Schizophrenia has been characterized by an impaired mentalizing. It has been suggested that distinguishing implicit from explicit processes is crucial in social cognition, and only the latter might be affected in schizophrenia. Two other questions remain open: (1) Is schizophrenia characterized by an hypo- or hyper attribution of intentions? (2) Is it characterized by a deficit in the attribution of intention or of contingency? To test these three questions, spontaneous mentalizing was tested in 29 individuals with schizophrenia and 29 control subjects using the Frith-Happé animations, while eye movements were recorded. Explicit mentalizing was measured from participants’ verbal descriptions and was contrasted with implicit mentalizing measured through eye tracking. As a group, patients made less accurate and less intentional descriptions of the goal-directed and theory of mind animations. No group differences were found in the attribution of contingency. Eye tracking results revealed that patients and controls showed a similar modulation of eye movements in response to the mental states displayed in the Frith-Happé animations. To conclude, in this paradigm, participants with schizophrenia showed a dissociation between explicit and implicit mentalizing, with a decrease in the explicit attribution of intentions, whereas their eye movements suggested a preserved implicit perception of intentions. Schizophrenia is characterized by impairments in several domains of social cognition, including Theory of Mind (ToM) or mentalizing124581011121415171819The other important issue is the distinction between implicit and explicit processing, which Frith and Frith (2008) have convincingly argued is of great relevance to the study of social cognition. Implicit mentalizing involves fast, automatic and relatively inflexible routines while explicit mentalizing rather reflects later, reflexive, decision-making and reporting processes2425262831Among the various paradigms designed to assess mentalizing in schizophrenia, we chose the Frith-Happé animations because it has been validated across a number of studies. Because participants are simply asked to report spontaneously what they have seen, this task does not require to solve a problem, as opposed to others classical ToM paradigms. In these animations, two triangles are moving according to random (R), goal-directed (GD) and ToM scenarios. In the R condition, the triangles drift and bounce independently like billiard balls whereas, in the GD condition, one triangle appears to act intentionally towards another triangle. Finally, in the ToM condition, the triangles seem to interact in a mentalistic manner, with one triangle trying to influence the mental states of the other (for example one triangle tries to trick the second). Individuals with schizophrenia typically provide less intentional and less accurate descriptions of GD and ToM scenarios than control participants13171819However, it remains unclear whether mentalizing deficits in schizophrenia are primary or whether they are a consequence of other cognitive impairments like executive functioning. In the present study, we chose to investigate the influence of one particular executive function, contextual processing because the deficit in contextual information integration has been considered as a central pathophysiological mechanism of cognitive impairment in schizophrenia353639This study also tested Frith’s (2004) hypothesis by recording eye movements on Frith-Happé animations to obtain a more implicit measure of mentalizing in schizophrenia. Although Frith-Happé stimuli elicit spontaneous, largely implicit and automatic mentalizing, putting the content of what is viewed into words is an explicit process. It requires an explicit recognition and an off-line verbalization of these mental states. Eyetracking can assess whether participants can focus attention on socially salient aspects of animations: for example, it has been recently shown that the impairment in false belief inference found in schizophrenia on an object displacement paradigm was related to a deficit in visual attention toward gaze orientation4344Comité de Protection des Personnes Ile-de-France XI) and was carried out in accordance with the approved guidelines. All participants received a complete description of the study in oral and written form. Written informed consent was obtained from each participant.29 individuals with schizophrenia and 29 control participants were recruited in this study. All participants had normal or corrected-to-normal vision. Exclusion criteria for both groups comprised substances or alcohol dependence within the past 6 months and current or prior history of untreated significant medical illness or of neurological illness. Two licensed psychiatrists confirmed all diagnoses in the schizophrenia group according to the DSM-IV-R criteria for schizophrenia. Patients were stabilized and were recruited from community mental health centers and outpatient clinics in the Versailles area. At the time of testing, all participants with schizophrenia were taking antipsychotics. The control group was screened for current or past psychiatric illness and participants were excluded if they met criteria for any axis I disorder of the DSM-IV-TR. The experiment was approved by the local medical ethics committee  and the two raters’ scores were averaged.Answers were evaluated according to three dimensions using the usual scoring criteria provided by Castelli kappa across the 4 categories: 0.82) and the two raters’ scores were averaged.The aforementioned intentionality scale mixes indices of contingency  and intentionality (whether the described actions have been done on purpose). For example, the fourth step of the scale is “deliberate action in response to other’s action” which involves both contingency and intentionality. This might be a problem, as intentionality and contingency have been characterized as two separate dimensions in schizophrenia222 and eye movement amplitude smaller than 0.1°. This algorithm is designed to avoid short fixations (less than 100 ms in duration).After the exclusion of blinks, fixations were automatically detected with the EyeLink software algorithm  according to the following thresholds: eye velocity below 30°/sec, eye acceleration below 8000°/secWe considered that the gaze fell within a triangle if it fell within a circle whose center was the barycenter of the triangle and whose radius was the distance between the barycenter and the furthest corner of the triangle plus 1°. Triangle time was calculated as the cumulative duration of gaze within either the blue or the red triangle, divided by the total length of the animation.We first ran repeated-measures ANOVAs on accuracy, intentionality and length of verbal descriptions with Group as the between-subject factor and Condition as the within-subject factor. We then ran a repeated-measures ANOVA on the number of described actions with Group and as the between-subject factor, and Condition, Intentionality, and Contingency as the within-subject factors. We finally ran repeated-measures ANOVA on fixation durations and triangle time with Group as the between-subject factor and Condition as the within-subject factor. Results were corrected for sphericity using the Greenhouse-Geisser (GG) method where appropriate.In a second step, we ran the same models as ANOVAs, adding covariates that might be confounding factors . This was done to test to what extent the results obtained might be due to such variables. The results of ANCOVAs are presented as Demographic, cognitive and clinical characteristics of the groups are shown in Three trials were excluded due to a lack of response . Barplots are presented in The ANOVA run on the length scale revealed a significant effect of Condition  = 69.7, p[GG] < 0.001) but no significant effects for Group  = 2, p = 0.16) and Group by Condition interaction  = 2, p[GG] = 0.14). Length was greater for GD than for R  = 240, p < 0.001) and greater for ToM than for R  = 363.6, p < 0.001) and GD  = 58.9, p < 0.001).The ANOVA run on the accuracy scale revealed a significant Group by Condition interaction  = 6.8, p[GG] = 0.002). The post-hoc tests revealed that Group was significant for GD  = 14.5, p < 0.001) and ToM  = 16.7, p < 0.001) but not for R  = 0.1, p = 0.76). Accuracy was lower for patients compared to controls for GD and ToM conditions. ANCOVAs  showed tFor controls, accuracy was not different between R and GD  = 2.3, p = 0.15), greater in R than ToM  = 23.5, p < 0.001) and greater in GD than ToM  = 19.1, p < 0.001). For patients, accuracy was greater in R than in ToM  = 113, p < 0.001) and GD  = 20.6, p < 0.001) and greater in GD than in ToM  = 16.1, p < 0.001).The ANOVA run on the intentionality scale revealed a significant Group by Condition interaction  = 4.5, p[GG] = 0.015). The post-hoc tests revealed that Group was significant for ToM  = 6.4, p = 0.015), GD  = 9.4, p = 0.003) but not for R  = 0.04, p = 0.84). For controls, intentionality was greater for GD than for R  = 169.2, p < 0.001) and greater for ToM than for R  = 224.6, p < 0.001) and GD  = 27.6, p < 0.001). For patients, intentionality was greater for GD than for R  = 107.4, p < 0.001) and greater for ToM than for R  = 164, p < 0.001) and GD  = 30.4, p < 0.001).Barplots are presented in The ANOVA run on the contingency/intentionality scale revealed a significant triple interaction between Condition, Intentionality, and Group  = 6.8, p[GG] = 0.002). This suggests that patients and controls differed in their descriptions of intentional actions, but not in the same way for R, GD and ToM animations. However, neither the triple interaction between Condition, Contingency, and Group  = 1.9, p[GG] = 0.16) nor the interaction between Contingency and Group  = 0.5, p = 0.47) were significant. This suggests that patients and controls did not differ in their descriptions of contingent actions. The quadruple interaction between Condition, Intentionality, Contingency and Group was significant  = 4.9, p[GG] = 0.011).The post-hoc tests revealed that Group was significant for intentional contingent actions in ToM  = 5.4, p = 0.024) and GD  = 12.1, p < 0.001) but not in R  = 0.1, p = 0.74). Group was also significant for intentional non-contingent actions in ToM  = 25.2, p < 0.001) but not in GD  = 2.5, p = 0.12) and R  = 2.3, p = 0.13). Group was not significant for mechanical non-contingent actions in ToM  = 0, p = 0.97), GD  = 0.6, p = 0.43) and R  = 0.9, p = 0.34). Group was not significant for mechanical contingent actions in ToM  = 0.2, p = 0.62), GD  = 1.9, p = 0.17) and R  = 0.3, p = 0.61). ANCOVAs  showed tThere was no significant correlation between verbal descriptions of Frith-Happé stimuli and clinical symptoms see .Barplots are presented in There was no fixation whose duration was below 100 ms (the shortest fixation duration was 199.8 ms). The ANOVA run on fixation duration revealed significant effects of Group  = 4.2, p = 0.045), Condition  = 36.1, p[GG] < 0.001) but no significant interaction between Group and Condition  = 2, p[GG] = 0.15). Fixation duration was lower for controls compared to patients. Fixation duration was greater for GD than for R  = 15.8, p < 0.001) and greater for ToM than for R  = 50.8, p < 0.001) and GD  = 30.1, p < 0.001). Correlation analyses  showed tThe ANOVA run on triangle time revealed a significant effect of Condition  = 234.7, p[GG] < 0.001) but no significant effects of Group  = 2.2, p = 0.14) and Group by Condition interaction  = 2, p = 0.15). Triangle time was greater for GD than for R  = 189.4, p < 0.001) and greater for ToM than for R  = 267.3, p < 0.001) and GD  = 169.2, p < 0.001).Finally, exploratory correlation analyses revealed no significant correlation between implicit mentalizing and clinical symptoms see , and no In this study, we used Frith-Happé animations to assess the ability to attribute intentions and contingency in schizophrenia. Explicit mentalizing ability was measured from participants’ verbal descriptions of the animations. Because little is known about how individuals with schizophrenia extract relevant cues when observing animated social agents, eye movements were recorded while participants were watching Frith-Happé animations. We examined whether participants with schizophrenia would show the same modulation of eye movements by the different types of animations as control participants, in the hope of obtaining some insight into implicit mentalizing processes.As in previous studies, individuals with schizophrenia differed from controls in the way they described the animations: they made less accurate and intentional description of GD and ToM animations. No group differences were found in the R condition, suggesting that this deficit was not just a general decrease in the ability to make verbal descriptions.et al. who found a normal attribution of intentions but an increased attribution of contingency in a transnosographical group of 22 deluded patientsWe found no evidence for hypermentalizing in schizophrenia, as patients did not attribute more intentions to triangles in any condition compared to controls. Neither did we find evidence for apophenia in schizophrenia, as patients did not attribute more contingency between the two moving triangles in any condition compared to controls. This last result is the opposite of the one reported by Blakemore It is worth noting that group differences in explicit mentalizing were not explained by cognitive control, verbal and performance IQ. However, contextual processing was associated with the accuracy of verbal description see , consist39The study did not show significant correlations between clinical symptoms and explicit measures of mentalizing. However, social cognition usually correlates moderately with disorganization and negative symptoms of schizophrenia (with r ranging between −0.2 to −0.32)The eyetracking results revealed that individuals with and without schizophrenia showed a similar modulation of eye movements in response to the different conditions of the Frith-Happé animations. First, both groups showed the same increase in fixation duration from R to ToM animations, consistently with previous studies424647Gaze was spontaneously directed to the intentional triangles (GD and TOM conditions) for longer durations than to the random ones, thus replicating the modulation of triangle time by the type of animation found in others studies464755One limitation to the present study came from the fact that the paradigm was not entirely implicit, as participants were required to focus their attention on the animations because they would be asked to describe them. In an entirely implicit design, participants would have been asked to perform a task unrelated to the description of animations, while eye movements were recorded. This study does not explore the stages between the visual processing of triangles and the interpretation of the scene. Another limitation was related to the DSM-IV-R diagnosis interviews of patients and controls that did not involve a structured instrument like the Structured Clinical Interview for Disorders. There was a discrepancy between the results on the length scale and the results on the contingency/intentionality scale. Although descriptions were as long in patients and controls, patients described fewer intentional actions in GD and ToM animations than controls. This decrease was not compensated by an increase in the description of the mechanical movements in these two conditions. There was a global reduction in the number of actions described in these two conditions: patients might have committed more repetitions, or described the triangles more on their physical appearance rather on their actions. The present results could thus also be interpreted according to the experiential  vs. narrative dichotomy, as patients were impaired in the verbal description of GD and ToM animations. Indeed, schizophrenia has been characterized by an uninformative speech with a low idea densityTo conclude, we found that patients with schizophrenia attributed fewer mental states to the Frith-Happé triangles but had a normal attribution of contingency between these two agents. This deficit in their ability to explicitly mentalize was dissociated from a preserved implicit mentalizing, as revealed by their eye movements.How to cite this article: Roux, P. et al. Preserved implicit mentalizing in schizophrenia despite poor explicit performance: evidence from eye tracking. Sci. Rep.6, 34728; doi: 10.1038/srep34728 (2016).


Soil urease, alkaline phosphatase and hydrogen peroxidase activities decreased with increasing years of continuous potato cropping. On average, the P–B rotation significantly increased (p <0.05) tuber yield by 19% and 18%, compared to P–C and P–L rotation respectively. P–L rotation also increased potato tuber yield compared to P–C, but the effect was lesser relative to P–B rotation. These results suggest that adopting potato–legume rotation system has the potential to improve soil biology environment, alleviate continuous cropping obstacle and increase potato tuber yield in semi–arid region.This study was conducted to explore the changes in soil microbial populations, enzyme activity, and tuber yield under the rotation sequences of Potato–Common vetch (P–C), Potato–Black medic (P–B) and Potato–Longdong alfalfa (P–L) in a semi–arid area of China. The study also determined the effects of continuous potato cropping (without legumes) on the above mentioned soil properties and yield. The number of bacteria increased significantly (p < 0.05) under P–B rotation by 78%, 85% and 83% in the 2, 4 and 7–year continuous cropping soils, respectively compared to P–C rotation. The highest fungi/bacteria ratio was found in P–C (0.218), followed by P–L (0.184) and then P–B (0.137) rotation over the different cropping years. In the continuous potato cropping soils, the greatest fungi/bacteria ratio was recorded in the 4–year (0.4067) and 7–year (0.4238) cropping soils and these were significantly higher than 1–year (0.3041), 2–year (0.2545) and 3–year (0.3030) cropping soils. Generally, actinomycetes numbers followed the trend P–L>P–C>P–B. The P–L rotation increased aerobic azotobacters in 2–year (by 26% and 18%) and 4–year (40% and 21%) continuous cropping soils compared to P–C and P–B rotation, respectively. Generally, the highest urease and alkaline phosphate activity, respectively, were observed in P–C (55.77 mg g Solanum tuberosum L.) is one of the staple food crops in the world. In northern China, potato has been the dominant crop, providing the main food source for the increasing human population 33]. Numb 14 days –36.4 titration method; urease activity was measured using phenol sodium hypochlorite colorimetric method; and phosphatase activity was measured using disodium phenyl phosphate colorimetric method [Hydrogen peroxidase activity was determined using KMnOc method .–1) was obtained by determining the tuber yield of each plot in each year at harvest. The tuber yield in the continuous potato cropping fields (CK) was determined in October 2011 and that of potato–legume rotation was determined in October, 2013.The tuber yield per hectare  with the rotation treatment as the fixed effect and replicate as random effect. Means comparison based on Duncan’s multiple range test (p<0.05). Due to significant year by treatment interactions for most of the variables evaluated in the study, the treatment effect was assessed separately for each year.The effect of continuous potato cropping on bacteria, fungi and actinomycetes are presented in Potato–legumes rotation had significant effect (p <0.05) on bacteria, fungi and actinomycetes numbers (ANs) depending on treatment . The numContinuous cropping had significant effect (p <0.05) on fungi/bacteria ratio . The resThe trend of fungi/ bacteria ratio under various crop rotation systems was not consistent in all the years of this study . The P–B4 CFU /g), which was significantly higher than the other years of continuous cropping by 505% on average. Response of both nitrifying and denitrifying bacteria to continuous potato cropping was not consistent  depending on treatment and cropping year . The treSoil urease, alkaline phosphatase and hydrogen peroxidase activities decreased gradually with increased years under continuous potato cropping . The lowSoil urease, hydrogen peroxidase and alkaline phosphatase activities were significantly increased in the 2–year cropping soils under the potato-legume rotation . The rot–1, which was significantly greater by 62% (13220 kg ha–1), 45% (14706 kg ha–1) and 37% (15553 kg ha–1) compared to 4, 4 nd 7 years, respectively. However, no significant differences (p <0.05) were observed in tuber yield between 3–7 years continuous cropping plots  in potato tuber yield depending on the treatment . The tubResults of the present study indicated significant positive correlations between BN and AA, NB (p<0.05), ANA, UR, AP (p<0.01), FN and AA, ANA, and AP (p<0.05) . SignifiSoil microorganisms are important components of the terrestrial ecosystems and their quantities can reflect not only the degree of energy metabolism, but also soil fertility status . HoweverReasonable rotation system is an important basis for preventing continuous cropping obstacles and improving crop production . As repoSoil microbial floras were shifted from fungi type to bacteria type by rotation of Common vetch and Medicago lupulina for 1 and 2–year continuous cropping, but fungi numbers did not increase by rotation of Longdong alfalfa at the same continuous cropping years. The amelioration was significant by rotation of Medicago lupulina and Longdong alfalfa for potato soil in 3 and 7–year continuous cropping and the improvement effect of Medicago lupulina was the best. For 4–year continuous cropping field, soil microbial floras were also shifted to bacteria type by rotation of these three legumes. However, the actinomycetes mycelium decreased for up to 4–year continuous cropping field by rotation of three legumes . TherefoMicrobial physiological group of nitrogen play an important role in nitrogen transformation in soil, including aerobic azotobacter, anaerobic azotobacter, nitrifying bacteria, denitrifying bacteria and ammonifying bacteria . In receAfter rotation with legumes, there was difference in physiological groups of microorganisms in potato continuous cropping soil. The aerobic azotobacters of 1 and 2–year potato continuous cropping soil have a very sensitive response to legumes and its number increases greatly by rotation of legumes compared with the continuous cropping soil. However, similar results were not observed in 3–year continuous potato cropping soil. Soil anaerobic nitrogen–fixing bacteria were significantly improved in all potato continuous cropping fields by rotation with Medicago lupulina and Common vetch. Ammonification can produce ammonia in the soil, which can be converted to nitrate by nitrifying bacteria. Denitrifying bacteria usually participate in the revivification of nitrate in the soil. The existence and activities of nitrifying and denitrifying bacteria in the soil play an important role in soil fertility and plant nutrition . Our resSoil enzymes have a close relation with soil microorganisms and its biodiversity, both of which can drive soil biological metabolic processes . The res3––N [Current study indicated a significant positive correlation between BN and AA, NB (p<0.05), ANA, UR, AP (p<0.01), FN and AA, ANA, AP (p<0.05). These findings showed that fungi and bacteria are the major soil microorganisms responsible for N cycling processes . Crensha3––N , 59. TheSoil microbial communities increased significantly after rotation with legumes. Number of bacteria and fungi/bacteria ratio was greatly improved by rotation of potato–Longdong alfalfa and potato–Black medic. Thus, obstacles to sustainable production of potato in the semi–arid region of Northwestern China due to continuous cropping can be reduced by adopting potato–legume rotation cropping.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 Fig(XLSX)Click here for additional data file.


Though parents’ physical activity (PA) is thought to be a predictor of children’s PA, findings have been mixed. The purpose of this study was to examine the relationship between pedometer-measured steps/day of parents’ and their children and potential moderators of this relationship. We also assessed the parent–child PA relationship as measured by questionnaires.Steps Count (SC)-T2 pedometers for four consecutive days. Parents reported their PA from the last seven days and their child’s usual PA. Hierarchical linear regressions were used to assess the parent–child PA relationships, controlling for covariates. Gender , gender homogeneity, weight status , weight status homogeneity, and socioeconomic status (SES) variables  were tested as potential moderators of this relationship. Partial r’s were used as an estimate of effect size.Six-hundred and twelve 7–8 year olds and one of their parents wore rpartial = .24). For every 1,000 step increase in parents’ steps, the children took 260 additional steps. None of the tested interactions were found to moderate this relationship. Using questionnaires, a relatively smaller parent–child PA relationship was found .Parents’ steps was significantly related to children’s steps (Physically active parents tend to have physically active children. Interventions designed to get children moving more throughout the day could benefit from including a parent component. Future research should explore the mechanisms by which parents influence their children, and other parent attributes and styles as potential moderators. Parental PA, on the other hand, was a weak predictor of child PA (r = .16). Several previous reviews have also reported mixed findings across studies for parental PA . Hetp = .08) [Several studies have examined the relationship between parents’ and their children’s objectively measured steps/day –14, acceTotal PA performed throughout the day, as well as higher intensity activity, are important for the health of young people , and thuAs illustrated in the IMPAP, the influence of parental variables on children’s PA can vary depending on different child and parent attributes. Examining moderators  is important because it can provide information about whom and under what circumstances specific interventions may be effective , 25. ThuThe purpose of our study was to examine the relationship between parents’ and children’s PA in a sample of 7- to 8-year-olds and one of their parents. Research question 1 examined the relationship between pedometer-measured steps/day in parents and their children. Research question 2 examined whether gender , gender homogeneity, weight status , weight status homogeneity, and SES variables  moderated this relationship. Research question 3 examined the parent–child PA relationship as measured by questionnaires. Consistent with Yao and Rhodes [This is a cross-sectional study of 7–8 year old children and parents living in Edmonton, Alberta, Canada  and the greater metropolitan area. The children were a part of the SHAPEs of Things to Come study, a longitudinal follow-up to the Spatial Health Assessment of Physical Environments (SHAPEs) study –34. In Sd = .15–.23).Of the children who participated, 92 % were 7- or 8-years-old. Because the parent–child PA relationship may differ by age we decided to use only the 7–8 year olds in this analysis, leaving a final sample size of 612 parent–child dyads . In this sample, 76 % of the parents had finished university or college, and 78 % of the families had a household income > $80,000/year. Using data from the baseline study (SHAPEs), those who were included in the current study did not significantly differ on age, unemployment rate, or parent-proxy reported PA from those who did not participate. Participants in our study did have significantly lower BMI’s, and lived in areas with higher household income and education levels; however the effects were small . Four days of monitoring was chosen because the literature at the time deemed this as an adequate period for determining habitual PA in children , 35, 36.Steps Count (SC)-T2 steps and activity time pedometers  were used to measure PA in children and parents. This pedometer, which is mechanically identical to the Walk for Life pedometer, has demonstrated good reliability and accuracy in adults [Unsealed n adults  and chiln adults . The SC-Parents reported their children’s PA using the CLASS survey, which has shown acceptable psychometric properties in 10–12 year olds . Parentsz-score was used to categorize children as non-overweight (≤1), overweight/obese (≥1.01). Parental height and weight were self-reported, which was used subsequently to calculate BMI and group individuals as non-overweight (<25 kg/m2), overweight/obese (≥25.01 kg/m2) [non-overweight category included participants who could be classified as “thin” (children n = 87) or “underweight” (parents n = 10) or “healthy”. A weight status homogeneity variable was created by coding parents and children with the same weight status  as 1 and those with a different weight status as 0.Weight and height were measured twice and recorded to the nearest .1 kg and .1 cm, respectively. If a discrepancy existed between the two measurements , a third measurement was taken and the three measurements were averaged. Children’s sex and age specific body mass index (BMI) z-scores were calculated using the World Health Organization’s (WHO) growth reference . The BMI1 kg/m2) . For botGiven the influence of season on PA , 45, theChildren’s date of birth was used to determine age in years. Parent gender was recorded for the parent who wore the pedometer. Gender homogeneity was created by coding parents and children with the same gender  as 1 and those with the opposite gender as 0. Household income was categorized into < $80,000 per year and > $80,000 per year for our analyses based on the approximate median income level in Alberta . Parent Area-level SES was calculated using families’ postal code data. GeoPinPoint™ Suite software  was usedAll analyses were conducted using IBM SPSS Statistics version 23. Outliers for the pedometer data were identified as days with <1,000 or >30,000 steps/day for children and <1,000 or >25,000 for adults were set as missing . Outlierp < .05) or marginally significant (p < .10) differences existed on parental BMI for parents’ and children’s steps/day. Importantly, families who participated in the initial assessment and those that returned the pedometers did not differ on parent self-reported leisure time MVPA  or children’s parental-proxy reported PA . We therefore assumed at least a partial missing at random mechanism and imputed all of the missing data  using multiple imputation in SPSS. This procedure uses the fully conditional specification method and imputes data using linear regression for continuous variables and logistic regression for binary variables. We used 100 iterations, which resulted in 100 separate datasets [Of the 28 variables included in this study, 83 % were missing on at least one value , children’s steps/day was entered as the criterion variable and parent’s steps/day and covariates as predictor variables. One by one we tested potential interactions including parent steps*child gender, parent steps*parent gender, parent steps*gender homogeneity, parent steps*child weight status, parent steps*parent weight status, parent steps*weight status homogeneity, parent steps*parent education, parent steps*household income, parent steps*area SES. In the models where the gender homogeneity and weight status homogeneity interactions were tested, these variables were also included as main effects. Before creating the interaction terms, the continuous variables  were centered on their mean [p < .01) to the interactions. For significant or near significant interactions, a simple slopes analysis was performed to determine the beta coefficients and p-values for each group. Beta coefficients for the simple slopes were calculated by hand using the pooled results [p-value (set at p < .05) was computed using the initial dataset . To address research question 3 (parent–child PA relationship as measured by questionnaires), children’s proxy-reported PA was entered as the criterion variable and parent self-reported leisure time MVPA and the covariates were entered as predictor variables. Coefficients were deemed significant at p < .05.To address research questions 1 (whether parents’ steps/day was related to children’s steps/day), a linear regression was run with children’s steps/day as the criterion variable and parents’ steps/day and covariates as predictor variables. Coefficients were deemed significant at eir mean . To cont results . The pooM = 9075, SD = 4832) took more steps than girls , t(1339) = 3.65, p < .001, d = .30. No significant differences existed in steps/day between mothers  and fathers , t(41870) = −.66, p = .51, d = .07.Sample characteristics are provided in Table r = .25, p < .001. The results from the linear regression analysis is presented in Table B = 0.26, p < .001), with small to medium sized effects . That is, for every 1,000-step increase in parents’ steps, children took approximately 260 additional steps. The model explained 8.8–15.4 % variance in children’s steps. The bivariate, unadjusted Pearson’s correlation between the parents’ and children’s steps was p < .01 level. However the interaction between parent steps and income , and parent steps and education  both approached significance. Specifically, in higher income households  the parent–child PA relationship was significant  and in lower income households it was not . Further, parents who had completed graduate school (n = 86) had a stronger parent–child PA relationship  than parents without a graduate degree .Table r = .15, p < .01. The results from the linear regression analysis of the parent–child PA relationship using subjectively measured PA is presented in Table B = 2.18, p < .01), with small sized effects . The model accounted for 1.8–5.2 % variance in children’s PA.The bivariate, unadjusted Pearson’s correlation between parents’ and children’s subjectively measured PA was The purpose of this study was to examine the relationship between pedometer-measured steps/day of parents and their children, and whether this relationship varied by gender , gender homogeneity, weight status , weight status homogeneity, parent education, household income, and area-level SES. We also assessed the parent–child PA relationship as measured by questionnaires. When PA was measured via pedometers, we observed a significant relationship between parents’ and children’s PA. Further, this relationship was stronger for higher income families and parents with a graduate degree, however the effects did not reach statistical significance. None of the other variables moderated this relationship. Using questionnaires, a relatively smaller parent–child PA relationship was found.r = .25, rpartial = .24). Several studies in recent years have assessed the parent–child PA relationship using pedometers in both children and parents and all have observed significant findings with at least one parent [We found a 260 step/day increase in the children’s steps/day for every 1000 step/day increase in the parents’ steps/day, which was a small to medium sized effect . Another study found father-daughter, father-son, mother-daughter, and mother-son step relationships on weekends, however the father’s steps was not related to his daughter’s steps on weekdays [Our study suggests that the parent–child PA relationship as measured by pedometers does not exhibit a same gender , child gender, or parent gender effect. The literature on gender specific parent–child PA relations using pedometers is quite mixed. For example, significant mother-offspring but not father-offspring step correlations were found in a sample of 8–18 year-old youth in France . Contrarweekdays . A limitweekdays  that assIn addition to testing differences by gender, and guided by the IMPAP, we also explored SES and weight status variables as potential moderators of the parent–child PA relationship. Correlations were higher for parents who had completed graduate school and for those who made > $80,000/year, but the results did not reach statistical significance. Weight status of the child and parent, and gender homogeneity were not effect modifiers. A few studies have examined SES moderators of the parent–child PA relationship. Jacobi and colleagues  observedvs subjective measures, yet the difference was not significant [A stronger child–parent PA relationship was found when PA was measured objectively using pedometers, compared to when measured subjectively using questionnaires. Similar to our study, a meta-analysis recently reported slightly higher effects when PA was measured using objective nificant . One expnificant . Thus, tnificant . In suppnificant . A finalIt is important to consider the potential reasons for why active parents have active children. Though this relationship is often described as parental “modeling” or observational learning , it is lcaused their children to take more steps themselves. To date, no studies have examined the parent–child PA relationship over time using an objective measure of PA in both parents and children. As such, longitudinal and experimental studies are needed to establish temporal precedence and determine if there is evidence of a causal relationship. Third, despite pedometers providing an objective measure of PA, they have limitations. Because the pedometers were unsealed and parents’ recorded theirs and their children’s steps each evening, they could have unknowingly increased their activity or made a recording error. Pedometers are also not able to provide an indication of missing wear time, and thus we had to rely on the logbooks to determine if the monitor was not worn. Further, reactivity could have occurred whereby the parents and children increased their activity level in response to wearing an activity monitor. Several studies have examined the patterns of PA across the week to determine if there is an initial increase in PA and then a leveling off. No evidence of reactivity was found with sealed and unsealed pedometers in both children and adults [Strengths of the study include the objective measure of PA used with both parents and children, the large sample size, and the examination of several moderators. Multiple imputation of missing data allowed us to retain a large sample, reduce biases, and to test several potential moderators. But, some limitations should also be acknowledged. First, because the participants were volunteers, and only 39 % of the participants from the baseline phase of the SHAPEs study completed follow-up phase, a self-selection bias could exist. Indeed, the parents in our sample were more educated and higher income earners than the general population in the region. Second, the study is cross-sectional and non-experimental; therefore, we cannot assume that the parents’ d adults , 57–59. d adults , 61. In d adults –64. Finad adults . When thd adults .Our findings demonstrate that active parents tend to have active children. Stronger parent–child PA relationships were observed with pedometers compared to questionnaires, which highlights the importance of using objective measures, and may help explain the mixed findings observed in the literature. Interventions designed to get children moving more throughout the day could benefit from including a parent component. Future research should explore the mechanisms by which parents influence their children, and other parent attributes and styles as potential moderators.


In solo synchronization-continuation (SC) tasks, intertap intervals (ITI) are known to drift from the initial tempo. It has been demonstrated that people in paired and group contexts modulate their action timing unconsciously in various situations such as choice reaction tasks, rhythmic body sway, and hand clapping in concerts, which suggests the possibility that ITI drift is also affected by paired context. We conducted solo and paired SC tapping experiments with three tempos  and examined whether tempo-keeping performance changed according to tempo and/or the number of players. Results indicated that those tapping in the paired conditions were faster, relative to those observed in the solo conditions, for all tempos. For the faster participants, the degree of ITI drift in the solo conditions was strongly correlated with that in the paired conditions. Regression analyses suggested that both faster and slower participants adapted theirtap timing to that of their partners. A possible explanation for these results is that the participants reset the phase of their internal clocks according to the faster beat between their own tap and the partners’ tap. Our results indicated that paired context could bias the direction of ITI drift toward decreasing. One of the essential characteristics of humans is coordination with others. Many studies have reported that people coordinate their actions both intentionally and unintentionally12et al. reported that ITI from 250 to 413 ms tended to decrease, while those from 513 to748 ms tended to increase1011121314et al. conducted an SC tapping experiment with a reference ITI of 500 ms; the results provided examples of undulating ITI time series with periods of approximately 50–100 sThe synchronization-continuation (SC) paradigm is often used to explore the ability to produce regular time intervals. In SC tapping tasks, participants are initially required to tap in synchrony with metronome beats, and then continue to tap at the same pace after the metronome has been turned off. Intertap intervals (ITI) in solo SC tapping tasks have been found to drift gradually away from the metronome intervalset al. conducted paired SC tapping experiments involving uncoupled (neither of participants in a pair could hear their partner’s beats), unidirectional coupling (only one of the participants in a pair could hear their partner’s beats), and bidirectional coupling (both of the participants in a pair could hear their partners’ beats) conditions171819202122The number of studies examining a paired version of the SC tapping task has increased recently. These studies focused mainly on the local timing correction process. For example, Konvalinka Therefore, previous studies show that ITI tends to decrease during paired SC tapping tasks; however, it is unclear how ITI drift behaves when participants intend both to maintain the reference tempo and synchronize their tapping with that of their partners for several minutes. In this study, we conducted solo and paired SC tapping experiments, to determine whether the behaviour of the ITI drift (1) changes according to tempo ; 800, 500, and 300 ms in ITI, respectively), and (2) differs between solo and paired conditions. In the experiment, participants were asked to tap with keeping the initial tempo as much as they could. The duration of each trial was 200 s, during which participants tapped in synchrony with the metronome beats for the first 10 s and continued to tap without the metronome beats for the remaining 190 s, in both the solo and paired conditions. In the paired conditions,participants were asked to synchronize their tapping with that of their partners, in addition to maintaining the reference tempo. The participants could hear their partners’ tapping but could not see it. The results suggested that tapping paces gradually accelerated in paired conditions, while tapping paces fluctuated around the initial paces in solo conditions . The que7F = 23.65, p < 0.001, and tempo, F = 10.02, p < 0.001, and a significant interaction between these factors, F = 3.88, p = 0.028. Post-hoc tests revealed significant differences between solo and paired conditions for all tempo conditions: 75 bpm: t(23) = 3.86, p = 0.001; 120 bpm: t(23) = 4.06, p < 0.001; 200 bpm: t(23) = 2.46, p = 0.022. In solo conditions, the mean ITI for the final 30 taps differed significantlybetween 75 and 200 bpm, t(23) = 4.47, p < 0.001, and between 120 and 200 bpm, t(23) = 4.53, p < 0.001. In the paired conditions, a significant difference was observed only between 120 and 200 bpm, t(23) = 2.68, p = 0.014.Although 26 people (13 pairs) participated in the experiments, one participant was unable to synchronize with the partner. We excluded the data for this pair of participants; therefore, the following results are based on data from the remaining 12 pairs. r (12) = 0.64, p = 0.03, and 200 bpm, r (12) = 0.79, p < 0.01, while a marginally significant correlation was observed for 75 bpm, r (12) = 0.56, p = 0.06. In slower participants, the correlation between the means for the solo and paired conditions was significant for 200 bpm, r (12) = 0.82, p < 0.01, while those for 75 and120 bpm were nonsignificant, r (12) = 0.41, p = 0.19 and r (12) = 0.23, p = 0.48, respectively.The results described above suggest that ITI tended to decrease in the paired conditions. This raised a question as to whether there would be a correlation between the degrees of ITI drift in the solo and paired conditions. Within each pair, participants could be divided into those who tended to tap faster (the faster participants) and those who tended to tap slower (the slower participants) in the solo conditions. Therefore, we examined correlations between means for the final 30 ITI in the solo and paired conditions, dividing the participants in each pair into the faster and the slower groups, based on means for the final 30 ITI in the solo conditions . In Fig.∆ITI) for the entire duration of each trial was affected by the interaction between participants in the pair. We set the ∆ITI(n), which was derived by subtracting the nth ITI from the (n + 1)th ITI for one participant , as the dependent variable and the ∆ITI (n − 1) and the ITIAsync (n), which was derived by subtracting the nth ITI for the partner from the nth ITI for the participant , as explanatory variables. Note that the ITIAsync (n) occurs immediately prior to the ∆ITI (n) because of its definition (see 2) for the ∆ITI(n − 1) were 0.41, 0.37, and 0.37 for 75, 120, and 200 bpm, respectively. Meanwhile, the mean R2 values for the ITIAsync (n) were 0.55, 0.45, and 0.36 for 75, 120, and 200 bpm, respectively. To determine which explanatory variable exerted the stronger effect on the ∆ITI (n), we conducted a two-way repeated measures ANOVA to compare R2 values between the ∆ITI (n − 1) and ITIAsync (n), with tempo and type of explanatory variable as factors. The ANOVA revealed significant main effects of tempo, F = 26.34, p < 0.001, and type of explanatory variable, F = 13.90, p = 0.001, and asignificant interaction between the two factors, F = 21.19, p < 0.001. Post-hoc tests revealed that R2 values for the ∆ITI (n − 1) and ITIAsync (n) differed significantly at 75 bpm, t(47) = 7.21, p < 0.001, and 120 bpm, t(47) = 3.40, p = 0.001, whereas not at 200 bpm, t(47) = 0.21, p = 0.65. A significant main effect of tempo was observed for both the ∆ITI (n − 1) and ITIAsync (n), F = 6.22,p = 0.003 and F = 30.09, p < 0.001, respectively. R2 did not differ significantly between faster and slower participants in each pair , ∆ITI (n − 1), and ITIAsync (n) * ∆ITI(n − 1), respectively. The results are summarized in 2 values were 0.59, 0.51, and 0.48 for 75, 120, and 200 bpm, respectively. In general, R2 increases slightly even when adding meaningless variables such as random numbers; therefore, we performed a permutation test using dummy variables composed via the randomly permutated ∆ITI (n − 1), to determine whether the increase in R2 was significant (2 were conducted separately for each tempo condition (a priori comparison), with regression model type (a): single, (b): multiple with dummy variables, and (c): multiple with the original∆ITI (n − 1) as the factor. The results revealed significant main effects of regression model type, F = 13.64, F = 24.10, and F = 44.04 for 75, 120, and 200 bpm, respectively (ps < 0.001). Post-hoc tests revealed significant differences between regression model types for each tempo condition: (a) versus (b): Cohen’s d = 0.05, 0.04, and 0.02 for 75, 120, and 200 bpm, respectively (ps < 0.01); (a) versus (c): Cohen’s d = 0.31, 0.49, and 0.70 for 75, 120, and 200 bpm, respectively (ps < 0.001). Regarding βs, as tempoincreased, the absolute value of β1 decreased and the absolute value of β2 increased  for the slower participant would be higher relative to that of the faster participant. To test this hypothesis, we compared β values between the faster and slower participants in each pair. Two-way mixed-design ANOVAs were performed on β1, β2, and β3 separately, with participant type (faster and slower participants) and tempo as factors. The results are shown in F = 0.71, F = 2.29, and F = 0.19 for β1, β2 and β3, respectively (ps > 0.05). The main effect of tempo was significant for β1 and β2, F = 20.81 and F = 15.41, respectively (ps < 0.001), and nonsignificant for β3, F = 2.66, p = 0.08. The interaction between the two factors was nonsignificant for all β values, F = 0.88, F = 0.66, and F = 0.63 for β1, β2, and β3,respectively (ps > 0.10).Correlation analysis suggested that the degrees to which ITI decreased in the paired conditions were comparable to those observed for the faster participants. The next question was which caused the decrease in the ITI in paired conditions, leadership of faster participants or interactive timing modulation between partners. Tap timing asynchrony between partners fluctuated around zero  and ITI ncreased . If the ITIAsync) exerted a significant effect on intrapersonal timing modulation (∆ITI), which indicated that participants adjustedtheir tapping timing to follow that of their partners. The R2 value for the ITIAsync (n) increased as the tempo decreased, which suggested that participants had a greater margin for which they adapt their timing to that of their partners in slower conditions. This was replicated in the multiple regression analyses: the relative importance of the ITIAsync (n) increased as tempo decreased (ITIAsync (n) indicated that participants prolonged subsequent ITI when their current ITIwere shorter than those of their partners, and vice versa. Therefore, ITI decrease appeared to have occurred not only because faster participants led slower participants unidirectionally but also via an interpersonal timing adaptation process. Although the number of studies involving paired tapping tasks has increased, none examined differences in ITI drift between solo and paired SC tapping tasks in which each trial lasts for several minutes. Many previous studies examining social interaction and interpersonal coordination demonstrated the modulation of human self-paced movement timing via interpersonal interaction16The purpose of this study was to examine the effects of tempo and number of participants on ITI drift. Our results indicated that the extent to which ITI drifted in a negative direction from the reference ITI for the paired conditions was greater than that for the solo conditions, within a tempo range of 75–200 bpm (the ITI range of 800–300 ms). Previous studies using solo SC tapping suggested that tapping pace increased with ITI of 300 msecreased  and 6. If noise is a potential cause of ITI drift in solo SC tasksf noise has a large low-frequency component and is ubiquitous in biological signals, such as walking293234f noise does not occur in a specific direction: most of the time series displayed a simple negative trend in the current study.In general, 1/ITIAsync (n) did not differ significantly between the faster and slower participants in each pair , which is a tendency to respond a few tens of milliseconds earlier than the metronome beat, is known as a typical behaviour in synchronization tasks involving metronome beats7Which neural correlates do underlie performance in the paired SC task? The cerebellum and basal ganglia are considered to make the greatest contribution to the production of the subsecond interval timing used in our study, in both paced and self-paced timing tasks1920In conclusion, our study revealed that the durations of ITI tended to decrease in pairs performing an SC tapping task. This is the first study to focus on ITI drift in a paired SC tapping task lasting several minutes. Phase resetting would have caused ITI decrease; however, further investigation is required to determine the underlying process. Drift has been regarded as an inherent property of central timekeeper75026 healthy adults  participated in the experiment. Only two participants reported musical expertise; they were amateur drummers who had played for 5 and 10 years. All participants provided informed consent for their participation in the experimental procedures, which were conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of the Graduate School of Arts and Sciences at the University of Tokyo.www.stonekick.com) on theexperimenter’s smartphone  and sampled at 1,000 Hz using the same analog-to-digital converter. Partitions were used to separate the participants in each pair from each other and the experimenter, to prevent visual contact and possible distraction resulting from the experimenter’s gaze.Two electric drum kits  were used to measure participants’ tap timing and produce sound feedback. Participants tapped WAVEDRUM sensors (with a 9.3 cm × 6.5 cm hard plastic board pasted on top for easy tapping) located in front of them, and the corresponding voltage was sampled at 1,000 Hz, using a 16-bit analog-to-digital converter . The monitor facing participants showed the time remaining in each trial, using a progress bar programmed via LabVIEW . Participants could hear the metronome beats through a speaker located beneath the monitor, and their drum sounds and those of their partners were played through the speakers. The metronome beats were presented using an application . These settings were intended to cover slow, medium, and fast musical tempo as well as replicate those used in previous studies; the ITI series in a solo 800 ms conditionshould remain the same or increase, while those in the solo 500 and 300 ms conditions should decrease8We preprocessed data to optimize tap onset detection, using the following procedure: We obtained the zero-mean voltage time series by subtracting the mean value from the original time series, and performed full-wave rectification. Data were passed through a 100–300 Hz second-order Butterworth band-pass filter. Filtered data were Hilbert transformed, and the instantaneous amplitude was calculated to obtain an envelope for the waveform. The tap onset times in the preprocessed data were defined as the times at which the data value was larger relative to that of the threshold, and the all values of the adjacent 50-ms window were smaller relative to that of the threshold. The threshold was set at a value equivalent to the percentile rank of 95% from the lowest of the preprocessed data. We also implemented an algorithm to ignore the prospects of tap onset that were close (<60% of the reference ITI) to actual onset, to avoid detectingunintended consecutive hits.Post-hoc Bonferroni tests were performed to compare the factors for which a significant effect was observed. All ANOVAs were performed using SPSS version 20.0 for Windows .We obtained the ITI time series by subtracting the tap onset time from the adjacent tap onset time . ITI werWe also calculated correlation coefficients for the relationships between the means for the final 30 ITI in the solo and paired conditions, classifying the participants in each pair into the faster and slower groups based on the final 30 ITI in the solo trials.∆ITI (n) and the explanatory variables were the ∆ITI (n − 1) and ITIAsync (n). These variables and the ITI were defined using tap timing, as follows:Single and multiple regression analyses were performed to determine whether and the extent to which the interaction between participants in a pair contributed to the modulation of their tap timing, and examine the extent of this relationship. The dependent variable was the ITI (n), t (n), ∆ITI (n), ITIAsync(n), and ITIPartner(n) denote the nth value of the ITI, tap timing, ∆ITI, ITIAsync, and ITI (n) for the partner, respectively. Note that the moment at which the ∆ITI (n) arises is the moment at which the (n + 2)th tap occurs (ITIAsync immediately before the ∆ITI (n) is n ; therefore, we sought to determine the extent of the increase in R2 in a comparison between multiple and single regression analyses. Weperformed multiple regression analysis using the ITIAsync(n) and randomly permutated the ∆ITI (n − 1) as a dummy variable 1,000 times in each trial (a permutation test), in addition to using the ITIAsync(n) and the original ∆ITI (n − 1). The means of the 1,000 adjusted R2 values obtained via the permutation test in each trial were compared with those obtained via single regression analysis using the ITIAsync(n) and multiple regression analysis using the original ∆ITI (n − 1). A two-way repeated measures ANOVA was performed, with tempo and regression model type as factors, to compare R2 values. Prior to the ANOVA, we calculated the positive square root ofR2 and performed z-transformation. In addition, to determine whether the partial regression coefficients differed between the faster and slower participants in each pair in the solo conditions, we performed a two-way mixed ANOVA, with tempo and participant group (the faster and the slower participants in each pair) as factors, for each β value. The level of significance was set at 5% for all statistical tests. A Greenhouse-Geisser correction was used to assess sphericity. All post-hoc tests for multiple comparisons were performed using the Bonferroni method.where p occurs ; therefo(n) is n . The R2 How to cite this article: Okano, M. et al. Paired Synchronous Rhythmic Finger Tapping without an External Timing Cue Shows Greater Speed Increases Relative to Those for Solo Tapping. Sci. Rep.7, 43987; doi: 10.1038/srep43987 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.Insufficient autophagy in podocytes is related to podocyte injury in diabetic nephropathy (DN). Advanced glycation end‐products (AGEs) are major factors of podocyte injury in DN. However, the role and mechanism of AGEs in autophagic dysfunction remain unknown. We investigated autophagic flux in AGE‐stimulated cultured podocytes using multiple assays: western blotting, reverse transcription–quantitative PCR, immunofluorescence staining, and electron microscopy. We also utilized chloroquine and a fluorescent probe to monitor the formation and turnover of autophagosomes. Mice of the db/db strain were used to model diabetes mellitus (DM) with high levels of AGEs. To mimic DM with normal levels of AGEs as a control, we treated db/db mice with pyridoxamine to block AGE formation. AGEs impaired autophagic flux in the cultured podocytes. Compared with db/db mice with normal AGEs but high glucose levels, db/db mice with high AGEs and high glucose levels exhibited lower autophagic activity. Aberrant autophagic flux was related to hyperactive mammalian target of rapamycin (mTOR), a major suppressor of autophagy. Pharmacologic inhibition of mTOR activity restored impaired autophagy. AGEs inhibited the nuclear translocation and activity of the pro‐autophagic transcription factor EB (TFEB) and thus suppressed transcription of its several autophagic target genes. Conversely, TFEB overexpression prevented AGE‐induced autophagy insufficiency. Attenuating mTOR activity recovered TFEB nuclear translocation under AGE stimulation. Co‐immunoprecipitation assays further demonstrated the interaction between mTOR and TFEB in AGE‐stimulated podocytes and in glomeruli from db/db mice. In conclusion, AGEs play a crucial part in suppressing podocyte autophagy under DM conditions. AGEs inhibited the formation and turnover of autophagosomes in podocytes by activating mTOR and inhibiting the nuclear translocation of TFEB. © 2018 The Authors.  Diabetic nephropathy (DN) is the leading cause of chronic and end‐stage renal disease worldwide Autophagy is an intracellular process that involves the degradation and recycling of defective cytosolic proteins and organelles via lysosomes Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates the growth and proliferation of cells. It is the most studied regulator of autophagy; it inhibits autophagy initiation by phosphorylation of autophagy‐related proteins Transcriptional regulation also plays a vital part in coordinating autophagy et al reported that the sera of DM patients and rodents with massive proteinuria caused autophagy insufficiency in cultured podocytes in vivo and in vitro.Podocytes exhibit a high level of constitutive autophagy, which is crucial for stress adaptation Further details can be found in the supplementary material, Supplementary materials and methods.The protocol of this study was approved by the Ethics Committee of Guangdong General Hospital . The study of patients was conducted according to the Helsinki Declaration. Renal tissues from biopsies were obtained with the informed written consent of patients. Normal kidney tissues distal from the tumor margin were obtained from patients with renal cell carcinoma after unilateral nephrectomy. These tissues were confirmed to be normal renal tissue by further pathological examination. Clinical characteristics of the patients are presented in the supplementary material, Table S1.m; Sigma‐Aldrich, St Louis, MO, USA) or an equal volume of dimethyl sulfoxide (Sigma‐Aldrich) as vehicle according to our method m Torin1 or an equal volume of dimethyl sulfoxide as vehicle for 3 h. The treatment time and concentration of CQ or Torin1 were based on the results of our preliminary experiment  To investigate the role of AGEs, cultured podocytes were exposed to 100 μg/ml AGE–bovine serum albumin  or BSA alone for 72 h unless otherwise noted . To study autophagosome formation, podocytes were treated with chloroquine  for 12 h. Three siRNAs targeting TFEB and control were used . The most effective one was selected for final experimentation . Podocytes were transfected with the most effective TFEB siRNA or negative control siRNA (scrambled siRNA), using Lipofectamine 2000  according to the manufacturer's protocol. To investigate the autophagic flux, podocytes were transfected with GFP‐mRFP‐LC3 adenovirus as we described previously Podocytes treated with BSA or AGE–BSA were used for ChIP assays. The Simple ChIP Enzymatic Chromatin IP Kit  was used following the manufacturer's protocol. Anti‐TFEB antibody  was used to pull down DNA–protein complexes, and goat IgG was used as a control. Purified DNA was quantified using quantitative PCR (qPCR); primer sequences  are listed in the supplementary material, Table S4.2 Spirit Twin; FEI).Transmission electron microscopy (TEM) was used to measure the width of podocyte foot processes and autophagic vacuoles. Kidney tissue specimens from humans or mice or cultured podocytes were fixed using 2.5% glutaraldehyde. Gradient dehydration using alcohol and post‐fixation using 1% osmium tetroxide were completed prior to embedding in resin (Epon 812). Ultrathin sectioning and double staining using uranyl acetate and lead citrate were employed to prepare TEM samples, which were examined using a transmission electron microscope . The width of podocyte foot processes was calculated as described previously Animal experimental procedures were undertaken according to the protocols of the Guangdong Academy of Medical Sciences. Eighteen male C57BL/KsJ db/db mice and six age‐matched non‐diabetic (db/m) mice were purchased from the Model Animal Research Center of Nanjing University .To investigate the role of AGEs in the autophagy in podocytes of db/db mice, we generated a DM model with normal levels of AGEs using pyridoxamine . This agent is a derivative of vitamin B6 and can reduce serum AGE levels by blocking AGE formation with no effect on the blood levels of glucose or lipids n = 6), db/db + PYR (n = 6) or db/db + Torin1 (n = 6) groups by body weight. PYR was given by gavage (400 mg/kg daily) for 12 weeks. Torin1 , an inhibitor of mTOR, was diluted to 2 mg/ml in 0.15 m NaCl, 5% polyethylene glycol 400, and 5% Tween 20 immediately before injection, as reported previously At 8 weeks of age, db/db mice were allocated randomly into db/db  after 6 h of fasting. AGE levels in the sera of experimental mice at 20 weeks were assessed using an OxiSelect™ Advanced Glycation End Product ELISA kit . Body weight was measured every week. For urine collection, once every 2 weeks individual mice were held in a metabolic cage for 24 h. Urinary levels of albumin and creatinine were measured using albumin ELISA  and creatinine kits  for mice, respectively, according to the manufacturers' instructions. Albuminuria was expressed as the ratio of albumin to creatinine. Mice were anesthetized  before killing, and kidney tissue was collected.7 Dynabeads M‐450  through the heart. The kidneys were removed and minced on ice into 1 mm3 pieces before digestion by collagenase A  and deoxyribonuclease I  at 37°C for 15 min with gentle agitation. The digested tissue was gently pressed through a 100 μm cell strainer  and then passed through a new strainer without pressing. The filtered tissue was transferred to a cooled tube and centrifuged at 200 × g for 5 min. The supernatant was discarded and the pellet was resuspended and transferred to a magnetic particle concentrator, which gathered the glomeruli that contained Dynabeads. The isolated glomeruli were washed at least three times with cold HBSS and then resuspended in lysis buffer  and homogenized in a Dounce homogenizer at 4°C. After protein extraction, the suspension was centrifuged at 14 000 × g for 10 min at 4°C; the pellet containing Dynabeads and insoluble material was discarded; and the supernatant was used for immunoprecipitation assays.db/db mice (n = 8) and db/db mice treated with PYR (n = 8) were anesthetized and then perfused with 8 × 10t‐test. Multiple comparisons were carried out using one‐way analysis of variance (ANOVA) for the cell culture experiments or two‐way ANOVA for the animal experiments. The Student–Newman–Keuls test was used for post hoc multiple comparisons. A two‐tailed p < 0.05 was considered significant.Results are reported as mean ± standard error of the mean (SEM). Statistical analysis was performed with SPSS v21.0 . All experimental procedures were repeated at least three times. Between‐group comparisons were made using Student's To explore podocyte autophagy in patients with DN, we utilized TEM to examine the number of autophagic vacuoles and immunofluorescent staining to observe microtubule‐associated protein 1 light chain 3  dots in podocytes.in vivo.We found fewer autophagic vacuoles and LC3 dots in podocytes from patients with DN compared with the control Figure A, B. WesTo understand the role of AGEs in the absence of high glucose levels in autophagic activity, we stimulated cultured podocytes with AGE–BSA. Under this condition, western blotting showed that the biosynthesis of LC3II protein and turnover of p62 were blocked, indicating inhibited autophagy , membrane fusion , lysosome biogenesis (Lamp1), and substrate degradation (Vps11)  Figure B, C. Mor) Figure D, E. TFE) Figure F. This aNuclear TFEB localization was decreased in podocytes from patients with DN Figure A and db/Atg9b, Lc3b, Lamp1, and Vps11  can recover TFEB activity through mTOR inhibition First, we evaluated these two mTOR inhibitors and further investigated the interplay between mTOR and TFEB. Rapamycin had no effect on TFEB distribution , whereas Torin1 recovered nuclear TFEB expression in db/db mice Figure I. These Insufficient autophagy in podocytes is a characteristic of DM patients and animals. Podocyte‐specific autophagy deficiency aggravates podocyte injury in DM mice et al found that sera from DM animals impaired autophagy in podocytes in vitroTagawa In STZ‐induced DM mice, staining for the autophagy marker LC3 in podocytes was found to be increased 4 weeks post‐STZ injection but decreased to below normal conditions 8 weeks post‐STZ injection We compared db/db mice with high or relatively normal AGE levels and also stimulated cultured podocytes with AGE–BSA or BSA. We monitored autophagic flux by multiple assays: western blotting, TEM, tandem probe GFP‐mRFP‐LC3, and RT‐qPCR. Our results demonstrate, for the first time, that AGEs blocked podocyte autophagic flux. Importantly, blockade of AGE accumulation in PRY‐treated db/db mice could recover podocyte autophagy. Because the glucose level in these PRY‐treated db/db mice remained high, this result implies the importance of AGEs in autophagy.in vitro. This suppression was confirmed further using the lysosomal inhibitor CQ and tandem probe GFP‐mRFP‐LC3. In db/db mice, expression of LC3II and beclin1 in kidney cortices and autophagosome number in podocytes were decreased. This downregulation of LC3II expression in db/db mice is not in accordance with a previous study suggesting upregulation of LC3 expression in STZ‐induced DM mice 4 weeks after STZ injection in vivo, 4 weeks of STZ injection may be too short for AGE accumulation. Conversely, db/db mice were under chronic hyperglycemia and had 1.7‐fold more AGEs than control mice . Our results are in accordance with data showing decreased LC3 expression in STZ‐induced DM mice 8 weeks after STZ injection Lamp1 and is consistent with Tagawa et al's report We studied further the role of AGEs in the formation and turnover of autophagosomes. AGEs decreased expression of the markers of autophagosome formation  The hyperactivation of mTOR in podocytes is a crucial step in the development of DN Besides post‐translational modulation by mTOR, autophagy can also be regulated by transcription factors such as TFEB TFEB is a therapeutic target for lysosomal‐storage diseases Recently, studies have unveiled the crucial role of mTOR as a TFEB regulator. However, the effect of mTOR on TFEB is significantly different. Brugarolas and co‐workers proposed that in Tsc2 knockout (mTOR constitutive activation) cells, TFEB is transported into the nucleus Nuclear localization of TFEB can be regulated by multiple serine/threonine kinases under different conditions, including mTOR, extracellular regulated kinase In conclusion, we unveiled a crucial role of AGEs in suppressing podocyte autophagy under DM conditions. We also found that TFEB is essential for autophagy and homeostasis in podocytes. AGEs inhibited the formation and turnover of autophagosomes in podocytes by activating mTOR and inhibition of the nuclear translocation of TFEB .XL and WS conceived the experiments. XZ, YC, and WS analyzed the data. XZ, YC, and XL wrote the manuscript. All the authors were involved in carrying out the experiments and had final approval of the submitted and published versions.Supplementary materials and methodsFigure S1. Role of AGEs in LC3II expressionFigure S2. AGEs block autophagosome formationFigure S3. Torin1 reduces mTOR activityFigure S4. Efficiency of TFEB interference and podocyte injury by TFEB knockdownFigure S5. The principle of the GFP‐mRFP‐LC3 fluorescent probeFigure S6. Effect of pyridoxamine (PYR) on blood levels of glucose, AGEs, and lipid parameters in experimental miceFigure S7. Change in TFEB expression in db/db mice and AGE‐induced podocytesFigure S8. DNA electrophoretogramFigure S9. Role of rapamycin in TFEB nuclear expressionFigure S10. The proposed role of AGEs in suppressing podocyte autophagyTable S1. Clinical characteristics in control subjects and patients with diabetic nephropathyTable S2. SiRNA sequencesTable S3. Antibody informationTable S4. Primers used for ChIP assaysTable S5. Primers used for RT‐qPCRAppendix S1. Supplementary materials and methodsClick here for additional data file.Figure S1. Role of AGEs on LC3II expression. (A) Cultured podocytes were treated with BSA or AGE‐BSA (AGE) for the indicated time. Western blotting indicated that AGE inhibited LC3II expression at 72 h  One way ANOVA, followed by post hoc Student–Newman–Keuls test. *P < 0.05 versus control.Figure S2. AGEs block autophagosome formation. AGEs decreased chloroquine (CQ)‐induced LC3II accumulation as shown by western blotting (A) and reduced CQ‐induced autophagosome accumulation on TEM (B) . One‐way ANOVA, followed by post hoc Student–Newman–Keuls test. *P <0.05 versus control, #P <0.05 versus CQ.Figure S3. Torin1 reduces mTOR activity. (A) Western blot assays show reduction of the p‐p70s6k to p70s6k ratio at 3 h in cultured podocytes by various Torin1 (TR) concentrations . One‐way ANOVA, followed by post hoc Student–Newman–Keuls test. *P <0.05 versus control. (B) Western blot assays of p‐p70s6k and p70s6k expression from the renal cortex showed that the p‐p70s6k to p70s6k ratio was increased in db/db mice . Two‐way ANOVA, followed by post hoc Student–Newman–Keuls test. *P <0.05 versus control, #P <0.05 versus db/db mice.Figure S4. Efficiency of TFEB interference and podocyte injury by TFEB knockdown. (A) Cultured podocytes were transfected with three siRNAs designed to target TFEB or scrambled siRNA. RT‐qPCR showed that TFEB mRNA expression was significantly downregulated by TFEB siRNA but not TFEB siRNA‐2 or TFEB siRNA‐3 . One‐way ANOVA, followed by post hoc Student–Newman–Keuls test, *P <0.05 versus control. (B) Western blot assay results showed that TFEB siRNA significantly reduced TFEB protein expression . One‐way ANOVA, followed by post hoc Student–Newman–Keuls test. *P <0.05 versus control. (C) Western blot assays showed that TFEB siRNA significantly reduced nuclear TFEB expression . One‐way ANOVA, followed by post hoc Student–Newman–Keuls test, *P <0.05 versus control. (D–F) TFEB siRNA significantly downregulated the expression of podocin (D) and synaptopodin (E) proteins, which are markers of intact podocytes, and (F) upregulated the expression of urokinase‐type plasminogen activator receptor (uPAR) a marker of podocyte injury.  Student's t‐test, *P <0.05 versus scrambled siRNA. (G) Cultured podocytes were transfected with and empty vector control or flag‐TFEB adenovirus (oTFEB). Expression of TFEB mRNA was significantly increased by oTFEB . One‐way ANOVA, followed by post hoc Student–Newman–Keuls test, *P <0.05 versus control. (H) Western blot assays show that TFEB protein expression was upregulated by oTFEB.Figure S5. The principle of the GFP‐mRFP‐LC3 fluorescent probe.Figure S6. Effect of pyridoxamine (PYR) on blood levels of glucose, AGEs, and lipid parameters in experimental mice. Serum AGEs (A), blood glucose (A), total cholesterol (B) and triglycerides (TRIGs) (C) were increased in db/db mice compared with db/m control mice. PYR reduced AGEs without affecting blood glucose, total cholesterol and TRIGs . Two‐way ANOVA, followed by post hoc Student–Newman–Keuls test, *P <0.05 versus db/m mice, #P <0.05 versus db/db mice. NS: no significance.Figure S7. Change in TFEB expression in db/db mice and AGEs‐induced podocytes. (A) TFEB expression in the nuclear and cytosolic fractions of mouse renal cortex was assayed by western blotting. Both PYR or torin1 reduced the expression of nuclear TFEB in db/db mice and increased cytosolic expression . One‐way ANOVA, followed by post hoc Student–Newman–Keuls test, *P < 0.05 versus db/m mice, #P <0.05 versus db/db mice. (B) Immunofluorescence staining of TFEB (red) in the renal cortex of db/db mice and db/m mice showed that TFEB was expressed not only in podocytes but also in other renal resident cells (dashed frame). TFEB expression in the renal tubules tended to increase under diabetic conditions. Scale bar = 20 μm. (C) Western blots of TFEB expression in cultured podocytes. After AGEs stimulation for 48 h, the nuclear TFEB and the cytosolic TFEB were respectively downregulated and upregulated, while the total TFEB was not changed significantly. Torin1 recovered nuclear TFEB expression, which was reduced in AGEs‐stimulated podocytes and did not affect total TFEB protein expression . One‐way ANOVA, followed by post hoc Student–Newman–Keuls test, *P <0.05 versus control, #P <0.05 versus AGE.Figure S8. DNA electrophoretogram. (A) Sonication effects were evaluated by agarose gel electrophoresis. The genomic DNA samples were ultrasonicated forming fragments with length of approximately 100–500 base pairs.Figure S9. Role of rapamycin on TFEB nuclear expression. (A) Cultured podocytes were treated with 5 nM rapamycin or an equal volume of the dimethyl sulfoxide vehicle for 24 h. Rapamycin did not affect nuclear TFEB expression as measured by western blotting . Student's t‐test, *P <0.05 versus control.Figure S10. The proposed role of AGEs in suppressing podocyte autophagy. (1) AGEs inhibit podocyte autophagy through mTOR activation. (2) AGEs suppress TFEB activity by inhibiting the nuclear translocation of TFEB and thus interrupting the translation of autophagy genes including Atg9b, LC3b, Lamp1 and Vps11. (3) AGEs‐induced mTOR activation decreases nuclear translocation of TFEB.Click here for additional data file.Table S1. Clinical characteristics in control subjects and patients with diabetic nephropathyClick here for additional data file.Table S2. SiRNA sequencesClick here for additional data file.Table S3. Antibody informationClick here for additional data file.Table S4. Primers used for ChIP assaysClick here for additional data file.Table S5. Primers used for RT‐qPCRClick here for additional data file.


The aim of the present investigation was to examine the acute effect of low and high levels of caffeine, −2) ingested 200 mL of water (W), coffee with low caffeine , or coffee with high caffeine  on three respective separate occasions. All sessions were performed at 09:00 in the morning in a counterbalanced, crossover manner, at least 5 days apart. Subjects remained in the laboratory while urine samples were collected every 60 min for 3 h post ingestion.Ten healthy adults , when compared to W (356 ± 53 mL) and LCAF (316 ± 38 mL). In addition, cumulative urinary osmotic excretion was significantly greater in the HCAF , as compared to the W (249 ± 36 mmol) and LCAF (177 ± 16 mmol) trials.Absolute caffeine consumption was 269 ± 45 and 537 ± 89 mg for the LCAF and HCAF, respectively. Coffee ingestion at the HCAF trial induced greater diuresis during the 3-h period (613 ± 101 mL, −1 in the form of coffee can induce an acute diuretic effect, while 3 mg kg−1 do not disturb fluid balance in healthy casual coffee drinking adults at rest.The data indicate that caffeine intake of 6 mg kg However, this is a standard amount not relative to body weight, which has been shown to effect caffeine metabolism , a naturally occurring methylxanthine also found in tea, chocolate, and energy drinks, acts as a competitive adenosine receptor antagonist to reduce fractional sodium reabsorption in both the proximal tubule and distal nephron , 15. Due−1 caffeine in the form of coffee . However, there was a slight increase in urinary sodium excretion  was investigated.However, few studies have specifically investigated the effects of caffeine in the form of coffee on fluid balance and electrolyte excretion. This is surprising considering coffee holds second position in consumption among all beverages after water . One stuxcretion . AlthougTo the authors’ knowledge, these are the only two studies directly investigating the diuretic effects of caffeinated coffee. The studies utilized different doses of caffeine  and reached different conclusions regarding diuresis. Consequently, it is important to elucidate fluid balance characteristics of coffee and to observe its dose–responses on hydration markers. The aim of this study was to compare the acute effects of low and high caffeinated coffee ingestion on fluid balance in habitual coffee drinkers at rest.−2) were recruited by local advertisement to participate in this study. Exclusionary criteria included smoking, specialized diets, participation in competitive sports, as well as hypertension, metabolic disease, gastrointestinal conditions, or recent surgical incidents. All participants were habitual coffee drinkers (1–3 cups per day). The protocol was approved by the Harokopio University Ethics Committee. Subjects provided informed written consent in accordance with the Declaration of Helsinki.Ten healthy adults , or 200 mL of instant coffee with high caffeine . The HCAF and LCAF trials were performed in a single blind fashion. The amount of fluid consumed during each trial did not change; however, the amount of coffee and concentration of caffeine in the 200 mL of fluid differed between treatments. This process was to ensure similar fluid balance at the onset of each trial. After ingesting coffee or water, volunteers rested in seating position in the laboratory for 3 h to allow for collection of urine samples.Study participants completed three trials on separate occasions at least 5 days apart in a counterbalanced, crossover manner. All sessions took place in the morning at 09:00 a.m. Participants refrained from ingesting foods or drinks with caffeine or other methylxanthines 24 h before each trial. After an overnight fast, they arrived at the laboratory, provided a urine sample and emptied their bladders. They consumed a standard breakfast composed of one slice of white bread with 5 g of butter and 10 g of sugar, within 5 min. Then they ingested 200 mL of water, 200 mL of instant coffee with low caffeine , while potassium and sodium via an electrolyte sensitive analyzer . Urinary osmotic or electrolyte excretion was determined by multiplying the volume and concentration of samples for each time-point. Cumulative values were also reported to present the overall effect over the 3-h timeline.Urine was collected at 60, 120, and 180 min after the test drink ingestion and UV was measured. If a volunteer needed to void outside of this structured time frame, this volume was added to the cumulative volume during the corresponding hour. The samples were analyzed fresh and in duplicate for osmolality and electrolyte content. Urine osmolality (UOsm) was measured P < 0.05 level.A two-way repeated measures ANOVA with Bonferroni correction was used to detect differences between trials. All statistical procedures were completed using JMP Pro 13. Data are presented as means ± SD of the means unless stated otherwise. Significance was set at the Volunteers consumed 269 ± 45 and 537 ± 89 mg of caffeine for LCAF and HCAF trials, respectively, representing a low and high dose of caffeine.P < 0.05). The cumulative UV (mL) during the 3-h period after the 200-mL bolus of either water or the two coffee trials is presented in Figure P < 0.05) when compared to LCAF and the W trial at the 2- and 3-h mark . UOsm was not different between treatments or time-points among trials (P < 0.05). However, cumulative urinary osmotic excretion was significantly higher during the HCAF trial than the LCAF and W trial at the 2- and 3-h time-points, without any differences between LCAF and W . Cumulative urinary sodium excretion at the 180-min time-point was significantly higher in HCAF as compared to the same time-point of water and LCAF (P < 0.05). Cumulative urinary potassium excretion was significantly higher at the 180-min time-point of HCAF as compared to the W and LCAF trial.Table k Figure ; no diff−1) resulted in an acute diuretic effect represented by higher cumulative UV 2 and 3 h after ingestion. UV and cumulative urinary osmotic excretion were higher 3-h after ingestion of 6 mg kg−1 of caffeine as compared to 3 mg kg−1 or water. This finding provides insight into the specific amount of caffeine relative to bodyweight and in the form of coffee that elicits a diuretic response. Participants during the HCAF treatment averaged 537 mg as compared to 269 mg of caffeine consumption in the LCAF treatment. A standard cup of coffee contains 95–165 mg of caffeine  induces an acute diuretic effect. This effect is not seen with lower doses (3 mg kg−1) or plain water. Therefore, habitual consumers of coffee not exceeding 3 mg kg−1 of caffeine should not worry about detrimental diuretic effects. However, once consumers approach 6 mg kg−1 of caffeine potential disruptions in fluid balance should be considered.Having considered study’s limitations, the results suggest caffeinated coffee in higher doses (6 mg kgThe protocol was approved by the Harokopio University Ethics Committee. Subjects provided informed written consent in accordance with the Declaration of Helsinki.SK, MY, and AG designed research; CB, AG, PG, and GA, conducted data collection and sample analysis; AS, JA, CB, and SK analyzed the data; AS, JA, and SK wrote the paper. SK was the principal investigator and had primary responsibility for the final content. All authors read, critically revised, and approved the final manuscript.SK was a scientific consultant for Quest Diagnostic and has active grants with Danone Research. AS is a scientific consultant for Gatorade Sports Science Institute. CB, AG, JA, PG, GA, and MY have no conflict to declare.