With the social development and lifestyle transition, increasing trends of overweight and obesity prevalence are commonly reported worldwide. Data focusing on overweight and obesity trends in rural residents and rural-to-urban migrants in China are limited. This study aims to assess the changes and related factors of overweight and obesity in Yi farmers and migrants in southwest China from 2007 to 2015, and to assess the disparities in prevalence changes.Pseudo-panel data was obtained from two cross-sectional studies conducted in Sichuan province, southwest China in 2007 and 2015. Standardized prevalence of overweight and obesity were evaluated by direct method using the 2010 national census population as the standard. Prevalence differences of overweight and obesity were calculated in each group and compared between groups to evaluate the disparity in prevalence changes. Generalized linear mixed model was performed to determine factors related to overweight/obesity.Standardized prevalence of overweight increased in both groups . Standardized prevalence of obesity increased only in Yi farmers (from 0.37 to 3.13%). Prevalence difference of overweight in Yi farmers was higher than that in Yi migrants (11.11 vs. 5.70), but prevalence differences of obesity were not significantly different between Yi farmers and migrants.Prevalence of overweight and obesity in Yi farmers, and prevalence of overweight in Yi migrants increased from 2007 to 2015. Yi farmers were developing overweight at a greater pace than migrants. More attention should be paid to spread health knowledge and encourage healthy lifestyle in Yi people, especially Yi farmers.The online version of this article (10.1186/s12889-018-5577-4) contains supplementary material, which is available to authorized users. It is well-established that high body mass index (BMI) is a major risk factor for non-communicable diseases (NCDs) including hypertension, diabetes, cardiovascular disease, and cancer –3. OverwMigrant studies provide valuable information about effects of transformed social environment and lifestyle behaviors on diseases. Previous studies demonstrated higher risk of obesity in rural-to-urban migrants compared with rural residents , 8. Yi pNot only for Yi people, China is now undergoing national-scale dramatic modernization and urbanization, with an increasing number of farmers migrating from rural to urban regions. But to our best knowledge, evidences for trends in overweight and obesity prevalence in rural-to-urban migrants and their rural counterparts in China are limited. Fortunately, Yi farmers and migrants provide a perfect opportunity for us to look into this issue. Our previous studies have demonstrated the higher prevalence of hypertension , dyslipiThe present analysis was based on the Yi Migrant Study. This is a study designed to evaluate cardiovascular risk factors in Yi rural and rural-to-urban migrant population in Liangshan Yi Autonomous Prefecture, Sichuan province, southwest China. Pseudo-panel data was obtained from two independent cross-sectional studies, which were carried out in 2007 and 2015, respectively. Individuals whose parents were both of Yi ethnicity were identified as Yi people. Yi farmers were defined as Yi people who had lived in villages since they were born, and Yi migrants were restricted as Yi people who reported being born in villages and then migrated to urban regions for more than 1 year prior to the surveys. Xichang city , Butuo, Zhaojue, Jinyang, Puge and Xide counties in 2007, Xichang city and Puge county in 2015 were selected as the study fields. Yi farmers were obtained using stratified cluster sampling method from each county. In the first stage, 1–4 townships isolated from the county seat were selected in each county. Then four villages were randomly selected from each township. In the last sampling stage, all Yi farmers aged 20–80 years in selected villages were enrolled. Because of the relative small number of Yi migrants, all of Yi migrants aged 20–80 years found in each county seat and Xichang city were enrolled into the surveys. Written informed consent was obtained from all study participants. Both two surveys were approved by the bioethical committee of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China.2). Both questionnaires and anthropometrical measurements were conducted by trained medical staff.In each survey period, standardized questionnaires were used to collect social-demographic characteristics  and lifestyles  by face-to-face interviews. The questions were translated by staff who mastered both Chinese and Yi language if the participants spoke Yi language only. Weight was measured in light clothing using an electronic scale  with an accuracy of 0.1 kg. Height was measured with barefoot by a fixed stadiometer  to the nearest 0.1 cm. The average of three height measurements was recorded. BMI was calculated as weight in kilograms divided by height in meters squared , moderate (middle or high school), and high (college or higher) according to the last educational institution attended. Personal annual income was categorized into three groups in accordance to tertiles. Smoking pack-years were calculated as the average packs of cigarettes smoked per day multiplied by the years of smoking duration. Current smokers were defined as those who smoked at least one cigarette per day for at least 6 months and were classified into three categories: light smokers (0–9 pack-years), moderate smokers (10–19 pack-years), and heavy smokers (20- pack-years). Current drinkers were defined as those who drank at least twice per month with intake of more than 640 ml (one bottle) beer or 100 ml liquor for at least 6 months and were classified into three categories: low-risk drinkers , moderate-risk drinkers , and high-risk drinkers  [According to WHO criteria , overweifemales) . Accordifemales) .2015 – P2007 and compared. P2015 and P2007 indicate the standardized prevalence in 2015 and 2007, respectively. The significance of disparity (PDfarmer vs. PDmigrant) was determined by standard error (SE) and 95% confidence interval using the formula: P < 0.05 was considered statistically significant.All analyses were conducted using SAS statistical software . Basic characteristics were described as means ± standard deviation (SD) for age and numbers (percentages) for categorical variables according to demographic group (farmer or migrant), sex, and survey period. Student’s t-test and Chi-square test were performed to assess the differences between periods. Covariance analysis for BMI was performed in order to adjust for age. Sex- and age-specific prevalence of overweight and obesity by six 10-year interval age groups  were calculated, and standardized prevalence were calculated based on the distributions of 2010 China census population, using the same age categories . Standar2 to 22.30 kg/m2 in males, from 21.56 kg/m2 to 22.42 kg/m2 in females). While in Yi migrants, the increase in BMI was only observed in females, of whom the adjusted BMI increased from 23.46 kg/m2 to 24.38 kg/m2.The basic characteristics of the study participants are illustrated in Table P < 0.0001), and the obesity prevalence increased from 0.48 to 2.96% (P < 0.0001). After adjustment for age and sex, the standardized prevalence markedly increased from 5.54 to 16.65%(P < 0.0001) for overweight and from 0.37 to 3.13% (P < 0.0001) for obesity. The increases in overweight and obesity were observed both in males and females. In Yi migrants, the crude prevalence of overweight increased from 29.27 to 36.57% (P = 0.0002), and the standardized prevalence increased from 27.91 to 33.61% (P = 0.0433). The changes of both crude and standardized prevalence of overweight were significant in females but not in males. While the changes of obesity prevalence in Yi migrants did not reach the statistical significance, for neither crude nor standardized prevalence.Figure p-values are available in Additional file The prevalence of overweight and obesity stratified by sex and age group are presented in Fig. farmer = 11.11, PDmigrant = 5.70, P = 0.0178), especially in males . In females, the PDs of overweight in Yi farmers were of no difference with Yi migrants. The PDs of obesity were not significantly different between Yi farmers and migrants, neither in males nor in females.The PDs in Yi farmers and migrants are shown in Fig. In multivariable-adjusted analyses, in both Yi farmers and Yi migrants, the risk of overweight/obesity was higher in 2015 than 2007 Figs.  and 5. AIn this study describing the changes of overweight and obesity, according to WHO criteria, we observed increases in standardized prevalence of both overweight and obesity in Yi farmers  and increase in standardized prevalence of overweight in Yi migrants (from 27.91 to 33.61%) from 2007 to 2015. In Yi farmers, the increases in overweight and obesity were evident in both gender and in young and middle-aged adults. While in Yi migrants, the changes of overweight were only apparent in females aged 20–39 and 50–59 years.2, obesity: BMI ≥ 28 kg/m2) [According to a nationwide survey conducted in 2007–2008, when Working Group on Obesity in China (WGOC) criteria was used (overweight: 24 ≤ BMI < 28 kg/m8 kg/m2) , the pre8 kg/m2) . In acco2, researchers found that age- standardized prevalence of obesity increased from 3.2 to 10.8% in males, and from 6.4 to 14.9% in females [2) increased from 8.0 to 17.1% in males and from 10.7 to 14.4% in females, and prevalence of obesity (BMI ≥27.5 kg/m2) increased from 2.9 to 11.4% in men and from 5.0 to 10.1% in women [The increases in BMI and overweight/obesity prevalence appear to be a worldwide concern. NCD Risk Factor Collaboration (NCD-RisC) pooled data of 19.2 million people from 200 countries to analyze the global trends of adults BMI from 1975 to 2014. By defining obesity as BMI ≥ 30 kg/mIt is widely accepted that the worldwide overweight and obesity epidemic are essentially attributed to sedentary lifestyles and high-fat energy-dense food intake . The decIn our study, prevalence of overweight increased both in Yi farmers and migrants, but PD between 2015 and 2007 in Yi farmers was greater than migrants. The results reflect the fact that Yi farmers were developing overweight at a more dramatic pace than their rural-to-urban counterparts. Additionally, the increasing prevalence of obesity was significant only in Yi farmers rather than Yi migrants. The rapid increases in overweight and obesity in Yi farmers can be partially explained by the improvement of income. Du et al.  found thWe observed relative low prevalence of overweight and obesity throughout the age groups in Yi farmers in 2007, especially male Yi farmers, in whom the obesity prevalence was zone except for adults at 30–39 years. Our previous study conducted in the same population demonstrated an extremely low prevalence of hypertension and stable blood pressure levels in all ages . In contNotably, we found that Yi migrants with longer residence in urban areas had higher risk of overweight/obesity compared with those recently arrived. Similarly, higher BMI and increased prevalence of obesity with longer-term migration have been reported both in transnational  and withOur study found that smoking was associate with lower risk of overweight/obesity in both Yi farmers and migrants. The data showed that in Yi farmers, heavy smokers (smoking exposure of 20 pack-years or more) had a 33% decreased risk to be overweight/obese when compared to never smokers. In Yi migrants, both moderate or heavy smoking (smoking exposure of 10 pack-years or more) were associated with lower risk. Previous studies identified that cigarette smoking could lead to weight loss by increasing the metabolic rate, decreasing metabolic efficiency or decreasing caloric absorption (reduction in appetite by nicotine) . HoweverThe relationship between alcohol drinking and overweight/obesity was controversial. Several previous studies reported nonlinear relationship between alcohol drinking and BMI, with light-to-moderate drinking being negatively associated with adiposity indicators . In our One of the strengths of our study lies in the collection of data from two population with same genetic background while in diverse living environment in two periods. The sample allows us to conduct comprehensive study in the changes of overweight/obesity as well as other non-communicable diseases, and to assess the association with changed environmental factors. Although our study was based on two independent cross-sectional surveys rather than a cohort, the results were reliable and comparable because: Firstly, the same sampling method and excluding criterions were used to obtain representative samples in the two surveys. Second, interviews and anthropometric measurements were carried out by well-trained personnel following standard method in the two surveys. Several limitations in our study deserve consideration. Firstly, due to the cross-sectional nature of our study, causality cannot be established. Secondly, we only used BMI to define overweight and obesity, which cannot distinguish fat and lean tissue or the body fat distribution . But as In conclusion, our study suggests increasing prevalence of overweight and obesity in Yi farmers, and increase in overweight in Yi migrants over the eight-year period. In spite of the relative low prevalence, Yi farmers were developing overweight at faster pace than Yi migrants. In order to contain the secular trends of overweight and obesity and to prevent obesity-related diseases, at the same time as socio-economic development, health knowledge should be disseminated and healthy life styles should be adhered, especially in Yi farmers.Additional file 1:Table S1. Sex and age specific prevalence of overweight and obesity in Yi farmers and migrants, 2007–2015. (DOCX 19 kb)


Nonalcoholic fatty liver disease (NAFLD) is a global problem and pediatric obesity has risen dramatically. Early NAFLD might progress to nonalcoholic steatohepatitis (NASH) or liver cirrhosis and significantly increase liver disease-related mortality. We looked for NAFLD predictors in children and adolescents.This community-based, cross-sectional study ran from December 2012 to September 2013 in southwestern Taiwan. Children <10 and >19 years old, with detected hepatic diseases, or who drank alcohol were excluded. The diagnosis of NAFLD was based on ultrasound: age, sex, anthropometric measurements, and laboratory data were evaluated for associated risks by using logistic regression analysis. Receiver operating characteristic (ROC) curves were used to determine cutoff values.P < 0.001). It indicated highly suspicion of NAFLD  when the WHtR for children and adolescents is above the cutoff value of 0.469.We enrolled one thousand, two hundred and ten children . Age, anthropometric measurements, and laboratory data were significantly higher in children with NAFLD. The association between NAFLD and the waist-to-height ratio (WHtR) was significant (adjusted odds ratio: 2.6; 95% confidence interval: 1.909-3.549; The WHtR might be a powerful index of the severity of pediatric NAFLD. Non-alcoholic fatty liver disease (NAFLD) is an emerging health problem associated with childhood obesity and adult metabolic syndrome (MetS) . NAFLD mLiver biopsy is the gold standard diagnostic tool for NAFLD, but it is expensive, invasive, risky, and infeasible in an annual health check-up. Ultrasound is a popular screening tool for patients with an abnormal liver function test, which varies without consistent values or upper normal thresholds in children. Body Mass Index (BMI) and waist circumference are anthropometric measurements widely used to evaluate the effects of obesity on metabovascular risk factors and NAFLD . ContrarThis study used a correlational cross-sectional design in which anthropometric measure and NAFLD were assessed concurrently in prepubertal children and adolescents. Ethical approval was provided by the institutional review board ethical committee .From December 2012 to September 2013 in Yunlin County, Taiwan, 12,348 residents underwent a community-based annual health checkup. We selected 10- to 19-year-olds for the study and obtained the informed consent from all of them. The exclusion criteria were: (1) < 10 or >19 years old; (2) incomplete health survey, anthropometric measurements, or laboratory data; (3) hepatitis B (HBV), hepatitis C (HCV), or both; (4) prior hepatic surgery; (5) habitual alcohol drinking; (6) liver cirrhosis, nodules, or tumors revealed by ultrasound scan. We finally enrolled 1210 participants  was calibrated and then was used to measure blood pressure (BP) with a standard procedure and appropriate cuff size. Every participant’s BP was measured twice and recorded. If the difference between first and second BP value was >10 mmHg, BP was measured a third time. The mean BP value was calculated in twice closest BP value and hypertension was defined as an average systolic or diastolic blood pressure level that was in the 95th percentile or greater based on at least three separate readings under guideline of the National High Blood Pressure Education Program (NHBPEP)  and 95% CIs). The independent variables included in the analyses were age groups, sex, and BMI categories.SPSS 19.0  was used for all analyses. Numeric variables were expressed as mean ± standard deviation (SD) and categorical variables as number (percent). Continuous data for the study group and comparison group were compared using Student’s y-axis and the false-positive rate (1−specificity) was plotted on the x-axis.The cutoff value for the risk score was determined by using the receiver operating characteristic (ROC) curve procedure. The ROC curve was plotted for NAFLD; sensitivity was plotted on the P < 0.001) with similar portion of smoking and betel nut chewing compared with non-obese group. Higher BMI , body weight , and waist circumference  were observed in obese group; however, the BP or laboratory data were insignificantly different.The data of 616 (50.1%) females and 594 (49.1%) males (mean (SD) age: 15.5 (2.8) years) were analyzed (Table P < 0.001)  of the participants were diagnosed with NAFLD Table . The per.8% of thP < 0.001) after final adjustments  predicting NAFLD was significantly higher than the WHR AUC   per standard deviation (SD), and the cutoff value was 0.469 for 10- to 19-year-olds.Although many studies have evaluated the correlation between the WHtR and central obesity, this is the first study to reveal a strong relationship between the WHtR and NAFLD in children and adolescents. The participants with a higher WHtR value had a 2.6-fold higher risk of NAFLD  in the autopsy study . The preWHtR is a better predictor for NAFLD than the WHR in some reasons: (1) more accurate sagittal and upper abdominal fat distribution; (2) hip circumference reflects different body components, such as fat, muscle, as well as bone mass. A WHtR threshold level of 0.5 has been proposed and recently validated . Weili aFeldstein and colleagues  reportedA liver biopsy has been a gold standard in the clinical diagnosis of NAFLD, but it is not well suited for screening or monitoring children because it is invasive and expensive, and it can cause complications in a pediatric physical examination. Ultrasonic sensitivity is acceptable, and interoperative differences can be reduced by experienced ultrasound users or by a review. A small regional study will limit the accuracy of the findings, but a large nationwide study will increase it. Nambiar and colleagues  had repoDespite some limitations, our study showed that the WHtR is strongly associated with fatty liver in children and adolescents. In addition to dietary education and exercise to prevent childhood obesity, using the WHtR simply as a primary screening tool should increase early detection of NAFLD and assess cardiometabolic risks before puberty.


Pinelliae Rhizoma (PR) is a commonly used Chinese medicinal herb, but it has been frequently reported about its toxicity. According to the traditional Chinese medicine theory, processing can reduce the toxicity of the herbs. Here, we aim to determine if processing reduces the toxicity of raw PR, and to explore the underlying mechanisms of raw PR-induced toxicities and the toxicity-reducing effect of processing. Biochemical and histopathological approaches were used to evaluate the toxicities of raw and processed PR. Rat serum metabolites were analyzed by LC-TOF-MS. Ingenuity pathway analysis of the metabolomics data highlighted the biological pathways and network functions involved in raw PR-induced toxicities and the toxicity-reducing effect of processing, which were verified by molecular approaches. Results showed that raw PR caused cardiotoxicity, and processing reduced the toxicity. Inhibition of mTOR signaling and activation of the TGF-β pathway contributed to raw PR-induced cardiotoxicity, and free radical scavenging might be responsible for the toxicity-reducing effect of processing. Our data shed new light on the mechanisms of raw PR-induced cardiotoxicity and the toxicity-reducing effect of processing. This study provides scientific justifications for the traditional processing theory of PR, and should help in optimizing the processing protocol and clinical combinational application of PR. Pinellia ternata (Thunb.) Breit., is a traditional Chinese medicinal herbShen Nong Ben Cao Jing , a book of 2,000 years ago. PR is commonly used in traditional Chinese medicine (TCM) prescriptions to manage cough, phlegm, vomiting and cancer2etc467921112Pinelliae Rhizoma (PR), the dried tuber of Chinese medicinal materials (CMMs) need to be processed to become decoction pieces before they are prescribed in the clinic or used for producing proprietary drugs. According to the TCM theory, processing can reduce the toxicity of the herbs15Because of the multiple chemical components and the multi-target nature of Chinese medical herbs19p < 0.01); PRZA group appeared body weight loss from day 4 (p < 0.05) and the severity was significantly less than that of raw PR group (p < 0.01)  . In addip < 0.01) (p < 0.05). Both raw PR and PRZA did not significantly affect the serum levels of ALT, AST, BUN and SCR . These results indicated that raw PR caused cardiotoxicity, and PRZA was less toxic than raw PR. No obvious liver and kidney toxicities were observed in both raw PR- and PRZA-treated rats.The serum levels of CK, CK-MB and LDH in raw PR group were all significantly higher than that in control group ( < 0.01) , indicatHistopathological damages were observed in the heart tissues of raw PR-treated rats with rupture and necrosis of the cardiomyocytes, and inflammatory cells infiltration, while, the damages were relatively mild in PRZA-treated rats . Liver aSix extracts from a random serum sample were continuously injected to the LC-MS system to evaluate the repeatability of the analytical method. Four common extracted ion chromatograms (EICs) shared by these injections were selected according to their different chemical polarities and m/z values. The relative standard derivations (RSDs) of these peaks were 4.04–14.51% for peak areas and 0.03–0.98% for retention times. These results indicated that our established LC-MS method was repeatable.The LC-MS system stability for the large-scale samples analyses was demonstrated by the test of pooled QC samples. The peak areas, retention times and mass accuracies of four selected EICs in four QC samples showed good system stability. RSDs of the four peaks were 4.84–14.08% for peak areas, 0.02–1.01% for retention times, and 0.12 × 10–04%–0.87 × 10–04% for mass accuracies. These data indicated that the LC-MS system was stable.2X = 0.138, R2Y = 0.937, Q2 = 0.753), there was obvious separation among the control, raw PR and PRZA groups. Top 200 significant ions were selected for metabolite identification. Among them, 10 significantly altered metabolites were identified and were shown in Typical base peak intensity (BPI) chromatograms of the serum samples were obtained for vehicle-, raw PR- and PRZA-treated rats . Based oSeven and eight significantly altered metabolites were identified in the serum samples of raw PR- and PRZA-treated rats, respectively . ProlineTo further understand the correlation between the candidate metabolites and the biological association networks, we performed bioinformatics analysis using the IPA software. A network was built based on the seven differential metabolites identified in the serum samples of raw PR-treated rats. The established network functions included amino acid metabolism, lipid metabolism, small molecule biochemistry, cell-to-cell signaling and interaction, as well as vitamin and mineral metabolisms. The canonical pathways included proline, uracil and lysine degradations, the TGF-β and mTOR signaling pathways, as well as serotonin biosynthesis . SimilarPrevious studies suggested that leucine up-regulation could activate mTOR signaling2123262829p < 0.05) (23p < 0.05), while, no significant difference in the protein expression level of TGF-β1 was observed between control and PRZA groups  that potentially involved in raw PR-induced cardiotoxicity, we examined the expressions of the critical proteins in these two pathways in rat heart tissues. Western blot analyses showed that raw PR significantly decreased the phosphorylation of mTOR  compared to the vehicle ( < 0.05) . Previou 0.05) 23, which sA groups . Studiesp < 0.01), suggesting that raw PR caused oxidative stress in rats. While the MDA content in PRZA group was lower than that in raw PR group (p < 0.01), and no significant difference was observed in control and PRZA groups, suggesting that processing reduced the oxidative stress-causing effects of raw PR or processing enabled the herb to possess free radical scavenging property. These findings further suggest that free radical scavenging may be responsible for the cardiotoxicity-reducing effect of processing.To verify the predicted molecular network function, free radical scavenging that may involve in the toxicity-reducing effect of processing, we determined the MDA contents in the serum samples of vehicle-, raw PR- and PRZA-treated rats. MDA is a classic biomarker of oxidative stress, which is produced by free radicals in the body. MDA is widely used as an indicator of the free radical levelTaken together, all above results suggest that inhibition of mTOR signaling and activation of the TGF-β pathway contributed to raw PR-induced cardiotoxicity, and free radical scavenging was, at least in part, responsible for the toxicity-reducing effect of processing. The characteristic metabolites and the metabolic regulatory networks of raw PR and PRZA were summarized in PR is toxic, it has irritant, teratogenic, carcinogenic, mutagenic, reproductive and organ toxicities. Many researchers have explored its organ toxicities, and found that PR could cause liver, kidney and heart toxicities. Intragastric administration of the water extract and the acid-water percolating liquid of PR could cause acute renal and liver damage and increase the activity of ALT and AST in mice3233According to the TCM theory, processing can reduce the toxicity of the herbs. The theory has been supported by modern studies35291115A total of 10 significantly altered metabolites were identified in the serum samples of vehicle-, raw PR- and PRZA-treated rats . Among tTwo characteristic metabolites leucine and 5-HT were only up-regulated in raw PR group. Leucine, one of the branched-chain amino acids, is commonly catabolized in cardiac muscle and highly effective in activating mTOR signaling213921Serotonin, also named as 5-HT, has growth-promoting effect on cardiac myocytes in physiological status2626Previous study has demonstrated that the toxicity mechanisms of raw PR-induced toxicity and the toxicity-reducing effect of processing were related to inflammation45etc. Studies suggested that PABA prohibits the generation of free radicals by inhibiting the nitric oxide formation3050Among the 10 significantly altered metabolites in the experimental groups, three metabolites kynurenine, PABA and tyrosine were only up-regulated in PRZA group. All these three metabolites have free radical scavenging properties282928in vivo. Previous studies suggested that the higher serum KMTB level, the more free radical generation5558Other five metabolites including KMTB, saccharopine, dihydrouracil, dhS1P and proline were identified to be altered in both raw PR and PRZA groups. Alterations of these metabolites might be responsible for the common toxic or therapeutic effects of raw PR and PRZA. Specifically, KMTB is a keto form of the sulfur-based amino acid methionine52In summary, we demonstrated that processing with ginger juice and alumen significantly reduced the cardiotoxicity of raw PR, which supported the TCM theory “processing can reduce the toxicity of raw PR”. Inhibition of mTOR signaling and activation of the TGF-β pathway contributed to raw PR-induced cardiotoxicity, and free radical scavenging might be responsible for the toxicity-reducing effect of processing, which shed new light on the mechanisms of raw PR-induced cardiotoxicity and the toxicity-reducing effect of processing with ginger juice and alumen. This study provides a scientific justification for the traditional processing theory, and should guide rational and safe clinical applications of PR by helping in optimizing its processing procedure and clinical compatibility. In addition, this study further suggests that metabolomics is a powerful approach for deciphering the complex mechanisms of herbal toxicities.LC-MS grade acetonitrile, methanol and formic acid (FA) were purchased from RCI Labscan Ltd. (Thailand). Leucine enkephalin (spectroscopic grade) and all chemical standards were purchased from Sigma-Aldrich . Milli-Q water was prepared using a Milli-Q system . Antibodies against phospho-AKT (Ser473), AKT, phospho-mTOR (Ser2448), mTOR and TGF-β1 were obtained from Cell Signaling Technology . Anti-GAPDH was purchased from Santa Cruz Biotechnology . Goat anti-rabbit IgG, goat anti-mouse IgG and protein markers were supplied by Bio-Rad .Raw PR , originaZingiber officinale Rosc. It was collected from the market  and authenticated in accordance with the monograph of Zingiberis Rhizoma Recens in CP2010 by the corresponding author. The contents of volatile oil and 6-gingerol in the fresh ginger were 0.29% (mL/g) and 0.07%, respectively .Zingiberis Rhizoma Recens (fresh ginger) is the fresh rhizome of Preparation of PRZA: soaked raw PR in water until the center of the cut surface was devoid of a dry core, boiled for 6 h after adding 12.5 kg alumen and 25 L freshly squeezed ginger juice for each 100 kg of raw PR, then took out and driedPreparation of raw PR and PRZA suspensions: raw PR (200 g) or PRZA powder (200 g) was suspended in 600 mL 0.5% sodium carboxymethylcellulose (CMC-Na) solution to prepare 0.33 g/mL raw PR or PRZA suspension9ad libitum. After one week of acclimatization, rats were randomly divided into three groups (n = 6), and daily intragastrically administered equal volume of the vehicle, raw PR or PRZA powder suspension at a dosage of 3 g/kg/day for 14 consecutive days9A total of 18 male SD rats were obtained from the Chinese University of Hong Kong. They were reared under standard laboratory conditions , and fed with commercial food and tap water Blood samples were collected from the retro-orbital venous plexus on day 15 and centrifuged at 3500 rpm for 10 min after standing for two hours at 4 °C. The serum was then transferred into new tubes and stored at −80 °C for further analysis. A portion of the collected serum was used for routine laboratory analyses of serum creatine kinase (CK), creatinine kinase-mb isoenzyme (CK-MB), lactate dehydrogenase (LDH), serum creatinine (SCR), blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and malondialdehyde (MDA) according to the manufacturer’s instructions of respective commercial test kits. Another portion of 200 μL of serum was added to 400 μL of acetonitrile, and the mixture was vortexed for 30 s. After centrifugation at 13,000 rpm for 10 min at 4 °C, the supernatant was pipetted out and lyophilized for LC-MS analyses. All rats were sacrificed following blood collection. Fresh cardiac, hepatic and renal tissues were obtained and fixed in 10% neutral buffered formaldehyde at 4 °C for paraffin embedment. Organ samples (4 μm) were sectioned and stained with H&E, and finally examined by light microscopy.3 C18 column  with a column temperature set at 35 °C. The flow rate was 0.45 mL/min, and the mobile phase was composed of 0.1% FA in acetonitrile and 0.1% FA in water. The following gradient program was used: 1% acetonitrile for 0–1 min; 1–70% acetonitrile for 1–10 min; 70–99% acetonitrile for 10–11 min; 99% acetonitrile for 11–13 min; re-equilibration step for 4 min. The sample injection volume was 2 μL.LC-TOF-MS analyses were performed by using an Agilent-1200 LC system coupled with an electrospray ionization (ESI) source  and an Agilent-6540 Q-TOF mass spectrometer. Separation of all samples were performed on an ACQUITY UPLC T2) flow rate, 8 L/min; gas temperature, 350 °C; pressure of nebulizer gas, 35 psig; capillary voltage, 4500 V; fragmentor, 145 V; skimmer voltage, 65 V; scan range, m/z 50–1400. All analyses were acquired using the instrument mass spray to ensure accuracy and reproducibility. Leucine enkephalin was used as the instrument reference mass (m/z 556.2771) at a concentration of 50 fmol/μL with the flow rate 40 μL/min. Data profile was recorded at a speed of 0.15 s/scan and the scanning delay of 0.01s during analysis.Mass detection was operated in both positive and negative ion modes with the following setting: drying gas  sample was analyzed at the beginning, the end, and randomly through the analytical run to monitor the stability of sequence analysis. The typical batch sequence of serum samples consisting of consecutive analysis cycles of 1 QC serum sample (at the beginning of the study) follow by 6 unknown serum samples. Meanwhile, samples were analyzed in a random order for a normal good practice. An identical sequence was repeated to complete the total set of injections  analyzed in less than 1 day per mode64The LC-MS raw data were exported by Agilent Mass Hunter Qualitative Analysis Software . The data of each sample were normalized to the total area to correct for the MS response shift between injections due to any possible intra- and inter-day variations. The sum of the ion peak areas within each sample was normalized to 10000. Multivariate analysis was performed using the Mass Profiler Professional (MPP) software B.12.00 . PCA analysis was used for classification of the differential metabolic phenotypes, and PLS-DA analysis was used for identify the differential metabolites.http://www.ingenuity.com), which enables the discovery, visualization and exploration of molecular interactions to identify the biological mechanisms, pathways and functions most relevant to the experimental datasets. The proof-of-knowledge based IPA can be used to find out the biomarkers, and further predict treatment-affected biological pathways and networks based on these biomarkers. We utilized IPA “metabolic analysis” module to analyze the identified metabolites in raw PR and PRZA groups; and comparisons of the canonical pathways/biological networks between the two groups were carried out using the “comparison” module in IPA software. The scores associated with the canonical pathways analyses were calculated using logarithm of the p-value (Fisher’s exact test). Significances for biological functions were then assigned to each network by determining a p-value for the enrichment of the metabolites in the network for such functions compared with the whole Ingenuity Pathway Knowledge Base as a reference set.The KEGG compound numbers and the matched identified metabolites in raw PR and PRZA groups were set up as the identifiers of two datasets. Each dataset was uploaded into the IPA system .p < 0.05 was considered statistically significant.Results of body weights, biochemical assays and Western blot analyses were presented as mean ± SD. These data were analyzed by one-way ANOVA followed by the Dunnett’s multiple comparisons using GraphPad Prism version 5.0 . How to cite this article: Su, T. et al. Metabolomics reveals the mechanisms for the cardiotoxicity of Pinelliae Rhizoma and the toxicity-reducing effect of processing. Sci. Rep.6, 34692; doi: 10.1038/srep34692 (2016).


The risk factors for recurrence and death after radiofrequency ablation (RFA) for hepatocellular carcinoma (HCC) remain poorly known. This study was aimed to study the 10-year overall survival (OS) of HCC treated by ultrasound (US)-guided RFA and the risk factors for recurrence and death.n = 348) and no recurrence groups (n = 62).Between June 2005 and June 2016, 1000 patients with HCC treated by US-guided RFA at 4 hospitals in China; among them, 525 patients met the criteria for radical ablation and 410 had high AFP levels before RFA treatment. Clinical and biochemical factors were tested for association with recurrence and survival. Patients were divided into the recurrence (P = 0.006), and AFP response  were independently associated with the risk of recurrence of HCC after RFA. The median survival of the patients with and without recurrence after RFA was 54 (95% CI 45–58) and 62 (95% CI 48–80) months, respectively .The 5- and 10-year survival rates were 66 and 35%, respectively. Tumor size , albumin levels , prothrombin time , and α-fetoprotein levels  were independently associated with mortality after RFA for HCC. Tumor size , HBV-DNA , AFP levels before treatment (HR = 2.172, 95% CI 1.256–3.756, Tumor size, albumin, prothrombin time, and α-fetoprotein levels were independently associated with mortality after US-guided RFA for HCC, while tumor size and HBV-DNA were independently associated with recurrence. Patients with recurrence had a poorer survival compared with those without. Hepatocellular carcinoma (HCC) is the fifth most common malignant tumor in the world , and theFor selected patients, radiofrequency ablation (RFA) is an alternative approach that achieves survival rates similar to those obtained with surgery, but with fewer risks –11. IndeHowever, according to the literature, the recurrence rate could be higher with RFA than with surgery . Shiina Therefore, the present study summarizes 1000 Chinese patients with HCC treated by ultrasound-guided RFA over 11 years. Among them, 525 patients were considered as having received curative RFA.This was a retrospective study of 1000 consecutive patients with HCC treated at the First Affiliated Hospital of Soochow University , the Changzhou Third People’s Hospital , the Xuhui District Center Hospital , and the People’s Hospital of Maanshan  between June 2005 and June 2016. This study was approved by the Ethics Committee of the People’s Hospital of Maanshan.Inclusion criteria were (1) histological diagnosis of HCC; (2) focal lesion with arterial enhancement and portal venous washout on CEUS, computed tomography (CT), or magnetic resonance imaging (MRI); and (3) complete ablation of the target lesions. Exclusion criteria were (1) portal vein tumor thrombus; (2) tumor margin close to portal vein branch by <1 mm; or (3) distant metastases. Finally, 525 patients met the criteria.The pretreatment assessment of each patient included renal and liver function, complete blood count (CBC), prothrombin time, ascites, AFP, HBV DNA, and electrocardiogram. Pretreatment image examination included ultrasound, CEUS, CT, and/or MRI of the abdomen.This study was in the field of interventional ultrasound. Therefore, according to the BCLC diagnostic criteria of HCC, patients with 2 typical vascular imaging presentations, or with 1 dynamic typical imaging presentation and AFP >400 ng/ml or confirmed by histopathological examinations were diagnosed with HCC. As the RFA was performed under ultrasound guidance, ultrasound was chosen as the main method to determine the maximum diameter of the tumor, locations, sizes, shapes, and extensions.The Esaote Mylab 90  and ACUSON S2000  were used for guidance, using the Sonovue ultrasound contrast agent . Intramuscular injection of 50 ml of dolantin and 1 ampoule of diazepam was performed before the operation. Fentanyl was used for sedation and analgesia during the operation under monitoring. Lidocaine was used for subcutaneous injection and liver capsule injection. The patients were placed in the supine position and were asked to cooperate with the clinicians during the puncture by breathing appropriately.Preoperative planning including evaluation of all comparative imaging studies and careful ultrasound scan was performed to identify the tumor size and target access. A Valleylab Cooltip  17-gauge single electrode with a 3-cm effective tip was used for lesions <2 cm in diameter. For tumors >2 cm, a 15.5-gauge Olympus Celon bipolar electrode T30 + T30/T40 + T40  was used, while being sure that the electrode tips distance inside the tumor was <25 mm –17. AttePatients underwent ultrasound or CEUS of the liver 1 month after RFA, as well as AFP and liver biochemistry assessment. Thereafter, surveillance of the ablated zone and residual liver was assessed by ultrasound and CEUS every 3 months.If AFP continued to increase during follow-up, MRI or CT examination was performed. Positron emission tomography (PET) was used for some patients. Patients showing changed in the RFA area, unclear boundaries, nodular enhancement in the arterial phase, and new nodules were considered with intrahepatic recurrence of HCC. The follow-up for the evaluation of local treatment effectiveness of RFA was performed according to the revised response evaluation criteria in solid tumors (mRECIST). Of the 525 cases of HCC patients with RFA treatment, 1 month after the procedure and immediate postprocedure, radiographic modified RECIST Criterion for evaluation of short-term efficacy. Local complete response rate was 92% (483 cases), partial response rate was 8% (42 cases), non response rate was 0%, and the rate of progression was 0%. In 42 cases of local partial response in postoperative patients after 1 month assessed by radiology and criteria for radical RFA again, receiving completely RFA ablation again, completely response rate 100%. Total ablation times is 567 .Follow-up was censored on 31st May 2016. Two patients were lost to follow-up as they did not respond to telephone follow-up. The data of these two patients were considered as censored data on their last available follow-up.9/L), serum AFP (<50 vs. 50–100 vs. 100–400 vs. >400 ng/ml), HBV virus load , and prothrombin time (<14.5 vs. >14.5 s). SPSS 16.0  was used for statistical analysis. Two-sided P values <0.05 were considered statistically significant.Continuous variables were presented as mean ± standard deviation. Categorical data were presented as proportions. Survival was analyzed using the Kaplan-Meier method and the log-rank test. A multivariate Cox analysis was used to determine the independent factors associated with recurrence and survival. For multivariate analysis, continuous variables were categorized: age (≤50 vs. >50 years), tumor size (≤2 vs. 2–3 vs. 3–5 vs >5 cm), total bilirubin (<17 vs. 17–34 vs. >34 μmol/L), serum albumin (<33 vs. 33–37 vs. 37–41 vs. >41 g/L), platelet count , 8 patients received surgical treatments for recurrent tumor, and 12 patients received liver transplantation.Figure P = 0.002), albumin levels , PT , and AFP levels  were independently associated with mortality after RFA for HCC.Table P = 0.0001) and HBV-DNA levels , AFP levels before treatment , and AFP response  were independently associated with the risk of recurrence of HCC after RFA.The 410 patients with HCC and high AFP levels before RFA treatment were categorized into the recurrence and no recurrence groups. Table In order to reveal the median survival time differences between recurrence and non recurrence group in HCC with the high preoperative level AFP, exclude 115 cases with normal preoperative AFP level , The 410 patients with HCC and high AFP levels before RFA treatment were categorized into the recurrence (348cases) and no recurrence groups (62 cases).P = 0.04).Figure In the present study, no death was attributed to RFA. Three patients had life-threatening complications requiring surgical interventions or blood transfusion. Among these three patients, one was with major thoracic hemorrhage, who recovered after the hematocele was cleared by surgery; one was with major abdominal hemorrhage, who recovered after percutaneous ultrasonography-guided gel injection to the bleeding site and blood transfusion; and the other patient was with subcutaneous hemorrhage accompanied with scrotum hematoma after RFA, who recovered after local pressuring and conservative treatment. In these three patients, the hemorrhage occurred at the early stage after RFA; their tumor size was relatively large, and the hemorrhage was caused by the repeated punctures during the overlapped ablation with a single electrode.Three patients were with subcutaneous metastases. All three patients received repeated preoperative 18G automatic biopsies to obtain specimens for multiple times.One patient suffered from hepatic infarction. This patient manifested postoperative fever and local pain. Ultrasound showed that there was no blood perfusion in the hepatic segments in the left liver lobe, and biopsy confirmed local infarction. The RFA probably damaged the small arteries, which induced ischemic necrosis of local intrahepatic bile ducts and gas accumulation.Eight patients were with biloma at the site of RFA. In these patients, repeated punctures damaged the small bile duct and induced a bile pool at the site of necrosis area caused by RFA. After the formation of the biloma, the CEA and CA199 in these patients increased to different degrees. None of these patients required specific treatments. Imaging examinations 6–12 months after RFA showed that the bilomas were absorbed, and CEA and CA199 were both decreased.No gastric-intestinal perforation was found in this study, which could be associated with the use of a straight rod ablation electrode, and the application of prophylactic measures for ascites during RFA. Two patients were with massive pleural effusion. One patient recovered after catheter drainage and the other recovered after fluid aspiration. Ascites were alleviated after conservative treatments.All patients were with different degrees of postoperative local pain. The body temperature and liver enzyme level of all patients were elevated at different degrees 24 h after operation.One patient was with minor local skin burn. This patient had received implantation of a metal graft due to femoral head necrosis. When the cool-tip skin electrode moved upward to the back, the overlapped ablation caused local skin reddening and swelling, as well as slight skin burn. The patient recovered without specific treatment.The risk factors for recurrence and death after RFA for HCC remain poorly known. Therefore, this study aimed to study the 10-year OS of HCC treated by ultrasound-guided RFA and the risk factors for recurrence and death. Results showed that tumor size, albumin, PT, and AFP levels were independently associated with mortality after RFA for HCC, while tumor size, HBV-DNA, AFP levels before, and AFP response were independently associated with recurrence. Patients with recurrence had a poorer survival compared with those without.The BCLC suggest that liver tumor diagnosis and treatment monitoring can be performed using liver ultrasound and especially CEUS , 19. LeeIn the present study, the 5- and 10-year survival rates were 66 and 35%, respectively, which were similar to that of previous studies  –24. Of cIn order to improve the OS after local tumor ablation, it is a necessity to identify the risk factors for OS and recurrence. The present study showed that tumor size, albumin, prothrombin time, and α-fetoprotein levels were independently associated with mortality after RFA for HCC, while tumor size and HBV-DNA were independently associated with recurrence. These results are similar to those of previous studies. Indeed, Shiina et al.  showed tLiver tumor recurrence involves complex mechanisms and studies show that after HCC resection, the overall 5-year postoperative recurrence rate is 50–81% –28. The The present study is not without limitations. The sample size was relatively limited and from only four centers. The selection criteria may have introduced a selection bias, limiting the generalizability of the results. Only a limited number of factors were studies. Future studies could explore additional factors that could be involved in survival and recurrence, such as inflammatory markers. Additional studies are still necessary to adequately predict the prognosis of HCC after ultrasound-guided RFA.In conclusion, tumor size, albumin, PT, AFP levels were independently associated with mortality after ultrasound-guided RFA for HCC, while tumor size, HBV-DNA, AFP levels before treatment, and AFP response were independently associated with recurrence. Patients with recurrence had a poorer survival compared with those without. Preoperative high level of AFP HCC patients with postoperative recurrence is an important clinical poor indicator in the median survival time.


During ECMO management, a mucus plug was removed by bronchoscopy daily and 18 L of water was removed using diuretics, resulting in weight loss of 24 kg. The patient was weaned from ECMO on day 5, extubated on day 16, and discharged on day 21. The fundamental treatment for obesity hypoventilation syndrome in morbidly obese patients is weight loss. VV-ECMO can be used for respiratory support until weight loss has been achieved.The mortality rate for respiratory failure resulting from obesity hypoventilation syndrome is high if it requires ventilator management. We describe a case of severe acute respiratory failure resulting from obesity hypoventilation syndrome (BMI, 60.2 kg/m However2 presented to a local hospital to undergo upper gastrointestinal endoscopy for epigastralgia. The patient's oxygen saturation was approximately 80%, and he was admitted to the general ward. His condition worsened, and blood gas analysis revealed the following: pH, 7.093; PaCO2, 115.3 mmHg; and PaO2, 96.7 mmHg on 100% oxygen. He was intubated and mechanically ventilated. His primary physician performed Swan–Ganz catheterization, which revealed the following: pulmonary capillary wedge pressure, 19 mmHg; pulmonary artery pressure, 51/28 (36) mmHg; and cardiac index, 4.78 L/min/m2. Echocardiography revealed an ejection fraction of 45.7% and tricuspid regurgitation pressure gradient of 29 mmHg without evidence of organic valvular disease. Chest radiographs showed bilateral pulmonary edema and consolidation. The diagnosis was ARF resulting from OHS combined with cardiac failure and pulmonary hypertension. The patient's relatively long, untreated clinical course, atelectasis, and hydrostatic pulmonary edema might have developed before his admission. After 9 days of conventional therapy, his respiratory function showed no improvement; therefore, the patient was transferred to our intensive care unit. On admission, he was sedated with a respiratory rate of 39 breaths/minute, heart rate of 96 beats/minute, and blood pressure of 124/66 mmHg. The patient required a driving positive pressure of 15 cm H2O to generate a 400 mL (6 mL/kg) tidal volume. The fraction of inspired oxygen was 1.0; arterial oxygen partial pressure (PaO2) was 69.8 mmHg; PaCO2 was 52.1 mmHg; and pH was 7.383. Chest X-ray showed an enlarged cardiac silhouette and diffuse pulmonary infiltrates suggesting congestive heart failure and atelectasis. Given the patient's age and single-system organ failure, VV-ECMO was emergently instituted. Cannulation was performed via the right internal jugular vein with 25-French drainage cannulae  for access and via the right femoral vein with 19-French cannulae (Medtronic) for return. The ECMO circuit was an adult ECMO bypass custom tubing pack consisting of a Rotaflow® centrifugal pump  and two gas exchangers . We used two oxygenators in parallel to enlarge the membrane surface area. ECMO was initiated at a blood flow of 5.7 L/min and sweep gas flow of 5.0 L/min in 100% oxygen per oxygenator. The pre- and postoxygenator pressures were monitored continuously throughout the ECMO run. The lungs were rested with pressure-controlled ventilation at an inspiratory pressure of 5 cm H2O, positive end-expiratory pressure (PEEP) of 10 cm H2O, and FIO2 of 0.3 to 0.6 during the ECMO run, permitting tidal volumes of 100 mL support. The patient was heparinized to maintain an activated partial thromboplastin time of 50 to 80 seconds. Because of clinical signs of pulmonary congestion, diuretic and inotropic therapies (milrinone and dobutamine) were initiated, with some improvement in the hemodynamic parameters. After initiation of ECMO, the patient's hemodynamic status was stabilized and he was successfully kept in a negative hydration balance every day. Furosemide was administered at 0.1 mg/kg/min, and a negative fluid balance of −17946 mL was achieved during ECMO. Maintaining this negative balance, his body weight decreased from 167 to 143 kg and his pulmonary congestion improved with a tricuspid regurgitation pressure gradient of 17 mmHg. We also performed bronchoscopy and removed sputum daily, which improved atelectasis, compliance, and oxygenation.A male patient in his mid-40s with a BMI of 60.2 kg/m2 flow to the oxygenator; therefore, we decided to wean him off ECMO. After weaning off ECMO, his respiratory condition worsened without a high PEEP, and his PaO2 decreased; therefore, we weaned PEEP slowly. We removed the mechanical ventilation 11 days later. Using a 2 L/min nasal cannula, the blood gas analysis results were as follows: pH, 7.415; PO2, 115 mmHg; and PCO2, 39.8 mmHg. The patient was discharged from the hospital 5 days after extubation with a final body weight of 125.2 kg.On day 5, we performed a 2-hour ECMO off-trial. The patient tolerated the trial without OMany studies have shown increased morbidity and mortality in obese patients in the intensive care unit –8. Obesi2, developed severe ARF on the fourth day of postsurgical intervention, and VV-ECMO was performed [2, developed severe ARF because of sepsis after treatment of necrotizing fasciitis, and VV-ECMO was indicated [2 elimination through the native lungs decreases in obese patients. In such cases, two oxygenators are necessary for VV-ECMO. Applying two oxygenators in parallel diverts the circuit blood flow into two oxygenators, which reduces the blood flow through each oxygenator, resulting in much more efficient oxygenation. Moreover, the total surface area of the membrane oxygenator is doubled, increasing the total amount of CO2 removal through the VV-ECMO system.Ull et al.  reportederformed . The secndicated . Extremendicated –14. HoweThe primary treatments in our case involved controlling the patient's water balance to a negative balance and performing daily bronchoscopy. The use of furosemide resulted in adequate weight loss, which is the indicated treatment for OHS. We believed that we could successfully manage his water balance because VV-ECMO stabilized his hemodynamic status. During VV-ECMO, we achieved lung rest by reducing the ventilatory pressure, which reduced the intrathoracic pressure and increased the left and right cardiac output. During VV-ECMO, oxygenated blood provided from the ECMO system mixes with deoxygenated blood from the native circulation in the right atria, and preoxygenated blood then travels to the pulmonary circulation. This oxygenated blood in the pulmonary circulation leads to pulmonary vascular dilatation, which in turns leads to decreased pulmonary vascular resistance, reduces septal displacement, and improves left ventricular function , 16.This allowed us to remove approximately 18 L of water, which treated his original obesity-related pulmonary hypertension. The secondary treatment in our case was daily bronchoscopy. We were able to perform bronchoscopy safely because the patient's oxygenation was controlled by the VV-ECMO. Frequent bronchoscopy during ECMO is required for bronchial toilet and to remove sputum that can cause atelectasis.2 after the start of VV-ECMO contributed to the decrease in the mean pulmonary artery pressure [The main limitation of this case report is that a pulmonary arterial catheter could not be used because of the high risk induced by extreme obesity; therefore, the pulmonary arterial pressure and cardiac output could not be measured directly. However, based on its physiological effects, we believe that VV-ECMO effectively reduced the intrathoracic pressure and pulmonary vascular resistance, which stabilized our patient's hemodynamic status and permitted the removal of large volumes of water from the body. Miranda et al. reported that initiation of VV-ECMO decreased the mean pulmonary artery pressure and increased the cardiac index and mixed venous oxygenation . Additiopressure .Obese patients are viewed as more complex to manage by ECMO. However, it is possible to use VV-ECMO for life-threatening respiratory failure if percutaneous cannulation is possible. The use of two membranes is also an important strategy for obese patients.In conclusion, VV-ECMO is a useful modality for managing severe ARF resulting from OHS because it may enable sufficient gas exchange and improve pulmonary circulation by reduction of pulmonary vascular resistance until diuretic weight loss is achieved.


Risk factor control after a coronary event in a recent European multi-centre study was inadequate. Patient selection from academic centres and low participation rate, however, may underscore failing risk factor control in routine clinical practice. Improved understanding of the patient factors that influence risk factor control is needed to improve secondary preventive strategies. The objective of the present paper was to determine control of the major risk factors in a coronary population from routine clinical practice, and how risk factor control was influenced by the study factors age, gender, number of coronary events, and time since the index event.A cross-sectional study determined risk factor control and its association with study factors in 1127 patients (83% participated) aged 18-80 years with acute myocardial infarction and/or revascularization identified from medical records. Study data were collected from a self-report questionnaire, clinical examination, and blood samples after 2-36 months (median 16) follow-up.p < 0.01) and obesity (p < 0.05) with increasing time since the event was observed.Twenty-one percent were current smokers at follow-up. Of those smoking at the index event 56% continued smoking. Obesity was found in 34%, and 60% were physically inactive. Although 93% were taking blood-pressure lowering agents and statins, 46% were still hypertensive and 57% had LDL cholesterol >1.8 mmol/L at follow-up. Suboptimal control of diabetes was found in 59%. The patients failed on average to control three of the six major risk factors, and patients with >1 coronary events (p < 0.001) showed the poorest overall control. A linear increase in smoking .Registered at ClinicalTrials.gov (ID The online version of this article (doi:10.1186/s12872-016-0387-z) contains supplementary material, which is available to authorized users. Over the recent years, there has been a decline in mortality rates worldwide  leaving Even though the abovementioned studies provide valuable data on the quality of secondary prevention, patient selection could potentially be a matter of concern. In EuroAspire IV  patient The reasons for unhealthy lifestyle and low risk factor control are complex and poorly understood and the identification of patient and healthcare factors of importance for coronary risk profile remains a public health priority . The oven = 28), psychosis (n = 18), drug abuse (n = 10), short life expectancy (n = 136), dead (n = 160), not able to understand Norwegian (n = 44), and other (n = 27). Of the remaining 1366 invited patients, 1127 (83%) participated in attending a clinical visit and completing a comprehensive questionnaire [The design, methods, and baseline characteristics of the NOR-COR Study have been described elsewhere . BrieflyThe study was conducted at two Norwegian hospitals (Drammen and Vestfold) with a total catchment of 380,000 inhabitants corresponding to 7.4% of the Norwegian population. The catchment area has a representative blend of city and rural districts and is representative of Norwegian geography, economy, age distribution, morbidity, and mortality . The carThe study was approved by the Regional Committee of Ethics in Medical Research. All patients signed a written informed consent prior to study participation.Medication and co-morbidity at the index event were registered from the hospital medical records. Cardiovascular medication, risk factors and study factors at follow-up were obtained from the self-report questionnaire, the clinical examination and blood-samples. All blood samples were analysed at Drammen hospital. Diet was assessed by a brief diet questionnaire including seven selected quantitative questions (the frequency of intake of different types of foods and beverages). These questions have been validated against intake of matching food groups . Time siSmoking: categorized as current, former or never.Overweight and obesity: Body weight was measured in light clothes without shoes . Height was measured using a wall fixed mechanical measuring rod . Overweight and obesity was defined as body mass index (BMI) >25 kg/m2 and >30 kg/m2, respectively. Waist circumference was measured with a non-stretchable tape . A waist circumference above 94 cm and 102 cm in men and above 80 cm and 88 cm in women was defined as central overweight and obesity, respectively.Physical activity: assessed by frequency , intensity , and duration . Low physical activity was defined as less than moderate activity level for 30 min of 2-3 times a week.Blood Pressure (BP) control: BP was measured after standard procedures using a Welch Allyn digital sphygmomanometer. Unfavourable BP control was defined as BP > 140/90 mmHg (>140/80 mmHg in diabetics).Blood-sugar control: assessed by HbA1c analysed - Tosoh G8, Ca, US. Unfavourable blood sugar control was defined as HbA1c ≥6.1% (non-diabetics) and >7.0% (diabetics) [abetics) .Low density lipoprotein (LDL) cholesterol: analysed - Architect ci16200, Ca, US. Elevated LDL cholesterol was defined > 1.8 mmol/l [8 mmol/l .Statistical analyses have been performed using SPSS version 21. Parametric descriptive statistics were applied. Binary logistic regression analysis was used to calculate odds ratios (ORs) for unfavourable risk factor control and adjusted for age, gender, number of coronary events, and time since the index event.General Linear Model (ANCOVA) was used to estimate marginal means for number of unfavourable risk factors  by age, gender and number of coronary events with all independents controlled as dummies simultaneously, and with time since event entered as a linear covariate.Baseline characteristics are presented in Table The prescription rate of recommended preventive drugs  was highThe proportion of unfavourable risk factors at follow-up was high Fig. . Of thosp < 0.001) and physical inactivity  were significantly more frequent in patients with low vs. high education, while overweight, unfavorably blood pressure, blood glucose and LDL cholesterol control were not.Current smoking  had the poorest overall risk factor control.The estimated marginal means for number of unfavourable risk factors  by gendep < 0.001), obesity (p < 0.001) and elevated HbA1c (p < 0.01) were significantly more frequent in the younger patients, while inadequate BP control (p < 0.001) was more frequent with increasing age. ORs for current smoking, low physical activity, and LDL >1.8 mmol/l were significantly higher in women compared to men. ORs for low physical activity, obesity, and elevated LDL cholesterol were significantly higher in patients with several coronary events. There were no significant differences in ORs between the four time groups since the index event, but for smoking (p < 0.01) and obesity (p < 0.05) the test for linear trend was statistically significant with reduction in risk factor control with increasing time since the event.Multi-adjusted odds ratios (OR) for unfavourable coronary risk factors at follow-up by age, gender, number of coronary events, and time since the index event are shown in Additional file Of the CHD patients included from a high income country with a well-developed health care system , the majThere are certain limitations of the study. First, the coronary risk factors and study factors were measured at one point in time and thus are prone to measurement and recall bias. Moreover, diet is calculated by a semi-quantitative measure, only. Our questions about physical activity have been validated , and we The latest EuroAspire Study  had simiWe found a higher use of anti-platelets (97% vs. 94%), and statins 93% vs. 86%), but lower use of beta-blockers (72% vs. 83%) and ACEI/ARBs (50% vs. 75%) compared with EuroAspire IV [%, but loLarge studies from different regions worldwide have also demonstrated that 30-80% of CHD patients had diabetes, were obese, and had LDL cholesterol and BP above the recommended targets –11. In tThe reasons for the low adherence to secondary prevention are complex and multi-factorial , 23. LowThe youngest patients had the highest proportion of unfavourable lifestyle factors, and this might have contributed to an early onset of CHD. The positive trend in acute myocardial infarction event rates and recurrences from 1994-2009 in Norway were mostly seen among patients older than 65 years, whereas less favourable trends were observed among younger patients, in particular women , 31. In Medical and psychosocial factors may act as barriers to lifestyle changes, treatment adherence and may moderate the effects of cardiac rehabilitation . The preThe majority of CHD patients from a routine clinical practice in a representative Norwegian population did not achieve control of the major coronary risk factors. The measured non-modifiable study factors had different impact on the risk factors, and the poorest overall control was found in patients with several coronary events. It is concerning that secondary prevention of CHD fails in a country with a well-developed health care system. Further knowledge about factors associated with poor risk factor control and strategies for implementation of these factors are strongly needed to improve secondary prevention. Even modest advances will provide major health benefits.


E. Coli O55:B5 and E. Coli O26:B6. The limit of detection (LOD) could reach around 5 ng/mL at optimized condition. The method was applied successfully to the determination of LPS levels in tap water and wastewater, and demonstrated sensitive, reproducible and reliable results.This study focuses on one of the key environmental threats, endotoxins, also known as lipopolysaccharides (LPS). A capillary electrophoresis method in combination with laser induced fluorescence (LIF) detection was developed for the analysis of endotoxins from 16 different bacterial strains. LPSs were derivatized with the amino-reactive fluorescent dye, fluorescein isothiocyanate (FITC), separated by capillary zone electrophoresis (CZE) under the optimized conditions with the use of 50 mM sodium tetraborate buffer (pH 9.30), and detected by LIF detector. To improve the sensitivity of CZE-LIF detection for the determination of trace amounts of endotoxins and to remove possible interference materials in environmental samples, a solid phase extraction (SPE) pre-concentration technique was applied successfully. The SPE targeted at polysaccharide moieties of LPSs and showed LPS enrichment effects too. CE migration time could also reveal the O-antigen chain lengths of LPSs. This CE method and SPE pretreatment showed linearity at 99.84%, and repeatabilities at 8.44% and 11.0% for endotoxins from  The outer membrane serves not only as a barrier for environmental stresses and various toxic substances, such as antibiotics, but also as nutrient transporter2. The term LPS usually refers to the purified form; while endotoxin refers to the LPS attached on the bacterial membrane4. LPS molecules which constitutes about 3.6% of a bacterial cell, occupy approximately three quarters of the surface area of the outer membrane of Escherichia coli5. LPSs are released from the bacterial surface during growth, cell division and lysis6. As such, endotoxins are universal toxic inflammatory agents in the environment.Endotoxins, also called Lipopolysaccharides (LPS), is the major constituent of the outer membrane of gram-negative bacteria8. LPS in the infected host’s blood stream induces high fever, leukocytosis, intravascular coagulation, multi organ failure, septic shock and even death9. Even 1–10 ng (10–100 EU)/kg body weight (intravenously) of endotoxin can induce fever10. It is also found that endotoxins can augment the toxicity of some chemicals such as microcystins12. Endotoxins have been considered as the main cause of pyrogenic reactions that might take place during the biotherapeutics administration13. In the Pharmacopoeia of the United States, China and the United Kingdom, a limit of 0.25 EU/mL has been established for injection water10. Four separate studies had indicated that endotoxin levels in surface water range from <1 to 1049 ng /mL10. So far, the risks associated with endotoxins in drinking water are not well-quantified. To date, there have been very few publications about the endotoxin levels in drinking water supplies, and no clear guideline values for endotoxin concentration have been established yet.Endotoxins are toxic because they can influence many cellular and humoral mediated systems15. However, large numbers of microorganisms are likely to be present in used water and secondary effluents. Therefore, removing these substances in reclaimed or reused water is extremely important16. During water treatment, the monitoring of endotoxin concentrations can promote the safety level. Endotoxin as typical toxic chemicals generated by microorganisms is one of the emerging contaminants. It has reportedly shown high activity in water treatment plant effluents ranging from 30 to 2,000 ng/mL17. Furthermore, it is difficult to sterilize the entire drinking water distribution system. Bacteria may enter the pipe network, attach to the pipe wall and become part of a biofilm. Such bacteria can pose a potential threat to public health if endotoxins are produced.Used water treatment and reuse are extremely important in the sustainable development of water resources18. Generally, the structure of the hydrophobic glucosamine-based lipid A is conserved during the biochemical synthesis20 and the lipid A part is responsible for the biological activity and toxicity of the LPS. The core polysaccharide region, which is usually identical for large groups of bacteria, consist of two or three 2-keto-3-deoxyoctulosonic acids (KDOs) and 8–15 monosaccharides21. The O-antigens, which is responsible for its antigenicity and serotype-specific immunogenicity, are composed of various numbers of repeating oligosaccharide subunits, due to different biosynthesis19. Endotoxin monomers have a molecular weight of approximately 10 to 20 kDa. However, due to its amphipathic structures, LPS molecules have strong tendencies to form aggregates into vesicles in aqueous solution with a micellar weight around one million daltons22. The smooth-type LPSs, containing all three components in the structure, usually can be isolated from the wild-type bacterial strains. The absence of the O-specific region (due to mutation) results in a shortened and more hydrophobic bacterial strain, with a “rough” colony morphology, are normally called rough-type LPSs21.Although the complex amphiphilic molecular structures of endotoxins vary among different bacterial strains, endotoxins share a common architecture typically consisting of three components, lipid A, core oligosaccharide and O-antigen polysaccharide. The polysaccharide chain is covalently bound to a lipid region, termed “lipid A” by Westphal and his associates22. The mechanisms for both rabbit pyrogen test and LAL test are attributed to the biologically reactive lipid A region, which is regarded as the “endotoxic” component of LPS and is responsible for the biological effects9. The in vivo rabbit pyrogen test suffers from ethical and economic problems22 and has gradually been replaced by the in vitro LAL test22, which is now the most popular method for the analysis of endotoxins. Limulus amoebocytes (cells found in the hemolymph of the horseshoe crab) can form an intracellular gel in the presence of endotoxins and turn the reaction medium yellow in the kinetic chromogenic LAL assay. Although LAL assay is extremely sensitive with detection limit as low as 100 picograms per milliliter23, it shows many shortcomings as well3. For example, temperature, pH, ions, and even reagent chemicals can influence the LAL results3. Besides, the non-specific methods would also give false positive results due to reactions with other microbial products that trigger the LAL reaction, such as peptidoglycan from gram-positive organisms, release of (1-3)-β-D-glucans9, and D-glucose polymers from cell walls24. Furthermore, the LAL method only provides the general activity of endotoxins in the sample, instead of the detailed structure or distribution profile25. LAL method measures endotoxin concentration indirectly, but endotoxins that possess diverse structures and originate from different bacterial serotypes and their mutants may introduce variations. These structural differences will directly affect the endotoxin reactivity and toxicity analyses in the LAL test26. Moreover, endotoxins have the ability to form agglutinate or micelles27. These micellar structures reduce their reactivity relative to LAL, rendering inaccurate LAL results in reflecting the true concentrations of endotoxins. A reliable analytical method for endotoxin analysis will serve as an useful tool in the monitoring of drinking water purification processes and water reclamation plants.There are two methods recognized by the Food and Drug Administration (FDA) for determining intact LPSs. They are rabbit pyrogen test (US Pharmacopoeia rabbit test) and Limulus amoebocyte lysate test (LAL-test)28, gas chromatography coupled with mass spectrometry (GC/MS)29 and capillary electrophoresis (CE)31. Among these techniques, CE provides rapid and highly efficient separation, and hence is especially suitable for the analysis of large molecules32. The analysis of LPS, either after O-deacylation or mild acid hydrolysis, has received considerable attention. However, to our knowledge, the application of CE with LIF detection for the analysis of intact LPS molecules has not previously been extensively studied. Given that LPS lack natural optically active groups in the chemical structure, detection by the commonly used UV absorbance detector proves challenging. Freitag and coworkers33 reported a CE with indirect UV-detection method, using a strongly UV-active buffer to obtain “negative” LPS peaks. However, the sensitivity of the UV absorbance detector is limited. Therefore, LIF detectors, being more sensitive by at least three orders of magnitude compared to the UV absorbance detection, are preferable for detecting trace amount of LPS34. Since LPS does not possess native fluorescence properties, chemical derivatization with a fluorescent dye is required. Fluorescein isothiocyanate Isomer 1 (FITC) is one of the most widely used fluorescent derivatizing agent for the analysis of amino-group containing analytes, primarily due to its high quantum yield (0.92).Other methods have been explored for quantifying endotoxins, such as enzyme-linked immune sorbent assayFor accurate, reliable and robust analysis of the endotoxins levels in water samples in complicated matrices, a sample preparation method is necessary. Few reports and commercial products were available so far. The sample preparation method serves mainly two purposes. Firstly, the sample pretreatment should be suitable for many different sample matrices. As a result, the pretreatment method in this study can be used to extract the targeted compounds, i.e. endotoxins, from other interference materials. Secondly, it will be helpful to enrich endotoxins during the preparation of the final injection solutions to increase the sensitivity of the analytical method. Solid phase extraction (SPE) can be employed to eliminate the influence of matrix components in real samples. So far, SPE study for endotoxins has not been extensively investigated. This work aimed at developing analytical methods for separating and determining endotoxins in water samples by CE coupled to laser induced fluorescent detection (LIF). In combination with the solid phase extraction method, the CE separation method could be used to determine endotoxin concentrations in both simple water matrices and complex water matrices.35. The derivatization reaction normally takes place for more than 12 hours due to the extremely slow kinetics.FITC is an amine-reactive fluorophore, which reacts with primary and secondary amino groups to form thiourea. This reaction is sensitive to pH and temperature and the optimum reaction condition is reported to be at pH 9.0, and 20–25 °C38. Although these amino groups appear to be present in sub-stoichiometric amounts, they make covalent bonding with FITC possible. Furthermore, since the lipid A and the core domain show the greatest similarity among various bacterial strains, labeling this part of the LPS molecule would constitute the most general strategy for LPS detection.Earlier studies had confirmed the presence of free amino groups in the form of ethanolamine and 4-amino-4-deoxy-L-arabinose residues  in both the core region and lipid A portion of the LPS moleculeInfluence of several parameters, including the composition, concentration and pH value of the running buffer, the length of the capillary were investigated in order to find the optimal separation conditions.4−) with cis-diol groups in the carbohydrate moiety within the LPS structure would enhance the electrophoretic mobilities of LPS39. Borate complexation provides the uncharged glycolipid with negative charges thus making them mobile in CE analysis40.The choice of the separation buffer plays an important role in the electrophoretic patterns of the analytes. Since the most commonly used buffers for the electrophoretic separation of carbohydrates are borate, this could also be applied for the separation of the LPS. Despite the fact that LPSs are neutral compounds, which makes them unsuitable for electrophoretic analysis, complexation of borate ions (B(OH)Since labeling reagent (i.e. FITC) is an amine reactive fluorophore, any buffer or buffer additives that contain amino groups such as Tris(hydroxymethyl)aminomethane (tris), Tricine and 1,4-diaminobutane (DAB), were tested and ruled out due to sensitivity loss and broad peak. Using borate buffer, separation performance of micellar electrokinetic chromatography (MEKC) with SDS (ranging from 10–40 mM) as surfactant was investigated. However, tailing peaks were observed and worse resolution of background FITC peaks with endotoxins was observed Figure . There mThe effect of sodium tetraborate concentration on the separation efficiency was evaluated in the range of 10–80 mM. In Fig. 40. Consequently, the effect of pH value of the separation buffer was investigated by varying the pH of the separation buffer from 8.50 to 10.0. Better resolution and longer migration times of analytes were observed  is commonly used as the standard endotoxins in LAL assay, LPS from Escherichia coli O55:B5 was also used for quantification by the CE-LIF method as reference value. Endotoxin levels were calculated relative to the level of the reference endotoxin.As there are many different bacteria strains in the environment and therefore numerous possibilities of LPS sources, it is impossible to confirm which bacteria generates certain particular LPS. Instead, it is more realistic to measure all LPSs and provide an estimated total LPS level, in a similar way as previous analytical methods of LPS, such as LAL. Since LPS from E. Coli O55:B5 with 12 different LPS concentrations in the range of 0.05 µg/mL to 100 µg/mL. For each concentration seven measurements were carried out. Good linear correlation was obtained with R2 = 0.9991. The LOD is about 50 ng/mL at a signal to noise ratio (S/N) of 3.To study the accuracy of quantification of LPS, a calibration curve was established using LPS from E. Coli O55:B5, with the samples stored in dark at room temperature for 20 days. The samples were analysed each day to obtain consecutive electropherograms. The results showed that FITC itself was not very stable in aqueous solution at room temperature as a significant decrease in fluorescence signal was observed with longer storage times. In contrast, after labelling with LPS, the product exhibited high stability in aqueous solution at room temperature. No obvious degradation was found after 20 days  method specifically designed for capturing polysaccharides can remove most interference substances and cross-validate the analytical method. For example, peptides or proteins can be labeled by FITC, but they will be removed during the SPE step; whereas polysaccharides can be captured by SPE, but they will not be labeled by FITC and therefore will not be detected by the CE-LIF method. Figure It is worth noting that the SPE pretreatment and CE-LIF method could prevent possible false positive results commonly associated with LAL assays that are due to the reaction with (1-3)-β-D-glucans as these interference substances will trigger the LAL reaction. In contrast, the CE-LIF method provides inherent discrimination of interfering substances including (1-3)-β-D-glucans Figure , due to As SPE targeted at the carbohydrate part of LPS, it is reasonable to note the limitation of this method, because LPSs with very short oligosaccharide chains may be lost during the SPE steps. Most wild strains of gram-negative bacteria can generate smooth type LPS. However, mutations of the wild strain bacterium can be induced by UV light or exposure to other mutagenic-causing compounds, resulting in R-type LPS with shorter polysaccharide chains. Ra, Rb, Rc, Rd and Re denote the different rough chemotypes of LPS, while each alphabetic increase means loss of several monosaccharides on the carbohydrate chain. Re-type LPS actually would have lost all O-antigen moiety and only core-oligosaccharide remains. The extreme case of Re-type LPS consists of lipid A carrying only two KDO residues. To investigate the applicability of the SPE method to LPSs with different lengths of carbohydrate chains, several R-type LPSs with shorter carbon chains were tested. The results indicated that the SPE method was suitable for extracting LPS with Rc or longer carbohydrate chains. In contrast, shorter carbohydrate chain LPSs may be lost during SPE as the Rd mutant LPS peak became very small after SPE Fig. . NeverthIt is also interesting to observe the long migration times for R-type LPSs in Fig. E. Coli O55:B5) in the range of 0.05–100 µg/mL. Combining SPE extraction and CE separation, the whole protocol showed good linearity (R2 = 0.9984). Analysis of 50 ng/mL LPSs from E. Coli O55:B5 and E. Coli O26:B6 by CE-LIF demonstrated good repeatabilities, with RSD of peak area at 2.82% and 2.94%, RSD of migration time at 0.33% and 0.40%, respectively (n = 6). Six LPS standards were subjected to six different SPE columns, and then the eluents were labelled with FITC and introduced into the CE system for analysis. The peak area RSD values acquired with the SPE procedure were 8.44% and 11.0% for LPSs from E. Coli O55:B5 and E. Coli O26:B6, respectively.Calibration was performed for LPS standard  were added to the raw effluent water sample, and the resulting spiked samples were subjected to SPE and derivatization procedures, and eventually subjected to CE-LIF analysis. Recoveries were calculated based on the calibration equation and the differences between the calculated concentration in the spiked samples and the known original concentrations. The recoveries determined for spiking 20 ppm and 60 ppm LPS standards were 105.0% and 83.4%, respectively. These results showed that the proposed SPE pretreatment and CE-LIF method could be used for identification and quantification of LPSs in water samples. To validate the repeatability of our testing methods, both inter- and intra-day analyses were conducted to ensure robustness of our method (Table n = 6) and peak area . For the inter-day tests , we report low standard deviations in both the migration time (average RSD = 0.44%) and peak area (RSD = 3.6%). Moreover, accuracy tests were executed to ascertain the calibration variance of our protocol. For the LPS standard concentration of 13.02 ppm, we obtained 6 readings  that gave us the desirable accuracy range of up to 91.3%. For the inter-day tests, our three-day tests yielded concentrations of standards with average RSD of 2.28%.Furthermore, spike-and-recovery experiments were then carried out in order to investigate the accuracy of the SPE pre-treatment and CE-LIF method. Known amounts of LPS standard -β-D-glucans, due to the specificity of the derivatizing agent. Furthermore, in this study, the combination of SPE extraction methods and the FITC labeling strategy furnishes significant advantages in terms of selectivity and sensitivity of the analysis method. The developed CE-LIF method has been tested with environmental water samples, and demonstrated sensitive, reproducible and reliable results.Escherichia coli 0111:B4 (Lot # 024M4019V), Escherichia coli 055:B5(Lot #025M4040V), Escherichia coli 026:B6 (Lot # 053M4060V), Escherichia coli 0127:B8 (Lot # 103M4051V), Salmonella enterica serotype enteritidis (Lot # 064M4035V), Pseudomonas aeruginosa 10 (Lot # 100M4101V), Salmonella enterica serotype typhimurium (Lot # 093M4088V), Escherichia coli J5  (Lot # 053M4112V), Salmonella typhosa (Lot # 063M4017V), Escherichia coli F583  (Lot # 055M4005V), Salmonella enterica serotype minnesota (Lot # 064M4015V), Escherichia coli 0128:B12 (Lot # 063M4014V), Klebsiella pneumoniae (Lot # 111M4038V), Escherichia coli K-235 (Lot # 031M4076V), Salmonella enterica serotype abortusequi (Lot # 104M4064V), and Serratia marcescens (Lot # 013M4078V) were purchased from Sigma Aldrich . Buffers were prepared with ultrapure water obtained from Merck Millipore . All other reagents were of analytical grade. Experiments were carried out using freshly prepared and filtered solutions.Sodium dodecyl sulfate (SDS), sodium tetraborate, and sodium hydroxide were obtained from Merck . Fluorescein isothiocyanate (FITC), (1-3)-β-D-glucans were purchased from Sigma - Aldrich . Lipopolysaccharides from All electrophoretic separations were performed on a Beckman Coulter PA 800 plus system , equipped with a solid-state laser induced fluorescence detector with excitation wavelength of 488 nm and emission wavelength of 520 nm. The 32 Karat version 9.1 software was used for data analysis, data acquisition and peak integration.et al.41 with slight modifications. Briefly, FITC was dissolved in 50 mM sodium tetraborate buffer (pH 9.30). LPS solution was prepared by dissolving LPS powder in ultra-pure water. Then, FITC was added to LPS solution in the mass ratio of 2: 1 and incubated in dark at 37 °C overnight. Normally the fluorescent dye is in excess amount to ensure complete labeling of LPS. FITC-labeled LPS was protected against light exposure throughout.LPS was labeled with FITC by a protocol similar to that of Skelly Solid phase extraction (SPE) was performed using the 96-well MassPREP™ HILIC (hydrophilic interaction chromatography) µElution plate  according to the manufacturer’s protocols. Briefly, 100 µL LPS standards or samples were constituted with 90% (v/v) acetonitrile. Then the sorbent was conditioned with 200 µL of HPLC grade water and equilibrated with 2 × 200 µL of 90% (v/v) acetonitrile. LPS samples were loaded on the sorbent without vacuum, followed by sample washing twice with 200 µL of 90% acetonitrile. Finally analytes were eluted with 100 µL of 1 mM Tris-citrate buffer.Fused-silica capillary were purchased from Polymicro Technologies Inc. . Capillaries with an internal diameter of 50 or 100 µm were used throughout. Prior to use, capillaries were rinsed with 1 M NaOH for 10 min, followed by rinsing with DI water for 10 min and separation buffer for 10 min. The samples were injected into the capillary by pressure injection for 5 s or 10 s under a pressure of 0.5 psi. The system was operated at a constant voltage of 30 kV. The sample storage plate temperature was set to be 16 °C and the separation temperature was set to be 25 °C. Separation buffer was refreshed after every five runs. Between runs, the capillary was conditioned by rinsing with 0.1 M NaOH for 2 min, ultra-pure water for 3.0 min and separation buffer for 5 min.All testing samples were collected in 100 ml glass bottles and were stored at 4 °C until analysis. Before analysis, the water samples were centrifuged at 3000 RPM for 5 min. The solid suspended particles retained at the bottom were discarded and the supernatant was used for analysis. Then SPE was performed using µElution plate. The eluents were labeled with FITC before CE-LIF analysis.Supplementary Information


AbstractEchinodermata: Echinoidea) obtained from samples collected in the Southern Ocean  during oceanographic cruises led over 150 years, from 1872 to 2015. Echinoids are common organisms of Southern Ocean benthic communities. A total of 201 species is recorded, which display contrasting depth ranges and distribution patterns across austral provinces and bioregions. Echinoid species show various ecological traits including different nutrition and reproductive strategies. Information on taxonomy, sampling sites, and sampling sources are also made available.This database includes over 7,100 georeferenced occurrence records of sea urchins  and for the following decades: 1955–1964, 1965–1974, 1975–1984, 1985–1994 and 1995–2012. They were compiled from different sources and transformed to the same grid cell resolution of 0.1° per pixel. We also provide future projections for environmental descriptors established based on the Bio-Oracle database . Project title: Species distribution modelling of Echinoids in the Southern OceanPersonnel: Salomé Fabri-Ruiz, Thomas Saucède, Bruno Danis, Bruno Davidwww.versoproject.be) funded by the Belgian Science Policy Office .SF-R’s work is supported by a PhD grant from French Ministry of Higher Education and Research. The project is a contribution to Biogeosciences laboratory (CNRS UMR6292) and contribution #15 to the vERSO project (ACC), the strongest and largest current of the planet that flows clockwise from west to east around Antarctica (STF), Sub-Antarctic Front (SAF), Polar Front (PF), Antarctic Divergence (AD), and separate water masses of different physical and biotic properties   . We checdatabase  and expeThe dataset includes historical data sampled in the Southern Ocean over a century and a half from the Challenger expedition to the most recent oceanographic campaigns led on the Kerguelen Plateau, in Adelie Land and around the Antarctic Peninsula Figure . All comDatasetName links the origins of occurrence records which are from academic collections , published articles, former databases , two areas characterized by a high species number (25–30) Figure , 2b. In Our knowledge of genus and species distributions is strongly biased by the quality of sampling effort. Figure Several areas have been little sampled including the waters close to the sea ice margin and deep oceanic basins, most records being concentrated in the first 400 meters Figure . Common Main biogeographic features of Southern Ocean echinoids is a constant decrease of genus richness southward whereas species richness decreases from 35°S to 60°S, increases from 60°s to 65°S, then decreases again southward until 70°S Figure  and hereth) they were downloaded from the Bio-Oracle database ; they were downloaded from the Bio-Oracle database (Environmental descriptors for the Southern Ocean were compiled from various sources but most of them come from the World Ocean Atlas  for currdatabase .Object name: Environmental_data_Southern_OceanFormat name: RasterDistribution: data.aad.gov.au/metadata/records/Environmental_data_Southern_Ocean doi: 10.4225/15/5949ba54ca33cPublication date of data: 2017-05-18Language: EnglishMetadata language: EnglishDate of metadata creation: 2017-05-18Hierarchy level: DatasetPageBreak