Tinnitus is a common medical symptom that can affect an individual’s emotional and functional quality of life. Psychological therapies are acknowledged as beneficial to people with tinnitus; however, such therapies are not always readily accessible. With their global reach, automated Internet-based interventions have the potential to reduce the disparity in access to psychological support that people with tinnitus currently experience. However, the evidence on the acceptability and efficacy of these interventions is lacking. Process evaluations that develop an in-depth understanding of how users experience these interventions provide an essential first step when evaluating complex psychological interventions.To describe the protocol for a study that will explore past, current, and new users’ reactions to and interactions with the Tinnitus E-Programme, an Internet-based intervention for the self-management of tinnitus.Two parallel mixed-methods studies will be carried out with 2 different populations. Study 1 will use an online survey to gather past and current users’ views of the program. Study 2 will recruit new program users to take part in an interview and complete a relaxation log to explore how well they were able to implement the skills they learned during the program in their everyday lives. The findings from both studies will be triangulated to develop an in-depth understanding of the program’s mechanisms of impact and identify any implementation or contextual factors that strengthen or impede its delivery and functioning.Study 1 is open for recruitment with a projected completion in June 2016 and Study 2 was completed November 2015. At the time of submission, 36 participants have been recruited to Study 1 and 12 participants have taken part in Study 2.Findings will inform the optimization of the Tinnitus E-Programme and guide future evaluation work to assess the program’s effectiveness as a therapy for people with tinnitus. Tinnitus (ringing in the ears) affects approximately 10%-18% of the population  and is cIn the absence of a definitive biomedical cure, current health care strategies focus on supporting people to manage their tinnitus to ultimately reduce the tinnitus percept and associated psychological distress . CurrentThere is evidence to suggest that Internet-based interventions are effective for reducing tinnitus distress and psychological comorbidity while improving quality of life -16. HoweTo evaluate the Tinnitus E-Programme, we are guided by the Medical Research Council’s guidance on developing and evaluating complex interventions  that empA useful first step when evaluating developed interventions is to carry out a process evaluation. This can provide information on the (1) implementation, (2) mechanisms of impact, and (3) contextual factors that influence the delivery and outcome of the intervention :Implementation is concerned with what is delivered in practice and the structures and resources required for successful implementation  and is tMechanisms of impact is concerned with how the intervention components—and a user’s interactions with them—lead to the desired changes in outcome . That isContext is concerned with how external factors may strengthen or impede the delivery and functioning of the intervention . Such exThis study will carry out a process evaluation of the Tinnitus E-Programme to further our understanding of the program’s mechanisms of impact and identify any implementation or contextual factors that strengthen or impede its delivery and functioning. Most process evaluations have been carried out on people who were recruited offline and are using the intervention for the first time as part of a research study. This reduces the findings’ external validity and relevance to real-world practice -32. ThisTo explore past, current, and new users’ reactions to and interactions with the Tinnitus E-Programme. The specific aims are to explore:The acceptability and usability of the program ;How users engage with the program ;Users’ perceptions of the processes and outcomes of the program ;User enactment of the relaxation skills learned in the program .The Tinnitus E-Programme  is a 10-Program content is delivered across 6 weekly modules, followed by a 4-week maintenance period where users are asked to continue the daily relaxation goals set in the previous period. No additional intervention content or support is delivered during this maintenance period. A recommended program structure is given; however, users have free choice regarding which components they access and in what order they access them. The express aim of the program is to reduce tinnitus distress, but the precise mechanisms by which this change should occur are not yet established.This research will adopt pragmatism  as its oConsistent with this approach, 2 parallel mixed-methods studies will be carried out with 2 different populations to evaluate the program from multiple perspectives. This design will allow triangulation of research data and methods that will generate and compare complementary perspectives and contexts. The intention is that the use of both qualitative and quantitative research methods and more than 1 study population will provide a more complete, in-depth, and valid understanding of the phenomenon than if only 1 method or population was used ,37. MixeStudy 1 will explore how past and current users react to and interact with the program in the real-world, outside of a research context. Due to technical limitations of the program, it is not possible to monitor actual program usage. Therefore, an online survey will be used to gain self-reports of how users interacted with the program, as well as users’ reactions to the program. A convergent mixed-methods design  will be Study 2 will recruit a cohort of individuals with tinnitus who have not previously used the Tinnitus E-Programme in an attempt to gather more in-depth, timely, and diverse views and experiences. Participants will complete the program for the first time and take part in a semistructured interview. Participants will complete a relaxation log to explore the extent to which they enacted the relaxation skills learned in the program and any barriers to doing so. An adapted version of an embedded mixed-methods design will be used  in whichThe findings of the 2 mixed-methods studies will be triangulated in an overall interpretation. This research has ethical approval from the University of Nottingham Research Ethics Committee (Reference Number: Q11122014 SoM NIHR RHA QEST).Participants in Study 1 will self-select, based on their own judgements of whether they meet the following inclusion criteria: (1) adults aged 18 years and over, (2) ability to read English, (3) access and ability to use the Internet, and (4) have visited the Tinnitus E-Programme website or used the program. Participants may have accessed the program anytime over the last 6 years since the program was launched. There will be no exclusions regarding length of time since starting the program in order to maximize recruitment for this very specific population. The program does not specify any inclusion criteria, as the intention is that it is suitable for everyone with tinnitus. In keeping with this, there are no exclusions regarding tinnitus duration, severity, or co-morbidities in an attempt to recruit all potential users.Past and current program users will be invited to take part in an online survey hosted on SurveyMonkey. Advertisements will be posted on the Tinnitus E-Programme website and online discussion forum, along with the participant information sheet. The survey will also be advertised via social media and national charities in an attempt to reach those who no longer interact with the program or website. Email invitations and the participant information sheet will also be sent to those who registered with the program website or online discussion forum. Sample sizes for similar descriptive online survey studies have been between 50-249 individuals -42; therThe initial survey design was informed by the study rationale, relevant literature, and the comprehensive intervention description developed previously . The surThe survey uses a mix of closed and open questions concerned with: (1) reasons for participating or not participating in the program, (2) how the program was used, (3) usability of the program, (4) acceptability of the individual program components, and (5) benefits derived from the program and its impact on tinnitus management. Demographic data will also be collected on gender, age, country of residence, whether English is their first language, presence of tinnitus, tinnitus duration, and tinnitus management strategies used previously or currently.To assess the acceptability and face validity of the survey, an initial set of survey questions were reviewed by a public and patient involvement (PPI) panel assembled for the purposes of this study. The panel included 4 people with tinnitus and/or hearing loss who were recruited from an established National Institute for Health Research (NIHR) Nottingham Hearing Biomedical Research Unit (NHBRU) PPI panel and 1 voluntary sector representative from the British Tinnitus Association who had experience in writing communication materials for people with tinnitus. Panel members were chosen from a wider established PPI panel, based on their availability and previous experience of reviewing research materials. A focus group was carried out with the PPI panel to gather initial feedback on a paper version of the draft survey. The focus group was attended by the first author and co-facilitated by the PPI manager at NHBRU and an external facilitator who was not involved in the study but was familiar with issues relevant to hearing research. Panel feedback focused on the relevance and ordering of the questions, language used, and appropriateness of question type . Following the focus group, the survey was uploaded onto SurveyMonkey, and this online version was circulated to the PPI panel via email for additional comments. The panel was satisfied with the online version and no further amendments were made.The final online survey was subsequently piloted with 3 Tinnitus E-Programme users recruited from the program’s online discussion forum. These participants completed the online survey and answered 4 additional questions about the survey length and relevance of the questions and closed-question answers. Informed consent was gained from these pilot participants who were told that their answers may or may not be used in the final analysis, depending on the outcome of the pilot.All 3 participants reported that the survey took less than 30 minutes and “just the right amount of time” to complete. One participant suggested that it would be helpful to add a free-text comments box next to some of the closed questions to allow people to clarify their answers. The same participant also suggested adding a “cannot remember” option for the questions regarding program usage  for those who used the program a long time ago. Both of these changes were made to the final survey. As these amendments were minor, the pilot data was retained for inclusion with the main study. A copy of the final survey can be found in The online survey is anonymous to encourage participation and only 1 submission per computer will be allowed. Participants will be given a 14-day period in which they can request to have their answers deleted. After this period, their answers will be downloaded onto university servers and cannot be deleted. Participants will be asked to provide a security word as part of the survey and will be asked to recite this for data identification purposes should they wish to withdraw their data from the study.Answers to closed questions will be analyzed in IBM’s SPSS Statistics 22 using descriptive statistics, including frequencies and percentages, and each statistic carried out on complete data only. Answers to the open questions will be analyzed separately using inductive thematic analysis  and analFirst, the 3 coders  will familiarize themselves with the data through repeated reading of the survey answers. Second, KG will utilize line-by-line coding, a technique from grounded theory , in whicThird, at least 1 other coder  will independently apply the coding manual to all transcripts to clarify ambiguous codes, remove duplicate codes, and identify data that did not fit the coding scheme. Coding will be compared and discussed between coders and subsequent modifications made to the coding manual. Fourth, coders will collectively organize these codes into overarching themes and the coding manual will be updated accordingly. The constant comparison method , a grounConsistent with a data-validation variant of the convergent mixed-methods design , the quaParticipants in Study 2 will self-select, based on their judgement of whether they meet the following inclusion criteria: (1) adults aged 18 years and over, (2) ability to read English, (3) access and ability to use the Internet, (4) have self-reported tinnitus, (5) reside in the United Kingdom, and (6) have not previously used the Tinnitus E-Programme. Again, as the program is meant to be suitable for all tinnitus users, participants were not excluded based on any tinnitus-related characteristics.A purposive sample of people will be chosen from the NHBRU research database, which includes approximately 900 UK residents with tinnitus who have agreed to be contacted about research. Maximum variation sampling  will be The procedure for Study 2 is illustrated in Interviews will be held no later than 8 weeks after the participant’s start date to explore how acceptable the 6-week timeline is and to ensure maximum recall of intervention experiences. Participants will be asked to complete a daily relaxation log on paper or online over the following 4 weeks. Email reminders will be sent to those who have not yet started the program, organized an interview date, or completed their relaxation log. Recruitment will cease once data saturation has been reached for the interviews; that is, when no new themes are emerging .An interview guide was developed that was informed by the literature, intervention coding, and study rationale. Specifically, the interview questions are concerned with how people used the program; reasons for any nonusage; experiences of using the program; expectations of the program; usability and acceptability of the program, as well as its individual components; benefits derived from the program; and suggested improvements to the program. The interview guide can be found in Demographic data—including gender, age, ethnicity, and tinnitus duration—will also be obtained. Interviews will be carried out by the first author, a health psychologist and PhD student experienced in qualitative interviewing who was not involved in the Tinnitus E-Programme’s development. Interviews will last no longer than 1 hour. Participants will be given the choice of being interviewed in person at the research unit, over the phone, or via video chat. In an attempt to be inclusive, those with severe or profound hearing loss will also be offered the option to be interviewed using text communication methods . The audio from the interviews will be recorded using a digital voice recorder and transcribed verbatim. The text from the textual communication methods will be saved electronically.The relaxation log will assess users’ enactment of the relaxation goals set by the program during the 4-week maintenance period. An online relaxation log will be created for each participant using Google Sheets, an Internet-based spreadsheet program. Google Sheets will be hosted on the NIHR Google Hub, a secure online file storage system. The relaxation log is in tabular format with 4 columns and 28 rows representing each day of the 4-week period. Users will be required to answer the following 3 questions each day:Did you practice the mind calming breathing exercise 3 or more times today?Did you practice any of the 30-minute relaxation exercises today?Did you use any other parts of the Tinnitus E-Programme today? If yes, please write which parts. If no, please write “no.”There is also a free-text comments box to write any other comments for each day. At the end of week 10, participants will be asked to answer 1 final open question: “Did you practice the recommended relaxation exercises every day? If not, could you tell us about some of the things that made it difficult to do so?”Participants will have the option of either accessing and completing their online log each day or printing and completing a paper copy. The completed paper copy may be posted or transferred onto the online log. Participants will be provided with instructions for completing their online relaxation log. Relaxation logs will be anonymous, identified only by a unique participant identification code. Each participant will be given a unique hyperlink to access their personal log, and only the participant and researcher will have access to this hyperlink.The interview data and open-question responses from the relaxation logs will be analyzed together using the same inductive thematic analysis strategy outlined in Study 1. The line-by-line coding will begin during data collection to help the interviewer to reflect and learn from previous interviews and refocus future interviews . The quaThe findings from the 2 mixed-methods studies will be triangulated  at the discussion-writing stage to produce an in-depth understanding of the program’s mechanisms of impact and identify any implementation or contextual factors that strengthen or impede its delivery and functioning. Triangulation will allow the findings from each study to be corroborated and validated ,37.At the time of manuscript submission, 36 participants have consented to take part in the online survey in Study 1. Thirty of these participants went on to answer questions about the program. For Study 2, 12 participant interviews have been completed and 6 relaxation logs submitted. Data collection for Study 2 was completed November 2015. Study 1 is open for recruitment and data collection will complete in June 2016.This protocol describes 2 mixed-methods studies to evaluate the Tinnitus E-Programme, an Internet-based intervention for tinnitus self-management. A process evaluation will explore past, current, and new users’ reactions to and interactions with the program.Ultimately, the findings of this research will provide the missing evidence-base that is necessary to guide future optimization and evaluation work for the program. First, the identification of any implementation or contextual factors that impede the delivery and function of the program will help us to decide which amendments need to be made to improve the program’s content, usability, and enactment for future users. Second, an in-depth understanding of the psychosocial context in which people with tinnitus interact with the program will provide insight into the circumstances in which the program works best and who is likely to benefit most from it. This can help guide decisions regarding appropriate research conditions and inclusion criteria for future evaluation studies. Third, understanding users’ perceptions of the outcomes of the program can guide evaluation choices regarding appropriate outcome measures. Finally, understanding the program’s mechanisms of impact can give us an understanding of how the program works and what makes it work. Such an understanding has wider implications for the management of tinnitus and can also inform the development of other Internet-based programs for people with similar conditions.This research has some limitations or challenges that need to be considered. As registration to the program is not mandatory and users can choose to complete it anonymously, we have no way of knowing how many people have previously used, or are currently using, the live program. This makes it difficult to reliably estimate the sample size and accurately assess external validity for Study 1. It is also not possible to track past and current users who did not register, making this target population potentially hard to reach and recruit. This limitation also means that convenience sampling was the only feasible sampling method, which may introduce a self-selection bias. The current program does not monitor actual program usage, which means that it will not be possible to validate whether participants actually used the program and their self-reported usage. However, the focus of this exploratory study is on the participants’ accounts of their usage and reasons for usage or any nonusage.Participants recruited to Study 1 are likely to represent a particularly motivated and satisfied group of users. Those who chose not to use the program, gained no benefit from the program, or no longer use the program are less likely to take part. Study 2 will provide more diversity as it will introduce a group of people to the dataset with different motivations . Once recruited, Study 2 participants will be encouraged to continue onto the interview, even if they did not complete or benefit from the program.In Study 1, there were no exclusions regarding length of time since completing the program to maximize recruitment for this very specific population. Some of the participants may have completed the program as long as 6 years ago, which may introduce a recall bias.There are also several strengths of this research. First, the proposed evaluation is being carried out by an independent research team who were not involved in the development of the program. This will minimize any biases that might be present during data collection, analysis, and interpretation. Second, 2 different populations—current or past and new program users—will be studied, allowing us to evaluate the program from 2 different but complementary perspectives and contexts. Combined with the use of mixed methods, this design will provide a more complete, in-depth, and valid understanding of users’ reactions to and interactions with the program. Finally, this study will explore users’ enactment of the relaxation skills learned in the program. This aspect of intervention implementation is more commonly explored in behavior change research where integrating new actions into everyday life is the ultimate outcome of interventions . Enactme


Introduction. While extensive scientific evidence exists on the tobacco epidemic, a lack of understanding of both policies and their appropriate way of implementation continues to hinder effective tobacco control. This is especially so in the developing countries such as India. The present study aims to understand current implementation practices and the challenges faced in mainstreaming tobacco control policy and program. Methods. We chose a qualitative study design to conduct the case analysis. A total of 42 in-depth interviews were undertaken with seven district officials in six districts of Andhra Pradesh. A conceptual framework was developed by applying grounded theory for analysis. Analysis was undertaken using case analysis approach. Results and Discussion. Our study revealed that most program managers were unfamiliar with the comprehensive tobacco control policy. Respondents have an ambiguous opinion regarding integration of tobacco control program into existing health and development programs. Respondents perceive lack of resources, low prioritization of tobacco control, and lack of monitoring and evaluation of smoke-free laws as limiting factors affecting implementation of tobacco control policy. Conclusion. The findings of this study highlighted the need for a systematic, organized action plan for effective implementation of tobacco control policy and program. Tobacco use is the world's leading preventable cause of death. It has been the cause of 5.4 million deaths annually and the toll is expected to account for 10% of all deaths globally by 2015 . To curbThe district tobacco control cell is responsible for overall planning, implementation, and monitoring of different activities and achievement of physical and financial targets planned under the program in the district. The NTCP outlines the involvement of district level health functionaries in implementation of various tobacco control policy measures which includes enforcement and monitoring of implementation of tobacco control laws . It alsoWhile extensive scientific evidence exists on the tobacco epidemic, a lack of understanding of both policies and their implementation continues to hinder effective tobacco control. This is especially so in the context of developing countries such as India . StudiesA qualitative study design was employed to conduct the case study analysis. Case studies are in-depth investigations of a single instance of a phenomenon in its real-life context. Program managers' perceptions and beliefs about tobacco control as a part of a health program constituted the case. Qualitative design helped us to explore in-depth understanding of the program managers' perspective regarding the success and challenges faced by them in implementation of tobacco control policy and program.Senior program heads responsible for different health programs like district tuberculosis control and reproductive and child health program.Senior program officers in charge of the district units of the National Rural Health Mission (NRHM), a flagship program of the Government of India that provides managerial and program support to ongoing health programs in the state of Andhra Pradesh in India.Divergent constituencies belonging to different health programs were chosen for this study so that differences in their approach towards the implementation of tobacco control in the districts could be captured meaningfully.The study was conducted in 6 districts of Andhra Pradesh. A total of 42 in-depth interviews were undertaken where seven district officials were interviewed from each district. A purposive sampling technique was employed with a conscious selection of certain subjects who could provide in-depth information on the topic of interest. We adopted a snowball approach asking each respondent at the end of the interview to recommend further respondents. The respondents were drawn from the health departments at the district level and were comprised of the following:A discussion guide was used as the research tool for this study. The discussion guide consists of several themes, questions, and also probes for further inquiry wherever required .Field investigators who had prior experience in collecting qualitative data were trained in collecting data in local language. They worked in close proximity with the investigating team. Audio tapes were used to record the interviews, wherever permission was granted. Summary sheets were used to capture nonverbal information shared by the respondent during the course of the interview. After the interviews were completed, transcripts were generated by translating from local language into English taking care of subtle nuances and the meaning of specific words. While the fieldwork was going on, the initial few transcripts were closely reviewed in the field to assess the relevance and comprehensiveness of the information obtained and corrections were made. The first author accompanied the investigator in the first few interviews to guide the investigation.The field notes of the pilot study were reviewed for accuracy and completion; changes were made to the guide and tested in a neutral district, following which the fieldwork was initiated in the intervention districts. The data obtained from the pilot transcript was used as the basis for generating new codes. The new codes were added to the previous codebook. The discussion guide used for this study listed down all the questions/themes that need to be addressed in interviews (which were transcribed by subject experts and used as transcripts). The data consist of taped recorded conversations and memos of field investigators. A conceptual framework was developed by applying Grounded theory for analysis, making an attempt to not only contribute to the existing knowledge but also highlight new emerging theories inductively. Grounded theory was considered appropriate in the present study as it applies a systematic procedure to generate theory or insights describing a phenomenon which is grounded in the views expressed by study participants . AnalysiWe adopted a “case analysis” approach in this study where we analyzed the data in 2 stages as follows.The process was initialized with organizing the interviews into groups based on their similarities in responses for each specific interview question as noted in Patterns of similarities and dissimilarities were searched across the cases. The transcripts were reviewed as multiple comparative cases; the coding was guided by the perspectives and experiences of one group of officials compared to the others as per a specific theme. The codes that generated from the data during analysis were revisited and revised and the codebook was updated accordingly. Similar codes clubbed together under families and themes were generated. Additional review of the coded data and reflection on the initial conceptualization led to refinement of the concepts used to describe the program manager's perspectives on the implementation of tobacco control in the districts. This provided more meaning to the data and helped in its interpretation.In describing the data, the terms “few,” “majority,” and “most” are used to represent approximately less than 40%, 40–80%, and more than 80%, respectively, of proportions of respondents in the study. ATLAS.ti 7 software was used for analysis.Informed consent was taken from the respondents. All respondents were asked if they are comfortable with the audio recording of their interviews, and care was taken not to intrude upon the schedule of the respondents by seeking prior appointments for the interview. A written permission was also obtained from the state department and each of the district administrations for the study. The study was conducted from March to April 2011. The study was approved by the Institutional Ethics Committee (IEC number 65/60).Case study analysis in health system research helps in understanding the current levels of knowledge and awareness of key stakeholders involved in decision-making processes. In the present case study analysis, intensive and rigorous two staged case by case analysis was conducted to explore in depth understanding and comparison of program manager's perceptions on each theme. The first level of “case analysis” methodology design enabled us to group similar and dissimilar responses to the interview questions and form cases as described in The findings are broadly categorized into four different themes and are discussed as follows.(1) Implementation of Tobacco Control Policy. Program managers highlighted several potential solution levers to strengthen the enforcement of existing tobacco control policy. Awareness generation among policy makers was the most common and cross cutting suggestion reported by the majority of respondents. A majority of them emphasized enhancing enforcement of tobacco control policy by including various information, education, and communication channels like television, media, advertisements, health warning, and health education. Respondents opined that multifaceted educational strategies in conjunction with community-based campaigns will bring substantial change in the trajectory of tobacco use.Public awareness should be improved. IEC, information, education and communication should be properly implemented… Then this program (NTCP) will be successful.A majority of program managers mentioned that involvement of policy makers will prioritize the implementation of tobacco control policy. Respondents mentioned that policy makers should be sensitized to ensure monitoring of tobacco control policy. Everyone (policy makers and community) should help and play their role in controlling tobacco epidemic… it is not only the people who are working in health programs or in tobacco control….Lastly, it was suggested by few program managers that changes in the fiscal policy and cost of tobacco products may also bring a visible difference in its use.Cost of tobacco products should be increased so that they (people) can stop (consuming it)….Tobacco control campaigns, which primarily utilized mass media to educate and inform the public, have been effective in promoting antismoking, thereby reducing tobacco prevalence in high-income countries  because the policy [No smoking policy] are just policies because people just read it and forget…. They [People] should themselves follow it first [Policy].The smoke-free policy in public places came into enforcement in India in 2008 as per l policy , 19. Ca(3) Integration of Tobacco Control Program into Existing Health Programs. Most of the cases view integrating tobacco control with other health and development programs to be a valuable tool. Respondents mentioned that this will also help in simplifying technical and financial support. For instance,The issue [Tobacco issue] has to integrate with NRHM, RNTCP, We [Stakeholder] have to tell them to do this [Tobacco control] program with that [NGO's own program] program, we have to give some instructions to them (NGO's).Few other cases were concerned that integrating tobacco control with other health programs would lead to dilution of the message as it would then become a low priority within the larger programs. Respondents stated the following:If we give (do) 10 programs likes that (together) it (tobacco control) gets diluted.It (tobacco control) should be a separate program not integrated.They (NRHM) are unable to do properly, so how can we (stakeholders) integrate with it?Thus, mixed reactions were observed on the effectiveness of integration of tobacco control program with other health programs. This is expected as the health programs that these program managers implement and overlook are different in design, size, and scope of implementation at the district level. The actual capacity and implementation of these programs also vary in different districts depending upon resources. Senior managers from NRHM  and RNTCP  continued to address the need for integration of tobacco cessation under large program. Many studies have revealed the importance of integrating tobacco control into health and development agendas  have to motivate them [Human resource and staff] and develop their skills and utilize them.Studies from developing  and deve(4.3) Lack of Monitoring and Interdepartmental Coordination. Another significant barrier as reported by a few respondents was lack of interdepartmental coordination and motivation within the government. Few respondents also revealed lack of regular monitoring to be a major factor responsible for limiting implementation of tobacco control policy. For instance,No monitoring and supervision is done, strict implementation is not there.Two respondents also reported existing corruption within the system and political interference during the implementation of the policy as a barrier for effective implementation. For example,There are many problems, like corruption.Once the policy [Smoke-free policy] is made then there should not be politician interference.Several cases urged for a need of adequate and comprehensive training in order to raise self-confidence and motivation amongst health care providers. Similar observations were made in other Indian studies which underscore the need of building capacity of health care providers and program implementers in tobacco control [ control , 30.The response of the cases on various aspects of implementation of tobacco control program was captured. It is clear from the number of positive responses received that there has been undoubtedly a lot of success achieved in implementation of tobacco control policy and program. On the other hand, various existing gaps and issues continue to hinder its successful implementation. This can be interpreted as negative responses. There are a very small percentage of cases that were neutral to certain issues of implementation of program. This clearly indicates that there exists a huge scope of improvement in implementation of tobacco control policy and program by overcoming the barriers. This is evident from a varied set of responses on each theme .The study is limited by the fact that the views of program managers interviewed may not be generalizable to the health system in different states in India. The study was conducted over a short time frame. The results of the study should be viewed as a “snapshot” pertaining to just one time point when the views of stakeholders on tobacco policy and program implementation were captured. However, despite the inherent limitations, stakeholder analysis is a vital tool in informing the design of health systems research.The findings of this study obtained from “case analysis” approach captured perceptions of various program managers on several aspects of implementation of tobacco control policy and program. Respondents perceive lack of intersectoral coordination and resources, low prioritization of tobacco control in health and development programs, and lack of monitoring and evaluation of implementation of smoke-free laws as limiting factors affecting successful implementation of tobacco control policy and program. Some of the potential solution levers as suggested by many program managers include employing dedicated staff, regular training, and both financial and nonfinancial incentives to raise their motivation.Based on the findings above, we highly recommend several actions to enable us to move towards significant reductions in the growing tobacco burden. First, we encourage central and state public health organizations to establish stringent and pragmatic goals which will help reduce tobacco use especially at the district level. At the same time, realistic planning needs to take into account existing capacity of the health system. An assessment of the health system implications and related resource costs will help in planning for proper implementation of tobacco control policy and program. This will also facilitate integration of the tobacco control program into broader national health plans.


We show possible scenarios for the occurrence of M ~ 7 interplate earthquakes prior to and following the M ~ 9 earthquake along the Japan Trench, such as the 2011 Tohoku-Oki earthquake. One such M ~ 7 earthquake is so-called the Miyagi-ken-Oki earthquake, for which we conducted numerical simulations of earthquake generation cycles by using realistic three-dimensional (3D) geometry of the subducting Pacific Plate. In a number of scenarios, the time interval between the M ~ 9 earthquake and the subsequent Miyagi-ken-Oki earthquake was equal to or shorter than the average recurrence interval during the later stage of the M ~ 9 earthquake cycle. The scenarios successfully reproduced important characteristics such as the recurrence of M ~ 7 earthquakes, coseismic slip distribution, afterslip distribution, the largest foreshock, and the largest aftershock of the 2011 earthquake. Thus, these results suggest that we should prepare for future M ~ 7 earthquakes in the Miyagi-ken-Oki segment even though this segment recently experienced large coseismic slip in 2011. A Mj9.0 great interplate earthquake occurred off the east coast of Tohoku district, northeast Japan, on March 11, 2011, with ruptures propagating over a distance of 400 km along the Japan TrenchThe deeper part of the Mj9.0 coseismic slip area is located off the east coast of Miyagi Prefecture, where Mj7.1–7.4 earthquakes have occurred with recurrence intervals of approximately 30–40 years, in 1897, 1936, 1978, and 2005315678The southern segments to the Miyagi-ken-Oki segment are known as Fukushima-ken-Oki and Ibaraki-ken-Oki segmentsOne of the characteristics of the 2011 earthquake is the sequence of seismic and aseismic events observed within the Mj9.0 source area prior to the mainshock. On March 9, 2011, 51 h prior to the Mj9.0 earthquake, a Mj7.3 interplate earthquake occurred within the Mj9.0 source area6More than 5 years have passed since the Mj9.0 earthquake. The post-seismic deformation rate is decreasing, and it has almost decayed by the time 2.5 years following the mainshockIn the Tohoku-Oki region, earthquakes having a similar magnitude as the 2011 earthquake occurred in 869 in the Jogan era and 1454 in the Kyotoku era as indicated by geological data of tsunami sediments1617et al.et al.et al.et al.Previous numerical simulations of the earthquake generation cycles of the 2011 Tohoku-Oki earthquake have shown that M ~ 9 earthquakes have occurred with a recurrence interval of 700–900 years2021In this study, we reproduce the observational characteristics prior to, during, and following the 2011 Mj9.0 earthquake including past Miyagi-ken-Oki earthquakes and foreshock, mainshock, largest aftershock, and afterslip by using realistic numerical simulation. We discuss the time interval between the Miyagi-ken-Oki earthquake immediately following the M ~ 9 earthquake and the M ~ 9 earthquake by using reasonable scenarios consisting of various observational characteristics. In addition, we investigate the scenario prior to the next M ~ 9 earthquake.The numerical simulations are based on the method used in previous studies22et al.We performed approximately 190 simulation runs by using various parameter sets. From the various simulations performed, we qualitatively reproduced the observational characteristics by using several frictional parameter sets, as shown in In each parameter set, two Mw9.1–9.2 earthquakes occurred with recurrence intervals of approximately 700–770 years  and 4a. The rupture initiation point differed among M ~ 9 earthquakes, occurring in the southern area near the SHL2 patch, the northern area near the SHL1 patch, and the shallower area. The M ~ 9 earthquake initiated from a point within the large coseismic slip area of each earthquake.A Mw7.0–7.2 aftershock occurred in the IBK patch within 2–50 days following each M ~ 9 earthquake. Furthermore, the afterslip following the M ~ 9 earthquake was distributed in the deeper side of the coseismic slip area. In addition, the afterslip in the MYG patch was less than 2 m during approximately three years . When weMw7.1–7.4 earthquakes occurred repeatedly in the MYG patch during interseismic periods of M ~ 9 earthquakes  and 4b wDuring the same 230-year period, Mw6.9–7.1 earthquakes occurred repeatedly in the IBK patch with average recurrence intervals of 39 years at a standard deviation of 12.3 years . In the During the last 200 years of the M ~ 9 earthquake cycle resulting from 19 simulations with five M ~ 7 patches , M ~ 7 eIn the SHL1 patch, two or three Mw7.2–7.4 earthquakes occurred during the interseismic period of the two M ~ 9 earthquakes, depending on the model . In the At the later stage of the interseismic periods of the M ~ 9 earthquake cycle, the locked state remained at the shallowest area. Moreover, transient slow slips occurred near the rupture initiation area at the end of each M ~ 9 earthquake cycle . These s2627The above characteristics were observed in many simulations using other frictional parameter sets such as those shown in The timing of the M ~ 7 foreshock differed for each cycle. One model, shown in 6A different model  showed a1Although we reproduced various characteristics, the M ~ 7 foreshock in the SHL1 patch that occurred 51 h prior to the Mj9.0 earthquake could not be reproduced by all models. In 15% and 30% of the simulations, the M ~ 7 foreshock in the SHL1 patch occurred within 4 days and 50 days prior to the M ~ 9 earthquake, respectively. For the simulations, parameter sets with a small radiation damping term of 0.3G/2β were used as those shown by red color in We reproduced the observational characteristics of the recurrence interval, coseismic slip, aftershock, and afterslip of the 2011 Mj9.0 earthquake and the recurrence interval of M ~ 7 earthquakes  along the Japan Trench. A range in recurrence intervals of M ~ 7 earthquakes was detected at the MYG, FKS, and IBK patches depending on slight differences in frictional parameters. Compared with past observations, the range of recurrence interval simulated in this study is considered to be acceptable.In this study, we generally reproduced coseismic slips of both M ~ 7 and M ~ 9 earthquakes in the Miyagi-ken-Oki segment with a simple model by using a circular patch. Similar to the observations of the 2011 Mj9.0 earthquake, a larger coseismic slip was observed in the MYG patch during the M ~ 9 earthquake than that in the M ~ 7 MYG earthquake. Nonetheless, the results showed that the time interval between the M ~ 9 earthquake and the subsequent MYG earthquake was shorter than the average recurrence interval observed in the later stage of the M ~ 9 earthquake cycle . Long-teTo understand the shorter time interval of the MYG earthquake immediately following the M ~ 9 earthquake , we compIn contrast, the stress increased at a low rate  varied with depth or strike and ranged from 1.0 km to 9.0 km. Hereafter, subscripts i and j denote subfaults modeled for the simulation. The time derivative of shear stress τ on the i-th subfault owing to slip at the j-th subfault is calculated asNumerical simulations were based on the method used by Nakata ij is the stress increment on a subfault i owing to unit slip on a subfault j, Vj is the slip rate of the j-th subfault in the plate convergence direction, Vpl,j is the plate convergence rate of the j-th subfault, and η denotes the seismic radiation damping termpl = 8.4 cm/yret al.et al.where KSpace-time variations in slip velocity are assumed to be an unstable slip with a frictional interface. Although we do not know the true friction law for the plate interface, we used a rate- and state-dependent friction law as an approximated mathematical model for large-scale frictional behavior on the plate interface23i for a given stress τi and a value of τsi (=τs*i + Δτsi). If we set the reference velocity V* at a slip velocity level below which the slip is sufficiently slow to be negligible, the value of τsi is the threshold level of stress τi required to cause significant slip velocity. Therefore, the value of τsi is analogous to the “strength as a threshold”pl in the calculation below; here, τs* (=μ*σ) represents the steady state strength with V = V*, and Δτs is the variation in strength from the steady state.s, which varies depending on the prior slip history. The parameters B (=bσ) and L control strength recovery and slip weakening. For slip weakening, B and L determine primarily the amplitude of strength variation Δτs and slip weakening distance Dc. These are the minimum characteristics of large-scale friction and are caused by various small-scale physical processes. From this perspective, frictional parameters A (=aσ), B (=bσ), and L in the rate- and state-dependent friction law are mathematical fitting parameters, which were assumed to be constant over the earthquake cycle in our simulations.32i/dt and obtained differential equations for slip rate V and strength Δτs. Assuming the initial values given below, differential equations were solved with an adaptive time step fifth-order Runge–Kutta algorithmpl, and strength was calculated as τs*i + Δτsi = Δτsi = −Biln(Vi/Vpl). For the boundary conditions, slip velocity was assumed to be constant and equal to Vpl outside the model region.To solve The frictional heterogeneity at the shallower part of the Miyagi-ken-Oki region was assumed to be slightly stronger  than the surrounding area  over a wide range of 150–170 km long along the strike and 8–22 km in depth. The extent of M ~ 9 rupture area was controlled by the larger value of L, which means larger slip weakening distance and fracture energy, in the shallower part of the Miyagi-ken-Oki region and in the surrounding background area of A–B < 0. In addition, we used five circular patches termed SHL1, SHL2, MYG, FKS, and IBK, as the past sources of the 1981, 2003, 1978 Miyagi-ken-Oki, and 1938 Fukushima-ken-Oki earthquakes and the 2011 Ibaraki-ken-Oki largest aftershock, respectively .How to cite this article: Nakata, R. et al. Possible scenarios for occurrence of M ~ 7 interplate earthquakes prior to and following the 2011 Tohoku-Oki earthquake based on numerical simulation. Sci. Rep.6, 25704; doi: 10.1038/srep25704 (2016).


Calprotectin, a heterodimer of S100A8 and S100A9 subunits, is associated with inflammatory disorders such as rheumatoid arthritis and cystic fibrosis. Although calprotectin levels are increased significantly in the gingival crevicular fluid (GCF) of periodontitis patients, its effects on periodontal ligament cells (PDLCs) remain largely unknown. The aim of this study was to evaluate calprotectin levels in the GCF of generalized aggressive periodontitis (AgP) patients and to investigate the effects of recombinant human calprotectin (rhS100A8/A9) and its subunits (rhS100A8 and rhS100A9) in PDLCs. Both the concentration and amount of crevicular calprotectin were significantly higher in the AgP group compared with healthy controls. In addition, the GCF calprotectin levels were correlated positively with clinical periodontal parameters including bleeding index, probing depth, and clinical attachment loss. rhS100A8/A9 promoted cell apoptosis, whereas rhS100A8 and rhS100A9 individually exerted little effect on apoptosis in PDLCs. rhS100A9 and rhS100A8/A9 increased the activation of nuclear factor-κB (NF-κB) by promoting the nuclear translocation of p65 in PDLCs, subsequently inducing expression of the pro-inflammatory cytokines IL-6, IL-8, TNFα, and COX2. Treatment with an NF-κB inhibitor partially reversed the rhS100A9- and rhS100A8/A9-induced upregulation of the pro-inflammatory cytokines. rhS100A9, and not rhS100A8, was mainly responsible for the pro-inflammatory role of calprotectin. Collectively, our results suggest that calprotectin promotes apoptosis and the inflammatory response in PDLCs via rhS100A9. These findings might help identify novel treatments for periodontitis. Periodontitis is an infectious disease that affects the tissues supporting the teeth and leads to eventual tooth loss Calprotectin is a heterodimeric calcium-binding protein consisting of S100A8 and S100A9 subunits in vivo. PDLCs reside in the periodontal ligament space and play a key role in forming the periodontal ligament and its fibers by secreting extracellular matrix components such as collagen and mineralized tissue Interestingly, human S100A8 is a potent and specific autocrine chemotactic factor in periodontal ligament cells (PDLCs); therefore, it might be an attractive therapeutic candidate for the treatment of periodontal disease In recent years the regenerative potential of PDLCs in tissue repair has received increasing attention The Ethics Committee of Peking University School of Stomatology approved the study protocol, and all clinical specimens were obtained from patients who provided written informed consent to use their tissues for research purposes .A total of 40 participants were included in the study. Of these, 20 patients <36 years old with generalized aggressive periodontitis were recruited from the Department of Periodontology, Peking University Hospital of Stomatology, China. The diagnostic criteria for generalized aggressive periodontitis were defined according to the classification proposed at the International Workshop for the Classification of Periodontal Diseases and Conditions in 1999 All subjects underwent a clinical periodontal examination including PD, BI, and CAL using a Williams periodontal probe at six sites of each tooth to determine their clinical periodontal status Two sites of each subject were selected for GCF sampling, one from the mesial buccal site of the right maxillary central incisor and the other from the mesial buccal site of the left mandibular first molar. GCF samples were collected using a paper strip  from healthy gingival crevices  for periodontally healthy controls and from periodontal pockets  in patients with generalized aggressive periodontitis. Supragingival plaques were removed from the interproximal surfaces using a sterile curette prior to the sampling. Paper strips were then inserted carefully into the crevice until mild resistance was felt and left there for 30 seconds.GCF samples were eluted from the strips by placing them in 300 µl of phosphate buffered saline (PBS). The calprotectin levels were then assayed using commercially available ELISA kits according to the manufacturer's instructions . The amount of calprotectin in each sample was calculated based on the dilutions as described previously Escherichia coli BAL21 (DE3) and their expression was induced using isopropyl β-D-thiogalactosidein . Next, a nickel column and a polymyxin B-agarose column  were used to purify the recombinant His-tagged proteins. The endotoxins contamination was <1 pg/µg protein, as measured using a Limulus amebocyte assay (Sigma-Aldrich). Recombinant proteins were analyzed on SDS-PAGE gels and stained using Coomassie brilliant blue. As shown in 2+ according to Vandal et al The methods used to produce and purify the recombinant proteins were described previously Primary normal human periodontal ligament cells were obtained from explant cultures using methods described previously To detect cell apoptosis, an annexin V-fluorescein isothiocyanate (FITC) kit  was used according to the manufacturer's protocol. Briefly, PDLCs were treated with rhS100A8, rhS100A9 or rhS100A8/A9, incubated for 48 h, trypsinized, and then re-suspended in the binding buffer containing 5 µL of annexin V-FITC and 4 µg/mL of propidium iodide after incubation in the dark at room temperature for 10 min. Apoptosis were then analyzed using fluorescence-activated cell sorting . At least 10,000 cells per sample were analyzed and annexin-V-FITC-positive cells were regarded to be apoptotic.4 PDLCs per well of a 24-well plate were incubated with rhS100A8, rhS100A9 or rhS100A8/A9 at a final concentration of 10−6 M for 48 h. They were then fixed with 4% paraformaldehyde (Sigma-Aldrich), stained with DAPI and visualized using a fluorescence microscope as described previously The morphology of PDLCs treated with calprotectin were evaluated using fluorescence microscopy after staining with 4′,6-diamidino-2-phenylindole (DAPI). Approximately 5×105′-TAC ATT TGC CGA AGA GCC-3′, human IL-8 forward 5′-TTT TGC CAA GGA GTG CTA AAG A-3′ and reverse 5′-AAC CCT CTG CAC CCA GTT TTC-3′, human TNFα forward 5′-CGA GTG ACA AGC CTG TAG CC-3′ and reverse 5′-TGA AGA GGA CCT GGG AGT AGA T-3′, human COX2 forward 5′-CTG GCG CTC AGC CAT ACA G-3′ and reverse 5′-ACA CTC ATA CAT ACA CCT CGG T-3′, human GAPDH forward 5′-ATG GGG AAG GTG AAG GTC G-3′ and reverse 5′-GGG GTC ATT GAT GGC AAC AAT A-3′. Real-time PCR was performed using a 7500 Fast Real-time RT-PCR System  according to the manufacturer's instructions. The relative expression of the targed gene was then normalized to GAPDH expression using 2−ΔΔCt method.Subconfluent cell monolayers were serum-depleted overnight in DMEM and were then treated with rhS100A8, rhS100A9 or rhS100A8/A9 alone or in combination with the NF-κB specific inhibitor pyrrolidine dithiocarbamate  for 12 h. Total RNA was isolated using RNA isolation reagent TRIzol  according to the manufacturer's instructions, and the concentration was determined spectrophotometrically. First-strand cDNA was synthesized using a first-strand cDNA synthesis kit  using oligo dT primers. The primers used were as follows: human IL-1β forward 5′-ATG GCT TAT TAC AGT GGC A-3′ and reverse 5′-GTA GTG GTG GTC GGA GAT T-3′, human IL-6 forward 5′-GTG AGG AAC AAG CCA GAG C-3′ and reverse −6 M rhS100A8, rhS100A9 and rhS100A8/A9 were added to the ELISA plates incubated with antibodies, aspirated and then washed with washing buffer. The optical density at 450 nm was then measured.The concentrations of IL-6 and IL-8 in cell culture supernatants were determined using ELISA  according to the manufacturer's instructions. Briefly, the supernatants of PDLCs treated with 104 cells/mL, and treated with rhS100A8, rhS100A9 or rhS100A8/A9 for 6 h. Immunofluorescent analysis of the p65 was then performed as described previously PDLCs were seeded on glass slides at a density of 5×10Nuclear PDLC extracts were prepared and EMSAs were performed as described previously p value <0.05 was considered to indicate statistical significance.Statistical analyses were performed using SPSS PASW version 18. Distribution of the variables was tested using the Shapiro-Wilk test. Continuous normally distributed variables were presented as means ± standard deviations (SD). The median (lower to upper quartile) was used to describe non-normally distributed data. Differences for age and calprotectin levels in the GCF between groups were analyzed using Student's t-test, whereas differences for clinical parameters between groups were analyzed using Mann-Whitney U-test. Gender was analyzed using chi-square test. Spearman's correlation tests were used to identify the associations between local clinical variables and the calprotectin levels. Pearson's correlation coefficient is used to identify the association between the age and GCF calprotectin level. Cell culture experiments were performed at least in triplicate, and the differences among groups were analyzed using one-way ANOVA. A two-tailed vs. 471±51.120 µg/mL, and 4,378±741.4 ng vs. 87.82±12.38 ng, respectively; p<0.05). The calprotectin levels in the GCF were moderately positively correlated with clinical periodontal parameters including BI, PD and CAL . Fewer apoptotic cells were observed in rhS100A8-treated cultures compared with rhS100A9-treated cultures. Because the recombinant S100A proteins used in these studies were derived from E. coli extracts, it is possible that the response was due to trace contamination with bacterial LPS. To exclude this possibility, an aliquot of the recombinant protein was heated at 100°C for 10 min to inactivate the protein; this abrogated its pro-apoptotic activity.Morphological changes in PDLCs treated with rhS100A8, rhS100A9 and rhS100A8/A9 were analyzed using fluorescence microscopy following DAPI staining . Viable p<0.05; The mRNA levels of IL-6, IL-8, TNFα and COX2 were increased significantly in cells treated with rhS100A9 and rhS100A8/A9 compared with the vehicle and heat-inactivated groups . IL-6 anTo determine the mechanism for the pro-inflammatory effects of rhS100A9 and rhS100A8/A9, indirect immunohistofluorescence was performed to detect the nuclear translocation of NF-κB-p65. The presence of red fluorescence in the nucleus, which is suggestive of NF-κB translocation, typically indicates its functional activation because it is otherwise retained in the cytoplasm as inactive complexes with IκB proteins. In untreated PDLCs, p65 was localized almost exclusively in the cytoplasm. In contrast, rhS100A9 and rhS100A8/A9 induced the nuclear translocation of p65, as indicated by the pink nuclear signal in the merged images caused by the overlapping blue (DAPI staining) and red fluorescence signals (NF-κB-p65 subunit) in the nucleus . rhS100ATo further assess the effects of rhS100A8, rhS100A9 and rhS100A8/A9 on NF-κB signaling, oligonucleotides containing the NF-κB-binding sequences were reacted with nuclear extracts. As shown in The present study revealed that calprotectin levels were significantly higher in the GCF of AgP patients compared with the control group, which is consistent with the previous studies in vitro and data revealed that rhS100A8/A9 had greater apoptosis-inducing effects than did rhS100A8 or rhS100A9 alone. Consistent with this, cytotoxic effects of rhS100A8/A9 in EL-4 cells were detected with relatively low concentrations in a standard medium, whereas rhS100A8 and S100A9 caused only marginal cytotoxicity at even higher concentrations S100A8/A9 was reported to have broad apoptosis-inducing activity in various cells including cancer cells such as AGS Anatomically, periodontal ligament cells are in close proximity to infiltrating inflammatory cells in vivo trials using anti-S100A9 might support this.The pro-inflammatory effects of rhS100A9 and rhS100A8/A9 in the current study were significantly greater than those of rhS100A8. Although there is a general consensus that the function of S100A8 or S100A9 is dependent on heterodimer formation, increasing evidence suggests that each subunit alone might exert specific effects. The present study revealed that treatment with rhS100A9 induced more apoptotic cells and a more significant upregulation of pro-inflammatory cytokines compared with rhS100A8. This suggests that S100A9 plays a more important role in the pro-apoptotic and pro-inflammatory actions of rhS100A8/A9 than does S100A8. Consistent with this, S100A9 exerts pro-inflammatory effects by triggering NF-κB activation in human THP1 cells, which is dependent on TLR-4 In conclusion, rhS100A8/A9 exerted PDLCs apoptosis, whereas rhS100A8 or rhS100A9 were less effective. In addition, rhS100A9 and rhS100A8/A9 amplified the pro-inflammatory cytokine response by activating NF-κB signaling in PDLCs; rhS100A8 was less potent. These results suggest that reducing local S100A9 or S100A8/A9 levels might have therapeutic potential for periodontitis.Figure S1SDS-PAGE analysis of recombinant S100A8 and S100A9 stained with Coomassie brilliant blue. Recombinant human S100A8 (lane 3) and S100A9 (lane 5) were expressed after induction with β-D-thiogalactoside but were absent in uninduced lysate (lane 2). Purified rhS100A8 and rhS100A9 were free from other contaminants (lanes 4 and 6).(TIF)Click here for additional data file.


Earthworms are important soil macrofauna inhabiting almost all ecosystems. Their biomass is large and their burrowing and ingestion of soils alters soil physicochemical properties. Because of their large biomass, earthworms are regarded as an indicator of “soil heath”. However, primarily because the difficulties in quantifying their behavior, the extent of their impact on soil material flow dynamics and soil health is poorly understood. Image data, with the aid of image processing tools, are a powerful tool in quantifying the movements of objects. Image data sets are often very large and time-consuming to analyze, especially when continuously recorded and manually processed. We aimed to develop a system to quantify earthworm movement from video recordings. Our newly developed program successfully tracked the two-dimensional positions of three separate parts of the earthworm and simultaneously output the change in its body length. From the output data, we calculated the velocity of the earthworm's movement. Our program processed the image data three times faster than the manual tracking system. To date, there are no existing systems to quantify earthworm activity from continuously recorded image data. The system developed in this study will reduce input time by a factor of three compared with manual data entry and will reduce errors involved in quantifying large data sets. Furthermore, it will provide more reliable measured values, although the program is still a prototype that needs further testing and improvement. Combined with other techniques, such as measuring metabolic gas emissions from earthworm bodies, this program could provide continuous observations of earthworm behavior in response to environmental variables under laboratory conditions. In the future, this standardized method will be applied to other animals, and the quantified earthworm movement will be incorporated into models of soil material flow dynamics or behavior in response to chemical substances present in the soil. Oligochaeta. They inhabit various types of soil ranging from 10 to 2000 individuals per meter squared, resulting in a large biomass of between 0.5 to 305 g dry mass m−22, CH4 and N2O Earthworms, the so-called ecosystem engineers, are important soil macrofauna belonging to the subclass Evaluation of the functional role of earthworms on soil ecosystems has been, however, insufficient because of methodological difficulties In addition to the importance of earthworms in ecosystem functions, they are also regarded as an important biological indicator of chemical toxicity in the soil. Ecotoxicology has traditionally used biological indicators to quantify or predict the changes in soil “health” or “quality” To date, automated systems have been used to track movement with high time resolution continuously using a web camera for nematodes, ants, honeybees, and drosophila, and to subsequently analyze the continuous image data Caenorhabditis elegans, have not considered changes in the extent of body stretch A number of automated tracking systems for a model worm, http://rsbweb.nih.gov/ij/version 1.46 for Windows) can provide a plug-in to manually track the two-dimensional positions and lengths of objects. Even when using this application, analyzing large data sets is labor intensive; therefore, a more reliable and automated quantification method is required for continuous image data.Although existing methods such as WormScan In the present study, we developed a high-throughput program that tracks two-dimensional coordinates of multiple points and body lengths of objects in continuously acquired image data from earthworms; to the program was able to process approximately 1 frame per second. Following user initialization, the program tracks the head, central position, and tail of a single earthworm. This program provides a means to track animals that change their body lengths over time both accurately and efficiently, though still with limited performance. The output was subsequently used to calculate the velocity of those points. Finally, in the future, the quantified behavior could be incorporated into soil material flow dynamic models or applied to measure their reactions to chemical inputs in the soil.Eisenia japonicaMetaphire hilgendorfiM. hilgendorfi is epigeic because it primarily inhabits the litter layer and E. japonica is polyhumic-endogeic because it mainly lives in the topsoil layer and has more inorganic content in the gut Two different species of earthworm were used in this study; E. japonica and M. hilgendorfi used in the experiment were 1.45 and 4.75 g, respectively. E. japonica and M. hilgendorfi were transferred from the plastic box to glass containers for network-camera recording inside an incubator, where the mean ambient temperature was set at 15°C and the ambient air humidity was saturated at the given temperature  with approximately 100 g of alluvial soil at 60% of its water holding capacity until the experiment. The weights of perature . A fiberperature . The netTo estimate local image distortions, checkerboard (8 rows and columns) was located for image capturing system on the same position as the glass container. The length of the diagonal lines were measured using a vernier caliper with a 0.1 mm precision and calculated the errors. The maximum error in mm was 0.2 mm that resulted the precision less than 0.1%.http://rsbweb.nih.gov/ij/version 1.46 for Windows) with an additional plugin, Manual Tracking . Currently manual tracking is a standard method to track animal behavior using image data. Second, we then used a newly developed program (MimizuTrack) that was compared with Manual Tracking in Fiji.Two methods were implemented: a manual tracking method and a new automated tracker. First we used a manual tracking method relies on the Fiji implementation of ImageJ  captured at 1 second intervals were converted to movie data, using a function in Fiji to convert from JPEG into.avi formatted data. Each coordinate per one second was tracked to obtain the velocity of the movement. We then handled the output data as ground truth for further evaluation since the data were input by expert users. The output data were used for evaluation of the program described in the next section. Our program was benchmarked against user input only. This was because no automated tracking system currently exists that is directly comparable to our tracker in this specific setting.The image data obtained were first processed to separate the objects (earthworms from the background). In the original image, the contrast between the earthworms and the background was highest in the red channel so only this component was used for the process. The procedures are done manually as an initial step, then the image data are processed using a program . The red image was then used for further analysis. Earthworms were assigned a value of 255 and the non-earthworm areas were eliminated by applying a value of 0.A threshold to extract the earthworm body was determined and a binary image with white for the object and black for the background was generated.The noise was eliminated by removing small fragments from the background.The rest of the image processing was carried out by our newly developed program. The procedures are shown in The tracking method is as follows;Thinning the earthworm body areasTo extract feature points, such as the head, tail, and center points, the program was tasked with identifying the end-points (tails) of the line segment corresponding to the worm body. That is as thin as possible while remaining fully connected and centered. Using binary images of the earthworm body, the program was able to obtain three feature points on each frame. First, the Hilditch algorithm is used to reduce the pixel region representing the earthworm to its skeletal form Extracting the head, tail, and center point of individuals (feature extraction)The next step was to determine the head, tail, and center point. The program extracted the feature points of the earthworm body by first identifying the head and tail using the different features between the two points , then the center point was determined. To initialize the program, the tail point was determined manually. By doing this, the program automatically calculated four-neighbor distances of the center line from the tail point and extracted the point at the furthest distance. The program defined this as the head point of the earthworm. Then, the program recognized the halfway point between the head and tail as the central point of the body. Using this process, the program could obtain the initial feature points of the earthworm . From the second frame, the program calculated new feature points using the previous ones. First, the program extracted new tail points based on the fact that the tail points of the earthworm move slower than the other points. Using this feature, the program recognized the edge points as new tail points with the shortest distance from the previous points. Both head and center points were obtained by the same procedure, which were extracted from the initial head and center points. These processes were carried out on all of the images. The program outputs positional data in each frame to a CSV file, allowing trajectories over time to be visualized.All statistical analyses were performed using SPSS 10.05 . Relationships between two variables  were assessed using standard bivariate correlation procedures. The correlation coefficient is shown in the text below and the figures.E. japonica and M. hilgendorfi respectively  were larger than those for the tail point . One reason for this was that the head moved more frequently while the tail stayed in the same position. Since the program tracks and determines the feature points by predicting the next steps, the program may fail to track the objects if the objects  move more broadly in the image-capturing interval (1 second). In the case of M. hilgendorfi, when they looped, coiled, or crossed their bodies, the program faced difficulties in recognizing the edges, and failed to detect the head positions.First, we were successful in tracking the three target points,  on a time scale of seconds, continuously followed by body length calculations using the newly developed program. We used a part of the obtained data for the evaluation of the program which shows typical movement of two species of earthworms every 150 seconds and 300 seconds for ectively –11. The japonica . In contM. hilgendorfi were due to the misestimation of head points by the automated program, in which case the head was attached to a part of the body. Otherwise, the automated program satisfactorily detected the peaks in terms of timing and absolute values. As shown in Table1, the mean velocities of the head and tail of E. japonica calculated by the automated program were 4.75±4.36 and 3.44±4.38 mm sec−1, respectively, while those from the manually calculated values were 4.81±4.29 and 3.56±4.44 mm sec−1, respectively. The mean velocities of the head and tail of M. hilgendorfi calculated by the automated program were 4.38±4.13 and 1.03±1.92 mm sec−1, respectively, while those from the manually calculated values were 5.09±4.43 and 0.92±2.21 mm sec−1, respectively. The differences between the automated and manually calculated values were 0.07±0.92 and 0.11±0.88 mm sec−1 for the head and tail of E. japonica, respectively and 0.71±2.49 and 0.11±0.80 mm sec−1 for those of M. hilgendorfi, respectively (Table2). The difference was largest in the head of M. hilgendorfi but the others matched quite well, with the differences being less than 0.2 mm sec−1. The tail points tended to move slower than the head points according to the calculated velocities, and the head point of M. hilgendorfi moved faster than that of E. japonica by 1.65±7.07 mm sec−1. The body lengths were calculated from the binary data, which were converted into a line (shown in Table3).Using their coordinates, the velocity of each point was calculated. The output data obtained with the automated program were further confirmed by comparing them with the manually acquired data  and 10. E. japonica and M. hilgendorfi were 167.02±13.62 and 199.28±10.05 mm, respectively, while the maximum and minimum lengths of E. japonica were 190.73 and 131.44 mm, respectively, and those of M. hilgendorfi were 220.38 and 145.33 mm, respectively. Therefore, the stretch from minimum to maximum was 59.19 and 75.05 mm for E. japonica and M. hilgendorfi, respectively. The extent of the stretchiness was in agreement with previously reported values The mean lengths of Caenorhabditis elegans (one of the model nematodes) M. hilgendorfi, when they coiled or crossed their bodies, the program faced difficulties in recognizing the edges, and failed to detect the head positions. To overcome such problems, one solution would be to obtain images at higher time resolutions so that the program could keep tracking more frequently moving objects without losing the target points because the higher time resolution is closer to continuous data. Increasing the contrast between the background and the objects for image analysis would also achieve more precise results. Furthermore, there would be alternative methods to detect feature points such as differentiating the shapes or brightness of head or tail, which will further improve the program.To date, there exists no method to process and extract such high time resolution image data  capturing three feature points along with tracking elastic objects such as earthworms, which move quickly and widely, although the program is still a prototype at this stage. A number of automated systems have been developed to track insects' movements, and our earthworm tracking system might be similar to that of E. japonica was synchronized; in contrast, those of M. hilgendorfi were not. In the case of M. hilgendorfi, the head and center points were synchronized but the tail point was not; it tended to stay still on the same coordinate. We recognize that the velocities were under non-soil conditions; therefore, earthworms in the soil will be much slower than our calculations indicate. However, the program picked up the features of each species when observed under the same conditions. We consider our newly developed program to be sufficient to measure earthworm activity; this method can be used to compare dynamic changes in these parameters between species or in response to environmental variables such as temperature, light, and moisture, as well as chemical contaminants.The velocity of each point of the different body parts of Image data is powerful, direct information that can be used to quantify physical information of changes in earthworm's activities. The intention of this study is to apply the program to quantify the physical parameters to indicate earthworm's activity from their movement speed or body stretchiness. It will be more beneficial if these data are combined with other data, such as data on gas emissions from the body, enzyme activities using molecular tools, or pathway of elements using isotopic data for mechanistic understanding. The method used in this study is artificial; a small Petri dish without soil as used. However, this study will advance our understanding of the basic behavior of earthworms depending on the alteration of their living environment. This basic behavior can be incorporated in models in terms of material flow belowground or reactions in response to chemical contaminant in soil Combining different kinds of studies will help to unravel or predict the complex processes in belowground systems We demonstrated that our newly developed program successfully tracks the movement of two species of earthworms that have different features in terms of body size, mass, and morphology as well as movement. Based on our data, the program reduced the time required for manual tracking 3-fold and the program could track the movement which is inherent in earthworm behavioral ecology studies. Large continuous image data sets can be immediately processed and the program can determine earthworm body parts along with their changes in elastic body lengths over time. In the future, quantified earthworm behaviors could be incorporated in material flow dynamic models in soil or in models of the response of earthworms to chemical inputs to soil.http://software.incf.org/) under the BSD license. Otherwise the program is open upon request to the following contact person; Assis. Prof. Toshifumi Kimura (email: kimura@shse.u-hyogo.ac.jp).The software “MimizuTrack” will be released on INCF Software Center (


OZ1 is mutated. No interaction of OZ1 with the chloroplast editing factors RIP2 and RIP9 was detected; however, OZ1 interacts with ORRM1, which binds to RIP proteins, allowing us to build a model for the chloroplast RNA editosome. The RNA editosomes that act upon most chloroplast C targets are likely to contain a PPR protein recognition factor, either RIP2 or RIP9, ORRM1, and OZ1. The organelle zinc finger editing factor family (OZ) contains 4 members in Arabidopsis, three that are predicted to be targeted to chloroplasts and one to mitochondria. With the identification of OZ1, there are now 4 nuclear-encoded protein families known to be essential for plant organelle RNA editing.C-to-U editing of transcripts in plant organelles is carried out by small (<400 kD) protein complexes called editosomes. Recognition of the proper C target for editing is mediated by pentatricopeptide repeat (PPR) containing proteins that recognize cis-elements. Members of two additional gene families, the RIP/MORF and ORRM families, have each been found to be required for editing of particular sets of Cs in mitochondria and/or chloroplasts. By co-immunoprecipitation of the chloroplast editing factor ORRM1, followed by mass spectrometry, we have now identified a member of the RanBP2 type zinc fingers (pFAM00641) protein family that is required for editing of 14 sites in chloroplasts and affects editing efficiency of another 16 chloroplast C targets. In yeast two-hybrid assays, OZ1 (Organelle Zinc finger 1) interacts with PPR site recognition factors whose cognate sites are affected when  Transcripts encoding chloroplast and mitochondrial proteins of flowering plants are profoundly affected by RNA editing. In Arabidopsis, over 600 genomically-encoded Cs are modified to Us in organelle transcripts, altering the encoded amino acids and creating stop and start codons. Pentatricopeptide proteins are known to bind to cis-elements near C targets of editing and chloroplast RNA editing also requires members of two additional protein families. Nevertheless, not all protein components of the editosome have been identified. We now report the discovery of a member of fourth gene family essential for chloroplast RNA editing: OZ1, member of a family of Arabidopsis RanBP2-type zinc finger proteins. Identifying all of the proteins in the RNA editosome is critical for understanding the mechanism behind the remarkable specificity of C-to-U editing. In vascular plants, specific cytidines are converted to uridines by RNA editing in the chloroplast transcripts –3. A typcis-element 5’ adjacent to the editable cytidine by a pentatricopeptide repeat (PPR) motif-containing protein [The composition of the molecular machines that carry out plant organellar RNA editing, the editosomes, is not yet fully understood. Editosomes are found between the 200 and 400 kD markers on size exclusion columns . Specifi protein –10. Reco protein ,12. Mult protein . The DYW protein  and is r protein –20. Atte protein ,21. Neve protein . Mutatin protein ,24.orrm1 mutants [In addition to the large PPR protein family that provides site-specific recognition, members of two other plant protein families have been identified as components of editosomes, the RIP/MORF family and the ORRM family ,25,26. AArabidopsis thaliana orrm1 mutants were complemented with an epitope-tagged ORRM1 protein. A candidate ORRM1-interacting protein, encoded by At5g17790, was identified in immunoprecipitates. Through analysis of mutant and silenced tissue, we demonstrated that the candidate protein is a novel chloroplast editing factor. The protein, which we have named OZ1 (Organelle Zinc finger 1), belongs to the RanBP2 type zinc finger protein family, and is required for editing of 14 sites in chloroplasts and affects editing efficiency of another 16 chloroplast C targets. OZ1 is a member of an Arabidopsis protein family that encodes three additional proteins predicted to be targeted to chloroplasts or mitochondria. Identification of OZ1 as a chloroplast editing factor implicates a previously unsuspected class of zinc finger-containing proteins as potentially involved in RNA editing or other aspects of plant organelle RNA metabolism.In order to identify components of chloroplast editosomes that contain ORRM1, matK C640, from 11% to 20%, following transfections of orrm1 protoplasts . Wild-type Arabidopsis, FLAG tag . TherefoCol negative control  is a more sensitive method to measure editing extent than bulk sequencing ,7, all csing PPE . The oz1oz1–1 and 15 other sites have significantly decreased editing . Although editing defects are massive, editing events on the same transcript are not all affected in the same pattern by oz1, hence the editing defects are unlikely to be a secondary effect caused by some change in a transcript itself. For example, ndhD C2 and ndhD C878 lost over 90% of the wild-type editing extent in oz1–1, but ndhD C383 is not affected at all. On the contrary, the sites recognized by the same PPR protein are largely affected in the same way by oz1–1 mutation. ndhD C878 and ndhB C467 share the same PPR recognition factor CRR28 and both of them lose over 90% editing in oz1–1 (ndhB C836 and ndhG C50 (controlled by OTP82) and editing of both rpoA C200 and clpP C559 (recognized by CLB19) are similarly affected. ndhF C290 ndhB C1481 and psbZ C50 are all recognized by OTP84, and in oz1–1, all sites exhibit mild defects in editing (5%-20%) (The assay data for the complete set of chloroplast sites is shown in in oz1–1 . Likewis(5%-20%) .OZ1-dependent sites and ORRM1-dependent sites to examine if these two factors participate in the same editing events. Indeed, editing efficiencies of the 14 OZ1-dependent sites are all severely affected in the orrm1 mutant. Many other sites mildly affected by the oz1–1 mutation are also orrm1-dependent  to transiently silence OZ1 expression in young Arabidopsis seedlings. To monitor the silencing efficiency, a GFP co-silencing marker harbored in the VIGS construct was used [ndhB C836 editing extent decreased from 97% in the untreated control to 47% in OZ1 silenced plants (P<0.01). rpoA C200 editing extent dropped from 74% in untreated control to 29% in OZ1 silenced plants (P<0.001). These results agree with the data from oz1–1 mutants, in which editing is abolished at both sites. The residual editing in the silenced plants is probably caused by incomplete depletion of OZ1 protein.Since a second T-DNA mutant in the coding region of was used . Agrobacoz1–1 mutant has a severely defective phenotype, the plants gradually recover some chlorophyll. In a 6-week- old oz1–1 plant, the old leaves remain pale yellow while the new leaves are light green  was included as a negative control. Monitoring of transfection efficiency of the YFP constructs by microscopy indicated expression of YFP in over 50% of the protoplasts.Although pigmentation has been partially recovered in light green leaves, plastid editing is still defective in those leaves compared to wild type Col . No obvicpYFP-transfected control. Introduction of 35S::OZ1 significantly increases the editing level for all the sites we tested. rpoA C200 increased from 3% to 21%, ndhB C836 from 19% to 31% and rps12-(i1) C58 from 3% to 15%. This confirms that the editing defects in the oz-1–1 mutant can be reduced by introduction of OZ1.RNA was extracted from protoplasts two days after the transfection and analyzed by PPE to examine the editing efficiency . No signoz1 mutant plant  assay was employed to examine the interaction between OZ1 and ORRM1. Both OZ1 and ORRM1 are plastid-targeted proteins, so the predicted transit peptide sequences were removed from each before cloning them into AD/BD fusion constructs. As shown in oz1–1 mutation on C targets controlled by OTP82 and CRR28  and transfected protoplasts. Confocal microscopic imaging revealed that OZ1-YFP is located in the chloroplasts at punctate loci . PreviouExcept for the zinc finger motif, no other annotated domain or motif was found in the OZ1 family. In order to find hidden uncharacterized motifs, motif scanning was performed using MEME against all four members to look for motifs between 15aa to 70aa. Five motifs were returned . The zinWe performed homology searches to determine whether orthologs of the four Arabidopsis OZ family members could be detected in other well-characterized plant genomes. We were able to identify putative orthologs for all 4 genes in poplar, grape, rice, and maize. Moss and Selaginella, which exhibit chloroplast RNA editing, also encode OZ-like proteins but with a lower similarity . We coulOZ1 was not available, we performed transient silencing, transient complementation, and stable complementation to verify the function of OZ1 in editing. Given that VIGS can only knock down gene expression, the editing level of the OZ1-dependent sites were reduced but not totally abolished. Introduction of a 35S::OZ1 construct into mutant protoplasts or into transgenic plants greatly increased the editing extents of the editing defective sites, demonstrating that the editing defects seen in the oz1 mutant can be attributed to loss of OZ1. The absence of OZ1 results in reduced editing efficiency at most of the affected C targets, but editing of rpoC1 C488 is increased. Possibly, the loss of OZ1 results in reduction of sequestration of an editing factor needed for rpoC1 C488 that is present in limiting amounts when OZ1 is present. If OZ1 is not needed for editing of rpoC1 C488, its loss could make available more of an unknown editing factor needed for efficient rpoC1 C488 editing.Because a second coding region mutant in oz1 mutant can survive on sucrose as a yellow seedling and then undergo sufficient chloroplast development to support autotrophic growth. Even in the green, fully photosynthetic leaves, the editing defects are still observed. Most of the sites at which editing is abolished in the oz1–1 mutant are in non-coding regions or in NADH dehydrogenase genes that are not needed in low light growth chamber conditions. The virescent phenotype could be largely due to the complete loss of editing of rpoA C200. Absence of editing at this site also occurs when the PPR editing factor gene CLB19 is mutated [oz1–1 is similar to the clb19 mutant. rpoA encodes a subunit of the plastid-encoded RNA polymerase (PEP); loss of editing at this particular site results in defective PEP and yellow seedling phenotype in early developmental stages. However, mutants survive defects in PEP and later partially recover because of the presence of the nuclear-encoded plastid polymerase that can remedy some of the impaired gene expression. The phenotype of oz1–1 differs from the previously characterized Ds insertion mutant var3 in At5g17790, which was found to exhibit variegated leaves [Although 14 chloroplast C targets have major loss of editing with nine sites showing no detectable editing by PPE, and editing efficiencies of 16 other editing sites are significantly altered, the  mutated , and thed leaves .OZ1 is clearly site-specific, because C targets that reside on the same transcript are differently affected. For example, ndhD C878 has a major loss of editing while ndhD C383 is barely affected. Which editing sites are affected is likely determined by the editing factors with which OZ1 interacts. C targets that share PPR recognition factors are similarly affected in the oz1 mutant. In Y2H assays, OZ1 binds to CRR28 and OTP82, PPR proteins that are required for editing of sites that also require OZ1. Furthermore, OZ1 interacts with ORRM1, and all 14 severely affected chloroplast sites are also affected in the orrm1 mutant. OZ1 interacts with RIP1, though the interaction is not as strong as that with ORRM1. We did not observe direct interaction of OZ1 with RIP2 or RIP9. However, ORRM1 can bind to RIP1 and RIP2. Previously we reported interaction between the RIP-RIP domain of ORRM1 and CRR28 and OTP82 [ndhD C878, drawn according to the yeast two-hybrid data, is shown in The action of nd OTP82 . Anothernd OTP82 . RIP2 annd OTP82 . Howevernd OTP82 . All of 2GDWICX2CX3NFARRX2CXRCX2-PRPEX2; pFAM00641) characterized in RAN binding protein 2 (RanBP2) and other nucleoporins. OZ1 contains two tandem RanBP2 zinc-finger domains while other members have various number of this domain. It is highly possible that other members of the OZ family have similar role in chloroplast or mitochondrial RNA editing. Such redundancy could explain the residual editing for some sites in the oz1 mutant Chloroplast RNA editing in vitro has been shown to be Zn2+ dependent [Three proteins that share high similarity with OZ1 are also predicted to be targeted to organelles according to Target P . OZ1 conependent . Zinc biependent ,23. The A. thaliana Columbia ecotype mutant SAIL_358_H03 in the OZ1 gene was obtained from the Arabidopsis Biological Resource Center (https://abrc.osu.edu/). After 3 days of stratification, the seeds were placed onto petri dishes containing Murashige-Skoog medium in a 25o room with 14 hour day length. Mutant plants and the wild-type siblings were then transferred into soil after 7 weeks on tissue culture medium. Leaves from 4 week- old and 8 week- old plants were collected for further analysis.The T-DNA insertional RecA-RRM construct was described previously. The OZ1 coding sequence was cloned using primer pair OZ1_F and OZ1_R from A. thaliana cDNA. The PCR product was first ligated to PCR8/GW/TOPO and then transferred into the destination vectors pSAT4a and pAUL13 [The ORRM1 coding sequence was cloned using primer pair ORRM1_1F and ORRM1_R_WO from previous constructs. The sequence was first cloned into PCR8/GW/TOPO , and then shuttled into a modified PBI121 vector with a 3XFLAG-strepII C-terminal tag. Alternatively, coding sequence of the ORRM1 mature form (without the predicted 54aa transit peptide) was fused to a N terminal 3XFLAG-strepII tag sequence and an artificial transit peptide sequence from RecA in an overlapping PCR using primer pairs RecA_F, RecA_R, 3FS_F, 3FS_R, ORRM1_163F and ORRM1_R. This chimeric gene was cloned into PCR8 vector first and then the PBI121 vector using LR ClonaseII . The C-terminal tag on the vector was eliminated by the endogenous stop codon of ORRM1 in the sequence. A d pAUL13  to creatAll oligonucleotides used in this study are shown in orrm1 or oz1 mutants were prepared following the protocol from Jen Sheen’s lab [oz1 mutant were used for protoplast preparation. 10 μg of plasmid DNA was used to transfect 2x104 cells. The transfected protoplasts were incubated in the dark at room temperature for 3 days (orrm1 complementation) or 1 day (oz1 complementation) before harvest.Protoplasts from en’s lab . Light gOZ1 gene-specific region was selected from the CATMA database [An database  and ampldatabase . 5 weeksorrm1 roots. The roots were first induced on Callus Inducing Medium (CIM) for 2 days and then infected with Agrobacteria in liquid media [oz1 mutant, a standard floral dip protocol was used to transform a heterozygous plant.The 3XFLAG-strepII-ORRM1ΔCTP and OZ1 constructs in PBI121 and PAUL13, respectively, were used to transform the Agrobacterium GV3101 strain. A standard root transformation protocol was followed to transform mutant id media . Roots wid media . Fully g2, 50mM Tris-HCl ph8.0, 150mM NaCl, 0.5%CHAPS). The final sample was prepared using SDS-PAGE sample prep kit .10 g of leaves of each line were ground in liquid nitrogen into fine powder. Total leaf protein was extracted using grinding buffer . The extract was cleared by 13,000 rpm centrifugation, 0.45 μm filtration and then 30 minutes incubation with unconjugated agarose beads  to minimize non-specific binding. 200 μl anti-FLAG agarose resins  were first blocked with 4%BSA before 2 hours incubation with around 30 μg pre-cleared protein extract. A washing step was done using washing buffer . The IP was eluted with elution buffer  followed by standard procedures of immunoblotting. α-FLAG-M2-HRP  was used to detect FLAG-tagged proteins. 10% of the IP samples were separated on a 10% polyacrylamide gel before being subjected to silver staining compatible to mass spectrometry. Co-purifying proteins with FLAG-tagged ORMM1 were identified by tandem mass spectrometry using a nanoLC-LTQ-Orbitrap instrument, followed by database searching with MASCOT against TAIR10 .DNA contaminants were removed from RNA samples by TURBO DNase . The cDNA was reverse transcribed from the RNAs using the pooled reverse primers as previously described . PCR proA. thaliana cDNA, respectively. PCR products were first cloned into PCR8/GW/TOPO and then pGADT7GW and pGBKT7GW destination vectors through homologous recombination by LR clonaseII . RIP1, RARE1, CRR28, OTP82, ORRM1 constructs produced for Y2H assays were previously described [600 0.5, 0.05, or 0.005. 10 μl of each dilution was spotted onto leucine-, tryptophan-, histidine-, adenine-deficient media plates. Growth results were collected after 3 days incubation at 30°C.Mature OZ1 coding sequence  was amplified using primer pair OZ1_100F and OZ1_R from cDNA and cloned into PCR8/GW/TOPO . Mature RIP2 and RIP9 coding sequences were amplified using primer pairs RIP2_133F and RIP2_R, RIP9_175F and RIP9_R from The first 100 codons of OZ1 was amplified from cDNA with primers listed in S1 Fig(A) Schematic diagram of the C- terminal and N-terminal epitope-tagged ORRM1 constructs. (B) C-terminal epitope-tagged ORRM1 fails to complement editing defect in a transient complementation assay. Editing extent is examined by PPE. (C) N-terminal tagged ORRM1 can enhance editing of matK C640 in orrm1 protoplasts. Star indicates significant difference (P<0.01) with the non-transfected control. Not transfected: untreated orrm1 protoplasts; ORRM1–3FS: orrm1 protoplasts transfected with a construct expressing C-terminal epitope-tagged ORRM1; RecA-RRM: orrm1 protoplasts transfected with a construct expressing RecA transit peptide fused with the RRM motif of ORRM1; RecA-3FS-mORRM1: orrm1 protoplasts transfected with this construct. E, edited product; U, unedited product; O, oligonucleotide.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.