The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor that regulates lipid catabolism and inflammation and is hepatocarcinogenic in rodents. It is presumed that the functions of PPARα in liver depend on cross-talk between parenchymal (hepatocytes) and non-parenchymal  fractions as well as inter-organ interactions. In order to determine how cellular composition and inter-organ interactions influence gene expression upon pharmacological activation of PPARα, we performed a meta-analysis of transcriptomics data obtained from mouse hepatocytes , mouse liver slices (containing both fractions), and mouse livers exposed to a PPARα agonist. The aim was to obtain a comprehensive view of common and model-specific PPARα-dependent genes and biological processes to understand the impact of cross-talk between parenchymal and non-parenchymal fractions as well as the effect of inter-organ interactions on the hepatic PPARα transcriptome.in vitro), mouse precision cut liver slices treated with Wy14643 for 24 h (ex vivo), and livers of wild type and Ppara knockout mice treated with Wy14643 for 6 h or 5 days (in vivo).To this end we analyzed microarray data of experiments performed in mouse primary hepatocytes treated with the PPARα agonist Wy14643 for 6 or 24 h  contains supplementary material, which is available to authorized users. Peroxisome proliferators, which include insecticides, herbicides, surfactants, phthalates, organic solvents, and hypolipidemic/anti-inflammatory fibrate drugs, were discovered as chemicals that upon administration to rodents increase the number of liver peroxisomes, stimulate fatty acid catabolism, and after chronic exposure cause hepatomegaly and hepatocarcinogenesis . The effApart from its profound role in regulation of lipid metabolism, PPARα exerts anti-inflammatory and anti-atherogenic effects, which may be beneficial in the treatment of several metabolic diseases associated with inflammation such as non-alcoholic steatohepatitis and atherosclerosis , 13. Thein vitro), precision cut liver slices  treated with Wy14643 for 24 h (ex vivo), and livers of wild type (WT) and Ppara knockout (KO) mice treated with Wy14643 for 6 h or 5 days (in vivo). The aim was to obtain a comprehensive view of common and model-specific PPARα-dependent genes and biological processes to understand the impact of the cross-talk between parenchymal and non-parenchymal fractions as well as the effect of inter-organ interactions on the hepatic PPARα transcriptome. In addition, we aimed to assess the performance of in vitro and ex vivo liver models to study the PPARα transcriptome in relation to the in vivo situation.Although PPARα is expressed in the liver parenchymal fraction (i.e. hepatocytes) and is not or very weakly expressed in the rodent non-parenchymal liver fraction  , 17, 24,Wy14643 was obtained from ChemSyn Laboratories . Recombinant human insulin (Actrapid) was obtained from Novo Nordisk . DMEM, fetal calf serum, calf serum, and penicillin/streptomycin/fungizone were obtained from Lonza Bioscience . Williams E medium supplemented with Glutamax, penicillin/streptomycin (pen/strep), D-glucose, phosphate buffered saline (PBS) were obtained from Invitrogen . Otherwise, chemicals were obtained from Sigma .Mouse hepatocytes were prepared and used in experiments have been described previously . Briefly2/80% O2 under continuous shaking. After 1 hour the media was replaced with fresh William’s E Medium in the presence or absence of Wy14643 (20 μM) dissolved in dimethyl sulfoxide (DMSO). After 24 h incubation, samples were snap-frozen in liquid nitrogen and stored in −80°C for RNA isolation. PCLS were obtained from four mice and were cultured independently per each experimental condition i.e. n = 4 for Wy14643 and n = 4 for DMSO.Precision cut liver slices (PCLS) were prepared and cultured as described previously . BrieflyIn vivo treatment with Wy4643 has been described previously [n = 5 per group). During chronic pharmacological activation of PPARα by Wy14643, WT and Ppara-KO mice were fed with Wy14643 for 5 days by mixing it in their food . Livers from the acute pharmacological activation of PPARα were collected at ~3 p.m. (ZT9) and livers from the chronic activation of PPARα were collected at ~11 a.m. (ZT5). Livers from all experiments were dissected and immediately frozen in liquid nitrogen for RNA isolation. All animal experiments were approved by the Ethical Committee for Animal Experiments of Wageningen University.eviously . BrieflyTotal RNA was extracted from primary hepatocytes using TRIzol reagent  and RNA was further purified using RNeasy micro columns .g, 8°C). Next, total RNA was isolated using the RNeasy Tissue Mini Kit  according to manufacturer’s protocols.Prior to RNA isolation, PCLS were homogenized with a tissue homogenizer Precellys 24 Bertin Technologies  using settings: 2x , and purified and DNAse treated using the SV Total RNA Isolation System .RNA concentration and purity were assessed spectrometrically using a Nano Drop ND-1000 spectrophotometer . RNA quality was assessed on an Agilent 2100 bioanalyzer . Only RNA samples that met quality criteria (i.e. RNA Integrity Number >8.0) were used for array hybridization experiments. For primary hepatocytes, purified RNA (500 ng) was used for one cycle cRNA synthesis . Hybridization of samples derived from primary hepatocytes was performed on Affymetrix Gene chip mouse genome 430 2.0 arrays. Purified RNA (100 ng) isolated from PCLS was labeled with the Ambion WT expression kit  and hybridized to Affymetrix Mouse Gene 1.1 ST array plate . RNA isolated from mouse livers (5 μg) was used for one cycle cRNA synthesis  and hybridized to Affymetrix Genechip mouse genome 430 2.0 arrays (wildtype and Ppara-KO treated with Wy14643 or vehicle for 6 hours as well as wildtype and Ppara-KO treated with Wy14643 or vehicle for 5 days). Hybridization, washing, and scanning of all Affymetrix Genechips was according to standard Affymetrix protocols. Scans of the Affymetrix arrays were processed using the Bioconductor package . The micExpression levels of probe sets were calculated using Robust Multiarray Averaging (RMA) with m-estimator summarization and remapped in Common Data Format (CDF) version 17.0.1 . Identifin vitro, ex vivo, and in vivo hepatic models. After correction for FDR ≤ 0.005 (Benjamini Hochberg), the GO terms were selected for further analysis and interpretation.The significantly up- and down-regulated genes by the treatments (i.e. Wy14643) were uploaded separately to the Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resource. In DAVID the Functional Annotation Clustering tool generated clusters of up- or down- overrepresented Gene Ontology (GO) terms , 32. TheVenn diagram analysis of the significantly affected genes identified in our study was performed using VENNY .Pearson’s correlation analysis was performed using IBM SPSS Statistics 20 and the results are presented as scatterplots using Microsoft Excel 2010.in vitro, ex vivo, and in vivo) treated with the synthetic PPARα agonist Wy14643 using Rank Products (RP) [in vivo hepatic gene expression elicited by Wy14643 and another common PPARα agonist fenofibrate were extremely similar  . The usein vitro and ex vivo) vs. the more complex models (i.e. in vivo), Venn diagram analysis of genes significantly regulated in each model was performed. The analysis showed that there was a substantial overlap between the “simpler” hepatic models and the in vivo models, Figure To determine the resemblance between “simpler” liver models  identified in vivo , an additional functional analysis, referred to as “PPARα-tailored analysis”, was performed. The incentive for this additional analysis was to annotate a higher number of genes than via GO analysis and assign genes to novel and more detailed functional categories. The “PPARα-tailored analysis” was performed gene by gene using the open access database GeneCards. GeneCards provides current genomic, proteomic, genetic, transcriptomic, and functional information on all known and predicted genes. The selection of genes for the “PPARα-tailored analysis” was rather subjective; we considered all significant genes that were overlapping between at least two hepatic models Figure A-B. Evenin vivo  , “cytoskeleton”, “endothelial functions”, “integrity of Golgi”, “vesicular trafficking”, “endocytosis”, and “neurotransmission”. Genes belonging to these categories were significantly upregulated almost exclusively in vivo, followed by ex vivo, and the least changes occurred in in vitro models. However, compared to GO analysis, the “PPARα-tailored analysis” annotated a higher percentage of the analyzed genes (i.e. ~50% vs. ~25% respectively) as well as identified additional and novel PPARα-related functional categories. A summary of the results of the ‘PPARα- tailored analysis’ is presented in Figure In general, the results of the “PPARα-tailored analysis” were in line with the statistical and GO analyses and revealed that the most significant changes occurred Biological processes identified in the meta-analysis were integrated into a simplified concept of hepatic PPARα signaling under not-inflamed conditions Figure . We propThe challenge in meta-analyses is to circumvent the variation of independent microarray experiments and extract biologically relevant information . The met2 method [To minimize the effects of the above-mentioned experimental differences, and to allow identification of biologically relevant significant genes, we applied the RP approach. The RP approach is a powerful statistical method to analyze microarray data generated from relatively few replicates, performed in different laboratories and/or using different platforms. Moreover, RP outperformed other statistical tests applied in meta-analyses such as t-based approach and Fisher’s Inverse χ2 method , 36, 37.In our study, we identified a variable number of significantly up- and down-regulated genes in different liver models. The number of significantly regulated genes corresponded with the complexity of the hepatic model determined by time of exposure, cellular composition, and the absence/presence of systemic effects (i.e. inter-organ interactions). Thus, the lowest number of significantly upregulated genes was found in primary hepatocytes and in this model very few genes were significantly downregulated. This finding contradicts with our previous analysis in which we identified more than 400 up- and down-regulated genes in primary hepatocytes after 6 and 24 h treatment with Wy14643 . This diin vivo[The very low number of downregulated genes in primary hepatocytes may be explained by the absence of the non-parenchymal fraction, which is an abundant source of pro-inflammatory factors . Althougin vivo.in vitro and ex vivo models for the in vivo situation, we performed a comparative analysis of the significantly altered genes in each model by means of Venn diagrams. In addition, Pearson’s correlation analysis was performed to compare the direction of changes in gene expression in in vitro and ex vivo models vs. the in vivo situation. The results of Venn diagram analysis showed substantial similarities between “simpler” liver models and the in vivo models, suggesting that primary hepatocytes and liver slices are relevant models to study pharmacological activation of PPARα in vivo. With regard to the results of Pearson’s correlation analysis, it revealed that almost all genes upregulated in vitro and ex vivo, were also upregulated in vivo. In contrast, only a small fraction of genes downregulated ex vivo was downregulated in vivo. These results indicate that primary hepatocytes and liver slices are valid liver models to study genes induced by activation of PPARα with Wy14643, but less suitable to study genes downregulated by activation of PPARα with Wy14643. This finding was unexpected and suggests that downregulation of genes by PPARα is model-dependent. Inasmuch as the cellular composition of liver slices is comparable to the liver in vivo, i.e. both models contain parenchymal and non-parenchymal cells [ex vivo.In order to evaluate the relevance of al cells , these din vitro and ex vivo models, suggesting that the presence of non-parenchymal cells does not affect the Wy14643-mediated regulation of genes involved in lipid metabolism. Genes related to lipid metabolism were induced most significantly (in terms of number and fold change) in vivo, indicating that lipid metabolism is more sensitive to regulation in vivo compared to in vitro and ex vivo models. However, it can’t be ruled out that our results are biased by the lack of standardization of conditions across all liver models in relation to differences in concentration of glucose, insulin, or other biomolecules, which could also affect lipid metabolism.In all liver models, lipid metabolism emerged as a dominant pathway regulated by PPARα , 27. Desin vivo, that could be assigned into novel functional categories not previously linked to PPARα, such as “ECM”, “cytoskeleton”, “endothelial functions”, “integrity of Golgi”, “vesicular trafficking”, and “endocytosis”. Given the key role of PPARα in regulation of hepatic lipid metabolism, we hypothesize that genes belonging to these novel categories might somehow be linked to transport, traffic, and catabolism of FA during activation of PPARα by Wy14643. It is possible that during pharmacological activation of PPARα, FA and TG carried by lipoproteins and albumin freely pass fenestrated hepatic endothelial cells [Next to the identification of processes related to different aspects of lipid catabolism, the “PPARα-tailored analysis” identified genes, mostly al cells , 44. In al cells , followeal cells . Next, tal cells . Interesal cells . Therefoex vivo and in vivo models  for 24 hours led to upregulation of 11 genes related to liver cancer  [Finally, in accordance with the known hepatocarcinogenic effect of peroxisomes proliferators in rodents , we foun24 hours , it can erpineI) . HowevererpineI) .Our observation that treatment of liver slices with Wy14643 did not alter genes related to cell proliferation conflicts with the finding that peroxisomes proliferators induced DNA synthesis in rodent co-cultures of hepatocytes with non-parenchymal cells , 53. A pex vivo and in vivo. However, the full spectrum of PPARα biology at the level of lipid metabolism, immunity, carcinogenesis, as well as novel aspects of PPARα signaling such as coagulation, vesicular trafficking and the extracellular matrix, seems to require systemic factors, as it was observed exclusively in vivo.In summary, this study provides a comprehensive picture of gene expression during pharmacological activation of PPARα by Wy14643 in different mouse hepatic models under not-inflamed conditions. PPARα-dependent regulation of many genes and processes involved in lipid metabolism is mostly independent of the presence of non-parenchymal cells or inter-organ interactions, as it was observed in all liver models. PPARα-dependent regulation of inflammatory genes requires the presence of non-parenchymal cells, because it was observed only in vitro and ex vivo systems turned out to be relevant liver models to study the PPARα transcriptome compared to the in vivo situation, liver slices behaved as an intermediate model between in vitro and in vivo, and thus represent a superior replacement for primary hepatocytes.Although A comparative analysis of gene expression by Wy14643 and fenofibrate in mouse liver. Global gene expression data obtained from samples representing livers of mice treated for 6 h with Wy14643 or fenofibrate were expressed as fold change calculated as ratio of gene expression in treatment group (n=4 or 5) vs. gene expression in control group (n=4). (PDF 624 KB)Additional file 1:PPARα tailored gene functional analysis (upregulation). Significantly upregulated genes that were overlapping between at least two hepatic models were selected for a functional analysis using an open access database GeneCards. Based on the provided information, the selected genes were grouped into functional categories and analyzed in each hepatic model i.e. primary hepatocytes treated with Wy14643 for 6 h or 24 h (PH 6H or PH 24 h), precision cut liver slices treated with Wy14643 for 24 h (PCLS 24 h), and livers of mice treated with Wy14643 for 6h or 5 days (L6h or L5d). Genes that were significantly altered are depicted in bold and underlined, red color indicates upregulation, black no change, and green downregulation of gene expression. Fold change was calculated as the average gene expression value in treatment group vs. control. (PDF 522 KB)Additional file 2:PPARα tailored gene functional analysis (upregulation). Significantly upregulated genes that were overlapping between at least two hepatic models were selected for a functional analysis using an open access database GeneCards. Based on the provided information, the selected genes were grouped into functional categories and analyzed in each hepatic model i.e. primary hepatocytes treated with Wy14643 for 6h or 24 h (PH 6H or PH 24 h), precision cut liver slices treated with Wy14643 for 24 h (PCLS 24 h), and livers of mice treated with Wy14643 for 6 h or 5 days (L6h or L5d). Genes that were significantly altered are depicted in bold and underlined, red color indicates upregulation, black no change, and green downregulation of gene expression. Fold change was calculated as the average gene expression value in treatment group vs. control. (PDF 391 KB)Additional file 3:PPARα tailored gene functional analysis (downregulation). Significantly downregulated genes that were overlapping between at least two hepatic models were selected for a functional analysis using an open access database GeneCards. Based on the provided information, the selected genes were grouped into functional categories and analyzed in each hepatic model i.e. primary hepatocytes treated with Wy14643 for 6 h or 24 h (PH 6H or PH 24 h), precision cut liver slices treated with Wy14643 for 24h (PCLS 24 h), livers of mice treated with Wy14643 for 6 h or 5 days (L6h or L5d). Genes that were significantly altered are in bold and underlined, red color indicates upregulation, black no change, and green downregulation. Fold change was calculated as the average gene expression value in treatment group vs. control. (PDF 430 KB)Additional file 4:


S. pneumoniae-related diseases. PPSV–23 was approved in 1988, while the extended use of PCV–13 was approved for adults aged 65 and older in June 2014. Despite these two vaccines being available, the recently launched national immunisation programme for the elderly only subsidised PPSV–23. The framework of the current immunisation programme lasts for five years. The elderly population eligible for the subsidised PPSV–23 shot for the 1st year are those aged 65, 70, 75, 80, 85, 90, 95 and ≥100. While from the 2nd year to the 5th year, those who will age 65, 70, 75, 80, 85, 90, 95 and 100 will receive the same subsidised shot.Currently in Japan, both 23-valent pneumococcal polysaccharide vaccine (PPSV–23) and 13-valent pneumococcal conjugate vaccine (PCV–13) are available for the elderly for the prevention of S. pneumoniae-related disease course pathways. The transition probabilities, utility weights to estimate quality adjusted life year (QALY) and disease treatment costs were either calculated or cited from literature. Cost of per shot of vaccine was ¥8,116  for PPSV–23 and ¥10,776 (US$98) for PCV–13. The model runs for 15 years with one year cycle after immunisation. Discounting was at 3%.We performed economic evaluations to (1) evaluate the efficiency of alternative strategies of PPSV–23 single-dose immunisation programme, and (2) investigate the efficiency of PCV–13 inclusion in the list for single-dose pneumococcal vaccine immunisation programme. Three alternative strategies were created in this study, namely: (1) current PPSV–23 strategy, (2) 65 to 80 (as “65–80 PPSV–23 strategy”), and (3) 65 and older (as “≥65 PPSV–23 strategy”). We constructed a Markov model depicting the Compared to current PPSV–23 strategy, 65–80 PPSV–23 strategy cost less but gained less, while the incremental cost-effectiveness ratios (ICERs) of ≥65 PPSV–23 strategy was ¥5,025,000  per QALY gained. PCV–13 inclusion into the list for single-dose subsidy has an ICER of ¥377,000  per QALY gained regardless of the PCV–13 diffusion level. These ICERs were found to be cost-effective since they are lower than the suggested criterion by WHO of three times GDP , which is the benchmark used in judging the cost-effectiveness of an immunisation programmne.The results suggest that switching current PPSV–23 strategy to ≥65 PPSV–23 strategy or including PCV–13 into the list for single-dose subsidy to the elderly in Japan has value for money. PPSV–23 was approved in 1988 [st year are those aged 65, 70, 75, 80, 85, 90, 95 and ≥100. While from the 2nd year to the 5th year, those who will age 65, 70, 75, 80, 85, 90, 95 and 100 will receive the same subsidised shot [Currently, in Japan, both PPSV–23 and PCV–13 are available for the elderly for the prevention of sed shot . CountriS. pneumoniae in Japan’s setting were accessed from PubMed database, Igaku Chuo Zasshi (Japana Centra Revuo Medicina) database, Ministry of Health, Labour and Welfare (MHLW) Grant System, and annual statistic reports published by the government. Igaku Chuo Zasshi, a Japanese medical bibliographic database, which contains over 10 million citations originating from Japan, comprehensively covers articles published in Japanese-language medical journals. Due to insufficient evidences from Japan, overseas’ reports from PubMed, Medline, The Cochrane Database of Systematic Reviews, Health Technology Assessment database, and The NHS Economic Evaluation Database regarding vaccine effectiveness, utility weights to estimate quality adjusted life year (QALY) and economic evaluation related to vaccines were used instead.We conducted a cost-effectiveness analysis with Markov modelling from payers’ perspective. We conducted a literature survey to define the alternative immunisation programmes and to construct the model. Studies pertaining to epidemiology and prognosis of relevant diseases caused by S. pneumoniae-related diseases. Vaccine uptake rates were assumed at 50.4% for all strategies, which was the same with the coverage rate of seasonal influenza vaccine in 2013 [The target population of the immunisation programmes to be evaluated are those aged 65 and older in 2014. In evaluating the efficiency of different PPSV–23 immunisation programmes, we set three different strategies with different ages to receive a shot of subsidised vaccine, namely: (1) current PPSV–23 strategy, (2) 65 to 80 (as “65–80 PPSV–23 strategy”), and (3) 65 and older (as “≥65 PPSV–23 strategy”). Age-specific populations were from demographic data . Current in 2013 .In order to investigate the cost-effectiveness of PCV–13 inclusion in the list for single-dose pneumococcal vaccine national immunisation programme, we made variations on the share of PCV–13 between the two pneumococcal vaccines from 10% to 100% with 10% interval, because it is unknown how doctors, vaccinees, and municipalities will choose between PPSV–23 and PCV–13. Ten levels of diffusion of PCV–13 were compared with current PPSV–23 strategy.Only single-dose subsidy was analysed and not the sequence of PCV–13/PPSV–23 or PPSV–23/PCV–13, this is mainly due to PPSV–23 immunisation being a newly-launched programme in Japan , 13. We S. pneumoniae-related diseases), bacteremia without pneumococcal pneumonia, bacteremia with pneumococcal pneumonia, meningitis, CAP caused by S. pneumoniae, neurological sequelae and death of or other than the related diseases  annual IPD incidence rates among persons age 65 and over 2.41 per 100,000) , (2) IPD0,000 ,  and 0.00004 QALYs; estimated ICER was ¥5,025,000  per QALY gained. For 65–80 PPSV–23 strategy, incremental cost and incremental effectiveness were both negative values, which indicated that 60–80 PPSV–23 strategy cost less but also gained less than current PPSV–23 strategy .Among 10 scenarios with different PCV–13 diffusion levels, scenarios with higher PCV–13 diffusion level resulted in larger vaccine cost, while it saved more treatment costs and gained more QALYs compared to current scenario. Reduced treatment costs did not offset vaccination cost, which means gained more QALYs with more costs. ICERs were ¥378,000  per QALY gained regardless of PCV–13 diffusion level .In one-way sensitivity analyses of ≥65 PPSV–23 strategy vs. current PPSV–23 strategy, the variables which were found to increase/decrease ICER more than ¥1,000,000  are as follows: (1) cost of per vaccine shot, (2) IPD incidence rate, (3) vaccine effectiveness of PPSV–23 in reducing IPD incidence rate, and 4) percentage of vaccine serotype causing IPD . In PCV– percentaThis study intends to address the following objectives: (1) to evaluate the efficiency of alternative PPSV–23 immunisation strategies compared to current PPSV–23 strategy, and (2) to investigate the cost-effectiveness of the inclusion of PCV–13 in the list of single-dose current pneumococcal vaccine national immunisation programme.Compared to the current PPSV–23 strategy, incremental cost and incremental effectiveness of 65–80 PPSV–23 strategy were both negative, which means that switching current strategy to 65–80 strategy was found to gain less QALYs than current PPSV–23 strategy (this outcome will not be considered in decision-making). Switching current PPSV–23 strategy to ≥65 PPSV–23 strategy was found to be favourable  compared to either of the suggested criterion by WHO of three times GDP  . MoreoveWe compared 10 scenarios (with 10 PCV–13 diffusion levels) to current PPSV–23 strategy. Results showed that PCV–13 inclusion in the subsidy list has value for money .Since there are only a few variables which will induce the ICER to go beyond ¥1,000,000  per QALY gained, we consider our results to be robust. In comparing ≥65 PPSV23 strategy with current PPSV23 strategy, the top four variables which have the biggest impact on ICER were cost per vaccine shot, IPD incidence rate, vaccine effectiveness, and percentage of vaccine serotype causing IPD. On the other hand, in comparing PCV–13 strategy with current PPSV–23, only cost per vaccine shot will change the ICER larger than ¥1,000,000  per QALY.This study has several limitations. In Japan, before the national immunisation programme was launched, some municipalities already provided subsidies to the elderly for single shot PPSV–23 from 2001 to September 2014 with a vaccine coverage of about 25% , if an eS. pneumoniae in our study compared to those in previous studies. All 10 scenarios with different levels of share of PCV–13 have favourable ICERs but were not cost-saving compared to current strategy, which was inconsistent with the results of previous studies. The inconsistency observed was due to high vaccination cost.Several studies have compared the cost-effectiveness of the use of either PPSV–23 or PCV–13 or substitution of PPSV–23 with PCV–13 among elderly. Different studies has shown that both PPSV–23 and PCV–13 were cost-effective, and PCV–13 has high value for money than PPSV–23 , 40–44. S. pneumoniae in Japan. We believe our results will provide useful results to policymakers.Regardless of these limitations, we make efforts on literature survey to find out the available data of epidemiology and prognosis of relevant diseases which were considered to reflect the current situation of diseases caused by Results of our analyses indicate switching the current strategy to ≥65 scenario or including PCV–13 into the list for single-dose subsidy to the elderly in Japan has value for money. A further budget impact analysis is awaited for well-informed policymakers.S1 Table(DOCX)Click here for additional data file.


Tail biting in pigs is a widespread problem in intensive pig farming. The tendency to develop this damaging behaviour has been suggested to relate to serotonergic functioning and personality characteristics of pigs. We investigated whether tail biting in pigs can be associated with blood serotonin and with their behavioural and physiological responses to novelty. Pigs (n = 480) were born in conventional farrowing pens and after weaning at four weeks of age they were either housed barren (B) or in straw-enriched (E) pens. Individual pigs were exposed to a back test and novel environment test before weaning, and after weaning to a novel object (i.e. bucket) test in an unfamiliar arena. A Principal Component Analysis on behaviours during the tests and salivary cortisol (novel object test only) revealed five factors for both housing systems, labeled ‘Early life exploration’, ‘Near bucket’, ‘Cortisol’, ‘Vocalizations & standing alert’, and ‘Back test activity’. Blood samples were taken at 8, 9 and 22 weeks of age to determine blood platelet serotonin. In different phases of life, pigs were classified as tail biter/non-tail biter based on tail biting behaviour, and as victim/non-victim based on tail wounds. A combination of both classifications resulted in four pig types: biters, victims, biter/victims, and neutrals. Generally, only in phases of life during which pigs were classified as tail biters, they seemed to have lower blood platelet serotonin storage and higher blood platelet uptake velocities. Victims also seemed to have lower blood serotonin storage. Additionally, in B housing, tail biters seemed to consistently have lower scores of the factor ‘Near bucket’, possibly indicating a higher fearfulness in tail biters. Further research is needed to elucidate the nature of the relationship between peripheral 5-HT, fearfulness and tail biting, and to develop successful strategies and interventions to prevent and reduce tail biting. Aberrant behaviours such as tail biting, i.e. the harmful oral manipulations of the group mates’ tails, can both reflect and contribute to health and welfare problems in pigs The experimental protocol followed during this study was approved by the Animal Care and Use Committee of Wageningen University (no. 2010055f) and then also adopted by the Animal Care and Use Committee of the University of Groningen, the Netherlands. Blood samples were taken near the home pens of the pigs and as quickly as possible to minimize stress. Pigs with severe tail wounds (i.e. tip of tail missing) were removed from the experiment and all pens (barren and enriched) received a jute sack from 8 weeks of age onward to keep tail biting in barren housing at an acceptable level.Pigs (n = 480) were born in 5 rounds at the experimental farm of TOPIGS Research Center IPG in Beilen (the Netherlands). Briefly, piglets were housed in a conventional (barren) farrowing pen with a sow crate. Tails and teeth were kept intact, but males were castrated. At four weeks of age piglets were transported to the experimental farm “De Haar” in Wageningen (the Netherlands). After weaning pigs were kept either barren (B) or enriched (E). The difference between E and B housing was the provision of wood shavings  and straw (1.5 kg daily) in E housing. B housed pigs received two handfuls of wood shavings daily, from six weeks of age onward. Additionally, from week 8 onward both B and E pens received a jute sack, to keep tail biting at an acceptable level in B pens. More details on housing of the pigs both pre- and post-weaning have previously been described see . Groups Four life phases were distinguished: one pre-weaning, and three post-weaning (1–3). These phases were, roughly, according to general production stages: piglet (0–4 weeks), weaner (4–8 weeks), grower (8–16 weeks), finisher (16–23 weeks). Pigs were, per phase, identified as tail biters and victims of being bitten based on home pen observations and tail damage scores, respectively Piglets (n = 480) were subjected to a back test at approximately 14 days of age   were, by litter, exposed to a novel environment (pNEe) at the age of 3.5 weeks see also . TestingPost-weaning, individual pigs  were subjected to a novelty test at 13 weeks of age see also . The ord9 cells/l) were counted using a Sysmex F-820 Counter .Blood samples were taken when the pigs were approximately 8 (T1), 9 (T2), and 22 (T3) weeks of age. The first  and last blood samples  were considered as basal samples. The second sample  was taken three days after a regrouping test, where pairs of pigs were, for another study, mixed with unfamiliar pigs during 24 h and then returned to their original home pen (see for more details Blood platelet serotonin level. Platelet serotonin (5-hydroxytryptamine or 5-HT) level was determined using a fluorimetric assay based on 9 platelets.Whole blood serotonin level. Serotonin in platelets represents >95% of 5-HT found in blood 9 cells/l) to obtain whole blood 5-HT. Whole blood 5-HT was expressed in nmol/ml blood.Blood platelet serotonin uptake velocity. Serotonin uptake velocity in blood platelets was measured by a radioactive uptake assay based on 9 platelets/min.Before (t = 0 min) and after  the start of the novelty test at 13 weeks of age, pigs  were allowed to chew on two cotton buds to obtain saliva samples to measure cortisol concentrations by a radioimmunoassay kit SAS version 9.2  was used for all statistical analyses. The effect of type of pig (see below) was considered per phase as tail biting behaviour observed in individual pigs was inconsistent throughout life resulting in different classifications per pig per phase The effect of housing (B/E) on serotonin measures was tested in a mixed model with a fixed effect of round (1–5) and random effect of pen (nested within round). Subsequently, the effect of type of pig with respect to tail biting  on serotonin measures was tested separately for both types of housing (B/E). At time of weaning piglets were classified as either a victim  or non-victim of tail biting . This classification in non-victim/victim (0/1) was tested as fixed effect in a mixed model, together with round (1–5) and the random effect of pen (nested within round). In each phase (1–3) post-weaning, pigs were classified as either a tail biter (at least twice involved in biting incident)  or a non-tail biter . In addition, all pigs were classified in each phase as either a victim (at least once a tail wound)  or a non-victim (no tail wounds) . Both 0/1 classifications were treated as fixed effects with two levels, i.e. the effects of ‘biter’ (non-tail biter = 0 versus tail biter = 1) and of ‘victim’ (non-victim = 0 versus victim = 1), respectively. These two fixed effects and their interaction were included in a mixed model, together with the fixed effect of round (1–5) and the random effect of pen (nested within round). Including the interaction between the effects of ‘biter’ and of ‘victim’ in the model made it possible to distinguish between the following four types of pigs: biter  , victim (‘biter’ = 0 and ‘victim’ = 1) , a combination of both, here referred to as biter/victim (‘biter’ = 1 and ‘victim’ = 1)  and pigs not involved in tail biting or being tail bitten, here referred to as neutral pigs (‘biter’ = 0 and ‘victim’ = 0) . A similar approach was used to test the effect of an overall classification of each pig. Pigs were classified as tail biter if they were at least once a tail biter in any of the phases . Similarly, pigs were classified in victims if they were at least once a victim in any of the phase . Including the interaction of the effects of ‘biter’ and ‘victim’ resulted in biters , victims , biter/victims  and neutrals . Serotonin measures were logarithmically transformed if necessary to approach normal distribution of residuals.A Principal Component Analysis (PCA) A mixed model was performed to test whether type of pig  with respect to tail biting, had an effect on the factors that were retained from the PCA. Similar to the analysis of serotonin measures, 0/1 classifications of pigs as ‘biter’ (non-tail biter = 0 versus tail biter = 1) and ‘victim’ (non-victim = 0 versus victim = 1) were included in the model as fixed effects, and their interaction was considered as well . Furthermore, the random effect of pen (nested within round) was included in the model. Round (1–5) was not included in the model, as variables were already corrected for the effect of round prior to PCA.To assess consistency over time  with respect to blood serotonin storage (both expressed in whole blood and in blood platelets), a general linear model per type of housing (B/E) with round (1–5) as fixed effect was run on raw data of serotonin variables to obtain residuals. Thereafter, Spearman’s rank correlation coefficients  were calculated between residuals of serotonin measures. A similar procedure was used to determine possible relationships between serotonin measures and factors obtained by the PCA. Spearman’s rank correlation coefficients were calculated between residuals of serotonin measures and factors.Housing significantly affected platelet 5-HT uptake velocity (determined at T3 only), where B housed pigs had higher velocities compared to E housed pigs (P<0.05) . No otheWhole blood 5-HT measured at T1 was significantly (P<0.001) positively correlated with whole blood 5-HT measured at T2  and T3 , and T2 and T3 were also significantly (P<0.001) correlated . Corresponding results were found for blood platelet 5-HT measures , where T1 was correlated with T2  and T3 , and T2 was also correlated with T3 .Considering a pig’s life as a whole, no significant main effects of ‘biter’ (i.e. non-tail biters versus tail biters) or ‘victim’ (i.e. non-victims versus victims), and no significant interaction between these effects  were found with respect to the blood 5-HT measures (data not shown). However, considering each phase of life separately did reveal relationships between type of pigs and serotonin measures. Victims at time of weaning had, when B housed post-weaning, lower whole blood and platelet 5-HT levels at T1 and T3 compared to non-victims . Tail biAlso when considering E housed pigs throughout life, no relationships were found between type of pigs (in terms of either significant fixed effects of ‘biter’ and ‘victim’ or their interaction) and 5-HT measures, although a tendency for an interaction between tail biters and victims was found with respect to platelet 5-HT measured at T3  (non-significant findings not shown). Victims at time of weaning tended to have a higher platelet 5-HT uptake velocity at T3 compared to non-victims when E housed post-weaning . Pigs idIn B housing, six factors were retained from the PCA . However, only five factors were retained from the PCA to maintain an equal number of factors for both B and E housing as in E housing five factors had an EV>1  and 6. TOverall, B housed pigs classified as tail biters had significantly lower ‘Near bucket’ scores compared to non-tail biters . Furthermore, tail biters tended to have a higher ‘Back test Activity’ compared to non-tail biters  and more specifically it tended to be so compared to victims and neutral pigs . No other significant (or tendencies to) relationships between the overall classification of pigs and PCA factors were found (data not shown). When considering phases of life separately, piglets with a tail wound at weaning had lower ‘Vocalizations & standing alert’ scores (P<0.05) and tended to have lower ‘Cortisol’ scores (P<0.10) . Tail biClassifying the E housed pigs once  with respect to tail biting and thereby combining all phases of life, did not reveal any relationships with the factors obtained from the PCA  (data not shown). Neither having a tail wound at the time of weaning  nor typeIn B housed pigs, ‘Vocalizations & standing alert’ was significantly positively correlated with platelet 5-HT levels determined at T1  and it tended to be so for T2  and T3 . Furthermore, also ‘Early life exploration’ tended to be positively correlated with platelet 5-HT levels determined at T1  and T2 . No other correlations were found between the factors retained from the PCA and 5-HT measures.In E housed pigs, ‘Back test activity’ was significantly positively correlated with whole blood 5-HT measured at T1  and T2 , and platelet 5-HT levels of T1  and T2 . Furthermore, ‘Cortisol’ tended to be negatively correlated with whole blood 5-HT levels at T2  and with platelet 5-HT levels at T2 .The main aim of our study was to explore whether tail biting in pigs is associated with behavioural and physiological responses to challenges, and also with blood serotonin. To our knowledge, this experiment is the first to provide evidence suggesting that tail biting in pigs is possibly linked to both fearfulness and the blood 5-HT system.Platelet serotonin (5-HT) uptake velocities were significantly higher in barren (B) compared to enriched (E) housed pigs. Except for one tendency for higher 5-HT platelet storage in B housed pigs than in E housed pigs, no housing effects on blood 5-HT levels were found. In a previous study in pigs Relationships between behaviours may be affected by the environment e.g. , which eClassifications of pigs according to performing and/or being the victim of tail biting at any phase of life, i.e. over the whole observation period, did not relate to peripheral serotonergic measures. Tail biting behaviour observed in individual pigs was, however, inconsistent throughout life In B housed pigs, whole blood and platelet 5-HT measured at T2 (i.e. week 9) were related to tail biting in phases 2 and 3 post-weaning. In E housing, associations between type of pig, with respect to tail biting and 5-HT measures were found at T3 (i.e. week 22) only. Additionally, 5-HT measures determined at T1 (i.e. week 8) were not significantly related to any type of pig in any phase, except for pigs that were victims of tail biting at time of weaning and subsequently B housed. It seems, therefore, plausible that, rather than suffering from a malfunctional blood 5-HT system from early life onward, pigs of both housing systems develop changes in their blood 5-HT system during life. Average blood 5-HT values determined at T1 and T2 were similar, most likely because the time span between the two samples was very short and decreases in 5-HT levels due to aging, as found in humans A fairly consistent relationship was observed in B housed tail biters with the factor ‘Near bucket’ (retained from a PCA) during a novel object test post-weaning, i.e. tail biting pigs spent or tended to spend less time near a metal bucket introduced during a novel environment test, and spent more time standing alert , being a tail biter in our study tended to be associated with a higher ‘Back test activity’  behaviours and serotonergic measures as both may fluctuate in time. Additionally, barren housed tail biters seemed more fearful after a challenging event. Taken together, considering both behavioural responses to challenging events and blood serotonergic measures in pigs may help in characterizing and identifying individuals at risk for developing damaging behaviours such as tail biting. Further research is needed to elucidate the nature of the relationship between peripheral 5-HT and tail biting, and to develop successful preventive strategies and interventions.


Randomized controlled trials (RCTs) are not always well reported, especially in terms of their methodological descriptions. This study aimed to investigate the adherence of methodological reporting complying with CONSORT and explore associated trial level variables in the Chinese nursing care field.In June 2012, we identified RCTs published in five leading Chinese nursing journals and included trials with details of randomized methods. The quality of methodological reporting was measured through the methods section of the CONSORT checklist and the overall CONSORT methodological items score was calculated and expressed as a percentage. Meanwhile, we hypothesized that some general and methodological characteristics were associated with reporting quality and conducted a regression with these data to explore the correlation. The descriptive and regression statistics were calculated via SPSS 13.0.In total, 680 RCTs were included. The overall CONSORT methodological items score was 6.34±0.97 (Mean ± SD). No RCT reported descriptions and changes in “trial design,” changes in “outcomes” and “implementation,” or descriptions of the similarity of interventions for “blinding.” Poor reporting was found in detailing the “settings of participants” (13.1%), “type of randomization sequence generation” (1.8%), calculation methods of “sample size” (0.4%), explanation of any interim analyses and stopping guidelines for “sample size” (0.3%), “allocation concealment mechanism” (0.3%), additional analyses in “statistical methods” (2.1%), and targeted subjects and methods of “blinding” (5.9%). More than 50% of trials described randomization sequence generation, the eligibility criteria of “participants,” “interventions,” and definitions of the “outcomes” and “statistical methods.” The regression analysis found that publication year and ITT analysis were weakly associated with CONSORT score.The completeness of methodological reporting of RCTs in the Chinese nursing care field is poor, especially with regard to the reporting of trial design, changes in outcomes, sample size calculation, allocation concealment, blinding, and statistical methods. The randomized controlled trial (RCT) is the most appropriate design for evaluating the effectiveness of a nursing intervention among eligible primary study designs for evidence-based practice As a set of evidence-based reporting guidelines for RCTs, the Consolidated Standards of Reporting Trials (CONSORT) For RCTs in various disciplines , the reporting quality has been assessed via CONSORT According to their total number of citations and impact factors (IFs) in the “Chinese S&T Journal Citation Reports 2012” Citations claiming to be RCTs in the titles and/or abstracts, according to the “Cochrane definitions and criteria for randomized controlled trials and controlled clinical trials” Controlled trials with two comparators.Trials of both nursing interventions  and non-nursing interventions .“Quasi-random” trials: when “random” was mentioned with a high-risk randomization method, such as alternation, assignment based on date of birth, case record number, or date of presentation.Irrelevant study reports: reports that are published in a summary form, reviews of randomized trials, and randomized trials on animals, even though they may be reported as “random.”Other study designs, such as cohort study and case series report.All citations from the CBM were allocated evenly to three units. By first screening titles and abstracts and then screening fulltexts, three groups consisting of two authors each selected eligible studies from these units of citations independently. When a disagreement arose in any of groups, another group would participate to arrive at an agreement. Then, two authors extracted relevant information and data from the included studies. A well-designed extraction form  was formWe implemented rigorous quality control throughout the study. Before selecting the eligible studies, investigators were trained to distinguish RCTs from irrelevant study designs using the definitions from the Centre for Evidence-Based Medicine, Oxford, UK (University of Oxford) Using SPSS 13.0, we performed a descriptive analysis, obtaining frequencies and percentages for categorical variables, and means, medians, standard deviations, and inter-quartile ranges (IQR) for continuous variables. Then, exploratory regression was conducted to examine whether general and methodological characteristics are associated with reporting quality, as measured by the CONSORT methodological items score. Univariate regression was performed for each potentially associated variable, and variables with P values lower than 0.25 were then included in a stepwise backward linear regression. Any P value lower than 0.05 was regarded as statistically significant. In this study, the sample sizes of the trials were included to power the exploratory linear regression.In total, 58,823 citations published in the five leading nursing journals from 1954 to June 2012 were retrieved via the “Journal Search Engine” function of the CBM disc. After screening abstracts and fulltexts, 5519 fulltexts that mentioned “random” were initially identified. Among them, 5502 trials described “randomization” in both abstracts and fulltexts, and 17 described “randomization” in only the fulltexts. Six hundred and eighty trials met the criteria for “details of randomization” and were included in the final analysis. Most of the included trials (99.9%) were published after the 2001 CONSORT . FurtherWhen screening abstracts rather than fulltexts, 97.9% of RCTs could be identified. Only two trials (0.3%) reported “randomized control trial” in the title. Overall, the mean CONSORT methodological score was 6.34±0.97 out of 17 sub-items. Additionally, in order to explore the influence of the score of “blinding ”, an analysis excluding data on blinding showed that the mean and SD of the CONSORT score were 6.28 and 0.94 respectively, which were similar to the mean score obtained while including data on blinding. No RCT reported descriptions and changes in “trial design,” changes in “outcomes” and “implementation,” or descriptions of the similarity of interventions for “blinding.” For the description of trial design, 99.7% of the studies used a parallel design while 0.3% used a crossover design. Sub-standard reporting was also found regarding the details of “settings of participants” (13.1%), “type of randomization sequence generation” (1.8%), calculation methods of “sample size” (0.4%), explanation of any interim analyses and stopping guidelines of “sample size” (0.3%), additional analyses of “statistical methods” (2.1%), and targeted subjects and methods of “blinding” (5.9%). In addition, no central randomization that implied allocation concealment was reported. Only two studies conducted allocation concealment through sequentiallynumbered opaque sealed envelopes. More than 50% of trials described randomization sequence generation, the eligibility criteria of participants, “interventions,” and definitions of “outcomes” and “statistical methods.” We explored the association of predictors, including general characteristics and methodological quality measurements, with CONSORT methodological score with a linear regression and found that publication year and ITT analysis were weakly associated with CONSORT score. Each predictor was associated with a small increase of 0.047 and 0.601 points to the overall CONSORT methodological score. The two predictors associated with CONSORT score  remained after the data on “blinding” were excluded, and their P values were 0.005 and <0.001 respectively. In this review, we investigated 680 RCT fulltexts using CONSORT methodological items and found that RCTs of nursing interventions in Chinese nursing care journals reported “trial design,” “participant settings,” changes in “outcome,” “sample size,” “allocation concealment mechanism,” “implementation,” “blinding,” and additional analyses of “statistical methods” poorly. The overall CONSORT score was low. Weak correlations between CONSORT score and two variables  were found.Concordant with our findings, Zhang et al. We found poorer reporting of blinding in 83.4% of non-pharmacological interventions, but no correlation was established via the regression analysis. Due to some methodological challenges, such as the complexity of the intervention The main strengths of this study were the comprehensive search strategy, determination of associations of CONSORT score with various predictors, and the well-conducted quality control assessment. However, this review had some limitations. First, nursing interventions are often far more complicated than drug interventions are. We did not investigate the reporting of items related to external validity. Second, as our eligible criteria was not restricted to any specific nursing intervention such as drug interventions, social and psychological interventions, traditional Chinese medicine interventions, or complex interventions, we did not use CONSORT extensions for social and psychological interventions, non-pharmacological interventions, herbal interventions, and CReDECI criteria In conclusion, the completeness of methodological reporting of RCTs in Chinese nursing care journals was very poor, especially in terms of trial design, outcome changes, sample size calculation, allocation concealment, blinding, and statistical methods. Authors are highly recommended to use the CONSORT criteria when submitting reports of RCTs. Editors should also present their peer-reviewed manuscripts under the guidance of CONSORT to ensure that journal articles contain the key clinical and methodological details that readers need. Initiatives recommended by Altman Appendix S1The total cites and impact factors in five Chinese nursing journals.(DOC)Click here for additional data file.Appendix S2Comprehensive electronic search strategy for CBM disc.(DOC)Click here for additional data file.Appendix S3Data extraction form.(DOC)Click here for additional data file.Appendix S4Number of RCTs in each publication year.(TIF)Click here for additional data file.Checklist S1PRISMA Checklist.(DOC)Click here for additional data file.


Chelotriton, but is unusually peramorphic; for many characters it is more peramorphic than all other caudates or even all other lissamphibians. Most noticeable are the position of the jaw joints far caudal to the occiput, the honeycombed sculpture on the maxilla, and the possible presence of a septomaxilla . Referral to a species would require a revision of the genus, but the specimen likely does not belong to the type species. A phylogenetic analysis of nonmolecular characters of Salamandridae, far larger than all predecessors, confirms the referral to Chelotriton. It further loosely associates the Oligocene Archaeotriton and the Miocene Carpathotriton with the extant Lissotriton, though the former may alternatively lie outside Pleurodelinae altogether. The Miocene? I. randeckensis may not belong to the extant Ichthyosaura. The Miocene “Triturus” roehrsi is found neither with the extant Ommatotriton nor with Lissotriton, but inside an Asian/aquatic clade or, when geographic distribution is included as a character, as the sister-group to all other European molgins. The main cause for discrepancies between the results and the molecular consensus is not heterochrony, but adaptations to a life in mountain streams; this is the most likely reason why the Paleocene Koalliella from western Europe forms the sister-group to some or all of the most aquatic extant newts in different analyses. We would like to urge neontologists working on salamandrids to pay renewed attention to the skeleton, not limited to the skull, as a source of diagnostic and phylogenetically informative characters.We describe an Oligocene newt specimen from western Germany that has gone practically unnoticed in the literature despite having been housed in the Museum für Naturkunde (Berlin) for a century. It is referable to the coeval  Salamandrina forms a third clade , and differing principally from the latter in having pustular rather than pit and ridge sculpture on dermal bone, reaching a somewhat larger size (adult snout-vent length about 100–140 mm), lacking ossification of carpals and tarsals except in largest individuals and by having the fourteenth or fifteenth vertebra as the sacrum, rather than the sixteenth or seventeenth in Tylototriton.“A common Middle [sic] Eocene-Upper [sic] Miocene salamandrid from Europe, closely related to living Tylototriton place the latter on a different growth line in graphical representation of skeletal ratios. Tylototriton is about equally distant from Brachycormus in growth line, suggesting that three genera are involved.” Chelotriton ogygius: ; this apThe bluntly pentagonal shape of the nasals which are about as wide as long  appears C. paradoxus from a broad range of localities  site of Randeck. It should be noted here, however, that   “TrituruCarpathotriton as well as the extant Neurergus, a clade formed by the extant Euproctus and Calotriton, and another formed by Ichthyosaura, Triturus and Ommatotriton.The crown-group of the European molgin clade is poorly resolved; it contains the extinct Our addition of character 98 appears to have counteracted the ecological signal that produced a most likely spurious clade of aquatic Eurasian molgins; unfortunately, this only led to a balance with other signals and therefore to a drastic loss of resolution.Chelotriton are sister-groups and are nested within Pleurodelini, and “Triturus” roehrsi stays far away from Ommatotriton and at least out of Lissotriton. Perhaps surprisingly, Lissotriton forms a paraphyletic series of relatives to the other European molgins, within which? Ichthyosaura randeckensis forms the sister-group to the rest.  in the others; Carpathotriton, if we ignore the shifting positions of the fragmentary taxa mentioned below, is either the sister-group to the European molgin clade or on the pleurodeline stem (between Archaeotriton and Brachycormus)–a position we would rather have expected for the much older Koalliella.Far from being close to acter 98 , Archaeoacter 98 . InspectTriturus” roehrsi,? Notophthalmus crassus,? N. robustus,? Taricha miocenica and Carpathotriton  prevent the backbone constraint , ), and a clade that unites all five OTUs of North American salamandrids, extant and extinct.When character 98 is added to the constrained analysis, 588 shortest trees are found . In their strict consensus , all of Koalliella, Brachycormus, “Triturus” roehrsi, Carpathotriton and Archaeotriton from the shortest trees reveals most of the constraint  ) clade, and all five Lissotriton species without any further resolution; the Adams consensus of the same pruned trees , and possibly even a septomaxilla . At the same time, the hyobranchial apparatus has not been affected by metamorphosis, indicating an aquatic lifestyle; this combination of peramorphosis and aquatic life is reminiscent of the Jurassic cryptobranchoid : f: fPachyt22. SR17: Posterolateral process of frontal absent (0); more or less triangular (1); robust (2) (ordered). We have reworded state 1 from “faint and thin” to delimit it more clearly, leading us to score Ommatotriton, Lissotriton montandoni and L. italicus (thin but long and not faint) as having state 2. The distinction between states 0 and 1 was uninformative in SR.Euproctus platycephalus has state 2, E. montanus state 1 , which forms the frontal part of the fronto-squamosal arch, is variably present in recent [= extant] moratus” . We haven though  . instead ,57; we hLissotriton vulgaris, as originally scored, but figs 24 and 25 of  The muscle is rather delicate in both Pleurodeles and Tylototriton. […] Increased strength characterizes the muscle of the remaining genera. The subarcualis rectus I is weakest in Paramesotriton and strongest in Hypselotriton and Pachytriton among these genera.” (ÖW: 108)–While the latter two are fully aquatic, Paramesotriton is not much less so, indicating to us that states 1 and 2 may not be fully correlated to a more aquatic lifestyle than seen in the taxa that have state 0.Hypselotriton is part of our Cynops OTU, using the “strongest” condition as a separate state would not have been parsimony-informative, so we have not separated it from state 2. State 2 occurs in those of our OTUs that ÖW (page 92) dissected, OTUs not mentioned there are scored as unknown.Because 53. V33-TL42-WÖ32: Mm. geniohyoideus and genioglossus unconnected (0); attached by dense connective tissue at anterior ends of ceratohyals (1).Taricha was scored 1. It seems to have state 0  character; given that the connection is present, we have scored state 1 for Pachytriton.TL (page 151) coded the condition of 54. TL39-WÖ28: M. basiradialis “rather small and weak to relatively well developed” (0); “very well developed and strong” (1) (quotes from WÖ: 127). We suspect that quantifying this character would have revealed more distinguishable states and thus more phylogenetic signal–we had no choice but to copy the scores of TL, which limit state 1 to Salamandrina and Chioglossa, while the condition is unknown in the outgroups.55. TL45-WÖ35: Mm. radioglossus and hyoglossus represented by a single, undifferentiated, unpaired muscle (0); two well-differentiated, well-developed muscles (1); a single hyoglossus and paired radioglossus (2); both greatly reduced (3) (unordered). Even among the first three states, no sequence for ordering is evident, so we have kept this character unordered.56. V34-TL44-WÖ34: M. rectus cervicis profundus inserts neither on the first basibranchial nor on the radii (0); on the caudal part of the first basibranchial (1); on both the first basibranchial and the radii (2) (ordered). This seems like a clear meristic character, so we have ordered it.57. TL47-WÖ37: M. subhyoideus not (0) attached to mandible (1). TL and WÖ distinguished two modes of attachment, but one is an uninformative autapomorphy of Chioglossa.Taricha is polymorphic (. The muscle is large and strong in Paramesotriton, Hypselotriton, and Pachytriton, and somewhat weaker in the other genera.” Following this statement from ÖW (page 105), we have scored this character for the taxa they treated; “Tylototriton” of ÖW included Echinotriton (they dissected both: ÖW: 92), and Hypselotriton is part of our Cynops OTU, which thus has states 0 and 1.77. ÖW3: Single head of M. depressor mandibulae: narrow (0); wide (1). This, too, refers to state 75(1), and it, too, is scored after ÖW (page 105): “The muscle […] in Pleurodeles and Tylototriton […] with narrow origins […] Triturus, Paramesotriton, and Pachytriton have a depressor mandibulae with a narrow origin […]. Euproctus, Notophthalmus, Neurergus, Taricha, Cynops, and Hypselotriton have a triangular muscle with a broader origin.” For the compositions of these taxa we have again relied on their list of dissected species (ÖW: 92).78. V32-TL40-WÖ30: Mm. recti abdominis profundus and superficialis differentiated and separate (0); not distinct (1).79. V20-TL8-WÖ8: Lungs well developed (0); weakly developed or absent (1).80. V21-TL9-WÖ9: Skin smooth (0); rough or keratinized on at least part of the body in some ontogenetic stages (1). This character was parsimony-uninformative before we added Ambystoma .Paramesotriton maolanensis, but not any other species of P.; given the deeply nested position of P. maolanensis, this is clearly an autapomorphy, and P. is ancestrally rough [State 0 is present in ly rough . We have81. GB34: Spur on the hindlimbs of adult males in the breeding season: present (0); absent (1). Without further commenting on this character, GB  ascribed state 0 to Tylototriton, “Euproctus”, “Triturus”, Neurergus, Notophthalmus, Taricha, and Hypselotriton (part of our Cynops OTU), leaving state 1 to Cynops sensu stricto, Paramesotriton and Pachytriton. While state 0 is correct for Euproctus, Calotriton has state 1 [Cynops. It is not at all clear which species of “Triturus” GB used to score this character; we have assumed that all species they used for their analysis of the phylogeny of “Triturus” have state 0, namely Ommatotriton, both of our Triturus OTUs, all five of our Lissotriton OTUs, and Ichthyosaura alpestris (GB: table V). state 1 . We have82. TL13-WÖ10: Eggs “small” (0); “medium” (1); “large” (2) (ordered). WÖ did not define these terms; with one exception, we have adopted their scores without changes or additions (other than those made by TL), except for ordering this potentially continuous character. The exception is Echinotriton: [Tylototriton, which they called “small” and which were so scored by TL and WÖ. This description makes state 2 more likely than 1 for Echinotriton.Echinotriton (E. chinhaiensis: 3.4 mm [ Table 1Table 1 p: 3.4 mm ) most li83. V22-TL18: Epidermal lining in anterior half of female cloacal chamber: present (0); absent (1).84. V23-TL19: Pseudopenis absent (0); present (1).Euproctus [Both states are found in uproctus .Calotriton, and indeed state 1 is explicitly stated to be present and illustrated by their source ; unciliated mucosa (1); keratinized epidermis (2) (ordered). As the original source of this character [haracter  made cleCalotriton and Salamandra) and the original scores of the same character (TL: page 129: state 1 –absence of cilia–scored for Triturus (T.) karelinii and Lyciasalamandra, with Neurergus and Mertensiella scored unknown and all other OTUs given state 0) contradict both each other and the statements by both of the cited sources: “Ciliated epithelium in the cloacal tube and/or anterior cloacal chamber of females” occurs in Lyciasalamandra, Pleurodeles and Salamandrina according to [Lyciasalamandra, Pleurodeles and Salamandrina, not mentioning any taxa as lacking cilia . It has not escaped our attention that, in the matrix of TL (page 129), T. (T.) karelinii is one line below Calotriton, while Lyciasalamandra is one line above Salamandra atra ; maybe TL put the intended score into the wrong line twice. Similarly, the comments on character TL17, including the statements about [Note that the original description of character TL17  as well as for Ambystoma and Dicamptodon , Lissotriton vulgaris, L. boscai, Ichthyosaura, Paramesotriton, Pachytriton and Chioglossa because [Neurergus because [We have scored state 0 for mptodon ; absent (1).87. TL21: Tubular glands secreting into caudal angle of cloaca absent (0); present (1).88. TL22: Male posterior ventral glands present (0); absent (1).89. TL14-HA1: No amplexus (0); ventral amplexus (1); dorsal amplexus (2); restraint of the female by the male using tail, hind limbs and jaws (3) (unordered). No evolutionary scenario is evident to us, so we have kept this character unordered.–State 3 is found in both Euproctus and Calotriton , Ommatotriton, Lissotriton  and Triturus marmoratus, all of which have state 0, Paramesotriton, which is polymorphic with states 0 and apparently 3, and Chioglossa, which has state 1, are scored after [Neurergus and by implication Pachytriton have state 0 according to [Pachytriton, are confirmed by HA, who also showed that Salamandra has states 1 and 2 while Notophthalmus shows states 2 and 0.ed after ; Neurergrding to . Many ofAmbystoma ; however, the phylogenetic tree in [Ambystoma as possessing state 0 only.States 0 and 2 are found in different species of 90. HA2: Sperm deposition: under female’s cloaca (0); in front of female’s snout (1); elsewhere (2) (unordered). In an additional state, “the spermatophore is deposited directly on or near the female’s cloaca”–apparently always on the female’s body rather than the bottom–after which it “is massaged into the female’s cloaca by the male’s hind feet” (HA: 398). This occurs only in Euproctus and Calotriton [Euproctus and Calotriton as inapplicable (“unknown”).lotriton , thus onDicamptodon courtship has not been observed. All that we know of courtship is that many spermatophores are deposited and that these resemble the spermatophores of Ambystoma ” (HA: 409). This excludes state 0 and makes state 1 quite unlikely; we have accordingly scored Dicamptodon as having state 2.“Tylototriton species, but unfortunately did not mention any species by name. Being unable to reconstruct the ancestral state for T., we have scored T. as having both of these states.HA reported that states 0 and 2 occur in different Neurergus and “all species of Triturus”–meaning T. (T.), T. marmoratus, Ommatotriton and all species of Lissotriton–are scored after [Neurergus, this agrees with the text of HA, but not with their table 10.2, which evidently contains an error.ed after  ; male turns 90° to brake female (1); pinwheel (2) (ordered). This character is inapplicable when deposition equals transfer, which is the case for taxa with states 89(3) and 90(0).Ambystoma (HA: 408); the phylogenetic tree in [Cynops, Paramesotriton and Pachytriton) is plesiomorphic for the clade. We have therefore scored Ambystoma as possessing state 0 only.State 0 as well as a different one–“courtship frenzies”–are found in different species of  tree in  confirmsDicamptodon (see character 90); we have accordingly scored partial uncertainty between states 0 and 1.The fine-tuned choreography of state 2 is unlikely for Neurergus has state 1 and additionally engages in a tail-nudging walk before spermatophore deposition [position ; this is92. HA4-AS1-AS3-AS4: Tail-fanning display absent (0); “abbreviated” (1); present, not “abbreviated” (2); fanning and flicking (3); fanning and whipping (4); whipping only (5) (ordered). Apparently inapplicable to Euproctus and Calotriton (HA: 403). HA described states 0 to 2 (mentioning but not quantifying state 1), AS described states 2 to 5 and tentatively suggested the ordering sequence. HA (fig 10.8A) illustrated a behavior they called “tail-fanning display” in Triturus (T.) cristatus, while AS and [T. (T.) cristatus; judging from AS, the behavior shown is “rocking”/“tail-snapping” (character AS3) and probably not homologous to fanning.e AS and  stated t93. AS2-AS5-AS6: Side-on display: absent (0); wave posture (1); lean-in display with cat-buckle posture (2); cat-buckle posture also occurs outside of lean-in display (3) (ordered). AS did not suggest the ordering of states 0 to 2, but the lean-in display seems to consist of the wave posture with additions and without subtractions (AS: 652–653).–Most likely inapplicable to Euproctus and Calotriton.94. AS7: Rocking/tail-snapping absent (0); present (1). Most likely inapplicable to Euproctus and Calotriton.95. AS10-AS12: Wiggling of upwards-bent tail tip  absent (0); present (1); present, additionally combined with flamenco (2) (ordered). The homology of the flamenco is somewhat speculative; we have followed AS in not including the posture sometimes seen in Triturus marmoratus, where the tail tip does not move.–Most likely inapplicable to Euproctus and Calotriton.96. AS11: Retreat display absent or rare (0); common (1). Retreat display can be difficult to recognize, so it may have been overlooked in many species where it is not common (AS: 655); we have evaded this problem by not distinguishing rare occurrences from absence.–Most likely inapplicable to Euproctus and Calotriton.97. AS16: Pushback: absent (0); present (1). This is only applicable to taxa with state HA3(1).98. New character: Geographic distribution: North America (0); Europe, Mediterranean, Iran (1); eastern Asia (2) (unordered). Different orderings would be appropriate for the Paleogene (1-0-2) and the Neogene (1-2-0), and yet another order (0-1-2) would be necessary for dispersal processes that, say, began in the Eocene and were completed in the Miocene; we have simplified this by making this character unordered.These characters occur in our sources, but are parsimony-uninformative for our taxon sample. To this list we have added a new character that was a promising candidate for inclusion but turned out to be parsimony-uninformative as well.Prearticular-angular not (0) fused (1) with articular. In both SR and V, this character was parsimony-uninformative , with state 0 being restricted to Salamandra. [Dicamptodon  and part of Ambystoma , this character is parsimony-uninformative for the current taxon sample.SR32-V18: amandra.  ; tall with thin crest forming straight horizontal dorsal margin (1). This character was an attempt to express the variation coded in characters 62–64, which vary (to some degree) independently of each other: for instance, Cynops pyrrhogaster has tall, thin, posterodorsally ascending neural spines that have a horizontal posterodorsal margin (B: fig 9).SR37: Fifth toe present (0); absent (1). As TL stated on page 149, state 1 is restricted to Salamandrina, making the character parsimony-uninformative.TL7-WÖ7: Rib protrusion absent (0); present (1). As TL stated on page 149, state 1 is restricted to Pleurodeles in their taxon sample, making the character parsimony-uninformative there. In our expanded sample, rib protrusion occurs in Echinotriton [Salamandrina has ribs of intermediate length–claims of rib protrusion have turned out to be false: [Pleurodeles, Echinotriton and Tylototriton; the first two have state 1, making state 0 an autapomorphy of Tylototriton [TL11-WÖ29: notriton ,45. Howee false: ; TL: 149totriton –the charCutaneous papilla projecting dorsally over base of tail absent (0); present (1). TL scored state 1 only for Mertensiella and Lyciasalamandra, both absent from our matrix. HA (page 398), contradicting TL, claimed state 1 also for Salamandra; if this is correct, state 1 would be an autapomorphy of S. in our matrix, so the character would still be parsimony-uninformative.TL12: Oviparity (0); ovoviviparity (1). State 1 occurs in Mertensiella, Lyciasalamandra and Salamandra; with the first two absent from our matrix, the character becomes parsimony-uninformative.TL15: Dorsal glands in male cloaca not (0) bifurcated (1). State 1 is limited to Lyciasalamandra and Salamandra, making the character parsimony-uninformative in our matrix.TL16: First hypobranchial does not (0) extend beyond caudal tip of second hypobranchial (1). As TL stated on page 150, state 1 is restricted to Chioglossa, making the character parsimony-uninformative.TL30-WÖ21: Distinct lengthening loop of M. rectus cervicis profundus absent (0); present (1). As TL stated on page 150, state 1 is restricted to Salamandrina, making the character parsimony-uninformative.TL33-WÖ22: Medial fibers of M. genioglossus inserting near tips of ceratohyals (0); medial fibers absent, lateral fibers well developed (1). State 1 is limited to Mertensiella, Lyciasalamandra and Salamandra, making the character parsimony-uninformative in our matrix.TL41-WÖ31: M. interradialis well developed (0); not well developed (1); absent (2) (ordered). We would thus have exchanged states 0 and 1 compared to our sources so as to be able to order this potentially continuous character.–Our state 0 is limited to Salamandrina, making this state uninformative. State 2 is limited to Mertensiella, Lyciasalamandra and Salamandra, making the character wholly parsimony-uninformative in our matrix.TL48-WÖ38: Zygosphene and zygantrum absent (0); present (1). Among salamandrids, state 1 occurs only in Salamandrina and in Chelotriton pliocenicus, which is not included in this matrix; the character is therefore uninformative.YZ9: Creep present. No other state occurs in the original sample, or probably in any salamanders with internal but not direct fertilization; the character is thus parsimony-uninformative.AS13: Tail-touching present. No other state occurs in the original sample, or probably in any salamanders with internal but not direct fertilization; the character is thus parsimony-uninformative.AS14: At least one spine on quadratojugal-quadrate bone: absent (0); present (1). This would have been an obvious character to add: state 1 occurs in Echinotriton [Chelotriton. Unfortunately, this character is inapplicable unless the quadratojugal-quadrate bone forms a substantial part of the ventrolateral skull margin, and it is only visible in ventral view in the Enspel and Randeck specimens of Chelotriton [Brachycormus where this bone is visible, so they would have to be scored as unknown; the same would apply to Salamandra, because S. “laticeps”–to which this character might be applicable–is likewise preserved in dorsal view. This means that state 0 could only be scored for Tylototriton, making the character parsimony-uninformative.New character: notriton ,45 and, notriton , the Enslotriton . MB.Am.4S1 DataOur analyses are coded in the PAUP block–executing the file repeats the constrained analysis with character 98 (geographic distribution) included, unless the PAUP block is modified or commented out first. To repeat an unconstrained analysis, replace “enforce = yes” by “enforce = no” in the line that begins with “hsearch”; to exclude character 98, add “exclude 98;” above that line.(NEX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigPhotograph taken by holding a camera to an ocular of a binocular microscope. Note that what looks like a pterygoid in the left orbitotemporal fenestra is an artefact of abrasion, as shown by comparison to MB.Am.45.1 and MB.Am.45.3.(TIF)Click here for additional data file.S4 FigThis figure differs from (PDF)Click here for additional data file.S5 FigNo taxa were pruned.(PDF)Click here for additional data file.S6 FigNo taxa were pruned.(PDF)Click here for additional data file.S7 FigThis figure differs from (PDF)Click here for additional data file.S8 FigThe same taxa were pruned as in (PDF)Click here for additional data file.


In the report, “Incidence of Notifiable Diseases Among American Indian/Alaska Natives — United States, 2007–2011,” multiple errors occurred in the text and table.9,061,675 cases were recorded (In the third paragraph, the first sentence should read “For 26 notifiable diseases examined for 2007–2011, a total of recorded .” The th10 times more often among AI/ANs than among whites; however, only 20 cases were reported among AI/ANs of a total of 112 cases reported during 2007–2011.” The second sentence should read, “The second largest difference was for tularemia, which was reported 7.7 times as often among AI/ANs.”In the fourth paragraph, the first sentence should read, “Of the 12 diseases with race information for >70% of records and for which rates were higher among AI/ANs than among whites, the largest difference was for hantavirus pulmonary syndrome, which was reported On page 17, the In the report, “Update on Progress in Selected Public Health Programs After the 2010 Earthquake and Cholera Epidemic — Haiti, 2014,” in the Figure on page 139, the data shown for “Eligible children receiving measles-rubella vaccination” pertain only to estimated coverage through routine immunization. Additional vaccinations were provided through special campaigns.


In the Surveillance Summary, “Abortion Surveillance — United States, 2010,” on page 19 in Table 2, under the column for abortions reported by residence, the following numbers, rates, and ratios of abortions should have been reported for Maine, Massachusetts, Pennsylvania, Rhode Island, Virginia, and Washington:μg/dL in >1 calendar year) were found among 2,210 (19%) of these adults.” The sixth sentence of the second paragraph should read, “A very high BLL measured in >1 calendar year was defined as a persistent very high BLL.” The second sentence of the third paragraph should read, “Among these adults, 2,210 (19%) had persistent very high BLLs, 1,487 (13%) had BLLs ≥60 μg/dL, and 96 had BLLs ≥60 μg/dL in >1 calendar year (Table 1).” On page 968, the last row header of Table 1 should read, “Total no. of adults with persistent very high BLLs .” On page 970, in the “What is added by this report?” section of the summary box text, the first sentence should read, “Data collected by the Adult Blood Lead Epidemiology and Surveillance program during 2002–2011 identified 11,536 adults with very high BLLs (≥40 μg/dL), of whom 19% had elevated BLLs recorded in >1 calendar year.” Finally, in the “What are the implications for public health practice?” section of the summary box, the first sentence should read, “The finding that many workers have harmful BLLs, some that are present for >1 calendar year, is of grave concern.”In the report, “Very High Blood Lead Levels Among Adults — United States, 2002–2011,” several errors occurred. On page 967, the ninth sentence of the first paragraph should read, “Persistent very high BLLs (≥40


Preference for beauty is human nature, as previous behavior studies have supported the notion of “beauty premium” in which attractive people were more easily to get promoted and receive higher salaries. In the present study, 29 males were recruited to participate in a three-person ultimatum game (UG) including a proposer, a responder and a powerless third player. Each subject, playing as the responder, had to decide whether to accept an offer from the allocator both for himself and a female third person. We aimed to elucidate how the facial attractiveness of the female subject affected the male subjects’ fairness and decision-making in social exchanges. Frontal feedback-related negativity (FRN) in response to four offers in an attractive-face condition revealed no significant differences between offers; however, when the companion was an unattractive female, an “unfair/fair” offer, which assigned a lower share to the responder and a fair share to the third player, elicited the largest FRN. Furthermore, when the third player was offered the smallest amount (“fair/unfair” offer), a larger FRN was generated in an attractive-face condition than unattractive-face condition. In the “unfair/fair” offer condition in which subjects received a smaller allocation than the third person, the beauty of their female counterparts attenuated subjects’ aversion to inequality, resulting in a less negative FRN in the frontal region and an increased acceptance ratio. However, the influence of the third player’s facial attractiveness only affected the early evaluation stage: late P300 was found to be immune to the “beauty premium”. Under the two face conditions, P300 was smallest following an “unfair/fair” offer, whereas the amplitudes in the other three offer conditions exhibited no significant differences. In addition, the differentiated neural features of processing facial attractiveness were also determined and indexed by four event-related potentials (ERP) components: N170, frontal N1, N2 and late positive potentials (LPPs). Social exchanges are not merely matters of goods and money; other nonmaterial factors, such as love, affection, esteem and social relationships, could be involved in economic activity. Correlations of earnings with physical appearance have been examined in multiple studies that have found that attractive persons are more likely to be hired and promoted and to earn higher salaries. The concepts of the “beauty premium” and the “plainness penalty” originated from Hamermesh and Biddle , who fouFurthermore, the two-person game is not always reflective of reality; social exchanges occur in multiple dimensions. In a three-person ultimatum game , a powerThis early negativity component, which is also called feedback-related negativity (FRN), is an event-related potential thought to originate in the dorsal anterior cingulate cortex (dACC). According to reinforcement learning theory, the subjective value of a forthcoming reward is coded in the midbrain dopaminergic system, causing a phasic activation of dopaminergic neurons. Negative prediction errors induced by unfavorable outcomes initiate phasic decreases in dopamine inputs and therefore give rise to increased ACC activities, which reflect a more negative FRN amplitude –10. ThusACC was also related to empathic resonance –30 and aThe social comparison of payoffs was also found to affect the late processing stage, indexed by a late positive potential (LPP) –38. ThisPrevious behavior experiments –47 showeAdditionally, we analyzed subjects’ ERPs during face presentation to measure whether they could distinguish between attractive and unattractive faces. Although brain-imaging studies have shown increased activity in the reward-related brain area when subjects perceive attractive faces compared to unattractive faces –55, ERP Twenty-nine right-handed male subjects participated in this study. They were all students at Zhejiang University and aged 18–27 years . They were all native Chinese speakers, had normal or corrected-to-normal vision, and did not have any history of neurological disorders or mental disease. All participants provided written informed consent to participate in this study, and the study was approved by the Neuromanagement Laboratory ethics committee at Zhejiang University. Data from three subjects were discarded, one for excessive recording artifacts and two for misunderstanding the rules of the game, resulting in twenty-six valid subjects for the final data analysis.attractive = 5.2, Munattractive = 2.11; t (19) = 14.516, p<0.001).The stimuli included 256 photos of Chinese females that were collected from the CAS-PEAL-R1 Face Database, photo pools of PKU and CAS and the Internet. The faces were unfamiliar to subjects and included no movie stars, singers or other celebrities. All faces were grey-processed using Photoshop software to ensure consistency in background, brightness, contrast, and color saturation and adjusted to a uniform size . All images were rated for attractiveness  by 20 male students. The ratings of the two categories of faces were compared by a paired t-test, wherein the attractiveness was significantly different , “unfair/unfair” offer , “fair/unfair” offer  and “unfair/fair” offer . In total, 256 offers and 256 photos were paired and distributed evenly in four blocks with eight conditions, 2 (face: attractive and unattractive) × 4 , each containing 32 trials.Subjects were seated comfortably in a dim, sound-attenuated and electrically shielded room. The experiment was introduced on paper handouts. The experimental stimuli were presented at the center of a computer screen at a distance of 100 cm from each subject’s face. A keypad was provided to the subjects to make their choices. Before the experiment formally began, each subject had 20 practice trials to become familiar with the experimental procedure.A single trial is illustrated in Scalp voltages were recorded  with a NeuroScan SynAmps2 Amplifier , using a 64-channel electro cap with Ag/AgCl electrodes, in accordance with the standard international 10–20 system. A cephalic (forehead) location was connected to the ground. The left mastoid was chosen for reference, and recorded EEGs were off-line re-referenced to the average of the left and right mastoids. For eye movement artifact correction, an electro-oculogram (EOG) was recorded from electrodes placed 10 mm from the lateral canthi of both eyes  and above and below the left eye . The electrode impedance was maintained below 5 kΩ during the recording.Post-hoc analysis was conducted using the Bonferroni correction.To analyze the behavioral data, repeated measures ANOVA was adopted to compare the acceptance ratio and reaction time across eight conditions, 2 (face: attractive and unattractive) × 4 . EEG data were analyzed using NeuroScan 4.3.1. EOG artifacts were corrected first, followed by digital filtering through a zero phase shift . The EEGs were segmented for 1000ms in each epoch, beginning 200ms before and continuing until 800ms after the onset of both the face and offer presentations. The entire epoch was then baseline-corrected by the 200ms interval prior to the stimulus onset. Trials that contained amplifier clipping, bursts of electromyography activity, or peak-to-peak deflection exceeding ±80 μV were excluded from the final average. To further improve the quality of the data, subjects with fewer than 15 trials were excluded, resulting in an average of 26 trials for each condition.During the face presentation, the EEG epochs were averaged for attractive and unattractive faces. During the offer presentation, the EEG epochs were separately averaged for face (attractive/unattractive face) × valence , resulting in eight conditions in total. ERPLAB toolbox 4.0.2.3 was used to generate topographic maps for each condition .Based on the visual observation of grand average waveforms and previous ERP studies on outcome processing –6, 26, tTo examine the neural processing of facial attractiveness, the mean amplitude of N170 , frontal N1 , frontal N2  and central-parietal LPP  were further analyzed for the ranges of 150-180ms, 110-130ms, 280-360ms and 450-650ms post-onset of face presentation, respectively. Two-way ANOVAs were conducted on the N2 and LPP components. The ANOVA factors were face (attractive and unattractive) and electrode sites. The degree of freedom of the F-ratio was corrected according to the Greenhouse-Geisser method.1, 25 = 13.021, p = 0.001) and offer . Generally, the acceptance ratio in the attractive-face condition was higher (p = 0.001) than that in the unattractive-face condition, as with the post-hoc comparisons between the two fair offers (“fair/fair” and “fair/unfair”) and two unfair offers (“unfair/unfair” and “unfair/fair”) assigned to subjects, with all of the four significant levels were below 0.001. The comparison between the “fair/fair” and “fair/unfair” offers was not significant (p = 0.368), and neither was the comparison between the “unfair/unfair” and “unfair/fair” offer conditions (p = 1.000), neither was the comparison between “unfair/unfair” and “unfair/fair” offers (p = 0.694).The ANOVA analysis for the acceptance ratio  revealed1, 25 = 4.659, p = 0.012), and the post-hoc comparison demonstrated that the acceptance ratio of the “unfair/fair” offer in the attractive-face condition  was higher than in the unattractive-face condition , p = 0.001. The comparisons between acceptance ratios of “unfair/unfair” offer in two face conditions was marginally significant (p = 0.065). Between the two face conditions, no other apparent differences were found regarding the acceptance ratios of “fair/fair” (p = 0.134) and the “fair/unfair” (p = 0.109) offers.The interaction effect also revealed a significant difference . The main effect of face was not significant . Post-hoc comparison showed that subjects responded quickest to the “fair/fair” compared to the “unfair/unfair” (p<0.001), “fair/unfair” offer (p = 0.001) and “unfair/fair” offer (p<0.001). The response time to the “fair/unfair” offer condition was not significantly different from those to the “unfair/unfair” (p = 1.000) and “unfair/fair” (p = 0.072) conditions.The ANOVA analysis of the response time  revealed1, 25 = 4.69, p = 0.005): The reaction time of the “fair/fair” offer was shorter (p = 0.009) in the attractive-face condition  than in the unattractive-face condition . While in the “fair/unfair” offer condition, subjects responded more rapidly in the attractive face-condition than in the unattractive-face condition (p = 0.04). No significant difference was found between two face conditions in the “unfair/unfair” (p = 0.122) and “unfair/fair” offers (p = 0.199). The acceptance ratio and reaction time of each condition are reported in The interaction effect revealed a significant difference  and electrode  but no main effect of face . The “fair/fair” offer generated the smallest FRN, and post-hoc comparisons revealed a marginally significant difference from the “unfair/unfair” offer (p = 0.053), and a significant difference compared with the “unfair/fair” offer (p = 0.001). Additionally, the amplitude of the FRN in response to the “fair/unfair” offer was more negative-going than the FRN for the “unfair/fair” offer (p = 0.008). However, this contrast did not produce a significant effect (p = 0.318) for the comparison between the “fair/unfair” and “unfair/unfair” offers.The ERP grand average waveforms at channel Fz during the presentation of an offer are depicted in 1, 25 = 8.83, p<0.001), while the interaction effect among face, offer and electrode was not significant . In the attractive-face condition, FRNs in response to four offers revealed no significant differences from one another. In the unattractive-face condition, the largest FRN was in response to “unfair/fair” offer, which revealed a remarkable difference from the other three offers . Additionally, in the unattractive-face condition, the FRN generated in response to the “unfair/unfair” offer was more negative than the FRN in response to the “fair/fair” (p = 0.006) and “fair/unfair” (p<0.001) offers. No other significant comparisons were found between the offers in the unattractive-face condition. Waveforms of FRN at Fz regarding the four offers in each face condition are presented in Moreover, the interaction effect between face and offer was significant (Fp = 0.047) in response to the “fair/unfair” offer in the attractive-face condition than in the unattractive-face condition. In response to “unfair/fair” offer, the comparison between the amplitudes of the two FRNs in two face conditions showed a reversed pattern in which the FRN was more positive (p = 0.001) in the attractive-face condition than in the unattractive-face condition (p = 0.182) offers, no significant differences was found between the two face conditions, while comparisons for “fair/fair” offer approached significance (p = 0.064).Additionally, the FRN was more negative  and electrode , but no main effect of face . Post-hoc comparisons indicated that the P300 amplitude was highest in the Pz electrode , and P300 for the “unfair/fair” offers was smaller than P300 for other three offers, i.e., the “fair/fair” (p<0.001), “unfair/unfair” (p<0.001) and “fair/unfair” (p = 0.007) offers. Apart from these differences, no other significant differences were found. The interaction effect between face and offer was not significant , and neither was the interaction effect among face, offer and electrode , which suggested that facial attractiveness had no effect on the late processing stage. The P300 amplitudes of each condition are reported in Regarding the central-parietal component P300 , we obse1, 25 = 5.318, p = 0.03) and frontal N2  were elicited by unattractive faces, and a more negative N170  and a more positive LPP  was elicited by attractive faces. The main effect of electrode  and the interaction effect between electrode and face  were significant for frontal N2. Unattractive faces elicited a more negative N2 than attractive faces at all selected electrodes. In attractive-face condition, N2 peaked highest at the Fz electrode  while in the unattractive-face condition, the most negative N2 was found at the FC2 electrode .The ANOVA analysis for N170, frontal N1, frontal N2 and parietal LPP  revealed1, 25 = 2.575, p = 0.095) nor the interaction effect between electrode and face was significant . For frontal N1 and N170, the main effect of electrode  both reached significance, while the interaction effects between electrode and face were not significant .However, for LPP, neither the main effect of electrode  and unfair offers (“unfair/unfair” and “unfair/fair”) assigned to the subjects themselves generated similar FRN amplitudes. A similar pattern was observed in a dictator game  when parIn addition to the differentiated FRN between all offers assigned to the responders in the two face conditions, the FRN pattern varied following advantageous (“fair/unfair”) and disadvantageous (“unfair/fair”) offers. As outlined above, the ACC was activated when the subjects experienced a sense of envy for others who possessed superior quality and ability , and incThis FRN pattern was found to be reversed when responders were in an advantageous (“fair/unfair”) condition. The ACC was also thought to code the subjects’ affective experience of empathy when perceiving the pain or distress of others  and FRN Another ERP component, P300, was thought to represent the motivational importance of reward , 67–68. Furthermore, in agreement with an ERP result from a social comparison task , the ampNotably, the pattern of the FRN effect outlined above was inconsistent with the pattern of the effect in acceptance ratios; in the attractive-face condition the “unfair/fair” offer was accepted more often and accompanied by a decreased FRN, whereas a higher FRN in response to the “fair/unfair” offer was not accompanied by a differentiated acceptance ratio. The apparent contradiction between the ERP pattern and behavior data was also reported in previous studies , 37, 71.The ERP data also illustrated how the brain responded differently to attractive and unattractive faces. LPP was elicited to a greater extent in response to attractive faces than to unattractive faces, which was in line with previous findings –57, 59. To summarize, in a three-person ultimatum game, the facial attractiveness of the third player modulated responders’ fairness in the early evaluation process, as indexed by FRN, whereas late P300 was immune to the effect of the “beauty premium” and was dominantly influenced by the comparative process. Behaviorally, increased altruism and attenuated envy towards the third players’ superior gain provoked subjects’ altruistic actions in a disadvantageous offer condition when the third player was attractive. In addition, the ERP associated with processing facial attractiveness was also examined. In addition to N2 and LPP, which were explored in previous studies, an earlier frontal N1 and N170 was found among the different neural responses to attractive and unattractive faces.S1 Fig(TIF)Click here for additional data file.S1 File(ZIP)Click here for additional data file.