The emergence of dissemination and implementation (D&I) science has driven a rapid increase in studies of how new scientific discoveries are translated and developed into evidence-based programs and policies. However, D&I science has paid much less attention to what happens to programs once they have been implemented. Public health programs can only deliver benefits if they reach maturity and sustain activities over time. In order to achieve the full benefits of significant investment in public health research and program development, there must be an understanding of the factors that relate to sustainability to inform development of tools and trainings to support strategic long-term program sustainability. Tobacco control programs, specifically, vary in their abilities to support and sustain themselves over time. As of 2018, most states still do not meet the CDC-recommended level for funding their TC program, allowing tobacco use to remain the leading cause of preventable disease and death in the USA. The purpose of this study is to empirically develop, test, and disseminate training programs to improve the sustainability of evidence-based state tobacco control programs and thus, tobacco-related health outcomes.This paper describes the methods of a group randomized, multi-phase study that evaluates the empirically developed “Program Sustainability Action Planning Training” and technical assistance in US state-level tobacco control programs. Phase 1 includes developing the sustainability action planning training curriculum and technical assistance protocol and developing measures to assess long-term program sustainability. Phase 2 includes a group randomized trial to test the effectiveness of the training and technical assistance in improving sustainability outcomes in 24 state tobacco control programs . Phase 3 includes the active dissemination of final training curricula materials to a broader public health audience.Empirical evidence has established that program sustainability can improve through training and technical assistance; however, to our knowledge, no evidence-based sustainability training curriculum program exists. Therefore, systematic methods are needed to develop, test, and disseminate a training that improves the sustainability of evidence-based programs.NCT03598114. Registered 25 July 2018—retrospectively registered. Demonstrating the effective implementation of a program is only the first step in influencing the health and wellness of a target population. For a population to experience the benefits of an implemented evidence-based intervention, the intervention must be sustained over time. Program sustainability is a complex process, often fraught with challenges –4. Reseapreventable cause of death and disease [As of 2013, 42.1 million—or one in five—adults in the USA smoke, leading to an estimated 480,000 deaths per year due to tobacco use . Accordi disease . Given tThe goal of this study is to increase capacity for sustainability among evidence-based tobacco control programs. Although all 50 states have implemented evidence-based tobacco control programs and policies, each program varies in its ability to support and sustain itself over time. Tobacco control funding directly correlates with US adult smoking rates in that states with more funding have lower smoking rates . BetweenThis study defines sustainability as the existence of adaptive structures and processes that enable a program to effectively implement and institutionalize evidence-based policies and activities over time . EmpiricThis study, funded by the National Cancer Institute, seeks to develop and evaluate the effectiveness of a program sustainability action planning training curriculum in increasing the capacity for sustainability among evidence-based tobacco control programs. The study will be directed by a multidisciplinary team of researchers and practitioners with expertise in: program sustainability assessment, tobacco control program and policy evaluation, planning and training, and dissemination and implementation science. This study team consists of researchers from the Prevention Research Center (PRC) at Washington University in St. Louis with collaboration from the Center for Public Health Systems Science (CPHSS) at Washington University in St. Louis and CDC’s Office on Smoking and Health (OSH). The Institutional Review Board of Washington University in St. Louis and the Protocol Review and Monitoring Committee approved all study procedures.The study involves three interconnected phases. Phase 1 consists of the development of a sustainability action planning training curriculum including the creation of an interactive workbook and design of a training workshop. Additionally, Phase 1 will focus on the development of surveys, interviews, and record abstraction protocols to collect programmatic, organizational, community-level, and funding data associated with program sustainability and institutionalization. Phase 2 is a group randomized, mixed methods study designed to assess the training curriculum’s effectiveness in increasing the capacity for sustainability among 24 statewide tobacco control programs. This involves two parallel study arms with 12 intervention states and 12 pair-matched comparison states. States will be paired together according to an analysis of programmatic factors, then randomly selected into intervention or comparison groups. Those programs placed in the intervention condition will receive numerous training strategies, including in-person sustainability action planning training workshops, ongoing technical assistance, and periodic communications regarding project progress as well as recent program sustainability and tobacco control news. State programs participating in the comparison condition will also be encouraged to create a sustainability plan, but they will receive neither training workshops nor technical assistance. Phase 3 will focus on both refining the training curriculum based on evaluation following implementation and actively disseminating all training materials to broader public health audiences. Figure The conceptual model for the study is driven by the theory of change , 19–21. The target audience for this study includes statewide tobacco control programs and their stakeholders, drawn from diverse organizations such as state and local health departments, community-based organizations, universities, and policymakers. All participants will be non-institutionalized adults ages 18 and over. The only exclusionary criterion is that participants must be involved in their state’s tobacco control program and must be selected by their state program manager as a stakeholder. Historically, tobacco control programs endure constant changes in funding and political support . SimilarThe study team will develop and deliver a series of in-person training workshops to educate stakeholders about program sustainability and facilitate their completion of a sustainability action plan. The study team will develop a workbook for each participant to use with the training curriculum. The workshop will elaborate upon previous trainings conducted by CPHSS and empirical recommendations –28 to inComplementary to these core principles, the study team set four goals to guide the development of the training curriculum workbook: (1) to offer an interactive guide for participants to return to as a sustainability planning resource; (2) to spark engaging, transparent, and collaborative discussions; (3) to communicate with a range of community-oriented organizations; and (4) to remain readable across print and digital formats. These goals, in combination with the experiential learning framework, will motivate the study team to design interactive activities throughout, including fill-in visualizations, journaling segments, and discussion prompts. In each section, the audience will apply educational material to the context of their program. The training workbook will mirror the format of the action planning workshop, and stakeholders will reference the workbook throughout the course of the training. In the following years, the workbook will serve as a resource encouraging proactive and accountable use of the action plan and accompanying sustainability planning resources.Technical assistance after the training will promote continuation of the action plan and provide troubleshooting for the intervention states. This is an essential step in the action planning process to help build each state program’s capacity for quality implementation of the action plan. Following each intervention, flexible and proactive technical assistance with clearly defined responsibilities and tailored messaging will strengthen communication between providers and recipients . ThroughFollowing recommendations from the advisory board and tobacco control experts, the study team will collect data primarily using record abstraction. This will help to alleviate the burden of an intensive, all-encompassing survey or interview. However, because it may be infeasible to collect all data points through program records, the study team has also developed a key informant interview tool to collect remaining information. The interviews will be conducted via a 15–20-min phone interview with state program managers or any other qualified surrogate. Additionally, all answers will be recorded, transcribed, and reviewed for completeness and accuracy. An online Qualtrics survey will be developed with identical questions for the convenience of state program managers that prefer not to complete a phone interview. Each interview or survey will be coded by assigned team members and checked for inter-rater reliability.State of Tobacco Control report [Healthy Americans report issued by Trust for America’s Health [Much of the record data for this study will come from annual state-level reports to the Office of Smoking and Health at the CDC. These reports address fulfillment criteria for the DP15-1509 funding announcement and describe the infrastructure, personnel, and activities of state tobacco control programs in detail. These funding announcements are a requirement of state programs, set by the CDC, to complete yearly reports of progress, goals, and challenges in order to receive federal funding. The study team has strategically timed this project to coincide with the annual release of these reports. In addition to the CDC reports, other data will be collected via secondary data sources, including the American Lung Association’s annual l report  and the s Health .Phase 1 also includes production of a codebook to organize and explain the programmatic and organizational data collected, as well as a record abstraction protocol. The project codebook will align each data point within four key areas  .The program sustainability framework was developed from previous research completed by the CPHSS team through an extensive literature review, concept mapping, and expert input , 33. TheTable As aforementioned, increased readiness and capacity for sustainability will mediate the effect of the workshop on sustainability success or institutionalization. Each mediating variable contributing to sustainability readiness consists of several factors . ProgramBest Practices for Comprehensive Tobacco Control Programs [Through a systematic literature review, the study team identified indicators of program sustainability that further characterized each key metric , 36–46. Programs  to detaiPrograms .While in initial development, these indicators were subject to several rounds of review by academic investigators on the team. In April of 2018, the study team assembled a panel of expert academics and practitioners to deliberate the list of candidate variables and discuss a strategy for collecting the data. This produced a final list of mediating variables. The study team then aligned these indicators with items annually reported by state tobacco control programs to the Office on Smoking and Health at the CDC.Phase 2 will include a longitudinal group randomized-effectiveness trial to determine differences in organizational and programmatic measures and program sustainability assessment scores between intervention and comparison groups. There will be three staggered recruitment stages with 12 intervention states and 12 paired comparison states. Participating intervention state programs will work closely with the study team to create and carry out their action plan through involvement in technical assistance activities. Although comparison states will receive neither the training curriculum nor technical assistance, they will be given resources to independently construct and carry out a sustainability action plan. Evaluation measures will occur at three time points  and will incorporate PSAT scores, program record abstraction, and key informant interview data.State tobacco control program selection and recruitment are based on four criteria: policy progress, resources, need, and previous participation in sustainability training. These characteristics were used to stratify the states when they were assigned to each condition (intervention or comparison). Stratification was important to help ensure the comparability of the grouped states.Tobacco control policy progress is operationalized as the American Lung Association’s (ALA) smoke-free score for each state . This ant tests of differences-in-means were conducted for the two groups using the changes in PSAT scores between pre- and post-intervention data. The standardized differences in t statistics yielded an effect size of 1.25, which is a large effect size according to Cohen’s criteria [The goal is to select three states from each quadrant per group, for a total of six per quadrant, with varying levels of need to build comparable and diverse groups. For example, from each quadrant, two states were selected with relatively low-smoking rates, two from the middle, and two with high-smoking rates. All states selected have not previously participated in sustainability action planning training. Based on a priori analysis, the study team estimated a range of effect size using data from a natural experiment. The data consists of pre- and post-PSAT averages from previous work with five tobacco control programs that received action planning training and five programs that did not. To determine effect size, criteria . Using tcriteria  and Chamcriteria , the effcriteria –51.State selection and enrollment will be staggered by three pairs at a time during the first two stages of recruitment and six pairs during the final stage over years two and three. The study manager will invite each of the chosen states’ program managers to participate in the study. The program managers will then select between five and 12 key program stakeholders or partners within their state to participate. After enrollment and baseline data collection, the paired states will be randomly assigned into intervention or comparison groups via simple random concealed allocation performed by the statistician. If any program declines to participate, a replacement will be selected from the same quadrant by identifying the state with the closest ALA scores and funding levels.In each intervention state, a primary group of tobacco-control stakeholders and study-team members will collaborate to identify and define programmatic aspects of the statewide initiative, including program mission and vision, target populations, strengths and weaknesses, and key partners within and outside of the program. Additionally, these states will participate in sustainability action planning that responds to an in-depth discussion of aggregate PSAT results by addressing particular framework domains. The workshops will consist of a two-day presentation at the state health department. On the first day, facilitators will guide states through defining their tobacco control program and reflecting on their PSAT scores. Participants will consult their scores to frame appraisals and activities addressing strengths and weaknesses of their program and guide the creation of their sustainability action plan. The second day consists of prompts related to creating specific, measurable, attainable, realistic, and time-sensitive (SMART) objectives; activities for participants to link areas of their program and evidence-based topics in sustainability; templates for determining actions and a timeframe; and guidance on creating clear outcomes. The creation of the sustainability action plan is the focal point of the training. The training curriculum will emphasize specific and concrete plans to increase the likelihood that plans are implemented , 25. IntEvaluation measures will be taken at three time points: pre-intervention (baseline) and at 1 and 2 years post-intervention. The study team expects that with the presence of an in-person, customized sustainability training curriculum will correspond with increases in established programmatic and organizational metrics of sustainability. Several process measures will be collected to assess the effectiveness of the training and support delivered. All measures will be collected via survey at the completion of each training component or tracked by review of programmatic records. Quantitative data collected will be analyzed using descriptive statistics while qualitative analysis of programmatic records will be conducted following the PSAT and program record abstraction procedures. This analysis will include dosage delivered, dosage received, and participant reactions. Dosage delivered is the number of hours of training and technical assistance provided to each state. Dosage received will be measured as the extent and frequency that state programs utilize their action plans and project resources and materials. All data will be collected via an evaluation form filled out by state program managers. First, state programs will indicate whether or not an action plan has been created and implemented . To describe frequency of use of their action plan, state programs will provide estimates of how often program personnel discuss and implement the action plan . To describe the extent of use, state programs’ evaluations will report S.M.A.R.T. objectives their stakeholder group determined at either training workshops  or independent meetings (comparison condition) as well as the specific steps this group outlined to achieve those objectives. At each time point, programs will note which steps they have completed under each S.M.A.R.T. objective. The resulting ratio of complete action plan steps to incomplete action plan steps will represent dosage received. This ratio measurement will allow comparisons between state programs with action plans of different lengths. Program managers will also indicate whether or not their state program has utilized project resources and materials . They will also describe their frequency of use of these resources and materials .In addition, participant reactions will be measured by the extent to which they felt that the objectives of the training and technical assistance were achieved. They will also be asked to report levels of satisfaction with, and their perceptions of the usefulness of, the training and technical assistance provided. This qualitative data will be collected through a post-training evaluation and will inform the study team of any limitations of the format and content as well as ways in which the training can be improved in the future. This feedback will assist the study team to modify the curriculum for later dissemination.Descriptive statistics, including frequencies and measure of central tendency and dispersion, will be calculated for both the intervention and comparison groups at each of the three data collection time points to assess baseline averages and changes. To incorporate the influence of each state’s distinctive characteristics, longitudinal regression analysis will be used to model the outcome for each of the dependent variables. This analytic model is described by the following equation:Y is the outcome variable and i = state and t = time point (1 or 2); PSATit = 0 is the baseline PSAT score, Gi signifies the group (intervention or comparison), Dit signifies dosage at time t, while (Git * Dit) represents an interaction between the group and dose terms; Xk,it represents a vector of programmatic, organizational, community, and funder variables, and uit is the error. This analysis will ultimately be used to test the hypothesis that the impact of the training is nonzero and positive.where A multivariate approach accounts for the numerous influences found in programmatic, organizational, community, and funder attributes. Additionally, data collection at different time points allows for measuring changes in these variables, and the influence these changes have on institutionalization outcomes. The level of adoption and implementation of action plans both from the training (intervention states) and from other initiatives (comparison states) will vary. This variance in dosage reception and delivery will be acknowledged through multivariate longitudinal analysis.Electronic versions of final workbook and curriculum materials;Instructional videos to support independent action planning work;Standardized reporting forms in partnership with the CDC-OSH to support ongoing data collection and fidelity to the training model;www.sustaintool.org;Technical assistance worksheets and activities posted on Webinars;Findings about the impact of the program sustainability action planning model and training curriculum on programmatic and organizational outcomes in high-visibility cancer, public health, and dissemination and implementation science journals and conferences;All lessons learned from the training program and a final curriculum product will be systematically disseminated to diverse audiences, since the methods and approaches are generalizable and adaptable to any public health initiative.The results of this study will be used in many different ways. Potential final dissemination strategies include:These final dissemination strategies will be provided to both intervention and comparison state tobacco control programs included in the study following its completion. Additionally, the materials will be generalizable and available for any public health program seeking to build capacity for sustainability.The study is currently in progress. The study team has completed certain phase 1 activities, including an extensive literature review, determination of key metrics and variables, and development of a program record abstraction protocol. The final development of the action planning workbook and other training materials is still ongoing and close to completion. Phase 2 of the study has begun. Recruitment and program record abstraction are in progress for the first round of three paired states.This study provides a potentially innovative approach to increase the sustainability capacity of tobacco control programs in a number of ways. First, this study will provide the first ever evidence-based program sustainability action planning model and training curriculum. By establishing this evidence-based method for action planning and technical assistance related to sustainability, the study team is supporting state tobacco control programs and other evidence-based public health initiatives to sustain their positive impact, despite tumultuous funding climates. Additionally, there are currently no available resources specifically designed to assist state tobacco control programs in meeting national requirements to “develop a sustainability plan” or “provide measures of execution activities as outlined in the plan” . The dev. The study team hopes to combat this by offering incentives to stay in the study, including use of action planning resources and access to the final dissemination products.The study has a few limitations. The main limitation is the possibility of state drop-out. Replacing participating programs will be difficult due to the longitudinal nature of the studyStaff turnover in state tobacco control programs is also a notable concern. Discussions with tobacco control consultants have described the seemingly unavoidable reality of key stakeholder turnover in states. Turnover is likely due to many factors, including lack of funding. This is why this study is particularly timely, as there is potential to acknowledge these hardships and create plans to improve. The study team, however, will monitor staff turnover and manage it by staying in close contact with the state program managers and OSH project officers.Finally, another important limitation to note is the lack of empirical literature related to technical assistance best practices, particularly within tobacco control programs. This makes it difficult to establish a uniform, evidence-based technical assistance protocol to apply to all states. However, through past experience of the team working with state programs and discussions with tobacco control consultants and a formative systematic literature review, the study team concluded that more flexible approaches for technical assistance may be the most efficient way to assist states, considering that every state is unique. Thus, the team will have technical assistance guidelines but will employ more tailored approaches for each state, based on need. The team hopes to gain insight on technical assistance best practices and describe lessons learned in the dissemination products, building on the small body of knowledge that exists for this topic area.This study has the potential to improve public health programs by introducing a rigorous evaluation for program sustainability and creating tools to improve sustainability over time. The benefits of program sustainability will not only benefit the state programs themselves, but also the health of state populations through the continuation of tobacco control initiatives shown to decrease tobacco-related disability and death. This study’s findings will contribute to the growing body of knowledge on how to mature and sustain activities over time, thereby achieving the full benefit of significant public health investment.


Certain immunoglobulins (Ig) are proposed to have protective functions in atherosclerosis.We tested whether serum levels of IgG and IgM autoantibodies against malondialdehyde low density lipoprotein (MDA-LDL) are associated with clinical coronary heart disease (CHD) and unfavorable plaque characteristics.NORDIL was a prospective study investigating adverse cardiovascular outcomes in hypertensive patients. IBIS-3 analyzed lesions in a non-culprit coronary artery with <50% stenosis using radiofrequency intravascular ultrasound (RF-IVUS) and near-infrared spectroscopy (NIRS). Imaging was repeated after a median of 386 days on rosuvastatin. Associations of antibodies with incident CHD and imaging parameters were assessed in the two sub-studies respectively.From 10,881 NORDIL patients, 87 had serum sampled at baseline and developed CHD over 4.5 years, matched to 227 controls. Higher titers of IgM anti-MDA-LDL had a protective effect on adverse outcomes, with odds ratio 0.29 . Therefore, the effect was explored at the lesional level in IBIS-3. 143 patients had blood samples and RF-IVUS measurements available, and NIRS was performed in 90 of these. At baseline, IgM anti-MDA-LDL levels had a strong independent inverse relationship with lesional necrotic core volume (p = 0.027) and percentage of plaque occupied by necrotic core (p = 0.011), as well as lipid core burden index (p = 0.024) in the worst 4 mm segment.Our study supports the hypothesis that lower circulating levels of IgM anti-MDA-LDL are associated with clinical CHD development, and for the first time relates these findings to atherosclerotic plaque characteristics that are linked to vulnerability. The role of anti-oxidized LDL antibodies in atherosclerosis has been extensively studied. IgM antibodies were hypothesized to be largely protective. There is little data linking antibody levels to plaque characteristics in patients and basic cross-sectional coronary angiography studies have not been conclusive.This study confirms that high levels of IgM antibodies against oxLDL are protective from coronary heart disease in a nested case control study within a large RCT. In addition, this study shows for the first time, that high levels of these IgM antibodies, and to a lesser extent total serum IgM, are associated with coronary plaque characteristics that reflect plaque stability . These novel findings postulate a mechanistic explanation of how IgM anti-oxLDL antibodies may exert their protective effects in patients with CHD.By linking low IgM antibody levels to clinical CHD and unfavorable plaque characteristics, our study may be useful in designing future immunotherapies for the ‘vulnerable plaque’ as well as in focusing patient selection for clinical cardio-protection trials and patient stratification in the clinic.Alt-text: Unlabelled Box1The immune system exerts both protective and pathogenic effects in atherosclerosis, with a fine balance between maintaining homeostasis and over-activation , 2. ImmuIn the quest to link the vulnerable patient to the rupture-prone plaque, it is now important to identify factors in the circulation that are related with both incident coronary events and negative characteristics of coronary plaques beyond just degree of arterial narrowing , 17. AltLimited work from randomized control studies as well as cohort studies suggested a link between IgM antibodies against various modifications of LDL and protection from incident cardiovascular events . HoweverWe first present findings from the NORDIL study  confirmiOur findings provide substantial support for IgM anti-MDA-LDL antibodies protecting not only from events in NORDIL but also at the level of the plaque in IBIS-3.22.12.1.1The NORDIL study has been extensively described , 22. It 2.1.2IBIS-3 was a prospective cohort study that was designed to determine the ability of rosuvastatin to decrease NC volume in coronary atherosclerosis. The study was conducted in the Erasmus MC, Rotterdam, the Netherlands from 2010 until 2013, and the population has previously been described in detail . The stu2.2Blood samples were drawn at randomization in NORDIL or prior to the procedures in IBIS-3, and were stored at −80 °C. After study completion, samples were transported under controlled conditions to the Vascular Sciences Section at Imperial College, London, UK. We measured total serum IgM and IgG and specific antibodies against MDA-LDL by ELISA, as previously described . Levels 2.320-MHz IVUS catheters  were used at a continuous motorized pullback speed of 0.5 mm/s . The IVUS images were analyzed offline for plaque burden and NC volume by an independent core laboratory , blinded for clinical and biomarker data. The IVUS grayscale and IVUS radiofrequency analyses, also known as IVUS virtual histology (IVUS-VH), were performed using pcVH 2.1 and qVH  software. The external elastic membrane and luminal borders were contoured for each frame . Extent and phenotype of the atherosclerotic plaque were assessed. Plaque volume was defined as the total volume of the external elastic membrane occupied by atheroma . Plaque 2.42 from a depth of approximately 1 mm2 perpendicular to the long axis of the catheter [NIRS was performed with the Infraredx system , at a pullback speed of 0.5 mm/s. The NIRS-system used acquires 1000 measurements per 12.5 mm of coronary artery of 1 to 2 mmcatheter . The corcatheter , 30. Bot2.5www.r-project.org), SAS V9.4 , or STATA V12 . Two-tailed p values of <0.05 were considered statistically significant. Further details on statistical methodology are provided in the Supplementary Methods.Continuous variables are presented as mean ± standard deviation (SD) or median ± interquartile range (IQR), depending on the normality of the distribution. Categorical variables are presented as numbers and percentages. Analyses were performed with R statistical software  versus 61·25 (6.30) years in the cases and controls respectively . There were no significant differences between the groups, with the exception of smoking rate (p < 0·0001), HDL-cholesterol (p = 0·04) and diabetes (p = 0·021).3.2There was a significantly higher median level of IgM anti-MDA-LDL in the controls  units) versus the CVD cases  units; p = 0·039) at baseline, whereas no relationship was demonstrated with IgG anti-MDA-LDL (p = 0·38) (Supplementary Table 3).As CVD may have a heterogeneous causal pathology, we focused on the relationship of antibodies against MDA-LDL with incident CHD events. After controlling for variables with significant inter-group variation  and randomized blood pressure treatments, as well as total IgG and IgM, immunoglobulin levels were log-transformed and divided into tertiles. The highest tertile of IgM anti-MDA-LDL was found to have a substantial protective effect on the development of CHD, with odds ratio (OR) 0·29  . No such3.3We examined samples from IBIS-3 to explore the putative protective effect of IgM anti-MDA-LDL antibodies at the level of the atherosclerotic plaque. Of the 241 patients eligible to start rosuvastatin treatment, 143 had both blood samples and matching baseline and follow-up RF-IVUS measurements available B. The pr3.4At baseline, IgM anti-MDA-LDL antibody levels had a strong inverse relationship with lesional NC volume (p = 0·027) and percentage (p = 0·011) . Thus, t3.54mm) in the non-culprit coronary artery segment using NIRS. The median maxLCBI4mm NIRS score was 308  in the lowest quartile of antibody levels, whilst those with the highest antibody levels had a much lower score  of 133·0 , p = 0·024 for trend   . There w, −0·21) . None of4Although several studies have reported links between higher serum IgM anti-oxLDL antibody level and lower incidence of CHD, as yet we have very little knowledge of whether high levels of these antibodies relate to different plaque characteristics, or whether protection is by some other means. We have provided this link herein, first demonstrating that higher levels of IgM anti-MDA-LDL independently confer protection from CHD in a well-characterized clinical endpoint-driven population, and secondly, connecting low IgM anti-MDA-LDL antibody levels with unfavorable plaque morphology in a well-characterized intravascular coronary imaging study.The NORDIL sub-study confirms the protective effect of IgM anti-MDA-LDL on clinical CHD, which has been recognized previously , 13, 31.Moreover, we have demonstrated here for the first time, a direct inverse association between anti-MDA-LDL antibodies and intravascular imaging (both IVUS and NIRS)-derived unfavorable coronary plaque characteristics. Our ability to make this association was heavily dependent on the sophisticated intracoronary imaging techniques used for plaque characterization. Previous work using conventional angiography did not find a relationship between anti-oxLDL antibodies and overall disease burden , althougOur most striking significant finding was that low levels of IgM anti-MDA-LDL antibodies were associated with greater coronary NC volume and lipid core burden of the worst affected 4 mm segment. Surprisingly, the antibodies performed far better than lipids in this respect, as levels of HDL- and LDL-cholesterol, ApoB and oxLDL did not relate significantly to plaque characteristics. Furthermore, the significance of associations for IgM anti-MDA antibodies was not affected by adjusting the data for levels of LDL- and HDL-cholesterol.The inverse baseline association between IgM anti-MDA-LDL antibodies and size of the NC is consistent with these antibodies being directly involved in modifying plaque biology. However, although the inverse association between IgM anti-MDA-LDL antibodies and NC percentage survived adjustment for total serum IgM, other IgM antibodies are no doubt also involved. Firstly, there was a significant link between total serum IgM and NC percentage. Secondly, adjustment for total serum IgM attenuated the inverse association between IgM anti-MDA-LDL antibodies and NC volume and LCBI measured by RF-IVUS and NIRS respectively. Thirdly, in vitro and preclinical mouse studies indicate a broad role for IgM antibodies in facilitating safe clearance of debris of various types, including modified lipoproteins, apoptotic cells, cholesterol crystals and microparticles , 34.In contrast to IgM anti-MDA antibodies and total serum IgM, IgG anti-MDA-LDL antibodies and total serum IgG did not relate to plaque characteristics. In previous work we demonstrated a strong inverse relationship of total serum IgG with major adverse coronary outcomes in the primary prevention hypertensive Anglo Scandinavian Cardiac Outcomes Trial (ASCOT) population . HoweverAs was the case in the core IBIS-3 study , our subThe clinical translational value of our findings, linking antibody levels to both favorable clinical outcomes as well as favorable plaque characteristics, would be in utilization as part of focused patient selection strategies in clinical trials for novel agents that target high risk populations , 40, as 5We confirm in our study that IgM anti-MDA-LDL antibodies confer protection from the development of clinical CHD, demonstrated in a hypertensive population. Importantly, we have shown for the first time that low levels of IgM anti-MDA-LDL antibodies, and to a lesser extent total serum IgM, predict worse NC and LCBI characteristics assessed directly by intracoronary imaging. These observations not only provide much needed clinical support for the protective role of humoral immunity in atherosclerosis proposed by preclinical studies, but also point to the possible use of low IgM anti-MDA-LDL antibody levels as a surrogate marker of unfavorable plaque characteristics.All publically available data is in the article and supplementary files.


Earthquake disaster insurance can effectively reduce the impact of earthquake disasters on rural households. Exploring rural households’ willingness to purchase earthquake disaster insurance in earthquake disaster areas provides an understanding of the motivations underlying the implementation of an insurance policy. However, few studies have examined the perspectives of rural households, in order to explore the correlations between the rural households’ livelihood capital, their disaster risk perception, and their willingness to purchase earthquake disaster insurance. A cross-sectional survey data including 241 rural households from the most severe disaster counties (cities) during the 5 • 12 Wenchuan earthquake was examined with regard to rural households’ livelihood and disaster risk perception, and ordinal logistic regression models were constructed to explore rural households’ willingness to purchase earthquake disaster insurance, as well as the driving mechanism behind this willingness. The results showed that 34.44% of rural households were very willing to purchase earthquake disaster insurance, and 7.05% of rural households were very reluctant to purchase earthquake insurance. Rural households’ livelihood capital and risk perceptions were the most important factors affecting their willingness to purchase earthquake disaster insurance. Rural households with higher scores on natural capital, physical capital, possibility, and worry were more likely to purchase earthquake disaster insurance. Specifically, keeping all other variables constant, every one unit increase in nature capital and physical capital corresponded to an increase in the odds of willingness to purchase earthquake disaster insurance by a factor of 0.14 and 0.06, respectively; every one unit increase in possibility and worry corresponded to an increase in the odds of willingness to purchase earthquake disaster insurance by a factor of 0.03 and 0.04, respectively. This study contributes to the current literature by increasing the understanding of the relationships between Chinese rural households’ livelihood capital and risk perceptions, and their willingness to purchase earthquake disaster insurance. China is a mountainous country; mountains cover 73.4% of the land area and are home to 45% of the population ,2. MountCatastrophe insurance is often considered to be an important part of residents’ preparedness for disaster prevention and for the construction of the government’s resilient disaster prevention and mitigation system ,16,17,18The third stage was from 1995 to 2001. In 1995, from the perspective of controlling and preventing the operating risks of insurance companies, the supervision and management institutions of China’s insurance industry required insurance companies to suspend earthquake insurance. In 1996, the People’s Bank of China stipulated that flood, earthquake, typhoon, and other catastrophe risks would be eliminated from their basic responsibilities in the new enterprise property insurance clauses, which were implemented on 1 July 1996. At the same time, in 2000 and 2001, the China Insurance Regulatory Commission (CIRC) continuously issued circulars on earthquake insurance, pointing out that “earthquake insurance can only be regarded as an additional insurance for enterprise property insurance and must not be taken as the main insurance to be covered alone”. Since then, as a special additional insurance, only flood disasters were covered by various insurance companies, while devastating catastrophes such as earthquakes and tsunamis were not covered by insurance companies. The fourth stage was from 2008 to the present. In 2008, an unprecedented earthquake occurred in Wenchuan; there were nearly 70,000 deaths in Sichuan and direct economic losses of nearly 845.2 billion Yuan resulted from this devastating earthquake. This impelled researchers, policymakers, and insurance professionals to rethink the issue of the establishment of earthquake catastrophe insurance [Disaster insurance plays a positive role in transferring the impact of disasters on residents; for this reason, it has remained a hot topic in academic circles ,22,23,24To date, research on the factors influencing the acquisition of disaster insurance has mostly focused on the correlations between risk perception, disaster experience, individual and family socio-economic characteristics, and willingness to purchase disaster insurance ,28,30,31Furthermore, many mountain settlements in China are areas where poverty and earthquake disasters are intertwined, as a result of the impact of disasters. In addition to the assistance from the government and society, the rural households’ own capability is also an important means to resist external shocks ,38,39,40Sichuan is a typical area in China where geological disasters and poverty are intertwined. Eight earthquake belts cross the four major poverty areas in Sichuan Province . For example, the Longmenshan earthquake belt crosses Wenchuan County and Lushan County, and these two counties successively experienced the Wenchuan earthquake and the Lushan earthquake. Since the 18th National Congress of the Communist Party of China, Sichuan has made outstanding achievements in the alleviation of poverty, and the poor rural population has decreased from 7.5 million to 1.71 million. However, the remaining poor population is primarily concentrated in the four major poverty areas, where there are frequent earthquake disasters; this makes it difficult to achieve rural revitalization and economic prosperity for the whole society. In the past 10 years, earthquake disasters have occurred frequently in Sichuan, resulting in considerable losses. The 5 • 12 Wenchuan earthquake, the 4 • 20 Lushan earthquake, and the 8 • 8 Jiuzhai Valley earthquake are world-famous. Among them, the most destructive earthquake since the founding of the People’s Republic of China, the Wenchuan earthquake, was the most typical earthquake, the direct economic losses reached 845.215 billion Yuan. At the same time, there are numerous mudslide and landslide disaster points around the earthquake belts, and unlike the low frequency of earthquake disasters, these geological disasters occur frequently, which is another factor contributing to the poverty of rural households in mountain settlements. In areas at high risk of earthquake disasters, it is crucial to establish a resilient disaster prevention system, and earthquake disaster insurance is one of the core aspects of the construction of a regional resilient disaster prevention system.Based on this, this study explores the relationships between household livelihood capital, risk perception, and willingness to purchase earthquake disaster insurance among farming households. The findings of this study will inform disaster prevention policies. Generally, this study attempted to answer the following two questions:(1) What are the characteristics of willingness to purchase insurance among rural households in the Wenchuan earthquake severe disaster areas?(2) What are the specific mechanisms of the relationships between household livelihood capital, risk perception, and willing to purchase earthquake disaster insurance among rural households?Hypothesis 1 (H1).The five types of livelihood capital of rural households will be significantly related to the willingness to purchase earthquake disaster insurance; the higher the score for the five types of livelihood capital, the stronger the willingness to purchase earthquake disaster insurance among rural households.Hypothesis 2 (H2).Risk perception will be significantly related to willingness to purchase earthquake disaster insurance. Respondents with a higher perception of possibility and worry will be more likely to purchase earthquake disaster insurance, while respondents with a higher perception of controllability will be less likely to purchase earthquake disaster insurance.Hypothesis 3 (H3).Respondents’ personal characteristics and household characteristics will be significantly related to their willingness to purchase earthquake disaster insurance; however, the directions of these relationships are unknown.Based on the literature, and with consideration to the actual situation in the research area (the Wenchuan earthquake zone), the following three hypotheses were proposed:2. In this area, there were 10 most severe disaster counties (cities), 41 severe disaster counties (cities), and 186 general disaster counties (cities). As of 12:00 on 18 September 2008, the 5 • 12 Wenchuan earthquake led to a total of 69,227 deaths, 374,643 injured persons, and 17,923 missing persons, becoming the most destructive earthquake since the founding of the People’s Republic of China, and the most deadly earthquake after the Tangshan earthquake.The 5 • 12 Wenchuan earthquake occurred on 12 May 2008 (Beijing time) at 14:28:04. The surface wave magnitude, the moment magnitude, and the earthquake intensity reached 8.0 Ms, 8.3 Mw, and 11 degrees, respectively. The earthquake affected more than half of China, as well as many other Asian countries and regions. The tremor was felt from Liaoning in the north, to Shanghai in the east and to Hong Kong, Macao, Thailand, Vietnam, and Pakistan in the west. Within China’s geographical area, the severe earthquake disaster area covered an area of more than 100,000 kmThe data for this study mainly came from a questionnaire survey conducted in September 2015 in the Wenchuan earthquake’s most severe disaster areas. The investigation largely focused on the situation of rural households in 2014 in terms of the livelihood capital, disaster risk perception, and behavioral strategies to avoid disaster. Each questionnaire took approximately half an hour to complete. This study selected sample counties (cities) from the 10 most severe disaster counties (cities) during the Wenchuan earthquake, using stratified random sampling.Specifically, according to the differences in economic development levels, the 10 most severe disaster counties (cities), namely Wenchuan County and Mao County in Aba Prefecture, Beichuan County, An County and Pingwu County in Mianyang, Mianzhu City and Shifang City in Deyang, Qingchuan County in Guangyuan, and Dujiangyan City and Pengzhou City in Chengdu, were randomly divided into two groups, and one county (city) was randomly selected from each group to represent the sample county (city). Beichuan County  and Pengzhou City  were selected as the sample counties. Next, taking full account the disaster severity of the sample counties (cities), together with the differences in economic and social development levels and ethnic distribution, this study selected Leigu Town in Beichuan Qiang Autonomous County and Longmenshan Town in Pengzhou City as the sample towns  . Among tThe aim of this study was to explore the driving mechanism behind rural households’ willingness to purchase earthquake disaster insurance from the perspective of rural households’ livelihoods and disaster risk perceptions. To achieve this goal, several key variables were measured. The key variables are described below.(1) Dependent variableThe dependent variable was rural households’ willingness to purchase earthquake disaster insurance, which was measured on a Likert scale ranging from 1–5 .(2) Independent (focus) variablesThe core variables of this study were rural households’ livelihood capital and disaster risk perceptions.(a) Livelihood capital measurementThe measurement of rural households’ livelihood capital was based on the sustainable livelihoods analysis framework from the Department for International Development (DFID); the livelihood capital was divided into human capital, natural capital, social capital, financial capital, and physical capital. With consideration to the research of the authors of ,45,46,47(b) Risk perception measurementThe current study adopted the psychological measurement paradigm of Slovic , which s(3) Control variablesIn order to test the robustness of the focus variables, several control variables that reflect the characteristics of the individual respondents  and the rural households  were added to the model. For information on the definition and measurement of variables please see The dependent variable in this study was an ordered multi-classification variable , and the independent variables included category variables and continuous variables. Therefore, an ordinal logistic regression model was constructed to explore rural households’ willingness to purchase earthquake disaster insurance and the driving mechanisms underlying this willingness. The formula is as follows:In Formula (1), p < 0.1), indicating that at least one independent variable was significantly correlated with the dependent variables in each model. In subsequent analyses, we focused only on the significance of the variables in the models.As can be seen from model 1, model 3, and model 4, consistent with part of research hypothesis H1, only the natural capital and physical capital were positively related with willingness to purchase earthquake disaster insurance; the results were robust. Based on this, model 4 and model 5 were primarily used to explain the results. Although the correlations between human capital, social capital, financial capital, and rural households’ willingness to purchase earthquake disaster insurance were positive, they were not significant. Specifically, keeping all of the other variables constant, every one unit increase in nature capital and physical capital corresponded to an increase in the odds of willingness to purchase earthquake disaster insurance by a factor of 0.14 and 0.06, respectively.Similarly, consistent with part of research hypothesis H2, risk perception was an important factor affecting households’ willingness to purchase earthquake disaster insurance. Possibility and Worry were positively related with willingness to purchase earthquake disaster insurance, and the results were robust. The correlation between Controllability and willingness to purchase earthquake disaster insurance among rural households was not significant. Specifically, keeping all other variables constant, every one unit increase in possibility and worry corresponded to an increase in the odds of willingness to purchase earthquake disaster by a factor of 0.03 and 0.04, respectively. Interestingly, inconsistent with H3, regardless of the model, the relationships between the respondents’ personal characteristics and household characteristics, and the household willingness to purchase earthquake disaster insurance were not significant.China is one of the most important mountain countries in Asia and has a very important position in the world. However, China is also one of the most earthquake-prone countries in the world. The extreme environment after the earthquake may lead to an increase in the prevalence of various infectious diseases, and seriously threaten the residents’ health in the disaster areas. In order to construct a sustainable mountain society, to help the residents of the disaster areas rationally respond to extreme environmental conditions, it is of great importance to explore the protection mechanism of catastrophe insurance and its influencing factors. Based on this, this study used survey data from the Wenchuan earthquake disaster area to analyze the characteristics of rural households’ willingness to purchase earthquake insurance, and to explore the relationships between livelihood capital, risk perception, and willingness of rural households to purchase earthquake disaster insurance. Compared with the existing research, the marginal contribution of this study was that econometric models were used to reveal the mechanisms underlying the willingness to purchase earthquake disaster insurance from the perspective of rural households’ abilities and risk perceptions. There were some similarities and differences between the current research results and the existing studies.This study found that livelihood capital is an important factor that influences the willingness to purchase earthquake disaster insurance, and that the amount of livelihood capital is directly related to the strength of the rural households’ response to the earthquake shocks; rural households’ with a strong resilience can recover quickly from the earthquake disaster, physiologically  and psychologically , and all get more relief. Specifically, there was a significant correlation between natural capital and physical capital of rural households and their willingness to purchase earthquake disaster insurance. However, the correlations between human capital, social capital, and financial capital, and willingness to purchase earthquake disaster insurance were not significant. This is an interesting finding. For rural households, physical capital is a symbol of family wealth. To improve quality of life, contribute to their children’s marriages, and because of other considerations, rural households need to convert many years of family income to family houses or other consumer durables. Hence, physical capital is the property that rural households want to specially protect when an earthquake occurs; if this kind of capital is more abundant, the household will be more willing to purchase earthquake disaster insurance. Land is the ultimate guarantee for the livelihood of rural households, and many rural households who do not go out to work depend on the land for living; because of the long-term dual division of urban and rural areas in China, many migrant workers are unable to enjoy the welfare of urban residents, so even rural households that go out to work will generally choose to go back home and rely on the land for living when they are old. Therefore, land capital is immovable, but it is a livelihood asset that rural households are particularly worried about when an earthquake disaster occurs. On the other hand, compared with physical capital and natural capital, human capital, social capital, and financial capital are movable, and even if an earthquake occurs, rural households do not need to worry too much about these capitals. This may be why they were not significantly related with willingness to purchase earthquake disaster insurance. Furthermore, the current study focused on how poverty and earthquake disasters are intertwined. Rural households’ human capital and financial capital are not high, while their social capital is highly homogenous, and the insufficiency of rural households’ own capability can also lead to their lack of willingness to purchase earthquake disaster insurance. In addition, when an earthquake occurs, which is considered to be a catastrophe characterized by “small probability and great loss”, it is generally not sufficient for rural households to rely only on their own capability to cope with the impact of the disaster, rather, they tend to expect the government to cover major disaster losses.There are similarities and differences between the current results and those by the authors of ,32,33,34In this study, the correlations between the control variables and willingness of rural households’ to purchase earthquake disaster insurance were not significant. This is consistent with research ,58, as Lp < 0.1, p < 0.05, and p < 0.01 as the criteria to determine the significance of variables. In fact, in some empirical studies of psychology, in order to avoid the influence of the excessive experiment-wise error rate on the model results, scholars often use p < 0.05 and p < 0.01 as the criterion to judge the significance of variables. By this standard, Nature capital in this study will become non-significant. Meanwhile, several other significant variables in the models will also become non-robust. Therefore, in future studies, the criterion of significance of variables can be further tightened.Although this study can provide a novel contribution to the existing research, there are still some deficiencies. For example, this study only focuses on rural households’ willingness to purchase earthquake disaster insurance and the factors influencing this willingness among the residents of the most severe disaster areas during the Wenchuan earthquake; thus, it is unknown whether the research results are applicable to the severe disaster areas and the general disaster areas. Furthermore, compared with landslides, mudslides, and other mountain disasters, earthquake disasters are characterized by low frequency and high destruction. Therefore, it is also unknown whether the results and conclusions of this study are generalizable to rural households in settlements at risk of landslide, mudslide, and other mountain disasters. In future research, the scope of the research can be further expanded to explore the relationships between rural households’ livelihood capital and disaster risk perception, and their willingness to purchase disaster insurance in settlements at risk of different types of disasters and with different degrees of threat risk. Meanwhile, this study does not explore the amount of money that rural households are willing to pay for insurance, which could also be further studied in the future. Moreover, similar to most studies, this study also uses Using survey data from the most severe disaster counties (cities) during the 5 • 12 Wenchuan earthquake, ordinal logistic regression models were constructed to explore the influence of livelihood capital and disaster risk perception on rural households’ willingness to purchase earthquake disaster insurance. The results showed the following:(1) Rural households’ livelihood capitals are important factors affecting rural households’ willingness to purchase earthquake disaster insurance. Rural households with higher scores on natural capital and physical capital were more likely to purchase earthquake disaster insurance. When keeping all other variables constant, every one unit increase in natural capital and physical capital corresponded to an increase in the odds of willingness to purchase earthquake disaster insurance by a factor of 0.14 and 0.06, respectively.(2) Respondents’ risk perception is also an important factor that affects households’ willingness to purchase earthquake disaster insurance. Rural households with higher scores on possibility and worry were more likely to purchase earthquake disaster insurance. When keeping all other variables constant, every one unit increase in possibility and worry corresponded to an increase in the odds of willingness to purchase earthquake disaster insurance by a factor of 0.03 and 0.04, respectively.(3) Interestingly, regardless of the model, the relationships between respondents’ personal characteristics, household characteristics, and household willingness to purchase earthquake disaster insurance were not significant.The results of this study also have important policy implications. For example, the study found a positive correlation between livelihood capital, disaster risk perception, and willingness to purchase earthquake disaster insurance. This suggests that, when implementing earthquake disaster insurance, the government should increase the amount of insurance compensation for earthquake disasters in a moderate way, thus providing policyholders with the funds they need to replace the property that is damaged or destroyed and enhancing rural households’ perception of disasters, so as to reduce barriers to the implementation of earthquake disaster insurance in severe earthquake disaster areas and to effectively reduce property loss of rural households.


TSE, in biological samples is important to develop antemortem tests for transmissible spongiform encephalopathies (TSEs). The real-time quaking-induced conversion (RT-QuIC) assay detects PrPTSE but requires dedicated equipment and relatively long incubation times when applied to samples containing extremely low levels of PrPTSE. It was shown that a microplate shaker with heated top (Thermomixer-C) accelerated amplification of PrPTSE in brain suspensions of 263K scrapie and sporadic Creutzfeldt-Jakob disease (sCJD). We expanded the investigation to include TSE agents previously untested, including chronic wasting disease (CWD), macaque-adapted variant CJD (vCJD) and human vCJD, and we further characterized the assays conducted at 42°C and 55°C. PrPTSE from all brains containing the TSE agents were successfully amplified using a truncated hamster recombinant protein except for human vCJD which required truncated bank vole recombinant protein. We compared assays conducted at 42°C on Thermomixer-C, Thermomixer-R (without heated top) and on a fluorimeter used for RT-QuIC. QuIC on Thermomixer-R achieved in only 18 hours assay sensitivity similar to that of RT-QuIC read at 60 hours (or 48 hours with sCJD). QuIC on Thermomixer-C required 24 hours to complete and the endpoint titers of some TSEs were 10-fold lower than those obtained with RT-QuIC and Thermomixer-R. Conversely, at 55°C, the reactions with sCJD and CWD on Thermomixer-C achieved the same sensitivity as with RT-QuIC but in shorter times. Human vCJD samples tested at higher temperatures gave rise to high reactivity in wells containing normal control samples. Similarly, reactions on Thermomixer-R were unsuitable at 55°C. The main disadvantage of Thermomixers is that they cannot track formation of PrP fibrils in real time, a feature useful in some applications. The main advantages of Thermomixers are that they need shorter reaction times to detect PrPTSE, are easier to use, involve more robust equipment, and are relatively affordable. Improvements to QuIC using thermal mixers may help develop accessible antemortem TSE tests.Detection of misfolded prion protein, PrP TSE, in affected organs and tissues [C, while not necessary to sustain life, must play an essential role in TSEs in that mice and cattle genetically modified to lack PrPC cannot be infected with a TSE agent [C remain elusive. During the progression of the diseases, normal α-helix-rich PrPC is converted to PrPTSE, a β-sheet-enriched relatively insoluble isoform that resists digestion with proteolytic enzymes. The cellular events that trigger this conversion as well as the mechanism by which PrPTSE propagates in vivo are still not fully understood.Transmissible spongiform encephalopathies (TSEs) or prion diseases are rare fatal infections characterized by the accumulation of abnormally folded prion protein, PrP tissues , 2. ExprSE agent , 4. The TSE, the only known protein biomarker of TSE infectivity [Iatrogenic transmissions of TSE infections from blood and tissues of asymptomatic donors are rare but have occurred and, thus, iatrogenic TSEs pose a demonstrated risk to public health –7. Detecectivity .TSE. Both methods rely on the ability of PrPTSE to catalyze in vitro the conversion of the normal prion protein to a misfolded amyloid form of the protein [TSE (“seed”) in a sample catalyzes the conversion of purified recombinant prion protein monomers (“substrate”) into fibrils using cycles of vigorous shaking to break up fibrils and create multiple new seeding units [TSE expressed as 50-percent seeding activity (SD50) in a manner analogous to other quantal calculations of 50-percent endpoint titers derived from ratios of infected versus uninfected animals or cell cultures exposed to serial dilutions of conventional viruses [Protein misfolding cyclic amplification (PMCA ) and rea protein , 10. In ng units . The out viruses .TSE by RT-QuIC can take 40–50 hours or longer [TSE concentration in the test materials, requiring longer times to detect low levels of the abnormal protein. Thus, while the recent improvements made RT-QuIC more suitable for diagnostic applications, its use to screen samples from blood and tissue donors will likely require shorter turnaround times. Thus, further reduction of reaction times is desirable. Caughey’s group first reported using Thermomixer-R for QuIC with 263K scrapie hamster brain seed amplified in tubes and detected by Western blot [TSE in infected samples while maintaining the high sensitivity and specificity that needed for future reliable antemortem tests using blood or other readily accessible biological fluids.One practical challenge of the current RT-QuIC assay is its relatively long lag time when tested at 42°C, presumably required to initiate assembly of fibrils and to propagate them to detectable amounts. In some cases, assaying biological materials containing minute amounts of PrPr longer –15. Recer longer –19. Howeern blot . The samern blot . Other iern blot . We inveAll brain homogenate samples used in this study were repository samples collected post-mortem. Human brain homogenate samples were prepared by the UK National Institute for Biological Standards and Controls. These sample are anonymized and are WHO reference reagents. We purchased the samples. Macaque brain homogenate was produced at FDA under FDA IACUC approval #2009–14. Deer brain homogenate was from an FDA IACUC-approved protocol #2013–21. Hamster brain homogenate was from an FDA IACUC-approved protocol #2014–14.We obtained human vCJD (Ru98/148), sCJD (Ru99/009) and normal human (Ru97/03) brain homogenates from the World Health Organization (WHO) Human TSE Biological Reference Materials collection at the CJD Resource Centre, National Institute for Biological Standards and Control, Potters Bar, UK , 23. TheWe prepared brain homogenates from hamsters infected with the 263K strain of scrapie agent and uninfected hamsters. We also prepared a pool of homogenized brains from three vCJD-infected cynomolgus macaques and a control preparation of uninfected macaque brain , 25. We E. coli cells expressing the two proteins were a generous gift of Byron Caughey . Briefly, we grew bacteria with pET41 vector containing the DNA sequence for each recombinant prion protein in Luria broth medium with kanamycin and chloramphenicol. We induced protein expression with the Overnight Express Autoinduction system 1  and prepared inclusion bodies by cell lysis with Bug Buster Master Mix or Bug Buster Plus Lysonase Kit . We purified recombinant prion proteins (recPrP) from inclusion bodies on a Ni-nitrilotriacetic acid Superflow resin chromatographic column  by gradient refolding in guanidine HCl followed by gradient elution in imidazole buffer using an AKTA Fast Protein Liquid Chromatography system . We dialyzed the eluted recPrP in 10-mM sodium phosphate buffer (pH 5.8) and filtered it through a 0.22-μm syringe filter (Millipore). We determined protein concentrations by measuring absorbance at 280 nm, using 1.44 mg/ml as the extinction coefficient for the proteins and adjusted concentrations to approximately 0.6 mg/ml before storing aliquots of protein at -80°C.We performed RT-QuIC assays according to a protocol previously described  except wWe prepared serial dilutions of each infected sample in dilution buffer (0.1% SDS with 1x N2 supplement in PBS) and initiated the seeded reaction with 2 μl of sample mixed with 98 μl of cocktail in each well of a 96-well black plate. The cocktail contained recombinant PrP (Ha-recPrP or BV-recPrP) in 10-mM phosphate buffer (pH 7.4) with 300-mM NaCl, 1-mM ethylenediaminetetraacetic acid tetrasodium salt (EDTA) and 10-μM Thioflavin T dye. Typically, 0.1 mg/ml of substrate is used in RT-QuIC. We found that using 0.05 mg/ml recombinant proteins did not affect reaction times or assay sensitivities. However, detection of human vCJD brain homogenate on Thermomixer-C required BV-recPrP at 0.1 mg/ml. We tested each sample in four wells and repeated each test at least three independent times. We then incubated each plate in a FLUOstar Omega plate reader  at 42°C. We also tested selected brain samples at 55°C, shaking at 700 rpm in cycles of one min on and one min off.Two commercial thermal mixers, Thermomixers-R and C , were fitted with adaptors to accommodate 96-well microplates. The two mixers differed in that Thermomixer-C was equipped with a heated top to reduce condensation of liquid in the wells; the Thermomixer-R has an unheated top. We prepared identical microplates containing serial dilutions of infected brain homogenates as described above. We assayed each sample in four replicate wells in at least three independent experiments. We set Thermomixers to shake at 700 rpm for one min on and one min off at 42°C or 55°C. We removed the microplates from the shakers at the indicated times and read the signals in a FLUOstar Omega fluorimeter as indicated above.10 dilutions of the brain homogenates using GraphPad Prism software .We calculated the average fluorescent signals from four-well replicates for each independent experiment, reported standard error of the mean (SEM) values, and plotted relative fluorescence units (rfu) versus log50) was the calculated 50% endpoint dilution at which at least one lower dilution had 100% positive replicate wells and at least one higher dilution had no positive replicates. We considered a well to be positive if the fluorescence at the end of the reaction exceeded the cut-off value for that instrument (see below). We averaged SD50 for each experiment and reported that value with the standard deviation in Tables 50 values.Seeding activity or seeding dose relative to 2 μl of sample was calculated independently from each experiment using the Spearman and Karber method , 27; the50 calculations.To determine the background for each instrument, we assayed three replicates (in four wells each) of normal human, hamster, and macaque brain homogenates, averaged the fluorescent signals from all these negative control wells, and calculated the standard deviation. The reaction cut-off values were calculated as the background plus three-times the standard deviation per each instrument. The background for RT-QuIC reaction at 60 hours was 43,000 rfu and the reaction cut-off was 64,000 rfu. The average background value for QuIC-R was 55,000 rfu and the reaction cut-off was 85,000 rfu. The average background value for QuIC-C was 21,000 rfu and the reaction cut-off was 33,000 rfu. We used these cut-off values to establish reactive/nonreactive wells for SD10 dilution of brain homogenates. After 12 hours of original RT-QuIC, only a few wells containing the highest concentrations of scrapie brain homogenate had become reactive. The RT-QuIC signals increased with time until 48 and 60 hours when the fluorescence reached plateau and the reactions were considered complete. QuIC on Thermomixer-R reactions were completed by 18 hours; prolonging the QuIC-R assay reaction times to 24 hours did not increase sensitivity  and a FLUOstar Omega fluorimeter  at 42°C. We first compared the two platforms by parallel tests using 263K scrapie hamster brain homogenate to seed truncated hamster recombinant prion protein (Ha-recPrP) substrate. sitivity . As cont50, as a measure of assay sensitivity, on the three platforms  while SD50 for QuIC-C differed from that for QuIC-R (p = 0.018).Next, we tested brains infected with other TSE agents. We also evaluated QuIC conducted on Thermomixer-C (abbreviated QuIC-C). latforms  and conflatforms . However50 of QuIC-C with CWD deer brain was 10-fold lower than those on the other two platforms  and reached the endpoint at 24 hours. Statistical analysis of SD50 confirmed that RT-QuIC and QuIC-C were different at 8 hours (p = 0.005) as were RT-QuIC and QuIC-C at 24 hours (P = 0.0042) but the two SD50 were statistically equivalent at the endpoint. Thus, although not as dramatic as results at 42°C, the trend confirmed that QuIC-C is faster than RT-QuIC when reactions are conducted at 55°C.Next, we seeded Ha-recPrP with CWD brain homogenate assayed with RT-QuIC, and analyzed the results at 8 hours, 24 hours  reached endpoint in 18 hours, and Thermomixer-C (heated-top shaker) in 24 hours compared to RT-QuIC which required 60 hours (48 hours with sCJD). When tested CWD and sCJD agents at 55°C, QuIC-C and RT-QuIC showed shortening of seeding times; however, QuIC-C was again faster than RT-QuIC. These results demonstrated the general applicability of Thermomixers to detect multiple TSE agents in the most extensive comparative study of QuIC assays on thermal mixers to date. The FLUOstar Omega fluorimeter, commonly used for RT-QuIC tests, is a highly versatile device, but it is relatively costly and, in our experience, it is not robust—many hours of vigorous shaking wears on the microplate transport system, requiring periodic refurbishing. Some of these issues can be addressed using a Thermomixer. Other investigators have used Thermomixers at 42°C and reported faster reaction times without reduced sensitivity , 20, 21.To explain differences in reaction times with the two test platforms, we verified that temperatures in the wells of the microplates in Thermomixers and FLUOstar Omega were those selected (42°C) and we considered differences in shaking conditions. Both Thermomixers shake plates in single orbits of 3-mm diameter. FLUOstar shakes in a double-orbital movement about 1.35 mm in diameter. This difference is probably important for fibril formation, but we could not rigorously verify effects of this parameter directly. In early studies with 263K hamster brain homogenate, we found that increasing the rate of shaking on the Thermomixer from 700 rpm (our standard conditions) to 1000 rpm did not significantly improve seeding activity, consistent with previously published data .TSE only with truncated BV-recPrP substrate. Truncated BV-recPrP also performed well as substrate with 263K scrapie hamster brain and CWD deer brain suspensions, but results with BV-recPrP were less reproducible than those with Ha-recPrP substrate, especially when samples contained low levels of brain homogenate. Thus, when possible, we used Ha-recPrP as the substrate. Full-length BV-recPrP has been proposed as a universal substrate for RT-QuIC [Truncated Ha-recPrP provided a reproducible and efficient substrate with all TSE brain suspensions tested except for human vCJD brain, which amplified PrP RT-QuIC . However50 values, while reducing assay times. To our knowledge, this is the first report of Thermomixer-C tested at a temperature higher than 42°C [50 when compared to the results at 42°C  On the other hand, Thermomixers are easier to operate, require no special software, are mechanically sturdier and are cheaper than the FLUOstar Omega. Although a fluorescence plate reader is still needed to read both QuIC-R and C reactions, we conclude that it is preferable to purchase one FLUOstar Omega and several thermomixers than multiple FLUOstar instruments. Thermomixers might offer a promising platform to develop rapid QuIC assays suitable to screen donors of blood and tissues with high throughput, an application for which tracing kinetics of fluorescent signals over time is not necessary.