Castanea sativa Miller) is mostly concentrated in Europe. Chestnut is recognized by its high content of antioxidants and phytosterols. This work aimed to evaluate the effects of dietary chestnut consumption over physiological variables of FVB/n mice. Eighteen FVB/n male 7-month-old mice were randomly divided into three experimental groups (n = 6): 1 (control group) fed a standard diet; 2 fed a diet supplemented with 0.55% (w/w) chestnut; and 3 supplemented with 1.1% (w/w) chestnut. Body weight, water, and food intake were recorded weekly. Following 35 days of supplementation, the mice were sacrificed for the collection of biological samples. Chestnut supplementation at 1.1% reduced abdominal adipose tissue. Lower serum cholesterol was also observed in animals supplemented with chestnut. There were no significant differences concerning the incidence of histological lesions nor in biochemical markers of hepatic damage and oxidative stress. These results suggest that chestnut supplementation may contribute to regulate adipose tissue deposition.The production of chestnut ( Castanea sativa is a species from the Fagaceae family mainly found in Mediterranean Europe [Castanea sativa Miller) has been used for several centuries as a food source in rural areas of Europe [n Europe . This spn Europe . Chestnuf Europe . Chestnuf Europe , and canf Europe . Additiof Europe . In the f Europe . Chestnuf Europe ,7 that if Europe , has higf Europe ,7. Moreof Europe . Chestnuf Europe . In addif Europe .Although the chestnut has never been studied as a whole, beneficial properties for health are attributed to its compounds. For example, acid linoleic plays an important role in preventing cardiovascular disease in adults and promotes brain and retinal development in children . The phoChestnut oil is rich in polyunsaturated fatty acid (PUFAs), including linoleic and oleic acids, and tocopherols, such as γ-tocopherol, which have been associated with multiple health benefits, as previously reviewed by Ramadan . Omega-3w/w) included in the diet of FVB/n mice.Taking into account these multiple reports on the hypocholesterolemic effects of the chestnut components, this study aimed to evaluate the physiological and potential toxicological changes associated with two different chestnut concentrations . These concentrations were calculated assuming that, during the chestnut season, an adult (70 kg) person consumes 150 g per serving or 450 g per week. For a 30-g mouse with a 5-g average daily food intake, this corresponds to 192.6 mg of chestnuts/week/mouse and the 0.55% (w/w) diet. The high-intake diet (1.1% w/w) was conceived to double the edible chestnut intake. The diets were prepared using an industrial mixer  and adding 5% (v/w) water to the mix to form new pellets (4.2 mm in diameter). The edible chestnut inclusion possibly added more carbohydrates and fiber but also fatty acids, phenolic compounds, and organic acids to the commercial feed. The base diet was prepared in the same method but without chestnut incorporation. All food lots were subsequently dried in an oven at 40 °C for 48 h and stored at 4 °C until further use.A maintenance diet, a commercial rodent feed , was used as a basis for the preparation of modified diets containing raw chestnut without the inner and the outer shell . The edible chestnuts were reduced to flour in a food processor and incorporated into two different modified diets and concentrations of 0.55%. and 1.1% (Mus musculus) were randomly divided into three different groups (n=6/group): Group 1, control group fed the standard diet without edible chestnut supplementation; group 2, fed a diet supplemented with 0.55% chestnut; and group 3, supplemented with 1.1% of chestnut. The well-being of the animals was checked weekly as well as the body weight of each animal and the water and food consumption. The animals were sacrificed 35 days after the beginning of the experimental procedures by intraperitoneal administration of ketamine  and xylazine , followed by cardiac puncture and exsanguination according to FELASA guidelines. Complete necropsies were performed. Heart, lungs, spleen, liver, thymus, kidneys, and abdominal and perirenal fat were collected and weighed in a precision balance .All experimental procedures were approved by the University of Trás-os-Montes and Alto Douro Ethics Committee (10/2013) and the Direção Geral de Alimentação e Veterinária (0421/000/000/2014). The animals were kept under controlled conditions of temperature (23 ± 2°C), light-dark cycle (12 h light/12 h dark), and relative humidity (50 ± 10%). Eighteen FVB/n 7-month-old male mice , aspartate aminotransferase (AST), creatinine, and cholesterol were determined by spectrophotometric methods using an autoanalyzer .The alkaline (pH > 13) comet assay was performed in mononuclear blood cells. A system of eight gels per slide was adopted by Guilherme et al. , in ordeFPG) were subtracted from the untreated GDI to quantify net enzyme-sensitive sites (NSSFPG).One hundred comets (50 comets per gel) were scored to obtain a genetic damage index (GDI) on a scale ranging between 0 and 400 arbitrary units. Scores obtained with FPG incubation  were stained with hematoxylin and eosin for observation under optical microscopy and histological analysis. In hepatic tissues, the presence of mitotic figures, intracellular inclusions, binucleated hepatocytes, tri- or multinucleated hepatocytes, focal necrosis and inflammation, cells with morphological changes suggestive of apoptosis, and hepatocellular vacuolar degeneration were recorded. Renal lesions were classified as non-suppurative interstitial nephritis, isolated cell necrosis, cell changes compatible with chronic nephropathy/tubular regeneration, and tubular accumulation of protein casts suggestive of proteinuria.2, and 0.5 mM of phenylmethylsulfonylfluoride (PMSF), prepared in ethanol to prevent protein degradation, pH 7.4), centrifuged  , and supernatants were collected. Total superoxide dismutase (SOD) activity was estimated according to the method described by Durak et al. [Liver and kidney samples were collected and stored at −80 °C until they were processed for oxidative stress analysis. The samples were homogenized in cold buffer solution .Body weight gain was calculated as previously . Statistp > 0.05, During the experimental work, no phenotypic or behavioral changes were observed among the experimental mice. Additionally, no mortality was observed during the experimental period. The mean body weight increased throughout the test in all groups and ponderal weight gains were not statistically different (p < 0.05) more food than controls (group 1), but there were no significant differences concerning water consumption (p < 0.005) as shown in Mice supplemented with edible chestnut consumed significantly  scores obtained from the comet assay without FPG were not statistically different between groups . When thp > 0.05)  .Mus musculus). Noh and colleagues previously evaluated the effects of the chestnut inner shell (Castanea crenata) on HepG2 (human liver cancer cell line) cells and in C57BL/6 mice treated with a high-fat diet. Inner shell extracts showed the ability to reduce oxidative damage caused by tert-butylhydroperoxide in HepG2 cells in vitro, as well as oxidative damage caused by carbon tetrachloride [Castanea sativa Mill) improved the liver and kidney function in diabetic Wistar rats, by reducing oxidative damage towards lipids and DNA and inhibiting protein glycation [This is the first experimental study to evaluate the effects of dietary supplementation with different edible chestnut concentrations in mice  in order to achieve results and, at the same time, apply the 3Rs  principle predicted for animal experiments. During the present experimental work, no mortality was recorded in any group and no physiological or behavioral changes were observed, supporting the idea that edible chestnut supplementation is well tolerated. In line with these findings, all groups showed positive ponderal weight gains. Experimental groups supplemented with edible chestnuts showed significantly higher feed intake, suggesting that the chestnut was highly palatable. Importantly, this increased food intake did not result in significant weight differences or obesity. Other studies from our group in which the diet of FVB/n mice was similarly manipulated with the addition of the polyphenols curcumin and rutin showed similar results . There wThe values obtained in the microhematocrit did not present statistically significant differences between the groups, in line with previous findings from another group . There wC. sativa), which accounts for over 50% of the total starch content in this kind of nut [fa/fa rats (obesity rat model)concluded that omega-3 PUFAs led to a reduction in cholesterol levels [The most interesting finding from the present study is the lower relative weights of peri-renal and abdominal adipose tissue in mice supplemented with 1.1% chestnut. This may be explained by the amount of digestion-resistant starch in chestnut  reduced oxidative stress in C57BL/6 mice exposed to a high-fat diet [C. sativa Mill) reduced DNA damage induced by streptozotocin in pancreatic β-cells, and associated this reduction with the high content of ellagic acid and its derivatives in chestnut. In fact, ellagic acid was already known to reduce DNA damage in Chinese hamster ovary cells through comet assay and cytofluorimetric analyses [The comet assay did not reveal significant increases in DNA damage in animals supplemented with edible chestnut, even when samples were pre-treated with FPG to reveal oxidative DNA-damage. In fact, there was a trend towards reduced oxidative DNA damage in animals that received edible chestnut. Based on these results, dietary supplementation with edible chestnut does not seem to cause damage to DNA and it may even reduce oxidative DNA damage, in line with the findings of Jovanović et al. , who shofat diet . Similarfat diet , who obsanalyses .We intend to carry out additional studies with higher doses of chestnut, different exposure times, and other types of chestnut preparations. In addition, our team intends to carry out an obesity model to assess the effects of chestnut on the animals’ body weight, cholesterol levels, and the percentage of white and brown fat.These experimental works suggest that an edible chestnut diet can be beneficial by reducing visceral fat and total blood cholesterol. Moreover, no toxic effects were observed in association with dietary edible chestnut supplementation, even when using doses estimated to be twice higher than those generally consumed by human populations.


Many studies have compared the performance of time series models in predicting pulmonary tuberculosis (PTB), but few have considered the role of meteorological factors in their prediction models. This study aims to explore whether incorporating meteorological factors can improve the performance of time series models in predicting PTB.We collected the monthly reported number of PTB cases and records of six meteorological factors in three cities of China from 2005 to 2018. Based on this data, we constructed three time series models, including an autoregressive integrated moving average (ARIMA) model, the ARIMA with exogenous variables (ARIMAX) model, and a recurrent neural network (RNN) model. The ARIMAX and RNN models incorporated meteorological factors, while the ARIMA model did not. The mean absolute percentage error (MAPE) and root mean square error (RMSE) were used to evaluate the performance of the models in predicting PTB cases in 2018.Both the cross-correlation analysis and Spearman rank correlation test showed that PTB cases reported in the study areas were related to meteorological factors. The predictive performance of both the ARIMA and RNN models was improved after incorporating meteorological factors. The MAPEs of the ARIMA, ARIMAX, and RNN models were 12.54%, 11.96%, and 12.36% in Xuzhou, 15.57%, 11.16%, and 14.09% in Nantong, and 9.70%, 9.66%, and 12.50% in Wuxi, respectively. The RMSEs of the three models were 36.194, 33.956, and 34.785 in Xuzhou, 34.073, 25.884, and 31.828 in Nantong, and 19.545, 19.026, and 26.019 in Wuxi, respectively.Our study revealed a possible link between PTB and meteorological factors. Taking meteorological factors into consideration increased the accuracy of time series models in predicting PTB, and the ARIMAX model was superior to the ARIMA and RNN models in study settings. Tuberculosis (TB) is a chronic communicable disease that severely threatens human health, ranking among the top ten causes of death worldwide. The World Health Organization (WHO) estimated that approximately 10 million people fell ill with TB around the world in 2019. Furthermore, there were an estimated 1.2 million TB deaths among HIV-negative people and 208 000 TB deaths among HIV-positive people [A time series is formed by recording the development process of a random event over time. Time series analysis plays a vital role in predicting trends by identifying the way in which health-related events change with time. The autoregressive integrated moving average (ARIMA) model is the most classic time series analysis model and has been widely applied to predict various infectious diseases, such as hepatitis B , hemorrhTime series analyses have been used to predict TB morbidity or mortality, but most were conducted in one city or one region and based on one or two models that did not incorporate meteorological factors , 12. Our2 for Xuzhou, 914.64 people/m2 for Nantong and 1424.43 people/m2 for Wuxi.Jiangsu Province is located on the eastern coast of China, with an area of 107 200 square kilometers. It governed 13 cities and had a permanent population of 80.7 million at the end of 2019. We randomly selected one city from northern, central, and southern Jiangsu and finally included Xuzhou, Nantong, and Wuxi as the study sites. The geographical locations of the three cities in Jiangsu Province are shown in Fig. https://10.249.6.18:8880/) operated by the Center for Disease Control and Prevention. The registry system is a particular virtual private network. For confidentiality, only authorized organizations can log in. We extracted the monthly reported number of pulmonary TB (PTB) cases in the study sites between 2005 and 2018. We also collected local meteorological factors at the same time from the China Meteorological Data Network (https://www.nmic.cn/). These meteorological factors included monthly average temperature , monthly average atmospheric pressure , monthly average wind speed , monthly average relative humidity , monthly precipitation , and monthly sunshine time .All newly diagnosed PTB cases in China are registered in an online surveillance system . The number of PTB cases predicted at time t  and root mean square error (RMSE) have been widely used to compare the performance of time series models , 17, thehttps://www.r-project.org/) to construct the RNN model. The significance level was set at 0.05.We used SPSS 25.0  to construct the ARIMA and ARIMAX models and the package “rnn” in R 3.6.3 12 for Nantong and Wuxi because (1) they had the smallest normalized BIC, (2) their residual series were demonstrated to be white noise, and (3) the parameters were all significant (P < 0.05)  or January to February (in Nantong and Wuxi) Fig. . Therefo12, ARIMA 12, ARIMA 12, ARIMA 12, ARIMA 12, and ARIMA 12 for Xuzhou, ARIMA 12, ARIMA 12, ARIMA 12, ARIMA 12, ARIMA 12, and ARIMA 12 for Nantong, and ARIMA 12, ARIMA 12, ARIMA 12, ARIMA 12, ARIMA 12, and ARIMA 12 for Wuxi, respectively. We then estimated the correlation between PTB and each meteorological factor at different lag times. The CCF plots showed that PTB was positively correlated with MAS (2-month lag), MAH (1-month lag) and MP (2-month lag) and negatively correlated with MST (1-month lag) in Xuzhou. PTB was positively correlated with MAT (0-month lag), MAP (1-month lag) and MAS (2-month lag) in Nantong and was positively correlated with MST (0-month lag) and negatively correlated with MAH (0-month lag) (P < 0.05) in Wuxi  for Xuzhou, ARIMA 12 with MAP (1-month lag) for Nantong and ARIMA 12 with MAH (0-month lag) for Wuxi. PTB cases in 2018 were predicted by the optimal ARIMAX model and are listed in Table The time series plots of the six meteorological factors in the three cities between 2005 and 2017 are shown in Additional file uxi Fig. . We incoP < 0.01), with MAS two months prior (P < 0.01), and with MAS three months prior (P < 0.01) and negatively correlated with MST two months prior (P < 0.01), with MAT three months prior (P < 0.01), with MP three months prior (P < 0.05), and with MST three months prior (P < 0.01). The number of PTB cases in the current month in Nantong was negatively correlated with MAS one month prior (P < 0.05), MAH one month prior (P < 0.05), MAS two months prior (P < 0.01), MAH two months prior (P < 0.01), MAS three months prior (P < 0.01), and MAH three months prior (P < 0.05). The number of PTB cases in the current month in Wuxi was positively correlated with MAT one month prior (P < 0.01), MAS one month prior (P < 0.01), MST one month prior (P < 0.05), MAS two months prior (P < 0.01), and MAS three months prior (P < 0.01) and negatively correlated with MAP one month prior (P < 0.01), MAH one month prior (P < 0.05), MAT three months prior (P < 0.05), and MAH three months prior (P < 0.05)  showed the best performance. To our knowledge, this is the first time series study to construct different models in different cities to explore the role of meteorological factors in predicting PTB.Although the notification rate of TB has declined at an annual rate of 3% between 2005 and 2017 , approxiThe ARIMA model, also known as the Box-Jenkins model, can analyze various types of time series data and is a commonly used model in time series analysis –6. UnlikConsidering that both ARIMA and ARIMAX are linear regression models, we also applied the RNN model, which has a strong nonlinear fitting ability. It can recognize the relationship between variables without any restrictions and has memory. This means that the RNN model uses as input not only current data but also its long-term experience. When constructing an RNN model, some parameters need to be determined artificially. In addition, since the initial weights and thresholds are random when training the RNN model, even for the same training set, the output of the model with the testing set will not be precisely the same. Therefore, we trained each RNN model with different parameters and compositions three times and compared their performance when using the testing set to determine the optimal RNN model. Finally, we found that the prediction performance of the RNN model was improved after incorporating meteorological factors.Mycobacterium tuberculosis transmission [M. tuberculosis, thereby reducing the risk of infection. Airflow usually occurs from high-pressure areas to low-pressure areas, so the correlation between PTB and atmospheric pressure may be related to wind speed, but further exploration is needed [M. tuberculosis [M. tuberculosis but also promotes the synthesis of vitamin D, which can protect people from TB to some extent [The possible link between PTB and meteorological factors may be attributable to the following reasons. First, the temperature can affect the indoor and outdoor activities of TB patients and other susceptible people. For example, during hot summers and cold winters, people tend to stay indoors, which will increase the probability of smission . Second,s needed . Third, rculosis , 24. Conrculosis . Fourth,e extent .The association between PTB and meteorological factors varied across regions , which mOur study has several limitations. First, the ARIMA, ARIMAX, and RNN models are all short-term prediction models; continuous data collection to update the models is essential for maintaining their prediction performance. Second, we incorporated all combinations of significant meteorological factors into the ARIMA model to construct the ARIMAX model, but we only incorporated four combinations of meteorological factors into the RNN model. In addition, the construction of the RNN model was based on monthly data, which may be insufficient for the RNN to reflect its predictive value. As the performance of the RNN model in this study was inferior to that of the ARIMAX model, its prediction performance needs further exploration. Third, we qualitatively evaluated only the linear correlation between PTB and meteorological factors based on monthly data. Considering that this relationship may be nonlinear and possess the lag time, we intend to apply the distributed lag nonlinear model to quantitatively evaluate it based on weekly or daily data in future studies. Fourth, most PTB cases are typically transmitted in dense indoor places, while all meteorological data in this study were derived from outdoor measurements, and indoor microclimates were not considered.The prediction performance of both the ARIMA and RNN models was improved after incorporating meteorological factors, and the ARIMAX model  had the best performance, indicating a potential link between PTB and meteorological factors. Taking meteorological factors into consideration may increase the accuracy of time series models in predicting the trend of PTB.Additional file 1: Table S1. Description of monthly meteorological factors in the three cities between 2005 and 2017. Table S2. Alternative ARIMA models for the three cities. Table S3. The Spearman rank correlation coefficients between the monthly number of PTB cases and meteorological factors in the three cities. Table S4. The detailed composition of the nine RNN models. Figure S1. ACF and PACF plots. Figure S2. Time series plots of the six meteorological factors in the three cities between 2005 and 2017. Figure S3. Epoch-error plots of the optimal RNN models of the three cities after three training cycles. File S1. R code.


From an epidemiological standpoint, quantifying the individual and the combined effect of lifestyle factors on uncontrolled blood pressure (BP) deserves further evaluation. We aimed to examine the individual and combined associations between unhealthy behaviors and uncontrolled hypertension among treated hypertensive adults. Cross-sectional analysis was conducted using data from CONSTANCES, an ongoing French population-based cohort study. Uncontrolled BP was defined as mean systolic BP ≥140 mmHg and/or mean diastolic BP ≥90 mmHg. Unhealthy behaviors were considered as heavy alcohol consumption, low or medium adherence to dietary recommendations, sedentary physical activity level, and overweight. A total of 10,710 hypertensive treated volunteer participants were included and 56.1% had uncontrolled hypertension; of them, 2.0%, 24.5%, 54.0% and 19.5% exhibited 0, 1, 2 or ≥3 unhealthy behaviors respectively. In men, there was an increased odds of uncontrolled hypertension with heavy alcohol drinking compared to light-or-never , with low as well as with medium adherence to dietary recommendations compared to high (p < 0.05 for both), and with overweight or obesity compared to a normal body mass index (p ≤ 0.001 for both). In addition, men reporting a combination of ≥3 unhealthy behaviors compared to none, had an increased odds of hypertension of 1.67 (95% CI 1.09–2.53). Unhealthy behaviors described as, heavy alcohol consumption, non-adherence to dietary recommendations and overweight are associated with uncontrolled hypertension, at the individual and combined level, and particularly in men. Improvement of modifiable lifestyle factors could offer considerable benefits in the management of hypertension. Despite the availability of efficient and well-tolerated medications and widespread public health efforts to treat individuals with hypertension, inadequate blood pressure (BP) control is frequently reported among treated hypertensive individuals, and contributes significantly to increased risk of cardiovascular disease (CVD), stroke and chronic kidney disease (CKD)2.Arterial hypertension is a global public health issue affecting more than 1 billion individuals worldwide and causing an estimated ten million deaths every year3. Within Europe, BP control rate among those treated reached 40% in England, 30% in Germany, 28% in Italy, 19% in Spain and 21% in Sweden4. Similarly, the estimated prevalence of hypertension in France is 31% and 51.3% of hypertensive treated patients are not controlled5.A number of epidemiological studies commonly reported a high prevalence rate of uncontrolled hypertension at the 140/90 mmHg threshold; in the United States, data analysis from the National Health and Nutrition Examination Survey (NHANES) found that among treated individuals around 45% had uncontrolled BP6, deficiencies of healthcare systems in their global approach to chronic diseases7 and low adherence to treatment including antihypertensive prescriptions and lifestyle changes8. In addition, factors such as socio-economic characteristics and poor lifestyle behaviors have been described as predictors of poor BP control9. Studies suggest that unhealthy lifestyle behaviors including heavy alcohol drinking, lack of physical activity, poor dietary habits and overweight may contribute to inadequate BP control among hypertensive treated individuals10. Alternatively, lifestyle changes were associated with decreased BP among hypertensive individuals;12 Appel et al., reported that behavioral interventions comprising increased physical activity, limitation of dietary sodium intake, reduced alcohol consumption, and weight loss, reduced systolic BP by 12.5 mmHg and diastolic BP by 5.8 mmHg12.A broad range of factors have been identified that contribute to poor BP control. These include, physician inertia (i.e. lack of therapeutic action when the patient’s BP is uncontrolled)14. This heterogeneity makes their promotion more challenging and justifies the need to conduct further studies evaluating their impact on different populations. In fact, these recommendations emphasize lifestyle changes based on intervention trials that were especially effective in pre- or hypertensive individuals; hence, the study of their effect in terms of improving BP control in hypertensive treated individuals remains necessary.Several lifestyle modifications or non-pharmacological approaches are widely recommended in worldwide guidelines for the management and prevention of hypertension. These interventions target dietary habits, salt intake, potassium intake, alcohol consumption, physical activity and weight. However the quantitative or qualitative targets for each of these measures differ across the guidelines15, which is designed to contribute to the development of epidemiologic research, serve as an opportunity to provide additional data on this subject. Hence, this ancillary study was conducted to evaluate the individual and collective relationship between unhealthy behaviors, particularly, heavy alcohol consumption, low physical activity, non-adherence to dietary recommendations, and overweight, with uncontrolled hypertension. We aimed to assess the quantitative extent to which modifiable lifestyle factors are determinants of uncontrolled hypertension in order to assess the magnitude of their effect in the management of hypertension, from a gender-based perspective.As such, from an epidemiological perspective, examining the quantitative effect of unhealthy lifestyle factors on uncontrolled hypertension, at the individual and combined level, warrants further evaluation. The French nationwide large population-based study, CONSTANCESTable Among the 10710 hypertensive treated participants 6003 had uncontrolled hypertension, reaching a prevalence of poor BP control of 56.1%. Uncontrolled hypertension was more prevalent in men than in women  and with increased age categories with the highest prevalence seen in those more than 65 years old (63.1%). After adjustment to age and gender, uncontrolled hypertension was more frequent in participants with lower education , living in couple , with diabetes  or with dyslipidemia . It was less common in those with history of CVD . As for lifestyle factors, those who are overweight or obesity and those with heavy alcohol consumption have a significantly higher prevalence of uncontrolled hypertension (p < 0.001 for both variables) while high dietary adherence is associated with lower frequency. Interestingly, inverse associations were seen with physical activity and smoking status, those with high-level physical activity (p = 0.008) had more often uncontrolled hypertension, whereas current smokers seem to have more often controlled hypertension (p < 0.001). Globally, 56.3% of the study subjects were receiving one anti-hypertensive medication, while 34.3% were on dual therapy and 9.4% were using three medications or more; those with uncontrolled hypertension were less likely to be receiving mono-therapy and more likely to be receiving dual or triple (or more) therapy (p = 0.001).Figure The association between uncontrolled hypertension and dietary adherence, physical activity, body mass index (BMI), alcohol consumption and the number of unhealthy behavior is described in Table Regarding dietary adherence, men reporting low or medium dietary adherence had a 1.26-fold (adjusted odds ration (ORa) 1.26, 95% confidence interval (CI) 1.04–1.53) and 1.41-fold  increase of the odds of uncontrolled hypertension compared to those with high dietary adherence. Similarly, the odds of uncontrolled hypertension increased by 1.25-fold (1.09–1.43) and 1.57-fold (1.35–1.83) in overweight and obese men, respectively, compared to a normal BMI. In addition, there was a significant association between alcohol consumption and uncontrolled hypertension (p = 0.003); men consuming alcohol heavily had an increase of the odds of uncontrolled hypertension compared to light/never drinkers by 1.34-fold . The association remained significant when dichotomizing the variable and comparing heavy drinking to moderate/light drinking . In addition there was a significantly increasing age-adjusted mean systolic BP across light, moderate and heavy drinking in both sexes: in women the mean systolic BP ± SD across categories was 134.6 ± 4.8, 135.5 ± 4.2 and 136.1 ± 4.2, respectively (p < 0.001) and in men it was 134.9 ± 4.6, 135.7 ± 4.0 and 136.4 ± 3.6, respectively (p < 0.001). As for physical activity, there was an unexpected inverse relationship between sedentary level physical activity and uncontrolled hypertension. Moreover, the prevalence of uncontrolled hypertension increased with the number of unhealthy lifestyle factors in men only (p < 0.001). Those reporting three or more unhealthy behaviors had 1.67-fold (1.09–2.53) increase of the odds of uncontrolled hypertension.Findings of our study show, from a population-based perspective, that modifiable unhealthy lifestyle factors such as heavy alcohol consumption, overweight and non-adherence to dietary recommendations were associated with increased risk of uncontrolled hypertension in hypertensive treated individuals. The association was significant in men only, but even after adjustment for socio-demographic characteristics and cardiovascular risk factors. Also, a dose-effect relationship was noticeable by increased odds of uncontrolled hypertension with higher number of unhealthy behaviors; compared to none, three or four unhealthy lifestyle factors had 1.7-fold increased odds of uncontrolled hypertension in men. To our knowledge, these results are among the few to estimate the quantitative extent to which individual and combined unhealthy lifestyle factors influence the risk of uncontrolled hypertension in pharmacologically treated patients.9. Similarly, the Framingham Heart Study reported that among treated subjects, increasing age, obesity and the presence of left ventricular hypertrophy were associated with lack of systolic BP control. The authors suggested that public health efforts should be directed at achieving goal BP levels especially in patients who are older, are overweight or have target organ damage16.Compared to a normal BMI, we found overweight and obesity to be strongly associated with uncontrolled hypertension in men, increasing its odds by 1.25-fold and 1.57-fold respectively. This association has been described elsewhere; one study in South Korea conducted on individuals being treated for hypertension and taking regularly their antihypertensive medications, found that overweight patients were less likely to have their BP under control compared with those whose body weight was normal 9. Other studies argued that moderate intensity aerobic exercise lowers BP in patients with hypertension and reduces the need for antihypertensive medication18. Although a dose-dependent relationship was not seen in our study, yet we found a protective relationship between moderate level physical activity and uncontrolled hypertension. Several aspects of the physical activity score we used could explain our divergent results. In fact we calculated a score that is not commonly used or is reproducible in the literature, which could have led to an inadequate estimation of physical activity level. Moreover, the score does not consider the metabolic equivalent (MET) of the reported physical activity, resulting in a distinct classification. Further studies are necessary to assess this aspect.There was no significant association between physical activity and uncontrolled hypertension in women. While in men, surprisingly, the multivariable analysis models found a weak but significant inverse association between physical activity and uncontrolled hypertension, whereby moderate and moderate-to-sedentary physical activity level compared to high level (as reference) were negatively associated with uncontrolled hypertension. In general, this is not in accordance with results of observational studies that reported a strong relationship between physical activity and BP control. Ham and Young, found that low physical activity (compared to high level) to be associated with poor BP control among hypertensive treated individuals19. On the other hand, most research studied the BP lowering effect of a DASH-diet in pre-hypertensive and hypertensive patients. For example, the DASH collaborative research group found that adopting a DASH-diet in patients with hypertension substantially lowers systolic and diastolic BP by 11.4 and 5.5 mmHg, respectively20, suggesting that such BP reductions can help in achieving adequate BP control. Our study demonstrated that non-adherence to dietary recommendation is associated with uncontrolled hypertension, while measuring the extent of the effect of the association. Accordingly, our findings suggest that lifestyle modifications involving the adoption of a DASH-style diet offer an important approach in the treatment of hypertension.Concerning adherence to dietary recommendations, low or medium adherence to the dietary approaches to stop hypertension (DASH)-diet was found to increase the odds of uncontrolled hypertension in men only. Few studies evaluated the association between a dietary approach and BP control in uncontrolled hypertensive individuals. One randomized controlled trial conducted on hypertensive patients with type 2 diabetes and uncontrolled hypertension, demonstrated that a DASH-diet combined with increased daily walking promotes a clinically relevant reduction in ambulatory BP monitoringet al. reported that heavy alcohol consumption defined as consumption of more than 60 g for men and 40 g for women during a single drinking session, was independently associated with poor BP control at the 140/90 threshold in a sample of hypertensive treated South Koreans9. In addition, a number of studies described an apparent and direct association between heavy alcohol intake and elevated BP22 that can result in exceeding recommended BP goals. One Japanese study found that in heavy drinkers, systolic and diastolic BP was 2.3/2.0 mmHg higher in heavy drinkers than in non-drinkers21. Differences between gender in drinking behavior, pattern and beverage choice could have influenced the lack of association in women. Nevertheless, an association between heavy drinking and high BP was once again demonstrated, supporting limitation of alcohol consumption recommendation, in the hypertensive treated population.We identified a strong association between heavy alcohol consumption and uncontrolled hypertension. Men who drank alcohol heavily had 1.34-fold increase in the odds of poor BP control. This association has been reported in previous studies. Ham 19. Importantly, with such BP reductions, more than half of the patients in the intervention group reached the recommended goals for daytime ambulatory BP monitoring19. BP reductions with a combination of lifestyle factors were discussed in previous studies on hypertensive patients, but there was no reference to the use of anti-hypertensive medications. For example, a systematic review of randomized controlled trials on patients with elevated blood pressure, reported BP reductions of 5.5 mmHg after a combination of interventions including physical activity, diet and weight loss, compared to 5.0 mmHg for improved diet, 4.6 mmHg for exercise and 3.5 mmHg for alcohol restriction23. In a recent separate analysis, we evaluated the extent to which unhealthy behavior influence the development of hypertension in the general population, and found that in both sexes, unhealthy behavior (as described in this study) significantly increased the odds of hypertension. We also reported that a combination of two and three-or-more unhealthy behaviors resulted in an increased odds of hypertension by 1.77-fold and 2.29-fold respectively in men, and by 1.71-fold and 2.14-fold respectively in women24. Our current study show that a combination of unhealthy behavior is independently associated with uncontrolled hypertension in hypertensive treated individuals. These findings along with the previous ones24 further support the important influence of lifestyle factors on high BP, in different populations and regardless of the presence of other risk factors. However, when comparing both studies, the magnitude of the effect of this association appears stronger on the development of hypertension than on BP control, suggesting that other factors should also be considered when evaluating determinants of BP control in individuals pharmacologically treated for hypertension.We found a dose-effect relationship between the number of unhealthy behaviors and uncontrolled hypertension; the likelihood of uncontrolled hypertension increased nearly linearly with 1, 2, 3 or more, unhealthy lifestyle factors, but reached statistical significance with 3 or more factors. Few epidemiological studies evaluated the role of modifiable lifestyle factors on BP control among treated individuals, or assessed their combined effect. In fact, collective unhealthy behaviors may have synergistic effect on BP control, emphasizing the importance of studying their combined effect. In patients with uncontrolled hypertension and type 2 diabetes, the combination of increasing physical activity and following a DASH-diet had a major reduction in systolic BP values of approximately 15 mmHg, as compared with a reduction of 3 mmHg in the control group25 and they were not particularly on unhealthy behaviors. Discrepancy between sexes could be explained by differences in lifestyle habits between men and women as well as to the influence of other confounding factors8 such as other socioeconomic factors , other diseases (chronic kidney disease) other behavioral factors  and adherence to anti-hypertensive medications. In addition, some data suggest that sex-related characteristics such as the level of sex hormones may influence the results26. Although further research is needed to clarify this difference, nevertheless adopting a global healthy lifestyle is important for prevention of cardiovascular diseases and should be encouraged in the general population14.Our study also pointed out gender differences as to these associations. Few observational studies evaluated the determinants of uncontrolled hypertension using a gender stratified analysis28, while others found no association29 and showed that office BP is not lowered by smoking cessation30. Further research can help yield more conclusive results. Nevertheless, smoking is an unhealthy behavior and a major risk of CVD and cancer; smoking cessation recommendations should be provided to all hypertensive individuals for the prevention of CVD including stroke, myocardial infarction and peripheral artery disease14.Lastly, age-and-gender-adjusted results found current smoking to be associated with decreased prevalence of uncontrolled hypertension. Epidemiological studies describe discrepancy with regards to effect of smoking on uncontrolled BP; some studies reported smoking to negatively influence BP control31, and certainly recent changes. Nevertheless, this is common in epidemiologic studies of cross-sectional design; prospective data from CONSTANCES can help in considering this point. Lastly, excessive salt intake is considered an unhealthy behavior, but we weren’t able to study its effect on uncontrolled hypertension because quantitative data on salt intake are not available since dietary habits were evaluated using a non-quantitative food frequency questionnaire.The main strength of our study is using data from CONSTANCES, which was designed adopting a population-based method including a large randomly selected sample of participants that ensures sufficient power. Moreover, BP measurements were collected following standardized protocols and data were gathered using different methods such as validated questionnaires and national databases, resulting in lack of missing information. Additionally, we explored the combined effect of unhealthy behaviors and performed an analysis stratified by sex. However, some limitations should be addressed. Reverse-causality bias is inherent in cross-sectional analyses, preventing the confirmation of a causal relationship between unhealthy behaviors and uncontrolled hypertension. In addition, our study might be subject to selection bias because of the selection effect related to voluntary participation, also because agricultural and self-employed workers were not included in CONSTANCES. Misclassification bias is also possible, since lifestyle behaviors were self-reported. Moreover, the time frame between recent medication adjustment and BP measurement was not taken into consideration. In fact, antihypertensive medication is a confounding factor in BP measurements, with epidemiologic data on BP often compromised by the effects of antihypertensive medicationsIn conclusion, findings of this study provide supportive evidence of the individual and combined effect of unhealthy behaviors on uncontrolled hypertension. Unhealthy lifestyle described as heavy alcohol drinking, non-adherence to dietary recommendations and overweight increased the likelihood of uncontrolled hypertension, which was further increased with a higher number of unhealthy factors. Our findings revealed that the associations were significant in men only, suggesting the presence of other factors influencing uncontrolled hypertension. Although further research is needed to clarify the reasons behind the gender-based differences, our findings contribute to epidemiologic data of utmost importance in the management of hypertension, especially in the presence of limited data on the effect of lifestyle factors on hypertension control. From a population-based perspective, our study advocates that public health strategies should promote improvement of modifiable behaviors through a multidisciplinary lifestyle changes approach, which could offer considerable benefits in the treatment and control of hypertension, particularly in men.http://www.constances.fr/index_EN.php) have been previously published32. Briefly, CONSTANCES is a prospective epidemiological cohort composed of randomly selected adult participants aged 18–69 years at inception affiliated with the French National Health Insurance Fund database  following a sampling scheme stratified on age, gender, socioeconomic status and region of France.Details concerning objectives and study design of the cohort CONSTANCES  and the Institutional Review Board of the National Institute for Medical Research and the local Committee for Persons Protection (Comité de Protection des Personnes).Volunteers who agreed to participate in the study had to fill self-administered questionnaires and were invited to attend to one of the 22 selected health-screening centers (HSCs) to benefit from a comprehensive health examination. They were also linked through French health administrative and national social databases. Through these different sources, social, demographic, health, behavioral, occupational, biological, and anthropometric data were collected. All the participants included in the CONSTANCES cohort have signed an informed consent form. This research follows the tenets of the Declaration of Helsinki and was approved by the National Data Protection Authority 24. Similarly, unhealthy behavior was defined like in the original study24. The lifestyle factors are presented also here.Lifestyle factors were assessed through validated self-administered questionnaires. They were described and classified based on a previous study evaluating the association between unhealthy behavior and risk of hypertensionAlcohol consumption was determined considering the quantity and type of alcoholic beverages consumed the previous week35. We subsequently defined alcohol consumption as never/light (0–3 glass/week (0–30 g/week) for men and 0–2 (0–20 g/week) for women), moderate (4–21 (40–210 g/week) glass/week for men and 3–14 (30–140 g/week) for women) and heavy drinkers (>21 glass/week (>210 g/week) for men and >14 (>140 g/week) for women)36. Heavy drinking was considered an unhealthy behavior.Physical activity was assessed through three questions that considered the frequency of transferring, leisure time activity and sports37. We assigned 0, 1, or 2 points for each question based on an escalating frequency of activity, then a score of 0–6 was calculated and physical activity level was classified as sedentary (0–2), moderately active (3–4) and highly active (5–6). Sedentary level was considered an unhealthy behavior37.Dietary assessment was done through a validated 52-items food frequency questionnaire (FFQ) from which a DASH score was constructed based on 8 food groups or nutrients for which consumption should be increased  or reduced 38. Consumption of each dietary component was divided into quintiles, and participants’ intakes were assigned 1–5 points according to a gender-specific intake ranking39. Component scores were summed, and an overall DASH score ranging from 8–40 was calculated. The DASH score was subsequently collapsed to tertiles for analysis; a higher tertile indicating a higher dietary quality, adherence to dietary recommendations was subsequently categorized into low, medium and high. We considered low/medium dietary adherence an unhealthy behavior.Body mass index  was calculated at the HSC, then categorized into three classes: normal (≤25 kg/m2), overweight (25 kg/m2<BMI <30 kg/m2), and obese (≥30 kg/m2). We considered overweight/obese (BMI >25 kg/m2) an unhealthy behavior.Accordingly, participants could exhibit 0 (none), 1, 2, 3, or 4 unhealthy behaviors.24 and they included the following. Education level was collected according to the International Standard Classification of Education (ISCED)40 and was then classified into three levels: High school diploma or less (≤13 years of education), undergraduate degree (14–16 years of education) and postgraduate degree (≥17 years of education). Marital status was categorized into couple life or single (including widowed or separated/divorced). Household monthly income was categorized into:<1000; 1000–2099; 2100–4199; ≥ 4200 euros per month.Covariates were defined and classified as the original analysis14. Chronic kidney disease was defined as known proteinuria or decreased renal function  for more than 3 months41, or a chronic kidney disease diagnosed by biopsy or renal ultrasound and confirmed by a nephrologist.Blood glucose, triglycerides and total cholesterol were measured by taking fasting blood samples at the HSC. Diabetes mellitus status was based on either receiving anti-diabetic medication or a fasting blood glucose concentration greater than or equal to 7 mmol/L. Dyslipidemia was defined as having a fasting plasma total-cholesterol or triglycerides level of ≥6.61 mmol/L (255 mg/dL) or >1.7 mmol/L (150 mg/dL) respectively. History of CV diseases was considered as any self-reported previous diagnosis of angina pectoris, myocardial infarction, cerebrovascular accident or peripheral artery diseaseDescriptive analysis was performed using counts and percentages or mean ± SD. Each characteristic was compared between subjects with controlled and uncontrolled hypertension using logistic regressions adjusted for age and sex. In addition, we compared characteristics of subjects according to the number of unhealthy behaviors using logistic regressions adjusted for age and stratified by sex. Also with logistic models, we estimated the association between uncontrolled hypertension and unhealthy behaviors. In a first step, models were adjusted for age, education and monthly income (model 1). In a second step, models were further adjusted for diabetes and dyslipidemia (model 2). Initially, we performed separate models for each unhealthy behavior using categorical variables and binary variables. Then, we examined the association between uncontrolled hypertension and the number of unhealthy behaviors (0- ≥ 3) independently associated with control of hypertension. General Linear Model was used to study age-adjusted mean systolic BP across ascending number of unhealthy factors. Adjusted odds ratios (ORa) were presented along with 95% confidence interval (CI), all statistical analyses were performed with SAS 9.4 (SAS Institute) and p ≤ 0.05 was considered significant.Supplementary information.


While studies of the left atrium (LA) have demonstrated associations between volumes and emptying fraction with atrial fibrillation (AF), the contribution of right atrial (RA) abnormalities to incident AF remains poorly understood.Assess the association between RA structure and function with incident AF using feature-tracking cardiovascular magnetic resonance (CMR).n = 368) were compared against AF-free (n = 2779). Cox proportional-hazards models assessed association between variables.This is a prospective cohort study of all participants in the Multi-Ethnic Study of Atherosclerosis with baseline CMR, sinus rhythm, and free of clinical cardiovascular disease at study initiation. RA volume, strain, and emptying fraction in participants with incident AF  and minimum volume index , and lower baseline RA emptying fraction , peak global strain , and peak free-wall strain  compared with the AF-free population. After adjusting for traditional cardiovascular risk factors and LA volume and function, we found RA maximum volume index  and minimum volume index  were independently associated with incident AF.Participants were aged 60 ± 10 yrs., 55% female, and followed an average 11.2 years. Individuals developing AF had higher baseline RA maximum volume index (mean ± standard deviation [SD]: 24 ± 9 vs 22 ± 8 mL/mIn a large multiethnic population, higher RA volume indices were independently associated with incident AF after adjustment for conventional cardiovascular risk factors and LA parameters. It is unclear if this predictive value persists when additional adjustment is made for ventricular parameters. Atrial fibrillation (AF) is the most common cardiac arrhythmia in the United States, with a lifetime risk of 1-in-4 for persons over 40 years of age and associated increases in morbidity and mortality . Much ofIn patients with subclinical heart disease, risk factors, including venous thromboembolism (VTE) and pulmonary embolism (PE) , obstrucCardiovascular magnetic resonance (CMR) is well-established for simultaneously assessing LA volume and strain as it potentially provides greater spatial resolution and excellent demarcation of endocardial and epicardial borders , 17. FeaIn this study, we sought to evaluate the role of altered RA structure and function, including peak global and free-wall strain, volumes, and emptying fraction (EF), to the development of AF in a large multiethnic population free of clinically-recognized cardiovascular disease at baseline. We hypothesized that increased RA size and reduced RA function (as assessed by EF and strain) are associated with greater risk of incident AF.MESA is a population-based cohort of participants free of clinically-evident cardiovascular disease at baseline. The MESA protocol has previously been described in detail . In brieIncident cases of AF were identified through December 2014. Cases were identified through MESA hospitalization surveillance, study electrocardiograms (ECGs), and for participants enrolled in fee-for-service Medicare, from inpatient and outpatient Medicare claims data. We excluded individuals who developed AF after coronary artery bypass or valve surgery or those who were found to have an AF claim occurring before their baseline MESA examination.The CMR protocol has previously been described in detail and was performed at time of enrollment . In brieRA function was assessed using multi-modality tissue tracking software , which has been previously described at length . This soRA EF was calculated based on RA volumes at different time points in the cardiac cycle, as follows:Strain measurement in 4-chamber cine images was automated within the software with segmentation of the borders into free-wall and septal distributions. Strain is computed as an average across the entire manually-defined cardiac contour. The zero-strain reference was set at RA end-diastole. Peak global strain refers to the maximum calculated strain during the cardiac cycle within the RA, which in these baseline measurements of a CVD-free population reflect RA reservoir strain. Free-wall strain refers to the RA lateral wall and RA roof, excluding the septal wall. Strain curves were computed for both peak global and free-wall longitudinal segments during the cardiac cycle and visually assessed for quality.Poor quality: Images were missing frames, out of plane, or out of sequence. Tracking was not possible in the MTT software as it was unable to propagate the manual contour. These were excluded from analysis.Fair quality: Images and tracking were acceptable but the cardiac cycle was incompletely captured or contrast was poor causing inaccurate tracking in part of the cardiac cycleGood quality: Images and tracking were without the above issues but required minor operator adjustmentsExcellent quality: Images and tracking required no operator adjustmentStudy quality was defined categorically from 1 to 4. A single operator was responsible for assigning quality across all studies. Definitions are as follows:Standardized questionnaires were used at baseline to collect information from participants about demographics and cardiac risk factors as previously described . We scremax) and RAVImin remained significantly associated with incident AF. RA EF, peak global and free-wall strain were not significant after this adjustment. In model 2, we made additional adjustments for LA maximum volume index, peak LA strain, and LA EF as obtained from previous studies [Our multivariate Cox models adjusted for these covariates as follows. In model 1, we included demographic characteristics  and traditional cardiovascular risk factors . RA volume index (RAVI studies .p = 0.04), passive LA EF , and peak LA strain . In the LV, LV mass was the strongest association with AF . For the RV, EF  and RV mass  were the most significant associations for AF.Additionally, we adjusted for LA, left ventricular (LV), and RV variables as obtained from and described in previous studies in MESA , 7, 21. max compared with above-median RAVImax, with matching on demographic and traditional risk factor covariates. P-value < 0.05 was considered significant. GraphPad Prism  was used to create the additional figure.Statistical analysis was performed with Stata software . Continuous variables are denoted here as mean ± standard deviation while categorical variables are denoted as numbers and percentages. Baseline participant characteristics and RA metrics were evaluated using the Student’s T-test for continuous variables and Chi-square test for categorical variables. ANOVA with Tukey’s post-hoc test was used to assess for variation in RA metrics by image quality. Cox proportional hazard models were used to assess the association of RA structure and function parameters with time to incident AF, with death, end of follow-up, and withdrawals treated as censored. Cox models were adjusted for significant predictors of AF selected from among the covariates listed above. Propensity score matching was used for validation of our results. We modeled the odds of AF for participants with a below-median RAVIWe evaluated the reproducibility of both RA volume and function metrics in MTT. Interobserver and intraobserver variability was assessed with 20 randomly-chosen cases. RA analysis was independently performed by two operators  while blinded to AF incidence and other clinical parameters. Intraclass correlation coefficient analysis was performed to evaluate variability.p = 0.25), nor was the proportion of developing AF among participants with CMR different from that in the whole MESA cohort (p = 0.37).All available CMRs from participants without AF at baseline were screened for inclusion (4967 participants). RA parameters were able to be obtained from CMRs of 3214 participants with fair or better quality. Of these, 3147 participants had AF data and comprised the study population. Of all included studies, 16% were of fair quality, 80% were of good quality, and 4% were of excellent quality. Participants were followed for an average of 11.2 ± 3.7 years. There were 368 participants who developed AF with a mean time to incident AF of 7.4 ± 3.7 years. The proportion developing AF in our participants is not significantly different than the proportion developing AF in all participants with CMR  and minimum volume indexed (RAVImin). RA structure and function in patients who did and did not develop AF are summarized in Table max was 6% higher and RAVImin is 9% higher in patients with incident AF . The RA EF was 4% lower in AF cases (p = 0.02). In parallel, baseline RA peak global strain and free-wall strain were 6% lower in AF cases (p < 0.001 and p = 0.049 respectively).RA volume was indexed by BSA yielding maximum volume indexed . Comparatively, the effects of LA metrics in the model were: LA maximum volume index (HR per SD: 1.46), LA passive EF (HR per SD: 0.79), LA peak strain (HR per SD: 0.82). Propensity score results yielded a similar direction. The top quantile of RAVImax was associated with a 3.5% higher incident AF (p = 0.007) at 1:1 matching for AF and non-AF participants. In adjusted quantile analyses of RAVImax, there was a notable graded increase in risk with increasing quantiles. Compared to the middle quintile, the 4th and 5th quintiles had, respectively: HR 1.4, p = 0.04 and HR = 1.7, p = 0.002. In contrast, risk did not appear to be mitigated with lower RA volumes and the lowest quintiles were not significantly different than the middle quintile.Larger RA structure as assessed by higher RAVI2DS2-VASc) and RA volumes is shown . RAVImin was not significant after adjustment . Further adjustment for LV mass had no effect on the model. Neither RAVImax nor RAVImin were significant after adjustment for demographics, traditional risk factors, and both LA and RV variables within the same model.We additionally created models adjusting for demographics, traditional risk factors, and RV variables previously identified as being associated with incident AF  Additio. RAVImaxmax, 0.89 for RAVmin, 0.91 for global strain, and 0.89 for free-wall strain. ICC for interobserver reproducibility were 0.93 for RAVmax, 0.85 for RAVmin, 0.85 for global strain, and 0.82 for free-wall strain.Reproducibility of RA was evaluated in 20 randomly selected subjects. Intraclass correlation coefficients (ICC) for intraobserver reproducibility were 0.93 for RAVThe sub-population of MESA who underwent CMR examination, as expected, was healthier than the sub-cohort who did not  have highlighted their potential role as predictors of AF .As with RA volume, literature on the relationship between RA function and incident AF is limited, the few existing studies having been conducted in symptomatic patients with cardiac disease. RA emptying fraction was not associated with development of AF in patients with a pathological atrial septal defect undergoing repair . In patiOur study has several limitations. A single reader was responsible for post-processing though reproducibility studies were excellent with multiple readers. Use of a single reader reduces variability in research as in this study but does not reflect general clinical usage. Our study had a higher rate of unusable or uninterpretable images than comparable studies featuring LA wall tracking. Examples of poor quality images which were excluded are shown (Additional file In this large, diverse population free of symptomatic cardiovascular disease at baseline, we found RA maximum and minimum volume indices measured using feature-tracking CMR had an independent positive predictive value for incident AF relative to conventional CVD risk factors and LA parameters. It is unclear if this predictive value persists when ventricular parameters are also taken into consideration. Our findings suggest RA structure among healthy individuals may play a role in the development of AF, and this association should be further investigated.Additional file 1. Correlation of AF risk factors and RA volume at study initiation. SD: Standard deviation; BMI: Body mass index. Association of risk factors for AF and RA volume indices is shown. RA volume was generally not significantly different for risk factors though demographic differences by sex and race are evident. Volume is indexed by body-surface area which may affect the significance risk factors due to collinearity.Additional file 2. Association of right atrial volume with incident AF after adjustment for right ventricle variables. EF: emptying fraction; EDM: end-diastolic mass. Models are also adjusted for demographics and traditional risk factors.Additional file 3. Baseline characteristics of patients who did and did not receive cardiac magnetic resonance imaging. Values are mean ± SD or %. CMR: cardiovascular magnetic resonance imaging; SD: Standard deviation; BMI: Body mass index; HDL: High density lipoprotein.Additional file 4. Variation of right atrial parameters at different quality levels, with 95% confidence intervals. Average and confidence intervals shown above for RA parameters at each quality level. RA: right atrium; RAVImax: RA maximum volume index, RAVImin: RA minimum volume index.Additional file 5. Examples of images from sequences excluded from the study for poor quality. Each of these representative images are taken from excluded sequences at RA end-diastole. Each image is from a different study site and was labeled as the long-axis image for that participant. Evident in these images are motion artifact and planes which do not completely capture the RA. Such images were numerous in our analysis, resulting in the exclusion rate.


Washington University polyomavirus (WUPyV) is a novel human polyomavirus detected in childwith acute respiratory infection in 2007. However, the relationship between WUPyV and respiratory diseases has yet to be established for lacking of a suitable in vitro culture system.To isolate WUPyV with human airway epithelial (HAE) cells, the positive samples were incubated in HAE, and then the nucleic acid, VP1 protein and virions were detected using real-time PCR, immunofluorescence and electron microscopy respectively.The result showed that WUPyV could replicate effectively in HAE cells and virions with typical polyomavirus characteristics could be observed. Additionally, the entire genome sequence of the isolated strain (BJ0771) was obtained and phylogenetic analysis indicated that BJ0771 belongs to gene cluster I.Our findings demonstrated clinical WUPyV strain was successfully isolated for the first time in the world and this will help unravel the etiology and pathogenic mechanisms of WUPyV in respiratory infection diseases. Betapolyomavirus in the Polyomaviridae family. It was first identified by Gaynor et al. in 2007 from a child with pneumonia using high-throughput sequencing [Washington University polyomavirus (WUPyV) is a small, nonenveloped virus, containing a circular double-stranded DNA genome that belongs to the genus quencing , 2. The WUPyV now has a worldwide distribution and the detection rate in children with acute respiratory tract infections (ARTIs) ranges from 0.4 to 16.4% . The maiHuman airway epithelial (HAE) cells are the primary target for many respiratory viruses in vivo. Compared with traditional cell lines, HAE cells reconstituted in vitro can provide an appropriate cellular environment and have been widely used in research about the etiology, pathogenic mechanisms, and potential treatment methods of respiratory viruses, including influenza viruses, parainfluenza virus, Nipah virus, bocavirus, and respiratory syncytial virus –17. The In this study, we successfully isolated a WUPyV strain (BJ0771) from a clinical sample for the first time in the world using ALI-HAE cells. The identity of the virus particles was confirmed by immunofluorescence and electron microscopy. We further explored the replication characteristics of WUPyV and obtained its whole genome sequence.Nasopharyngeal aspirates (NPAs) of hospitalized children with ARTI (≤14 years) were collected from the Beijing Friendship Hospital (China). WUPyV-VP1 gene fragments were detected by real-time PCR using the TaqMan Gene Expression Master Mix  according to the manufacturer’s protocol. The following primers were used: forward primer: 5′-CCAATGGTACTGTGCCTCATGT-3′, reverse primer: 5′-CCATGATTCAATGCTGTACTTTTCA-3′, and probe: FAM-ATTCCAGTTCTGAAACACCCAGGGCAAG-TAMRA. The PCR products were confirmed by sequencing.2 incubator. Once the cells reached 80–90% confluence, they were digested with trypsin and plated at a density of 3 × 105 cells/well on type IV collagen-coated Transwell plates , and the culture medium for both the apical and basolateral sides were renewed every other day. Once the cells formed tight junctions, the medium was replaced with HAE 3D medium (BEGM + DMEM + additives) and cultured for 5 days, then HAE cells were cultured at an ALI for 4–6 weeks until the cells were well differentiated into a 3D pseudostratified structure.Primary HAE cells were kept in our laboratory. ALI-HAE cells were cultured according to methods previously established in our laboratory . Briefly2 incubator. Then, 200 μL of medium was added to the apical layer following a 2 h incubation at specific time points post-infection for harvesting the virus. Nucleic acids were extracted from both the apical washes and the basal medium for real-time PCR, and then a replication curve was constructed.One of WUPyV-positive samples with a Ct value less than 20  was selected for virus isolation. The positive samples were added to the apical layer at a density of 100 copies/cell . After 2 h of incubation, HAE cells were rinsed twice with PBS, and maintained in a 37 °C, 5% COE. coli. The protein was then purified through a Ni column  and used to immunize rabbits twice, and finally the serum was obtained. HAE cells were infected with harvested WUPyV at 100 copies/cell, then fixed with 4% paraformaldehyde for 30 min at 14 days post infection (dpi) and permeabilized with 0.2% Triton X-100 for 2 h. After blocking with 5% bovine serum albumin (BSA) for 1 h, cells were incubated with primary antibodies diluted in 1% BSA overnight at 4 °C . Then cells were incubated with secondary antibodies at 37 °C for 2 h . Nuclei were counterstained with DAPI . Confocal images were recorded using a confocal microscope .Rabbit antiserum against WUPyV VP1 developed in our laboratory was used in immunofluorescence analysis. The WUPyV VP1 gene (GenBank accession no. ABQ09289) was ligated to the pET-30a vector  and the recombinant histidine-tagged VP1 was expressed in BL21 9 copies/μL. The supernatants were then observed by transmission electron microscopy .HAE cells were incubated with harvested WUPyV at 100 copies/cell and the supernatants were collected when the viral genome copy number reached 10For complete genome sequencing of WUPyV, six pairs of primers containing overlap were designed according to the WUPyV reference strain B0 (GenBank accession number: EF444549), as shown in Table From Apr. 2017 to Mar. 2018, 76 (5.96%) WUPyV positive samples were detected among 1276 hospitalized children. WUPyV positive samples with a Ct value less than 20 were selected to infect HAE, and a strain of WUPyV (BJ0771) was isolated from a 3-year-old child who was diagnosed with asthmatic bronchopneumonia in Beijing Friendship Hospital, China. The patient was hospitalized in November, 2017, with symptoms of fever, cough, wheezing, runny nose and vomiting.9 copies/μL. Additionally, the viral genome was detected in the basolateral medium at 2 dpi, and the viral load increased gradually with time until 30 dpi. The viral loads in the basolateral medium were ~ 3 log less than the copy numbers released from the supernatant over the course of infection , with a nearly 400-fold increase in virus particles in the apical washes, and finally reached a plateau around 10 dpi and then maintained a level of about 10β-tublin [β-tublin-specific fluorescence in both the experimental and control groups, indicating that HAE cells had differentiated into ciliated cells. In WUPyV-infected HAE cells, VP1-specific fluorescence was observed in both the cytoplasm and nucleus, with the nucleus appearing enlarged and round in shape. By contrast, no immunoreactivity for VP1 was detected in mock cells, and their nuclei showed no morphological changes  strain in genetic distance. The sequence of BJ0771 did not show any significant deviations, with ~ 99.3% and ~ 99.0% identity at the nucleotide and amino acid levels, respectively, compared with the selected reference strains. The whole genome sequence of BJ0771 was deposited in the GenBank database under accession no. MT066199 , the complete genome was sequenced using six pairs of primers with a partial overlap. The WUPyV genome is a circular double-stranded DNA of 5229 nucleotides (nt) in length, composed of two early regions encoding the large and small tumor antigens (LT and ST), and three late regions encoding the structural proteins . Thirty WUPyV reference genome sequences were downloaded from the GenBank database for phylogenetic analysis. A phylogenetic tree was constructed using the maximum likelihood method and MEGA 7.0 software. WUPyV strains could be divided into three main clusters (I–III). Strain BJ0771 belonged to cluster I, which was closest to the WUPyV199 Fig. .Fig. 4PAlpha-, Beta-, Gamma-, and Deltapolyomavirus. To date, a total of 102 polyomaviruses have been reported around the world, 13 of which are human polyomaviruses (HPyVs), including BKPyV and JCPyV identified in the early 1970s, and 11 types of HPyV detected since 2007, including KIPyV, WUPyV, and MCPyV [The polyomavirus family contains four genera, nd MCPyV . HPyVs cnd MCPyV , with twnd MCPyV . Second,nd MCPyV . WUPyV hnd MCPyV . Howevernd MCPyV , prompti+ cells , CD68+ cells (macrophage/monocyte lineage), and MUC5AC+ cells (goblet cells) in the lung tissue by immunohistochemistry [To date, there have been no cell lines available for the proliferation of HPyVs from clinical samples, with the exception of JCPyV and BKPyV . Much ofhemistry , 29, ind9 copies/μL), indicating that WUPyV could cause a persistent infection. Viral nucleic acids were detected in the basolateral chamber, suggesting that WUPyV could break through the respiratory epithelial barrier, being released into the basal medium from the surface of pseudostratified cells. The ability to break through this cell barrier is crucial for virus release and disease development, as the progeny viruses then have the opportunity to infect other cells with the same receptors.In this study, ALI-HAE cells cultured on Transwell plates were used for clinical sample inoculation, and a WUPyV strain (BJ0771) was successfully isolated from a 3-year-old child with respiratory infection. The replication curves showed that WUPyV grew rapidly in the early stage, with viral loads reaching a plateau at 6 dpi. Despite the growth rate slowing after day 6, viral loads remained at a constant high level  in the HAE cell system. Electron microscopy provided evidence that complete WUPyV virions could be released. These two experimental techniques confirmed that HAE cells could produce infectious WUPyV.Moens et al.  exploredAt present, international classification standards for WUPyV have not been published. Previous studies indicated that WUPyV could be divided into three main clusters (I–III) and several sub-clusters (Ia–Id and III a–IIIc) , 31, butIn summary, using the ALI-HAE cell system, WUPyV strain BJ0771 was successfully isolated in vitro for the first time in the world, and the replication kinetics and morphological characteristics of the virus were clarified. Additionally, phylogenetic analysis of its whole genome showed that BJ0771 belongs to cluster I. In future research, we will focus on the cellular receptors, cell tropism, and pathogenicity of WUPyV, and further explore the possibility of WUPyV culture on passaged cell lines.


P = 0.003, P = 0.004). Frequencies of genotypes TT of rs2296188 and rs2296283 were declined in patients compared with healthy controls . CC genotype of rs7324510 and rs9582036 was negatively correlated with SLE risk . Distribution of GG, GC, GG + GC genotypes of rs9554322 were different between SLE patients and healthy controls . Moreover, frequency of TC genotype of rs7324510 was higher in SLE patients with lupus headache  and frequency of TC genotype of rs9943922 was lower in patients with cylindruriain . Frequencies of allele C of rs7324510 and allele T of rs9943922 were decreased in SLE patients with cylindruria and hypocomplementemia, respectively . However, frequency of allele C of rs9554322 was increased in SLE patients with pyuria . In addition, SLE patients carrying GG, GC, CC genotypes for rs9554322 had higher levels of serum sVEGFR-1. In conclusion, serum sVEGFR-1 was elevated in SLE patients and may be a disease marker. VEGFR1 gene polymorphisms related to risk of SLE in a Chinese Han population.Correlation between soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) concentration, VEGFR1 gene polymorphisms and systemic lupus erythematosus (SLE) risk remains unclear. The present case–control study comprised 254 SLE patients, 385 other rheumatic diseases patients and 390 healthy controls. Serum levels of sVEGFR-1 were detected by enzyme-linked immunosorbent assay. Seven VEGFR1 genetic variants  were genotyped by KASP. Serum levels of sVEGFR-1 were up-regulated in SLE and positively correlated with disease activity. Furthermore, serum sVEGFR-1 presented a distinctive elevation in SLE in comparison with other rheumatic diseases. Frequencies of allele T of rs2296283 and allele G of rs9554322 were significant lower in SLE patients ( Gender difference also exists in SLE. It is known that occurrence of the disorder in women is more frequent than men, with a ratio of approximately 6:12. The aetiology of SLE has remained elusive, but genetic predisposition, environmental triggers and hormonal factors are demonstrated to involve in. A higher concordance rate in monozygotic twins than dizygotic twins and the high sibling recurrence risk ratio both support a strong heritability of SLE3. In the past decade, a variety of genome-wide association studies (GWAS) have recognized over 40 SLE susceptibility loci existing in exons and introns5.Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with a heterogeneous organ involvement that hinges on multiple autoantibodies production. Considering the worldwide estimation of incidence of SLE, there was a regional variation of the disease. European countries had a lower incidence of SLE, whereas Asia, Australia and the Americas had higher incidenceVEGFR1 gene is located in chromosome 13q127. It was originally found to be expressed on vascular endothelial cells8, and then discovered expressed on smooth muscle cells9, monocytes10, trophoblasts11, mesangial cells12 and osteoblasts13. sVEGFR-1 competes with signaling receptors of VEGF (VEGFR-1/VEGFR-2) by capturing their ligands14. It has a negative role in angiogenesis after binding to the VEGF. However, sVEGFR-1 interacts with endothelial cells components, showing an angiogenesis effect. Therefore, sVEGFR-1 exists multiple effects on vessel growth progression and may be a new therapeutic target for VEGF-mediated pathological signaling. In systemic sclerosis (SSc) patients, lower serum level of sVEGFR-1 was detected in comparison with healthy controls15. In rheumatoid arthritis (RA) patients, expression of sVEGFR-1 was increased, correlating with VEGF concentration16. Serum level of sVEGFR-1 in patients with osteoarthritis (OA) was higher compared with that in non-arthritic controls16. VEGFR1 genetic variant associated with RA disease activity17. Collectively, sVEGFR-1 plays important roles in inflammatory and autoimmune diseases. To date, relationship of sVEGFR-1 and lupus is limited. What is the expression profile of sVEGFR-1 in lupus, if VEGFR1 gene polymorphisms relate to SLE risk needs to be discussed.Soluble vascular endothelial growth factor receptor-1  is a nature vascular endothelial growth factor receptor (VEGFR) competitor. The human P < 0.001) and AS (P < 0.001). In addition, the hypocomplementemia, proteinuria, arthritis and rash were the four dominant clinical characteristics in SLE patients . Other clinical information was contained in Table The demographical characteristics of the patients with SLE and controls are shown in Table P = 0.015, Fig. s = 0.557, P < 0.001, Fig. P < 0.001, Fig. P = 0.001, P = 0.021, P = 0.012, P = 0.017, respectively, Fig. P < 0.001, Fig. Concentration of sVEGFR-1 in patients with 61 SLE was higher in comparison with that in 94 healthy subjects (17.738 7.604–26.286) vs 12.115 (8.655–12.115) ng/ml, 6.286 vs VEGFR1 gene affect SLE, seven SNPs were analyzed between SLE patients and healthy controls. No deviation from the HWE test was observed in patients and controls for each polymorphism . Similarly, frequency of rs9554322 allele G was significantly associated with SLE . As for frequencies of VEGFR1 genotypes, the most significant difference was observed in rs2296283, by which the genotype TT was dramatically decreased in SLE patients compared with healthy controls . There were decreased frequencies of TT + TC in patients with SLE as compared with healthy controls . Frequencies of all rs9554322 genotypes  were lower in SLE group . For rs2296188 and rs9943922, frequencies of TT were both lower in SLE patients when compared with controls . Moreover, we found marginal differences of genotype CC in rs7324510 and rs9582036 in SLE patients compared with healthy controls . Frequencies of rs9554320 genotypes, although not statistically significant, were lower in SLE patients as compared with healthy subjects (data not show).To investigate whether polymorphisms of 2 = 0.521; D′ = 0.716, r2 = 0.221; D′ = 0.700, r2 = 0.131) . Proportions of CTC and TTA haplotypes were fewer in SLE patients . The other haplotypes were not figured out statistical significance . For rs9943922, TC genotype was different in patients with cylindruria as compared with patients without this feature . Distribution of CC, CA, AA genotypes of rs9582036 and rs9554320 was different between SLE patients with and without pyuria . In addition, C allele frequency of rs7324510 was lower in SLE patients with hypocomplementemia comparing to those without . T allele frequency of rs9943922 polymorphism was different between SLE patients with cylindruria and those without . It was found that the C allele frequency of rs9554322 was increased in SLE patients with pyuria . No significant association was found between the other SNPs and clinical, laboratory manifestations of SLE  correlated with risk of SLE. It is worthy that rs9554322 and rs9582036 was the first time found to relation with SLE. Genotypes of GG, GC and GG + GC in rs9554322 were significantly lower in patients than in healthy controls, suggesting that rs9554322 polymorphisms may negatively correlate with the risk of SLE in Chinese Han population . Genotype CC of rs9582036 was related to SLE risk (OR = 0.508). It is possible that regulatory, structural or quantitative polymorphisms at the VEGFR1 locus may affect VEGF signaling pathway and enhance susceptibility to some angiogenic conditions. In our study, rs2296283 located in the functional 3′-UTR region and other three SNPs  were within the introns. In a Polish study, there was no statistic difference in allele or genotypes frequencies of rs2296283 between RA patients and healthy controls17. On the contrary, we obtained a statistical difference in the frequencies of genotype TT and TT + TC for rs2296283 in SLE patients . Compared with the Polish study, we further observed statistic differences in genotype TT of rs2296188, TT of rs9943922 and CC of rs7324510 , which were encoded in the introns. Therefore, rs2296283 located in the functional region of VEGFR1 gene may influence the mRNA translation and stability through regulating polyadenylation, miRNA-mRNA and protein-mRNA interactions. However, further study needs to verify the effect of rs2296283 within the functional region. The significance of SNPs located in non-coding region of VEGFR1 (introns) were not clear. Several studies showed that polymorphisms in non-coding region of VEGFR1 possibly silenced or enhanced the transcriptional activity of sVEGFR-128. Thus, it is postulated that polymorphisms in the non-coding region may implicate in selective splicing of RNA and promote the transcriptional activity of target protein. It is well known that SNP is the predominant pattern in the genomic DNA sequence variation. The majority of SNPs affected the gene expression instead of the protein composition. In agreement with previous studies, our results exhibited that six of seven SNPs  were not related with sVEGFR-1 levels. Several GWAS studies indicated that although most of SNPs did not possess the direct ability to change the gene expression, these SNPs sometimes served for the functional counterparts which regulated gene expression and protein assemble30. Thus, we hypothesized that the six SNPs selected in this study may not directly influence sVEGFR-1 concentration in SLE patients. On the other side, serum levels of sVEGFR-1 were different among rs9554322 genotypes (P = 0.029). SLE patients with rs9554322 GG genotype had higher sVEGFR-1 protein levels, suggesting that VEGFR1 rs9554322 G/C genetic variant may contribute to abnormal sVEGFR-1 serum levels. However, what is the exact role of polymorphisms in VEGFR1 gene, sVEGFR-1 protein expression needs to be discussed and whether the selected SNPs in the current study can affect VEGFR1 gene, sVEGFR-1 protein expression needs to be conducted by functional study in the future.Concerning There are several limitations in this study. First, the sample size in our study is relatively limited, where cases were recruited from two hospitals. Considering the large number of Chinese Han population, larger scale and multi-center studies are needed in the future. Second, the clear mechanism of sVEGFR-1 involves in SLE pathogenesis needs to discuss.VEGFR1 gene polymorphisms related to risk of SLE in a Chinese Han population.In summary, the present study showed that serum levels of sVEGFR-1 were elevated in SLE patients, may be a disease marker, and 31, 1987 ACR revised criteria for RA32, Osteoarthritis Criteria Subcommittee of the American Rheumatism Association (ARA) criteria for OA33, 1977 ARA criteria for gout34, American-European classification criteria for SS35 and Modified New York criteria for AS36. The SLE disease activity index (SLEDAI) was calculated to evaluate the disease activity of SLE patients37. Based on the SLEDAI score, the disease activity of SLE patients were divided into less-active period (SLEDIA < 10) and active period (SLEDIA ≥ 10). Age and sex matched healthy controls were selected from Jiangyang district center for disease control and prevention in Luzhou, having no history of SLE and other inflammatory autoimmune diseases. All the participants were Chinese Han origin. The Medical Ethics Committee of Southwest Medical University approved our study protocol. Written informed consent was obtained from each subject. Blood samples were collected from patients and healthy controls.Case–control studies were conducted for sVEGFR-1 serum levels and gene polymorphisms. A total of 254 SLE patients, 385 other rheumatic diseases patients and 390 healthy controls were recruited in the present study. Test for serum levels of sVEGFR-1was performed in two stages. The discovery set included 61 SLE and 94 healthy controls, and the validation set comprised another independent 100 SLE and 385 SLE-free disease controls . The study of gene polymorphisms consisted of 254 SLE and 390 healthy controls. All patients were recruited from the Department of Rheumatology and Immunology, Affiliated Hospital of Southwest Medical University and Affiliated Minda Hospital of Hubei Minzu University, classified by 1997 American College of Rheumatology (ACR) revised criteria for SLEVEGFR1 gene was performed. Based on the 1,000-genome project (https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/), all candidate SNPs complied with following screening criteria: pairwise tagging of HapMap population with r2 ≥ 0.8; a minor allele frequency (MAF) ≥ 5%; Chinese Han Beijing (CHB) ethnicity. At last, seven SNPs including rs2296188, rs9943922, rs2296283, rs7324510, rs9554322, rs9582036, rs9554320 were selected.A systemic search for previous literature about VEGFR1 genotyping reactions were completed by Gene Company using KASP . Information of KASP primers  was listed in Supplementary Table Peripheral blood was collected from ulnar veins in the fasting and clearheaded state. Samples were centrifuged and serum was stored at – 80 °C until analysis. Genomic DNA was extracted utilizing TIANamp Blood DNA kits  in line with manufacturer’s instructions. sVEGFR-1 protein levels of SLE patients and control groups were assessed by enzyme‐linked immunosorbent assay (ELISA) kits  in accordance with the manufacturer’s protocol. All samples were measured in duplicates and plates were read automatically at an absorbance of 450 nm using LT-4000MS reader . Concentration was calculated on the basis of a linear standard curve. The detection limit was 0.039 ng/ml.VEGFR1 haplotypes were assessed by using online software SHEsis . Statistical power was assessed (https://biostat.mc.vanderbilt.edu/wiki/Main/PowerSampleSize). P value lower than 0.05 was significant.Data was performed by Statistical Package for the Social Sciences . Categorical data were expressed as frequency and percentage. According to Shapiro–Wilk test, measurement data were expressed by mean ± standard deviation (SD) when it was normally distributed or median (inter-quartile range) when it was not normally distributed. For comparison of genotype and allele distribution between cases and controls, chi-squared or Fisher’s exact test were used as appropriate. Odds ratio (OR) and 95% confidence interval (CI) were analyzed by logistic regression model. Relationship between two variables was evaluated using Spearman’s rank test. Area of receiver operating characteristic (ROC) curve evaluated the specificity and sensitivity of predictive power of sVEGFR-1 in SLE. Hardy–Weinberg equilibrium (HWE) of genotypes in patients and healthy controls was estimated by chi-squared test. HaploView 4.1 software was used to analyze linkage disequilibrium (LD). Supplementary Table 1.Supplementary Table 2.