The purpose of this systematic review was to apply the Reach, Efficacy, Adoption, Implementation and Maintenance (RE-AIM) framework to evaluate the extent to which rural physical activity interventions in the U.S. have reported on dimensions of internal and external validity and to offer suggestions for future physical activity interventions for rural U.S. populations.Previous reviews of rural physical activity interventions were focused on intervention effectiveness and had reported overall mixed findingsn = 13), efficacy/effectiveness (n = 10), adoption (n = 21), implementation (n = 9), and maintenance (n = 8).Pubmed, PsychINFO, CINAHL, PAIS, and Web of Science were searched through February 2019 to identify physical activity intervention studies conducted in rural regions in the U.S. with adult populations. Titles, abstracts, and full texts of articles were reviewed against inclusion and exclusion criteria. Data extraction from included articles included a summary of study details, rural classification system used, and the presence or absence of a total 61 RE-AIM indicators, including reach , population/census-based definitions , Rural Urban Continuum Codes , Rural Urban Commuting Area codes , the 2014 Alabama Rural Health Association classification system  and the U.S. Office of Management and Budget classification system . Individual studies reported between 14.8 to 52.5% of total RE-AIM indicators. Studies reported 15.4 to 84.6% indicators for reach; 20.0 to 70.0% indicators for efficacy/effectiveness; 4.8 to 47.6% indicators for adoption; 11.1 to 88.9% indicators for implementation; and 0 to 25.0% indicators for maintenance.A total of 40 full-text articles representing 29 unique studies were included. Classifications of rurality included self-statements by authors (We found an overall poor reporting of components related to external validity, which hinders the generalizability of intervention findings, and a lack of consistency in the definition of rurality. Future research should focus on balancing factors of internal and external validity, and should aim to develop a greater understanding of how rurality influences health and behavior to provide contextual knowledge needed to advance the translation of physical activity interventions into practice in rural communities and reduce rural health disparities.CRD42019116308.The review protocol was registered with PROSPERO:  Rural areas cover about 97% of land in the United States (U.S.) and include about 20% of the population, or around 60 million residents . Rural rSeveral reviews have assessed the effectiveness of physical activity interventions among rural populations and have reported mixed findings –8. ResulAlthough the literature shows there are promising components to existing physical activity interventions for rural residents, previous reviews have cited mixed findings and high risk of bias among included studies as major limitations, and have thus been unable to draw strong conclusions regarding the effectiveness of physical activity interventions in rural settings –8. FurthThe RE-AIM framework can be applied to evaluate the internal and external validity of interventions , 15. TheA comprehensive review of the internal and external validity of physical activity interventions in rural populations is currently lacking. This gap in the literature is coupled with a lack of certainty regarding the effectiveness of physical activity interventions in rural populations, which may be due to the poor quality of studies and high risk of bias, which limits the ability to draw strong conclusions as shown in Cai and colleagues’ review . This la.This systematic review is registered with the PROSPERO international prospective register of systematic reviews (registration number CRD42019116308) at the Centre for Reviews and Dissemination, University of York, UK, and adheres to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) reporting guidelines . The PRI1Study inclusion criteria are described in Table The following five electronic databases were searched for articles: Pubmed , PsychINFO , CINAHL , PAIS , and Web of Science . The search was limited to original research articles published in English from each database’s inception through October 9, 2018 and updated on February 10, 2019. The search strategy was developed in consultation with a health sciences librarian and included the following search concepts: 1) rural population, rural health services, or rural health; 2) exercise, physical activity, walking, jogging, bicycling, or recreation; and 3) intervention studies, health promotion, or wellness programs . Citation details for all articles  were downloaded and loaded into a single file. Duplicate articles were identified using Endnote X9, reviewed, and removed from the database. Two coders (NB and PS) independently completed initial screening of titles and abstracts, separately. The full texts of the remaining articles were then independently reviewed against inclusion and exclusion criteria by two coders (NB and PS). The inter-rater reliability, which was calculated using Cohen’s κ, was .82, indicating a high degree of agreement . Disagren = 13), efficacy/effectiveness (n = 10), adoption (n = 21), implementation (n = 9), and maintenance (n = 8), which are described in Table A coding tool adapted from a previous systematic review using the RE-AIM framework  was usedtion n = 1, implemOur search yielded 2710 articles after the exclusion of duplicates , and 51.7% (n = 15) of studies included exclusively women. Studies ranged in duration from 5 weeks to 96 weeks . In-person intervention settings included churches , a mix of various community locations , worksites , medical practices/clinics , participants’ homes, , community or recreation centers , or were unreported . Out of the 10 interventions that were not delivered in person, a total of 6 interventions (20.7%) used telephone-based delivery, 2 interventions (6.9%) were website-based, and 2 interventions (6.9%) were SMS message-based. Classifications of rurality included self-statements by authors (with no description of a standardized classification system) , population/census-based definitions , Rural Urban Continuum Codes (RUCC) , and Rural Urban Commuting Area (RUCA) codes . Additionally, one study used the Alabama Rural Health Association 2014 classification system to define six counties in Alabama as rural [n = 20) studies reported a significant improvement in at least one physical activity, exercise, or fitness outcome, and 9 (31.0%) interventions reported no significant improvements in any physical activity, exercise, or fitness outcome.Studies included in this review are described in Additional file as rural , and oneas rural . Most  out of a total of 61  RE-AIM indicators. Studies reported 2–11 (Median = 7) indicators out of 13 indicators  for reach; 2–7 (Median = 3) out of 10 indicators  for efficacy/effectiveness; 1–10 (Median = 5) out of 21 indicators  for adoption; 1–8 (Median = 4) out of 9 indicators  for implementation; and 0–2 (Median = 0) out of 8 indicators  for maintenance. The number of indicators reported by each included article is shown in Table 2n = 29), when companion articles (n = 10) were included in the synthesis, studies (N = 40) reported 10 to 39 (16.4 to 63.9%) out of a total of 61 RE-AIM indicators. Studies reported 3–12 indicators (23.1 to 92.3%) out of 13 indicators for reach; 3–9 indicators (30.0 to 90.0%) out of 10 indicators for efficacy/effectiveness; 2–15 indicators (9.5 to 71.4%) out of 21 indicators for adoption; 1–7 indicators (11.1 to 77.8%) out of 9 indicators for implementation; and 0–4 indicators (12.5 to 50.0%) out of 8 indicators for maintenance.Compared to the number of RE-AIM indicators reported by individual studies , and Rural Urban Commuting Area (RUCA) codes . Most inHowever, we do not suggest that the solution moving forward is to select one of the existing definitions of rurality as the standard rurality classification to be used among public health researchers. Existing common definitions of rurality are based on factors such as population size, density, proximity, degree of urbanization, adjacency and relationship to a metropolitan area, principal economic activity, economic and trade relationships, and work commutes . These dThe current review makes a unique contribution to the literature on the examination of the internal and external validity of physical activity interventions in rural adults in the U.S. In a previous paper, Umstattd Meyer and colleagues (2016) assessed and outlined gaps in the evidence-base for an ecological model of active living for rural populations . While UAdditional strengths of this current review include an exhaustive search strategy, developed and conducted in consultation with a trained librarian, and well-defined enumeration of inclusion and exclusion criteria. Despite study strengths, there are limitations that should be noted. First, inclusion criteria were restricted to articles published in English and studies conducted in the U.S., and interventions targeting older adults (or study samples with a mean age > 65 years old) were excluded. Inclusion and exclusion criteria were kept similar to a previous review to aid comparisons , and futIn sum, this systematic review provides information relevant to physical activity promotion in rural populations in the U.S. The poor reporting of components related to external validity, such as adoption and maintenance, may be indicating that improving factors such as representativeness or sustained intervention at the setting and staff level are currently not prioritized among public health researchers. This may be contributing to the limited widespread dissemination of effective physical activity interventions among rural populations; therefore, we recommend that researchers focus on balancing factors of internal and external validity and reporting these dimensions rigorously. Furthermore, we encourage researchers to continue testing strategies for increasing physical activity among rural populations, given the finding that many included interventions did not improve physical activity. Lastly, while there may not be a universally-accepted or standardized definition of rurality, steps need to be taken in order to facilitate the comparison of studies across rural settings. Therefore, we encourage researchers to elucidate the concept of rurality by further exploring factors influencing the health and behavior of rural residents, and to use that contextual knowledge when selecting intervention participants and settings.Additional file 1. PRISMA Checklist. Description: Completed PRISMA checklist.Additional file 2. Database search strategies. Description: Full individual search strategies for Pubmed, PsychINFO, CINAHL, PAIS, and Web of Science.Additional file 3. Characteristics of original studies included in review. Description: The citation details, sample characteristics, location/setting, rurality classification, and summary of findings of included articles.


China has undergone rapid socioeconomic transition accompanied by lifestyle changes that are expected to have a profound impact on the health of its population. However, there is limited evidence from large nationwide studies about the relevance of socioeconomic status (SES) to risk of diabetes. We describe the associations of two key measures of SES with prevalent and incident diabetes in Chinese men and women.The China Kadoorie Biobank study included 0.5 million adults aged 30–79 years recruited from ten diverse areas in China during 2004–2008. SES was assessed using the highest educational level attained and annual household income. Prevalent diabetes was identified from self-report and plasma glucose measurements. Incident diabetes was identified from linkage to disease and death registries and national health insurance claim databases. We estimated adjusted ORs and HRs for prevalent and incident diabetes associated with SES using logistic and Cox regression models, respectively.At baseline, 30,066 (5.9%) participants had previously diagnosed (3.1%) or screen-detected (2.8%) diabetes among 510,219 participants included for cross-sectional analyses. There were 480,153 people without prevalent diabetes at baseline, of whom 9544 (2.0%) had new-onset diabetes during follow-up (median 7 years). Adjusted ORs (95% CIs) for prevalent diabetes, comparing highest vs lowest educational level, were 1.21  in men and 0.69  in women; for incident diabetes, the corresponding HRs were 1.27  and 0.80 , respectively. For household income, the adjusted ORs for prevalent diabetes, comparing highest vs lowest categories, were 1.45  in men and 1.26  in women; for incident diabetes, the HRs were 1.36  and 1.06 , respectively.Among Chinese adults, the associations between education and diabetes prevalence and incidence differed qualitatively between men and women, whereas higher household income was positively associated with diabetes prevalence and incidence in both sexes, with a stronger relationship in men than in women.The online version of this article (10.1007/s00125-019-4896-z) contains peer-reviewed but unedited supplementary material, which is available to authorised users. The prevalence of diabetes in China has increased markedly in the past few decades. The proportion of Chinese adults estimated to have diabetes was 0.9% in 1980, 2.5% in 1994 and 10.9% in 2013 [Evidence from developed countries that have completed the epidemiological transition shows that non-communicable diseases (NCDs) are initially more common in population subgroups of high socioeconomic status (SES) and then, with increasing development, become more common in lower SES groups . HoweverDetailed information about the study design, survey methods and population of the CKB has been reported previously . BrieflyEthical approval for this study was obtained from the University of Oxford, the Chinese Centre for Disease Control and Prevention and the local Centres for Disease Control and Prevention in the ten study regions. All participants provided written informed consent.We assessed participants’ SES status in two ways: (1) the highest level of school education an individual attained ; and (2) total household income in the previous year .2 and <28 kg/m2) and obesity (≥28 kg/m2) [We obtained covariates from the baseline questionnaire, including demographic characteristics , health-related behaviours , personal and family medical history . Regular active smoking was defined as current smoking on most days or more. Regular passive smoking was defined as exposure to other people’s tobacco smoke for 4 or more days per week. Regular consumption of alcohol was defined as drinking alcohol monthly or more frequently. Regular consumption of fresh fruit, vegetables and fish was defined as consumption of these foods on four or more days per week. A range of physical measurements were undertaken by trained technicians using a standard protocol and calibrated instruments, including BMI, waist and hip circumference, fat percentage and systolic and diastolic BP. Physical activity was estimated by summing the metabolic equivalent task (MET) h per day spent on work, commuting, housework and non-sedentary recreational activities . Economi8 kg/m2) .A 10 ml non-fasting blood sample was collected from study participants at baseline and fasting time was recorded. Plasma glucose levels were tested on-site using the SureStep Plus meter . Participants with plasma glucose levels from 7.8 mmol/l to less than 11.1 mmol/l were invited to return for a fasting plasma glucose test on the following day. Participants were asked at baseline: ‘Has a doctor ever told you that you had diabetes?’, with those reporting ‘yes’ defined as having self-reported previously diagnosed diabetes. Screen-detected diabetes was defined among participants without self-reported diabetes on the basis of any of: (1) random plasma glucose level ≥7.0 mmol/l and a fasting time ≥8 h; (2) random plasma glucose level ≥11.1 mmol/l and a fasting time <8 h; and (3) fasting plasma glucose level ≥7.0 mmol/l. Prevalent diabetes includes both self-reported previously diagnosed diabetes and screen-detected diabetes.http://apps.who.int/classifications/icd10/browse/2016/en).The vital status of participants was obtained periodically from local death registries based at China’s Disease Surveillance Points system, checked annually against local residential records and health insurance records and confirmed with street committees or village administrators. Incident diabetes was identified from linkage to disease and death registries and national health insurance databases, collecting details of diagnoses resulting in, or during, any hospital admission via individuals’ unique national ID. We defined incident diabetes using the E10-E14 codes from the Tenth Revision of the International Classification of Diseases (We excluded 2672 (0.5%) participants with missing, implausible or extreme values for all variables, leaving 510,219 participants for cross-sectional analyses of prevalent diabetes  was used to perform the analyses.We performed further analyses to investigate the impact of a composite measure of educational level and household income on incident diabetes. We categorised participants into four groups: (1) low educational level and low income; (2) low educational level and high income; (3) high educational level and low income; and (4) high educational level and high income. Low educational level was defined as having education at primary school level or below; low household income was defined as having annual household income <20,000 Chinese yuan. Additional sensitivity analyses were done after excluding individuals with baseline CHD, stroke or TIA, or cancer, to reduce potential reverse causality. R software    Fig.  and 4.Tan = 480,153) by SES were similar to those of people included for cross-sectional analyses (ESM Tables pinteraction < 0.0001 between sex)  , except for educational level and incident diabetes in women (p = 0.047). After additional adjustment for BMI, the associations between SES and incident diabetes were markedly attenuated and no longer statistically significant at the 5% level for any comparison between the highest and lowest SES groups  Fig. . The adjes) Fig. . The adjups Fig. . Furtherups Fig. .Table 3Spinteraction = 0.035) but not in women (pinteraction = 0.99) and was more pronounced in less economically developed geographic areas  than in more developed areas (Eastern) in both sexes (pinteraction = 0.017 in men and pinteraction = 0.010 in women) . Compared with income, educational level is probably more strongly linked to an individual’s cognitive functioning and health-promoting behaviours, and plays a greater role in the onset of diseases, while income is more strongly related to the progression of diseases . This suA key strength of this study is that it is the first nationwide study to describe contemporary associations between SES and diabetes incidence in China. The large and diverse population sample permits the investigation of potential differences in the association between SES and diabetes in settings where economic development and epidemiological transition may be at different stages. Reviews of medical records in about 1000 incident cases identified a positive predictive value of 97% based on ADA diagnostic criteria  and mediOne of the key study limitations is potential under-ascertainment of both prevalent and incident diabetes. The diabetes prevalence in the CKB was slightly lower than in contemporaneous nationally representative surveys in which diabetes diagnosis was based on a combination of self-report, fasting blood glucose, glycated haemoglobin measurement and oral glucose tolerance tests , 28. It In summary, we found that among Chinese adults in mainland China, educational level was positively associated with prevalent and incident diabetes in men, but inversely associated with diabetes in women. Household income was positively associated with prevalent and incident diabetes in both sexes. The findings of this study provide useful information for identifying priority groups for prevention of diabetes and to allow evaluation of the effect of policies that influence health inequalities in China. Health and social policies that aim to reduce socioeconomic and geographic inequalities in diabetes burden should give priority to primary and secondary prevention of overweight and obesity in Chinese adults and take account of potentially different associations with SES in men and women. Future research is needed to identify effective approaches to reducing inequalities by SES in diabetes incidence and to investigate whether SES also affects the risk of developing diabetic complications in China.ESM(PDF 1284 kb)


Multimorbidity is an emerging public health priority. Physical activity (PA) is recommended as one of the main lifestyle behaviours, yet the benefits of PA for people with multimorbidity are unclear. We assessed the benefits of PA on mortality and life expectancy in people with and without multimorbidity.Using the UK Biobank dataset, we extracted data on 36 chronic conditions and defined multimorbidity as (a) 2 or more conditions, (b) 2 or more conditions combined with self-reported overall health, and (c) 2 or more top-10 most common comorbidities. Leisure-time PA (LTPA) and total PA (TPA) were measured by questionnaire and categorised as low -min/week), moderate (600 to < 3000 MET-min/week), and high (≥ 3000 MET-min/week), while objectively assessed PA was assessed by wrist-worn accelerometer and categorised as low (4 min/day), moderate (10 min/day), and high (22 min/day) walking at brisk pace. Survival models were applied to calculate adjusted hazard ratios (HRs) and predict life expectancy differences.491,939 individuals  had a median follow-up of 7.0 (IQR 6.3–7.6) years. Compared to low LTPA, for participants with multimorbidity, HR for mortality was 0.75 (95% CI 0.70–0.80) and 0.65 (0.56–0.75) in moderate and high LTPA groups, respectively. This finding was consistent when using TPA measures. Using objective PA, HRs were 0.49 (0.29–0.80) and 0.29 (0.13–0.61) in the moderate and high PA groups, respectively. These findings were similar for participants without multimorbidity. In participants with multimorbidity, at the age of 45 years, moderate and high LTPA were associated with an average of 3.12  and 3.55  additional life years, respectively, compared to low LTPA; in participants without multimorbidity, corresponding figures were 1.95  and 1.85 . Similar results were found with TPA. For objective PA, moderate and high levels were associated with 3.60  and 5.32  life years gained compared to low PA for those with multimorbidity and 3.88  and 4.51  life years gained in those without. Results were consistent when using other definitions of multimorbidity.There was an inverse dose-response association between PA and mortality. A moderate exercise is associated with a longer life expectancy, also in individuals with multimorbidity.The online version of this article (10.1186/s12916-019-1339-0) contains supplementary material, which is available to authorized users. The average life expectancy continues to increase in most of developed countries; however, this longevity does not necessarily translate in better health . The ageTo our knowledge, only two studies to date have assessed the relationship between PA and mortality in people with multimorbidity, with inconsistent findings. The first study showed that, in people with multimorbidity, high PA levels were related to a significantly lower risk of mortality, compared to being physically inactive . ConversWe aimed to investigate the association between PA and all-cause mortality and to estimate the effects of PA on life expectancy in people with and without multimorbidity, using both subjective and objective measures of PA in a large contemporary cohort from the UK.n = 2416) and also excluded participants with no data for the self-reported PA (n = 8256), leaving 491,939 participants for analysis.This research has been conducted using the UK Biobank Resource (Application Number 14146). UK Biobank is a prospective study designed to improve the prevention, diagnosis, and treatment of chronic diseases in people aged between 38 and 73 years recruited from 22 sites across the UK. This study included 502,611 participants with baseline measures collected between 2006 and 2010 , and datThe UK Biobank collected self-reported medical information based on physician diagnosis . Three sMortality data were obtained from the National Health Service (NHS) Information Centre for participants from England and Wales and the NHS Central Register for participants from Scotland. Data for survivors were censored on 31 January 2016 for England and Wales and 30 November 2015 for Scotland.PA was measured both subjectively and objectively. Two questionnaires were used for subjectively reported (self-reported) PA: 1) leisure-time PA (LTPA), which included five activities undertaken in the last 4 weeks—walking, light DIY (do-it-yourself), heavy DIY, strenuous sports, and other exercises, and (2) a modified version of the International Physical Activity Questionnaire, which assessed total physical activity (TPA) , includi leisure-Other relevant covariates, including sociodemographic ) and lifestyle  factors, were also extracted , TPA , and objective PA . Spearman’s rank correlation was used to assess the agreement between the two continuous subjective PA measures and between the continuous subjective and objective PA measures. We used Cox proportional hazards regression, with time since baseline assessment as the start of follow-up, to model the association between PA and all-cause mortality in individuals with and without multimorbidity. Hazard ratios (HR) and corresponding 95% confidence intervals (95% CI) were calculated, and Schoenfeld’s residuals were used to verify the proportional hazards assumption. Four incremental models were fitted: model 1—unadjusted; model 2—adjusted for age and sex; model 3—additionally adjusted for sociodemographic factors; and model 4—additionally adjusted for lifestyle factors. The lowest PA group was the reference category. The calculation of years of life lost (i.e. difference in life expectancy) involved a two-step process using flexible parametric survival models with age as time scale. First, residual life expectancy was estimated as the area under the survival curve up to 100 years old, conditional on surviving at ages 45 to 100 years old . Second, the differences in years of life were calculated as the difference between the areas under two survival curves [GitHub yc244.We estimated the prevalence of multimorbidity, with the pattern of comorbidities illustrated using heat maps. Baseline characteristics stratified by multimorbidity status were summarised using numbers, proportions, and medians with interquartile range (IQR). We created an additional category for each sociodemographic and lifestyle covariate to account for missing data. For each PA measure, participants with complete PA data were included in the analyses, i.e. complete-case analysis for LTPA  or “low-impact” (e.g. hypertension and asthma) conditions. Therefore, we performed two additional analyses to account for the severity of the conditions. Firstly, we used participants’ self-reported overall health rating (UK Biobank Data-Field 2178), as a proxy of severity, and categorised participants into four groups: good health without multimorbidity (reference), poor health without multimorbidity, good health with multimorbidity, and poor health with multimorbidity; participants with missing health rating were excluded from analyses (n = 2324). Secondly, we used the 10 most common comorbidities [We grouped self-reported PA measures in tertiles and calculated HR (95% CI) and differences in residual life expectancy. Additionally, the PA measures were investigated as continuous variables using a log base of 1.1 ) to represent a 10% increase in total PA. Due to the time difference between the baseline and objective PA measurements, we further analysed the association between objective PA and mortality taking the time at objective PA assessment as the start of the follow-up. Model 1 was unadjusted, model 2 was adjusted for age at objective PA measurement and sex, and model 3 was further adjusted for ethnicity and socioeconomic status. Adjustments for other lifestyle factors were not possible as they may have changed between the baseline measurement and objective PA measurement. We also recalculated the HR and corresponding 95% CI for the association between objective PA and mortality; instead of the tertiles being based on the overall acceleration, we based the tertiles on the time accumulated above 250 mbidities  and caten = 96,622). Most participants were white (94.3%), with a median (IQR) age of 58 years (50–63). Participants with multimorbidity were mainly older compared to participants without , lived in deprived areas , were overweight or obese , had slightly lower consumption of alcohol , and spent more time in sedentary behaviour   and between both subjective and objective PA measures . When categorised into low, moderate, and high PA levels, more than double of the participants with multimorbidity were in the low PA group than in the high PA group . We found similar baseline characteristics when accounting for the severity of the conditions, top-10 comorbidity, and the subsample of participants who undertook the objective PA. Participants who completed the objective PA measurements were generally from higher affluent areas, more educated, in employment, and less likely to be current smokers or obese compared to non-participants , asthma (11.6%), cancer (8.2%), depression (5.6%), and diabetes (4.2%). The most prevalent comorbidities include chronic kidney disease and hypertension (70%), diabetes and hypertension (64%), and myocardial infarction and angina (42%)  occurred in participants with multimorbidity. Self-reported PA showed an inverse dose-response association with mortality for all participants, independent of multimorbidity status and adjustments for confounders  participants had multimorbidity. Using time since baseline assessment as the start of the follow-up, the median follow-up was 6.9 (range 3.8–9.1) years with 306 participant (0.3%) deaths. The amount of time spent walking ‘at a brisk pace for exercise’ (> 250 mg) was 4, 10, and 22 min/day in the low, moderate, and high volume PA groups. In the fully adjusted model, the risk of mortality in participants with multimorbidity was 51% lower in the moderate PA group  and 71% lower in the high PA group,  compared to the low PA group  log-transformed objective PA, (4) alternative definitions of multimorbidity, accounting for overall health rating, or top-10 conditions  lower than participants without multimorbidity; the corresponding figure at the age of 65 years was 4.54  years Fig. . In sensUsing LTPA, in participants aged 45 years with multimorbidity, moderate and high PA were associated with an average of 3.12  and 3.55  additional life years gained, respectively, compared to low PA; corresponding figures in those without multimorbidity were 1.95  and 1.85  additional life years gained, respectively  and 5.32  additional life years gained, respectively, compared to the low PA group; corresponding values for those without multimorbidity were 3.88  and 4.51  additional life years, respectively , using LTPA, showed that in participants with multimorbidity (n = 571), being physically active was related to a 35% (95% CI 16–50%) lower risk of mortality, compared to being inactive [n = 16,091) found that LTPA had a minimal effect on the multimorbidity-mortality relationship. The models were stratified by PA levels, and the number of chronic conditions was the exposure variable. Results showed that for each additional chronic condition, the association with mortality remained strongly significant, regardless of the amount of PA . However, for those who performed ≥ 8000 MET-min/month, the multimorbidity-mortality relationship was much smaller , suggesting that people with multimorbidity would gain a benefit only if they report extremely high levels of PA [Only two studies have assessed the relationship between self-reported PA and mortality in people with multimorbidity. The first study from Spain Additional file 2Table S1. Number of participants with comorbidity. Table S2. Number of participants by the total number of chronic conditions.Table S3. Most to least prevalent chronic conditions by overall health rating and multimorbidity status. Table S4. Participant characteristics by overall health status and multimorbidity. Table S5. Number of participants with the top-10 comorbidity. Table S6. Characteristics by participants with and without top-10 comorbidity. Table S7. Participant characteristics who undertook the objective physical activity by multimorbidity status. Table S8. Participants characteristics by completion of the objective physical activity measurements. Table S9. Self-reported physical activity in tertiles and years of life gained at the age of 45 years by multimorbidity status. Table S10. Association between continuously measured (log-transformed) physical activity and mortality. Table S11. Association between physical activity and mortality for participants with good or poor overall health rating and multimorbidity status, and years of life gained at the age of 45 years. Table S12. Association between physical activity and mortality for participants with and without top-10 comorbidity, and years of life gained at the age of 45 years. Table S13. Association between objective physical activity in tertiles of time accumulated above 250 mg of total acceleration and mortality. Figure S1. Most to least prevalent chronic conditions used to define multimorbidity . Figure S2. Association between self-reported physical activity in tertiles and mortality. Figure S3. Association between objective physical activity and mortality, taking the time at objective PA assessment as the start of the follow-up in participants with and without multimorbidity. (DOCX 2092 kb)


A high prevalence of periodontitis has been reported in rheumatoid arthritis (RA) patients, although the strength of this association, its temporal link and the possible relationship between the severity of periodontitis and RA disease activity remain unclear. The objective of this work was to investigate whether periodontitis is associated with RA and whether periodontitis severity is linked to RA disease activity.This case–control study included 187 patients diagnosed with RA and 157 control patients without inflammatory joint disease. RA disease activity and severity were evaluated by the Disease Activity Score 28, the Simplified Disease Activity Index, the Clinical Disease Activity Index, rheumatoid factor, anti-citrullinated protein antibody titers, the erythrocyte sedimentation rate, C-reactive protein, presence of extra-articular manifestations and type of RA therapy. Exposure severity was assessed by the following periodontal parameters: plaque index, bleeding on probing, probing pocket depth and clinical attachment levels. Sociodemographic variables and comorbidities were evaluated as confounding variables. Outcome and exposure variables were compared by both parametric and nonparametric tests, and possible associations were assessed through regression analysis with a calculation for the adjusted odds ratio (OR).p < 0.001). Compared with controls, all parameters related to periodontal status  were significantly worse in RA patients (p < 0.001). Periodontitis severity was significantly associated with RA disease activity (p < 0.001), showing in an ordinal logistic regression model an association between periodontal severity and disease activity with an adjusted OR of 2.66 .A significant association was demonstrated between periodontitis and RA with an adjusted OR of 20.57  contains supplementary material, which is available to authorized users. Rheumatoid arthritis (RA) is a systemic autoimmune chronic disease that causes inflammation and the proliferation of synovial membranes in di-arthrodial joints. When uncontrolled, RA destroys articular structures, resulting in functional disability, decreased quality of life and reduced life expectancy, mainly due to an increased risk of cardiovascular events . RA is aPorphyromonas gingivalis, a Gram-negative, anaerobic bacterium, by releasing a specific deaminase is able to induce protein citrullination, which through a process of mimicry might stimulate ACPA formation in RA patients [Periodontitis is a chronic inflammatory disease characterized by the destruction of the periodontal ligament and alveolar bone, which if untreated can lead to tooth loss. Periodontitis is considered one of the most prevalent chronic inflammatory noncommunicable diseases , 6. Recepatients , 14. In patients , 16.The objective of this case–control study was to investigate the link between RA and periodontitis, to assess whether RA disease activity is associated with periodontitis severity and to determine the degree to which this association is affected by shared risk factors.RA patients (cases) were consecutively included from those outpatients attending the Department of Rheumatology of the Hospital Universitario de Canarias in Tenerife from January to September 2016 following a design stratified by the use of biologics (see later). RA patients aged 18 years and older fulfilling the 2010 ACR/EULAR classification criteria  were incAll enrolled subjects were informed about the objectives and characteristics of this study and signed a written informed consent previously approved by the ethics committee of Hospital Universitario de Canarias.For all RA patients the following parameters were recorded: 28-joint Disease Activity Score using the erythrocyte sedimentation rate (ESR) (DAS28) or C-reactive protein (DAS28-CRP) , the SimPatients’ periodontal status was evaluated for cases and controls using the following parameters  were recorded for all patients. In RA patients, any history of therapy with glucocorticoids, synthetic disease-modifying antirheumatic drugs (sDMARDs) and bDMARDs was also recorded.Anthropometric and socioeconomic variables were also recorded including BMI, smoking status, stress by the Perceived Stress Scale (PSS-14) categorized into high stress yes > 28 points) or no (≤ 28 points) , 28 and  points oThis was an observational, case–control study of RA patients treated in a single rheumatology department who were assessed for the presence and severity of periodontal disease.Cases were subjected to a routine medical examination. The patient’s and care provider’s global assessments of disease activity were based on a 100-mm visual analog scale (VAS), and the RA functional index was assessed by the HAQ. Disease activity was calculated by means of DAS28, DAS28-CRP, SDAI and CDAI scores, and the medications used were logged from the patient files and by asking the patient during medical examination. Blood samples were tested for plasma RF and CRP using an immunoturbidimetric assay  and for ACPA , with a positive value established as that exceeding 25 U/ml in both serological tests and 3 mg/l in the CRP test.A full oral and periodontal examination was carried out by two experienced periodontists masked to the patient’s diagnosis. A kappa test showed 70% inter-examiner concordance. Queried dental visits and symptoms of oral sicca were also recorded.Full mouth PPD and CAL measurements were registered using an UNC-15 periodontal probe (six sites per tooth), excluding third molars and implants. Full mouth periapical X-ray scans were taken to confirm the periodontal diagnosis when it was not clear. Although subjects were informed on their periodontal status and advised to seek periodontal therapy when appropriate, no periodontal therapy was rendered as part of this investigation.t test and one-way ANOVA with Dunnett’s post test. The Mann–Whitney U test and Kruskall–Wallis test were used for nonparametric continuous variables and a chi-squared test for categorical variables, using Fisher’s exact test in the comparison of 2 × 2 tables with expected values < 5. The degree of relationship between the categorical ordinal variables was measured with Kendall’s tau-b correlation coefficient.For sample size calculation we used the DAS28 score as the main outcome variable, with the assumption of the difference between RA patients exposed (with periodontitis) versus nonexposed (without periodontitis) reported by Mikuls et al. . A samplp = 0.340). The linear predictor of cumulated probabilities was assessed, maintaining the sign of the thresholds and changing the sign of coefficient estimations of the different covariates [The study of the relationship between periodontitis (exposure) and RA (outcome) was carried out with a logistic regression model examining the odds ratio (OR) and 95% confidence intervals (CIs). Additionally, these values were adjusted for possible confounders (covariates) such as age, sex, sociodemographic index, annual dental prophylaxis, tobacco use, BMI and comorbidities. In RA patients, the relationship between periodontitis severity (classified as Level 0 + 1 and Level 2) and RA disease activity levels  was studied with an ordinal logistic regression model taking into account the information from the previous covariates. The coefficients of the different covariates and factors verified the test of parallel lines .In total, 380 patients were screened. Three RA patients and 12 controls decided not to participate after reading the informed consent. Twenty-one controls did not attend their appointments for periodontal evaluations. Table p = 0.001)  received steroids with a mean daily dose of 5.47 ± 4.59 mg of prednisone, for a mean time of 3.79 ± 5.09 years.Ninety-nine patients (52.94%) received sDMARD as monotherapy, mainly methotrexate (79.14%), while only 12.3% of patients received two or more sDMARDs (9.6% methotrexate and leflunomide). The remaining 56 patients (29.95%) were treated with a bDMARD. Almost half of RA patients (p < 0.001). Severe periodontitis (Level 2) was significantly higher in RA patients (44.92%) than in controls (12.1%) (p < 0.001) (Table Different degrees of periodontitis (Level 1 + 2) were observed in 97.33% of RA patients and 66.24% of patients in the control group (p < 0.001). After adjusting for possible confounders such as age, gender, socioeconomic status, annual prophylaxis, osteoporosis, smoking habit, stress, BMI, hypertension and myocardial infarction, the multivariable analysis shows an adjusted OR of 20.57 . When this association was studied with respect to periodontitis Level 1 or Level 2, the adjusted OR was 14.78 (95% CI 4.23–51.68) and 81.01 (95% CI 18.59–353.07), respectively. As is shown in Table Table p = 0.002)  compared to controls, both in absolute numbers  .Of the RA patients with high disease activity, 64% presented severe periodontitis compared with 30% of patients in remission Fig. . When RAp = 0.012), after adjusting for confounding variables (Table p = 0.48). In the linear regression analysis, statistically significant increases of 0.24 and 0.19 in the DAS28 and DAS28-CRP scores, respectively, were found for each millimeter of periodontal attachment loss in RA patients  , an association was observed between periodontitis severity and RA disease activity with respect to patients in remission, with an OR of 2.66  than patients with Level 2 periodontitis  (p = 0.0046).No association was observed between the immunological characteristics of RA (presence of RF or anti-CCP) and the presence of periodontitis. The severity of periodontitis in RA patients was not associated with the use of either sDMARDs, bDMARDs or steroids. Interestingly, when we performed a retrospective subanalysis of disease activity for the 2 years prior to the periodontitis evaluation in the 56 RA patients treated with bDMARDs, we observed that those with periodontitis Level 0 or 1 had a significantly lower mean disease activity   and pleuritis (p = 0.025). There was no association between functional disability, as measured by the HAQ score, and the presence of periodontitis in RA patients.In general, no association was found between periodontitis severity and the presence of extra-articular manifestations. Nevertheless, statistically significant differences were observed in patients with severe periodontitis compared to those without or with mild cases vis-à-vis the presence of rheumatoid nodes  and 81.01 , respectively. While initial studies did not demonstrate any association between periodontitis and RA [This study confirms a very strong and independent statistical association between RA and the presence of periodontitis. When this association was studied categorizing by periodontal severity as mild (Level 1) and severe Level 2), consistent adjusted ORs were obtained: 14.78  with respect to controls 12.1%) was found, which confirms other studies (44.96–51% vs 24.6–26%) .1% was f. Using a2% with rWe additionally observed that there was a higher number of pockets ≥ 5 mm, tooth loss and clinical attachment loss in moderate-to-high activity RA patients, compared to patients in remission and controls. Moreover, a higher prevalence of severe periodontitis was found in ERA patients compared to established RA patients (60% vs 41.45%), which is in agreement with previous reports , 40. ThiOur data support that the severity of periodontitis in RA patients is not related to the use of sDMARDs, bDMARDs or steroids. In this regard, previous studies have revealed discrepancies concerning the effects of bDMARDs on the severity of periodontitis in RA patients , 41–46. We have found no relationship between periodontitis and the presence of RF in RA patients. Although there is evidence suggesting a possible association between RF and periodontitis , 47, ourInterestingly, we observed a not previously described statistical significant association between severe periodontitis and the presence of rheumatoid nodes. This fact could be explained by the higher frequency of these complications in uncontrolled high-activity RA patients.There are both some limitations and strong points to this study. The selection of a degenerative articular pathology as a control group may have impacted the results, although other studies have also selected similar controls , 12. TheThis study has demonstrated a significant association between RA and periodontitis, and more specifically between periodontitis severity and RA disease activity. These results suggest an independent relationship between severe periodontitis and RA in spite of common shared risk factors and other confounding factors affecting both diseases. Further well-designed prospective intervention studies are needed to elucidate how periodontitis may influence the development and progression of RA.Additional file 1:Periodontal section. (DOC 25 kb)Additional file 2:Table S1. Disease activity in early and established RA patients. Table S2. Linear regression model of clinical attachment level and RA disease activity. (DOC 59 kb)Additional file 3:Figure S1. Relationship between RA disease activity assessed by different indexes and periodontitis severity. Figure S2 Periodontal parameters in patients with RA in relation to their clinical activity. (DOC 399 kb)


Tendinopathy is prevalent in athletic and many occupational populations; nevertheless, the pathogenesis of tendinopathy remains unclear. Tendon-derived stem cells (TDSCs) were regarded as the key culprit for the development of tendinopathy. However, it is uncertain how TDSCs differentiate into adipocytes, chondrocytes, or osteocytes in the degenerative microenvironment of tendinopathy. So in this study, the regulating effects of the degenerative tendon microenvironment on differentiation of TDSCs were investigated. TDSCs were isolated from rat Achilles tendons and were grown on normal and degenerative (prepared by stress-deprived culture) decellularized tendon slices (DTSs). Immunofluorescence staining, H&E staining, real-time PCR, and Western blot were used to delineate the morphology, proliferation, and differentiation of TDSCs in the degenerative microenvironment. It was found that TDSCs were much more spread on the degenerative DTSs than those on normal DTSs. The tenocyte-related markers, COL1 and TNMD, were highly expressed on normal DTSs than the degenerative DTSs. The expression of chondrogenic and osteogenic markers, COL2, SOX9, Runx2, and ALP, was higher on the degenerative DTSs compared with TDSCs on normal DTSs. Furthermore, phosphorylated FAK and ERK1/2 were reduced on degenerative DTSs. In conclusion, this study found that the degenerative tendon microenvironment induced TDSCs to differentiate into chondrogenic and osteogenic lineages. It could be attributed to the cell morphology changes and reduced FAK and ERK1/2 activation in the degenerative microenvironment of tendinopathy. Tendinopathy is common in athletes and people in many occupations involving repetitive work. It is associated with focal tendon tenderness, activity-related pain, and decrease of strength and movement in the affected position. The pathogenesis of tendinopathy remains elusive. There still is a big challenge to prevent tendinopathy and to develop effective treatment for it .Kannus and Jozsa analyzed the histological changes in tendon tissue of 891 tendinopathy patients in 1991 . It was Repetitive mechanical loading has generally been considered to be the etiology of tendinopathy . In vivoRecent discoveries confirmed that complete removal of external stress, also known as stress deprivation, could lead to the degeneration of tendons as well –14. ClinIn recent years, an extracellular matrix (ECM) from decellularized tendons was isolated, which harbors the biochemical cues of tissue microenvironment including growth factors, proteins, glycosylaminoglycans, and proteoglycans. Besides, they maintain the native ultrastructure and mechanical strength of the tendon tissue to the greatest extent, which provide ideal scaffolds for in vitro study , 18, 19.g for 15 minutes to obtain cell pellets. After cell counting, the cells were inoculated in a 25 cm2 culture flask with the density of 500 cells/cm2 in DMEM supplement with 10% FBS, 100 U/ml penicillin , and 100 mg/ml streptomycin . Approximately 10 days later, TDSC colonies were formed and were isolated by local digestion with 0.25% trypsin .Approval was obtained from the Institutional Animal Care and Use Committee of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, prior to performing the study. Achilles tendons were harvested from Sprague-Dawley rats  . After the tendon sheaths were removed, the core portions were minced into small pieces. Then the pieces were digested with 4 mg/ml dispase  and 3 mg/ml collagenase I  in phosphate-buffered saline (PBS)  solution for 3 hours at 37°C. After adding Dulbecco's Modified Eagle's Medium (DMEM)  supplemented with 10% FBS , cell suspension was centrifuged at 300To verify the stemness of the cells, TDSCs at passage 2 were incubated with FITC-CD44 (1 : 1000) , FITC-CD90 (1 : 8000) (BioLegend), FITC-CD45 (1 : 1000) (BioLegend), and PE-CD106 (1 : 1000) (BioLegend) in the dark at 4°C for 20 minutes. After being washed three times with PBS, TDSCs were analyzed by a BD FACSCanto II flow cytometer .2 density, and adipogenic, chondrogenic, and osteogenic induction media  were added. The media were changed every other day. Two weeks later, TDSCs were fixed with 4% paraformaldehyde  at room temperature for 15 minutes. After being washed with deionized water, cells were stained with Oil Red O , Safranin O (Solarbio life science), and Alizarin Red S (Solarbio Life Science). The stained cells were verified by inverted light microscope .TDSCs were inoculated in a six-well plate with 500 cells/cm2 in air for up to 4 weeks. The medium was changed every other day. The stress-deprived tendons were collected as degenerative tendons hereafter in this study.Rat Achilles tendons were harvested and maintained under stress-deprived conditions, based on a previously reported study with some modifications . In brieμM calcein AM and 4 μM ethidium homodimer-1 at 37°C for 0.5 h. After being washed with PBS, the cells within the depth of 70 μm of the tissue surface were observed using confocal laser scanning microscopy .Normal and degenerative tendons were stained with live/dead staining working solution  composed of 2 μm thickness and stained with hematoxylin and eosin  to observe cell morphology and cell number. Histological samples were imaged using an upright microscope .Normal and degenerative tendons were fixed with 4% paraformaldehyde, dehydrated with 30% sucrose and embedded with OCT , and frozen at −80°C before section. Cryosections were cut at 5 μm. After removing the OCT by washing with PBS 3 times for 30 minutes, tendon slices were frozen in liquid nitrogen for 2 minutes and thawed in normal saline for 10 minutes at 37°C for 5 cycles. Then the tendon slices were incubated in 200 μg/ml RNase and 400 μg/ml DNase  for 4 hours at 37°C, followed by washing with PBS at room temperature with gentle agitation. Finally, each side of the DTSs was sterilized with UV radiation for 2 hours for further usage.DTSs were prepared according to the published protocols with some modifications . In brieNormal and degenerative DTSs were incubated with DAPI at room temperature for 15 minutes, followed by washing with PBS 3 times. The cell nucleus was observed by an inverted fluorescence microscope . Total DNA of normal and degenerative DTSs was extracted by TIANamp Genomic DNA Kit  following the manufacturer's instructions. DNA content was measured with an ultraviolet spectrophotometer .μm glued onto the V-shaped silicon-nitride cantilevers with a nominal spring constant of 0.06 N/m were employed. Cyclic load-displacement curves were recorded at 6 different sites on the sample surface. In all cases, the DTSs were immersed into pH 7.4 PBS solution.The elastic modulus of normal and degenerative DTSs was measured by atomic force microscope (AFM) . Oxide-sharpened, square-based pyramidal silicon-nitride tips with a borosilicate glass sphere at a radius of 5 2. At selected time points, TDSCs were incubated with DMEM plus 10% (v/v) CCK8 at 37°C for 2 h, then 200 μl supernatant was used to measure the absorbance. The absorbance at 450 nm with a reference wavelength of 630 nm was recorded using a microplate reader .Cell viability was detected using a Cell Counting Kit-8 (CCK8) assay , according to the manufacturer's instructions. Briefly, TDSCs were inoculated on normal and degenerative DTSs with a density of 1000 cells/cmTDSCs on normal and degenerative DTSs were fixed with 4% paraformaldehyde at room temperature for 15 minutes, followed by washing with PBS 3 times, then treated with PBS containing 0.1% Tween-20 (Sigma) and 1% goat serum (Thermo) for 15 minutes, and washed with PBS 3 times. Finally, the TDSs were incubated with Flash Phalloidin Green 488 (1 : 20) (BioLegend) and DAPI (Sigma) at 4°C in the dark for 30 minutes. Photos were taken by an inverted fluorescence microscope .T method (2T−ΔΔC).Total RNA was isolated using an RNA purification kit  according to the manufacturer's instructions. Reverse transcription (RT) was performed using a PrimeScript RT Reagent Kit . Real-time polymerase chain reaction (PCR) was carried out with SYBR Premix Ex Taq  (Takara). PCR amplification and fluorescence detection were performed using a LightCycler® 96 System . The primers used in this study were listed in Table S. GAPDH was used as an internal control. The results were presented as the calculated comparative expression ratios of the target sample to control group for each sample using the Cβ-actin (Santa Cruz Biotechnology) primary antibodies overnight at 4°C. After being washed with TBST (Keygentec), the PVDF membranes were incubated with secondary antibodies  at room temperature for 30 minutes, followed by washing with TBST. Finally, the PVDF membranes were covered with 3,3-diaminobenzidine (DAB) for the display of specific protein bands.TDSCs were lysed in lysis buffer containing protease and phosphatase inhibitors . Protein concentration was quantified by BCA kit (Keygentec), and an equal amount of protein was loaded in each lane. Constant voltage electrophoresis was carried out with 10% polyacrylamide gels. Then the proteins were transferred to polyvinylidene fluoride (PVDF) membranes . PVDF membranes were blocked with 3% BSA (Sigma) and hybridized with ERK1/2 , phospho-ERK1/2 (Thr202/Tyr204) (Santa Cruz Biotechnology), FAK (Santa Cruz Biotechnology), phospho-FAK (Tyr397) (Santa Cruz Biotechnology), and t-tests. Differences among more than two experimental groups were evaluated by one-way ANOVA. Differences were considered significant at P < 0.05.All individual experiments were performed at least three times, with three replicates. Data were expressed as means ± standard deviation (SD). The significance of differences between two groups were determined using unpaired Student's TDSCs, from rat Achilles tendon, showed cobblestone-shaped morphology  and had The degenerative tendon was produced by stress-deprived culture of rat Achilles tendons ex vivo for 4 weeks. Live/dead staining showed that almost all cells have good viability within a fresh tendon ; howeverA repetitive freeze/thaw procedure with nuclease treatment on normal and degenerative tendon tissues was applied to remove the resident cells and to produce extracellular matrix scaffolds of normal and degenerative tendons. After decellularization, the number of cells in normal Figures  and degeThe compressive elastic modulus can be used to represent the matrix stiffness of tendon tissue. The matrix stiffness of degenerative DTS was 20.98 ± 9.57 kPa, significantly lower than that of normal DTS (36.79 ± 7.39 kPa) , which iDuring the culture period of 14 days, TDSCs were well attached to the surfaces and maintained good viability on normal Figures  and degeF-actin staining was performed to investigate the alteration of TDSC cytoskeleton organization between normal and degenerative DTSs. TDSCs were aligned, with disorganized actin filaments on normal DTSs Figures . HoweverIn order to understand the effects of degenerative tendon microenvironment on the differentiation of TDSCs, the expressions of tenogenic, chondrogenic, and osteogenic markers were detected. Tenocyte markers, COL1, SCX, and TNMD, were highly expressed on normal DTSs than on degenerative DTSs, especially COL1 and TNMD . On the β1, FAK, and ERK at the mRNA level was lower on degenerative DTSs than on normal ones, and the differences of integrin β1 and FAK were significant  plays an important role in mediating mechanotransduction pathways. It mediates the interactions between cell-cell and cell-matrix. Furthermore, FAK affects intracellular signals through the extracellular signals. Extracellular signal-regulated kinase 1/2 (ERK1/2) is a well-known downstream signaling molecular of FAK and a force-activated protein kinase . The FAKThe morphology of cells was determined by the cytoskeleton, a structure subject to extracellular stress. The cytoskeleton connects with the ECM through focal adhesions (FA). Therefore, the actin cytoskeleton is continuously stabilized and destabilized by FAK-regulated processes. Several studies showed that when ADSCs or MSCs grew on substrates of different stiffness, the spreading area increased and more F-actin stress fibers formed on the substrate with higher stiffness , 28. NevMechanical loading constantly acts on tendons under physiological conditions. The differentiation of TDSCs toward tenocytes and nontenocytes was reported to be affected by the mechanical loading. When loaded with a 4% stretch, TDSCs highly expressed tenocyte-related markers COL1 and TNMD, while nontenocyte markers LPL, Sox9, Runx2, and Osterix were not affected [Finally, besides the biophysical cues, the degenerative tendon experienced a series of biochemical changes, including the growth factor or ECM protein loss during the stress-deprived culture period. Tuan et al. found that the soluble fraction of tendon ECM could enhance the differentiation of ADSCs into tenogenic lineage , which iIt is important to understand the role of TDSCs in the pathogenesis of tendinopathy and the mechanisms contributing to their erroneous differentiation for the management of tendinopathy. This study demonstrated that TDSCs undergo erroneous differentiation toward chondrogenic and osteogenic lineages on degenerative DTSs. The underlying mechanism might be attributed to the degenerative microenvironment with the decrease of matrix stiffness, which resulted in the change of cell morphology and the reduced activation of FAK and ERK1/2.


P = 0.008). BS adds significant survival benefits in addition to TKIs and WBRT, especially for patients with EGFR-mutant NSCLC and the largest BM over 1 cm.The role of brain surgery (BS) on the survival of patients with non-small-cell lung cancer (NSCLC) and brain metastases (BM), particularly those with epidermal growth factor receptor (EGFR) mutations under tyrosine kinase inhibitors (TKIs) is yet to be defined. We aimed to investigate whether BS could improve the survival of patients in addition to the combination of TKIs and whole brain radiotherapy (WBRT). A cohort of 1394 NSCLC patients between 2011 and 2016 was retrospectively studied. One hundred patients with BM receiving TKI + RT were enrolled. Forty patients (40%) received TKI + BS + RT, and 60 patients (60%) received TKI + RT. Survival time was calculated from the date of BM diagnoses to the date of death or last follow-up. With a median follow-up of 25.6 months , the median survival after BM was 18.2 months  in the TKI + BS + RT group and 11.8 months  in the TKI + RT group. Cox proportional hazards regression model for the patients with the largest BM over 1 cm showed that TKI + BS + RT group was associated with improved survival relative to TKI + RT group (HR, 0.49; 95% CI, 0.29 to 0.83;  Lung cancer remains lethal in all nations: 5-year OS is below 20% everywhere in Europe, in the range 15–19% in North America, and as low as 7–9% in Mongolia and Thailand2. The latest U.S. study utilized the National Cancer Data Base to identify 457481 patients with non-small-cell lung cancer (NSCLC) diagnosed between 2010 and 2012. The median and 1-, 2-, and 3-year OS for these patients with BM were 6 months and 29.9%, 14.3%, and 8.4% respectively3.Thirty years ago, the median overall survival (OS) after a diagnosis of brain metastasis (BM) for patients with lung adenocarcinoma was 73 days5. However, a U.S. study of 2142 patients with stage I to IV NSCLC found that EGFR mutations in tumors from ever smokers represented 40% of all mutations detected and those from men represented 31%6. A prospective study of 1482 patients confirmed the EGFR mutation frequency of 51.4% overall in tumors from Asian patients with adenocarcinoma7. Now it is mandatory to detect EGFR mutation prior to treatment. EGFR tyrosine kinase inhibitors (TKIs) have shown a response rate of 70–80% with improved progression-free survival (PFS) and OS than those obtained with standard chemotherapy in patients harboring EGFR mutations8. The use of any first- or second-generation EGFR-TKIs alone for the treatment of intracranial involvement in patients with EGFR mutant-positive lung adenocarcinoma showed a favorable cerebral response rate of more than 50%10.In the epoch of target therapies, screening for specific mutations to guide treatment is necessary. The predictive factors for epidermal growth factor receptor (EGFR) mutations are the female sex, never-smoker status, adenocarcinoma histology, and East Asian racial origin11. With the evolution of sophisticated radiation technology, most clinicians integrate radiotherapy (RT) into the comprehensive treatment of patients under TKIs in order to maximize the therapeutic effects13. The life expectancy in EGFR-mutant patients has been significantly prolonged16. Because of an aging population and advances in the treatment of NSCLC, patients are living longer and are more likely to experience distant metastases. A recent Taiwanese study reported that EGFR mutation was a predictor for subsequent BM17. Patients with EGFR-mutated NSCLC have a higher cumulative incidence of BM18. They can be surgical candidates because they have already demonstrated the proclivity to longevity despite their cancer diagnoses.The optimal treatment of BM is debatable. Classic therapeutic options include local therapies such as whole-brain radiation therapy (WBRT), stereotactic radiosurgery and surgical resection, EGFR-TKIs, and chemotherapy. Systemic chemotherapy is considered futile in the treatment of intracranial involvement due to the blood-brain barrier (BBB), which includes efflux pumps on brain capillaries19. However, improvement in neurosurgical techniques such as microneurosurgery, use of neuronavigation, intraoperative imaging, and cortical and subcortical mapping, along with concurrent progress in neuroanesthesia, has substantially decreased surgical morbidity and mortality20. Surgical resection remains an important tool for treating BM from NSCLC, particularly in patients with one large or symptomatic lesion21. Patients who received TKIs after a diagnosis of stage IIIB or IV lung cancer and WBRT were reimbursed by the Bureau of National Health Insurance of Taiwan22. Herein, we analyzed the factors affecting the prognosis for the patients with EGFR-mutant NSCLC BM under both TKIs and WBRT. The efficacy of brain surgery (BS) will be scrutinized in this research article.Some may consider novel targeted agents  are potential alternatives to surgical resection since the survival benefit of surgery seems limited to the subgroup of patients with controlled systemic disease and good performance statusThe present study (KMUHIRB-E(II)-20180185) was approved and conducted under compliance of the Institutional Review Board (IRB) regulations of Kaohsiung Medical University Hospital. All patients provided written informed consent prior to RT and/or BS. Patient information was anonymized and de-identified before analysis. All data were analyzed anonymously and retrospectively.We sorted 1394 NSCLC patients in the data base of a tertiary university hospital and retrospectively recruited one hundred consecutive patients with pathologically proven lung adenocarcinoma who had received both WBRT and TKIs between January 1, 2011 and June 14, 2016. Their BM was diagnosed by either brain imaging or cytology. The inclusion criteria were positive EGFR mutations, the diagnosis of BM, the use of TKIs, and WBRT. The exclusion criteria were a history of malignancies other than lung cancer, prior brain irradiation, or EGFR-TKI resistance mutation, or incomplete WBRT.23. All patients started taking EGFR-TKIs once the diagnosis of stage IIIB or IV lung cancer with EGFR mutation was established. Some patients received BS which was performed prior to WBRT. BS was recommended at the discretion of neuro-surgeons after discussion with each patient. All patients with or without BS had WBRT. For WBRT, three-dimensional conventional radiotherapy was done by a 2100 C/D linear accelerator . Boost plans were generated by intensity-modulated radiotherapy either with an Eclipse, version 8.6  or Hi-Art helical tomotherapy unit, version 2.2.4.1 . Our RT schedule was 30 Gy in 10 fractions or 37.5 Gy in 15 fractions. Some patients had a boost to BM of 45 Gy in 10 fractions, or 45 Gy in 15 fractions. The decision whether to boost BM was made after discussion with each patient.All the patients underwent pretreatment workups comprising a physical examination, a history review, chest radiography, bronchoscopy with a tumor biopsy, chest computed tomography (CT), brain magnetic resonance imaging (MRI) or CT, and routine laboratory studies. The tumor stage was classified according to the seventh edition of the American Joint Committee on Cancer (AJCC) Cancer Staging Manual and Handbook24.The following variables were collected: age, sex, stage, initial clinical Tumor and Nodal classification, extracranial metastases, histological grading, smoking history, EGFR mutation, Eastern Cooperative Oncology Group (ECOG) performance status at the time of BM, number of BM, size of largest BM, whether the patient was symptomatic from BM, mean dose of ionizing radiation delivered, name of EGFR-TKI, number of lines of TKI, mean duration of TKI use, and number of lines of chemotherapy. The date of initial cancer diagnosis, the date of BM diagnosis, RT treatments, systemic therapy, most recent follow-up, and death were documented. In addition, a disease-specific Graded Prognostic Assessment (ds-GPA) was calculated for each patient to determine whether the cohorts shared similar prognostic featurest-test or chi-squared test. Besides, we performed univariate analyses and a multivariate Cox proportional hazards regression to examine all collected variables. We calculated the estimated risks of death using hazard ratios (HR) with 95% confidence intervals (CIs). The level of statistical significance was set at P < 0.05; all reported P values were two-tailed. The analyses used the SPSS software package, version 19.0 for Windows .The primary end point was the survival after a diagnosis of BM was established. We calculated the survival from the date of BM diagnoses to the date of death from any cause or until the date of the last follow-up. And then we assessed the survival after a diagnosis of BM by Kaplan–Meier methods and used the log-rank test to compare time-to-event distributions. We stratified the data set and compared outcomes by P = 0.001). Patients who received BS were more likely to have EGFR mutation in exon 19  and were less likely to have EGFR mutation in exon 21 . One patient has EGFR mutation in both exon 19 and 21. There was no significant difference in terms of age, gender, stage, initial clinical Tumor and Nodal classification, extracranial metastases, histological grading, smoking history, ECOG performance status at the time of BM, number of BM, whether the patient was symptomatic from BM, mean RT dose, number of lines of TKI, mean duration of TKI use, ds-GPA and number of lines of chemotherapy .All patients started having EGFR-TKI  once the diagnosis of stage IIIB or IV lung cancer with EGFR mutation was established. Twelve patients had afatinib; 57 patients had erlotinib; 64 patients had gefitinib; and 5 patients had osimertinib. Thirty-seven (37%) patients had more than one line of TKIs due to disease progression or intolerance of side effect. The median duration of TKIs use was 14.4 months . The median duration of TKIs use was 14 months  in the TKI + BS + RT group and 14.4 months  in the TKI + RT group. The mean duration of TKIs use were 18 ± 14 months and 20 ± 18.5 months for patients with and without BS respectively (P = 0.026).After a median follow-up of 25.6 months (95% CI 18.6 to 35.7), the median survival after BM was 15.1 months  for the 100 patients in this study. The median survival after BM was 11.2 months  for the 147 patients irrespective of treatment. Specifically, the median survival after BM was 18.2 months  in the TKI + BS + RT group and 11.8 months  in the TKI + RT group. The mean survival after BM were 21.9 ± 14.8 months and 15.6 ± 14.5 months for patients with and without BS respectively , exon 19 mutation (P = 0.048) and single BM relative to more than 3 BM (P = 0.01) were associated with improved OS (Table P = 0.134). EGFR mutation in exon 19 was not an independent favorable prognostic factor . Table P = 0.017) and single BM relative to more than 3 BM .Univariate analysis suggested that BS was a favorable prognostic factors for longer survival . Clinical nodal classification 0–1 relative to 2–3  and the use of erlotinib  were also beneficial.In order to identify potential differences in the benefits of BS in patients by the size of largest BM, we selected 72 patients with the largest BM larger than 1 cm. Thirty-six (50%) patients had BS. Cox regression analysis revealed that BS was a strong favorable prognostic factor for longer survival . Female  and single BM relative to more than 3 BM  were two independent favorable prognostic factors.In order to identify potential differences in the benefits of BS in patients with EGFR mutation, we selected 85 patients with mutation in exon 19 or 21 or both. Thirty-four 40%) patients had BS. Cox regression analysis revealed that female, BS and single BM were favorable prognostic factors for longer survival . In addition, the rate of cerebral recurrence was distinctly higher in the non-WBRT group29.The mainstay of treatment for BM consisted of surgical resection, RT, or a combination of these modalities. A prospective randomized study showed that patients with cancer and a single BM who received BS plus RT lived longeret al. concluded that the survival of NSCLC with synchronous solitary M1 was more similar to stage III than other stage IV NSCLC and advocated for BS30. In this large retrospective study of 4832 patients, 64% of patients had BM. Operation conducted at both primary and metastatic sites  was an independent prognostic factor for longer survival. For accessible tumors with diameter of more than 3 cm, BS still carries the advantages of obtaining histological diagnosis, providing immediate symptomatic relief by removal of local mass effect and source of edema, and decreasing the length of steroid use21.Toffart et al., however, EGFR-TKIs were not available28. In the era of EGFR mutations and TKIs, we seek to compare survival trends that are likely to be attributable to combined treatment, especially BS plus TKIs and WBRT. We investigated one hundred patients who received TKIs as a first-line therapy for advanced EGFR-mutant NSCLC in a tertiary cancer center. Because the administration of EGFR-TKI has limited penetration across BBB, combined RT would provide better outcome31. According to Burel-Vandenbos et al., BM occurring during the course of TKI, despite good control of extracranial disease, is possibly due to insufficient concentration of TKI in cerebral spinal fluid32 although raising TKI doses might increase the possibility of drug intolerance. Soon et al. conducted a meta-analysis from 2008 to July 2014, and reported there was a better 2-year OS  for patients with upfront WBRT compared with TKI alone33.At the time of the studies of Patchell 32, therefore combination therapy is required. We hypothesized that BS would be beneficial prior to WBRT. After controlling for significant covariables in a multivariable model, EGFR mutation in exon 19  or the TKI + BS + RT group was not associated with improved survival relative to the TKI + RT group . However, for the 72 patients with the largest BM over 1 cm, multivariate analysis showed that the TKI + BS + RT group was associated with improved survival relative to the TKI + RT group . For the majority of the patients with EGFR mutation in exon 19 or 21, Female and single BM relative to more than 3 BM were two strong independent favorable prognostic factors.However, NSCLC is a radioresistant malignancy and 30 Gy of WBRT may not be sufficient to sterilize the metastatic brain lesionsThe present study demonstrated the advantage of BS, especially for patients with stage IV EGFR-mutant NSCLC who had the largest BM over 1 cm. To the best of our knowledge, this is the first study to confirm the advantage from combination therapy of TKI + BS + RT. Until recently, there was no prospective randomized trial regarding the addition of BS to TKIs and WBRT.The limitations of the current study are the inherent biases in retrospective studies. The pitfalls include limited patient numbers, possible selection bias from surgical intervention, incomplete records of post-operative complications, cognitive evaluation and intracranial control. This study enrolled a real-world population of NSCLC patients, including sicker patients who were not eligible for a clinical trial. Although the patients were not randomized, fundamentally, similar characteristics existed between TKI + BS + RT and TKI + RT groups. In the current study, we focused on prognostic features, molecular markers, and survival change from the addition of BS to the combination of TKIs and WBRT.BS prior to WBRT adds significant survival benefits in addition to the combination of TKIs and WBRT, especially for patients with EGFR-mutant NSCLC who had the largest BM over 1 cm. This observational study on BS outcome needs to be interpreted with some caution because there are potential confounding factors. The result should be cautiously applied. Further prospective study is warranted.


Capsular warning syndrome (CWS) is a rare clinical syndrome, which is defined as a recurrent transient lacunar syndrome. The mechanism and clinical management of CWS remain unclear. The aim of the study was to discuss the clinical characteristics of CWS and evaluate the different outcome between rt-PA and no rt-PA therapy.January 2013 and December 2018. The clinical characteristics of CWS were analyzed. Patients were divided into two groups: rt-PA and no rt-PA groups. The therapeutic effects and prognosis of these two groups were analyzed. A good prognosis was defined as 3-month modified Rankin Scale (mRS) ≤ 2.The present multicenter retrospective study involved three medical centers, and the clinical data were collected from patients with CWS between P > 0.05). A good prognosis was observed in 61 (84.72%) patients after 3 months, in which 23  patients were from the rt-PA group and 38  patients were from the no rt-PA group (P > 0.05). After 3 months of follow-up, two patients had recurrent ischemic stroke.Our study included 72 patients, 27 patients were assigned to rt-PA group, 45 in no rt-PA group. Hypertension and dyslipidemia were the most common risk factors. The mean number of episodes before irreversible neurological impairment or the symptoms completely disappeared was five times (range: 3–11 times). A total of 58 (80.55%) patients had acute infarction lesions on the diffusion weighted imaging (DWI). The most common infarct location was the internal capsule , followed by the thalamus and pons. The difference in therapeutic effects between the rt-PA, single and double antiplatelet groups was not statistically significant (The most effective treatment of CWS remains unclear. Intravenous thrombolysis is safe for CWS patients. Regardless of the high frequency of infarction in CWS patients, more than 80% patients had a favorable functional prognosis. The term ‘Capsular Warning Syndrome’ (CWS) was first proposed in 1993 by Donnan et al. This was defined as having at least three stereotyped episodes of motor lacunar syndrome (MLS) or sensorimotor lacunar syndrome (SMLS) within 24 h, with complete recovery between episodes, which involved two of three body parts  or more, without cortical symptoms ), numbers (%), or mean ± standard deviation (SD), and categorical variables were expressed as frequencies and percentage. Second, patients were classified into two groups: no rt-PA group and rt-PA group. Then, the demographic characteristics and prognosis were compared between these two groups in the univariate analysis. Continuous variables were compared using Student’s The EMRs of 4213 patients were retrospectively reviewed. Among these patients, 3370 patients had AIS and 843 patients had TIA. The EMRs were manually reviewed to identify patients affected by CWS. After reviewing the patient’s history, clinical characteristics, risk factors and neuroimaging, it was found that 417 AIS patients had fluctuating presentations, while 171 TIA patients had fluctuating presentations . The clinical characteristics of CWS patients are presented in Table 00 g/day.The median NIHSS score at admission was 8 (range: 2–12). A total of 59 patients (81.94%) had elevated blood pressure when they were admitted to the stroke ward, and their mean systolic pressure was 167.75 mmHg. Furthermore, 32 (44.44%) patients had pure motor syndrome, 24 (33.33%) patients had sensory motor syndrome, 12 (16.67%) patients had pure sensory syndrome, and 4 (5.56%) patients had ataxic syndrome. The mean number of episodes before irreversible neurological impairment or the symptoms completely disappeared was five times (range: 3–11). The mean duration of episodes was 24 min (range: 2–50). After CWS, permanent neurological impairment occurred in 52 (72.22%) patients, and the symptoms completely disappeared in 20 patients (27.78%).A total of 28 (65.28%) patients had hyperlipidemia on admission. The carotid artery ultrasonography revealed that 41 (56.94%) patients had mild carotid artery plaque. All patients were subjected to brain computed tomography (CT) and 24-h electrocardiogram (ECG) monitoring on admission. No arrhythmia was found during the ECG monitoring, and the echocardiography did not reveal any cardiac embolic sources in these patients. The cranial CT revealed no acute cerebral infarctions on admission. The intracranial and extracranial vasculature were evaluated through computed tomography angiography (CTA) in all patients, which was conducted within 5 days after admission. The cranial CTA revealed no severe stenosis or dissection in all patients.A brain magnetic resonance imaging (MRI) was performed at 12–48 h after admission for all patients, and a total of 58 patients had an acute infarction on the diffusion-weighted imaging (DWI). All the ischemic lesions found in the MRI were subcortical, the size of the lesion was < 15 mm, and the most frequent infarct area was the internal capsule , while other locations were in the thalamus , pons , midbrain , and striatum .P > 0.05). There were no significant group differences in the percentage of hypertension, diabetes and hyperlipidemia (P > 0.05).A total of 27 (37.50%) patients were treated with rt-PA on admission, these patients were treated with antiplatelet therapy (aspirin or clopidogrel) after 24 h, when hemorrhage was excluded by the CT examination. A total of 45 patients were treated with antiplatelet therapy, among these patients, 30 patients were treated with 300 mg of clopidogrel, followed by 75 mg/day of the same, and aspirin (100 mg/day) plus high-dose atorvastatin (40 mg/day), 12 patients were treated with 200 mg of aspirin plus high-dose atorvastatin, and three patients were treated with clopidogrel plus high-dose atorvastatin on admission. The baseline characteristics of patients in the rt-PA group and no rt-PA group were compared . Furthermore, 10 patients in the no rt-PA group had a complete recovery . Moreover, 36 patients in the no rt-PA group had acute infarctions in the DWI . The patients who receive rt-PA therapy appeared to have a higher percentage of complete recovery, but the difference in therapeutic effects among the rt-PA, single and double antiplatelet groups were not statistically significant  had a good outcome after 3 months. Among these patients, 23 patients were from the rt-PA group, and 38 patients were from the no rt-PA group . Furthermore, 2 patients had recurrent ischemic stroke . Compared with the baseline, the NIHSS score after 3 months decreased in both two groups. There was no significant difference in the 3-month prognosis between the two groups  patients after 3 months. Among these patients, 23 patients  were from the rt-PA group and 38  patients were from the no rt-PA group. There was no difference in functional outcome at 3 months between the rt-PA and no rt-PA groups. In general, intravenous thrombolysis is safe for CWS patients, and no bleeding complications have been reported. Therefore, intravenous thrombolysis is an alternative treatment for CWS within 4.5 h after the onset of symptoms. Due to the small number of patients, the effect of rt-PA in CWS must be further confirmed in clinical studies.Some limitations of the present study merit consideration. The present study updates some information about CWS. Since the present study was a multicenter and retrospective study, CWS is a rare clinical disease, and the number of included patients was small, these may have affected the results of the present study. A total of seven patients were followed up by face-to-face interview. The recollection of these patients on the functional outcome after 3 months of onset might lead to some recollection bias. In the future, there may be a need to further expand the number of patients and further explore the mechanism of CWS, in order to find the best treatment.In conclusion, CWS is a rare clinical syndrome, which describes recurrent stereotyped lacunar transient ischemic attacks clustered within a short period of time and is associated with a high risk of developing a completed stroke. The mechanism of CWS had not been fully elucidated. Various treatments are available and being used for CWS. However, the optimal treatment remains unclear. Intravenous thrombolysis is an alternative treatment for CWS within 4.5 h after the onset of symptoms. Regardless of the high incidence of infarction in CWS patients, most patients had a good prognosis after 3 months.


The mortality-to-incidence ratio (MIR) is a marker that reflects the clinical outcome of cancer treatment. MIR as a prognostic marker is more accessible when compared with long-term follow-up survival surveys. Theoretically, countries with good health care systems would have favorable outcomes for cancer; however, no report has yet demonstrated an association between gallbladder cancer MIR and the World’s Health System ranking.We used linear regression to analyze the correlation of MIRs with the World Health Organization (WHO) rankings and total expenditures on health/gross domestic product (e/GDP) in 57 countries selected according to the data quality.p = 0.01 and p = 0.030, respectively).The results showed high crude rates of incidence/mortality but low MIR in more developed regions. Among continents, Europe had the highest crude rates of incidence/mortality, whereas the highest age-standardized rates (ASR) of incidence/mortality were in Asia. The MIR was lowest in North America and highest in Africa . Furthermore, favorable MIRs were correlated with good WHO rankings and high e/GDP  contains supplementary material, which is available to authorized users. Gallbladder cancer (GBC) and extra-hepatic duct cholangiocarcinoma are rare diseases with age standardized incidence rates of around 2 to 3 per 100,000 populations in both gender separately worldwide . GBC hasSalmonella [Helicobacter infections [The known risk factors of GBC are gallbladder disease  15]. I. ISalmonfections  increaseThe only treatment that provides a good outcome for GBC is early curative resection. The 5-year mortality rate is around 50% in cases with peri-muscular connective tissue involvement , regardlTechnical and equipment improvements suggest that health care systems may be able to improve early lesion detection. Better socioeconomic conditions can prevent delays in surgical cholecystectomy, thereby avoiding the extra-gallbladder spread of GBC. We considered that the mortality-to-incidence (MIR) ratio for GBC would be low in a country with a good health care system, as a similar concept has recently been confirmed for prostate and colon cancers –23. The http://gco.iarc.fr/). The WHO rankings are the World Health Organization’s ranking of the health systems based on an index of factors including health, responsiveness, and fair financial contribution. The health expenditure and life expectancies were obtained from the World Health Statistics 2015 of WHO.The data acquisition protocol was described previously . In brieThe GLOBOCAN 2012 database contains information for 184 countries. We excluded countries that lacked WHO ranking data (22 countries) or that had a low availability level of data . Ultimately, 57 countries were used for our analyses. The MIR was defined previously as the ratio of the crude rate of mortality to the incidence .P values < 0.05 were considered statistically significant. Scatter plots were produced using Microsoft Excel 2010.The method used for statistical analysis was described previously . We evalThe incidence/mortality numbers, crude rates, ASR, and MIRs are listed in Table The data for 57 selected countries are summarized in Table p = 0.031, Additional file 2 = 0.176, p = 0.001; R2 = 0.083, p = 0.030, respectively, Fig. The association between crude rate/age-standardized rate of incidence/mortality and the WHO ranking or e/GDP is illustrated in Additional file In this study, we analyzed the correlation of incidence, mortality, and MIRs for GBC with WHO rankings and total expenditures on health/GDP. A correlation between MIR and health care disparities was confirmed previously for many cancers , 23. OurThe best treatment choice is early diagnosis, so screening programs and high probability population for GBC have been established in some studies , 26. The2 = 0.176, p = 0.001; R2 = 0.083, p = 0.030, respectively, Fig. The WHO ranking and e/GDP showed no significant correlations with the crude rate and ASR of incidence/mortality, except for the crude rate of incidence. We found an association between a higher e/GDP and a higher crude rate of incidence for GBC in our analysis. We believe that the higher e/GDP countries have better or more frequent screening programs, which lead to more GBC diagnoses. Furthermore, the favorable MIRs of 57 countries are significantly associated with good WHO ranking and high e/GDP  could not be fully analyzed in our study, and these issues may add some bias to our study. Third, the use of MIR for predicting disease outcome has many limitations, since MIR was calculated from the cross-sectional data of mortality and incidence for a certain period causing different patients calculated in the incidence and mortality. MIR would not substitute for prognostic data from long-term follow up or from a cohort study. Forth, we excluded countries with relatively poor or unknown data quality which changes the distribution of countries according to the regions or continents. We analyzed the main results without country selection, the conclusion remains unchanged. The favorable MIRs of all countries were significantly associated with good WHO ranking and high e/GDP  incidence, and (B) mortality; the ASR of (C) incidence, and (D) mortality. Figure S2. The association between the total expenditures on health/GDP and the crude rates of (A) incidence, and (B) mortality; the ASR of (C) incidence, and (D) mortality. Figure S3. The (A) World Health Organization rankings (N = 142) and (B) total expenditures on health/GDP (N = 139) are significantly associated with the MIR in gallbladder cancer under investigation without country selection. (DOCX 739 kb)