Infant feeding experiences are important for the development of healthy weight gain trajectories. Evidence surrounding milk feeding and timing of introduction to solids is extensive; however, the impact of the method of introducing solids on infant growth has been relatively underexplored. Baby‐led weaning (where infants self‐feed family foods) is proposed to improve appetite regulation, leading to healthier weight gain and a reduced risk of obesity. However, the evidence is mixed and has methodological inconsistencies. Furthermore, despite milk being a large part of the infant diet during the period infants are introduced to solid foods, its influence and interaction with introductory style have not been considered. The aim of this study was to explore growth among infants aged 3–12 months according to both style of introduction to solid foods and milk feeding; 269 infants were weighed and measured, and body mass index (BMI) computed. The results showed that overall, infants who were spoon‐fed (compared with self‐fed) at introduction to complementary feeding (CF) had greater length (but not weight or BMI). However, when milk feeding was accounted for, we found that infants who were both spoon‐fed and fully formula fed had greater weight compared with spoon‐fed, breastfed infants. There was no significant difference in weight among self‐fed infants who were breastfed or formula fed. The results highlight the importance of considering infant feeding as a multicomponent experience in relation to growth, combining both milk feeding and method of CF. This relationship may be explained by differences in maternal feeding style or diet consumed. Early feeding experiences are important in relation to growth and appetite regulation in infancy and later life.There is an association between spoon‐feeding and length, but this association does not increase over time, suggesting a longer length may lead to an infant being spoon‐fed.Infants who are spoon‐fed and fully formula fed are heavier than those spoon‐fed and breastfed, but no difference in weight is seen for self‐fed babies whether they are breastfed or formula fed.A responsive feeding approach is likely to foster healthy weight gain trajectories and appetite regulation. Infants who are both spoon‐fed and bottle fed may have less opportunity to self‐regulate their intake of food.1Feeding experiences during the first year of life are recognised as playing an important role in laying down the foundations of weight and eating behaviour  approach, where infants self‐feed family foods, has been proposed to promote healthier weight gain trajectories than a traditional spoon‐feeding approach. Research with mothers who follow BLW highlights a perception that infants have greater control over their intake of food and therefore demonstrate greater satiety responsiveness (regulation of food intake), supporting a healthy weight  study—a trial that randomised mothers to either a baby‐led approach or traditional feeding approach using research‐led clinical measurements of infant growth—found no difference in infant weight at 12 or 24 months. However, adaptations made to the specific baby‐led feeding approach encouraged may differ from how many families practice BLW in the community. Families were encouraged to offer their infants protein at every meal, with an emphasis on good fats, as there was concern that infants may not self‐feed sufficient calories  study, infants who were breastfed throughout the first year had a reduced risk of overweight compared with those who discontinued breastfeeding , were excluded from participation.Participants were from the SHIFT  study in the United Kingdom, which studied the impact of different aspects of infant feeding upon weight and growth of infants aged 3–12 months in the United Kingdom. Data were collected between February 2016 and November 2017. Participants are mother–infant dyads from South Wales and South West of England. All infants in the study were born 2.2z‐score (WAZ), length‐for‐age z‐score (LAZ), body mass index‐for‐age z‐score (BMIZ), and weight gain velocity (WAZV) were the primary outcome measures.Infant weight and length were measured by the lead researcher using SECA® calibrated scales and SECA length measuring mat. Mothers self‐reported infant birth weight. Weight‐for‐age Mothers completed a self‐report questionnaire including infant and maternal demographic background , milk feeding at birth (breast or formula milk) and at time of questionnaire , infant age at commencement of complementary feeding, and method of introducing complementary foods.always, often, sometimes, rarely, and never).To measure the method of introducing complementary foods, participants were asked to estimate their infant's degree of spoon‐feeding at the introduction to complementary feeding. This approach was used as self‐definition of feeding approach , which varies in reliability  were placed in community halls, family centres, and on social media. Interested participants contacted the lead researcher for further study information. Contact was also made with leaders of local baby groups for permission to attend the group and give a short presentation about the research. Participants were given a study information and consent form, and if they met the inclusion criteria, a suitable time for measuring their baby and completing the background questionnaire was agreed.Some participants chose to have measurements taken of their baby at just one time point, and these measurements formed a larger cross‐sectional data set. However, participants, particularly those whose baby was as the start of the period of introducing complementary foods (around 4–7 months) were invited to have their baby measured again in at least 4 months' time (towards the end of the complementary feeding period) to form part of a longitudinal data set.2.4z‐scores were calculated using the Anthro software package, which uses the World Health Organization  versus predominantly spoon‐fed . This was based on similar categorisation used in previous BLW research  and one for infants in the longitudinal data set. Where infants had two sets of measurements , only one of these sets of measurements was included in the cross‐sectional data set. A decision was made to use the second set of measurements, as this gave a more balanced range of ages across the cross‐sectional data set. Fewer parents were available for measurements when their baby was towards the end of the complementary feeding period (10–12 months) due to practicalities of returning to work.t test was used to examine any differences in birth weight z‐score between those who went on to choose a self‐feeding or spoon‐feeding approach. Multivariate analysis of covariance (MANCOVA) was used to examine the difference in WAZ, LAZ, BMIZ, and WAZV between feeding groups. Partial correlations explored associations between infants' age at time of measurement and growth. For infants with two measurements over time, a repeated measures analysis of variance was used to explore differences in WAZ, LAZ, and BMIZ over time.The association between maternal and infant demographic background and feeding approach was explored. A The combined impact of milk feeding and complementary group upon growth was explored through separate models for self‐fed and spoon‐fed infants, following analyses used in similar studies , with 95% power with alpha at.05. The second, for the repeated measures analyses, indicated that a sample size of 106 was required to detect a small effect size with 95% power, at the.05 alpha level.For the main sample size, we conducted two separate power calculations  to examine sample size. The first indicated that a sample size of at least 210 would be required to detect a small effect size  and the British Psychological Society Code of Research Ethics .3SD] = 9.62; range: 26–59). There were two sets of twins giving a total of 267 mothers. Mean age of the mothers was 31.7 . For further maternal demographic details, see Table n = 4) or maternal education (n = 2), giving 263 complete data sets available for analyses, which include these variables.Two hundred and sixty‐nine infants were weighed and measured. For the infants, 134 were male and 135 were female. Mean age was 42.5 weeks . For feeding approach at the start of complementary feeding, 109 infants were classified as self‐feeding (40.5%) and 160 as spoon‐fed (59.5%). For feeding approach at the time of measurement, 182 infants were predominantly self‐feeding and 86 were being spoon‐fed.Mean age of starting complementary foods was 23.5 weeks  or maternal education, X2(4) = 2.28, p = .68, and feeding approach.No significant associations were found for either maternal age (X2(2) = 46.2, p < .000. Infants who self‐fed started complementary foods at a significantly later age (Mdn = 26 weeks) than spoon‐fed infants . Therefore, milk feeding and timing of introduction to solid foods were controlled for throughout.A significant association was found between milk feeding and complementary feeding approach. Infants who self‐fed were more likely to be breastfed at the time of the questionnaire, and spoon‐fed infants were more likely to be formula fed, 3.1t test found no significant difference in infant birth weight z‐score between mothers who went onto follow a self‐fed or spoon‐fed approach, t(269) = 0.20, p = .84. Infants who self‐fed had a mean birth weight z‐score of 0.29 (SD: 11) and spoon‐fed a mean z‐score of 0.32 (SD: 0.97).A 3.2F = 3.34, p = .07, BMIZ, F = 0.29, p = .59, or WAZV (change in weight for age z‐score from birth to age at time of measurement), F = 1.38, p = .24, was found according to feeding approach. However, LAZ significantly differed between the groups, F = 6.25, p = .01. The spoon‐fed infants had the greatest mean LAZ , and the self‐fed infants were the shortest .A MANCOVA examined differences in WAZ, LAZ, BMIZ, or WAZV between feeding groups. For feeding approach at the start of introduction to complementary foods, no significant difference in WAZ, To explore whether growth changed over the period of introduction of solid foods, potentially due to length of exposure to introduction methods, partial correlations between each growth outcome and the length of time exposed to solids (in weeks) were undertaken .N = 109), no significant associations seen with WAZ , LAZ , or BMIZ  and length of time exposed to solids. Similarly, among infants who were spoon‐fed (N = 160), no significant associations were found for WAZ , LAZ , or BMIZ  and length of time exposed to solids.Two separate analyses were conducted to explore this association in self‐fed and spoon‐fed infants. Among infants who self‐fed (3.3n = 101) were measured twice: one at the start of complementary feeding and one towards the end with at least 16 weeks between measurements. Mean infant age at first measure was 24.9 weeks (SD: 8.12), and mean infant age at second measure was 51.7 weeks (SD: 7.47).A further subset of infants  = 0.03, p = .86, and interaction effect between time and approach group was also not statistically significant, F = 1.79, p = .18. For LAZ, the between‐subject effect was not significant, F = 0.22, p = .64, and interaction effect between time and approach group was also not statistically significant, F = 0.12, p = .73. For BMIZ, the between‐subject effect was not significant, F = 1.003, p = .32, and interaction effect between time and approach group was also not statistically significant, F = 3.34, p = .07.Changes in WAZ, LAZ, and BMIZ were examined for the two approach groups over time. One hundred and one infants had two measures (30 self‐fed and 71 spoon‐fed): one at around 6 months and one at around 12 months. Table 3.4z‐score, and timing of solids introduction.Within each feeding approach group, differences in growth between breastfed and formula‐fed infants were examined. Any breastfeeding  was compared with full formula feeding Table . Any breF = 0.04, p = .84, LAZ, F = 0.002, p = .88, or BMIZ, F = 0.007, p = .93, based on whether the infant had received any breast milk (N = 94) or were fully formula fed (N = 14). However, a significant difference in WAZ was seen among the spoon‐fed infants, F = 4.26, p = .04. Fully formula‐fed infants (N = 81) were found to be significantly heavier  than infants who had received any breastfeeding . No significant differences were seen for LAZ, F = 0.28, p = .59, or BMIZ, F = 2.32, p = .13, between full formula feeding and any breastfeeding.Within the self‐fed group, no significant growth differences were found in WAZ, 4This study explored the impact of method of introducing complementary foods upon growth at 3–12 months, considering further how this might interact with milk feeding method. It found no significant differences in weight or BMI between infants introduced to complementary foods via spoon‐feeding or self‐feeding but found that infants who were spoon‐fed were significantly longer than those who were self‐feeding. However, no significant differences were found for current method of introduction. Moreover, although no significant difference was seen in the growth of infants who were self‐feeding according to milk type, infants who were both spoon‐fed and fully formula fed were significantly heavier than those spoon‐fed alongside being breastfed. The findings have important considerations for how responsive feeding may be a key aspect of growth in the latter part of infancy.First, infants who were following a traditional complementary feeding approach were longer than those that were self‐feeding. It is possible that method of complementary feeding is affecting length gains due to differences in infant diet. Spoon‐fed infants are more likely to consume fortified infant cereals when compared with self‐feeding infants, especially when introduced to solid foods at an earlier date  is more “controlling.” This may be affecting weight, although research examining the longer term impact of responsive feeding in infancy is sparse. However, a large body of literature suggests that for older children, a responsive feeding style is associated with the most positive weight outcomes. For example, in a 2015 systematic review of the literature, the authors concluded that restrictive and controlling feeding practices were associated with higher child BMI at ages 4–12 years , and thus, the length of time for mothers to recall earlier information  was variable, potentially affecting reliability. However, infant feeding data are likely to be accurately recalled within a time period of 3 years (Li, Scanlon, and Serdula , so variLimitations aside, this study explored a so‐far under‐researched area, highlighting a possible cumulative effect of bottle feeding and spoon‐feeding. It is the first study in the United Kingdom considering method of introducing solid foods to provide prospective and longitudinal measurements. The findings have important implications for the responsive feeding literature and considering infant weight to be a consequence of a cumulation of experiences rather than just one aspect in isolation.The authors declare that they have no conflicts of interestSWJ was responsible for the study design, data collection, data analysis, draft writing, and critical revisions. ML was responsible for the study design and critical revisions. AB was responsible for the study design, data analysis support, draft writing support, and critical revisions.


Depth information is necessary for perceiving the real size of objects at varyingvisual distances. To investigate to what extent this size constancy present inanother vertebrate class, we addressed the two questions using pigeons: (a)whether pigeons see a corridor illusion based on size constancy and (b) whetherpigeons prioritize pictorial cues over motion parallax cues for size constancy,like humans. We trained pigeons to classify target sizes on a corridor. Inaddition, we presented a dynamic version of corridor illusion in which thetarget and corridor moved side by side. Target speed was changed to manipulatemotion parallax. With the static corridor, pigeons overestimated the target sizewhen it was located higher, indicating that pigeons see a corridor illusion likehumans. With the dynamic corridor, the pigeons overestimated the target sizedepending on target position, as in the static condition, but target speed didnot affect their responses, indicating that the pictorial precedence alsoapplies to pigeons. In a follow-up experiment using the same stimulus, weconfirmed that humans perceive object size based on pictorial cues. Theseresults suggest that size constancy characteristics are highly similar betweenpigeons and humans, despite the differences in their phylogeny and neuralsystems. Animals with eyes are faced with the issue of how to reconstruct three-dimensionalrepresentations from two-dimensional retinal images to function adequately in theirenvironments . Humans make use of a variety of cuesincluding binocular disparity, motion parallax, and pictorial depth . Which dPapio papio)to show a Go response when two pictures of humans on a screen were of differentsize. In probe trials in which two persons of the same size were presented, theyshowed more Go responses in the corridor background condition than in no-corridorbackground condition. Pan troglodytes) totouch the larger of two objects on a screen. They found that the chimpanzees chosethe farther one on a corridor background when two objects of identical size werepresented and that the accuracy declined when the smaller object was presented on afarther point and the perceived difference in size of two objects diminished. Thesestudies suggest that size constancy using pictorial cues is shared at least in theorder primates.Object size perception is affected by visual depth cues . As viewColumba livia) are known to experience the Ponzoillusion, another size constancy illusion induced by via two converging lines . To this aim, we produced a dynamicversion of the corridor illusion. A classic corridor illusion stimulus uses a staticperspective corridor picture as background, and objects are perceived larger thenearer they are to the vanishing point of the corridor. In our dynamic corridorillusion, by contrast, the background corridor and objects moved sinusoidally sideto side so that faster points appeared nearer.Previous animal studies using multiple depth cues suggest an additive effect on thecomputation of depth information .For invIn this study, we first trained pigeons to classify target sizes into two categories in static and dynamic corridor conditions, respectively. The targetposition in the static condition and the target position and speed in the dynamiccondition were constant throughout training. We then manipulated target height in the static condition to assess the classic corridor illusion inpigeons. We also investigated cue selectivity in size constancy in pigeons bymanipulating target height  and speed  in thedynamic condition. We predicted that, if size constancy occurs in pigeons, theywould overestimate target size when the target appears in a higher position in the static condition. We also predicted that, if cueselectivity is similar across species, pigeons would overestimate target size whenthe target is presented in a higher position and moving faster and underestimate size when it is presented lowerdown and moving slower . Finally, we conducted thesame experiment with humans to confirm the precedence of pictorial over motion depthcues in our dynamic corridor illusion, as shown in previous studies .Six male pigeons participated . All hadparticipated in several operant experiments but were naïve to sizediscrimination tasks. They were individually housed and maintained at 85% to95% of their free-feeding weights. Water and grit were freely available inthe home cage. The experiments were conducted with the approval of theanimal experiment committee of the Graduate School of Letters, KyotoUniversity (No. 16-34).The experiments were conducted in six identical operant chambers(35 × 35 × 35 cm) installed with a 15-in. LCD monitor  and touch-sensitive frame . The monitor resolution was1,024 by 768 pixels. The monitor refresh rate was set to 60 Hz based on thefact that pigeons discriminate motion stimuli at this rate , althougA corridor background was composed of 5 white line squares of different sizesradially (80 to 500 pixels) and 32 radial lines connecting the squares. ThelinPigeons were trained on a size classification task . A trialThe pigeons were first trained on the largest and smallest targets (25 and 55pixels) without the corridor background in the static condition. Afterreaching at least a 90% correct rate, the probability of primaryreinforcement was lowered from 100% to 25%, and the number of trialsincreased from 96 to 360. Then, the dynamic conditions were inserted on thehalf of trials by increasing the movement width in four steps. Thebackground was gradually faded in in five steps by manipulating the lineluminance. Finally, six target sizes were presented in the static anddynamic conditions in one session, in random order.The pigeons advanced to test sessions after scoring above 85% correct on twoconsecutive training sessions. Each test session consisted of 360 trials ofwhich 72 were probe trials. Half of the probe trials were the staticcondition in which the target appeared at a higher (PF) or lower position(PN) than in the training condition (±20 pixels). The other half of the testtrials presented dynamic stimuli in which the target appeared at a higher(PF) or lower (PN) position (±20 pixels) and moved slower (MF) or faster. Thus, there were four probe conditions of dynamicstimuli: Pictorial-Far-Motion-Far (PFMF), PFMN, PNMF, andPictorial-Near-Motion-Near (PNMN). The pigeons were always reinforced inprobe trials, regardless of which response icon they pecked. The pigeonscompleted 20 test sessions in which accuracy on training trials exceeded80%. Between test sessions, at least one training session was conducted toprevent degradation of performance due to the addition of probe trials.RP = proportion of “LARGE”icon choice and x = target size. Within four freeparameters, β3 corresponds to PSE. Linear mixedmodel (LMM) was fitted to the shift of PSE between the probe and trainingsessions, using the “lmer” function from the R package “lme4” (std) were calculated as effect sizefor each fixed effect of LMM. One-sample t test wasconducted to analyze if PSE shifted significantly from training for eachcondition of static and dynamic stimuli. To test whether adding the motionparallax cue improved size constancy, amounts of illusion were calculatedfor static and dynamic conditions, respectively. The amount of illusion isthe difference of PSE shift between PF and PN for the static condition andbetween PFMF and PNMN for the dynamic condition. LMM was fitted to amount ofillusion with the stimulus dynamicity as a fixed and subject ID as a randomfactor.For each training and test condition, the proportion of “LARGE” icon choiceswas fitted with a sigmoidal function as a function of target size, and thepoint of subjective equality (PSE) was calculated. e “lme4” .The anaThe subjects were trained between 39 and 66 sessions before advancing to testsessions; for each bird, mean accuracy on the final three training sessions was86.7% (84.5%–89.2%). Their performances did not deteriorate following insertionof probe trials in test sessions (85.8%–90.0%).F = 63.118, p = .000,βstd = 0.794. PSE shifts in PN were significantly above 1.0,t(5) = 3.526, p = .017,d = 1.439. PSEs in PF condition were smaller than thosein training but the difference was not significant,t(5) = 2.169, p = .082,d = 0.886. These perceptual biases are consistent with thecorridor illusion in humans and other primates  = 39.591, p = .001,βstd = 0.668. Neither the effect of the motion parallax cuenor the interaction with the pictorial cue was significant—main effect,F = 2.847, p = .152,βstd = 0.130; interaction, F = 0.165,p = .701, βstd = 0.071. PSE shifts in PFMNcondition were significantly below 1.0, t(5) = 3.504,p = .017, d = 1.430. In the remainingthree conditions, PSE values did not differ significantly fromtraining—PFMF, t(5) = 2.516, p = .053,d = 1.027; PNMF, t(5) = 2.196,p = .079, d = 0.897; PNMN,t(5) = 1.959, p = .108,d = 0.800. These results indicate that size constancyin pigeons is driven mainly by pictorial depth cues, while motion parallaxcues have little effect, consistent with findings in humans . Amounts of illusion in the dynamiccondition  were notdifferent from those in the static condition (0.168: 0.052),F = 0.980, p = .368,βstd = 0.221, indicating that motion parallax does notcontribute to size constancy in pigeons.Experiment 1 demonstrated that pigeons prioritize pictorial cues over motion parallaxfor size constancy, as observed in humans . In ExpeSix adult humans including one of the authors (Y. H.) participated . They had normal or corrected-to-normal vision,and five participants were naïve to the purpose of the study. They wereinformed about the purpose of the study and gave verbal consent inaccordance with the ethical guidelines in the Declaration of Helsinki.The experiments were controlled by a personal computer running MATLAB with the Psychtoolbox extensions . StimuliThe stimulus configuration was almost the same as that of the dynamiccondition in Experiment 1 , and the other was alwaysthe same size (the sample). The participant was instructed to adjust thesize of the comparison circle (range was 15–60 pixels) to match the samplecircle in image size using up and down arrow keys on a keyboard. The time toadjust was not limited. After the perceived sizes of both circles appearedidentical, the participant pressed the enter key, and after 3 s, the nexttrial started. All participants completed 80 trials: 20 trials for eachstimulus condition .t test was conducted toanalyze if perceived size differed from physical size for each conditionseparately. To test the species difference directly, LMM was fitted to thepooled data of PSE shifts in the pigeon experiment and perceived sizes inthe human experiment, with pictorial cue, motion parallax cue, and speciesas fixed factors and subject ID as a random factor.We calculated the perceived size as the size of the sample circle divided bythe size of the adjusted comparison circle. Perceived size means werecalculated for each condition individually. LMM was fitted to the perceivedsize means with pictorial and motion parallax cues as fixed factors andsubject ID as a random factor, using the “lmer” function from the R package“lme4” . One-samF = 52.108,p = .000, βstd = 0.924; motion cue,F = 18.386, p = .008,βstd = 0.611. The interaction between cues was notsignificant—F = 2.833, p = .153,βstd = 0.299. Perceived size in PFMF was significantly below thephysical size ,t(5) = 10.549, p = .000,d = 4.306. Perceived size in PFMN was not significantlydifferent from physical size (0.995: 0.065), t(5) = 0.182,p = .863, d = 0.074. Perceived sizes inPNMF and PNMN were significantly above the physical sizes—PNMF, 1.049: 0.041,t(5) = 2.946, p = .032,d = 1.203; PNMN, 1.096: 0.033, t(5) = 7.038,p = .001, d = 2.873. These results suggestthat size constancy in humans depends more on pictorial cues than motionparallax cues, as found in previous studies  = 74.578,p = .000, βstd = 0.781; motion cue,F = 15.652, p = .003,βstd = 0.517. Neither the effect of species nor the interactionwith other factors was significant—species, F = 1.578,p = .238, βstd = 0.063; Species × Pictorial,F = 0.822, p = .386,βstd = 0.037; Species × Motion, F = 1.649, p = .228, βstd = 0.323;Species × Pictorial × Motion, F = 1.823,p = .207, βstd = 0.253. These findings indicatethat the extent to which pictorial and motion parallax cues contribute to sizeconstancy is not different between pigeons and humans.We also tested whether the effects of pictorial and motion parallax depth cueswere different between the two species. A statistical analysis with pooled datafrom the two experiments revealed significant effects of pictorial and motionparallax cues—pictorial cue, The first aim of the present study was to assess a corridor illusion in pigeons. InExperiment 1, we found that size constancy using pictorial depth cues occurred inpigeons, similar to humans and other primates . This reOur finding appears consistent with previous reports that pigeons see a Ponzoillusion, which is a size illusion induced by two simple converging lines , 1993. FThe present study also investigated the relative effects of pictorial and motionparallax depth cues on the corridor illusion in pigeons. From Experiments 1 and 2,it is clear that both pigeons and humans depend more on pictorial depth cues thanmotion parallax depth cues for size constancy. When pictorial and motion parallaxcues signified contradicting depth directions, both species perceived the objectsize on the basis of pictorial cues, consistent with human studies in whichparticipants judged the object size , or thePrevious studies in pigeons and baboons demonstrate additive effects of pictorialdepth cues . In contmotion parallax is best thought of as providing information about therelative rotation of an object or scene with respect to the observer, andthe previous empirical evidence makes it clear that the human visual systemdoes not make any strong assumption about the stability or even rigidity ofobjects over time.Thus, humans discard that assumption when motion parallax cuescontradict other depth cues and perceive an illusionary motion in trick artworks, and absence of statisticalsignificance does not mean the absence of effect itself. Second, changes in movingspeed of the target were smaller in Experiment 1 (±8 pixels) than Experiment 2 (±40pixels). The manipulation of motion parallax in the pigeon experiment might havebeen too small to change the perceived size. Furthermore, other differences inexperimental settings could affect the behaving results .We found a species difference in the effect of motion parallax cues on the corridorillusion: significant in humans but not in pigeons. We feel that this does not trulyreflect a species difference in size constancy, for some reasons. First, whenanalyzing the pooled data from Experiments 1 and 2, we found no significantinteraction between species and motion parallax. We acknowledge that this point isweak for the evidence of species similarity because it might be due to the smallsample size (As mentioned earlier, we found no significant effect of motion parallax in pigeons.However, this does not mean that pigeons are insensitive to motion parallax cue.Motion parallax cues are important for pigeons given that their lateral position oftheir eyes rules out use of binocular disparity cues , and theOne limitation in our study is that motion parallax was represented by simpletranslational movements of external objects, not synchronized with self-movements.This leaves ambiguity about depth structure from motion parallax: The backgroundgrid appears to be a concave corridor but sometimes appears to be a convex truncatedpyramid. It is known that fixational eye movements related to self-movements areimportant for disambiguating depth order from motion parallax . We do nThis study investigated the presence of size constancy in pigeons and itscharacteristics in relation to depth cue selectivity. The results showed thatpigeons see a corridor illusion on the basis of size constancy with pictorial depthcues. As in primates, motion parallax had little effect on size constancy inpigeons. These findings suggest that size constancy characteristics are highlysimilar in pigeons and primates including humans, despite the differences in theirphylogeny and neuronal visual processing systems.Click here for additional data file.Supplemental material, IPE911408 human data for Dynamic Corridor Illusion inPigeons: Humanlike Pictorial Cue Precedence Over Motion Parallax Cue in SizePerception by Yuya Hataji, Hika Kuroshima and Kazuo Fujita in i-PerceptionClick here for additional data file.Supplemental material, IPE911408 pigeon data for Dynamic Corridor Illusion inPigeons: Humanlike Pictorial Cue Precedence Over Motion Parallax Cue in SizePerception by Yuya Hataji, Hika Kuroshima and Kazuo Fujita in i-Perception


This study aims to measure serum vascular endothelial growth factor (VEGF) levels in a sample of Jordanian patients and to determine their relationship with the different stages of diabetic retinopathy. It also explores the correlation between VEGF concentrations and different biochemical and demographic findings. N = 62) and patients with DM type 2 affected by DR (N = 105). DR patients were further subclassified into nonproliferative (N = 41) and proliferative (N = 64). Basic laboratory tests were measured to correlate with VEGF levels. Irisin, a hormone linked to diabetic retinopathy was also measured and correlated with VEGF.  A total of 167 adults participated in the study. Participants were divided into two main categories: patients with diabetes mellitus (DM) type 2 without diabetic retinopathy (DR) , and a significant negative correlation with high-density lipoprotein (HDL) levels, age, and irisin.  In this cohort of Jordanian diabetics, serum VEGF concentrations strongly correlated with the presence and stages of diabetic retinopathy, suggesting it as an appropriate indicator for diabetic retinopathy early detection and management in this society. VEGF levels also significantly correlated with HbA1c, HDL, and irisin levels. Further studies are encouraged to explore these relationships in other ethnic groups and with different diabetic complications. Diabetes mellitus (DM) type 2 is characterized by a chronic hyperglycemic state which eventually leads, if uncontrolled, to damage in specific organs including eyes, kidneys, and peripheral nerves . OphthalAdvanced DR occurs when fragile new blood vessels form on the surface of the retina over time . These aDue to the high and rising rates of DR caused by DM type 2, it is essential to develop strategies to detect and manage this complication as early as possible. One of these strategies is the search for molecules that are associated with DR. Hence, extensive research was and is currently being conducted to investigate possible molecules linked to DR , 9.Vascular endothelial growth factor (VEGF) has emerged as an important factor associated with DR development and an important prognostic indicator of the disease , 11. VEGFew regional studies and many international studies have revealed a significant difference in serum VEGF levels between patients with DM type 2, with or without DR , 20. A mSerum VEGF has also been studied in relation to other biochemical and demographic factors, such as glycosylated hemoglobin A1c (HbA1c), high-density lipoprotein (HDL), low-density lipoprotein (LDL), body mass index (BMI), total cholesterol (TC), triglycerides (TGs), and age without conclusive results –25. ReceHere, we aim to measure serum VEGF levels in a sample of Jordanian DM type 2 patients and determine the difference in VEGF levels among different stages of DR and investigate the correlation between VEGF concentrations and different biochemical and demographic findings including irisin.This study has been conducted according to the Declaration of Helsinki (1964) and its amendments. The study has been approved by Jordan University Hospital (JUH) Institutional Review Board (IRB). Written informed consent has been obtained from all study participants.α of 0.05 and power of 90%. Recruitment of participants and sample collection was from March to December 2016 at the Ophthalmology Clinic at JUH. Participants were divided into two main categories: patients with DM type 2 not affected by DR as a control group (N = 62) and patients with DM type 2 who are affected by DR (N = 105). DR patients were further subclassified into two categories: NPDR (N = 41) and PDR (N = 64). Whole blood samples were collected into plain tubes. Samples were centrifuged at 1,600 ×g for 15 min at 4°C. Serum has been kept at −80°C for a period less than a month.A total of 167 adults participated in this study. Sample size was determined by power analysis and sample size software with The quantitative measurements of VEGF in human serum samples were performed using a commercial enzyme linked immunosorbent assay (ELISA) kits for VEGF  per the manufacturer's instructions. The absorbance from each sample was measured by a spectrophotometric microplate reader at a wavelength of 450 nm . The sensitivity of the VEGF assay is typically less than 1 pg/mL and the linear range of the standard is 12.5–400 pg/mL. For irisin, the quantitative measurements in human serum samples have been performed using ELISA kit  per manufacturer's instructions. Absorbance from each sample has been measured in duplicate by the same spectrophotometric microplate reader at wavelength of 450 nm. The sensitivity of the assay is 6.8 ng/mL and the linear range of the standard is 6.8–96.1 ng/mL.2) <18.5, 18.5–24.5, 25–29.9, and ≥30 have been considered underweight, normal, overweight, and obese, respectively. Basic laboratory tests including HbA1c, HDL, LDL, TGs, and TC have been measured and recorded at the hospital laboratories.Anthropometric measurements have been obtained using standard protocols and techniques. Body mass index (BMI) has been calculated as weight in kilograms divided by the square of height in meters (BMI = weight (kg)/(height (m))2) and used as a measure of obesity. Participants with BMI  was used to compare more than two groups, and spearman correlation analysis was used to investigate associations. Probabilities of less than 0.05 were considered statistically significant.GraphPad PRISM 5 Statistical software and EXCEL Microsoft Office Professional Plus 2016 were used for statistical analyses. Continuous variables were expressed as mean ± SD. Associations of circulating VEGF levels with patients' laboratory findings and Irisin levels have been investigated. Unpaired The demographic and clinical parameters of DR patients and controls are displayed in N = 62) and DR participants (N = 105) were 60 ± 17.4 pg/mL and 192 ± 66.3 pg/mL, respectively. VEGF results were further analyzed for DR patients per their classification. NPDR patients (N = 41) had a mean of (133 ± 36.5 pg/mL), while PDR patients (N = 64) had a mean of (229 ± 52.0 pg/mL) (p < 0.001). Unpaired t-test demonstrated statistically significant differences among each two groups (p < 0.001).One hundred and sixty-seven blood samples were analyzed by ELISA for VEGF concentration . The means of VEGF serum concentrations for controls (0 pg/mL) . One-wayp < 0.001). Unpaired t-test demonstrated statistically significant differences among each two groups (p < 0.001).VEGF: vascular endothelial growth factor. DR: diabetic retinopathy. PDR: proliferative diabetic retinopathy. NPDR: nonproliferative diabetic retinopathy. Mean concentration of VEGF ± SD is shown. The difference is statistically significant among the different groups of patients , and a significant negative correlation with age , HDL , and irisin  levels. No significant correlation was found with TCs, TGs, BMI, or LDL levels . Spearman correlation analyses revealed significant positive correlation with HbA1c  patients and 1699 DM patients without DR . The anaThis study also uncovers a statistically significant correlation between VEGF levels and different biomarkers such as HbA1c, HDL, and Age. We found a significant positive correlation with HbA1c, a finding reported in previous studies where HbA1c was significantly correlated with the severity of DR . This isRegarding irisin, we found a negative relationship between levels of VEGF and this myokine. Irisin is a hormone which has recently been investigated as a potential biomarker for DR and was found to have lower concentrations in patients with DR compared to DM type 2 without retinopathy , 28. IriThe present study has the intrinsic limitation of being a case-control study where associations and differences are concluded, but causation is not investigated. Association of VEGF with other diabetic complications has not been explored in this project. Such relationships are expected to be intriguing as well. Also, measuring VEGF levels in samples of DM type 2 patients with and without DR involvement using multiple ELISA kits might explain the variation in serum VEGF levels in the literature and should be explored as a future direction. Up to our knowledge, this is the first study to document serum VEGF levels in the Jordanian population and establish a link with DR in such a cohort. Also, this is the first attempt to explore the relationship between VEGF and irisin.We conclude that, in this cohort of Jordanian diabetics, serum VEGF levels strongly correlated with the presence and stages of DR, showcasing this biomarker as an appropriate indicator for DR early detection and management in this ethnic group. Serum VEGF concentrations were also found to be statistically correlated with some biochemical  and demographic findings (age). Further studies are encouraged to explore these relationships in other societies and with other diabetic complications.


Syria has experienced war since 2011, leaving over 80% under the poverty line and millions displaced. War and its retaliations have significantly impacted the mental health of Syrians. This study evaluates the post-traumatic stress disorder (PTSD), and the severity of the mental distress caused by war and other factors such as low social support. This study also evaluates other variables and compares the findings with those of multiple studies on Syria and refugees.This is a cross-sectional study that included people who lived in Syria in different governorates. Online surveys were distributed into multiple online groups and included the Kessler 10 (K10) scale which screens for anxiety and depression, the Screen for Posttraumatic Stress Symptoms (SPTSS) tool, the Multidimensional Scale of Perceived Social Support, and questionnaires on demographic and war-related factors.r = 0.623) was found between SPTSS and K10 scores.Our study included 1951 participants, of which, 527 (27.0%) were males and 1538 (78.8%) between the age of 19 and 25. Among participants, 44% had likely severe mental disorder, 27% had both likely severe mental disorder and full PTSD symptoms, 36.9% had full PTSD symptoms, and only 10.8% had neither positive PTSD symptoms nor mental disorder on the K10 scale. Around 23% had low overall support. Half of the responders were internally displaced, and 27.6% were forced to change places of living three times or more due to war. Around 86.6% of the responders believed that the war was the main reason for their mental distress. Those with high SPTSS and K10 scores were found to take more days off from work or school due to negative feelings and having somatic symptoms. Moreover, the number of times changing places of living due to war, educational level, and being distressed by war noise were the most prominent factors for more severe PTSD and mental distress. No differences in PTSD and mental disorder prevalence were noted in participants living in different governorates or among different types of jobs. A strong significant correlation (The conflict in Syria has left the population at great risk for mental distress which was higher compared to Syrian refugees elsewhere. Many measures with an emphasis on mental health are needed to help the people against a long-term avoidable suffering. Warfare has acute and chronic effects on the community and is considered one of the most mentally stressful events that can be experienced by someone. The indirect effects of warfare can be disastrous as they can continue for years after the settling of the conflict. The aftermath of war is challenging, from shortage of resources such as food, water, fuel and medical equioment, to displacement and disease outbreak . Since tThe damage to the medical infrastructure and the loss of medical staff have greatly affected the health system in Syria. Mental health in Syria has been neglected for decades, with no efforts to reduce the stigma on mental health, and few psychologist and psychiatrist available. This has caused the mental health sector to suffer greatly compared with other medical specialties during the war. The education sector has also been significantly injured after many facilities being destroyed by the conflict with the destruction of the Syrian social structure, most children had to grow up in a war-burdened environment that scarred their normal development . These dMany Syrians have fled the country and sought asylum in different countries. Although most of them lived in a relatively safer environment after facing grave dangers to reach their destination, they suffered mentally as they were far from their homeland, family members, friends, and usual work environment, all while experiencing bad living conditions in case they were living in camps. People who left the conflict zone have also developed mental disorders that could be different from those who have stayed . FurtherThis is a cross-sectional study conducted in Syria from 3/3/2019 to 24/3/2019. Online surveys in Arabic were distributed to participants from several Syrian governorates. Only participants who lived in Syria at the time of the survey and answered the demographic and K10 questions were enrolled. The questionnaires were posted online twice each day at 10 AM and 10 PM in social media groups with different topics such as educational, cuisine, market, entertainment, cultural, musical etc. Any participant who spoke Arabic, lived in Syria at the time of the study, and answered the required questions was included.The informed consent was taken online for using and publishing the data before carrying on with the survey. Confidentiality was assured and no identity-revealing questions were asked. This method was approved by Damascus University.Our study protocol and ethical aspects were reviewed and approved by Damascus University deanship in Damascus, Syria.There is no suitable validated SES measurement to use in the Syrian population as we cannot ask directly about the monthly salary due to cultural norms that make it inappropriate. In addition, there are huge differences between the normal wage in Syria and other countries. Therefore, SES was assessed through three criteria; 1) the level of education, 2) the profession of the participant or that of the working family member/partner, 3) the monthly income adequacy of the individual who is the main source of income in the household. SES was then divided into four different categories: lower, upper-lower, middle, and upper.Kessler 10 is typically used in screening surveys to measure mental distress –19. An AAn Arabic version of the Multidimensional Scale of Perceived Social Support (MSPSS) was used to measure the social support that the individual receives from their social network, including friends, family, and the significant other , 21. TheThe Screen for Posttraumatic Stress Symptoms (SPTSS) tool was used; a self-reporting screening method for PTSD that is very concise, suitable, easy to understand and does not focus on specific traumatic events. SPTSS is not meant to be a definitive diagnostic tool for PTSD. It measures three PTSD clusters over the previous 4 weeks; avoidance with 7 items, hyper-hyper-arousal with 5 items, and re-experience with 5 items. Each question has 5 potential responses, ranging from “Not at all” to “More than once every day”, with scores ranging from 0 to 4. However, when calculating the final score for each cluster, the first two scores represent 0 and the other three scores represent 1. Scoring 3 or more on avoidance cluster, 2 or more on re-experience, and 1 or more on excessive hyper-arousal is an indication of PTSD for that specific cluster. The scores and measures are validated in Arabic , 23. ThiSubjects were asked basic demographic questions such as gender, age, educational level, governorate of current living, and whether they had consanguineous parents. Subjects were also asked a few questions, both directly and indirectly, about war, including changing place of living due to war, losing someone close, and being distressed from war noises. Finally, subjects were asked if they had any chronic medical condition.This study defined third-degree consanguineous parents as having parents who were first cousins, and fourth-degree consanguineous parents as having parents who were second cousins, or second cousins once removed who is either a child of one’s second cousin, or one’s parent’s second cousin.Data were processed using IBM SPSS software version 26 for Windows . Chi-square was used with nominal variables such as in Table  while onP values were considered statistically significant. Bonferroni correction was used to reduce type 1 error when comparing multiple variables in Tables P = α\m where α in our study is 0.05 and m is the number of hypotheses.Through the same software, odds ratios (ORs) and the 95% confidence intervals for the groups were calculated using the Mantel–Haenszel test. Values of less than 0.05 for the two-tailed Overall, 2173 filled in the survey. However, 198 were excluded as they did not answer all the questions, or their replies were invalid. Another 24 participants refused to be enrolled despite their initial consent when initiating the survey. The study included 1951 responders with 527 (27.0%) being males, and 1538 (78.8%) participants being within the age range of  years.Among responders, 19.2% (CI 95%: 17.5–21%) were likely to be well, 19.5% (CI 95%: 17.8–21.3%) had a mild mental disorder, 16% CI 95%: 14.9–18.1%) had a moderate mental disorder, and 44.7% (CI 95%: 42.6–47.0%) had a severe mental disorder according to K10 scale. Other characteristics of the subjects are demonstrated in  met the criteria for having symptoms of avoidance, 64.4 (CI 95%: 62.4–66.5%) for hyper-arousal, 58.9% (CI 95%: 56.9–61.2%) for re-experience, and 36.9% (CI 95%: 35.0–39.1%) for full PTSD symptoms.P < 0.05). Regression model is demonstrated in (Table According to Table in Table .Table 5P = 0.219). However, disturbance of K10 scores among governorates is demonstrated in .There was no statistically significant difference when comparing different K10 results with governorates of current living (P < 0.001) were found between K10 scores and higher numbers of days being unable to work, study, or manage daily activities because of the negative feelings (r = 0.110), more frequent visits to a health professional because of the negative feelings (r = 0.110), and more frequent physical problems being attributed to the negative feelings (r = 0.180). K10 scores were not correlated with the type of work (P = 0.357) and are demonstrated in . However, P-value was higher than 0.05 with other LM questions.When comparing the governorate of current living and MSPSS, we did not find a statistically significant difference P < 0.001). When regressing the previous two variables from Table P < 0.001) with an R2 of 2.3%. Regression model is demonstrated in (Table According to Table in Table .P = 0.357) and they are demonstrated in .Comparing SPTSS clusters with each variable is demonstrated in Table . SPTSS t in Fig. . When coP < 0.05). When using the same method on hyper-arousal, distress from war noise, number of times changing places of living, and education were statistically significant (P < 0.05). When using the same method on re-experience, distress from war noise, losing someone due to war, educational level were statistically significant (P < 0.05). All regression models are demonstrated in . The mean score of K10 was 32.1 (±9.2) in participants with hyper-arousal symptoms, 22.6 (±7.9) in participants without hyper-arousal symptoms, and t = − 22.76 (P < 0.001). The mean score of K10 was 32.3 (±9.2) among participants with re-experience symptoms, and 23.5 (±8.2) in participants without re-experience symptoms, and t = − 21.78 (P > 0.001).When using independent t-test, the mean score of K10 was 32.9 (±23.9) in participants with avoidance symptoms, 23.9 (±8.4) in participants without avoidance symptoms, and t = − 22.69 ((Table ). When using Pearson correlation, higher K10 and SPTSS symptoms scores were significantly associated with lower family, friends, and significant other support. Family support score was significantly (P < 0.001) correlated with K10 score (r = − 0.195), avoidance score (r = − 0.236), arousal score (r = − 0.195), and re-experience score (r = − 0.192). Friend support score was also significantly (P < 0.001) correlated with K10 score (r = − 0.171), avoidance score (r = − 0.257), arousal score (r = − 0.214), and re-experience score (r = − 0.192). Finally, significant other support was significantly (P < 0.001) correlated with K10 score (r = − 0.160), avoidance score (r = − 0.233), arousal score (r = − 0.177), and re-experience score (r = − 0.157).When using one-way ANOVA, MSPSS scores were significantly associated with worse K10 results and SPTSS clusters which are demonstrated in Table . When usP = 0.061), avoidance symptoms (P = 0.0190), hyper-arousal symptoms (P = 0.034), and re-experience symptoms (P = 0.043). Fewer subjects declared they could work but had to cut down on what they did which was statistically insignificant (P > 0.05). Furthermore, more frequent visits to health professionals because of what was felt was associated with total PTSD symptoms (P = 0.063), avoidance symptoms (P > 0.05), hyper-arousal symptoms (P = 0.022), and re-experience symptoms (P = 0.022). More subjects also declared that having physical health problems being the main cause of the negative feelings was associated with more SPTSS symptoms (P < 0.0001), avoidance symptoms (P = 0.003), hyper-arousal symptoms (P < 0.0001), and re-experience symptoms (P < 0.0001).When using one-way ANOVA and independent t-test, more days were declared being unable to work, study, or manage daily life because of the negative feelings among subjects with more SPTSS symptoms  which is weak. SES was the most contributing factor to social support.Although only 23.2% of our sample had a low total support, the high prevalence of PTSD, and mental disorders suggest other factors being involved besides low support levels as (P < 0.0001).The literature indicates that social support was a preventive factor for the development of PTSD for men and women. Furthermore, the incidence of post-traumatic stress increased in those with a low social support. For many who have experienced trauma in their lives, social support was a preventive factor for the development of PTSD . This coHowever, after a long period of exposure to trauma, the impact of social support as a protective factor may be mitigated . A studyBeing fearful, easily angered, nervous, having difficulty sleeping or staying asleep, absence of hope for the future, and spells of terror or panic were some of the characteristics that the Syrian refugees experienced at Alzatary Camp in Jordan . SimilarMoreover, war has affected university students , and prer < 0.3 with P < 0.001), other measurements are required to boost mental health in the society. National-wide programs are needed to increase awareness, and humanitarian assistance is required to benefit from international experts in mental health. Financial assistance is also needed as the deteriorating financial situation is a strong contributing factor to the suffering.The stigma of mental health in Syria is very common, and only a few practicing psychiatrists and psychotherapists exist. As social support was only weakly but significantly correlated with lower K10 and SPTSS scores  that the population had faced. Moreover, geographical characteristics appear to influence the psychological wellbeing of displaced populations. Most studies showed that severe mental disorders were more common in cities compared to rural areas , 52. HowThis study did not consider the mental background of participants, which could have aggravated the symptoms of PTSD or biased the questionnaire. This study was online which made it difficult to determine the population at risk. Moreover, responders who had an internet connection and were willing to do the questionnaires are probably in a better mental condition than those who are truly severely affected, and do not have internet connection or the will to do the survey. Finally, most of the responders were university students with potentially higher SES than the normal population. For all the previous reasons, this study might have underestimated the true prevalence of distress amongst the general population.SES could not be accurately determined since asking about the salary is inappropriate in the Syrian culture. There is a huge difference in living costs in Syria, where people can live of a lower income compared to other countries in the region. SPTSS is based on DSM-IV, not V. However, it can resemble ICD-11.The Syrian conflict has caused severe mental distress in the Syrian society. Efforts and interventions to improve the psychological wellbeing of the Syrian population are needed to ensure the people are prepared for the reconstruction of their country when the situation improves. The results of this study reflect the underlying disaster that made people severely mentally impaired. The social support had a relative weak effect, meaning that resources should be re-allocated to other aspects of care. Syrian people have serious concerns about financial and psychiatric aspects of their lives and require various measures to ameliorate their situation. These results also emphasises on the importance of security either economically or in terms of personal safety which are more important in the long term than support from family, friends, and the significant other as higher mental distress is seen in those who remained in Syria compared to refugees in camps.This study suggests that internally displaced people, and even those who were not displaced in the conflict, experienced more severe mental disorder than the Syrian refugees in most countries. The number of times changing places of living due to war and being distressed from war noise have contributed the most to the high PTSD, anxiety, and depression burden. More than 60% of the population suffered from PTSD and severe mental disorders. Having a high educational background was associated with less severe mental disorders and PTSD. Moreover, a higher number of times changing places of living due to war, a lower educational level, and being distressed from war noise were associated the most with sever PTSD and mental distress. No significant differences in mental disorders, or PTSD were noted among participants from different governorates or with different job types. Many other variables have contributed to these findings altogether which indicate the need for addressing multiple issues. Females and young participants suffered more on the psychiatric aspect.


Myocardial remodelling is important pathological basis of HF, mitochondrial oxidative stress is a promoter to myocardial hypertrophy, fibrosis and apoptosis. ECH is the major active component of a traditional Chinese medicine Cistanches Herba, plenty of studies indicate it possesses a strong antioxidant capacity in nerve cells and tumour, it inhibits mitochondrial oxidative stress, protects mitochondrial function, but the specific mechanism is unclear. SIRT1/FOXO3a/MnSOD is an important antioxidant axis, study finds that ECH binds covalently to SIRT1 as a ligand and up‐regulates the expression of SIRT1 in brain cells. We hypothesizes that ECH may reverse myocardial remodelling and improve heart function of HF via regulating SIRT1/FOXO3a/MnSOD signalling axis and inhibit mitochondrial oxidative stress in cardiomyocytes. Here, we firstly induce cellular model of oxidative stress by ISO with AC‐16 cells and pre‐treat with ECH, the level of mitochondrial ROS, mtDNA oxidative injury, MMP, carbonylated protein, lipid peroxidation, intracellular ROS and apoptosis are detected, confirm the effect of ECH in mitochondrial oxidative stress and function in vitro. Then, we establish a HF rat model induced by ISO and pre‐treat with ECH. Indexes of heart function, myocardial remodelling, mitochondrial oxidative stress and function, expression of SIRT1/FOXO3a/MnSOD signalling axis are measured, the data indicate that ECH improves heart function, inhibits myocardial hypertrophy, fibrosis and apoptosis, increases the expression of SIRT1/FOXO3a/MnSOD signalling axis, reduces the mitochondrial oxidative damages, protects mitochondrial function. We conclude that ECH reverses myocardial remodelling and improves cardiac function via up‐regulating SIRT1/FOXO3a/MnSOD axis and inhibiting mitochondrial oxidative stress in HF rats. In recent years, ECH has been extensively studied in nervous system and tumour, and it has been reported to possess a variety of pharmacological effects, such as antioxidant, anti‐inflammatory, anti‐apoptosis and anti‐tumour properties.SIRT1 is closely related to oxidative stress, and SIRT1/FOXO3a/MnSOD signalling axis is the most important antioxidant axis and responsible for suppression of mitochondrial oxidative stress, MnSOD distributes widely in mitochondria and primarily responsible for clearing of mitochondrial ROS, thus inhibiting oxidative stress, compounds which activate this antioxidant axis can inhibit oxidative stress.22.12. Once the cells reach 70%‐80% confluence, ISO  group cells are incubated with medium containing 10uM ISO  for 24h, ECH group cells are pre‐treated with 50uM ECH  for 30min prior ISO exposure. Control cells (Ctrl) are treated nothing.AC16 cells are purchased from BeNa Culture Collection. The cells are maintained in high glucose Dulbecco's modified Eagle's medium  supplement with 10% foetal bovine serum are  in T‐75 flasks . Conditions are maintained at 37℃ in a humidified atmosphere containing 5% CO2.2Mitochondria are isolated and purified of AC‐16 cells using Mitochondria Isolation Kit  according to the manufacturer's instructions. Protein concentrations are determined by BCA protein assay kit (Bio tech). Mitochondrial ROS level is detected by luminal  using an Elisa kit , it reacts with ROS and causes emission of photons, which is a band spectrum having a peak at about 460nm, and Relative Light Unit (RLU) is detected by chemiluminescence measuring instrument and converted into RLU/μg mitochondrial protein.2.3Mitochondrial DNA oxidative damage is evaluated by detecting the content of 8‐Hydroxy‐2‐deoxyguanosine (8‐OHdG). Purified mitochondria in cells are isolated using mitochondria isolation kit as above (Sigma). Protein concentrations are determined by BCA protein assay kit (Bio tech). Then, they are dissociated to determine the level of 8‐OHdG using an Elisa kit  according to the manufacturer's instructions. The absorbance of the colour change is measured in spectrophotometer at the wavelength of 450 nm. The concentration of 8‐OHdG in the samples is then determined by comparing the OD of the samples to the standard curve, and converted into ng/ml.2.4Mitochondria are isolated and purified of AC‐16 and protein concentration is determined as mentioned above. Then the mitochondria are resuspended at 1mg/ml concentration, the MMP is measured using JC‐1 assay kit according to the manufacturer's instructions. The fluorescence intensity is measured at excitation wavelength: 490nm, slit 5nm, emission wavelength: 590 nm, slit 5nm with a spectrophotometer instrument,2.5Flow cytometry is used to assess cellular apoptosis. An Annexin V‐FITC/PI apoptosis detection kit  is used to detect apoptotic and necrotic cells according to the supplier's instructions. Early stage of apoptosis, late stage of apoptosis or necrosis, cellular debris and viable cells are identified as annexin V (+)/PI (−), annexin V (+)/PI (+), annexin V (−)/PI (+) and annexin V (−)/PI (−), respectively. Fluorescence intensities are analysed using a flow cytometer . Each experiment is repeated 3 times.2.62, and then tested in a flow cytometer at 488 nm excitation. The fluorescence intensity represents the level of intracellular ROS.After treatment, cells are trypsinized, washed twice with PBS. The cells are then resuspended in PBS, stained with DCFH‐DA  at the final concentration of 1 μM and incubated for 1 h at 37℃ in an incubator with CO2.7After treatment, purified mitochondria are isolated and sonicated on ice and are treated with 1% streptomycin sulphate to precipitate mitochondrial nucleic acids, protein concentration are determined. The carbonyl protein content is tested using an Elisa kit  based on derivatization of the carbonyl group with dinitrophenylhydrazine (DNPH), according to the instructions of the kit, OD is measured by a microplate reader and converted into nmol/mg protein by comparing to the standard curve.2.8After treatment, cells are collected and lysed, mitochondria are isolated and purified and cracked by ultrasound, protein concentration is determined by BCA protein assay kit (Bio tech), lipid peroxidation is determined by the level of malondialdehyde (MDA) via the TBARS assay, using a kit  and MDA level is measured following the manufacturer's protocol. OD is detected at 586 nm by a microplate reader and converted into μmol/g protein by comparing to the standard curve.2.9Young male Sprague‐Dawley rats weighing 120‐140g are acquired from Xi'an Jiao tong University Laboratorial Animal Center . The investigation conforms to The Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health , and the experimental protocols were approved by the local authorities . Rat model of HF is established as described previously by us.2.10Animals are anaesthetized by an intraperitoneal injection of 300 mg/kg of chloral hydrate. The SONOS‐2500 ultrasound system with an ultrasound transducer of 7.5 MHz  is used to make echocardiographic examinations. The left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end‐diastolic dimensions (LVIDd), left ventricular end‐systolic dimensions (LVIDs), heart rate (HR), interventricular septal thickness in diastole (IVSTd), left ventricular posterior wall thickness in diastole (LVPWTd) are measured as described previously by us.2.11Rat is weighed before anaesthetization, fresh heart is isolated and wash out blood with 0.1mol/L PBS, cut away vessels and other redundant tissue and the heart is weighed, then HW/BW(mg/100g) is calculated.2.12The hearts of rats are dissected and fixed in 4% paraformaldehyde, embedded in paraffin and sectioned into 5 μm‐thick slices. Haematoxylin‐Eosin (HE) staining is used to observe the pathological changes and Masson's trichrome is used to evaluate collagen fibres of rats myocardium tissue. The cardiac collagen volume fraction (CVF) and cardiomyocytes cross‐sectional area is measured with Image‐Pro Plus 6.0 .2.13Myocardial tissue from the left ventricle is collected and the TUNEL assay is carried out as described previously by us2.143 in volume and fixed again in 2.5% glutaraldehyde at 4℃ for 2h, then is washed with 0.1mol/L PBS, fixed in 1% osmic acid, dehydrate with acetone, embedded with epoxy resin, ultrathin slices which thin about 800nm are made, stained with uranyl acetate and lead citrate, and then are observed under a transmission electron microscope . To quantify the protective effect of ECH in mitochondrial morphology, the mean mitochondrial circumference, mean mitochondrial cross‐sectional area, percentage of degeneration and percentage of vacuolization in mitochondria are analysed statistically with Image‐Pro Plus 6.0 .Fresh heart tissue is fixed in 2.5% glutaraldehyde at 4℃, tissue is then cutted into 1mm2.15‐ΔΔCT method.Total RNA is prepared from left ventricular tissue with Trizol reagent . cDNA is synthesized using the TUREscript 1st Strand cDNA Synthesis Kit . Specific primer sequences of collagen I used for real‐time PCR is as follows: forward: 5′‐GTCGTATCCAGTGCGTGTC‐3′, revers: 5′‐GTGGAGTCGGCAATTGCA‐3′. GAPDH forward 5'‐GGC AAG GTC ATCCCA GAG CT‐3', reverse 5'‐CGC CTG CTT CAC CAC CTT CT‐3'.Real‐Time quantitative PCR (qPCR) is performed with SYBRPremix Ex Taq  . The relative level of mRNA is calculated by normalizing to GAPDH, according to the 22.16Total protein of left ventricular tissue are extracted on ice using cold RIPA Buffer  with Protease Inhibitor Cocktail (Sigma‐Aldrich). The concentration is determined by BCA method. Procedures of Western blot are described previously.2.17Purified mitochondria of rat myocardium are isolated using Mitochondria Isolation Kit  according to the manufacturer's instructions, mitochondrial ROS, MMP, oxidative damage of mtDNA, carbonylated protein content and lipid peroxidation level in mitochondria are all detected using the same methods as cell experiments described above. Of note, ROS in myocardial tissue is detected with DCFH‐DA fluorescent probes, fluorescence intensity is detected with enzyme‐labelling measuring instrument, the results are represented as RLU/μg mitochondrial protein.2.18P < .05 is considered significant.All data are presented as means ± SEM. One‐way ANOVA is used to compare differences among multiple groups, followed by Tukey post hoc test for significance. All statistics are determined using SPSS15.0 software . A probability value of 33.1Untreated cells are set as Ctrl group. Firstly, as Figure 3.2As shown in Figure Results of HE staining indicates that the myocardial hypertrophy, arranged disorder or degeneration necrosis, inflammatory cell infiltration and interstitial hyperaemia and oedema in HF rats, and ECH significantly alleviates these damages, myocardial hypertrophy and arranged disorder are relieved, the degeneration necrosis and inflammatory infiltrate are reduced, as shown in Figure 3.3Transmission electron micrographs results show that the damage of myocardial ultrastructure could be attenuated by ECH, as Figure 3.4Western Blot analysis demonstrates that the protein expression level of SIRT1, FOXO3a and MnSOD are significantly down‐regulated in HF rat, however, ECH obviously up‐regulates the decreased protein expression. The representative protein bands of immunoblotting and statistical analysis results are showed in Figure 3.5In this part, mitochondrial ROS, MMP, oxidative damage of mitochondrial DNA, carbonyl protein content and lipid peroxidation level in mitochondria in left ventricular myocardial tissue of rats are all measured, and the results are consistent with cell experiments, that is to say, ECH decreases mitochondrial ROS, protects MMP, relieves the damage degree of mtDNA, reduces carbonyl protein content and lipid peroxidation in mitochondrial, as Figure 4This study demonstrates that ECH exerts effective roles in the prevention of myocardial remodelling and improvement of heart function via up‐regulating SIRT1/FOXO3a/MnSOD signalling axis and inhibiting mitochondrial oxidative stress.Cistanche tubulosa. it is widely studied and has been proved possessing kinds of pharmacological activities in recent years, such as anti‐tumour, anti‐apoptosis, anti‐ageing, neuroprotective action, and so on. Studies have proved that the pharmacological actions of ECH mainly depend on its property of antioxidant and anti‐inflammatory activity,Mitochondria are major site of generation of ROS as a by‐product of oxidative phosphorylation, by their proximity to ROS, mitochondrial proteins, lipids and mtDNA are believed to be primary targets of oxidative damage after excessive emission of mitochondrial ROS,In normal conditions, mitochondrial ROS is mainly cleared by MnSOD which distributes in mitochondria and protects against mitochondrial oxidative damage. Ang II significantly decreases MnSOD expression of cardiomyocytes and induces myocardial hypertrophy, however, resveratrol could increase the expression of MnSOD and inhibit myocardial hypertrophy induced by Ang II.The expression of MnSOD is regulated by FOXO3a, which is a transcription factor and binds in the gene promoter region of MnSOD and leads to the promotion of gene transcription.Long‐term chronic ischaemia can cause myocardial hibernation, these hibernating myocardial cells typically show a loss of contractile filaments, there are excess glycogen granules in the cytosol, sarcoplasmic reticulum and transverse tubules are lost,The previous study also detected myocardial hibernation in a non‐ischaemic heart failure pig model by pacing the LV free wall with high‐frequency,Of note, cardiomyocyte apoptosis is well known to be an important pathologic changes in heart failure, and the present study indeed confirms that large amount of apoptotic cells in HF rats and ECH significantly reduces the apoptosis via regulating SIRTI/FOXO3a/MnSOD pathway and inhibiting mitochondrial oxidative stress, as mentioned above.Above all, ECH reverses myocardial remodelling and improves heart function via up‐regulating SIRT1/FoxO3a/MnSOD signalling axis, and thus inhibits mitochondrial oxidative stress and reduces hibernating myocardium. Thus, it is suggested that ECH is a potential drug for prevention or treatment of myocardial remodelling and HF.The authors declare that they have no competing interests.JW‐Z and YJ‐N designed the study, planned and performed experiments, analysed data and drafted and revised the manuscript. JD, XL and QL helped to perform experiments, administrated experiments, collected and analysed data. JL‐Z validated data, supported software and revised the manuscript.Supplementary MaterialClick here for additional data file.


Task‐based functional neuroimaging methods are increasingly being used to identify biomarkers of treatment response in psychiatric disorders. To facilitate meaningful interpretation of neural correlates of tasks and their potential changes with treatment over time, understanding the reliability of the blood‐oxygen‐level dependent (BOLD) signal of such tasks is essential. We assessed test–retest reliability of an emotional conflict task in healthy participants collected as part of the Canadian Biomarker Integration Network in Depression. Data for 36 participants, scanned at three time points  were analyzed, and intra‐class correlation coefficients (ICC) were used to quantify reliability. We observed moderate reliability , within occipital, parietal, and temporal regions, specifically for conditions of lower cognitive complexity, that is, face, congruent or incongruent trials. For these conditions, activation was also observed within frontal and sub‐cortical regions, however, their reliability was poor (median ICC < 0.2). Clinically relevant prognostic markers based on task‐based fMRI require high predictive accuracy at an individual level. For this to be achieved, reliability of BOLD responses needs to be high. We have shown that reliability of the BOLD response to an emotional conflict task in healthy individuals is moderate. Implications of these findings to further inform studies of treatment effects and biomarker discovery are discussed. They report an average ICC value of 0.397 across 90 studies in their meta‐analysis, which reflects poor reliability. Independent analyses of 11 commonly used tasks revealed ICCs of <0.3, again indicating rather poor test–retest reliability  protocol , incomplete scan sequence (n = 1), severe ghosting or other data quality issues (n = 6). For n = 5, task‐based fMRI data was missing at one of the three time‐points or participants withdrew. In addition, for six participants the behavioral data were either not useable  (n = 1) (n = 3) or did not meet accuracy threshold (see below) (n = 2). This resulted in one site having only one participant contribute to the sample, so this participant was removed from further analysis. Hence, data were analyzed for 36 participants at three time points: weeks 0, 2, and 8. Mean time elapsed between week 0 and week 2 testing was 14.2 (±1.7) days; between week 2 and week 8 it was 42.5 (±3.6) days.Fifty‐nine healthy control participants were recruited at academic healthcare centers across Canada as a subset of participants in the first Canadian Biomarker Integration Network in Depression study  and displayed on a projection screen. Participants viewed the screen via a mirror attached to a head‐coil. Stimuli were presented for 1 s and participants were instructed to identify the facial emotional expression via button press, as quickly and accurately as possible. Inter‐stimulus intervals, during which participants were instructed to look at a projected fixation cross, varied between 3 and 5 s and were jittered. Images were counterbalanced for equal numbers of congruent and incongruent presentations, in two consecutive “runs.” Participants completed the “runs” in the same order in each of their scan sessions to avoid between‐session variance associated with order. Each “run” comprised 74 stimuli and lasted 6 min 35 s. Prior to scanning, participants practiced the task outside of the scanner, to demonstrate understanding of task requirements.The emotional conflict task  and accuracy. For the analysis of RT data error trials, post‐error trials , and trials were RT exceeds two standard deviations above or below the trial type mean were not included. Commission error threshold was set at 25% per run, and the threshold of total allowable errors (combined omission and commission errors) was set at 30% per run. For the analysis of accuracy, trials were RT exceeded two standard deviations from the trial type mean and post‐error trials were included. For the neuroimaging analysis of all trials , all trials were included, regardless of RT or accuracy. Trial types analyzed included all face trials, which included error and post‐error trials, incongruent trials, congruent trials, incongruent minus congruent trials, fear minus happy face trials, fear minus happy word trials and iI minus cI trials which represent an incongruent trial being preceded by an incongruent trial (iI) minus an incongruent trial being preceded by congruent trial (cI). The main purpose of the “all faces” condition was to serve as control condition, rather than a main contrast of interest.2.3Data were acquired at the University Health Network, Toronto, including Centre for Addiction and Mental Health; St. Joseph's Healthcare Hamilton; Providence Care Hospital, Kingston; the Mathison Centre for Mental Health Research & Education in the Hotchkiss Brain Institute, Calgary and Djavad Mowafaghian Centre for Brain Health, University of British Colombia, Vancouver. Three models of scanners were used across the clinical sites and included a Discovery MR750 3.0T , Signa HDxt 3.0T  and a MAGNETOM Trio . Each participant was scanned at the same site, using the same scanner across all three time points. Acquisition parameters are described in detail elsewhere . A sequence of fixed preprocessing and analysis steps were applied to the fMRI data as described by Churchill and colleagues . (d) Slice‐timing correction (TIMECOR) with Fourier interpolation via AFNI's 3dTshift; (e) Spatial smoothing using AFNI's 3dBlurToFWHM to smooth fMRI images at FWHM = 6 mm in x,y,z directions allowing for different intrinsic reconstructed smoothing levels at each site: To match the spatial smoothing across MRI scanners at different sites, we used the 3dBlurToFWHM module in AFNI to smooth the fMRI images to the smoothness level of FWHM = 6 mm in three directions . Since the FWHM should reflect the spatial structure of the noise, we first regressed out the BOLD response modeled using the canonical hemodynamic response and then used the resultant residual image as the “blur master”. The “blur master” controls the process of smoothing on the original image. Blurring is applied to both the original image and the residual until the smoothness of the residual reaches the desired FWHM = 6 mm. Using a smoothing kernel with a FWHM ≈2× (in‐slice voxel width) has previously been shown to provide major improvements in prediction and reproducibility for group analysis of an fMRI motor task  Neuronal tissue masking: Neuronal tissue masking was performed by estimating a probabilistic mask to reduce the variance contribution of non‐neuronal tissues in the brain . This step uses the first part of the PHYCAA+ algorithm developed by Churchill and Strother (http://nitrc.org/projects/phycaa_plus). (h) Calculation of nuisances regressors to be regressed‐out from the data concurrently via multiple linear regression: Temporal trends were modeled using a second‐order Legendre polynomial basis set, head motion effects on time‐series were modeled using participant motion parameter estimates (MPEs) obtained from MOTCOR, (Step 3). To obtain motion parameter regressors (MOTREG) per fMRI session, we performed PCA on the six MPE time‐courses, and used the largest‐variance principal components, which preserved 85% of the variance, as motion regressors. This allowed us to maximize the amount of head motion variance accounted for, while minimizing loss of power and collinearity effects due to unnecessary parameterization.Dicom images were converted to nifti, using mricron (Strother  to estimNative_processed‐fMRI) but then transformed into normalized space (sNORM_processed_fMRI) to allow participants' results to be combined. For this, all scans were aligned to the MNI template (4 mm resolution) using FSL's FLIRT transformations. Subsequently, each participant's scans were transformed to MNI space. For more comprehensive discussions of these pipeline steps and their relative importance, see , using a two‐way random effects model for absolute agreement, and performed in SPSS v25. ICC is described in detail in Section Statistical analyses of the behavioral data were completed using SPSS 25 . Demographic data were analyzed using one‐way analyses of variances (ANOVA) for age and education and with chi‐square tests for sex and handedness. Behavioral data were analyzed using the Shapiro–Wilk test, to assess normality of the continuous variables, that is, RT and accuracy. Data were not normally distributed, and, therefore, nonparametric statistical analyses were conducted. Comparisons of paired samples were completed with the related‐samples Wilcoxon signed rank test. Friedman's test was used for within‐group analyses across all three time‐points. For within group comparisons, Wilcoxon signed rank tests or a sign test were conducted. To correct for multiple comparisons, Bonferroni corrections were applied, and adjusted 2.4.3First‐level analyses for individual runs were completed using FSL FEAT v.6.00. Data were filtered with a high pass filter with a cut‐off of 100 Hz. Images were FILM prewhitened and a normal linear search with 3° of freedom (only translation) to the standard was applied to resample the images to the space of the 2 mm MNI 152 T1 standard brain.https://www.fil.ion.ucl.ac.uk/spm/, a higher‐level fixed‐effect model was constructed for each condition, separately for each time point, that is, weeks 0, 2, and 8, using the COPE images generated above. The model included regressors representing different trial types of the task: (a) all face trials, (b) congruent trials, (c) incongruent trials, (d) happy faces; (e) fear faces. Additionally, contrasts reflecting high and low conflict resolution trials, that is, incongruent (high conflict resolution) minus congruent (low conflict resolution) trials to measure the emotional Stroop condition, fear minus happy face trials, and iI minus cI trials were generated. It is thought that the comparison of “high conflict resolution > low conflict resolution” identifies regions associated with “conflict resolution” whereas the contrast “low conflict resolution > high conflict resolution” would identify regions implicated either in the “generation of conflict” or the “monitoring of conflict.” Second‐level analyses across participants  were conducted in SPM12, using random‐effects analyses with the above contrasts. For all conditions and contrasts tested, a cluster‐level threshold to control for multiple comparisons was set at p < .05—family‐wise‐error (FWE)‐corrected, with a cluster size of 10 or more voxels.Using Statistical Parametric Mapping 12 :Test–retest reliability of brain activation was assessed using intraclass correlation coefficients (ICC)  were treated as fixed effects. ICCs can be interpreted as a ratio of variance Bartko, . ICCs aphttps://masi.vuse.vanderbilt.edu/workshop2012/index.php/Challenge_Details) and available via SPM, provided by Neuromorphometrics, Inc. (http://neuromorphometrics.com/) under academic subscription. Exploring reliability across the whole‐brain is relevant when few anatomical or functional constraints have been established a priori  or overall accuracy across time, and neither RT nor accuracy changed over time for congruent or incongruent trials, all The ICCs of the behavioral measures are listed in Table 3.23.2.1pFWE‐corrected = .05) for the “all faces trials” contrast  was observed in the fusiform gyrus and the middle temporal gyrus, as well as the right precuneus and the left post‐central gyrus for congruent faces (see Table pFWE‐corrected = .05) was observed within frontal and sub‐cortical regions such as the right precentral gyrus and the left cingulate gyrus and the left insula. In these areas, ICC values were 0.4. Poor reliability was observed for significant activation within the right thalamus, pyramis, insula, the right postcentral gyrus and the right superior frontal and right superior temporal gyri, as well as the left insula and the left parahippocampal gyrus.Significant activation (3.2.3pFWE‐corrected = .05) for incongruent faces  was observed within the right supramarginal gyrus and the right inferior parietal lobule.For the fear face minus happy face contrasts, significant activation (pFWE‐corrected = .05) was observed within right superior and middle temporal gyri.For the 3.2.5incongruent minus congruent contrast, significant activation (pFWE‐corrected = .05) was observed for the right inferior parietal lobule, the left precuneus and the left cingulate, as well as within the left medial and precentral gyri and the inferior frontal gyrus, bilaterally. Median ICC values within these regions were ≤0.1  minus cI  contrast, no significant results were obtained when using FWE‐corrected threshold. To facilitate ICC analyses, reported here are the results from an uncorrected threshold, puncorrected = .001, observed within bilateral cingulate gyri. However, median ICC values were poor . For congruent trials only, the pattern was similar and higher ICC values were observed within visual regions, including bilateral cuneus , bilateral occipital fusiform gyri (ICC = 0.5), bilateral calcarine cortex (ICC = 0.5), bilateral lingual gyri (ICC = 0.5), bilateral inferior occipital gyri , bilateral superior occipital gyrus . The pattern was similar for incongruent trials; ICC values between 0.5 and 0.6 were observed for the cuneus , bilateral occipital fusiform gyri (ICC = 0.5), bilateral calcarine cortex , bilateral lingual gyri (ICC = 0.5), bilateral inferior occipital gyri (ICC = 0.5) and bilateral superior occipital gyrus (ICC = 0.5).The voxel‐wise ICC estimates across the three time‐points for the “The voxel‐wise ICC estimates across all three time‐points for all cognitively more demanding contrasts were poor, ICC values ≤0.2.4The aim of this study was to examine the reliability of the BOLD signal for an emotional conflict task as a prerequisite to assessing the task's suitability to establish biomarkers of treatment response in clinical populations. Comparing across three time‐points, weeks 0, 2 and 8, we observed moderate reliability  within occipital, parietal and temporal regions, specifically for conditions of lower cognitive complexity, such as all faces, and congruent or incongruent trials relative to baseline. Activation was also observed within frontal and sub‐cortical regions for the same conditions, but the median ICC values were poor. We did not observe “good” or “excellent” reliability for any regions. Median ICC values of 0.5 and 0.6 were also calculated for the lingual gyri, cuneus and occipital fusiform gyri for less cognitively demanding conditions, whereas poor reliability was observed for contrasts demanding more cognitive processing when using the neuromorphometrics template in exploratory analyses.Our findings are consistent with previous reports . Activation in BA44 has previously been linked to selective response suppression in response‐inhibition tasks, such as a go/no‐go task  regions such as Brodmann area (BA) 44 and BA47, dorso‐lateral and anterior PFC regions such as BA46 and BA10, in addition to subcortical structures, such as the cingulate gyri, the insula and the parahippocampus/amygdala  may not be regions related to task‐relevant activation . Noble and colleagues, using resting state fMRI, reported similar observations, stating that the most reliable edges are not necessarily the most informative ones, and vice versa . Likewise, Plichta et al.  reportedn = 36) that represents at least a comparable number of participants to those examined in previous studies. Noble and colleagues (2017) scanned 12 participants on four occasions, Nord et al.  for neuroimaging analysis in CAN‐BIND and he is the Chief Scientific Officer of ADMdx, Inc., a neuroimaging consulting company.Drs. Appendix S1: Supporting InformationClick here for additional data file.