In India, community health workers’ (CHW) effectiveness in providing home-based neonatal care (HBNC) has been well documented. The nature of challenges faced and strategies adopted while providing HBNC services need to be studied in-depth.A qualitative study to understand the challenges faced and strategies used by Sakhis (women CHW) while providing services as part of a HBNC program implemented by a non-profit organization. Data consisted of 20 in-depth interviews and three focus group discussions (FGD) with Sakhis.Sakhis negotiated with the community to start working as a CHW. They faced challenges while changing behaviors at individual level and also while bringing about a change in harmful normative practices that increased chances of maternal and neonatal mortality. Managing crises at the time of deliveries and facilitating a safe delivery was the most critical challenge faced by many Sakhis.The key strategies used by Sakhis included: proactively and persistently providing services even when they faced resistance from the woman or her family; evolving contextually suitable counseling techniques and tactics to bring about behavioral change; balancing compliance to traditional practices and promoting HBNC; defying traditional practices and assisting the woman in times of an emergency to save lives. Having on-call support from supervisors and cultivating a good working relationship with health providers facilitated effective service provision by Sakhis.CHWs having a strong sense of commitment can develop strategies to address challenges and provide HBNC services effectively if they also have strong supervisory support.The online version of this article (doi: 10.1186/s12905-018-0511-6) contains supplementary material, which is available to authorized users. Community health workers (CHW) play a crucial role in ensuring availability of basic healthcare services. They are at the core of the public health strategies to ensure access to primary health care for all populations. The CHW are derived from the community they serve and hence are usually the point of contact for various health needs, especially emergency care. The government of India (GOI) introduced CHW called accredited social health activist (ASHA) in 2005, who have been trained to provide basic Maternal and Child Health (MCH) services during pregnancy and childbirth, and monitor the health of infants.A landmark study by Bang et al.  demonstrStudies have examined various aspects of ASHAs’ work since their introduction in the public health system in 2005. These suggest that ASHA are primarily functioning as link workers and are accepted as service providers by the community . An evalThe performance of these roles is affected by contextual factors such as existing power structure within a community, proscriptive gender norms around women’s roles, difficult geographic terrain that limits ability of the CHW to link community to healthcare services, and programmatic factors such as incentives received under different schemes . PersonaThe processes involved in a CHW’s work in the community have received lesser attention in comparison to the focus on her performance. A qualitative study showed that outreach and health services provided by CHW are embedded in the process of developing a relationship of trust with the community . It has The study was conducted with CHWs called Sakhis who have worked in a MCH program initiated by Ambuja Cement Foundation (ACF) in 2005. The Sakhi program was initiated in response to the felt need of the community following participatory process involving key opinion leaders in the community. The program’s objective was to mitigate the challenge of high infant and maternal mortality in the three blocks, namely, Jiwati, Korpana and Rajura, of Chandrapur district in the state of Maharashtra, India. The structure and conduct of the program was largely based on the home based neonatal care model adopted by Bang et, al. The Sakhis were recruited from the community and underwent a series of training sessions interspersed with field practice and continuous supportive supervision provided on an as needed basis and through bi-monthly group meetings.A qualitative exploratory study that was guided by the grounded theory approach was conducted. The reiterative process of conceptualization comprised of several rounds of observations and interactions with stakeholders including program staff and some Sakhis to understand the evolution of the program and the nature of their work in the community. A conceptual framework was evolved on the basis of this information, which guided further exploration in the study Fig. . Key conAdopting a purposive selection strategy Sakhis with at least 2 years of experience were recruited from each of the three blocks in which the program was implemented. Overall, 35 Sakhis participated in the study Table . In-deptThe interviews and FGD were conducted by three of the authors who are proficient in Marathi, the local language, and the transcripts were prepared from audio-records by a translator proficient in both languages. Data analysis was initiated after the authors had conjointly read few interviews and all FGDs and developed mutually agreed upon code definitions. Subsequently, at least two authors coded each interview and FGD using NVivo and then again conjointly identified emerging themes.The ethics committee of the Narotam Sekhsaria Foundation approved the study.Many Sakhis shared that in the initial days of their work they had to be patient and work consistently for people to accept them as service providers. Some faced challenges from their own family members who perceived that a lay woman taking up the role of a paraprofessional was not consistent with the prescribed gender roles in the community. Many reported that they started working against the family’s wish, but the family gradually accepted their role after they started getting recognition from the community.“Some people in our community were against my work as it was unusual according to them. They asked why I was working, why I was going out. They also told me to discontinue my job as I was going alone for the training for 10 days… I stopped working for some period. Then Sir (ACF supervisor) came and convinced them.”Another Sakhi shared that at times it took as long as 2 years for the community to start unconditionally cooperating with them. The resistance continued in the form of women not giving information about pregnancy because their families did not trust the Sakhi.A few Sakhis shared that some communities did not accept them for various reasons such as their limited literacy and their work being unconventional for a woman. The extent to which community’s pressure could impede the functioning of Sakhis can be seen in the experience shared by a Sakhi:“Some women used to give information, but some used to say, ‘Why is she asking? What is she going to do with all this information? Is she going to give us food?’ Even after this, we didn't deter, didn’t fear.”It appeared from the data that the Sakhis were able to create an identity and work in the community as a result of two main strategies that were adopted concurrently. One was of calling for community meetings involving key opinion leaders. The support to Sakhis from ACF as an institution was explained by the supervisors in these meetings, which helped to legitimize their role as a CHW. Persuading the leaders of the need for Sakhi’s work also helped in getting their endorsement for HBNC work, which in turn led to the community opening up to the Sakhi. The other strategy involved Sakhis proactively visiting families to identify pregnant women. Many families did not appreciate the Sakhi visiting and providing information about mother and child care, yet the Sakhis continued to visit them repeatedly and did not exclude them from their services, which ultimately led to acceptance by the family. The spirit of persistence can be seen in a Sakhi’s sharing:The Sakhis shared several social norms and cultural practices followed by some communities, which increased the risk of morbidity and mortality for the mother and neonate. Some examples of peculiar practices included the community forcing the woman to conduct her delivery on her own by secluding her, keeping the new born child and the woman in separate rooms and forbidding the mother from seeing the baby for 7 days after birth, and not allowing the mother to step out of her room out of fear of being possessed by evil spirits.“they (mothers-in-law) thought we gave nonsensical advice, and said that they had given birth to seven odd children but had never needed a doctor, never went to any maternity home, never took an injection” and hence did not want the daughter-in-law to take care of health according to Sakhi’s advice. Sakhis made efforts to change views of all members including mother-in-laws, women in the family, and also actively engaged with men as they were reluctant to engage in discussions because of the gendered stereotype restricting men’s engagement in care of women during pregnancies. The strategy of focusing on family members and men was beneficial. It was evident from a Sakhi’s experience, in which people from the community disallowed her to assist a woman during delivery because they believed that the woman should be left alone at the time of delivery. In this situation, however, the husband dared to defy the community norm and allowed the Sakhi to enter the secluded room and assist his wife.A review of the data for how Sakhis facilitated a change in these practices showed that, in addition to conducting group-based educational activities that targeted change in entire community, the Sakhis meticulously conducted their intervention by treating the “family” as a unit of intervention. This appeared to be because resistance manifested most during home-visits made by Sakhis, and usually arose from many members of the family who dissuaded the pregnant woman even if she was willing to seek help from Sakhi. For instance, while explaining how family members thwarted their efforts to help the pregnant woman, a Sakhi shared that “I used to go prior to starting of her (pregnant women’s) labour pains. After the delivery, if I wanted to touch the baby I had to burst an egg on my head and then I had to take bath. I had to return home by the route outside the village. I was not allowed to go home by the road, which passed through our village.”It can be seen from this sharing that the Sakhi followed the community norm in order to provide services. Overall, it was evident from the data that the grip of traditional cultural practices typically started loosening when the community witnessed a Sakhi using her HBNC tools to conduct a safe delivery, or when she saved the life of a mother or neonate in a critical situation.The Sakhis also encountered situations where the community threatened families and the Sakhi with sanctions such as ostracizing them or forcing them to go through cleansing practices for breaching traditional practices. The Sakhis addressed challenges emanating from such practices by relying on their knowledge and familiarity of the community’s culture. They used it to contextualize their messages and formulate explanations and counter-arguments to change myths and misconceptions associated with traditional practices. In many situations that warranted immediate action, Sakhis appeared to have judiciously balanced compliance to prevalent norms and their aim of providing services. In some situations they also risked the ire of the community by taking steps for preventing mortality, and in this process endured humiliating treatment by the community. The delicate balancing of the community’s expectations and performing their role can be seen in how a Sakhi addressed a difficult situation:The Sakhis’ narration of various situations showed that they embedded counseling in routine interactions with pregnant women and families. Offering analogies that suited the social background of the women stood out as the most used method of convincing and facilitating a change in behavior. It was also evident that the Sakhis provided targeted counseling on specific issues that were relevant for a selected member of the family. For instance, a counseling interaction with a mother-in-law would be primarily focused on garnering her support for allowing the pregnant daughter-in-law to take medications. As shared by a Sakhi, in this situation the mother-in-law would be convinced by using an analogy – the need to take medication for better health would be compared with the need to change farming practice. The explanation offered was that as farmers had changed their approach to adopting modern methods of farming to get better yield, there was also a need to change the approach to giving medication for better health of pregnant woman.A predominant misconception encountered by many Sakhis was that families believed using health care services was uneconomical because the pregnant woman would be advised to stop working in the farms. The strategy used to address this issue was of making the family re-examine the belief by providing information about potential costs and savings in seeking healthcare services. For example, to convince a family to spend on food for the pregnant woman and allow her to take rest, the Sakhi would first make the family evaluate the cost of care if the mother or child fell sick. Then she would help the family to understand how this potential cost was higher than the earnings lost because of the woman not working in the field.“It is your responsibility to give her medicines” in the presence of the pregnant daughter-in-law, and on the other hand tell the pregnant woman “I will take you to the medical camp only if you take tablets”. Another tactic was to innovatively use available material to ensure that their messages could be followed by women with low literacy. An illustration of this can be seen in the report of a Sakhi helping the woman to follow the right practice of feeding the neonate:“I had given her a piece of coal and told her to (make a) mark on the wall every time, when she fed the baby. She was illiterate. I told her to feed the baby for at least 10-12 times a day regularly. She used to write with the help of coal on the wall, the number of feedings. I used to see her in the evening everyday (to check if the child was fed adequately)”.The essential element that appeared to have led women and their family members to change their health seeking behavior was the assurance from Sakhis that they were available round-the clock to anyone in need.The data showed that Sakhis had cleverly developed certain tactics to ensure that women followed their advice. One such tactic involved gentle confrontation and conditional provision of services. For example, a Sakhi shared that she would assertively tell the mother-in-law Many vivid descriptions by Sakhis showed that they perceived providing assistance during deliveries as a complex event riddled with challenges posed by the social context, and not just a process involving use of their HBNC knowledge and skills. In some crises situations the Sakhi had to make judgments and decisions without much support. A challenge faced by almost all Sakhis was of arranging for a vehicle to take the woman to the hospital for delivery. The strong commitment of Sakhis and their ability to manage crisis without panicking was evident in the experiences of crisis management. For instance, in one situation the Sakhi could not get the government’s free ambulance service even after trying to get it from various nearby health facilities, and therefore paid from her own pocket to take the woman to the district hospital. Another Sakhi assisted the woman’s delivery at home and also detached the placenta applying knowledge gained from training, even though it was not her responsibility.“If you don’t want to help, don’t go but let me go and see. I will not harm your baby. It will be my responsibility, if anything goes wrong”. When Sakhis assisted during deliveries at home, they often had to overcome their fear of being blamed by the family if the outcome of the delivery was fatal for the mother or the child. The fear experienced by a Sakhi who reached a house when a woman had delivered but not received any help is evident from the experience shared by her:“Her mother stayed nearby, when we tried calling her relatives, nobody came near. They said that they will not touch her. And said that you do whatever you want to do by yourself… And I said (to myself) that I will be blamed if something worst happened to that woman”.At times the community’s resistance and non-cooperation also posed a challenge. In one such experience shared, when a community was not willing to assist a woman in a complicated delivery and neither allowing the Sakhis to intervene, two Sakhis shared the risk in defying the community by telling them dai  of the community, as facilitated by the ACF supervisors, the approach adopted was a clear de-lineation of the roles. When the dai was present for deliveries, the Sakhis focused primarily on providing post-partum services and using specific HBNC techniques needed for averting mortality. With dai and Anaganwadi workers, the Sakhis attempted to bring about a change in practices by taking a supportive stance. For example, in a village, Sakhis coaxed Anganwadi workers to identify and report cases of severely and moderately malnourished children and also assured them of support in providing care to the children.The data showed that one of the main challenges faced by Sakhis was to negotiate a space and role for themselves in the established system of both formal and informal health providers. With the The overall approach to working with formal providers appeared to be focused on developing a cordial working relationship. A Sakhi shared that doctors did not trust them initially and questioned their presence at the time of delivery, but accepted them when they witnessed the Sakhi using her skills. In this process, possessing the HBNC kit appeared to have helped the Sakhis in partnering with the doctors. This was because some doctors were not aware of the HBNC and hence didn’t know how to use it, and in case of some, equipment was not available or was dysfunctional in their facility. Some Sakhis also shared experiences in which they had to insist with a resistant doctor or nurse to be allowed to use HBNC tools for resuscitating the child after the doctor had given up hope of revival. However, as reported by the Sakhis, such encounters usually resulted in getting the provider’s appreciation, and helped them to develop a strong working relationship. The partnership in the relationship with the doctors was reflected in many experiences which showed that doctors had started allowing Sakhis inside the labor room at the time of delivery, some had shared their personal contact number and made themselves available to Sakhis at any time, others gave referral notes to mothers to seek follow-up care from Sakhis in the village, and at times some even went beyond their mandate to support the Sakhi in managing crises, which contributed to saving many lives of mothers and newborn.“We used to report each and everything. I used to tell Bhau (supervisor) about everything. And if I didn’t understand anything, I used to ask information regarding the same. We used to get information from Sakhi in other villages and vice versa. If something we did was wrong, we used to correct it by knowing from Sakhis in other villages”.Most Sakhis also considered availability of any-time support from the supervisor as vital to their effective functioning in the community. They requested for such support by calling the supervisors and asking them to be present with them in difficult situations or sought guidance for specific problems, which, as Sakhis emphasized in a FGD, was always obliged to by the supervisors. Other experiences also reflected that association of supervisor and Sakhi was not limited to getting technical information and guidance. In many difficult situations such as when the family compelled the Sakhi to discontinue her work or doubted her integrity because she would be away for long duration of time for her work, the supervisors visited their homes and vouched for the genuineness of Sakhi’s work. Overall, the narratives revealed that Sakhis had developed a bond with their supervisors which helped them to function effectively, regardless of the difficulties they faced.Many Sakhis opined that the quality and approach of their training had helped them build their skills. They asserted that they were able to use the HBNC kit because it was made available during the training, in which they were oriented to the kit and then asked to practice for a few weeks along with their supervisors, and then trained again in refresher training sessions. Along with the training, Sakhis found on-the-job support provided by their supervisors in the initial period as most valuable and attributed their skill development and confidence to this supervision. Another source of support for many Sakhis was the bi-monthly meetings conducted by their supervisors. How these meetings served the purpose of getting information and clarifying doubts can be seen in a Sakhi’s description of a typical meeting:The CHW are the fulcrum of the HBNC model that is in the process of being scaled up across the country. We conducted an in-depth inquiry to understand the challenges faced by CHW and their mitigation strategies in providing HBNC. Even though the experiences shared by Sakhis in our study were in a particular geographic and temporal context, several insights that can guide the scale-up process were acquired.As reported in earlier studies, , 8 our sThe Sakhis had ingeniously developed messages and used several techniques such as developing analogies for bringing about a change in health behaviors. They were able to make their messaging compatible to the socio-cultural background of the people and present it in a manner acceptable to the communities. Using these techniques and making their services available round-the-clock, the Sakhis reached out to the most marginalized and hard to reach communities. This illustrates how CHW can use their competence drawn from their aptitude and experience of working with communities to adapt the knowledge they have received as part of their training. In other words, the CHW can bring in valuable additions from their personal competence, which otherwise may be seen as a limiting factor by program planners.The narratives about deliveries reaffirmed that CHWs can effectively use HBNC skills to avert neonatal mortality. They often actively facilitated safe deliveries even in crises situations. It is noteworthy that the crises were usually due to social and contextual factors that created obstacles for the Sakhi and not due to a lack of knowledge and skills of Sakhis. With strong commitment, confidence and support from supervisors, they were able to surmount several difficulties, guide families, and make critical decisions on their behalf. It is most likely that they could overcome their fear of being blamed and use HBNC tools and techniques because they had established their credibility through a long process of engagement with community and other providers. It also reflects that it is not adequate for CHW to possess only HBNC skills; they need to be prepared for crises situations and have a repertoire of different skills to mitigate these situations.An important insight from this study is also that a good working relationship between the formal health system and CHW can be developed to meet the objective of averting maternal and neonatal mortality. In case of Sakhis it appeared to have grown over a period of time as doctors started understanding and recognizing the potential of Sakhis. This shows that in Indian context where a significant gap exists between the formal health-care system and the community, nurturing partnerships between CHW and formal health care providers in their day-to-day work can go a long way in closing this gap.A key learning from this study is that CHW can nurture a relationship with the community if they receive on-going support in not only developing HBNC knowledge and skills, but also in addressing challenges they face in delivering HBNC services. Such support can be provided by creating platforms such as bi-monthly meetings where CHW get opportunity to validate their experiences and learn from their peers. In other words, consistent and committed high-quality supervision can be a key determinant of CHW’s performance.The Sakhi experience shows that their genuine interest in improving the lot of the women in their community led them to develop solutions and negotiate with community members and family to achieve the desired outcomes. In the process their belief in themselves grew and of the health ecosystem in them also grew. The role of a supportive network would be crucial as they rely mainly on the supervisors when faced with operational and clinical difficulties. While we show the effectiveness of the Sakhi program, it may not be extended to similar instances of ASHAs. But we do show that if there is no improvement in areas where ASHAs work, it would be of interest for the system to conduct a similar inquiry to tease out problems that can be solved. Our model can thus be used as a comparison to other areas that utilize CHWs in similar capacities. Our current work reiterates the belief of public health managers in CHWs as the last mile connectivity for maternal and child health.


Given the astounding rates of diabetes and related complications, and the barriers to providing care present in Indigenous communities in Canada, intervention strategies that take into account contextual factors such as readiness to mobilize are needed to maximize improvements and increase the likelihood of success and sustainment. As part of the national FORGE AHEAD Program, we sought to develop, test and validate a clinical readiness consultation tool aimed at assessing the readiness of clinical teams working on-reserve in First Nations communities to participate in quality improvement (QI) to enhance diabetes care in Canada.A literature review was conducted to identify existing readiness tools. The ABCD – SAT was adapted using a consensus approach that emphasized a community-based participatory approach and prioritized the knowledge and wisdom held by community members. The tool was piloted with a group of 16 people from 7 provinces and 11 partnering communities to assess language use, clarity, relevance, format, and ease of completion using examples. Internal reliability analysis and convergence validity were conducted with data from 53 clinical team members from 11 First Nations communities (3–5 per community) who have participated in the FORGE AHEAD program.r = 0.45, p < 0.05.The 27-page Clinical Readiness Consultation Tool (CRCT) consists of five main components, 21 sub-components, and 74 items that are aligned with the Expanded Chronic Care Model. Five-point Likert scale feedback from the pilot ranged from 3.25 to 4.5. Length of the tool was reported as a drawback but respondents noted that all the items were needed to provide a comprehensive picture of the healthcare system. Results for internal consistency showed that all sub-components except for two were within acceptable ranges (0.77–0.93). The Team Structure and Function sub-component scale had a moderately significant positive correlation with the validated Team Climate Inventory, The testing and validation of the FORGE AHEAD CRCT demonstrated that the tool is acceptable, valid and reliable. The CRCT has been successfully used to support the implementation of the FORGE AHEAD Program and the health services changes that partnering First Nations communities have designed and undertaken to improve diabetes care.NCT02234973. Date of Registration: July 30, 2014Current ClinicalTrial.gov protocol ID The online version of this article (doi:10.1186/s12913-017-2175-6) contains supplementary material, which is available to authorized users. The burden of diabetes on both families and the healthcare system in Canada is growing at an astonishing pace with prevalence rates that have more than doubled in the last decade . In 2010It has been suggested that barriers to optimal diabetes care are different and sometimes more pronounced in Indigenous communities in Canada than those experienced in non-Indigenous communities due to geographic isolation, cultural differences and the disjointed healthcare provided by a combination of federal government, provinces and territories , 11. GivWhen evaluating readiness relative to the healthcare setting in Indigenous communities in Canada, a valuable assessment is possible when all individuals involved in the prevention and treatment process participate . The results of these evaluations can be used to guide care and maximize health improvements . Holt etDetermination of health teams’ degree of readiness is advantageous with regards to maximizing the success of interventions through the identification of available resources and the present knowledge of a health issue . A low rTransFORmation of IndiGEnous PrimAry HEAlthcare Delivery (FORGE AHEAD): Community-driven Innovations and Strategic Scale-up Toolkits is a 5-year national research program that partners with Indigenous communities in Canada to improve chronic disease care and access to available resources by developing and evaluating community-driven, culturally-relevant primary healthcare models through a quality improvement (QI) process [The  process . The pro process , 22; andwith communities rather than on communities is consistently recognized as a critical factor in research with Indigenous partners [The objective was to develop a validated Clinical Readiness Consultation Tool (CRCT) to assess the readiness of clinical teams working on-reserve in Indigenous communities before they engage in QI efforts to enhance diabetes care in Canada. A consensus approach  within tpartners , 26, 27.partners .FORGE AHEAD partners with 11 First Nations communities across 6 provinces  and three isolation levels .A literature review was conducted in March 2014 using PubMed to identify existing clinical readiness tools published prior to January 2014. Key search terms included “Indigenous” or “First Nations” or “Aboriginal AND “healthcare” or “primary healthcare” or “chronic disease” AND “clinical readiness”, or “clinical change readiness” or “organizational readiness” or “organizational change” or “change management”. With no tools found in the Canadian Indigenous domain, the search was expanded internationally, included all populations, and examined grey literature for key content. A total of 13 PubMed articles and four grey literature articles/sources were found. Inclusion criteria included use in Indigenous populations, a health domain tool, and restricted to clinicians (versus patients).Members of the Working Group which consisted of experts in the fields of readiness, Indigenous research, survey development and chronic disease care/epidemiology assessed the literature to identify the most appropriate tools for use within FORGE AHEAD. From the review of literature, the Chronic Care Model , ExpandeThe aforementioned existing tools  were considered for the FORGE AHEAD program; however the following drawbacks were identified: i) reliance on costly resources external to the community for tool implementation and/or interpretation, ii) not tailored to the unique Canadian setting and First Nations primary healthcare setting, iii) not focused on type 2 diabetes, iv) lack of a qualitative component where Indigenous team members’ can add information regarding their insights and perceptions regarding diabetes care, and, v) no built-in mechanism to integrate individual perceptions to be used to support clinical readiness at a small group level. Therefore, the FORGE AHEAD Program aimed to develop and test a CRCT relevant to diabetes care in the Canadian Indigenous context that could be applied by primarily using existing internal community resources without the need for external interviewers and transcription. The ABCD-SAT (Version 2) was selected by the Working Group as the basis for conceptualizing and developing the CRCT. The overall goal of the Working Group was to develop a comprehensive, user-friendly, understandable, and stand-alone questionnaire that could be administered and used by community-based facilitators  in First Nations communities within the FORGE AHEAD Program to assess readiness to adopt chronic disease interventions.Adapting the ABCD-SAT into the CRCT involved a thorough review by Working Group members over a series of six rounds from January to July 2014. Each of the components and sub-components as well as the individual items that encompass the sub-components of the ABCD-SAT were reviewed for relevance to the context of Canadian First Nations primary healthcare delivery systems, redundancies, cultural relevance, and language. One item was added on cultural competence and two questions about registries were removed. Type 2 diabetes specific examples were inserted to enhance clarity in most items. All items were revised to reflect the Canadian First Nations context and the language was adjusted for improved clarity and ease of understanding.In June 2014, 15 people from 7 provinces comprised of 3 co-investigators and 12 Indigenous and non-Indigenous community members from 11 partnering communities received the pilot CRCT. This group was selected given their prior involvement in the planning and development of the FORGE AHEAD Research Program proposal. Community members included family physicians, nurses, health managers and administrators, health promoters, and adult educators. The draft CRCT was distributed electronically along with a feedback form  completed the CRCT at three time points: pre, during, and post program implementation. For this study, only pre-program implementation data was used to avoid a learning effect bias.α) which measures whether a group of items is related to a single construct or sub-component [α but an α greater than 0.9 may also be indicative of a redundant item. An α greater than or equal to 0.7 is considered satisfactory (34). Sub-components with a single item were excluded from the internal consistency analysis. Overall α for the total scale (CRCT) was not calculated because each sub-component is intended to measure a distinct factor. Missing data was handled through pairwise deletion  of cases. Descriptive statistics were generated to evaluate the score distribution and proportion of missing data per item. Items with high levels of missing data (>10%) were identified and considered for further analysis and interpretation.The internal reliability assessment was based on the correlations between the individual items that make up the sub-components, relative to the variances . The intomponent . The higAs part of FORGE AHEAD, participants also completed the Team Climate Inventory (TCI) to examine team functioning of clinical teams working on-reserve. The TCI  has been validated  and usedThe 27-page CRCT  completed the feedback form for an overall review and response rate of 53.3%. The CRCT took an average of 118 min to complete and ranged from 53 to 240 min. Based on the five-point Likert scale feedback form, average respondent scores were: appropriate language use for First Nations health clinics in Canada (3.75); questions clearly written (3.25); questions are relevant to health clinics in First Nations communities (4); format of the tool is appropriate (4.5); and, helpfulness of examples to complete the clinical readiness assessment (4). The 12-point Likert scale of the CRCT was maintained based on the qualitative feedback. Analysis of the written answers to open-ended questions revealed that although the questionnaire was long, respondents found that all the questions were important and necessary to obtain a comprehensive understanding of the current services available in communities. Some respondents noted that the language was geared for higher education/reading and comprehension skills. Revisions to the tool were made following the pilot to improve clarity to address this concern.Of the 53 people who completed the CRCT, 87% were female and 62% were Indigenous   to calculate sub-component 3) Table . Two subα < 0.7, a low level of correlation (<0.4), and where removal of the item did not affect the total α  were assessed by the Working Group members based on the internal consistency results and the theoretical concepts to ensure acceptability of the level of consistency and validity. Of the 68 items, only one was found to increase the reliability of the tool if removed. The Appointment and Scheduling sub-component had three items and removing the third item (Is it routine practice for the diabetes community based activities and programs to be planned or scheduled ahead of time?) improved the internal consistency of the sub-component (from 0.66 to 0.69). The two-item Patient Access sub-component was retained given that information related to patients’ barriers to access are an important aspect of chronic disease care. Two items in the Communication and Cooperation subcomponent were removed due to redundancy (α = 0.91),  and .The CRCT items with r = 0.45, p < 0.05 , which demonstrated adequate internal consistency for standard scale development criteria. Based on a rigorous review guided by theoretical and statistical principles, only three out of 74 items were removed.Examination of the CRCT response data also demonstrated good acceptability of the tool with a high completion rate for all items except 8 that were left unanswered by more than 10% of the participants. Given the relatively high number of items in the CRCT, the proportion of unanswered items represents a small number. In addition, all items with higher proportions of missing data were those asking about the regional health planning and development, organizational commitment, or existing QI strategies at the health center. Given the fact that participants had a wide range of professional roles, this could be due to difficulty in understanding those questions and/or less knowledge about the issues that may have been more external to their role. In fact, of the four Health Directors/Program Managers who completed the CRCT, all four answered all the questions about regional health planning and development, organizational commitment and existing QI strategies. Reliability analysis demonstrated an acceptable As expected, the TSF sub-component of the FORGE AHEAD CRCT showed evidence of convergent validity when compared to the validated TCI tool. This suggests that the sub-component of the CRCT intended to examine team functioning measures the same construct as the TCI. The moderate level of correlation is acceptable and can partly be explained by the fact that the TSF and TCI are structured differently with varying scales and types of questions. While the CRCT instrument has demonstrated validity and reliability, there are limitations. The small sample size did not allow for a confirmatory factory analysis to test whether the data collected from First Nations communities in Canada fits with the hypothesized measurement of readiness using the CRCT. Future confirmatory factor analysis studies with larger samples are suggested to better establish the validity of the FORGE AHEAD CRCT. Also, the psychometric properties of convergent validity for the entire CRCT could not be evaluated due to the non-existence of validated tools that measure a similar concept. As such, only the TSF sub-component was tested for convergent validity against the validated TCI.The FORGE AHEAD Program is still in its implementation phase and as such, complete data are not yet available on the recruitment and retention of partnering communities, community and clinical team members QI initiatives, clinical outcomes, and cost analyses. Data on the use of the CRCT to facilitate QI initiatives and the impact of the CRCT on health-related outcomes will be available upon completion of the program. Detailed process evaluations including stakeholder interviews will inform further revisions to the CRCT as well as best practices for the successful implementation and scale-up of the tool. Process evaluations will also highlight how the CRCT enabled understanding and relationship building among healthcare team members working in Indigenous communities and between researchers and community stakeholders.In conclusion, the validated CRCT has been successfully used to support the implementation of the FORGE AHEAD Program and the health services changes that partnering First Nations communities have designed and undertaken to improve diabetes care.Additional file 1:FORGE AHEAD: Clinical Readiness Consultation Tool – Community Feedback Form. The Community Feedback Form was used during the pilot of the CRCT and was includes a qualitative (open-ended) section for suggestions/comments and a five-point Likert scale to gather responses on the following: (1) appropriate language use for First Nations health clinics in Canada, (2) clarity of questions, (3) relevance of questions to health clinics in First Nations communities, (4) appropriate format for the tool, and, (5) helpfulness of examples provided for each question to complete the clinical assessment tool. (DOCX 43 kb)Additional file 2:FORGE AHEAD: Clinical Readiness Consultation Tool. The 27-page tool has 4 main sections: 1) 1-page introduction describing the background, confidentiality, benefits, risks, reimbursement, consent, and contact information; 2) 1-page brief instruction ; 3) general information (brief 8-item demographic profile); and 4) 5 main components and sub-components of healthcare systems important in chronic disease care. (DOCX 92 kb)


A low contact resistance is an imperative requirement to consider graphene as a candidate material for electronic and optoelectronic devices. Unfortunately, measurements of contact resistance in the literature do not provide a consistent picture, due to limitations of current graphene technology, and to incomplete understanding of influencing factors. Here we show that the contact resistance is intrinsically dependent on graphene sheet resistance and on the chemistry of the graphene-metal interface. We present a physical model of the contacts based on ab-initio simulations and extensive experiments carried out on a large variety of samples with different graphene-metal contacts. Our model explains the spread in experimental results as due to uncontrolled graphene doping and suggests ways to engineer contact resistance. We also predict an achievable contact resistance of 30 Ω· RC  is an imperative requirement for the industrial adoption of graphene in electronics3 and for the adoption of other two-dimensional materials, which often rely on the use of graphene-metal interfaces4.Low and reproducible metal-graphene contact resistance RC is found in the literature for the same metal12. Measurements of graphene-metal contact resistance for different metals  via transfer-length and four-probe methods are strongly dependent on factors such as deposition temperature and process conditions, in addition to intrinsic factors such as metal work function, number of graphene layers, back-gate voltage5. In addition, photoresist residues are generally an issue that leads to high contact resistance in experimental devices. A reduced contact resistance has been reported in the case of contacts to graphene edges or defects, and has been attributed to stronger covalent bonding of graphene and metal or to a reduction of the bonding distance, which would entail a larger orbital overlap compared to van der Waals contacts16.However, graphene contact fabrication technology is not yet mature and fully reproducible, and therefore a broad range of experimental values of 24. In the model proposed by Xia et al.17, electrons first tunnel through the graphene-metal interface and then transfer from the graphene region under the metal to the graphene channel. Ji et al.18 used this concept in a systematic study of the contact resistance, including both single-sided and double-sided contacts for different graphene-metal systems. However, they compute graphene-metal tunnelling with the Wentzel–Kramers–Brillouin approximation, that is inadequate for high transmission, and their study is limited to a single geometry. Similar ab-initio studies only investigate lateral in-plane transmission, and therefore completely neglect the critical issue of vertical transport through the heterointerface22.Theoretical work has provided insightful contributions into the physics of graphene-metal contactset al.23 showed that the nickel-graphene contact resistance is independent of the orientation of graphene and of the contact area, in agreement with experimental observations, but the actual resistance calculation is roughly approximated. Liu et al.5 further analysed the impact of molecular orbitals involved in the contact on transmission, finding that the conductance of the metal-graphene-metal junction is affected not only by the interfacial binding, but also by which molecular orbitals are involved and their symmetry, and that contact resistance decreases with the increase of the contact area at low bias voltage.The contact geometry affecting the formation of covalent bonds at the interface certainly plays a role. Stokbro et al.24 carried out a systematic first-principles study on contact resistance between several metals and graphene, observing the dependence of contact resistance on edge termination, contact area and point defects on the contact region. As in the pioneering work by Matsuda et al.16, they identify three categories of graphene-metal contacts on the basis of the interaction strength. Metals weakly interacting with graphene (i.e. Au and Ag) are very sensitive to the atomistic configuration at the contact region: edges without chemical terminations, small contact length and point defects result in decreased contact resistance. Strongly interacting metals (i.e. Ni and Pd) show small sensitivity of the contact resistance to those factors. Finally, metals like Pt and Cu, with an intermediate strength of interaction, exhibit a slight dependence of the contact resistance on the details of the atomistic configuration of the contact. Matsuda et al.16 have shown that the different interaction strength can affect the contact geometry and give rise to “edge” configurations.Ma et al.20 conducted an insightful investigation on graphene suspended between Al contacts and singled out charge transfer at the leads and into the freestanding section as a determining factor, but their study was limited to a single metal and did not pursue a stringent comparison with experiment. Such a comparison is in general lacking in previous theoretical work, especially rigorously translating the obtained results in terms of a model at a higher level of abstraction which can only allow comparison with experiments and full validation of the models.Finally, Barraza-Lopez For this reason, we have performed a comprehensive theoretical and experimental study of graphene-metal contacts. An in-depth theoretical investigation based on ab-initio simulations helps us in understanding the nature of the graphene-metal interface at the most fundamental level. This in turn enables us to develop a simple analytical model of the graphene-metal contact based on few parameters, and to devise ways of experimental validation. Finally, a large variety of different graphene-metal contacts were fabricated and experimentally tested in order to validate the main hypotheses of our model and approach. Edge contacts are not considered in the model, because the experimental structures are sufficiently large to assume the graphene edge under the metal to be infinitely far away from the actual graphene-metal edge.We aim to gain insights into the main mechanisms at play when contacting graphene with different metals. To this end we investigate a specific, but realistic geometry of a graphene-metal interface, and perform a detailed analysis of the electrostatic potential at the graphene-metal interface. In Fig. In order to properly consider transport across the graphene-metal heterointerface, as in ref. z transport direction (approximately because of the ABC stacking) thus avoiding artefacts due to the presence of induced dipoles at the graphene-metal interface. Two metal islands are connected by a graphene sheet, placed in a side-contact configuration with the fragment geometries taken from separated metal and graphene components. The only free geometric parameter is the graphene-metal distance, which is optimised at the DFT level. In order to study the effect of the distance d between the two metal islands, three systems with different d have been considered, as illustrated and described in Fig. Our simulated structure Fig.  is appro25, a plane wave basis set, a gradient-corrected exchange correlation functional (Perdew-Burke-Ernzerhof (PBE))26, and ultrasoft pseudopotentials (US-PPs)27 in scalar relativistic form, we include dispersion corrections  and we follow an analysis similar to that proposed in ref. We perform first-principles DFT calculations with the Quantum Espresso package29.To compute transport, we cut the system as illustrated in Fig. G (and therefore the contact resistance RC = G−1) is obtained within the Landauer-Buttiker linear response theory31q is the elementary charge, h is Planck’s constant, k is Boltzmann’s constant, T is the temperature, T(E) is the trasmission coefficient as a function of energy, EF is the Fermi energy and f the Fermi-Dirac function.The conductance 20: (i) the evolution of the Dirac point energy ED(z) along the transport direction, which determines the number of electronic states available for transport ; (ii) the potential barrier and intermixing of orbital states at the interface between the two materials.Transmission is affected by two main factors32. This procedure is illustrated in Fig. WG (loc) and WM (loc), respectively, are also derived as a by-product of this analysis. It is an important validation of the proposed procedure that the local work functions so estimated in the interacting system in points of the graphene or the metal far from the contact region coincide with those of the free fragments.We extract the energy of the Dirac point at each given point of the graphene in the interacting system from a comparison of the background electrostatic potential evaluated in the interacting system with the electrostatic potential in isolated grapheneED(z) along the longitudinal direction. In Fig. ED(z) extracted from the electrostatic potential along the z-axis for x = y = 0 at the middle of carbon-carbon double bonds of graphene, for the short, medium and long structures , for the four considered metals. The curvature and the decay length of ED(z) are related to the sign and amount of charge redistribution at the interface. As apparent from the difference in curvature for the different systems, electrons are transferred from metal to graphene in the case of Cu, Ni, Pd but from graphene to the metal in the case of Pt. Moreover, also the decay length of ED(z) is very different for the different metals. This is due to an interfacial charge redistribution occurring also in the z-direction, determined by the interplay of charge donation from graphene to the metal and back-donation into the graphene electronic edge states36.This analysis also allows us to obtain the complete profile of the Dirac point energy x and z directions, which can be taken as useful descriptors of interfacial charge redistribution. The x-dipole is related to the graphene-metal charge transfer and the difference in electrostatic potential between graphene underneath the metal contact and bulk graphene, although the net result of this complex charge and electrostatic potential redistribution also depends on the change in the metal work function due to graphene adsorption33. It is important to note that such x-dipoles in our systems are rather different from those obtained on extended graphene-metal contact of identical geometry33, as reported in Table x-dipole is compensated by introducing a dipole correction34. The z-dipole instead is related to the other quantity affecting transmission as apparent from Fig. ED(z) from the contact.The system thus develops a dipole moment along both x and z directions into a single quantity: ΔEFcont, i.e. the difference between the Fermi energy and the Dirac point energy at the graphene-metal interface  to negative (n-doped) at a distance between the Pd plane and the graphene plane of about 2.56 Å. We then use this quantity in the analysis of computational results in the next section.We merge the effects of the dipole in the We consider two different systems (MS and SS) and different cut points to which we apply the semi-infinite graphene lead  is shown as a function of the energy for different metals, the main impact of the structure (MS or SS) and of the choice of the cut point on the profile of T(E) is a shift in energy: T(E) is zero for the Dirac point energy of the cut point, which corresponds to zero density of electronic states in the graphene semi-infinite lead. The graphene Dirac-point energy moves to the Fermi energy as one moves away from the contact, because graphene is undoped and the electric field is progressively screened , which is computed as in Eq. (39 and which only depends on the difference between the Fermi energy and the Dirac-point energy at the cut point (ΔEF), that is the symmetry point of the structure  shown in Fig. vF is the Fermi velocity in graphene and the denominator between square brackets is the ballistic transmission coefficient of a graphene sheet39.We can see that the ratio Since interband tunnelling in graphene is favoured by the absence of a gap and does not limit transmission, the other relevant parameter for the measured contact resistance is the Dirac-point energy in graphene at the cut point et al.40 — based on only two parameters that can be extracted through ab-initio simulations: Teff and ΔEFcont. The value of ΔEF depends on the electrostatics of the structure or of the actual device which the contact is part of.We therefore have defined a simple physical model of the contact — even simpler than that described by Chaves RC as:In detail, we can express EF). This prediction can be used as a means to validate or to falsify the model. Let us highlight that experiments in the literature46, have shown the dependence of graphene-metal contact resistance on the back-gate voltage (and therefore on Dirac-point energy), but have never pointed to the mentioned linear dependence and to the related physical model.Our model predicts that the measured contact resistance is a linear function of the graphene sheet resistance in the case of diffusive transport, when both are extracted using the transfer-length method on a relatively large structure , gold (150 nm), nickel/gold (25 nm/125 nm), palladium (150 nm), platinum/gold (25 nm/125 nm). Spacing between the contacts is varied from 5 μm to 30μm in order to extract both the contact resistance and the sheet resistance using the Transfer-Length Method (TLM). Electrical measurements were carried out on TLM structures under a varying back-gate bias Vbg which enables tuning of the Dirac point energy  contact resistance is obtained as RGmin/Teff. As can be seen, contact resistance can be as low as 30 Ω · μm for nickel contacts, which would be very desirable for high performance graphene-based FETs45.From this understanding we can draw as one main conclusion that we can optimise the contact resistance by adjusting the Dirac-point energy of graphene near the metal via the back-gate voltage Fig. , thus tu42. After the transfer, the PMMA support layer was removed by immersing the sample in a bath of acetone overnight and successively annealing the sample for one hour in an atmosphere consisting of argon (95%) and hydrogen (5%) at a temperature of 450 °C. A positive tone photoresist was spin-coated on the substrate at a speed of 2500 rpm for 50 s and soft baked at 110 °C for 90 s to achieve a thickness of 1.4 μm. Photolithography was done to define patterns on the resist followed by an oxygen (O2) plasma etch to etch graphene in the unprotected region and photoresist was removed by placing the sample in a bath of acetone overnight. At this step the channel region of the devices is defined. Using the same photoresist but in an image reversal lithography process, source-drain contacts were defined. Various metals were deposited on different samples by evaporation, followed by removal of excessive metal using lift-off process in AZ 100 solution which is kept at 70 °C.The process steps of fabrication started with the cleaning of the samples in bath of acetone to remove photoresist used as a protection layer during dicing, followed by iso-propanol and de-ionized water rinse. Graphene growth on copper foil was performed in a NanoCVD rapid thermal processing tool using the method described in ref. μm). A linear fit of the data is performed and the contact resistance RC and sheet resistance Rsh are extracted. By multiplying the extracted values by the channel, width the contact resistance in Ω μm and sheet resistance in Ω/□ are obtained. The extracted values give a ± error value (taking the upper limit and the lower limit while fitting the scattered data points for extracting RC and Rsh) which is seldom reported in the literature  is measured by applying a varying back-gate voltage and a fixed voltage between drain and source (50 mV). This measurement procedure is repeated for varying contact separation and the total device resistance (Ω) is plotted as a function of the contact separation (Supporting information


Drosophila neurons and glia, a model that recapitulates many of the characteristics of TDP-43-linked human disease including protein aggregation pathology, locomotor impairment, and premature death. We report that such expression of hTDP-43 impairs small interfering RNA (siRNA) silencing, which is the major post-transcriptional mechanism of retrotransposable element (RTE) control in somatic tissue. This is accompanied by de-repression of a panel of both LINE and LTR families of RTEs, with somewhat different elements being active in response to hTDP-43 expression in glia versus neurons. hTDP-43 expression in glia causes an early and severe loss of control of a specific RTE, the endogenous retrovirus (ERV) gypsy. We demonstrate that gypsy causes the degenerative phenotypes in these flies because we are able to rescue the toxicity of glial hTDP-43 either by genetically blocking expression of this RTE or by pharmacologically inhibiting RTE reverse transcriptase activity. Moreover, we provide evidence that activation of DNA damage-mediated programmed cell death underlies both neuronal and glial hTDP-43 toxicity, consistent with RTE-mediated effects in both cell types. Our findings suggest a novel mechanism in which RTE activity contributes to neurodegeneration in TDP-43-mediated diseases such as ALS and FTLD.Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two incurable neurodegenerative disorders that exist on a symptomological spectrum and share both genetic underpinnings and pathophysiological hallmarks. Functional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in ~40% of FTLD cases, but the cascade of events leading to cell death are not understood. We have expressed human TDP-43 (hTDP-43) in  Drosophila neurons or glial cells, which results in toxic cytoplasmic accumulation of TDP-43, causes broad expression of retrotransposons. In the case of glial hTDP-43 expression, the endogenous retrovirus (ERV) gypsy causally contributes to degeneration because inhibiting gypsy genetically or pharmacologically is sufficient to rescue the phenotypic effects. Moreover, we demonstrate that activation of DNA damage-mediated programmed cell death underlies hTDP-43 and gypsy mediated toxicity. Finally, we find that hTDP-43 pathology impairs small interfering RNA silencing, which is an essential system that normally protects the genome from RTEs. These findings suggest a novel mechanism in which a storm of retrotransposon activation drives neurodegeneration in TDP-43 mediated diseases such as ALS and FTLD.Functional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in ~40% of FTLD cases, and mutations in TDP-43 are causal in a subset of familial ALS cases. Although cytoplasmic inclusions of this mostly nuclear protein are a hallmark of the disease, the cascade of events leading to cell death are not understood. We demonstrate that expression of human TDP-43 (hTDP-43) in  After transcription, an RTE-encoded reverse transcriptase generates a cDNA copy, and this cDNA is inserted into a new genomic location at the site of double stranded DNA breaks created by an endonuclease activity encoded by the RTE . Unrestrlt brain –12. Morelt brain –20, incllt brain . One or TDP-43 is a member of the hnRNP family that homodimerizes to bind single stranded RNA and DNA with UG/TG-rich motifs . This plFunctional abnormality of TDP-43, an aggregation-prone RNA binding protein, is commonly observed in a spectrum of neurodegenerative diseases that spans motor neuron deterioration and progressive paralysis in ALS to dementia and cognitive decline in FTLD . 90% of Animal models of TDP-43 related disorders–and neurodegenerative disorders in general–have taken advantage of the concentration dependence of low complexity domain protein aggregation . Most ande novo genomic insertions in adult brain tissue [de novo insertions per cell is still hotly debated [in vivo, no reports have probed the functional impact of TDP-43 pathology on the natural mechanisms of RTE suppression employed by somatic tissue such as the brain, and no studies have investigated the toxic effects of endogenous RTE activation on nervous system function.The RTE hypothesis investigated here is motivated by a series of prior observations. First, as mentioned above, LINE 1 RTEs are expressed in some somatic tissue , 4 and cn tissue –12, alth debated , 49. Sec debated . Moreove debated –57 and r debated –61. More debated , 55, 58  debated . HoweverDrosophila melanogaster [Drosophila transgenic model which afforded the means to examine whether RTE activation causally contributes to TDP-43 mediated toxicity and cell death. We found that several hallmarks of TDP-43-induced degeneration are the result of activation of the gypsy ERV, an RTE that is structurally related to HERV-K, and that this activation leads to DNA damage mediated cell death. Moreover, we uncovered an inhibitory effect of TDP-43 expression on small interfering RNA (siRNA) mediated silencing, leading to broad activation of a panel of RTEs. These findings strongly suggest a broad impact of TDP-43 pathology on general RTE activity.The destructive capacity of RTEs has been extensively documented in many other biological contexts, including a wealth of seminal data from nogaster –64. To tDrosophila. As with other animal models, including mouse, rat, fish, and C elegans, such expression reproduces many neuropathological hallmarks of human disease, likely via interference with endogenous protein(s) function [Drosophila, there is an endogenous putative ortholog of TDP-43, TBPH. Null mutations in TBPH in flies are lethal [Drosophila [In order to determine whether RTEs contribute to TDP-43 pathological toxicity, we implemented an established animal model in which hTDP-43 is transgenically expressed in function , 65, 66.e lethal , as is te lethal , 66, 68.e lethal  and an ee lethal –71. Toxiosophila –75. We tELAV > hTDP-43) or pan-glial (Repo > hTDP-43) hTDP-43 compared with control flies that carried the hTDP-43 transgene alone with no Gal4 driver (hTDP-43 / +). We generated two independent sequencing libraries for each genotype from a population of animals that were 8–10 days post-eclosion. We generated a total of ~900 million reads, or about 150 million reads per sample (Repo > hTDP-43) and neuronal (ELAV > hTDP-43) expression of hTDP-43 caused differential expression of a number of cellular transcripts  relative to controls (hTDP-43 / +) but no significant effect was observed with pan-neuronal expression of hTDP-43 (ELAV > hTDP-43)  , and 1E.gypsy RTE as a candidate of interest to test the functional impact of loss of endogenous RTE suppression in response to hTDP-43 expression for several reasons. First, although gypsy was not the most abundantly expressed RTE in the RNA seq data, this element is known to be one of the most active natural transposons in Drosophila melanogaster, and is responsible for a high fraction of the spontaneous mutations that have been identified. Second, we have previously documented that gypsy is capable of replicating and generating de novo insertions in brain during advanced age [gypsy is an ERV with functional similarity to HERV-K, which is expressed in some ALS patients [gypsy function. We began by performing quantitative RT-PCR (qPCR) for both ORF2 (Pol) and ORF3 (ENV) of gypsy on head tissue of flies expressing either pan-neuronal (ELAV > hTDP-43) or pan-glial (Repo > hTDP-43) hTDP-43. Because disease risk is age dependent and symptoms in ALS patients are progressive, we also examined the compounding effects of age. At two relatively young ages (2–4 and 8–10 days post-eclosion) we observe a dramatic increase in expression of both ORFs  in a similar manner to genetic controls that do not express hTDP-43 . These analyses , where g or glia . Taken tgypsy ENV glycoprotein [gypsy levels above that seen in wild type flies at any time point with either qPCR or immunolabeling . In brains from animals 3 days post eclosion, we observe regional puncta with a variable intensity and spatial location  neurons that constitute the mushroom body, results in little to no increase in TUNEL labeling  and ELAV-Gal4 (neurons), does not differ with age, suggesting that divergent age effects on expression level cannot account for the observed differences in toxicity and impact on physical health  even whpression . Thus, tt models . As is tt models  as well t models , implyingypsy RTE itself can actively replicate and generate de novo insertional mutations in brain tissue during aging [gypsy silencing might in fact account for a portion of the physiological toxicity observed with hTDP-43 expression in glia. To test whether the Drosophila ERV gypsy causally contributes to the harmful effects of hTDP-43, we used a previously published inverted repeat (IR) “RNAi” construct [gypsy ORF2 (gypsy(IR)) that is sufficient to reduce the expression of gypsy by approximately 50% in head tissue of 28-day old animals (gypsy(IR) substantially ameliorates the lifespan deficit induced by glial hTDP-43 expression (Repo > hTDP-43 + GFP(IR); gypsy(IR) nor the GFP(IR) constructs, when expressed alone under Repo-Gal4  does not rescue the lifespan deficit exhibited by animals expressing hTDP-43 in neurons  lifespan rescue is consistent with our observation that gypsy expression is induced specifically when TDP-43 is expressed in glia, lending credence to the conclusion that gypsy is causally participating in the resulting degenerative phenotype We of course cannot rule out the possibility that the gypsy-RNAi construct may also impact gypsy family RTEs that share sequence homology to gypsy. The conclusion that RTEs contribute to TDP-43 toxicity is further supported by the mild but significant lifespan extension that we observe with pharmacological inhibition of the reverse transcriptase activity that is essential for all RTE replication ), the Drosophila ortholog of chk2, which is sufficient to significantly reduce levels of endogenous loki mRNA in head tissue of 28-day old animals (loki(IR) with hTDP-43 is able to fully rescue the lifespan deficit caused by hTDP-43 expression in glia ; loki(IR) constructs when expressed individually under Repo-Gal4 or ELAV-Gal4 or present without a Gal4 driver  that was so effective in suppressing hTDP-43 toxicity in survival analyses (Repo > hTDP-43 + loki(IR)) and found that this was sufficient to abolish the dramatic accumulation of TUNEL-positive nuclei induced by glial expression of hTDP-43 ) also significantly reduces the TUNEL labeling observed in the CNS of these animals (loki(IR) and gypsy(IR) as co-expression of an unrelated UAS-(IR) construct with hTDP-43 in glia (Repo > hTDP-43 + GFP(IR)) does not significantly alter the number of TUNEL positive cells compared to brains of flies expressing hTDP-43 alone under Repo-Gal4 (loki(IR), and gypsy(IR) constructs with hTDP-43 under Repo-Gal4 also does not significantly reduce the expression of hTDP-43  leads to precocious gypsy expression in Drosophila head tissue and this is accompanied by rapid age-dependent neurophysiological decline [Drosophila nervous system in vivo.The major post-transcriptional RTE silencing system available in somatic tissue such as the brain is the siRNA pathway –92. siRN decline . We therRepo-Gal4 driver ) with a GFP transgenic reporter. By selecting an effective GFP(IR), we were able to generate substantial silencing of the GFP reporter . To test4 driver  or in mu4 driver . Brains 4 driver . What wes of age . Brains s of age . Indeed,reporter . As withreporter . In contwith age . Taken tDcr-2 or Ago2, the two major effectors of siRNA-mediated silencing in Drosophila [Dcr-2 , suggesting that down-regulation of Dcr-2 and Ago2 likewise cannot explain the later elevation of gypsy expression observed in these genotypes , cytoplasmic foci for modulating mRNA translation that materialize in response to cellular stress , 101–104ectively . This diR enzyme . Our fingypsy we demonstrate that this RTE has a causal impact on cell death and physiological decline in the animal’s health. We also identify DNA damage-induced cell death, mediated by Chk2 activity, as a major contributing factor in the toxicity of TDP-43 both at the cellular and organismal level. Importantly, gypsy is not the only RTE whose expression we found to be increased in response to hTDP-43 expression. We in fact observe a panel of RTEs that exhibit elevated expression, with some variation in this profile when hTDP-43 is expressed solely in neurons or glia. This is consistent with the observation that knocking down gypsy expression only partially suppresses the toxicity of TDP-43, whereas blocking loki (chk2) expression leads to a near complete suppression of the effects of hTDP-43 expression on cell death and lifespan reduction. The involvement of DNA damage-induced cell death suggest that gypsy and likely other RTEs may be successfully or abortively inserting into genomic DNA, although we are mindful of the fact that increased levels of RTE proteins and RNAs may themselves be cytotoxic, as is observed with the Alu RTE in macular degeneration [Unregulated RTE expression is known to be highly toxic in other biological contexts for a number of reasons, including accumulation of toxic RNAs, creation of harmful mutations, and accumulation of DNA damage. In the case of neration .gypsy ERV (or HERV-K)—as the major contributing factor in hTDP-43-related neurophysiological deterioration. This conclusion is supported by our RNA-seq data which shows a broad and general increase in RTE expression in head tissue of Drosophila expressing either neuronal or glial hTDP-43, as well as a pronounced reduction specifically in antisense siRNAs which target the RTEs we observe to exhibit increased expression in response to hTDP-43 expression. In accordance with this notion, we have previously shown in Drosophila that mutation of Ago2, a major effector protein of the siRNA system, results in activation of several different RTEs in brain tissue and causes rapid age-related cognitive decline and shortened lifespan [In the case of HERV-K, it has recently been shown  that TDPlifespan .Drosophila, the human genome contains more than one type of functional RTE. In addition to HERV-K, the human genome contains on the order of 100 fully active copies of L1 RTEs, and a far higher number of non-autonomous elements that replicate in trans by capitalizing on the protein machinery encoded by L1s [Like d by L1s . Moreoved by L1s –57, and d by L1s –18, incld by L1s . Previoud by L1s , 18. Ourd by L1s , 45–47.,w(IR) and GMR-Gal4, were backcrossed into our in-house wild type strain, the Canton-S derivative w1118 (isoCJ1) [OK107-, ELAV-, and Repo-Gal4 lines [gypsy(IR) [GMR-Gal4, Gal80ts, w(IR), GFP(IR), and tdTomato lines were acquired from the Bloomington Drosophila Stock Center , and the loki(IR) line was acquired from the Vienna Drosophila Resource Center [All transgenic fly stocks used, with the exception of (isoCJ1) , for at l4 lines , as welll4 lines  and gypsypsy(IR)  lines, ae Center  . RNA-Seq libraries were constructed using the NuGEN Ovation Drosophila RNA-Seq kit including DNase treatment with HL-dsDNase (ArcticZymes Cat. # 70800–201) and cDNA fragmentation using the Covaris E220 system according to manufacturer specifications. After amplification, library quality was measured using the Agilent Bioanalyzer system and quantity was determined using Life Technologies Qubit dsDNA HS Assay kit (for use with the Qubit 2.0 Fluorometer). Prior to sequencing, pooled libraries were quantified using the Illumina Library Quantification kit  from Kapa Biosystems and a 1.2 pM loading concentration was used for PE101 on the Illumina NextSeq500 platform. Small RNAs were cloned using TruSeq (Illumina) approach with modifications described in Rozhkov 2015 . BrieflyDrosophila dm3 genome with STAR [gypsy element, reads were mapped to the gypsy consensus sequence (GenBank accession: M12927) using Bowtie [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE85398).RNA-seq libraries were run on an Illumina NextSeq (paired end 101). Reads were mapped to the ith STAR  allowingg Bowtie  with up Reads mapped to ribosomal RNAs were removed from each library. For the remaining reads, expression abundance estimation and differential expression analysis were performed using the TEtranscripts package . Reads fThe ortholog pairs of fly and human were predicted with the Drosophila Integrated Ortholog Prediction Tool (DIOPT), which integrates the ortholog predictions from 11 existing tools . The paiMale flies were used for all lifespan assays since the majority of glial-expressing hTDP-43 flies that escape their pupal cases are male. Flies were housed 15 to a vial with a total of 75 flies per genotype and flipped into fresh food vials every other day. All vials were kept on their side in racks for the duration of the experiment. Lifespan experiments were performed blind.Tenofovir disoproxil fumarate , Zidovudine , and Stavudine  were prepped as solutions using dimethyl sulfide (DMSO) as solvent. Standard fly food was melted and cooled to a liquid, and NRTI solutions were added just before solidification to give final concentrations of 0 , 1, 5, 10, and 15 μM of each NRTI in a total volume of 0.2% DMSO, then stored at 4°C for a maximum of 2 weeks until use. Lifespan was monitored in all male flies with 20 flies per vial for a total of 100 flies monitored per genotype per treatment. Lifespans were performed as above.ad libitum for 24 hours. Measurements were initiated one hour following the switch to experimental solutions and monitored for a total duration of 24 hours. Experimentally treated solutions were prepared to match the concentrations of each NRTI treatment used in solid food for lifespan analysis, as well as an additional solution of high concentration for each NRTI (100 μM) and a solution of vehicle alone control (0.2% DMSO). Displacement due to evaporation was controlled for by subtracting measurements from fly-less CAFE chambers with the vehicle solution set up in parallel to the experimental assays. CAFE assays with untreated solution were also set up in parallel and controlled for evaporation.To quantify and determine the consistency of feeding across treatments, Capillary Feeder (CAFE) Assays  were perLocomotion behavior was assayed using the classic Benzer counter current apparatus as in Benzer, S., 1967 , with thAct5C (assay ID Dm02361909_s1), Dcr-2 (assay ID Dm01821537_g1), Ago2 (assay ID Dm01805433_g1), TARDBP (assay ID Hs00606522_m1), TBPH (assay ID Dm01820179_g1), and loki (assay ID Dm01811114_g1). All custom TaqMan probes were designed following the vendor’s custom assay design service manual and have the following assay IDs and probe sequences: gypsy ORF2 , gypsy ORF3 , and DCV . For genomic QPCR, DNA was isolated from 2–4 day old flies . After RNAase treatment, equal input levels of DNA were used for each PCR reaction, and each reaction was performed in triplicate. The CT values for Gypsy ORF2 were normalized to those for Actin within each sample. The values shown Tissue preparation, cDNA synthesis and qPCR were performed as previously described  using thet al. 2013 [Dissection, fixation, immunolabelling, and confocal imaging acquisition were executed as previously described . The ENVal. 2013 , 78. ForRepo > GFP or OK107 > GFP) and kept consistent across all subsequent brains imaged. The GFP signal of the median 10 optical sections of the appropriate structures  was calculated using ImageJ software, as previously described [The gain on the confocal microscope was set using the positive control  was used. The same dissection, fixation, and penetration and blocking protocol used for antibody staining was followed [Repo > hTDP-43) and kept consistent across all subsequent brains imaged. A projection image was generated using the middle 50 optical slices from the z-stack image of the whole brain. This projection image was then thresholded using the maximum entropy technique  via thewhite+.Flies of the appropriate age and genotype were placed at -70°C for 25 minutes and then kept on ice until immediately prior to imaging. Imaging was performed using a Nikon SMZ1500 stereoscopic microscope, Nikon DS-Vi1 camera and Nikon Digital Sight camera system, and the Nikon NIS-Elements BR3.2 64-bit imaging software package. The experiment was designed such that each group is balanced for the number of mini-white transgenes and heterozygous for genomic Drosophila heads were removed, the cuticle removed and the brains fixed overnight in 2% paraformaldehyde and 2% glutaraldehyde in 0.1 mol/L PBS. Samples were rinsed in distilled water and post-fixed for one hour in 1% osmium tetroxide in 1.5% potassium ferrocyanide in distilled water. Next, the samples were dehydrated in a graded series of ethanol and the final 100% ethanol was replaced with a solution of absolute dry acetone . The samples were then infiltrated with agitation for one hour in an equal mixture of acetone and Epon-Araldite resin, followed by infiltration with agitation overnight in 100% resin. Samples were transferred to embedding capsules with the posterior head facing towards the bottom of the capsule and the resin was polymerized overnight in a vented 60°C oven. Thin sections were made from the mushroom body region and collected on Butvar coated 2mm x 1mm slot grids (EMS) and the sections were counterstained with lead citrate. Thin sections were imaged with a Hitachi H700 transmission electron microscope and recorded on Kodak 4480 negatives that were scanned with an Epson V750 Pro Scanner at 2400 DPI. 3 individual brains processed for Repo / +, 4 individual brains processed for Repo > hTDP-43; many images collected of each brain.For qPCR data, the p-values of all data sets with only two groups were calculated using an unpaired t-test. Where an effect of age for more than two time points within one genotype was determined, a one-way ANOVA was performed, and where multiple ages and genotypes are represented a two-way ANOVA was performed; the results are reported in the figure legends. All pairwise comparisons for qPCR reported in the figures were corrected using the Bonferroni method for multiple comparisons. For both the locomotion data and the GFP quantification, p-values were reported using the Sheffé method; ANOVA results are reported in the figure legends. The CAFE assay is similarly reported, with pairwise comparisons between treatments being made using one-way ANOVA with Tukey’s Multiple Comparisons test. Survival analyses for the lifespan curves were performed using the Kaplan-Meier method, and the Gehan-Breslow-Wilcoxon test were used to compare survival curves. All pairwise comparisons for lifespan curves were corrected using the Bonferroni method. Sample sizes were selected based on standard practices in the literature. No randomization was employed in this study.S1 FigRepo>TDP-43 heads.Among the KEGG gene set annotated as functionally related to ALS pathways. Although this is by no means a complete list of genes that have been implicated in ALS in the literature, it represents a set of gene pathways with known involvement in ALS. 20 of the ALS KEGG gene set contain clear Drosophila orthologs and a significant fraction of these (11/20) are identified as differentially expressed in our RNAseq from (PDF)Click here for additional data file.S2 Figgypsy ORF3 (Env) as detected by qPCR on whole head tissue of flies expressing (A.1) neuronal hTDP-43 (ELAV > hTDP-43), or genetic controls: (A.2) ELAV / + and (A.3) hTDP-43 / +. gypsy ORF3 transcript levels display an increase by 21–23 days post-eclosion that drops back down by 40–42 days post-eclosion, regardless of genotype. In all cases transcript levels have been normalized to Actin, and the aged cohort  are represented as a fold change over an appropriate young (2–4 day) cohort that has been processed in parallel. Unpaired t-tests have been used to calculate p-values for each aged cohort with its matched young cohort, while p-values comparing aged cohorts within genotypes have been calculated using the Bonferroni method for multiple comparisons. For all three genotypes a one-way ANOVA shows a significant effect of age on gypsy ORF3 transcript levels between the aged cohorts . N = 5 for all groups. (A.4). qPCR of whole head tissue reveals that the presence of the hTDP-43 transgene alone with no Gal4 driver results in elevation of gypsy ORF2 transcript levels. N = 6 for both groups. Quantitative genomic PCR (A.5) reveals that the wild type, Elav-Gal4 and Repo-Gal4 lines have comparable levels of gypsy DNA copy number, and (A.6) mRNA expression levels. The UAS-hTDP-43 parental line exhibits marginally higher levels of gypsy genomic DNA (A.5), although this cannot explain the difference between expression in ELAV > hTDP-43 vs Repo> hTDP-43. (B) Quantification of immunoreactive puncta averaged across the central 10 optical slices of brains of flies expressing hTDP-43 in glial cells (Repo > hTDP-43) and genetic controls (Repo / + and hTDP-43 / +) aged to 10 days and whole-mount immunostained using the gypsy ENV monoclonal antibody in a separate experiment from Repo / + and hTDP-43 / +; p = 1.0000), but that 10 day old flies that express hTDP-43 in glial cells (Repo > hTDP-43) display significantly more ENV immunoreactive puncta than flies carrying the UAS-hTDP-43 transgene  or Repo-Gal4 alone . Displayed as means + SEM; N = 8 for all groups. (C) Projections through whole-mount brains immunolabeled with a gypsy ENV monoclonal antibody demonstrate that gypsy expression turns on post-developmentally, with very little gypsy expression immediately following eclosion (0 Days) in genetic controls (hTDP-43 / + and Repo / +) and in flies expressing hTDP-43 in glia (Repo > hTDP-43). Expression turns on stochastically at 3 days post-eclosion only in the CNS of flies expressing glial hTDP-43. Replicates of 3-day old Repo > hTDP-43 brains illustrate the variability of gypsy expression at this early time point. N = 2 for all 0 Day groups; hTDP-43 / +, 3 Day, N = 3; Repo / +, 3 Day, N = 4; Repo > hTDP-43, 3 Day, N = 7.(A) Transcript levels of (PDF)Click here for additional data file.S3 FigOK107-Gal4, even when the animals are aged to 30 days post-eclosion. Mushroom bodies marked by co-expression of GFP, shown in green; TUNEL staining shown in red. OK107 > GFP, 5 Day, N = 3; OK107 > GFP + hTDP-43, 5 Day, N = 5; OK107 > GFP, 30 Day, N = 5; OK107 > GFP + hTDP-43, 30 Day, N = 4. (B) Full length human TDP-43 (green) can be detected by immunolabelling in the brains of flies expressing glial hTDP-43 under the Repo-Gal4 driver at 21–23 days post-eclosion, and co-localizes with Repo (red) immunoreactivity (left). Repo / + (N = 4); Repo > hTDP-43 (N = 4). Immunoreactivity for a disease-specific phosphorylated isoform of the protein (pSer409) can also be readily detected and co-localizes with Repo (right). Repo / + (N = 7); Repo > hTDP-43 (N = 4). A 63x blow-up is shown in the pop-out. (C) Both the full-length (left) and disease specific (right) isoforms of hTDP-43 (green) are mainly observed in the cytoplasm and vacate the nucleus . Arrowheads indicate the hTDP-43-filled cytoplasm of a cortical glial cell wrapped around several neuronal nuclei in the neuropil of flies expressing glial hTDP-43. For full length hTDP-43 antibody, Repo / + (N = 6), Repo > hTDP-43 (N = 13); for pSer409 phosphorylated hTDP-43 antibody, Repo / + (N = 4), Repo > hTDP-43 (N = 9). (D) qPCR of whole head tissue demonstrates that transcript levels of hTDP-43 diminishes under Repo-Gal4 from 2–4 days to 8–10 days. Transcript levels normalized to Actin and displayed as fold change relative to 2–4 day old flies (means + SEM). N = 6 for all groups. (E) A similar effect of age on hTDP-43 expression is observed in neurons under ELAV-Gal4, and continues to drop off by 40–42 days post-eclosion. A one-way ANOVA shows a significant effect of age (p < 0.0001). N = 6 for all groups.(F) qPCR of whole head tissue demonstrates that expression of hTDP-43 does not effect levels of the Fly ortholog, TBPH, regardless of cell type of expression. Transcript levels normalized to Actin. N = 4 for the hTDP-43 / + group, N = 5 for all other groups.(A) TUNEL staining reveals very little apoptotic activity when hTDP-43 is expressed in the mushroom body under (PDF)Click here for additional data file.S4 FigHowever, NRTI administration extends lifespan in flies expressing hTDP-43 in glia. (A) qPCR on head tissue demonstrates that expressing an IR directed against gypsy ORF2 (gypsy(IR)) in neurons (ELAV > gypsy(IR)) or glia (Repo > gypsy(IR)) effectively inhibits age-dependent elevation of gypsy transcript levels, and results in an ~2.5-fold reduction at 28 days post-eclosion. gypsy transcript levels from head tissue of young (2–4 day) and aged (28 day) flies was normalized to Actin and displayed as fold change in 28 day old flies of each genotype relative to each respective young cohort (displayed as means + SEM). A one-way ANOVA shows a significant effect of genotype (p = 0.0182). N = 2–3 biological replicates generated from heads of 5 mL of flies for each group. (B) Expression of gypsy(IR), loki(IR), and GFP(IR) individually in glial cells under the Repo-Gal4 driver does not significantly alter lifespan. (C) Expression of gypsy(IR) and loki(IR) individually in neurons under the ELAV-Gal4 driver does not significantly alter lifespan. (D) The presence of each of the IR constructs alone without any Gal4 driver (gypsy(IR) / +; loki(IR) / +; GFP(IR) / +) only moderately effects lifespan. (E) The capillary feeder (CAFE) assay demonstrates that neither the final concentration of vehicle (0.2% DMSO) alone, any of the final concentrations of D4T used in solid fly food in the lifespan analysis  CAFE assay performed as in N = 9–12 for all AZT groups. (G) Lifespan analysis shows that the NRTI Stavudine (D4T) partially suppresses the lifespan deficit induced by glial expression of hTDP-43 (Repo > hTDP-43) when supplied in solid fly food at final concentrations of either 5 μM, p = 0.0450; or 10 μM, p = 0.0186; in comparison to vehicle alone control (0 μM). (H) Lifespan analyses performed as in (D) for the NRTI Zidovudine (AZT). AZT partially suppresses the lifespan deficit exhibited by flies expressing hTDP-43 in glia (Repo > hTDP-43) when supplied at 5 μM (p = 0.0245) in comparison to a vehicle alone control (0 μM).(PDF)Click here for additional data file.S5 FigELAV > loki(IR)) and glial (Repo > loki(IR)) expression of an IR directed against loki (loki(IR)) effectively blocks the age-dependent elevation of loki transcript levels, resulting in an ~2-fold reduction at 28 days post-eclosion. A one-way ANOVA shows a significant effect of genotype (p = 0.0039). N = 3–4 biological replicates. (B) Co-expression of GFP(IR) with hTDP-43 under Repo-Gal4 (Repo > hTDP-43 + GFP(IR)) does not significantly alter the number of TUNEL-positive nuclei detected compared to hTDP-43 expression alone under Repo-Gal4 (Repo > hTDP-43). N = 8 for Repo > hTDP-43 and N = 9 for Repo > hTDP-43 + GFP(IR); data normalized to Repo > hTDP-43. (C) qPCR for hTDP-43 expression (TARDBP) on whole head tissue demonstrates that co-expression of each of the IR constructs with hTDP-43 under Repo-Gal4 (Repo > hTDP-43 + GFP(IR), Repo > hTDP-43 + gypsy(IR), and Repo > hTDP-43 + loki(IR), respectively) does not significantly reduce hTDP-43 expression levels compared to hTDP-43 expression alone under Repo-Gal4 (Repo > hTDP-43) Fold change is displayed as the mean fold change relative to Repo > hTDP-43, while p-value represents the p-value of a two-tailed Student’s t-test in comparison to Repo > hTDP-43. N = 4 for all groups.(A) An equivalent analysis as described for (S5A) demonstrates that neuronal ((PDF)Click here for additional data file.S6 Figgypsy cannot be explained by hTDP-43- or age-dependent effects on siRNA effector molecules.Loss of suppression of  (A) Schematic representation of the experimental design. (B) Representative images demonstrating that turning off w(IR) expression post-developmentally rescues red pigmentation of the Fly eye. N = 5 for all groups. (C) qPCR of whole head tissue demonstrates that reduced expression of (C.1) Dcr-2 and (C.2) Ago2 cannot account for loss of suppression of gypsy in flies expressing glial hTDP-43 (Repo > hTDP-43) at either 2–4 or 8–10 days post-eclosion. Transcript levels normalized to Actin and displayed as fold change relative to flies carrying the hTDP-43 transgene with no Gal4 driver (hTDP-43 / +) at 2–4 Days (means + SEM). For Dcr-2, a two-way ANOVA reveals an effect of age (p = 0.0006) but no effect of genotype (p = 0.1081); for Ago2, a two-way ANOVA also reveals an effect of age (p = 0.0258) but no effect of genotype (p = 0.1591). N = 8 for all groups. (D) qPCR of whole head tissue demonstrates that age-dependent changes in expression of Dcr-2 (top) and Ago2 (bottom) cannot account for age-dependent loss of suppression of gypsy in flies expressing neuronal hTDP-43  or genetic controls: (D.2) ELAV / + and (D.3) hTDP-43 / +. All data analyzed as in (S1A.1-S1A.3); one-way ANOVA shows an effect of age across almost all groups . N = 8 for all groups.(PDF)Click here for additional data file.S7 Figgypsy exhibit elevated levels of expression in the RNAseq data , there is a selective decrease in anti-sense relative to sense stranded siRNAs. (A) scatter plots of log2Fold change in RNAseq vs log2Fold change in small-RNA seq. 3S18, mdg3 and data see , and als(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableA: Abundance of gene-derived transcripts in head tissue of flies expressing pan-neuronal hTDP-43 compared to controls. B: Abundance of transposon-derived transcripts in head tissue of flies expressing pan-neuronal hTDP-43 compared to controls. C:Fly orthologs of ALS KEGG gene set.(XLSX)Click here for additional data file.S3 TableA: Abundance of gene-derived transcripts in head tissue of flies expressing pan-glial hTDP-43 compared to controls. B: Abundance of transposon-derived transcripts in head tissue of flies expressing pan-glial hTDP-43 compared to controls.(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.