Tripneustes is one of the most abundant and ecologically significant tropical echinoids. Highly valued for its gonads, wild populations of Tripneustes are commercially exploited and cultivated stocks are a prime target for the fisheries and aquaculture industry. Here we examine Tripneustes from the Kermadec Islands, a remote chain of volcanic islands in the southwest Pacific Ocean that mark the boundary of the genus’ range, by combining morphological and genetic analyses, using two mitochondrial (COI and the Control Region), and one nuclear (bindin) marker. We show that Kermadec Tripneustes is a new species of Tripneustes. We provide a full description of this species and present an updated phylogeny of the genus. This new species, Tripneustes kermadecensis n. sp., is characterized by having ambulacral primary tubercles occurring on every fourth plate ambitally, flattened test with large peristome, one to two occluded plates for every four ambulacral plates, and complete primary series of interambulacral tubercles from peristome to apex. It appears to have split early from the main Tripneustes stock, predating even the split of the Atlantic Tripneustes lineage. Its distinction from the common T. gratilla and potential vulnerability as an isolated endemic species calls for special attention in terms of conservation. Being widely distributed, easily accessible for sampling, and intensely studied since the 19th century, tropical sea urchin species are prime model taxa for the study of speciation and biogeography in the marine environment4.The upsurge of molecular genetic data being generated over the past two decades has facilitated major advances in our understanding of speciation and biogeography across the tree of life. In particular, data from widely distributed genera and species can (1) provide independent evidence for genetic boundaries between cryptic species, (2) facilitate estimations of divergence times, (3) depict species divergence patterns through phylogenetic reconstructions, and (4) allow the examination of the geography of speciationTripneustes are some of the most abundant and common circumtropical echinoids5. The genus consists of several largely allopatric species that vary in their levels of molecular and morphological divergence. Of those, Tripneustes gratilla  is the most widespread, ranging from the Western Indian Ocean to the Eastern Pacific6. Apart from its role as an ecological keystone species in many tropical regions7, T. gratilla is unique in being one of the most commercially valued echinoid species to date and is a prime target for the fisheries and aquaculture industry8. In some localities, such as Oahu Island in Hawaii, T. gratilla are also being propagated as agents of environmental control to regulate the expansion of invasive alien seaweeds . At present, T. gratilla is being cultured at numerous locations throughout the species range12. Consequently, native populations are under growing pressure from cultivated fishery stocks and face the risk of being obliterated by either intentional release  or accidental leakage from near shore facilities. This risk is particularly eminent for small endemic populations and species that are difficult to recognize morphologically.Sea urchins of the genus Tripneustes is a distinct clade, endemic to the RS13. This clade was likely formed at a time when gene flow between the RS Tripneustes and the wider Indian Ocean population was constricted. Considering the large morphological variability observed for Tripneustes across its vast range, it seems probable that the genus may contain additional highly localized endemic species, particularly around the periphery of its range.A recent study, combining molecular genetics , morphological, fossil, and ecological evidence demonstrated that the Red Sea (RS) Tripneustes, the Kermadec Islands represent a remote area close to the southern Pacific boundary of the genus’ range. This chain of volcanic islands in the southwest Pacific Ocean is located ca. 1,000 km northeast of New Zealand and stretching for 2,600 km towards Tonga  (under the name Tripneustes variegatus)21. Although several studies on the phylogeography of Tripneustes have now been published, covering most of this genus’ presumed range23, only a single study24 so far has utilized DNA sequence data on Tripneustes from the Kermadec Islands. Based on the mitochondrial cytochrome c oxidase subunit 1 (COI) gene the latter authors found that seven individuals of Tripneustes sampled at the Kermadec Islands were represented by a single haplotype that is shared by most other Tripneustes populations in the east Indian and Pacific oceans. Consequently, they identified the Kermadec Tripneustes as the common Indo-Pacific T. gratilla24.Previous studies have identified the Kermadec Tripneustes by combining novel genetic data  and the first in-depth morphological analyses of Kermadec Tripneustes to resolve the taxonomic status and reconstruct the phylogeny of Tripneustes from this remote location. We then use these data to explore Tripneustes evolutionary history.Here we re-examine Kermadec 6. In the present case, however, pedicellariae offer few clues to the placement of the Kermadec Tripneustes population . Fortunately, however, gross morphology, tuberculation and ambulacral plating offer good evidence for the placement of this population  is the best match in terms of corona shape, but differs strongly in tuberculation pattern and ambulacral plating (compare13). Exceptionally large specimens of the East Pacific T. depressus can also have a similarly flattened shape, but usually , show strong support for the monophyly of Kermadec Tripneustes . Phylogenies based on the novel CR marker are congruent with the results obtained from COI  An Indo-Pacific cluster that includes T. g. elatensis, T. depressus and T. g. gratilla; (2) an Atlantic cluster comprising of T. ventricosus haplotypes; and (3) a cluster including only Kermadec Tripneustes  and Maximum Likelihood (ML) analyses produced congruent phylogenies for all clades and subclades in all datasets used and thus only BI inferred topologies are presented supplemented by the corresponding bootstrap values of the ML analysis and posterior probabilities support values of the BI analysis. Both mitochondrial markers; a portion of the tes Figs . Rooted sus Fig. . It thusCOI Fig. . Analysilla Fig. . The mitork Fig. . Similartes Fig. , none ofTripneustes, results of the nuclear bindin analyses were similar to the results inferred from the mitochondrial markers. The resulting nuclear bindin tree recovered Kermadec Tripneustes as a strongly supported clade  with strong nodal support , similar to the divergence within the Atlantic T. ventricosus, the RS T. g. elatensis and T. g. gratilla clade A   and the nuclear bindin gene . Zigler and Lessios23 reported no evidence of diversifying selection for bindin in Tripneustes, asserting its suitability as a phylogenetic marker for this group. Following the results obtained from the first two markers and the arising contradiction to previously published COI data, we analyzed a longer stretch of the COI gene and, in addition, added a novel marker sequence, for which we recently developed primers located adjacent to the control region (CR) . The non-coding mitochondrial control region was chosen due to its high mutation rate and assumed selective neutrality. DNA extractions, primer usage, and Polymerase Chain Reaction (PCR) amplifications and cloning of the first two markers were performed according to the protocols described in Bronstein et al.13. To amplify a longer stretch of the COI gene we designed a new forward primer (TripCOI6+: 5′ AACATGCAACTAAGACGATGA 3′), that was used with the reverse primer COI1a23 to produce a 1347 bp amplicon. PCR reactions using this primer combination were performed in 50 µl containing 2.0 μl DNA (10–15 ng), 5 μl 10X High Fidelity PCR Buffer, 2.0 μl of 50 mM MgCl2, 1.0 μl of 10 mM dNTPs, 0.2 μl of each 50 mM forward and reverse primers, and 0.2 μl Platinum®Taq High Fidelity polymerase. PCR conditions were 2 min at 94 °C, 5 cycles of 94 °C for 15 s, 60 °C for 30 s, 68 °C for 90 s, followed by 35 cycles of 94 °C for 15 s, 50 °C for 30 s, 68 °C for 90 s. For amplification of the CR we designed a new set of primers, the forward primer CR15fwd (5′ TACACATCGCCCGTCACTCT 3′) and the reverse primer CR08rev (5′ TTAACGGCCAAGCGCCTTT 3′) that generated an amplicon of ca. 590 bp. PCR reactions were performed in 25 µl containing 1.0 μl DNA (10–15 ng), 2.5 μl 10X TopTaq PCR Buffer, 2.0 μl of 50 mM MgCl2, 1.0 μl Q-Solution, 0.5 μl of 10 mM dNTPs, 0.25 μl of each 10 mM forward and reverse primers, and 0.25 μl TopTaq DNA polymerase. PCR conditions were 3 min at 94 °C, 35 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 60 s, followed by a final elongation step at 72 °C for 10 min.Two DNA marker sequences were initially analyzed: the mitochondrial bindin sequences were cloned prior to sequencing  containing 13 T. kermadecensis n. sp., 23 T. g. elatensis and 34 T. g. gratilla sequences plus an additional 14 T. ventricosus and 6 T. depressus with Lytechinus variegatus serving as outgroup (see Tables COI dataset (448 bp long) comprised 13 sequences generated in the present study plus 326 Tripneustes COI sequences from the literature  including parts of both exons and the intron. The CR alignment comprises 22 sequences and has a length of 446 bp. Alignable sequences from each marker  were concatenated from a subset of specimens for which all three markers were sequenced from the same individual. The concatenated alignment comprises 19 sequences with a total length of 2828 bp.Phylogenetic analyses were based on the re Table . Our ful29 and AliView v.1.1830. Median-Joining networks were calculated for the 448 bp COI dataset with PopArt31 applying the default settings. For the other datasets positions with gaps were removed using TrimAl v.1.432 and identical sequences identified using DAMBE v.6.0.133. Phylogenetic reconstructions were performed using both the Maximum Likelihood (ML) method and Bayesian Inference (BI). ML analyses were all performed with RAxML v.7.2.634 while BI analyses were conducted with MrBayes v.3.2.235. Partitions and optimal substitution models were determined in PartitionFinder v.1.1.136 using the Bayesian Information Criterion (BIC) metric for model selection37. The best-fitting substitution models for the 1126 bp COI datasets were GTR + G and HKY + G  for ML and BI, respectively. For the 531 bp COI datasets best-fitting substitution models were GTR + G and HKY + I  for ML and BI, respectively. The bindin gene was partitioned by 1st and 2nd exons, the intron, and codon positions. The best fitting model for the bindin dataset was GTR + G for the ML analyses and HKY + G for the BI analyses. The best-fitting substitution models for the 446 bp CR dataset were GTR + G and K80 + I, for the ML and BI analyses respectfully. For the concatenated analysis, the best-fitting model for the ML analysis was GTR + G . For the BI analysis, best models were HKY + G for CR, COI and the bindin intron, and HKY for the two bindin exons.Raw sequences were edited and aligned manually using BioEdit v.7.1.3th tree. Convergence was assessed according to the average standard deviation of split frequencies <0.01. The runs were also visually checked by plotting generations vs. likelihood scores in Tracer v.1.638 to assess whether the two runs had converged and when the stationary phase was reached. In a conservative approach, the first 25% of trees were discarded as burn-in and a 50% majority rule consensus tree was calculated from the remaining 75,000 trees.RAxML tree reconstructions were carried out using 100 random starting trees. Branch support was computed based on 1000 bootstrap replications. Analyses in MrBayes were calculated for each of the datasets applying the respective model parameters. The analyses were run for 10 million generations each , sampling every 10039) genetic distances within and between species/clades were calculated based on the bindin dataset using MEGA v.6.0.640.Uncorrected mean p-distances and Kimura two-parameter  were noted for each specimen. The skeletal components  of selected specimens were assessed following removal of the soft tissue. This was done using enzymatic digestion of the soft tissue with Enzyrim  following the manufacturers protocol. Measurements of both the corona (height and diameter) and peristome were performed using digital Vernier calipers to the nearest 0.1 mm. Pedicellariae and elements of the lantern were observed and measured under a Scanning Electron Microscope (SEM)  at the Central Research Laboratories of the Natural History Museum in Vienna, Austria. Pedicellariae and lantern elements were coated with gold prior to SEM examinations and visualized using the secondary electron images, while tubefeet spicules were not sputter-coated and examined in low vacuum mode employing back scatter electron images.To account for potential ontogenetic variation, Kermadec ZooBank LSID: urn:lsid:zoobank.org:act:19A5CD2E-6D57-4F03-BDF7-AA1A3E5D4B80 .Auckland War Memorial Museum, Auckland, New Zealand (AIM MA) specimen AIM MA73563 (isolate nr. Ker907), a cleaned corona, spines and lantern, collected by J. David Aguirre and Libby Liggins on 31/10/2015 at a depth of 0 to 10 m. A tissue sample (tubefeet) has been deposited together with the holotype.Two cleaned coronas  NHMW-Geo 2017/0016/0001 [formerly MA 121530.10]; isolate Ker903 and Ker910 respectively) and five intact, formalin-fixed specimens preserved in ethanol  116558 ; Naturhistorisches Museum Wien, 3. Zoologische Abteilung, Sammlung Evertebrata Varia, Vienna, Austria (NHMW-EV) NHMW-EV 20452 and 20453 ); collection details as for holotype.Tripneustes were abundant in this habitat of rock spurs that had small areas of horizontal rock, to near vertical rock walls (0–10 m) leading to a base of sand (starting at 11 m and gently sloping deeper).West of Meyer Islands (S 29°14′39.06″ W177°52′46.56″), near Raoul Island, Kermadec Islands, New Zealand. Three intact, formalin-fixed specimens preserved in ethanol  and tissue (gonad) samples  from various sites in the Kermadec Islands; for details on localities and voucher specimens see Supplementary Table Named after the type region, the Kermadec Islands, a subtropical island arc in the South Pacific Ocean, ca. 800–1,000 km NE of New Zealand’s North Island.Tripneustes are referred to as “Lamington urchin” based on the superficial similarity with coconut-covered chocolate cakes, termed “Lamington cake”.Locally species of Tripneustes with ambulacral primary tubercles occurring on every fourth plate ambitally; flattened test ); large peristomal opening (ca. 25%TD) without sunken margin; presence of one to two plates for every four ambulacral plates occluded from perradial suture ; primary series of interambulacral tubercles continuous from peristome to apex; tubercles large; secondary interambulacral tuberculation reduced above ambitus.A species of Corona large, 94.4 mm in horizontal diameter; circular in outline. In profile corona low, bun-shaped with 46.9 mm test height; ambitus low, at about one third of test height Fig. .Apical disc hemicyclic, with oculars I and V insert. All genital plates, as well as the insert oculars bear secondary tubercles along the periproctal margin, forming a distinct circle around the periproct. Remaining plate surface covered with scattered miliary spines and numerous granules (pedicellariae attachment sites). Gential pores circular and located in outer half of the plates, about their own diameter from the margin. Madreporic pores minute, forming a well-defined, slightly raised patch.Periproct large , circular; densely covered with polygonal scales; anus almost central.25). Plate occlusion and enlargement of plates in between leads to rather irregular zigzagging of the perradial suture , with comparatively narrow attachment areas, whereas oral ones have distinctly wider attachment areas and approach type P3.Ambulacra trigeminate, compounded in echinoid style; pores forming three distinct vertical series, the innermost of which are almost straight, while the other two are distinctly wiggling. Primary tubercles occurring on every other plate close to the peristome, every third plate in the outer half of the oral surface, and on every fourth plate ambitally Figs  and 4C. Interambulacra fully tuberculate on oral side with up to five subequal tubercles per plate in the subambital region. At the ambitus, the size of the enlarged secondary tubercles diminishes rapidly, with almost none occurring beyond six plates above the ambitus. The primary tubercle columns, in contrast, persist as large, prominent tubercles with areoles taking up most of the plate height. Primary and secondary tubercles with well-developed areoles; secondary tuberculation very dense, especially so in the ambulacra.Peristome large , circular, with prominent buccal notches (without distinct tag) that extend to the third interambulacral tubercle; peristomal margin barely sunken Fig. . Buccal Lantern of typical camarodont type with large foramen magnum and fused epiphyses; rotulae rectangular with poorly differentiated triangular part adaxially; compasses showing short bifurcations distally, which are comparatively flattened and wide Fig. . AuricleT. ventricosus.Primary spines up to 15.5 mm in length; almost straight sided in proximal half and slightly tapering to a blunt point distally; bases very high  and slightly constricted below milled ring; in cross-section spines consist of dozens of wedges forming concentric circles. Microsculpture of wedges in primary and secondary spines saw-tooth-like, minute Fig. , very siTridentate pedicellariae apparently absent; globiferous pedicellariae Fig.  abundantSpicules in tubefeet mainly of large, C-shaped type; dumb-bell shaped spicules present, but deeply embedded in skin and usually not apparent in BSE-SEM images.Aboral coloration of naked corona light pink to light violet; darker along perradial and interradial sutures; outer margin of apical disc traced by a distinct reddish-pink line; oral side less colored, white along the perradial and interradial sutures, as well as the pore zones, only the area adjacent to the interambulacral primary tubercles more intensely colored.Living animals black with dense cover of white spines Fig. ; spine pCorona large, reaching up to 130 mm in horizontal diameter, with test height ranging from 46.7 to 61.2%TD . Apical disc usually hemicyclic, with oculars I and V insert. In the largest specimen (TD = 127.7 mm), however, oculars II and IV are also insert. In the smallest specimen studied Fig. .In the smallest specimen examined all plates reach the perradial suture, in moderate (>80 mm TD) to large sized specimens, in contrast, numerous ambulacral plates are occluded from the perradial suture, typically those located immediately below and/or above the plate bearing the ambulacral primary tubercles, forming a “pseudo-polyporous” arrangement.Peristome Fig.  large, rCompasses of lantern show short bifurcations distally at all examined growth stages, with incision of distal end only increasing slightly during growth Fig. .Primary spines of up to 9 mm length in the smallest and up to 16 mm length in the largest specimen examined. Color is usually white, but sometimes shows a pinkish tinge.Spicule shape is slightly variable both within an individual and between individuals. At the base of the tubefeet mainly medium-sized, C-shaped spicules occur, typically with blunt or swollen ends Fig. . Their sTripneustes depressus from the Eastern Pacific differs from T. kermadecensis n. sp. by its more prominent interambulacral tuberculation with multiple enlarged secondary tubercles present even a few plates below the apex and presence of ambulacral primary tubercles on every second  to third  plate; only rarely are there three plates in between adjacent primary tubercles ambitally. Occluded plates are less common than in T. kermadecensis n. sp. Detailed morphological descriptions for T. depressus, however, have not been published so far and further material needs to be studied to gain a good understanding about its variability.Tripneustes gratilla gratilla differs from T. kermadecensis n. sp. by its higher test ; ambulacral primary tubercles on every second  to third plate ; regular zigzagging at perradial suture and absence of occluded plates . It differs from the latter by its ambulacral primary tubercles on every second  to fifth plate ; regular zigzagging at perradial suture and absence of occluded plates; much sparser interambulacral tuberculation, with primary interambulacral tubercles typically only occurring on every second plate aborally; smaller areoles of primary tubercles, typically taking up only half of plate height; larger peristome , with deeper buccal notches, and narrower auricles.Tripneustes ventricosus from the Caribbean and Atlantic Ocean differs from T. kermadecensis n. sp. by its higher test ; ambulacral primary tubercles on every second  to third plate ; regular zigzagging at perradial suture, except at ambitus, where every third plate is strongly widened perradially to accommodate a second column of enlarged tubercles .les Fig. ; plates les Fig. ; interam43 defines species based on their ability to interbreed, while the phylogenetic species concept attempts to delimit species boundaries based on phylogenetic reconstructions, mostly using mitochondrial DNA haplotypes  indicating that Kermadec Tripneustes represents a novel species.As species of COI and CR) as well as the nuclear marker (bindin) unambiguously resolve T. kermadecensis n. sp. as a strongly supported monophyletic clade  reflects the strong divergence of T. kermadecensis n. sp. from its IWP congeners , is a common mode of speciation with a distinct geographic signature53. Peripheral locations and particularly islands, such as the Hawaiian Archipelago, Lord Howe Island, and now the Kermadec Islands, should be expected to harbor endemic species whose sister taxa are widespread in the Indo-Pacific. The marine biodiversity of the Kermadec Islands is still actively being discovered and described . Covering 620,000 km2 the Kermadec Ocean Sanctuary would be one of the world’s largest protected areas harboring over 150 species of fish, 39 species of seabirds, 35 species of whales and dolphins, and numerous other marine species unique to this area. Identifying T. kermadecensis as a new species in this area highlights the gaps in our current knowledge about this remote and unique oceanic region. In an era of global change and increased anthropogenic pressure, protection of pristine ocean environments, such as the Kermadec Islands, is paramount in our attempts to maintain and protect marine biodiversity.Identifying cryptic speciation has far reaching implications on conservation efforts, particularly for highly localized endemic species. Fortunately, the described population of Supplementary PDF File


Because the ability to infer other people’s emotions from their facial expressions is critical for many aspects of social communication, deficits in expression recognition are a plausible candidate marker for ASD. However, previous studies on facial expression recognition produced mixed results, which may be due to differences in the sensitivity of the many tests used and/or the heterogeneity among individuals with ASD. To ascertain whether expression recognition may serve as a r = .74).This study tested 46 individuals with ASD and 52 age- and IQ-matched typically developing (TD) participants on the Films Expression Task, which combines three key features of real-life expression recognition: naturalistic facial expressions, a broad range of simple and complex emotions, and short presentation time. Test-retest reliability was assessed in 28 individuals who did not participate in the main study and revealed acceptable reliability (ICC p = 1.1 × 10− 10), with an effect size (Cohen’s d = 1.6), more than twice as large as the mean effect size reported in a previous meta-analysis . The ASD group also had significantly increased mean reaction times overall  and on correct trials . However, whereas 63% of people with ASD showed severe deficits  performed normally (within one standard deviation of the mean).Case-control comparisons showed highly significant mean group differences for accuracy  contains supplementary material, which is available to authorized users. These findings remain unchanged when adolescents below the age of 18 years were excluded  = 7.9, p = 1.4 × 10−10)  = 65.0, p = 2.3 × 10−12) and RTs  but there were no significant effects of sex ) or group by sex interactions . This finding indicates that the overall case-control difference cannot be attributed to differences in the slightly higher male:female ratio in the ASD than those in the control group.Given previous reports of sex differences on several social-cognitive tasks in the typical population and in ASD , we perfNext, we explored whether in the ASD group, the expression recognition impairments reported above were driven by specific problems in identifying complex emotional expressions. To do so, we separately analysed trials with simple vs. complex target emotions  = 70.7, p = 3.8 × 10−13) and a significant effect of emotion category  = 5.9, p = .016) on response accuracy, such that on average, both groups were better at recognizing simple than complex emotions. The group x emotion category interaction was not significant F = .14, p = .7)  × 2 (emotion category) repeated-measures ANOVA revealed the above reported significant main effect of group (p > .09). In the TD group, there was a marginally significant relationship between accuracy scores and FIQ (r(52) = .26, p = .057), such that participants with higher intelligence also had higher expression recognition scores.Next, we tested whether age, verbal, performance, or full-scale IQ were related to accuracy or reaction time  on the Films Expressions Test. In the ASD group, we found no significant relationships between expression recognition variables and age or IQ  for whom AQ data was available  = − .48, p = .0002. However, when the ASD and TD groups were considered separately, there were no significant relationships in either group (ASD r(25) = .09, p = .66, TD r(28) = .03, p = .88). This suggests that the negative correlation for the overall sample could be explained by the performance differences between the two groups.Finally, we investigated correlations with ASD symptoms in a subset of correct trials: overall r(53) = .53, p = .00003) and in the TD group (TD r(28) = .48, p = .012). Although a similar pattern was found in the ASD group, it did not reach statistical significance (ASD r(25) = .29, p = .16).A somewhat different picture emerged for RTs. We found that longer reaction times on correct trials were associated with more ASD symptoms. This was significant overall, i.e., when both groups were collapsed  = .54, p = .000026). When the TD and ASD groups were analysed separately, similar but non-significant patterns were found (TD: r(28) = .31, p = .10, ASD: (r(25) = .29, p = .16).Likewise, for overall RTs, we found a significant relationship with ASD symptoms when both groups were collapsed  in terms of symptom severity. To begin to address this, we conducted correlation analyses between severity of expression recognition deficits and ASD symptom severity, as measured by the AQ. When the ASD and TD groups were combined, we found both lower accuracy in expression recognition and higher RTs to be moderately related to greater severity of autism traits. However, whereas accuracy scores were not sensitive to the severity of ASD symptoms within the ASD and TD groups, respectively, longer reaction times on correct trials were significantly related to greater ASD symptoms in the TD group. A similar relationship was found in the ASD group with moderate correlation coefficients which, however, did not reach statistical significance. These correlation analyses should be viewed as preliminary, because AQ scores were only available in about half of the participants. As a consequence, the sample sizes were small when split by group, which reduced our power to detect a significant effect. In addition, the AQ is a composite measure of a range of autism-related traits, including features such as intolerance of changes that may not be expected to relate to expression recognition. This may have diluted potentially higher associations with more specific social communicative impairments.In sum, our findings revealed that the majority of individuals with ASD had severe deficits in expression recognition deficits but also that a sub-sample of individuals with ASD had no behavioural impairments on this task. This indicates that expression recognition may more likely serve as a stratification as opposed to diagnostic marker, subject to further substantiation that subgroups with/without expression recognition deficits also differ in symptom severity or adaptive behavior. One implication of the current findings is that intervention programmes that specifically target expression recognition may be valuable for a substantial proportion of people with this disorder.Several potential limitations of the current study as well as implications for future research should be considered. First, we deliberately chose a task that incorporated several factors crucial for expression recognition in real-life situations, including a wide range of simple and complex target emotions, and relatively short presentation times. As a consequence, could factors unrelated to expression recognition per se have contributed to the deficits observed in some of the ASD participants, for example, comprehension of the target words or other cognitive impairments/ anomalies?If difficulties in comprehending some of the emotion words accounted for performance impairments, one would expect larger group differences in trials with complex target emotions than basic emotions. However, separate analyses, split by emotion target (simple vs complex), showed significant impairments in the ASD group on trials with basic as well as complex emotions. Basic emotion words are typically understood by the age of 6 years or earlier. Also most of the complex emotions used here are typically understood by age 8  facial expressions of emotions? This alternative explanation seems unlikely for two reasons. Several previous studies reported no deficits in rapid visual processing in ASD , 36. ForThe present finding raises the question of what may account for the differences between people with ASD with/without expression recognition deficits. This question cannot be settled here but we discuss potential factors and outline some avenues for future research. A likely possibility is that (some) people with ASD have problems in the neurocognitive mechanisms that represent facial expression information and/or multi-modal expression information. One hypothesis that recently gained prominence is the notion that expression recognition deficits in ASD may be linked to the high frequency of comorbid alexithymia , 42, estTaken together, our findings show highly significant mean case-control differences in facial expression recognition, with one of the largest effect sizes ever reported in the expression recognition literature of ASD. This suggests that problems with expression recognition are more widespread than currently thought, likely owing to the more naturalistic character of the tasks used here. Nevertheless, we also highlight important variability in expression recognition skills among individuals with this condition and showed that a minority of people with ASD had no behavioural impairments. This finding indicates that the Films Expression Test may serve as a valuable tool to study expression recognition as a candidate stratification marker for ASD.Additional file 1:Target emotion words, split by age of acquisition (AoA) norms. (DOCX 15 kb)Additional file 2:Descriptive statistics and contrasts for main variables of interest. (DOCX 19 kb)


ADP‐induced aggregometry in whole blood classified 74%  of critically ill patients as poor responders (n = 43), and 65% (49–79%) responded poorly according to the vasodilator‐stimulated phosphoprotein phosphorylation (VASP‐P) assay. Although the plasma levels of clopidogrel active metabolite normally exceed the inactive prodrug ∼30‐fold, the parent drug levels even exceeded those of the metabolite 2‐fold in critically ill patients. The half‐life of pantoprazole was several‐fold longer in these patients compared with reference populations. The inverse ratio of prodrug/active metabolite indicates insufficient metabolization of clopidogrel, which is independently confirmed by the ∼5‐fold increase in half‐life of pantoprazole. Thus, high‐risk patients may benefit from treatment with alternative platelet inhibitors.Only limited data are available regarding the treatment of critically ill patients with clopidogrel. This trial investigated the effects and the drug concentrations of the cytochrome P450 (CYP450) activated prodrug clopidogrel ( WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?☑ Only limited data are available on the effects and on drug concentrations of clopidogrel in critically ill patients. High on treatment platelet reactivity (HTPR) occurs in –30–40% of stable patients.WHAT QUESTION DID THIS STUDY ADDRESS?☑ We hypothesized that HTPR is frequent in critically ill patients and measured drug concentrations of clopidogrel, its active metabolite, and the similarly metabolized pantoprazole to identify potential mechanisms.WHAT THIS STUDY ADDS TO OUR KNOWLEDGE☑ Approximately 70% of critically ill patients were insufficiently treated with clopidogrel. The cytochrome‐dependent activation of clopidogrel to its active metabolite is substantially reduced during critical illness. Likewise, the half‐life of pantoprazole, which is also metabolized via cytochrome enzymes, is increased approximately 5‐fold compared with healthy volunteers, indicating a substantially reduced activity of cytochrome enzymes.HOW THIS MIGHT CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE☑ The results of our trial indicate substantially altered PKs of cytochrome‐dependent drugs in critically ill patients. To optimize treatment, trials in critically ill patients should be included in the drug approval process.Clopidogrel is a cytochrome (CYP)2C19‐dependent prodrug, which after activation irreversibly inhibits the P2Y12 ADP receptor on platelets.The nonresponsiveness to pharmacological platelet inhibition is termed “high on treatment platelet reactivity” (HTPR),Current guidelines recommend the use of acid‐suppressive drugs in ventilated patients to prevent stress ulcers and gastric bleeding.In a small study in patients undergoing successful cardiopulmonary resuscitation who received a loading dose of 600 mg clopidogrel because of a percutaneous coronary intervention, bioavailability of clopidogrel and consequently platelet inhibition were reduced compared with stable patients.Supplementary Figure S1). Five patients participated in both groups. TableForty‐three clopidogrel‐treated patients and 16 pantoprazole‐treated patients admitted to three medical ICUs participated in this study between November 15, 2012, and September 29, 2016 (P < 0.001).P = 0.001). Four patients had platelet counts <80*109/L and/or required therapy with extracorporeal membrane oxygenation and, after exclusion of these, the HTPR rate increased to 82% .Whole blood aggregometry classified 32 of 43 clopidogrel‐treated patients  59–87%) as having HTPR according to the predefined cutoff of 46 U. ADP‐induced aggregation was significantly higher compared with patients with stable coronary artery disease  assay showed that 65% (95% CI = 49–79%) of patients had HTPR with a Platelet Reactivity Index (PRI) > 42% Figure.18 Compan = 1) and all intermediate (n = 5) metabolizers were found to have HTPR. However, also extensive, rapid, and ultrarapid metabolizers were regularly tested with HTPR (Supplementary Table S1). The PRI correlated well with serum cholinesterase levels .According to VASP‐P and aggregometry, all poor .The ratio of clopidogrel active metabolite to clopidogrel concentrations was median 0.6 (quartiles: 0.3–2.0) 2 h after intake of clopidogrel . Similarly, patients with measurable pantoprazole trough concentrations had significantly higher miRNA‐130b concentrations compared with patients in whom pantoprazole was not detectable 24 h after the last dose (8.2 (7.9–9.5) vs. 18.8 (10.4–33.1) copies/µL; P = 0.01; Supplementary Figure S3). The miRNA concentrations did not correlate with platelet function assays or markers of inflammation. Levels of miRNA‐130b and miRNA‐223 correlated well with each other .Plasma concentrations of miRNA‐130b or miRNA‐223 did not correlate with plasma concentrations of clopidogrel, its active metabolite, or PK parameters of pantoprazole. Given the sparse blood sampling and the high variability of PKs in critically ill patients, we also dichotomized patients by measurable trough level concentrations of clopidogrel active metabolite. Intriguingly, miRNA‐130b concentrations were significantly lower in patients with no measureable trough levels when compared with patients with quantifiable plasma concentrations 24 h after the last clopidogrel intake (median = 5.0 (quartiles = 2.3–7.9) vs. 8.9 (4.0–17.6) copies/µL; This trial in critically ill patients provides several clinically important findings. The majority of patients in the ICU who had previously been receiving a regular 75 mg dose of clopidogrel had high “on treatment” platelet reactivity. To clarify the mechanism, we investigated drug concentrations of clopidogrel. Generation of clopidogrel active metabolite was markedly reduced when compared with data from healthy volunteers or other patient groups.Although rates of HTPR vary between studies and patient populations, overall, ∼30–40% of patients respond poorly to clopidogrel treatment.Noteworthy, ADP‐induced whole blood aggregometry and VASP‐P assay supported the diagnosis of HTPR in 70% of patients. In critically ill patients treated with prasugrel, an even greater discrepancy between the two testing systems was reported (65% in ADP‐induced whole blood aggregometry vs. 26% in VASP‐P assay).The PKs of clopidogrel are well characterized in healthy individuals and in less sick patients. After being absorbed, clopidogrel is activated by CYP enzymes and the levels of clopidogrel active metabolite normally exceed that of clopidogrel up to 50‐fold, depending on the genetically determined metabolizer status.To disentangle overlapping poor absorption and metabolism, we determined the PKs of intravenous pantoprazole, which is also metabolized by CYP2C19. We hypothesized that a reduced activity of CYP enzymes should result in an increased half‐life. The half‐life for extensive metabolizers was ∼7 h and for rapid metabolizers ∼4 hours, which is 5‐fold and 4‐fold longer when compared with healthy volunteers with the same genotype.It was demonstrated previously that miRNA‐130b may be a negative regulator of CYP enzymes.In contrast with stable patients with acute coronary syndromes,The population included in this trial were indeed critically ill, as reflected by the high mortality of ≥40%. Although the concept of inflammation‐induced “phenoconversion” of CYP enzymes with a consecutively reduced activity has been examined in other patient populations,There are a number of limitations that need to be considered. First of all, to minimize blood loss in anemic critically ill patients, we only performed sparse blood sampling in 37 of 43 clopidogrel‐treated patients. Due to the noninterventional character of the trial, only reference to external control groups simultaneously treated at our center can be made, which, however, is deemed adequate for PK comparisons and because of the large body of published data on HTPR rates with the applied assay systems in different cardiovascular patients, including our previous trials. The trial included a single poor metabolizer and only five intermediate and four ultrarapid metabolizers. However, this was due to chance as we did not power the study to include a certain number of patients with each genotype. The overall sample size was small. However, the observed differences are large and the 95% CIs for HTPR are narrow. Therefore, we deem the sample size to be sufficient. The miRNA‐223 levels were measured in serum samples, which may have limited its discriminative power. Direct comparisons of miRNA‐223 levels to other trials should not be performed.In conclusion, the number of patients responding poorly to clopidogrel treatment is extraordinarily high in critically ill patients. The PKs of clopidogrel and pantoprazole demonstrate that a reduced activity of CYP2C19 enzyme is at least in part responsible, and whose activity may be downregulated by miRNAs. The critically ill may benefit from treatment with alternative P2Y12 inhibitors.www.clinicaltrials.gov NCT02285751).The Independent Ethics Committee of the Medical University of Vienna and the competent authorities approved the study, which complied with the principles set forth in the Good Clinical Practice guideline and the Declaration of Helsinki. The trial was registered in publicly available databases  or pantoprazole  treatment, were included in the trial. Exclusion criteria included allergies or hypersensitivities to the trial drugs, active bleeding, known coagulation disorders, or intake of other antiplatelet drugs except for acetylsalicylic acid. Included patients were on long‐term clopidogrel treatment for coronary artery disease or received daily doses of 40 mg intravenous pantoprazole as a prophylaxis for stress ulcers.Blood samples were drawn before patients received the daily dose of 75 mg clopidogrel . Samples were then drawn again 2 and 24 h postdrug administration. A more detailed PK analysis was obtained from 6 subjects taking 75 mg clopidogrel and in 16 subjects receiving 40 mg pantoprazole intravenously. In those patients, blood samples were obtained at the following time points: 0, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, and 24 h. Clopidogrel tablets had to be crushed and/or dissolved in 0.9% sodium chloride solution, if they were administered via a nasogastric tube. Pantoprazole was infused as an intravenous bolus. Blood samples were drawn using existing central venous or arterial lines. Plasma samples were harvested after centrifugation at 2000 g for 10 min at 4 °C, and aliquots (500 µL) stored at ‐80 °C.Supplementary Material).The primary end point of the trial was ADP‐induced whole blood aggregometry. Whole blood aggregation was determined using the Multiple Electrode Aggregometry on the Multiplate Analyzer . ADP‐induced platelet aggregation was performed, as explained previously .The VASP‐P assay was measured by an enzyme linked immune assay, as described previously . These miRNAs were chosen based on their association with cytochrome enzymes and platelet aggregation.The concentrations of miRNA‐130b and miRNA‐223 were quantified in serum by quantitative polymerase chain reaction using components of the ThrombomiR kit  that is based on a previously published methodwww.saps3.org on trial day 1. Clinical data was obtained from chart review.The Sequential Organ Failure Assessment (SOFA) score and the Simplified Acute Physiology Score (SAPS) III were calculated using the official SAPS 3 Score Calculation Sheets from Initially, we planned to screen up to 100 patients to identify 36 patients with HTPR for inclusion in a subsequent randomized trial. However, we terminated recruitment early on in that trial because the prevalence of HTPR was very high and physicians were reluctant to randomize patients.The sample size calculation for pantoprazole was based on previous publications demonstrating a half‐life of 1.27 ± 0.41 h in extensive metabolizers. A sample size of six per subgroup (either extensive or rapid metabolizers) should suffice to show a twofold prolongation in half‐life .Baseline characteristics, demographics, results of platelet function tests, and PKs are presented by descriptive statistics. Nonparametric Spearman correlations were calculated. Commercially available statistical software was used . The influence of genotypes on PKs was compared using nonparametric Spearman correlation.M.H. is employed at TAmiRNA. J.S.M. received lecture fees from Astra Zeneca, Daiichi Sankyo, Bayer, Eli Lilly, and Roche.This work was supported by the Austrian Science Funds (grant number SFB54P04).C.S. wrote the manuscript. B.J. and C.S. designed the research. B.J., C.S., E.H., P.S., G.H., W.S., J.S.M., M.S., R.S.‐P., T.S., and M.H. performed the research. B.J. and C.S. analyzed the data.Supporting InformationClick here for additional data file.


Increasing attention has focused on the emotional dysregulation that can result from adverse childhood experiences among those who commit sexually violent crimes. While studies confirm a relationship between child maltreatment and anger the research is limited and it is unclear how anger and child maltreatment effect the use of violence during the commission of the sex crime.This study examined the relationship between childhood maltreatment, anger and violent behavior by reviewing the records of 571 adult male offenders convicted of sexual assault or child molestation. The aims of the present study were to 1) examine differences in anger levels between those offenders who engaged in verbal or physical violence or used a weapon during the commission of their crime; 2) explore differences in anger levels for those sex offenders who experienced childhood abuse  and those who were not; 3) examine whether there were differences in anger between rapists and child molesters and 4) assess whether anger either mediated or moderated the relationship between childhood abuse and the use of violence in the commission of the crime.Overall we found that sex offenders who were rated as being angrier were more likely to have used violence in the commission of their crime and were more likely to be abused as children. Further, while these relationships held for both rapists and child molesters independently, rapists were found to be angrier than child molesters. Finally, anger neither mediated nor moderated the relationship between an offender’s adverse childhood and committing a violent sex crime.These results suggest that anger should be target in intervention and prevention programs with violent sex offenders. The United States has one of the highest rates of reported sexual violence of any industrialized country Stewart, . An averCriminal sexual behaviors by definition contain an element of aggression and coercion  who were housed in either a prison-based sex offender treatment facility or the general population of a state prison  and excellent level of agreement between researchers on pervasive anger scores (ICC = .924). All procedures were approved by the university and department of corrections institutional review boards.The data for this study was gathered from a larger study of offenders convicted of sex crimes  on a pervasive anger measure scored by trained clinicians  at the time they entered prison. Offenders came from diverse racial and ethnic backgrounds including 234 Whites (41 %), 240 African Americans (42 %), 88 Latinos (15.4 %), 3 Asian Pacific Islanders (.5 %), and 4 were of Other (.7 %) ethnicity. The ethnicities of two offenders were unknown (.4 %). The sample consisted of individuals with an index offense conviction of rape (n =124), child molestation (n = 438), or both rape and child molestation (n = 9). Of this sample, 463 offenders victimized females, 86 offenders victimized males, and 18 offenders victimized both males and females. There were eight files missing victim gender. When looking at offender’s prior histories and convictions, 284 offenders had a history of prior charges and/or convictions, while 278 offenders did not have a prior history, while nine offenders’ prior histories were unknown.Offenders ranged in age from 18 to 74  Angry person who easily loses temper or anger directed at multiple targets in multiple situations; (2) Consistent pattern of verbal aggression against both males & females; (3) Assaults against males and frequent (2 or more occasions) physical fights with males; (4) Offender reports preoccupation with aggressive fantasies that include thoughts of beating, killing, etc.; and (5) Offender has been cruel to animals, which includes having beaten, tortured or killed them. This measure was designed at the treatment center and was scored by the treating clinician. Each of the aforementioned items were coded dichotomously (yes/no) to represent the presence or absence of this trait based upon the treating clinician’s judgment. An offender’s total pervasive anger score represented the number of anger items endorsed by the clinician.Three different types of violence curing the commission of the crime were assessed: verbal aggression, physical aggression and weapon usage. All information was retrieved and coded from the offenders’ prison records. Verbal abuse was dichotomously (yes/no) coded if the offender threatened the victim during the commission of the offense. Physical violence was also dichotomously (yes/no) coded if the index crime involved slapping, punching, or hitting, while weapon usage was dichotomously (yes/no) coded if the offender utilized a weapon during the commission of a crime.Childhood abuse was categorized into four types consisting of physical abuse, sexual abuse, psychological abuse  and neglect. Information was retrieved from the offender’s prison files.SD = 1.30 (range 0–5). Further, 39.1 % (n = 223) of the sample reported a history of childhood abuse. Within the sample, 30 % (n = 171) offenders used verbal violence to threaten their victim(s), 21.4 % (n = 122) offenders used physical violence, 15.3 % (n = 87) offenders used a weapon, and 380 offenders did not use any act of violence to threaten or harm their victim(s).Descriptive statistics for the dependent measures are presented in Tables t (262.36) = −7.02, p < .01, two tailed, d = .67; physical aggression t (158.80) = −7.56, p < .01, two tailed, d = .83; and weapon usage t (109.24) = −6.05, p < .01, two tailed, d = .75 such that those who used verbal aggression, physical aggression or who used weapons were rated higher on perceived anger.To test whether there were group differences in perceived anger scores between those who used verbal aggression, physical force or who used a weapon during the commission of the crime a series of t-tests were conducted. Significant differences in anger were found between those who used verbal aggression t (166.86) = 5.95, p < .01, two tailed, d = .65; such that MSO-R were rated significantly higher on pervasive anger than MSO-CM. Further, we found that MSO-R were more likely to engage in verbal aggression, χ2 =  = 86.86 p = .00, phi = −.40; physical aggression, χ2 =  = 140.15 p = .00, phi = −.50; and weapon usage, χ2 =  = 164.18 p = .00, phi = −.55 than MSO-CM.To test whether there were group differences in perceived anger scores between MSO-R and MSO-CM a separate t-test was conducted. We found significant differences in pervasive anger scores between offenders t (92.08) = −2.00, p = .05, two tailed, d = .38; and physical aggression t (114.82) = −3.55, p < .01, two tailed, d = .66; such that those who used verbal aggression and physical aggression during the commission were rated significantly higher on pervasive anger. There was no significant difference in pervasive anger scores for MSO-R who used a weapon t (122) = −1.30, p = .198, two tailed, d = .23.Next we tested whether offender type (MSO-R and MSO-CM) impacted differences in pervasive anger scores by type of violence used during the commission of the crime. To do this we conducted a separate series of t-tests for MSO-R and MSO-CM. For the MSO-R group we found significant differences in pervasive anger scores between those who used verbal aggression t (112.40) = −4.68, p < .01, two tailed, d = .60; physical aggression; t (48.96) = −3.66, p < .01, two tailed, d = .64 and weapon use t (19.84) = −3.46, p < .01, two tailed, d = .91; where those who used verbal and physical abuse and used weapons were rated higher on the pervasive anger measure.Among the MSO-CM group, significant differences were found in pervasive scores between those who used verbal aggression M = 1.11, SD = 1.42) and without a history of abuse  = −2.70, p = .01, two tailed, d = .25).In order to explore the relationship between childhood abuse and anger an independent sample t-test was conducted to compare the Pervasive Anger scores for the population of MSO who have a history of childhood abuse and those who do not (yes/no). There was a significant difference in scores for offenders with a history of abuse  for the population of offenders convicted of sex crimes as a whole was further investigated using a Pearson product–moment correlation coefficient where childhood abuse was measured as a continuous variable, with those experiencing multiple types receiving higher scores. There was a small, positive correlation between the two variables, M = 2.02, SD = 1.56) and offenders convicted of rape without a history of abuse  = −2.72, p = .01, two tailed, d = .50). Further, using abuse as a continuous variable there was a small, positive correlation between the two variables, r = .22, n =120, p < .05, with high levels of pervasive anger associated with childhood abuse for offenders convicted of rape. Among MSO-CM there was no significant difference in anger scores for offenders convicted of child molestation with a history of abuse  and without a history of abuse  = −1.88, p = .06, two tailed, d = .19) and further there was no significant correlation between pervasive anger and childhood abuse assessed continuously, r = .09, n = 410, p > .05.The relationship between childhood abuse and anger for MSO-R and MSO-CM independently was further investigated. There was a significant difference in anger scores for MSO-R with a history of abuse  = 2.78, p = .006; as with the mediator variable of anger, and the outcome variable of violence . Therefore pervasive anger does not act as a mediator between a sex offender’s history of abuse and committing a violent sex crime, but maltreatment produces anger and anger produces violence in sex offenders.To test the hypothesis that pervasive anger mediates the relationship between an individual’s childhood history of abuse for offenders convicted of rape and/or child molestation and committing a violent sex crime, a mediation model was tested. In Step 1 of the mediation model Sexual Violence was regressed on childhood abuse and was not found to be significant R2 = .00, F  = .01, p = .91. In step two, when pervasive anger was added into the equation, both anger and childhood abuse accounted for a significant amount of variance in committing a violent sex crime, R2 = .11, F  = 33.01, p < .01. In step three, the interaction term between childhood abuse and pervasive anger was added to the regression model and did not account for a significant proportion of the variance in committing a violent sex crime, ΔR2 = .113, F  = 22.619, p = .207. Therefore, pervasive anger was not found to moderate the relationship between a sex offender’s history of abuse and committing a violent sex crime, but an offender’s total pervasive anger alone does predict violence.Utilizing Baron and Kenny’s  method aAn emerging issue in the field of sex offender theory and treatment is whether emotional and other affective states function as antecedents for offenses  a total of 571 offenders convicted of sexual assault, child molestation, or both received a score from the treating clinician on the pervasive anger measure limiting the results to a much smaller sample of MSO. Offenders were not matched based on any criteria nor was a control sample used. Because offenders were not matched based on any criteria there is an increased chance that factors other than childhood abuse  influenced the offender’s anger score. In addition, the measure of pervasive anger was not validated. This was a measure comprised of 5 clinician rated items pertaining to the general construct of anger. This has both advantages and drawbacks. Specific concerns regarding self-report measures are their vulnerability to lying, manipulation, and self-presentation biases (Kroner et al. There are several limitations to the current study. Since these analyses were based upon archival review, the data available in the files were often not complete. As with any archival study of this nature, the data was not collected for research purposes, but rather was data maintained in prison files for sentencing, treatment, and management purposes. Thus the validity and accuracy of the results are dependent upon what information was entered and coded from the offenders file. Further in many instances the history of childhood abuse was not officially documented but rather reported by the offender. While there is a concern for potential fabrications, research suggests that the overall bias is in the opposite direction. Individuals are more likely to minimize or deny their adverse childhood experiences (Brewin et al. The assumption that individuals convicted of sex crimes are likely to have problems with the experience and expression of anger is reflected in the widespread use of anger management strategies in sexual offender treatment programs (Marshall and Eccles, Few studies confirm anger as a mediating or moderating factor for violent behavior among offenders convicted of rape and/or child molestation leaving much room for further exploration. Given the current lack of epidemiological, prevention, and intervention research examining expressive forms of aggression in sexual offending (Smallbone and Milne,


P > 0.05). Several other blood metabolites  were also similar between groups. This very low DCAD during the prepartum period may severely compromise animal physiology unnecessarily, with little advantage over normal calcium concentrations at parturition, when compared with a less negative DCAD (−53 mEq/kg DM). Feeding a less negative DCAD ration (−53 mEq/kg DM) did not decrease plasma Ca levels right after parturition compared to a DCAD ration of −143 mEq/kg DM, reinforcing the lack of benefit of a more negative DCAD.During the periparturient period, subclinical hypocalcemia  is a potential problem for the dairy cow; consequently, its prevention is essential for success of fertility and productive performance. Dietary cation–anion difference (DCAD) has been defined as the difference in milliequivalents of cations  and anions  per kilogram of dry matter (DM) and has a direct impact on blood acid–base metabolism. Diets rich in K and Na induce metabolic alkalosis, interfering with tissue sensitivity to parathyroid hormone, and diets rich in Cl and S  cause metabolic acidosis, reducing the risk of hypocalcemia. Consequently, the use of anionic salts has become a popular method to prevent hypocalcemia in dairy cattle. Monitoring diets with anionic salts can be done by measuring urine pH, with optimal values between 6.2 and 6.8 for Holstein cows. The objective of this report is to present a herd case investigation involving a dairy farm feeding a very low DCAD (−143 mEq/kg DM), expecting improved Ca homeostasis. The diet of −143 mEq/kg (urine pH 5.2–5.8) was changed to a diet with −53 mEq/kg DM (urine pH 6.2–6.8). Blood samples were taken at the time of calving for 10 cows that calved before and then for 10 cows that calved after changing the diet. Cows with extremely low DCAD had Ca concentrations of 2.11 ± 0.22 mmol/l and cows with a more moderated DCAD, 2.11 ± 0.16 mmol/l ( On October 1, 2015, the University of Missouri dairy herd initiated a more systematic cow health monitoring program. The dairy consisted of 200 Holstein lactating cows, milked twice a day, and fed a total mixed ration meeting or exceeding the NRC requirements  in regarAfter this urine pH evaluation, the ration showed a theoretical dietary cation–anion difference (DCAD) of −143 mEq/kg DM (Low DCAD diet), which was consistent with the urine pH obtained. In addition, each cow at parturition received a subcutaneous injection of 500 ml of a commercial product based on Ca borogluconate 23% solution . There has not been a case of clinical hypocalcemia in this dairy during the last 11 months. The last clinical case was diagnosed and treated on November 2014. In addition, the incidence of other periparturient disorders was within reported targets  Tables  and 2. CBefore beginning the new dietary approach, urine pH was tested four more times. The results are shown in Table On March 15, 2016, the prepartum diet was changed to the new formulation . From that time until this case report was completed, no cases of clinical hypocalcemia were diagnosed.Between March 23 and April 10, 2016, a blood sample was obtained from 10 cows within 6 h of parturition before receiving the application of subcutaneous Ca to assess the same plasma metabolites as described previously. Results of plasma metabolites are reported in Table ntervals  between ntervals  was 2/10Calcium is a major mineral that plays a central role in muscle contraction, blood coagulation, enzyme activity, neural excitability, hormone secretion, cell adhesion, and an essential structural component of the skeleton. To maintain a constant concentration of Ca, endocrine control mechanisms have evolved, which primarily consist of the interaction of three major hormones: parathyroid hormone (PTH), calcitonin, and vitamin D , 7.During the periparturient period, hypocalcemia is a potential problem in the dairy cow, as a result of the sudden drain of Ca to colostrum at the onset of lactation. A cow affected with clinical hypocalcemia <1.25 mmol/l) may present with nervousness and staggering, but usually becomes recumbent and is unable to rise. If blood Ca concentration is not restored quickly, death may occur. 10–50% of cows may develop subclinical hypocalcemia (1.25–2.0 mmol/l) up to 10 days postpartum, affecting organs that have smooth muscle function, such as the uterus, rumen, and the abomasum 5 mmol/l . These dEventually, Ca homeostasis is mediated primarily by PTH, which stimulates bone Ca resorption and renal Ca reabsorption. However, high DCAD or diets rich in K and Na, using the equation (Na + K) – (Cl + S), interferes with tissue sensitivity to PTH , 8. As ar2 = 0.95) between urinary pH and net acid excretion by cows fed the diets containing anionic salts makes the urinary pH evaluation a useful tool to assess the degree of metabolic acidosis imposed by dietary anionic salts  the dairy was milking twice a day with intermediate milk production (32 kg/cow/day), consequently, the drainage of Ca through the mammary gland is lower than cows milked three times a day . In the Supporting the data presented in this current case report, a study conducted in Canada  demonstrIn another study, it was also demonstrated that cows fed either a diet with a DCAD of −120 mEq/kg DM or a diet with a DCAD of −98 mEq/kg DM had similar concentrations of plasma Ca at parturition and higher than a control group (DCAD +220 mEq/kg DM). In addition, no clinical cases of hypocalcemia were reported . A prepaBy feeding a less negative DCAD ration (−53 mEq/kg DM) compared to a more negative DCAD ration (−143 mEq/kg DM), the plasma Ca concentrations right after parturition remained the same within reference intervals and were not statistically different before and after changing the prepartum feed management approach. Plasma P, Mg, Na, K, Cl, albumin, globulins, blood urea nitrogen creatinine, and GGT also remained within reference intervals. This report reinforces the lack of benefit of reducing DCAD beyond −100 mEq/kg DM and putting prepartum dairy cows at higher risk of an uncompensated metabolic acidosis.This case report is a description of the normal routine of handling of a commercial dairy herd.PM obtained urine and blood samples, formulated the new diet, conducted the statistical analysis, and wrote the manuscript. SP obtained urine and blood samples and helped in the writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In recent years, elements of the modern environment  have been criticized for having an increasingly sexualized or appearance focus, which has been suggested to be detrimental to girls’ development. The current study examined the impact of an appearance-focused Internet game on young girls’ body image and career cognitions and aspirations. Eighty British girls aged 8–9 years were randomly assigned to play an appearance-focused or a non-appearance focused game for 10 minutes. Girls in the appearance-focused game condition displayed greater body dissatisfaction compared to the control condition. Type of game did not impact girls’ perceived capacity to do various jobs. However, girls who played the appearance-focused game reported a greater preference for feminine careers compared to the control group. This provides preliminary evidence that appearance-focused Internet games may be detrimental to young girls’ body image and aspirations. Internet games should be included in our consideration of influential messages for young girls. Report of the APA Task Force on the Sexualization of Girls , which refers to the process by which girls internalize an observer’s perspective on their bodies and learn to treat themselves as objects to be valued primarily on their appearance. A sizeable body of research has demonstrated that self-objectification is associated with a host of negative consequences in adolescent girls and women, including disordered eating  have been suggested to be increasingly grown up and often have an appearance or sexualized focus. This concern has been reported in the American Psychological Association’s of Girls  as well n Bailey . Both reThe current study begins to address this gap in the literature by exploring the impact of Internet games on young girls’ body image and career aspirations. Despite being a common form of entertainment for young children, Internet games have not received any empirical investigation as to their potential impact. Here we investigate the impact of playing an Internet game that has an “appearance-focus”—the female character changes her outward physical appearance (clothes and hairstyle) in order to be appealing to a male character. The impact of brief exposure to this game on girls’ body dissatisfaction, self-objectification, and career aspirations and preferences is investigated.Children are being exposed to appearance-focused messages and sexualized portrayals at an early stage of their socio-emotional development. This is concerning as even very young children may be influenced by this exposure. From as young as 3 years old, children are able to verbally describe themselves. Over the following years, children begin to compare themselves to others, although this is initially only in relation to one person at a time Smolak . Also frCinderella, The Little Mermaid) suggested that many films contain 10 or more body image-related messages , whereas the control condition were exposed to films clips with no appearance-related messages . Perhaps surprisingly, results indicated that exposure to the appearance-focused clips did not negatively impact on girls’ body dissatisfaction, with the authors suggesting that very young children may adopt the persona of attractive characters with whom they identify rather than comparing themselves directly to the characters as is thought to be the case with slightly older children. Other experimental research has focused on the impact of sexualized toys. Dittmar et al. , that could result in lowered aspirations and expectations. Previous research has largely focused on the impact of consuming traditional media images and engaging in doll play. However, Internet game play is likely to encompass important elements of both of these. Not only do Internet games contain numerous images, they are also likely to encourage some sort of character identification or adoption that may be influential. This medium provides a powerful and fertile environment for empirical research.The current study aimed to examine the influence of an appearance-focused Internet game on the body image and career aspirations of 8–9 year old girls. It was hypothesized that girls who played an appearance-focused game would report increased body dissatisfaction and self-objectification compared to girls who played a control game. In addition, following Sherman and Zurbriggen , it was Participants were 80 girls aged between 8–9 years, with a mean age of 8.43 (SD = .50) and were in Year 4 according to the U.K. National Curriculum. They were recruited from six primary schools in the southwest of England. Most of the schools were larger than the national average in size, had a lower than average number of pupils eligible for free school meals, and had a majority of White British students. The sample were all English speaking and were predominantly White (90%). Of the total participants, 47.5% of the girls were in the appearance focused-exposure condition, and 52.5% were in the non-appearance focused-exposure condition (control condition).Following approval by the Research Ethics Committee at the University of the West of England and from Head teachers of all schools, a letter of introduction outlining the study and accompanying consent form was sent home to parents of all girls in Year 4. Consent forms were returned by 85 parents  and of these, 80 girls were available to partake in the study (five girls absent on the days of the experiment).The experimental sessions were conducted at school during normal class time and took approximately 25 min per child to complete. The study was introduced to the girls as looking at what sorts of games girls like to play and what they would like to be when they grow up. The girls were told they would be asked some questions before and after playing a game on a laptop. Before starting, girls also provided verbal assent to participate. It was emphasized that this was not a test, that there were no right or wrong answers, and the answer formats were explained carefully for each section. Each question was read aloud by the researcher, and responses were entered on a laptop.Friv for ten minutes, after reading the simple online instructions. In the experimental (appearance-focused) condition the girls played a game called Dream Date Dress Up that requires players to memorize an image of a male character’s “dream date”. Girls were then required to change the clothes and hairstyle of a female character to match this image. At the end, children received a score based on how well their final character matched the boy’s “dream date”. In the control condition the girls played a game called Penguin Diner that involves a penguin character working as a waiter in a café serving customers. The Dream Date Dress Up game was chosen as it was typical of the numerous “dress-up/make-over” games that were prevalent on the Friv website, and also explicitly contained the message that a girl’s value comes from her physical appearance . The Penguin Diner game was chosen as the control game as it did not contain any human figures (to elicit appearance-based comparisons), the penguins had no apparent gender, and the game appeared equally appealing and engaging to young girls.Participants were randomly assigned to one of two experimental conditions (“appearance-focused” game and “control” game). Prior to playing the game, researchers collected data in relation to demographics and Internet use at home. Participants then played one of two games from the games site After playing the game the game for 10 min, participants completed self-report measures of self-objectification, body dissatisfaction, and career cognitions. At the end of the study girls were thanked and given a small gift, such as a sticker or piece of stationery, for their participation., 1–2 times a week, Hardly ever). Participants were then asked to indicate what they used the Internet for: , Watching videos , Playing games, Searching for information using Google or Bing, Sharing things (such as making YouTube videos), Other, , as well as to share any rules their parents set about when they could use the Internet or what they could look at whilst on the Internet. Finally, girls were asked specifically whether they played on the Internet games website Friv .A number of questions about Internet usage were developed. First, participants were asked whether they used the Internet at home and how often they go on the Internet each week Using the Child Figure Rating Scale (Tiggemann and Wilson-Barrett State self-objectification was measured using the Modified Twenty Statements Test (TST) .Almost all of the girls (96.3%) indicated that they used the Internet at home. Of these girls, 13.0% indicated they used the Internet every day, 33.8% used the Internet “most days”, 42.9% used the Internet 1–2 times a week, and the remaining 10.4% of girls indicated they “hardly ever” used the Internet. One third of the girls (33.3%) reported that they were allowed to use the Internet “whenever they like”, with the remainder (66.6%) reporting they could only use the Internet “with their parents’ permission”. The most common activities to do on the Internet were: playing games (reported by 81.8% of girls who used the Internet at home), searching for information (81.8%), watching videos (77.9%), doing homework (64.9%), talking to friends (23.4%), and sharing photos or videos (14.3%). When asked specifically whether they played on the games website t(77) = 1.33, p = .18, frequency of Internet use, t(75) = 1.27, p = .21, frequency of exposure to the Friv website, t(78) = .66, p = .51, nor for enjoyment of the experimental and control games, t(78) = 1.79, p = .08.An independent t-test confirmed no difference between the experimental and control groups for age, t (78) = 2.10, p = .02, d = .46. There was no significant difference between the current figure selected by girls in the two game conditions, t (78) = −0.05, p = . 48. However, girls in the appearance game condition selected significantly thinner figures for their ideal figure, t (78) = −2.15, p = .02, d = −.48, and their ideal future figure, t (78) = −1.77, p = .04, d = −.39, compared to girls in the non-appearance game condition.Independent sample t-tests were conducted to assess the effect of game type on girls’ figure rating preferences (see Table t (78) = .18, p = .43, see Table An independent samples t-test was conducted to assess for differences on state self-objectification by condition. There was no significant effect of condition on state self-objectification, F = 73.04, p < .001, partial η2 = .48. Comparisons indicate that participants reported that boys, F  = 86.28, p < .001, partial η2 = .53, and other girls, F  = 75.34, p < .001, partial η2 = .49, could do significantly more jobs than they could do themselves  = 16.43, p < .001, partial η2 = .17. However there was no significant main effect for condition, F  = 1.06, p = .31, partial η2 = .01.The means and standard deviations for the career endorsement by actor, career gender stereotype, and condition are reported in Table F  = 23.40, p < .001, partial η2 = .23. To explore this interaction, analysis was conducted separately for endorsements of feminine and masculine occupations. There was a significant effect of actor on endorsements for both feminine occupations, F  = 34.75, p < .001, partial η2 = .47, and masculine occupations, F  = 56.75, p < .001, partial η2 = .59. Participants reported that they could do fewer masculine jobs than boys, t (79) = −10.58, p < .001, and other girls, t (79) = −6.75, p < .001. Participants also reported that they could do fewer feminine jobs than boys, t (79) = −3.41, p < .001, and other girls, t (79) = −7.57, p < .001. Participants reported that boys could do more masculine jobs than other girls, t (79) = 5.33, p < .001. Conversely, participants reported that other girls could do more feminine jobs than boys, t (79) = −4.44, p < .001. Hence participants endorsed traditional gender expectations about career potential. In addition, participants reported that they were generally less able than both other boys and other girls of doing the occupations listed.The significant effect for actor was qualified by a significant interaction between actor and occupation, F  = .30, p = .74, partial η2 = .008, nor occupation and condition, F  = .06, p = .80, partial η2 = .001, were statistically significant. Moreover, the three way interaction between actor, condition, and occupation was not significant, F  = 1.52, p = .22, partial η2 = .04. Therefore, contrary to hypothesis, playing the appearance-focused game had no impact on cognitions about possible careers.Critically for our hypotheses, none of the interactions involving condition were significant. Neither of the two-way interactions between actor and condition, could do each of the jobs listed by Sherman and Zurbriggen  = 1.83, p = .04, d = .40, see Table t (78) = .43, p = .67.In addition to reporting on whether they rbriggen  girls ints < 1.46 and all ps > .148.In our analysis of career potential, we replicated Sherman and Zurbriggen  and we mF = 3.04, p = 0.15, ηp2 = .17.In our other analyses, we conducted multiple t-tests to target each of our hypotheses directly. However, this increases the chance of finding significance by chance. A MANOVA across these variables confirmed a significant difference between the control and appearance-focused game group, λ = .83, Dream Date Dress Up) for a brief period of 10 min demonstrated heightened body dissatisfaction compared to girls who played an appearance-neutral Internet game (Penguin Diner). This body dissatisfaction was expressed via a preference for a thinner ideal body now as well as a preference for a thinner ideal body in the future. Our identification of a potential contributor to childhood body dissatisfaction is both important and concerning given the established links between early body dissatisfaction and later serious psychological outcomes such as dieting, disordered eating  in order to be appealing to the boy character. It is possible that the playing of such dress-up and make-over games encourage girls to focus on their outward physical appearance and highlights a perceived discrepancy between their own appearance and bodies and that of the culturally prescribed thin ideal. Further research to replicate and expand the current findings with young girls will be critical in order to fully appreciate the impact of engaging in this type of interactive game at a young age, as well as the longer term impact as children move into early adolescence when engagement with other life simulation video games  and social networking services  rapidly increases  may serve to strengthen traditional gender-role stereotypes when it comes to girls thinking about their ideal future careers.Interestingly, although playing the appearance-focused game did not impact on what occupations girls The present study was the first to investigate the impact of playing appearance-focused Internet games on young girls’ body image and career aspirations. Given the pervasiveness and the popularity of Internet-based play for young children  is extremely common and popular with girls of this age Kafai , the finThe present study has made an important first step in investigating the impact of appearance-focused Internet games on the body image and career aspirations of young girls. Girls who played an appearance-focused Internet game for 10 min expressed greater body dissatisfaction compared to girls who played an appearance-neutral game. In addition, the appearance-focused game contributed to an increased preference for traditionally feminine occupations. The study illuminates that exposure to particular types of Internet games may be detrimental to girls, not only in terms of how they come to view their bodies, but also in terms of their future aspirations, and that these effects may be apparent at a much earlier age than previously thought. Internet games should be included in our consideration of influential sources of appearance-based messages for young girls.


P < 0.05). A positive relationship between serum UA and CdB was found in Chinese men after adjusting for the estimated glomerular filtration rate (eGFR), current smoking status, diabetes, dyslipidemia, hypertension and body mass index and in participants with eGFR > 60 mL/min per 1.73 m2. Further, the odds ratio of hyperuricemia increased with increasing CdB quartiles (P for trend < 0.05) in men. In conclusion, CdB was positively related to the serum UA level and to hyperuricemia in Chinese men but not in Chinese women.Few studies have investigated the association between serum uric acid (UA) and cadmium exposure. Our previous study revealed a significantly higher blood cadmium (CdB) level in the Chinese population compared to populations in other countries. To determine whether CdB in Chinese adults is associated with serum UA and hyperuricemia, 2996 participants from the cross-sectional SPECT-China study were recruited. CdB was measured by atomic absorption spectrometry. Hyperuricemia was defined as a serum UA concentration ≥416.4 μmol/L for men and ≥356.9 μmol/L for women. Regression analyses were used to analyze the association of CdB with serum UA and hyperuricemia. We found that the median CdB level was higher in men with hyperuricemia (2.40 μg/L) than in men without hyperuricemia (1.98 μg/L,  Increased production or decreased excretion of UA causes hyperuricemia2. Previous studies have indicated that hyperuricemia is associated with cardiovascular diseases3 and metabolic diseases such as diabetes4, hypertension5 and dyslipidemia6. In past decades, the prevalence of hyperuricemia has increased to 21% and 13% in the US and Chinese general populations, respectively. Although this trend may be related to the increasing prevalence of adiposity and hypertension4, environmental factors cannot be ignored.Serum uric acid (UA) is the final enzymatic product when the body breaks down purine7. Occupational exposure is mainly from industrial processes. Smoking tobacco and contaminated food such as vegetables and rice are the main sources of general cadmium exposure7. Blood cadmium (CdB) levels vary by region, age and ethnicity7. Previous studies have confirmed the pathogenic role of cadmium exposure in renal damage7, bone destruction8 and cancer10. Recent research has focused on the role of cadmium as an important environmental endocrine disruptor11. Epidemiological studies have linked cadmium exposure to metabolic diseases such as diabetes12, obesity13 and thyroid disease14, although the results have not been consistent16. We also found a relationship between cadmium exposure and prediabetes in our previous work17. However, the relationship between cadmium exposure and hyperuricemia remains unknown.Cadmium is a toxic metal with negative effects on health18, cadmium mainly accumulates in the kidney and liver of human bodies, which may lead to elevated plasma uric acid levels according to several animal studies20. Furthermore, as mentioned above, cadmium exposure is associated with metabolic diseases and thus may prompt the occurrence of hyperuricemia and gout22. Epidemiological evidence of a relationship between cadmium exposure and hyperuricemia is scarce. A cross-sectional study from the National Health and Nutrition Examination Survey (NHANES) showed no relationship between them2. Nevertheless, our previous study revealed that the CdB level (median of 1.70 μg/L) was much higher than that reported in other countries17, and approximately 17% of subjects still had a CdB level higher than 5.0 µg/L. No study has ever explored this association in the Chinese population at the current CdB level, which differs from the level in the US. Hence, using data from a population-based investigation called the Survey on Prevalence in East China for Metabolic Diseases and Risk Factors (SPECT-China) in 2014, we aimed to explore the relationships between the CdB and serum UA levels and hyperuricemia in the general Chinese population.Due to its long biological half-lifeP < 0.05). The median CdB level was 2.40 (0.68–4.61) μg/L higher in men with hyperuricemia than in men without hyperuricemia (P < 0.05), but the CdB levels in women showed no significant difference between individuals with and without hyperuricemia. Additionally, the median blood lead (PbB) levels were comparable between different serum UA levels.The characteristics of the study population, categorized by sex and hyperuricemia status, are provided in Table 2) and smokers, the serum UA level remained positively associated with CdB . However, no relation between the CdB and serum UA levels was observed in women in either the crude or the fully adjusted model. Furthermore, PbB was analyzed as an independent variable, and we found no correlation between PbB and serum UA levels in either gender.Linear regression modeling of the data showed that a higher CdB level was associated with a higher serum UA concentration  in men after adjusting for PbB (Table P for trend < 0.05). After adjusting for eGFR, current smoking status, PbB, diabetes, dyslipidemia, hypertension and BMI, the ORs of Q3 and Q4 CdB for hyperuricemia were 1.82  and 1.61 , respectively (P for trend < 0.05). Higher ORs for the CdB levels in Q3  and Q4  were observed in participants with relatively normal renal function . After we excluded smoking participants, a marginal significance for CdB as a risk factor for hyperuricemia remained (P for trend = 0.08). In women, the CdB levels were still not related to hyperuricemia (P for trend > 0.05).In multivariate-adjusted logistic regression analyses Table , the CdB2), a positive relationship between CdB and hyperuricemia remained.We explored the association between CdB and UA in Chinese adults. Our study revealed that CdB was positively associated with serum UA levels and hyperuricemia in Chinese men but not in women. This association was independent of PbB, eGFR, current smoking status, diabetes, dyslipidemia, hypertension and BMI. Furthermore, in male participants with relatively normal renal function  is associated with hyperuricemia and gout51. Moreover, there is still a link between relatively lower PbB and hyperuricemia52. Thus, we regarded PbB as an important confounding factor. Moreover, we evaluated the relationship between PbB and UA levels in our participants, but there was no significant relationship in either men or women.Cadmium and lead (Pb) are two toxic metals that are widely distributed in the environment. They share similar population exposure routesThis study is the first exploration of the relationship between CdB and hyperuricemia in different genders in the Chinese population. Homogeneity and strict quality control were guaranteed because the same trained staff was used. Furthermore, we considered PbB to be a confounding factor when exploring the association between CdB and UA levels.2 53. Second, we used the blood cadmium levels rather than urinary cadmium. Urinary cadmium reflects lifetime cadmium exposure, but for relatively low cadmium exposure levels, blood cadmium levels may be more appropriate38. It would have been ideal if we could detect both. Third, this study did not include information on food intake. A high serum UA level is usually associated with an intake of large amounts of food that is high in purines2. It is reasonable that the CdB levels are parallel with the serum UA levels in participants with large daily food intakes. Furthermore, we could not determine the causal relationship between CdB and hyperuricemia in this cross-sectional study.There are some limitations of this study. First, using a cystatin C-based formula to adjust for the GFR estimates is required in healthy populations with normal renal function, which was not available to us, but the CKD-EPI equation applied in our study was confirmed to be more accurate than the Modification of Diet in Renal Disease Study equation, particularly for censoring numerical estimates greater than 60 mL/min per 1.73 mIn conclusion, CdB was positively associated with serum UA levels and hyperuricemia in Chinese men but not in women. This study indicated that cadmium exposure may confer a risk for hyperuricemia, which was not attributed solely to cadmium toxicity-induced renal dysfunction. However, in cases of relatively normal renal function, the CdB level was still positively related to serum UA. Further study is needed to demonstrate causality and elucidate the underlying mechanisms. In addition, efforts to reduce cadmium exposure in adults are warranted.55. The sampling method was described in detail in our previous study23. A total of 2996 subjects were enrolled in our final study after excluding participants with missing values for UA (n = 3429) and CdB (n = 474). Before the data collection, written informed consent was provided by all participants. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2008. The study protocol was approved by the Ethics Committee of Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine.Our data (n = 6899) were from the SPECT-China study55. Body weight, height and the calculation of BMI were calculated consistently with the previous study23. Waist circumference and blood pressure were measured by strict adherence to the standard procedure21. Current smoking was defined as having smoked at least 100 cigarettes in one’s lifetime and currently smoking cigarettes17.The questionnaires about demographic characteristics, medical history and lifestyle risk factors and anthropometric data were constructed by the same trained staff as previously described55. Serum UA levels were measured using the uricase method with a Beckman Coulter AU 680 (Germany). The coefficient of variation was between 1.2% and 2.7%. Serum creatinine (Scr) was measured using a kinetic-rate Jaffe method with a Beckman Coulter AU 680 (Germany), and we converted the Scr levels to the estimated glomerular filtration rate (eGFR) according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation: (1) for a woman with Scr ≤0.7 mg/dL, eGFR = 144 × (Scr/0.7)−0.329 × (0.993)age; (2) for a woman with Scr >0.7 mg/dL, eGFR = 144 × (Scr/0.7)−1.209 × (0.993)age; (3) for a man with Scr ≤0.9 mg/dL, eGFR = 141 × (Scr/0.9)−0.411 × (0.993)age; and (4) for a man with Scr >0.9 mg/dL, eGFR = 141 × (Scr/0.9)−1.209 × (0.993)age. The value of eGFR is reported in units of mL/min per 1.73 m2 of body surface area17.Venous blood samples were drawn, processed and shipped as previously described17. Standard curves were established, and quality control materials were tested before the samples were measured. Two quality control personnel participated in the process control. Outliers were detected by duplicate runs. The detection limits for blood cadmium and lead were 0.01 µg/L and 0.1 µg/L, respectively. The inter-assay coefficient of variation for cadmium was 10%.Cadmium and lead levels in blood samples were tested using graphite furnace atomic absorption spectrometry17.Fasting plasma glucose (FPG), total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), insulin and glycated hemoglobin (HbA1c) were assessed with the methods used previously2. The definitions of overweight, obese, diabetic and hypertensive in this study have been previously described21. Dyslipidemia was defined as described previously56.Hyperuricemia was defined as a serum UA concentration ≥416.4 μmol/L and ≥356.9 μmol/L for men and women, respectivelyP value < 0.05 for a two-tailed test indicated a significant difference. The specific statistical methods for continuous variables and categorical variables were described in detail in a previous study17.The IBM SPSS Statistics software, version 22 , was used for data analysis. Analyses were performed separately for men and women due to major gender differences in serum UA concentrations. A B) and standard errors. The full model included PbB, eGFR (which incorporates age and serum creatinine level), current smoking status, diabetes, dyslipidemia, hypertension and BMI.The association of CdB (an independent variable) with serum UA levels (a dependent variable) was assessed by linear regression analysis. The results were expressed as unstandardized coefficients , current smoking status, PbB, diabetes, dyslipidemia, hypertension and BMI. PbB, eGFR, and BMI were entered as continuous measures. Data were expressed as odds ratios (ORs) .2 or less2. Moreover, smokers are at high risk of cadmium exposure17, and previous studies have indicated an association between smoking and increased purine catabolism57. Thus, we performed another subgroup analysis excluding current smokers. The regressions were performed by the same strategy as in the above analyses.Because hyperuricemia is known to be associated with kidney dysfunction and the kidneys are the most important target organs for cadmium exposure, we performed subgroup analyses that excluded participants with an eGFR of 60 mL/min per 1.73 m