Education and training are two essential ingredients for a successful career. On one hand, universities provide students a curriculum for specializing in one’s field of study, and on the other, internships complement coursework and provide invaluable training experience for a fruitful career. Consequently, undergraduates and graduates are encouraged to undertake an internship during the course of their degree. The opportunity to explore one’s research interests in the early stages of their education is important for students because it improves their skill set and gives their career a boost. In the long term, this helps to close the gap between skills and employability among students across the globe and balance the research capacity in the field of computational biology. However, training opportunities are often scarce for computational biology students, particularly for those who reside in less-privileged regions. Aimed at helping students develop research and academic skills in computational biology and alleviating the divide across countries, the Student Council of the International Society for Computational Biology introduced its Internship Program in 2009. The Internship Program is committed to providing access to computational biology training, especially for students from developing regions, and improving competencies in the field. Here, we present how the Internship Program works and the impact of the internship opportunities so far, along with the challenges associated with this program. Academic training is an important aspect of career building. Students pursuing higher education, in particular, are encouraged from early-career stages to explore opportunities that will assist in both choosing a career path as well as building their skill set. Few things boost a student's career as much as hands-on experience in a field of interest. An internship provides such an experience and is an increasingly important route for students of Science, Technology, Engineering, and Math (STEM) to gain new skills, as well as for employers to find new talent. While internships are commonplace in industry , fewer oBenefits of an internship are not limited to the participating student. The labs they work at stand to benefit as well, with the integration of good research practices from other research environments into their own. Bringing ideas together to unite research efforts promotes an inclusive and accessible scientific culture for all. Furthermore, talented and highly motivated students constitute an invaluable potential for group leaders looking for students to undertake projects in their labs. On a global level, knowledge sharing across nations encourages scientific and technological innovation, which in return impacts economic growth.Securing an internship in an established lab can be challenging. Since funding capacity and opportunities are limited, internships in academia are typically scarce. While these challenges are prevalent across the world, the barriers to maximizing career potential are exacerbated for students based in developing nations . This crIn this article, we describe the Internship Program’s operation and impact. The article also discusses the associated challenges that need to be addressed and overcome in order to further develop the initiative and continue to build the next generation of computational biology researchers.The Internship Program is aimed at improving accessibility to high-quality research for students from developing regions. To this end, the EIC strives to manage the internships in an efficient and transparent manner , providiThe EIC then decides on an operational timeline and opens a call for applications requesting documents such as a cover letter, recommendation letters, certificate of enrollment at an academic institution, and a curriculum vitae. The team mobilizes its principal source of outreach, the Regional Student Groups (RSGs), to circulate the call to as many students as possible . In addiApplications are received through a submission system tailored for the Internship Program by the Web Committee. The applications received before the deadline are reviewed by a review committee—formed by the SC members—based on the criteria provided by the PI and the overall quality of the application. Recommended applicants are forwarded to the PI, who then conducts interviews to select a final candidate. On average, the committee receives over 40 applications for each call, of which up to five are forwarded to the PI.The overall process of recruitment takes around two months. While deciding the start date of the internship, PIs are advised to take into account the interview procedure and the visa application process, which may take a substantial amount of time for students from developing countries.Since the beginning of the Internship Program, eight students from developing nations have benefited from research stays in labs across Europe and Australia . All theWhile the main beneficiary of an internship is the participating student, the home and host labs also tend to benefit from the student’s internship experience. The student acquires valuable research skills and builds new contacts, which could open new doors for their future success. Many of those new skills could be transferred to peers at their home institution upon return from the internship. In a similar fashion, the host lab not only gains new perspectives that come with having a visitor in the lab but also possibly a new collaborator over time. In addition, living abroad even for a short period of time provides insights into new cultures, which is useful when one is collaborating across nations and attending international conferences.The most obvious reward of participation in an internship is the opportunity to hone skills that a smaller or less-established lab cannot provide. Internships help students gain exposure to the current research trends, as well as hands-on access to the latest equipment and facilities that may be unavailable at the home institution of the student. For example, Mohammed Rehan, currently an assistant professor at the King Fahd Medical Research Center in Saudi Arabia, attributes a brand new perspective on his graduate work to the internship in Professor Burkhard Rost's Lab at the Technical University of Munich. Rehan, who spent three months of 2010 in Munich, profited from the discussions at group meetings and guidance from colleagues, which helped shape his project back in India. “The acquired knowledge helped me in how to present my work, asking new questions and designing new projects,” he recollected.The experience can also expose students to parts of our field they might not have had access to otherwise. Rohit Thakur had the opportunity to work with Dr. Alex Bateman’s group at the European Bioinformatics Institute (EBI) while he was a student at the Vellore institute of Technology in India. The internship led him to discover the breadth of research being carried out in the field of computational biology at a premier research organization. Currently a Marie Sklodowska-Curie research fellow at the University of Leeds in the United Kingdom, Rohit praises his experience as an opportunity that helped establish his research interests and acquire interdisciplinary proficiency in computational biology and programming.Rohit’s experience also exemplifies another benefit of the program in that it expands career opportunities by making connections that may not have been possible otherwise. Kaur Alasoo, Dr. Reinhard Schneider’s lab intern in 2010 at European Molecular Biology Laboratory Heidelberg, gratefully mentioned his internship experience and emphasized the positive outcome of the program that helped to shape his future opportunities. He added, “The amazing five months are now over, but I am sure that they will keep influencing my life for many years to come…[I] probably will start my PhD in two years…[T]his internship has changed me a lot. It has encouraged me to more eagerly look for opportunities and pursue my research interests.”Besides the direct benefit of being involved in a cutting-edge research project, being at a well-established research institution carries additional advantages. Academically, these secondary benefits include events such as seminars, symposia, workshops, as well as the diverse everyday interactions that are commonplace at major research institutions. Such events, along with the quality of the research itself, serve as great sources of motivation and inspiration for students, particularly those from developing nations where there may be logistical constraints to the organization of research-oriented events, such as limited funding for science. The secondary benefits are also social; the experience of carrying out research at an institution abroad helps students appreciate the cultural diversity in the country they visit, which contributes to their personal development.In concert with the benefits to the students, the Internship Program creates a unique opportunity for the host lab because the intern will contribute to the diversity of the research group. The internship facilitates interaction between the intern and the graduate students in the host organization and creates an environment for mutual scientific and cultural interchange. The peers have a first-hand and up-to-date perspective on the country of the intern and discuss pressing scientific and sociopolitical issues. In fact, the impact of having an intern from abroad is not only limited to the peers working at the host institution. The peers from other research groups at the host institution also benefit from such intercultural interactions, potentially igniting a reconsideration of stigmas that might be attached to certain countries.Furthermore, upon completion of the internship, when students return to their home institution, they will undoubtedly share some of the knowledge they have gained abroad. For example, Dedan Githae, another past intern of the Rost lab who participated in the Internship Program from Kenya, refers to his internship as a stimulating and inspiring research environment that provided him transferable knowledge. In his words, participating students “get to learn new and effective skills from leading research groups in developed countries, leading to development and improvement in quality of research back in their own countries. This is not only effective but leads to establishing contacts for the future.”The scientific and cultural exchange between the intern and the host group could also bring novel research questions to attention and initiate collaboration. As a result, it could help PIs to extend their professional and personal network to otherwise less-accessible places to bridge the scientific gap between developed and developing nations.Dr. Alex Bateman, a host PI in 2014, expressed his appreciation of the program, saying, “I was delighted to take part in the ISCB-SC internship program to help give a hand up to excellent students from around the world.” The participating PIs were also satisfied with the enthusiasm of the students: “The students who joined our group through this Internship Program were very active and eager to learn new things,” were the words of Dr. Reinhard Schneider, who hosted two interns in 2010 and 2015.Recently, the outreach activities and social impact of a research group have become increasingly important during the evaluation of grant applications. To this end, involvement in the Internship Program provides a valuable opportunity for PIs to reach out to students from developing countries and contribute to the training of the next generation of researchers in the field of computational biology. As a result, this could strengthen the social impact of the research group and center.From a logistics perspective, the main advantage of this program for a hosting lab is that the EIC facilitates what is otherwise a time-consuming process of finding a motivated and talented student. As outlined above, the EIC acts as an intermediary, advertising the position, evaluating applicants, and providing a list of qualified candidates according to the requirements specified in the offer. Furthermore, the vast reach of the SC network highly increases the probability of finding a suitable candidate for the host lab. Dr. Bateman notes the effective role of the SC in the Internship Program by saying, “The ISCB Student Council made the process very simple and preselected the most able students for me to interview.”In the long run, the experience of students positively influences computational biology research worldwide and has already had social and scientific impact on their host and home institutions. This includes the transfer of current computational techniques to their research environment, as well as introducing good practices from the host to the home lab, such as task management, resource allocation, and social interactions. Furthermore, participating students pursuing their careers as researchers are likely to participate in initiatives of a collaborative nature in the future, such as the Internship Program . AdditioThe Internship Program has been a rewarding yet challenging initiative to sustain since its inception. The EIC works to liaise between the students from developing nations and research groups from developed nations with the aim of improving equality of opportunity and increasing diversity in the field of computational biology. The Internship Program relies on the support of PIs and the members of the SC for hosting and coordinating the internship opportunities for students from developing countries, placing certain challenges on its sustainability.Increasing the visibility of the Internship Program among PIs has been one of the major challenges since the establishment of the program. In an effort to publicize the program, the SC posts information about the Internship Program on its website , and theDespite these efforts, the most effective form of publicity that the program has received so far has been presentations during the annual Intelligent Systems for Molecular Biology (ISMB)  meeting The outreach of the Internship Program could be further increased by organizing workshops, seminars, and career sessions for students across different countries in an effort to reach out to non–ISCB-affiliated potential PIs and interns. During these events, participants would be encouraged to both look out for potential internship positions (if they are eligible) and inform the group leaders they know about the program. To achieve this, the SC aims to leverage the globally spread RSG networks, which typically organize local events and can promote the program among both students and group leaders ,16. In aWhile it is important for the SC to reach a broader community to increase the internship offers, finding the most suitable candidates is also an essential component. Besides securing the best candidate, the SC also strives to balance the participants based on their gender and country of origin. However, the internship offers have seen varying demand from students across different countries. This can be partly attributed to geographical barriers, such as the narrow reach of the RSGs in certain continents like Africa , hinderiOn the other hand, the timing of the internship also has a strong effect on the number of students who apply for the position. Internships that span the break between academic years, such as summer months, usually receive more applications than those taking place during the rest of the year because the students cannot accommodate an internship during the academic year. Therefore, the EIC tries to organize internships during the summer to maximize the number of applicants.Among all the challenges that the Internship Program experiences, the financial and bureaucratic burdens of hosting internships are arguably the toughest. The program lacks financial independence and depends entirely on the hosting institution’s funding to support the internship applicants. This may imply allocating budget for stipends for the duration of the internship, travel expenses, and in many cases, expensive visa fees. Consequently, the scarcity of funds could prevent some group leaders interested in participating in the Internship Program. Furthermore, having adequate funds for the aforementioned expenses does not warrant the participation of a PI in the program. Restrictions imposed by funding agencies on how funds can be used or stipulations in some national grants on supporting only students from the host country preclude participation in the program.Another major challenge that the program faces is associated with tightened visa rules that hamper the movement of people between countries, especially from developing countries. These restrictive rules involve complicated entry requirements, unclear application procedures, and increased transaction costs, which introduce substantial hindrances for both students and PIs and, more importantly, discourage movement and cooperation . TherefoThe EIC comprises volunteers of the SC, including both full-time students and early-career researchers, who oversee the coordination of the Internship Program in their spare time. Consequently, during peak times of the Internship Program  when an internship is announced and the selection process takes place, the program-related tasks tend to be demanding for the committee members. This creates a workload imbalance for the volunteers, requiring more time commitment during certain months and less-intensive dedication for the rest of the year. To tackle this issue, the EIC usually establishes ad hoc evaluators for each internship position, by which volunteers from other committees or RSGs participate in assessing applications—determining the eligibility of the applicant, interpreting official documents issued by the home institution , performing quality checks, and performing skill evaluation. The EIC also tries to optimize its communication workload from applicants by providing a clear definition of the requirements and answers to potential (frequently asked) questions students might have.In an attempt to provide more benefits to the community, as well as to motivate volunteers, the EIC is working on introducing new initiatives, such as building a resource portal for computational biology education. The portal will be an open source online knowledge base dedicated to bioinformatics and computational biology researchers across the globe. The resource is planned to contain various tutorials and exercises ranging from basic to advanced bioinformatics topics and algorithms and the latest journal articles and books, and it will provide an interface to submit personalized materials  to assist researchers. The goal of this portal is not to replicate the efforts of ISCB Education Committee  and the The Internship Program plays a key role in bridging the gap in computational biology research between developing and developed countries. The program relies on the host labs to sponsor students from developing nations, and the SC strives to minimize the effort and time commitment from a PI for recruiting an intern by streamlining the entire pre-internship process, from advertising the position and collecting applications to screening the applications. The breadth of the SC and affiliated RSG network ensures that the calls for applications reach the countries where these RSGs are located . The extTo ensure the continuity and success of the Internship Program, the SC aspires to engage more PIs and students to contribute to the program. Ideally, the EIC would like to increase the number of positions per year steadily, but this depends on resources, such as the availability of funded positions, the number of students interested in applying for them, and volunteers dedicated to their handling. Currently, the SC leverages various ISCB conferences , SC’s syThe support of an influential professional body such as the ISCB, which plays a major role in advancing the field of computational biology, becomes crucial in taking the Internship Program initiative forward. The ISCB has helped establish the SC, and its leadership continues to provide guidance, support, and financial aid, as well as promote various SC activities. Gauging the potential of the Internship Program, the ISCB has recently announced that it will provide funding to support travel costs for internship positions offered by ISCB-affiliated PIs  through The SC also seeks to increase the number and expand the diversity of students benefiting from the program by trying to accommodate the needs of students across different countries. Accordingly, the SC tries to offer internship opportunities during the largest interval in the academic calendar, facilitating the participation of students who are enrolled in courses during the fall and spring semesters. To expand the reach of the program to countries where French rather than English is the second language, the SC looks forward to reaching out to PIs from French-speaking developed countries as potential hosts.The Internship Program has a well-defined organizational structure that has seen success over the past eight years, placing eight students from developing nations as interns in four research labs across Europe and Australia. The internship positions kindly offered by a number of PIs have created a monumental impact on the careers of the participating students, prompting the SC to ensure the continuity of the Internship Program. However, various factors put its sustainability in jeopardy, including scarce funding, difficulties in advertising, limited participation, and bureaucracies associated with international travel for the interns. Some of these roadblocks are more easily addressed than others. To address the lack of participation, more host labs are needed to step forward and contribute to initiatives like the Internship Program, and on the other hand, students must realize the importance of exposure beyond classroom lectures and actively look for opportunities to broaden their skills. The SC is dedicated to launching the careers of more students from developing countries, and together with the support and encouragement from sponsors and international research organizations, the initiative holds great potential to shape the future of the field of computational biology.


The aim of this proof-of-concept pilot study was to assess the usefulness of a mentoring support program with a goal to improve hope for recovery in people with an eating disorder.Significant improvements (p ≤ 0.05) in hope for recovery were found for the mentees in the following domains: social relationships (p = 0.027), romantic relationships (p = 0.032), family life (p = 0.047), work (0.003) and overall scores (p = 0.003). There were no significant findings for any of the areas for the mentors. Despite this, mentoring programs that focus on improving hope may provide a valuable adjunct support for those in treatment for an eating disorder.The online version of this article (10.1186/s13104-017-3026-6) contains supplementary material, which is available to authorized users. Mentoring in the context of eating disorders, is a relationship between “a person with a lived experience of an eating disorder who has recovered” (mentor) and a person with an eating disorder (mentee) , 2. MentThe pilot study was advertised, in eating disorder treatment facilities and online through relevant websites. Fifty-four individuals responded, see Fig. The mentoring program was an adjunct to individuals’ eating disorder treatment. As no mentoring programs have assessed the promotion of hope, a proof-of-concept (POC) study was considered. Proof-of-concept studies are considered when the objective of the study is to obtain an initial evaluation of the potential benefit of a treatment or program . ParticiThere were two types of measures used in the study: (1) those to assess the benefits of the program and; (2) those to monitor the health and well-being of the participants during the study.The primary outcome measure was hope. Hope was measured using the validated Domain Specific Hope Scale . This scThe secondary outcome measures included quality of life, distress and the mentoring relationship. Quality of life was assessed with the validated self-report questionnaires; SF-12  and the Distress was assessed with the Kessler Psychological Distress Scale (K10) . The K10The perception of the mentoring relationship was assessed using the validated match characteristic questionnaire (MCQ) . The MCQThese measures were assessed at baseline (before the program started) and at completion of the program except for the MCQ, which was assessed post program only.The following were used to monitor the participants well-being during the study: 12-item short form survey (SF-12)  and the t test comparing baseline and the post treatment results using Microsoft Excel. Standard scoring methods were applied to the validated questionnaires.All outcomes (except for the MCQ) assessing the benefit of the program were analysed using a paired http://www.mentoringevaluation.com. This scores each of the domains. The Australian scoring tool was used and provides a population average for comparison against the mentors only. As the mentees could not be compared to any means there were no comparisons. The MCQ scoring was done only post program.The MCQ was scored using the tool available from Intent to treat was applied for the incomplete questionnaires by carrying baseline over to post-program scores. Outcome measures to assess the health and well-being of the participants were subjected to inferential statistical analysis.The dyad who corresponded via email only discontinued the program 2 weeks after commencement, as the mentee returned to hospital (eating disorder related). This couple did not complete any further questionnaires. In addition one mentee did not complete any of the post program questionnaires.Medians and standard deviations for the data for assessing benefit are reported (see Tables Population averages were only available for the mentors for the MCQ and the mentors were generally similar to the population norms (see Additional file There were no significant results for the mentors. The mentors’ outcome measures remained stable from pre to post program completion.There was a significant increase in hope for the mentees from baseline to post program. The authors found no mentoring and eating disorder trials that assessed hope however there is considerable qualitative research that shows that hope is an important aspect in the recovery process from an eating disorder –10. PrevWhile improvement in hope was seen for the mentees as a group, specifically the two participants with severe and enduring AN had small increases in hope from baseline to post program. Hope is an important factor in the recovery process from longstanding AN. These individuals are often resistant to traditional treatments with high levels of impairment in most aspects of life with poor outcomes . MentoriOne of the concerns of a mentoring program for individuals with an eating disorder is maintaining safety. The study results show that the mentors remained stable during the program and although non-significant in all aspects, except for reported hope domains, the mentees, on average, improved or were stable in all study outcomes, except the EDQoL psychological domain for which there was a small decrease. The reason for the decrease in the psychological EDQoL domain is unknown. The mid-point EDQoL results for mentees also showed a small average rise so it seems unlikely that the end of the program influenced quality of life. Examination of the individual answers to the psychological domain questions did not indicate a specific decrease in any one aspect of psychological QoL. The program can be viewed as ‘safe’ for both mentors and mentees.Interest exceeded expectations and resources indicating that individuals with an eating disorder are interested in a mentoring program. Given this, it is feasible that a larger study could recruit the mentee participant numbers needed. Further given the increase of hope seen and the promising results that indicate program safety, future efficacy randomised controlled trials are warranted.The mentoring support program showed promise in improving hope. The program appears ‘safe’ for both mentors and mentees. Further research involving a larger sample size is needed to replicate these findings.It is important to note that the program had no comparator intervention so whilst the results are promising it is not within the scope of this study to determine its efficacy.


The RE-AIM (Reach Effectiveness Adoption Implementation Maintenance) planning and evaluation framework has been applied broadly, but users often have difficulty in applying the model because of data collection needs across multiple domains and sources. Questions in the more common “who, what, where, how, when, and why” format may be an effective guide to ensure that individual participants, organization staff, and the perspectives of the setting are considered in planning and evaluation. Such a format can also help users in typical community and clinical settings to identify which outcomes are most valued and to focus limited measurement resources. Translations of RE-AIM that are easy to understand and apply are needed for application in real-world community and clinical settings where research and evaluation resources are limited. The purpose of this article is to provide simplified, pragmatic, user-centered and stakeholder-centered recommendations to increase the use of RE-AIM in community and clinical settings and in translational research. The RE-AIM (Reach Effectiveness Adoption Implementation Maintenance) framework was first developed to help make research findings more generalizable by encouraging scientists and evaluators to balance internal and external validity when developing and testing interventions . The goaThe RE-AIM framework encourages planning strategies that can reach the most people who have health disparities, effectively achieve and maintain positive health outcomes, be widely adopted by diverse settings and staff, be consistently implemented at a reasonable cost, and be sustained among varied settings by staff members with a range of expertise . From anThe Diabetes Health Connection, which included interactive technology and health coach support for patients with diabetes, provides an example of how RE-AIM can be applied to a programmatic intervention . The stuAlthough RE-AIM has been applied for varied public health issues and intervention targets, challenges arise when considering the planning and evaluation of translational projects in typical clinical or community settings where interventions may be complex and multileveled. Furthermore, primary data collection across all RE-AIM indicators is challenging given available resources. For example, some users have reported that doing a “full RE-AIM application” is overly burdensome or not possible. In such cases, a pragmatic application of RE-AIM may be warranted . We feelEven with substantial funding, literature reviews on studies reporting use of the RE-AIM framework have generally produced only incomplete applications of the model. Kessler et al reviewed 42 National Institutes of Health (NIH) grant applications that used the RE-AIM framework and found that only 10% proposed to address all 5 RE-AIM dimensions . Within The purpose of this article is to provide and discuss the use of a series of a familiar set of “who, what, when, where, how, and why” pragmatic questions based on the RE-AIM framework to guide the planning and evaluation of intervention strategies  when evaluation resources are limited. We also provide decision points to guide how RE-AIM information can be pragmatically gathered and used in planning programs and evaluating relevant outcomes. Following this, and to make more concrete pragmatic uses of RE-AIM, we provide example of pragmatic uses of RE-AIM from the literature ,10 for bWe present the key pragmatic planning questions and measuring suggestions for stakeholders to consider for each RE-AIM dimension . Users aWho is (was) intended to benefit and who actually participates or is exposed to the intervention? This is reach at the level of patients, clients, or participants. It is important to consider not only how many persons participate out of those intended or targeted, but also the characteristics of those who take part compared with those who do not. Given the impact of health disparities, participation levels of various key underserved and vulnerable subgroups are especially important.What are (were) the most important benefits you are trying to achieve and what is (was) the likelihood of negative outcomes? “What” refers to the effectiveness and individual-level maintenance components of RE-AIM at the patient, client, citizen, or participant level. Responses to this question include defining the key or desired outcomes and the impact on different subgroups . Additional outcomes include quality of life indicators and any negative effects or unintended consequences from the program.Where is (was) the program or policy applied (and who applied it)? The issue of participation is also related to the settings level. Called adoption in the RE-AIM framework, participation is critical to understand the number and types of organizations, clinics, or agencies that initiate a program or policy. This participation involves considering the characteristics of the settings that are approached or targeted to participate compared with those that actually do. It also involves understanding barriers to and facilitators of adoption as well as how the program or policy fits with organizational priorities and existing workflow. These issues are also important at the suborganizational level, particularly in identifying which staff members try the new compared with those who are resistant or wait for others to attempt adoption first (on first .How consistently is (was) the program or policy delivered, what adaptations to the original plans were made, and how much does (did) it cost? “How” refers to the delivery or implementation of the intervention. How will the program or policy be delivered, managed, or enforced? How will you track adaptations or changes to the program in different settings, by different staff members, or over time? And how will you ensure that such changes do not reduce effectiveness?How questions also include the organizational resources that are required and how much the policy or program costs to implement. Different organizations and decision-makers are concerned about different types of costs . Many organizations are concerned with short-term or medium-term return on investment, but this often means different things to different people . At miniWhen will (did) the initiative become fully operational, how long do results last, and how long will (was) the initiative sustained? “When” refers to organizational, setting, or staff level maintenance. This dimension can be operationalized as the sustainability of the policy or program and includes consideration of many factors, including but not limited to resource availability; alignment of the policy or program with organizational mission, objectives, and goals; and integration into job descriptions and performance evaluations. The timing of results is often important to decision-makers, and the end goal of most policies and programs is sustainability so that activities become institutionalized or normalized as part of the way of doing business (business .Why will (did) the results come about? “Why” questions are concerned with the underlying reasons for intervention’s success or failure and apply across RE-AIM dimensions. Why did they come about? Often why questions are approached via interviews or other qualitative approaches, such as focus groups with users, and in combination with other measures and as ways to probe outcomes .We provide examples of practical application of RE-AIM to issues of planning and outcome evaluation. The “translated,” less jargon-based RE-AIM questions above may be especially helpful in comparing 2 or more alternative programs or actions.Planning application. Finlayson et al provide a helpful illustration of how RE-AIM can be used to help design a program, in their case, a program to prevent falls among people with multiple sclerosis (MS) (http://re-aim.org/re-aim-as-a-planning-tool/). www.re-aim.org) provided initial estimates on most of these issues, but “why” interviews could have provided additional information for planning. A score from 1 through 10 is provided for each dimension, based on the extent to which the various issues within that dimension are addressed (sis (MS) . The Intddressed .Evaluation example. Burke et al used the RE-AIM framework to evaluate an academic–community partnership approach to address childhood obesity ( obesity . They id obesity .CHAMP was delivered at both university and community (YMCA) locations  and included staff from both organizations . Twenty-Implementation (How) of the CHAMP intervention strategies was more than 90%, and assessment of parent comprehension of intervention objectives was used as an indirect assessment of receipt of intervention components directed at the home (66% of content correctly identified) . Burke and colleagues also provided information on the cost of implementation across personnel, supplies, and dissemination with a total of cost of approximately $140,000 across 2 years . Finally, qualitative feedback from participants indicated that the children found the program engaging and fun to participate in (Why). Participants also indicated that providing fun exercise experiences helped distract the children from the intensity of the activities; showing that a variety of healthy food options helped improve dietary intake; and engagement in the program helped the children feel better about themselves  .Although RE-AIM is less complex and more intuitive and understandable than many evaluation models and systems ,14, it iThese challenges in applying dissemination and evaluation models in applied or even research settings are not unusual or specific to RE-AIM. In many ways, this use of RE-AIM for decision-making is parallel to environmental impact assessment procedures or community-informed system dynamics planning  in that This practical application or translation of RE-AIM can be used to evaluate different approaches to a given problem or issue and for various programs, policies, and content areas. Figure www.re-aim.org). We encourage their use and hope that in the future there will be sufficient reports of use of this approach to enable summarizing of results and lessons learned from such applications.In summary, the use of who, what, where, how, when, and why questions based on the RE-AIM model should be useful in both community and clinical settings with limited resources. All of these questions and the RE-AIM framework and tools have been placed in the public domain (


Preventing zoonotic diseases requires coordinated actions by government authorities responsible for human and animal health. Constructing the frameworks needed to foster intersectoral collaboration can be approached in many ways. We highlight 3 examples of approaches to implement zoonotic disease prevention and control programs. The first, rabies control in Ethiopia, was implemented using an umbrella approach: a comprehensive program designed for accelerated impact. The second, a monkeypox program in Democratic Republic of the Congo, was implemented in a stepwise manner, whereby incremental improvements and activities were incorporated into the program. The third approach, a pathogen discovery program, applied in the country of Georgia, was designed to characterize and understand the ecology, epidemiology, and pathogenesis of a new zoonotic pathogen. No one approach is superior, but various factors should be taken into account during design, planning, and implementation. For example, in developing countries where canine rabies is still endemic, a rabies prevention program focused primarily on preventing human deaths by increasing access to vaccines for postexposure prophylaxis (PEP), with little or no simultaneous investment in vaccination of dogs, will undoubtedly save lives but is not as cost-effective as investing in mass canine vaccination aimed at eliminating disease from the primary reservoir  but also on the nature of the disease . The firAn example of the use of a comprehensive (umbrella) approach to program implementation is the Rabies Prevention and Control Program implemented in 2015 in Ethiopia. The program involves collaboration and partnership between the Ethiopian Public Health Institute, the Ministry of Livestock and Fisheries, Addis Ababa Urban Agriculture Bureau, and the US Centers for Disease Control and Prevention (CDC) directed toward priority zoonotic diseases identified by the Ethiopian government in September 2015. At the conclusion of the Ethiopia joint zoonotic diseases prioritization workshop, rabies was identified as the priority disease . Many questions remain about monkeypox virus, including the extent and nature of human-to-human transmission , the precise zoonotic reservoir(s) of the virus, and ecologic determinants of disease incidence  obligations of rapidly detecting, responding to, and controlling public health emergencies to enhance global health security. Minimizing the threat posed by zoonotic diseases is one goal of GHSA. The conceptual framework of One Health provides a model on which to build programs to successfully detect, prevent, and control zoonotic diseases  requires constant refinement and modification consistent with local capacities and needs. For a comprehensive program implementation, as described for rabies in Ethiopia, piloting the broad-based integrated system at several distinct locations was determined to be the key first step so that system gaps or inconsistencies could be addressed and costs estimated before nationwide implementation. In DRC, the program for monkeypox detection and control was built step-by-step on a platform of surveillance, with next steps determined by needs and gaps identified through evaluation of data and surveillance performance. Technical capacities were augmented through ongoing program enhancements and research activities. In Georgia, gaps in scientific knowledge about an emerging pathogen drove the initiation of integrated human, livestock, and wildlife disease surveillance and enhancement of laboratory and research capacity.The lessons learned through the design and implementation of these programs are continuously derived from persons working at different levels of all contributing institutions. Most significantly, the work should focus on eliminating solely vertical program elements  . ProgramFinally, an indispensable element of GHSA zoonotic disease prevention programs is training of the future workforce. Not just in the animal health sector, where sizable gaps are evident, but also training must be performed to ensure that human public health workers appreciate and know about the importance of veterinary medicine and animal health in controlling zoonotic diseases and that young, university-based scientists have the training and experience necessary to address questions and problems posed by endemic and emerging zoonotic diseases. The training of future public and animal health professionals is a huge component of all 3 programs described in this report.Achieving the end goal of an effective, fully integrated program for preventing and controlling zoonotic diseases has many possible approaches. The 3 described here differ in their disease-specific context, but all were equally affected by the situation, the resource base, and the initial technical capabilities of the GHSA partner country in which the program was created. The suggested approaches for zoonotic disease program implementation have limitations. Scientific evidence is scant to support 1 approach over another. There is a need for increased zoonotic disease program evaluation and subsequent publication of empirically based recommendations for program design and implementation based on the identified strengths and weaknesses of various approaches. In the interim, national governments and partners can use the approaches we suggest as a guide during the program design phase when they consider suitable approaches for their specific context and settings.


P < 0.001). The area under the ROC curve (AUC) for the BCP was 0.86 (95% CI: 0.78–0.94). The best cutoff BCP for differentiating between benign and malignant LNs was determined as 36.7%. All the 16 LNs (20.3%) with a BCP lower than 27.9% were diagnosed as benign tissues. Our study suggests that elastography is a feasible technique that may safely help to predict LN metastasis during EBUS-TBNA. We found a clear BCP cutoff value to help define positive and negative LNs.This study aimed to semi-quantitatively evaluate the elastographic imaging color distribution of mediastinal and hilar lymph nodes (LNs), and explored its utility in helping define malignant and benign LNs for lung cancer patients. We prospectively collected patients who underwent preoperative mediastinal staging of suspected lung cancer by EBUS-TBNA. We analyzed the elastography color distribution of each LN and calculated the blue color proportion (BCP). The LN elastographic patterns were compared with the final EBUS-TBNA pathological results. A receiver operating characteristic (ROC) curve was constructed to evaluate the diagnostic value of BCP. We sampled and analyzed 79 LNs from 60 patients. The average BCP in malignant LNs was remarkably higher than that in benign LNs (57.1% versus 30.8%,  PET-CT has been proven to be more efficient than CT in distinguishing benign from malignant mediastinal LNs. However, several research groups have provided evidence to indicate that PET-CT was insufficient to replace mediastinoscopy for mediastinal staging in patients with lung cancer3, as long as some controversies were unresolved4. In recent years EBUS-TBNA has emerged as a revolutionary method to estimate and manage both benign and malignant diseases with higher diagnostic yield, which might also replace traditional mediastinoscopy in patients with potentially resectable non-small cell lung cancer (NSCLC). Elastography is a new technique that has been utilized recently in the diagnostic procedures of focal lesions in breast, thyroid, pancreatic and prostate gland tissues8.The accurate classification of lymph nodes (LNs) as benign or malignant is essential for lung cancer staging. In addition to systematic LN dissection during surgery and pathological diagnosis afterwards, there are several other minimally invasive or noninvasive methods available to detect LN metastasis before surgery, such as computed tomography (CT), endobronchial/endoscopic ultrasound transbronchial needle aspiration (EBUS/EUS-TBNA), positron emission tomography-CT (PET-CT), B-mode morphological ultrasound and elastography. Early studies applied chest CT for nodal staging, using a measurement of 1.0 cm as the threshold beyond which LNs were defined as enlarged and suspected to contain metastasis. However, the results of these studies varied, with pooled sensitivities of 57%, specificity of 82%, positive predictive value of 56% and negative predictive value of 83%15, the quantitative method of evaluating elastography’s role in differentiating benign and malignant mediastinal and hilar LNs is still not well delineated. We conducted this study to investigate the utility of elastography in diagnosing hilar and mediastinal LNs during EBUS-TBNA, and to identify a threshold to distinguish between benign and malignant lesions based on pathological results of EBUS-TBNA and the color distributions of elastographic images, using a newly developed semi-quantitative method.EUS elastography was reported to be a highly sensitive and specific method for detecting malignant involvement of pancreatic lesions and LNs. This led to utilization of elastography in EBUS. Usually, LNs containing neoplastic components are of higher cellularity and vascularity, which would cause more areas of the tissue to be relatively stiff, compared to normal ones. Yet with only some small-scale papers published to date on the topic of EBUS elastographyThe baseline characteristics of all the LNs evaluated in our study were shown in Table P < 0.001) than the malignant LNs.The AUC for the BCP was 0.86 (95% CI: 0.78–0.94). Using the Youden index (sensitivity + specificity-1), the best cutoff BCP for the differentiation of benign and malignant LNs was 36.7%. Using this cutoff value , sensitivity, specificity and accuracy were 92.3%, 67.5% and 78.5%, respectively. This cut off value showed a better efficiency compared with the above EBUS features. As shown in Table Only six participants in the patient cohort underwent surgery, because most patients were diagnosed with late stage lung cancer, and some patients were only admitted to the outpatient department. In these six cases, four showed coincidence of diagnosis between EBUS-TBNA and a postoperative pathological diagnosis or EBUS elastography BCP results. Two cases showed a discrepant diagnosis between EBUS-TBNA and a postoperative pathological diagnosis, with the EBUS-TBNA showing negative but postoperative pathological diagnosis showing positive LNs. However, the EBUS elastography BCP results indicated that these specimens were malignant .16. Conventionally, B-mode and power Doppler imaging methods were employed to predict the possibility of cancer metastasis during EBUS scanning. Elastography is a new noninvasive EBUS modality which is hypothesized to be able to predict mediastinal and hilar nodal metastasis based on stiffness of tissue.The accurate staging of lung cancer is essential to determining appropriate treatment options. Among the different methods of preoperative staging, real-time EBUS-TBNA has been used more and more widely in clinical practice over the last decade. The recent edition of the ACCP Guidelines for Lung Cancer (3rd edition) also recommended needle-based methods as first-line approaches for invasive mediastinal staging of NSCLC15 and most of which used subjective method to classify the elastography imaging color distribution15. Furthermore, only one published paper gave clear correlated pathological information of detected LNs based on elastography color distributions15, and only a few papers involved preoperative PET-CT results15. Our data confirmed that real-time elastography during EBUS offered high sensitivity and specificity of detecting metastasis in mediastinal and hilar LNs using a firstly reported semi-quantitative method to classify the elastography color distribution. With the help of a widely used software, we were able to get the quantitative BCP and gave a clear BCP threshold to distinguish the benign and malignant LNs. Our study also provided more detailed information on disease subtypes correlated with elastography BCP. We also involved various EBUS features and SUV value in predicting malignant nodal metastasis and compared the diagnostic efficiency with our semi-quantitative method. We showed that the BCP cut off value had a better efficiency compared with the EBUS features and SUV value.To the best of our knowledge, only limited published papers have evaluated elastography’s utility during EBUS10. Our study involved a thorough evaluation of stiffness in the targeted LN and a semi-quantitative analysis of the image color distribution. Takahiro Nakajima et al. analyzed the color distribution of 49 LNs for 21 patients using “stiff area ratio”. Their results showed that when using a cutoff value of 0.311 for stiff area ratios, the sensitivity and specificity for predicting metastatic disease were 81% and 85% respectively. The stiff area was histologically compatible with metastatic distribution in surgically harvested LNs17. Our study showed similar results, but we enrolled a larger patient population (60 patients) and gave more detailed information on the color distribution of LNs with different diseases. Jiayuan Sun et al. utilized qualitative and quantitative methods to evaluate the ability of EBUS elastography to differentiate between benign and malignant LNs during EBUS-TBNA. The authors used a software and transformed the image to gray scale, in which the values varied from 255  to 0 ; and the target was contoured later. The mean gray value inside the target was calculated and could reflect the stiffness of the targeted LN, which was similar to our method. The authors showed that SCLC showed a lower gray value compared to NSCLC (201.33 versus 196.37)15, which was similar to our result that SCLC showed a lower BCP compared to NSCLC. However, the previous study didn’t give a clear mean gray value for adenocarcinoma or squamous carcinoma subtypes, while we showed in our present study that lung squamous carcinoma had a highest BCP of 71.6%, and the average BCP for adenocarcinoma was 61.9%.Takehiro Izumo and his research team defined the color distribution of LN elastographic imaging according to three types: Type 1, predominantly non-blue, Type 2, partly blue, partly non-blue , and Type 3, predominantly blue. The LNs that were classified as Type 1 were benign in 24/24 (100%), and Type 2 were benign in 6/14 (46.9%), while Type 3 were malignant in 35/37 (94.6%). The authors concluded that elastography is a noninvasive technique that can be performed reliably and may be helpful in the prediction of nodal metastasis during EBUS-TBNA. However, their definitions of elastography types were generally subjective and might not be replicable by other scholars. In this study, we chose a widely used software to semi-quantitatively analyze the color distribution of the LN elastography images, and the methods were easier to learn and manipulate. Rozman and his colleagues reported that when judging malignant LNs at a strain ratio ≥8, the accuracy of EBUS elastography for malignancy prediction was 86.3% . They concluded that strain ratio was more accurate than conventional B-mode EBUS modalities for differentiating between malignant and benign LNs. However, the strain ratio was calculated by two selected round “target regions” in a LN, which might not be able to precisely predict the stiffness of the whole LN and could miss the real aspiration region of the EBUS-TBNAIn our data, there were three cases with BCP less than 36.7% on the elastographic images, but still contained metastasis. Two of these LNs were diagnosed as SCLC, and the third was diagnosed as poorly differentiated NSCLC. We hypothesized that these tumors were generally considered to be highly aggressive, and even without a large amount of colonization they could still spread further. This may also indicate that the blue area in the LN imaging was the real area that contained metastasis, especially when the primary tumor is highly aggressive. We suggest that further research is needed to precisely examine the metastasis in the blue area of elastographic images.P < 0.001, 31.1% versus 57.1%). The pathological subtype with highest average BCP was shown to be lung squamous cell carcinoma (71.6%).Our data also showed that the BCP of LNs containing benign diseases was higher than that of normal LNs containing lymphatic tissue . These results may reveal that some diseases, such as pulmonary infection and granulomas, could lead to the elevation of LN stiffness, which may be caused by a higher density of cells and vessels. These LNs seemed to contain metastasis if assessed solely by elastography. However, compared to the benign diseases, the BCP in benign LNs was still remarkably lower than the malignant LNs  of the LN was found on a chest CT scan or an increased 18 [F]-fluorodeoxyglucose (FDG)uptake was shown [standardized uptake value (SUV) max > 2.5], both of which indicated metastasis based on the patient’s medical history and other examinations, a corresponding EBUS-TBNA was taken for the LN. Patients without a suspected primary lung cancer on PET-CT scan were excluded from this study. This study was approved by the institutional review board of the First Affiliated Hospital of Zhejiang University . We confirmed that all EBUS-TBNA and pathological examination procedures were performed in accordance with relevant guidelines and regulations. All enrolled patients gave written informed consent before the EBUS-TBNA procedure.21. The area in a LN presenting one or more of these features was designated as the sampling area for EBUS-TBNA. When multiple LNs satisfying these criteria were found in the same station, we chose the biggest LN as the target.All EBUS-TBNA procedures were performed by the same bronchoscopist (Zhou An). We applied local anesthesia to the pharynx of every patient with 10 ml 4% lidocaine spray, then inserted the convex probe EBUS  through the patient’s oral route, with instillation of 2–4 ml aliquot doses of 2.0% lidocaine when needed. The bronchoscopist performed scanning at an ultrasound frequency of 7.5 MHz, and the strain elastography was performed using the ELST mode in the processor EU-ME2 PREMIER PLUS . The enlarged target LNs on CT scan or LNs with high SUV value were identified by conventional B-mode EUBS and then elastography was performed on these LNs. Previous studies reported that LNs had the following sonographic characteristics on B-mode and vascular patterns on power Doppler indicated probable malignancy: a short-axis diameter larger than 1 cm, round shape, distinct margin, heterogeneous echogenicity and abundant blood flow with varying vessel diameters and tortuosityWe performed elastography on each LN that was sampled for EBUS-TBNA. By comparing the elasticity of the targeted tissues with the surrounding tissues, the scanned area was reconstructed and translated into a color signal, which was then overlaid on the B-mode imaging. The colors that stood for hard, intermediate and soft tissues were blue, green and yellow/red, respectively. The complete spectrum from blue to red encoding was applied to each elastographic image and showed the calibration of relative elasticity of the scanned LN area. The B-mode and elastographic images were simultaneously displayed on the monitor side by side. The scanned area was adjusted to a maximal depth of 4.0 cm, with an aim of including the whole pre-determined targeted LN and variable portions of the surrounding tissue. After elastography evaluation, TBNA with negative pressure was done 10–20 times in the blue color abundant area of the targeted LN, under real-time EBUS guidance. The corresponding histology and cytology specimens were collected. The final diagnosis of EBUS-TBNA sampled specimens was done by pathologists who did not know the results of the EBUS elastography, and the specimens were usually viewed about 1 day after the EBUS-TBNA procedure.22. The blue color proportion (BCP) in the targeted LN was calculated by dividing the pixel value of the blue area by the pixel value of the whole LN. To achieve this, we used the following image processing steps in Photoshop: (1) open the “Histogram” under the “window” menu bar option, and open the raw and binary image of a LN, (2) the borders of a whole targeted LN were manually selected with “quick selection tool”, then the pixel value was shown in the “Histogram” and recorded in an Excel data sheet, (3) the blue area of the same LN was selected using the “magic wand tool”, and the pixel value was also recorded from the “Histogram” window, and (4) the BCP of this LN was calculated by dividing the pixel value of the blue area by that of the whole LN. Example images of the above processing procedures were shown in Fig. We collected all images of the sampled LNs after every EBUS-TBNA procedure. We analyzed the color distribution of LNs, and used Photoshop  for the image processing which was similar to previous studiesa sheet,  the blueStatistical analysis was performed with SPSS 20.0 software . We constructed a receiver operating characteristic (ROC) curve and the area under the ROC curve (AUC) with 95% CI was determined to define the cutoff BCP for differentiating between benign and malignant LNs. The sensitivity and specificity of the cutoff BCP in diagnosing malignant or benign LNs were calculated separately with the final diagnosis of LNs. All statistical tests were two-sided and P < 0.05 was considered statistically significant.The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.


Background: Large-scale food fortification (LSFF) of commonly consumed food vehicles is widely implemented in low- and middle-income countries. Many programs have monitoring information gaps and most countries fail to assess program coverage.Objective: The aim of this work was to present LSFF coverage survey findings  from 18 programs  conducted in 8 countries between 2013 and 2015.Methods: A Fortification Assessment Coverage Toolkit (FACT) was developed to standardize the assessments. Three indicators were used to assess the relations between coverage and vulnerability: 1) poverty, 2) poor dietary diversity, and 3) rural residence. Three measures of coverage were assessed: 1) consumption of the vehicle, 2) consumption of a fortifiable vehicle, and 3) consumption of a fortified vehicle. Individual program performance was assessed based on the following: 1) achieving overall coverage ≥50%, 2) achieving coverage of ≥75% in ≥1 vulnerable group, and 3) achieving equity in coverage for ≥1 vulnerable group.Results: Coverage varied widely by food vehicle and country. Only 2 of the 18 LSFF programs assessed met all 3 program performance criteria. The 2 main program bottlenecks were a poor choice of vehicle and failure to fortify a fortifiable vehicle .Conclusions: The results highlight the importance of sound program design and routine monitoring and evaluation. There is strong evidence of the impact and cost-effectiveness of LSFF; however, impact can only be achieved when the necessary activities and processes during program design and implementation are followed. The FACT approach fills an important gap in the availability of standardized tools. The LSFF programs assessed here need to be re-evaluated to determine whether to further invest in the programs, whether other vehicles are appropriate, and whether other approaches are needed. The former should achieve higher coverage levels at the population level, assuming legislation standards are followed .Large-scale food fortification (LSFF)Despite being widely practiced, many LSFF programs in lower-resource settings have not been able to demonstrate impact . This isThe WHO has published a general guidance document on fortification practices  and an uIn 2013, the Global Alliance for Improved Nutrition developed and operationalized the Fortification Assessment Coverage Toolkit (FACT) to support coverage assessments in both population-based  and targeted  fortification programs . The tooThe fortification program activities in countries in which FACT surveys were implemented are shown in In all surveys, the instruments collected data on household- and individual-level variables. In 5 surveys  households and women of reproductive age were assessed. For these surveys, data were collected on demographics; socioeconomic status; education levels within the household; housing conditions; recent infant and child mortality; water, sanitation, and hygiene practices; food security; women’s dietary diversity; and coverage and consumption of fortified food vehicles. For 3 surveys  households and caregivers with children in the first 2 y of life were assessed. These surveys collected the same data as above, as well as data on child health, infant and young child feeding practices, and maternal and child anthropometric measurements. All survey modules  were taken or adapted from validated guidelines where available , 20.Ethical clearance to conduct the coverage surveys was obtained in each setting from a national or academic institutional review board. Consent to participate was obtained from the primary survey respondent on the basis that participation in the survey was voluntary. Oral consent was obtained in 5 countries , and written consent was obtained in 3 countries . At least 2 attempts were made to conduct the survey at each selected household.In all surveys, data were collected by trained interviewers under the supervision of experienced field supervisors. All interviewers and field supervisors were trained before the surveys and were supervised by dedicated technical personnel during implementation. The survey instruments were pilot-tested in each setting to ensure that the language and wording of questions were clear, and that question-skip logic and response options were appropriate to the setting. In 5 countries  data were collected with the use of paper forms. In these surveys, data quality was ensured by interactive checking  during data entry, as well as batch checking . In 3 countries  data were collected with the use of mobile devices by using interactive checking to ensure data quality. A description of the sampling schemes used in each coverage survey is shown in In this article, we used 3 indicators of risk to assess the relations between coverage and vulnerability. The risk indicators were poverty, poor women’s dietary diversity, and rural residence. These indicators were selected on the basis that they were associated with poor micronutrient status and highlighted the more marginalized subgroups within the population , 22. Pov1) consumption of the vehicle—the household consumes the vehicle; 2) consumption of the fortifiable vehicle—the food vehicle used by the household is processed industrially and hence is well suited to large-scale fortification; and 3) consumption of the fortified vehicle—the vehicle used by the household is fortified. Each stage depends on all of the preceding stages being true. All stages must be true for a member of the population to be effectively covered.Three measures of coverage were assessed while following the Tanahashi coverage framework . This frIf, e.g., a coverage assessment finds that 90% consume the vehicle, 20% consume the vehicle in a fortifiable form, and 18% consume the fortified vehicle, then the key program bottleneck is that the vehicle is consumed in a nonfortifiable form. For an LSFF program of wheat flour, then, this might mean that production of wheat flour is dominated by small-scale milling and that wheat flour is not a good choice of vehicle.1) raw coverage (RC)—the proportion of all households that were covered ; 2) met need (MN)—the proportion of households defined as vulnerable that were covered (this is a measure of how well the program addresses vulnerability); and 3) coverage ratio (CR)—the ratio of the coverage in vulnerable households to the coverage in households considered to be not vulnerable.Three summary statistics were calculated for each measure of coverage: The CR ranged between 0 and positive infinity. CR values <1 indicated that coverage favored nonvulnerable population groups. CR values >1 indicated that coverage favored vulnerable population groups. A CR of 1 indicated equitable coverage between vulnerable and nonvulnerable population groups. Further details on the RC, MN, and CR statistics are published elsewhere , 20.1) the point estimate of RC  should be ≥50% ; 2) theThe standards associated with the criteria for the RC, MN, and CR measures can be modified or reasonable alternative criteria could be formulated. Results for the RC, MN, and CR measures are therefore presented to enable the reader to apply modified or alternative criteria. The criteria were applied for each vehicle to the highest Tanahashi coverage stage for which results were available . The priFortification status for all food vehicles assessed in each country setting was determined by brand identification  and by quantitative laboratory analyses . For quantitative analyses, food specimens were collected at the household or market level, depending on what was logistically feasible in each country setting. Specimens were shipped to reference laboratories for quantitative analyses. Households were classified as consuming a fortified or nonfortified vehicle based on the laboratory results. In cases in which a brand could not be determined in a household or a specimen was not collected, the household was classified as nonfortified in the analyses.m PSUs were sampled with replacement (where m is the number of PSUs in the survey sample). Observations within selected PSUs were also sampled with replacement with the same within-PSU sample size that was achieved in the survey. A total of r = 400 bootstrap replicates were used. The resulting estimate consists of the 2.5th (lower 95% CI), 50th (point estimate), and 97.5th (upper 95% CI) percentiles of the distribution of the statistic across all replicates.Survey data were analyzed with the use of the R language for data-analysis and graphics (version 3.2.2), the R-AnalyticFlow scientific workflow system (version 3.0.1), and SAS (version 9.4). Summary statistics were calculated with the use of bootstrap estimation techniques that consisted of a set of within-primary sampling unit (PSU) survey samples that were sampled with replacement and with a probability proportion to PSU population size with the use of a roulette wheel  algorithm . For eacCharacteristics of the survey populations and survey response rates (defined as the proportion of the target sample size achieved) are shown in RC for each measure of coverage for wheat flour, maize flour, and edible oil at the household level are shown in The FACT project fills an important void in the availability of simple, cost-effective tools that fortification programs can use to assess and diagnose program coverage. Results from the analyses highlight the importance of adequate program design and appropriate monitoring activities to ensure program success.Only 2 of the 18 LSFF programs assessed met all program performance criteria used in the current analyses. This finding is consistent even if lower thresholds are considered for the program performance assessments. For programs with low RC, the results also indicate that coverage was not concentrated in vulnerable population groups . The main reasons for programs failing to meet the criteria were a poor choice of vehicle  and failure to fortify a fortifiable vehicle. These 2 reasons alone account for the principal bottlenecks in the 16 programs that did not meet the 3 performance criteria used in the analyses. Poor selection of a food vehicle is a failure of program planning and design. Because LSFF is not intended to change population dietary patterns, there is nothing that can be done during program implementation to increase program coverage in such instances. Failure to fortify a fortifiable vehicle may be a problem of program design  or a problem of compliance or enforcement of fortification. Failing to cover vulnerable population groups may be a problem of access, affordability, or the fact that these at-risk groups do not consume the respective fortified food vehicles. Further assessments of these programs are required to determine whether the existing programs need strengthening, whether other food vehicles should be considered, and whether other interventions to deliver micronutrients are required.The edible oil program in Côte d’Ivoire met the 3 program performance criteria used in the current analyses. For cost and logistical reasons, the assessment was only conducted in the capital city of Abidjan; therefore, conclusions about the rest of the country cannot be drawn from the current work. Further coverage assessments in rural and other urban areas outside of Abidjan would be needed to fully assess equity of fortification coverage in this country. South Africa’s maize flour fortification program met the program performance criteria used in these analyses in the 2 regions surveyed. These regions were selected for surveying because they are the 2 provinces with the highest population density and represent the most diverse areas of the country . Even thPlanning of effective LSFF programs needs to be informed by detailed investigations of patterns of production, distribution, and consumption, and requires the selection of vehicles with the potential for high coverage in the population. Without this due diligence, programs rely largely on chance to achieve impact. The capital-intensive startup phase of these programs means that this is a gamble made with high stakes, as we have reported in other contexts . The maiResults from these analyses also highlight the importance of having multiple strategies to address micronutrient needs in the population. LSFF programs by design are not intended to be a panacea for micronutrient malnutrition in the population, and complementary strategies are needed to address specific population groups whose needs may be higher or who for various reasons may not access these fortified staples . Many co1) the development of a standardized toolkit to assess program coverage; 2) a peer-review process that reviewed and refined the research approach; 3) the use of standardized and validated indicators to assess vulnerability; and 4) the use of program coverage assessments that were conducted in the overall population and in vulnerable subpopulation groups. As for limitations, household coverage estimates do not capture foods purchased and consumed outside of the households, such as snacks and restaurant meals. This may result in underestimating the potential coverage of fortification interventions in the population. This article reports on estimates that are common to and available from all surveys. Although not presented in this article, most of the surveys also assessed individual-level consumption of foods made with the use of the respective vehicles, which generated information on individual-level coverage and consumption . Further development of the FACT methods should systematically include or be linked to market-level assessments. Assessing fortification status is challenging to conduct in the field. Quantitative analyses were used to determine the fortification levels of food vehicles, but are costly and time-consuming, and fortificants and micronutrients are subject to degradation over time because of storage conditions and length of time from collection to analysis. Having field-friendly tools to determine the presence of fortificants for all major food vehicles  need to be developed. Mobile photometers are available that can measure multiple micronutrients in flour  and oil 1) the selection of appropriate food vehicles before programs are started, and 2) routine monitoring of the fortification process to ensure that fortification occurs at the desired level. Where vehicles were chosen that have little potential for population impact, the use of funding to support such programs should be reconsidered. The second issue can and should be improved during the course of the program.The FACT project successfully developed and operationalized a program-ready tool for carrying out fortification coverage assessments of LSFF programs. The results identified 2 major areas that programs need to focus on: LSFF has been demonstrated to be a highly cost-effective intervention strategy to address micronutrient needs in the population ; however, this can only be achieved when the necessary activities and processes during program design and implementation are followed. A number of the programs that were assessed have high potential for impact based on the consumption of fortifiable vehicles, a potential that can only be achieved with substantially improved compliance with fortification . For oth


Specifically, product formation in nanolitre-sized cultivation vessels is sensed and translated into the formation of catalase as a reporter protein. Exposure to hydrogen peroxide leads to oxygen gas formation and thus to a density shift of the cultivation vessel. Exploiting Archimedes’ principle, this density shift and the resulting upward buoyancy force can be used for batch-wise library sampling. We demonstrate the potential of the method for both, screening and selection protocols, and envision a wide applicability of the system for biosensor-based assays.The ability of whole cells to catalyse multistep reactions, often yielding synthetically demanding compounds later used by industrial biotech or pharma, makes them an indispensable tool of synthetic chemistry. The complex reaction network employed by cellular catalysts and the still only moderate predictive power of modelling approaches leaves this tool challenging to engineer. Frequently, large libraries of semi-rationally generated variants are sampled in high-throughput mode in order to then identify improved catalysts. We present a method for space- and time-efficient processing of very large libraries (10 Frequently, analysis and sorting of the samples occur in a consecutive fashion in which samples are analysed one after the other, and then a mechanical operation is applied to separate a potentially positive variant from the rest. As a result, the throughput remains limited. Methods enabling high throughput at reasonable costs are therefore in high demand5.Screening and selection procedures are key strategies in the discovery or optimization of whole cell biocatalysts in high-throughput campaigns. In both cases, changes in the concentration or the activity of the product need to be read out. For screening assays, improved strains are typically identified by a colour change, or an increased fluorescence or luminescence signal, while in the case of selection assays, positive variants show an elevation of the growth rate over the background of negative variants9.We exploited Archimedes’ principle, which relates the buoyancy force of an immersed object to its density, to identify positive variants in biocatalyst screening and selection assays. By changing the buoyancy force of a cultivation vessel with a monoclonal microcolony in response to a desired change, such vessels can be made to ascend from a background population and float, allowing for strictly parallel analysis and sorting of strain variants at very high rates. As cultivation vessels, we used monodisperse hydrogel-based nanolitre reactors (nLRs), with a precisely adjustable diameter between 200 to 500 µm, as microcompartments for controlled expansion of single cells into monoclonal microcolonies (approx. 10 to 100 µm colony diameter), analysis, and recovery of positive variants−1 for an nLR containing mostly water and cells10 to less than 0.9 g mL−1. Different possibilities were considered (see Listeria seeligeri)11 in Escherichia coli MDS42 from plasmid pCat led to liberation of molecular oxygen at rates of approx. 9 pL min−1 cell−1  and primarily expanded towards the inner core of the nLRs, possibly following the density gradient of the hydrogel12  colonies. When reducing the contact time and concentration of H2O2 to the minimum necessary for buoyancy separation, all colonies could be regrown after the separation process  and the catalase expression level can be exploited to make nLRs ascend see Fig. .E. coli EcNR1 variants14, each metabolizing d-lactose at a different rate and thus displaying different maximum specific growth rates  were treated with H2O2. Positive and negative nLRs separated within one minute. The nLRs containing fast-growing strains had also been fluorescently labelled, thereby allowing counting by large-particle flow cytometry6. The results indicated a 267-fold enrichment of the fast-growing over the slow-growing variants (from 0.2% to 55% of occupied nLRs) and a recovery of 62% , incubated the nLRs to generate microcolonies, and subjected them to buoyancy separation. Microscopic inspection before and after separation indicated the absence of large microcolonies in the buoyancy negative bottom fraction  secreted by whole cell biocatalysts17. First, a collection of biocatalysts with different B2 production capacities was generated. For this, E. coli BW23474 was transformed with plasmids containing either the synthetic B2 biosynthesis operon ribDBECA , the operon ribDBECAPL (no recombinant promoter), or the operon ribBECA  uracil; see Supplementary Fig. 2 to levels of up to 170 µM (pB2_ribDBECA), 72 µM (pB2_ribDBECAPL), and 33 µM (pB2_ribBECA)  lead to differences in the expression level of a marker protein. We used a riboswitch-based control elementsee Fig. .Figure 32 levels into different catalase amounts was constructed. The biosensor cells use the B2 transporter RibM for facilitated B2 uptake18 and convert it to flavin mononucleotide (FMN) by the endogenous enzyme RibF. Elevated B2 levels thus lead to elevated FMN levels, which lead in turn to the increased expression of Cat-GFP, a fusion of the catalase enzyme to the N-terminus of the superfolder green fluorescent protein (sfGFP), via an FMN-sensitive hammerhead ribozyme8  were mixed with a background of 363,000 nLRs containing 79,000 E. coli microcolonies of the poorly performing strains carrying the plasmids pB2_empty, pB2_ribBECA, and pB2_ribDBECAPL. The resulting mixture was subjected to buoyancy separation and the buoyancy-positive top fraction consisting of 549 nLRs was isolated. Microscopic analysis revealed that 472 were fluorescently labelled and therefore carrying the potent B2 producer (pB2_ribDBECA). The results indicate a 95-fold enrichment (from 0.8% to 79%) and a recovery of 66%  by the catalase protein renders such tools suitable for buoyancy separation. Using a simple lab-demonstrator, we analysed and isolated 107 samples within one minute, hence exceeding the throughput at which standard flow-cytometric sorters operate by at least one order of magnitude. We further argue that the capacity of the method can be adapted by changing the diameter of the customized separator such that scaling up to 109 analysed microcolonies per batch would require only a small adaptation  selection and (II) screening. We showed that either differences in metabolic activity or expression level of a marker protein can be used to produce differential buoyancy forces, resulting in a clear-cut separation of diversely performing whole cell biocatalysts. This suggests good compatibility with the existing toolbox of biological sensing elements including RNAtion see . Furthertion see . The nLRL. seeligeri was isolated from plasmid pAHA1  was modified using the CRMAGE31 strategy. Briefly, an oligonucleotide pool (oligonucleotide ID 8) introducing changes within the wild-type RBS of lacZ was used for MAGE13. The pool contained 18 different oligonucleotides, all targeting the RBS of the lacZ gene, but each of them encoding a different RBS-sequence . The gRNA for targeting Cas9 to the wild-type RBS sequence was designed and checked for off-target effects with the Cas9 Online Designer (COD)32 and the resulting oligonucleotides encoding the gRNA sequence (oligonucleotides ID 6 and ID 7) were annealed and ligated into BsaI-linearized pCRISPR plasmid to obtain plasmid pCRISPR_lacZ. Chromosomal modifications were performed in strain E. coli EcNR1, previously transformed with plasmid pCas9. Briefly, a 3 mL culture was grown in LB-Luria broth , supplemented with 20 µg mL−1 chloramphenicol, and incubated at 32 °C until an OD600 of approx. 0.6 was reached. Cells were then heat-shocked for 15 min at 42 °C to induce expression of the λ-Red genes. Next, the induced cells were made electrocompetent by washing three times with ice-cold ddH2O and resuspended in a volume of 50 µL. The lacZ RBS oligonucleotide pool was then added to a final concentration of 2 µM and the cells were electroporated in 1 mm gap cuvettes  at 1.8 kV and 4 to 6 ms pulse-length. Cells were recovered by addition of 3 mL LB-Luria broth supplemented with 20 µg mL−1 chloramphenicol, again induced at 42 °C and made competent for one further electroporation with the oligonucleotide pool where 100 ng of the pCRISPR_lacZ plasmid was also added. Cells were then recovered overnight in LB-Luria broth, supplemented with 50 µg mL−1 kanamycin (added 1 h after electroporation) and plated the next day on LB agar plates with with 50 µg mL−1 kanamycin. To identify clones with the correct RBS modification, the genomic region containing the lacZ RBS was amplified by colony PCR using Taq DNA polymerase and primers ID 9 and ID 10 and the PCR product was Sanger sequenced using DNA oligonucleotide ID 9. For selected strains, the plasmid pCas9 was removed from the cells by repetitive cultivation without antibiotic selection . The strains were transformed with the plasmid pCat to enable catalase-based separation, resulting in 18 different strains E. coli EcNR1_XX [pCRISPR_lacZ + pCat] (where XX represents the ID of the RBS variant).The RBS of the E. coli BW25113 served as a chassis for the construction of the B2 auxotrophic biosensor. The strain was engineered using plasmid-based homologous recombination33. Briefly, a part of the chromosomal gene for the riboflavin synthase RibC was replaced by the gene for the riboflavin transporter RibM (PnuX) from Corynebacterium glutamicum18. Plasmid pKO3_ribM contained a 2.5 kbp fragment amplified from the genome of E. coli JM101 encoding for the ribC gene. While deleting 247 bp of the central part of ribC, the ribM gene was integrated into that sequence together with a RBS to allow for ribM translation. The plasmid was used to transform E. coli BW25113 and homologous recombination was performed according to the protocol provided by Link et al.33 but additionally 10 µM B2 was added to the medium. The resulting strain E. coli BW25113 ΔribC::ribM is a B2 auxotroph but can grow with externally supplied B2 taken up via RibM. Correct insertion of the ribM gene was verified by colony PCR using primers ID 11 and ID 12 and replica plating on LB-Miller agar plates with and without B2.tac promoter. The translation of the resulting mRNA is controlled by an aptazyme consisting of FMN aptamer 21A8, an optimized connecting linker and a hammerhead ribozyme. To adapt it for the expression of the catalase, plasmid pRSG_2A1 was linearized by cutting between the gene for the aptazyme and sfgfp with the restriction enzyme NsiI. The catalase gene was PCR-amplified from plasmid pCat using primers ID 13 and ID 14, digested with NsiI and ligated into the linearized pRSG_2A1, thus generating a plasmid containing a translational fusion of the catalase to the N-terminus of sfGFP (Cat-GFP). Correct clones were identified by testing for their fluorescence properties and catalase activities and confirmed by Sanger sequencing (primers ID 15 and ID 16). Next, the cat-gfp gene together with its regulatory elements was isolated by restriction digestion with NdeI and HindIII and cloned into plasmid pSEVA281, linearized with the same enzymes, to generate plasmid pSense_cat-gfp. The plasmid was then used to transform the strain E. coli BW25113 ΔribC::ribM yielding the final B2 biosensor.The previously constructed expression system pRSG_2A1 was used for regulation of catalase expression. Briefly, plasmid pRSG_2A1 contains a sfGFP gene under transcriptional control of the P2 biosensor, the strain E. coli BW25113 ΔribC::ribM [pSense_cat-gfp] was grown in LB-ΔRib broth . Culturing was continued until the cells became B2 limited and did not grow anymore even in the presence of excess nutrients . Then, the B2 limited cells were supplemented with glycerol to a final volume fraction of 20% and stored as frozen stocks at −80 °C.Prior to its application as B8. Single droplets were generated by laminar-jet breakup with a Var D encapsulator  from sodium alginate suspensions (20 g L−1 in ddH2O) inoculated with a precisely adjusted concentration of bacterial cells (see results). Inoculation was either done from a liquid pre-culture, where the number of colony-forming units (CFUs) per volume was estimated by measuring the optical density at a wavelength of 600 nm (1 OD600 unit = approx. 5 × 108 CFUs mL−1) using a BioPhotometer , or from frozen-stocks of the strains, where the number of CFUs was measured by plating and colony counting on agar plates beforehand. The desired number of cells were added to one part of sterile ddH2O in a 50 mL centrifugation tube and filled up with four parts of sodium alginate (25 g L−1 in ddH2O). When fluorescently labelled nLRs were required, fluorescein-alginate  or a ROX-silica-particle suspension  was added to a final volume fraction of 0.5% together with the cells to the water. After mixing by inverting the tube several times, the suspension was transferred into a 24 mL or 60 mL syringe and connected to the syringe pump of the encapsulation device. Depending on the desired size of the nLRs, one of the following encapsulator operation mode was selected: Production of nLRs with 460 µm diameter (approx. 50 nL volume): 0.7 kHz with 3.3 mL min−1 alginate flow-rate and a nozzle diameter of 150 µm. Production of nLRs with 230 µm diameter (approx. 6 nL volume): 3.75 kHz with 2.0 mL min−1 alginate flow-rate, coaxial-nozzle with an outer diameter of 350 µm and operated at an airstream with a pressure of 30 mbar. Reactors were gelled in nLR hardening buffer for 20 min and briefly rinsed with nLR wash buffer before transferring them into growth medium.Nanolitre reactors (nLRs) were synthesized as described previouslyE. coli strain MDS42 [pCat] was grown from frozen stocks in 5 mL LB-Miller broth (supplemented with 20 µg mL−1 chloramphenicol) in a pre-culture until mid-exponential phase (OD600 = 0.6 to 1), encapsulated into nLRs with a diameter of approx. 460 µm and incubated (100 g L−1 wet nLRs) in Erlenmeyer shake-flasks in OB broth . Incubation was performed at 27 °C (specific growth rate of approx. 0.44 h−1) or 30 °C (approx. 0.54 h−1) on an orbital shaker . The induction of catalase gene expression was initiated after 18 h of incubation with 0 to 200 ng mL−1 anhydrotetracycline. After 22 h incubation was stopped, nLRs were washed several times with nLR wash buffer and stored at 4 °C in the same buffer until separation.For initial characterization of the buoyancy properties, E. coli EcNR1_XX [pCRISPR_lacZ + pCat] strains containing the different lacZ RBS variants were grown from frozen stocks in two consecutively inoculated pre-cultures (both supplemented with 20 µg mL−1 chloramphenicol and 50 µg mL−1 kanamycin): (I) in LB-Miller broth and (II) in CDM (see Supplementary Methods for composition) supplemented with 4 g L−1 D-glucose. Culturing was performed in 800 µL of medium in Nunc DeepWell 96-well plates. After encapsulation, nLRs were incubated (50 g L−1 wet nLRs) in Erlenmeyer shake-flasks containing CDM supplemented with 4 g L−1 D-lactose, 20 µg mL−1 chloramphenicol and 50 µg mL−1 kanamycin at 30 °C on an orbital shaker . In cases where the precise number of occupied nLRs was important, nLRs were incubated for 5 h under conditions described above, isolated into nLR wash buffer and the embedded microcolonies (consisting of approx. 1 to 10 cells) were stained with the DNA intercalating green fluorescent dye SYTO 9 . Next, monoclonally occupied nLRs were isolated by large-particle flow cytometry (based on the fluorescence of the contained microcolonies), the sorted nLRs were then transferred to the same medium again and incubation was continued. If co-incubation of different strains was desired, batches of counted nLRs were mixed prior to further incubation. Catalase expression was induced with 5 ng mL−1 anhydrotetracycline after a total incubation time of 43 h. After 48 h incubation was stopped, nLRs were washed several times with nLR wash buffer and stored at 4 °C in the buffer until separation.The 2 producer strains harbouring the different B2 overproduction plasmids were grown from frozen stocks in 5 mL LB-Miller broth (supplemented with 50 µg mL−1 kanamycin) in a pre-culture until mid-exponential phase (OD600 = 0.6 to 1). The biosensor was directly inoculated from a fresh vial of a B2 limited frozen stock (see above). Both strains were co-encapsulated in nLRs and incubated (100 g L−1 wet nLRs) at 37 °C on an orbital shaker  in LB-ΔRib broth supplemented with 20 µg mL−1 kanamycin. At this stage of incubation, only the B2 producer strains can form microcolonies within the nLRs (due to their endogenous B2 synthesis) while the B2 auxotrophic biosensor does not grow. After incubation for 7 h, B2 production (encoded on the plasmid) was induced with 2.5 ng mL−1 anhydrotetracycline and expression of the Cat-GFP marker was induced with 20 µM isopropylthiogalactoside (IPTG). The nLRs were incubated for an additional hour and then removed from the medium by sieving. Next, the nLRs were dispersed in paraffin oil (100 g L−1 wet nLRs) supplemented with 20 g L−1 Abil EM90  and 1 g L−1 Tween 20 as emulsifiers and using a paddle mixer operated at 1,000 rpm for 30 s. The emulsion was then incubated in 50 mL aliquots in polystyrene containers with screw caps  at 37 °C on an orbital shaker . After 22 h, the nLRs were recovered by decanting the oil and washing the remaining slurry (containing the nLRs) several times with nLR wash buffer until all the oil was removed. The nLRs were then stored at 4 °C in the buffer until further use.B2 in water) was spotted on the dish. Depending on the experiment, typically 10 to 25 nLRs were spotted in one place and therefore an aqueous volume of 40 to 100 µL was accumulated per spot. Separation was initiated by quickly adding an approximately equal volume (40 to 100 µL) of diluted H2O2 (3.5% in nLR wash buffer) to each of the spots . The gas liberation process was followed by bright field microscopy and nLRs with positive buoyancy were counted in the focal plane at the top while nLRs with negative buoyancy were counted at the focal plane at the bottom of the droplet. For each condition, normally 5 to 10 spots were assayed in this way, adding to roughly 60 to 250 analysed microcolony-occupied nLRs per condition.For initial characterization of the buoyancy properties, experiments were performed in small scale (10 to 25 nLRs at a time) and the separation was performed and evaluated directly under the microscope. After incubation and isolation of the nLRs from growth medium, monoclonal nLRs displaying the desired phenotype  were distributed over rectangular Nunc OmniTray plates using large-particle flow cytometry. With each sorted nLR, approx. 4 µL of sheath-fluid  were transferred through the outlet at the top into the sampler chamber of the device containing approx. 500 mL of nLR wash buffer . Next, the mixture was allowed to stand until the nLRs had settled on the bottom of the sampler. Then, 200 mL of diluted H2O2 (2% in nLR wash buffer) was slowly pumped in from the feeder through the porous bottom of the sampler chamber. Note that the final concentration of H2O2 in the sampler was thus <2%. Within 10 to 20 seconds, positive nLRs  started to float and readily rose to the top of the suspension. Due to the continuous supply of H2O2 to the system the liquid volume in the separator device slowly increased. This led to an outflow of the liquid over the upper rim of the inner tube of sampler into the collector, initially filled with 50 mL of nLR wash buffer. This liquid flow supports the removal of floating nLRs from the inner part of the device. Note that the nLRs are no longer exposed to high concentrations of H2O2 once they entered the collector filled with H2O2-free wash buffer. After approx. 60 s the process was completed and the recovered fraction was removed from the collector with a cell strainer , washed with an excess of nLR wash buffer and stored at 4 °C in the same buffer (see Supplementary Fig. Separation of large numbers of nLRs was performed in a custom-made separator device inspired by the design of a fish trap (see Supplementary Fig. Supplementary InformationSupplementary Video 1: Oxygen production and capturing in nanolitre reactors.Supplementary Video 2: Demonstration model for buoyancy separation.


A total of 52 schools (n = 8679 students) participated in the experimental implementation phase of the project P.A.T.H.S. . After completion of the Tier 1 Program, students were invited to respond to the Subjective Outcome Evaluation Form (Form A) to assess their views of the program, instructors, and perceived effectiveness of the program. Based on the consolidated reports submitted by the schools to the funding body, the research team aggregated the consolidated data to form a “reconstructed” overall profile on the perceptions of the program participants. Results showed that high proportions of the respondents had positive perceptions of the program and the instructors, and roughly four-fifths of the respondents regarded the program as helpful to them. The present study provides additional support for the effectiveness of the Tier 1 Program of the P.A.T.H.S. Project in Hong Kong.