These tandem duplicates may have evolved new functions , possibly involved in the biosynthesis of novel compounds, and/or were selected to improve the metabolic flux of specific bio-synthetic steps that alter the dosage of certain endpoint metabolites. Future studies are needed to further investigate the possible role of tandem duplications in having modified metabolite levels and composition in wild and cultivated blueberry. Our analyses also revealed that high bush blueberry, a tetraploid, likely arose from the hybridization of two distinct parents, possibly allopolyploidy, based on the sequence divergence, unique transposable element insertions, and sub-genome expression patterns. Our analyses revealed that the sub-genomes in high bush blueberry may be controlling a distinct set of genetic programs . The dominantly expressed sub-genome in most surveyed tissues becomes the lowest expressed during fruit development. This observation is similar to findings in allopolyploid wheat where developmental and adaptive traits were shown to be controlled by different sub-genomes.
For example,10 liter drainage pot cell type- and stage dependent sub-genome expression dominance was observed in the developing wheat grain. We argue that both high bush blueberry and hexaploid wheat, each now with high-quality reference genomes, make excellent systems to further investigate these underlying mechanisms of sub-genomes dominance. sub-genomes dominance has far-reaching implications to numerous research areas including breeding efforts. For example, marker-assisted breeding needs to target the correct set of dominant homoeologs given the trait in polyploids that exhibit sub-genomes dominance. Thus, we anticipate that this genome, combined with improved insights into sub-genomes dominance, will greatly accelerate molecular breeding efforts in the cultivated high bush blueberry.Cardiovascular disease remains the leading cause of mortality in the United States, and after decades of decline, is rising coincident with the increase in obesity, insulin resistance, and diabetes that characterize cardiometabolic risk. Notwithstanding hereditary predisposition, reduction in identified, modifiable lifestyle risk factors can reverse CMR and CVD. It is estimated that 45.4% of all cardiometabolic deaths in the United States due to heart disease, stroke, and diabetes are associated with sub-optimal intakes of 10 dietary factors. Fewer than 1% of American children and adolescents meet full recommended metrics of heart healthy nutrition, falling especially short of recommended intake in the categories of fruits, vegetables, fiber and essential fatty acids.
Intensive pediatric lifestyle interventions for obesity are effective in achieving significant reduction in body mass index but do not elicit stable changes in nutrition habits in children and adolescents. These studies suggest a critical need for developing innovative tools to improve diet quality in youth. We have previously shown that twice daily consumption for two weeks of a whole food based nutrient bar composed of a blueberry, dark chocolate, red grape, and walnut matrix, soluble and insoluble fiber, with supplemental vitamins, minerals and essential long chain fatty acids, significantly increased high density lipoprotein cholesterol , due primarily to a 28% increase in large HDL particles, in generally healthy and insulin sensitive lean and overweight adults. In a subsequent 2 mo study of the effects of the nutrient bar on CMR markers in individuals across a range of BMIs, only those with low inflammation at baseline as assessed by high sensitivity C-reactive protein < 14.3 nmol/L responded comparably to those in the earlier trial, with not only increased HDL cholesterol and large HDL particles but also a trend toward increased high molecular weight adiponectin and a decrease in other CMR factors at 2 weeks, sustained at 2 months . In particular, a shift in low density lipoprotein particle subfractions toward a less atherogenic profile was evident in the non-inflamed group . Although the participants with overweight or obesity and CRP > 14.3 nmol/L did not show this response, they did experience an upward trend in adiponectin by 2 months. These results suggest that there may be a continuum of metabolic responsiveness to this nutrient supplement that is slowed in the face of the chronic low-level inflammation commonly observed with obesity and insulin resistance. A 6 month study of this nutrient bar in obese adolescents with non-eosinophilic asthma showed improved lung function at 2 months, but favorable movement in cardiometabolic biomarkers only began to emerge at 6 months.
It is not known whether more sensitive biomarkers of early metabolic change may be capable of detecting short-term effects of the nutrient bar supplementation in persons at CMR. Past metabolomics studies in obese adolescents and adults have identified strong positive associations between baseline levels of branched chain, aromatic, sulfur, and gluconeogenic amino acid metabolites and parameters of inflammation and insulin resistance. Similarly, elevated levels of specific ceramide species have been shown to associate with inflammation, dyslipidemia and insulin resistance in both adult and adolescent obesity. The relative sensitivity of these biomarkers to reflect moderate changes in dietary intake of polyphenols, essential lipids, fiber and vitamin/minerals, remains incompletely understood. In the present randomized, controlled, non-blinded trial, a two month intervention with exercise and nutrition counseling alone or with nutrient bar supplementation was performed in a high CMR cohort of adolescent /parent adult caretaker family units to determine 1) cross-sectional relationships in both adolescents and adults between traditional CMR biomarkers and amino acid and ceramide metabolites and 2) longitudinal changes within groups in the same CMR biomarkers following the lifestyle +/- nutrient bar intervention.This study was approved by the University of California Benioff Children’s Hospital Oakland Human Subjects Review Board, under approval number 2011022 entitled: The Impact of a Nutritional Supplement on Weight and Metabolic Health in a Parent-Child Intervention . All participating adolescents signed an assent document and all parent /adult legal guardian participants signed a written consent for their own participation and a separate consent for their participating adolescent. The study cohort was recruited from the UC San Francisco Benioff Children’s Hospital Oakland weight management program .
Teen participants were eligible for inclusion if they had a BMI greater than the 95th percentile, were between 14 and 18 years of age, fluent in English , and willing to eat the nutrient bar twice daily, after having tasted a sample, with one or both PACs also willing to participate. PAC inclusion criteria were the same except that there was no adult weight threshold. Exclusion criteria for both Teens and PACs included the diagnosis of diabetes mellitus or hypertension , and use of glucocorticoid, weight loss, insulin-sensitizing, lipid lowering, or anti-hypertensive medication. With Institutional Review Board approval, PAC consent for self and adolescent, and adolescent assent, 17 dyads and 1 triad were enrolled in May and June, 2011. One INT PAC dropped out before study measures were initiated but the rest of the family unit continued participation. All family units were seen within two weeks of the baseline group counseling session and within 2 weeks of the final session on the Clinical and Translational Research unit for pre-post blood testing. Participants were randomized at the baseline visit 1.25:1 to INT:CONT groups that met on separate days in the late afternoon after school and work for eight identical lifestyle counseling sessions in July and August 2011; 30 minutes of weekly group nutrition counseling followed by 30 minutes of supervised group exercise. In addition,25 liter pot the INT group was given a one week supply of nutrient bars at each visit through the seventh week and advised to return wrappers at the following session. Bar composition has been previously described , and was designed to supplement prevalent nutrient deficiencies in the American diet up to daily recommended intake , including 515 mg polyphenols, 4 gm whey protein, 9 gm of total fiber and 200 mg of long chain omega 3 fatty acid docosahexaenoic acid . Most vitamins and minerals added to the whole food matrix of the bar are in amounts representing 10% to 50% of their corresponding recommended daily intake, with the exceptions of vitamin C and vitamin D both added above RDI. Additional vitamin C serves as an antioxidant preservative for other bar ingredients, notably the omega 3 fatty acid DHA. Additional vitamin D addresses the prevalent deficiency in persons at high CMR, and the relatively low blood levels in the study cohort . Consumption of 2 bars each day was advised, with the first to be eaten before noon and the second in either the afternoon or evening, together with a minimum of 8 ounces of water with each bar due to the high fiber content. Compliance with eating the supplement and home lifestyle adherence were evaluated with phone call or text communication to each participating family every other day. Pre-post change in 25 hydroxy vitamin D levels served as an objective measure of compliance with the nutrient bars.Assessment of physical , behavioral and activity, and metabolic status was conducted at baseline and study completion on all Teen and PAC participants.Anthropometric and clinical evaluation: All anthropometric measures were performed in duplicate in the clinical research center and if not within 10% agreement, were repeated a third time. The reported measure is an average of the two closest numbers. Height was measured with a stationary stadiometer.
Weight was measured using a digital electronic scale and the BMI and waist to height ratio were calculated. Waist circumference was measured at end expiration to the nearest mm with a Gulick II Plus tape midway between the lowest border of the rib cage and the upper border of the iliac crest.Blood pressure and Resting Heart Rate: Each was measured in triplicate after 5 minutes sitting quietly with readings taken at least one minute apart. An automatic digital blood pressure monitor was used with cuff size adjusted for arm size. Traditional CMR biomarkers: Fasting blood samples were drawn and processed in the UCSF Benioff Children’s Hospital Oakland Clinical Research Center and sent to ARUP Diagnostic Laboratories for: standard lipid profile [total triglyceride, total cholesterol, and cholesterol within HDL and LDL , glucose, insulin, 25 hydroxy Vitamin D level and CRP. TG to HDL ratio and non-HDL were calculated. Fasting insulin and glucose were used to calculate the Homeostasis Model Assessment of Insulin Resistance Index according to the formula: fasting insulin x fasting glucose /22.5. Lipoprotein particle subclasses were analyzed by an ion mobility procedure that sensitively and directly measures concentrations of lipoprotein particle subfractions. High molecular weight adiponectin was measured by solid-phase sandwich ELISA . Metabolomic analyses: 1) Targeted analyses of 42 amine-containing metabolites consisting of 20 major amino acids, and secondary metabolites of arginine and cysteine whose levels are sensitive to inflammation and oxidative stress were performed on stored samples preserved at -70o Fahrenheit. Briefly, plasma was acidified with 5% perchloric acid containing 8 stable isotope internal standards. Acid-soluble supernatant was used for strong-cation exchange solidphase extraction to capture cationic amine- containing metabolites. Extracted metabolites were further derivatized with isopropylchloroformate. Derivatives of metabolites were resolved using Agilent 1260 ultra-high pressure liquid chromatography and eluted with a gradient of water and isopropanol . An Agilent 6490 triple quadrupole mass spectrometer was used to detect resolved analytes and quantify them using authenticated external and internal standards. 2) Sphingolipidomics by electrospray tandem mass-spectrometry by validated techniques was used to identify sphingolipid metabolites, including ceramides.De-identified baseline and study completion data points, paired by participant study ID, were entered into SPSS . Descriptive analyses of the study cohort were summarized and results for Teens and PACs, as well as for combined CONT and INT groups, were compared by unpaired Student t-test. Continuous physical and metabolic variables were tested for normality by examining the skewness, kurtosis and the Shapiro Wilk tests and transformed as necessary before analysis. Most of the variables in our data were normally distributed. Log transformations were conducted for the continuous physical and metabolic variables that were skewed to make them as normal as possible. The Shapiro-Wilk tests show that all of the transformed variables except two are approximately normal. The measures of metabolite concentrations used for principal component analysis were Z-transformed to render them normally distributed on the same scale with mean of zero and standard deviation of one. Pre-post change in absolute metabolite levels were compared by repeated samples paired t-test. Baseline z-scores of metabolites were subjected to principal component analysis without rotation. PCA is an unsupervised analysis that aims to decrease the complexity of data by reducing variables to a smaller number of principal components . A direct oblimin rotation was used and 6 factors before the bend in the scree plot , and eigenvalues >1 were retained. Component scores for each participant were calculated with a standardized scoring coefficient.