Leymus chinensis (Poaceae) and Setaria viridis (Gramineae) and significantly reduced the relative abundance and aboveground biomass of the Suaeda corniculata (Chenopodiaceae). Although elevated temperature and N addition can affect species composition or suppress the development of AMF, AMF are likely to play a vital role in increasing plant diversity and productivity. Notably, AMF might reduce the threat of climate change induced degradation of temperate meadow ecosystems.To examine the influence of elevated temperature and nitrogen (N) addition on species composition and development of arbuscular mycorrhizal fungi (AMF) and the effect of AMF on plant community structure and aboveground productivity, we conducted a 5-year field experiment in a temperate meadow in northeast China and a subsequent greenhouse experiment. In the field experiment, N addition reduced spore population diversity and richness of AMF and suppressed the spore density and the hyphal length density (HLD). Elevated temperature decreased spore density and diameter and increased the HLD, but did not affect AMF spore population composition. In the greenhouse experiment, AMF altered plant community composition and increased total aboveground biomass in both elevated temperature and N addition treatments; additionally, AMF also increased the relative abundance and aboveground biomass of the grasses  Arbuscular mycorrhizal fungi (AMF) play a vital role in the survival and development of plants in terrestrial ecosystems by improving growth and increasing productivity12457810Global climate change induced by human activities influences the development and species composition of AMF. Elevated temperature increases the allocation of carbon to AMF and hyphae length131513As one of the major threats to ecosystem stability, nitrogen (N) deposition reduces plant species number2315232728Many studies demonstrated that plant-induced alterations of the soil affect the structure of the plant community3031333436−2 yr−1.The Songnen grassland is located at the eastern edge of the Eurasian grassland and is the largest and most typical meadow steppe in ChinaTo better understand the effects of elevated temperature and N addition on the development and species community composition of AMF communities and their feedbacks on the aboveground plant community, a 5-year elevated temperature and N addition field experiment and a greenhouse experiment were conducted. We hypothesized that (1) N addition and elevated temperature would affect the development and species composition of AMF in the field experiment and that (2) AMF would alter the composition of the plant community and increase plant diversity and productivity under the elevated temperature and N addition conditions in the greenhouse experiment because of AMF can reduce the negative of N addition and elevated temperature on plant growth.P < 0.05) and 10% (P < 0.05), respectively, and increased the hyphal length density (HLD) by 27% (P < 0.001). The N addition treatment decreased the AMF HLD 14% , but no interactive effect on HLD was observed  or evenness (E)  and 25% (P < 0.05), respectively, compared with control, but had no impact on E , but a significant interactive effect of elevated temperature × N addition on species richness was observed (P < 0.05).The species composition of AMF community was significantly affected by the elevated temperature and N addition treatments. Elevated temperature had no impact on AMF species richness, diversity (act on E . No inteP > 0.05). In N addition treatment, AMF significantly altered plant community composition, the mycorrhizal benefits of plant diversity, richness and evenness were 16.3 33.8 and 11.7, respectively, and significant differences between AM and NM were observed . Compared to control, the mycorrhizal benefits on plant diversity, richness and evenness all significantly increased  both in elevated temperature and N addition treatments. Significant interactive effects of N addition × AMF on plant diversity, richness and evenness were observed, and significant interactive of elevated temperature × AMF on plant diversity and evenness were also observed. No interactive effects of elevated temperature × N addition × AMF on plant species diversity, richness and evenness were detected , respectively; but the abundance of S. viridis and S. corniculata significantly decreased by AMF and the mycorrhizal benefits were −9.6 (P < 0.05) and −28.6 (P < 0.05). In N addition treatment, AMF had no impact on the abundance of S. viridis (P > 0.05) and C. virgata (P > 0.05), and AMF decreased abundance of S. corniculata (P < 0.05) and the mycorrhizal benefit was −20.3 whereas AMF increased the abundance of L. chinensis and L. davurica and the mycorrhizal benefits of them were 25.0 (P < 0.05) and 16.7 (P < 0.05). Significant interactive effects of N addition × AMF on the abundances of L. chinensis and S. corniculata were observed, no significant interactive effects of elevated temperature × N addition, elevated temperature × AMF, and elevated temperature × N addition × AMF were detected  and 258.5 (P < 0.01), respectively, but significantly decreased the biomass of C. virgata (P < 0.05) and S. corniculata (P < 0.05). In elevated temperature treatment, aboveground biomass of L. chinensis, S. viridis, L. davurica significantly increased by AMF, the mycorrhizal benefits of them were 366.0 (P < 0.01), 374.4 (P < 0.01) and 57.9 (P < 0.05), respectively, AMF decreased aboveground biomass of C. virgata and S. corniculata significantly. In the N addition treatment, AMF increased aboveground biomass of L. chinensis, S. viridis and L. davurica, the mycorrhizal benefits were 51.1, 47.4 and 186.8, respectively, whereas the aboveground biomass of S. corniculata decreased significantly. No significant interactive effects of elevated temperature × N addition, N addition × AMF, or elevated temperature × N addition × AMF on the aboveground biomass of L. davurica and C. virgata were detected  and 124.8 (P < 0.01), respectively, whereas decreased the proportion of total plant from C. virgata and S. corniculata and the mycorrhizal benefits were −13.7 (P < 0.05) and −77.2 (P < 0.05), respectively. Compared to control, the mycorrhizal benefits of L. chinensis, S. viridis and L. davurica significantly increased, but the mycorrhizal benefits of S. corniculata decreased. In the N addition treatment, AMF also increased the proportion of total plant from L. chinensis and S. viridis, the mycorrhizal benefits were 133.7 (P < 0.01) and 259.5 (P < 0.01), whereas decreased the proportion of total plant from L. davurica and S. corniculata and the mycorrhizal benefits were −35.7 (P < 0.05) and −78.4 (P < 0.05), respectively. The mycorrhizal benefits of L. chinensis, S. viridis and L. davurica in elevated temperature and N addition treatments significantly increased  compared to control, but the mycorrhizal benefits of S. corniculata decreased, significantly. Additionally, significant main effects of AMF and interactive effects of AMF × N addition on the proportions of L. chinensis and S. corniculata to total biomass were observed  affected the development of AMF and determined the composition of AMF spores population. Although the effect of elevated temperature on the composition of the AMF has been examined in many field studies of different ecosystems202823In the current study, N addition significantly decreased the species diversity and richness of the AMF spore population. These findings are consistent with earlier studies that show that N fertilization decreases the spore richness and abundance of AMF2324232324In our study, significant interactive effects of elevated temperature × N addition on AMF spore density and diameter and species richness were observed. However, we did not detect significant interactive effects of elevated temperature × N addition on plant diversity, richness or aboveground productivity21536L. chinensis and S. viridis) with the addition of N in the greenhouse experiment  in the elevated temperature and N addition treatments in the greenhouse experiment. The relative abundance of ecreased  with AMFL. chinensis and S. viridis increased significantly with AMF, whereas the proportion of biomass from S. corniculata decreased significantly. These results are consistent with early findings in a semiarid herbland in South AustraliaL. chinensis, S. viridis and L. davurica and negative mycorrhizal benefits of C. virgata and S. corniculata were detected. In the Songnen grassland, the species in the grass family  are the most important forages, whereas the species in Chenopodiaceae family  are weeds that livestock do not like to eat. Therefore, these results suggest that AMF can significantly improve the productivity of forage and reduce the productivity of harmful weeds, which would sustain livestock production. Furthermore, the plant species S. corniculata is a typical indicator of degradation in the Songnen grassland, and our results indicate that that AMF can reduce the risk of grassland degradation under conditions of climate change by suppressing the growth of S. corniculata.A large number of studies have demonstrated that AMF can improve aboveground productivity15413Elevated temperature and N addition treatments, proxies for global climate change, altered the spore population composition and suppressed the development of AMF. However, AMF reduced the negative effects of elevated temperature and N addition on plant diversity and productivity and increased the contribution of grasses thereby increasing the productivity of grassland. Thus, AMF have the potential to reduce the degradation of grasslands caused by elevated temperature and N addition.Leymus chinensis ; Chloris virgata , Setaria viridis , Lespedeza davurica  and Suaeda corniculata  are the main companion species.The experiment was conducted at the Songnen Grassland Ecological Research Station , Northeast Normal University, Jilin Province, in northeast China. The grassland is situated at the eastern edge of the Eurasian steppe and is characterized as Eurasian continental meadow steppe. Mean annual precipitation is approximately 400 mm, 90% of which occurs from May to October. Annual average air temperature is 4.9 °C, and annual average land surface temperature is 6.2 °C. The soil in the study area is a soda-saline soil, and the soil pH was 8.2, with 3–4% organic matter in the soil surface layer. The vegetation of the experimental site is dominated primarily by −2 yr−1−2 yr−1) was added as an aqueous pulse on the first day of May each year. In the control and elevated temperature plots, an identical amount of water (without N) was added to account for that added in N addition condition. The experiment began in May 2006 and was terminated in September 2010.The experiment was a completely randomized block factorial experiment with two factors: elevated temperature and N addition. There were four treatments: control (C), elevated temperature (T), N addition (N), and elevated temperature plus N addition (T + N), with 6 replicates each. The size of the plots was 2 m × 3 m. The plots in the elevated temperature treatment were all heated continuously by infrared radiators  that were suspended 2.25 m over the plot center. In each control or N addition plot, one ‘dummy’ heater of the identical shape and size was installed to mimic the shading effects of the infrared radiator. The heaters in the elevated temperature treatment were set for approximately 2000 W of radiation output. In northern, temperate grassland ecosystems, the saturation rate for N deposition is approximately 10 g mThree soil cores  were randomly collected and were mixed to create a composite sample at the end of the experiment. Fresh soil samples were sieved (2-mm) to remove roots and debris for AMF spore isolation.et al.The extraradical hyphae from the homogenized soils were extracted and stained with trypan blue using the method of Jakobsen http://invam.caf.wvu.edu/) and Schüβler’s Glomeromycota Species List (http://www.lrz.de/~schuessler/amphylo/). The slides are maintained at Northeastern Normal University. A sporocarp was counted as a single spore. The spore density is the number of AM fungal spores in 20 g of soil. The average spore diameter was calculateed for each soil sample.The spores were extracted using wet sieving and sucrose centrifugationH′) and evenness.The species richness (SR) = number of AM fungal taxa found in 20 g of soil and the relative abundance (RA) =  × 100. The number of spore population and the density were also used to calculate species richness (mean number of AMF species per 20 g of soil), diversity  was homogenized and used to inoculate the soils with AMF in the greenhouse experiment. The seeds of one dominant species L. chinensis and four companion species C. virgata, S. viridis, S. corniculata and L. davurica were collected from the Songnen meadow steppe and were stored in a refrigerator at <4 °C before use.The soil used in this experiment was collected from the grassland experimental sites. The soil was sterilized 2 times using high-pressure steam for 2 h at 121 °C per time. When the first sterilization completed, the soil were homogenized and then sterilized again. To isolate the AMF spores, we collected the soil (500 g) from the field plots under treatment for 5 years with elevated temperature and N addition. The isolated AMF spores were cultured using 60Co γ-rays and was used for the non-AMF (NM) treatment. To minimize differences in the rhizosphere microbial communities of AMF and non-AMF treatments, 10 ml filtrates free from mycorrhizal propagules from the inoculum were added to the non-AMF treatments and 10 ml deionized water were added to AMF treatments. The pots were 34 cm (in diameter) × 23 cm (in depth) and filled with 2.5 kg (dry weight) soil. The seeds of the five species were surface disinfected in 10% (v/v) hydrogen peroxide for 5 min, rinsed five times with deionized water, and germinated at 20 °C. After 48 h, the germinated seeds were added to each pot, and the density of these five species was thinned based on their densities in the field and was kept consistent for all treatments. The densities of these species were L. chinensis 15 plants per pot, S. viridis 10 plants per pot, C. virgata 7 plants per pot, S. corniculata 5 plants per pot and L. davurica 5 plants per pot.In the greenhouse experiment, the four treatments were identical to the treatments in field experiment, with control, elevated temperature, N addition, and elevated temperature plus N addition. Each treatment was also replicated 6 times. Soil samples were collected from each treatment in the field experiment and were homogenized to use as AMF inocula in the greenhouse experiment. The sterilized soil was placed into pots, and 200 g of AMF inoculum (approx. 6000 spores) from the similar treatment (from the field experiment) was added to the soils in the AMF treatment (AM). An identical amount of the inoculum mix was sterilized using 10 k Gy −2 S−1 (06:00–20:00) daily. The relative humidity was 40–60%. In the control and N addition treatments, the temperature settings were 22 °C from 06:00–10:00, 25 °C from 10:00–15:00, 22 °C from 15:00–20:00, and 22 °C from 20:00–06:00. In the elevated temperature and elevated temperature plus N addition treatments, the temperature in each time period was elevated 3 °C compared with the control and N addition treatments. The air temperature in the phytotrons was adjusted and monitored every 10 seconds, and the temperature matched the mean summer temperature from 2000–2011 in the Songnen meadow grassland−2 yr−1) was added to the pots of N addition and elevated temperature plus N addition treatments. The pots were irrigated every 2 days; the soil water content was 50–60% of field capacity.The experimental pots were placed in phytotrons  at Northeast Normal University. In the phytotrons, light intensity was 350 μ molH), richness and evenness. The mycorrhizal benefit was calculated according to the following formula. The mycorrhizal benefit = [(AM/NM) − 1] × 100.The plant species number and density of each species was observed and recorded daily at 14:00. The plants were harvested after twelve weeks of growth. The shoots and roots were removed from the pot and were washed with deionized water. The roots were cut into 1-cm segments and thoroughly mixed. A sub-sample of 0.5 g was cleared with 10% (w/v) KOH at 90 °C for 2 h and then stained with trypan blue. Mycorrhizal colonization  was estiP = 0.05. The data were all tested for normality and homogeneity of variance before analysis.The statistical analyses were conducted using the SPSS statistical software package 7 . The spore density counts and estimates of species richness were square root transformed. Other data were analyzed without transformation. Spore density and AM fungal diversity index results are shown as arithmetic mean values with standard errors. A two-way ANOVA was used to test the effects of elevated temperature, N addition and their interaction with the AM fungal HLD, spore density, species richness, diversity, and evenness in the field experiment, and a three-way ANOVA was used to test the feedback of AMF on the community composition and productivity of the plant community in the greenhouse experiment. Treatment means were compared by Tukey at How to cite this article: Zhang, T. et al. Response of AM fungi spore population to elevated temperature and nitrogen addition and their influence on the plant community composition and productivity. Sci. Rep.6, 24749; doi: 10.1038/srep24749 (2016).


Cognitive psychology research has suggested an important role for executive functions, the set of skills that monitor and control thought and action, in learning mathematics. However, there is currently little evidence about whether teachers are aware of the importance of these skills and, if so, how they come by this information. We conducted an online survey of teachers' views on the importance of a range of skills for mathematics learning. Teachers rated executive function skills, and in particular inhibition and shifting, to be important for mathematics. The value placed on executive function skills increased with increasing teaching experience. Most teachers reported that they were aware of these skills, although few knew the term “executive functions.” This awareness had come about through their teaching experience rather than from formal instruction. Researchers and teacher educators could do more to highlight the importance of these skills to trainee or new teachers. One challenge for cognitive psychology research is to reveal the processes and mechanisms that are important for successful learning. In the past two decades the learning of mathematics, in particular, has received increasing attention. Psychologists have identified a number of skills that are related to successful mathematics achievement. These include both domain-specific skills such as knowledge of number facts and conceptual understanding , 52% taught in primary or junior schools (age 6–11), and 40% taught in secondary schools (age 11–18). Teachers who completed the survey also varied in years of teaching experience: 1% were in training, 29% had 1 to 3 years experience, 35% had 4 to 10 years experience, and 34% had more than 10 years experience. Just over half of respondents (54%) taught mathematics as their main subject or specialism. In the UK, all foundation and primary school teachers are required to teach mathematics, but in secondary school mathematics is only taught by specialists. Our sample had a higher proportion of primary school teachers (52% vs. 33%), and within secondary schools, a higher proportion of mathematics specialist teachers (87% vs. 14%) compared to the national population  to 4 (extremely important). Four items referred to mathematics-specific (MS) skills, four items referred to executive function (EF) skills, and four items referred to other skills (OS) .Teachers completed a Mathematics Skills Questionnaire in which they rated the importance of 12 basic skills for mathematics learning on a 5-point Likert scale from 0  = 12.9, p < .001. MS items (M = 3.05) were rated as more important than EF items , or OS items , which did not differ (p = .128).Teachers' responses were explored with a series of analysis of variances (ANOVAs) and follow-up Bonferroni-corrected F = 3.69, p = .029, and domain, F = 13.35, p < .001, and the interaction approached significance, F = 2.08, p = .086. Teaching experience had a significant effect on importance ratings for the MS items, F = 4.62, p = .012, and EF items, F = 5.92, p = .004, but not OS items, F = 2.52, p = .09. For MS skills, teachers with 1 to 3 years experience gave lower importance ratings (M = 2.77) than teachers with either 4 to 10  or more than 10 years experience , who did not differ (p = .99). In contrast, for EF skills, only teachers with more than 10 years experience (M = 3.10) gave higher ratings than those with less than 3 years experience , whereas ratings from teachers with 4 to 10 years experience (M = 2.86) did not differ from either less (p = .133) or more experienced (p = .412) teachers.We then explored whether participant characteristics  affected responses for each domain. A two-way ANOVA was conducted with domain  and years of teaching experience  as factors. There were significant effects of teaching experience, F = 16.30, p < .001, and school type, F = 4.91, p = .009, which were modified by a significant interaction, F = 2.59, p = .039. School type did not significantly impact importance ratings for EF items, F = 2.82, p = .065, or OS items, F = 1.51, p = .227, but did affect ratings for MS items, F = 10.32, p < .001. Teachers in secondary schools rated MS skills as less important (M = 2.76) than teachers in primary schools  or foundation stage , whose ratings did not differ (p = .276). This is likely to reflect differences in the nature of mathematics content across these age groups.A second two-way ANOVA was conducted with domain  and school type  as factors.F = 5.92, p = .004, and skill, F = 9.28, p < .001. Teachers rated inhibition skills (“Focus on relevant information and avoid distractions”) as more important than working memory skills . They also rated shifting skills (“Be able to think flexibly”) as more important than both working memory items (p = .001 and p = .009 respectively). They did not differentiate between inhibition and shifting skills (p = .999) or between the working memory items (p = .999). There was no interaction between skill and teaching experience, F < 1.We next explored ratings within the EF domain to discover which individual skills were considered most important and whether this differed for teachers with different levels of experience . Teachers who had more years of teaching experience were more likely to report that they knew a little or a lot about the role that executive function skills play. In keeping with this, the majority of teachers reported that they had learnt this from their own teaching experience (63%), rather than during their initial teacher training or later professional development (16%).Only 18% of teachers reported that they had heard of the term Our findings give a picture of UK teachers' knowledge and understanding of the role of executive function skills in mathematics learning. Teachers rated all of the skills in our survey as somewhat important, but differentiated between the specific skills that we explored. It is clear that, even without formal instruction in this topic, the majority of teachers are aware that skills such as holding and manipulating information in mind (working memory), ignoring distractions (inhibition), and thinking flexibly (shifting) are important, even if they have never heard the term “executive functions.” This demonstrates that the importance of these skills is apparent in the classroom as well as in the laboratory. Our findings are based on a small-scale convenience sample and it would be helpful to replicate these effects with a larger sample that is fully representative of the population of teachers.Within the domain of executive functions, it is interesting to note that teachers identified inhibition and shifting skills as being more important than working memory. This is based on responses to individual items and thus this pattern should be validated in further research with more extensive questionnaires. However, it is interesting to note that this pattern is somewhat in contrast to research evidence, which has tended to focus on the role of working memory in mathematics learning , ratings of the importance of executive function skills only increased after more than 10 years of teaching experience. Given these results, it is important to consider whether teacher education courses provide adequate training in understanding the importance of these skills.Teachers in our survey who were aware of the importance of executive function skills reported that they had learnt this through their own teaching experience and not during their training. This is in keeping with research showing that student teachers fail to learn about child development during their training (McDevitt & Ormrod, Why might cognitive psychology research be undervalued in teacher education? Laski et al.  found th


New regulations by the Swiss Federal Food Safety and Veterinary Office provide for the monitoring of breed health by Swiss breeding clubs. In collaboration with the Swiss Bernese Mountain Dog Club, the purpose of this study was to investigate the causes of death in purebred dogs registered by the club and born in 2001 and 2002.P < 0.00). The cause of death was unknown in 89/381 dogs (23.4 %). For the remaining dogs, the most frequent causes of death were neoplasia , degenerative joint disease , spinal disorders , renal injury , and gastric or mesenteric volvulus . However, large numbers of dogs were diagnosed with neoplasia without histopathologic or cytologic confirmation. Dogs with neoplasms had a shorter median survival than dogs with other disorders. The shortest median survival (6.8 years) was found for dogs with renal injury.Of a total of 1290 Bernese mountain dogs (BMDs) born in 2001 and 2002 in Switzerland, data was collected from owners and veterinarians using a questionnaire designed for this study from 389 dogs (30.2 %). By the end of the study, 381/389 dogs (97.9 %) had died. The median life expectancy of all dogs was 8.4 years . Female dogs had a significantly longer median survival  than male dogs  (Findings of this study confirm a high prevalence of neoplasia and associated low life expectancy in BMDs. The results underline a need for more widespread precise diagnostics and further research on malignant tumours in this breed to improve overall breed health. Previous studies have demonstrated a relatively low life expectancy and high incidence of neoplasia in the Bernese mountain dog (BMD) –4. IndeeSeveral authors explain the low life expectancy in the BMD by the high incidence of neoplastic diseases , 7. IndeRecent new regulations have been introduced by the Swiss Federal Food Safety and Veterinary Office (FSVO) to restrict breeding of animals with inherited defects, which are associated with pain or suffering . This neData of purebred BMDs, born in Switzerland in 2001 and 2002 and registered with the Swiss Bernese Mountain Dog Club, were collected. Of a total of 1290 dogs, owners of 402 dogs (31.2 %) had participated in a previous study evaluating disease prevalence in BMDs , based oThe questionnaire was modified from that used in the previous study to include only data relevant to the cause of death. The main body of the questionnaire identified the animal by name and by the 15-digit microchip number from the Animal Identity Services AG (ANIS), and served to classify diseases both by the affected organ system and the pathophysiologic disease process involved. In addition, veterinarians were asked to formulate a definitive, suspected or symptomatic diagnosis in a free-text field. Check boxes are available to identify the types of examinations conducted during consultation, treatments given and the clinical outcome. Each questionnaire was signed by both the owner and the veterinarian, confirming consent to study participation. The questionnaire was made available in German, French and English and was sent with a letter explaining the study aims to all owners of the 206 dogs in March 2012. Two months later, a reminder was sent to owners not responding to the initial contact and an attempt was made to contact those owners by telephone. A final renewed attempt to contact nonresponding owners was made in June 2013. The study was submitted for approval to the Cantonal Veterinary Services of the Canton of Berne and was determined exempt from the need for review, according to the Swiss Federal Welfare Act of December 16th 2005 and adheres. The study adheres to ARRIVE guidelines for reporting animal research.Data collected from the completed questionnaires, the internal surveillance of the Swiss Bernese Mountain Dog Club and the ANIS Company were combined with data from the previous study  using a The reliability of diagnoses was categorized as poor (cause of death reported by the owner without veterinary data or cause of death unknown), moderate , high  or excellent . No distinction in reliability of diagnoses was made between primary-care and referral veterinarians.www.ncss.com, and MedCalc Statistical Software version 15.4, MedCalc Software bvba, Ostend, Belgium; https://www.medcalc.org; 2015). Descriptive statistics were used to evaluate survival based on sex, reliability of diagnoses, organ system, disease process and specific diagnoses. Normality was evaluated using D’Agostino-Pearson tests. Kaplan-Meier survival analyses were performed to estimate and compare life expectancy based on sex, reliability of diagnoses, organ system, disease processes and specific diagnoses. Comparison of survival curves between dogs with different characteristics  was performed using log rank tests. Hazard ratios for the event death for specific dichotomous variables were calculated using the logrank test calculations of hazard rates derived from the cumulative hazard function of the Kaplan-Meier curves [P = 0.05 throughout.Data analysis was performed using statistical software  and 220/389 (56.6 %) were born in 2001 and 2002, respectively. There were 227/389 (58.4 %) female  and 162/389 male  animals.At the end of the study, 381/389 dogs were dead and 8/389 were alive . No distinction was made between dogs that died naturally and those euthanised. Data was available for 196 dogs from the previous study  and a fuP = 0.575). For all 389 dogs, females had a significantly longer life expectancy than males (P < 0.001) (Table n = 310), significantly more females than males were neutered (P < 0.001). A significant difference in survival was found between the groups based on sex and gonadectomy status (P = 0.005) (Table P = 0.225), between intact males and castrated males ; P = 0.376), or between intact females and spayed females ; P = 0.221). However, a significant difference was found between castrated males and spayed females ; P = 0.001), castrated males having a higher hazard than spayed females , 8.03–8.47) and 8.40 years (interquartile range (IQR), 6.89–9.66), respectively. No difference in life expectancy was found between dogs born in 2001 and those born in 2002 , moderate in 56/381 dogs (14.7 %), high in 91/381 dogs (23.9 %) and excellent in 51/381 dogs (13.4 %). The poor reliability category included 89 dogs for which the cause of death was unknown. The median life expectancy was 9.24 , 8.17 , 7.18  and 7.17  years for diagnoses with poor, moderate, high and excellent diagnoses, respectively.n = 222), degenerative joint disease (n = 16), spinal disease (n = 13), renal injury (n = 12), and gastric or mesenteric volvulus (n = 7).Specific causes of death recorded in more than 5 dogs were neoplasia , acute or chronic kidney injury (n = 12) and anaemia (n = 5). All degenerative processes were attributed to degenerative joint disease and/or cruciate ligament failure. Mechanical processes were due to gastric or mesenteric volvulus. Immune-mediated processes were most frequently immune-mediated haemolytic anaemia (4 of 5 cases). Only 2 cases were attributed to inflammatory/infectious or metabolic/toxic processes, respectively, and a single case was attributed to trauma. No cause of death was attributed to idiopathic, congenital or alimentary disease processes. For disease processes with at least 5 cases, the shortest survival was associated with immune-mediated processes, followed by neoplastic processes  (Table emia n = . All degn = 4), leukaemia (n = 4), fibrosarcoma (n = 2), haemangiosarcoma (n = 2), mast cell tumour (n = 2), transitional cell carcinoma (n = 1) and chondroma (n = 1). The remainder of neoplasms (unconfirmed on cytology or histology) were multiorgan tumours (n = 45), lung tumours (n = 29), splenic tumours (n = 14), liver tumours (n = 6), oropharyngeal tumours (n = 3), nervous system tumours (n = 2), intestinal tumours (n = 2), and 1 each of tumours involving the urinary bladder, heart base, hypophysis, mediastinum, nasal cavity, prostate, spine, and axial skeleton.Specific neoplasms diagnosed in the 222 dogs that died due to a neoplastic process were HS in 23/222 dogs (10.4 %), lymphoma in 15/222 dogs (6.8 %) and osteosarcoma in 11/222 dogs (5.0 %). Other tumours diagnosed on cytology or histology were sarcoma not further differentiated . Male dogs had a relative risk of neoplasia of 1.20  compared to females (P = 0.030), and an odds ratio of 1.58  compared to females (P = 0.033). Moreover, male dogs with neoplasia had a shorter survival than female dogs with neoplasia (Table P = 0.010) and an odds ratio of 3.36  compared to females (P = 0.009). However, no significant difference in survival was detected between male and female dogs with HS, lymphoma or osteosarcoma. The median survival was 7.17 years  for dogs with HS, 7.32 years  for dogs with lymphoma, and 8.74 years  for dogs with osteosarcoma.A cause of death due to neoplasia was attributed to a significantly greater proportion of male dogs  than female dogs   and median 8.40 years) life expectancy of BMDs in the present study was higher than that reported in most studies. However, life expectancy of BMDs appears to have increased over time from 5.7 years  and 6.8  years an0 years lThe low life expectancy in the BMD has been attributed to a high prevalence of neoplasia, reported to be between 26.1 % and 55.1 % , 19, 22,Female BMDs in the present study had a higher life expectancy than males, corroborating findings of a previous study in which female BMDs were reported to live about 1 year longer than males . AlthougThe most frequently diagnosed neoplasia in the present study was HS . This was similar to previous investigations in Dutch and Norwegian BMD populations, in which HS accounted for 15.3 % , respectOf dogs with confirmed HS, the median survival of 7.2 years was somewhat higher than the previously reported 6.3 years . PreviouWith regards to non-neoplastic disease processes, dogs with symptomatic conditions included 13 dogs that were euthanised or died with paraparesis, paraplegia or tetraparesis. The median life expectancy of these dogs was 8.4 years, suggesting that at least some of these dogs may have suffered from degenerative myelopathy, previously described in older dogs of this breed . HoweverIn contrast to 1 previous study , cardiovThe reason for a short lifespan in the BMD is likely multifactorial but significant factors include the high prevalence of tumours, as only few other breeds, including flat-coated retrievers and boxers, exhibit a comparable high prevalence –4. FurthIn the present study, information regarding the cause of death that was purely based on information acquired from dog owners was considered of low diagnostic reliability. Interestingly, dogs with higher longevity were more frequently classified in the lowest quality of diagnoses. In contrast, dogs that died at an early age were classified in the highest categories. This observation may suggest that dog owners and veterinarians are more willing to invest time and money in the work-up and diagnosis of diseases in dogs presenting at a younger age. However, it is also possible that far more detailed data was transmitted by veterinarians when dogs were euthanized at a younger age.To the authors’ knowledge, this is the first study to investigate only purebred BMDs with pedigree born in a single country and representing a large proportion of all dogs born within the same period. As the vast majority of dogs  in the present study had died by the end of the study, bias due to right-censored data was minimal in the present study. Major limitations of the study include a relatively large proportion of owners who chose not to participate or could not be located, creating possible responder bias. In addition, slightly over 20 % of dogs died or were euthanised without any specific diagnosis, and the diagnosis of neoplasia was mostly made without histologic confirmation. Moreover, a large proportion of diagnoses were of poor or moderate reliability. Possible reasons include unwillingness of owners to confirm diagnoses in cases of presumed poor prognosis, and unwillingness to perform post-mortem examinations in cases in which no specific antemortem diagnosis was evident. For owners unwilling to perform necropsy for emotional and/or spiritual reasons, minimally invasive digital imaging, such as X-Ray, Ultrasound, CT and MRI in combination with fine needle aspiration and needle biopsy, may constitute a promising tool to improve post-mortem diagnoses . In addiBesides analyses on the causes of death, the collection of health histories from birth to death, as well as clinical and genetic material for analyses of specific diseases may be valuable to more precisely assess breed health. Further studies, prospectively collecting health data from defined health populations or cohorts may enable breeding clubs to more accurately assess breed health.This study confirms the high prevalence of neoplasia in BMDs. Although the prevalence may be overestimated due to the lacking histological or cytological confirmation, neoplasia is an important factor for the low life expectancy in BMDs.The diagnostic accuracy leaves much to be desired and further research on malignant processes with a high diagnostic standard is necessary to improve breed health.ANIS, Animal Identity Services AG; BMD, Bernese mountain dog; HS, Histiocytic sarcoma


There is limited and controversial information regarding the potential impact of depression on glycemic control. This study aims to evaluate the association between depression and poor glycemic control. In addition, the prevalence of depression and rates of poor glycemic control were determined.ESSALUD in Peru. The outcome of interest was poor glycemic control, evaluated by glycated hemoglobin (HbA1c: < 7% versus ≥ 7%), whereas the exposure of interest was depression defined as 15 or more points in the Patient Health Questionnaire‐9 tool.Cross‐sectional study performed in the endocrinology unit of two hospitals of Poisson regression models with robust standard errors reporting prevalence ratios (PR) and 95% confidence intervals (95% CI) adjusting for potential confounders.The association of interest was evaluated using SD: 4.8), and 7.1 (SD: 6.8) years of disease were analyzed. Only 31 participants  had moderately severe or severe depression, whereas 70  had good glycemic control. Depression increased the probability of having poor glycemic control  after adjusting for several potential confounders.A total of 277 participants, 184 (66.4%) males, mean age 59.0 (There is an association between depression and poor glycemic control among type 2 diabetes patients. Our results suggest that early detection of depression might be important to facilitate appropriate glycemic control and avoid further metabolic complications. Worldwide, there are an estimated 382 million people living with type 2 diabetes (T2D), mostly aged between 40 and 59 years, and causing 5.1 million deaths and at least US$ 548 billion in health care expenditures  in Peru: Hospital Guillermo Almenara Irigoyen  and Hospital Alberto Sabogal Sologuren  between February and September 2014. Both are third‐level hospitals with specialized units and services. HGAI attends 80,000 patients yearly, whereas HASS has 78,000 patients annually , were invited to participate. Illiterate patients and non‐Spanish speakers were excluded from the study.The outcome of interest was poor glycemic control, assessed using HbA1c. Data were collected from clinical records up to three months before the date of interview as HbA1c reflects average plasma glucose over that previous time in a single measure , developed by Kroenke et al.  and vali et al.,  was usedet al., Sociodemographic variables included gender , age (<60 or ≥60 years), education level , work status (no or yes), place of birth , and hospital settings (HGAI or HASS). In addition, clinical history variables were evaluated such as time of disease, in years, and categorized in <5, 5–9, or ≥10 years; hospital status, evaluated as outpatients or inpatients; last year number of hospital admissions, categorized in 0, 1, 2, or ≥3; as well as hypertension diagnosis (no or yes), retinopathy diagnosis (no or yes), and diabetic foot diagnosis (no or yes). Finally, laboratory results were also assessed including total cholesterol, defined as <200 or ≥200 mg/dL, according to the Adult Treatment Panel III  or during current hospitalization (inpatients). Patients were recruited using a non‐probabilistic technique, consecutively as they presented in the consulting room or in the hospitalization area. After oral informed consent, a face‐to‐face questionnaire was applied containing detailed information regarding sociodemographics, clinical history, and information regarding depressive symptoms. The most recent laboratory results, including glycated hemoglobin (HbA1c), total cholesterol levels, and 24‐hour protein in urine values, were obtained from clinical records.et al., Sample size was calculated using the statistical package Power and Sample Size . Using a 5% significance level and 80% of power, 272 participants were required to find a strength of association ≥ 2.5, assuming that 73.6% of patients has poor glycemic control (HbA1c <7%)  for statistical analysis. First, a description of the study population was performed using proportions to compare population characteristics according to the presence of depression and glycemic control, our variables of interest. Comparisons between variables were performed using Chi‐squared test of Fisher's exact test accordingly. Second, prevalence and 95% confidence intervals (95% CI) of the variables of interest were calculated. Given the cross‐sectional nature of the study, as well as potential high prevalence of the outcome of interest  accepted participation and were then evaluated. Of them, 28 (9.2%) were excluded from further analyses because of incomplete information or data with inconsistencies. Sociodemographic characteristics of those included and excluded from analysis were not different (data not shown). Thus, 277 participants, 184 (66.4%) males, mean age 59.0 (SD: 4.8), and 7.1 (SD: 6.8) years of disease were analyzed.P = 0.04), place of birth (P = 0.02), and hospital setting (P = 0.01) were associated with depression. Detailed information of the population characteristics according to the presence of depression is shown in Table Based on PHQ‐9 results, 31 participants  presented moderately severe or severe depression. Only being currently working (P = 0.03), hospital setting (P < 0.001), time of disease (P = 0.002), number of hospital admissions in last year (P < 0.001), retinopathy diagnosis (P = 0.02), total cholesterol (P = 0.01), and 24‐hour proteins in urine (P < 0.001). Information is presented in Table Seventy participants  had good glycemic control based upon levels of glycated hemoglobin. In bivariable model, factors associated with adequate glycemic control were: age (P = 0.01). Depression increased the probability of having poor glycemic control  after adjusting for several potential confounders . Nevertheless, previous results from longitudinal studies showed that diabetes distress linked specifically to diabetes and its management, but not clinical depression or depressive symptoms seems to be associated with glycemic control (Fisher et al., et al., et al., There are several cross‐sectional studies reporting that depression is a factor associated with poor glycemic control among patients with T2D (Lustman et al., et al., et al., et al., Prevalence of depression among T2D patients is high when compared with general population (Huang et al., et al., et al., et al., et al., et al., et al., et al., On the other hand, rates of poor glycemic control are similarly consistent with previous international (Koro et al.,The present study benefits from using a validated instrument to assess depression, imposing a cutoff to better detect participants with depression, as well as the large number of confounders including in the models created utilizing a hierarchical approach to disentangle potential impact of these covariates. However, this study has several limitations. First, the study design, cross‐sectional in nature, can only determine association and not causality. Although we used regression models and adjusted for several confounders, longitudinal studies, with repeated participants' measures, are needed to corroborate our findings. Second, the HbA1c value was not obtained at the same time as the mental health assessment; however, we believe that the potential impact of this can be negligible as glycated hemoglobin reflects mean plasma glucose over the previous two to three months (World Health Organization, In conclusion, there is an association between depression and poor glycemic control among T2D patients. Our results suggest that early detection of depression might be important to facilitate appropriate glycemic control and thus avoid further metabolic complications.The authors declare they have no conflict of interest.


Great efforts have been made to develop fluorescent probes for pyrophosphate (PPi) detection. Nucleus staining with fluorescence microscopy has been also widely investigated. But fluorescent probes for PPi detection with high sensitivity in water medium and nucleus staining with low–cost non–precious metal complexes in living cells are still challenging. Herein, we report simple terpyridine–Zn(II) complexes for selective nanomolar PPi detection over ATP and ADP in water based on aggregation induced emission (AIE) and intramolecular charge transfer (ICT). In addition, these terpyridine–Zn(II) complexes were successfully employed for nucleus staining in living cells. These results demonstrated simply obtained terpyridine–Zn(II) complexes are powerful tool for PPi detection and the development of PPi–related studies. Inspired by these results, we proposed a strategy for PPi detection by combination of its coordination affinity with Zn2+ and AIE as illustrated in Anions play important roles in a variety of biological and environmental processes. Pyrophosphate (PPi) anion has received a lot of attention due to its considerable role in cellular metabolic processes and several diseases such as ATP hydrolysis and calcium pyrophosphate dihydrate crystal deposition disease12346789101112131516181920Besides, fluorescence imaging cellular nucleus with luminescent dyes has also been widely utilized in biological research2627283031322+, known as coordination–driven self–assembly39404142434546Recently, terpyridine derivatives and their Zn(II) complexes have emerged as one–photon and two–photon dyes used in bioimaging due to their rich photophysical properties353637CZtpyZn for the detection of PPi, its fluorescence response to PPi was first studied in HEPES buffer . CZtpyZn exhibited weak orange emission at 570 nm. However, upon addition of PPi dissolved in water, the emission showed a blue shift to 515 nm with green emission, accompanied with significant enhancement of intensity (CZtpyZn) . On the other hand, the Job plot verified this binding stoichiometry (CZtpyZn was found to be 5.37 nM (S/N = 3), indicating CZtpyZn is a highly sensitive probe for the detection of PPi.To assess the possibility of ntensity . The greCZtpyZn) . Good liCZtpyZn) , suggesthiometry , in whicCZtpyZn, we further studied its ability of detecting PPi in more complicated environment, especially in the presence of other competing nucleotide phosphates such as ATP, ADP and AMP. As illustrated in 32−, SO42−, HSO4−, Br−, I−, NO3−, CH3COO−, SO32−, H2PO4−, HPO42− and PO43− led to negligible changes for the emission of CZtpyZn (10 μM). Nucleotide phosphates including ATP, ADP and AMP also induced less effects on the emission compared with PPi. The competitive experiments showed that all anion solutions displayed green emission after subsequent addition of 0.4 equivalent (4 μM) PPi, even though weak yellow emission was observed in presence of ATP and ADP before addition of PPi  in water. Then, interestingly, as shown in CZtpyZn mainly lighted up the nucleus of HeLa cells, while very weak emission in the cytoplasm. Although the emission intensity of CZtpyZn was weaker than that of DAPI, CZtpyZn stained cells with same region as DAPI  level, which is far beyond the LOD of in water . We foun as DAPI . Low cyt as DAPI . In addied cells . The coned cells . TherefoAMtpyZn) was also facilely synthesized and developed for the detection of PPi in HEPES buffer  and imaging cells  of PPi with AMtpyZn was determined to be 9.72 nM (S/N = 3). We also employed AMtpyZn for cell imaging. However, AMtpyZn displayed different cell staining behavior from that of CZtpyZn, which lighted up a whole cell  complexes can be manipulated by modifying their molecular structure.To manipulate the staining behavior of terpyridine–Zn(II) complexes, another new amino–modified terpyridine–Zn(II) complexes  complexes bearing two terpyridine ligands were not capable of staining nucleus but only cytoplasm37CZtpyZn and AMtpyZn bearing one terpyridine ligand and liable leaving group (Cl− anions) are capable of entering into cellular nucleus probably because of their coordination with phosphates in nucleus, especially PPi anions. This suggested that PPi anions not only induced strong emission of CZtpyZn and AMtpyZn but also PPi anions were helpful for their uptake and entry into cellular nucleus. The different staining behavior is ascribed to the structure difference between carbazole group and amino group. The carbazole–based compounds are widely studied owing to its donor ability, good planarity, rich photophysical properties and excellent biocompatibility5051As mentioned, In summary, we successfully developed simple terpyridine–Zn(II) complexes for nanomolar PPi detection in water medium based on AIE and ICT. Nanoaggregates were confirmed in the detection processes. The carbazole–modified terpyridine–Zn(II) complex has a LOD of 5.37 nM and the amino–modified terpyridine–Zn(II) complex has a LOD of 9.72 nM. These results demonstrated that anion–induced aggregation emission is a powerful strategy for anion detection in water. Furthermore, taking advantage of the high sensitivity towards PPi, we employed them in cell imaging and found that the carbazole–modified terpyridine–Zn(II) complex mainly lighted up the nucleus and the amino–modified terpyridine–Zn(II) complex stained a whole cell. We believe the simply obtained and low–cost terpyridine–Zn(II) complexes have great potential applications in PPi–related studies such as cell imaging and diagnosis.1H NMR spectra were carried out using a Bruker Avance 500 MHz instrument with tetramethysilane (TMS) as an internal standard. HRMS spectra were recorded using a Thermofisher Q–Exactive instrument. Emission spectra were measured by Lengguang Tech. Instruments (F97PRO) with Xe lamp as the light source. UV–vis absorption spectra were obtained on a PerkinElmer Lambda 950 spectrophotometer. The scanning electron microscope (SEM) images were carried out on a Hitachi S–4800 scanning electron microscope. TEM image was recorded on JEM–2100 at 200 kV. The solid state quantum yield was measured using a calibrated integrating sphere on a Quantaurus–QY spectrophotometer. Particle size was determined with Malvern Nano ZS. Confocal Laser Scanning Microscope experiments for cell imaging were carried out with ZEISS LSM 510 according to the literature procedureAll materials were purchased from commercial suppliers and used as received. Cell lines were obtained from the lab of Prof. Zhigang Xie in Changchun Institute of Applied Chemistry Chinese Academy of Sciences. 2  in CH3OH (5 mL) was added into a solution of the carbazole–modified terpyridine ligand2Cl2 (8 mL). The solution was stirred at 40 °C for 3 h. Then the precipitate was filtered, washed with water, methanol, and diethyl ether. The pure product was obtained after dried under vacuum. Yield: 47.0 mg, 60%. 1H NMR , δ (ppm): 9.21 , 9.03 , 8.86 , 8.47 , 8.37 , 7.89 , 7.73 , 7.56 , 7.35 , 4.58 , 1.40 . ESI–HRMS (m/z): found 525.0824 for [M–Cl]+ .The synthetic route is shown in 1H NMR , δ (ppm): 8.97 , 8.84 , 8.34 , 8.21 , 7.87 , 6.89 , 3.49 , 1.18 . ESI–HRMS (m/z): found 479.0980 for [M–Cl]+ .AMtpyZn was prepared using the same procedure as CZtpyZn, but the carbazole–modified terpyridine ligand was replaced by the amino–modified terpyridine ligandHow to cite this article: Chao, D. and Ni, S. Nanomolar pyrophosphate detection and nucleus staining in living cells with simple terpyridine–Zn(II) complexes. Sci. Rep.6, 26477; doi: 10.1038/srep26477 (2016).


Takifugu obscurus, a species of anadromous fish, experiences several salinity changes in its lifetime. Cadmium (Cd) is a toxic heavy metal that can potentially induce oxidative stress in fish. The present study aimed to detect the combined effects of Cd  and salinity  on juvenile T. obscurus. Results showed the juveniles could survive well under different salinities; however, with Cd exposure, the survival rates significantly decreased at 0 and 30 ppt. At 15 ppt, tolerance to Cd increased. Cd exposure clearly induced oxidative stress, and the responses among different tissues were qualitatively similar. Salinity acted as a protective factor which could reduce the reactive oxygen species and malondialdehyde levels. In addition, salinity could enhance the antioxidant defense system, including superoxide dismutase, catalase and glutathione. Na+/K+–ATPase activity significantly decreased under Cd exposure in gill, kidney and intestine. These findings indicated that Cd could moderate the adaptability of juvenile T. obscurus to high salinity and low salinity played a protective role upon Cd exposure. Thus, the role of salinity should be considered when evaluating the effect of heavy metals on anadromous and estuarine fishes.Obscure puffer  With the increase in freshwater contamination by numerous natural and industrial chemical compounds, Cd has been considered an important global issue because of its persistence and capacity for bioaccumulation1Takifugu obscurus, an ecologically and economically important fish in the Yangtze River in China, is a species of anadromous fish. T. obscurus migrates to freshwater rivers to reproduce during the spawning season from February to May. Newly hatched larvae remain in freshwater for several months and then move to the sea for one or two years until sexual maturity is reached. After approaching maturity, the fish return to freshwater rivers to spawn14T. obscurus a useful model species for studying osmoregulation+/K+–ATPase is an important membrane protein that provides the driving force for ion regulation and mediates whole-body osmoregulation among aquatic organisms. Therefore, salinity significantly affects Na+/K+–ATPase activities17Obscure puffer Crassostrea virginica) tissue and shell were found to be 35 and 42 times higher than those of Gulf of Mexico sediment samplesGracilaria lemaneiformis were exposed to different concentrations of Cd  for 28 days, the Cd concentrations in G. lemaneiformis were increased 2.32, 3.42, 8.65 and 10.15 times respectively compared to those in the control −1 , the T. obscurus, which preys on shellfish and plankton with enriched Cd, is still probably damaged by Cd. Although the independent effects of Cd and salinity on T. obscurus have been investigated16T. obscurus juveniles as a bioindicator in the present study to investigate the potential interactions between waterborne Cd exposure and environmental salinity. We considered this study to be meaningful and proposed the following three hypotheses: (1) exposure to Cd may moderate the adaptability of juveniles to high salinity, which will be determined by observing and recording the death of juveniles daily and the changes in Na+/K+–ATPase activities; (2) sublethal waterborne Cd exposure may induce oxidative stress under different salinity levels, which will be evaluated by measuring ROS and MDA levels in various tissues of juveniles and the antioxidant enzyme activities ; (3) certain salinity may reduce Cd damage, which will be evaluated by comparing survival rates, ROS levels and oxidative stress parameters among the juveniles cultured under different concentrations of Cd ) and salinity (0 and 15 ppt).The concentrations of Cd in oyster (−1 (−1). With increasing exposure time, the survival rates declined sharply under Cd concentrations >10 mg L−1 at all salinity treatments. After exposed to Cd (5 and 10 mg L−1) for 96 h, the survival rates were higher at salinity of 15 ppt (70% and 70%) than 0 (60% and 10%) and 30 ppt (0% and 0%). Meanwhile, the survival rates at different salinity levels without Cd exposure exceeded 90%. The Cd EC50 values of survival rates at 15 ppt were higher than at other salinities , kidney (p = 0.006), intestine (p = 0.022) and liver (p < 0.001) (The response of SOD activities is illustrated in < 0.001) .Salinity itself did not significantly affect CAT activities without Cd exposure . After ep < 0.001), kidney (p = 0.039), muscle (p < 0.001) and liver (p < 0.001)  . A signi< 0.001) .+/K+–ATPase activities significantly increased at 15 ppt than at 0 ppt without Cd exposure in the gill, kidney and intestine. However, after exposed to Cd for 96 h, these activities significantly decreased at 15 ppt (+/K+–ATPase activities exhibited a significant decrease under Cd exposure at 0 ppt. Cd significantly affected Na+/K+–ATPase activities in the gill (p = 0.015), kidney (p = 0.003), intestine (p = 0.002) and liver (p < 0.001), but only an interaction was noted between Cd and salinity in the gill (p = 0.039) (Nat 15 ppt . In the = 0.039) .T. obscurus to high salinity (30 ppt); (2) sublethal waterborne Cd exposure (5 mg L−1) would induce oxidative stress under different salinity levels; and (3) salinity acted as a protective factor in reducing Cd damage.The present study supports the following three hypotheses: (1) Cd exposure will moderate the adaptability of T. obscurus survival at different salinities. T. obscurus can tolerate a wide salinity variation and live well in sea, river and estuary water. However, with industrial development, Cd pollution in water is considerably more serious, thereby possibly inducing combined effects with salinity on fish.The contamination of Cd in aquatic systems is a consequence of industrial, agricultural, and other anthropogenic activities. Cd can accumulate in fish tissues and may become toxic for fish after tissue metal accumulation reaches a substantially in high level, depending on metal type, exposure duration, fish species, and water chemistry22T. obscurus could survive well at different salinities. However, upon Cd exposure of juveniles, the survival rates decreased significantly at high concentrations of Cd  at 0 and 30 ppt. As exposure time was prolonged, the survival rates continued to decline with each salinity level. After 96 h of exposure, in the lower concentration of Cd (5 mg L−1), the survival rate still reached 70% at 15 ppt , hydrogen peroxide (H2O2) and hydroxyl radical (HO•−)T. obscurus21T. obscurus is first reported in this study.The oxidative stress in response to Cd in fish has been the focus of numerous studies262729p < 0.05). However, the increase in MDA at 15 ppt was less than that at 0 ppt which indicated that the injury was lighter at higher salinity. The significant decreased of MDA levels were detected at 15 ppt after exposed to Cd, while it was not observed in ROS. These findings may be attributed to significantly higher SOD, CAT and GSH at 15 ppt than at 0 ppt upon Cd exposure; thus, the excess ROS were cleared.Aerobic organisms are known to continuously produce ROS even when they simply maintain a basal level of metabolism. In this study, Salinity did not exert significant effect on ROS . We also33T. obscurus. The results showed that antioxidative enzymes of fishes were more active in salinity treatment than in freshwater. We also observed that SOD activity in each tissue was higher than CAT activity under Cd exposure at 15 ppt, especially in the gill and liver. It is speculated that the increase in SOD may lead to the accumulation of H2O2, which will restrain the function of CAT4244T. obscurus. A previous study speculated the mechanism of salinity protection against heavy metal zinc uptake in Fundulus heteroclitus2+ ion in total zinc concentration; and salinity also increased the concentration of potentially competitive cations, such as Ca2+ which competes with the Zn2+ uptake mechanism in the gills. These mechanisms may explain the changes in ROS, MDA and antioxidant enzymes in our study, but the defense of fish itself should also be taken into consideration.Aquatic animals possess an evolutionary-conserved antioxidant defense system for the removal of excess ROS. This defense system is composed of antioxidative enzymes and non-enzymatic antioxidants3839+/K+–ATPase is a membrane protein that facilitates the active exchange between two extracellular K+ ions and three intracellular Na+ ions as one ATP molecule is hydrolyzed. In this process, crucial K+ and Na+ concentration gradients between the cytosol and extracellular fluid are maintained+/K+–ATPase activity following salinity changes17+/K+–ATPase activity of juvenile T. obscurus in gill, kidney and intestine+/K+–ATPase activities of the gill, kidney and intestine were significantly inhibited at 15 ppt. This finding suggested that most of the energy was used to resist against free radicals caused by Cd, and the ability of osmotic regulation was diminished. Thus, we can explain why juveniles could not tolerate high salinity (30 ppt) when exposed to low Cd concentration (5 mg L−1). In freshwater, Na+/K+–ATPase activity in the liver decreased significantly after 96 h of exposure to Cd, as the liver is an important organ of body that functions in detoxification. However, muscle cells cannot synthesize more Na+/K+–ATPase to maintain ion homeostasis in the cytoplasm. Overall, Na+/K+–ATPase activities of tissues were inhibited by Cd under different salinities. Certain salinity can induce high Na+/K+–ATPase activities in T. obscurus+/K+–ATPase activities are negatively correlated with oxidative stress49The main function of transmembrane ATPase is to provide energy for ion transportation across membranes and maintain ion homeostasis in the cytoplasm. NaT. obscurus could tolerate high salinity (30 ppt); however this adaptability was moderated by Cd exposure. Sublethal Cd exposure significantly induced oxidative stress in T. obscurus. When fish were exposed to Cd for 96 h, ROS and MDA levels decreased, and the antioxidant defense system  was enhanced at 15 ppt in five tissues which suggested that salinity could reduce the damage caused by Cd to T. obscurus and enhanced the antioxidant defense system. Thus, within the suitable salinity and sublethal range of waterborne Cd, salinity played a protective role for juveniles under Cd exposure. Previous studies confirmed that Cd2+ is more toxic to Photobacterium phosphoreum, Daphnia magna and Carassius auratus in alkaline environments (pH = 9) than in acidic environments (pH = 5)−1 as CaCO3 for nitrifying and 400 mg L−1 as CaCO3 for saprophytic bacteriaT. obscurus is a useful animal model to assess the risk of various pollutants in anadromous and estuarine fishes at different salinities.In summary, juvenile All experiments were approved by and carried out in accordance with the guidelines of the Institutional Animal Care and Use Committees (IACUC) of Nanjing Normal University, Nanjing, China.T. obscurus juveniles  were obtained from a Yangzhong obscure puffer hatchery , transferred to our laboratory and maintained in an aquarium containing 100 L of freshwater. No Cd was present in the tissues and diets of the fish. The rearing conditions in the laboratory were similar to those of the fish farm . About 500 juveniles were placed in three aquariums. The commercial fish diet was supplied to satiation twice a day, without any residue left. Half of the water was renewed daily to maintain good water quality. After 7 days, healthy juveniles were selected to assess the actual effect of Cd and salinity on survival and biochemical responses in juveniles.The −1 . The three salinities were as follows: 0, 15 and 30 ppt. Therefore, 5 × 3 combinations of treatment were established, and each treatment had three replicates. For ease of expression, the following Cd concentrations were used to indicate CdCl2 concentrations. Ten juveniles were placed in an aquarium filled with 30 L of test solution and continuously aerated. To acclimate to the designated salinity, we added refined industrial salt and ensured that water salinity increased 3 ppt every 6 hours until reached our pre-set salinities; the juveniles continued to adapt to 3 days in each salinity and then add different concentrations of CdCl2. Cd concentrations and salinity levels were monitored daily to ensure relatively constant metal and salinity exposures. Cd concentrations were analyzed using a flame atomic absorption spectrophotometer−1. The number of dead juveniles was recorded and removed daily. After 96 h of exposure, one fish was randomly selected from each of these aquariums . The juveniles were euthanized with a lethal dose of NaOH-neutralized MS-222. The biochemical analysis was performed on individual. Samples of gill, kidney, intestine, muscle and liver were rapidly removed by dissection, rinsed with cold physiological saline (0.6% NaCl), dried in filter paper, frozen in liquid nitrogen, and stored at −80 °C. For ROS detection, a slice of each tissue was measured immediately after rinsing and drying.We used a full-factorial design for Cd and salinity. The five concentrations of Cd were as follows: 0, 5, 10, 20 and 50 mg L+/K+–ATPase) activities, ROS, MDA and GSH levels in supernatants were measured using the Diagnostic Reagent Kits according to the manufacturer’s instructions . Protein concentrations were estimated using Diagnostic Reagent Kit (Coomassie protein assay dye).The samples were placed in a Dounce homogenizer with 0.6% physiological saline solution and then homogenized. Subsequently, each tissue was centrifuged at 3000 rpm for 10 min at 4 °C to eliminate cellular debris and cartilage fragments. Supernatants were removed and used for biochemical parameter assays. Enzyme  with a significance level of 0.05 to determine the effects of Cd exposure, salinity as well as their interaction on ROS, MDA, SOD, CAT, GSH, and Na+/K+–ATPase values (tested separately) in each tissue. Before ANOVA, we had conducted a homogenous test and passed it (p > 0.05). The post-hoc test of holm-sidak method was employed for multiple comparisons. All statistical analyses  were carried out using SigmaPlot 11.0.All data were expressed as treatment mean ± standard error (SE). The ECHow to cite this article: Wang, J. et al. Combined effects of cadmium and salinity on juvenile Takifugu obscurus: cadmium moderates salinity tolerance; salinity decreases the toxicity of cadmium. Sci. Rep.6, 30968; doi: 10.1038/srep30968 (2016).


Drosophila melanogaster (D. melanogaster) eggs with HHP. Approximately 75% of the surviving flies showed significant morphological abnormalities from the egg to the adult stages compared with control flies (p < 0.05). Some eggs displayed abnormal chorionic appendages, some larvae were large and red, and some adult flies showed wing abnormalities. Abnormal wing phenotypes of D. melanogaster induced by HHP were used to investigate the mutagenic mechanisms of HHP on organism. Thus 285 differentially expressed genes associated with wing mutations were identified using Affymetrix Drosophila Genome Array 2.0 and verified with RT-PCR. We also compared wing development-related central genes in the mutant flies with control flies using DNA sequencing to show two point mutations in the vestigial (vg) gene. This study revealed the mutagenic mechanisms of HHP-induced mutagenesis in D. melanogaster and provided a new model for the study of evolution on organisms.Although High hydrostatic pressure (HHP) as an important physical and chemical tool has been increasingly applied to research of organism, the response mechanisms of organism to HHP have not been elucidated clearly thus far. To identify mutagenic mechanisms of HHP on organisms, here, we treated  Drosophila melanogaster (D. melanogaster). D. melanogaster has been used as a powerful genetic model for approximately one century because many genes and signal pathways are evolutionarily conserved between humans and D. melanogasterD. melanogaster also provide an excellent model for studying the response mechanisms of stress resistance in organismsD. melanogaster with abnormal wings from mutant populations induced by HHP, and we investigated the mechanism for this mutagenesis using DNA microarray, RT-PCR and DNA sequencing technologies. Our objectives may facilitate identifying and characterizing the mechanisms of HHP mutagenesis in organisms and elucidate the molecular underpinnings for the application of HHP to the evolution of organisms.High hydrostatic pressure(HHP), a physical parameter, which can produce aunique effect on biological systems, has caused great interest of interdisciplinary researchers such as biophysicists and biochemists123569D. melanogaster. To determine whether HHP induced mutagenic effects in D. melanogaster, we treated D. melanogaster eggs using a pressure of 60 MPa for 20 min at room temperature. Approximately 75% of the flies that survived the HHP treatment showed significant morphological abnormalities from the egg stage to the adult stage compared with control flies (p < 0.05). The eggs displayed abnormal chorionic appendages, the larvae were large and red, the pupae had melanotic tumors, and the adult flies showed wing abnormalities. Normally, the wing is oval shaped, with a length two times than that of the abdomen and five longitudinal veins and two cross veins as well as established wing margins. However, the abnormal wings of D. melanogaster treated with HHP showed degradation and obvious modifications in size, shape, vein patterning, and margin formation   . To inve < 0.05) . The cel < 0.05) . The mol < 0.05) . In addind Nedd4 . To confnd Nedd4 . Most  and scalloped (sd) genes, which are involved in wing development. The sequencing results indicated that the sd gene sequence remained unaltered; however, two point mutations, four bases deletion and an insertion sequence occurred in the vg gene of D. melanogaster. The first point mutation was a transversion from C to T at nucleotide 1892 in complete cDNAs of vg gene  used in this study originated from the Institute of Genetics and Cytology, Northeast Normal University, Changchun (China). The flies were maintained according to the report by Sørensen’s. et al.D. melanogaster eggs were collected from healthy flies by placing a fresh food large plate in the big box for 1 h. These eggs were transferred into sterile bags (300 eggs/bag). Subsequently, each bag with eggs was exposed to pressure of 60 MPa for 20 min at room temperature. The control eggs were maintained under normal atmospheric pressure (0.1 MPa).For HHP experiments, To screen mutant strains, the flies that survived the HHP treatment were observed with electron microscope and stereomicroscope, and screened different mutants at all stages from eggs to adult flies.DNA microarray analyses were performed according to the protocols of Affymetrix and the methods described previously27http://sas.ebioservice.com/.Data analysis and statistical analyses were performed using the SBC Analysis System from the website Total RNA was isolated from the control and mutant flies using RNAiso Plus . RT-PCR analyses were performed according to previously published protocols29Genomic DNA was extracted from individual fly using a unique method in its own right. Polymerase chain reaction (PCR) was performed in a volume of 22 uL that contained 1 μL of template DNA (100 ng/μL), 0.4 μL of DNA polymerase , 2.0 μL of 10 × buffer, 1 μL of dNTPs (2.5 mM each), and 0.4 μL of each primer (10 μM), and 16.8 μL sterilized double distilled water. The conditions for PCR were as follows: denaturation for 5 min at 94 °C followed by 45 sec at 94 °C, annealing for 30 sec at 59 °C, and extension for 1 min at 72 °C. This procedure was repeated for 35 cycles with a final 7 min extension at 72 °C. The amplified products were separated on 2% agarose gels. The PCR products were excised from the gel, purified with the AxyPrep DNA gel extraction kit, and sequenced.How to cite this article: Wang, H. et al. Molecular Mechanisms for High Hydrostatic Pressure-Induced Wing Mutagenesis in Drosophila melanogaster. Sci. Rep.5, 14965; doi: 10.1038/srep14965 (2015).


Xmn1‐HBG2, HMIP‐2, and BCL11A, but the effect of these loci on the hematological phenotype in SCD, has so far not been investigated. In a cohort of individuals with SCD in Tanzania , HbF levels were positively correlated with hemoglobin, red blood cell (RBC) indices, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH), and negatively with white blood cell (WBC) and platelet counts . We subsequently assessed the contribution of the three HbF modifier loci and detected diverse effects, including a reduction in anemia, leukocytosis, and thrombocytosis associated with certain HbF‐promoting alleles. The presence of the ‘T’ allele at Xmn1‐HBG2 led to a significant increase in hemoglobin (P = 9.8 × 10−3) but no changes in cellular hemoglobin content. Xmn1‐HBG2 ‘T’ also has a weak effect decreasing WBC (P = 0.06) and platelet (P = 0.06) counts. The BCL11A variant (rs11886868‐‘C’) increases hemoglobin (P = 2 × 10−3) and one of the HBS1L‐MYB variants decreases WBC values selectively (P = 2.3 × 10−4). The distinct pattern of effects of each variant suggests that both, disease alleviation through increased HbF production, and ‘pleiotropic’ effects on blood cells, are involved, affecting a variety of pathways. Am. J. Hematol. 90:E1–E4, 2015. © 2014 Wiley Periodicals, Inc.Fetal hemoglobin (HbF) is a recognized modulator of sickle cell disease (SCD) severity. HbF levels are strongly influenced by genetic variants at three major genetic loci,  Gγ globin chain of HbF (termed Xmn1‐HBG2) and clusters of variants in regulative elements for two hematopoietic transcription factors, BCL11A and MYB. BCL11A acts as a repressor of γ globin gene expression, whereas genetic variation near MYB  affects the levels of HbF indirectly by altering the kinetics of erythropoiesis HMIP‐2 locus influences average volume (MCV), hemoglobin content (MCH), and number (RBC) of erythrocytes HMIP‐2 affects hemoglobin (Hb), hematocrit (HCT), mean corpuscular hemoglobin concentration (MCHC) BCL11A variants have generally weaker pleiotropic effects, on RBC, MCV, and MCH Sickle cell disease (SCD) is an inherited hemoglobin disorder caused by the Glu6Val mutation in the β globin chain. It has a devastating impact in Sub‐Saharan Africa, where it is highly prevalent and a significant cause of childhood mortality BCL11A, HMIP‐2, and Xmn1‐HBG2, in 726 Tanzanian SCD patients, who have minimal disease intervention such as regular blood transfusion or hydroxyurea therapy.We wanted to know how the natural variability in HbF levels and the known genetic HbF modifiers influence the hematological phenotype of SCD patients. For this purpose, we studied general blood cell parameters, peripheral HbF levels, and genotype at the three main HbF modifier loci, The Muhimbili Sickle Cell Cohort has been described previously 0 thalassemia genotype) and HbF quantification were carried out by High Performance Liquid Chromatography . Hematological parameters  were measured with an ABX Pentra 60 Analyzer . Mean/median values for our cohort are shown in the Supporting Information Table SI.Confirmation of diagnosis , aged 5–43 years .Patients were excluded if they were on hydroxyurea therapy, younger than 60 months of age, tested malaria‐positive, had pain, fever, or had been hospitalized 30 days before or after study, or were lacking alpha thalassemia (3.7 deletion) data. Only patients with Hb SS or HbS/βA and B) present at HMIP‐2 in individuals of African descent (Table rs11886868 (for BCL11A), rs9389269 (for HMIP‐2B), and rs7482144 , or by PCR fragment sizing  Genetic variants were selected from ten SNPs genotyped at the three main HbF modifier loci, resulting a set of four, one effectively tagging HbF‐associated genetic variability at each locus nt Table . They wea priori in all regression models. WBC, MPV, and HbF were log‐transformed. The genetic association analysis of the four tag markers with hematological parameters was repeated with ln[HbF] as a covariate, to test for the dependence of genetic effects on HbF levels.Multiple linear regression  was used to test for association of genetic markers with hematological parameters and HbF, as well as for the influence of HbF on blood cell parameters. Alpha‐thalassemia status (3.7 deletion), age  and sex were included BCL11A, HMIP, and Xmn1‐HBG2, on the hematological phenotype of Tanzanian SCD patients.We detected a significant influence of HbF levels and of variants at three major HbF modifier loci, P = 5.49 × 10−6), and negatively with WBC  and platelet counts . Hb gains with higher HbF were accompanied by increases in MCV  and MCH , while RBC was unchanged (P = 0.97).HbF levels associated positively with hemoglobin  had by far the largest allelic effect on Hb , with a tendency towards increased RBC , but no change in MCV and MCH values (P > 0.1). When adjusting for the influence of HbF levels, the Hb‐increasing effect of rs7482144‐‘T’ remained large and significant  suggesting independence of this relationship from HbF levels. BCL11A (rs11886868‐‘C’) also had a significant effect on Hb , but this ceased to be significant after adjusting for HbF levels (P > 0.1). The two HbF‐increasing variants at HMIP‐2  are uncommon in our population and no significant effect on Hb was detected. However, the HMIP‐2B variant had a significant positive effect on MCV and MCH and the HMIP‐2A variant had a negative effect on WBC , which was not diminished when adjusting for HbF.HbF levels were strongly influenced by all four variants tested, confirming previous findings rs11886868: Beta = 0.19, P = 0.002, rs7482144: Beta = 0.79, P = 9.8 × 10−3), suggesting that they contribute independently and therefore beneficial effects might add their effects when occurring in the same individual. Accordingly, patients with HbF‐promoting alleles at both loci (one at Xmn1‐HBG2 and either one or two at BCL11A) had Hb levels of up to 8.5 g/dl on average, compared with 7.3 g/dl for patients lacking any such allele  were, in general, similar to that of HbF itself: Hb, MCV, and MCH were increased when the loci were analyzed jointly, using a summary score. However, individual analysis of the four variants revealed distinct patterns of effects, suggesting that diverse biological mechanisms are involved. The ‘T’ allele at Xmn1‐HBG2 is a component of the ‘Arab‐Indian’ β globin gene cluster haplotype Xmn1‐HBG2‐T allele, Hb was raised, but MCV and MCH were not, probably due to the direct [34], ‘pancellular’ effect of β globin cluster variants on HbF production.Effects of the known genetic HbF modifiers (HbF‐increasing variants at BCL11A (rs11886868‐C) had a significant effect on Hb, which was HbF‐dependent. The HbF increase due to this allele (Beta) was small, but as it is highly prevalent in this population , it created an overall significant impact. Five patients had HbF increasing alleles at both BCL11A and Xmn1‐HBG2 loci, resulting in maximum Hb levels  and we detected no effect on Hb. HMIP‐2B (rs9389269) does influence MCV and MCH, a finding that resembles pleiotropic HMIP‐2 effects observed in nonanemic individuals HMIP‐2A, but not HMIP‐2B, has a striking effect on WBC, independent of HbF. HMIP‐2 variants have been reported to influence the WBC count in healthy populations L11A rs1186868‐C hWe believe that the significant effects of the three modifier loci on general blood traits we have shown represent a combination of both, disease amelioration through HbF modification and pleiotropic effects, and that the mechanisms underlying both phenomena are diverse and gene‐specific. To explore this further, we will increase the power provided by our cohort by recruiting more patients and by broadening the scope of biological systems tested. Inclusion of additional hematological data in future analysis is expected to account for part of the background variability, thus improving our ability to detect more subtle genetic effects.The authors thank the patients and staff of Muhimbili National Hospital, Muhimbili University of Health and Allied Sciences (MUHAS), Tanzania, Hematology Outpatient Unit and staff of King's College Hospital, London, and members of Professor Thein's Molecular Hematology group, King's College London. We extend our special thanks to J. Mgaya, H. Mariki of the Muhimbili Wellcome Programme, MUHAS and H. Rooks from Professor Thein's Molecular Hematology group. The sponsors of this study are nonprofit organizations that support science in general. They had no role in gathering, analyzing, or interpreting the data. Siana Nkya Mtatiro is a PhD candidate at Muhimbili University of Health and Allied Sciences and this work is submitted in partial fulfillment of the requirement for the PhD.S.L.T., S.M., S.N.M., S.E.C., and J.M. designed the study. S. M and S.N.M. designed and performed the genotyping assays. B.M. performed the initial analysis. S.N.M., S.M., S.L.T., and S.E.C. wrote the manuscript and all authors commented on the drafts of the manuscript.Supporting InformationClick here for additional data file.


The familial predilection toward autoimmunity is in part due to genetic risk factors, and recent studies have greatly expanded our understanding of the complex genetic architecture underlying autoimmune disease. Technical advances and the assembly of large patient cohorts have resulted in rapid progress in the field, which does not appear to be slowing. Studies which have included multiple ancestral backgrounds have demonstrated differences in risk factors between ancestral backgrounds, as well as some similarities –3. Also,In the current issue, a series of papers highlight some exciting topics within autoimmune disease genetics. M. G. Zavala-Cerna et al. explore genetic associations between PAD4 and rheumatoid arthritis in a Mexican cohort. The PAD4 enzyme is implicated in posttranslational protein modification characteristically targeted by rheumatoid arthritis-associated autoantibodies. B. N. Frederiksen et al. examine genetic polymorphisms underlying type I diabetes and islet cell autoimmunity, finding both age- and disease-stage relevant differences in association. This study illustrates the complex ways in which genetic factors can influence disease, and it is likely that this complexity occurs in many different autoimmune diseases. Genetic risk alleles may depend on other factors such as age, disease stage, and environment to influence risk of disease. Discovering these relationships will greatly improve our understanding of autoimmune disease pathogenesis. C. E. Weckerle et al. report a familial aggregation study looking at circulating levels of tumor necrosis factor alpha, a cytokine which is elevated in lupus patients , in unafS. A. Zavaleta-Muñiz et al. study polymorphisms in the IL6 gene with regard to susceptibility to rheumatoid arthritis. A. Zóka et al. provide a comprehensive review of the alterations in the immune system which are related to type I diabetes. H. C. Chai et al. examine polymorphisms in genes within the Toll-like receptor and type I interferon pathways in systemic lupus erythematosus patients from a South Asian population, extending our knowledge of these susceptibility genes to an additional world population. While one issue cannot be comprehensive, the studies included in this issue provide an overview of some of the current frontiers in the genetics of autoimmune disease.Timothy  B.  NiewoldTimothy  B.  NiewoldGeorge  N.  GoulielmosGeorge  N.  GoulielmosShervin  AssassiShervin  Assassi


Hum Brain Mapp 36:4104–4115, 2015. © 2015 The Authors Human Brain Mapping Published by Wiley Periodicals, Inc.This functional magnetic resonance (fMRI) study was designed to investigate changes in functional patterns of brain activity during creative ideation as a result of a computerized, 3‐week verbal creativity training. The training was composed of various verbal divergent thinking exercises requiring participants to train approximately 20 min per day. Fifty‐three participants were tested three times (psychometric tests and fMRI assessment) with an intertest‐interval of 4 weeks each. Participants were randomly assigned to two different training groups, which received the training time‐delayed: The first training group was trained between the first and the second test, while the second group accomplished the training between the second and the third test session. At the behavioral level, only one training group showed improvements in different facets of verbal creativity right after the training. Yet, functional patterns of brain activity during creative ideation were strikingly similar across both training groups. Whole‐brain voxel‐wise analyses  revealed that the training was associated with activity changes in well‐known creativity‐related brain regions such as the left inferior parietal cortex and the left middle temporal gyrus, which have been shown as being particularly sensitive to the originality facet of creativity in previous research. Taken together, this study demonstrates that continuous engagement in a specific complex cognitive task like divergent thinking is associated with reliable changes of activity patterns in relevant brain areas, suggesting more effective search, retrieval, and integration from internal memory representations as a result of the training.  In view of the rapidly increasing complexity of the world around us “creativity is more important now than ever before” Runco, , p. 658 Research from the psychometric research tradition has indeed revealed some factors that may unfold beneficial effects on creativity, among the most important being positive affect ) ) [cf. Fink et al., z‐standardized fluency and originality scores of the CE task.Verbal creativity was assessed by means of verbal idea generation tasks  [Urban and Jellen, tin” or “umbrella”). In the control task, participants worked on the Instances Task (IT) [Wallach and Kogan, BOLD response was measured during the performance of the AUT, which requires the participants to generate unusual and original uses of given conventional everyday objects , resulting in a total number of 40 trials. At each time point a different set of AUT and IT stimuli was used. As shown in Figure z‐standardized scores for AUT fluency and AUT originality. This composite measure was significantly (P < 0.01) associated with the total score of the full‐length verbal imagination subscales of the BIS, a widely used and proven psychometric measure of verbal creativity . This finding supports the validity of the AUT during fMRI assessment.Creative task performance during fMRI assessment was quantified via ideational fluency  and originality of ideas in the AUT. The originality rating procedure of the AUT responses was similar as for the psychometric tests outside the scanner. Again four raters evaluated the ideas of the participants on a four‐point rating scale ranging from “0”  to “3” . The raters received lists of all (nonredundant) responses of the participants in an alphabetical order and were hence blind to the relationship of responses and experimental groups. For each trial we computed a top‐1 score , which then was averaged across all 20 trials. Inter‐rater agreement was satisfactory (intraclass correlation: 0.75). For further analyses we computed a composite measure of AUT creativity for each time point of assessment by aggregating the Imaging was performed on a 3T Siemens Magnetom Skyra tomograph  using a 32‐channel head coil. BOLD‐sensitive T2*‐weighted functional images were acquired using a single shot gradient‐echo EPI pulse sequence . To record the verbal response of the participants, a MR compatible microphone was used . Visual stimuli were presented using the Software Presentation .Functional MRI data analysis was performed using SPM8 software . Preprocessing steps included motion correction, slice time acquisition correction, and spatial normalization into the standard space . Finally, the functional data were smoothed using a Gaussian filter of 8 mm. A high‐pass filter with a cut‐off frequency of 1/128 Hz was employed to remove low frequency drifts.t‐contrasts between both experimental conditions  for each time point  were computed. These contrasts were then entered into a random effects one‐sample t‐test for each training group  and time point of assessment. All reported activations were corrected for multiple comparisons by means of AlphaSim  equivalent to P < 0.05. Following this approach the smoothness of each contrast was estimated individually and the minimum cluster size ranged between 25 and 28 voxels .For each participant all three time points were entered into one design matrix and linear CreaTrain on verbal creativity was analyzed both with respect to AUT performance during fMRI assessment  and with respect to psychometric test performance outside the scanner . For each dependent variable separate repeated measurement ANOVAs with the factors TIME  and experimental GROUP  were computed.The efficacy of F  = 1.64, P = 0.20), suggesting that the mere generation of instances to given adjectives was not significantly modulated by the training  = 3.42, P = 0.04, 2pη = 0.06. Subsequent paired t‐tests separately for both training groups revealed significant differences only in TG2 from t2 to t3 (t (28) = −2.16, P = 0.04), suggesting an increase in AUT performance right after the training in this group  revealed no significant findings  = 4.58, P = 0.01, 2pη = 0.08), and as shown in Figure t‐tests revealed a significant difference only in TG2 between t1 and t3 (t (28) = −2.22, P = 0.04), indicating an increase in verbal creativity in this group. With respect to word fluency, both groups tended to increase their performance right after the training  = 2.67, P = 0.07, 2pη = 0.05).The findings for the composite measure of verbal creativity obtained outside the scanner were strikingly similar. The ANOVA again revealed a significant TIME by GROUP interaction  = 6.41, P = 0.01, 2pη = 0.11), indicating generally higher figural creativity scores in TG 2 than in TG 1  = 6.12, P = 0.003, 2pη = 0.11, indicating that in TG1 more creative individuals showed increases in verbal creative task performance right after the training, while less creative individuals showed comparatively strong decreases.Finally, we examined whether or to which extent individual differences in baseline trait creativity have an impact on the training effects. For this reason, for each creativity measure  3‐way repeated measurement ANOVAs with TIME and GROUP and the mean BIS verbal creativity score (obtained prior to the training study) as continuously distributed between subjects factor were performed. A significant interaction between TIME, GROUP and BIS creativity was observed only in the CE task, Prior to the training (t1), the AUT was associated with stronger activation than the IT in a left‐lateralized brain network involving the calcarine sulcus and regions of the inferior temporal and occipital gyri (see Table k = 30) involving the left SMG and the left IPC  prior to the training (at t1), apart from a small activation cluster k = 0 involviTo provide a formal test of the neurophysiological effects of the creativity training, time‐related changes of the contrast estimates (AUT > IT) in functionally defined ROIs were investigated. The ROIs were defined on the basis of a combined mask including all significantly (de‐)activated voxels as revealed by the whole brain AUT vs. IT contrasts across all time points and both groups, hence representing relevant brain areas associated with AUT task performance. This procedure resulted in 10 clusters, for which separate TIME  by GROUP  repeated measurements ANVOAs were performed.F  = 4.68, P = 0.01, 2pη = 0.08; right: F  = 4.28, P = 0.02, 2pη = 0.08), suggesting general increases in AUT (relative to IT) activation as a function of time. Inspection of Figure t(23) = −3.16, P = 0.004). The relevant interaction between TIME and GROUP, however, was not significant  = 2.03, P = 0.14, 2pη = 0.04). In addition to the significant TIME effects in the bilateral SMG, brain activation in the left posterior MTG tended to increase from t1 to t3  = 2.69, P = 0.07, 2pη = 0.05).The ANOVAs yielded significant main effects of TIME for the SMG bilaterally  = 2.64, P = 0.08, 2pη = 0.05). A significant TIME by GROUP interaction was found in the left AG  = 3.77, P = 0.03, 2pη = 0.07, suggesting comparatively strong activation increases in TG1 from t2 to t3  = 4.19, P = 0.046, 2pη = 0.08), and a tendency toward more pronounced AUT deactivations in the right inferior parietal cortex  = 3.24, P = 0.08, 2pη = 0.06).As was to be expected on the basis of the whole brain voxel‐wise analyses, both groups tended to decrease AUT (relative to IT) activation in the left MTG see Fig.  right afF  = 4.76, P = 0.03, 2pη = 0.09), and a trend toward significance in the left SMG  = 3.03, P = 0.09, 2pη = 0.06), in each case indicating a positive association between brain activation and AUT performance at the post‐test.To assess whether or to which extent any time‐related changes in brain activation patterns were influenced by the success of training, for each ROI TIME  by GROUP  ANCOVAs were computed in additionally considering AUT performance right after the training  as continuously distributed between subjects factor. The ANCOVAs yielded no significant interactions involving AUT performance. There was, however, a significant main effect of AUT performance in the left pMTG  has consistently been found to show stronger activation during the generation of creative/original ideas (AUT) than during the performance of tasks involving lower creativity‐related demands [Cousijn et al., Besides the effects in the left IPC and the left MTG, creative ideation was generally also strongly associated with brain activity in the visual cortex. At first glance, these effects appear to be somewhat puzzling since the majority of studies highlighted the role of prefrontal brain regions as being particularly relevant for different creativity‐related task demands [Arden et al., Among the most important strengths of this study is the fact that we applied a longitudinal research design including three time points of assessment and two training groups, which received the training time‐delayed. Moreover, brain activation was measured during performance of well‐known creative ideation tasks, which hence extends findings from previous resting‐state training or intervention studies. Finally, we also recorded oral responses during performance of the scanner tasks, which enabled us to monitor and evaluate task performance  and relate it to brain activation. The employed longitudinal research design does also provide some information on the stability of fMRI effects. It was found that the activation patterns of the AUT vs. IT contrasts were fairly stable: On the one hand across groups, when we compare the activation patterns of the TG1 and TG2 at t1 (prior to the training), and on the other hand as a function of time, when we look at the TG2 which showed similar activation patterns at t1 and t2, or at the TG1 which showed quite similar activation at t2 and t3. Generally, the activation patterns of the TG2 were somewhat more pronounced than those of the TG1. Also, only the TG2 showed significant improvements in behavioral measures of verbal creativity. Supplemental analyses with the untransformed fluency and originality scores indicated that both measures showed decreases between t1 and t2 in both trainings groups, possibly indicating that the second time point of assessment was generally more challenging or exhausting for the participants. Another reason for failing significant behavioral training effects in TG1 may also include the different time points of the training . In addition to this, we should also pay attention to the fact that the assessment of reliable and valid behavioral performance indicators during imaging is per se highly challenging, not least due to the fact that participants are required—unlike in the natural environment—to be creative while lying supine in the scanner. In light of this unclear picture of behavioral results in the first training group, it seems even more striking that both training groups showed a highly similar pattern of results at the level of the brain.Finally, it is noteworthy that this study revealed fMRI effects specifically in a network of brain regions that was previously shown as being particularly relevant for verbal creative thinking demands [such as the left IPC and the left MTG: e.g., Benedek et al.,