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Dietitians working at evacuation shelters conduct weighed food records (WFRs) for multiple days for dietary assessment. Because the menus in evacuation shelters do not change much from day to day, this study examined whether one- and two-day WFRs are sufficient for dietary assessment at shelters and identified dietary components that can influence the number of assessment days. Overall, 26 WFRs were collected from 10 shelters in Kumamoto Prefecture, and the amounts of energy; protein; vitamins B 1 , B 2 and C and salt were calculated. Correlation analysis and paired sample tests were conducted to examine significant differences between 'one- and two-consecutive- or nonconsecutive-day WFRs' and 'three-consecutive-day WFRs', which were set as the standard in this study. Additionally, the coefficients of variation (CV) for the categories by meal and dish were calculated to examine the variables that affected the large variations. As a result, one-day WFRs had significant positive correlations with the standard; thus, it could be used for the triage of shelters requiring nutrition assistance as a substitute for three-day WFRs. Two-consecutive-day and nonconsecutive-day WFRs showed a stronger correlation with the standard compared with the one-day WFRs. For energy and nutrients and dish categories, ready-to-eat foods had larger CV than boxed meals or foods from hot meal services. Whenever the meals included ready-to-eat foods, a two-nonconsecutive-day WFR is recommended considering large between-day variations. Salty soup or beverages affected the variation of some nutrients. Our result would help municipalities to consider the number of WFRs during emergency.

Padres Preparados, Jóvenes Saludables was a Latino family-based obesity prevention intervention implemented from 2017 to 2020 across eight programs in-person only, in a blended format (online/in-person), and online only. The intervention aimed to enhance father parenting skills to improve adolescent diet and activity behaviors. Mothers were encouraged to attend. Factors associated with participation were explored using a mixed-methods, qualitative (focus group/individual interviews by Zoom) and quantitative (process evaluation) design. Eleven focus group and 24 individual interviews were completed after participation with 24 fathers, 27 mothers, and 40 adolescents with responses not sorted by delivery method before analysis. Binomial logistic regression models examined associations between fathers' program completion and predictor variables of delivery characteristics, father demographic characteristics, and family attendance patterns. Parents were married (96% fathers, 76% mothers), had low income, a high school education or less (68% fathers, 81% mothers), and had lived in the United States a mean of 19 years. Parents were motivated to participate to improve health, and to be involved with and improve communication with their child. Common barriers to participation were work and life priorities and programmatic factors including scheduling conflicts and technological issues. Participation was greater for fathers attending sessions in-person compared with online only (OR = 11.6). Fathers were more likely to participate if they attended sessions with family members vs. not attending with family members (OR = 7.2). To maximize participation, findings suggest involving multiple parents/caregivers and adolescents, addressing contextual and programmatic barriers, and promoting benefits of better health and relations with family members.

Objective: To evaluate the conditional effect of time spent in the kitchen on the association between frailty status and healthy diet among older women., Design: Secondary analysis of an online cross-sectional survey conducted in January 2023., Participants: Six hundred Japanese women (aged ≥ 65 years)., Main Outcome Measure(s): Frailty status evaluated using the Kihon Checklist (25 affirmative questions assessing daily functions, weight status, and mental condition); healthy diet assessed by the days of consuming ≥ 2 meals that include staple, main and side dishes in a meal (SMS meal) in a day; and time spent in the kitchen., Analysis: Moderation analysis was used to evaluate the conditional effect of time spent in the kitchen on frailty status and SMS meal intake. Chi-square tests for independence were used to evaluate the differences in the Kihon Checklist items by frailty status., Results: Spending longer time in the kitchen indicated more frequent SMS meal intake and the trend was stronger among older women with frailty than those with robustness. All items except for 1 item regarding weight status (P = 0.15) were significantly associated with frailty status (P < 0.001)., Conclusions and Implications: Further studies are needed to evaluate the causal relationship between frailty status, healthy diet, and kitchen use., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Gratitude for food is a concept associated with various aspects of healthy and sustainable diet and originates from Buddhism and Shintoism in Japan. Against this background, a scale specifically targeting adults is essential for boosting studies concerning gratitude for food. This study aimed to adapt the Gratitude for Food Scale (GFS), originally developed for Japanese children, to Japanese adults and to examine the reliability and validity of GFS for adults (GFS-A). This cross-sectional study used baseline and one-week follow-up data from a longitudinal survey. The analysis included 1800 Japanese adults at baseline and 1380 at the one-week follow-up based on completion of self-administered questionnaires in February 2023. Construct validity was confirmed using exploratory and confirmatory factor analyses. Further, Spearman's and intraclass correlation coefficients and Cronbach's alpha were calculated to confirm criterion validity and reliability (test-retest and internal consistency). Participants' mean (SD) age was 40.2 (11.2) years (women: n  = 900, 50.0%) at baseline. The analysis revealed a one-factor structure comprising five items with good model fit. Criterion validity was confirmed by the correlation coefficients between the total GFS score and the gratitude questionnaire and the sub-score of awareness and appreciation for food, which represents a subscale of the expanded mindful eating scale. Moreover, test-retest reliability and internal consistency were confirmed. From this study's results, we can see that the developed GFS-A is an appropriate scale with good reliability and validity for measuring Japanese adults' gratitude for food., Competing Interests: Declaration of conflicting interestsThe authors have no conflicts of interest to declare.

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 B 1 , B 2 , and C were calculated for each meal. Energy and nutrient contents were compared between the categorical groups., The 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 B 1 , B 2 , and C content was significantly lower than that of meals with marks on other checkboxes in the same category., Shelters 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.

Background: We have suggested "Revised Nutritional Reference Values for Feeding at Evacuation Shelters" (Revised RV) as a daily nutritional recommendation for meals served at evacuation shelters where poor diets had been reported. Since there are no meal examples to satisfy the Revised RV, our objectives were, for the future meal provision, to develop nutritionally adequate meal plans using the foods served at shelters in the past and to examine if the Revised RV could be met by changing combination of foods available., Methods: In this case study using secondary data, we analyzed food weights of 86 meals served and recorded at 12 shelters after the heavy rains in July 2020. We obtained these data from Kumamoto Prefecture that was damaged and asked us dietary assessment for nutrition assistance. Foods were classified into 3 types according to the check mark in the record sheets: food aid (commercial packaged food), boxed meal, and hot meal service. We counted serving frequency of each food and analyzed nutritional differences by their combinations. Menus were devised by choosing foods that were served more frequently or were more nutritious among those served at shelters. The target values for one meal were set at 1/3 of the Revised RV for energy, protein, vitamins B 1 , B 2 , and C, and salt., Results: None of the meals served in the shelters satisfied the target. We created 2 menus using food aid only: (#1 curry doughnut, milk with long shelf-life, and orange jelly) and (#2 salmon rice ball, ham and cheese sandwich, and vegetable juice); 1 menu by combination of boxed meal and food aid: (#3 boxed meal and vegetable juice); and 2 menus by combination of hot meal service and food aid: (#4 chicken meatball soup, packaged tofu, soy sauce, preprocessed white rice, and bottled green tea) and (#5 bamboo shoots rice, chicken and vegetable miso soup, and bottled green tea). Planned menus generally contained more energy, protein, and vitamins and less salt than the meals served. Their vitamin C contents were especially higher., Conclusion: Nutritionally adequate meals could be planned by changing the combination of foods available in shelters., (© 2023. The Author(s).)

Picky eaters are at risk of an unbalanced diet, which is critical for women of reproductive age. A sensory profile, which is a potential factor in picky eating, has not been well researched. This study assessed the differences in sensory profile and dietary intake according to the picky eating status among female Japanese undergraduate college students. Cross-sectional data were obtained from the Ochanomizu Health Study conducted in 2018. The questionnaire included items regarding demographic characteristics, picky eating status, sensory profile, and dietary intake. Sensory profile was assessed using the Adult/Adolescent Sensory Profile questionnaire, and dietary intakes were calculated using a brief-type self-administered diet history questionnaire. Among the 111 participants, 23% were picky eaters and 77% were non-picky eaters. The age, body mass index and household status did not differ between the picky eaters and non-picky eaters. Being a picky eater was associated with higher scores on sensory sensitivity and sensation avoiding, and lower thresholds for taste and smell, touch, and auditory stimuli than being a non-picky eater. Of the picky eaters, 58% and 100% were at a high risk for folate and iron deficiencies, respectively, compared to 35% and 81% of non-picky eaters. Nutrition education for picky eaters in reproductive age to increase vegetable dishes comfortably in their diet is suggested to prevent anemia during their future pregnancy., Competing Interests: Declaration of competing interest There are no conflicts to report., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest None., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yui Kawasaki reports financial support was provided by the Asahi group foundation., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Community-based programs may contribute to Latino pediatric obesity prevention; however, attendance remains low. This study aimed to identify factors associated with Latino father/male caregiver attendance in family-based adolescent obesity prevention programs conducted as part of a randomized controlled trial. The study used cross-sectional data from 137 fathers in the program, involving eight weekly sessions to improve their children's energy balance-related behaviors. Evaluation data were collected at baseline and post-intervention. Fathers in a waitlisted control group attended sessions after post-intervention data collection. Attendance patterns were identified using cluster analysis: consistently high attendance (50% of fathers), declining attendance (19%), and non-attendance (31%) clusters. Multinomial logistic regression was used to examine associations between attendance patterns and variables. Compared to consistently high attendance, fathers in the non-attendance cluster were more likely to have been assigned to the control group (odds ratio [OR] = 7.86, p < 0.001), tended to have lower household income (OR = 2.50, p = 0.098), and attended the program at a site where the program occurred weekdays (OR = 0.35, p = 0.094). Maintaining contact during waiting periods and providing sessions online or during weekends could reduce non-attendance by enhancing convenience for low-income fathers working multiple jobs.

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., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Family income may influence nutrient and food group intakes among preschool children, thus increasing risk of nutrient deficiencies. This study compared nutrient and food group intakes and diet quality among a U.S. sample of children 2-5 years of age by family income to poverty ratio (PIR) based on National Health and Nutrition Examination Survey (2011-2018) data ( n = 2249). Regression analyses were used to compare dietary intakes based on proxy-reported 24-h dietary recalls and Healthy Eating Index-2015 total scores by low PIR versus mid-high PIR levels adjusted for covariates. PIR levels varied by child race/ethnicity and household reference person's sex, marital status, and education. More than half of the children in the low PIR group (56%) were reported to have received WIC benefits in the past year. Marginally lower calcium ( p = 0.008) and lower fiber intakes, lower total HEI-2015 scores, and lower intakes of fruits and whole grain foods (all p < 0.007) were observed among children in low PIR households compared to mid-high PIR households. However, iron intakes were higher ( p = 0.0003) among children in low PIR households compared to mid-high PIR households. Lack of other differences in nutrient and food group intakes may be attributable to WIC benefits.

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 >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.

Background: 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., Objective: 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., Design: 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/setting: 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., Main Outcome Measures: 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., Statistical Analysis: 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., Results: 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 P < 0.01) or total carotenoid intakes (β = .31 and P < 0.01). 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., Conclusions: 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., (Copyright © 2021 Academy of Nutrition and Dietetics. Published by Elsevier Inc. All rights reserved.)

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 < 0.05) and reporting lower vs. higher household income (OR: 3.2, p < 0.05). Adjusted odds of reporting food insecurity were significantly higher for mothers perceiving their neighborhood was unsafe vs. safe (OR: 4.1, p < 0.01) and reporting lower vs. higher home availability of fruit and vegetable (OR: 5.5, p < 0.01). Dyad discordance in reporting food security status occurred in 24% of the dyads. Adjusted odds of dyad discordant reports of food insecurity status were significantly higher for dyads reporting discordant responses regarding previous nutrition education (OR: 3.4, p < 0.05) and higher home fruit and vegetable accessibility (OR: 3.1, p < 0.05) compared to dyads reporting concordant responses. Among the 28 dyads who reported discordant nutrition education participation, 21 reported that fathers had never participated but mothers had participated more than once., 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.

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 2 -10 4 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.

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.

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.

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.

Although it is generally accepted that cellular differentiation requires changes to transcriptional networks, dynamic regulation of promoters and enhancers at specific sets of genes has not been previously studied en masse. Exploiting the fact that active promoters and enhancers are transcribed, we simultaneously measured their activity in 19 human and 14 mouse time courses covering a wide range of cell types and biological stimuli. Enhancer RNAs, then messenger RNAs encoding transcription factors, dominated the earliest responses. Binding sites for key lineage transcription factors were simultaneously overrepresented in enhancers and promoters active in each cellular system. Our data support a highly generalizable model in which enhancer transcription is the earliest event in successive waves of transcriptional change during cellular differentiation or activation., (Copyright © 2015, American Association for the Advancement of Science.)

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(+) naïve 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.

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.

CAGE (cap analysis gene expression) and RNA-seq are two major technologies used to identify transcript abundances as well as structures. They measure expression by sequencing from either the 5' end of capped molecules (CAGE) or tags randomly distributed along the length of a transcript (RNA-seq). Library protocols for clonally amplified (Illumina, SOLiD, 454 Life Sciences [Roche], Ion Torrent), second-generation sequencing platforms typically employ PCR preamplification prior to clonal amplification, while third-generation, single-molecule sequencers can sequence unamplified libraries. Although these transcriptome profiling platforms have been demonstrated to be individually reproducible, no systematic comparison has been carried out between them. Here we compare CAGE, using both second- and third-generation sequencers, and RNA-seq, using a second-generation sequencer based on a panel of RNA mixtures from two human cell lines to examine power in the discrimination of biological states, detection of differentially expressed genes, linearity of measurements, and quantification reproducibility. We found that the quantified levels of gene expression are largely comparable across platforms and conclude that CAGE and RNA-seq are complementary technologies that can be used to improve incomplete gene models. We also found systematic bias in the second- and third-generation platforms, which is likely due to steps such as linker ligation, cleavage by restriction enzymes, and PCR amplification. This study provides a perspective on the performance of these platforms, which will be a baseline in the design of further experiments to tackle complex transcriptomes uncovered in a wide range of cell types.

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.

Background: Cap 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., Methodology: In 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., Conclusions: We 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.