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Aim This study aimed to explore differences in body mass index (BMI) trajectories among youth exposed or not exposed to maternal gestational diabetes mellitus (GDM) and understand whether these associations differ across life stages. Methods Data from 403 mother/child dyads (76 exposed; 327 not exposed) participating in the longitudinal Exploring Perinatal Outcomes among Children (EPOCH) study in Colorado were used. Participants who had two or more longitudinal height measurements from 27 months to a maximum of 19 years were included in the analysis. Life stages were defined using puberty related timepoints: early childhood [27 months to pre-adolescent dip (PAD, average age 5.5 years)], middle childhood (from PAD to age at peak height velocity (APHV, average age 12.2 years), and adolescence (from APHV to 19 years). Separate general linear mixed models, stratified by life stage, were used to assess associations between GDM exposure and offspring BMI. Results There was not a significant association between exposure to GDM and BMI trajectories during early childhood (p = 0.27). Participants exposed to GDM had higher BMI trajectories compared to those not exposed in middle childhood (males: p = 0.005, females: p = 0.002) and adolescent (p = 0.02) periods. Conclusions Our study indicates that children who are exposed to GDM may experience higher BMI trajectories during middle childhood and adolescence, but not during early childhood. These data suggest that efforts to prevent childhood obesity among those exposed in utero to maternal GDM should start before pubertal onset.

OBJECTIVES Maternal prepregnancy BMI (ppBMI) and an infant’s rapid weight gain (RWG) are each associated with increased risk for childhood obesity. We hypothesized that ppBMI and RWG interact to further raise childhood obesity risk. METHODS Mother-infant dyads (n = 414) from the Healthy Start Study, an observational prebirth cohort, were included. RWG was defined as a weight-for-age z score increase of ≥0.67 from birth to 3 to 7 months. Body composition was measured by air displacement plethysmography at age 4 to 7 years. General linear regression models were fit to characterize associations between ppBMI, RWG, and their interaction with the outcomes of childhood BMI-for-age z score and percent fat mass (%FM). RESULTS A total of 18.6% (n = 77) of offspring experienced RWG. Maternal ppBMI and RWG were both positively associated with offspring BMI z score and %FM. RWG amplified the association between ppBMI and BMI z score, especially among females. Females exposed to maternal obesity and RWG had an average BMI at the 94th percentile (1.50 increase in childhood BMI z score) compared with those exposed to normal ppBMI and no RWG (average childhood BMI at the 51st percentile). RWG had a weaker effect on the association between ppBMI and %FM. Adjustment for breastfeeding status or childhood daily caloric intake did not significantly alter findings. CONCLUSIONS Rapid infant weight gain interacts with maternal ppBMI to jointly exacerbate risk of childhood obesity. Pediatric providers should monitor infants for RWG, especially in the context of maternal obesity, to reduce future risk of obesity.

Background: Hispanic women have lower breast cancer incidence rates than non-Hispanic white (NHW) women. To what extent genetic versus nongenetic factors account for this difference is unknown.Methods: Using logistic regression, we evaluated the interactive influences of established risk factors and ethnicity (self-identified and identified by ancestral informative markers) on breast cancer risk among 2,326 Hispanic and 1,854 NHW postmenopausal women from the United States and Mexico in the Breast Cancer Health Disparities Study.Results: The inverse association between the percentage of Native American (NA) ancestry and breast cancer risk was only slightly attenuated after adjusting for known risk factors [lowest versus highest quartile: odds ratio (OR) =1.39, 95% confidence interval (CI) = 1.00–1.92 among U.S. Hispanics; OR = 1.92 (95% CI, 1.29–2.86) among Mexican women]. The prevalence of several risk factors, as well as the associations with certain factors and breast cancer risk, differed according to genetic admixture. For example, higher body mass index (BMI) was associated with reduced risk among women with lower NA ancestry only [BMI 30: OR = 0.65 (95% CI, 0.44–0.98) among U.S. Hispanics; OR = 0.53 (95% CI, 0.29–0.97) among Mexicans]. The average number of risk factors among cases was inversely related to the percentage of NA ancestry.Conclusions: The lower NA ancestry groups were more likely to have the established risk factors, with the exception of BMI. Although the majority of factors were associated with risk in the expected directions among all women, BMI had an inverse association among Hispanics with lower NA ancestry.Impact: These data suggest that the established risk factors are less relevant for breast cancer development among women with more NA ancestry. Cancer Epidemiol Biomarkers Prev; 26(5); 692–701. ©2016 AACR.

Supplemental Table 1. Relationship between percent Native American ancestry and ER positive breast cancer among postmenopausal non-Hispanic Whites and US Hispanics. Supplemental Table 2. Relationship between percent Native American ancestry and ER negative breast cancer among postmenopausal non-Hispanic Whites and US Hispanics. Supplemental Table 3. Relationship between risk factors and ER positive breast cancer among postmenopausal non-Hispanic Whites and US Hispanics according to percent Native American ancestry. Supplemental Table 4. Relationship between risk factors and ER negative breast cancer among postmenopausal non-Hispanic Whites and US Hispanics according to percent Native American ancestry.

Background When evaluating the impact of environmental exposures on human health, study designs often include a series of repeated measurements. The goal is to determine whether populations have different trajectories of the environmental exposure over time. Power analyses for longitudinal mixed models require multiple inputs, including clinically significant differences, standard deviations, and correlations of measurements. Further, methods for power analyses of longitudinal mixed models are complex and often challenging for the non-statistician. We discuss methods for extracting clinically relevant inputs from literature, and explain how to conduct a power analysis that appropriately accounts for longitudinal repeated measures. Finally, we provide careful recommendations for describing complex power analyses in a concise and clear manner. Methods For longitudinal studies of health outcomes from environmental exposures, we show how to [1] conduct a power analysis that aligns with the planned mixed model data analysis, [2] gather the inputs required for the power analysis, and [3] conduct repeated measures power analysis with a highly-cited, validated, free, point-and-click, web-based, open source software platform which was developed specifically for scientists. Results As an example, we describe the power analysis for a proposed study of repeated measures of per- and polyfluoroalkyl substances (PFAS) in human blood. We show how to align data analysis and power analysis plan to account for within-participant correlation across repeated measures. We illustrate how to perform a literature review to find inputs for the power analysis. We emphasize the need to examine the sensitivity of the power values by considering standard deviations and differences in means that are smaller and larger than the speculated, literature-based values. Finally, we provide an example power calculation and a summary checklist for describing power and sample size analysis. Conclusions This paper provides a detailed roadmap for conducting and describing power analyses for longitudinal studies of environmental exposures. It provides a template and checklist for those seeking to write power analyses for grant applications.

OBJECTIVE The metabolic phenotype of youth-onset type 2 diabetes (T2D) differs from that of adult-onset T2D, but little is known about genetic contributions. We aimed to evaluate the association between a T2D genetic risk score (GRS) and traits related to glucose-insulin homeostasis among healthy youth. RESEARCH DESIGN AND METHODS We used data from 356 youth (mean age 16.7 years; 50% female) in the Exploring Perinatal Outcomes Among Children (EPOCH) cohort to calculate a standardized weighted GRS based on 271 single nucleotide polymorphisms associated with T2D in adults. We used linear regression to assess associations of the GRS with log-transformed fasting glucose, 2-h glucose, HOMA of insulin resistance (HOMA-IR), oral disposition index, and insulinogenic index adjusted for age, sex, BMI z score, in utero exposure to maternal diabetes, and genetic principal components. We also evaluated effect modification by BMI z score, in utero exposure to maternal diabetes, and ethnicity. RESULTS Higher weighted GRS was associated with lower oral disposition index (β = −0.11; 95% CI −0.19, −0.02) and insulinogenic index (β = −0.08; 95% CI −0.17, −0.001), but not with fasting glucose (β = 0.01; 95% CI −0.01, 0.02), 2-h glucose (β = 0.03; 95% CI −0.0004, 0.06), or HOMA-IR (β = 0.02; 95% CI −0.04, 0.07). BMI z score and in utero exposure to maternal diabetes increased the effect of the GRS on glucose levels. CONCLUSIONS Our results suggest that T2D genetic risk factors established in adults are relevant to glucose-insulin homeostasis in youth and that maintaining a healthy weight may be particularly important for youth with high genetic risk of T2D.

No reports examine the relationship between in-utero exposure to gestational diabetes mellitus (GDM), offspring epigenetic age acceleration (EAA), and offspring insulin sensitivity.Using data from a cohort study, we examined associations between GDM in-utero exposure and offspring EAA at approximately 10 years of age, using separate regression models adjusting for offspring chronological age and sex. We also examined associations between EAA with updated homeostasis model assessment of insulin sensitivity and secretion (HOMA2-S and HOMA2-β) measured at approximately 10 and 16 years of age, using mixed linear regression models accounting for repeated measures after adjustment for offspring chronological age and sex.Compared to unexposed offspring (n = 91), offspring exposed to GDM (n = 88) had greater EAA or older extrinsic age compared to chronological age (β-coefficient 2.00, 95% confidence interval [0.71, 3.28], p = 0.0025), but not greater intrinsic EAA (β-coefficient -0.07, 95% CI [-0.71, 0.57], p = 0.93). Extrinsic EAA was associated with lower insulin sensitivity (β-coefficient -0.018, 95% CI [-0.035, -0.002], p = 0.03) and greater insulin secretion (β-coefficient 0.018, 95% CI [0.006, 0.03], p = 0.003), and these associations persisted after further adjustment for measures of maternal and child adiposity. No associations were observed between intrinsic EAA and insulin sensitivity and secretion, before or after adjustment for measures of maternal and child adiposity.In this study, children exposed to GDM experience greater extrinsic EAA, which is associated with lower insulin sensitivity and greater insulin secretion. Further studies are needed to determine the directionality of these associations.

The limitations of self-report measures of dietary intake are well-known. Novel, technology-based measures of dietary intake may provide a more accurate, less burdensome alternative to existing tools. The first objective of this study was to compare participant burden for two technology-based measures of dietary intake among school-age children: the Automated-Self-Administered 24-hour Dietary Assessment Tool-2018 (ASA24-2018) and the Remote Food Photography Method (RFPM). The second objective was to compare reported energy intake for each method to the Estimated Energy Requirement for each child, as a benchmark for actual intake. Forty parent–child dyads participated in two, 3-d dietary assessments: a parent proxy-reported version of the ASA24 and the RFPM. A parent survey was subsequently administered to compare satisfaction, ease of use and burden with each method. A linear mixed model examined differences in total daily energy intake between assessments, and between each assessment method and the Estimated Energy Requirement (EER). Reported energy intake was 379 kcal higher with the ASA24 than the RFPM (P = 0·0002). Reported energy intake with the ASA24 was 231 kcal higher than the EER (P = 0·008). Reported energy intake with the RFPM did not differ significantly from the EER (difference in predicted means = −148 kcal, P = 0·09). Median satisfaction and ease of use scores were five out of six for both methods. A higher proportion of parents reported that the ASA24 was more time-consuming than the RFPM (74·4 % v. 25·6 %, P = 0·002). Utilisation of both methods is warranted given their high satisfaction among parents.

We studied arterial stiffness in youth and young adults with T1D and potential longitudinal mediators of racial and ethnic disparities. SEARCH for Diabetes in Youth Study participants (n=2317; 14% Hispanic, 12% non-Hispanic Black, 74% non-Hispanic White) had CV risk factors (body size, lipid profile, blood pressure, A1c, urinary creatinine, pulse) measured 2-6 times from ∼9 months up to ˜years after T1D diagnosis. Arterial stiffness was measured with pulse wave velocity (PWV) at ˜years T1D duration (˜18 years of age) . For each risk factor, we summarized the longitudinal data as mean, max, slope, and area-under-the-curve (AUC) . Using split test/replication datasets, we a) identified the summary statistic for each risk factor most associated with PWV using flipped multivariate general linear models, and b) used the resulting statistics to construct a risk score predicting PWV with a stepwise general linear model. The final score included AUC for BMI, triglycerides and creatinine; heart rate slope; max mean arterial pressure; and age and T1D duration at PWV measurement, explaining 35% of variability in PWV at 18 years. PWV varied by race and ethnicity (Figure - left) . Adjustment for the risk score partially attenuated this disparity (Figure - right) . These data suggest CV risk factors from T1D diagnosis onward are related to racial and ethnic disparities in arterial stiffness, but other factors are likely involved. Disclosure K.A.Sauder: None. S.M.Marcovina: None. E.J.Mayer-davis: None. A.K.Mottl: Advisory Panel; Bayer AG, Board Member; Bayer AG, Research Support; Alexion Pharmaceuticals, Inc., Aurinia, Bayer AG, Boehringer Ingelheim International GmbH, Pfizer Inc. C.Pihoker: None. K.Reynolds: Research Support; Amgen Inc., Merck & Co., Inc., Novartis Pharmaceuticals Corporation. A.S.Shah: None. E.M.Urbina: Advisory Panel; Astellas Pharma Inc. L.E.Wagenknecht: None. S.R.Daniels: None. D.Dabelea: None. D.H.Glueck: None. K.K.Harrall: None. R.Dagostino: Consultant; Aetion, Inc., AstraZeneca, Biogen, Bristol-Myers Squibb Company, Daiichi Sankyo, Merck & Co., Inc. L.M.Dolan: None. A.Lane-cordova: None. A.D.Liese: None. E.Lustigova: None. F.Malik: n/a. Funding National Institutes of Health

Unhealthy diet, physical inactivity, and social disconnection are important modifiable risk factors for non-communicable and other chronic diseases, which might be alleviated through nature-based community interventions. We tested whether a community gardening intervention could reduce these common health risks in an adult population that is diverse in terms of age, ethnicity, and socioeconomic status.In this observer-blind, randomised, controlled trial, we recruited individuals who were on Denver Urban Garden waiting lists for community gardens in Denver and Aurora (CO, USA), aged 18 years or older, and had not gardened in the past 2 years. Participants were randomly assigned (1:1), using a randomised block design in block sizes of two, four, or six, to receive a community garden plot (intervention group) or remain on a waiting list and not garden (control group). Researchers were masked to group allocation. Primary outcomes were diet, physical activity, and anthropometry; secondary outcomes were perceived stress and anxiety. During spring (April to early June, before randomisation; timepoint 1 [T1]), autumn (late August to October; timepoint 2 [T2]), and winter (January to March, after the intervention; timepoint 3 [T3]), participants completed three diet recalls, 7-day accelerometry, surveys, and anthropometry. Analyses were done using the intention-to-treat principle (ie, including all participants randomly assigned to groups, and assessed as randomised). We used mixed models to test time-by-intervention hypotheses at an α level of 0·04, with T2 and T3 intervention effects at an α level of 0·005 (99·5% CI). Due to potential effects of the COVID-19 pandemic on outcomes, we excluded all participant data collected after Feb 1, 2020. This study is registered with ClinicalTrials.gov, NCT03089177, and data collection is now complete.Between Jan 1, 2017, and June 15, 2019, 493 adults were screened and 291 completed baseline measures and were randomly assigned to the intervention (n=145) or control (n=146) groups. Mean age was 41·5 years (SD 13·5), 238 (82%) of 291 participants were female, 52 (18%) were male, 99 (34%) identified as Hispanic, and 191 (66%) identified as non-Hispanic. 237 (81%) completed measurements before the beginning of the COVID-19 pandemic. One (1%) participant in the intervention group had an adverse allergic event in the garden. Significant time-by-intervention effects were observed for fibre intake (p=0·034), with mean between-group difference (intervention minus control) at T2 of 1·41 g per day (99·5% CI -2·09 to 4·92), and for moderate-to-vigorous physical activity (p=0·012), with mean between-group difference of 5·80 min per day (99·5% CI -4·44 to 16·05). We found no significant time-by-intervention interactions for combined fruit and vegetable intake, Healthy Eating Index (measured using Healthy Eating Index-2010), sedentary time, BMI, and waist circumference (all p0·04). Difference score models showed greater reductions between T1 and T2 in perceived stress and anxiety among participants in the intervention group than among those in the control group.Community gardening can provide a nature-based solution, accessible to a diverse population including new gardeners, to improve wellbeing and important behavioural risk factors for non-communicable and chronic diseases.American Cancer Society, University of Colorado Cancer Centre, University of Colorado Boulder, National Institutes of Health, US Department of Agriculture National Institute of Food and Agriculture, Michigan AgBioResearch Hatch projects.

Adiposity is an established risk factor for pediatric nonalcoholic fatty liver disease (NAFLD), but little is known about the influence of body composition patterns earlier in life on NAFLD risk.We aimed to examine associations of body composition at birth and body composition trajectories from birth to early childhood with hepatic fat in early childhood.Data were from the longitudinal Healthy Start Study in Colorado. Fat-free mass index (FFMI), fat mass index (FMI), percentage body fat (BF%), and BMI were assessed at birth and/or ∼5 y in1200 children by air displacement plethysmography and anthropometrics. In a subset (n = 285), hepatic fat was also assessed at ∼5 y by MRI. We used a 2-stage modeling approach: first, we fit body composition trajectories from birth to early childhood using mixed models with participant-specific intercepts and linear slopes (i.e., individual deviations from the population average at birth and rate of change per year, respectively); second, associations of participant-specific trajectory deviations with hepatic fat were assessed by multivariable-adjusted linear regression.Participant-specific intercepts at birth for FFMI, FMI, BF%, and BMI were inversely associated with log-hepatic fat in early childhood in models adjusted for offspring demographics and maternal/prenatal variables [back-transformed β (95% CI) per 1 SD: 0.93 (0.88, 0.99), 0.94 (0.88, 0.99), 0.94 (0.89, 0.99), and 0.90 (0.85, 0.96), respectively]. Whereas, faster velocities for BF% and BMI from birth to ∼5 y were positively associated with log-hepatic fat [back-transformed β (95% CI) per 1 SD: 1.08 (1.01, 1.15) and 1.08 (1.02, 1.15), respectively]. These latter associations of BF% and BMI velocities with childhood hepatic fat were attenuated to the null when adjusted for participant-specific intercepts at birth.Our findings suggest that a smaller birth weight, combined with faster adiposity accretion in the first 5 y, predicts higher hepatic fat in early childhood. Strategies aiming to promote healthy body composition early in life may be critical for pediatric NAFLD prevention.This study was registered voluntarily at clinicaltrials.gov as NCT02273297.

Objective: The metabolic phenotype of youth-onset type 2 diabetes (T2D) differs from that of adult-onset T2D, but little is known about genetic contributions. We aimed to evaluate the association between a T2D genetic risk score (GRS) and traits related to glucose-insulin homeostasis among healthy youth. Research Design and Methods: We used data from 356 youth (mean age 16.7 years, 50% female) in the EPOCH cohort to calculate a standardized weighted GRS based on 271 single nucleotide polymorphisms (SNPs) associated with T2D in adults. We used linear regression to assess associations of the GRS with log-transformed fasting glucose, 2-hour glucose, homeostasis model of insulin resistance (HOMA-IR), oral disposition index, and insulinogenic index adjusted for age, sex, body mass index (BMI) z-score, in utero exposure to maternal diabetes, and genetic principal components. We also evaluated effect modification by BMI z-score, in utero exposure to maternal diabetes and ethnicity. Results: Higher weighted GRS was associated with lower oral disposition index (b=-0.11; 95% CI: -0.19, -0.02) and insulinogenic index (b=-0.08; 95% CI: -0.17, -0.001), but not with fasting glucose (b=0.01; 95% CI: -0.01, 0.02), 2-hour glucose (b=0.03; 95% CI: -0.0004, 0.06), or HOMA-IR (b=0.02; 95% CI: -0.04, 0.07). BMI z-score and in utero exposure to maternal diabetes increased the effect of the GRS on glucose levels. Conclusions: Our results suggest that T2D genetic risk factors established in adults are relevant to glucose-insulin homeostasis in youth and that maintaining a healthy weight may be particularly important for youth with high genetic risk for T2D.

Objectives: Epigenetic age acceleration (EAA) occurs when DNA methylation-predicted age is older than chronological age. EAA is associated with glucose intolerance in adults. In-utero exposure to gestational diabetes (GDM) is associated with glucose intolerance in offspring. It is not known if GDM predicts EAA in offspring or if offspring EAA is associated with insulin sensitivity and secretion. Methods: We examined the association between exposure to GDM in-utero and EAA, computed at average age 10.5 years, and EAA and measures of insulin sensitivity (HOMA-2S) and secretion (HOMA-2β) at average age 10.5 and 16.7 years in 209 girls and 209 boys in the Colorado EPOCH (Exploring Perinatal Outcomes Among Children) cohort. We calculated EAA using the Horvath, Hannum, and Levine calculators. Separate general linear mixed models adjusted associations for chronologic age and sex. Results: Exposure to GDM was associated with EAA using the Hannum (beta-coefficient 1.39, p=0.009) and Levine (1.92, p=0.024) but not the Horvath calculator (-0.01, p=0.69). EAA by the Hannum calculator predicted lower insulin sensitivity and higher insulin secretion (Table). Conclusions: We conclude that GDM predicts offspring EAA, and EAA is associated with decreased insulin sensitivity and increased insulin secretion. Associations are dependent upon the EAA calculator used. Disclosure C. Kim: None. K. K. Harrall: None. D. H. Glueck: None. B. Needham: None. D. Dabelea: None. Funding National Institutes of Health (R01DK076648, R01GM121081, R01DK068001307, M01RR00069, 5UG3OD023248)

AIMS/HYPOTHESIS: We previously showed that intrauterine exposure to gestational diabetes mellitus (GDM) increases selected markers of adiposity in pre-pubertal adolescents. In the present study, we examined these associations in adolescence, and explored whether they are strengthened as the participants transition through puberty. METHODS: Data from 597 individuals (505 unexposed, 92 exposed) participating in the longitudinal Exploring Perinatal Outcomes among Children (EPOCH) study in Colorado were collected at two research visits when the participants were, on average, 10.4 and 16.7 years old. Adiposity measures included BMI, waist/height ratio, and visceral and subcutaneous adipose tissue (as determined by MRI). Separate general linear mixed models were used to assess the longitudinal relationships between exposure to maternal GDM and each adiposity outcome. We tested whether the effect changed over time by including an interaction term between exposure and age in our models, and whether the associations were explained by postnatal behaviours. RESULTS: Compared with unexposed participants, those exposed to maternal GDM had higher BMI (β = 1.28; 95% CI 0.35, 2.21; p < 0.007), waist/height ratio (β = 0.03; 95% CI 0.01, 0.04; p = 0.0004), visceral adipose tissue (β = 4.81; 95% CI 1.08, 8.54; p = 0.01) and subcutaneous adipose tissue (β = 35.15; 95% CI 12.43, 57.87; p < 0.003). The magnitude of these differences did not change over time and the associations did not appear to be explained by postnatal behaviours. CONCLUSIONS/INTERPRETATION: Our data provide further evidence that intrauterine exposure to maternal GDM is associated with increased offspring adiposity, an effect that appears early in life and tracks throughout adolescence. Efforts to prevent childhood obesity following intrauterine exposure to maternal GDM should target the prenatal or early life periods.

OBJECTIVE: It is unclear how childhood adipose tissue deposition influences sex hormone profiles in later adolescence. DESIGN: Prospective cohort study PARTICIPANTS: Children (n=418) with a mean age of 10.5 (1.5) years at visit 1 and 16.7 (1.2) at visit 2 in the Exploring Perinatal Outcomes among Children (EPOCH) Study. MEASUREMENTS: We used reverse-scale Cox proportional hazard models to assess associations between pubertal dehydroepiandrosterone (DHEA), testosterone (T), and estradiol (E2) and childhood-to-puberty rate of change in visceral (VAT) and subcutaneous adipose tissue (SAT). Models stratified by sex and adjusted for childhood adiposity, maternal factors, birthweight, and pubertal onset, and then further adjusted for insulin, luteinizing hormone (LH), leptin and hepatic fat fraction. RESULTS: Among boys, more rapid accumulation of either VAT or SAT was associated with lower testosterone at visit 2 (HR 0.86, and 0.96, respectively, both p

Objective Adiposity, particularly visceral adipose tissue (VAT), predicts adverse cardiovascular risk factor profiles in children as well as adults. Although endogenous sex steroids likely influence VAT in adults, such an association has not been established in youth. The association between childhood and adolescent sex steroids with adiposity, specifically VAT, was examined before and after adjustment for other hormone changes. Methods These analyses examined longitudinal associations between sex steroids (testosterone, estradiol, dehydroepiandrosterone [DHEA]) and magnetic resonance imaging assessments of VAT in 418 children, 49% of whom were non‐White, at approximately 10 years old at Visit 1 (V1) and 17 years old at Visit 2 (V2). Linear mixed effects models adjusted for maternal education, household income, child caloric intake, physical activity, fasting insulin and leptin, and hepatic fat fraction. Differences in associations by race and pubertal stage were also assessed. Results At V1, mean body mass index (BMI) for boys was 19.1 (4.7) kg/m2 and for girls was 18.5 (4.1) kg/m2. At V2, mean BMI for boys was 23.7 (5.5) kg/m2 and for girls was 23.6 (5.7) kg/m2. For each ng/dl (0.035 nmol/L) increase in testosterone at V1, there was a 0.25 cm2 increase in concurrent and future VAT in non‐White (p = 0.04) but not White girls (p = 0.78). Higher levels of testosterone and DHEA at V1 were associated with greater concurrent and future VAT at V2. These associations were consistent regardless of pubertal stage. In boys, higher testosterone predicted higher future VAT but lower concurrent VAT. Estradiol and DHEA did not predict future VAT in boys. In girls, DHEA predicted future subcutaneous adipose tissue (SAT), and no sex steroids predicted future SAT in boys. Conclusions Testosterone levels predict VAT in boys and girls, and DHEA predicts VAT in girls, even after adjustment for other hormone changes.

OBJECTIVES: To explore the longitudinal association of neonatal adiposity (fat mass percentage) with BMI trajectories and childhood overweight and obesity from ages 2 to 6 years. METHODS: We studied 979 children from the Healthy Start cohort. Air displacement plethysmography was used to estimate fat mass percentage. Child weight and recumbent length or standing height were abstracted from medical records. Overweight and obesity were defined as BMI levels ≥85th percentile for age and sex. Mixed-effects models were used to examine the association between neonatal fat mass percentage and BMI trajectories from age 2 to 6 years. We tested for effect modification by sex, race and/or ethnicity, and breastfeeding duration. We estimated the proportion of children classified as overweight or obese at specific levels of neonatal fat mass percentage (mean ± SD). RESULTS: The mean neonatal adiposity level was 9.1% ± 4.0%. Child BMI levels differed by neonatal adiposity. Each SD increase in neonatal adiposity resulted in a 0.12 higher overall BMI level between ages 2 to 6 years (95% confidence interval: 0.03 to 0.20; P < .01), and this association was not modified by offspring sex, race and/or ethnicity, or breastfeeding duration. Increasing neonatal adiposity was associated with an increasing proportion of childhood overweight and obesity by age 5 years (P = .02). CONCLUSIONS: We provide novel evidence that higher neonatal adiposity is significantly associated with higher overall BMI levels and an increased likelihood of overweight or obesity from ages 2 to 6 years. Because various prenatal exposures may specifically influence offspring fat accretion, neonatal adiposity may be a useful surrogate end point for prenatal interventions aimed at reducing future childhood overweight and obesity.

Among adults, higher T predicts lower VAT in men and higher VAT in women, and higher E2 predicts greater SAT in women. The degree to which the association between adiposity and sex steroids is bidirectional is controversial. Using data from a cohort of adolescents, we examined whether sex steroid levels in early puberty influenced VAT and SAT deposition in adolescents. EPOCH participants (n=418, 209 girls and 209 boys) underwent sex steroid measures at visit 1 (mean age 10 years) and magnetic resonance imaging (MRI) assessments of VAT and SAT at visit 1 and visit 2 (mean age 17 years). Using mixed models, we examined whether T and E2 at visit 1 were associated with overall VAT and SAT levels from visit 1 to visit 2. Models adjusted for maternal education and income as well as children’s race, pubertal status, caloric intake, physical activity levels, insulin, leptin, and MRI-measured hepatic fat fraction. Among girls, each unit increase in T was associated with a higher overall VAT (0.16, 95% confidence interval [CI] 0.04, 0.28) and SAT (0.33, 95% CI 0.33, 1.52) levels. Each unit increase in E2 was associated with overall higher SAT (0.56, 95% CI 0.28, 0.83) levels. Among boys, undetectable T was associated with higher VAT (5.12, 95% CI 1.90, 8.36) and lower SAT (-18.85, 95% CI -36.23, -1.46) levels. We conclude that endogenous T and E2 during the pubertal transition significantly influence adolescent adipose tissue deposition in both boys and girls. Future investigations should examine how the use of exogenous sex hormones impacts adipose tissue deposition during this critical period. Additional follow-up is needed to determine how adolescent fat deposition impacts subsequent glucose tolerance in early adulthood. Disclosure C. Kim: Research Support; Spouse/Partner; Apple. K.K. Harrall: None. D.H. Glueck: None. D. Dabelea: None. Funding National Institutes of Health (R01DK068001)

Aim: We explored the feasibility of collecting and analyzing human microbiome data in a longitudinal randomized controlled trial of community gardening. Methods & materials: Participants were randomly assigned to gardening (N = 8) or control (N = 8). Participants provided stool, mouth, hand and forehead microbiome samples at six timepoints. Analyses combined mixed models with Qiita output. Results: Participant satisfaction was high, with 75% of participants completing evaluations. While no microbial effects were statistically significant due to small sample size, the analysis pipeline utility was tested. Conclusion: Longitudinal collection and analysis of microbiome data in a community gardening randomized controlled trial is feasible. The analysis pipeline will be useful in larger studies for assessment of the pathway between microbiota, gardening and health outcomes. This study was funded by the University of Colorado Boulder Population Center (CUPC, J Litt, PI), through the National Institute of Child Health & Human Development of the NIH under award number P2CHD066613-06 and the Center for Microbiome Innovation at the University of California San Diego. We also received supplemental funding through the Clinical & Translational Research Center (CTRC) to cover all laboratory costs (J Litt, PI). M Gascon received a fellowship from the Societat Econòmica Barcelonesa d'Amics del País (SEBAP) in 2018, Barcelona (Catalonia), for her research stay at the University of Colorado to conduct the statistical analysis for this work. DH Glueck was supported, in part, by R01GM121-81 and R25 GM111901

The following review article presents clinical and experimental features of alcohol-induced liver disease (ALD). Basic aspects of alcohol metabolism leading to the development of liver hepatotoxicity are discussed. ALD includes fatty liver, acute alcoholic hepatitis with or without liver failure, alcoholic steatohepatitis (ASH) leading to fibrosis and cirrhosis, and hepatocellular cancer (HCC). ALD is fully attributable to alcohol consumption. However, only 10–20% of heavy drinkers (persons consuming more than 40 g of ethanol/day) develop clinical ALD. Moreover, there is a link between behaviour and environmental factors that determine the amount of alcohol misuse and their liver disease. The range of clinical presentation varies from reversible alcoholic hepatic steatosis to cirrhosis, hepatic failure, and hepatocellular carcinoma. We aimed to (1) describe the clinico-pathology of ALD, (2) examine the role of immune responses in the development of alcoholic hepatitis (ASH), (3) propose diagnostic markers of ASH, (4) analyze the experimental models of ALD, (5) study the role of alcohol in changing the microbiota, and (6) articulate how findings in the liver and/or intestine influence the brain (and/or vice versa) on ASH; (7) identify pathways in alcohol-induced organ damage and (8) to target new innovative experimental concepts modeling the experimental approaches. The present review includes evidence recognizing the key toxic role of alcohol in ALD severity. Cytochrome p450 CYP2E1 activation may change the severity of ASH. The microbiota is a key element in immune responses, being an inducer of proinflammatory T helper 17 cells and regulatory T cells in the intestine. Alcohol consumption changes the intestinal microbiota and influences liver steatosis and liver inflammation. Knowing how to exploit the microbiome to modulate the immune system might lead to a new form of personalized medicine in ALF and ASH.

Objective: To describe and evaluate recruitment approaches for a randomized controlled trial (RCT) of community gardening in Denver, Colorado. (ClinicalTrials.gov: NCT03089177). Methods: We used community and staff feedback to adapt our recruitment approach from year 1 to year 2 of a multi-year RCT to address health behaviors related to cancer prevention. In year 2, we added a full-time recruitment coordinator, designed and implemented a tracking spreadsheet, and engaged advisory committee members, local garden leaders, and health partners in planning and outreach. Screening and consent rates, staff time and costs for years 1 and 2 are compared. Results: In year 1, recruitment methods yielded 136 initial contacts, 106 screenings and 64 consented participants. In year 2, enhanced staffing and outreach yielded 257 initial contacts, 193 screenings, and 123 consented participants. Personal referrals, health fairs, NextDoor, and fliers yielded the highest percentage of consented participants. School and community meetings yielded the lowest yield for potential participants. Spanish-speaking participants were mostly recruited by direct methods. Compared to year 1 recruitment, which required 707 h of staff time and cost $14,446, year 2 recruitment required 1224 h of staff time and cost $22,992. Average cost for retained participants was $226 (year 1) and $186 (year 2). Discussion: Those planning pragmatic clinical trials with recruitment in multi-ethnic communities can use the results from this study to understand the efficacy of techniques, and to budget costs for recruitment. While our culturally-tailored recruitment methods cost more, they provided more effective and efficient ways to reach recruitment goals. Keywords: Community-level, Clinical trial, Recruitment, Health behavior change, Cancer prevention, Gardens

Whether changes in visceral adipose tissue (VAT) in childhood predict adolescent androgens is not known. We examined the longitudinal relationship between MRI-assessed visceral adipose tissue (VAT) in childhood and future testosterone (T) among 418 EPOCH participants (209 girls and 209 boys) who were, on average 10.5 years at visit 1 and 16.7 years at visit 2. We used reverse Cox proportional hazards models in which hazard ratios less than 1 indicate greater risk for low (undetectable) sex hormones consistent with pre-puberty. Models explored the associations of baseline VAT, and change in VAT from visit 1 to visit 2, with risk of low sex hormone levels at visit 2, adjusted for covariates selected through backwards regression (race, self-reported Tanner stage, maternal income, insulin and leptin levels). All analyses were stratified by sex. In addition, due to different patterns of fat deposition in white and non-white adults, we tested for effect modification by race. In boys, the association between VAT at visit 1 and T at visit 2 differed by race; more VAT at visit 1 was associated with lower risk of low T at visit 2 among non-white boys (HR 1.02, 95% CI 1.007, 1.04) but not white boys (HR 1.00, 95% CI 0.98, 1.01). In contrast, greater fat accumulation between visits was associated with higher risk of low T at visit 2 (HR 0.90, 95% CI 0.83, 0.96), independent of race and other covariates. Among girls, neither baseline VAT levels, nor VAT accumulation between visits predicted risk of later low T concentrations. We conclude that greater VAT accumulation in childhood is associated with increased risk for low T levels, suggesting delayed puberty, in boys. These associations suggest that fat deposition may influence sex hormone profiles even in early childhood. Disclosure C. Kim: None. K.K. Harrall: None. D.H. Glueck: None. D. Dabelea: None. Funding National Institutes of Health

BACKGROUND: Intrauterine exposure to gestational diabetes (GDM) is associated with increased adiposity; however, not all offspring exposed to GDM exhibit excess adiposity. OBJECTIVES: Examine whether optimal diet and activity behaviors in infancy, childhood, and adolescence modify the association between GDM exposure and adiposity. METHODS: In 564 offspring (84 exposed to GDM), we assessed breastfeeding (maternal recall), dietary intake (food frequency questionnaire), physical activity (3-day recall) and adiposity (BMI, waist-to-height ratio, visceral and subcutaneous adipose tissue, subscapular-to-triceps skinfold ratio) at 10.4 (SD 1.5) and 16.7 (SD 1.2) years. Optimal behaviors were defined as ≥6 breastmilk months, Healthy Eating Index score ≥60, and daily vigorous activity >1 hour. Linear mixed models assessed the association between GDM exposure and adiposity among those with optimal versus suboptimal health behaviors, adjusting for sex, race/ethnicity, age, and pubertal status. RESULTS: GDM exposure was associated with increased skinfold ratio, visceral and subcutaneous adipose tissue among those with 0.10). GDM exposure was associated with increased BMI and subcutaneous adipose tissue among those with >1 hour vigorous activity (p0.30). CONCLUSIONS: The association of GDM exposure with excess adiposity is attenuated in offspring with more optimal diet and activity behaviors in infancy, childhood, and adolescence.

Gene co-expression analysis has proven to be a powerful tool for ascertaining the organization of gene products into networks that are important for organ function. An organ, such as the liver, engages in a multitude of functions important for the survival of humans, rats, and other animals; these liver functions include energy metabolism, metabolism of xenobiotics, immune system function, and hormonal homeostasis. With the availability of organ-specific transcriptomes, we can now examine the role of RNA transcripts (both protein-coding and non-coding) in these functions. A systems genetic approach for identifying and characterizing liver gene networks within a recombinant inbred panel of rats was used to identify genetically regulated transcriptional networks (modules). For these modules, biological consensus was found between functional enrichment analysis and publicly available phenotypic quantitative trait loci (QTL). In particular, the biological function of two liver modules could be linked to immune response. The eigengene QTLs for these co-expression modules were located at genomic regions coincident with highly significant phenotypic QTLs; these phenotypes were related to rheumatoid arthritis, food preference, and basal corticosterone levels in rats. Our analysis illustrates that genetically and biologically driven RNA-based networks, such as the ones identified as part of this research, provide insight into the genetic influences on organ functions. These networks can pinpoint phenotypes that manifest through the interaction of many organs/tissues and can identify unannotated or under-annotated RNA transcripts that play a role in these phenotypes. Electronic supplementary material The online version of this article (doi:10.1007/s00335-016-9656-5) contains supplementary material, which is available to authorized users.

Background People with type 2 diabetes (T2D) have impaired exercise capacity, even in the absence of complications, which is predictive of their increased cardiovascular mortality. Cardiovascular dysfunction is one potential cause of this exercise defect. Acute infusion of vitamin C has been separately shown to improve diastolic and endothelial function in prior studies. We hypothesized that acute vitamin C infusion would improve exercise capacity and that these improvements would be associated with improved cardiovascular function. Methods Adults with T2D (n = 31, 7 female, 24 male, body mass index (BMI): 31.5 ± 0.8 kg/m2) and BMI-similar healthy adults (n = 21, 11 female, 10 male, BMI: 30.4 ± 0.7 kg/m2) completed two randomly ordered visits: IV infusion of vitamin C (7.5 g) and a volume-matched saline infusion. During each visit peak oxygen uptake (VO2peak), brachial artery flow mediated dilation (FMD), reactive hyperemia (RH; plethysmography), and cardiac echocardiography were measured. General linear mixed models were utilized to assess the differences in all study variables. Results Acute vitamin C infusion improved diastolic function, assessed by lateral and septal E:E’ (P P = 0.92), or VO2peak (P = 0.33) in any participants. Conclusion Acute vitamin C infusion improved diastolic function but did not change FMD, forearm reactive hyperemia, or peak exercise capacity. Future studies should further clarify the role of endothelial function as well as other possible physiological causes of exercise impairment in order to provide potential therapeutic targets. Trial registration NCT00786019. Prospectively registered May 2008

Objectives: Studies suggest that rheumatoid arthritis (RA)-related autoimmunity is initiated at a mucosal site. However, the factors associated with the mucosal generation of this autoimmunity are unknown, especially in individuals who are at-risk for future RA. Therefore, we tested anti-cyclic citrullinated peptide (anti-CCP) antibodies in the sputum of RA-free first-degree relatives (FDRs) of RA patients and patients with classifiable RA. Methods: We evaluated induced sputum and serum from 67 FDRs and 20 RA subjects for anti-CCP-IgA and anti-CCP-IgG, with cut-off levels for positivity determined in a control population. Sputum was also evaluated for cell counts, neutrophil extracellular traps (NETs) using sandwich ELISAs for protein/nucleic acid complexes, and total citrulline. Results: Sputum anti-CCP-IgA and/or anti-CCP-IgG was positive in 17/67 (25%) FDRs and 14/20 (70%) RA subjects, including a portion of FDRs who were serum anti-CCP negative. In FDRs, elevations of sputum anti-CCP-IgA and anti-CCP-IgG were associated with elevated sputum cell counts and levels of NET complexes. Anti-CCP-IgA was associated with ever-smoking and elevated sputum citrulline levels. Conclusions: Anti-CCP is elevated in the sputum of FDRs, including seronegative FDRs, suggesting the lung may be one site of anti-CCP generation in this population. The association of anti-CCP with elevated cell counts and NET levels in FDRs supports a hypothesis that local airway inflammation and NET formation may drive anti-CCP production in the lung and may promote the early stages of RA development. Longitudinal studies are needed to follow the evolution of these processes relative to the development of systemic autoimmunity and articular RA. This article is protected by copyright. All rights reserved.

Background: Hispanic women have lower breast cancer incidence rates than non-Hispanic white (NHW) women. To what extent genetic versus nongenetic factors account for this difference is unknown. Methods: Using logistic regression, we evaluated the interactive influences of established risk factors and ethnicity (self-identified and identified by ancestral informative markers) on breast cancer risk among 2,326 Hispanic and 1,854 NHW postmenopausal women from the United States and Mexico in the Breast Cancer Health Disparities Study. Results: The inverse association between the percentage of Native American (NA) ancestry and breast cancer risk was only slightly attenuated after adjusting for known risk factors [lowest versus highest quartile: odds ratio (OR) =1.39, 95% confidence interval (CI) = 1.00–1.92 among U.S. Hispanics; OR = 1.92 (95% CI, 1.29–2.86) among Mexican women]. The prevalence of several risk factors, as well as the associations with certain factors and breast cancer risk, differed according to genetic admixture. For example, higher body mass index (BMI) was associated with reduced risk among women with lower NA ancestry only [BMI 30: OR = 0.65 (95% CI, 0.44–0.98) among U.S. Hispanics; OR = 0.53 (95% CI, 0.29–0.97) among Mexicans]. The average number of risk factors among cases was inversely related to the percentage of NA ancestry. Conclusions: The lower NA ancestry groups were more likely to have the established risk factors, with the exception of BMI. Although the majority of factors were associated with risk in the expected directions among all women, BMI had an inverse association among Hispanics with lower NA ancestry. Impact: These data suggest that the established risk factors are less relevant for breast cancer development among women with more NA ancestry. Cancer Epidemiol Biomarkers Prev; 26(5); 692–701. ©2016 AACR.

The family Anelloviridae includes human and animal torque teno viruses (TTVs) with extensive genetic diversity. The antigenic diversity among anelloviruses has never been assessed. Using torque teno sus virus (TTSuV) as a model, we describe here the first investigation of the antigenic relationships among different anelloviruses. Using a TTSuV genotype 1a (TTSuV1a) or TTSuV1b enzyme-linked immunosorbent assay (ELISA) based on the respective putative ORF1 capsid antigen and TTSuV1-specific real-time PCR, the combined serological and virological profile of TTSuV1 infection in pigs was determined and compared with that of TTSuV2. TTSuV1 is likely not associated with porcine circovirus-associated disease (PCVAD), because both the viral loads and antibody levels were not different between affected and unaffected pigs and because there was no synergistic effect of concurrent PCV2/TTSuV1 infections. We did observe a higher correlation of IgG antibody levels between anti-TTSuV1a and -TTSuV1b than between anti-TTSuV1a or -1b and anti-TTSuV2 antibodies in these sera, implying potential antigenic cross-reactivity. To confirm this, rabbit antisera against the putative capsid proteins of TTSuV1a, TTSuV1b, or TTSuV2 were generated, and the antigenic relationships among these TTSuVs were analyzed by an ELISA and by an immunofluorescence assay (IFA) using PK-15 cells transfected with one of the three TTSuV ORF1 constructs. The results demonstrate antigenic cross-reactivity between the two genotypes TTSuV1a and TTSuV1b but not between the two species TTSuV1a or -1b and TTSuV2. Furthermore, an anti-genogroup 1 human TTV antiserum did not react with any of the three TTSuV antigens. These results have important implications for an understanding of the diversity of anelloviruses as well as for the classification and vaccine development of TTSuVs.

Porcine Torque teno virus (TTV), a single-stranded circular DNA virus, has been incriminated in swine diseases recently. Multiple infection with porcine TTV species 1 (PTTV1) and species 2 (PTTV2), each consisting of two types (PTTV1a and 1b) or subtypes (PTTV2b and 2c), in a single pig had been reported by our group previously. The present study described three novel assays for quantitation and differential detection of porcine TTV. First, we developed two SYBR green-based real-time PCR assays to quantify viral loads of two porcine TTV species, respectively. The PTTV1- and PTTV2-specific real-time PCR primer sequences were selected to target conserved regions identified by multiple alignments of ten available porcine TTV full-length genomes. Furthermore, by coupling the two singleplex PCR assays, a duplex real-time PCR assay followed by melting curve analysis was established for simultaneous detection and differentiation of PTTV1 and PTTV2. In addition, a type-specific duplex nested PCR was also developed to simultaneously detect and distinguish between the two types, PTTV1a and 1b, in PTTV1 species. These assays provide rapid and practical tools for molecular diagnosis of species- or type-specific porcine TTV.

The authors used a randomized study to evaluate a research experience in equivalent introductory biology courses at a 4-yr and a 2-yr institution. They found that unique barriers exist at 2-yr institutions that preclude student engagement. Students, instructors, and support staff needed a more comprehensive implementation strategy., Early research experiences must be made available to all undergraduate students, including those at 2-yr institutions who account for nearly half of America's college students. We report on barriers unique to 2-yr institutions that preclude the success of an early course-based undergraduate research experience (CURE). Using a randomized study design, we evaluated a CURE in equivalent introductory biology courses at a 4-yr institution and a 2-yr institution within the same geographic region. We found that these student populations developed dramatically different impressions of the experience. Students at the 4-yr institution enjoyed the CURE significantly more than the traditional labs. However, students at the 2-yr institution enjoyed the traditional labs significantly more, even though the CURE successfully produced targeted learning gains. On the basis of course evaluations, we enhanced instructor, student, and support staff training and reevaluated this CURE at a different campus of the same 2-yr institution. This time, the students reported that they enjoyed the research experience significantly more than the traditional labs. We conclude that early research experiences can succeed at 2-yr institutions, provided that a comprehensive implementation strategy targeting instructor, student, and support staff training is in place.

Anelloviruses are a group of single-stranded circular DNA viruses infecting humans and other animal species. Animal models combined with reverse genetic systems of anellovirus have not been developed. We report here the construction and initial characterization of full-length DNA clones of a porcine anellovirus, torque teno sus virus 2 (TTSuV2), in vitro and in vivo . We first demonstrated that five cell lines, including PK-15 cells, are free of TTSuV1 or TTSuV2 contamination, as determined by a real-time PCR and an immunofluorescence assay (IFA) using anti-TTSuV antibodies. Recombinant plasmids harboring monomeric or tandem-dimerized genomic DNA of TTSuV2 from the United States and Germany were constructed. Circular TTSuV2 genomic DNA with or without introduced genetic markers and tandem-dimerized TTSuV2 plasmids were transfected into PK-15 cells, respectively. Splicing of viral mRNAs was identified in transfected cells. Expression of TTSuV2-specific open reading frame 1 (ORF1) in cell nuclei, especially in nucleoli, was detected by IFA. However, evidence of productive TTSuV2 infection was not observed in 12 different cell lines transfected with the TTSuV2 DNA clones. Transfection with circular DNA from a TTSuV2 deletion mutant did not produce ORF1 protein, suggesting that the observed ORF1 expression is driven by TTSuV2 DNA replication in cells. Pigs inoculated with either the tandem-dimerized clones or circular genomic DNA of U.S. TTSuV2 developed viremia, and the introduced genetic markers were retained in viral DNA recovered from the sera of infected pigs. The availability of an infectious DNA clone of TTSuV2 will facilitate future study of porcine anellovirus pathogenesis and biology.

Porcine Torque teno virus (TTV) has a single-stranded circular DNA genome and is currently classified into a new genus Iotatorquevirus with two species in a newly established family Anelloviridae. Viral DNA of both porcine TTV species (TTSuV1 and TTSuV2) has a high prevalence in both healthy and diseased pigs worldwide and multiple infections of TTSuV with distinct genotypes or subtypes of the same species has been documented in the United States and in Europe. However, the prevalence of specific TTSuV antibodies in pigs remains unknown. In this study, the putative ORF1 capsid protein from TTSuV2 isolate PTTV2c-VA was expressed in Escherichia coli. The purified recombinant ORF1 protein was used as the antigen for the development of Western blot and indirect ELISA to detect TTSuV2-specific IgG antibodies in pig sera. The results revealed a relatively high rate of seropositivity to TTSuV2 in conventional pigs from different sources but not in gnotobiotic pigs. Overall, pigs with undetectable TTSuV2 viral load were more likely to have a lower anti-TTSuV2 antibody level. An analysis of 10 conventional pigs during a 2-month period showed that decreased viral loads or presumed virus clearance were associated with elevated anti-ORF1 IgG antibody levels. Interestingly, porcine circovirus associated disease (PCVAD)-affected pigs had a significantly lower level of TTSuV2 antibody than PCVAD-unaffected pigs (p