Se), and monthly consumption rate limits (CRmm) for each species were also calculated. THg and Se were analyzed by atomic absorption spectrophotometer , while MeHg was determined by Trace Ultra gas chromatograph connected with a PolarisQ MS . None of the analyzed fish had Hg levels above the European Community regulatory limits, while most large predators had MeHg levels over the threshold concentration set by US EPA. The estimated weekly intakes of THg and MeHg exceeded in many cases the PTWIs and the Se estimated daily intakes were provided from 0.71% to 2.75% of the RDA. Se:Hg molar ratios above 1 and positive HBVSe index suggested that Se in fish could be enough to alleviate the potential toxic effect of Hg. However, high-risk groups as children should consume fish in moderation because a large consumption pattern, especially of swordfish and tunas, might be of concern for health.Total mercury (THg), methylmercury (MeHg), and selenium (Se) concentrations were measured in various commercially important fish species. The benefit–risk binomial associated with these chemicals was assessed in children through the probability of exceeding the provisional tolerable weekly intakes (PTWIs) of the contaminants and the Se recommended dietary allowance (RDA). The Se:Hg molar ratios, selenium health benefit values (HBV Se) index for each fish species, (4) evaluate the health benefit/risk caused by the fish consumption in children, comparing the estimated intakes with Hg and MeHg provisional tolerable weekly intakes (PTWIs) and with Se recommended dietary allowance (RDA), and finally, (5) derive consumption limit recommendations for noncancer health effects (CRmm).Fish has been acknowledged as a healthy addition to any diet, providing high-quality proteins, vitamins, and numerous other important nutrients , therebyn = 15) of about 0.1–0.2 kg of muscle tissue were obtained. After collection, the specimens, separated by species and disposed in polythene bags, were carried to the laboratory. The dissection was operated from the dorsal surface of each animal. Fish identification was carried on the basis of illustrated taxonomic keys [3 solution (10%) overnight and rinsed many times with ultrapure water prior to use.A total of 152 specimens from the Mediterranean area and belonging to different fish species  were purmic keys . For eac4 , cysteine , NaBPh4 , CuSO4 , NaCl , and sodium acetate . Adequate amounts of deionized water were used to prepare daily solutions of these reagents. THg and Se standard  solutions were made by dissolving suitable pure quantities in acidified water (HNO3 0.3%), while methylmercury chloride was obtained from Alfa Aesar,  (99.4%). Stock solutions were prepared by dispersing apposite quantities of salt in deionized water. Methylmercury chloride  and ethylmercury chloride  standard stock solutions were prepared by dissolving the appropriate amounts of pure compounds in methanol. Standard solutions of phenylated organomercury compounds  in hexane at different concentrations were prepared by reacting different amounts of the above standard stock solutions with NaBPh4 at pH 3. All standard solutions were stored at 4 °C, away from light before use, and the working standard solutions for each individual mercury species were prepared daily. Certified reference materials (CRMs) were Lobster Hepatopancreas (TORT-3) provided by the National Research Council of Canada . Glassware was rinsed with ultra-pure water, decontaminated overnight in 10% (v/v) nitric acid solution, and rinsed again.All solvent used   were of pesticide analysis grade and all acids used (nitric acid (65%) , hydrochloric acid (37%) , sulfuric acid (96%) , and glacial acetic acid  were of analytical reagent grade. The reagents used were NaBH2SO4–HNO3 (1:1). The sample solution was then cooled and diluted with double distilled water according to the method recommended by the official Italian agencies [4. For the quantification of organic Hg (MeHg), the protocol described by Ipolyi et al. [w/v) was added to the sample, and the mixture was centrifuged (2400 rpm for 10 min). The supernatant was extracted twice with toluene and the combined organic extract was subjected twice to back-extraction with a 1% (v/w) cysteine aqueous solution. After acidification of the collected cysteine extract with H2SO4 (0.1 M), the derivatization reaction was carried out by adding 1 mL of saturated CuSO4 solution and 0.2 mL of 1% (v/w) NaBPh4 aqueous solution in the presence of n-hexane. After 20 min of agitation, the organic phase was separated and analyzed using a Trace Ultra gas chromatograph connected with a PolarisQ MS . A SPB-608 capillary column   was utilized. One μL of the sample was injected in splitless mode at an injection temperature of 250 °C. The transfer line temperature was at 280 °C temperature program, 50 °C × 1 min and then increased at a rate of 20 °C min−1 to 280 °C and held for 10 min. Detector temperature was designed at 240 °C. Helium (99.99%) was used as a carrier gas at a flow rate of 1.0 mL min−1. Electron impact ionization was performed with an electron energy of 70 eV. A mass range from m/z 50–350 was recorded in the full-scan mode to check for spectral interferences, while the SIM setup was MeHgPh: m/z = 292.00, 294.00, and 279.00; EtHgPh: m/z = 279.00, 306.05, and 308.10. The dwell time was 100 ms. Reporting data were expressed on a wet weight basis.The extractive analytical procedure and the instrumental conditions to determine total mercury (THg) and selenium (Se) concentrations have been described in detail elsewhere . Brieflyagencies . THg andi et al.  was folln = 3)  were in accordance with certified values , (% recovery = 96–101%). The limits of detection  and of quantification  are the following: LODs: THg: 5 ng g−1 wet weight, MeHg: 0.03 ng g−1 wet weight, Se: 1 ng g−1 wet weight; LOQs: THg 13 ng g−1 wet weight, MeHg: 0.12 ng g−1 wet weight, Se 3.6 ng g−1 wet weight.Accuracy and precision were proved by using TORT-3 Lobster Hepatopancreas . Replicate analyses (−1), and BW is children’s body weight (26.2 kg) [−1 BW week−1; MeHg: 1.3 µg kg−1 BW week−1) [−1 in children of 4–8 years old) [Estimated daily intakes (EDIs) of Hg, MeHg, and Se through fish consumption in children (age: 3.0–9.9 years old) were determined using the subsequent equation:26.2 kg) . The res week−1)  and withars old) .−1) was calculated individually for each fish species dividing Se and Hg concentrations by their respective molecular weights . The selenium health benefit value (HBVSe) was calculated using the molar concentrations of two elements according to the following equation [The molar ratio (Se:Hg)  (g day−1) for non-carcinogenic effects was computed using the following equation [−4 mg kg−1 day−1) determined by the US EPA [−1). The maximum allowable daily fish consumption rates (CRlim) were transformed to the allowable number of fish meals per month (CRmm)  in accordance with the following equation:The maximum allowable daily fish consumption rate  . If the hildren) .p ≤ 0.05 was adopted.The Kruskal–Wallis test was carried out to check whether the levels of total mercury, methylmercury, and selenium varied significantly among different fish species. The level of significance set at −1; MeHg: 0.02–0.55 μg g−1). The proportion of MeHg relative to THg, which is expressed as a percentage, ranged from a minimum of 66.7% in European anchovy to a maximum of 92.9% in Atlantic bonito, indicating that in fish muscle tissue MeHg represents the bulk of total Hg, as documented in numerous other studies [As can be seen in  studies .−1), Atlantic bluefin tuna , and albacore  showed greater concentrations than the others pelagic carnivorous fish, Atlantic bonito , chub mackerel , and Atlantic mackerel , which are species with similar ecology  . Concentrations statistically lower were found in gilthead seabream  and European seabass , both omnivorous species, followed by European hake  and by the smallest pelagic zooplanctivore species such as European anchovy  and European pilchard . Among the benthic fishes, piscivorous species such as sandy ray , longnose skate  and shagreen ray  showed consistent Hg levels, whereas more moderate concentrations were in turbot  and common sole  . In general, organisms dwelling during their life in strong contact with sediments and from where they mainly feed are more readily exposed to the greater quantities of Hg that accumulate in sediments than other fish, confirming either the considerable process of sedimentation of this metal in marine depths or the importance of feeding patterns on Hg accumulation process in fish. However, independently from complex mercury dynamics in aquatic ecosystems, the studied species exhibited Hg values within the European Commission Regulation [−1, except for predatory species for which the accepted tolerance level raises to 1 μg g−1. Furthermore, looking to MeHg, the most dangerous mercury compound, a more stringent guideline value (0.30 μg g−1) was fixed by the US EPA [The key of interpretation for this large interspecific difference in THg and MeHg levels  is in the complexity of the interactions involving biological , ecological , and environmental factors , which affect the bioaccumulative process of Hg in marine biota . Furthergulation , which e−1 to 0.58 μg g−1 were significantly lower than those of THg and MeHg (p < 0.02). Furthermore, lower overall variability of Se concentrations  compared to those of THg and MeHg was observed among various species, coherent with the homeostatic regulation of this essential element in the organism [−1 wet wt.) and red mullet (1.73 µg g−1 wet wt.), levels higher than those in large predators such as Atlantic bluefin tuna (1.05 µg g−1 wet wt.). Conversely, Olmedo et al. [−1 wet wt.) and tuna (0.57 µg g−1 wet wt.) contain higher levels of Se compared to other fish species (0.004–0.35 µg g−1 wet wt.). Azad et al. [−1 wet wt.), followed by the demersal species (0.43 µg g−1 wet wt.) and benthopelagic fish group (0.30 µg g−1 wet wt.). In our study, the pelagic fish such as Atlantic bluefin tuna (0.58 µg g−1), albacore (0.52 µg g−1), and swordfish (0.44 µg g−1), had significantly higher concentrations of Se  compared either to the Scombridae family fish  or to clupeids and in remaining pelagic species  having levels equal or below 0.30 µg g−1. For benthic fish, Se concentrations ranged from 0.28 µg g−1 up to 0.47 µg g−1 with a greater enrichment in the different elasmobranch species  with respect to the other two considered fishes . However, despite the fluctuation of Se content, the statistical analysis did not highlight any significant differences in concentrations between the considered fish categories .Regarding Se, the concentrations from 0.15 µg gorganism  and withorganism . Literatorganism  found ino et al.  note thad et al.  instead,−1 BW week−1 and of 1.3 µg kg−1 BW week−1 for THg and MeHg, respectively, have been established by EFSA [−1 BW week−1), followed by Atlantic bluefin tuna (5.07 µg kg−1 BW week−1) and albacore (4.27 µg kg−1 BW week−1). Moreover, the consumption of Atlantic mackerel (1.79 µg kg−1 BW week−1), chub mackerel (2.09 µg kg−1 BW week−1), Atlantic bonito (2.78 µg kg−1 BW week−1), and of the benthic species (1.79–3.78 µg kg−1 BW week−1) determined an exposure rather high but within the safe level, while eating fish as gilthead seabream (1.49 µg kg−1 BW week−1), European seabass (1.29 µg kg−1 BW week−1), European hake (0.80 µg kg−1 BW week−1) or anchovy (0.50 µg kg−1 BW week−1) and sardine (0.30 µg kg−1 BW week−1) led to low or moderate Hg intakes. The scenario was different for MeHg because the weekly intakes were close (gilthead seabream: 1.29 µg kg−1 BW week−1) or higher than MeHg PTWI in all cases (1.39–5.47 µg kg−1 BW week−1), except for consumption of European seabass: 1.09 µg kg−1 BW week−1, European hake: 0.60 µg kg−1 BW week−1, and clupeids: 0.20–0.40 µg kg−1 BW week−1, which were within the safe level. With respect to Se, a micronutrient of fundamental importance for many bodily processes, an adequate intake via diet for all age groups is desirable. However, as with all essential elements, low or moderate Se intakes are necessary to sustain life but excessive intakes can produce toxicity. A dietary Se excess can result in selenosis whose symptoms include gastrointestinal upsets, hair loss, white blotchy nails, fatigue, and irritability [−1 BW day−1 to 0.82 µg kg−1 BW day−1 provided from 0.71% to 2.75% of the recommended dietary allowance (RDA) [The estimated THg and MeHg exposures from fish consumption by children are described in  by EFSA  as the atability , while ace (RDA) , with AtDietary Se intake may determine a positive effect on the toxicological outcomes of Hg exposure; contemporarily, estimates of Se intake alone may not adequately reflect the health risk/benefit of Se if its relationship with Hg is not carefully pondered. A lot of studies have, in fact, confirmed that Se not only moderates the uptake of Hg but counteracts its toxicity in a multitude of animal species, including fish and humans ,51. The Se) provides a more trustworthy key to evaluate Hg exposure risks [Within deep-sea fish, the Se:Hg molar ratio variation was moderately large, with the common sole (3.95) and shagreen ray (3.73) exhibiting the highest values, followed by the remaining fish species having similar molar ratios (3.14–3.16). It is clear that the observed decrease in values of Se:Hg molar ratio as it moves along the food chain from planktivorous to omnivorous and carnivorous species is dependent on a combination of several factors   but veryre risks .Se showed a moderate inter-species variation with the lowest values in clupeids  and the greatest in Atlantic bluefin tuna (6.47), albacore (5.89), Atlantic bonito (5.22), and in the elasmobranch group (4.67–5.35), while in the remaining species, including the common sole, turbot, European hake, European seabass, chub mackerel, and swordfish, the values ranged from 3.32 to 3.92  suggest that a precautionary principle must prevail even when the HBVSe index is positive. This assumption becomes more realistic especially when measuring positive but low HBVSe values. In our study, modest values of this index have been calculated for all species, but especially for sardine (1.89), revealing the possibility of a potential health hazard as a result of their consumption. The obtained data corroborate previous studies reporting beneficial HBVSe and favorable Se:Hg ratios in almost all seafood, except top predators for which contrasting findings were observed. In sharks, for example, Kaneko and Ralston [Se index either negative [Se or Se–HBV and healthy molar ratios [Analysis of our data in terms of HBV to 3.92 . Looking Ralston  and Olme Ralston  presenteet al. 20 suggest negative . In swornegative  measurednegative  and Olmenegative  found far ratios ,57,58,59mm) is shown in With regard to Hg, establishing restrictions on the consumption of fish and other aquatic species for the general population, but especially for sensitive sub-groups of the population such as pregnant women and children, allows to reduce exposure and, at the same time, to reap the nutritional benefits provided by this food. To protect and help these most vulnerable population groups to make informed choices about the type of fish to consume, the United States Food and Drug Administration and the United States Environmental Protection Agency  have creSe as a tool of health risk assessment because the studies on the interactions between Se and Hg involve non-human models and consequently the exact molar ratio at which neutralizing effects of Se over MeHg occurs in human is unknown [The analysis of uncertainty is a usual component of risk assessment because there are many variables, including per capita consumption, consumption frequency, metal concentration, consumer body weight, exposure time period, etc. that can greatly affect the interpretation of results. Consequently, when discussing human exposure, uncertainties and limitations need to be acknowledged. In our case, more accurate information on the consumption of each individual fish by children and body weight at each age are necessary parameters to reduce the uncertainty associated with exposure. There are limitations even when using HBV unknown . Moreove unknown . However unknown ,64.Se is not particularly high. This precautionary principle must predominate also in the light of the results relative to the maximum allowable fish consumption rates. The application of this parameter linked to non-carcinogenic health effects suggests that monthly consumption of swordfish, Atlantic bluefin tuna, albacore, and Atlantic bonito should be extremely moderate and this advice should be extended to these high-trophic level pelagic species and to other specific species (see sandy ray and longnose skate) in consideration of the low safe consumption frequency calculated. These results highlight the importance to develop guidelines on the amounts, types, and frequency of fish consumption that currently do not exist in Italy, unlike other countries where bans, advice, and recommendations for vulnerable population groups, such as young children and women during the reproductive period, have already been in vogue for some time.There is wide scientific evidence supporting the benefits and potential harms of consuming fish, and this problem becomes even more important when it involves harmful chemicals such as mercury and high-risk groups such as children for whom various factors  contribute to creating critical windows of vulnerability that can determine lifetime consequences . Informa


Nitrosomonas mobilis, an ammonia-oxidizing bacterium with high stress tolerance, often forms aggregates mainly in wastewater treatment systems. Despite the high frequency of aggregate formation by N. mobilis, its relationship with survival currently remains unclear. In the present study, aggregates were formed in the late stage of culture with the accumulation of nitrite as a growth inhibitor. To clarify the significance of aggregate formation in N. mobilis Ms1, a transcriptome analysis was performed. Comparisons of the early and late stages of culture revealed that the expression of stress response genes (chaperones and proteases) increased in the early stage. Aggregate formation may lead to stress avoidance because stress response genes were not up-regulated in the late stage of culture during which aggregates formed. Furthermore, comparisons of free-living cells with aggregates in the early stage of culture showed differences in gene expression related to biosynthesis  and motility and adhesion . Biosynthesis genes for growth were up-regulated in free-living cells, while motility and adhesion genes for adaptation were up-regulated in aggregates. These results indicate that N. mobilis Ms1 cells adapt to an unfavorable environment and grow through the division of labor between aggregates and free-living cells.Bacteria change their metabolic states to increase survival by forming aggregates. Ammonia-oxidizing bacteria also form aggregates in response to environmental stresses.  Bacteria form appropriate microstructures that increase survival in response to various environmental changes. The aggregates of multiple bacteria are considered to be the main form in natural environments and engineered systems .Ammonia-oxidizing bacteria (AOB) are often observed in aggregates rather than as free-living cells  between aggregates and free-living cells.Since N. mobilis Ms1 isolated from nitrifying granules was cultured in a batch mode with mineral medium containing 2.1 mM NH4Cl as previously reported (–1), MgSO4·7H2O (0.4 g L–1), CaCl2·2H2O (0.073 g L–1), KCl (0.038 g L–1), KH2PO4 (0.034 g L–1), FeCl2 (0.002 g L–1), EDTA (0.0043 g L–1), MnCl2·4H2O (0.1 mg L–1), CoCl2·6H2O (0.024 mg L–1), NiCl2·6H2O (0.024 mg L–1), CuCl2·2H2O (0.017 mg L–1), ZnCl2 (0.068 mg L–1), Na2WO4·2H2O (0.033 mg L–1), Na2MoO4 (0.024 mg L–1), and H3BO3 (0.062 mg L–1). Cells were grown at 28°C without shaking in triplicate 500-mL cultures under dark conditions. All analyzed cells were prepared by inoculating cells from the early exponential phase culture into fresh medium and then cultivation for 2 weeks (early stage) or 8 weeks (late stage). During the culture, ammonium concentrations were measured once every 2 to 4 d using the indophenol method  and then dried on a slide glass. Cells were stained with the SYTOX Green nucleic acid stain (Thermo Fisher Scientific) and observed using a fluorescence microscope .g for 30 min and resuspended in 10 mL of mineral medium. After sonication at 30% amplitude for 3 min, aggregates were collected by trapping on a filter with a pore size of 5 μm (Merck). Free-living cells were collected by passage through a filter with a pore size of 5 μm and trapping on a filter with a pore size of 0.2 μm (Merck).Free-living and aggregated cells were separated by filtering to compare gene expression. Approximately 500 mL of the culture was centrifuged at 4,800×N. mobilis Ms1, qPCR was performed targeting the glnA gene of DNA extracted from each sample. The glnA gene exists as one copy in the N. mobilis Ms1 genome. Therefore, the cell number matched the copy number of the glnA gene in extracted DNA. The primers NSMM_glnA_f (5′-GGCCATCAAGGGTGGCTATT-3′) and NSMM_glnA_r (5′-TCCACAGGGATGCCAAGTTC-3′) were used. To prepare a standard sample of the glnA gene, PCR was performed using TB Green Premix Ex Taq II . Based on the concentration of the PCR product and length of the glnA gene, the copy number of the amplified glnA gene was calculated. The PCR product was diluted to create a standard sample. qPCR was performed by TB Green Premix Ex Taq II (Tli RNaseH Plus) and Thermal Cycler Dice Real Time System II (Takara Bio). qPCR used the following thermal profile: an initial denaturation step was conducted at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s, annealing at 60°C for 30 s, and elongation at 68°C for 30 s, with a melting curve analysis. The R2 value for qPCR was >0.99 . Ribosomal RNA was removed from extracted total RNA using the Ribo-Zero rRNA Removal Kit . The library was then prepared using the SureSelect Stranded Prep Kit (Agilent Technologies). The mRNA library was sequenced with Illumina HiSeq3000 under the condition of paired End 100 bp. Raw sequence data are available in the DDBJ Sequenced Read Archive under the accession number DRA007360.Biological triplicates of the culture were prepared for the transcriptome analysis. To lyse cells in the culture, 100 μL of 15 mg mLfraction 0.5, and Similarity fraction 0.8 to the N. mobilis Ms1 genome sequence (Accession numbers FMWO01000001–FMWO01000112)  <0.05 were defined as DEGs. Furthermore, DEGs were mapped to the pathway provided by the Kyoto Encyclopedia of Genes and Genomes (KEGG).The quality of the reads was improved using CLC genomics workbench (CLC Bio) by trimming the rest of the adapter, low quality reads (limit 0.05), short reads (>15 bp), and one base from the 5′ and 3′ ends. Reads were also mapped under the conditions of Mismatch cost 2, Insertion cost 3, Deletion cost 3, Length N. mobilis Ms1 during cultivation, N. mobilis Ms1 was cultured for 8 weeks with the addition of 2 mM NH4Cl. The timing of the addition of NH4Cl was shown in 4Cl was added seven times and nitrite gradually accumulated , early culture aggregates (early Agg), late culture free-living cells (late FL), and late culture aggregates (late Agg). Among 3,254 N. mobilis Ms1 genes, the reads obtained were mapped at 3,152 genes. The overall metabolic status of the four groups was compared by clustering using read count data from the four groups of biological triplicates. As a result, late FL and late Agg were found to have similar metabolic states. There were only three DEGs  between late FL and late Agg. All three genes were up-regulated in late Agg; however, their functions currently remain unknown. Based on the results of the clustering analysis, four groups  were roughly divided into three groups. Cells were expected to have similar metabolic states in the late stage of culture even if cell morphologies differed. In contrast, since early FL and early Agg exhibited completely different transcriptional states, morphological differences in the early stages of culture were expected to reflect different metabolic states and survival strategies.A transcriptome analysis of Based on the overall metabolic status of the four groups, the two groups described below were compared. Comparisons of early FL and early Agg was performed to reveal gene expression that represented differences in morphology. Comparisons of early Agg and late Agg were performed to reveal gene expression, which represented differences in the culture period.Comparisons of early FL and early Agg revealed the significantly increased expression of 72 genes in early FL and of 251 genes in early Agg. Comparisons of early Agg and late Agg revealed the significantly up-regulated expression of 174 genes in early Agg and of 43 genes in late Agg. The number of strongly expressed genes was higher in early Agg.Strongly expressed genes were classified into functional clusters  . In compGene expression differences between early Agg and early FL and between late Agg and early Agg are summarized in atpH, atpG, and atpC), which were classified into energy production and conversion. In addition, several ribosomal proteins  were classified into translation, ribosomal structure, and biogenesis. Biosynthesis-related gene expression was higher in early FL when morphology was different. In contrast, comparisons of early Agg and late Agg showed that genes up-regulated in late Agg included ribosomal proteins , which were classified into translation, ribosomal structure and biogenesis. The operon encoding ribulose-1,5-bis-phosphate-carboxylase/oxygenase (RuBisCO), which is the carbon fixation pathway of N. mobilis Ms1, was up-regulated in late Agg. When the early and late stages were compared, the expression of the biosynthetic system increased in the late stage.Comparisons of early FL and early Agg revealed that genes up-regulated in early FL included ATP synthases , which are essential genes for ammonia oxidation, were strongly expressed in all groups. However, no significant differences were observed in expression. Previous AOB studies reported that amoA and amoC mRNAs were conserved under substrate starvation conditions (Nitrosomonas europaea transitioned from ammonia- to oxygen-limited steady-state growth. bioRxiv 765727). These transcriptional states suggest that the expression of genes encoding AMO and HAO, the most important enzymes for energy conversion, are always maintained. AOB recovers quickly from stress by maintaining the expression of the most important enzymes, even during stress, and N. mobilis Ms1 showed similar expression profiles.amoE1 and amoE2 were expressed in a different manner than the other ammonia oxidation-related genes. The amoE gene is harbored by beta-proteobacterial AOB and is considered to be derived from amoD duplication. The function of amoE currently remains unknown. However, structural predictions suggest that it interacts with ammonia oxidation and electron transport , pilus genes , chemotaxis genes , and two-component genes (fleS and yrbD). The EPS biosynthetic gene NSMM150022, which is involved in cell-cell adhesion  and proteases  in early Agg. In contrast to the accumulation of the growth inhibitor nitrite in the long-term culture, the increased expression of stress response genes was not confirmed in the late stage of culture. Instead, the expression of stress response genes was increased in the early stage of culture. This result may be explained by assuming that N. mobilis Ms1 is always exposed to stress, and that aggregate formation leads to stress protection. In the early stage of culture, cells may be exposed to stress, leading to the need for adaptation. In contrast, in the late stage of culture, since many cells form aggregates and may avoid stress, there may be no need for a stress response.Comparisons of early Agg and late Agg revealed the up-regulation of genes encoding refolding proteins, including chaperones (cas) and restriction-modification systems  in early Agg. In addition, the expression of transposons and conjugation genes  was up-regulated in early Agg. Based on these results, early Agg may uptake many foreign genes. Horizontal gene transfer mechanisms are generally related to the mechanisms of stress resistance, such as the acquisition of antibiotic resistance and bioremediation ability  Transcriptome Analysis of the Ammonia-Oxidizing Bacterium https://doi.org/10.1264/jsme2.ME19148Supplementary Material 1Supplementary Material 2


Orrù, Elena Antelmi, Angela Mammana, Andrew G. Hughson, Giovanna Calandra‑Buonaura, Anna Ladogana, Giuseppe Plazzi, Pietro Cortelli, Byron Caughey and Piero Parchi, was originally published electronically on the publisher’s internet portal on 27 April 2020 without open access.With the author(s)’ decision to opt for Open Choice the copyright of the article changed on 26 May 2020 to © The Author(s) 2020 and the article is forthwith distributed under a Creative Commons Attribution 4.0 International License (The original article has been corrected.


However, it could self-restore within 107 days after undergoing four stages, at which Planctomycetes became dominant with a relative abundance of 64.62%. Network analysis showed that anammox bacteria  were positively related to some functional bacteria such as Nitrosomonas, SM1A02, and Calorithrix. Canonical correspondence analysis presented a strong correlation between the microbial community and influencing factors during the recovery phase. With the increase of nitrogen loading rate, the decrease of free nitrous acid and the synergistic effects, heme c content, specific anammox activity (SAA), NRR, and the abundance of dominant genus increased correspondingly. The increase of heme c content regulates the quorum sensing system, promotes the secretion of extracellular polymeric substances, and further improves SAA, NRR, and the relative abundance of the dominant genus. This study highlights some implications for the recovery of the CANON reactor after being exposed to an alkaline shock.Responses of a microbial community in the completely autotrophic nitrogen removal over nitrite (CANON) process, which was shocked by a pH of 11.0 for 12 h, were investigated. During the recovery phase, the performance, anaerobic ammonia oxidation (anammox) activity, microbial community, and correlation of bacteria as well as the influencing factors were evaluated synchronously. The performance of the CANON process deteriorated rapidly with a nitrogen removal rate (NRR) of 0.13 kg·m Moreover, correlation among the microbial communities in the CANON system in response to alkaline shock remains to be studied in depth. In addition, pH is found to inhibit microbial communities in two ways: (1) pH inhibits the microbial activity by changing the characteristics of microbial habitats [A completely autotrophic nitrogen removal over nitrite (CANON) process, a combination of partial nitritation and anaerobic ammonia oxidation (anammox), is highly sensitive to key environmental parameters, such as nitrite habitats . AlthougIn this study, we hypothesised that synergy effects among the bacteria associated with the nitrogen cycle would improve the performance of the CANON process after applying transient alkaline pH shock. To test this hypothesis, the CANON process was exposed to an alkaline shock of pH 11.0 for 12 h. The nitrogen removal performance, activity indicators , and signalling molecule), variation of the microbial community structure, correlation among the microorganisms, and the correlation between the microbial community and the influencing factors in the CANON reactor were investigated during the whole recovery process. This study is aimed at broadening our current knowledge of the underlying mechanisms of the microbial community in response to an alkaline shock and encouraging technical regulations optimising the CANON process for a maximal efficiency during the recovery process.−1, respectively [−1, respectively.A pilot-scale upflow anaerobic filter (UAF) reactor  was usedectively . The ino−3·d−1, respectively. Then, it was exposed to a transient alkaline shock of pH 11.0 for 12 h. The recovery phase mainly consisted of four stages: accommodation stage (days 1–37), anammox recovery stage (days 38–68), CANON recovery stage (days 69–100), and steady stage (days 101–107), according to the nitrogen removal performance of the CANON reactor and a previously reported method by Zhang et al. [4+-N and NO2−-N for approximately 1 h at a flow rate of 1600 mL·min−1. The influent concentrations of NH4+-N and NO2−-N were decreased to 250 mg·L−1 to avoid the substrate inhibition [The CANON process was cultivated using synthetic wastewater, and the mineral medium was supplied according to our previous study . After sg et al. . The opehibition . Next, tSAA, an important parameter to evaluate the performance of the anammox pathway , and hem4+-N), nitrite (NO2−-N), nitrate (NO3−-N), and total nitrogen (TN) were analysed based on standard methods [Sludge and water samples were withdrawn from the CANON process on days 1, 37, 53, 63, 75, 85, and 107, in order to investigate the EPS concentration, SAA, heme c content, and signalling molecule. Sample collection was conducted in triplicate. Concentrations of EPS and heme c were determined based on the previous reports by Ma et al.  and Berr methods .To investigate responses of microbial communities in the CANON process to the exposure of an alkaline shock, sludge samples collected from the reactor on days 1, 37, 63, and 107 were subjected to DNA extraction, and quality control was also performed according to our previous study . Next, thttps://www.rstudio.com/). Pairwise Spearman's correlation coefficients among 12 genera, which have an average abundance higher than 1%, were calculated by SPSS Statistics 22  . Furthermore, network analysis based on Spearman's correlation coefficient (r) > 0.6 or <-0.6, and the corresponding p value < 0.05 between two genera was performed and visualised by Gephi software  [Canonical correspondence analysis (CCA) was conducted to further explore the relationship between microbial communities and influencing factors using the “vegan” package in RStudio environment (hi.org/) .−1 to 367.08 mg·L−1, which was much higher than inhibition thresholds of AOB and anammox bacteria after being exposed to alkaline shock of pH 11.0 for 12 h [4+-N and NO2−-N were adjusted according to for 12 h . Thus, tfor 12 h  also foufor 12 h , 36. Sub−3·d−1, respectively, within 9 days. This phenomenon was similar to the lag stage in the start-up of the anammox process [−3·d−1. To improve the nitrogen removal performance of the CANON system during the recovery stage (days 69–100), DO was maintained at 0.2–0.8 mg·L−1. As expected, the NRR increased to 0.82 kg·m−3·d−1 and NRE was at around 72%. Simultaneously, the stoichiometric ratios of ΔTN/ΔNH4+-N, ΔNO3−-N/ΔNH4+-N, and ΔNO3−-N/ΔTN gradually increased from 1.29, 0.10, and 0.10, respectively, on day 1, to their theoretical value , as described in the relevant studies [μg·L−1) on nitrogen removal performance was more significant (p < 0.05) than that of the FA fluctuation on days 62–68. Previous studies report the FNA inhibition of AOB activity to be 100 μg·L−1 [μg·L−1 [μg·L−1).On days 38–52, the nitrogen removal performance of the anammox process recovered slowly with an increase of nitrogen removal efficiency (NRE) from 32% to 49.67%. Afterwards, the NRE and NRR increased rapidly to 86.44% and 0.18 kg·m process . On days−3·d−1, respectively. The sensibility, low biomass yield, and slow growth rate of anammox bacteria were the main challenges for the application of the CANON process [At the steady stage (days 101–107), the water quality of effluent and stoichiometric ratios became stable, and the nitrogen removal performance was even better than the initial level where the averages of NRE and NRR increased to 83.28% and 0.80 kg·m process . Recover−1 VSS·d−1 and 0.50 μmol·g−1 VSS, respectively, on day 1. It was clear that approximately 98% of anammox activity was lost compared to the activity in the initial phase. In the meantime, signalling molecule content was nearly zero, which was the quickest response to the transient alkaline shock. The signalling molecule was easily affected by the pH and quickly degraded under alkaline conditions [−1 VSS on day 1. This was likely because some microbial members excreted excessive EPS. Subsequently, the signalling molecule-based quorum sensing (QS) system could not regulate EPS production normally after being adversely influenced by the extreme alkaline shock [Shifts in SAA, heme c, EPS, and signalling molecule content of the CANON process are shown in nditions . It implne shock , 40. Prene shock , 42 have−1 VSS·d−1, 3.03 μmol·g−1 VSS, and 143.65 mg·g−1 VSS, respectively. Although these values were similar to the findings in previous studies [p < 0.01) after the alkaline shock in this study. The NLR, NRR, and NRE reached the maximum values, and the CANON system remained stable.Significant increases of SAA, heme c, and the signalling molecule were observed on days 53–63, 37–53, and 53–63, respectively, during which the corresponding NRE were being recovered from 53% to 85%. This meant the anammox bacteria and AOB activities were recovered gradually. Moreover, the recovery time of heme c was shorter than that of the other parameters. Heme c is an important component of hydrazine synthesis, hydroxylamine oxidoreductase, and hydrazine oxidase in purified anammox cells . It is d studies , 46, theA shift in the microbial community structure of the CANON process after alkaline shock was investigated by amplicon sequencing. The OTU number and diversity index increased gradually as the CANON process resumed .  FigureBacillus, belonging to Firmicutes, was the dominant genus with a relative abundance of 67.98% after the alkaline shock. Bacillus is a robust model organism for biofilm formation [Bacillus can not only contribute to O2 removal [Bacillus survived after alkaline shock. The accommodation stage (days 1–37) in this study was analogous to the cell lysis stage in the start-up of the anammox process [Thermomonas was capable of utilising the organic products from cell lysis [Candidatus Jettenia, Kuenenia, and Brocadia) gradually increased from 0.13% to 7.52%. After that, AOB (Nitrosomonas) together with anammox bacteria  became the dominant genera as aeration began. The relative abundances of AOB and anammox bacteria (Candidatus Jettenia+Kuenenia+Brocadia) at the enhancement stage increased quickly from 2.57% and 7.52% to 3.05% and 53.30%, respectively. Excess proliferation of NOB was not detected throughout the experiment. The good performance of the CANON process could be explained through microbiological perspectives.At the genus level , Bacilluormation . Bacillu removal , nitrate removal , and org removal  but can  removal . All the process . Thermomll lysis ; therefoCandidatus Jettenia, Candidatus Kuenenia, Candidatus Brocadia, Nitrosomonas, SM1A02, and Calorithrix. They were positively related to each other (p < 0.05). Limnobacter, one genus of module B, showed a significant negative correlation with Candidatus Jettenia, Candidatus Brocadia, Nitrosomonas, and Calorithrix (p < 0.05). Additionally, Bacillus was negatively correlated with all members in module A. CCA was further applied to determine the correlation of microbial communities and the influencing factors  with the influencing factors of SAA, NLR, NRR, heme c, EPS-PS, and FNA. Nitrosomonas was positively correlated with EPS, EPS-PN, SAA, NLR, NRR, heme c, EPS-PS, and FNA. However, Bacillus was only positively correlated with EPS-PN.Network analysis is an effective analytical approach for further exploring the correlation among the dominant microbial taxa  in the C factors  during tLimnobacter, Bacillus, and the dominant genus in network module A. As shown in Candidatus Jettenia, Brocadia, and Kuenenia) oxidised NH4+-N to N2 with NO2−-N and CO2, which were produced by AOB (Nitrosomonas) and DNB , respectively. These served as the electron acceptors and inorganic carbon sources under anaerobic conditions [SM1A02 has the anammox ability and might be a novel anammox strain; therefore, it has the same synergistic effects with Nitrosomonas and Calorithrix [Nitrosomonas can utilise NH4+-N and DO and create anaerobic conditions for anammox bacteria such as SM1A02 and Calorithrix [Calorithrix provides a substrate of NH4+-N to anammox bacteria, SM1A02, and Nitrosomonas by dissimilatory NO3−-N reduction to NH4+-N [Limnobacter competed with other functional bacteria for acetate, O2, and NO3−-N [Limnobacter deteriorated the resistance of the microbial community to adverse influences [Bacillus was eliminated in long-term anaerobic conditions with the increase of NLR and therefore was negatively correlated with module A [The abovementioned results can be explained by 1) the impact of NLR-led influencing factors, (2) synergistic effects of the dominant genus in the network module A, and (3) antagonistic effect of nditions , 58. As orithrix –61. In aorithrix , 60. Cal the impao NH4+-N , 44, 45.d NO3−-N  and consfluences , 63 and module A . The syn−3·d−1 after the system underwent four stages . During the entire recovery process, heme c content responded the fastest to the transient alkaline shock among all tested parameters and increased gradually, followed by the signalling molecule. Both parameters improved the AOB and anammox activity and regulated the EPS secretion. Firmicutes, a type of spore-forming bacterium, was dominant at the initial stage, and subsequently at the later period, Planctomycetes became the dominant phylum with a relative abundance of 64.62%. Network analysis clearly revealed positive relationships among anammox bacteria , AOB (Nitrosomonas), SM1A02, and DNB . Additionally, there was a strong correlation between microbial communities and the influencing factors during the recovery phase. With the increase of NLR, decrease of FNA, and synergistic effects of dominant microbial members, heme c content, SAA, NRR, and the relative abundance of the dominant genus increased correspondingly. The increase of heme c content regulates the quorum sensing system, promotes the secretion of EPS, and further improves SAA, NRR, and the relative abundance of the dominant genus. The response of the microbial community to environmental change gives an insight into the recovery of the CANON process after being exposed to an alkaline shock and provides technical regulations for its engineering application.The CANON process was found to rapidly deteriorate after an alkaline shock of pH 11. However, it could be self-restored with the final NRR of 0.80 kg·m


Emotional empathy is defined as one of the basic concepts in social perception. The present study aimed at presenting a new model of emotional empathy, determining structural relation of perspective taking, emotional comparison, Feminine traits, self-monitoring, and emotion regulation with emotional empathy of the students. In this study, 503 students of Tabriz University were evaluated using Balance Emotional Empathy Scale (BEES), Interpersonal Reaction Inventory (IRI), Cognitive Emotion Regulation Questionnaire (CERQ), Mindfulness Attention Awareness Scale (MAAS), Bem Sex Role Inventory (BSRI), revised Self-Monitoring scale (RSMS), Emotional Comparing Scale (ECS). Fitness indexes of the structural model of emotional empathy were determined using LISREL.8.54 software. Mean age of the subjects was 21.53±2.41. There was a direct structural relationship between emotional empathy and feminine traits, emotional comparison, perspective taking, adjusted emotion regulation, and unadjusted cognitive emotion regulation, respectively. There is an indirect relationship between emotional empathy and sensitivity to the expressive behavior of others .  According to this preliminary study, constructs of emotional comparison, sensitivity to the expressive behavior of others, and emotion regulation play a significant role in emotional empathy or emotional perception. The significance of social comparison was discussed in the model of emotional empathy.Emotional empathy, Feminine traits, Emotional comparison, Perspective taking, Self-monitoring, Cognitive Emotion regulation, Mindful


Various wearable devices for objectively evaluating motor symptoms of patients with Parkinson’s disease (PD) have been developed. Importantly, previous studies have suggested protective effects of physical activity in PD. However, the relationships between conventional clinical ratings for PD and three-axis accelerometer measures of physical activity  are still unclear, particularly for METs. In the current study, we sought to elucidate these relationships on a daily basis, and to clarify optimal predictors for clinical states on a 30-min basis.Patients who were hospitalized for adjustment of drugs or deep brain stimulation were enrolled. Using waist-worn three-axis accelerometers, PAL and METs parameter data were obtained and compared with UPDRS-3[On] and symptom diary data. We extracted data from the patients’ best and worst days, defined by the best and worst UPDRS-3[On] scores, respectively. Thus, 22 data sets from 11 patients were extracted. We examined the correlations and produced scatter plots to represent the relationships, then investigated which METs parameters and activity patterns were the best predictors for “On” and “dyskinesia”.Rho: − 0.799 for UPDRS-3[On], 0.803 for On hours [p < 0.001]). Scatter plots suggested that PAL tended to have higher values in patients with involuntary movement. However, METs parameters focusing on higher METs seemed to alleviate this tendency. We clarified that “time over 2.0 METs” and “time over 1.5 METs” could be predictors for “On” and “dyskinesia” on a 30-min basis, respectively . The specificity and sensitivity of the optimal activity pattern for “On” were 0.858 and 0.621.The parameter “mean METs value within the 95–92.5 percentile range on a day ” exhibited the strongest correlation with conventional daily clinical ratings (This study suggested feasible activity patterns and METs parameters for objective evaluation of motor symptoms on a 30-min or daily basis. Three-axis accelerometer measures focusing on higher METs may be appropriate for evaluating physical activity. Further larger-scale studies are necessary to clarify the validity, reliability, and clinical utility of these objective measures. Parkinson’s disease (PD) is one of the most common age-related neurodegenerative disorders, characterized by various motor symptoms including slowness of movement (bradykinesia) as a main symptom, tremor (particularly at rest), and rigidity . AlthougThe current gold standard for evaluation of PD motor symptoms involves subjective evaluation for a certain period with a symptom diary , 4 and oNew methods and technologies using accelerometers or gyroscopes for the objective assessment of motor symptoms through different computational methods have been developed to overcome these limitations . PreviouIn recent years, evidence suggesting the protective effects of physical activity against PD has accumulated . A meta-Methods]) in various conditions among a variety of PD patients. Furthermore, we also investigated which METs parameters and activity patterns were the best predictors for the “On” and “dyskinesia” states on a 30-min basis.Thus, despite the potential utility of evaluating motor symptoms using three-axis accelerometer measures of physical activity and examining these measures in PD patients, their relationships with the conditions of motor symptoms are currently unclear, particularly for METs. Therefore, the current observational study sought to clarify the relationships on a daily basis between conventional clinical ratings  and three-axis accelerometer measures  were recruited from the Neurology Department at Nagoya Medical Center, Japan, between June 2017 and March 2018. Eligible participants had a diagnosis of idiopathic Parkinson’s disease. Other inclusion criteria were as follows: a) absence of severe cognitive impairment ; b) written consent was granted by the patients’ free will. Patients who met any of the following criteria were not included in the study: a) heart failure, COPD or acute diseases such as infectious disease that restrict exercise; b) the research director or research co-workers determined that participation in this study was not appropriate . We did not apply any cut-off points regarding physical activity or severity of PD to patients. Our study was conducted in a hospitalized setting to confirm patients’ conditions in more detail and in the same environment. The study protocol was approved by the ethical committee at Nagoya Medical Center. Written informed consent was obtained from all participants in accordance with the declaration of Helsinki.Data analysis]) to identify relationships  with conventional clinical ratings (UPDRS-3 [On] and On hours in symptom diary) on a daily basis.Study I: To screen three-axis accelerometer measures  are the best predictors of the “On” and “dyskinesia” clinical states on a 30-min basis.Study II: To investigate which specific METs parameters  exhibited the largest change between the best and worst days.Data analysis) for “On”.In addition, we conducted activity pattern analysis to investigate the specificity and sensitivity of the defined activity patterns and their combinations  was used for assessment of PAL and METs parameters . The monUPDRS-3 [On]  was evalBecause our protocol required hospitalization, we did not include healthy controls. Each data set  was obtained on a daily basis. In drug adjustment patients, 2–3 opportunities (days) of evaluation were conducted on a daily basis before and after drug adjustment, and in DBS patients, 2–5 opportunities (days) on a daily basis were conducted for each setting of DBS. To reflect various conditions of patients and ensure the same number of data sets (2 days) in all patients, we extracted and analyzed data sets from the patients’ best and worst days, defined by UPDRS-3 [On] scores. Evaluation using conventional clinical ratings and three-axis accelerometer measures were also performed on the same days. Evaluation was stopped when the patient requested discontinuation, and data from those days were not included. Patients underwent rehabilitation as usual.METs determined by this three-axial accelerometer have been reported to be closely correlated with METs calculated using energy expenditure measured by indirect calorimetry , 26. BasThe processing flow of the obtained METs data is described in Fig. In study I, as METs parameters, each mean value within a range of 2.5 percentiles (from the 100th to 70th percentile) was calculated. Likewise, the area/time/peaks over specific METs values (every 0.5 METs from 1.5 to 3.0 METs) and percentile values (every 2.5 percentiles between the 100th and 90th percentile) were examined. In study II, area/time/peaks over specific METs value (every 0.5 METs from 1.5 to 2.5 METs) and percentile values (every 2.5 percentiles from the 100th to 90th percentile) on a 30-min basis were examined.Criterion I: at least one peak greater than 2.5 METs with four or more consecutive epochs over 2 METs.Criterion II: four or more peaks greater than 2.5 METs.In the activity pattern analysis, the process of developing the definitions of criteria are described in Supplementary Fig. Criterion for “tremor”: “tremor” was determined by a combination of 90 epochs or more over 1.5 METs and clinical information of resting tremor (sub-score of UPDRS-3 ≥ 2). Tremor is included as “Non-On”.Exclusion criterion: we excluded peaks over 4 METs.Continuous locomotive activity for more than 1 min is counted as just one peak.As this pilot study was exploratory in nature, although no formal sample size estimation was performed, a sample of 11 participants was anticipated to be appropriate for a pilot study.Z-scores and nonparametric Wilcoxon signed-rank tests. In study II, the area under the receiver operating characteristic curve (AUROC) was used to assess model performance. In the analysis of “dyskinesia” in study II, the data from the only day on which the patient showed subjective dyskinesia were used in one patient. Statistical analysis was conducted using easy R version 1.40. The significance level was set to p < 0.05.In study I, Spearman’s rank-order correlations were calculated between objective evaluation items measured with the three-axis accelerometer and conventional clinical ratings. We produced scatter plots of the primary results. Changes from the worst to best day of the three-axis accelerometer measures showing stronger correlations in study I were compared using SD: 18.6 ± 19.6 and 8.6 ± 5.1 [hours], 30.5 ± 20.0 and 1.8 ± 2.6 [hours], p < 0.01 and p = 0.013, Table Eleven patients (mean age: 67.1 [56–81] years) completed repeated assessments more than twice during hospitalization. Ten patients provided data in symptom diaries (mean recorded time: 11.7 [10.0–12.0] hours). UPDRS-3 [On] and On hours were significantly lower and longer, respectively, on the best day compared with the worst day , but only on the worst day for the parameter “area over 92.5 percentile value” (0.623 [p = 0.054] and 0.691 [p < 0.05] with On hours).Twenty-two data sets were obtained from 11 patients. A stronger (anti) correlation was observed in the parameter “mean METs value within the 95-92.5 percentile range on a day ” with both UPDRS-3 and On hours . Plots of patients exhibiting involuntary movement were located among the higher values of PAL in Fig. Next, we calculated the changes between the worst and best day in the measures showing stronger correlations in study I. Among them, the changes were largest in “area over 92.5 percentile value”  may provide an objective severity measure for PD on a daily basis. The highest percentile values  were not strongly correlated with conventional ratings. This may be because some patients tended to have more peaks over relatively high METs values (≥4 METs), even on their worst day . Moreover, possible artefacts were not excluded, since we could not deny the possibility that higher METs might be caused by some activities, such as loss of balance or pulsion phenomenon. The results should be interpreted with caution because of the small sample size , and the absence of a healthy control group. Since we did not collect “tremor” state in the symptom diary, it is currently unclear whether dyskinesia is distinguishable from tremor based on only the data from our device.In study I, 95–92.5 percentile value exhibited the strongest correlation with conventional daily clinical ratings. Scatter plots suggested that objective measures focusing on higher METs  may be appropriate for evaluating physical activity. In study II, the optimal predictors for “On” and “dyskinesia” were “time over 2.0 METs” and “time over 1.5 METs”, respectively. The defined activity patterns may be feasible predictors for “On”. Further larger-scale studies are necessary to clarify the validity, reliability, and clinical utility of these objective measures.Additional file 1: Figure S1. Changes between worst and best day of three-axis accelerometer measures. †; p < 0.01, ‡; p < 0.001. Every measure was standardized using Z-scores, then compared. In the three axis accelerometer measures showing stronger correlations in study I, the largest changes were observed in “area over 92.5 percentile value ”. The mean Z scores on the best and worst day are connected with a red line.Additional file 2: Figure S2. Dot-plots of “time over 1.5 and 2 METs” and application of their cut-off values. Int, intermediate; Pt, patient. (A) Data were obtained from 10 patients, with 392 segments (dots). As a candidate for a cut-off value of “time over 2.0 METs”, 10 (epochs) were considered (red dashed line). (B) Data were obtained from 10 patients , with 398 segments (dots). We considered 114 (epochs) as a candidate for the cut-off value of “time over 1.5 METs” (blue dashed line). Supplementary fig. 2B showed that patients with resting tremor also had values over the cut-off values. The application of the cut-off value for patient 11  suggests increased dyskinesia time on the best day.Additional file 3: Figure S3. Production process and criteria of activity patterns to distinguish “On” from “Non-On”. (A) ROC curve of “time over 2 METs” and “peaks over 2.5 METs”. To develop the criteria, we focused on the better predictors “time over 2 METs” and “peaks over 2.5 METs” for “On” , and a relatively higher percentage and larger number were observed on the worst day, suggesting that some PD patients on the worst day tended to have more peaks over 4 METs. On the basis of these results, we developed the criteria for exclusion of peaks ≥4 METs. (C) Criteria for activity patterns to distinguish “On” from “Non-On”. The definitions of the criteria for “On”, “Non-On” and “tremor (resting tremor)” . The criteria for dyskinesia are also described for reference. Regarding “tremor” (the sub-score of resting tremor in UPDRS-3 ≥ 2), we focused on “time over 1.5 METs”. A cut-off value of 90 epochs was determined, because patients with resting tremor showed milder elevation of baseline of METs compared with dyskinesia (reference) and almost all patients with bradykinesia did not have values over 90 epochs  using three-axis accelerometer measures (percentile) on a 30-min basis with ROC curve analysis in study II.Additional file 6: Table S3. Specificity and sensitivity of the defined activity patterns and their combination.


A submillisecond-response and light scattering-free polymer-network liquid crystal (PNLC) for infrared spatial light modulators is demonstrated. Our new liquid crystal host exhibits a higher birefringence, comparable dielectric anisotropy, and slightly lower visco-elastic constant than a commonly employed commercial material, HTG-135200. Moreover, the electro-optical performance of our PNLCs with different monomer concentrations, cell gaps, and liquid crystal (LC) hosts is compared and discussed from four aspects: operating voltage, hysteresis, relaxation time, and light scattering loss. The temperature effect on hysteresis is also analyzed. Potential applications of PNLCs for laser beam steering and spatial light modulators especially in the infrared region are foreseeable. In the visible region, to achieve 2π phase change demands dΔn = λ/2, which can be achieved using a high Δn and low viscosity nematic LC [n and longer wavelength (λ) is needed. This will dramatically increase the response time because the response time of a LC device is proportional to d2. To reduce response time, a multi-layer approach [Spatial light modulators, such as liquid-crystal-on-silicon (LCoS) ,2,3, havmatic LC ,18. As tapproach ,20 has bapproach  for achid region ,24,25,26n of the LC host, cell gap, and domain size. A LC material with large Δε and high Δn is desirable because it enables a thinner cell gap to be used, which in turn helps lower the required voltage. Reducing the monomer concentration will lead to a lower Typically, a PNLC precursor consists of ~93% nematic LC host, ~6% reactive mesogen monomer, such as RM257, and ~1% photo-initiator. Upon UV curing, submicron polymer network domain size is formed to constrain LC molecules. Thus, submillisecond response time can be achieved due to small LC domain size and strong anchoring energy provided by the networks. However, the tradeoffs include high operation voltage, hysteresis, double relaxation, and light scattering loss. In a PNLC, the operating voltage is determined by several factors, such as dielectric anisotropy  for making PNLCs and characterize their electro-optical performance, including λ = 1.06 ε helps to lower the threshold voltage  are measured at from Vth ,30:(1)Vtγ1/K11) from the free relaxation time. In Equation (1), d = 8 n at each temperature is obtained from the measured phase retardation by applying a 1 kHz square-wave AC voltage to the LC cell. n at λ = 1.06 μm, where dots represent the measured data and solid line is the fitting curve with Haller’s semi-empirical equation [To measure equation :(2)Δn=ΔnT = 0 K, S is the order parameter, and β is a material constant. The obtained In Equation (2), The visco-elastic coefficient (ormation . By measequation :(3)γ1K11A is a proportionality constant, A and In Equation (3), λ = 1.06 μm, a He-Ne laser at λ = 632.8 nm, and a tunable Argon ion laser . The experimental results are shown in To investigate the device performance at different wavelength, the birefringence dispersion of these two LC hosts should be measured. The probing beams we employed include a diode laser at λ* is the mean resonance wavelength. Once these two parameters are determined, the birefringence at any wavelength of interest can be calculated from Equation (4). The obtained G and λ* values are also listed in In Equation (4), G is a proportionality constant and π phase change at a reasonable voltage. However, the cell gap merely satisfies 2π phase change will lead to a very high Vπ2. To lower Vπ2, we choose a slightly thicker cell gap to achieve about 2.2π phase change, which corresponding to d ≈ 10 to 12 μm, depending on which LC host is employed. If the cell gap is too thick, then the light scattering loss will increase proportionally.To fabricate transmissive-mode PNLCs, we first prepared precursors by adding different amounts of reactive mesogen RM257 (Merck) and 0.5 wt% photo-initiator Irgacure 819 to M1 and HTG LC hosts. Next, we filled each precursor into ~10 μm and 12.15 μm homogeneous LC cells (glass substrates) whose inner surface was deposited with a thin indium-tin-oxide (ITO) electrode and then overcoated with a polyimide alignment film. Then, a UV lamp , λ, to achieve 2π phase change using a transmissive PNLC, the cell gap (d) is determined by the effective birefringence Δeffn of the PNLC composite asFor a given wavelength n LC host helps to increase the Δeffn of PNLC, which in turn enables a thinner cell gap to be used. By substituting Equation (6) into Equation (5), From Equation (6), a high Δeffn and large Δε help to lower λ = 1.06 μm. The measured ε than HTG, under the same cell gap and monomer concentration, the Vπ2 than the corresponding HTG host, as clearly shown by comparing PNLC-1 with HTG-1, PNLC-2 with HTG-2, and PNLC-3 with HTG-3. The reason is that M1 has a 10% higher birefringence, while not compromising its dielectric anisotropy and viscosity. Our employed LC cells have a small variation in cell gap, but the overall trend of VP curves is consistent with our analysis. In Equation (7) shows that high ΔIn a polymer-stabilized LC system, when the applied voltage is removed, the process of relaxing back to the original state sometimes cannot be simply described by a single exponential decay because of the sophisticated interaction between LC and polymer network. Usually, the relaxation process involves two steps, starting with a fast decay from the submicron LC domains and then followed by a slow relaxation caused by the electrostriction effect of polymer network . Such a equation :(8)δ(t)=A, B) and A/(A + B) stands for the degree of double relaxation. For example, when A/(A + B) = 1 , it means double relaxation does not exist because of the vanishing second term. On the other hand, a larger B leads to a smaller A/(A + B), indicating a stronger double relaxation. In Equation (8), the first term indicates the fast relaxation process, and the second term represents the slower one. Here, , To investigate the hysteresis behavior at elevated temperatures, we chose PNLC-4 to conduct the experiment. An elevated temperature helps suppress hysteresis because the interaction between the LC molecules and polymer network is weaker due to the lower viscosity. ε decreases as the electric field frequency increases. This phenomenon is known as dielectric relaxation [For a high laxation , and a claxation . To inveλ = 1.06 μm. In all, the measured transmittance curves of 4 samples with different monomer concentration and cell gap are consistent with the theoretical analysis based on Equation (10). A more detailed theoretical analysis based on different models has been discussed in [In practical applications, high transmittance is critical. To analyze the scattering loss, we scan the transmittance of PNLC 1–4 from 800 to 1200 nm. The scattering loss of PNLCs can be analyzed by the Rayleigh–Gans–Debye model . In thisussed in . The oscλ = 1.06 μm. So far, we have investigated the From Next, we compare the electro-optical performance of PNLC-4 with HTG-4 because they have the same monomer concentration and cell gap; the only difference is different LC hosts. As  process . As demo process , low temπ phase change by using a 50% thinner cell gap because the incident light traverses the PNLC layer twice. As a result, its Finally, it is worth mentioning that our above PNLCs are designed for transmissive mode operation. If we use a reflective LCoS, then we can obtain 2n M1 mixture is not limited to PNLCs but it is also applicable to polymer-stabilized blue phase liquid crystals. Potential applications of PNLCs for laser beam steering and spatial light modulators especially in the IR region are foreseeable. In conclusion, we have developed a new LC mixture, designated as M1, which is a promising host for PNLCs to lower the operating voltage because of its high


When considering the “beauty-is-good” stereotype, facial attractiveness should facilitate empathy for pain. On the other hand, having in mind the “threat value of pain” hypothesis, pain cues would be more salient, and thus, its processing would not suffer influence by facial attractiveness. The event-related potential (ERP) allows investigating if one of these theories could predict individuals’ responses regarding the perception of pain or attractiveness in others’ faces. We tracked 35 participants’ reactions to pictures depicting more and less attractive faces displayed in a painful and non-painful condition. Each participant completed the following two tasks when presented the images of faces: (1) the Pain Judgment Task, in which participants should rate the pain levels, and (2) the Attractiveness Judgment Task, in which participants should rate the attractiveness. Results showed that participants exhibited differences rating more and less attractive faces in the non-painful pictures, but not in the painful pictures. These results were observed in P3 and LPC amplitudes in the Pain Judgment Task, as well as in N170 and P2 amplitudes in the Attractive Judgment Task. Our results suggested that both explicit and implicit empathic pain processing inhibited the processing of attractiveness perception. These findings supported the “threat value of pain” hypothesis. Besides, in the Attractive Judgment Task, the N170 and P2 amplitudes for more attractive painful pictures were larger than those for more attractive non-painful pictures. In contrast, no significant difference was found between the amplitudes for painful and non-painful, less attractive pictures. Our findings suggest that explicit facial attractiveness processing for more attractive face images potentiates the implicit empathy for pain processing, therefore partly supporting the “beautiful-is-good” stereotype. When individuals witness others’ pain or injuries, they usually can identify the pain and relate to these experiences as if they were their own2. This competency is thus called empathy for pain4. According to the “threat value of pain” hypothesis8, the perception of another’s pain activates the individual’s survival mechanisms, meaning that, they may experience withdrawal and avoidance like when exposed to real or perceived danger. Therefore, the ability to recognize others’ pain helps individuals to avoid possible hazards and promotes empathic behavior9.Empathy refers to the ability to understand and share others’ feelings11, plays an important role in interpersonal interactions in daily life. Attractive faces can be considered rewarding stimuli, eliciting positive emotional reactions. Those activate brain areas associated with the processing of positive emotions and rewarding, such as the nucleus accumbens and the ventral tegmental area12. According to the “beauty-is-good” stereotype13, people with higher physical attractiveness are deemed by others as possessing positive personality attributions, such as sociability, intelligence, and morality15.Physical attractiveness, as a symbol of biological quality signaling fertility and health16. However, another study revealed that physical attractiveness inhibited empathy for children in need, especially when a victim’s need was obviously severe17. A functional magnetic resonance imaging study18 indicated that the modulation of physical attractiveness on pain empathy varied between male and female participants: there were greater activations in regions associated with empathy for pain processing, such as the insula and anterior cingulate cortex, to less attractive men and to more attractive women.One study showed that physical attractiveness facilitates empathic responses, observing a greater empathic response to pain in more attractive people, as compared to pain in less attractive people20. According to the “threat value of pain” hypothesis, others’ pain, serving as warning signals to avoid or escape8, would elicit negative emotional reactions24, thus suppressing the perception of physical attractiveness, as evidenced by decreased attractiveness ratings and neural responses.On the other hand, whether the perception of physical attractiveness in others could be influenced by pain remains unclear. Previous studies have shown that perceptions of facial attractiveness could be influenced by emotions in the faces, such that negative emotions suppressed attractiveness assessments as compared to positive and neutral emotional faces26. The present study aimed to explore the interplay between others’ pain and physical attractiveness as perceived by participants. More specifically, we examined (1) whether empathy for pain can be influenced by others’ attractiveness and (2) whether perceptions of others’ attractiveness can be influenced by others’ pain. Therefore, four categories of facial stimuli were adopted, i.e., non-painful and painful facial stimuli with either low or high physical attractiveness. Each participant completed two tasks. In the first, we applied the Pain Judgment Task, in which participants were asked to judge whether facial stimulation was painful or non-painful. In this way, others’ pain would be processed explicitly, while attractiveness would be processed implicitly. The second task consisted of the Attractiveness Judgment Task, in which participants were asked to assess whether the presented face was attractive or non-attractive. In this task, attractiveness processing would be explicit processed and pain processing would be implicit.Both others’ pain  and attractiveness  could be processed prioritized by the human attention system13, thus capturing more attention resources. Therefore, we hypothesized that both of the explicit and implicit processing of others’ pain would be potentiated by physical attractiveness processing, as reflected by heightened empathic neural responses to faces with high physical attractiveness. As predicted by the “threat value of pain” hypothesis, the perception of others’ pain and injure is a potential threat to the self, apparently provoking observers’ threat-detection system and possibly activating a general aversive response7. Therefore, we hypothesized that both of the explicit and implicit processing of others’ attractiveness would be suppressed by processing of others’ pain, as reflected by the decreased processing of facial attractiveness caused by pain cues present in the faces.As predicted by the “beauty-is-good” stereotype, more attractive faces are perceived as being morally good and as having better personalitiesF1,34 = 105.18, p < 0.001, ηp2 = 0.76), with participants accurately judging painful and non-painful pictures (Painful: 5.60 ± 1.36 vs. Non-painful: 2.02 ± 1.21). Nevertheless, pain intensity ratings were modulated neither by “attractiveness” nor by the interaction (p > 0.05 for both comparisons).As shown in the bottom panel of Fig. F1,34 = 108.90, p < 0.001, ηp2 = 0.76) and by “pain” , indicating that participants judged more attractive models as more attractive than less attractive models (More attractive: 5.86 ± 1.04 vs. Less attractive: 3.73 ± 0.93), non-painful pictures as more attractive than painful pictures (Non-painful: 4.97 ± 1.01 vs. Painful: 4.62 ± 0.97).The attractiveness ratings were significantly modulated by “attractiveness”  and “pain” , indicating that the participants felt negatively toward the less attractive models, relative to the more attractive models (Less attractive: 5.58 ± 0.66 vs. More attractive: 5.14 ± 0.82), and felt negatively toward the painful pictures, relative to the non-painful pictures (Painful: 5.75 ± 0.80 vs. Non-painful: 4.97 ± 0.68). Emotional reactions were also significantly modulated by the interaction between “attractiveness” and “pain” , indicating that (1) the difference between less and more attractive models was greater for non-painful pictures than for painful pictures , and that (2) the difference between painful and non-painful pictures was larger for more attractive models than for less attractive models .Participants’ emotional reactions were modulated by “attractiveness” .As shown in the top panel of Fig. Grand average ERP waveforms to painful and non-painful pictures with high and low attractiveness in the Pain Judgment Task, as well as scalp topographies of dominant waves, are shown in Fig. F1,34 = 4.77, p = 0.036, ηp2 = 0.11), with N1 waves showing more negativity toward the less attractive faces than toward the more attractive faces (Less attractive: −3.84 ± 3.82 μV vs. More attractive: −3.60 ± 3.80 μV). N2 amplitudes were significantly modulated by “pain” , indicating that painful pictures elicited significantly greater N2 amplitudes than non-painful pictures (Painful: −2.62 ± 5.19 μV vs. Non-painful: −2.09 ± 5.15 μV).N1 amplitudes were significantly modulated by “attractiveness” . Although for the non-painful pictures, P3 and LPC amplitudes to the more attractive faces were larger than to the less attractive faces , no significant difference of P3 and LPC amplitudes was observed between the more attractive and less attractive faces for the painful pictures . Another direction of comparison of P3 amplitudes revealed that, for the less attractive faces, P3 amplitudes to the painful pictures were significantly larger than to the non-painful pictures , whereas no significant difference was observed between the painful and non-painful pictures for the more attractive faces .P3 and LPC amplitudes were significantly modulated by the interaction between “attractiveness” and “pain” , where participants judged the more attractive faces with less speed and accuracy than the less attractive faces .As shown in the middle panel of Fig. F1,34 = 5.33, p = 0.027, ηp2 = 0.14). For the painful pictures, ACCs to the more attractive faces were lower than to the less attractive faces , whereas no significant difference was observed between the more attractive and less attractive faces for non-painful pictures . These results suggest that participants tended to judge more attractive faces with pain cues as less attractive.ACCs were significantly modulated by the interaction between “attractiveness” and “pain” , with amplitudes being more negative to the more attractive faces than to the less attractive faces . N170 and P2 amplitudes were also significantly modulated by the interaction between “attractiveness” and “pain” . For the non-painful pictures, amplitudes to the more attractive faces were more negative than to the less attractive faces , whereas no significant difference was observed between the more attractive and less attractive faces for the painful pictures . Another direction of comparison revealed that, for the more attractive faces, amplitudes to the painful pictures were significantly or marginally larger than to the non-painful pictures , whereas no significant difference was observed between the painful and non-painful pictures for the less attractive faces .Averaged ERP waveforms and scalp topographies of each condition are shown in Fig. F1,34 = 6.76, p = 0.014, ηp2 = 0.17), with painful pictures eliciting significantly greater amplitudes than non-painful pictures . LPC amplitudes were only modulated by “attractiveness” , such that LPC amplitude was higher for the more attractive faces than for the less attractive faces (More attractive: 10.08 ± 4.92 μV vs. Less attractive: 9.31 ± 4.14 μV).N2 and P3 amplitudes were significantly modulated by “pain” , and faces29. Interestingly, this effect was not observed when assessing more attractive faces. One possible explanation for those findings would be that the implicit attractiveness processing of more attractive faces could attenuate the empathic responses to others’ pain. Therefore, when individuals pay attention to the pain cues, empathy for pain could be reduced towards people with more attractive faces. Previous studies had found that empathic responses to others’ facial pain might be sensitive to some physical features, for instance, when participants assessed images of faces ethnically different than themselves, their empathy for pain was reduced30. The same happened regarding tasks involving trustworthy and untrustworthy faces31. It might be possible that own-race images or trustworthy faces are perceived as closer to oneself. Previous work has demonstrated that the perceived closeness to another individual enhances empathic responses32. Somehow, the more attractive models may be perceived as non-similar to the participants and the facial features may be more or less prioritized based on the implicit-explicit processing line of reasoning33. Thus, participants may exhibit inhibited empathic responses to more attractive models’ pain when they instructed to pay attention to the pain cues in the stimuli. However, we acknowledge that alternative explanations for these findings may be possible. As more attractive faces elicited larger P3 amplitudes than less attractive faces in later components34. Thus, enlarged P3 amplitudes for more attractive non-painful faces decreased the difference between painful and non-painful more attractive pictures. Above all, the results of the Pain Judgment Task suggest that explicit empathic processing to others’ facial pain inhibits the implicit attractiveness processing, which supports the “threat value of pain” hypothesis.We observed significant P3 amplitude differences between painful and non-painful pictures of less attractive models, which is consistent with findings of previous ERP studies on empathy for pain. Those studies have found larger amplitudes in the processing of painful images compared to non-painful, using images of hands and feet  and ERP amplitudes to painful pictures were not influenced by the implicit attractiveness processing. Hence, these results suggest that explicit empathic processing to others’ facial pain inhibits the implicit attractiveness processing, which more supports the “threat value of pain” hypothesis20. Our findings demonstrated a significant effect on behavioral and neural responses of pain on attractiveness. Participants exhibited decreased accuracy in judging the more attractive painful pictures, suggesting that pain cues decrease the perception of attractiveness, especially to more attractive faces. In addition, attractiveness ratings to the more attractive faces showed a decrease in painful situations than did the less attractive models. These results indicated that pain decreased perceptions of attractiveness on a behavioral level.Consist with previous findings that individuals’ facial attractiveness was influenced by their expressions, negative emotions decreased perceptions of attractiveness35, N170, P2, and LPC amplitudes were modulated by “attractiveness”, with more attractive faces eliciting a negative deflection in N170 and P2 components and a positive deflection in LPC components relative to less attractive faces. Similarly to previous studies29, we also found a main effect of “pain” in N2 and P3 amplitude in the Attractive Judgment task, with painful pictures eliciting more negative N2 and more positive P3 waves than non-painful pictures. These results may suggest that N2 and P3 are sensitive to others’ pain cues, independently of task demands. As frontal N2 to others’ painful faces were suggested in relation to affective components of empathy for pain28, and as P3 over the posterior parietal cortical area have been thought to link to a cognitive evaluation component of empathy29, it appears that processing resources of evaluation of others’ pain were recruited automatically in these time windows even though the pain cues in the Attractive Judgment Task were unrelated to task goals.In line with previous ERP studies31 suggested that the N170 and P2 components were involved in the automatic coding of both facial identity and emotional state, such as pain. Thus, our results suggested that implicit pain processing decreased the distinguishing automatic coding process of others’ attractiveness in these time windows, supporting the “threat value of pain” hypothesis7.More importantly, N170 and P2 components were modulated by the interaction between “pain” and “attractiveness”; that is, for the non-painful pictures, N170 and P2 waves to the more attractive faces were more negative than to the less attractive faces, whereas no difference was found between the more and less attractive faces for the painful pictures. Previous ERP findings13, more attractive faces are perceived as being morally good and as having better personalities, thus, this would increase the observers’ empathy for pain. Under this perspective, our findings partly supported the “beauty-is-good” stereotype13.Interestingly, another aspect of the comparison between “pain” and “attractiveness” revealed that for more attractive faces, N170 and P2 amplitudes of painful pictures were larger than non-painful pictures, whereas no significant difference was observed between the painful and non-painful pictures for less attractive faces. These results suggest that explicit facial attractiveness processing for more attractive faces enhanced the implicit empathy for pain. Based on the “beauty-is-good” stereotype7. That is, both attractive faces35 and pain cues36 automatically capture our attention faster and hold it longer. When offered simultaneously, however, individuals would automatically allot more attention to pain cues. On the other hand, the explicit facial attractiveness processing for more attractive faces enhanced the implicit empathy for pain processing. These results partially support the “beauty-is-good” stereotype13.The findings of both tasks in the present study indicate that empathy for pain and facial attractiveness may not be processed independently of each other. The perception of facial attractiveness could be suppressed by empathy for pain, regardless of the task, which strongly supports the “threat value of pain” hypothesisDespite possible implications, several limitations of the present study should be addressed. Firstly, all face images were gray scale: whether these responses related to daily life requires further investigation. Secondly, both female and male prototypes were involved in the study: it may be that the effect is different for male and female faces. Future research should include gender in the experimental design.7. Furthermore, explicit facial attractiveness processing for more attractive faces enhanced the implicit empathy for pain processing, which partly supported the “beautiful-is-good” stereotype13.To investigate the association between empathy for pain and the attractiveness, this study employed the Pain Judgment Task and the Attractiveness Judgment Task use ERPs. The results suggested that both explicit and implicit empathic pain processing inhibited the processing of others’ attractiveness, thus supporting the “threat value of pain” hypothesisThirty-five adults  from the Chongqing Normal University, Chongqing, China, participated in this study as paid volunteers. None of the participants had been previously diagnosed with a psychiatric, medical, or neurological disorder. All participants were right-handed, aged 18–24 years , and in possession of normal or corrected-to-normal vision. All participants signed informed consent after receiving a complete description of the study. All participants gave their free and informed consent to the study before the experiment in accordance with the Declaration of Helsinki, and all procedures were approved by the Chongqing Normal University research ethics committee. The procedures were performed in accordance with ethical guidelines and regulations.38. The database comprised photos of 30 more attractive faces  and 30 less attractive faces . Each face was transformed to depict pain by penetrating the model’s cheek with a syringe needle, and a non-painful version was created by touching each model’s face with a soft object (Q-tip), using the software “Adobe Photoshop CS6” , attractiveness levels , emotional valence , and arousal  of pictures were assessed by 42 undergraduate students (they did not participate in the experiment). In addition, emotional appearance of the faces  were assessed by 30 undergraduate students (they did not participate in the experiment). The descriptive statistics and statistical analysis of pictures were summarized in Supplementary Material, S. Table The participants were seated in a quiet room with an ambient temperature of about 20 °C. They were instructed to participate in two experimental tasks: (1) the Pain Judgment Task and (2) the Attractiveness Judgment Task. The order of these two tasks was counterbalanced between participants. For both tasks, the order of stimulus presentation was randomized, using the E-Prime (3.0) program. Electroencephalography (EEG) data were recorded during these two tasks.In the Pain Judgment Task , participants were instructed to rate the picture attributes of pain intensity  and attractiveness levels , as well as their subjective emotional reactions , based on 9-point Likert scales. The experimental procedure of this task was illuminated in Supplementary Material, S. Figure EEG data were recorded from 64 scalp sites, using tin electrodes mounted on an actiCHamp system . The electrode at the right mastoid was used as a recording reference, and that on the medial frontal aspect was used as the ground electrode. All electrode impedances remained below 5 kΩ.39. Continuous EEG signals were band-passed filtered (0.01–40 Hz) and segmented using a 1000 ms time window. Time windows of 200 ms before and 800 ms after the onset of stimuli were extracted from the continuous EEG, and the extracted window was baseline-corrected by the 200 ms time interval prior to stimuli onset. EEG epochs were baseline-corrected by a 200 ms time interval prior to stimuli onset. EEG epochs were also visually inspected, and trials containing significant noise from gross movements were removed. EOG artifacts were corrected via the independent component analysis (ICA) algorithm40. These epochs constituted 5 ± 2.4% of the total number of epochs.EEG data were pre-processed and analyzed via MATLAB R2014a  and the EEGLAB toolbox31, dominant ERP components were identified as follows: N1 ; N2 ; P2, P3 and LPC ; N170 . Amplitudes of N1, N2, P2 and P3 components were calculated as the mean amplitudes with a latency interval of peak ± 10 ms at electrodes displaying maximal responses. In addition, LPC amplitudes were measured at latency intervals of 400–600 ms.After confirming scalp topographies in both single-participant and group-level ERP waveforms and previously reported studiesSubjective ratings of pictures, including ratings of pain intensity, attractiveness and subjective emotional reaction, were compared using repeated measures analysis of variance (ANOVA) with factors of “attractiveness” (more attractive vs. less attractive) and “pain” (painful vs. non-painful). If any main effect or interaction effect was observed, post hoc comparisons were performed.Behavioral data, including reaction times (RTs) and accuracies (ACCs), and electrophysiological data (peak latencies and amplitudes of dominant ERP components) were compared separately for the Pain Judgment task and for the Attractiveness Judgment task. Subjective ratings to stimuli, behavioral data and electrophysiological data were compared via a repeated measures ANOVA using the within-participants factors, “attractiveness” (more attractive vs. less attractive) and “pain” (painful vs. non-painful). If any main effect or interaction effect was observed, post hoc comparisons were performed.p > 0.05). Relevant results have been summarized in Supplementary materials, S. Table In addition, a separate analysis with a factor for “order” (Pain Judgment Task First vs. Pain Judgment Task Second) was taken into account the possible order effect with which the tasks were performed. However, none significant comparisons were found in all dependent variables (all This research was approved by the Chongqing Normal University research ethics committee. All participants had signed informed consent after being given a complete description of the study. The ethics committee approved this consent procedure.Supplementary materials.