Search

Latino early adolescents have a high prevalence of overweight and obesity. Interventions to address healthy eating among Latino youth frequently focus on fruit and vegetable (FV) intake. Reflection spectroscopy assessed skin carotenoid (SC) levels has been proposed as an easy, noninvasive method to evaluate FV intake, but validation studies involving ethnically diverse youth are lacking.This study aimed to assess the association between reflection spectroscopy-measured SC scores and self-reported FV intake among low-income, urban, Latino early adolescents, controlling for potential confounding factors.This study was a cross-sectional secondary analysis of baseline data from a community-based intervention program (Padres Preparados, Jóvenes Saludables) involving Latino fathers and adolescents to improve paternal parenting practices and youth energy balance-related behaviors.Participants were 195 low-income, Latino early adolescents (aged 10 to 14 years). Data were collected in the Minneapolis/St Paul metropolitan area from 2017 to 2020 during fall or winter months.SC scores were measured using reflection spectroscopy, usual intakes of FV and carotenoid compounds were estimated based on the assessment using 24-hour dietary recalls.Multivariable linear regression analyses were used to estimate associations of SC scores and each dietary component and potential confounding factors after assessing variables for inclusion in the analyses.The mean SC score was 225 ± 95. The mean FV and total carotenoid intakes were 3.3 ± 0.5 servings/day and 8,360 ± 786 μg/day, respectively. Higher SC scores were observed among youth who had higher FV (β = .37 and P0.01) or total carotenoid intakes (β = .31 and P0.01). SC scores measured during fall were higher than scores measured during winter. Study participants with higher home FV availability and accessibility had higher SC scores.Findings supported using SC score as a potential easy-to-use indicator of FV intake among Latino youth with consideration of seasonal variation and home FV availability and accessibility.

The consumption of meals prepared away from home (FAFH) in the U.S. has been commonly linked to overall poorer diet quality; however, less is known about the relationship with health parameters. The aim of this cross-sectional study was to assess the diet quality and health parameters of adults who reported the frequency of obtaining or eating FAFH using a combination of four 2-year cycles of National Health and Nutrition Examination Survey (NHANES, 2011–2018) data. Data from 14,999 adults aged 20 to 65 years were used to examine the associations between diet and health outcomes and the weekly frequency of FAFH. Multivariable regression models were used to compare Healthy Eating Index-2015 (HEI-2015) scores by FAFH frequency, adjusting for sociodemographic variables. Adults with more frequent FAFH meals were more likely to be younger, male, lower-income, and unmarried. Total HEI-2015 scores and component scores for greens and beans, total and whole fruits, whole grains, saturated fats, and added sugars were significantly lower in those consuming >2 FAFH meals/week vs. ≤2 FAFH meals/week. BMI and waist circumference tended to be higher for women having more frequent FAFH meals. More frequent FAFH meals among adults were associated with poorer diet quality and weight outcomes, building on results from earlier studies.

Adolescent obesity prevention programs focusing on Latino fathers may be useful to address Latino adolescent obesity. Adolescent obesity has become an urgent issue because of the coronavirus disease 2019 pandemic, with limited ability to deliver prevention programs in-person. This study aimed to assess the feasibility of a community-based, adolescent obesity prevention program for Latino father-adolescent dyads delivered remotely, adapted from the in-person Padres Preparados, Jóvenes Saludables program. A quasi-experimental design was used to assess the feasibility of the remotely delivered program based on criteria adapted from other feasibility studies of community-based health promotion programs for Latino families. Father-adolescent dyads were recruited at two sites in a Midwestern state during 2020; mothers were also encouraged to participate. Recruitment met the feasibility criteria (65 families expressed interest between August and October) with 26 families participating in this study. The retention ratio (percentage of participants who completed a post-session survey to those who completed the baseline survey) among fathers was acceptable (77%), while a lower percentage of adolescents were retained (68%). The session attendance ratio (number of sessions attended of the eight total sessions offered) was higher among adolescents (88%) and lower among fathers (68%) compared to the criteria. Satisfaction ratings (≥88%) and completeness of data collection at both baseline and post-session survey (≥76%) were acceptable. Favorable results were obtained for parent outcomes, while adolescent outcomes were not favorably changed. This remotely delivered adolescent obesity prevention program was feasible for Latino fathers; however, additional engagement with adolescents may be needed.Adolescent obesity became a more urgent issue because of the coronavirus disease 2019 pandemic with less physical activity under lockdown, more food insecure families and limited in-person access to programs. Our study concluded that a remotely delivered version of a community-based, adolescent obesity prevention program for Latino father–adolescent dyads (the Padres Preparados, Jóvenes Saludables program) was feasible based on participation, satisfaction and parent outcomes. Additional engagement in activities to improve adolescent health behaviors may be needed to improve the overall effectiveness of the program.

To maintain the health of evacuees, meals provided at emergency shelters should be monitored and improvement measures should be taken where necessary. Time and human resources are limited during devastating disasters; thus, the authors have developed the Dietary Assessment Sheets for Evacuation Shelters. This study examines whether the quality of meals can be assessed using simple check items on the Dietary Assessment Sheets.A total of 28 Dietary Assessment Sheets for 98 meals provided 16-19 days after the heavy rains in Kumamoto in July 2020 at 12 shelters were used in the analysis. Dietitians working for shelters were requested to fill the Dietary Assessment Sheets and food record sheets provided by the Prefectural Government. If any subitem was checkmarked, it was considered "checked," and if left blank, it was considered "not checked" and divided into categorical groups. Based on the food record sheets and meal photos, contents of energy, protein, and vitamins BThe study revealed that nutritionally poor meals were characterized as those comprising only "Grain dishes," those that were not "Boxed meals," and those involving "Self-Defense Forces," "Dietitians," or "Others." Their energy, protein, and vitamin BShelters that provide these meals should be given priority for nutrition assistance due to their poor diets. The results imply that the Dietary Assessment Sheets could serve as a simple tool to determine the shelters that require nutritional assistance.

Certain types of community-based social activities improve the health issues of older adults; however, the present patterns of participation in community activities remain unknown. This study aims 1) to identify community-dwelling older adults' patterns of participation in community-based activities and 2) to evaluate the relationships between social support, self-efficacy, self-rated health, and the patterns of participation in community activities. This cross-sectional study used data collected from 146 older adults aged ≥65 years who participated in community events in Japan in 2018. Cluster analysis was used to identify subjects' patterns of participation in community activities. In the multinomial logistic regression model, the participation pattern (dependent variable), and social support and self-efficacy (independent variables), were included, adjusting for age, sex, and years of residency. Three participation pattern clusters were identified: diverse activities (58%), municipal events (30%), and senior citizen club (12%). The proportion of participants reported themselves healthy were 93%, 88%, and 78% for diverse activities, municipal events, and senior citizen club clusters, respectively. Compared to those in the senior citizen club cluster, older adults in the diverse activities cluster were more likely to have self-efficacy (adjusted odds ratio (aOR): 1.19, p = .041) and social support (aOR: 2.35, p = .018), while participants in the municipal events cluster were associated with only social support (aOR: 3.29, p = .022). Increasing social support and self-efficacy may promote seniors' participation in diverse community activities, which would be beneficial for their healthy aging. Further studies are needed to evaluate the causal relationships.

The consumers' Sustainable and Healthy Dietary Behaviors (SHDBs) links sustainable behaviors related to eating (e.g., choosing, cooking, eating, and disposing of foods) with the level of "dietary behavior." It was hypothesized that SHDBs will vary depending on consumers' meal preparation habits, such as food selection, cooking frequency, and skill. This study examined (1) some of the behavioral patterns of SHDBs and (2) independent predictors of each pattern. In total, 508 Japanese adults who completed cross-sectional self-administered questionnaire surveys in December 2021 were included in the study. Principal component analysis (PCA) was used to identify the behavioral patterns of the 30-item SHDBs developed previously by the authors. Additionally, independent predictors of each component score were analyzed using multiple linear regression models, which included demographics, psychological factors concerning SHDBs (pros and cons for the decisional balance of SHDBs and number of environmental issues of interest), and meal preparation habits. Participants' median age was 41 (18-59) years (women: n = 259, 51.0%). Five SHDB patterns with several independent predictors were identified: amalgamation of all SHDBs, healthy food choices but unsustainable cooking, sustainable disposal, avoiding plastic products, and sustainable behaviors regarding expiry date. The multiple linear regression analysis demonstrated the independent predictors of the amalgamation of all SHDBs, such as being a man (β = -0.105), greater pros (β = 0.134), higher cooking frequency (β = 0.443) and skill (β = 0.244). Greater pros and/or smaller cons were associated with four of the five SHDB patterns, including an amalgamation of all SHDBs, healthy food choice and unsustainable cooking, avoiding disposal, and avoiding plastic products. Sustainable and healthy diet education must be implemented to ensure the adoption of all SHDBs.

Frequency of consuming foods prepared away from home has been associated with lower diet quality among adults and adolescents in several earlier studies. Nutrient and food group intake and Healthy Eating Index (HEI)-2015 scores were compared among a U.S. nationally representative sample of adolescents (12–19 years, n = 3975) by frequency of consuming food prepared away from home ≤2 times/week (n = 2311) versus &gt, 2 times/week (n = 1664) using National Health and Nutrition Examination Survey (NHANES) data from 2011–2018. Regression analyses were used to compare intakes among adolescents by frequency of eating meals prepared away from home adjusting for covariates. Older (16–19 years) vs. younger (12–15 years) adolescents and those from homes with higher vs. lower family income to poverty ratios had greater frequency of eating meals prepared away from home. Intakes of nutrients of concern for adolescents including choline, vitamin D, potassium, magnesium, fiber, phosphorus, folate, iron, and total HEI-2015 scores and component scores for total vegetables, and greens and beans were lower among adolescents who consumed meals prepared away from home more vs. less often. However, no differences were noted among food group intakes considered good sources of nutrients of concern such as total fruit, total vegetables, whole grains, and total dairy, except for beans and peas by frequency of eating foods prepared away from home. Greater frequency of eating foods prepared away from home was associated with lower diet quality among adolescents in a nationally representative sample of U.S. adolescents.

Objective:To investigate the influence of general parenting dimensions on adolescents’ energy balance-related behaviours (EBRB) and its interactions with behaviour-specific parenting practices based on Darling and Steinberg’s contextual model of parenting style.Design:Multiple linear regression analyses and the Hayes PROCESS procedure to analyse self-reported cross-sectional survey data.Setting:In-person survey.Participants:Latino early adolescents and their fathers (n 225 dyads) recruited using convenience sampling from metropolitan areas of north-central USA.Results:Both paternal parenting dimensions of warmth and autonomy granting were positively associated with adolescents’ fruit intake, vegetable intake and physical activity. Coercive control was positively associated with adolescents’ sugary drink intake and sweets/salty snack intake. These associations were predominantly mediated by the parenting practices of setting expectations/allowances, role modelling, and managing availability and accessibility for corresponding EBRB. After adjusting for parenting practices, paternal warmth was inversely associated with adolescents’ screen time, paternal autonomy was positively associated with sugary drink intake, and both paternal warmth and autonomy granting were positively associated with adolescents’ fast food intake. In addition, positive associations between fathers’ parenting practices and adolescents’ sugary drink intake were significantly stronger among those who perceived high v. low levels of paternal warmth and autonomy granting.Conclusions:Paternal warmth and autonomy granting showed mixed effects on adolescents’ EBRB, and coercive control showed undesirable relationships with adolescents’ dietary intake via interactions with behaviour-specific parenting practices. Lifestyle intervention programmes for Latino adolescents need to consider incorporating paternal parenting education components.

Alkali-heat DNA extraction, a rapid and economical method, was evaluated for use in the detection of Shiga toxin-producing Escherichia coli in food using real-time PCR assays. Alkali-heat DNA extracts led to highly sensitive detection (102-104 CFU/mL) of stx and O-antigen genes in beef liver, ground beef, sliced pork, cheese, lettuce, radish sprouts, tomato, and spinach, equivalent to the sensitivity obtained using a commercial DNA extraction kit that utilizes proteinase K lysis, and silica membrane purification. Although there were differences in DNA concentration and purity between DNA extraction methods, the sensitivity of real-time PCR assays was similar. These results indicate that alkali-heat DNA extraction is a viable method when testing food products with real-time PCR assays for the presence of stx and O-antigen genes.

Background Food security status has been assessed as a representative score for households; however, different members in the same household may perceive and report food insecurity differently. A high prevalence of food insecurity has been reported among Latino households, therefore understanding differences in reporting food insecurity by Latino father-mother dyads may improve accuracy of assessment and plans to address food insecurity. This study aimed to 1) determine demographic characteristics and/or food-related factors associated with perceptions of food security status among Latino father-mother dyads, and 2) identify factors associated with discordance in perceptions of food insecurity between dyads. Methods Baseline data were used from a community-based, youth obesity prevention program among Latino families (n = 106 father-mother dyads). Food security was assessed with a 2-item food insecurity screen. Logistic regression models were used to evaluate associations between reporting food security status and predictor variables for fathers, mothers, and dyad-discordant responses. Results Food insecurity was reported by 39% of fathers and 55% of mothers. Adjusted odds of reporting food insecurity were significantly higher for fathers perceiving their neighborhood was unsafe vs. safe (OR: 3.7, p OR: 3.2, p OR: 4.1, p OR: 5.5, p OR: 3.4, p OR: 3.1, p Conclusions Differential factors were associated with reporting food security among Latino father-mother dyads. Nutrition education for fathers that improves awareness of home food supplies and a better understanding of how food accessibility influences maternal perceptions may improve dyad discordance in reporting household food security.

Background: Food assistance and nutrition education are strategies used to address the high prevalence of food insecurity among U.S. Latino households. Assessment of food security status is necessary to implement these strategies. However, similarities and differences in perceptions of food security status have not been reported among Latino fathers and mothers who may have different home food acquisition and preparation responsibilities. This study aimed to 1) determine demographic characteristics and/or food-related factors associated with perceptions of food security status among Latino fathers and mothers, and 2) identify those factors correlated with concordance in perceptions of food insecurity between fathers and mothers.Methods: Baseline data were used from a Latino-family, community-based program to improve paternal parenting practices and youth energy balance-related behaviors (Padres Preparados, Jóvenes Saludables), including 106 father and mother couples. Food security was assessed with two questions from the U.S. Department of Agriculture 18-item Household Food Security Survey. Logistic regression models were used to evaluate associations between food security and predictor variables in fathers or mothers and between couple discordant responses in food security status and predictor variables.Results: The prevalence of household food insecurity was reported by 39% of fathers and 55% of mothers. Food security status was associated with neighborhood safety (odds ratio (OR): 3.7, p < 0.05) and household income (OR: 3.2, p < 0.05) in fathers, and with neighborhood safety (OR: 4.1, p < 0.01) and home fruit and vegetable availability (OR: 5.5, p < 0.01) in mothers. Couple discordance in reporting food security status occurred in 24% of the couples and was related to higher discordance among couples where fathers reported a lower frequency of participation in nutrition education than mothers (OR: 3.4, p < 0.05) and different perceptions of home fruit and vegetable accessibility (OR: 3.1, p < 0.05).Conclusions: Among Latino fathers and/or mothers, factors associated with perceptions of food insecurity differed, possibly related to fathers versus mothers having access to information about different household aspects related to food insecurity. Nutrition education for fathers may support an improvement in couple discordance in assessment of food security.

Alkali-heat DNA extraction, a rapid and economical method, was evaluated for use in the detection of Shiga toxin-producing Escherichia coli in food using real-time PCR assays. Alkali-heat DNA extracts led to highly sensitive detection (10

Acculturation may influence diet and health outcomes among Asian adults in the U.S., therefore associations were examined between acculturation and diet and health indicators among a population-based sample of U.S. Asian adults. Asian adults (20–65 years) (n = 1401) participating in the 2011–2016 National Health and Nutrition Examination Survey were categorized as less (n = 825) and more acculturated (n = 576) based on an acculturation scale (country of birth, length of U.S. residence, language spoken at home). Food group and nutrient consumption, Healthy Eating Index-2015 (HEI-2015) scores and health indicators were compared by acculturation level using multivariable regression models adjusted for covariates. More versus less acculturated participants were more likely to have higher family incomes and more education. More versus less acculturated participants also had lower HEI-2015 component scores for total vegetables, greens and beans, and added sugars and saturated fats (indicating less conformity to guidelines), lower intakes of dietary fiber, higher intakes of added sugars and solid fats, and higher body mass index. No other associations were observed between acculturation and health and disease indicators. Findings can be used by public health professionals to implement programs tailored to more acculturated U.S. Asian adults to improve diet and health.

Seafood provides nutritional benefits, but is not commonly consumed by most Americans. Regression analyses were used to examine associations between self-reported seafood intake frequency, diet quality and health indicators among U.S. adults (20–65 years) based on data from the National Health and Nutrition Examination Survey (NHANES 2011–2016) (n = 11,478). A low percentage (14 %) consumed seafood ≥8 times/month. The proportion consuming seafood ≥8 times/month was higher among foreign-born compared to U.S.-born adults and those with more education and higher incomes. Individuals consuming seafood intake ≥8 times/month had higher Healthy Eating Index-2015 scores, greater intakes of protein, cholesterol and alcohol, certain vitamins and minerals, most monounsaturated and polyunsaturated fatty acids, fruits, vegetables and total meat, and lower intakes of carbohydrates, total sugars, saturated fatty acids, dairy, refined grains, and solid fats and added sugars than those consuming seafood

Background Latino family-centered, community-based intervention studies to prevent childhood obesity have primarily involved mothers; therefore, little information about father participation is available. A Latino father-focused program to prevent adolescent obesity was conducted with low participation from some fathers. Objective The study aim was to determine factors associated with fathers’ attendance in an adolescent obesity prevention program. Study Design, Settings, Participants Data were from 147 fathers participating in the Padres Preparados, Jovenes Saludables program, a randomized controlled trial to prevent Latino adolescent (10-14 years) obesity. Eight weekly (2-hour) sessions were conducted in the Minneapolis/St. Paul metropolitan area (2017-2020). Participants were randomly assigned to intervention or waitlist control groups after baseline data collection. Measurable Outcome/Analysis The program facilitator recorded father presence or absence at each session. Attendance data were used in cluster analysis to identify attendance patterns. Predictor variables were measured at baseline with surveys and via anthropometric measures, including demographics, socioeconomic status, adolescent weight status, adolescent weight concerns, food-related activities with adolescents, family meal frequency, food assistance program participation, family stress, number of children, neighborhood safety, assigned group, site, and session season. Multinomial logistic regression analyses were used to examine associations between attendance patterns and predictor variables after assessing appropriateness for inclusion in regression models. Results Three attendance patterns were identified for all fathers: consistently high attendance (52%), declining attendance (18%), and non-attendance (30%). Compared to fathers with a consistently high attendance pattern, fathers with a non-attendance pattern were more likely to be assigned to the waitlisted control group (OR: 7.9 [95% CI: 2.6-23.9]; P Conclusion Maintaining contact with control families during waiting periods and providing online sessions to increase flexibility for low-income families could address Latino father non-attendance.

Laser-capture microdissection was used to isolate external germinal layer tissue from three developmental periods of mouse cerebellar development: embryonic days 13, 15, and 18. The cerebellar granule cell-enriched mRNA library was generated with next-generation sequencing using the Helicos technology. Our objective was to discover transcriptional regulators that could be important for the development of cerebellar granule cells-the most numerous neuron in the central nervous system. Through differential expression analysis, we have identified 82 differentially expressed transcription factors (TFs) from a total of 1311 differentially expressed genes. In addition, with TF-binding sequence analysis, we have identified 46 TF candidates that could be key regulators responsible for the variation in the granule cell transcriptome between developmental stages. Altogether, we identified 125 potential TFs (82 from differential expression analysis, 46 from motif analysis with 3 overlaps in the two sets). From this gene set, 37 TFs are considered novel due to the lack of previous knowledge about their roles in cerebellar development. The results from transcriptome-wide analyses were validated with existing online databases, qRT-PCR, and in situ hybridization. This study provides an initial insight into the TFs of cerebellar granule cells that might be important for development and provide valuable information for further functional studies on these transcriptional regulators.

Uncaging promoter and enhancer dynamics In order to understand cellular differentiation, it is important to understand the timing of the regulation of gene expression. Arner et al. used cap analysis of gene expression (CAGE) to analyze gene enhancer and promoter activities in a number of human and mouse cell types. The RNA of enhancers was transcribed first, followed by that of transcription factors, and finally by genes that are not transcription factors. Science , this issue p. 1010

Scientific Data, 4, ISSN:2052-4463

Human blood monocytes comprise at least 3 subpopulations that differ in phenotype and function. Here, we present the first in-depth regulome analysis of human classical (CD14(++)CD16(-)), intermediate (CD14(+)CD16(+)), and nonclassical (CD14(dim)CD16(+)) monocytes. Cap analysis of gene expression adapted to Helicos single-molecule sequencing was used to map transcription start sites throughout the genome in all 3 subsets. In addition, global maps of H3K4me1 and H3K27ac deposition were generated for classical and nonclassical monocytes defining enhanceosomes of the 2 major subsets. We identified differential regulatory elements (including promoters and putative enhancers) that were associated with subset-specific motif signatures corresponding to different transcription factor activities and exemplarily validated novel downstream enhancer elements at the CD14 locus. In addition to known subset-specific features, pathway analysis revealed marked differences in metabolic gene signatures. Whereas classical monocytes expressed higher levels of genes involved in carbohydrate metabolism, priming them for anaerobic energy production, nonclassical monocytes expressed higher levels of oxidative pathway components and showed a higher mitochondrial routine activity. Our findings describe promoter/enhancer landscapes and provide novel insights into the specific biology of human monocyte subsets.

CD4(+)CD25(+)FOXP3(+) human regulatory T cells (Tregs) are essential for self-tolerance and immune homeostasis. Here, we describe the promoterome of CD4(+)CD25(high)CD45RA(+) naive and CD4(+)CD25(high)CD45RA(-) memory Tregs and their CD25(-) conventional T-cell (Tconv) counterparts both before and after in vitro expansion by cap analysis of gene expression (CAGE) adapted to single-molecule sequencing (HeliScopeCAGE). We performed comprehensive comparative digital gene expression analyses and revealed novel transcription start sites, of which several were validated as alternative promoters of known genes. For all in vitro expanded subsets, we additionally generated global maps of poised and active enhancer elements marked by histone H3 lysine 4 monomethylation and histone H3 lysine 27 acetylation, describe their cell type-specific motif signatures, and evaluate the role of candidate transcription factors STAT5, FOXP3, RUNX1, and ETS1 in both Treg- and Tconv-specific enhancer architectures. Network analyses of gene expression data revealed additional candidate transcription factors contributing to cell type specificity and a transcription factor network in Tregs that is dominated by FOXP3 interaction partners and targets. In summary, we provide a comprehensive and easily accessible resource of gene expression and gene regulation in human Treg and Tconv subpopulations.

During mammalian embryogenesis, hematopoietic stem and progenitor cells (HSPCs) originate from mesoderm-derived endothelial cells in the aorta-gonad-mesonephros (AGM) region and placenta (PL). Later, HSPCs expand in fetal liver (FL) and migrate to bone marrow (BM) shortly before birth. Understanding global transcriptional regulation governing HSPC emergence from embryonic stem/induced pluripotent stem cells is necessary to devise clinical applications, such as novel transplantation approaches. In this study, to assess transcriptional dynamics during development, we performed cap analysis of gene expression on 10 developmental murine HSPC populations isolated from the AGM region, PL, FL, and BM and identified 15,681 transcripts across HSPC ontogeny. We performed microarray analysis of AGM-derived HSPCs at 9.5 and 10.5 days postcoitum (dpc) and identified 40 differentially expressed genes, 23 confirmed as significantly changed by real-time polymerase chain reaction. We conclude that a transcriptional switch point occurs in HSPC ontogeny between 9.5 and 10.5 dpc in the AGM region.

In development, epigenetic mechanisms such as DNA methylation have been suggested to provide a cellular memory to maintain multipotency but also stabilize cell fate decisions and direct lineage restriction. In this study, we set out to characterize changes in DNA methylation and gene expression during granulopoiesis using 4 distinct cell populations ranging from the oligopotent common myeloid progenitor stage to terminally differentiated neutrophils. We observed that differentially methylated sites (DMSs) generally show decreased methylation during granulopoiesis. Methylation appears to change at specific differentiation stages and overlap with changes in transcription and activity of key hematopoietic transcription factors. DMSs were preferentially located in areas distal to CpG islands and shores. Also, DMSs were overrepresented in enhancer elements and enriched in enhancers that become active during differentiation. Overall, this study depicts in detail the epigenetic and transcriptional changes that occur during granulopoiesis and supports the role of DNA methylation as a regulatory mechanism in blood cell differentiation.

BackgroundCap analysis of gene expression (CAGE) is a 5' sequence tag technology to globally determine transcriptional starting sites in the genome and their expression levels and has most recently been adapted to the HeliScope single molecule sequencer. Despite significant simplifications in the CAGE protocol, it has until now been a labour intensive protocol.MethodologyIn this study we set out to adapt the protocol to a robotic workflow, which would increase throughput and reduce handling. The automated CAGE cDNA preparation system we present here can prepare 96 'HeliScope ready' CAGE cDNA libraries in 8 days, as opposed to 6 weeks by a manual operator.We compare the results obtained using the same RNA in manual libraries and across multiple automation batches to assess reproducibility.ConclusionsWe show that the sequencing was highly reproducible and comparable to manual libraries with an 8 fold increase in productivity. The automated CAGE cDNA preparation system can prepare 96 CAGE sequencing samples simultaneously. Finally we discuss how the system could be used for CAGE on Illumina/SOLiD platforms, RNA-seq and full-length cDNA generation.

Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.