The synthesis and self-assembly of two new phenylacetylene macrocycle (PAM) organogelators were performed. Polar 2-hydroxyethoxy side chains were incorporated in the inner part of the macrocycles to modify the assembly mode in the gel state. With this modification, it was possible to increase the reactivity of the macrocycles in the xerogel state to form polydiacetylenes (PDAs), leading to a significant enhancement of the polymerization yields. The organogels and the PDAs were characterized using Raman spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM). The self-assembly of molecular building blocks is an increasingly popular method for the preparation of new semiconducting materials. Rational design of building blocks and their assembly using non-covalent interactions can provide control over the size, shape and electronic properties of the resulting nanoarchitectures –3. It isRecently, many research groups used this strategy to design several types of π-conjugated monomers capable of hydrogen bonding to create nanowires –23, nanoPAM2 and PAM3, In order to take a step further toward a better comprehension of the self-assembly of PAMs and to increase the polymerization yield, we decided to introduce substituents on the inside of the macrocycles, more precisely a polar 2-hydroxyethoxy group. Such a polar group proved to be useful to increase the intermolecular interactions between PAMs through hydrogen bonding in the solid state –41. We hPAM2 and PAM3 possess some structural dissimilarity. First, PAM2 possesses two 2-hydroxyethoxy chains and two amide groups in the benzamide configuration while PAM3 contains four of each. It is important to mention that the alcohol chains are positioned for synthetic ease. The synthesis and gelation properties of PAM1 are reported in the literature [PAM1 was used in this study as a control molecule because of the absence of 2-hydroxyethoxy chains in its structure, providing a reliable comparison.terature . PAM1 waPAM2 and PAM3 is shown in 1 in excellent yield (93%). Then, 2-hydroxyethoxy chains were installed using K2CO3 to obtain compound 2 in good yield (61% yield). Amidation using diisopropylcarbodiimide (DIC) and 6-chlorohydroxybenzotriazole (6-ClHOBt) as coupling agents was then performed, followed by protection of the hydroxy moiety with TBS in order to facilitate the further synthetic steps by increasing the solubility of the compound (57% yield over 2 steps). Then, by using a standard Castro–Stephens–Sonogashira coupling, TMS protecting groups were installed (73% yield). After basic removal of the alkyne protective group, the half-macrocycle 5 was obtained by Castro–Stephens–Sonogashira coupling with previously reported 3,5-diiodooctylbenzene [6 which can be deprotected using potassium hydroxide and directly used in modified Eglinton ring closing reaction in pseudo-high dilution to afford hydroxy-protected PAM. Deprotection was directly performed without purification using TBAF to afford PAM2 in good yields (88% yield). Same approach was used for PAM3. Starting from previously synthesized compound 3, the half-macrocycle 9 was obtained by standard Castro–Stephens–Sonogashira coupling with dialkyne 8 in good yield (59%). It is noteworthy that compound 8 was obtained from oxidative deprotection of compound 7. After installing TMS-protected alkynes with good yields , selective removal of TMS using potassium hydroxide and ring closing reaction under modified Eglinton conditions were performed to afford a TBS protected PAM. Deprotection of TBS using TBAF was then realized without purification to afford PAM3 in rather low yield. We hypothesized that this low yield can be due to the increased steric hindrance for compound 10 (2 OTBS groups) compared to compound 6 (1 OTBS group), impairing the macrocyclization reaction and leading to a significant amount of oligomerized product. It is noteworthy that purification of PAM3 was realized by precipitation in methanol. Standard column chromatography was not effective due to the low solubility of the macrocycle after deprotection of the hydroxy-containing side chains.The synthetic pathway toward lbenzene  in good PAM2 and PAM3 have been studied and compared to those of PAM1. For each PAM, a precise quantity was mixed with the solvent in a capped glass tube. After sonication to breakdown the aggregates, the mixture was heated until a clear solution was obtained. Then, the solution was allowed to slowly cool at room temperature and the formation of the gel was confirmed in each case by the tube inversion method [PAM1, PAM2 and PAM3 in different solvents are summarized in Table S1 . The DSC analysis was carried out at temperatures ranging from 298 to 223 K. In both cases, a very sharp exotherm attributable to the crystallization of supercooled solvent was observed  than for PAM1, which has an exotherm at 267 K. This result suggests that the gel of PAM2 might be less organized than that of PAM1. By heating the gel from 223 to 298 K, an endotherm was observed in both cases, which is closely related to the melting point of free cyclohexane [In order to investigate the crystallization temperature of solvents within the gel and to compare the influence of the 2-hydroxyethoxy side chains on the thermal stability of the gel, two gel samples were prepared with lohexane .PAM2 only since PAM3 did not present any gelation properties. For SEM analysis, a gel sample was allowed to dry at room temperature on a metallic substrate to form a xerogel. Then, gold was sputtered on the sample prior to imaging. SEM images of PAM2 are shown in PAM1 and other phenylacetylene macrocycles, a dense network of nanofibrils was formed during the gelation process [To explore the morphology of the structures created during the gel formation, scanning electron microscopy (SEM) was performed on  process –48. Thes process –50.PAM2  was performed on actogram ,39. InstPAM2 in cyclohexane (10 mg/mL) was performed for 24 h under UV light (254 nm). The resulting blue material was purified using size-exclusion chromatography (SEC) (Bio-Beads SX-1) to remove all traces of the starting macrocycle. To our surprise, an increase of the polymerization yields was observed every time a polymerization was achieved with PAM2. In several attempts, a maximum yield of 30%  was obtained after purification, representing a two-fold increase compared to the best result obtained for the same PAM scaffold (≈15% yield). This result suggests that the diyne units in PAM2 are in a more suitable orientation than PAM1 to undergo a topochemical polymerization, although gelation properties and XRD result seem to suggest otherwise. It is also possible that the apparent yield increase comes from a better solubility of the PDA obtained from PAM2 compared to that obtained from PAM1, although quantitative measurements of the solubility have not been conducted because of the small quantity of materials prepared.Despite this unexpected finding, irradiation of a xerogel sample of PAM2 reacted during irradiation, Raman spectroscopy was performed on the resulting PDA (−1) is present. The final PDA presented bands at 1470 and 2100 cm−1, which could be respectively associated to alkene and alkyne moieties of PDA. It should be noted that these two bands are also present in the Raman spectrum of PAM2 due to partial photo-induced polymerization during spectrum acquisition. This demonstrates the high reactivity of PAM2 under UV irradiation. Further research on the resulting nanoarchitectures is currently underway to establish if covalent nanotubes have been formed and to study their properties.Given the observed polymerization of diyne units, UV–vis spectroscopy was performed to confirm the appearance of polydiacetylene. As shown in ting PDA . As expePAM2 has been transformed exclusively into nanotubes or nanorods and attempts to disperse individual molecules in organic solvents failed. Considering the strict structural parameters needed to undergo a topochemical polymerization and the one-dimensional nature of the starting xerogel of PAM2, it is unlikely – but not impossible – that the final blue materials can be made of other nanoarchitectures than the nanotubular or nanorods one. Nonetheless, no conclusive remarks can be made and more analysis will have to be performed in order to gain better insight about the final structure of the PDA obtain from PAM2.To assess the presence of nanotubes or nanorods, HRTEM analysis has been performed on the purified blue materials. Although one-dimensional features can be observed at different locations on the grid  did not affect the gelation properties while the incorporation of four side chains and four amide groups significantly alter the solubility of the resulting PAM (PAM3). Topochemical polymerization on self-assembled PAM2 to yield PDA-based materials proved to be much more efficient than for the 2-hydroxyethoxy-free analogues (PAM1). Characterization is currently underway to investigate the precise nature of the nanoarchitectures formed.In summary, the synthesis and self-assembly of two new phenylacetylene macrocycles were performed. To improve the self-assembly properties in the gel state and further yield to better topochemical polymerization yield, the incorporation of 2-hydroxyethoxy side chains was accomplished. The gelation properties demonstrated that the incorporation of two of these chains (File 1Experimental part.


Psychotic experiences are prevalent in community samples and are highly correlated with depressive symptoms. This study aimed to investigate the longitudinal associations between psychotic experiences and depressive symptoms between adolescence and young adulthood.Prospective cohort study with a 6 year follow-up in a community sample of 7632 adolescents and young adults. Depressive symptoms were assessed with the Short Moods and Feelings Questionnaire and psychotic experiences with a semi-structured clinical interview at 12 and 18 years. Longitudinal and cross-sectional associations were investigated with regression and structural equation models.Depressive symptoms and psychotic experiences were associated at each time-point  and there were longitudinal within-phenotype associations . There was an across-phenotype association between psychotic experiences at 12 and depressive symptoms at 18 r = 0.139 , but no association between depressive symptoms at 12 and psychotic experiences at 18 r = −0.022 .Longitudinal across-phenotype associations were substantially weaker than cross-sectional associations or within-phenotype longitudinal associations. Whilst psychotic experiences at 12 years were associated with a small increase in depression at 18 years, depression at 12 years was not associated with psychotic experiences at 18 years once across-phenotype cross-sectional and within-phenotype longitudinal associations were accounted for. This suggests that the biological mechanisms underlying depression at this age do not increase subsequent risk of psychotic experiences once they resolve. Psychotic experiences (PEs) are reported by approximately 5–7% of individuals in community samples The strong correlation between PEs and depressionWhilst there is evidence from longitudinal studies to support the hypothesis that PEs are associated with later depression In an elegant study Structural equation modelling (SEM) minimizes the problem of residual errors by modelling associations between latent traits rather than observed variables, and also allows examination of associations whilst allowing for the correlation between phenotypes. To our knowledge there has been only one other study We hypothesised that PEs and depression would not be associated over time, once the effect of persistent PEs or depression had been accounted for. Our aims were: i) to use SEM to estimate the extent to which depression and PEs are associated over time whilst accounting for cross-sectional (across-phenotype) and longitudinal (within-phenotype) effects, and minimising residual error, and ii) to compare our results using an SEM approach with those when using standard regression models.http://www.bristol.ac.uk/alspac/) participants. All pregnant women resident in the former Avon Health Authority in South West England with an estimated delivery date of between 1st April 1991 and 31st December 1992 were invited to participate. The children of 15,247 pregnancies were recruited. Of this sample of 15,458 foetuses, 14,701 were live births and alive at 1 year The study sample consists of ALSPAC  of participants with data on at least one measure of; depressive symptoms (DS) or PEs at 12 or 18 years. The dataset represents approximately 52% of the original cohort. See Missing data patterns were examined. Multiple imputation in the Stata statistical package PEs were assessed using a semi-structured interview (PLIKSi) that has been described elsewhere The average Kappa values for inter-rater reliability were 0.72 and 0.83 for the 12 year and 18 year interviews respectively. Test-retest agreement at 18 years was 0.74.Self-reported DSs were assessed using the Short Moods and Feeling Questionnaire (SMFQ) We adjusted for gender and two proxy measures of social class . Marital status was dichotomised into never married and married presently or previously. Maternal education was dichotomised into lower  and higher .Logistic regression models were used to investigate the cross-sectional association between PEs and DSs at each time-point, and the longitudinal association between DSs at 12 years and PEs at 18 years, adjusted for PEs at 12 years, DSs at 18 years, and confounders. Linear regression was used to investigate the association between PEs at 12 years and DSs at 18, adjusted for DSs at 12 years, PEs at 18 and other confounders. PEs were coded as 1 for PE suspected or definitely present detected during the clinical interview or a positive response to a stem question and 0 for no PE detected during the clinical interview or a no response to a stem question. If a stem question was positively responded to but not detected during the clinical interview it was still coded as 1.SEM comprises of a measurement model employing confirmatory factor analysis, and a structural element In order to enable identification of the measurement model the PEs and DSs factors at 12 and 18 were allowed to co-vary, but the indicators and errors were not. To enable scaling of the factors, the variance of each was set to 1A maximum likelihood estimator (with robust standard errors) was used because it is robust to non-normally distributed continuous variables.Since the period of assessment for each outcome at 18 years was different (see Methods) the SEM model was repeated but only including those with PEs at 18 that had occurred within the previous year  because the range of model fit diagnostics available in Mplus for imputed data is limited.We tested associations between factors with either DS18 or PE18 as the primary outcome, in order to test the conditioning effect of the other primary outcome factor. The results of this analysis are shown in Compared to those excluded because of missing data, study participants were less likely to be male (51.3% versus 47.9%) or have a mother who was unmarried (19.1% versus 14.3%) or with lower education (64.6% versus 57.3%) at their birth .Mean DS score increased between 12 (mean SMFQ score = 3.44) and 18 years (mean = 5.76) by approximately 0.6 of a standard deviation. The proportion of those experiencing suspected or definite PEs was smaller at 18 years  than at 12 years  .Using linear and logistic regression on observed data, there was strong evidence that PEs and DSs were associated cross-sectionally, both at 12 and 18 years. DSs at age 12 were associated with PEs at age 18, even after adjusting for baseline PEs, DSs at age 18, and potential confounders. Similarly, PEs at age 12 were associated with DSs at age 18, even after adjusting for baseline DSs, PEs at age 18, and potential confounders see .In the confirmatory factor analysis, the standardised loadings of each indicator on each factor were high see . The corIn the SEM analysis using latent constructs of PEs and DSs, cross-sectional associations at each time-point and longitudinal within-phenotype associations were strong see . HoweverOur hypothesis that there would be no across-phenotype association between psychotic experiences and depression over time once the effects of persistent psychotic experiences and depression had been accounted for was supported for one pathway but not the other.With standard regression modelling we found strong evidence that PEs at age 12 were associated with depressive symptoms at age 18 even after adjusting for concurrent depressive symptoms at age 12 and persistence of PEs at age 18. We also observed a similarly strong relationship between depressive symptoms at age 12 and PEs at age 18.However, when using an SEM approach we found little evidence that those who had high depressive symptom scores at 12 were more likely to experience PEs at 18 if their depressive symptoms had resolved by this age, whereas those with PEs at 12 were slightly more likely to experience depressive symptoms at 18 even if their PEs had resolved by 18 years. The most likely explanation for the association we observed between depressive symptoms at 12 and PEs at 18 when using standard regression modelling, inconsistent with our findings when using SEM, is measurement error resulting from the combination of: i) the association between depressive symptoms at 12 and depressive symptoms at 18, and ii) the strong cross-sectional association between PEs and depressive symptoms at both 12 and 18 years.These findings lead to three main conclusions. Firstly, the method of statistical analysis used is important when dealing with repeated measures of strongly correlated data. As we have observed here it is possible to come to different conclusions about whether depressive symptoms at age 12 increase risk of PEs at age 18 independently of the effects of persisting depression when using standard regression techniques compared to using an SEM approach. Regression models with observed data cannot adequately deal with the problem of correlation between outcomes and exposures. Secondly, early adolescent depression is not associated with increased risk of developing PEs once the co-existence of depression and PEs is accounted for. It is perhaps not unexpected that depression is associated with PEs; for example increased feelings of self-consciousness that occur in depression could manifest as paranoid ideation or delusions at their extreme. Our findings suggest that disrupted biological  mechanisms underlying depression do not exert long-term effects on risk of PEs once they resolve. Thirdly, there is evidence that early adolescent PEs do infer a slightly increased risk of later depression. One possible explanation for this is that PEs may cause long-term problems with loss of trust in others, impaired self-confidence, and social isolation, and if these persist even if PEs do not, they could increase the risk of subsequent depression.One previous study of adolescents Whilst we observed evidence of an association between PEs at baseline and depression at follow-up, three other studies did not In order to explain the continuities and discontinuities of psychopathology over time homotypic and heterotypic theories of continuity have been proposed Not all PEs arise within the context of depression, and there are likely to be important differences in the pathogenic mechanisms underlying these two phenotypes in community samples. Depression scores increase during adolescence whilst frequency of PEs decreases, consistent with this belief. It is possible that PEs arising outside the context of depressive symptoms index aetiological mechanisms underlying schizophrenia more strongly than PEs arising within the context of depression. We are not able to test this hypothesis here, though if it were possible to identify individuals for whom PEs were more likely to reflect mechanisms of interest for disorders such as schizophrenia, this would have important implications for research in this field.Our study has three major strengths. First, the use of a semi-structured interview to assess PEs and the advantage of detailed repeated measures data on both PEs and depressive symptoms. Second, the large population sample has enabled more precise estimation of associations between depression and PEs than previous studies to date. Third, the use of SEM has allowed us to examine both within and across-phenotype relationships of PEs and depressive symptoms over time whilst minimising problems arising from correlated data.There are also a number of limitations. First, selection bias resulting from sample attrition associated with factors associated with the outcome, the exposure, or both is a concern. However attrition from the ALSPAC cohort is similar to that from other similar sized cohortsIn the context of these limitations it is important for future studies with data on both PEs and depressive symptoms, measured repeatedly over time, to further investigate their complex relationship during adolescent development. Our findings demonstrate the importance of using appropriate statistical methods to deal with problems arising from strong co-morbidity of phenotypes if the aim is to understand more about the long-term relationships between these phenotypes, and their influence on progression to clinical disorder over time.Figure S1Post hoc analyses.(DOCX)Click here for additional data file.Figure S2Structural equation model and coefficients.(TIF)Click here for additional data file.Table S1Comparison of variables in the ALSPAC cohort and the study dataset.(DOCX)Click here for additional data file.Table S2Pattern of missing data.(DOCX)Click here for additional data file.Table S3Factor loadings.(DOCX)Click here for additional data file.Appendix S1Unusual experiences and psychotic experiences questions.(DOCX)Click here for additional data file.


Failure to consume an adequate diet or over consumption during adolescence can disrupt normal growth and development, resulting in undesirable weight change. This leads to an increase in unhealthy weight control practices related to eating and exercise among both adolescent girls and boys to meet the societal ‘ideal’ body shape. This study therefore aims to examine the longitudinal changes in eating attitudes, body-esteem and weight control behaviours among adolescents between 13 and 17 years; and, to describe perceptions around body shape at age 17 years. A total of 1435 urban South African black and mixed ancestry boys and girls, who had data at both age 13 and 17 years from the Birth to Twenty cohort were included. Data were collected through self-administered questionnaires on eating attitudes (EAT-26), body esteem and weight control behaviours for either weight loss or muscle gain attempts. Height and weight were measured at both time points and BMI was calculated. Black females had a higher BMI (p<0.001) and an increased risk of developing eating disorders as well as significant increase in the prevalence of weight loss practices between the ages 13 and 17 years. At age 17 years both Mixed ancestry adolescents had lower body-esteem compared to black adolescents. The prevalence of possible eating disorders was 11% and 13.1% in early and late adolescents respectively. Males and females shared similar opinions on normal silhouettes being the ‘best’, ‘getting respect’ and being the ‘happiest’, while the obese silhouette was associated with the ‘worst’ and the ‘unhappiest’, and the underweight silhouette with the “weakest”. Black females had a higher BMI and an increased risk of developing eating disorders. Adolescent females engaged more in weight loss practices whereas, males in muscle gain practices indicating that Western norms of thinness as the ideal are becoming more common in South Africa. Nutritional needs are greater during adolescence than at other times during childhood, and over-consumption or failure to consume an adequate diet during this period can disrupt normal growth and development, resulting in undesirable weight change In most high-income countries (HICs) Research by Mchiza and others in an urban adolescent sample (Cape Town) of South African girls found that body image dissatisfaction was greater in white than black African girls. In addition, black African girls experienced less pressure from family and peers to change their current body shape Some studies have shown that increasing age during adolescence is strongly associated with heightened emphasis on appearance and body shape Because of limited longitudinal information on eating attitudes, body image perception and weight control behaviors in South African black and mixed ancestry adolescents, we examined changes in these parameters between early (13 years) and late (17 years) adolescent boys and girls. Secondly, we assessed differences between 17-year-old boys and girls in their perceptions of female body silhouettes.Data for this study were obtained from the Birth-to-Twenty longitudinal birth cohort study A trained research team collected socio-demographic and anthropometric data, and interviewer-assisted questionnaires on eating attitudes, body esteem and weight control behaviors on the adolescents at two time points: at age 13 years  and four years later at age 17 years . A total of 1 435 participants had complete data at both 13 and 17 years (boys = 690 and girls = 745). This study only included black African and mixed ancestral (children born to white European and black African parents) participants from the cohort and those who had data at both time points. Adolescents belonging to other racial groups (i.e. white and Indian) were excluded because of the small sample sizes. This study is presented in two-phases: A cross- sectional study at age 13 and 17 years, and a longitudinal study that examines the changes that took place between age 13 and 17 years. Ethics approval was obtained from the University of the Witwatersrand Human Research Ethics Committee. Written informed consent was obtained from the participants’ legal guardians/caregivers, and written assent from the participants themselves.2 (m2)), and an international age and gender BMI cut-off were used to define overweight, obesity nd grade i.e. <2SD) Height and weight were collected at both time points. Height was measured using a portable stadiometer  and recorded to the nearest millimeter, and weight was measured using a digital scale to the nearest 100 grams. Participants’ height and weight measurements were used to calculate BMI (weight (kg)/heightThe EAT-26 questionnaire was completed in order to measure eating attitudes Body-esteem was measured using a body esteem scale All participants were asked a number of questions about their attempts to change their weight. Girls and boys were asked the following questions: “During the past year have you done anything to try to lose weight?”, “During the past year have you done anything to try to gain muscle?” If participants answered positively they were asked to give reasons, which included; health and cosmetic reasons e.g. to look better, clothes too tight, too fat, unhappy with self, and want to be a model. They were further asked about the methods they used to lose weight, and their responses were categorized into three groups: (i) healthy weight control behaviors  At 17 years of age girl and boy participants were also asked to select a female body silhouette from a series of 8 randomly arranged body silhouettes STATA  was used for analysis. Skewness and kurtosis tests for normality were applied to all numerical variables. A kurtosis of 3 was used to define a normal distribution and parametric tests were conducted. Data with a kurtosis greater than 3 were considered to be skewed and were subjected to non-parametric methods. None of the data was transformed. Student t-tests were used to determine differences in normally distributed numerical variables between the binary variables (e.g. gender and ethnicity). Associations between categorized weight–control behaviors and other characteristics  were assessed with chi-squared tests.Multi-nominal logistic regression models were fitted to identify determinants of weight control behaviors, eating attitudes and body esteem. We ran several bivariate models and identified variables that were significantly associated with the outcomes at the 5% level and included them in the multinomial regression.This study results are presented in three parts: Part 1 examines the cross-sectional data at ages 13 and 17 years, part 2 examines longitudinal changes in those who had complete data at both time points, and part 3 presents the results of the participants’ perceptions of the female silhouettes at age 17 years.The prevalence of underweight across the four groups at age 13 years  was 2.1%In the whole group, 11% of the participants reported an EAT-26 score >20, which is indicative of being at risk of developing an eating disorder, however there were no sex or ethnic differences between the groups. There was also no difference between the genders or ethnic groups for those adolescents categorized as low, average or high body esteem. Significantly more girls than boys engaged in weight loss practices (p<0.05), and conversely, significantly more boys than girls engaged in muscle gain practices (p<0.05).A significant proportion of 13 year olds engaged in unhealthy weight control behaviors (boys 37% and girls 39.5%), with the majority (58%) engaging in healthy weight control habits.The prevalence of underweight among the 17 year-old adolescents ranged from 2.7–8.6% in the girls and 6.2–10% in the boys . OverweiFor the whole sample at age 17 years, 13.1% had an EAT-26 score >20, and there was no difference in EAT-26 score or the proportion of adolescents with an EAT-26 score >20 between the gender or ethnic groups. Significantly more mixed ancestry boys and girls had low body esteem compared to their black African counterparts (both p = 0.001), however there were no differences within the ethnic groups. Significantly more black African girls than boys engaged in weight loss practices (p = 0.001) and significantly more boys than girls, in both ethnic groups, engaged in muscle gain practices (both p = 0.001). In addition, there was a difference between the black and mixed ancestry boys with regard to participation in muscle gain practices. For the whole group, 14% of the boys and 30.8% of the girls engaged in unhealthy weight control behaviors, however there were no significant differences between the gender and ethnic groups for weight control behaviours.The frequency of the various reasons given for the weight control behaviors reported by the adolescents at 13 and 17 years of age was determined . The desAt 13 years of age, there was no significant association between body esteem, eating attitudes and weight control behaviors, and ethnicity and BMI. At 17 years old, girls were more likely than boys to engage in unhealthy weight control behaviours  irrespective of ethnicity and BMI. At age 17 years girls of mixed ancestry origin were 36% less likely to be at risk of developing eating disorders (EAT26>20) compared to their black African peers, irrespective of BMI .The comparisons between the 17-year-old boys’ and girls’ perceptions of female body silhouettes are presented in We have shown in the cross-sectional component of our study a high prevalence of overweight and obesity among Black African girls, reaching 27.5% in the 17 year old girls. In both age groups, the prevalence of weight loss attempts was higher in girls, and muscle gain attempts were higher in boys, when compared to the opposite sex. At age 17 years both mixed ancestry boys and girls had lower body esteem than their black African peers, and the longitudinal data confirmed an increase in the prevalence of low body esteem in the mixed ancestry boys and girls with age. In addition, the desire to look better was the most common reason reported for engaging in weight control behaviors for all adolescents. Longitudinally, the prevalence of healthy weight control behaviors increased in the black African adolescent boys, but decreased in the mixed ancestry boys, while in the black African girls there was an increase in the prevalence of weight loss practices.In low-middle income countries (LMICs), there has been a reduction in the prevalence of underweight and an increasing concern around the increase in the prevalence of overweight and obesity in childhood and adolescence Our study showed an increase in muscle gain practices among the boys and weight loss practices among girls. This may be linked to the socio-cultural transition occurring in South Africa with adolescent boys working towards being leaner and more muscular, and girls wanting to be leaner to meet the societal expectations of an ideal body. Results from studies conducted in high-income countries (HICs) observe a similar trend with men engaging in muscle gain practices to achieve a male ideal V-shaped figure and girls engaging in weight loss practices to attain an ideal lean body shape with an emphasis on slim hips, bottom, and thighs The prevalence of low body esteem increased between 13 and 17 years of age in both the mixed ancestral boys and girls, and at the age of 17 years the prevalence was higher in the mixed ancestral group compared to their black African peers. This could be as a result of the mixed ancestral adolescents not feeling like they belong to a particular ethnic group resulting in a lack of identity. Our findings suggest that adolescents become more aware of themselves and their bodies, resulting in more negative perceptions, thoughts and feelings about their own body with increasing age. Previous studies have found that when persons of different cultural backgrounds internalize the Western norms of thinness as the ideal, a greater degree of disordered eating is observed Our study found that late adolescent boys and girls associated an obese silhouette with negative attributes such as the “worst, clumsy and unhappy”. This suggests that there is a degree of social stigma associated with female obesity among males and females in urban South Africa. This indicates a greater pressure for adolescent girls to be thinner in order to meet the social expectations associated with leanness including beauty and attractiveness. Previous international studies have shown that weight stigma invokes psychological stress that might lead to depression, low self-esteem and body dissatisfaction Our study also found that an underweight silhouette was associated with weakness, which might reflect an association with HIV/AIDS and tuberculosis (conditions that are very common in South Africa). The study has several strengths. We present longitudinal data and therefore we are able to illustrate the longitudinal change of psychosocial behaviors during adolescent maturation. Few studies in South Africa have included boys in their samples, thus our data on boys is unique and of value in helping to understand societal changes in adolescent attitudes. Further, the tools used in this study  have been validated both internationally and within South Africa. This study also explored multiple methods i.e. EAT-26, body esteem and weight control behaviors, to assess adolescents’ behavior.This study has several limitations; 1) the study only included black African and mixed ancestral participants due to the small numbers of responses from the other ethnic groups. It would be of value to investigate eating attitudes, body esteem and weight control behaviors across all the ethnic groups in South Africa. 2) The EAT-26 and body-esteem tool were self-reported and thus were dependent on participants’ honesty and accuracy. Social desirability bias may occur during the interviews due to interactions with the participants who may then over-report desirable behaviors and underreport undesirable behaviors. Recall bias may also occur in the interview setting due to the immediacy of the response required and the respondent alone has to judge whether the information they have recalled is relevant to the question and how best to respond.Black African girls had a higher BMI and an increased risk of developing eating disorders than girls of mixed ancestral origin. More adolescent girls engaged in weight loss practices than boys, whereas more boys engaged in muscle gain practices than girls. Weight control behaviors are prevalent among South African adolescents. In addition, low body esteem was prevalent in mixed ancestry adolescents. Adolescent boys and girls of both ethnic groups shared similar views on many of the female body silhouette attributes, which are similar to those expected in a western community.


DNA origami used as a platform to study the bleaching behavior of four commonly used fluorophores in cw-STED nanoscopy. Superresolution microscopy is currently revolutionizing optical imaging. A key factor for getting images of highest quality is – besides a well-performing imaging system – the labeling of the sample. We compared the fluorescent dyes Abberior Star 488, Alexa 488, Chromeo 488 and Oregon Green 488 for use in continuous wave (cw-)STED microscopy in aqueous buffer and in a durable polymer matrix. To optimize comparability, we designed DNA origami standards labeled with the fluorescent dyes including a bead-like DNA origami with dyes focused on one spot and a DNA origami with two marks at a designed distance of ∼100 nm. Our data show that all dyes are well suited for cw-STED microscopy but that the optimal dye depends on the embedding medium. The precise comparison enabled by DNA origami nanorulers indicates that these structures have matured from the proof-of-concept to easily applicable tools in fluorescence microscopy. Especially, commercially available superresolution systems, which are fully and automatically aligned and easy to use, pave the way for superresolution microscopy to become a standard technique in biological and medical research.In the last few years, substantial technical developments took place,4 Here we focus on Stimulated Emission Depletion (STED) microscopy, which has already shown to improve resolution by almost two orders of magnitude5 and is compatible with 3D live imaging of cells, tissue and even transgenic animals.8 In STED microscopy, the diffraction limited excitation spot is overlayed with a doughnut-shaped spot of a longer wavelength, featuring a local minimum of zero intensity at the very center. The doughnut-shaped light immediately de-excites fluorophores back into the electronic ground state S0, thereby effectively switching them off. In the very center, where the de-excitation intensity is zero, however, the fluorophores are not switched off, giving rise to a fluorescence signal which is constrained to a subdiffraction-sized spot. By scanning this spot through the sample, a superresolution image is obtained.Nowadays, there are several, in many regards, complementary techniques to break the diffraction barrier.9 Due to the higher requirements of the fluorophore, care has to be taken to collect as many photons as possible before photobleaching. Besides optimizing conditions like scanning speed, the excitation and STED intensities and the filter settings, this includes the choice of the best fluorophore for the applied settings.For getting first class superresolution images, in addition to a well-adjusted microscope, the quality and composition of the sample is of crucial importance. Because the signal is collected from a smaller area than that in conventional microscopy, the labeling of the sample has an extensive influence on the obtainable image.11 Since in STED, unlike in PhotoActivated Localization Mikroscopy (PALM), Stochastic Optical Reconstruction Microscopy (STORM) and related superresolution techniques,16 mostly more than one fluorophore is excited simultaneously, single-molecule bleaching experiments do not reflect the experimental conditions normally applied. Fluorescent beads like YellowGreen-beads are a useful tool for roughly checking if the microscope is properly adjusted, but the signal intensities are much higher than in most real-world samples. Furthermore, they are limited concerning the choice of dyes and control over the exact number of dyes, and the fluorophores are not exposed to the embedding medium, which has a substantial influence on the behavior of the fluorophore. Using antibody-stained samples like actin or single antibodies immobilized on a surface, as is now commonly done in many labs17 due to the absence of a better alternative, does not allow a fair comparison either, because the number of fluorophores attached to the antibody is not precisely defined and varies in most cases between 3 and 8.However, systematic studies on the photophysical behavior of fluorophores under STED conditions, although crucial for the successful implementation of STED microscopy, are to our knowledge missing. One possible reason for this shortcoming might be the lack of an adequate test specimen. For a precise comparison, such a sample has to fulfill several requirements: a well-defined number of fluorophores have to be located in a subresolution-sized area, and be exposed to the embedding medium.18 as a platform for studying the properties of different fluorophores under cw-STED conditions. The idea behind the assembly of a DNA origami is that a long strand of DNA, called a scaffold, can be forced into basically any three-dimensional shape by the addition of short (app. 50 nucleotides long) complementary single-stranded oligonucleotides. These structures can be used as molecular breadboards where small molecules like fluorophores or biotins can be precisely arranged.Therefore, we decided to use DNA origami20 Such DNA origami nanorulers have also been used to validate approaches for counting molecules and as comparison samples for biological structures.21This approach has already demonstrated its potential in the construction of two- and three-dimensional superresolution nanorulers.23 where fluorescent molecules can be bound within a small area of about 300 nm2. A different DNA origami was then used to test the results obtained with the bead like DNA origami: a nanoruler based on a twelve-helix-bundle construct (12HB) as published in 2012 in Science by Derr et al.24 was labeled with 2× 17 dyes at a distance of 100 nm. This nanoruler allows the simple illustration of the resolving power of the microscope.Here, we use a modified DNA origami design for the comparison of four fluorescent dyes which are commonly used for continuous-wave (cw)-STED microscopy using an excitation wavelength of 491 nm and a STED wavelength of 592 nm. This is probably the most commonly used combination for cw-STED at the moment, because it allows for live cell imaging using common and bright fluorescent proteins like Yellow Fluorescent Protein YFP. Because this wavelength combination is also commercially available, the performance of these dyes is of particular importance for biological and medical labs. To screen conditions for cw-STED microscopy, we designed a bead-like DNA origami which offers a practically free choice of dyes and a defined number of fluorophore molecules per mark. The bead DNA origami is based on a rectangular DNA origami structure,STED measurements were performed on a home-built STED microscope. For excitation, a cw laser diode  at a wavelength of 491 nm was focused using a 1.4-NA objective lens . The fluorescence was collected using the same lens and separated from the excitation beam using a custom-made dichroic mirror . It was filtered using a 535/70 bandpass filter  and detected using an avalanche photo diode  with a multimode optical fiber (diameter 62.5 μm) serving as a confocal pinhole.Stimulated emission was accomplished using a cw solid state laser  at 592 nm. Excitation beam and STED beam were overlayed using a custom made dichroic mirror . A doughnut-shaped laser beam was produced using a helical phase ramp .25Sample scanning was realized using a scan head consisting of two galvanometric mirrors . Control over both the hardware and detection was performed using the software Imspector.Unmodified as well as biotinylated DNA oligonucleotides in HPLC purification grade were purchased from MWG Eurofins (http://www.eurofinsgenomics.eu/). Fluorescently labeled oligonucleotides in the PAGE purification grade were purchased from IBA . Prolong Gold Antifade® (PGA) was purchased from Invitrogen . All other chemicals were purchased from Sigma Aldrich .All DNA origamis were functionalized with biotin anchors on the bottom side of the structure. The bead DNA origami carries 6 biotin modifications, the 12HB 5 biotins.2 using a thermal ramp from 95 °C to 4 °C over 2 hours. Excessive staple strands were removed by filtration using an Amicon filter system . The sample was recovered in 3 minutes at 3k rcf.Biotinylated bead DNA origamis were assembled with a 10 fold excess of the staple strands with respect to the scaffold strand  in Tris-acetate-EDTA (TAE) buffer  containing 11 mM MgCl2 using a fast folding method (2 hours at 47 °C).26 Excessive staple strands were removed by filtering with an Amicon filter system . The sample was recovered in 3 minutes at 3k rcf.Biotinylated 12HBs were assembled with a 10 fold excess of staple strands with respect to the scaffold strand  in TAE buffer containing 16 mM MgCl2) and incubated for 2 hours at 37 °C with the complementary oligonucleotide . Samples were purified with an Amicon filter system by filtering three times  with TAE + 11 mM MgCl2. The samples were recovered in 3 minutes at 3k rcf.External labeling was accomplished by mixing the unmodified DNA origamis (approx. 25 μL) with 70 μL of a buffer , incubation of 0.25 mg mL–1 neutravidin for 15 minutes, washing with PBS containing 100 mM MgCl2). Measurements in buffer (e.g. PBS + 100 mM MgCl2) were performed in home-made flow chambers featuring a #1.5 coverslip and a volume of approximately 25 μL sealed with twinsil®  after incubation and washing of the sample.All measurements were performed on bovine serum albumin (BSA)/biotin–neutravidin surfaces (incubation of 1 mg mLe.g. surfaces were dried before application of the embedding media). The embedded samples were dried overnight at room temperature and sealed with twinsil®. Samples in mowiol were prepared the same way as in PGA.For the embedding in PGA, samples were immobilized on #1.5 coverslips and embedded according to the manufacturer's protocol  was added to the surface and incubated for 2 minutes. The surface was then washed five times with TAE buffer to remove unbound samples. The images were recorded in the tapping mode using high dense carbon ultra-short cantilevers . The JPK Data Processing Software was used for analyzing the images.All measurements were performed on a NanoWizard® 3 ultra AFM  in TAE. A freshly cleaved mica surface  was incubated with a 10 mM NiClConfocal bleaching was minimized by adjusting the parameters of the confocal measurement to less than 5% confocal bleaching per scan.Parameters for the confocal measurements were 0.05 ms per pixel scanning speed, 50 nm pixel size for all but Chromeo 488 in Mowiol, Oregon Green 488 in PBS and Oregon Green 488 in Mowiol (100 nm pixel in size).The excitation power was adjusted to 1000 ± 150 photon counts per integrated spot. The excitation power was 3 μW for Alexa 488 in PBS, Abberior Star 488 in PBS; 6 μW for Alexa 488 in PGA, Chromeo 488 in PBS and PGA and Oregon Green 488 in PGA; 9 μW for Alexa 488 in Mowiol, Abberior Star 488 in Mowiol and PGA and Oregon Green 488 in PBS; 12 μW for Chromeo 488 in Mowiol and Oregon Green 488 in Mowiol; laser power was measured in the aperture of the objective lens.All STED measurements were performed under the same conditions to ensure comparability. The scanning speed was 0.05 ms per pixel; the pixel size was 25 nm. The STED laser was turned to full power for all experiments. Full STED power measured in the aperture was 370 mW.Monte Carlo simulations were performed using custom-made Labview software for two two-dimensional Gaussian functions with a given FWHM at a given distance. To ensure comparability, the simulation parameters were chosen according to the real measurement parameters: FWHM was set to 180 nm and 90 nm for the confocal and STED simulations, respectively. The distance was set to 100 nm, the pixel size to 25 nm and the number of photons was 1000 photons per spot. The simulations underlie shot-noise but no background.18 in a slightly modified way that minimizes distortion.23 In a small area of about 300 nm2, the 3′ ends of the staple strands facing the top of the DNA origami were extended by a 24 nucleotide sequence, which serves as an anchor for binding complementary fluorophore-modified oligonucleotides, as can be seen in To compare the performance of different fluorophores for cw-STED microscopy, we designed a so-called bead DNA origami, which is based on a rectangular structure originally described by Paul Rothemund17 the number of fluorophores bound is well defined with the bead DNA origamis, and can be adapted to match experimental conditions.The advantage of the bead DNA origami is the predetermination of the number of fluorophores and the exposition of the fluorophores to the solvent. Unlike in conventional fluorescent beads, the fluorophores are directly accessible to different embedding media, therefore also allowing for the comparison of their influence on the photophysical properties. In contrast to fluorophore-labeled antibodies, which are so far the method of choice for comparing different dyes,For specific binding on the surface opposite to the fluorophore-carrying surface, the lower side of the DNA origamis was labeled with biotin, which binds to a BSA–neutravidin covered surface and therefore gives rise to a predominant direction with the fluorophores facing upwards into solution. In Next, we scrutinized the bead DNA origamis by STED microscopy.To compare the bleaching behavior of these dyes under STED conditions, we took serial images of the respective fluorophore-labeled DNA origamis: after a confocal scan, a STED image of the same region with a maximal STED power was recorded, followed by a second confocal scan.The residual fluorescence intensity in the second confocal image was compared to the first, pre-STED scan. Therefore, spots were detected in the first confocal image, the brightness was determined by spot integrated statistics and compared to the relative spot integrated brightness at the same position in the last confocal scan. Care was taken so that no bleaching could occur in confocal imaging, so all the intensity loss was due to the STED scan.In addition to comparing different fluorophores, we also used different embedding media which are common in (superresolution) fluorescence microscopy: PBS  is a water-based buffer often used for monitoring biological processes, whereas Mowiol and PGA are polymers which allow for the long-term storage of the samples. Oxygen-depleted buffers lead to pronounced blinking; therefore, we did not pursue this approach any further.The intensity of the STED laser was set to the maximum power to achieve the highest resolution obtainable with these settings. The excitation intensity was adapted so that the detected fluorescence intensity in the STED images was the same for all samples. Apparently, the excitation intensity is a key factor for bleaching in STED imaging and therefore has to be tightly controlled when comparing different fluorophores and the dependence of the embedding medium.27 As shown in We compared four different dyes often used in STED microscopy, namely Alexa 488, Abberior Star 488, Chromeo 488 and Oregon Green 488.As can be seen in e.g. PGA for the embedding of fluorescently labeled samples.As discussed before, bleaching strongly scales with the excitation intensity in STED imaging. Generally, it has to be adjusted to obtain a reasonable signal-to-noise ratio in the STED image and therefore strongly depends on the sample and especially the fluorophore density per pixel. In this example, about 7 fluorophores were located in a circular area with a diameter of 20 nm, as determined by comparing the brightness of single fluorophores immobilized on the surface. This number mimics a sparsely labeled immunostained sample. For samples more densely labeled, as is the case for most applications, the number of fluorophores might be higher. For rather dim samples, however, we conclude that if there are no other restrictions on the choice of fluorophore (such as membrane permeability or labeling strategy) the optimal fluorophore depends on the embedding medium: the photostability of Alexa 488 and Abberior Star 488 is clearly the best in PBS whereas Oregon Green 488 bleaches least in PGA and Chromeo 488 is hardly affected by the medium. Therefore, for the imaging of a biological specimen in buffer, Alexa 488 and Abberior Star 488 should be preferred. Oregon Green 488 is less photostable in PBS but as it can penetrate the cell membrane, it should definitely be considered for live cell experiments. Also for the use in embedded samples, our data recommend the use of Oregon Green 488 along with PGA as a medium. Chromeo 488 shows a moderate photostability in all media tested in this study. This consistency might be useful for comparative studies. Additionally, all dyes tested here were least photostable in Mowiol. Therefore, our data recommend the use of 2 can be arranged in different distances along a 12 helix bundle (12HB)  see . With thTherefore, we labeled the 12HB with two external labeling sites with an intermark distance of 100 nm using Alexa 488 and Abberior Star 488, respectively. 29 The software automatically detects the structures, rotates them such that the 12HBs are oriented horizontally and sums vertically over the intensity of all pixels. The resulting one-dimensional intensity histogram is fitted with the sum of two Gaussian functions. The distance between the maxima of the Gaussian functions then provides the distance between the two spots. The distance distribution  STED or other superresolution microscopy.The results indicate that such DNA origami structures can represent a general platform for testing and quantitatively compare new fluorophores, with regard to different conditions like STED intensities, pulse length, wavelength, embedding media


Epigenetics of asthma and allergic disease is a field that has expanded greatly in the last decade. Previously thought only in terms of cell differentiation, it is now evident the epigenetics regulate many processes. With T cell activation, commitment toward an allergic phenotype is tightly regulated by DNA methylation and histone modifications at the Th2 locus control region. When normal epigenetic control is disturbed, either experimentally or by environmental exposures, Th1/Th2 balance can be affected. Epigenetic marks are not only transferred to daughter cells with cell replication but they can also be inherited through generations. In animal models, with constant environmental pressure, epigenetically determined phenotypes are amplified through generations and can last up to 2 generations after the environment is back to normal. In this review on the epigenetic regulation of asthma and allergic diseases we review basic epigenetic mechanisms and discuss the epigenetic control of Th2 cells. We then cover the transgenerational inheritance model of epigenetic traits and discuss how this could relate the amplification of asthma and allergic disease prevalence and severity through the last decades. Finally, we discuss recent epigenetic association studies for allergic phenotypes and related environmental risk factors as well as potential underlying mechanisms for these associations. The term epigenetics was coined by C.H. Waddington in the 1950’s to describe means in addition to genetics to explain cell differentiation ,3.The purpose of this review is to provide allergy/immunology professionals and researchers with a broad, yet easy-to-follow, review of the epigenetic regulation of asthma and allergic disease. The main focus will be on DNA methylation and histone modifications, their relevance in the process of allergic sensitization, their impact on disease heritability and association with environmental exposure and allergy phenotype. MicroRNA, which constitute a distinct epigenetic mechanism, are beyond the scope of this review. Their role in allergic disease has been reviewed recently elsewhere .DNA methylation was the first epigenetic mechanism recognised and the one that is most extensively studied. De novo methylation occurs in response to various cellular stressors and signal by DNA methyltransferases (Dnmt3a and Dmnt3b) which add a methyl group to position 5 of cytosine residues at a CpG site Figure . CpG sitThe palindromic nature of a CpG site is important as it ensures replication of the methylation pattern with each cell division . With DNConversely, a CpG site can be demethylated by oxidation of the methyl group. Physiologically, this process is initiated by the enzyme Ten-eleven translocation (TET) dioxygenase which gives rise to 5-hydroxymethylcytosine and then to 5-formylcytosine and 5-carboxylcytosine . These mThe mechanism by which DNA methylation is associated with gene silencing is still not fully understood. Earlier studies reported that methylation could directly limit the access to transcription factors (TF) . AlthougChromatin, the complex of DNA and nucleic proteins in the nucleus, is another central target of epigenetic modifications. Transcriptionally inactive heterochromatin is packed densely whereas active euchromatin is less condensed Figure . The corCore histones have long N-terminal tails protruding from the nucleosome which can undergo posttranslational modifications that alter their interaction with DNA and nuclear proteins. The standard way of reporting those modifications is by naming the histone, followed by the amino acid, and the modification. For example, H3K4me1 would denote single methylation (me1) of lysine 4 (k4) on histone 3 (H3). Research has shown a strong relation between covalent histone modifications and gene expression . As a geWhile epigenetic changes have been coined as the hallmark of cell differentiation, their importance in other processes is now coming to light. Of note, T cell activation and skewing, which could be viewed as a certain type of cell differentiation, is governed in great parts by epigenetic changes which insure that the clone of a T cell will retain its phenotype  .Th2 skewing is triggered by simultaneous TCR and IL4 receptor activation, which leads to the phosphorylation of STAT6 and expression of Th2 master regulator GATA-3 and Th2 signature cytokines, including IL-4. Th1 differentiation is similarly triggered by simultaneous TCR and IL-12 receptor activation, phosphorylation of STAT4 and expression of Th1 master regulator TBET and Th1 signature cytokine INF-γ, with silencing of Th2 cytokines.In resting CD4 T cells, both IL-4 and IFN-g genes are methylated . Upon alThe main Th2 genes are positioned in the Th2 locus control region (LCR) on chromosome 5 which forms a chromatin hub that interacts with GATA-3 Figure  23]. GA. GA23]. The exact mechanism of DNA demethylation of Th2 genes is still incompletely understood, possibly due to the only recent discovery of the TET enzymes which are responsible for physiological DNA demethylation. In a mouse study, GATA-3 was insufficient to induce DNA demethylation of the RAD50 DHS site 7 in the Th2 LCR, although the process was shown to be dependent on STAT6 . InteresInterestingly, the GATA-3 promoter has been shown to keep its repressive histone modification despite TH2 activation and present a bivalent state with both repressive and activating histone modification . This suT regulatory cells are a subset of T cells which suppress the inflammatory response and thus play an important role in immune tolerance to self and exogenous antigens. Their function and number in tissue has been shown to inversely correlate with allergic phenotypes and their importance in allergic disease has been well described .FOXP3 is the master regulator for regulatory T cells (Treg)  which caIn the thymus, tTregs are induced by TCR engagement with self-peptide major histocompatibility complex with specific strength and duration. The subsequent NF-κB signaling induces permissive histone modification (H3K4me1) at the CNS3 and potentially initiates chromatin remodeling in the FOXP3 locus through the c-Rel subunit . In paraBesides Tregs, activated T cells also express FOXP3 upon TCR engagement . Howeverscrufy mice, but had maternal-fetal conflict and inflammatory disease at the mucosal interface, suggesting a specific role for pTregs in acquired tolerance to exogenous antigens [In contrast to thymic-derived Tregs, generation of Tregs from peripheral naïve T cells is favored by suboptimal TCR stimulation in the presence of TGF-β . TGF-β pantigens . The genantigens . While iantigens .Development of allergy and asthma is determined by interplay between environmental and inherited factors, the later accounting for over half of the risk . InteresGenetics also fail to explain the sudden rise in allergies and asthma as even with significant selection pressure, any change in population genetics would necessitate multiple generations to occur. Epigenetic changes on the other hand can be induced much more rapidly with various environmental exposures and, like with genetics, these changes can be passed down from parents to offspring. There are several ways in which epigenetics can influence phenotype inheritance, including gene imprinting, in utero modifications and transgenerational inheritance.Asthma and atopy are complex genetic traits meaning that the phenotype is the result of the interaction of multiple genes each with their own Mendelian pattern of inheritance. The expected result is that no clear pattern of inheritance would be discernable, with risk from both parents being very similar overall. In reality, the risk for allergy and asthma inherited from the mother is up to 5 fold greater than the paternal risk -51.The discrepancy in parental risk could be explained in part by parental imprinting. Parental imprinting is a process by which some genes are epigenetically silenced during gametogenesis in a parent-of-origin-specific manner, which results in only one allele being expressed for the imprinted loci. The best example probably consists of polymorphisms of FcϵR1-β which are only associated with atopy when the risk allele is inherited from the mother in multiple cohorts ,52,53.A more recent study showed that maternal but not paternal atopy predicted the expression profile of 18 cytokines and chemokine in the airway mucosal fluid of newborns . HoweverIt has been well described that through development, with the shift from pluripotent stem cells to well differentiated specialized cell types, chromatin becomes increasingly repressed by histone modifications and less activated by permissive histones . However6), which was not exposed to the diet, actually exhibited a further increase in fur pigmentation, with normal color returning only with the second generation off therapy (F7). Mice from the fifth generation had been weaned and put on normal control diet before mating, showing the effect of the diet did not take place in utero. Rather, it suggests that germ cells exposed to excess methyl donors within the developing F5 females retained a memory of the methyl donor effect, manifested in F6 mice [In their canonical experiment with the agouti mouse model, Morgan et al., fed mice with a methyl-donor rich diet that favored the methylation of the agouti gene, which codes for signalling peptide in mice, which affects coat colour pigmentation. Not only did this influence their immediate offspring’s fur color, but throughout five generations with the exact same diet, the fur coat would get darker and darker showing transgenerational transmission of the phenotype, which is not only inherited but augmented . The exp F6 mice .In utero diet supplementation with methyl donors has similarly been shown to increase allergic disease in a mouse model . The F1 Transgenerational inheritance of epigenetic traits is extremely interesting from an epidemiologic point of view as it provides a new hypothesis for the persistently increasing prevalence of allergy and related disorders. If the recent increase had been due to a change in environment, prevalence should have increased at once and remained stable as long as this environment remained the same. However, if the new environment induces epigenetic changes, a transgenerational amplification of the atopic phenotype would be expected even with stable exposure Figure . FurtherAt this stage, most of the epigenetic literature on asthma and allergy consists of association studies -88. As wFigure An exciting aspect of epigenetics is that it sheds a new light on previous genetic association studies. Since epigenetic marks have the potential to silence a gene, it is to be expected that it can modulate the effect of an underlying polymorphism on disease risk. For example, IL-4R single-nucleotide polymorphism (SNP) rs3024685, which is not associated with asthma on its own, carries a significant risk for the disease when controlled for IL4-R methylation .Similarly, the 17q12-21 which carries 2 polymorphisms associated with maternally inherited risk for asthma was found to be highly methylated in adult males . In factGenetic polymorphisms can also in turn affect epigenetics regulation of gene expression at a given locus ,75. MoraAlthough our understanding is still superficial, there is an increasing amount of literature looking into the mechanisms by which allergic phenotypes and environmental exposures could be associated with specific epigenetic changes.In lung biopsies, the HDAC: HAT ratio has been shown to be lower in asthmatic samples and to correct with treatment -96. ThisEnvironmental tobacco smoke supresses the expression and activity of HDAC2 and HDAC3 ,105. ExpAlthough a diet rich in methyl-donor nutrients has been shown to promote DNA methylation and to induce an allergic phenotype in mice, the same has yet to be shown for humans . MaternaEpigenetics is an exciting new field in allergy and asthma research that has strongly evolved in the last decade. Recent studies shed a new light on the pathogenesis of this complex group of disease, not only with regards to gene-environment interaction but also with regards to the model of inheritance and its epidemiological implications. The field is still at its infancy stage and more work needs to be done to dissect the epigenome of asthma and allergy and to better understand its underlying mechanisms.DNMT: DNA methyltransferase; HAT: Histone acetyltransferase; HDAC: Histone deacetylase; TET: Ten-eleven translocation; DHS: DNAse I Hypersensitivity site; TSDR: Treg-specific demethylated region; LCR: Locus control Region; TCR: T cell receptor; SNP: Single nucleotide polymorphism.The authors declare that they have no competing interests.PB performed litterature review and wrote the manuscript. NK reviewed the manuscript. Both authors read and approved the manuscript.


In this report we have employed multiparametric phosphoflow cytometry, as well as a novel generalization of the Overton algorithm from one- to two-dimensional unimodal distributions to simultaneously monitor the effect of low level Hg2+ intoxication on activation of ERK and several upstream elements of the BCR signaling pathway in WEHI-231 B cells. We have found that, after exposure to low levels of Hg2+, only about a third of the cells are sensitive to the metal. For those cells which are sensitive, we confirm our earlier work that activation of ERK is attenuated but now report that Hg2+ has little upstream effect on the Btk tyrosine kinase. On the other hand, we find that signaling upstream through the Syk tyrosine kinase is actually augmented, as is upstream activation of the B cell signalosome scaffolding protein BLNK.It has been suggested that environmental exposures to mercury contribute to autoimmune disease. Disruption of BCR signaling is associated with failure of central tolerance and autoimmunity, and we have previously shown that low levels of Hg Mercury is a potent immunotoxicant. Early experiments demonstrated that exposure to high or moderate levels of mercury induced immune cell death through either necrosis or apoptosis. Fortunately, exposure to high levels of mercury is no longer common, but large segments of the population continue to be exposed to low levels. In contrast to higher level exposures, low level exposure to mercury is not linked to immune cell death, but rather to immune disorders characterized by immunoproliferation and autoimmunity , 2.2+), and so low-level Hg2+ exposure is now a source of concern [2+, so that over time mercury burdens in organs such as brain or spleen normally encompass both inorganic and organic species, even under conditions where exposure has been limited to organic mercury [2+ produced as a result of the metabolism of organic mercury, but not residual organic Hg, is the mercury species directly responsible for mediating an autoimmune inflammatory response. In other words, with respect to autoimmune disorders and inflammatory processes Hg2+ is the active metabolite [Today in the US most exposure to mercury is the result of consumption of seafood contaminated with organic mercury. It has also been reported that high fructose corn syrup, a major constituent of most processed foods, often contains surprisingly high levels of inorganic mercury  of Hg2+ with autoimmunity suggests that Hg2+ could potentially interfere with signal transduction pathways in immature B cells in a manner so as to compromise central tolerance.In principle, autoimmune disorders might result from a genetic defect in, or environmental interference with the control of central tolerance, the mechanism whereby the immune system neutralizes self-reactive immature lymphocyte clones. In immature B cells the B cell receptor (BCR) complex signaling pathway is of critical importance for this process , 11. In 2+ interferes with the BCR signaling pathway in triggering growth arrest and apoptosis [2+ attenuates activation of ERK1/2 during BCR signaling [2+ acts on an upstream element of the BCR signal transduction pathway to initially suppress ERK activation and subsequently growth arrest and apoptosis after BCR engagement of an agonist ligand [WHEI-231 is a well-characterized mouse B cell line that exhibits many of the characteristics of immature B cells. In particular, in contrast to mature B cells for which stimulation of the BCR is associated with mitogenesis and increased proliferation, WEHI-231 in a process similar to immature B cells undergoes growth arrest and apoptosis in response to BCR stimulation , 14. We poptosis . Furtherpoptosis . We haveignaling , thus sut ligand .2+ intoxicated (exposed) and BCR stimulated WEHI-231 cells we have gained insight into the mechanism whereby Hg2+ at low exposure levels interacts with upstream elements of the BCR signal transduction pathway in B cells.Phosphoflow cytometry is a relatively new technology which has been shown to be far superior to traditional western blotting technology in investigating phosphorylation dependent signaling pathways in immune cells, and in particular B cells –21. In t2 (≥99.5% pure) was obtained from Aldrich Chemicals .HgClμM 2-mercaptoethanol, and 1X antibiotic antimycotic solution  in a humidified 5% CO2 atmosphere. Cells were passaged twice a week at a concentration of 1 × 105 cells/mL. Cell viability was monitored visually by Trypan blue exclusion.WEHI-231 cells were obtained from American Type Culture Collection . They are maintained in RPMI 1640 containing L-glutamine  and supplemented with 10% fetal bovine serum , 50 Polyclonal goat affinity-purified antibody to mouse immunoglobulin mu chain was purchased from MP-Biomedicals-Cappel, Solon, OH. Fluorescently labeled antibodies used for cytometric analysis of phosphorylated ERK1/2, Syk, BLNK, and Btk were purchased from BD Biosciences .6 cells/mL in the presence or absence of varying concentrations of HgCl2 in polypropylene tubes. Subsequently, 1 × 106 cells were added to tubes containing goat anti-mouse immunoglobulin specific to the mu chain. Cells were incubated for the specified period of time at 37°C. At the end of stimulation , the cells were fixed by adding an equal volume of 4% paraformaldehyde, pH 7.4, and incubating for 10 minutes at 37°C. The cells were diluted in Dulbecco's phosphate-buffered saline, pH 7.4, pelleted by centrifugation, resuspended in 90% methanol, and then incubated on ice for at least 30 minutes to permeabilize the plasma membrane. Following permeabilization the cells were pelleted to remove the methanol and then washed twice in sample buffer . The cells were resuspended in an antibody cocktail containing fluorescently labeled antibodies specific for the phosphorylated residues of ERK1/2, Syk, BLNK, or Btk according to the manufacturer's instructions. The cells were incubated for 1 to 2 hours on ice and then washed 3 times with sample buffer. The cells were kept in the dark at 4°C until being analyzed on a Beckman-Coulter Cyan ADP flow cytometer.WEHI-231 cells were counted, washed in serum-free RPMI medium, and resuspended at 3 × 10For all experiments, 10,000 events were acquired per sample and the data were initially analyzed using Sumit  and/or FlowJo  software. Additional data analysis and graphing was done using Excel  and Sigma Plot  software. For any fluorescently labeled cell population the Mean Fluorescence Index (MFI) is defined as median fluorescence intensity of the population.2+ on BCR signaling. Accordingly, we first employed phosphospecific flow cytometry to investigate the kinetics of BCR activation of ERK 1/2 in control WEHI-231 cells. ERK1/2 are serine/threonine kinases which in B cells are central to the BCR signal transduction pathway and upon BCR signaling become activated by dual phosphorylation of a canonical threonine/tyrosine motif [μg/mL) to stimulate the BCR. At timed intervals the cells were probed with a fluorescently labeled antibody specific to dually phosphorylated ERK 1/2 (anti-pERK), or else an isotype control antibody. The level of ERK 1/2 activation was then determined by phosphospecific flow cytometry. In In our previous investigation of the effect of mercury on BCR signal transduction we employed western blotting technology to show that ERK activation was impaired . Howeverne motif . In FiguThe kinetics of ERK activation resulting from BCR stimulation are perhaps more easily appreciated in σ is the standard deviation of the distribution, and n the number of independent measurements. For the distributions shown in σ's are of the order of the MFIs, but since each distribution represents about 10,000 cells, the standard errors are of the order of 1% of the MFIs.Generally speaking, because of the large number of cells examined, population differences in MFI determined by flow cytometry tend to be highly significant. For instance the standard error of the median for a normally distributed variable is given by 1.25 Another way of appreciating the significance of the MFI measurements in μg/mL to stimulate the BCR. This initial 25 μg/mL figure was based upon our previous experience with this system [s system . However2+ on ERK and upstream elements of the BCR pathway during signaling, so for control purposes we measured the level of activated ERK and Syk (pERK and pSyk) which was induced by Hg2+, alone, in the absence of specific BCR signaling, to those induced by anti-mu through binding to the BCR. Accordingly WEHI-231 cells were treated with  Hg2+, and, at various time points after addition of Hg2+, cells were stained with different fluorescently labeled anti-pERK and anti-pSyk. We have previously shown that treating WEHI-231 cells with concentrations of 5 μg/mL HgCl2 for 10 minutes results in cellular burdens of Hg2+ of the order of 1 ng/107 cells, and this cellular burden maximally impacts BCR signaling [ in vitro Hg2+ burdens are not toxic to WEHI-231 cells (NS), while similar in vivo Hg2+ burdens are not toxic to mouse spenocytes [2+. Five minutes later at the 15 minute mark these cells were likewise assayed for pERK and pSyk by phosphospecific flow cytometry. Untreated cells were also labeled with isotype controls to pERK and pSyk antibodies and assayed by flow cytometry.The tyrosine kinase Syk is an important element of the BCR signaling pathway upstream of ERK. During BCR signaling, activation of ERK is known to be dependent upon activation Syk, and, like ERK, Syk activity is increased by tyrosine phosphorylation , 23. In ignaling . We haveenocytes . pERK an2+, which are displayed as line plots. Compared to anti-mu induced BCR signaling, Hg2+ alone has little effect on ERK or Syk.The results for pERK and pSyk are shown in Figures 2+ on BCR signaling upstream of ERK. BCR signaling is a complex process that is still not fully understood. However it is generally accepted that shortly after antigen engagement of the BCR complex that multimolecular cluster formation begins and assembly of the B cell receptor signalosome ensues [We next investigated the effect of Hge ensues , 25. Fore ensues .2+ for 5 minutes and then stimulated or not with anti-mu. At timed intervals cells were fixed and levels of activated ERK (pT202/pY204), Syk (pY436), Btk (pY551), and BLNK (pY84) simultaneously determined by multicolor phosphospecific flow cytometry. In each plot cells which have been preincubated with Hg2+ are compared to cells which have not. The results are expressed as the difference between the MFI of cells which have or have not been treated with anti-mu as a function of time after addition of anti-mu.One of the advantages of phosphospecific flow cytometry is that it provides the ability to simultaneously monitor multiple elements of a phosphorylation dependent signaling pathway in individual cells. Accordingly in Figures 2+ attenuates the normal ERK signal realized during BCR signal transduction, while 2+ actually enhances the strength of the Syk signal. On the other hand BTK appears unaffected, as Hg2+ does not seem to have any appreciable effect on either the magnitude or kinetics of the BTK response. Finally phosphorylation of BLNK, like that of Syk, is enhanced by Hg2+.2+ depresses and augments the activation of ERK and Syk, respectively, it is difficult to discern if there are differences in kinetics. To evaluate whether Hg2+ altered the kinetics of ERK or Syk activation during BCR signaling we replotted the data of Figures 2+. This figure demonstrates that under control conditions the kinetics of ERK activation is in phase with that of Syk activation. In 2+. Here it is clear that although Hg2+ may decrease the magnitude (phosphorylation) of the ERK response while increasing the magnitude (phosphorylation) of the Syk response, exposure has little effect on the phase relationship between activation of the two kinases during BCR signal transduction.Although Figures 2+ on BCR signal transduction was then essentially determined by comparing these numbers among differentially treated cell populations. However the initial data reduction to MFIs means that Although we had earlier pointed out that one of the advantages of phosphospecific flow cytometry is that it potentially permits analysis of signaling events on the single level, we did not take advantage of this in the analysis accomplished in 2+ on ERK and Syk activity during BCR signaling but forego initial reduction to MFIs in order to preserve single cell information. Accordingly y-axis) and pERK (x-axis) for the control population of WEHI-231 cells which have not been treated with Hg2+ or anti-mu. Here the number of cells expressing any particular pSyk, pERK value pair is proportional to the density of the plot in that region. Since 2+. Each histogram is divided into quadrants, and the percent of the cell population falling into each quadrant is indicated.In low, pERKlow and only 0.18% pSykhi, pERKhi. Utilizing the identical quadrant settings, upon anti-mu stimulation this changes after 5 minutes to 73.06% pSykhi, pERKhi and 5.18% pSyklow, pERKlow. As we already determined in 2+, the profile of pSyk and pERK is altered after anti-mu stimulation of the BCR from what is found for cells which are not intoxicated. This is reflected in 2+ intoxicated cells the percentages of cells in each of the quadrants do not drastically differ from those of cells which are not intoxicated, but nevertheless the bulk of the distribution seems to move to higher pSyk and lower pERK values.In 2+ exposure on the population shift that occurs simultaneously with respect to cellular levels of pERK and pSyk after BCR stimulation, and which has been visualized as two-dimensional histograms. Although we are working with two-dimensional histograms, just as for one-dimensional histograms, the effect of Hg2+ can be analyzed by subtracting the histogram for nonintoxicated cells  has little direct effect on ERK. However, at these levels Hg2+ interferes with BCR signal transduction so that activation of ERK is attenuated [, and 4(a) are in complete support of these previous findings. Since there is a direct connection between suppression of BCR signals in immature B cells and increased mature autoimmune B cell clones [2+ attenuation of ERK activation implies that Hg2+ intoxication may permit some self-reactive B cell clones to escape central tolerance and contribute to autoimmunity or autoimmune disease upon maturation.We have previously shown that intoxication of WEHI-231 B cells with low and nontoxic levels of Hgtenuated . These el clones , to the 2+ seem to have little direct effect on ERK implies that the target of Hg2+ is upstream of ERK, and that signaling through at least some elements of the BCR signal transduction pathway upstream of ERK should also be attenuated. Syk is one such possible element. It is a central tyrosine phosphokinase operating upstream of ERK in the BCR signal transduction pathway, and as such is necessary for the integrity of the BCR signal and the eventual elimination of self-reactive immature B cell clones [2+ intoxicated WEHI-231 B cells utilizing western blot technology was inconclusive as to whether Hg2+ intoxication attenuated Syk activation [2+ enhances the activation of Syk.The fact that in both of these experiments and in our earlier work low levels of Hgl clones , 23. Ourtivation . However2+ intoxication on their activity during BCR signaling  signaling. In particular, in T cells TCR cross-linking leads to the transient activation of ERK, much like BCR cross-linking leads to the activation of ERK in B cells. We have found that in T cells intoxicated with low levels of Hg2+ ERK activation is attenuated during TCR signaling [2+ is associated with attenuation of ZAP-70, as well as LAT, the scaffolding protein homolog of BLNK [On the other hand, the finding that Syk activation is enhanced, while downstream activation of ERK is attenuated by Hgignaling . In T ce of BLNK .2+ dependent attenuation of ERK activation is correlated with enhancement of Syk activation, these experiments only speak of what is happening on a population level. The question remains whether this correlation strictly holds on the cellular level. Utilizing the identical dataset used to generate While Figures 2+. Again, most cells still seem to be responding to anti-IgM as in 2+ intoxication leads on average to measurably lower pERK and higher pSyk levels. To assess the effect of Hg2+ intoxication on BCR signaling on the cellular level, we have utilized a generalized Overton algorithm to subtract the histogram in 2+ intoxicated populations after BCR signal induction than in control populations. Likewise the negative areas of the histogram (represented by darker hues) represent cellular positions which become less populated as a result of Hg2+ intoxication. They essentially represent areas of the histogram from which cells (in the control) will move from as a result of Hg2+ exposure.When one-dimensional histograms are subtracted it is common practice to display only the one-dimensional histogram of the resulting positive cells. However for the two-dimensional subtracted histogram in 2+ intoxicated cells suppressed ERK activity is associated with enhanced Syk activity. Significantly, in 2+ intoxicated cells respond to BCR cross-linking with depressed ERK and enhanced Syk activation when compared to control cells. The remaining 62.4% of Hg2+ intoxicated WEHI-231 cells respond to BCR cross-linking by enhancing pERK and pSyk, just as they would as if they had not been exposed to Hg2+.In 2+ augments activation of Syk while it simultaneously attenuates downstream activation of ERK is at first glance a bit puzzling. However a recent report suggests that in B cells there are two functionally and topologically distinct ERK activation pathways [2+ was to depress the calcium signal by interfering with an upstream regulator of Icrac channels, then it would be possible that ERK activation through this pathway could still be attenuated, even though upstream signaling through Syk was augmented. In any event it seems clear that Hg2+ initially interacts with a B cell target or targets upstream of Syk, possibly including Icrac channels.The overall finding that Hgpathways . Althougpathways , 23, but2+, signal transduction initiated by the BCR is altered. Analysis on the single cell level shows that while activation of ERK is suppressed, upstream activation of Syk is enhanced and phosphorylation of BLNK is increased. On the other hand, activation of Btk is unaffected. As WEHI-231 is a model for immature B cells, suppression of ERK activation in these cells suggests that low level exposures to Hg2+ may also interfere with central tolerance in primary immature B cells. We suggest that interference with central tolerance may be one of the mechanisms connecting low level mercury intoxication to autoimmune disease.After exposure of WEHI-231 B cells to low levels of Hg