High sedentary time has been considered an important chronic disease risk factor but there is only limited information on the association of specific sedentary behaviors on weekdays and weekend-days with body composition. The present study examines the prospective association of total sedentary time and specific sedentary behaviors during weekdays and the weekend with body composition in young adults.A total of 332 adults  were followed over a period of 1 year. Time spent sedentary, excluding sleep (SED), and in physical activity (PA) during weekdays and weekend-days was objectively assessed every 3 months with a multi-sensor device over a period of at least 8 days. In addition, participants reported sitting time, TV time and non-work related time spent at the computer separately for weekdays and the weekend. Fat mass and fat free mass were assessed via dual x-ray absorptiometry and used to calculate percent body fat (%BF). Energy intake was estimated based on TDEE and change in body composition.r ≤ 0.34). Associations between body weight and specific sedentary behaviors were less pronounced and significant during weekdays only (r ≤ 0.16). Nevertheless, decrease in SED during weekends, rather than during weekdays, was significantly associated with subsequent decrease in %BF . After adjusting for PA and energy intake, results for SED were no longer significant. Only the association between change in sitting time during weekends and subsequent %BF was independent from change in PA or energy intake , while there was no significant association between TV or computer time and subsequent body composition.Cross-sectional analyses showed a significant correlation between SED and body composition (0.18 ≤ The stronger prospective association between sedentary behavior during weekends with subsequent body composition emphasizes the importance of leisure time behavior in weight management. Pregnant women, women who were planning on getting pregnant and those planning to change their use of contraceptive medications in the following 2 years were also excluded. The study protocol was approved by the University of South Carolina Institutional Review Board and all participants signed an informed consent prior to data collection.The present study uses baseline through one-year follow-up data from a prospective, observational study, which has been described in detail elsewhere 2) was calculated using the average of 3 measures obtained at each measurement visit. Baseline measures were used to classify participants as normal weight (BMI < 25 kg/m2) or overweight/obese (BMI ≥ 25 kg/m2) Total fat mass (FM) and fat free mass (FFM) were assessed via dual X-ray absorptiometry  and percent body fat (BF) was calculated (FM/body weight). Due to the limitations of diet reports Anthropometric measurements were obtained every three months by trained and certified research staff. Body weight (kg) and height (cm) were measured with participants in surgical scrubs and bare feet to the nearest 0.1 kg and 0.1 cm, respectively. Body mass index  were determined using the SenseWear Mini Armband . The armband is worn on the upper arm and combines measurements of near body temperature, skin temperature, heat flux, galvanic skin response, and accelerometry. Several studies have examined the validity of the armband in various populations and previous research indicated accurate estimates of energy expenditure, physical activity (PA) and sleep in free-living adults During periods of non-wear time participants reported their activities. Using individual resting metabolic rate (RMR), energy expenditure during non-weartime was estimated based on the Compendium of PA Information on specific sedentary behaviors was obtained via self-report. Specifically, participants were asked about time spent sitting, watching TV and non-work related computer use during a typical weekday and weekend-day. Average time spent in various sedentary behaviors for the entire week was calculated using a 5:2 ratio for weekdays and weekend-days.Statistical Analysis. In order to be included in the analysis, valid data needed to be available for at least 3 measurement time points, including baseline and 12-month follow-up. Upon confirmation of normal distribution of the data differences between compliant and non-compliant participants were examined via ANOVA and Chi-square tests for categorical variables. Differences between weekdays and weekend were examined via paired-sample t-tests. Correlation analyses were used to examine the cross-sectional association between objectively determined sedentary time, self-reported sedentary behavior and body composition. The strength of the association was defined as weak (0.1 > r > 0.3), moderate (0.3 ≥ r ≥ 0.5) or strong (r > 0.5) for positive and negative trends 3.p = 0.01). Average armband wear-time for compliant participants was 23.1 ± 0.6 hours/day for a total of 50.2 ± 3.3 days over the one-year observation period. p = 0.01). Participants spent 66.2 ± 8.5% of their waking hours in sedentary pursuits. Normal weight participants displayed lower sedentary time and more time in MVPA (p < 0.01). Sedentary time during waking hours was lower during the weekend compared to weekdays (p < 0.01) due to longer sleep duration; there was no difference in light PA and MVPA between weekdays and the weekend. Self-reported time spent at specific sedentary behaviors did not differ between normal weight and overweight obese participants. Sitting time and time spent at the computer was significantly higher on weekdays compared to weekends (p ≤ 0.01). TV time, on the other hand, was significantly higher on weekends compared to weekdays (p < 0.01).A total of 332 adults  provided valid data throughout the 1-year observation period. Two thirds (66.6%) of the sample were white/Caucasian with a majority (86%) having a college degree. Baseline descriptive characteristics did not differ between compliant and non-compliant participants, except for a higher prevalence of college graduates in compliant compared to non-compliant participants . The correlations between weekday and weekend behaviors were strong for objective measurements, moderate for sitting time but only weak for computer time and TV time (r = 0.4) and change in sitting time (r = 0.3). Accordingly, change in objectively determined sedentary time was not significantly correlated with change in specific sedentary behaviors.Correlations between total sedentary time and time spent at specific sedentary behaviors were generally weak. Objectively determined sedentary time was significantly correlated with sitting time during weekdays and weekends. The correlation between objectively measured sedentary time and TV time as well as time spent at the computer, however, was only significant during weekends . Correla TV time . CorrelaCross-sectional analyses between body composition and sedentary time showed a low to moderate association between body composition and total sedentary time, particularly in overweight/obese . The corOver the 1-year observation period, participants experienced a weight gain of 1.0 ± 3.4 kg, which was associated with an increase of 0.7 ± 2.5 %BF. Weight change did not differ between normal weight and overweight groups, but change in %BF was more pronounced in those who were normal weight at baseline. Change in objectively determined sedentary time and specific sedentary behavior did not differ between normal weight and overweight/obese participants.β = 0.04; p = 0.01). This association remained after additionally adjusting for MVPA and EI.Regression analyses did not show a significant prospective association between change in sedentary time and body weight at 12-month follow-up after adjusting for baseline body weight . Baselinβ = 0.13; p = 0.04). No significant associations were observed between change in body composition and specific self-reported sedentary behaviors at 1-year follow-up. Analyzing men and women separately revealed similar results as those shown for the total sample.There was also an association between change in body weight and total sedentary time at 1-year follow-up in overweight/obese participants  during weekdays. The greater freedom for conscious choices during weekends, on the other hand, provides more options for a reduction in sedentary time by engaging in various forms of recreational activities of light intensity or structured MVPA in a potentially conscious effort to control body weight. The fact that people tend to engage in more active behaviors during the morning hours Of additional interest is the weak association between specific self-reported sedentary behaviors and body composition, which emphasizes the need for a comprehensive assessment of overall sedentariness when examining the health risks associated with excess sedentary time. Even though TV time has been commonly used as indicator for sedentary behavior, this behavior appears to contribute only a small proportion to total sedentary time, particularly in light of the high amount of occupational sedentary time Besides the concerns regarding measurement accuracy with either method additional limitations should be considered as well when interpreting the findings of this study. The present study did not provide information on the duration or number of bouts of sedentary pursuits. Interruptions of prolonged sedentary bouts, however, have been shown to affect various health outcomes, independent of total sedentary time Overall, results of the present study emphasize the need for a comprehensive approach in weight management, including PA and diet. Even though the rise in occupational sedentary time has been of particular concern in modern society


Because of unreliable self-report, accelerometry is increasingly used to objectively monitor physical activity (PA). However, results of accelerometric studies vary depending on the chosen cutpoints between activity intensities. Population-specific activity patterns likely affect the size of these differences. To establish their size and stability we apply three sets of cutpoints, including two calibrated to a single reference, to our accelerometric data and compare PA estimates.1402 German adolescents from the GINIplus and LISAplus cohorts wore triaxial accelerometers (Actigraph GT3x) for one week  at the hip. After validation of wear, we applied three sets of cutpoints for youth, including the most common standard  and two calibrated to a single reference,  to these data, estimating daily sedentary, light, moderate, vigorous and moderate-to-vigorous PA . Stability of differences was assessed by comparing Romanzini’s two sets of cutpoints.Relative agreement between cutpoints was closer for activity of lower intensities (largest difference for sedentary behaviour 9%) but increased for higher intensities . Romanzini’s uniaxial and triaxial cutpoints agreed no more closely with each other than with Freedson’s.Estimated PA differed significantly between different sets of cutpoints, even when those cutpoints agreed perfectly on another dataset (i.e. Romanzini’s.) This suggests that the detected differences in estimated PA depend on population-specific activity patterns, which cannot be easily corrected for: converting activity estimates from one set of cutpoints to another may require access to raw data. This limits the utility of accelerometry for comparing populations in place and time. We suggest that accelerometric research adopt a standard for data processing, and apply and present the results of this standard in addition to those from any other method. Physical activity (PA) is a major protective factor for most noncommunicable diseases , 2 and iBecause accelerometry is not vulnerable to reporting bias as are self-reports, it is an increasingly popular technique for assessing PA under field conditions. Accelerometers measure accelerations, which are then converted to unitless counts which are summed per unit time (epoch). Activity intensity  in each epoch is defined by applying cutpoints that have been observed, in calibration studies, to best indicate metabolic demands , 9 in suComparisons of different accelerometric protocols  and device types have become increasingly common as accelerometry moves into the mainstream of research. , 17, 18 This study specifically addresses these concerns by applying three sets of cutpoints to the same dataset with the same data-cleaning protocol and comparing their estimates of activity in each intensity, quantified both as minute-by-minute agreement and as estimated total minutes. One set of cutpoints, created by Freedson et al,  is amongmarie.standl@helmholtz-muenchen.de) and the ethics committees and acceptance of a data transfer agreement from the legal department of the Helmholtz Zentrum München.Both the GINIplus and LISAplus studies were approved by local Ethics Committees of Bavaria and West-Rhine Westphalia, and by written consent from participating families. This study sampled adolescents from two different population-based German birth cohorts: GINIplus and LISAplus, born between 1995 and 1999 in the regions of Munich and Wesel. Accelerometry was done between 2011 and 2014, and subjects were 15.6 (SD 0.5) years old at the time of accelerometry. Details on study design and cohort selection are published elsewhere.  We do noAccelerometry participants were recruited from the entire 15-year followup of GINIplus and LISAplus that lived in Munich and Wesel, which is all of GINIplus but only 64% of LISAplus, since those LISAplus participants living in the study centers Bad Honnef and Leipzig were not approached for accelerometry. For more details on followup see  and 21]21]. Of tth and 90th percentiles in a subset of this cohort:[Accelerometry protocol has been described at . Subjects cohort: more thaSampling rate was set to 30 Hz and the measured accelerations stored at 1 Hz after conversion into activity counts. Data were summed over 60-second epochs. Data filtering was set to default  as recommended by ActiGraph. ActiLife software was used for initialization of accelerometers  and for download of data. The 60-second epoch length chosen was the most common in two recent reviews: Cain et al  found thActivity counts per minute when the subject was awake were assigned to the four intensity levels—sedentary, light, moderate, and vigorous physical activity—using Freedson’s (2005) commonly-used uniaxial cut-points for children  for the All statistical analyses used SAS 9.2. All graphics were created using Excel. All analyses were limited to validated recording time where the subject reported being awake and out of bed.th, 95th percentiles. To establish the significance of differences, linear or log-linear models were fit to compare each pair of algorithms’ estimate of number of minutes in activity of each intensity. Only the largest (i.e. least significant) p-value is shown. These differences are then visually presented using Bland-Altman plots. [Mean minutes in each activity intensity for each subject, according to each cutpoint, was calculated and then summarized across subjects. These summary results are presented as mean (standard deviation), 5n plots.  RomanzinInteralgorithm agreement on the intensity of each minute’s activity is presented using cross-tabulations, without statistical tests. Each minute was classified into the four intensities by each of Romanzini’s cutpoints, and we present the percent of minutes that fell into each possible combination of estimated intensities. Romanzini’s cutpoints are compared to Freedson’s in Daily activity for 1402 Germans  was accelerometrically monitored over 4–7 days per subject for an average of 14.7 hours per day.  DetailedThe three cutpoints agreed well on total estimated sedentary behaviour  and pubIn a single dataset, we found that adolescents’ estimated moderate and vigorous physical activity differed by up to 50% based on the cutpoints used for data handling. This difference was highly statistically significant and would be clinically relevant if it were real and not artefactual. Furthermore agreement was no better with a pair of algorithms calibrated to the same test dataset  than witS1 TablePercent of time . If no minutes fell into that category (DOC)Click here for additional data file.S2 TablePercent of time .If no minutes fell into that category .Freedson’s algorithm for children from Freedson P; Pober, D; Janz, KF Calibration of accelerometer output for children. Med Sci Sports Exerc. 2005;37(11(Suppl)):523–30. Romanzini’s triaxial algorithm from Romanzini M; Petroski, EL; Ohara, D; Dourado, AC; Reichert, FF. Calibration of ActiGraph GT3X, Actical and RT3 accelerometers in adolescents. European Journal of Sport Science. 2014;14(1):91–9. 10.1080/17461391.2012.732614.(DOC)Click here for additional data file.S3 TableCounts per minute (bottom of category).Freedson’s cutpoints from Freedson P; Pober, D; Janz, KF Calibration of accelerometer output for children. Med Sci Sports Exerc. 2005;37(11(Suppl)):523–30. Romanzini’s cutpoints from Romanzini M; Petroski, EL; Ohara, D; Dourado, AC; Reichert, FF. Calibration of ActiGraph GT3X, Actical and RT3 accelerometers in adolescents. European Journal of Sport Science. 2014;14(1):91–9.(DOC)Click here for additional data file.S1 FigFreedson’s uniaxial  algorithm for children from Freedson P; Pober, D; Janz, KF Calibration of accelerometer output for children. Med Sci Sports Exerc. 2005;37(11(Suppl)):523–30.Romanzini’s triaxial algorithm from Romanzini M; Petroski, EL; Ohara, D; Dourado, AC; Reichert, FF. Calibration of ActiGraph GT3X, Actical and RT3 accelerometers in adolescents. European Journal of Sport Science. 2014;14(1):91–9. 10.1080/17461391.2012.732614.(TIF)Click here for additional data file.S2 FigFreedson’s uniaxial  algorithm for children from Freedson P; Pober, D; Janz, KF Calibration of accelerometer output for children. Med Sci Sports Exerc. 2005;37(11(Suppl)):523–30.Romanzini’s uniaxial  algorithm from Romanzini M; Petroski, EL; Ohara, D; Dourado, AC; Reichert, FF. Calibration of ActiGraph GT3X, Actical and RT3 accelerometers in adolescents. European Journal of Sport Science. 2014;14(1):91–9. 10.1080/17461391.2012.732614.(TIF)Click here for additional data file.


Aegla constitute the most threatened freshwater decapods of South America, occupying pristine habitats with narrow distribution. Using phylogenetic comparative methods, we addressed: Is the variability of habitat physicochemical parameters and tissue ADS phylogenetically structured? How do environmental features correlate with ADS? How do they vary among species? Several physicochemical parameters of water, as well as metals in sediments, were measured in ten aeglid species’ habitats. Additionally, metal accumulation and ADS parameters  were evaluated in hepatopancreas. Water conductivity and pH showed phylogenetic signal, while all other physicochemical traits demonstrated plastic variability. Metals were present at natural concentrations, which are corroborated by the relative stable GSH/GSSG ratio, and by their absence of correlation with bioaccumulation levels and MTLP, both phylogenetically structured. However, metal variability across species’ niches is associated with ACAP, a potential biomarker tool. Thus, the physiological sensitivity of aeglids is environmentally driven but also  phylogenetically constrained, unraveling the importance of systematic framework for cross-species investigations and future monitoring strategies of these conspicuous freshwater animals.The antioxidant defense system (ADS) protects organisms against the potential oxidative stress induced by environmental features, underlying processes of habitat diversification. The anomurans  It provided an explosion of various biochemical cascades, offered more energy for cell signaling networks, and underpinned the formation of various secondary gene products that regulates expression and modifies the organism physiology3. The higher energy availability promoted the diversification of structures and functions associated with homeostasis when facing variations in environmental parameters, such as water temperature, dissolved O2 content, conductivity, salinity and metal concentrations8.Oxygen is considered a selective pressure in driving the evolution of aerobiosis in Eukarya9. However, the emergence and evolution of an antioxidant defense system (ADS) have protected the metabolic pathways against ROS effects, thus avoiding an imbalance of the oxidative status at sub-cellular level, with consequent damage to biomolecules. An oxidative stress condition would lead to negative effects at both systemic and ecosystem levels, such as impairment in metabolism, growth rate, immunocompetence, longevity and reproduction13. In fact, biological distribution and tolerance to environmental changes and anthropogenic stressors are associated with the maintenance of the oxidative status1.Environmental features are related to aerobic generation of energy and oxidation processes, producing reactive oxygen species (ROS) such as peroxides, superoxides and hydroxyl radicals, highly destructive due to their capacity to oxidize lipids, proteins and nucleic acids1. This is particularly aggressive to endemic fauna or species with narrow distribution because of their reduced capability of dispersion and/or more restricted physiological mechanisms16. In this context, freshwater anomurans Aegla constitute an interesting model to evaluate the environmental and phylogenetic correlates of inter-specific variability of ADS. Aegla is endemic to Neotropical region, with 85 described species for temperate and subtropical freshwater habitats19, with well-established phylogenetic relationships20. Additionally, aeglids constitute the most threatened decapod taxon present in freshwater habitats of South America: almost 60% of species are under threat, with 72% of the Brazilian representatives21.The combination of biotic and abiotic factors constrains the distribution of life, while contaminants can threaten organisms by diminishing population sizes and even promoting extinction of entire populations or speciesAegla species are being threatened by stressing conditions resulting from human activities, especially those associated with agriculture, urbanization and deforestation of riparian forests, a condition that is aggravated considering their high endemism and fragmented geographical distribution22. In fact, they are considered as environmental indicators of water quality24. It is not known whether the concerning with aeglid conservation started in 195926 is only a consequence of such ecological-geographical constraints, as historically proposed. It could be also due to a reduced capacity of ADS in protecting aeglids against environmental stressors, including those associated with human activities. Furthermore, aeglid physiology has rarely been investigated; studies are mainly focused on metabolic and osmoregulatiory aspects31. Indeed, investigations performed were conducted in a species-specific way, with a complete lack of comparative approaches.Aegla species comprising three evolutionary groups 20. We have measured several physicochemical parameters  of the water at the sampling site of each Aegla species. Additionally, we have measured the concentrations of metals  in sediments, since the trophic niche of aeglids is explored at the bottom, where accumulate these chemicals. We also characterized metal accumulation and the ADS in hepatopancreas, the biotic site of metal detoxification. Antioxidant defenses were characterized based on metallothionein-like proteins (MTLP) concentration, antioxidant capacity against peroxyl radicals (ACAP), and the glutathione system (GSH-GSSG). These parameters are involved in ROS scavenging during exposure to environmental stressors.Aiming to test for the role of environmental features in driving antioxidant defense variability, as well as for the influence of phylogenetic relationships on such physiological variation, we have sampled 10 Aegla species is unprecedented in Metazoa’s physiology and toxicology, and could underpin future management strategies for this remarkable group of freshwater animals. The environmental and phylogenetic correlates are discussed in the evolutionary context of the clades C, D and E20, with regard to some representative Aegla species from southern Brazil.Based on the information generated, and using phylogenetic comparative methods, we have addressed three main questions: Is the variability of habitat physicochemical parameters and tissue ADS phylogenetically structured? How do environmental features correlate with ADS? How do they vary among species? The phylogenetic comparative evaluation of ADS in The aeglid species were collected under permits of the Brazilian Ministry of Environment .Aegla species, representative of three evolutionary groups 20, were sampled by the end of the summer of 2016. They were collected at different localities of Rio Grande do Sul (RS) and São Paulo (SP) states, in southern and southeastern Brazil, respectively  were captured using traps or manually collected by turning over the rocks against a steel-mesh builder’s sieve. They were kept in plastic gallons containing aerated local water and immediately transferred to our mobile laboratory. Hepatopancreas was dissected, split into two aliquots, and stored in liquid nitrogen. Samples were then transferred to our laboratory at the Universidade Federal do Rio Grande - FURG , and stored in ultrafreezer (−80 °C).2 content and pH of water from each collecting site were measured using a portable multiparameter analyzer . Water was collected (500 mL), in duplicate, for further laboratorial analyses of total organic carbon , ion concentration and alkalinity: for TOC, samples were acidified  and analyzed using a TOC analyzer ; Cl− concentration was measured according to Zall et al. (1956)32; Na+, Ca2+ and K+ concentrations were measured in filtered (0.45-µm mesh filter) samples using a flame spectrophotometer . Measurement accuracy and standard curves were obtained using standard solutions , which are tracked to reference material of the National Institute of Standards and Technology . Sulfate concentration was measured as described by Tabatabai (1974)33. Alkalinity was measured according to the method described by APHA (1989)34.During field collections of species, temperature, conductivity, dissolved O3, 3 mL HCl, and 3 mL HF). For sample digestion, plastic tubes were kept in a closed system (oven with forced air circulation) at 60 °C, for 24 h. After complete digestion, samples were evaporated to 1 mL and diluted (10×) with high purity deionized water (resistivity of 18.2 MΩ/cm). Metals concentrations were determined using an Atomic Absorption Spectrometry with Graphite Furnace . The limits of detection and quantification of the method employed ranged from 0.001 to 0.03 µg/g and 0.004 to 0.10 µg/g, respectively, depending on the metal analyzed. Data on metal concentration were normalized considering the amount of TOC in sediments, since TOC is considered as being a major matrix for complexing metals. Sediment samples were decarbonized in a desiccator using 37% HCl35. Total organic carbon (%) was measured using an elementary analyzer (CHNS Perkin Elmer 2400 Series II). For TOC, the limits of detection and quantification of the method employed corresponded to 0.08% and 0.26%, respectively. All procedures and analyses were performed in quadruplicate.Since the trophic niche of aeglids is associated with the water/bottom sediment interface, sediment samples were collected (in quadruplicate) for metal quantification. Samples were analyzed for the total concentration of Mn, Fe, Zn, Cd, Cr, Cu, Ag and Pb. Sediment sample (1.0 g) was placed in plastic tubes, followed by addition of acids  of the National Institute of Standards and Technology . Certified reference material  was also analyzed following the same procedures adopted for sample analysis. Analytical results of the quality control procedures showed good agreement with the certified values, with recoveries ranging from 91.3 to 97.5% for metals and from 95.5 to 99.4% for TOC.36. Hepatopancreas samples (0.25 g) of each aeglid species (N = 4 per species) were dried (dry weight) and completely digested with 65% HNO3  at 60 °C for 24 h. High purity deionized water (resistivity of 18.2 MΩ/cm) was employed to dilute samples and standard solutions. A multi-element  stock solution at 1 g/L  was employed to prepare the standard solutions. Metal concentrations in digested samples were analyzed using the Atomic Absorption Spectrometry with Graphite Furnace . The quality assurance and quality control procedures for metal quantification were based on regular analyses of blanks and spiked matrices, like for metal concentration in sediments. Measurement accuracy and standard curves were also obtained using standard solutions, and certified reference material  was also analyzed. It was processed following the same procedures adopted for sample analysis. Analytical results of the quality control procedures showed good agreement with the certified values with recoveries ranging between 93.5 and 98.6%. All procedures and analyses were performed in quadruplicate.Hepatopancreas was selected because of its key role in metal detoxification in crustaceans39. Therefore, ACAP is considered a multidimensional measure of ADS with high ecological relevance, showing significant predictive capacity of environmental effects on redox status40. Hepatopancreas samples  were homogenized (1:20 w/v) on ice in a buffer solution containing tris(hydroxymethyl)aminometano-hydrochloride , ethylene diamine tetraacetic acid , MgCl2 (5 mM), and phenylmethylsulphonyl fluoride  using an ultrasonic homogenizer  Sample homogenates were centrifuged  at 4 °C for 20 min. The supernatants were collected for ACAP analysis, following procedures described by Amado et al.39. To standardize measurements and enable inter-specific comparisons, all supernatants were diluted until achieve the same final concentration of total proteins (1.5 mg/ml). Protein concentration was measured using a commercial reagent kit based on the Biuret method . Measurements were performed at 550 nm using a microplate reader . Peroxyl radicals were generated by thermal decomposition (35 °C) of 2,2′-azobis (2 methylpropionamidine) dihydrochloride (ABAP). Reactive oxygen species (ROS) generated that were not counteracted by antioxidants present in sample homogenate reacted with 2′,7′-dichlorofluorescin diacetate (H2DCF-DA). The substrate fluorescence was monitored  using a microplate reader . Antioxidant capacity against peroxyl radicals was determined by calculating the difference between the fluorescence areas obtained for the same sample in the presence and absence of ABAP. Results were relativized to the fluorescence measured in samples without ABAP. Data were expressed as the inverse of the relative area (1/relative area). Therefore, high values indicated higher ACAP.Antioxidant capacity against peroxyl radicals (ACAP) combines various components that act specific- and individually, providing a general indicator of health and susceptibility to oxidative stress, especially against peroxyl radicals42. As a result, metal availability is regulated and the potential toxicity is reduced by scavenging hydroxyl (·OH) and superoxide (O2−) radicals45. Hepatopancreas samples  were homogenized (1:5 w/v) on ice in a buffer solution (pH 8.6) containing tris(hydroxymethyl)aminometano , sucrose (500 mM), PMSF (0.5 mM) and β-mercaptoethanol (0.01%) using the ultrasonic homogenizer. After an ethanolic fractionation, a partially purified metallothionein fraction was obtained. The concentration was determined spectrophotometrically (412 nm), based on the reaction of sulfhydryl groups present in the sample homogenate with 5-5-dithio-bis 2-nitrobenzoic acid (DTNB) and using GSH as standard, following Viarengo et al.46. Data were expressed as μmol GSH/mg wet tissue weight.Metallothionein-like proteins (MTLP) show low molecular weight (6–7 kDa) with no catalytic activity. They contain high content of aromatic amino acids and cysteine with thiol (-SH) groups, enabling chemical binding to metals48. This antioxidant is present in its reduced (GSH) and oxidized (GSSG) forms, being the GSH/GSSG ratio a significant marker of oxidative stress49. Such system has a key role in oxy-reduction reactions linked to protein synthesis, cell division and metabolism, regulating the cellular redox status through reduction of disulfide groups and ROS scavenging. Once used, GSH levels are restored by synthesis of new molecules or mainly by the conversion of GSSG to GSH, which is catalyzed by glutathione reductase50. Homogenates of hepatopancreas samples  were prepared as described above for ACAP measurements. Quantification of GSH and GSSG concentration was performed using a commercial reagent kit  based on the reaction of GSH and GSSG with DTNB. The GSH content was determined subtracting the GSSG concentration from the total glutathione concentration measured; GSH and GSSG concentrations were calculated based on standard curves built with solutions of GSH (0.5–50 μM) and GSSG (0.5–25 μM), respectively. Data were normalized by the content of total proteins in sample homogenates and expressed as μmol GSH or GSSG/mg protein.Glutathione is a tripeptide (γ-L-glutamyl-L-cysteinyl-glycine), being considered as one of the most important non-enzymatic water-soluble antioxidant present at high concentrations (0.1–10 mM) throughout the phylogenetic treeFor quality control purposes, all analyses were performed in duplicate, with standard curves being built for every batch of samples analyzed. Only standard curves showing standard deviation <5% from the expected values were accepted.Aegla suggests that “species” might not be statistically independent units, requiring the inclusion of a phylogenetic component to the comparative analyses since degrees of freedom can be overestimated when using conventional analyses54. Also, overall physiological variability usually driven by environmental factors could be constrained by the phylogenetic history, misleading comparative and evolutionary interpretations if the historical relationships are neglected57.Shared ancestry during the evolution of 20 proposed for Aegla, we used a set of analytical methods to evaluate the environmental and phylogenetic correlates of ADS variability. This phylogeny contains 64 aeglid species and subspecies, and was constructed using the partial sequences of the mitochondrial genes 12 S, 16 S, COI and COII, as well as the complete sequence of ribosomal 28 S, incorporating 5601 nucleotides evaluated by maximum likelihood. We have pruned such tree to match the species for which we have traits available, which varies from 9 (ADS system) to 15 (pH and temperature) species, as a standard practice in phylogenetic comparative studies58. The genetic distance among species was maintained exactly as in the original tree. Aegla rosanae is not present in the phylogeny here assumed.Assuming the molecular phylogenyI coefficient60. Moran’s I ranges between −1 and +1, being significant positive values descriptors of similarities between related species, while significant negative values demonstrate differences between closely related species. Such autocorrelation analysis was performed using Phylogenetic Analysis in Macroecology application61 in four phylogenetic distance classes, but the Moran’s I was showed for the first class only (species level). Ancestral states were reconstructed only for those environmental traits with significant phylogenetic signal, using a Maximum Likelihood method under Brownian Motion model of evolution51.The phylogenetic pattern of environmental and ADS traits were evaluated employing an autocorrelation analysis, using Moran’s phytools package62 after scaling and centralizing the data, while the positive and negative scores for each trait were displayed in the Aegla’s phylogeny using adephylo63. For those parameters correlated with the two main eigenvectors and with explained variance higher than 60%, we have tested for each one hypotheses of co-variation between ADS and environmental traits, as well as the total metal concentration with tissue accumulation, using a pGLS model . The pGLS considers phylogenetically correlated residual variation among species, and is traditionally used in cross-species studies64, assuming an O-U model of evolution with the selection strength better estimated. The procedure used the nlme65 and ape packages66.Aiming to analyze the multivariate comparative data involved in the ADS variability, we applied a phylogenetic principal component analysis (pPCA) to reduce the number of variables and to detect a pattern of correlation among them, considering that trait variability changes with phylogenetic distance among species. For sake of simplicity, all physicochemical and ADS parameters were analyzed together in 67, setting the minimum significance level at P = 0.05. Figures were made using QGIS 2.14.3 (http://www.qgis.org), R 3.3.3 (https://www.r-project.org), or SlideWrite Plus 7.0.All statistical analyses were implemented using the R platformAll data generated and analyzed are included in the article [and into Supplementary Information (SI) file].Aegla. Water conductivity and pH were phylogenetically correlated, while all other physicochemical traits demonstrated a more plastic variability. Metal concentrations in sediments were below their current quality criteria established by the Brazilian environmental regulation68 and the United States Environmental Protection Agency69. Furthermore, such natural concentrations are corroborated by the relative stable GSH/GSSG ratio of ≈10%, and by the lack of correlation between metal concentration and bioaccumulation levels or MTLP. In a multivariate analysis, ACAP and MTLP were correlated to the eigenvector 1, which explained 44.1% of total variance: ACAP is a potential biomarker whose variability was driven by some metal concentrations and water conductivity, while MTLP, interestingly, showed phylogenetic signal and was not related to metal availability.In the present study, individual variability of ADS was incorporated into an intra-specific pattern, the inter-specific variance was phylogenetically corrected, and the environmental correlates of the ADS inter-specific variability were explored in three evolutionary groups of A. plana and A. inermis, and up to 24.1 °C for A. platensis, while dissolved O2 content varied between 6.5 and 10.7 mg/L for A. violacea and A. rosanae, respectively. Alkalinity ranged from 7.5 mg/L CaCO3 for A. violacea to 52.5 mg/L CaCO3 for A. longirostri, while pH varied from 6.1 for A. violacea up to 7.5 for A. camargoi and A. leptodactyla. Total organic carbon concentration ranged between 2.1 mg/L for A. violacea and 19.4 mg/L for A. platensis. Sulfate concentration varied between 24.1 mM for A. violacea and 28.5 mM for A. camargoi and A. leptodactyla. Conductivity values ranged between 7.8 μS/cm and 96.0 μS/cm for A. perobae and A. longirostri, respectively. See Table 2+, Na+, K+ and Cl- concentrations.The water physicochemical parameters measured from each species’ habitat were consistent with those compiled from the literature for other aeglids Table . Illustr2 content, TOC concentration and alkalinity in water, as well as for metal concentrations (see S1 file for details) in sediments . A similar result was found for the accumulation of Cu, Cd and Zn in hepatopancreas . On the other hand, variations in water pH, conductivity, sulfate concentration and accumulation of Ag, Cr, Fe, Mn and Pb in hepatopancreas  were correlated with the phylogenetic relationships: closely related aeglid species — particularly those sampled from the clades C, D and E — tend to occupy similar habitats concerning water pH and conductivity, but with dissimilar sulfate concentrations, and manifest similar accumulation levels of the aforementioned metals in the hepatopancreas.Regarding the inter-specific variability of physicochemical traits, it was plastic for temperature, dissolved O21. It is noteworthy that pristine habitats with low temperatures, high levels of dissolved O2 and near-neutral pH characterize niches explored by aeglid species, axiomatically suggesting the role of environmental factors in driving their biological distribution8. However, a quantitative comparative evaluation with a formal phylogenetic incorporation is unprecedented, but it is now explored. Habitat temperature is not correlated with the phylogenetic position, neither structured within the particular clades investigated; the non-significant phylogenetic signal demonstrates the impossibility of predictions about thermal similarities or dissimilarities among closely related species. Results indicated that clade C, whose geographic distribution of some species reaches northernmost latitudes (i.e. aeglids sampled in São Paulo state), show nominally the lowest temperature variability  when compared to the clade D (22.3 ± 0.9 °C) and clade E (17.8 ± 2.7 °C) species. The same case is observed for dissolved O2 concentration, which varies significantly among seasons, as for A. leptodactyla of southern Brazil . As the present data and those compiled from literature correspond to the summer season, the non-discrimination of temperature and dissolved O2 among clades can be derived from the marked summer conditions observed in niches of subtropical species. In fact, average habitat temperature tends to increase at this season homogenizing putative temporal variability. Furthermore, water alkalinity, total organic carbon and concentrations of K+, Na+, Cl−, and Ca2+ were not phylogenetically structured, with significant variation among the species here evaluated.The relationship between environmental factors and distribution and abundance of aeglids is well documented by the macroecological literatureAegla phylogeny, water conductivity was 28.2 μS/cm, decreasing at the origin of clade C (22.7) but increasing at the onset of lineages D (34.5 μS/cm) and E (39.7 μS/cm)  and pH (6.1–7.9) were dependent on the phylogenetic relationships, being similar among closely related aeglid species Fig. , differe8. This could have affected the geological nature of the hydrographic basins and driven the aeglid physiology. Differently from temperature and dissolved O2, pH and conductivity do not appear to demonstrate significant seasonal variations, as demonstrated for pH in the case of A. franciscana 73, and for conductivity in the case of A. leptodactyla and A. camargoi 70.Aeglid macroecology is strongly affected by water conductivity, since the dispersion of these decapods along South America reflects orogenetic processes associated with geoclimatic events, marine ingressions, tectonic uplifts and glacial refuges68 and the United States Environmental Protection Agency69; the other metals  are not regulated by both agencies. It is important to note that the threshold effect concentrations for the several metals analyzed are the same for both agencies: Cu, 35.7; Cd, 0.6; Cr, 37.3; Pb: 35.0; and Zn: 123.0 mg.kg−1. Furthermore, none metal concentration was correlated with the respective accumulation in hepatopancreas across species of the clades C, D and E . These findings indicate that these aeglid habitats seem not to be contaminated with metals associated with anthropogenic activities, and that their natural availability depends on the nature of rocks and minerals into the hydrographic basins, even in the absence of significant human inputs74.Regarding metals evaluated in sediments for each aeglid habitat from southern Brazil (see SI file for details), concentrations of most metals analyzed  were lower than their respective current quality criteria established by the Brazilian environmental regulations75. Metal accumulation depends on the balance between metal uptake and excretion, as well as the particular metabolic mechanisms of metal detoxification76. Therefore, analysis of metal accumulation under a phylogenetic perspective could predict the physiological processes involved in animal tolerance to metal exposure, suggesting that closely related aeglid species would show similar tolerance and metabolic pathways of detoxification for each metal. These findings point out the phylogenetic framework as an important tool for future monitoring strategies in Aegla species.Metal concentration in aeglid niches is not related to the phylogenetic relationships of the species here evaluated. However, accumulations of Ag, Cr, Fe, Mn and Pb in hepatopancreas are correlated with phylogeny, which means that closely related species share similar levels of bioaccumulation. Similarly, Cd and Zn efflux rates are differentially constrained in some lineages of arthropods, annelids, mollusks and chordates, with variation in bioaccumulation across species being driven by the ability to excrete these metals77. We investigated here three parameters related to ADS: metallothionein-like proteins (MTLP), antioxidant capacity against peroxyl radicals (ACAP), and glutathione system (GSH-GSSG). They are important non-specific scavengers of ROS generated by changes in environmental conditions, such as temperature, salinity, UV radiation, pH, dissolved O2, and metals80. Also, MTLP are related to metal homeostasis due to their ability to bind and transfer metals43, protecting cells against oxidative stress79.Antioxidant defense system (ADS) protects organism against the potential biological damage associated with oxidative processes induced by environmental stressors. It is important to note that these oxidative processes axiomatically underlie those related to habitat diversification and occupation of niches with different abiotic compositionsA. plana to 3.1 ± 0.2 μmol GSH/mg in A. longirostri . However, the GSH/GSSG ratio was 10% in both cases. Regarding the inter-specific ADS variability in the sampled species of the clades C, D and E, only MTLP is phylogenetically structured : closely related species tend to show dissimilar concentrations  of total variance  with ADS traits  into a phylogenetic multivariate analysis (pPCA) for the sampled aeglid species, the two main eigenvectors retains 62.9% (PCA. longirostri (96 μS/cm), tend to show higher ACAP values (2.1), while niches with lower water conductivity (≈9 μS/cm) tend to be occupied by species with lower ACAP values (≈0.16), such as A. plana. The same pattern was observed for the metals listed above, with the habitat of A. longirostri showing higher metal concentrations. Interestingly, MTLP variability was not correlated with any metal concentration , while GSH mobilization is also not dependent on dissolved O2 content and Cl− and Cd concentrations .Using pPCA results as a guidance for pGLS models of correlated variability, ACAP in hepatopancreas appear to be associated with water conductivity  and Fe, Cu, Mn, and Zn concentrations . Species sampled from habitats with higher water conductivity, such as Aegla species in regions affected by human activities is historically reported as a consequence of their high sensitivity to contaminants81, although physiological measurements have never been evaluated so far. Here, the natural levels of metal concentration in sediments are corroborated by the relative stable GSH/GSSG ratio observed: the balance of ≈10% suggests the non-variability in the intracellular pool of oxidized/reduced glutathione, possibly reflecting a constant redox state. However, the metal variability across species’ niches is associated with ACAP, indicating it as a potential biomarker. On the other hand, the inter-specific variation of MTLP is not correlated with metal availability, revealing a phylogenetically structured incapacity of mobilization: closely related aeglids show dissimilar MTLP values, being the evolutionary pattern driven by another metal not measured in the present study. Thus, ACAP and MLTP illustrate that the historically assumed physiological sensitivity of aeglids is environmentally driven but also phylogenetically constrained, at least for the Aegla species here evaluated.The absence of Aegla, demonstrating the tendency of niche conservatism and retention of historical states, while most manifest a plastic inter-specific variability. In this context, metal accumulation and ACAP appear to be interesting biomarkers in aeglids: metal tissue accumulation under a phylogenetic perspective enable predictions about metal tolerance among closely related species, while environmental disorders/variabilities can be detected by changes in ACAP. Thus, the incorporation of phylogenetics to evaluate the environmental correlates of ADS parameters retrieves a still cryptic evolutionary history of aeglid physiology, unraveling a potential framework for future monitoring strategies of this conspicuous freshwater group.In summary, our findings show that some environmental traits and antioxidant parameters are phylogenetically correlated in Supplementary Information


We included 84 patients with EC and 99 with GCC. GCC tissue was more poorly differentiated than EC tissue and more invasive, with more histomorphologic variation. Lymph node metastasis was more frequent in GCC than in EC. The Helicobacter pylori infection rate was higher but not significantly with GCC than EC. Survival was shorter with lymph node metastasis. We found a statistically significant trend for progressive increase of TLR4 expression from normal mucosa to inflammation in GCC. GCC in this high-risk area displays clinicopathologic characteristics different from those of EC and different from those of gastroesophageal junction carcinomas in other countries, although this was not analyzed statistically. Increased TLR4 expression in gastric cardia lesions may be associated with GCC tumorigenesis.The incidence of gastric cardia cancer (GCC) is high in China. However, the clinicopathological characteristics and the carcinogenesis of GCC are unclear. Toll-like receptor 4 (TLR4) is an important innate immunity receptor and has a role in non-GCC (NGCC). We compared the clinicopathological characteristics of GCC patients from a high-risk area in China to esophageal cancer (EC) patients. Immunohistochemistry for TLR4 was performed in 201 histological samples of normal gastric cardia mucosa ( Gastric cancer is the fifth most common malignancy, with an estimated 952,000 new cases in 2012 worldwide [Generally, gastric cancers can be classified into two categories: gastric cardia cancer (GCC) arising in the area of the stomach adjacent to the gastroesophageal junction (GEJ) and non-GCC (NGCC) arising from more distal regions of the stomach . The incCancer Incidence in Five Continents, Vol. X (CI5X) and “GLOBOCAN 2012 v1.0, cancer incidence and mortality worldwide: IARC CancerBase No. 11” [From the  No. 11” , 10, Col No. 11” , the incVol. X CIX and “GLEC and GCC used to be treated as a single disease because of the coincidence of the two cancers, similar clinical symptoms, and limited techniques to distinguish them. Since the 1990s, with widespread use of endoscopy and a new classification by the World Health Organization (WHO), GCC is diagnosed as a disease different from EC in high-risk areas in China; however, the tumorigenesis, pathogenesis, development, and prognosis of GCC from high-risk areas are poorly understood. Although GCC has the same geographic distribution as EC in China , previouHelicobacter pylori (H. pylori) infection has been confirmed as an important factor for distal gastric cancer [H. pylori infection and the related chronic inflammation may contribute to the high incidence of GCC in the Chaoshan high-risk area [H. pylori recognition and they initiate inflammatory pathways that may acquire oncogenic potential [c cancer . Its relc cancer –29. Our otential –32. Tollotential . The relotential –38. HoweIn the present study, we compared the clinicopathologic features of GCC and EC in the Chaoshan high-risk area in China. TLR4 expression was evaluated by immunohistochemistry in normal gastric cardia mucosa, chronic gastric cardia inflammation, and GCC, to better understand the potential role of TLR4 in gastric cardia carcinogenesis. Despite different classifications and definitions of cancers originating around the GEJ , 40, we We included 84 patients with EC  and 99 with GCC . Patients with EC were younger than those with GCC. Although more GCC than EC patients were male, the difference in the male : female ratio between the two groups was not considerable .P = 0.001) (P < 0.001) and exhibited a wider histopathological spectrum. All 84 EC tumors (100%) were squamous cell carcinoma. In contrast, 75.8% of GCC tumors were tubular adenocarcinoma; the remainder were mucinous carcinoma (16.2%), adenosquamous carcinoma (4%), small-cell undifferentiated carcinoma , squamous cell carcinoma , or a tumor of neuroendocrine phenotype . Representative histology images are in Figures The median tumor size was larger with GCC than that with EC  . For 66 = 0.001) . RepreseP < 0.001). In addition, a higher proportion of patients with GCC showed lymph node metastasis and deep infiltration , a virulence factor that may damage the gastric mucosa and cause inflammation and cell death. Almost all of the East Asian H. pylori strains are CagA positive. Detecting CagA can be used to detect CagA-positive H. pylori infection [H. pylori infection between EC and GCC groups (P = 0.59) (A significantly higher proportion of patients with GCC than EC showed advanced-stage disease (nfection . We foun = 0.59) .P = 0.731) (P = 0.029) .Survival did not differ among patients who received surgery alone and both surgery and adjuvant therapy, so we chose all 84 EC patients and 99 GCC patients who underwent surgery for survival analysis. The mean survival was shorter with EC than that with GCC  but not significantly (= 0.731) . The cum= 0.029) . The cumP = 0.392 and P = 0.678, resp.).The 3-year survival rate was better for patients with EC than that with GCC (40.5% versus 34.3%), but the 5-year survival rate was poorer with EC than that with GCC (22.6% versus 25.3%). Neither of these differences was statistically significant (P < 0.05) (In  < 0.05) .In the present study, we compared clinical-pathological features between EC and GCC and described for the first time TLR4 expression restricted to the gastric cardia epithelium. Patients with GCC were significantly older and had a higher male : female ratio than patients with EC, although the difference in the latter variable was not considerable. 92.92% (92/99) of GCC patients were older than 50 years, and 72.62% (61/84) of EC patients were older than 50 years, indicating middle age and elderly people are high-risk group for both GCC and EC, especially for GCC. The mean age for GCC increased compared to the mean age of patients in the 1980s and 1990s . It mighH. pylori infection, although the difference did not reach statistical significance. The GCC group had more histological variants than the EC group. GCC included adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, small-cell undifferentiated carcinoma, and neuroendocrine carcinoma. In contrast, in ECs, all tumors were squamous epithelial cell carcinoma. The most common location for EC in our sample was the middle thoracic part of the esophagus, whereas in the Western countries, EC originates mainly from the lower part of the esophagus, and the most common histological type is adenocarcinoma [GCC tumors were, on average, larger and more poorly differentiated, were of higher pathological stage, and were more likely to have lymph node metastasis and deeper invasion than EC tumors. Patients with GCC were more likely to have arcinoma , 45. SurAlthough different studies have shown some shared genetic risk factors between EC and GCC from high-risk areas in China , 46–48, Huang et al. performed a study of the clinical and pathological features of GEJ carcinomas in Chinese and US patients. In terms of their data from the United States, patients with GCC in our group showed larger tumor size (5.92 ± 2.22 cm versus 3.5 ± 2.2 cm), lower 3-year (34.3% versus 43%) and 5-year (25.3% versus 28%) survival, and higher disease stage than the US patients . CompareH. pylori infection is the primary risk factor for distal gastric carcinomas in the Chinese population [H. pylori infection [H. pylori infection may contribute to the high incidence of GCC and esophageal squamous cell carcinoma in the Chaoshan region [H. pylori gastritis via the epithelial precursor lesions. It is believed that a virulent bacterium in a genetically susceptible host is associated with more severe chronic inflammation, and this long-term inflammation may lead to cancer [Adenocarcinoma of the distal esophagus and Barrett's esophagus-related diseases remain uncommon in China. A large-scale longitudinal clinical and histological data was analyzed on 5401 esophageal cancer (EC) patients diagnosed during 10-year period 2002–2011) at Henan Taihang Mountain high risk area in China. All 217 esophageal adenocarcinoma (EAC) patients from these 5401 EC patients were examined, and EAC was relatively rare and accounted for approximately 5% of all esophageal cancers. Only 10 out of 217 (4.6%) EAC cases were detected to have any evidence of Barrett's esophagus 02–2011 a, 23. Patnfection . Our pren region , 56. Gaso cancer , 57. ObsH. pylori infection and cancer [Previous studies suggested that TLR4 expression might be the link between d cancer , 37, 58,d cancer . In the d cancer . TLR4 exd cancer . Confirmd cancer , 62–64. d cancer . ConsideIn summary, this study showed that GCC carcinomas are biologically different from EC carcinomas in the Chaoshan high-risk area in China, although they share genetic risk factors and similar geographic aggregation. GCC in this high-risk area displays different characteristics from those of GEJ carcinomas in developed countries as well. We detected TLR4 expression in gastric cardia epithelial cells and demonstrate a progressive increase in TLR4 expression from normal gastric cardia tissue, gastric cardia inflammation, and GCC, which suggests that TLR4 plays a role in GCC carcinogenesis.All surgical pathology reports with a final diagnosis of EC and GCC were collected from the Tumor Hospital and the First Affiliated Hospital of Shantou University Medical College in China. Not all patients underwent radiotherapy and chemotreatment postoperatively. All cases were divided into EC and GCC groups based on the location of the tumor epicentre. Inclusion criteria were (1) surgical resection of tumors with lymph node dissection and (2) for GCC, the center of cancer within 2 cm of the GEJ on the gastric side. The GEJ was as defined by the WHO . Most ofμL extracted DNA, 4 μL primer, 3 μL of 10x PCR buffer, 0.3 μL AmpliTaq DNA polymerase, and 3 μL dNTP. The amplification cycle consisted of an initial denaturation at 94°C for 3 min, followed by 34 cycles at 94°C for 30 s, 50°C for 45 s, 72°C for 1 min, and a final extension at 72°C for 10 min. PCR products were analyzed by electrophoresis on a 2% agarose gel stained with ethidium bromide.A total of 127 gastrointestinal mucosal tissue samples were collected from 83 patients with GCC and 44 with EC in the Tumor Hospital of Shantou University Medical College in China. Tissue DNA was extracted using a commercially available kit  according to the manufacturer's instructions . The priμm. Each sample was deparaffinized in xylene, rehydrated in a graded ethanol series, then preincubated with 3% hydrogen peroxide for 10 min. Antigen retrieval was performed by microwave heating. Following incubation in 10 mmol/L citrate buffer for 20 min, sections were incubated with primary antibody for TLR4  at 4°C overnight, then horseradish peroxidase-conjugated goat antirabbit IgG antibody at 37°C for 30 min, and counterstained with haematoxylin. Images were captured under a Leica IM50 microscope .Immunohistochemical (IHC) staining involved the Envision Labeled Peroxidase System . Paraffin-embedded samples were sectioned at 4 IHC slides were evaluated by two experienced pathologists in a blinded manner. Staining intensity score (0–3) was considered according to a subjective evaluation of the intensity of marked cells . The overall staining intensity (0–3) was multiplied by the proportion of positive cells (0–100%), and all values were added to generate a final score ranging from 0 to 300 .Inflammation in the gastric cardia tissue was graded as normal, mild, and severe according to the updated Sydney System . The nort-test for trend was used to evaluate the trend in increase or decrease in expression. All statistical analyses involved the use of SPSS v16 . P < 0.05 was considered statistically significant.Differences in frequencies between the two groups were analyzed by using chi-square or Fisher's exact test, as appropriate. Survival was estimated by the Kaplan-Meier method with a log-rank test. Cox regression analysis was used to identify risk factors for overall survival.


Patients with tumor size >5 cm were less likely to gain CA1st. Procedure-related complications were recorded and no procedure-related death occurred. 22 (10.4%) complications occurred with 8 (3.8%) being major ones. Tumor characteristics  do not significantly influence complication rates. After a median follow-up of 41.0 (ranging 25.0–63.5) months, the median RFS and OS was 14.0 months (95% CI: 9.254–18.746) and 41.0 months (95% CI: 33.741–48.259) respectively. Multivariate analysis identified two significant prognosticators  of RFS and five significant prognosticators  of OS. In conclusion, MWA provides high technique effectiveness rate and is well tolerated in patients with HCC as a first-line treatment.This retrospective study aimed at evaluating the long-term outcomes and prognostic factors of microwave ablation (MWA) as a first-line treatment for hepatocellular carcinoma (HCC). 221 consecutive patients receiving MWA in our center between October 11, 2010 and December 31, 2013 were enrolled. Technique effectiveness was evaluated one month post-ablation. Initial complete ablation (CA1 Hepatocellular carcinoma (HCC) is one of the most common cancers and the second leading cause of cancer death worldwide1456656911In recent years, MWA is gaining momentum in the clinic. As the number of HCC patients receiving MWA keeps increasing, great variance in the progression-free survival (PFS) and overall survival (OS) after MWA has been observed among individual patients. In order for prognosis predication and patient stratification, there is a need to investigate prognosticators of patients with HCC receiving MWA. This study aimed at evaluating long-term outcomes and complications of HCC patients receiving MWA as an initial treatment and identifying clinicopathologic characteristics that significantly impact patients’ RFS and OS.The protocol of this study conformed to the ethical guidelines of the World Medical Association Declaration of Helsinki and was approved by the Institutional Ethics Committee of Renji Hospital . The medical records of HCC patients who received MWA in Renji Hospital  from October 11, 2010 to December 31, 2013 were retrieved and reviewed. Informed consents from patients to allow the review and analyses of their medical records were obtained. The flowchart of patient enrollment of this study is shown in 9/L, INR < 2.0) and renal (creatinine <2.0 mg/dL) functions.Patients inclusion criteria: (1) HCC patients who received ultrasound-guided percutaneous MWA as an initial anticancer treatment; (2) Total number of tumor lesions ≤3; (3) Largest single tumor diameter ≤10 cm; (4) For patients with multiple tumors (2 or 3), no more than one lesion >5 cm; (5) ECOG (Eastern Cooperative Oncology Group) performance status (PST): 0–1; (6) Child-Pugh score A or B; (7) Adequate hematologic (platelet count >40 × 10Patients exclusion criteria: (1) Patients who received anticancer treatment before MWA, such as hepatic resection, sorafenib, radiofrequency ablation or transcatheter arterial chemoembolization (TACE); (2) Patients who received laparoscopic MWA or intraoperative MWA rather than percutaneous MWA as initial treatment ; (3) Patients with portal vein, hepatic vein or inferior vena cava invasion, extrahepatic metastases, or malignancies of other tissue-of-origin; (4) Patients with signs of decompensated cirrhosis such as clinical hepatic encephalopathy and refractory ascites.HCC was diagnosed according to the recommendations by the European Association For The Study Of The Liver (EASL)HCC were staged according to the Cancer of the Liver Italian Program (CLIP) stageFor each patient, treatments were allocated based on patient will and clinicopathological characteristics, which were assessed by the HCC Expert Team in Renji Hospital. This team comprised hepatologists, liver surgeons, interventional radiologists and oncologists.It should be noted that although a substantial part of patients included in our study were candidates for liver resection or liver transplantation according to mainstream guidelines5It is also noteworthy that in our institute, the initial treatment options for patients beyond Milan criteria (single tumors ≤5 cm or 3 nodules ≤3 cm) included TACE, MWA, RFA, liver resection, liver transplantation and sorafinib . There wAll MWA procedures were performed percutaneously guided by real-time ultrasound (MyLab Twice scanner or HM1498XS1 scanner) using a 3.5 Mhz probe. The selection of local or general anesthesia was based on tumor number, size and location. General anesthesia was performed in patients with tumors ≥3 cm in diameter, multiple tumors, or tumors adjacent to nerve-rich areas such as abdominal wall or major hepatic vessels. Vital signs were monitored throughout the procedures.MWA was performed with a 2450 MHZ MTC-3C microwave generator , which has a 25 cm cooled-shaft electrode probe (15-gauge) with a 1.5 cm expandable tip. Power output was set at 80 to 100 watts.During ablation procedure, complete coverage of the tumor region by hyperechoic under real-time ultrasound was regarded as a measure of complete ablation. In non-risk areas (definition shown below), at least 0.5 cm of the normal hepatic parenchyma surrounding the tumor was ablated as an ablative margin to guarantee complete tumor destruction. For tumors in risk areas (definition shown below), the width of ablative margin was narrowed in the intervals between tumor and adjacent tissues to reach a balance between the need of an ablative margin and the avoidance of heat injury to adjacent tissues.To treat the majority of tumors within 3 cm in diameter, single ablation plus needle-tract ablation with one electrode was usually sufficient, but for those highly irregular ones, multiple ablations were applied so as to guarantee treatment efficacy.For tumors ranging 3–5 cm in diameter, the strategy of multiple overlapping and needle-tract ablations with one electrode were harnessed. The electrode was inserted until the distal margin of the lesion and then was withdrawn every 1.0–1.5 cm to repeat the ablation.For tumors larger than 5 cm, multi-electrode, multi-tract and multiplanar ablation strategy was used in which two electrodes were inserted parallelly  through the same intercostal space to ablate tumor and then repeated the processes through the next intercostal space until the entire tumor was ablated. Detailed ablation parameters were shown in Ablation strategy  were discussed at an interdisciplinary meeting prior to each MWA procedure and were dependent mainly on tumor characteristics and patient general conditions.After ablation, patients were monitored for a couple of hours in a recovery unit and then were sent back to the ward. A complete panel of blood chemistry including liver and renal functions was examined pre- and post-ablation.Based on previous literatures1515It should be noted that in our study, patients with tumors of the following characteristics had already been excluded because what they received were laparoscopy-assisted or intraoperative MWA: tumors protruding from liver surface, contacting or adhering to diaphragm or abdominal viscera.For tumors in risk areas, specific ablation technique and parameters were used. Optional routes of electrode insertion were carefully considered on ultrasound scrutiny and a route with the least possibility of injuring adjacent tissues or vessels was selected. For tumors adjacent to large vessels, the ablation power was set at relatively low level  to prevent vessel injury whereas the ablation time  was elonst (complete ablation at first MWA) and regarded as primary technique effectiveness. Otherwise, an additional session of MWA was performed and the patients were reevaluated one month later. If CA was achieved by this time, it was recorded as CA2nd and regarded as secondary technique effectiveness. Otherwise, the treatment was defined as incomplete ablation (IA2nd) and technically failing.Contrast-enhanced computed tomography (CT) or magnetic resonance (MR) was performed one month after MWA. The ablation was considered complete if the ablation zone completely covered the tumor and if there was no irregular enhancement at the treatment margin, which was recorded as CA1nd or IA2nd, the complications following the initial and the second sessions of MWA were all recorded for subsequent analysis.According to the recommendations by the International Working Group on Image-Guided Tumor AblationContrast-enhanced CT or MRI and blood chemistry was performed one month after MWA, every 3 months for the first year, and every 4–6 months thereafter.st or CA2nd), local tumor progression (LTP) was defined as the reappearance of enhancement within or along the margin of the ablation zone on follow-up imagines. Intrahepatic distant recurrence (IDR) was defined as the occurrence of HCC within the liver at locations not contacting the ablation zone. Extrahepatic recurrence (ER) was defined as metastases outside the liver.Tumor progression and recurrence were defined according to recommendations by the International Working Group on Image-Guided Tumor AblationRecurrence-free survival (RFS) was calculated from the day of initial MWA to the day of either the earliest event  or last follow-up without an event. Overall survival (OS) was calculated from the day of initial MWA to the day of death  or last follow-up.st or CA2nd included: liver transplantation in 4 patients, hepatic resection in 4 patients, TACE in 86 patients, TACE plus sorafinib in 4 patients, MWA in 20 patients, RFA in 32 patients, and Chinese traditional medicine in 7 patients. Detailed treatment strategies after tumor progression/recurrence were shown in Treatment strategies after tumor recurrence for patients of CA1p values < 0.15 on univariate analyses were included in multivariate analysis . All statistical analyses were conducted using SPSS 22.0 . p < 0.05 was considered as statistically significant.Chi-square test or Fisher’s exact test were used to analyze the correlations between tumor characteristics and technique effectiveness, as well as correlations between tumor characteristics and incidence of complications. among patients with difference were analyzed with. Median OS (mOS) and median RFS (RFS) were calculated using the Kaplan-Meier method and compared by the log-rank test. Variables with According to the patient inclusion and exclusion criteria, a total of 221 patients were included in this study finally , of whicst was gained in 201 (90.95%), CA2nd in 8 (3.62%) whereas the other 12 (5.43%) suffered from technique failure (remaining incomplete ablation [IA2nd] after two sessions of MWA). No patients died within 30 days of MWA. Chi-square test or Fisher’s exact test were performed to investigate whether tumor characteristics  can impact technique effectiveness. The results showed that patients with tumor > 5 cm are more likely to suffer from IA2nd and less likely to gain CA1st compared with those with tumor ≤5 cm in size , 170 had suffered from tumor progress or recurrence by the last follow-up, of which 33 were LTP, 120 were IDR, and the other 17 were ER. The median RFS (mRFS) and median OS (mOS) of the total cohort was 14.0 months (95% CI: 9.254–18.746) and 41.0 months (95% CI: 33.741–48.259), respectively. The 1- and 2-year RFS rates were 35.9% and 15.3% respectively, and the corresponding OS rates were 87.1% and 63.2% respectively.Of the 209 patients with primary or secondary technique effectiveness  and AFP 20–400 ng/ml  were significant favorable prognosticators of RFS , 14 months (95% CI: 0.000–30.631) and 12 months (95% CI: 4.079–19.921) respectively (p < 0.001)  .p < 0.001), three lesions also showed an unfavorable trend albeit without statistical significance . Compared with AFP > 400 ng/ml, AFP ≤ 20 ng/ml  and AFP 20–400 ng/ml  were significant favorable prognosticators (p < 0.001) and 3–5 cm  were significant favorable prognosticators. Compared with LTP, IDR  was significant favorable prognosticators whereas ER  was significant unfavorable prognosticators. Besides, GGT ≤ 50 U/L  and ALK ≤ 110 U/L  were significant favorable prognositcators of OS relative to GGT > 50 U/L and ALK > 110 U/L, respectively  complications were observed during follow-up, of which 8 (3.8%) were major complications . All theTo date, only a few studies1517191519202121152021Our results showed that lower levels of AFP and GGT were significant favorable predictors of RFS on both uni- and multivariate analyses . As for st, CR2nd and IA2nd were 43.0 months (95% CI: 34.526–51.474), 14.0 months (95% CI: 0.000–31.631) and 12.0 months (95% CI: 4.079–19.921), respectively (p < 0.001)  . This isst and 3.6% for CA2nd). Although tumors >5 cm are not recommended for local ablative therapies in mainstream guidelinesst was gained in 81.8% of these patients, much higher than the reported initial complete response rate  of HCC patients receiving repetitive TACESecond, our study revealed that MWA can provide complete tumor ablation in the majority of tumors >5 cm  did not significantly affect RFS, OS and complication rates and should not be regarded as a contraindication to MWA treatment. Controversies long exist regarding the influence of “risk areas” on treatment efficacy of MWA. A previous studyp < 0.001), lending support to a previous assumption that LTP might be another form of incomplete ablation, in which the residual tumor cells get into a dormant state following ablation and afterwards become the source of recurrenceForth, the mOS of patients with LTP  was significant shorter than that of patients with IDR   of patients in our study had HBV infection, the scenario of antiviral treatments they received may alos impact both RFS and OS and thus should be taken into analyses. However, these data are missing in our study.In conclusion, our study analyzed long-term outcomes and prognosticators of RFS and OS in HCC patients receiving MWA as an initial treatment. Our results suggest that MWA provides high technique effectiveness rate and is well tolerated in patients with HCC. We identified levels of AFP and GGT as independent prognosticators of RFS and identified tumor number, tumor size, AFP, GGT and recurrence type as independent prognosticators of OS, which may guide patient selection and prognosis prediction and hold the potential of changing clinic practice of MWA for HCC. These results need to be validated in larger patient cohorts, and prognostic nomograms need to be developed in the future to provide reliable and convenient systems for the prediction of RFS and OS in HCC patients receiving MWA.How to cite this article: Wang, T. et al. Microwave ablation of hepatocellular carcinoma as first-line treatment: long term results and prognostic factors in 221 patients. Sci. Rep.6, 32728; doi: 10.1038/srep32728 (2016).


RDD does not usually threaten life and spontaneous remission is frequently observed. However, unusually in our case the patient passed away within 1 month of confirmed diagnosis.As Rosai–Dorfman Disease presents generally nonspecific symptoms, differential diagnosis can be of great learning curve for physicians. Additionally,  A 38‐year‐old male was admitted with a fever (39°C), abdominal and back discomfort, and extensive lymphadenopathy. Laboratory tests revealed progressive thrombocytopenia and leukocytosis, platelet count of 84 × 109/L and peak WBC of 46.52 × 109/L. CT scanning uncovered enlarged lymph nodes of the neck, axillary (28/23 mm), hilar (21/24 mm), subcarinal (19/18 mm), paraaortic (30/29 mm), mesenteric (41/35 mm) and curvatura minor gastritis (26/22 mm). Additionally, abdominal ultrasound revealed free fluid in the abdomen combined with an enlarged liver (157 mm) and spleen (157/56 mm). Sterile blood culture ruled out the possibility of a bacterial or fungal infection. An excisional biopsy of an axillary lymph node was performed with no complications. Microscopic examination of the specimen revealed a diffuse proliferation of histiocytes, fibrosis with inflammatory infiltration of the node's capsule and lymphocytes engulfed in the cytoplasm of the histocyte‐like cells (emperipolesis), typical pathology of Rosai–Dorfman Immunohistochemical stain analysis revealed histiocytes staining strongly for S‐100 protein, CD68 protein, and expansion of sinusoids with an uncontrolled expression of plasma cells, lymphocytes and histiocytes with large oval nuclei. Despite the disease having relatively good prognosis, the patient was dead within a month of confirmed diagnosis, occurring in <3% of Rosai–Dorfman disease cases. The patient's condition progressively deteriorated prior to death. Enlargement of lymph nodes, acute circulatory deficiency, and acute respiratory failure was treated with Dexamethasone and Methylprednisolone. The cause of death was due to suspected respiratory infections resulting from therapy‐based immunosuppressants and initial chemotherapy treatment. No autopsy was carried out due to the patients family declining. As observed in our patient, common symptoms of Rosai–Dorfman disease are nonspecific and can be manifested in malignant and nonmalignant diseases, such as lymphomas, Langerhans cell histiocytosis None declared.DB: primary surgeon of the excisional biopsy; involved in critical analysis of report. ZB: wrote the report. SA: wrote the report. NT: surgeon of the excisional biopsy of lymph nodules. RR: involved in the final approval of report.


Intestinal bile salts (BSs) may be implicated in NEC development. We hypothesized that fecal BS levels are higher in preterm infants at risk for NEC.We compared the composition and concentration of fecal BSs in ten preterm infants who developed NEC (Bell’s Stage ≥ II) with twenty matched control infants without NEC. Conjugated and unconjugated fecal BSs were measured after birth (T1) and twice prior to NEC . Data are presented as medians and interquartile ranges.+4 weeks and ~1010 g. Age of NEC onset was day 10 (8–24). T1 was collected 2 (1–3) days after birth. T2 and T3 were collected 5 (5–6) days and 1 (0–2) day before NEC or at corresponding postnatal ages in controls. The composition of conjugated BSs did not differ between the two groups. Total unconjugated BSs were 3-fold higher before NEC compared to controls at corresponding ages (0.41 μmol/g feces (0.21–0.74) versus 0.14 μmol/g feces (0.06–0.46), p < 0.05).GA and BW were similar in all preterms: ~27Fecal BS concentrations are higher in preterm infants who develop NEC compared to infants without NEC. Further study is needed to determine the predictive value of fecal BSs in the development of NEC. It remains unpredictable whether or not a preterm infant will develop NEC, despite well-characterized epidemiological and clinical risk factors such as low gestational age (GA), low birth weight (LBW), formula feeding, and antibiotic treatment . Early sin utero, and BSs can be detected in meconium [It is thought that the accumulation of intestinal bile salts (BSs) is implicated in the development of NEC. BSs are the main organic constituents of bile and act as biological detergents that facilitate intestinal absorption of dietary fats. The so-called primary BSs, i.e. cholate and chenodeoxycholate, are synthesized from cholesterol in the human liver and are conjugated with either the amino acid glycine or taurine. The synthesis of BSs already starts meconium . Intestimeconium ,4. Intrameconium . Previoumeconium . Intestimeconium . In agremeconium . Furthermeconium .The foregoing data support the putative role of high intestinal BSs in NEC development. We hypothesized that high levels of fecal BSs might serve as early parameters to identify infants with imminent NEC development. Prospectively, we determined fecal BS concentrations and compositions in preterm infants at risk for NEC. Subsequently, we compared BS parameters of infants who later developed NEC with GA and BW-matched infants who did not develop the disease.We obtained written, informed parental consent in all cases. The study was approved by the ethics review board of University Medical Center Groningen.This study was a case-control study and part of a large, prospective observational cohort registered with the Dutch Trial Registry . The study was performed in the NICU of the Beatrix Children’s Hospital of the University Medical Center Groningen (UMCG) between October 2012 and February 2014. For the purpose of the cohort study, preterm infants with a GA below 30 weeks, a BW below 1000 grams, or a combination of a GA below 32 weeks and a BW below 1200 grams were eligible for enrollment within the first 48 hours after birth. Exclusion criteria consisted of congenital intestinal diseases or abdominal wall defects. One hundred infants were included in the cohort study. For the present study, we selected all infants with NEC (n = 10) and matched two controls from the same cohort to each infant who developed NEC, using GA and BW, in descending order of importance, allowing for a maximum deviation of 10%.We collected all data prospectively. Neonatal data included gender, GA, BW, twin or singleton, antenatal treatment with steroids, preterm prelabor rupture of membranes >24 hours, Apgar scores at 1, 5, and 10 minutes, antibiotic treatment during first 48 hours after birth, prolonged antibiotic treatment (> 48 hours after birth), hemodynamically significant patent ductus arteriosus (hsPDA), ibuprofen treatment for hsPDA, time to onset of enteral feeding and type of enteral feeding. In addition, we recorded the need for mechanical ventilation, inotropics, fluid resuscitation, and red-blood cell transfusion during the study period as well as mortality data. Data are in ®, Nutricia, Zoetermeer, the Netherlands) was added after the first week when feeding volume was minimally 80 mL/kg/day. Parenteral nutrition was started immediately after birth; amino-acids from the date of birth and fat emulsions thereafter.Enteral feeding consisted of own mother’s milk, preterm formula, or a combination if own mother's milk was not (sufficiently) available in 21 infants. Nine patients (1 NEC and 8 controls) had been included in the Early Nutrition Study , a RCT comparing effects of pasteurized donor milk or preterm formula during the first 10 days of life in addition to mother's own milk in very low birth weight infants (< 1500 g.) [The primary outcome of this study was a combination of fecal BS concentrations and composition in preterm infants who did, or did not, develop NEC. NEC diagnosis was defined by the presence of either pneumatosis intestinalis or portal venous gas, or both according to the modified Bell’s staging criteria (Bell’s Stage ≥ II). The diagnosis was established by the attending radiologist and later confirmed independently by five neonatology consultants. We defined the time of NEC onset as the first abdominal X-ray after clinical suspicion of the disease. All preterm infants with NEC were treated conservatively with nil per os, gastric decompression, and broad-spectrum antibiotics until radiographic signs of NEC resolved and clinical signs stabilized. Surgery was performed when a perforation was suspected or when the infant deteriorated clinically despite conservative treatment.Fecal samples were collected from the diaper in sterile tubes within 48 hours after birth and subsequently twice weekly on every first and fourth day calculated from birth, until either NEC Bell’s Stage ≥ II occurred, or the infant was transferred to another hospital, or reached the age of 36 days. If the infant did not produce feces on the predetermined day of feces sampling, the next feces produced was collected (up op two days after the predetermined sampling day). In controls, selection of feces samples was adjusted according to the closest corresponding time point of feces samples from the infants who ultimately developed NEC. Fecal samples were stored at -80°C until batch analysis of unconjugated and conjugated BSs.measured BSs. The lower limit of detection of unconjugated BSs varies between 0.3 and 0.5 nmol dry weight feces, but is slightly dependent on the BS species . Fecal conjugated BS composition was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This system consists of a Shimadzu liquid chromatography system  coupled to a Sciex API-3200 triple quadrupole mass spectrometer with an electrospray ionization (ESI) source . In short, about 50 mg of freeze-dried homogenized feces was weighed exactly and extracted with 2 mL of alkaline methanol for two hours at 80°C. After cooling down to room temperature, 8 mL of demineralized water was added and the tubes were vortexed. After adding 250 μL internal standard solution to 1 mL of sample solution, the samples were prepared for analysis using solid phase extraction (SPE). After SPE, the samples were dried using N2 at 40°C, and the samples were redissolved in 500 μL 50% methanol. Finally, the samples were filtered with 0.2 μm spin filters. Ten μL of sample was injected into the LC-MS/MS system. The BS species we evaluated were glycocholate (GC), taurocholate (TC), glycochenodeoxycholate (GCDC), and taurochenodeoxycholate (TCDC). Data are in Fecal BS concentrations were measured in an aliquot of freeze-dried homogenized feces and determined by capillary gas chromatography (GC) as methylester-trimethylsilyl derivatives on a Hewlett Packard gas chromatograph (HP 5880 A) as previously described . GC methp < 0.05. All analyses were performed using SPSS 22.0 software for Windows .All data of fecal BSs are presented as medians and interquartile ranges (IQR) unless specified otherwise. Either odds ratios ) or the Mann-Whitney test was used to analyze differences in clinical characteristics, as appropriate. The Median Test (one-tailed) was used to compare levels of fecal unconjugated BS concentrations and of percentages of conjugated BS profiles between the two groups at the three different points in time (independent variables). The Wilcoxon signed Rank Test (two-tailed) was used to compare median levels of fecal BS concentrations within subgroups (dependent variables). Cut-off values for NEC prediction were investigated with receiver operating characteristics (ROC) analyses. The level of significance for statistical analyses was set at Ten preterm infants developed NEC during the study period at a median age of 10 (4–30) days after birth. Bell’s Stage II occurred in two infants and Bell’s Stage III in eight. Seven infants underwent surgery. In one infant surgery was indicated but not performed due to the infant’s poor clinical condition. Three out of the ten infants who developed NEC died as a consequence of the disease. A total of 76 fecal BS samples were available for analysis of preterm infants: 25 samples of infants who ultimately developed NEC and 51 samples of control infants. Unconjugated BSs could be measured in 59 samples (24 NEC and 35 controls), and conjugated BSs could be measured in 69 samples (22 NEC and 47 controls). In all infants, the first feces sample (T1) was collected at a median of 2 days after birth (IQR 1–3). In all infants, the two consecutive samples taken immediately prior to the diagnosis of NEC, T2 and T3, were collected at a median of 5 days (IQR: 5–6) and 1 day (IQR: 0–2) before onset of NEC, respectively. In NEC infants, the last feces sample was collected at a median (IQR) of 1.5 (0–3) day before NEC occurred. In controls, median (IQR) day of life (DOL) at T2 was 5 (4–21.5) days, compared to 7.5 (4–20) in the NEC infants (NS). Median (IQR) DOL at T3 was 11.5 (8–24) in controls and 12 (8–12) in NEC infants (NS).p = 0.02). In the feces samples at T3, one day prior to NEC, total unconjugated BSs were two-and-a half times higher in the NEC group than in the controls (0.29 μmol/g feces (0.19–0.46) versus (0.13 μmol/g feces (0.05–0.37), p = 0.07). When the fecal samples of T2 and T3 were analyzed together, we found that total unconjugated BSs were significantly increased in infants developing NEC (median 0.41 μmol/g feces (0.21–0.74) versus 0.14 μmol/g feces (0.06–0.46), p = 0.03).Total BS concentrations were not significantly different in the first feces samples after birth (T1) between the NEC group and the controls . At T2, p = 0.01, p < 0.05).We observed no statistically significant decrease in total fecal unconjugated BSs between any of the time points in the infants developing NEC. The decrease in total BSs was statistically significant in the controls between T1 (0.86 μmol/g feces (0.55–1.41) and T2 (0.16 μmol/g feces (0.05–0.99), p = 0.06). Of unconjugated BSs, chenodeoxycholate (CDC) was the only unconjugated BS species that showed a trend toward significance between the NEC group and the controls. Unconjugated CDC concentrations were higher in the infants developing NEC in the week prior to NEC (0.18 μmol/g feces (0.13–0.43) versus 0.08 μmol/g feces (0.03–0.28), p = 0.02, The ROC curve of total BSs at T2 and T3 illustrated that total unconjugated BS > 0.13 μmol/g feces tended to predict NEC with 93% sensitivity and 47% specificity (area under curve (AUC) 0.74. 95% CI 0.57–0.91; Our aim was to determine whether fecal BS analysis could, potentially, provide an early marker for the development of NEC in preterm infants. We demonstrated a significantly higher level of total fecal BSs in the week before NEC started to develop compared to corresponding samples in GA and BW matched controls. Yet, the differences in the composition of conjugated fecal BSs at any of the three time points were not statistically significant between the NEC group and the controls. The difference in fecal BS could also not be attributed to major differences in specific, individual unconjugated BS. Apparently, the decrease was quantitative more that qualitative, and the variation in individual BS concentrations precluded statistical significance. Taking into consideration the sensitivity and specificity of fecal BSs for the development of NEC, it seems impossible at this stage to predict the onset of NEC using fecal BS analysis in preterm infants.Our results are in accordance with previous analyses of total BS concentrations in neonates, i.e. with respect to the highest levels for total BSs in meconium . In our We acknowledge several limitations to our study and results. Obviously, due to its size, our sample lacked the power to discriminate between a normal and a slow albeit “NEC-predisposing” decay pattern of fecal BSs. A substantial larger sample size  would be required for sufficient power. Secondly, measurement of fecal BSs on one particular day does not allow one to predict whether and if so when NEC will occur: serial measurements from birth until NEC onset should be obtained to assess the decay of fecal BSs. Thirdly, higher levels of fecal BSs, or a slower decrease in fecal BSs, may help to identify preterm infants with an increased risk of developing NEC, but it remains unknown how we can reduce the risk of NEC in these particular infants. Finally, no data on the fecal microbiome, that would affect bile salt composition, were provided.The applied GC methodology allows for reliable and fast quantification of many BSs in a way that may more or less be routinely incorporated in clinical care for preterm infants. Yet, we could not detect either conjugated or unconjugated BS in some feces samples. Upon its consistency upon reanalysis, it is tempting to speculate that—apart from any methodological issues—variations in the developing microbiome of the immature intestine in preterm infants is responsible for these variations. We did not choose any (un)conjugated at forehand. Rather, these particular BS species appeared to be present upon analysis by our methodology. Interestingly, HC and HDC are usually not detected in appreciable amounts in (adult) humans. The presence of these BS species in some of the samples is not clarified. The observation that they are found both in NEC and in non-NEC feces pleads against an important pathophysiological role in the development of NEC.Despite predictive clinical risk factors NEC remains a multifactorial disease with rapid unpredictable onset of intestinal damage for which powerful predictive biochemical markers are urgently needed, but these are, as yet, unavailable ,17. The In preterm infants who develop NEC, the levels of fecal BSs are higher in the week preceding NEC than in controls. Serial measurement of fecal BSs may help to identify preterm infants who are vulnerable to developing NEC.S1 File(SAV)Click here for additional data file.S2 File(SAV)Click here for additional data file.S3 File(SAV)Click here for additional data file.