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Carbohydrates are the most abundant biomolecules in nature, and specifically, polysaccharides are present in almost all plants and fungi. Due to their compositional diversity, polysaccharide analysis remains challenging. Compared to other biomolecules, high-throughput analysis for carbohydrates has yet to be developed. To address this gap in analytical science, we have developed a multiplexed, high-throughput, and quantitative approach for polysaccharide analysis in foods. Specifically, polysaccharides were depolymerized using a nonenzymatic chemical digestion process followed by oligosaccharide fingerprinting using high performance liquid chromatography-quadru-pole time-of-flight mass spectrometry (HPLC-QTOF-MS). Both label-free relative quantitation and absolute quantitation were done based on the abundances of oligosaccharides produced. Method validation included evaluating recovery for a range of polysaccharide standards and a breakfast cereal standard reference material. Nine polysaccharides (starch, cellulose, beta-glucan, mannan, galactan, arabinan, xylan, xyloglucan, chitin) were successfully quantitated with sufficient accuracy (5-25% bias) and high reproducibility (2- 15% CV). Additionally, the method was used to identify and quantitate polysaccharides from a diverse sample set of food samples. Absolute concentrations of nine polysaccharides from apples and onions were obtained using an external calibration curve, where varietal differences were observed in some of the samples. The methodology developed in this study will provide complementary polysaccharide-level information to deepen our understanding of the interactions of dietary polysaccharides, gut microbial community, and human health. U.S. Department of Agriculture Agricultural Research Service (USDA-ARS) (NACA) [58-8040-0-014]; National Institutes of Health (NIH) Published version This project is funded by the U.S. Department of Agriculture Agricultural Research Service (USDA-ARS) (NACA #58-8040-0-014). Funding from the National Institutes of Health (NIH) is also acknowledged.

The ASN Board of Directors appointed the Nutrition Research Task Force to develop a report on scientific methods used in nutrition science to advance discovery, interpretation, and application of knowledge in the field. The genesis of this report was growing concern about the tone of discourse among nutrition professionals and the implications of acrimony on the productive study and translation of nutrition science. Too often, honest differences of opinion are cast as conflicts instead of areas of needed collaboration. Recognition of the value (and limitations) of contributions from well-executed nutrition science derived from the various approaches used in the discipline, as well as appreciation of how their layering will yield the strongest evidence base, will provide a basis for greater productivity and impact. Greater collaborative efforts within the field of nutrition science will require an understanding that each method or approach has a place and function that should be valued and used together to create the nutrition evidence base. Precision nutrition was identified as an important emerging nutrition topic by the preponderance of task force members, and this theme was adopted for the report because it lent itself to integration of many approaches in nutrition science. Although the primary audience for this report is nutrition researchers and other nutrition professionals, a secondary aim is to develop a document useful for the various audiences that translate nutrition research, including journalists, clinicians, and policymakers. The intent is to promote accurate, transparent, verifiable evidence-based communication about nutrition science. This will facilitate reasoned interpretation and application of emerging findings and, thereby, improve understanding and trust in nutrition science and appropriate characterization, development, and adoption of recommendations.

BACKGROUND: Branched-chain fatty acids (BCFAs) are rumen-derived fatty acids comprising ∼2% of bovine-milk fatty acids. BCFAs possess anti-inflammatory properties and enriching the BCFA content of bovine milk may provide human health benefits. OBJECTIVE: We determined whether forage content impacts the BCFA content of milk from Holstein cows and identified fatty acid phenotypes in high vs. low BCFA–containing milks. METHODS: Holstein cows (n = 62), fed for 67 d in a crossover design, consumed a diet with high forage and low concentrate (HF:C) and a diet with low forage and high concentrate (LF:C). Milk samples were collected at the end of each treatment period and fatty acid content determined. Paired t-tests, 1-factor ANOVA, sparse partial least-squares discriminant analysis (sPLSDA), and Pearson's correlation analysis were used to analyze the data. RESULTS: The total milk fatty acid concentration for cows fed the HF:C diet was greater than that of cows fed the LF:C diet (4.2 ± 0.7 g/100 mL vs. 3.9 ± 0.9 g/100 mL). sPLSDA demonstrated separation of the dietary treatments, with BCFAs and odd-chain fatty acids as primary determinants. Total BCFA content in milk fat was elevated by HF:C intake compared with LF:C intake (1.80 vs. 1.68%). Quintile separation of high vs. low BCFA milks resulted in 4 groups: HF:C /low BCFAs, HF:C /high BCFAs; LF:C /low BCFAs, and LF:C /high BCFAs. Milks from the high BCFA quintiles had lower palmitic acid content (29.6% vs. 34.4%) but higher oleic acid content than milks from the low BCFA quintiles (19.7% vs. 17.0%). Some cows were identified as high BCFA producers or low BCFA producers regardless of diet. CONCLUSIONS: BCFA content of milk is diet-sensitive but variation in responses exists. The potential to produce milk with high BCFA content and lower SFA content needs further study.

OBJECTIVES: Branched chain fatty acids (BCFA) are rumen-derived fatty acids comprising about 2% of bovine milk fatty acids. BCFA possess anti-inflammatory properties and enriching the BCFA content of bovine milk may provide human health benefits of milk consumption. In this work, we determined whether high vs low forage diets impact the BCFA content of milk from Holstein cows and identified fatty acid phenotypes in high vs low BCFA-containing milks. METHODS: In this study, 62 Holstein cows were fed for 70 days, in a cross-over design fashion, either a high forage: concentrate (HF: C) diet or a low forage: concentrate (LF: C) diet. At the end of the 70-day feeding period, cows were switched to the alternate feeding arm. Milk samples were collected prior to the start of the first feeding period and at the end of each treatment period. Milk fatty acid content was determined by fatty acid methyl ester analysis. Paired t-tests, one-way ANOVA, and sparse partial least squares discriminant analysis (sPLSDA) were used to analyze the data. RESULTS: The total milk fatty acid concentration at the end of HF: C diet period was greater than that of the LF: C diet (4.2 ± 0.9 g/100 mL vs 3.95 ± 0.9 g/100 mL). sPLSDA demonstrated clear separation of the dietary treatments, with BCFA and odd-chain fatty acids as primary determinants. Total BCFA content was elevated by HF: C intake (1.86 ± 0.13%) vs LF: C intake (1.77 ± 0.14%). Quintile separation of high vs low BCFA milks resulted in 4 groups (n = 12) HF: C/low BCFA, HF: C/high BCFA; LF: C/low BCFA, and LF: C/high BCFA. The total BCFA contents of the low BCFA milks were not different regardless of forage content (1.70 ± 0.06% and 1.61 ± 0.04%) and were lower than the high BFCA milks (2.04 ± 0.11% and 1.98 ± 0.11%). Milks from the high BCFA quintiles had lower saturated fatty acid (SFA) content but higher oleic acid content than milks from the low BCFA quintiles. Of the 62 cows, 5 cows were identified as high BCFA producers and 6 cows as low BCFA producers regardless of the LF: C or HF: C diets. CONCLUSIONS: The data demonstrate that the BCFA content of milk is diet-sensitive but variation in cow responses suggest factors such as genotype or rumen microbiome composition may play significant roles. The potential to produce milk with high BCFA content and lower SFA content needs further study. FUNDING SOURCES: USDA-ARS Projects 3062–53000-001–00D, 5090–31000-025–00D, 80–8040-05–01-0000–0000.

While great interest in health effects of natural product (NP) including dietary supplements and foods persists, promising preclinical NP research is not consistently translating into actionable clinical trial (CT) outcomes. Generally considered the gold standard for assessing safety and efficacy, CTs, especially phase III CTs, are costly and require rigorous planning to optimize the value of the information obtained. More effective bridging from NP research to CT was the goal of a September,2018transdisciplinaryworkshop. Participants emphasized that replicability and likelihood of successful translation depend on rigor in experimental design, interpretation, and reporting across the continuum of NP research. Discussions spanned good practices for NP characterization and quality control; use and interpretation of models (computational through in vivo) with strong clinical predictive validity; controls for experimental artefacts, especially for in vitro interrogation of bioactivity and mechanisms of action; rigorous assessment and interpretation of prior research; transparency in all reporting; and prioritization of research questions. Natural product clinical trials prioritized based on rigorous, convergent supporting data and current public health needs are most likely to be informative and ultimately affect public health. Thoughtful, coordinated implementation of these practices should enhance the knowledge gained from future NP research.

OBJECTIVES: To discuss good practices and criteria for optimal design and interpretation of pre-clinical and clinical natural product (NP) research in order to increase benefit from our investment in NP clinical trials (CT). Background: Large, randomized, controlled CT often fail to reject the null hypothesis or show rigorous evidence of benefit. This includes recent large, NIH-supported CT of nutrients such as vitamin D and selenium, and of botanical dietary supplements. Negative and positive outcomes may be equally important for public health, but because large CT cost at least $20 M each, plus opportunity costs, it is important that CT designs maximize the yield of actionable information regardless of outcome. METHODS: Experts and stakeholders from academia, government and the private sector collaboratively developed a broadly attended workshop in which good practices to enhance rigor, reproducibility and translational relevance were discussed. RESULTS: N/A. CONCLUSIONS: Critical issues in CT design include product identity, reproducibility and pharmacology (where feasible), power to test a primary outcome significant to consumers, and placebo controls. When basing a CT on traditional use or prior in vitro or in vivo studies, similarity of product (e.g., source identity, methods of preparation, form and intake), health outcome and population (e.g., age, sex, genetics, diet and environment), require careful consideration. Appropriate controls for known types of in vitro assay interference (e.g., aggregation, membrane disruption, protein denaturation) are imperative. Compounds with limited bioavailability, or activity only at concentrations above those achievable by ingestion, are likely poor candidates for dietary CT. Translational validity of model systems should be carefully assessed. Appropriate analyses (e.g., p-curve and meta-regression methods) should be used to obtain bias-corrected effect size estimates, and to identify research areas where the evidence base may be weaker than published findings suggest. Finally, CT prioritization should consider expected impact on public health, and whether known NP causal mechanisms of action are such that useful information, e.g., on product bioavailability or biological activity, are generated even if the completed CT fails to reject the null hypothesis. FUNDING SOURCES: NIH, FDA, USDA.

Rising CO2 levels may induce nutritional deficits (protein, minerals, and vitamins) in the highest rice-consuming countries., Declines of protein and minerals essential for humans, including iron and zinc, have been reported for crops in response to rising atmospheric carbon dioxide concentration, [CO2]. For the current century, estimates of the potential human health impact of these declines range from 138 million to 1.4 billion, depending on the nutrient. However, changes in plant-based vitamin content in response to [CO2] have not been elucidated. Inclusion of vitamin information would substantially improve estimates of health risks. Among crop species, rice is the primary food source for more than 2 billion people. We used multiyear, multilocation in situ FACE (free-air CO2 enrichment) experiments for 18 genetically diverse rice lines, including Japonica, Indica, and hybrids currently grown throughout Asia. We report for the first time the integrated nutritional impact of those changes (protein, micronutrients, and vitamins) for the 10 countries that consume the most rice as part of their daily caloric supply. Whereas our results confirm the declines in protein, iron, and zinc, we also find consistent declines in vitamins B1, B2, B5, and B9 and, conversely, an increase in vitamin E. A strong correlation between the impacts of elevated [CO2] on vitamin content based on the molecular fraction of nitrogen within the vitamin was observed. Finally, potential health risks associated with anticipated CO2-induced deficits of protein, minerals, and vitamins in rice were correlated to the lowest overall gross domestic product per capita for the highest rice-consuming countries, suggesting potential consequences for a global population of approximately 600 million.

Background Associations between air pollution and morbidity/mortality from cardiovascular disease are recognized in epidemiologic and clinical studies, but the mechanisms by which inhaled fibers or particles mediate the exacerbation of atherosclerosis are unclear. Objective and methods To determine whether lung inflammation after inhalation of a well-characterized pathogenic particulate, chrysotile asbestos, is directly linked to exacerbation of atherosclerosis and the mechanisms involved, we exposed apolipoprotein E–deficient (ApoE−/−) mice and ApoE−/− mice crossed with CD4−/− mice to ambient air, NIEHS (National Institute of Environmental Health Sciences) reference sample of chrysotile asbestos, or fine titanium dioxide (TiO2), a nonpathogenic control particle, for 3, 9, or 30 days. Results ApoE−/− mice exposed to inhaled asbestos fibers had approximately 3-fold larger atherosclerotic lesions than did TiO2-exposed ApoE−/− mice or asbestos-exposed ApoE−/−/CD4−/− double-knockout (DKO) mice. Lung inflammation and the magnitude of lung fibrosis assessed histologically were similar in asbestos-exposed ApoE−/− and DKO mice. Monocyte chemoattractant protein-1 (MCP-1) levels were increased in bronchoalveolar lavage fluid and plasma, and plasma concentrations correlated with lesion size (p < 0.04) in asbestos-exposed ApoE−/− mice. At 9 days, activator protein-1 (AP-1) and nuclear factor-κB (NF-κB), transcription factors linked to inflammation and found in the promoter region of the MCP-1 gene, were increased in aortas of asbestos-exposed ApoE−/− but not DKO mice. Conclusion Our findings show that the degree of lung inflammation and fibrosis does not correlate directly with cardiovascular effects of inhaled asbestos fibers and support a critical role of CD4+ T cells in linking fiber-induced pulmonary signaling to consequent activation of AP-1– and NF-κB–regulated genes in atherogenesis.

We hypothesized that normal human mesothelial cells acquire resistance to asbestos-induced toxicity via induction of one or more epidermal growth factor receptor (EGFR)–linked survival pathways (phosphoinositol-3-kinase/AKT/mammalian target of rapamycin and extracellular signal–regulated kinase [ERK] 1/2) during simian virus 40 (SV40) transformation and carcinogenesis. Both isolated HKNM-2 mesothelial cells and a telomerase-immortalized mesothelial line (LP9/TERT-1) were more sensitive to crocidolite asbestos toxicity than an SV40 Tag-immortalized mesothelial line (MET5A) and malignant mesothelioma cell lines (HMESO and PPM Mill). Whereas increases in phosphorylation of AKT (pAKT) were observed in MET5A cells in response to asbestos, LP9/TERT-1 cells exhibited dose-related decreases in pAKT levels. Pretreatment with an EGFR phosphorylation or mitogen-activated protein kinase kinase 1/2 inhibitor abrogated asbestos-induced phosphorylated ERK (pERK) 1/2 levels in both LP9/TERT-1 and MET5A cells as well as increases in pAKT levels in MET5A cells. Transient transfection of small interfering RNAs targeting ERK1, ERK2, or AKT revealed that ERK1/2 pathways were involved in cell death by asbestos in both cell lines. Asbestos-resistant HMESO or PPM Mill cells with high endogenous levels of ERKs or AKT did not show dose-responsive increases in pERK1/ERK1, pERK2/ERK2, or pAKT/AKT levels by asbestos. However, small hairpin ERK2 stable cell lines created from both malignant mesothelioma lines were more sensitive to asbestos toxicity than shERK1 and shControl lines, and exhibited unique, tumor-specific changes in endogenous cell death–related gene expression. Our results suggest that EGFR phosphorylation is causally linked to pERK and pAKT activation by asbestos in normal and SV40 Tag–immortalized human mesothelial cells. They also indicate that ERK2 plays a role in modulating asbestos toxicity by regulating genes critical to cell injury and survival that are differentially expressed in human mesotheliomas.

In cultured cells, palmitic acid (PA) and oleic acid (OA) confer distinct metabolic effects, yet, unclear, is whether changes in dietary fat intake impact cellular fatty acid (FA) composition. We hypothesized that short-term increases in dietary PA or OA would result in corresponding changes in the FA composition of skeletal muscle diacylglycerol (DAG) and triacylglycerol (TAG) and/or the specific FA selected for β-oxidation. Healthy males (N = 12) and females (N = 12) ingested a low-PA diet for 7 days. After fasting measurements of the serum acylcarnitine (AC) profile, subjects were randomized to either high-PA (HI PA) or low-PA/high-OA (HI OA) diets. After 7 days, the fasting AC measurement was repeated and a muscle/fat biopsy obtained. FA composition of intramyocellular DAG and TAG and serum AC was measured. HI PA increased, whereas HI OA decreased, serum concentration of 16:0 AC (P < 0.001). HI OA increased 18:1 AC (P = 0.005). HI PA was associated with a higher PA/OA ratio in muscle DAG and TAG (DAG: 1.03 ± 0.24 vs. 0.46 ± 0.08, P = 0.04; TAG: 0.63 ± 0.07 vs. 0.41 ± 0.03, P = 0.01). The PA concentration in the adipose tissue DAG (µg/mg adipose tissue) was 0.17 ± 0.02 in those receiving the HI PA diet (n = 6), compared to 0.11 ± 0.02 in the HI OA group (n = 4) (P = 0.067). The relative PA concentration in muscle DAG and TAG and the serum palmitoylcarnitine concentration was higher in those fed the high-PA diet.

Advanced age is a major risk factor for atherosclerosis, but how aging per se influences pathogenesis is not clear. Insulin-like growth factor-1 receptor (IGF-1R) promotes aortic vascular smooth muscle cell (VSMC) growth, migration, and extracellular matrix formation, but how IGF-1R signaling changes with age in VSMC is not known. We previously found age-related differences in the activation of Akt/FOXO3a and ERK1/2 pathways in VSMC, but the up-stream signaling remains unclear. Using explanted VSMC from Fischer 344/Brown Norway F1 hybrid rats shown to display age-related vascular pathology similar to humans, we compared IGF-1R expression in early passages of VSMC and found a constitutive activation of IGF-1R in VSMC from old compared to young rats, including IGF-1R expression and its tyrosine kinase activity. The link between IGF-1R activation and the Akt/FOXO3a and ERK pathways was confirmed through the induction of IGF-1R with IGF-1 in young cells and attenuation of IGF-1R with an inhibitor in old cells. The effects of three kinase inhibitors: AG1024, LY294002, and TCN, were compared in VSMC from old rats to differentiate IGF-1R from other upstream signaling that could also regulate the Akt/FOXO and ERK pathways. Genes for p27kip-1, catalase and MnSOD, which play important roles in the control of cell cycle arrest and stress resistance, were found to be FOXO3a-targets based on FOXO3a-siRNA treatment. Furthermore, IGF-1R signaling modulated these genes through activation of the Akt/FOXO3a pathway. Therefore, activation of IGF-1R signaling influences VSMC function in old rats and may contribute to the increased risk for atherosclerosis.