DNA methylation at birth and lateral ventricular volume in childhood: a neuroimaging epigenetics study.

BACKGROUND: Lateral ventricular volume (LVV) enlargement has been repeatedly linked to schizophrenia; yet, what biological factors shape LVV during early development remain unclear. DNA methylation (DNAm), an essential process for neurodevelopment that is altered in schizophrenia, is a key molecular system of interest. METHODS: In this study, we conducted the first epigenome-wide association study of neonatal DNAm in cord blood with LVV in childhood (measured using T1-weighted brain scans at 10 years), based on data from a large population-based birth cohort, the Generation R Study (N = 840). Employing both probe-level and methylation profile score (MPS) approaches, we further examined whether epigenetic modifications identified at birth in cord blood are: (a) also observed cross-sectionally in childhood using peripheral blood DNAm at age of 10 years (Generation R, N = 370) and (b) prospectively associated with LVV measured in young adulthood in an all-male sample from the Avon Longitudinal Study of Parents and Children (ALSPAC, N = 114). RESULTS: At birth, DNAm levels at four CpGs (annotated to potassium channel tetramerization domain containing 3, KCTD3; SHH signaling and ciliogenesis regulator, SDCCAG8; glutaredoxin, GLRX) prospectively associated with childhood LVV after genome-wide correction; these genes have been implicated in brain development and psychiatric traits including schizophrenia. An MPS capturing a broader epigenetic profile of LVV - but not individual top hits - showed significant cross-sectional associations with LVV in childhood in Generation R and prospectively associated with LVV in early adulthood within ALSPAC. CONCLUSIONS: This study finds suggestive evidence that DNAm at birth prospectively associates with LVV at different life stages, albeit with small effect sizes. The prediction of MPS on LVV in a childhood sample and an independent male adult sample further underscores the stability and reproducibility of DNAm as a potential marker for LVV. Future studies with larger samples and comparable time points across development are needed to further elucidate how DNAm associates with this clinically relevant brain structure and risk for neuropsychiatric disorders, and what factors explain the identified DNAm profile of LVV at birth.

Maternal cognitive functioning and psychopathology predict quality of parent-child relationship in the context of substance use disorder: A 15-month longitudinal study.

This longitudinal study aimed to investigate the role of maternal cognitive functioning and psychopathology in parent-child relationship quality during residential treatment for mothers with Substance Use Disorder (SUD), in order to identify factors that may enhance or limit intervention effects.We assessed cognitive functioning (Esame Neuropsicologico Breve-2 [ENB-2]) and psychopathology (Symptom Checklist-90 Revised [SCL-90-R]) in 60 mothers diagnosed with SUD (Mage = 30.13 yrs; SD = 6.79) at treatment admission. Parent-child relationship quality was measured during free-play interactions using the Emotional Availability Scales every three months from admission (Child Mage = 17.17m; SD = 23.60) to the 15th month of the residential treatment.A main effect of maternal psychopathology and an interaction effect of time and cognitive functioning were found. More maternal psychopathology predicted lower mother-child relationship quality. Mothers with higher cognitive functioning presented a better treatment trajectory, with an increase in mother-child relationship quality, whereas mothers with lower cognitive functioning showed a decrease in relationship quality after initial improvement.These findings suggest that maternal psychopathology and cognitive functioning may influence the treatment of parent-child relationships in the context of SUD, although causality is not yet established. Implications for assessment and intervention are discussed.

The quality of qualitative research.

In this contribution, we discuss criteria for the quality of qualitative research. We consider reliability and validity as specifications of the comprehensive requirement for 'intersubjective replicability', with which qualitative research should comply. In the data collection phase, 'argumentative' reliability generally must suffice; in the data analysis phase, attention must also be given to 'technical' reliability. Validation of qualitative research has to take place via three approaches: 'communicative', 'critical' or 'empirical' validation. This clarifies the relative validity or 'authenticity' of qualitative research.

Transitioning from childhood into adolescence: A comprehensive longitudinal behavioral and neuroimaging study on prosocial behavior and social inclusion.

Acting prosocially and feeling socially included are important factors for developing social relations. However, little is known about the development of neural trajectories of prosocial behavior and social inclusion in the transition from middle childhood to early adolescence. In this pre-registered study, we investigated the development of prosocial behavior, social inclusion, and their neural mechanisms in a three-wave longitudinal design (ages 7-13 years; NT1 = 512; NT2 = 456; NT3 = 336). We used the Prosocial Cyberball Game, a ball tossing game in which one player is excluded, to measure prosocial compensating behavior. Prosocial compensating behavior showed a linear developmental increase, similar to parent-reported prosocial behavior, whereas parent-reported empathy showed a quadratic trajectory with highest levels in late childhood. On a neural level we found a peak in ventral striatum activity during prosocial compensating behavior. Neural activity during social inclusion showed quadratic age effects in anterior cingulate cortex, insula, striatum, and precuneus, and a linear increase in temporo-parietal junction. Finally, changes in prosocial compensating behavior were negatively associated with changes in ventral striatum and mPFC activity during social inclusion, indicating an important co-occurrence between development in brain and social behavior. Together these findings shed a light on the mechanisms underlying social development from childhood into adolescence.

Epigenome-wide change and variation in DNA methylation in childhood: trajectories from birth to late adolescence.

DNA methylation (DNAm) is known to play a pivotal role in childhood health and development, but a comprehensive characterization of genome-wide DNAm trajectories across this age period is currently lacking. We have therefore performed a series of epigenome-wide association studies in 5019 blood samples collected at multiple time-points from birth to late adolescence from 2348 participants of two large independent cohorts. DNAm profiles of autosomal CpG sites (CpGs) were generated using the Illumina Infinium HumanMethylation450 BeadChip. Change over time was widespread, observed at over one-half (53%) of CpGs. In most cases, DNAm was decreasing (36% of CpGs). Inter-individual variation in linear trajectories was similarly widespread (27% of CpGs). Evidence for non-linear change and inter-individual variation in non-linear trajectories was somewhat less common (11 and 8% of CpGs, respectively). Very little inter-individual variation in change was explained by sex differences (0.4% of CpGs) even though sex-specific DNAm was observed at 5% of CpGs. DNAm trajectories were distributed non-randomly across the genome. For example, CpGs with decreasing DNAm were enriched in gene bodies and enhancers and were annotated to genes enriched in immune-developmental functions. In contrast, CpGs with increasing DNAm were enriched in promoter regions and annotated to genes enriched in neurodevelopmental functions. These findings depict a methylome undergoing widespread and often non-linear change throughout childhood. They support a developmental role for DNA methylation that extends beyond birth into late adolescence and has implications for understanding life-long health and disease. DNAm trajectories can be visualized at http://epidelta.mrcieu.ac.uk.

Fathers' sensitive parenting enhanced by prenatal video-feedback: a randomized controlled trial using ultrasound imaging.

BACKGROUND: The aim of this study was to evaluate an interaction-based prenatal parenting intervention program aimed at promoting parental sensitivity and involvement in expectant fathers using ultrasound images: Prenatal Video-Feedback Intervention to Promote Positive Parenting (VIPP-PRE). METHODS: In this randomized controlled trial, 73 first-time, healthy expectant fathers were enrolled. Participants were randomly assigned to the VIPP-PRE intervention (n = 39) or a dummy intervention (n = 34). Parental sensitivity was coded from video-recorded 10-min interactions with an infant simulator at a prenatal pretest and with fathers' own infant at a postnatal posttest. Prenatal and postnatal involvement was assessed via an application on participants' smartphones. RESULTS: Fathers receiving VIPP-PRE demonstrated increased sensitivity across the perinatal period, relative to fathers receiving a dummy intervention. Fathers' involvement with the infant increased significantly from the prenatal to postnatal period, regardless of the intervention. CONCLUSIONS: Prenatal video-feedback using ultrasound imaging of the unborn child has the potential to promote the quality of parenting in an important, but understudied, population and period: men in the transition to fatherhood. Future research should examine the long-term effectiveness of VIPP-PRE and its effectiveness in increasing parenting quality in at-risk families. IMPACT: This study identifies a brief and focused prenatal intervention using assisted interactions between the father and his baby by means of ultrasound imaging as a promising strategy to improve sensitive fathering in the early postnatal phase. Our study shows that pregnancy provides a window of opportunity for promoting prenatal involvement and bonding in expectant fathers, with potential long-term benefits for the future father-child relationship. Ultrasound measures are currently used to monitor fetal growth and development, but our results suggest that they may also create an opportunity for stimulating father-infant interaction to promote postnatal caregiving quality.

Socioemotional profiles of autism spectrum disorders, attention deficit hyperactivity disorder, and disinhibited and reactive attachment disorders: a symptom comparison and network approach.

Children with autism spectrum disorders (ASDs), attention deficit hyperactivity disorder (ADHD) and disinhibited and reactive attachment disorders (RAD/DAD) often experience socioemotional problems. Elucidating a clear picture of these profiles is essential. Strengths and Difficulties Questionnaires (SDQs) were analysed from cohort of children with ASD (n = 1430), ADHD (n = 1193), and RAD/DAD (n = 39). Kruskal-Wallis Tests and network analytic techniques were used to investigate symptom profiles. Children with ASD experienced more emotional problems, peer problems and fewer prosocial behaviours. Children with ADHD and RAD/DAD had higher levels of hyperactivity and conduct problems. Overall, ASD and ADHD networks were highly correlated (rs = 0.82), and we did not observe a statistically significant difference in terms of global Strength.

Improving parenting, child attachment, and externalizing behaviors: Meta-analysis of the first 25 randomized controlled trials on the effects of Video-feedback Intervention to promote Positive Parenting and Sensitive Discipline.

Improving parenting, child attachment, and externalizing behaviors: Meta-analysis of the first 25 randomized controlled trials on the effects of Video-feedback Intervention to promote Positive Parenting and Sensitive Discipline (VIPP-SD). VIPP-SD combines support of parental sensitive responsiveness with coaching parents in sensitive limit setting. Here, we present meta-analyses of 25 RCTs conducted with more than 2,000 parents and caregivers. Parents or children had various risks. We examined its effectiveness in promoting parental cognitions and behavior regarding sensitive parenting and limit setting, in promoting secure child-parent attachment, and reducing externalizing child behavior. Web of Science, MEDLINE, PubMed, and recent reviews were searched for relevant trials (until May 10, 2021). Multilevel meta-analysis with META, METAFOR, and DMETAR in R took account of the 3-level structure of the datasets (studies, participants, measures). The meta-analyses showed substantial combined effect sizes for parenting behavior (r = .18) and attitudes (r = .16), and for child attachment security (r = .23), but not for child externalizing behavior (r = .07). In the subset of studies examining effects on both parenting and attachment, the association between effect sizes for parenting and for attachment amounted to r = .48. We consider the way in which VIPP-SD uses video-feedback an active intervention component. Whether VIPP-SD indeed stimulates secure attachment through enhanced positive parenting remains an outstanding question for further experimental study and individual participant data meta-analysis.

Exploring everyday state attachment dynamics in middle childhood.

The current study explored dynamics of secure state attachment expectations in everyday life in middle childhood, specifically state attachment carry-over and reactivity to experiences of caregiver support in the context of stress. In two independent samples (one community sample, N = 123; one adoption sample, N = 69), children (8-12 years) daily reported on their state attachment for respectively 14 and 7 consecutive days. Additionally, they reported daily on their experiences of distress and subsequent experiences of caregiver support. Results in both samples indicated that secure state attachment on a day-to-day basis is characterized by a significant positive carry-over effect, suggesting that state attachment fluctuations are (partially) self-predictive. In Study 1, experiencing no support following distress significantly related to intraindividual decreases in secure state attachment; in Study 2, experiencing effective support during distress related to intra-individual increases in secure state attachment. Taken together, the current studies provide novel and important insights into how state attachment temporally evolves on a day-to-day basis in middle childhood.

Prenatal predictors of childhood anxiety disorders: An exploratory study of the role of attachment organization.

Childhood anxiety disorders (CAD) are a common childhood mental disorder and understanding early developmental pathways is key to prevention and early intervention. What is not understood is whether early life stress predictors of CAD might be both mediated by infant cortisol reactivity and moderated by infant attachment status. To address this question, this exploratory study draws on 190 women recruited in early pregnancy and followed together with their children until 4 years of age. Early life stress is operationalized as maternal depression measured using the Structured Clinical Interview for the DSM, Childhood Trauma Questionnaire, Parenting Stress Index, and antenatal maternal hair cortisol concentrations. Infant cortisol reactivity was measured at 12 months together with the Strange Situation Procedure and CAD assessed at 4 years of age using the Preschool Age Psychiatric Assessment. There was no direct association between attachment classification and CAD. Furthermore, infant cortisol reactivity neither mediated nor attachment moderated the association of early life stress predictors and CAD. However, only for infants with organized attachment classifications, higher maternal antenatal depression, and hair cortisol were associated with a higher risk of CAD.

Exploring the meaning of unresolved loss and trauma in more than 1,000 Adult Attachment Interviews.

Unresolved states of mind regarding experiences of loss/abuse (U/d) are identified through lapses in the monitoring of reasoning, discourse, and behavior surrounding loss/abuse in response to the Adult Attachment Interview. Although the coding system for U/d has been widely used for decades, the individual indicators of unresolved loss/abuse have not been validated independently of the development sample. This study examined the psychometric validity of U/d, using individual participant data from 1,009 parent-child dyads across 13 studies. A latent class analysis showed that subsets of commonly occurring U/d indicators could differentiate interviewees with or without unresolved loss/abuse. Predictive models suggested a psychometric model of U/d consisting of a combination of these common indicators, with disbelief and psychologically confused statements regarding loss being especially important indicators of U/d. This model weakly predicted infant disorganized attachment. Multilevel regression analysis showed no significant association between ratings of unresolved other trauma and infant disorganized attachment, over and above ratings of unresolved loss/abuse. Altogether, these findings suggest that the coding system of U/d may have been overfitted to the initial development sample. Directions for further articulation and optimization of U/d are provided.

Maternal sensitivity in early childhood and body mass index in adolescence: A population-based study on the role of self-regulation as a mediator.

Maternal sensitivity has been implicated in various aspects of child health and development, including overweight. However, long-term effects, the role of paternal sensitivity and the explanatory pathways are unclear. This study examined whether maternal sensitivity in early childhood is prospectively associated with adolescent body mass index and whether children's self-regulation mediates this relation. Data from 540 children and their mothers were available from a large cohort study in the Netherlands. Maternal sensitivity was assessed at child ages 1, 3, and at 4 years paternal sensitivity was also included. Children's self-regulation skills were observed at age 3, eating behaviour was assessed at 10 years, and child BMI was measured at 13 years. Longitudinal structural equation modelling was applied. The cross-sectional association between maternal sensitivity and child self-regulation was significant, while lower levels of self-regulation and higher levels of food responsiveness and restrained eating predicted a higher child BMI at 13 years. Furthermore, a direct association of paternal sensitivity at 4 years with BMI at 13 years was found, but only in girls. Maternal sensitivity was not directly associated with child BMI after adjusting for covariates. Our findings showed the importance of self-regulation in the early years for subsequent weight development. Nevertheless, as self-regulation could not explain the relationship between parenting and child weight, research should focus on the contribution of other contextual factors, such as feeding styles and the social environment, to this relationship.

Longitudinal changes in DLPFC activation during childhood are related to decreased aggression following social rejection.

Regulating aggression after social feedback is an important prerequisite for developing and maintaining social relations, especially in the current times with larger emphasis on online social evaluation. Studies in adults highlighted the role of the dorsolateral prefrontal cortex (DLPFC) in regulating aggression. Little is known about the development of aggression regulation following social feedback during childhood, while this is an important period for both brain maturation and social relations. The current study used a longitudinal design, with 456 twins undergoing two functional MRI sessions across the transition from middle (7 to 9 y) to late (9 to 11 y) childhood. Aggression regulation was studied using the Social Network Aggression Task. Behavioral aggression after social evaluation decreased over time, whereas activation in the insula, dorsomedial PFC and DLPFC increased over time. Brain-behavior analyses showed that increased DLPFC activation after negative feedback was associated with decreased aggression. Change analyses further revealed that children with larger increases in DLPFC activity from middle to late childhood showed stronger decreases in aggression over time. These findings provide insights into the development of social evaluation sensitivity and aggression control in childhood.

In defense of unresolved attachment: re-modelling intergenerational transmission of attachment.

Intergenerational transmission of attachment is one of the core hypotheses of attachment theory. How parents or other caregivers look back on their childhood attachment experiences is suggested to shape their infants' attachments. In the current paper, we show that a new twist to correspondence analysis (Canonical Correlation Analysis [CCA]) of cross-tabulated attachment classifications with oblique rotation Correspondence Analysis (CA) may uncover the latent structure of intergenerational transmission showing the unique role of parental Unresolved representations in predicting infant Disorganized attachments. Our model of intergenerational transmission of attachment supports predicted associations between parental and infant attachments. Despite growing skepticism about the validity of parental Unresolved trauma and infant Disorganized attachment, we come to an evidence-based statistical defense of these generative clinical components of attachment theory awaiting a substantive experimentum crucis.

Development of the virtual-VIPP and a systematic review of online support for families during the COVID-19 pandemic.

During the COVID-19 pandemic lockdowns made it impossible for parenting coaches to reach the families without digital means of communication. Several studies were initiated to transform existing parenting interventions into hybrid or fully online versions and to examine their feasibility, acceptability and efficacy. We present one such transformation in detail, the Virtual-VIPP which is based on Video-feedback Intervention to promote Positive Parenting and Sensitive Discipline (VIPP-SD). Furthermore, we report a systematic review of 17 published trials with online versions of parenting programs. Overall, online parenting interventions seem feasible to implement, are well-received by most families, and to show equivalent effects to face-to-face approaches. Careful preparation of technicalities and monitoring of fidelity are prerequisites. Advantages of online parenting interventions are their potentially broader reach, more detailed process documentation, and better cost-utility balance. We expect that online parenting interventions are here to stay, but their efficacy needs to be rigorously tested.

Epigenome-wide associations between observed maternal sensitivity and offspring DNA methylation: a population-based prospective study in children.

BACKGROUND: Experimental work in animals has shown that DNA methylation (DNAm), an epigenetic mechanism regulating gene expression, is influenced by typical variation in maternal care. While emerging research in humans supports a similar association, studies to date have been limited to candidate gene and cross-sectional approaches, with a focus on extreme deviations in the caregiving environment. METHODS: Here, we explored the prospective association between typical variation in maternal sensitivity and offspring epigenome-wide DNAm, in a population-based cohort of children (N = 235). Maternal sensitivity was observed when children were 3- and 4-years-old. DNAm, quantified with the Infinium 450 K array, was extracted at age 6 (whole blood). The influence of methylation quantitative trait loci (mQTLs), DNAm at birth (cord blood), and confounders (socioeconomic status, maternal psychopathology) was considered in follow-up analyses. RESULTS: Genome-wide significant associations between maternal sensitivity and offspring DNAm were observed at 13 regions (p < 1.06 × 10-07), but not at single sites. Follow-up analyses indicated that associations at these regions were in part related to genetic factors, confounders, and baseline DNAm levels at birth, as evidenced by the presence of mQTLs at five regions and estimate attenuations. Robust associations with maternal sensitivity were found at four regions, annotated to ZBTB22, TAPBP, ZBTB12, and DOCK4. CONCLUSIONS: These findings provide novel leads into the relationship between typical variation in maternal caregiving and offspring DNAm in humans, highlighting robust regions of associations, previously implicated in psychological and developmental problems, immune functioning, and stress responses.

Maternal anxiety during pregnancy and newborn epigenome-wide DNA methylation.

Maternal anxiety during pregnancy is associated with adverse foetal, neonatal, and child outcomes, but biological mechanisms remain unclear. Altered foetal DNA methylation (DNAm) has been proposed as a potential underlying mechanism. In the current study, we performed a meta-analysis to examine the associations between maternal anxiety, measured prospectively during pregnancy, and genome-wide DNAm from umbilical cord blood. Sixteen non-overlapping cohorts from 12 independent longitudinal studies of the Pregnancy And Childhood Epigenetics Consortium participated, resulting in a combined dataset of 7243 mother-child dyads. We examined prenatal anxiety in relation to genome-wide DNAm and differentially methylated regions. We observed no association between the general symptoms of anxiety during pregnancy or pregnancy-related anxiety, and DNAm at any of the CpG sites, after multiple-testing correction. Furthermore, we identify no differentially methylated regions associated with maternal anxiety. At the cohort-level, of the 21 associations observed in individual cohorts, none replicated consistently in the other cohorts. In conclusion, contrary to some previous studies proposing cord blood DNAm as a promising potential mechanism explaining the link between maternal anxiety during pregnancy and adverse outcomes in offspring, we found no consistent evidence for any robust associations between maternal anxiety and DNAm in cord blood. Larger studies and analysis of DNAm in other tissues may be needed to establish subtle or subgroup-specific associations between maternal anxiety and the foetal epigenome.

Effects of oxytocin and vasopressin administration on human fathers' sensitive and challenging parenting: A randomized within-subject controlled trial.

This randomized, double-blind, placebo-controlled within-subject study examined the effects of intranasal administration of oxytocin and vasopressin on fathers' sensitive and challenging parenting behaviors. Furthermore, we examined the moderating role of fathers' early childhood experiences. The sample consisted of 70 fathers with their 2- to 12-month-old infants. All fathers were assigned to each of the three experimental sessions (oxytocin, vasopressin, and placebo), on three separate days, with random order and intervening periods of one to two weeks. Sensitive and challenging parenting behaviors (CPB) were observed during a 10-minute free play task. Results showed no effects of vasopressin administration on paternal sensitivity. Fathers in the oxytocin condition were less sensitive than fathers in the placebo condition, and this effect was moderated by fathers' own childhood experiences: Fathers who reported higher levels of experienced parental love withdrawal were less sensitive in the oxytocin condition as compared to the placebo condition, whereas fathers with less experienced parental love withdrawal showed no difference in sensitivity between the oxytocin and placebo condition. No effects were found of oxytocin and vasopressin administration on fathers' CPB. Our results, although partly unexpected, are largely in line with previous literature showing that oxytocin administration can exert negative effects in individuals with adverse childhood experiences.

The latent structure of the adult attachment interview: Large sample evidence from the collaboration on attachment transmission synthesis.

The Adult Attachment Interview (AAI) is a widely used measure in developmental science that assesses adults' current states of mind regarding early attachment-related experiences with their primary caregivers. The standard system for coding the AAI recommends classifying individuals categorically as having an autonomous, dismissing, preoccupied, or unresolved attachment state of mind. However, previous factor and taxometric analyses suggest that: (a) adults' attachment states of mind are captured by two weakly correlated factors reflecting adults' dismissing and preoccupied states of mind and (b) individual differences on these factors are continuously rather than categorically distributed. The current study revisited these suggestions about the latent structure of AAI scales by leveraging individual participant data from 40 studies (N = 3,218), with a particular focus on the controversial observation from prior factor analytic work that indicators of preoccupied states of mind and indicators of unresolved states of mind about loss and trauma loaded on a common factor. Confirmatory factor analyses indicated that: (a) a 2-factor model with weakly correlated dismissing and preoccupied factors and (b) a 3-factor model that further distinguished unresolved from preoccupied states of mind were both compatible with the data. The preoccupied and unresolved factors in the 3-factor model were highly correlated. Taxometric analyses suggested that individual differences in dismissing, preoccupied, and unresolved states of mind were more consistent with a continuous than a categorical model. The importance of additional tests of predictive validity of the various models is emphasized.

Maternal trauma but not perinatal depression predicts infant-parent attachment.

Understanding if maternal depression is a predictor of infant-parent attachment classification is important to furthering knowledge about the early pathways and predictors of socio-emotional development. Yet few studies that have utilised the Strange Situation Procedure, the gold standard for measurement of infant-parent attachment, have examined antenatal depression as a predictor of attachment, and none has also included a measure of maternal trauma. This study uses data on 224 women recruited in early pregnancy and followed up until 12 months postpartum. Maternal depression was measured in pregnancy using the Structured Clinical Interview for the DSM and repeat Edinburgh Postnatal Depression Scale as well as Stressful Life Events scale across pregnancy and postpartum including items on domestic violence. A past history of trauma was measured using the Childhood Trauma Questionnaire. Attachment was measured using the Strange Situation Procedure (SSP) at 12 months postpartum. We found that maternal depression was not associated with insecure or disorganized attachment. However, a maternal history of childhood trauma and current domestic violence both predicted insecure-avoidant attachment at 12 months, whereas increased number of stressful life events prior to conception and in pregnancy was associated with insecure-resistant attachment. Neither trauma, past or current, nor depression predicted disorganized attachment. In the first study to have included measures of antenatal depression, maternal childhood trauma, and current stressful events as predictors of infant attachment measured using the SSP, we found maternal experiences of past and current trauma but not depression were significant predictors of infant-parent attachment security.