To the implementation fidelity literature e.g., , 18), th, th18]),Three phases of an innovation processes: adoption, implementation, continuation;Five domains of determinants influencing the innovation process: socio-political, organization, professional, program, patient;The implementation strategy.The conceptual framework described by Wierenga et al. (2013)  was usedThe organizations participating in our study received support to implement the RSE program during a 3-year period (2013–2015), which we defined as the implementation phase. The continuation phase  started after the program period (January 2016). We used Wierenga et al.’s classification of the determinants  for the description of the identified trajectories during the implementation phase. Based on Hoekstra et al. (2017) , we assuThe analyses were based on data from the Rehabilitation, Sports and Active Lifestyle (ReSpAct) study, which is a multicenter longitudinal study designed to evaluate the RSE program on organizational- and patient-level , 22. SurImplementation fidelity was assessed at 17 locations consisting of 12 rehabilitation centers and 5 rehabilitation departments of hospitals. Inclusion criteria for these organizations were (1) willingness to implement and continue the RSE program, (2) willingness to support the ReSpAct-study, (3) being involved in the implementation of the RSE program during the whole program period.n = 2).The program coordinators  initiated and coordinated the implementation process in the participating organizations on a national level. Information about organizations’ adoption process and implementation strategy was obtained from logbooks of the program coordinators  provided information about the implementation process in their organization. Inclusion criteria for professionals were (1) being actively engaged in the implementation of the RSE program, (2) working at the location of the organization that received financial incentive for implementing the program. All professionals meeting the inclusion criteria were asked to participate in the study by filling in a survey at three points in time. In each organization, a project leader and at least one counselor had been appointed to implement and execute the program. In some situations, one professional fulfilled multiple roles .All adult patients participating in the RSE program were asked by the counselors to participate also in the linked ReSpAct-study . Data frPatients receive an intake session with a rehabilitation professional  to discuss their interests in participation in sports and exercise activities during their rehabilitation treatment.Patients take part in sports and exercise activities during rehabilitation.Patients are referred to the Sports Counseling Center (SCC) at the end of their rehabilitation treatment.Patients receive tailored advice on active lifestyle during a face-to-face consultation at the SCC by using motivational interviewing (MI) to initiate a behavioral change .Patients are provided with four telephone-based counseling sessions initiated by counselors working in the SCC to further stimulate patients in maintaining an active lifestyle after rehabilitation.The counselors working in the SCC collaborate with sports and exercise providers in the community.The evidence-informed RSE program consists of six core components :Patientsn = 17) received support to implement the RSE program in their daily routines. The support consisted of national and regional meetings for involved professionals, advice and support from program coordinators, financial incentives, provision of promotion material, and educational courses in MI.The organizations . The survey contained questions about the extent to which the core components of the RSE program were implemented in the organization. Completing the survey took approximately 30 to 40 min. Surveys were adapted to the role of the professional  indicating that the survey included questions associated with the tasks of the professionals.n = 17), a total fidelity score was calculated by counting the number of questions dichotomized into ‘yes’ and dividing it by the maximum score . Total fidelity scores are presented in percentages in which higher scores are associated with a more complete implementation of the core components of the RSE program.Implementation fidelity was measured by calculating a total fidelity score (%) for each organization at each time point T0, T1, T2) . The fid, T2 . ThInformation about professional  and organizational characteristics  was derived by collected data from the surveys filled in by professionals at the start (T0) and end (T2) of the program period. Questions were closed-ended, answered on a 4 or 5 Likert scale.Qualitative data from interviews conducted with rehabilitation professionals and program coordinators were used to verify the quantitative data and identify additional relevant characteristics. Researcher FH (first author) invited rehabilitation professionals  for a semi-structured interview. During these interviews, open questions were posed about perceived facilitators and barriers to the implementation and continuation of the RSE program in their organization. The interviewer probed for the organization’s plans and procedures concerning the implementation process.Researcher FH invited the program coordinators for two separate interviews. In the first interview, open questions were asked about their experiences with the program’s implementation in each participating organization. The second interview focused on program coordinators’ perceptions of factors influencing the implementation process. Further details about the data collection procedures in the interview rounds are described in Hoekstra et al. (2017) .Information about patients’ outcomes was collected by survey data from patients enrolled in the ReSpAct-study. The baseline measurement took place between 3 and 6 weeks before the end of the outpatient rehabilitation treatment and the follow-up measurement took place 14 weeks after the end of outpatient rehabilitation. Patients’ physical activity behavior at baseline and follow-up were measured with the adapted version of the Short QUestionnaire to ASses Health enhancing physical activity (SQUASH) , 31. TheData analyses consisted of four main steps. Firstly, an agglomerative hierarchical cluster analysis based on Ward’s method  with a sU tests were performed to assess differences between the clusters of organizations in implementation determinants in order to externally validate the clusters and explain differences between the clusters. These determinants were related to the professionals  and the organization  measured at the start (T0) and end of the program period (T2). Determinants were selected using the outcomes of a previous qualitative study on perceived facilitators and barriers to the implementation of the RSE program ).first tension that arose concerned the dichotomy between implementing according to the nationwide evidence-informed protocol and according to the local organization’s health promotion vision. The former is an implementation fidelity requirement [The uirement , whereasuirement . Our finuirement . A changuirement , 46. Theuirement , 18. Basuirement , 48.second tension we came across was the balancing between physicians’ active engagement and management’s buffering of scarce physician resources. On the one hand, engagement of the most influential professionals, i.e., physicians, helps implementation, and sustenance [The stenance , 49. On stenance , the extthird tension involves the balancing between the choice for a high fidelity implementation and for a cost-efficient implementation strategy. The stable high fidelity organizations feature a strategy aimed at high fidelity implementation, while our data suggest that the ‘moderate and improving fidelity’ organizations stressed cost-efficient implementation. Consequently, the moderate and improving fidelity organizations achieved comparatively lower levels of implementation fidelity, yet these organizations showed higher continuous improvement in implementation fidelity over time. Apparently, an incremental implementation trajectory was more affordable and moderate fidelity achieved over the study’s time span, did not result in lower patient outcomes than in the stable high fidelity cluster. On the contrary, the routinization of the program components was lower in the moderate and improving fidelity organizations suggesting less promising results regarding the sustainability of the program fidelity on the longer term. These insights are relevant for investors who want to implement and scale up their health promotion programs. They should make decisions about how they want to invest their money and how to deal with a trade-off between quality and efficiency?The The results showed no significant differences in changes in patient outcomes between the stable high fidelity and moderate and improving fidelity organizations. This is in contrast with the review of Durlak and DuPre (2008) . A possiThe study’s major strength is its longitudinal design including multisource data  based on mixed methods . We collected detailed information about the implementation process within each participating organization from different perspectives  creating a rich and unique dataset. As a result, we were able to apply triangulation techniques making our findings relevant for both implementation research and practice.The study’s procedure for calculating implementation fidelity has both merits and limitations. The measurement of the total fidelity scores at the organizational level enabled the identification of variety in implementation fidelity trajectories. Clustering of the emerging trajectories enabled the exploration of associations with both the organizational and the aggregated professional- and patient-level factors, following a multiple case study logic .As to the limitations, firstly, the measurement method for these fidelity scores relied on self-constructed items. Moreover, in calculating the total fidelity scores we dichotomized each item and weighed each item equally, but we have no way of knowing whether each item is equally important. Further research is necessary to gain more insight into the reliability and validity of this measurement method.A related limitation concerns the influence of missing items on the total fidelity scores. Any missing item was conservatively counted as ‘zero,’ which means that the observed fidelity scores may have been somewhat lower than the real scores. This conservative measure seems legitimate: to the extent that an organizations’ professionals do not participate in the program’s evaluation, they can be regarded as less engaged with the program.n = 2) prohibited an analysis of its characteristics. Nevertheless, the deviating fidelity trajectory in these two organizations reflects real-world phenomena that deserve further study. The steep decrease and increase in fidelity scores over time underscore the dynamic complexity of implementing programs in multidisciplinary settings.A final limitation is that the third cluster’s small sample size