This effect was driven by patients with recurrent MDD , and we detected no differences between first episode patients and controls. Age of onset ⩽21 was associated with a smaller hippocampus  and a trend toward smaller amygdala  and larger lateral ventricles . Symptom severity at study inclusion was not associated with any regional brain volumes. Sample characteristics such as mean age, proportion of antidepressant users and proportion of remitted patients, and methodological characteristics did not significantly moderate alterations in brain volumes in MDD. Samples with a higher proportion of antipsychotic medication users showed larger caudate volumes in MDD patients compared with controls. This currently largest worldwide effort to identify subcortical brain alterations showed robust smaller hippocampal volumes in MDD patients, moderated by age of onset and first episode versus recurrent episode status.The pattern of structural brain alterations associated with major depressive disorder (MDD) remains unresolved. This is in part due to small sample sizes of neuroimaging studies resulting in limited statistical power, disease heterogeneity and the complex interactions between clinical characteristics and brain morphology. To address this, we meta-analyzed three-dimensional brain magnetic resonance imaging data from 1728 MDD patients and 7199 controls from 15 research samples worldwide, to identify subcortical brain volumes that robustly discriminate MDD patients from healthy controls. Relative to controls, patients had significantly lower hippocampal volumes (Cohen's  Widely available structural magnetic resonance imaging has led to hypotheses of (para-)limbic circuits being involved in MDD, but still the exact pattern of structural brain alterations associated with MDD remains unresolved, perhaps due to small sample sizes, disease heterogeneity and the complex interactions between clinical characteristics and brain morphology. Therefore, in the current study we meta-analyzed structural magnetic resonance imaging data of a very large sample (n=8927) to identify subcortical brain volumes that robustly discriminate MDD patients from healthy controls.With a lifetime prevalence of >16%,5 Hippocampal volume reduction (one of the most extensively studied regions) in MDD has been one of the most widely replicated findings.3 However, the association between MDD and hippocampal volume reduction is likely complex, and hippocampal volumes may be smaller, on average, in patients with recurrent MDD compared to patients early in the course of adult-onset MDD.6 Other factors may modulate the effect size of morphological changes in the hippocampus in MDD, including disease severity, childhood maltreatment, age of onset, antidepressant medication and illness duration.7Many studies have found structural alterations in various subcortical brain regions in MDD. To date, volumetric differences have not always been consistent and are poorly replicated for some regions; moreover, sample sizes are often small, limiting the power to detect subtle brain differences. Meta–analyses represent useful tools to identify the most robust findings across studies, and indicate that morphological changes in MDD are regional rather than global.et al.,8 Frodl et al.9 and Hamilton et al.10), but a greater number of episodes and a family history of MDD have been associated with smaller amygdala volume . Other brain regions have been less extensively investigated, including prefrontal areas, thalamus and striatum.Amygdala volume abnormalities in MDD are inconsistently reported and may depend on the illness phase, medication use and family history of MDD. A current first episode and antidepressant use has been associated with enlarged amygdala volume  consortium. This is an international collaboration currently evaluating 15 research samples from six different countries worldwide, including neuroimaging data from 1728 MDD patients and 7199 healthy individuals. The primary aim of our ENIGMA-MDD Working Group is to identify imaging markers that robustly discriminate MDD patients from healthy controls using an individual participant data (IPD)-based meta-analysis. This IPD-based meta-analytic approach applied to data from our consortium provided us with the opportunity to plan Here, we investigated subcortical gray matter, lateral ventricle and total intracranial volume (ICV) alterations in MDD patients compared to healthy individuals by performing the largest meta-analysis to date. As additional exploratory analyses, we examined potential modulating effects of demographic, clinical and methodological characteristics on morphological differences in MDD patients.The ENIGMA-MDD Working Group includes 15 international samples with neuroimaging and clinical data from MDD patients and healthy controls (participating sites are shown in 14 Image acquisition parameters and software descriptions for each sample are given in http://enigma.ini.usc.edu/protocols/imaging-protocols/). Further details on image exclusion criteria and quality control can be found in Structural T1-weighted magnetic resonance imaging brain scans were acquired at each site and analyzed locally using the fully automated and validated segmentation software FreeSurfer.d-metric computed from the t-statistic of the diagnosis indicator variable from the regression models. Throughout the manuscript we report uncorrected P-values with a significance threshold determined by Bonferroni correction for testing nine regions of interest (P=0.05/9=5.6 × 10−3).We examined patient versus control group differences within each sample using multiple linear regression models, where the mean ((left+right)/ 2) region of interest volume was the outcome measure and a binary indicator of diagnosis  was the predictor of interest. All models were controlled for age, sex and ICV. Additional covariates were included whenever necessary to control for scanner differences. Effect size estimates were calculated using Cohen's 15) and stage of illness (first and recurrent episode patients). Further, we investigated whether symptom severity at the time of scanning was associated with brain changes using the 17-item Hamilton Depression Rating Scale (HDRS-1716) and the Beck Depression Inventory (BDI-II17).To explore the influence of sex on subcortical brain volume differences between patients and controls, we performed diagnosis-by-sex interaction effects within each sample. To further investigate the sources of regional brain changes in MDD, we performed separate stratified meta-analyses comparing age of onset . The meta-analysis of symptom severity scores was an exception: severity scores were treated as continuous variables, so effect sizes were in terms of Pearson's r, a partial-correlation after removing nuisance variables . The final meta-analyzed Pearson's r was estimated following the same inverse variance-weighted random-effect meta-analysis model used for all other meta-analyses. See All regression models and effect size estimates were fit at each site separately and a final Cohen's We tested whether mean age of each sample , magnetin=1728) compared with all healthy controls (n=7199) across nine brain structures : d=−0.14 ; P-value=4.60 × 10−4, % difference=−1.25). Case-control differences for all structures are listed in In our primary analysis, we assessed case-control differences between all MDD patients (ructures . Only men=583) and recurrent episode patients (n=1119) and compared to healthy controls . Recurrent episode patients showed lower mean hippocampal volume than controls ; P-value=1.12 × 10−5, % difference=−1.44). Full meta-analyzed recurrence status differences are listed in We examined how the current stage of a depressed patient may relate to brain volumes by splitting the sample into first episode (controls . We did n=541) showed significantly lower mean hippocampal volumes than controls ; P-value=2.31 × 10−4, % difference=−1.85). In addition, we found lower amygdala volume ; P-value=0.033, % difference=−1.23) and higher mean lateral ventricle volume ; P-value=0.009, % difference=5.11) in early onset patients compared to controls, but neither survived correction for multiple comparisons. Although sample sizes were too small to split first and recurrent episode patients into early and late onset groups, only about half (57%) of the early onset MDD patients had a recurrent episode, and the percentage of recurrent episode patients did not moderate the result of smaller hippocampal volumes in early onset MDD patients (P=0.54), suggesting that this effect is at least partly independent of recurrence status. Patients with a late age of onset  showed no detectable brain volumetric differences compared to controls. Full age of onset effect sizes are listed in We examined how the age of onset modulates volumetric brain changes . Patientn=667) and BDI-II (n=667) questionnaires. Full effect sizes of severity scores are available in We did not detect any significant associations between symptom severity at study inclusion and brain volumes using the HDRS-17 , but this was not significant after correcting for multiple comparisons. We also found an influence of percentage of patients taking antidepressants on hippocampal effect size estimates , but again this was not significant after correction for multiple comparisons. Finally, we found a near-significant effect of antipsychotic drug use on mean caudate volume , implying that caudate volume of depressed patients was greater as the percentage of patients taking antipsychotic drugs was higher . On the basis of the final meta-analyzed effect sizes found in this study, to detect mean differences in hippocampal volumes between MDD patients and controls, a study in the general MDD population at large would require 802 subjects per group to have 80% power to detect a difference at P-value=0.05. When focusing analyses on recurrent MDD patients and controls, 545 subjects per group would be needed to provide 80% power to detect a difference in hippocampal volume at P-value=0.05. When focusing analyses on early age of onset MDD patients (⩽21 years) and controls, 394 subjects per group would be needed to have 80% power to detect a difference in hippocampal volume at P-value=0.05. See With 1728 MDD patients and 7199 controls, we were able to detect brain volume differences as small as Cohen's This worldwide effort to identify subcortical gray matter, lateral ventricle and total ICV alterations associated with MDD using an IPD-based meta-analytic approach showed robust reductions in hippocampal volume (1.24%) in MDD patients compared with healthy controls. These hippocampal volume reductions were mainly present in recurrent and/or early onset (⩽21 years) MDD, whereas hippocampal volume reductions were absent in first episode patients and less pronounced in patients with later age of onset (>21 years) MDD. Furthermore, smaller amygdala volume and larger lateral ventricles volume were found in early onset MDD, but neither survived correction for multiple comparisons.6 This robust finding of smaller hippocampal volume is often linked to the ‘neurotrophic hypothesis of depression'. This proposes that elevated glucocorticoid levels associated with chronic hyperactivity of the hypothalamic–pituitary–adrenal axis in MDD may induce brain atrophy via remodeling and downregulation of growth factors including brain-derived neurotrophic factor.19 This process may preferentially target the hippocampus, a major site in the glucocorticoid negative feedback loop of the hypothalamic–pituitary–adrenal axis with high expression of glucocorticoid receptors.20 In animal models of depression, stress-induced increases in glucocorticoid levels may result in regression of dendritic processes, inhibition of neurogenesis and loss of neurons.21 We only observed hippocampal volume deficits in recurrent MDD and not in first episode patients. There is also direct evidence from longitudinal studies that hippocampal volume progressively decreases during the course of the disease, beyond levels expected from normal aging.22 We also found that hippocampal volume reductions were more pronounced in early onset patients (⩽21 years), consistent with prior findings of smaller hippocampal volume in early onset depression.23 Several factors, including early life stress, temperamental low effortful control and family history of depression have been linked to early onset depression,26 as well as smaller hippocampal volume.28Our finding of smaller hippocampal volume in MDD is in line with previous retrospective meta-analyses of aggregated data.29 and smaller hippocampal volumes have been found to predict a protracted response to antidepressant treatment over weeks31and a more severe long-term illness course,32 so morphological hippocampal alterations may represent risk markers for depression, recurrence and chronicity. As we only observed hippocampal volume reduction in recurrent patients and no subcortical volume alterations in first episode patients, the current finding of smaller hippocampal volume in early onset depression may reflect a longer illness duration and/or greater number of episodes in patients with early onset MDD instead of a premorbid vulnerability factor. Nonetheless, as only about half (57%) of the early onset patients in our meta-analysis had a recurrent episode and as the percentage of recurrent episode patients did not moderate the result of smaller hippocampal volumes in early onset patients , we conclude that early disease onset is (in part) independently associated with lower hippocampal volumes. Unfortunately, for many of the samples complete information on the exact number of episodes and duration of each episode was not available and simply calculating disease burden as current age minus age of onset is unsatisfactory, given the relapsing and remitting nature of MDD. Clearly, there is a continued need for longitudinal studies tracking hippocampal volume changes over the disease course, to further elucidate whether hippocampal abnormalities result from prolonged duration of chronic stress , represent a vulnerability factor for MDD, or both.Patterns of abnormal hippocampal development may predate adolescent depression onsetet al.,8 Frodl et al.,9 Kronenberg et al.11 and Hickie et al.33). Our meta-analysis suggested smaller amygdala volume in early onset MDD, but this effect was not significant after correction for multiple comparisons. This is consistent with a previous retrospective meta-analysis of aggregated amygdala volume data by Hamilton et al.,10 which also failed to detect a reliable difference in amygdala volume between depressed patients and controls. However, prior meta-analyses have shown larger amygdala volume in samples with a higher proportion of patients taking antidepressants.10 We could not replicate this result in the current meta-analysis.Prior structural neuroimaging studies in depression have yielded conflicting results regarding alterations in amygdala volume  rather than widespread. Reduced hippocampal volume is not specific to MDD, as it has been observed in other psychiatric disorders, such as schizophrenia, posttraumatic stress disorder, borderline personality disorder and obsessive-compulsive disorder.34 Interestingly, IPD-based meta-analytic studies of both schizophrenia35 and bipolar disorder , using highly similar protocols, reported more widespread alterations in subcortical volumes. Generally, detrimental effects of schizophrenia and bipolar disorder on subcortical brain volumes appear greater than those in MDD, although a study design directly comparing patients with these disorders is clearly needed to resolve this.Furthermore, our controlled meta-analysis could not replicate findings of smaller caudate and putamen volumes observed in prior smaller meta-analyses.et al.,11 Hickie et al.36 and Frodl et al.37), but some studies found positive38 as well as inverse associations39 with, for example, amygdala and hippocampal volumes. Our meta-analyses, based on the HDRS-17 (n=667) and BDI-II (n=667) questionnaires, found no evidence of association between symptom severity and subcortical gray matter volumes. As severity of an entire depressive episode is not fully characterized by depression severity at study inclusion and given the predictive value of hippocampal volume,32 this result awaits further investigation in prospective longitudinal treatment designs.The current study is the first to meta-analyze the relationship between symptom severity at study inclusion and subcortical volumes in MDD patients. Previous studies have yielded conflicting results; most studies found no correlations between depressive symptom severity and brain volume .We found that an increase in the percentage of patients taking antipsychotic drugs was associated with larger caudate volume in depressed patients relative to controls. This is in line with prior observations of caudate enlargement following antipsychotic treatment in patients with schizophrenia and can occur as early as 3 weeks post-exposureA key strength of our study is the IPD meta-analytic approach, which increased the power to detect small effects by combining data from 8927 individuals, while ensuring low methodological heterogeneity by standardizing brain segmentation techniques and statistical models across all participating samples. Moreover, this IPD-based approach allowed us to systematically investigate the effects of clinical characteristics such as recurrence status, age of onset and severity of symptoms on brain alterations in MDD patients.44 we limited the meta-analysis with respect to severity of symptoms to a selection of studies using the same instrument, which considerably reduced the sample size. Finally, we restricted our analyses to subcortical gray matter, lateral ventricle and total ICVs and did not include cortical measures. As efforts such as the current one require significant work in harmonization and quality control by all sites, the current study is only a first step toward identifying robust brain volume alterations in MDD patients. Because prior studies have shown a potentially important role of especially the anterior cingulate cortex,45 the important next step within our consortium will be examining cortical brain alterations associated with MDD.Nonetheless, our study has some limitations. First, we could not directly examine the influence of remission of MDD on brain volume. In most studies, the majority of patients were acutely depressed at the time of scanning, yielding groups of remitted patients that were too small to properly investigate how remission relates to brain volume. However, the proportion of MDD patients in remission at each site had no moderating effect on differences in brain volume when examined in a meta-regression analysis see . Moreove37 learning and memory deficits36 and worse treatment response46 in MDD, suggest that the hippocampus is a prime target region for future research aimed at further unraveling the pathophysiology of MDD and improving treatment.Despite these limitations, results of this first initiative of the ENIGMA-MDD working group clearly indicate a key role of the hippocampus in the pathophysiology of MDD, showing robust hippocampal volume reductions particularly in recurrent and early onset MDD. Brain changes in other subcortical regions in MDD were less evident. Our findings together with the observations of associations between smaller hippocampal volume and executive impairments,


Rangifer tarandus caribou from the Rivière-aux-Feuilles herd in northern Québec (Canada) with GPS telemetry collars and studied their space use. We measured contemporary  and historical  variations in freshwater-ice availability and evaluated the concurrent responses of caribou to these changes.Freshwater lakes and rivers of the Northern Hemisphere have been freezing increasingly later and thawing increasingly earlier during the last century. With reduced temporal periods during which ice conditions are favourable for locomotion, freshwater bodies could become impediments to the inter-patch movements, dispersion, or migration of terrestrial animals that use ice-covered lakes and rivers to move across their range. Studying the fine-scale responses of individuals to broad-scale changes in ice availability and phenology would help to understand how animals react to ongoing climate change, and contribute to the conservation and management of endangered species living in northern environments. Between 2007 and 2014, we equipped 96 migratory caribou Ice had a positive influence on caribou movement rates and directionality, and caribou selected ice and avoided water when moving across or in the vicinity of large water bodies. When ice was unavailable, caribou rarely swam across (6 % of crossings) and frequently circumvented water bodies for several km. Although ice phenology did not change significantly during our study, climate projections indicated that ice availability could decrease considerably before the end of the century, generating a ~28 % increase in distance travelled by caribou during the early spring and fall migrations.We demonstrated that ice availability influenced the movements of a migratory arctic ungulate. Warmer air temperatures in the Arctic will undoubtedly modify the phenology of ice forming on freshwater lakes and rivers. If migratory caribou are unable to adjust the timing of their migrations, they could be forced to circumvent unfrozen water bodies more frequently and over broader areas, which may increase the distance, time, and energy they use to reach wintering areas. The long-term conservation of wide-ranging species will ultimately depend on our ability to identify the fine-scale behavioural reactions of individuals to broad-scale changes in climate and land use.The online version of this article (doi:10.1186/s40462-016-0079-4) contains supplementary material, which is available to authorized users. Climate is changing rapidly , and theUrsus maritimus  reveals that caribou in Northern Québec are initiating their spring and fall migrations earlier. This response may allow caribou to avoid a potential mismatch between the timing of migration and freshwater ice availability. Assuming caribou are unable to adjust the timing of their migrations further , delaysCaribou in Northern Québec seemed to favour ice substrates during migrations and winter displacements. Their movements in water were slower and more sinuous than their movements on ice. The relatively long “pauses” caribou often made on lake shores could have allowed them to assess the quality  of the ice before crossing . CaribouNeovison vison) spend 2.7 times more energy swimming compared to running [Caribou swim at the water surface using quadrupedal paddling, which is amongst the least efficient types of swim in vertebrates . Few com running . To cove running . It is pR.t. platyrhynchus) increased their displacement distances following rain-on-snow events, which are predicted to occur more frequently during warm winters. In Northern Québec, we revealed a link between caribou movements and inter-annual variations in ice availability. Caribou avoided water and selected ice in years with long and short ice-availability periods, respectively, suggesting that caribou were able to respond to ice availability and, indirectly, prevailing climatic conditions. Ice availability could thus be one of many drivers explaining the distribution of caribou following climate change. Predicting future migration routes of caribou, however, remains challenging [The movements of large herbivores are influenced by a suite of environmental and anthropogenic factors, including the type of substrate , topograllenging .Antilocapra americana) and mule deer (Odocoileus hemionus) of the Greater Yellowstone Ecosystem  to migrate through relatively narrow bottlenecks surrounded by topographic and anthropogenic features. Although Northern Québec is much less disturbed than Wyoming [Other landscape features, notably anthropogenic disturbances, also have the potential to disrupt ungulate migrations . Berger  Wyoming , 26, the Wyoming . Hydroel Wyoming . Along tThe dataset supporting the conclusions of this article is available in the Dryad Digital Repository (DOI:10.5061/dryad.4k275).


Negative urgency , is a personality trait that has been linked to problematic involvement in several risky and impulsive behaviours, and to various forms of disinhibitory psychopathology, but its neurobiological correlates are poorly understood. Here, we explored whether inter-individual variation in levels of trait negative urgency was associated with inter-individual variation in regional grey matter volumes. Using voxel-based morphometry (VBM) in a sample (n = 152) of healthy participants, we found that smaller volumes of the dorsomedial prefrontal cortex and right temporal pole, regions previously linked to emotion appraisal, emotion regulation and emotion-based decision-making, were associated with higher levels of trait negative urgency. When controlling for other impulsivity linked personality traits  and negative emotionality per se (neuroticism), these associations remained, and an additional relationship was found between higher levels of trait negative urgency and smaller volumes of the left ventral striatum. This latter finding mirrors recent VBM findings in an animal model of impulsivity. Our findings offer novel insight into the brain structure correlates of one key source of inter-individual differences in impulsivity. •We identified brain structure correlates of rash impulsivity (negative urgency).•Urgency was inversely linked to dorsomedial frontal & right temporal pole volumes.•Controlling for neuroticism, urgency also linked to lower ventral striatal volumes. The personality trait of “impulsivity” has long been implicated as an important risk and maintaining factor across a diverse range of risky behaviours and mental health problems . It is iA notable feature of the UPPS model is that it has drawn attention to emotion-related impulsigenic traits that were largely neglected previously. In particular, one facet of impulsivity, negative urgency, refers specifically to the disposition to act rashly in the context of intense negative emotional states . After cRecent structural magnetic resonance imaging (MRI) studies have found that inter-individual variation in a number of behavioural traits – including personality traits – can be predicted from the local structure of grey matter as assessed by voxel-based morphometry (VBM) . Here, wOne-hundred and fifty-two healthy right-handed participants, primarily undergraduate and graduate university students , underwent structural magnetic resonance imaging (MRI) after providing written informed consent and screening for MRI contra-indications, including a history of neurological or psychiatric disorders. The study was approved by the Cardiff University School of Psychology Research Ethics Committee.agree strongly) to 4 (disagree strongly). Higher mean scores reflect higher levels of impulsivity. It has excellent internal consistency, with Cronbach's alpha level typically exceeding 0.8 . An extensive body of evidence supports the validity of the negative urgency scale. The scale has repeatedly emerged as uni-dimensional, scores converge across self-report and interview assessment methods  for negative urgency were calculated and used in all analyses. The other traits from the UPPS  were also scored and used as covariates.disagree strongly) to 5 (agree strongly). In this study, Cronbach's alpha for Neuroticism was 0.85, indicating excellent internal consistency.Trait neuroticism (the tendency to experience negative emotions) was measured using the neuroticism scale of the Big Five Inventory (BFI) 44-item version . This se0, 1 mm isotropic resolution) were acquired with 168 or 172 slices depending on head size.MRI scans were obtained on a 3 T GE HDx signa MRI scanner at the Cardiff University Brain Research Imaging Centre (CUBRIC) fitted with an 8-channel head coil. High resolution T1-weighted fast spoiled gradient (FSPGR) coronal scans  into grey matter, white matter and cerebrospinal fluid tissue classes using unified segmentation. Grey matter images were then normalised to a grey matter population template, generated from the complete image set using the diffeomorphic anatomical registration using exponentiated lie-algebra (DARTEL) registration method . This nohttp://www.mccauslandcenter.sc.edu/mricro/mricron/). Results were visualized using NeuroElf (www.neuroelf.net).Voxel-based multiple regression analysis  was carried out using SPM8 with voxel-wise grey matter volume (GMV) as the dependent variable. Age and gender (known predictors of brain volume) were added as nuisance covariates to the GLM . Negativ1.a.Associations with grey matter volume in the dmPFC , based on the VBM study of personality by b.Associations with grey matter volume in the right temporal pole , based on lower levels of volumes in this region reported in individuals with co-occurring cocaine dependence and personality disorder .Correlations between negative urgency scores and grey matter volumes were assessed using a region of interest (ROI) based approach, with ROIs sampled using 10 mm diameter spheres  correction based on Gaussian Random Field Theory (RFT) . We then looked for any other regions throughout the brain that showed associations with negative urgency . To test for potential sex differences in the association between regional grey matter volume and negative urgency, we additionally formally tested for an interaction between gender and negative urgency scores in SPM using a full factorial model  with the factor of gender, the covariates of negative urgency and age and the global factor of intracranial volume for proportional global scaling.2.To assess the amount of unique variance captured by negative urgency, we repeated the regression after including mean [lack of perseveration and lack of planning] and sensation-seeking (as well as age and gender) as nuisance covariates. An averaged score combining (lack of) perseveration and (lack of) planning was used due to the high correlation between these two measures , consistent with previous findings that (lack of) perseveration and (lack of) planning are correlated facets of (low) conscientiousness .3.We then repeated the ROI analyses from (1) in the subset of participants  who had completed both the UPPS and the BFI-44 neuroticism scales, after including mean neuroticism score from the BFI-44 as a covariate of no interest. This analysis allowed us to examine the relation between regional grey matter volume and levels of emotion-driven rash impulsivity, controlling for inter-individual differences in the tendency to experience negative emotions per se.Statistical analysis was then carried out in the following stages.Mean scores on the UPPS traits, including negative urgency, together with mean levels of trait neuroticism are reported in 3) and in the right temporal pole , such that higher levels of negative urgency were associated with lower GM volumes in these regions    . There wrrected) .No positive associations were observed between negative urgency and grey matter volumes in either of the ROIs.3).Both the relationship between negative urgency and dmPFC volume and the relationship between negative urgency and right anterior temporal pole volume remained significant after controlling for the other UPPS traits. The same associations with negative urgency were seen within the dmPFC when controlling for levels of neuroticism in the subset of individuals who had completed the BFI-44 in addition to the UPPS  , after controlling for age and gender (3). The relationship between negative urgency and grey matter volume in the right temporal pole remained significant after additionally controlling for the other UPPS traits .The whole brain analysis revealed a significant negative association between urgency and grey matter volumes in the right temporal pole    .In this study we provide new evidence on the unique brain structure correlates of inter-individual variation in dispositions towards rash, negative emotion-linked impulsivity (negative urgency) in a sample of healthy volunteers. In line with our a priori predictions, we found significant negative associations between trait urgency and grey matter volume in regions of the dmPFC, and in the right anterior temporal pole, such that individuals reporting higher levels of negative urgency  showed smaller grey matter volumes in these brain regions. While some previous studies have linked variation in dmPFC volumes or cortical thickness to broad personality measures of impulsivity , ours isAmongst the traits delineated in the UPPS model , negativRecent research suggests that negative urgency may be of even broader significance to understanding risk for emotional disorders — with negative urgency acting as a prospective predictor of depression, as well as to risk for externalizing disorders . It is nIn addition to a relation with relatively smaller dmPFC grey matter volume, both the ROI-based and whole brain analysis revealed an association between lower grey matter volume in the right temporal pole and increased levels of negative urgency. Lower grey matter volume or thickness in the temporal pole has previously been reported in individuals with attention deficit/ hyperactivity disorder , cocaineTheories of negative urgency conceptualize the trait in terms of inter-individual differences in emotion regulation , emotionThe role of the dmPFC in emotion regulation and decision making appears to be part of a broader role for this region in higher-level emotional appraisal . The dmPWe did not find a significant association between urgency and grey matter volume of other regions linked to emotion regulation, in particular the ventromedial prefrontal cortex . A recenThe negative association between trait urgency and grey matter volume in the left ventral striatum, after controlling for levels of neuroticism is of particular note. Smaller ventral striatal volumes have been reported in a number of clinical externalizing disorders associated with markedly high levels of negative urgency, including alcoholism  and nicoThere are some limitations that should be considered when interpreting our results. First, as with all cross-sectional studies, there is no indication as to whether our correlations imply causal relationships. There are genetic influences on both negative urgency  and greySecond, the cellular basis of the factors underlying VBM findings associated with structural MRI are unknown, as is the relationship between brain structure and brain activity as measured by functional MRI see . AdditioFinally, our sample was not evenly matched for sex, with approximately twice as many females as males. Previous research has found that relations between negative urgency and risk outcomes do not differ by sex , but theThis study examined the brain structure correlates of rash, negative emotion-based impulsivity, demonstrating that relatively smaller grey matter volumes in the dmPFC, temporal pole and ventral striatum are associated with higher levels of negative urgency. The regional associations we found point to the importance of understanding the contributions of variation in both subcortical structures that likely mediate the overt behavioural manifestations of impulsivity, and within cortical regions that mediate higher-level cognitive processes, such as self-reflective emotional appraisal, to inter-individual variation in impulsivity, the latter of which has until recently been relatively neglected, particularly in animal models of impulsivity. Illuminating the neurobiological variation underpinning inter-individual variation in negative urgency can potentially offer novel insight into factors promoting resilience to problematic risky behaviours and diverse forms of emotional disorder.


Glaucoma is the second leading cause of irreversible blindness and a neurodegenerative disease with a complex pathogenesis. Increasing evidence suggests that oxidative stress and mitochondrial dysfunction have crucial roles in most neurodegenerative diseases such as glaucoma. The conventional clinical treatment for glaucoma is lowering the intraocular pressure (IOP). Some patients have normal IOP, whereas other patients appear to obtain adequate control of IOP after filtration surgery or medication. However, these patients still experience progressive visual field loss. Vision field loss in glaucoma is attributed to retinal ganglion cell (RGC) apoptosis. Many recent researches demonstrated that the link between mitochondrial dysfunction and oxidative stress was a major cause of RGCs apoptosis. How oxidative stress leads to RGCs apoptosis in glaucoma is unclear but may involve the neurotoxic effects of oxidative stress-induced mitochondrial dysfunction and/or damage from reactive oxygen species (ROS). Investigations are needed concerning the mitochondria as effective targets for potential therapeutic interventions to maintain mitochondrial function and reduce oxidative stress, and thereby delay or stop RGC loss and prolong visual function. The mitochondria-targeted antioxidant Szeto-Schiller (SS) peptide is a candidate molecule. Szeto-Schiller-31 (H-D-Arg-Dmt-Lys-Phe-NH2) is an attractive mitochondria-targeted antioxidant that can protect the mitochondria and RGCs against oxidative damage. Therefore, we suggest SS-31 as a novel neuroprotective ophthalmic drug for protecting RGCs in glaucoma. Glaucoma is increasingly recognized as a chronic neurodegenerative disorder characterized by optic nerve head cupping and visual field defects caused by damage to and apoptosis of RGCs. Glaucoma treatment focuses on lowering IOP via pharmacotherapy or surgery. Epidemiological investigators have recently reported that lowering IOP alone cannot prevent the progressive loss of visual field in glaucoma patients. There is improved knowledge about the mechanism of optic nerve damage. As a consequence, the concept of neuroprotection has been increasingly proposed. Additional neuroprotective strategies may offer novel treatments to prevent RGCs loss and disease progression. Increasing IOP is a major risk factor in glaucoma; however, other concomitant factors also significantly affect the eye such as oxidative stress caused by ROS , increasOxidative stress and mitochondrial dysfunction in glaucomaIn brief, oxidative stress is an imbalance between processes that generate ROS and processes that remove them. More than two decades ago, oxidative stress was first proposed as a contributor of glaucoma pathogenesis . Many clThe mitochondria are normally protected from oxidative damage, owing to mitochondrial antioxidant systems, a multilayer network; however, if ROS production exceeds the antioxidant capacity of mitochondria, these cells experience oxidative damage . With thA pathological feature of glaucoma is apoptosis of RGCs. Caspase mediation activates the proteolysis cascade, which can trigger the apoptosis of RGCs by different stimuli ,17. CaspBased on the aforementioned evidence, we can understand that mitochondrial dysfunction in combination with oxidative stress orchestrates the apoptosis of RGCs by activating different stimuli. Mitochondrial dysfunction is responsible for cytotoxic events in RGCs apoptosis. Enhancing mitochondrial function could prolong RGCs survival. Oxidative damage to mitochondria leads to hyperpolarization of mitochondrial membrane potential, which causes mitochondrial dysfunction and releases stimulative apoptotic compounds. In addition, mitochondria are major generators of ROS and scavenge ROS to reduce ROS-evoked apoptosis signaling. We suggest that a targeted mitochondrial antioxidant could be a viable therapeutic intervention.Retinal ganglion cells require high energy. They rely on the mitochondria for survival and function. Pre-existing congenital or acquired mitochondrial dysfunction can increase the vulnerability of RGCs to stress from risk factors . AlteratEvaluation of the hypothesisHazel Szeto and Peter Schiller discovered a group of small synthetic peptides, called SS peptides, which are limited to fewer than 10 amino acid residues and contain alternating aromatic residues and basic amino acids. There are three peptide analogues in this series: SS-02, SS-31 and SS-20. Each molecule carries a 3+ net charge at physiological pH . These pCompared to general antioxidants such as vitamin E, brimonidine, and other biological antioxidants, mitochondrial-targeted peptides show more potential therapeutic neuroprotective effects in some research. General antioxidants tend to be poorly cell-permeable and quite susceptible to the mitochondria. They have an unsatisfactory curative effect because they do not reach the relevant sites of free radical generation within the mitochondria. Vitamin E and coenzyme Q are mitochondrial-targeted antioxidants, and they are very lipophilic. They are taken up into mitochondria by exploiting the potential gradient across the IMM . HoweverIncreasing evidence supports SS-31 as superior to the other SS peptides analogues such as SS-02 and SS-20. A few studies have shown that SS-31 can scavenge mitochondrial ROS in a dose-dependent manner because it possesses a tyrosine residue . HoweverThe peptide SS-31 can inhibit MPT, reduce mitochondrial ROS production, and prevent mitochondrial swelling in isolated mitochondria . In addiIn the field of ophthalmology, Huang and colleagues  assessedGlaucoma is a multifactorial optic neuropathy characterized by progressive degeneration of RGCs. Oxidative stress occurs in glaucoma and contributes to RGCs degeneration. Mitochondria have a central role as generators and as targets of oxidative stress, and there has been growing evidence of mitochondrial dysfunction in the pathogenesis of glaucoma . We sugg


Loud sound exposure exacerbates aminoglycoside ototoxicity, increasing the risk of permanent hearing loss and degrading the quality of life in affected individuals. We previously reported that loud sound exposure induces temporary threshold shifts (TTS) and enhances uptake of aminoglycosides, like gentamicin, by cochlear outer hair cells (OHCs). Here, we explore mechanisms by which loud sound exposure and TTS could increase aminoglycoside uptake by OHCs that may underlie this form of ototoxic synergy. Mice were exposed to loud sound levels to induce TTS, and received fluorescently-tagged gentamicin (GTTR) for 30 min prior to fixation. The degree of TTS was assessed by comparing auditory brainstem responses (ABRs) before and after loud sound exposure. The number of tip links, which gate the GTTR-permeant mechanoelectrical transducer (MET) channels, was determined in OHC bundles, with or without exposure to loud sound, using scanning electron microscopy. We found wide-band noise (WBN) levels that induce TTS also enhance OHC uptake of GTTR compared to OHCs in control cochleae. In cochlear regions with TTS, the increase in OHC uptake of GTTR was significantly greater than in adjacent pillar cells. In control mice, we identified stereociliary tip links at ~50% of potential positions in OHC bundles. However, the number of OHC tip links was significantly reduced in mice that received WBN at levels capable of inducing TTS. These data suggest that GTTR uptake by OHCs during TTS occurs by increased permeation of surviving, mechanically-gated MET channels, and/or non-MET aminoglycoside-permeant channels activated following loud sound exposure. Loss of tip links would hyperpolarize hair cells and potentially increase drug uptake via aminoglycoside-permeant channels expressed by hair cells. The effect of TTS on aminoglycoside-permeant channel kinetics will shed new light on the mechanisms of loud sound-enhanced aminoglycoside uptake, and consequently on ototoxic synergy. Aminoglycoside antibiotics, like gentamicin and tobramycin, are clinically-essential antibiotics for treating life-threatening Gram-negative bacterial infections in premature babies or patients with cystic fibrosis, Gram-positive infections like tuberculosis, and protozoal infections , are more susceptible to aminoglycoside-induced cytotoxicity than other cochlear cells, particularly at the base of the cochlea most sensitive to high frequency sound. Once these sensory cells are lost, they cannot be regenerated, leading to life-long hearing loss and deafness. Thus, extensive efforts are underway to ameliorate and prevent aminoglycoside-induced hair cell death.Aminoglycosides can rapidly cross the blood-labyrinth barrier (BLB) into cochlear tissues and fluids  injected with fluorescently-conjugated gentamicin  dissolved in phosphate buffered saline  within 30 min of cessation of noise exposure on the 3rd day, to track gentamicin uptake as previously described  for 6 h/day for 3 days  and xylazine (13 mg/kg) and placed on a heating pad in a sound-proof, electrically isolated chamber. Needle electrodes were placed subcutaneously below the test ear, at the vertex, and with a ground on the claw. Each ear was stimulated separately with a closed tube sound delivery system sealed into the ear canal. ABR threshold was defined as the sound level at which the minimum identifiable ABR response occurred.Mice were assessed by auditory brainstem response (ABR) measurements to determine the degree of TTS and time course of hearing recovery. Briefly, ABRs to 1 ms rise-time tone burst stimuli at 4, 8, 12, 16, 24 and 32 kHz, with 5 dB steps, were recorded and thresholds obtained for each ear, using an in-house system described previously  as described previously  at 86 dB SPL for 18 h over 3 days did not induce TTS Figure . ProlongExposure to prolonged WBN at 86 dB SPL which did not induce TTS Figure  did not All OHCs in cochleae with 96 dB SPL-induced TTS exhibited greater GTTR fluorescence compared to OHCs from equivalent regions in control cochleae Figure . GTTR flstria vascularis .Scanning electron microscopy of OHCs with or without prolonged exposure to WBN at 91 dB SPL, revealed OHCs with a classic “V” or “W” stereociliary bundle Figures . In OHCsin vivo; (ii) higher levels of sound exposure (96 dB SPL) increase the degree of TTS and widen the region of GTTR uptake; and (iii) tip link survival in the region of increased GTTR uptake is reduced significantly. In addition, pillar cell uptake of GTTR is not significantly increased after loud sound exposure, suggesting that endolymph levels are statistically unchanged, and that increased OHC uptake of GTTR after loud sound exposure is hair cell-specific. These findings are clinically relevant as they provide additional evidence for a synergistic ototoxicity of loud sound exposure and aminoglycoside treatment  moderate-to-loud sound level exposures (91 and 96 dB WBN for 6 h per day over 3 consecutive days) that induce TTS also increase OHC uptake of ototoxic aminoglycosides (GTTR) in vivo, it may contribute to the recovery of auditory function from TTS in the 4 kHz region after 96 dB SPL WBN exposure. To test this hypothesis, it will be necessary to test whether the extent of tip-link loss and regeneration correlates with the degree of TTS and recovery observed in this region. Experiments to verify this are ongoing.The findings have two additional implications. The first is that loss and regeneration of stereociliary tip links may explain loud sound-induced TTS. In organ of Corti explants, stereociliary tip links are regenerated 24–48 h after ablation, restoring functional kinetics and aminoglycoside permeability to MET channels  current; and (3) via hyperpolarization of OHCs by activating outward potassium current , may provide another route for the sound-enhanced aminoglycoside uptake by OHCs (Luk et al., These mechanisms are dependent on aminoglycoside entry into the scala tympani, which has been demonstrated (Tran Ba Huy et al., in vivo by hair cells, delipidation of cochlear tissues to unmask GTTR fluorescence will remove endosome-associated GTTR fluorescence, and alternative unmasking steps should be utilized to further test this hypothesis (Myrdal et al., Hair cells are also able to endocytose aminoglycosides into the apical cytoplasm (Hiel et al., This study reveals one possible contributor to loud sound-induced TTS is the loss of tip links, for which tip link regeneration may underlie the functional recovery associated with TTS. Furthermore, this study shows increased OHC uptake of GTTR during TTS that may contribute to the synergistic ototoxicity of noise and aminoglycosides. Finally, the apparent discrepancy between tip link loss and enhanced GTTR uptake may be explained by the activation of one or more additional routes of aminoglycoside entry into hair cells during TTS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


While recent integrative analyses of copy number and gene expression data in breast cancer have revealed a complex molecular landscape with multiple subtypes and many oncogenic/tumour suppressor driver events, much less is known about the role of DNA methylation in shaping breast cancer taxonomy and defining driver events.Here, we applied a powerful integrative network algorithm to matched DNA methylation and RNA-Seq data for 724 estrogen receptor (ER)-positive (ER+) breast cancers and 111 normal adjacent tissue specimens from The Cancer Genome Atlas (TCGA) project, in order to identify putative epigenetic driver events and to explore the resulting molecular taxonomy. This revealed the existence of nine functionally deregulated epigenetic hotspots encompassing a total of 146 genes, which we were able to validate in independent data sets encompassing over 1000 ER+ breast cancers. Integrative clustering of the matched messenger RNA (mRNA) and DNA methylation data over these genes resulted in only two clusters, which correlated very strongly with the luminal-A and luminal B subtypes. Overall, luminal-A and luminal-B breast cancers shared the same epigenetically deregulated hotspots but with luminal-B cancers exhibiting increased aberrant DNA methylation patterns relative to normal tissue. We show that increased levels of DNA methylation and mRNA expression deviation from the normal state define a marker of poor prognosis. Our data further implicates epigenetic silencing of WNT signalling antagonists and bone morphogenetic proteins (BMP) as key events underlying both luminal subtypes but specially of luminal-B breast cancer. Finally, we show that DNA methylation changes within the identified epigenetic interactome hotspots do not exhibit mutually exclusive patterns within the same cancer sample, instead exhibiting coordinated changes within the sample.Our results indicate that the integrative DNA methylation and transcriptomic landscape of ER+ breast cancer is surprisingly homogeneous, defining two main subtypes which strongly correlate with luminal-A/B subtype status. In particular, we identify WNT and BMP signalling as key epigenetically deregulated tumour suppressor pathways in luminal ER+ breast cancer, with increased deregulation seen in luminal-B breast cancer.The online version of this article (doi:10.1186/s13148-015-0159-0) contains supplementary material, which is available to authorized users. ERBB2, CCND1, ZNF218 or EMSY, as well as deletion of tumour suppressors like TP53 and PTEN. The largest study to date, performed by the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), suggests that many more putative driver events exist, with many of these being specific to certain breast cancer subtypes [Large-scale integrative analyses of copy number and gene expression data in breast cancer have revealed a complex molecular landscape with many putative driver events –4. The mAnother mechanism that can lead to deregulation of gene expression in cancer is aberrant DNA methylation. In particular, changes in DNA methylation patterns occurring in the vicinity of gene promoters have long been recognized as a mechanism of tumour suppressor inactivation or oncogene activation –8. In spThus, we here aimed to perform a comprehensive analysis of the integrative DNA methylation gene expression landscape of breast cancer. In doing so, we wanted to address the following key questions. First, how does the molecular taxonomy of breast cancer look like when viewed from the perspective of DNA methylation-driven gene expression changes? Second, are their specific signalling pathways which are epigenetically deregulated and how does this epigenetic deregulation vary across breast cancer subtypes? Third, do putative epigenetic driver events targeting specific gene modules or signalling pathways exhibit a mutually exclusive pattern of deregulation within individual tumours, similar to what is observed at the copy number level ? In ordeHAND2 was causally implicated in endometrial carcinogenesis. In fact, it was shown how DNA methylation-induced suppression of HAND2 inactivates the progesterone receptor signalling pathway, which is the key tumour suppressor pathway in this type of cancer. Integration of DNA methylation data with a human interactome was also shown to be a fruitful approach leading to novel insights in other contexts. For instance, DNA methylation changes occurring in ageing do not happen randomly in the context of protein interaction networks [The feasibility of the FEM algorithm to uncover driver events in cancer was demonstrated by us previously , 12. Indnetworks , but tennetworks . Given tGiven that the process of cellular differentiation is dictated by the specific activation/deactivation of signalling pathways and that this is largely controlled by epigenetics, we reasoned that putative epigenetic drivers of cancer could be identified by integrating DNA methylation with gene expression data in the context of a functional gene network, which incorporates pathway-level information, such as that provided by a comprehensive high-quality human protein interactome . In ordeP values were always smaller than 0.5, indicating that their non-significance could be due to other factors such as the unmatched nature of the independent DNA methylation (DNAm) and messenger RNA (mRNA) data. Indeed, scatterplots of t-statistics of differential methylation  between the TCGA and validation sets revealed strong agreement at both DNAm and mRNA levels for all FEM modules  and with 5 exhibiting simultaneous hypermethylation and underexpression  . The topion Fig. . The FSTion Fig. .Table 2GiCluster R package [k = 2, i.e. there was no statistical evidence for more than two clusters. We observed that the two main clusters were strongly correlated with luminal subtype status .Next, we decided to study the prognostic significance of the identified FEM modules. For the two predicted clusters Fig. , we consB-MYB activation, p53 mutation, PIK3CA activation and stemness, supporting their association with a highly proliferative and poor-outcome ER+ breast cancer  .To further assess the significance of the FEM modules, we clustered their FEM deviation scores in a clustering analysis which also included activity estimates of various signalling pathways and proliferation indices, as recently estimated by Gatza et al. . All ninOne of the most interesting insights to have emerged from TCGA studies is that functional cancer driver events targeting a common signalling pathway often occur in a mutually exclusive fashion within the same tumour . We askeTo investigate whether patterns are coordinated or mutually exclusive, we constructed for each FEM module a binary representation matrix over the significantly differentially expressed and differentially methylated genes in the FEM and across all ER+ cancers . Each biFinally, to show that there is indeed a difference in the level of mutually exclusive patterns exhibited by DNA methylation and copy number, we compared the average path length distance of top-ranked differentially methylated genes to top-ranked amplified/deleted genes, using the TCGA data for which both DNA methylation and Affy SNP data is available. Focusing on the same number of top-ranked genes by differential methylation or copy number change, we observed that the average shortest path length distance between top-ranked differentially methylated genes was indeed smaller than for the correspondingly top-ranked amplified/deleted genes  DNA methylation and gene expression set. Remarkably, integrative clustering of DNA methylation and matched gene expression over the significant genes within these FEMs only revealed two main clusters. The same result was obtained after clustering over all genes exhibiting anti-correlative patterns between differential DNAm and mRNA expression, indicating that the relative homogeneity of the epigenetic landscape of ER+ breast cancer was not due to us using the FEM algorithm. Importantly, the two main clusters correlated very strongly with luminal-A/B subtype status. Indeed, our work clearly shows that whereas the luminal-B and luminal-A subtypes do not differ in terms of the specific FEMs , they do however differ significantly in terms of the absolute levels of DNA methylation and gene expression deviation from the normal reference. Specifically, luminal-B cancers invariably showed higher levels of promoter DNA methylation and correspondingly lower levels of gene expression, than luminal-A cancers. Our result thus extends a previous observation by a recent study reporting higher levels of promoter DNAm in luminal-B breast cancer : our stuThe relative homogeneity of the integrative epigenomic-transcriptomic landscape of ER+ breast cancer contrasts with the corresponding integrative landscape at the copy number level. Indeed, the recent METABRIC study, which performed an integrative clustering of copy number and transcriptomic changes for genes exhibiting significant correlations, revealed considerable heterogeneity with at least 10 integrative subtypes across all breast cancers. An implication of this, as we have seen, is that unsupervised clustering of DNA methylation-induced gene expression changes appears to discriminate luminal-A from luminal-B tumours with a much higher accuracy than what appears possible based on copy number data. This is interesting because it suggests that DNA methylation changes explains much more of the homogeneity in the transcriptomic differences observed between luminal-A and luminal-B breast cancers, whereas copy number changes may explain more of the heterogeneous differences. Thus, from a purely epigenetic perspective, our data suggests that ER+ breast cancer consists of only two main epigenetic subtypes, which differ mainly in the level of epigenetic deregulation, and not in the specific deregulated genes or signalling pathways.CAV1 and which was strongly enriched for many members of the WNT signalling pathway. Many members of this module, including WIF1, WNT1, SFRP1, FZD9, and FZD8 were hypermethylated and underexpressed in both luminal-A and luminal-B breast cancers but exhibiting larger deviations in DNAm and mRNA expression in the luminal-B subtype. One potential interpretation of this is that luminal-A and luminal-B breast cancers are epigenetically similar entities but with the higher proliferation rate of luminal-B cancers causing increased deviations from the normal epigenetic landscape. The moderate significant correlation of the FEM module score with the proliferation marker PCNA partially supports this. On the other hand, the WNT signalling pathway has been implicated as a key pathway underlying breast cancer development and progression [WIF1 [SFRP1 [Illustrating this surprising homogeneity is a FEM module, centred around gression –27. Indegression –23. In tgression , 28. Ouron [WIF1  and SFRP1 [SFRP1 . Hyperme1 [SFRP1 . Thus, eFSTL1 appears to play a tumour suppressor role in two other hormone-related women cancers  [Besides WNT signalling, another important FEM mapped to the TGF-β/BMP signalling pathways. Among the 18 genes which mapped to the WNT signalling pathway, 7 exhibited simultaneous hypermethylation and underexpression. Among these, FST and FSTL1 are known to bind activin members of the TGF-beta superfamily and function to control cellular proliferation . Hyperme cancer) .FSTL1 module, whereas TGF-β family members TGFB1 and TGFB3 were significantly overexpressed. Interestingly, a recent study showed that BMP7 significantly inhibited the TGF-β1-activated epithelial-mesenchymal transition (EMT)-related genes in breast cancer cells, resulting in a significant reduction in TGF-β1-triggered cell growth and cell metastasis, suggesting that the BMP7 signalling axis could be a promising pathway for therapeutic intervention in breast cancer [Many bone morphogenetic proteins, including BMP2, BMP6, and BMP7, were also simultaneously hypermethylated and underexpressed in the same FEM t cancer . BMP6 hat cancer –36, whict cancer , 36. Epit cancer .CCL11 FEM module was significantly enriched for chemokines and chemokine receptors, which were mostly hypomethylated and overexpressed. Chemokines have been reported to influence the metastatic potential of breast cancer [LEP FEM module contained several G protein-coupled receptors (GPCRs), which could play a role in mediating the effects of chemokines. The MME FEM module was significantly enriched for genes in endothelin pathways. Previous research has found that endothelin signalling plays a crucial role in cell differentiation, proliferation and migration processes [EDN2 to be hypomethylated and overexpressed, whereas EDN3 was hypermethylated and underexpressed. Finally, the PROC FEM module was significantly enriched for genes involved in the fibrin clot clotting cascade. Coagulation and fibrinolysis are known to serve as haemostatic elements that can facilitate the metastatic potential of breast cancer [Among the other inferred FEM modules, it is worth highlighting the following. The t cancer . Interesrocesses . Consistt cancer . In summAnother interesting observation of our study is the absence of a mutually exclusive pattern of DNA methylation changes within FEM modules. Whereas copy number changes within signalling pathways have been shown to occur in a mutually exclusive fashion , the samThe recent observation that DNAm changes may mediate functional changes primarily by reorganizing the patterns of transcription factor binding  also raiIn summary, our work demonstrates that a key component of the integrative epigenomic-transcriptomic landscape of ER+ breast cancer is surprisingly homogeneous. Specifically, we have shown that from the perspective of proximal promoter DNA methylation changes that drive gene expression alterations, luminal-B and luminal-A are characterized by the same epigenetically deregulated hotspots but with luminal-B tumours exhibiting more aggravated levels of DNA methylation and gene expression change from the normal state. Our data point towards WNT and BMP signalling as key epigenetically deregulated pathways underlying both luminal subtypes, specially luminal-B breast cancers.limma package [Illumina HiSeq 2000 RNA Sequencing Version2 Level 3 data for breast invasive carcinoma samples was acquired from the TCGA data portal. Samples were filtered to include only 724 ER-positive breast cancers and 111 adjacent normal breast tissue samples. RNA-Seq data included gene-normalized values for 20,531 genes. Normalized values equal to 0 were substituted by the minimum non-zero positive value. Values were then log2-transformed in order to regularize the dynamic range. Inter-array normalization using the quantile method was then performed using the  package .Expression data for 13 normal breast tissue samples and 110 ER-positive breast cancer samples encompassing 13,262 genes was obtained from Yu et al. .n = 1992 samples) were acquired from the European Genome-phenome Archive at the European Bioinformatics Institute. We used only the 774 ER-positive breast cancer samples from the METABRIC discovery data set, given that the validation set exhibited significantly lower cellularity. The expression data matrix included 24,924 genes.Normalized Illumina HT-29 v3 expression data of the METABRIC project , and all patients gave written informed consent. Data are available on GEO under accession number GSE69914. Data underwent a quality control procedure as implemented by us previously [Illumina 450K methylation data from  were dowThe Functional Epigenetic Module (FEM) algorithm is a functional supervised algorithm which is aimed at identifying functionally related genes which exhibit both differential DNA methylation and differential expression in relation to some phenotype of interest , 12. Theg, we thus obtain two statistics of association, one at the DNAm level (tgD) and another at the mRNA level (tgR). The statistics are inferred using an empirical Bayesian method [Because the statistics of differential DNA methylation are constructed at the gene level, there are many possibilities on how to do this in the case of the Illumina 450K beadarrays as there are many probes mapping to a given gene. As shown in Jiao et al. , probes n method .g and h an average integrated statistic according to the following rule:H(x) defines the Heaviside function.We then assign to any given edge in the network, specified by genes Inference of hotspots/modules on the maximally connected subnetwork of the full PPI then proceeds using a spin-glass algorithm, as described in detail in . Very brlimma package. Statistics of one data type (mRNA) were then scaled to ensure equal variance of statistics of both data types, as described previously [We performed a system-level integrative analysis of DNA methylation and gene expression data using our previously published FEM algorithm , 12, in eviously , 12.P values in the validation set were obtained for each FEM by counting the number of permutations where the modularity score (average edge weights of the FEM module) is larger than the observed one.As validation set, we used the Illumina 450K methylation data (Germany) and Affymetrix mRNA expression data from Yu et al. . These iThe second validation compares the differential methylation and expression statistics between discovery and validation sets for all genes within a FEM module. A scatterplot of these statistics should yield a significant positive correlation. This validation tests for consistency in the directional patterns of change but does not assess the hotspot nature of the FEM modules in the validation set.iCluster R package [k  and for different values of the sparsity parameter λ . The optimal clustering solution was obtained at POD = 0.0086 corresponding to k = 2 and λ = 0.1.Matched DNAm and mRNA data for 463 ER+ breast cancers from the TCGA and for all FEM genes which were either significantly differentially methylated or differentially expressed, or both (241 genes), were used as input to a joint latent variable framework for integrative clustering using the  package . The optg denote such a gene and s denote a tumour sample. We then compute Z-statistics asD and R stand for DNA methylation and mRNA expression, respectively, and where μ and σ denote the mean and standard deviation, respectively, as estimated over the normal samples. A complication arises from the fact that the two Z-statistics are not directly comparable due to intrinsic factors related to each data type. Thus, when constructing an integrated score, in order to avoid biasing the score to one data type, we need to introduce a scaling factor α, which will be data-type specific. To estimate this scaling factor, we compute the standard deviation of DNAm and mRNA Z-statistics over all genes (i.e. not just FEM genes) and samples, quantities we denote by σZ(D) and σZ(R). We then define the scaling factor as the ratio of the standard deviations obtained from the DNA methylation and mRNA expression Z-statistics,In order to be able to quantify the deviation in DNAm and mRNA expression of FEM module genes from the values in the normal reference tissue, we devised for each module a sample-specific FEM score. Construction of this score requires samples with both DNAm and mRNA expression data, so this score computation was restricted to the matched 82 normal samples and 463 ER-positive breast cancers. Deviations were calculated using the mean and standard deviation of each gene across normal samples. Given a FEM module, we only use those genes which gave rise to the significance of the module in the first place, i.e. we include only genes that are significant at both differential DNAm and differential mRNA levels, and which also exhibit an anti-correlation (i.e. hypermethylated and underexpressed or hypomethylated and overexpressed in ER+ breast cancer). Thus, let s is calculated asm is the number of genes in the FEM module passing the requirements mentioned above.Finally, the FEM score of the FEM module in sample The iCluster algorithm over all significantly anti-correlated FEM genes  in ER+ breast cancer resulted in only two clusters. Centroids at the DNAm and mRNA level were constructed by averaging the corresponding values of the samples in each cluster. For each gene, its DNAm and mRNA profile was centred before performing the averaging per cluster. With the centroids thus defined, i.e. two centroids for each of the DNAm and mRNA data, independent ER+ samples can then be classified using a nearest-centroid classification rule. Specifically, for an independent ER+ mRNA or DNAm data set, each of the 99 genes was first centred to mean zero. Samples were then classified using a nearest-centroid criterion, with a Spearman rank correlation coefficient defining the distance metric. For data sets with matched DNAm and mRNA data (i.e. Fleischer set), we thus obtained two independent classifications, one from the DNAm and another from the mRNA data.Z-statistics for each gene in each tumour sample, by comparing the DNAm and mRNA level in the sample to the normal tissue samples. Specifically, we estimated the mean and variance for each gene across the normal samples and then computed the Z-statistic deviation score. Assuming a Gaussian distribution for the normal samples, we thus obtained two P values (one for DNAm and another for mRNA expression) for each gene in each tumour sample. We then assigned a value of 1 to the corresponding matrix entry if both P values were less than 0.1 and if there is an anti-correlative pattern (i.e. hypermethylated and underexpressed or hypomethylated and overexpressed); otherwise, we assigned a value of 0. The relaxed threshold was chosen because (i) the FEM algorithm infers modules without imposing specific thresholds on the statistics of differential expression and methylation and (ii) this allowed us to assess the patterns in more genes within FEMs. For each FEM, we next estimated the average Manhattan distance (MHD) between the rows (significant genes) of this binary matrix. This distance was then compared to the distance obtained by independently permutating the samples within each row, using a total of 1000 permutations to derive a null distribution. An overall P value was then obtained by counting the number of permutations with an average MHD more extreme than the observed one.To determine whether DNAm-associated mRNA expression changes of FEM genes within FEM modules occur in a coordinated or mutually exclusive manner within individual tumour samples, we first constructed a binary representation matrix for each FEM module using only genes that are differentially methylated and differentially expressed and which exhibit an anti-correlative pattern. The binary matrix was obtained by calculating To objectively assess the level of coordination or mutual exclusivity of DNAm changes, we decided to compare these changes to those at the copy number level. Thus, we compared the distribution of the shortest paths of top-ranked differentially methylated genes to that of the top-ranked amplified/deleted genes, estimated in the context of the same PPI network used in the FEM algorithm. However, genes were ranked according to differential DNA methylation between normal and ER+ breast cancer without using FEM. We selected the top 100 and top 200 ranked differentially methylated genes, as well as the top-ranked 100 and 200 genes according to copy number change using the matched TCGA breast cancer data. The shortest paths were then estimated between every pair of genes in the top-ranked list, and both the distribution and average compared between DNAm and copy number.


Animal and human clinical studies have demonstrated the ability of dietary food proteins to modulate endogenous lipid levels during abnormal lipid metabolism (dyslipidemia). Considering the susceptibility of proteins to gastric proteolytic activities, the hypolipidemic functions of proteins are possibly due, in part, to their peptide fragments. Food-derived peptides may directly modulate abnormal lipid metabolism in cell cultures and animal models of dyslipidemia. The peptides are thought to act by perturbing intestinal absorption of dietary cholesterol and enterohepatic bile acid circulation, and by inhibiting lipogenic enzymatic activities and gene expression in hepatocytes and adipocytes. Recent evidence indicates that the hypolipidemic activities of some peptides are due to activation of hepatic lipogenic transcription factors. However, detailed molecular mechanisms and structural requirements of peptides for these activities are yet to be elucidated. As hypolipidemic peptides can be released during enzymatic food processing, future studies can explore the prospects of combating metabolic syndrome and associated complications using peptide-rich functional food and nutraceutical products. Human lifestyle is gradually shifting towards an increase in the consumption of high energy diet (over-nutrition) and decrease in physical activity (sedentary lifestyle) . This shde novo lipogenesis by inhibiting hepatic fatty acid synthase mRNA expression and enzymatic activity, with concomitant decrease observed in the levels of hepatic lipids  and liver weight in OLEFT rats with non-alcoholic fatty liver disease [DGA1 mRNA compared to treatments with proteins or amino acid mixture of similar amino acid composition [The consumption of food proteins has been found to modulate endogenous lipid profiles during dyslipidemia . Soy β-c disease . Hypolip disease , chicken disease , soy pro disease  and ferm disease  in diffe disease . Althoug disease . There i disease . Althoug disease , indicat disease . Furtherposition . These fEscherichia coli, and the resulting peptides demonstrated lipid-lowering activities when fed to rats [The physiological processes that constitute metabolic dyslipidemia  have been modulated by dietary food protein hydrolysates and peptides in cellular systems and animal models. The mechanisms of hypolipidemic activity of food protein hydrolysates and peptides have been discussed . The pep to rats . This wo to rats ,21,22,23The intestine is thought to be a major site of action of hypolipidemic peptides. Dietary cholesterol is incorporated into micelles with PL and bile salts prior to intestinal uptake . Food pri.e., resistant to gastric proteolysis) was found to be important for gastric hypolipidemic activity [Endogenous cholesterol is metabolized in hepatocytes to fat-soluble bile acids, which are secreted in the duodenum as water-soluble bile salts, and later reabsorbed through enterohepatic circulation . This paactivity . Bulky aactivity .in vitro and by decreasing lipid accumulation (pre-adipocyte differentiation) in human subcutaneous adipose tissue culture [aP2), an adipogenic marker gene, and decrease the expression of preadipocyte factor-1 mRNA (Pref-1) with concomitant increase in adiponectin secretion in cultured adipocytes [in vivo to ascertain if they are responsible for the physiological effects of the dietary proteins. The direct interaction of peptides with adipocytes is plausible considering earlier reports of anti-obesity effects (decreased body/adipose tissue masses) and molecular events  triggered by dietary hydrolysates of salmon protein and casein (compared to intact casein) in white adipose tissue of Wistar Hannover GALAS rats and C57BL/6J mice, respectively [Peptides are known to exert hypolipidemic effects in adipose tissues by inhibiting fatty acid synthase (FAS), pre-adipocyte differentiation and elevating expression of PPARγ co-activator-1α, uncoupling protein-1, carnitine palmitoyltransferase-1/2, and medium chain and long chain acyl-CoA dehydrogenases, which promote fatty acid oxidation; see review . The α a culture . Peptide culture . Similaripocytes . The effectively ,34.in vitro inhibition of the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR), which catalyzes the rate-limiting step of endogenous cholesterol biosynthesis [Srebf1 gene, respectively, in diet-induced hyperlipidemic animals [FATP4, ACS, CPT1 and ACOX in hepatocytes [in vitro in the study) towards inhibition of de novo lipogenesis.Hepatocytes play a central role in lipid metabolism, and food protein hydrolysates and peptides have demonstrated several effects on hepatic lipogenic genes and proteins resulting in the modulation of endogenous lipid profiles. As recently reviewed , food peynthesis ,35. This animals ,36. More animals . Furtheratocytes . The PPAatocytes . The lipatocytes . Despitede novo lipogenesis and activation of lipid uptake and degradation in hepatocytes. Moreover, it is also possible that hypolipidemic peptides within the hydrolysates can interact with one another to induce pronounced hypolipidemic effects. Although hydrophobicity is a desirable peptide functionality for some hypolipidemic activities , a hydrophilic peptide fraction  was found to retain the activity of its parent soy protein hydrolysate by decreasing TG synthesis and apoB100 secretion in cultured hepatocytes [It is important to note that the effects on transcription factors that control lipid metabolism were mostly observed with food peptide mixtures (protein hydrolysates or fractions), which makes it challenging to identify particular bioactive principles or decipher the structural basis of activity of the peptides. One possible mechanism involved in the modulatory effects could be due to the direct interaction of peptides with the transcription factors of lipid metabolism. Recently, binding modeling assay showed that dipeptide WE bound PPARα through H-bonding with its Glu286, Asn219 or Met220 residues leading to conformational changes that increased the proximity of a coactivator peptide . In addiatocytes . Intestiin vitro, cellular and animal studies. Therefore, human clinical studies are needed to validate bioactivity in order to make conclusive statements on the application of hypolipidemic food-derived peptides.Based on current literature, it is expected that the hypolipidemic effects of dietary food proteins could be due to the peptides released after enzymatic hydrolysis in the gastrointestinal tract. These effects could be centrally mediated by direct interaction of the peptides with transcription factors and enzymatic activity related to endogenous lipid metabolism especially in hepatocytes and adipocytes. Moreover, physical disruption of dietary cholesterol absorption and enterohepatic bile acid reabsorption by the peptides can play key roles in endogenous cholesterol homeostasis. The hypolipidemic peptides hold promise for use in suppression of hepatic lipid synthesis and accumulation especially in health conditions such as hepatic steatosis . Most evidence presented to date is based on


Probiotics are live microorganisms that confer nutrition- and health-promoting benefits if consumed in adequate amounts. Concomitant with the demand for natural approaches to maintaining health is an increase in inclusion of probiotics in food and health products. Since probiotic bacteria act as reservoir for antibiotic resistant determinants, the transfer of these genes to pathogens sharing the same intestinal habitat is thus conceivable considering the fact that dietary supplements contain high amounts of often heterogeneous populations of probiotics. Such events can confer pathogens protection against commonly-used drugs. Despite numerous reports of antibiotic resistant probiotics in food and biological sources, the antibiogram of probiotics from dietary supplements remained elusive.Here, we screened five commercially available dietary supplements for resistance towards antibiotics of different classes. Probiotics of all batches of products were resistant towards vancomycin while batch-dependent resistance towards streptomycin, aztreonam, gentamycin and/or ciprofloxacin antibiotics was detected for probiotics of brands Bi and Bn, Bg, and L. Isolates of brand Cn was also resistant towards gentamycin, streptomycin and ciprofloxacin antibiotics. Additionally, we also report a discrepancy between the enumerated viable bacteria amounts and the claims of the manufacturers.This short report has highlighted the present of antibiotic resistance in probiotic bacteria from dietary supplements and therefore serves as a platform for further screenings and for in-depth characterization of the resistant determinants and the molecular machinery that confers the resistance.The online version of this article (doi:10.1186/s12937-015-0084-2) contains supplementary material, which is available to authorized users. Probiotics are live microorganisms usually bacteria or yeasts, that confer health-promoting benefits to the host if consumed in adequate amounts. These benefits range from improvement of intestinal health  and immuin vitro and/or in vivo among Lactobacilli and from Lactobacilli to pathogens [Widespread use of probiotic bacteria in conjunction and in close association with antibiotic use or rather misuse, can over time establish a reservoir of antibiotic resistant genes in probiotic bacteria . While iathogens  and viceathogens . These rWhile the detection of antibiotic resistance in probiotic strains from food and biological sources have intensified, such reports from dietary supplements have remained somewhat elusive. This is surprising considering the fact that probiotic dietary supplements contain high amount and often a heterogeneous population of probiotic bacteria both of which, are conditions that encourage the trafficking of resistant genes. Therefore, we hypothesize that antibiotic resistant probiotics may be present in dietary supplements.Lactobacilli. We note that Lactobacilli constitute the majority of the probiotic bacteria population in the dietary supplements (Table 6 CFU) were cultured overnight in MRS broth for enrichment of probiotic bacteria after which the overnight culture was adjusted to uniform concentrations of 7×106 CFU/mL of bacteria by spectroscopy (OD690nm) prior to antibiotic susceptibility tests using commercial antibiotic discs and according to the manufacturer’s instructions -aph(2”), ant(6), and aph(3’)-IIIa respectively [blaZ genes which have also been previously found in Lactobacilli [Lactobacilli. Although probiotic bacteria (predominantly Lactobacilli) of all products were able to grow in the presence of vancomycin -aph”, ant(6)cin Fig. , this is residue . Notablyobacilli . Althougobacilli  and biolobacilli  are wide’-aph”, aobacilli , 16. Furobacilli  constituL. reuteri DSM 17938 whereby two resistant plasmids were successfully removed from the parent L. reuteri (ATCC 55730) [In summary, this is a pilot study that reports the present of antibiotic resistance in probiotic bacteria in dietary supplements thus expanding the screening of antibiotic resistance to include this largely unexplored but increasingly popular group of functional foods. This study also serves as a platform for further screenings and in-depth characterization of the resistant determinants and the molecular machinery that confers the resistance. In particular, resistant probiotics can be isolated and identified by a combination of biochemical tests and 16S rRNA gene sequencing analysis and the antibiotic resistant genes characterized using degenerate primers and/or a motif-based identification approach . ManufacC 55730)  while no


Drosophila and some other insects such as aphids. Identification of the DEAD-box protein Vasa (Vas) as a conserved germline marker in flies and aphids suggests that they share common components for assembling the germ plasm. However, to which extent the assembly order is conserved and the correlation between functions and sequences of Vas remain unclear. Ectopic expression of the pea aphid Vas (ApVas1) in Drosophila did not drive its localisation to the germ plasm, but ApVas1 with a replaced C-terminal domain (HELICc) of Drosophila Vas (DmVas) became germ-plasm restricted. We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation. Similarly, HELICc of the grasshopper Vas could be recruited to the germ plasm in Drosophila. Nonetheless, germ-plasm localisation was not seen in the Drosophila oocytes expressing HELICcs of Vas orthologues from aphids, crickets, and mice. We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species. This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.Formation of the germ plasm drives germline specification in  It was initially identified in the fruit fly Drosophila melanogaster in late 1980s23In Metazoans, germ cell specification is critical for establishing the germline lineage. For acquiring germline identity, cells can recruit germline determinants from maternal germ plasm, typically preformed in an uncellularised egg chamber, or receive signals from neighbouring cells to induce the expression of germline genesDrosophila oogenesis, maternal Vas is localised to the germ (pole) plasm in the posterior end of developing oocytes. The localisation of Vas is found to be dependent on the predeposition of Oskar (Osk) protein in the posterior end of the oocyte after stage 977vas hypomorphic mutants fail to form pole cells and abdominal segmentsvas-null females exhibit aberrant ovarioles with atrophied germaria, fewer egg chambers, or mispatterned oocytesosk and nanos in the germ plasm101112gurken translation in the oocyte15gurken and mei-P26 for anteroposterior/dorsoventral patterning in oocytesDuring Drosophila Vas, abbreviated as DmVas to distinguish it from other Vas proteins mentioned in this study, is composed of 661 amino acids. Similar to other DEAD-box helicases, DmVas contains conserved helicase signature motifs that compose the ‘helicase core’, but sequences flanking this region are less conserved  simulations further demonstrated that these residues played a decisive role in maintaining the integrity of the HELICc structure. Sequences located N-terminal to the helicase core, by contrast, were found essential to pole cell formation and posterior development. Altogether, our results unveil critical connections between sequences and functions of DmVas during oogenesis and early embryogenesis.At first, we expressed ApVas1, a pea aphid orthologue of DmVas, in Drosophila and the pea aphid, respectively, displays highly conserved features in their helicase core domains DEXDc and HELICcDrosophila female germline by using a maternal tubulin 67c promoter82829Alignment of DmVas and ApVas1, which are germline markers in Drosophila, the germ plasm localisation of Vas is dependent on the pre-deposition of Osk in the posterior pole of the oocyte731Drosophila oocyte, we performed domain-swapping analyses replacing various lengths of N-terminal ApVas1 sequences with N-terminal DmVas sequences (1–320) was still not posteriorly localised to the oocyte , surprisingly, was localised to the germ plasm of Drosophila oocyte  and Osk did not produce positive signals . Moreover, no positive signals were detected in cells co-expressing Vls and various DmVas constructs : a polypeptide composed of DmVas residues 446–661 failed to interact with OskDrosophila, we preformed co-immunoprecipitation experiments. Our results showed that both DmVas460–621/HELICc and DmVas460–661 could be co-precipitated with Osk in the ovary extract  of bcd mRNA brings the transcript to the anterior cortex29460–621/HELICc with Osk 7vas mutant embryos  further hindered pole cell formation; practically, pole cells were not or barely formed , was not localised to the germ plasm of oocyte (Drosophila. Because a pea aphid homolog of Drosophila osk has not yet been identified41osk-independent machinery for assembling the germ plasm and (2) ApVas1 lacked the sequences recognised by Osk or any other factors associated with Osk. To test the aforementioned hypotheses, we performed domain swapping between ApVas1 and DmVas and identified the sequences responsible for the germ plasm localisation of DmVas. We found that the previously identified Osk-interacting motif (OIM)in vivo. Without OIM, for example, the HELICc domain alone was still localised to the germ plasm in oocytes, though localisation was postponed from stage 9 to 10 of oogenesis  presented crystal structure of the N-terminal LOTUS domain of Osk and, via GST-pull down assay, demonstrated that it could interact with the helicase core of DmVas, a polypeptide region containing both DEXDc and HELICcDrosophila  identified four EMS-induced mutations in HELICc that could disrupt germ plasm localisation of VasTo understand how Gln527 participated in the Osk–Vas interaction in redicted ’. Gln527f site 2 ’. The farm plasm ”’ suggesosk mutant background46In addition to germ plasm localisation, Gln527 and the residues 460–469 were involved in the nuage localisation of DmVas because the Q527A substitution and the truncation of residues 460–469 disabled the nuage localisation of HELICc ”. Howevevas mutants, deletion of the first 157 amino acids led to a rescue of all abdominal segments but only recovered part of the pole cells  were PCR amplified from pPvas:egfp-vasvas were RT-PCR amplified from ovarian lysates. The CDS encoding the HELICc of mvh was obtained through PCR amplification of the testis cDNA (a gift from Dr. Yung-Hao Ching). The QuikChange Lightning Site-Directed Mutagenesis kit (Agilent Technologies) was used to generate amino acid substitutions in the HELICc of DmVas.Oregon R was used as the wild-type strain. The The ovary and embryo immunostaining protocols used was previously described50osk CDS was PCR amplified from the EST clone LD24944. The vls CDS was obtained through RT-PCR amplification of the RNA extracted from the Drosophila ovary.The yeast strain Y187 was used. Yeast culturing and the colony lift assay were performed according to the Clontech yeast two-hybrid handbook. Yeast transformation was performed using the Frozen-EZ Yeast Transformation II kit (Zymo Research). The full-length DmVas CDS (1–661) and other truncated fragments  were PCR amplified and subcloned into pAS2-1 (Clontech) for generating GAL4 DNA-binding domain fusion proteins. The full-length Osk and Vls CDSs were PCR amplified and subcloned into pACT2 (Clontech) for generating GAL4 activation domain fusion proteins. The How to cite this article: Wang, S.-C. et al. Germ plasm localisation of the HELICc of Vasa in Drosophila: analysis of domain sufficiency and amino acids critical for localisation. Sci. Rep.5, 14703; doi: 10.1038/srep14703 (2015).