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Aims/hypothesis: This is the first study to examine the association between variants of the glucagon-like-peptide-1 receptor gene (GLP-1R) and metabolic characteristics among youth. We explored separate associations of three GLP-1R polymorphisms (rs10305420, rs6923761, and rs1042044) with BMI trajectories and markers of glucose-insulin homeostasis., Methods: Mixed models examined associations between GLP-1R polymorphisms and trajectories of BMI. Linear models examined associations of GLP-1R polymorphisms with glucose and insulin concentrations across OGTT, insulin sensitivity (HOMA2-IR), insulin secretion (insulinogenic index and HOMA2-%B), and beta cell function (oral disposition index)., Results: Rs10305420 and rs6923761, but not rs1042044, were associated with growth and metabolic characteristics in early life. Rs6923761 genotype GG was associated with faster BMI growth velocity, when compared to carriers of the minor allele (difference in velocity [95% CI]: 0.16 kg/m2/year [0.07, 0.24] at age 10), which led to significantly higher average BMI by age 16 (average difference [95% CI]: 1.29 kg/m2 [0.22, 2.37]). Rs10305420 CC and rs6923761 GG genotypes had higher HOMA2-IR (β[95% CI]: 1.19%[1.06, 1.32] and 1.13%[1.01, 1.26], respectively) compared to minor allele carriers. Rs10305420 CC had higher HOMA2-%B (β[95% CI]: 1.09%[1.01, 1.17]), and higher stimulated insulin secretion at 30-minutes (β[95% CI]: 27.62 uIU/mL [3.00, 25.24]) and 120-minutes (β[95% CI]: 18.94 uIU/mL [1.04, 36.84), when compared to carriers of the minor allele., Conclusions: GLP-1R polymorphisms were associated with faster BMI growth across development, and lower estimated insulin sensitivity and higher compensatory insulin secretion during adolescence. GLP-1R polymorphisms should be considered in future pediatric studies of genetic susceptibility for obesity and diabetes., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. See the journal About page for additional terms.)

Researchers often aim to assess whether repeated measures of an exposure are associated with repeated measures of an outcome. A question of particular interest is how associations between exposures and outcomes may differ over time. In other words, researchers may seek the best form of a temporal model. While several models are possible, researchers often consider a few key models. For example, researchers may hypothesize that an exposure measured during a sensitive period may be associated with repeated measures of the outcome over time. Alternatively, they may hypothesize that the exposure measured immediately before the current time period may be most strongly associated with the outcome at the current time. Finally, they may hypothesize that all prior exposures are important. Many analytic methods cannot compare and evaluate these alternative temporal models, perhaps because they make the restrictive assumption that the associations between exposures and outcomes remains constant over time. Instead, we provide a tutorial describing four temporal models that allow the associations between repeated measures of exposures and outcomes to vary, and showing how to test which temporal model is best supported by the data. By finding the best temporal model, developmental psychopathology researchers can find optimal windows for intervention.

Tracking trajectories of body size in children provides insight into chronic disease risk. One measure of pediatric body size is body mass index (BMI), a function of height and weight. Errors in measuring height or weight may lead to incorrect assessment of BMI. Yet childhood measures of height and weight extracted from electronic medical records often include values which seem biologically implausible in the context of a growth trajectory. Removing biologically implausible values reduces noise in the data, and thus increases the ease of modeling associations between exposures and childhood BMI trajectories, or between childhood BMI trajectories and subsequent health conditions. We developed open-source algorithms (available on github) for detecting and removing biologically implausible values in pediatric trajectories of height and weight. A Monte Carlo simulation experiment compared the sensitivity, specificity and speed of our algorithms to three published algorithms. The comparator algorithms were selected because they used trajectory information, had open-source code, and had published verification studies. Simulation inputs were derived from longitudinal epidemiological cohorts. Our algorithms had higher specificity, with similar sensitivity and speed, when compared to the three published algorithms. The results suggest that our algorithms should be adopted for cleaning longitudinal pediatric growth data., (© 2024. The Author(s).)

We give examples of three features in the design of randomized controlled clinical trials which can increase power and thus decrease sample size and costs. We consider an example multilevel trial with several levels of clustering. For a fixed number of independent sampling units, we show that power can vary widely with the choice of the level of randomization. We demonstrate that power and interpretability can improve by testing a multivariate outcome rather than an unweighted composite outcome. Finally, we show that using a pooled analytic approach, which analyzes data for all subgroups in a single model, improves power for testing the intervention effect compared to a stratified analysis, which analyzes data for each subgroup in a separate model. The power results are computed for a proposed prevention research study. The trial plans to randomize adults to either telehealth (intervention) or in-person treatment (control) to reduce cardiovascular risk factors. The trial outcomes will be measures of the Essential Eight, a set of scores for cardiovascular health developed by the American Heart Association which can be combined into a single composite score. The proposed trial is a multilevel study, with outcomes measured on participants, participants treated by the same provider, providers nested within clinics, and clinics nested within hospitals. Investigators suspect that the intervention effect will be greater in rural participants, who live farther from clinics than urban participants. The results use published, exact analytic methods for power calculations with continuous outcomes. We provide example code for power analyses using validated software., (© 2024. The Author(s).)

Aims: Determining diabetes type in children has become increasingly difficult due to an overlap in typical characteristics between type 1 diabetes (T1D) and type 2 diabetes (T2D). The Diabetes Study in Children of Diverse Ethnicity and Race (DISCOVER) programme is a National Institutes of Health (NIH)-supported multicenter, prospective, observational study that enrols children and adolescents with non-secondary diabetes. The primary aim of the study was to develop improved models to differentiate between T1D and T2D in diverse youth., Materials and Methods: The proposed models will evaluate the utility of three existing T1D genetic risk scores in combination with data on islet autoantibodies and other parameters typically available at the time of diabetes onset. Low non-fasting serum C-peptide (<0.6 nmol/L) between 3 and 10 years after diabetes diagnosis will be considered a biomarker for T1D as it reflects the loss of insulin secretion ability. Participating centres are enrolling youth (<19 years old) either with established diabetes (duration 3-10 years) for a cross-sectional evaluation or with recent onset diabetes (duration 3 weeks-15 months) for the longitudinal observation with annual visits for 3 years. Cross-sectional data will be used to develop models. Longitudinal data will be used to externally validate the best-fitting model., Results: The results are expected to improve the ability to classify diabetes type in a large and growing subset of children who have an unclear form of diabetes at diagnosis., Conclusions: Accurate and timely classification of diabetes type will help establish the correct clinical management early in the course of the disease., (© 2023 John Wiley & Sons Ltd.)

Using data from a longitudinal cohort of children, we examined whether epigenetic age acceleration (EAA) was associated with pubertal growth and whether these associations were mediated by adiposity. We examined associations between EAA at approximately 10 years of age with pubertal growth metrics, including age at peak height velocity (PHV), PHV, and sex steroid levels and whether these associations were mediated by measures of adiposity including body mass index (BMI) and MRI-assessed visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT). Children (n = 135) with accelerated EAA had higher PHV (β 0.018, p = 0.0008) although the effect size was small. The association between EAA and age at PHV was not significant (β  - 0.0022, p = 0.067). Although EAA was associated with higher BMI (β 0.16, p = 0.0041), VAT (β 0.50, p = 0.037), and SAT (β 3.47, p = 0.0076), BMI and VAT did not mediate associations between EAA and PHV, while SAT explained 8.4% of the association. Boys with higher EAA had lower total testosterone (β  - 12.03, p = 0.0014), but associations between EAA and other sex steroids were not significant, and EAA was not associated with sex steroid levels in girls. We conclude that EAA did not have strong associations with either age at onset of puberty or pubertal growth speed, although associations with growth speed were statistically significant. Studies with larger sample sizes are needed to confirm this pattern of associations., (© 2024. The Author(s).)

Background: Infant feeding patterns have been linked with obesity risk in childhood, but associations with precise measures of body fat distribution are unclear., Objective: We examined associations of infant feeding practices with abdominal fat and hepatic fat trajectories in childhood., Methods: This study included 356 children in the Healthy Start Study, a prospective prebirth cohort in Colorado. Infant feeding practices were assessed by postnatal interviews and categorized as any human milk <6 mo compared with ≥6 mo; complementary foods introduced ≤4 mo compared with >4 mo; soda introduced ≤18 mo compared with >18 mo. Abdominal subcutaneous (SAT) and visceral adipose tissue (VAT) areas and hepatic fat (%) were assessed by magnetic resonance imaging in early and middle childhood (median 5 and 9 y old, respectively). We examined associations of infant feeding with adiposity trajectories across childhood using linear mixed models., Results: In the sample of children, 67% consumed human milk ≥6 mo, 75% were introduced to complementary foods at >4 mo, and 81% were introduced to soda at >18 mo. We did not find any associations between duration of any human milk consumption and childhood adiposity trajectories. Early introduction to complementary foods (≤4 mo) was associated with faster rates of change for SAT and VAT during childhood (Slope [95% CI]: 15.1 [10.7,19.4] cm 2 /y for SAT; 2.5 [1.9,2.9] cm 2 /y for VAT), compared with introduction at >4 mo (5.5 [3.0,8.0] cm 2 /y and 1.6 [1.3,1.9] cm 2 /y, respectively). Similarly, early introduction to soda (≤18 mo) was associated with faster rates of change for all 3 outcomes during childhood (Slope [95% CI]: 20.6 [15.0,26.1] cm 2 /y for SAT, 2.7 [2.0,3.3] cm 2 /y for VAT, 0.3 [0.1,0.5] %/year for hepatic fat) compared with delayed introduction (5.4 [2.8,8.0] cm 2 /y, 1.7 [1.3, 2.0] cm 2 /y, -0.1 [-0.2,0.0] %/y, respectively)., Conclusions: The timing of introduction and quality of complementary foods in infancy was associated with rates of abdominal and hepatic fat accrual during childhood. Experimental studies are needed to assess underlying mechanisms., Competing Interests: Conflict of interest The authors have no conflicts of interest to disclose., (Copyright © 2023 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

Background: Prenatal exposures to certain poly- and perfluoroalkyl substances (PFAS) are associated with reduced humoral responses to some childhood immunizations., Objective: We estimated associations between prenatal PFAS exposure and child antibody titers for measles, mumps, rubella (MMR), and varicella after immunization., Methods: We measured serum antibody titers of 145 children (4-8 y old) enrolled in the Healthy Start cohort in Colorado, whose mothers had PFAS quantified mid-pregnancy (2009-2014). We used linear and logistic regression models to assess the relationship between five PFAS detected in > 65 % of mothers and continuous or non-high-censored ("low") antibody titers and quantile g-computation to evaluate the overall effect of the PFAS mixture., Results: Median concentrations of individual PFAS were at or below the median reported among females in the United States. After receiving two vaccine doses, seropositive levels of antibodies were detected among most (93%-100%) children. Each log-unit increase in perfluorononanoate was associated with 2.09 [95% confidence interval (CI): 1.13, 3.87] times higher odds of a low measles titer, and each log-unit increase in perfluorooctanoate was associated with 2.46 (95% CI: 1.28, 4.75) times higher odds of a low mumps titer. Odds ratios for all other PFAS were elevated, but CIs included the null. Each quartile increase in the PFAS mixture was associated with 1.35 (95% CI: 0.80, 2.26) times higher odds of a low measles titer and 1.44 (95% CI: 0.78, 2.64) times higher odds of a low mumps titer. No significant associations were observed between PFAS and varicella or rubella antibodies. In stratified analyses, associations were negative among female children, except for perfluorohexane sulfonate and varicella, whereas they were positive among males., Discussion: Some prenatal PFAS were associated with lower antibody titers among fully immunized children. The potential for immunotoxic effects of PFAS requires further investigation in a larger study, because exposure is ubiquitous globally. https://doi.org/10.1289/EHP12863.

Context: Previous studies have shown that exposure to maternal gestational diabetes mellitus (GDM) is associated with increased offspring body mass index (BMI) and risk for overweight or obesity., Objective: This study aimed to explore differences in BMI trajectories among youth exposed or not exposed to maternal 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 2 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 = .27). In middle childhood, participants exposed to GDM had higher BMI trajectories compared to those not exposed (males: P = .005, females: P = .002) and adolescent (P = .02) periods., Conclusion: 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., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Effortful control, or the regulation of thoughts and behaviour, is a potential target for preventing childhood obesity., Objectives: To assess effortful control in infancy through late childhood as a predictor of repeated measures of body mass index (BMI) from infancy through adolescence, and to examine whether sex moderates the associations., Methods: Maternal report of offspring effortful control and measurements of child BMI were obtained at 7 and 8 time points respectively from 191 gestational parent/child dyads from infancy through adolescence. General linear mixed models were used., Results: Effortful control at 6 months predicted BMI trajectories from infancy through adolescence, F(5,338) = 2.75, p = 0.03. Further, when effortful control at other timepoints were included in the model, they added no additional explanatory value. Sex moderated the association between 6-month effortful control and BMI, F(4, 338) = 2.59, p = 0.03, with poorer infant effortful control predicting higher BMI in early childhood for girls, and more rapid increases in BMI in early adolescence for boys., Conclusions: Effortful control in infancy was associated with BMI over time. Specifically, poor effortful control during infancy was associated with higher BMI in childhood and adolescence. These findings support the argument that infancy may be a sensitive window for the development of later obesity., (© 2023 World Obesity Federation.)

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 We examined arterial stiffness in individuals with type 1 diabetes, and explored whether differences between Hispanic, non-Hispanic Black (NHB), and non-Hispanic White (NHW) individuals were attributable to modifiable clinical and social factors. Methods and Results Participants (n=1162; 22% Hispanic, 18% NHB, and 60% NHW) completed 2 to 3 research visits from ≈10 months to ≈11 years post type 1 diabetes diagnosis (mean ages of ≈9 to ≈20 years, respectively) providing data on socioeconomic factors, type 1 diabetes characteristics, cardiovascular risk factors, health behaviors, quality of clinical care, and perception of clinical care. Arterial stiffness (carotid-femoral pulse wave velocity [PWV], m/s) was measured at ≈20 years of age. We analyzed differences in PWV by race and ethnicity, then explored the individual and combined impact of the clinical and social factors on these differences. PWV did not differ between Hispanic (adjusted mean 6.18 [SE 0.12]) and NHW (6.04 [0.11]) participants after adjustment for cardiovascular risks ( P =0.06) and socioeconomic factors ( P =0.12), or between Hispanic and NHB participants (6.36 [0.12]) after adjustment for all factors ( P =0.08). PWV was higher in NHB versus NHW participants in all models (all P <0.001). Adjustment for modifiable factors reduced the difference in PWV by 15% for Hispanic versus NHW participants; by 25% for Hispanic versus NHB; and by 21% for NHB versus NHW. Conclusions Cardiovascular and socioeconomic factors explain one-quarter of the racial and ethnic differences in PWV of young people with type 1 diabetes, but NHB individuals still experienced greater PWV. Exploration of pervasive inequities potentially driving these persistent differences is needed.

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., (© 2023. The Author(s).)

Background: 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., Methods: 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., Findings: 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 p>0·04). 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., Interpretation: 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., Funding: 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., Competing Interests: Declaration of interests JRH owns controlling interest in Connecting Health Innovations (CHI), a company that has licensed the right to his invention of the dietary inflammatory index (DII) and for which JRH and the University of South Carolina have secured a federally registered trademark for the DII. The CHI aims to develop computer and smartphone applications for patient counselling and dietary intervention in clinical settings. CHI owns all derivative products, including the energy-adjusted DII (E-DII). All CHI-related activity occurred outside the submitted work, which includes royalties paid to CHI. JRH confirms that the subject matter of this manuscript did not have any direct bearing on that work, nor has that activity exerted any influence on this project. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

The objective of the study was to investigate whether adverse and benevolent childhood experiences were associated with trajectories of sleep quality throughout pregnancy. The study was conducted at obstetrics and gynecology clinics in the Rocky Mountain region of the USA. The participants of the study were pregnant individuals ( N = 164). Sleep quality was measured with the Pittsburgh Sleep Quality Index at three gestational time points, and adverse childhood experiences (ACEs) and benevolent childhood experiences (BCEs) were assessed once. Multilevel models were conducted to examine the trajectory of sleep quality across gestation in relation to ACEs and BCEs. Sleep quality was similar in early to mid-pregnancy, with a worsening of sleep quality late in pregnancy, following a quadratic trajectory. Higher levels of ACEs predicted poorer prenatal sleep quality ( b = 0.36, SE = 0.13, p = .004) throughout pregnancy, while higher levels of BCEs predicted better sleep quality ( b = -0.60, SE = 0.17, p < .001) throughout pregnancy. Examination of ACEs subtypes revealed that childhood maltreatment predicted poor sleep quality ( b = 0.66, SE = 0.18, p < .001), while childhood household dysfunction was not significantly associated ( b = 0.33, SE = 0.21, p = .11). Associations remained after covarying for socioeconomic status and current stressful life events. Both adverse and benevolent childhood experiences predict sleep health during pregnancy. Prevention and intervention strategies targeting resilience and sleep quality during pregnancy should be implemented to promote prenatal health and well-being., Competing Interests: Conflict of Interest The authors declare no competing interests.

Aims: No reports examine the relationship between in-utero exposure to gestational diabetes mellitus (GDM), offspring epigenetic age acceleration (EAA), and offspring insulin sensitivity., Methods: 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., Results: 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., Conclusions: 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., (© 2022 The Authors. Diabetic Medicine published by John Wiley & Sons Ltd on behalf of Diabetes UK.)

Background: 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., Objectives: 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., Methods: 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 in >1200 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., Results: 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., Conclusions: 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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society for Nutrition.)

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.

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., (© 2021 by the American Diabetes Association.)

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/m 2 and for girls was 18.5 (4.1) kg/m 2 . At V2, mean BMI for boys was 23.7 (5.5) kg/m 2 and for girls was 23.6 (5.7) kg/m 2 . For each ng/dl (0.035 nmol/L) increase in testosterone at V1, there was a 0.25 cm 2 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., Competing Interests: The authors declared no conflict of interests. This work was presented in part at the 2019 Scientific Sessions of the American Diabetes Association., (© 2021 The Authors. Obesity Science & Practice published by World Obesity and The Obesity Society and John Wiley & Sons Ltd.)

The purpose of this study was to compare activPAL algorithm-estimated values for time in bed (TIB), wake time (WT) and bedtime (BT) against self-report and an algorithm developed by van der Berg and colleagues. Secondary analyses of baseline data from the Community Activity for Prevention Study (CAPS) were used in which adults ≥ 18 years wore the activPAL for seven days. Mixed-effects models compared differences between TIB, WT, and BT for all three methods. Bland-Altman plots examined agreement and the two-one-sided test examined equivalence. activPAL was not equivalent to self-report or van der Berg in estimating TIB, but was equivalent to self-report for estimating BT, and was equivalent to van der Berg for estimating WT. The activPAL algorithm requires adjustments before researchers can use it to estimate TIB. However, researchers can use activPAL's option to manually enter self-reported BT and WT to estimate TIB and better understand 24-hour movement patterns., Competing Interests: Declaration of Conflicts of Interest Kate Lyden is the Chief Science Officer at VivoSense, Inc. She previously worked as an independent consultant for several wearable device manufacturers, including PAL Technologies.

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., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2020 by the American Academy of Pediatrics.)

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.

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 behaviours 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, and subscapular-to-triceps skinfold ratio) at 10.4 (SD, 1.5) and 16.7 (SD, 1.2) years. Optimal behaviours 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 behaviours, 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 <6 breastmilk months (all Ps < .05), but only associated with increased skinfold ratio among those with >6 breastmilk months (P = .01). GDM exposure was associated with increases in all adiposity measures among those with Healthy Eating Index scores <60 (P < .01), but not those with scores >60 (P > .10). GDM exposure was associated with increased BMI and subcutaneous adipose tissue among those with >1 hour of vigorous activity (P < .05) but not among those with <1 hour of vigorous activity (P > .30)., Conclusions: The association of GDM exposure with excess adiposity is attenuated in offspring with more optimal diet and activity behaviours in infancy, childhood, and adolescence., (© 2019 World Obesity Federation.)

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., (© 2019 The Author(s).)

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 oestradiol (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 .96, respectively, both P < .0001), independently of race/ethnicity, LH, leptin and hepatic fat fraction. Among boys, more childhood VAT was associated with lower testosterone in adolescence (HR 0.98, P = .003), but this association did not persist after adjustment for leptin or insulin. No associations were observed between either fat measure and oestradiol or DHEA in boys. In girls, no associations between childhood fat or fat accumulation and sex hormones were observed., Conclusions: More rapid accumulation of fat is associated with lower testosterone in boys. These associations suggest that fat growth influences androgen profiles in adolescent boys. Since fat accumulation is a modifiable risk factor, the study results provide a possible intervention target and time period for improving adult health., (© 2019 John Wiley & Sons Ltd.)

We performed a meta-analysis to evaluate the femoral neck and lumbar spine bone mineral density (BMD) in adults with type 1 diabetes (T1D) compared with controls. Adults with T1D have modestly lower BMD at femoral neck and lumbar spine than adults without diabetes., Introduction: Fracture risk is four to sixfold higher in adults with T1D. Since BMD is one of the major contributors for fracture risk, we performed a meta-analysis to evaluate differences in femoral neck and lumbar spine BMD between adults with T1D and controls., Methods: MEDLINE, Ovid, and the Cochrane library and abstracts from various scientific meetings were searched. Studies reporting the femoral neck and/or lumbar spine BMD in adults (age > 20 years) with T1D in comparison with people without diabetes were selected. General linear mixed models were used to assess differences in BMD at femoral neck and lumbar spine between subjects with T1D and controls adjusting for age, sex, and dual x-ray absorptiometry (DXA) instruments., Results: Sixteen studies met the inclusion criteria. The femoral neck BMD was modestly lower in adults with T1D compared to controls (-0.055 g/cm 2 ; 95% CI: -0.065, -0.045). There were no differences in lumbar spine BMD between adults with T1D and controls (0.0062 g/cm 2 ; 95% CI -0.04, 0.016). However, in a sensitivity analysis, lumbar spine BMD was modestly lower in adults with T1D compared to controls (-0.035 g/cm 2 ; -0.049, -0.02). Studies using Lunar DXA instruments have reported higher lumbar spine and femoral neck BMD compared to studies using Hologic DXA instruments., Conclusion: Femoral neck and lumbar spine BMD were modestly lower in adults with T1D compared to controls. However, this modest reduction in femoral neck and lumbar spine BMD cannot explain much higher observed fracture risk in adults with T1D.

Objective: 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 of 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 samples from 67 FDRs and 20 RA patients for IgA anti-CCP and IgG anti-CCP, with cutoff levels for positivity determined in a control population. Sputum was also evaluated for cell counts, neutrophil extracellular traps (NETs) using sandwich enzyme-linked immunosorbent assays for protein/nucleic acid complexes, and total citrulline., Results: Sputum was positive for IgA and/or IgG anti-CCP in 14 of 20 RA patients (70%) and 17 of 67 FDRs (25%), including a portion of FDRs who were serum anti-CCP negative. In the FDRs, elevations of sputum IgA and IgG anti-CCP were associated with elevated sputum cell counts and NET levels. IgA anti-CCP was associated with ever smoking and with elevated sputum citrulline levels., Conclusion: Anti-CCP is elevated in the sputum of FDRs, including seronegative FDRs, suggesting that the lung may be a 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., (© 2017, American College of Rheumatology.)

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 <25 versus >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 ., (©2016 American Association for Cancer Research.)

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.

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., (© 2014 T. D. Wolkow et al. CBE—Life Sciences Education © 2014 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

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.

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<0.01). This is the first study to establish essential serodiagnostic tools for investigation of TTSuV seroprevalence and infection dynamics, which will help elucidate the potential pathogenicity of TTSuV infection in pigs., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)