The two breast measurements were moderately correlated (Spearman correlation coefficient = 0.65). Age, BMI, marital and working status were independently associated with bust line and total breast area. In the multivariable analyses, the most pronounced effects were observed for BMI (24.2 cm difference in bust line and 39.4 cm2 in breast area comparing women with BMI ≥30 kg/m2 to BMI <20 kg/m2). Ethnicity was a positive predictor for total breast area, but not bust line.Breast size as a risk factor of breast cancer has been studied extensively with inconclusive results. Here we examined the associations between breast size and breast cancer risk factors in 24,353 Asian women aged 50 to 64 years old enrolled in a nationwide mammography screening project conducted between October 1994 and February 1997. Information on demographic and reproductive factors was obtained via a questionnaire. Breast size was ascertained as bust line measured at study recruitment and total breast area measured from a mammogram. The average bust line and total breast area was 91.2 cm and 102.3 cm Apart from being a potential marker of health and fertility from an anthropological point of view, breast size has been shown to affect women’s quality of life and well-being in many ways – socially, psychologically and physically3. For example, several studies on sport and exercise participation in adolescent girls revealed large breasts to be a cause of physical strain and a major reason why they do not take part in physical activity5. Back problems are also more common among women who are more well-endowed6. In terms of breast health, breast size has been studied extensively as a risk factor of breast cancer, but the results are inconclusive7.Breast development occurs during puberty in girls, after which the enlarged breasts are retained throughout adulthood. Breast size in adult women is associated with perceived femininity and is sexually important in many cultures8. Several genome-wide association studies have also identified common genetic variants associated with breast size10. Asian women are typically less well-endowed12 than women of Caucasian ancestry. Several demographic, reproductive and lifestyle factors have been suggested to influence breast size, but most of these links are anecdotal in nature. Few studies have examined the associations rigorously14, and fewer have been performed in Asian populations. Here, we aim to identify non-genetic determinants of breast size using a large cohort of women in Southeast Asia. In addition, we examined how the candidate predictors of breast size affects the different tissue components that make up the breast.Breast size is a highly heritable trait. A twin study previously estimated the heritability of bra cup size to be 56%15. From 166,600 women identified to be eligible in a comprehensive population registry for randomization to breast screening or observation, 69,473 women were randomly chosen and offered free mammography screening. Women who consented to take part in the study were interviewed by a nurse or study coordinator and asked to complete a questionnaire regarding their demographic and anthropometric measures as well as family and reproductive history before being invited for a one-time mammogram examination. Participation rate (eligible women who took up the invitation for free mammography screening) was 41.7%.The study population was made up of Singaporean women aged 50 to 64 years old on 1 October 1994 who enrolled in the Singapore Breast Cancer Screening Programme (SBCSP). SBCSP was a prospective nationwide mammography screening project conducted between October 1994 and February 1997, which has previously been described in detailEligible participants were first asked five questions: 1) Have you ever been diagnosed with breast cancer? 2) Have you ever been diagnosed with other cancer (except non-melanoma skin cancers)? 3) Have you had a mammogram done in the past one year? 4) Have you had a breast biopsy in the past one year? 5) Are you likely to be pregnant? Women were excluded if they answered “Yes” to any of the five questions. Of 28,672 women who returned questionnaire data, mammographic density measurements were available for 24,363 participants (85.0%). Mammograms were either unavailable, or the image quality of the scanned mammogram was too poor for the accurate assessment of mammographic density for 4,309 women. Ten additional women were excluded due to missing information on bust line, weight or height. The final analytical dataset consisted of 24,353 participants.16. Briefly, original film mammograms from SBCSP were retrieved and digitized between February 2012 through February 2013 using the 2905 Laser Film Digitizer , with a sampling pitch of 50 micrometers and a gray-scale contrast resolution of 12 bits. Total mammographic breast area and dense area were then estimated using a fully-automated thresholding method described by Li et al.17 for mammograms from each breast before mean values were computed. Mean nondense area was derived by taking the difference between mean total breast area and mean dense area. The concordance correlation coefficients for total and absolute dense breast area measured by Li et al.’s method and the gold standard method (Cumulus) were 0.95 (95% CI: 0.94–0.95) and 0.85 (95% CI: 0.83–0.87), respectively.Bust line is measured as the chest circumference of the fullest part of a woman’s breasts at study entry. The mean of two measurements of the bust line were taken. Total breast area, dense area and nondense area were measured from mammograms taken from the medio-lateral oblique view Fig. . Dense a2) was computed based on self-reported measures of height and weight.Information on self-reported demographic , reproductive and hormonal factors (number of children [“How many deliveries (of at least 6.5 months or 28 weeks of gestation) have you had?”], ever breastfeeding , oral contraceptive use , ever hormone replacement therapy use ) were obtained from the SBCSP questionnaire at the time of breast size ascertainment (mammography). Participants were also asked about family history of breast cancer  in the questionnaire. Body mass index  from the multivariable adjusted analyses. The transformation takes the logarithm of the absolute value plus 1. If the original value was negative, the sign was preserved by multiplying the transformed value by −1. All of the statistical tests were 2-sided, and the differences were considered statistically significant at a P-value of <0.05.Spearman correlation coefficients from non-parametric rank-based tests between bust line, total breast area, nondense area and dense area were calculated and correlations were visualized using scatter diagrams. To correct for non-normality, total breast area and dense area were square-root transformed , two-thirds reported ever breastfeeding. Six in ten women had never taken oral contraceptives. The ever use of hormone replacement therapy (HRT) was low at 13.6% . Less than 3% of the women reported that a first-degree relative have had a breast cancer. Women in the SBSCP study had, on average, a mean bust line of 91.2 cm (standard deviation [SD] = 8.1) at study entry. The average difference between two bust line measurements used to compute the mean bust line was −0.047 (0.44) cm. The mean total breast area, nondense area and dense area captured on mammograms were 102.3 (20.8), 81.3 (24.3) and 21.0 (13.2) cm2, respectively.The characteristics of the 24,353 SBCSP participants included in our study are described in Table The highest correlation was found between total breast area and nondense area (Spearman correlation coefficient = 0.80), followed by total breast area and bust line (Spearman correlation coefficient = 0.65) Fig. . Nondens2 in total breast area and 53.1 cm2 in nondense area between women with BMI ≥30 kg/m2 compared to women with BMI <20 kg/m2), ethnicity , and number of children  . Age ethnicity and menopausal status remained positively associated with total breast area and nondense area with similar effect sizes (Z scores), but not bust line. Number of children was not independently associated with bust line, although having three or more children significantly decreased total breast area. In a subset of 21,073 women with children, older age at first birth remained significantly associated with smaller bust line, smaller total breast area, smaller total nondense area and bigger dense area in the multivariable model . In this large cohort of Singaporean women invited for screening mammography, fatty tissue reflected as nondense area constituted a large proportion of the total breast area captured on mammograms. Variables found to remain significantly associated with bust line, total breast area, and nondense area included BMI, marital status, and working status. Age, ethnicity, and number of children were significant predictors of breast area and nondense area, but not bust line.7. The main advantage of determining breast size from mammograms over other methods is the objectivity and a common unit of measurement (cm2). “Vanity sizing”, a phenomenon where different bra manufacturers follow different measurements for identically labelled bra sizes, makes it difficult to generalize study-specific findings to other populations18. Bust line, which uses a formal unit of measurement (cm), is arguably more objective than bra cup or band size, but is subjected to confounding by chest circumference below the bust line. Given the low correlation between bust line and total breast area found in our study, it is therefore difficult to compare between studies that look at different measures of breast size. Nonetheless, the general relationship between factors and breast size remains similar.Breast size ascertainment is not an exact science. Previous studies have used self-reported breast size, bra cup size, chest circumference and breast size measured from mammograms as approximations of breast size19. Marriage rates in European screening mammography populations vary, ranging between 50–75%21. Seventy percent of the women in this study (50 to 64 year old) had at least three children, while the proportion was less than a third of the attendees of screening mammography in Sweden between 1988 to 199721. Only 32.5% of the Singaporean women in this study were gainfully employed, compared to ~80% of the women described in the Swedish screening mammography study21. Prevalence of HRT use is also known to be lower in Asian women compared to Caucasian women22.This study, which comprises women aged 50 to 64 years old recruited from 1994 to 1997, is made up of an Asian population of ethnic Chinese, Malay, Indian and Eurasian women with distinct demographic differences from Western screening mammography populations that have previously been studied. For example, 80% of women in this study were married, a much larger proportion than the ~65% marriage rate observed for predominantly White screening mammography populations in North America14. Firstly, the strongest predictor of breast size was BMI, which has been shown previously to share a significant genetic component with breast size in Caucasian women8. Secondly, according to a European population-based study conducted on women attending breast screening, one in five women experienced an increase in breast size after menopause14, which corroborates our finding on the association between higher age and larger breast area.Our findings on determinants of breast size in Singaporean women agree with what has been published in literature in Caucasian populations25. Breast enlargement and loss of firmness are the most common changes experienced by women post-pregnancy25. A German study estimated an increase of ~100cc regardless of the initial breast volume before pregnancy23. Breast size and stiffness changes were not associated with breastfeeding26. To our knowledge, the effect of pregnancy and lactation on breast size many years after childbirth has not been examined. In our study, while number of children was not an independent predictor of bust line after adjusting for other factors, having three or more children was significantly associated with smaller total breast area. In addition, older age at first birth among women with children was associated with reduced breast size. This finding is in contrast to the enlargement effect observed directly after pregnancy by other studies25. Future studies investigating longitudinal changes in breast size over a woman’s life time will be needed to clarify these conflicting results.It is commonly known that childbearing can change breast size and appearance27. Dense tissue is associated with an increase in breast cancer risk, while nondense tissue is associated with a decrease in breast cancer risk28. It is likely that the hypothesized association between breast size and breast cancer risk pursued by many studies7 is an interplay between age, BMI, dense and nondense components of the breast. While ethnic variations in percent mammographic density, absolute dense and nondense area have been reported for a comparable Southeast Asian study29, breast size differences among ethnic groups have not been reported. However, Mariapun et al. observed that adjusted nondense area  was significantly lower in Chinese women compared to their Malay and Indian counterparts29. In a study of ethnic differences in percentage body fat (which influences proportion of nondense tissue) in Singapore, Deurenberg-Yap showed that for the same age, gender and BMI, Indians have a higher percentage body fat (and hence larger proportion of nondense tissue) than Malays, who in turn have a slightly higher percentage body fat than Chinese30. After controlling for age and gender, Chinese, Malays and Indians were all shown to have considerably higher percentage body fat for the same BMI than Caucasians30, which is consistent with the corresponding breast cancer risks observed for different ethnic groups in Singapore and Asians in general compared to Caucasians31.Total mammographic breast area is made up of dense and nondense components, which are known to be associated with breast cancer risk in different directionset al. have previously published a study on the determinants of mammographic features  in a subset of 803 Chinese women in the SBCSP study32. We have then expanded the study by retrieving, digitizing and measuring mammograms for more than 24,000 participants, and is now one of the largest Asian study on the determinants of breast size and breast composition. We compared two different measures of breast size in this study (bust line and breast area on mammogram) and also examined the proportions of dense and nondense components.A strength of the study is the size of the study population. Heng 33. In addition, the mammograms used in this study were collected before the adoption of digital mammography and development of methods to measure volumetric breast density. Hence, volumetric breast size, which has the advantage of taking into account breast thickness (i.e. variation due to different levels of compression), was not examined34. Information on previous breast augmentation or reduction surgery was not collected.While it is of merit to derive objective breast size measurements from mammograms as a large number of women will invariably get a mammogram taken for screening or diagnostic purposes, a resulting limitation is that the study population will be largely restricted to older women who are of mammography screening age. The use of a mediolateral oblique (angled) view, instead of a cranial-caudal (from above) view, may also impact the results. However, mammographic density measurements obtained from the two different views have been shown to be highly correlated (r > 0.8)Although measurements of breast size, total breast area and bust line were only moderately correlated, the determinants of breast size found for Singaporean women were similar to those observed for Western populations. Local ethnic differences in breast size, measured as total breast area on a mammogram, persisted even after adjusting for BMI and other factors.Supplementary File


Daphnia magna (three different clones). Microplastic particles of ~40 μm were supplied at a low concentration (1% of the food particles) leading to an average of ~30 particles in the digestive tract which reflects a high microplastic contamination but still resembles a natural situation. Neither increased mortality nor changes on the morphological  or reproductive parameters were observed for adult Daphnia. The analyses of juvenile Daphnia revealed a variety of small and rather subtle responses of morphological traits . For adult Daphnia, alterations in expression of genes related to stress responses  as well as of other genes involved in body function and body composition (i.e. SERCA) were observed already 48h after exposure. We anticipate that the adverse effects of microplastic might be influenced by many additional factors like size, shape, type and even age of the particles and that the rather weak effects, as detected in a laboratory, may lead to reduced fitness in a natural multi-stressor environment.Microplastic particles are ubiquitous not only in marine but also in freshwater ecosystems. However, the impacts of microplastics, consisting of a large variety of synthetic polymers, on freshwater organisms remains poorly understood. We examined the effects of two polymer mixtures on the morphology, life history and on the molecular level of the waterflea  Microplastic particles, most often defined as particles of less than 5 mm in size, are ubiquitous in the marine environment –3, and cTo date several potential harms of microplastic particles were described. The release of harmful substances from microplastic particles into the digestive tract is postulated to be one of the major threats . The oriDaphnia magna at the morphological, life history and molecular level. Three clonal lines were used to further test if possible responses differ among clones. These D. magna clones were exposed to a large range of polymers commonly detected in freshwater ecosystems [Daphnia genome is available [D. magna is primarily a filter feeder, they also graze on sedimentary algae. The latter feeding mode is used especially in the littoral zone of lakes or small ponds [D. magna was already shown in laboratory experiments [2, max: 0.22 micro- and macroplastic particles/m2, median: 0.04 micro- and macroplastic particles/m2, reviewed in [Daphnia were exposed to a considerable low concentration of microplastic particles to food particles (1% microplastic particles in the food). This led to an ingestion rate which reflects a high microplastic contamination but might still resemble a natural situation and therefore allows for a realistic assessment of the effects of microplastics.The aim of this study was to establish baseline knowledge on the effects posed by environmentally relevant concentrations of microplastics on the freshwater cladoceran waterflea osystems –13, 18 bosystems . Since ovailable  and funcll ponds  where hieriments . Until neriments ) or perferiments ). Curreniewed in ). In thiD. magna were chosen from an available large collection of laboratory clones. No ethical approval is required for invertebrate use in toxicity testing. The clones originated from three different sites in Europe but all clones are established laboratory clones which have been raised in the lab during the last 6–20 years. K34J was isolated in 1998 from a former fishpond north of Munich, Germany. These fishponds were used as organic waste water treatment by a waste water treatment plant since 1929. BL2.2 originated from a small pond (Oud Meren) within a small park in Leuven, Belgium and is in culture since 1997. BL2.2 coexisted with backswimmers (Notonecta sp.) and fish. Max4 was hatched in 2010 from resting eggs originating from sediments from the Camargue/Tour de Valat Nature reserve, France). Predatory tadpole shrimps (Triops cancriformis) hatched from the same sediment. For none of the three locations data about potential plastic contamination is available. However, the highest probability for a contamination with plastic particles might exist for the fishponds near Munich which received waste water treatment plant effluent. The clones were cultured in an artificial medium based on ultrapure water, phosphate buffer, salts and trace elements [Daphnia were fed with the green algae Scenedesmus obliquus ad libitum.Three different clones of elements  at 20°C D. magna clones  were exposed to two different mixtures of microplastic particles, each consisting of four polymers. The tested polymers were an assortment of commonly used, non-buoyant plastic types, which are ranked among the 40 most toxic polymers [Daphnia medium and their concentrations were quantified as described in the The three polymers . All tespolymers , 18 and polymers –12. The polymers , 18. A dDaphnia to an amount of microplastic particles resembling environmental conditions we quantified the number of algae particles during standardized feeding  in the water column using sound analytical methods are rare 0 μm in t. The sam feeding  as well  feeding . In cont feeding  and a si feeding . The preiewed in ). Nevertiewed in ), in fisiewed in ), from eiewed in ). Howeveth brood, as well as number of juveniles produced and their morphological parameters.Effects of microplastic particles were assessed through two approaches : (I) EvaDaphnia were fed with the green algae S. obliquus at a concentration of 1 mg Carbon/L every second day. A full water exchange was performed on the third day. On the sixth day, when animals were observed to have reached primiparity, plastic exposure was started. Primiparity is a well-defined and easy to observe time point which enables a standardized experiment by using age synchronized animals. The start of the plastic exposure was initiated by transferring the Daphnia into a jar prepared 24h prior the transfer with fresh artificial medium, food and the corresponding plastic mixtures , and stock suspensions of microplastic particles 24h prior the transfer  was added. The animals were homogenized using a plastic pistil, which was thereafter rinsed with an additional 500 μl of Trizol. Samples were stored at -80°C until further processing. RNA was isolated from Trizol-preserved samples according to the manufacturer’s protocol  with the following modifications: (i) the DNase step was omitted (presence of DNA was excluded), (ii) introduction of a second drying step, (iii) elution step performed twice using half amounts of RNase-free water at the end. Extracted RNA was aliquoted in 3 tubes and stored at -80°C. Total RNA concentration and quality of the isolated RNA were assessed using a NanoDrop ND-1000 spectrophotometer .Daphnia  according to manufacturer instructions. In order to exclude contamination of samples with genomic DNA, a TATA-box fragment was amplified for each sample using an additional DNA positive control originating from three adult ribed in ). ResultDaphnia and co-regulating the production of male offspring [D. magna to environmental stress [Difference in expression level was measured based on seven candidate genes: Two genes involved in general stress response ; one geffspring . Increasl stress .2+ transporter, thus playing an important role in cell calcium signaling pathways [Daphnia to the presence of predators [D. magna qPCR assays [Eight putative housekeeping genes were included as possible reference genes: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase (SDH), Syntaxin 16 (Stx16), TATA box binding protein (TBP), ubiquitin conjugating enzyme (UBC), alpha Tubulin (aTub), Actin (Act) and Sarco(endo)plasmic reticulum calcium ATPase (SERCA). SERCA is a Capathways , 58, andredators . All theR assays , 60–62. R assays  and qBasR assays . SimilarPrimers for Act, aTub, Flot, GAPDH, GST, HSP60, JHE, MetA, MetB, SDH, UBC were obtained from , 60, 65.2 ≥0.993 and an efficiency between 92–106%, except the primers of aTub and MetA for which the efficiencies were 88.4 ± 1.5% and 87.1 ± 3%, respectively.Standard curves were produced for each of the genes to examine the efficiency of the primers and their dynamic range. For each gene, a standard curve with a fivefold dilution series from a cDNA mixture (from each clone and each treatment) was performed in three technical replicates. Standard curves were run in a Thermocycler  using the following protocol: 98°C for 2 min, 98°C for 2 s, 58°C or 60°C (according to the primer temperature) for 7 s, all repeated 45 times. After every run, a melting curve analysis was performed in order to test specificity of the amplified products. The specificity of the amplicons was obtained by heating from 65°C to 95°C, in steps of 5°C. Standard curves were produced for all genes using qbase+ 2.6 . Single technical replicates were removed when the value varied more than one cycle from the other two. Best amplification rates were yielded at a dilution of the cDNA stock of 1:100. Primer temperature and primer concentration were optimized to RThe qPCR reactions were run in technical duplicates, including a non-template control on the CFX Connect Real Time System . For the qPCR reactions, 10 μl of the SensiFAST SYBR NO-Rox Kit  was mixed with 8.4 μl of diluted cDNA , the corresponding amount of forward (F) and reverse (R) Primer (400 nM), and sterile water to obtain a final volume of 20 μl. The same conditions were applied in the thermal cycling program as for the efficiency screening. After every run a melting curve analysis was performed by heating from 65°C to 95°C, in steps of 5°C.All raw data were exported from the software  and further processed with the software qbase+ 2.6 . All statistical analyses were performed using qbase+ 2.6. Gene expression data were analyzed for each clone individually given clonal differences in the expression stability of reference genes. Differences of quantitation cycle (Cq) values were normalized to the reference genes and log10 transformed . This inDaphnia were carrying the 3rd and 5th brood. This resulted in a time of exposure of 20–22 days, depending on the duration necessary to produce the 5th brood. Measurements were made using an image analyzing system . Recorded parameters were body length (distance between the upper edge of the eye and base of the tail spine), body width  and tail spine length  were recorded from five randomly chosen neonates of each replicate from the 1st, 3rd and 5th brood, similar to the adults.The morphological parameters of the adults were recorded 48h after primiparity  were performed –72. PERMDaphnia and the neonates produced in the 1st, 3rd and 5th clutch as well as the number of produced offspring were tested for differences between the control group and animals fed with plastic mix A or plastic mix B, respectively. For the adults, PERMANOVA tests were conducted over the entire experimental period, using three different time points  as an additional fixed factor. For the neonates as well as the number of produced offspring, PERMANOVA tests were conducted over the entire experimental period using PERMANOVA with the three different time points  as an additional fixed factor and by nesting the neonates of each replicate. To compensate for size-dependent differences, analyses of body width and tail spine length of both adults and juveniles were performed using body length as a covariate. The effect of the covariate was always significant. The homogeneity of regression slopes was checked by the interaction term of the fixed factor and the covariate and was non-significant for all tests. Figures showing the body width and tail spine length of the neonates were prepared using the ratio of the body width and the tail spine length to body length in percent to compensate size-dependent differences. For figures visualizing nested data, the five individuals of each replicate were averaged to represent the nested individuals in the figures. The error bars give the 95% confidence interval which was calculated by SPSS .The body length, body width and tail spine length of adult The reference gene stability tests showed a varying gene expression among the potential reference genes between the three clones and no common reference genes could be identified due to the high interclonal differences. Therefore, normalization of gene expression was performed for each clone separately . Differences in body length were not detected between the control and plastic mix treatment groups , S4 Filemiparity , S4 Filemiparity , S4 Filerd clutch) and clone Max4 (1st clutch), but larger for clone K34J (5th clutch) and clone Max4 (3rd clutch) in the plastic mix B treatment . Howevefter 96h , 74). Foarticles , 76. Addd fibers . ContrarD. magna adults were affected by microplastics, offspring showed a variety of significant but small differences in body length, body width and tail spine length. Strong responses, such as increased mortality or malformations after exposure of D. magna to nano-polystyrene [D. magna among the clones showed inconsistent results. However, this cannot be regarded as influence of the microplastic particles, as the reaction of the control treatments of the corresponding clones showed a comparable high variation. The above mentioned observations correspond to frequently described results from Daphnia ecotoxicity tests of pharmaceuticals with low acute toxicity, where only subtle effects occur after chronic exposure or the effects are only observed in future generations [Although neither morphology nor life history of ystyrene , were noerations , 78.Daphnia. [Daphnia survival if considered in an ecological context.Changes in offspring body size but also the elongation of tail spines are a common phenotypic plastic defenses of Daphnia. , 79, 80.Daphnia. . Hence, Daphnia suggest that only some of the assessed parameters were affected and that occurring effects are small and subtle. The gene expression data indicate an increased stress level of adult Daphnia of some clones after only 48h exposure to both plastic mixtures, although also here the observed changes were rather small. In addition, similar to the morphological data, the interclonal variation in gene expression between the three clones was high, as likewise described in other Daphnia studies evaluating differences in gene or protein expression [D. magna after 24h exposure to NO3 coated silver nanoparticles at a concentration of 1/4 LC25 [T. japonicus to environmental toxicants (4-nonylphenol and 4-t-octylphenol), indicating that the downregulation of HSP70 can be utilized as a sign of stress [Daphnia to rapid microevolution, leading to the acquisition of toxicant resistance. This was suggested in another study [D. magna, cadmium accumulation and expression of HSP70 in these clones. Varying susceptibility to plastic exposure within the three clones could be due to increasing persistence of microplastic particles in the environment over the last few decades and resulting adaptation to related compounds. However, no record of microplastic particles exists for the field sites where clones were isolated from. Only for the origin of K34J, a fishpond used for organic treatment of waste water by a nearby waste water treatment plant, a contact with microplastic particles can be anticipated. Nevertheless, genes from the HSP family could remain a proxy for stress responses in D. magna. In addition to general stress genes, the gene GST was downregulated in BL2.2. As GST is an enzyme which removes reactive oxygen species from cells [Mytilus galloprovincialis) to virgin as well as pyrene treated polyethylene and polystyrene microparticles revealed an inhibition of anti-oxidant responses [2O2 or trace metals , it rems  or environmental pollutants. In contrast to many other studies microplastic particles were supplied in amounts resulting in low ingestion rates. While low, these rates are more realistic considering currently published contamination levels in freshwater systems.To the best of our knowledge, this is to date the first study exposing D. magna clones tested here were severely affected by microplastic exposure; neither an increased mortality nor malformations of the adults or the juveniles were detected. Nevertheless, several small and subtle but also inconsistent effects were detected on both the morphological and the molecular level. It is possible that these subtle effects might be due to natural variation or random effects and therefore D. magna species does not respond to the used microplastic particles. Nevertheless, we cannot exclude that environmental concentrations of microplastics will pose a threat to Daphnia, as the toxicity might be influenced by many additional factors like size, shape, type and even age of the particles.None of the three 2 levels and temperature, etc.) on the other hand. To conclude, further research  are necessary to assess the effects of microplastic particles, as superficially weak effects, as observed in this laboratory environment, could have an impact at both the individual or population level in nature.Our study sets a baseline for future studies of microplastic particles examining more complex scenarios; concerning the shape, size, additive content or adsorbed chemical compounds in an environmental multi-stress context, with natural stress on one hand  and the joint effect of anthropogenic stress Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.


Pelagostrobilidium paraepacrum ‘blooms’ inshore and; 3) environmental factors may differentially impact the distributions of individual ciliates (i.e. OTUs) rather than the community as a whole as OTUs tend to show distinct biogeographies . Together, these data show the complexity underlying the spatial distributions of marine protists, and suggest that biogeography may be a property of ciliate species rather than communities.Although protists  provide an important link between bacteria and Metazoa in food webs, we do not yet have a clear understanding of the spatial scales on which protist diversity varies. Here, we use a combination of DNA fingerprinting (denaturant gradient gel electrophoresis or DGGE) and high-throughput sequencing (HTS) to assess the ciliate community in the class Spirotrichea at varying scales of 1–3 km sampled in three locations separated by at least 25 km—offshore, midshelf and inshore—along the New England shelf. Analyses of both abundant community (DGGE) and the total community (HTS) members reveal that: 1) ciliate communities are patchily distributed inshore (i.e. the middle station of a transect is distinct from its two neighboring stations), whereas communities are more homogeneous among samples within the midshelf and offshore stations; 2) a ciliate closely related to  Microbial eukaryotes, or protists, play a critical role in plankton food webs by linking picoplankton  and Metazoa (copepods and fish) –3). ReceWe focus on ciliates, one of the most diverse protist clades in marine systems , 7. In p2) for a relative long period of time (weeks) as observed by remote sensing [Mesodinium rubrum [Lohmaniella oviformis, have been reported to form patches of high abundance at small scales (1-10s of km2) [The spatial scale of variation in ciliate diversity is not well known. Like many microbial eukaryotes, ciliates exhibit boom-bust cycles (i.e. rapid growth interspersed with quiescent periods) related to their relatively short generation time, the availability of pulses of nutrients, the presence of predators, and other environmental conditions . A typic sensing , 23, shom rubrum , 24. Oth of km2) , 26, the of km2) .To have a better understanding of the spatial scale of variation of oligotrich and choreotrich ciliates, we use a combination of DGGE and HTS to assess the diversity patterns at spatial scales of 1–2 km from three areas that are at least 25 km apart. We hypothesized that ciliate communities will be the similar in samples taken close together and that beyond some minimum distance, the community membership changes. We also expected to find strong assemblage changes related to the distance from shore as previously observed , 17, 19 th to the morning of the 9th July 2012 (around 1 hour between stations during the 12 hour transit inshore from the shelf break). No permits were required for the field sites as the locations are neither privately-owned nor protected, and the studied organisms did not include endangered species. At each location, the first two stations were separated by 1 km, and the third station was 2 km away from the second station , midshelf , and inshore  and stored at 4°C until DNA extraction. Our sampling protocol resulted in 54 samples: 3 sampling locations, each with 3 stations, times 3 depths, times 2 size fractions. For extraction, we used a modified phenol-chloroform method .The DNA from the filters was amplified under conditions aimed at minimizing PCR recombination artifacts. The amplifications were carried out using primers specific to ciliates within the Spirotrichea class , whCGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC) was added to the reverse primer for DGGE. Each depth layer and size fraction  were run on an independent DGGE gel—i.e. each gel had six stations . Using the Dcode Universal Mutation System  DGGE setup, 6% acrylamide gels with a denaturant gradient from 35 to 55% (100% denaturant corresponds to 7 M urea and 40% deionized formamide) were run at 245 V for 5 minutes followed by an incubation at 45 V for at least 15 hours. Gels were stained for 30 min in 200 mL of TAE buffer with 20 μL of SYBR Gold  and photographed using Kodak molecular imaging software . We previously confirmed the robustness of our DGGE primers and methods by replicating several gels using PCR reactions run on different days [A 39 base length GC clamp  followed standard protocols for the GS FLX Titanium instrument . The raw reads are available under the BioProject PRJNA339757 and Sequence Read Archive SRP082687. We analyzed the data following recommendations from previous studies , 33, 34.-150. For the phylogenetic approach, we aligned our sequences with Mafft E [For DGGE, the brightest bands (i.e. phylotypes) and all common phylotypes were excised from the gels, amplified by 10 cycles of PCR, and sequenced by the Sanger method (GenBank accession numbers KX714479-KX714518). The resulting phylotypes from DGGE and the OTUs from HTS were compared to our curated list of morphospecies sequences from GenBank and DGGE phylotypes from previous studies , 17, 19. Mafft E  and buil Mafft E  with the Mafft E ). OTUs achao1; [chao1 was used to estimate the ‘maximal’ or expected total diversity in each sample. Dissimilarity matrices were calculated using Fast Unifrac [We used EstimateS v9  to calcuchao1; ) for div Unifrac   varied mainly by depth layer and distance from shore . All surPelagostrobilidium paraepacrum  and is identical to the sequence observed with DGGE  dominated at stations 34 and 36, comprising nearly 100% of the read numbers throughout the water column. In contrast, the intervening station 35 is composed from 4 to 6 abundant taxa, including OTU329. We also found some OTUs whose distribution varied by depth layer rather than location. For example, while the OTU2165 is observed throughout the water column, OTU3046 and OTU2101 are present only at the surface. Other taxa are present only below the photic zone such as the OTU2594, while two taxa  onto a phylogenetic tree . At the Clustering analysis using Unifrac unweighted distance and principal coordinate analyses (PCoA) discriminates assemblages (clusters of sample sharing a similar community) by location  off the New England coast lead to two main insights: 1) ciliate communities are patchily distributed on the shelf at scales of 1–3 km; and 2) ciliate communities and abundant OTUs do not share the same biogeographies.Ciliate communities show a complex distribution related to their distance from shore and from each other. Communities sampled across large spatial scale  clustered by location but did not show any clear clustering at small spatial scales  when sampled offshore and midshelf while communities are patchier at the same spatial scales when sampled at inshore stations . The samPelagostrobilidium paraepacrum  are known to bloom in small-scale patches (13–170 m) and throughout the water column [Monospecific blooms in marine systems are well-known, particularly for phytoplankton such as diatoms , dinoflar column –26. Thesr column  while otr column , physicar column .Ciliate communities clustered distinctly based on distance from shore . , 56. 6, Strombidium chlorophyllum and phytoplankton grazers such as S. siculum. Other taxa (e.g. OTU1970) have complex patterns that may be related either to parameters not measured here or they may reflect neutral processes [By contrast, individual taxa (i.e. OTUs) show specific biogeographies related to depth layers that are not observed at the whole-community level. These distinct biogeographies are best illustrated by Venn diagrams  and by mrocesses , 58. Ourrocesses , as OTUsWe used DGGE and HTS to quantify patterns of ciliate community composition across the New England continental shelf. The most important insight of our study is that the biogeographic patterns of communities and individual taxa (i.e. OTUs or phylotypes) appear to be decoupled: whole communities vary by distance from the shore, while the distributions of individual lineages are more closely linked to depth strata. This suggests that to understand the distribution and ecology of communities, we need to have a better knowledge of each member of the community, including the rare species that are only seen in HTS.S1 FigA total of 100 (11±8 for nanosize and microsize) and 500 reads  was subsampled for A and B, respectively. The diversity indices show the same pattern using H’ index  and the Choa1 diversity estimator .(DOCX)Click here for additional data file.S2 FigIn contrast, there is evidence of a bloom at all depths in two non-adjacent inshore samples (i.e. stations 34 and 36). Each OTU has an unique identifier, follow by the number of reads and occurrence of this OTU during the cruise , followed by the best BLAST result .(DOCX)Click here for additional data file.S3 FigIn black, HTS OTUs with more than 1,000 reads were considered to simplify the tree. In orange are the OTUs from DGGE analyses See . OTU wit(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S6 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableSome abundant OTUs are related to the distance to the shore and to the depth (e.g. OTU329 mainly observed inshore or OTU2594 mainly observed within the surface layers in the offshore location) as observed in (DOCX)Click here for additional data file.


Past research has demonstrated negative associations between exposure to stressors and quality of interpersonal relationships among children and adolescents. Nevertheless, underlying mechanisms of this association remain unclear. Chronic stress has been shown to disrupt prefrontal functioning in the brain, including inhibitory control abilities, and evidence is accumulating that inhibitory control may play an important role in secure interpersonal relationship quality, including peer problems and social competence. In this prospective longitudinal study, we examine whether changes in inhibitory control, measured at both behavioral and neural levels, mediate the association between stress and changes in secure relationship quality with parents and peers. The sample included 167 adolescents  who were first recruited at age 13 or 14 years and assessed annually three times. Adolescents’ inhibitory control was measured by their behavioral performance and brain activities, and adolescents self-reported perceived stress levels and relationship quality with mothers, fathers, and peers. Results suggest that behavioral inhibitory control mediates the association between perceived stress and adolescent’s secure relationship quality with their mothers and fathers, but not their peers. In contrast, given that stress was not significantly correlated with neural inhibitory control, we did not further test the mediation path. Our results highlight the role of inhibitory control as a process through which stressful life experiences are related to impaired secure relationship quality between adolescents and their mothers and fathers. Though adolescence can be a difficult transitional period in which risks to both physical and mental health, including depression, substance abuse, and suicide increase , adolescExtant research has shown that children and adolescents with stressful life experiences tend to have negative relational experiences—with peers as well as parents. With respect to peer relationships, children who have been rejected by their peers are more likely than non-rejected children to have been exposed to multiple chronic stressors, including financial strain, living with a single parent, violence in the home, parental divorce, and family illness . FurtherTo date, how stressful life experiences may be related to interpersonal relationships is not clearly understood. Within the neuroscience literature, previous research has demonstrated that stress is associated with impaired structure and function of the prefrontal cortex (PFC), a brain region that contributes to self-regulation capabilities see . While sRecent research is beginning to clarify the effects of these structural changes due to stress, suggesting impairments in executive functions, including in inhibitory control, attention, and memory. For example, children exposed to chronic stress via poverty display multiple self-regulatory deficits. In a behavioral study, children who experienced chronic poverty were rated by both parents and teachers as weaker in inhibitory control and delaying gratification . SimilarSuch consequences may manifest themselves within the quality of interpersonal relationships. For instance, In the current longitudinal study, we aimed to investigate whether inhibitory control may be an explanatory process of the detrimental effects of stress on interpersonal relationships. Specifically, we hypothesized that earlier perceived stress is related to later secure interpersonal relationship quality over time via inhibitory control abilities (after controlling for baseline levels of the mediator and outcome). Past research thus far has primarily focused on objective indicators of stress , 14 or 15 years of age at Time 2 , and 15 or 16 years of age at Time 3 . Adolescents primarily identified as 80% Caucasian, 13% African–American, and 7% other. Mean family income was $25,000–$34,999 per year at Time 1 and Time 2 and $35,000–$49,999 per year at Time 3. At Time 1, 157 families participated. At Time 2, 10 families were added for a final sample of 167 parent–adolescent dyads. However, 24 families did not participate at all possible time points for reasons including: ineligibility for tasks (n = 2), declined participation (n = 17), and lost contact (n = 5) during the follow-up assessments. We performed attrition analyses using general linear model (GLM) univariate procedure to determine whether there were systematic predictors of missing data. Results indicated that rate of participation (indexed by proportion of years participated to years invited to participate) was not significantly predicted by demographic variables  or study variables at Time 1 .The current community sample included 167 adolescents  from southwestern Virginia, the United States of America. Adolescents were 13 or 14 years of age at Time 1 . The current study used data from Time 1, Time 2, and Time 3; each assessment was approximately 1 year apart from the previous one. Data collection took place at the university’s offices where adolescents and their primary caregivers were interviewed by trained research assistants. Both parents and adolescents received monetary compensation for their time. All procedures were approved by the institutional review board of the university with written informed consent and assent from all participants.Perceived stress was assessed using adolescent self-report at Time 1 using the 10-item perceived stress scale PSS;  that hasThe short version of the Inventory of Parent and Peer Attachment was utilized at Time 1 and Time 3 to determine the degree of adolescents’ perceived security in their relationships with parents and peers IPPA; . Adolesct(153) = 69.58, p < 0.001, as well as accuracy for correct responses, t(153) = -15.47, p < 0.001. These results showed that accuracy was lower and reaction time was higher  for interference compared to neutral trials. A behavioral inhibitory control factor score was calculated using two indicators from this task: (1) the difference in accuracy of the neutral and interference trials  and (2) the intra-individual variability in reaction time or the intra-individual standard deviations across correct response latency trials in the interference condition. These two indicators were significantly correlated . Higher scores for the accuracy difference and lower scores for the intra-individual variability indicated better cognitive control. Confirmatory factor analysis (CFA) was performed to produce a factor score using standardized scores of these two indicators. At both waves, the models were fully saturated  with significant loadings for the two indicators .At Time 1 and Time 2, adolescents’ inhibitory control was captured by the MSIT , using aFunctional neuroimaging data were acquired on a 3T Siemens Tim Trio MRI scanner with a standard 12-channel head matrix coil. Echo-planar images (EPIs) were collected using the following parameters: slice thickness = 4 mm, 34 axial slices, field of view (FoV) = 220 mm × 220 mm, repetition time (TR) = 2 s, echo time (TE) = 30 ms, flip angle = 90 degrees, voxel size = 3.4375 mm × 3.4375 mm × 4 mm , 64 × 64 grid, and slices were hyperangulated at 30 degrees from anterior–posterior commissure. The structural scan was acquired using a high-resolution magnetization prepared rapid acquisition gradient echo sequence with the following parameters: TR = 1200 ms, TE = 3.02 ms, FoV = 245 mm × 245 mm, and 192 slices with the spatial resolution of 1 mm × 1 mm × 1 mm.p < 0.001.Data were pre-processed and analyzed using SPM8 . Functional images were corrected for head motion using a six-parameter rigid-body transformation, realigned, and normalized to a Montreal Neurological Institute (MNI) template using parameters derived from a segmented anatomical image coregistered to the mean EPI. The resulting image was spatially smoothed using an 8 mm full-width at half-maximum Gaussian kernel. Each participant’s preprocessed imaging data were whitened and analyzed using a GLM that included a boxcar regressor for each condition of interest, six motion-parameters as nuisance regressors, and a high-pass filter with cutoff at 128 s. Temporal autocorrelations were estimated using an autoregression AR(1). A subsequent second-level random effects analysis was conducted on individual interference minus neutral contrasts. Spherical regions-of-interest (ROI), 6 mm in radius and centered at peak voxels in the second-level analysis, were extracted using a family-wise corrected (FEW) threshold of neural inhibitory control factor scores created for Time 1 and Time 2. At each wave, CFA was performed to produce a factor score using standardized scores of two ROIs. At both waves, the models were fully saturated  with significant loadings for two indicators .Regions-of-interest values were considered based on (1) regions known to be engaged by inhibitory control related to interference and error processing  and (2) SD from the mean. In these cases (n = 7), values were winsorized to retain statistical power and attenuate bias resulting from elimination (ps > 0.10). Thus, they were not included as covariates in the main analyses.For all study variables, descriptive statistics were examined to determine normality of distributions and outliers. Skewness and kurtosis were examined for all variable distributions and acceptable levels were skewness less than 3 and kurtosis less than 10 . Outliermination . MultivaThe hypothesized model was tested via structural equation modeling (SEM) in MPlus 7.4 . The ana2 goodness of fit test, root mean square error of approximation (RMSEA), and confirmatory fit index (CFI). RMSEA values of less than 0.05 were considered a close fit while values less than 0.08 were considered a reasonable fit (ad hoc methods (The mediation model included paths (a) from perceived stress at Time 1 to changes in inhibitory control from Time 1 to Time 2, (b) from changes in inhibitory control to secure relationship quality with mother, father, and peers from Time 1 to Time 3, and (c) from perceived stress at Time 1 to changes in secure relationship quality with mother, father, and peers. We also estimated correlations among the three secure relationship quality outcomes. Overall model fit indices were determined by χable fit , and CFIable fit . We calcable fit . These Cable fit . Full inable fit  was used methods . In ordeTable 1. We first tested the effects of perceived stress at Time 1 on secure relationship quality for mothers, fathers, and peers at Time 3 after controlling for their respective relationship qualities at Time 1  via behavioral inhibitory control at Time 2 after controlling for behavioral inhibitory control at Time 1 . The original model was a fully saturated model, with χ2 = 0, df = 0, p = 0, CFI = 1.00, RMSEA = 0. The direct effects from perceived stress at Time 1 to the secure mother relationship quality residualized change score  and secure father relationship quality residualized change score  were not significant. The effect from behavioral inhibitory control to the secure peer relationship quality residualized change score was also not significant . For model parsimony, we constrained these paths to zero.Correlations and descriptive statistics for all study variables can be found in 2 = 2.25, df = 3, p = 0.52, CFI = 1.00, RMSEA = 0 . Higher levels of perceived stress at Time 1 were associated with lower inhibitory control residualized change scores . In turn, lower inhibitory control residualized change scores were related to lower secure relationship quality residualized change scores for adolescents and their mothers  and fathers . The indirect effects of perceived stress at Time 1 on the secure mother relationship quality residualized change score  and the secure father relationship quality residualized change score , through the behavioral inhibitory control residualized change score, were significant. For secure peer relationship quality, higher levels of perceived stress at Time 1 directly predicted a lower secure peer relationship quality residualized change score . Given that the behavioral inhibitory control residualized change score was not significantly associated with the secure peer relationship quality residualized change score, the indirect effect of perceived stress on the secure peer relationship quality residualized change score through the behavioral inhibitory control residualized change score was not estimated. In addition, the secure mother relationship quality residualized change score significantly correlated with that of the secure father relationship quality residualized change score  and the secure peer relationship quality residualized change score . However, the correlation between the secure father relationship quality residualized change score and the secure peer relationship quality residualized change score was not significant .The final model showed an excellent fit, χneural inhibitory control at Time 2 after controlling for neural inhibitory control at Time 1 . The original model was a fully saturated model, with χ2 = 0, df = 0, p = 0, CFI = 1.00, RMSEA = 0. Perceived stress at Time 1 did not predict the neural inhibitory control residualized change score  or the three secure relationship quality residualized change scores . The predictive effects of the neural inhibitory control residualized change score was only significant for the secure father relationship quality residualized change score , but not for the secure mother relationship quality residualized change score  or the secure peer relationship quality residualized change score . Given that perceived stress at Time 1 was not related to the neural inhibitory control residualized change score, we did not further test the indirect effects of perceived stress at Time 1 on the three secure relationship quality residualized change scores via the neural inhibitory control residualized change score. In this model, the secure mother relationship quality residualized change score significantly correlated with that of the secure father relationship quality residualized change score  and the secure peer relationship quality residualized change score . However, the residual covariance between the secure father relationship quality residualized change score and the secure peer relationship quality residualized change score was not significant .We then tested the effects of perceived stress at Time 1 on the secure relationship quality for mothers, fathers, and peers at Time 3 after controlling for their respective levels at Time 1  via 2(10) = 9.31, p = 0.503 for behavioral inhibitory control model and Wald χ2(10) = 7.50, p = 0.677 for neural inhibitory control model. Therefore, results suggested that the associations among perceived stress, behavioral/neural inhibitory control, and secure relationship quality were comparable between males and females.To test whether the associations among perceived stress, inhibitory control, and secure relationship quality may vary by sex, we performed an omnibus multiple groups Wald test for behavioral inhibitory control model and neural inhibitory control model, respectively. The Wald test compared all regression paths between males and females in the original saturated model. In both tests, multiple groups analyses did not reveal any significant sex differences: Wald χWe reran the models with level scores  and tested the effects of Time 1 perceived stress on Time 3 secure relationship quality with mother, father, and peers via Time 2 behavioral/neural inhibitory control. Overall, the results on the level of these variables are highly similar to our original results using residualized change scores, confirming that the results of the residualized change score model are robust. Detailed results for behavioral/neural inhibitory control follow.behavioral inhibitory control, the original model was a fully saturated model, with χ2 = 0, df = 0, p = 0, CFI = 1.00, RMSEA = 0. The effect from behavioral inhibitory control to secure peer relationship quality was not significant . For model parsimony, we constrained this path to zero. The final model showed an excellent fit, with χ2 = 0.22, df = 1, p = 0.64, CFI = 1.00, RMSEA = 0. Higher levels of perceived stress at Time 1 were associated with lower inhibitory control at Time 2 . In turn, lower inhibitory control was related to lower secure relationship quality for adolescents and their mothers  and fathers  at Time 3. The indirect effects of perceived stress on secure mother relationship quality  and secure father relationship quality , through behavioral inhibitory control, were significant. For secure peer relationship quality, higher levels of perceived stress at Time 1 directly predicted lower secure peer relationship quality . Given that behavioral inhibitory control was not significantly associated with secure peer relationship quality, the indirect effect of perceived stress at Time 1 on secure peer relationship quality at Time 3 through behavioral inhibitory control at Time 2 was not estimated.For neural inhibitory control at Time 2. The original model was a fully saturated model, with χ2 = 0, df = 0, p = 0, CFI = 1.00, RMSEA = 0. Perceived stress at Time 1 did not predict neural inhibitory control , however, it predicted significant lower secure relationship quality with mothers , fathers , and peers . The predictive effects of neural inhibitory control were not significant for secure mother relationship quality , secure father relationship quality , or secure peer relationship quality . Given that perceived stress at Time 1 was not related to neural inhibitory control at Time 2, we did not further test the indirect effects of perceived stress at Time 1 on secure relationship quality at Time 3 via the neural inhibitory control at Time 2.We then tested the effects of perceived stress at Time 1 on secure relationship quality for mothers, fathers, and peers at Time 3 via The present study sought to elucidate the underlying role of inhibitory control in explaining the association between stress and secure interpersonal relationship quality in adolesence. Results demonstrated indirect paths via adolescent behavioral inhibitory control such that perceived stress was related to lower inhibitory control which in turn was related to lower secure relationship quality between adolecents and their mothers and fathers. These findings were obtained despite controlling for baseline levels of inhibitory control and secure relationship quality. Our data suggested a direct link between adolescents’ perceived stress and their secure relationship quality with peers, such that higher perceived stress predicted poorer secure relationship quality with peers; however, the effects of stress on secure peer relationship quality was not mediated by behavioral inhibitory control. In contrast to the findings of behavioral inhibitory control, stress was not significantly correlated with neural inhibitory control. Thus, we did not test the indirect effects of perceived stress on secure relationship quality via neural inhibitory control. Finally, the pattern of findings was not moderated by sex, indicating that the associations among stress, behavioral and neural inhibitory control, and secure relationship quality were comparable between males and females.The indirect effect of perceived stress on adolecent’s secure mother and father relationship quality via behavioral inhibitory control supported our hypothesis. In line with previous research in adults indicating an association between stress and impaired executive functioning related to self-regulation capabilities , our datBased on previous literature emphasizing the importance of self-regulation on interpersonal relationship quality , we expeOur results demonstrating that inhibitory control was not directly associated with secure peer relationship qualilty appear to be inconsistent with previous research showing significant links between executive functions (including inhibitory control) and peer problems in early childhood through middle adolesence . HoweverWe found a significant direct link between perceived stress and secure relationship quality with peers. While no prior research to our knowledge has focused on perceived stress, objective stressful life events have been documented in the literature as a risk factor for negative peer relationships . Thus, obehavioral inhibitory control to be more strongly related to general perceived stress than neural inhibitory control. Prior studies involving adopted or foster children and adolescents with early-life stress defined by neglectful and abusive care revealed that these children showed impairements in neural inhibitory control compared to those who did not experience such early adversity  within adolesence, given that stress exposures during adolescence can have more potent effects on the brain than when those exposures occur in adulthood – due to rapid brain development and increased plasticity of developing systems relative to adulthood . From a Past research has rarely investigated the link between inhibitory control and positive developmental outcomes such as secure interpersonal relationship quality. Our longitudinal analyses suggest that adolescents with poor behavioral inhibitory control are likely to show poor secure relationship quality with both mothers and fathers over time. This association may be expected given that flexible inhibitory control promotes social affective skills , ultimatThe identification of inhibitory control as a mechanism in the stress-secure interpersonal relationship quality association is beneficial in how it may inform intervention work for children and adolescents who are especially prone to chronic stress. Interventions that teach and practice skills related to inhibitory control may help protect against negative parent–adolescent relationships later on.This study was carried out in accordance with the recommendations of Virginia Tech Institutional Review Board for the Protection of Human Subjects, Office of Research Compliance with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Virginia Tech Institutional Review Board for the Protection of Human Subjects.TH conceived the study, participated in data collection, participated in statistical analyses and interpretation of the data, and drafted the manuscript. ML performed statistical analyses and interpretation of the data and drafted the manuscript. DM performed statistical analyses and interpretation of the data and critically revised the manuscript. JL participated in statistical analyses and interpretation of the data and helped to draft the manuscript. KD-D critically revised the manuscript. BK-C participated in the design of the study and statistical analyses. JK-S conceived the study, participated in its design and coordination, and drafted the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rapid DNA identification is the use of a rugged, field-deployable system to generate short tandem repeat (STR) profiles in law enforcement, military, immigration, and homeland security applications. A performance verification study was conducted on the ANDE Rapid DNA identification system using FlexPlex27, a highly multiplexed, 27 locus assay that generates data for the expanded CODIS core loci and all additional STR loci required for international databasing. The assay contains 23 autosomal loci , three Y-chromosomal loci , and Amelogenin. Study results demonstrate that the instrument is reliable, reproducible, accurate, robust, and ready for a large scale, comprehensive developmental validation by NDIS-participating laboratories. The additional loci in the FlexPlex assay allow for improved STR profile sharing globally, increase the power of discrimination for identification matches, and improve the effectiveness of kinship analyses.The online version of this article (doi:10.1007/s00414-017-1567-9) contains supplementary material, which is available to authorized users. Short tandem repeat (STR) analysis is applied in a wide range of disciplines, including law enforcement, paternity testing, disaster victim identification, and the military. Based on a series of advances over the past two decades, STR analysis in the laboratory is now well-established, and it is not surprising that the utility of STRs is broadening beyond the laboratory. Perhaps two extensions of the reach of STR analysis are most important in this regard: Rapid DNA identification and expanded sets of core STR loci.Rapid DNA identification can be defined as the fully automated generation and interpretation of STR profiles  in less than 2 h in a ruggedized, field-deployable system by nontechnical users. The potential impact of rapid DNA technology is evidenced by the fact that the Department of Defense, the Federal Bureau of Investigation (FBI), and the Department of Homeland Security have collaborated to develop a series of requirements for rapid DNA Identification systems . FurtherIn parallel with these developments, several countries have modified and expanded their sets of core STR loci used for databasing and matching against casework, military, and other samples. For example, the FBI has announced the expansion of the US Combined DNA Index System (CODIS) core loci from 13 to 20, with a goal of reducing the likelihood of adventitious matches  and incrKinship analysis using STRs is based on classical Mendelian genetics. The closer the biological relationship between two individuals, the more alleles they are likely to share. First-degree relatives are those that share approximately 50% of their genetic material—individuals that have parent-offspring and sibling relationships. Paternity testing is the most common kinship application of STR technology. An offspring inherits two alleles at each STR locus, one from each parent—these are “obligate alleles” in the sense that the genome of the offspring must contain these alleles . Because of obligate alleles, it is generally straightforward to establish parental-child relationships with an assay which targets the CODIS core 13 loci. In contrast, Butler and colleagues  showed tWe have developed a multiplex assay which interrogates 27 loci, termed FlexPlex27 Fig.  and adapThe initial ANDE instrument  performs laser-based detection of four fluorescent dyes and is compatible with the PowerPlex 16 System (Promega Corporation) . To accoWhen operating the ANDE System, five buccal swabs were loaded into each chip which was then inserted into the instrument, and all steps were completed by following onscreen instructions. The instrument automatically processes the swabs to purify the DNA, amplify the purified DNA to generate labeled STR fragments, and separate and detect the STR fragments. The on-board ANDE Expert System automatically performed allele calling and profile interpretation. The total processing time is approximately 90 min per run.Buccal swab samples were collected using ANDE swabs as follows. Donors were instructed to press the swab gently against the inside of one cheek, move it in an up-and-down motion six times, and repeat with the other cheek (6 swipe protocol). The swab head is rotated during the collection to ensure that buccal cells are collected around the entire surface area. Other swab types can be used in the system; the advantage of the ANDE swab is that it contains a 2-D barcode and RFID chip in the swab cap that simplifies sample tracking. For concordance purposes, samples processed on the ANDE instrument were also subjected to conventional laboratory testing. The study was approved by an Institutional Review Board, and informed consent was obtained from all individual participants included in the study.Three buccal swabs were collected from each of ten unique donors and processed on three unique ANDE 6C instruments. Concordance, signal strengths, and heterozygote peak height ratios of the three replicate profiles from each donor were compared.Buccal swabs were collected in triplicate from five unique individuals using a 1 swipe, 3 swipe, and 6 swipe protocol. Each set of swabs was processed on a unique ANDE 6C instrument. The resulting profiles from the 15 replicates of each swabbing protocol were assessed for signal strength and heterozygote peak height ratios.−4 and spiked on swabs. The quantity of DNA per species spiked on each swab is as follows. Mouse (Mus musculus), Ferret (Mustela putorius furo), Horse , and Dog (Canis lupus familiaris): 1000 ng of genomic DNA. Orangutan (Pongo pygmaeus), Gorilla (Gorilla gorilla), and Chimpanzee (Pan troglodytes): 500 ng of genomic DNA. Lactobacillus plantarum, Staphylococcus aureus, and Streptococcus pneumoniae: 100 ng of genomic DNA.Duplicate buccal swabs were spiked with DNA from each of ten unique species and processed on ANDE 6C. Genomic DNA from each of ten species was prepared as a 50-μl solution of TEPurified DNA from a male and a female donor was quantified using a Nanodrop 2000C Spectrophotometer (Thermo Fisher Scientific). DNAs from each donor were mixed in ratios of 19:1, 5:1, 1:1, 1:5, and 1:19 yielding a total of 1 μg of DNA in 50 μl of TE-4. Each mixture was pipetted onto an ANDE swab and processed in duplicate. Each profile was reviewed manually and all alleles assigned to either the major or minor donor.Two buccal samples from each individual were collected in the presence of ten unique potentially inhibitory substances: mint, gum, toothpaste, mouthwash, blood, beer, tea, tobacco dip, cigarette, and coffee. The potential inhibitors were consumed or used by the donor immediately prior to buccal swab collection. For example, gum was chewed for approximately 5 min just before standard buccal swab collection was performed. The only exception was that 10 μl of blood from the same donor as the buccal sample was pipetted directly onto the buccal swab following collection and prior to the run.Fourteen buccal swab samples were collected from each of two unique donors. Immediately after buccal swab collection, one set of swabs was stored in a protective clear plastic tube containing desiccant and the other set was stored in the standard protective clear plastic tube (not containing desiccant). For each set of 14 samples, two samples from each donor were processed immediately (fresh), after 1 day of storage at 22 °C, 1 day of storage at 4 °C, 2 days of storage at 22 °C, 2 days of storage at 4 °C, 7 days of storage at 22 °C, and 7 days of storage at 4 °C.Runs were made with the following three sample loading configurations: blank/blank/blank/blank/blank, sample/blank/sample/blank/sample, and blank/sample/blank/sample/blank. Each loading configuration was performed in duplicate. Buccal swab samples were collected following the standard protocols, and blank swabs were new swabs removed from the packaging and placed directly into the chip.Two hundred twenty unique donor samples (44 chip runs) were processed to determine first pass success, concordance, signal strength, and peak height ratio. The first pass success rate was determined by evaluating the number of samples with all CODIS core 20 loci or all FlexPlex27 loci passing on the first run (including fully integrated Expert System interpretation). Concordance was determined by comparing the allele calls generated by ANDE 6C with allele calls of the same donor generated by an outside laboratory using conventional methods.Precision and resolution were analyzed based on 76 runs (380 samples). Inter-run precision was calculated by determining the standard deviation of the fragment sizes (in bases) of the allelic ladder fragments. Resolution was calculated as previously described , with R All Rapid DNA Identification was performed within a previously described  chip, a The ANDE 6C instrument is based on the previously described ANDE 4C  instrumeNote that all ANDE chips, whether for buccal swab samples , caseworAn allele table listing all passing allele calls for all samplesA .png file (electropherogram) for rapid output visualizationAn .xml file for upload to Combined DNA Index System (CODIS)An .fsa file to permit review with conventional software packagesThe ANDE 6C Expert System is based on the NDIS-approved 4C Expert System , 15, witOne swab collected from each unique donor from the following studies was sent for conventional laboratory STR testing: reproducibility, sensitivity, stability, mixtures, inhibitors, stability, contamination, and accuracy. DNA was extracted from the buccal swabs using the BioSprint 96 Robotic Workstation (Qiagen). Approximately 0.5 to 2.0 ng of the extracted DNA was amplified using the PowerPlex Fusion 6C and PowerPlex 21 Systems (Promega) and detected using an Applied Biosystems® 3500 Genetic Analyzer (Life Technologies). The data was analyzed using Genemapper® ID-X, version 1.1 (Life Technologies), and interpreted by two qualified analysts. Additional validated techniques such as reinjection, reextraction, quantification, and increased cycle numbers were employed for samples that did not initially pass technical specifications for reporting.Profile storage and management was performed with ANDE Data Management Software (ADMS). Data generated on multiple ANDE systems was transferred to ADMS through a local area network. The ADMS stores profile data in an onboard database and allows individual profiles to be viewed, exported, and deleted. The software also allows DNA databases to be created, imported, exported, and deleted.Comparisons between profiles may be made by exporting to a central database or locally using ADMS, which allows automatic searching and matching of newly imported profiles against profiles that are stored in database. The match between a new profile and a profile in the database is determined by first calculating the following at each locus:Number of loci available for matching. A locus is defined as available for matching when it has allele calls in both the sample profile and the database profile.Number of loci that match. A locus is classified as a match when both alleles of the sample profile and the database profile are identical.Number of loci with drop-ins or drop-outs. A locus is classified as a drop-in or drop-out when one of the alleles matches, but other allele does not match.Number of mismatched loci. A locus is classified as a mismatch when both alleles of the locus do not match.A final result of match, possible match, or mismatch is assigned based on the match criteria as defined by the jurisdiction.Kinship analysis was performed implementing eDNA software . The kinship module has been validated to calculate likelihood ratio, paternity index-PI, and combined relative index-CRI, and STR profiles from two individuals when compared to calculate the likelihood of the main hypothesis  versus the likelihood of alternate hypothesis . eDNA software uses established kinship formulas , 17 and STR profiles from a male and female donor generated with the FlexPlex27 assay are shown in Fig. Sensitivity of the system was assessed studying the impact of 1, 3, and 6 swipes collection protocol on signal strength and peak height ratio. Full CODIS core 20 profiles were generated for all 45 samples processed on three different ANDE 6C instruments, and all profiles were concordant with those generated by conventional testing. There was no apparent difference in the signal strengths between samples collected with a 6 swipe and 3 swipe protocol, and there was a decrease in signal strength for samples collected with a 1 swipe protocol , Ferret (Mustela putorius furo), Horse , Dog (Canis lupus familiaris), Lactobacillus plantarum, Staphylococcus aureus DNA, and Streptococcus pneumoniae did not contain any called peaks. Profiles generated from primates Orangutan (Pongo pygmaeus), Gorilla (Gorilla gorilla), and Chimpanzee (Pan troglodytes), which possess genetic similarities with humans, have no called alleles, but a significant number of alleles labeled in red warning boxes. Alleles in red warning boxes are not considered as passing by the Expert System and are not included in the database-compatible output file.Specificity of the FlexPlex assay to human samples is essential to ensure that interpretation of STR profiles derived from human subjects is not complicated by cross-reactivity from non-human samples. A set of 10 relevant organisms including bacteria, domestic animals, and primates were evaluated in duplicate, and none yielded a passing profile. Profiles generated for Mouse  generated using the ANDE System may be utilized for databasing matching or for kinship analysis using local databases. Rapid DNA technology and related legislation and policies have advanced in parallel over the past decade and promise to have a major impact on societal safety.Supplementary Table 1(XLSX 17 kb)Supplementary Table 2(XLSX 10 kb)Supplementary Table 3(XLSX 16 kb)