Brief Summary of Minutes of Annual Meeting

Meeting was called to order on Feb. 8 and Feb. 9, 2018 at 8:30 a.m. MST at the University of Arizona.

Welcome and Introductions (Feb. 8) – Participants were welcomed by the host and Meeting Chair (Jennifer Teske, University of Arizona).

Executive session (Feb. 8) – Dr. Yanhong Liu was elected by the members present to be the chair of the next annual meeting, 2019. She will coordinate next year’s W4002 meeting in Davis, CA; targeted for some time in February 2019. Dr. Richard Bruno, Ohio State University, was elected as secretary.

Dr. Milan Shipka, our administrative advisor, joined the meeting, updated the project’s renewal, and gave the guidance for annual report submission.  

Each W3002 Investigator attending the meeting gave an oral progress report in the following order:

• Bruno Richard, Ohio State University – “Anti-inflammatory mechanisms of green tea involving the gut-liver axis in obese models of NASH”
• Frank Duna, University of Arizona – “Gut feelings about obesity and diabetes”
• Ingolf Gruen, University of Missouri – “Functionality of Azadirachta Indica A. Juss”
• David Dallas, Oregon State University – “Digestion of milk proteins in infants and bioactive peptide discovery”
• Hord Norman, Oregon State University – “Nitrate and nitrite exposure includes differential effects on cognitive behavior and oxygen consumption during exercise in zebrafish”
• Ji-Young Lee, University of Connecticut – “Novel effect of astaxanthin on the modulation of histone deacetylases and metabotypes of hepatic stellate cells for the prevention and therapy for liver fibrosis”
• David Sands, Montana State University – “Global Nutrition: Why is it so difficult?”
• Barry Shane, University of California, Berkeley – “Genetic influences on folate status bio-markers”
• Connie Weaver, Purdue University – “Berries and bone project”
• Janos Zempleni, University of Nebraska – “Delivery and alterations of microbial signals by bovine milk exosomes in non-bovine species”
• Jen Teske, University of Arizona – “Sleep and obesity: using pre-clinical sleep studies to improve health”
• Yanhong Liu, University of California, Davis – “Anti-inflammatory and antioxidant properties of phytochemicals”
• Wendy White, Iowa State University – “Modeling the dose effects of soybean oil in salad dressing on the bioavailability of carotenoids and fat-soluble vitamins in salad vegetables”

At the end of each presentation, a short discussion took place.

The meeting adjourned on Feb. 9, 2018, at 11 AM.

Members of the W3002 Multistate project have been vastly productive during the past reporting period as evidenced by the dissemination of new products, technologies, and knowledge through peer-review publications, book reviews, presentations at symposia, conferences, and in the media. Project objectives are listed below along with scholarship activities from each of the represented institutions.

1. Determine the bioavailability (absorption, distribution, metabolism, elimination) of nutrients and other food components.
2. Evaluate the bioactivity of nutrients and other food components in order to elucidate their underlying protective mechanisms.

Kansas State University (Brian Lindshield). We collected nutrition, economic, and women's empowerment baseline information from small-holder (<5 hectares) maize Guatemalan farmers through questionnaires before distributing GrainPro Bags or plastic drums. We plan to administer the same survey again next year. We also produced some newly formulated corn-soy and sorghum soy fortified blended foods that we plan to assess the nutritional efficacy of in the near future. Outputs: we published 5 new peer reviewed journal articles since our last meeting/report (May, 2017-Feb, 2018). We hope to have all MFFAPP project manuscript published this year. Outcomes: An application to have the sorghum-cowpea fortified blended food that we developed has been submitted for inclusion on the USAID approved product list.

Montana State University (David C. Sands). Background: The problem of developing food crops with higher nutritional value is that the selection of nutritional value often conflicts with selection for traits necessary for high agronomic yield. This is a problem with our field crops - peas, potatoes, wheat, oat and barley. We have developed rapid selection parameters for potatoes that are lower in glycemic index than the main commodity varieties. We now have 7 cultivars being tested for agronomic production and of these we will choose several for human glycemic index testing. One low glycemic cultivar has been selected for its higher lysine level and this is being crossed with other cultivars.  Similarly, a series of high lysine tomato cultivars have been similarly selected and these are in the F2 cross stage however it may be that they have a lysine excreting endophyte. We have developed a high throughput screening platform for the oilseed crop, Camelina sativa yielding four lines that are higher, much higher in the omega-3: omega-6 fatty acid ratio than canola, corn, soy, or safflower oils. A food processing company in Illinois is now growing this specialty crop for use of its high omega, low glucosinolate oil and it anticipates production of 40,000 acres. Proatina a naked oat is being developed for use as a high protein/low glycemic substitute for rice because of its nutritional value compared to rice (2.5x higher in protein, much lower glycemic index, absence of arsenates, and because of its low pesticide demand and dryland habit it is a more ecologically sustainable to produce. A large specialty milling company has contracted to increase production of Proatina to 20 million pounds. The market target is the global community of Type II diabetics, especially those that use rice as a food staple. Progress: This year’s research continued focus on development of new crop cultivars specifically selected for enhanced nutritional traits. This was the first year when some of our improved nutrition cultivars were produced in field scale amounts by processing and marketing concerns aimed at the nutritional food markets. These include high lysine wheats, low glycemic potatoes, a high protein-low glycemic naked oat (Proatina), and a high omega 3 selection of Camelina sativa. In each case the economic outcome and output based on the nutritional value-added aspect of the new crop had to make up for the trade-off in yield. Nutritional selection reached our milestones of commercial product interest, development and field production. The increased consumer demand for nutritionally improved crops has been prompted by basic research and physiology that defined what was needed in human nutrition and health promoting diets.

The Ohio State University (Richard Bruno). Under Aim 1, the OH station has completed a clinical trial in humans that has defined the altered metabolism, pharmacokinetics, and elimination of a novel metabolite of alpha-tocopherol, i.e. α-carboxyethyl hydroxychromanol (a-CEHC), in patients with metabolic syndrome compared with healthy adults. Reduced generation and elimination of α-CEHC occurred without affecting the flux of its upstream metabolism (α-CMBHC) to α-CEHC. The findings of this investigation demonstrate that individuals with metabolic syndrome have lower α-tocopherol bioavailability, and their lower bioavailability occurs without upregulating P450-mediated metabolism of α-tocopherol to generate α-CEHC. That individuals with metabolic syndrome have substantially less generation of α-CEHC, suggests insufficient hepatic α-tocopherol status. Consequently, this milestone supports higher dietary α-tocopherol requirements for individuals with metabolic syndrome, and provides foundational basis for future efforts to validate vitamin E metabolites as novel biomarkers to assess vitamin E status. Under Aim 2, the OH station has completed preclinical studies in obese mouse models of nonalcoholic steatohepatitis (NASH) to assess the mechanism by which green tea extract (GTE) mitigates NFkB-dependent liver injury. Studies were performed in wild-type and loss-of-function TLR4-mutant mice to define the extent to which GTE protects against NASH in a TLR4-dependent manner. GTE reduces hepatic NFkB activation and histological evidence of NASH to the extent lowered in TLR4-mutant mice regardless of GTE supplementation. GTE in WT mice also decreased hepatic TLR4 and MyD88 expression. Although unaffected by genotype, GTE decreased circulating levels of endotoxin (a ligand for TLR4) and upregulated expression levels of intestinal tight junction proteins. These findings support that GTE ameliorates NASH through a mechanism involving the gut-liver axis. Specifically, GTE likely limits NFκB activation in a TLR4-dependent manner at the liver, but intestinal-level benefits that limit endotoxin translocation are apparently independent of intact TLR4 signaling. This milestone establishes evidence in preclinical models that GTE is effective in managing NASH through a mechanism involving the gut-liver axis, and our long-term goal to study GTE in humans to alleviate metabolic endotoxemia in relation to NASH risk.

Oregon State University (Emily Ho). Zinc and chronic disease: Recently we have found that zinc status is compromised with age.  Zinc deficiency causes immune cells to be sensitized to inflammation and also occurs in older animals and humans.  We have also examined the impact of zinc deficiency on developmental outcomes, gut microbiota, oxidative stress and inflammation induced by arsenic. For these studies we have developed a zebrafish and rodent models to examine the interaction among zinc and arsenic.  Zinc deficiency exacerbated disturbances on the microbiome in rodents and neurobehavior in zebrafish. Plant-derived phytochemicals and cancer: We have found that sulforaphane, a chemical found in cruciferous vegetables inhibits histone deacetylases, alters DNA methylation and histone methylation, lincRNA expression and has anti-cancer properties in the prostate. We have found unique metabolomic signatures in plasma of human subjects fed broccoli sprouts.  This work may help define new biomarkers of intake and novel mechanistic targets of bioactives derived from broccoli. Outputs: 1) Identify new risk factors in prostate cancer and offer novel dietary modifications to reduce the incidence of prostate cancer; 2) Establish low cruciferous vegetable intake as a risk factor for the development of prostate cancer by altering histone modifications and cell proliferation pathways; 3) Gain knowledge of the mechanisms behind the health benefits of micronutrients and phytochemicals such as zinc and compounds derived from cruciferous vegetables; 4) Establish low dietary zinc as risk factor for inflammatory processes, DNA damage and cancer risk and identify new biomarkers for human zinc deficiency; 5) Establish function of zinc and changes in zinc metabolism with development and aging.

Oregon State University (Norman Hord). Background: Dietary exposure to nitrates and nitrites is associated with cardiovascular health benefits and improved athletic performance while, in the context of processed meats consumption, increased gastrointestinal cancer risk. Humans concentrate nitrate from dietary or endogenous sources in the salivary glands, which is then reduced to nitrite, swallowed, and absorbed. Circulating nitrite acts as a reservoir for nitric oxide (NO) with its reduction to NO potentiated in acidic or hypoxic areas, such as contracting skeletal muscle. NO is an important signaling molecule with a short half-life that regulates cardiovascular function, cellular energetics, and neurotransmission. Progress: We had previously demonstrated, using two female rat models (young rats and ovariectomized rats), that dietary nitrate has no effect on bone growth, bone loss or the community structure of the gut microbiome. We extend the knowledge in this field with data described below by quantifying human exposure to dietary nitrate and nitrite and data from a zebrafish model informing how dietary nitrate may improve exercise performance by lowering the oxygen cost of physical activity (manuscript in preparation). Project 1 demonstrates the variability in the concentration of dietary nitrate in cultural food patterns and dietary supplements. Nitrate and nitrite content in foods is lacking from nutrient databases which limits the ability to study health-related epidemiological associations. Therefore, we estimated human nitrate and nitrite intakes from cultural meal patterns, foods and dietary supplements in order to determine the potential exposure range from available foods. Examination of prototypical daily meal patterns from four cultures showed meal patterns with the greatest nitrate and nitrite concentration were those with an abundant amount of leafy greens and root vegetables, such as the Japanese and Chinese diet, while concentrations in the American and Indian diet were considerably lower. Furthermore, consumption of one serving of a nitrate-rich food or supplement can exceed the World Health Organization Adequate Daily Intake (ADI) for nitrate (0-3.7 mg/kg body weight/day or 222 mg/day for a 60kg adult). The results for project 2 described below demonstrate improved muscle performance in zebrafish in response to exercise in a swim test. Inorganic nitrate improves exercise performance by reducing the oxygen cost of exercise. Nitrate is reduced into nitrite and NO in vivo, which is favored under acidic and hypoxic conditions. We hypothesized that nitrate-induced changes in mitochondrial function would stimulate changes in the utilization of metabolic fuels for energy production. Adult zebrafish were treated with sodium nitrate (606.9 mg/L) or control water for 21 days. Nitrate treatment significantly increased both blood and whole-body levels of nitrate and nitrite, demonstrating that nitrate was uptaken and reduced into nitrite. Nitrate reduced the oxygen cost of exercise during a 2-hour strenuous graded exercise test. There were no significant differences between mitochondrial function between control and nitrate-treated zebrafish, as evidenced by OROBOROS analysis of isolated mitochondria after treatment. Metabolomics analysis of whole fish illustrated that nitrate altered metabolites related to NO homeostasis, and stimulated glycolysis, ketogenesis, and fatty acid oxidation in zebrafish.   

Oregon State University (David Dallas). Background: Human milk evolved to provide nourishment, immunoprotection and support for the development of a commensal-dominant gut microbiome for term infants. Milk proteins are carriers of encrypted bioactive peptides with antimicrobial, immunomodulatory, anti-hypertensive, calcium delivery and gut barrier enhancement functions. These peptides must be released during infant digestion to be biologically relevant. Preterm infants have poor protein digestion and a structurally and functionally immature digestive system. Little evolutionary adaptation to provide optimal nutrition to significantly preterm infants is likely, as these infants rarely survived prior to advances in modern medical care. Therefore, bioactive peptides that evolved to be released within the term infant may be missing in the preterm infant. Differential or sub-optimal release of these bioactive peptides in early premature infants could conceivably alter health outcomes in multiple ways. Though research evidence has shown that the premature infant gut is structurally immature and protease production is decreased, what remains unknown is how these changes impact the overall breakdown of human milk proteins and release of bioactive peptides in the premature infant. There is, therefore, a critical need to determine how prematurity affects protein digestive function, the release of bioactive peptides in the infant gut, and how that ultimately impacts the infant. Our long-term research goal is to identify the optimal protein nourishment for preterm neonates to improve developmental outcomes. Our overall objective of our several funded projects is to examine the release of peptides in the digestive tract of infants, determine their functions and determine which peptides are missing in the preterm infant gut. These projects have potential for significant impact in that the identification of the degree to which digestion and the release of bioactive peptides is impaired for preterm infants will inform ways in which mother's milk and donor milk could be augmented, for example, with enzymatic supplementation or bioactive peptide supplementation to improve infant gut health. Progress: 1) We have determined the peptides released in the stomach of term and preterm infants; 2) We have determined which milk and gastric proteases are responsible for peptide release; 3) We have identified hundreds of released peptides that are homologous with known functional peptides; 4) We have established a database of known bioactive milk peptides from across literature that is open to all. Short-term outcomes: In one year of W-3002 support, 2 postdoctoral scholars, 2 graduate students, 6 undergraduates and 1 faculty research assistant were supervised. Eight manuscripts were published and twelve research presentation were given. Our research includes collaborations with many other scientists, including neonatologists in Oregon at Oregon Health & Sciences University and Randall Children’s Hospital.

Purdue University (Connie Weaver). Blueberries and other fruits with high polyphenolic content have been associated with reducing postmenopausal bone loss.  We determined the effective dose in an OVX animal model and are now conducting a clinical trial in postmenopausal women.  With collaborators, we are exploring antioxidant and anti-inflammatory mechanisms.

University of Arizona (Frank Duca). Background: The role of the gut microbiota in the development of metabolic disease is becoming increasingly recognized. Recent work has shown that the small intestinal microbiota, an often overlooked microbial site, can impact intestinal sensing pathways that regulate energy and glucose homeostasis of the host. However, the mechanisms upon which alterations in small intestinal microbiota impact the gut-brain signaling axis is poorly understood. Progress: 1) We have assessed the role of prebiotics, a non-digestible carbohydrate in improving gut-brain neuronal signaling that regulates food intake and energy homeostasis, 2) We have demonstrated that prebiotics alter the small intestinal microbiota, which is associated with changes in small intestinal chemosensory machinery. Short-term outcomes: In one year of W-3002 support, 4 visiting scientists, graduate students, and undergraduate students were supervised. Three presentations were delivered and 3 abstracts were submitted to internationally recognized conferences. Our research includes collaborations with 3 other scientists both within and outside the University of Arizona

University of Arizona (Jennifer Teske). The laboratory focuses on the negative impact of poor sleep and low physical activity to health. We investigate the relationship between sleep curtailment due to environmental noise and metabolism in a rodent model. We expanded these studies to include female rodents to test sex-specific effects and establish a rodent model of sleep deprivation-induced weight gain for females. These studies revealed that noise exposure reduces sleep by reducing the total time spent asleep and sleep quality indicated by an increased number of awakenings. Moreover, sleep loss due to noise exposure caused weight gain and increased feeding. Activities: Performed validation studies to determine sex-specific effects of noise exposure on sleep, wake, feeding, and weight gain in rodents. Demonstrated that the combination of consumption of a hedonic diet plus sleep disruption worsens metabolic outcomes than either alone. Milestones: To test whether noise-induced sleep loss exacerbates hedonic feeding behavior and weight gain in male and female rats. Short-term outcomes: 1) Surgery training and proficiency provided for students at all levels and international scholars; 2) Hands on anthropometrics, energy expenditure and intake training and estrous cycle determination for students at all levels and international scholars; 3) Grant writing and presentation skills training for oral and posters for students at all levels; and 4) Mentored student in proposal writing. Outputs: we have published 6 publications, got 2 grants, and presented 3 oral presentations, 4 poster presentations at different scientific conferences, and given one seminar. Long-term outcomes: Jennifer Teske received $1.3 million in funding for her research, provided research experiences for 42 individuals (4 high school students, 20 undergraduate students, 13 master’s degree student, 2 international scholars and 3 doctoral students) and provided employment for 2 employees.

University of California, Berkeley (Barry Shane). We have continued studies on the metabolic and nutritional effects of common polymorphisms in human folate-related genes that have been shown to influence disease risk. We continue to evaluate genetic risk factors for neural tube defects and to identify putative modifier genes which influence folate and vitamin B12 status, homocysteine levels, and methylation potential using a number of mouse strains and a cohort of students at Trinity College, Dublin. Vitamin B12 deficiency affects 10-15% of individuals over age 65. Circulating vitamin B12 levels can be used to diagnose deficiency but this test has significant false positive and false negative rates. We conducted genome-wide association studies (GWAS) in which we resolved total serum vitamin B12 into the fractions bound to transcobalamin and haptocorrin: two carrier proteins with very different biological properties. We replicated reported associations between circulating vitamin B12 levels and a common null variant in FUT2. This allele determines the secretor phenotype in which blood group antigens are found in non-blood body fluids. Vitamin B12 bound to haptocorrin (holoHC) remained highly associated with FUT2 rs601338 (p.Trp154Ter). Transcobalamin bound vitamin B12 (holoTC) was not influenced by this variant. HoloTC is the bioactive the form of the vitamin and is taken up by all tissues. In contrast, holoHC is only taken up by the liver. Using holoHC from individuals with known FUT2 genotypes, we demonstrated that FUT2 rs601338 genotype influences the glycosylation of haptocorrin. We then developed an experimental model demonstrating that holoHC is transported into cultured hepatic cells (HepG2) via the asialoglycoprotein receptor (ASGR).

University of California, Davis (Yanhong Liu). Background: Excessive production of reactive oxygen species (ROS) renders oxidative damage to lipid membrane, protein and DNA of host cells which may lead to cell death and tissue injury. The risk of oxidative damage is high at the epithelial layer of gastrointestinal tract due to frequent exposure to environmental pathogen. Consumption of oxidized food/feed constituents (i.e. oxidized fat) and the insult from environmental stress factor (i.e. heat-stress) also heightens the production of ROS in enterocytes and compromises barrier function of gut epithelium in animals and humans. Progress: 1) We have assessed the antioxidant activities of several mint-derived essential oils with different types of in vitro models; 2) we have conducted an animal trial to evaluate the antioxidant capacities of inorganic selenium and organic selenium using pig as model; 3) we have evaluated a pig model for oxidative stress research in human. Short-term outcomes: In one year of W-3002 support, 10 visiting scientists, graduate students, undergraduate students were supervised. Two review papers and 6 meeting abstracts that were related to this research area were published. Eight presentations were delivered and 4 manuscripts were submitted to peer-reviewed journals. Our research includes collaborations with 4 other scientists.

University of California, Davis (Peng Ji). Background: Iron fortification has been commonly practiced in most developed countries, even though the prevalence of iron-deficient anemia is low. A follow-up study in humans has shown that iron fortification in infants born with iron-replete status has long-term negative effect on cognitive development. We use neonatal piglets as translational model to study the impact of iron over-supplementation in early life on systemic iron homeostasis, CNS oxidative stress and social cognition. Progress: 1) We have performed two studies that evaluated different dose of iron fortification in preweanling piglets on CNS and peripheral iron accumulation, transcriptional adaptation of iron regulatory proteins, CNS lipid peroxidation, and hippocampal metabolomics; 2) We also performed sociability and radio maze behavioral test to assess impact of iron fortification on social and spatial cognition. 3) We conduct in vitro cell culture work to study the regulatory mechanism of cellular iron homeostasis. The results of our studies suggested iron fortification significantly increase CNS iron deposition that is associated with increased lipid peroxidation. Similarly, hippocampal metaoblomics results highlighted pathways associated with purine metabolism were interrupted. Genes encoding iron regulatory proteins demonstrate a coordinated change in the manner to inhibit further iron uptake. Piglets that received high iron supplementation had suboptimal sociability in comparison with non-supplemented control group. The results are novel, and we are working on manuscripts of the study. The cell culture work is still in pilot tests to optimize culture, treatment, and testing conditions. Some preliminary results have validate that iron treatment significantly increased intracellular iron content in gut epithelial cells.

University of Connecticut (Ji-Young Lee; Sung Koo). Endotoxin tolerance is a phenomenon where exposure of innate immune cells to lipopolysaccharide (LPS) induces a refractory state to subsequent endotoxin exposures. The goal of this study was to investigate if Spirulina platensis extract (SPE) induces an endotoxin tolerance-like state. We used splenocytes and peritoneal macrophages from C57BL/6J mice fed a high fat/high sucrose (HF/HS) control or a HF/HS diet containing 0.25% (w/w) SPE for 16 weeks for ex vivo LPS stimulation and endotoxin tolerant macrophages to evaluate the effects of SPE on endotoxin tolerance. Cells from SPE-fed mice displayed significantly less expression of pro-inflammatory genes than those from control mice. Endotoxin tolerant (ET) macrophages were produced in vitro by incubating RAW 264.7 macrophages with low-dose LPS to determine the energy phenotype of naïve, SPE-treated, and ET macrophages. Compared to naïve macrophages exposed to a high-dose LPS (100 ng/mL) for the first time, ET macrophages showed significantly less pro-inflammatory gene expression after LPS stimulation, which was also observed with SPE treatment. Consistently, nuclear translocation of p65 was markedly reduced in both ET and SPE-treated macrophages upon LPS stimulation with increase in nuclear protein levels of p50 and B-cell lymphoma 3- encoded protein. In conclusion, the anti-inflammatory effect of SPE is at least partly attributable to the induction of an endotoxin tolerance-like state in macrophages, which shares common characteristics of macrophage endotoxin tolerance.

Activation of hepatic stellate cells (HSCs) is critical for liver fibrosis development. Previously, we showed that astaxanthin (ASTX), a xanthophyll carotenoid, has anti-fibrogenic effects in LX-2 cells, a human HSC cell line. We sought to determine the effect of ASTX on HSC activation, and to identify molecular mediators that are critically involved in the processes. ASTX prevented the activation of mouse primary HSCs, as evidenced by attenuated induction of procollagen type I a1. In human primary HSCs, ASTX also inhibited transforming growth factor b1 (TGFβ1)-induced fibrogenic gene expression. Among 11 classical histone deacetylases (HDACs), difference in HDAC9 mRNA levels between quiescent and activated HSCs was most evident while ASTX significantly decreased the expression of HDAC9 and its transcriptional regulator myocyte enhancer factor 2 (MEF2). ASTX decreased HDAC9 protein as well. In the activated HSCs, ASTX significantly reduced mRNA of HDAC9 and MEF2. Human primary biliary cirrhosis livers showed significantly higher HDAC9 mRNA and protein levels than normal livers, and other liver pathologies also exhibited induced HDAC9 expression. HDAC9 knockdown in LX-2 cells decreased TGFβ1-induced fibrogenic gene expression. In conclusion, ASTX inhibits HSC activation and facilitates HSC inactivation, which is attributable to its inhibitory action on HDAC9 expression.

We previously showed that polyphenol-rich blackcurrant extract (BCE) showed a hypocholeseterolemic effect in mice fed a high fat diet. As direct cholesterol removal from the body via the intestine has been recently appreciated, we investigated the effect of BCE on the modulation of genes involved in intestinal cholesterol transport using Caco-2 cells as an in vitro model. BCE significantly increased protein levels of low-density lipoprotein receptor (LDLR) without altering its mRNA, which consequently increased low-density lipoprotein uptake into Caco-2 cells. This post-transcriptional induction of LDLR by BCE was markedly attenuated in the presence of rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1). In addition, BCE altered genes involved in cholesterol transport in the enterocytes, including apical and basolateral cholesterol transporters, in such a way that could enhance cholesterol flux from the basolateral to apical side of the enterocytes. Indeed, BCE significantly increased the flux of LDL-derived cholesterol from the basolateral to the apical chamber of Caco-2 monolayer. LDLR protein levels were markedly increased by anthocyanin-rich fraction, but not by anthocyanin-free fraction. In conclusion, the mTORC1-dependent post-transcriptional induction of LDLR by BCE anthocyanins drove the transport of LDL- derived cholesterol to the apical side of the enterocytes. This may represent a potential mechanism for the hypocholesterolemic effect of BCE.

We previously showed that the organic extract of a blue-green alga, Spirulina platensis (SPE) had potent anti-inflammatory effects in macrophages. We investigated the contribution of the anti-inflammatory effects of SPE in macrophages to adipogenesis/lipogenesis in 3T3-L1 adipocytes. 3T3-L1 preadipocytes were treated with 10% conditioned medium from LPS-stimulated RAW 264.7 macrophages (CMC) or LPS-stimulated but SPE-pretreated macrophages (CMS) at different stages of adipocyte differentiation. The expression of adipocyte differentiation markers were significantly repressed by CMC, while the repression was attenuated by CMS. Oil Red O staining confirmed that adipocyte maturation in CMS-treated cells, but not in CMC-treated cells, was equivalent to that of control cells. In lipid-laden adipocytes, CMC promoted the loss of lipid droplets while CMS had minimal effects. Histone deacetylase 9 mRNA and protein levels were increased during adipocyte maturation, which were decreased by CMC. In conclusion, by cross-talking with adipocytes, the anti-inflammatory effects of SPE in macrophages promoted adipocyte differentiation/maturation, at least in part, by repressing the activation of NF-kB inflammatory pathways, which otherwise can be compromised in inflammatory conditions. Endotoxin tolerance is a phenomenon where exposure of innate immune cells to lipopolysaccharide (LPS) induces a refractory state to subsequent endotoxin exposures, resulting in a lack of pro-inflammatory cytokine and chemokine production.

We evaluated whether SPE induces endotoxin tolerance-like state to exert its anti-inflammatory effect and to determine its effect of macrophage energy phenotype. Splenocytes and resident peritoneal macrophages from mice fed a HF/high sucrose (HF/HS) diet containing SPE displayed significantly less expression of pro-inflammatory genes than those from control mice. We produced endotoxin tolerant (ET) macrophages in vitro by incubating RAW 264.7 macrophages with low-dose of LPS. Compared to naïve macrophages exposed to a high-dose LPS for the first time, ET macrophages showed significantly less pro-inflammatory gene expression after LPS stimulation, which was also observed with SPE treatment. Consistently, nuclear translocation of p65 was markedly reduced in both ET and SPE-treated macrophages upon LPS stimulation. Both SPE-treated and ET macrophages had increased mRNA and nuclear protein levels of p50 and B-cell lymphoma 3-encoded protein, which play a critical role in endotoxin tolerance. Both SPE-treated and ET macrophages had similar energy phenotype in that they relied more on glycolysis than mitochondrial respiration for energy generation compared to naïve cells. In conclusion, the anti-inflammatory effect of SPE is at least partly attributable to the induction of an endotoxin tolerance-like state in macrophages, which shares common characteristics of macrophage endotoxin tolerance.

University of Illinois, Urbana-Champaign (Juan Andrade). Background: Our research focuses on developing technologies or approaches to improve nutrition in vulnerable populations in the US and abroad. Efforts have been focused on: 1) characterization of legume-based protein nanoaggregates to improve stability and delivery of fat-soluble bioactives and nutrients; and, 2) characterization of lipid-based nutrition supplement (LNS) to prevent and control severe acute malnutrition (SAM) and parasitic infection. Outputs: 1) Characterization of legume-based protein nanoparticles. A legume-based nanoparticle system can be advantageous for the delivery of fat soluble compounds that can address nutrition gaps due to its enhanced stability and bioavailability. Nanoaggregates containing vitamin D were characterized in terms of stability and bioavailability in animal models. We found that dispersion of vitamin D in nanoaggregates enhances its oral bioavailability as evaluated in rats. 2) Lipid-based nutrition supplement (LNS) characterization. We evaluated the addition of essential oils into an LNS designed for the Indian context. We found that oregano essential oil bioactives were capable of reducing infection of Cryptosporidium using an absorptive cell model. We also found that encapsulation of bioactives into cyclodextrin particles allows for the feasible, target delivery of bioactives as encapsulation enhances delivery, resist digestion conditions, and do not affect sensory attributes of LNS.

University of Maine (Dorothy Klimis-Zacas). Atherosclerosis is a chronic inflammatory, progressive disease of the large arteries that can lead to CVD and stroke. Angiogenesis is the formation of new capillary blood vessels from existing ones and endothelial cell migration and proliferation contribute to the development of angiogenesis; critical in the early stages of atherosclerosis. Wild blueberries (Vaccinium angustifolium) are rich in anthocyanins (ACNs) and phenolic acids (Phen) having an exceptional ranking for antioxidant capacity compared to other berries and fruits. During 2017-2018 we continued to investigate the effect of ACNs and Phen fractions and their combination on endothelial cell migration and angiogenesis and mechanisms thereof, by exploring relevant biomarkers of cell migration and angiogenesis such as RhoA, Rac1 GTPases, AKT, VEGF and eNOS and their gene expression, critical for cell morphology, cytoskeleton integrity, cell permeability, angiogenesis and cell migration. Preliminary results document a differential response of the above bioactive compounds on endothelial cell migration and relevant proteins and their gene expression, based on type of fraction and its concentration. Studies on angiogenesis also document a concentration-depended effect, critical concentrations at which angiogenesis is modulated and differential response based on the different fractions (ACNs or Phen). ACNs seem to inhibit HUVEC migration and angiogenesis while PAs promote this process. Another project initiated in 2017, targets the role of Red Raspberries (Rubus idaeus) on endothelial function (vasoconstriction and vasodilation) and obesity-induced inflammation by assessing pro-inflammatory markers and their gene expression in hepatic and adipose tissues in an animal model of the Metabolic Syndrome (MetS), the Obese Zucker Rat (OZR). Preliminary results support the role of Red Raspberries in normalizing the endothelial dysfunction and attenuating inflammation associated with MetS. Outputs: Results from the period covered in this report were disseminated in form of a poster at the International Conference of Polyphenols and Health in Quebec, Canada (October 2017), at the WBANA Health Summit in Bar Harbor, Maine (September, 2017) in form of an oral presentation, at the Berry Health Benefits Symposium in Pismo Bay, California (March, 2017) in form of a poster, which received second price in the poster competition, and results will be presented at the American Society for Nutrition Meetings, in Boston, MA (April, 2018). Additionally, several manuscripts are in preparation to be submitted by Summer 2018. Other outputs will be presentations at invited lectures and through the media (newspaper articles, blogs and interviews). A provisional application to EFS for a patent is pending to commercialize the findings related to the role of phenolics on cell migration and angiogenesis.

University of Massachusetts (Yeonhwa Park; Hang Xiao). We have studied various bioactive food components to improve bioaccesibility and bioavailability and determined the bioactivites of food bioactivities using various models. We have tested compounds, such as piceatannol, 3,3'-Diindolylmethane, β-Carotene, and nobiletin during this period.

University of Missouri (Ingolf Gruen). Neem (Azadirachta indica) is an evergreen tree cultivated in various parts of the Indian sub-continent. It has been in use over centuries in the Indian folk medicine for its therapeutic value. Given the prominent role it has played in curing diseases of the villagers over centuries, it has been hailed as a “divine” tree, a “village dispensary” and “ nature’s drugstore” .Today, extensive research has shown that it may have anti-cancer, anti- diabetic, anti-inflammatory, anti-ulcerogenic, and anti-microbial effects. However, there are significant gaps in the scientific literature about the compounds contributing to its medicinal potential. In this study, select flavonoids, namely flavonols: myricetin, quercetin and kaempferol are quantified by a HPLC method in Neem powder and leaves as well as green and black tea leaves for comparison. It was observed that Neem dry powder had significantly higher (p< 0.05) flavonol content at 11.146 ±1.09 mg/g than green and black tea 5.782 ± 0.723 mg/g and 5.599 ± 0.484 mg/g respectively. The tea infusions of the plant materials also yielded similar results. The total phenolics were found to be the highest in green tea infusion because of its high flavan-3-ol content, followed by black tea, Neem powder and Neem tea cut leaves with values of 174.55 ± 12.85, 106.85 ± 6.511, 55.60 ± 7.90 and 87. 35 ± 7.42 mg/g respectively. DPPH and FRAP anti-oxidant assays yielded contradictory results which can be explained by the very principle of these assays. It was also observed that the particle size of ground tea leaves significantly influenced the yield of flavonols, phenolics and anti-oxidant assay. In addition, neem is extremely bitter, in large part due to its limonoid content, making it unpalatable and limiting its potential use in dietary supplements or foods alike. Bitterness reduction, especially of foods and beverages containing phytonutrients, is one of the biggest challenges in the food industry. The objective of this experiment of our overall study was to apply two adsorbent based strategies, namely solid phase extraction (SPE) and Amberlite XAD-16 (AMB) resin, to achieve de-bittering of neem tea and to determine the effects of the de-bittering on the bio-active, color, and volatile properties. The solid SPE treatment completely removed the flavonol, quercetin, from neem tea while in Amberlite XAD-16 treated tea (AMB) it was only insignificantly (p > 0.05) reduced. We also observed decreases in total phenolic content and consequently anti-oxidant activities after de-bittering. A 62% mean reduction of limonoid aglycones indicated diminished levels of bitterness. The loss of phenolics lead to a visually appreciable color change in the treated teas. The de-bittering also leads to a loss of sesquiterpenes, ketones and acids from neem tea. In conclusion, we found that while SPE cartridges were more efficient in removing bitterness, they caused a greater reduction in bio-active compounds than AMB XAD-16 resins, which may ultimately affect the health properties of neem tea.

Over the last year, in the context of this project, we initiated an investigation into the bioactive compounds and the resulting bitterness of cacao and chocolate, specifically our goal is to identify and quantify the complex mixture of important bitter compounds within the three compound classes that are known to be important to cocoa--methylxanthines, flavan-3-ols, and diketopiperazines. Chocolate is made from the fermented, dried, and roasted seeds of the Theobroma cacao tree. It is now understood that cacao, also known in English-speaking countries as cocoa, contains a variety of compounds, including well-known flavonoid polyphenols, the consumption of which has positive impacts on human heart health and blood pressure, cancer reduction, LDL cholesterol reduction, and insulin resistance improvements, as numerous in vitro, in vivo, and observational studies have confirmed. In important research published in 2017, performed, in part, by Harvard's Chan School of Public Health, a participant observational study, composed of men and women between the ages of 50 and 64, led to the determination that chocolate consumption is inversely correlated with atrial fibrillation (AR). This is an important finding, as AR is associated with multiple significant health problems such as "higher risk of stroke, heart failure, cognitive decline, dementia and mortality." A persuasive case has, therefore, been made that cacao is a healthful addition to a balanced diet. However, despite the strong evidence for the healthful qualities of cacao, which has led to an overall increase in consumer purchases of more high-cacao-content chocolate than in previous decades, sales figures still show that there is an unwillingness by many Americans to consume higher-cacao-content chocolate, which tends to be more bitter, a taste modality not readily appreciated by most. Therefore, if the bitterness of cacao could be minimized, higher-cacao-content and lower-sugar chocolate confection sales could capture an even larger segment of the conventional and healthy snack-food market by achieving the elusive combination of being both tasty and healthy. The key is to better grasp the causes of bitterness in cacao, which are complex and still not well understood. What has long been known is that the methylxanthine, theobromine, is an important contributing bitter compound in cacao, with its name even being derived from the genus of the cacao tree, Theobroma. Similarly, the related methylxanthine, caffeine, which is well known as an important bitter compound in coffee, has also been found to impart bitterness in cacao and chocolate. Certain flavan-3-ols, which are a subclass of the aforementioned healthy flavonoids, and which are found in black tea, are also bitter and present in cacao at levels above the detection threshold, especially the compound (-)-epicatechin. After project initiation, cocoa beans were obtained from Ghana, Madagascar, and Peru and have been roasted according to a standard roasting profile by chocolate manufacturer Patric Chocolate, LLC. Methods have been developed and validated, and standard curves have been established for the cocoa bitter compounds: Caffeine, Theobromine, Catechin, Epicatechin, and selected Diketopiperazines (DKPs), including cyclo(L-Pro-L-Val) which according to the most recent literature available is considered the most important DKP in regards to cocoa bitterness. The bitter compounds caffeine, Theobromine, Catechin, and Epicatechin were found in all samples. While a preliminary analysis of the data showed, as expected, some variation, we still need to analyze the data statistically. We were not able to replicate the finding reported by Stark (Stark T, Bareuther S, Hofmann T. Molecular definition of the taste of roasted cocoa nibs (Theobroma cacao) by means of quantitative studies and sensory experiments. J Agr Food Chem. 2006;54(15):5530-9) that the diketopiperazine cyclo(L-Pro-L-Val) is an important bitter compound! While we were able to positively identify cyclo(L-Pro-L-Val) in a very dark roasted cocoa using LC-MS-MS, which then allowed us to also detect and quantify this diketopiperazine in other roasts, the concentration of cyclo(L-Pro-L-Val) is apparently considerably lower, i.e. only about 5-10% of that reported by Stark, which put its concentration at or below its sensory threshold. However, since Stark's sample was not well characterized, i.e. no information was given on roast profile or how many samples were analyzed, a direct comparison of results is difficult. On the other hand, we were able to detect this diketopiperazine even in unroasted samples, indicating that DKPs are formed already during the fermentation stage of the beans.

Neem (Azadirachta indica) is an evergreen tree cultivated in various parts of the Indian sub-continent. It has been in use over centuries in the Indian folk medicine for its therapeutic value. Extensive research has shown that it may have anti-cancer, anti-diabetic, anti-inflammatory, anti-ulcerogenic, and anti-microbial effects. However, neem is extremely bitter, in large part due to its limonoid content, making it unpalatable and limiting its potential use in dietary supplements or foods alike. The objective of this experiment was to apply two adsorbent based strategies, namely solid phase extraction and Amberlite XAD-16 (AMB) resin, to achieve de-bittering of neem tea and to determine the effects of the de-bittering on the bio-active, color, and volatile properties. The solid phase extraction treatment completely removed the flavonol, quercetin, from neem tea while in Amberlite XAD-16 treated tea (AMB) it was only insignificantly reduced. We also observed decreases in total phenolic content and consequently anti-oxidant activities after de-bittering. A 62% mean reduction of limonoid aglycones indicated diminished levels of bitterness. The loss of phenolics lead to a visually appreciable color change in the treated teas. The de-bittering also leads to a loss of sesquiterpenes, ketones and acids from neem tea. In conclusion, we found that while solid phase extraction cartridges were more efficient in removing bitterness, they caused a greater reduction in bio-active compounds than AMB XAD-16 resins, which may ultimately affect the health properties of neem tea.

University of Nebraska, Lincoln (Janos Zempleni). Background: Virtually every cells produces and secretes exosomes (nanoparticles) loaded with cargos such as various species of RNAs, proteins and lipids. Exosomes play essential roles in cell-to-cell communication. The transfer of exosomes cargos from donor cells to receptor cells alters gene expression and metabolism in receptor cells. We have made the paradigm-shifting discovery that exosomes and their cargos are not only obtained through endogenous synthesis but also from dietary sources such as bovine milk and chicken eggs. Progress: 1) We have assessed the microRNA cargos in chicken egg yolk, the bioavailability of chicken egg exosomes and their microRNA cargos in humans, and the effects of exosomes and cargos in peripheral blood mononuclear cells. 2) We have conducted a comprehensive analysis of the bioavailability and distribution of fluorophore-labeled milk exosomes and microRNAs in mice. 3) We have developed an exosome and cargo tracking (ECT) mouse. 4) We have assessed storage stability and microRNA cargos  in human milk, bovine milk and infant formulas by RNA-sequencing analysis and qRT-PCR. 5) We have assessed the role of glycoproteins on the surface of milk exosomes in absorption and distribution of bovine milk exosomes in mice. 6) We have characterized the composition of exosome-defined, AIN-93G-based diets developed in our laboratory. 7) We demonstrated that depletion of milk exosomes and their RNA cargos elicits phenotypes such as impaired fecundity, aberrant purine metabolism, altered immune function, impaired spatial learning and memory, and changes in the gut microbiome. 8) We have generated preliminary data suggesting that bovine milk exosomes deliver a large load of microbial RNAs to hosts. Short-term outcomes: In five years of W-3002 support, 21 visiting scientists, postdoctoral fellows, graduate students, undergraduate students, and staff were supervised. Fourteen papers, six reviews, one book chapter, 15 meeting abstracts and five outreach publications (newspapers, magazines) were published, and 33 presentations were delivered. A public website was created that disseminates information about dietary microRNAs (Shu et al.). Our research includes collaborations with one scientist from this W-3002 group and 13 other scientists. We have filed a provisional patent application Extracellular Vesicles and Methods of Using, on 3/15/2017 (62/471,572).

Iowa State University (Wendy White). Background: Based on the bioavailability of carotenoids and fat-soluble vitamins measured in our human study, we developed statistical models to predict the relation between the amount of soybean oil in salad dressing and the absorption of: 1) alpha-carotene, beta-carotene, and lycopene in salad vegetables; and 2) retinyl palmitate, the major form of vitamin A absorbed after the intestinal metabolism of alpha- and beta-carotene. For all carotenoids and fat-soluble vitamins studied, we provided important new information regarding the optimal amount of vegetable oil needed to promote their absorption from fresh vegetables. We evaluated the effects of increasing amounts of vegetable oil in salad dressing on the absorption and bioactivity of alphacarotene and beta-carotene. Bioactivity was measured as the retinyl palmitate (vitamin A) formed through the intestinal metabolism of these nutrients. This project has provided extensive training for the Principal Investigator in the form of four summers of self-study in the use of statistical models to predict the intestinal absorption of nutrients. Outcomes: A manuscript describing the results was published in the American Journal of Clinical Nutrition, the top-ranked nutrition journal worldwide. The manuscript was selected to be the focus of an editorial published in the same issue of that journal. The study results were disseminated via a university press release, which was then covered by national and international media outlets, including LinkedIn, the Daily Mail (United Kingdom), Men's Health, Men's Fitness, AARP Magazine, New Indian Express (India), KPCC Radio (Southern California), NutraIngredients, and Science Daily. The study findings were presented at the annual meeting of the American Society for Nutrition in conjunction with the Experimental Biology '17 conference in Chicago, IL.

Abelilla, J. J., Y. Liu, and H. H. Stein. 2018. Digestible indispensable amino acid score (DIAAS) and protein digestibility corrected amino acid score (PDCAAS) in oat protein concentrate measured in 20- to 30-kilogram pigs. J. Sci. Food Agri. 98:410-414.

Aguilar-Lozano A, Baier SR, Grove R, Shu J, Giraud D, Mercer KE, Cui J, Badger TM, Adamec J, Andres A, Zempleni J. Concentrations of purine metabolites are elevated in human fluids from adults and infants and in livers from mice fed diets depleted of bovine milk exosomes and their RNA cargos (submitted).

Ahluwalia, A., Gladwin, M., Coleman, G.D., Hord, N. G., Howard, G., Kim-Shapiro, D., Lajous, M., Larsen, F., Lefer, D.J., McClure, L.A., Nolan, B.T., Pluta, R., Schechter, A., Wang, C.-Y., Ward, M.H., and Harman, J.L. (2016) Dietary nitrate and the epidemiology of cardiovascular disease: Report from a National Heart, Lung, and Blood Institute Workshop, Journal of the American Heart Association J Am Heart Assoc. 2016 Jul 6;5(7). doi: 10.1161/JAHA.116.003402.

Bae M., Y. Park, J. Lee. Comprehensive review on food components with anti-fibrotic activity for the prevention of liver fibrosis. J Nutr Biochem 2018; 55:1-11. Epub ahead of print on November 16, 2017.

Bai, L. L., F. Wu, H. Liu, S. Zhang, L. Liu, X. S. Piao, Y. H. Liu, P. A. Thacker, and F. L. Wang. 2017. Effects of dietary calcium levels on growth performance and bone characteristics in pigs in grower-finisher-translational phase. Anim. Feed Sci. Technol. 224:59-65.

Bai, M., H. Liu, K. Xu, B. Zou, R. Yu, Y. Liu, W. Xing, H. Du, Y. Li, and Y. Yin. 2017. Effects of dietary coated cysteamine hydrochloride on pork color in finishing pigs. J. Sci. Food Agric. doi: 10.1002/jsfa.8647.

Bailey RL, Weaver CM, Murphy S.  Using the Dietary Reference Intakes to assess intakes in Research:  Successful Approaches.  Van Horn L, ed.  Academy of Nutrition and Dietetics, Chicago IL, 2017.

Bauer, PV, Duca FA, Waise TM, Dranse HJ, Rasmussen BA, Puri A, Rasti M, O’Brien CA, Lam TK. Microbiota in the upper small intestine alters ACSL3-dependent fatty acid sensing pathway to influence whole-body glucose homeostasis. Cell Metab 2017 (in press).

Beaver LM, Truong L, Barton CL, Chase TT, Gonnerman GD, Wong CP, Tanguay RL, and Ho E. (2017) Combinatorial effects of zinc deficiency and arsenic exposure on zebrafish (Danio rerio) development.  PLoS One. 2017 Aug 24;12(8):e0183831. doi: 10.1371/journal.pone.0183831.

Beaver, L. M., Kuintzle, R., Buchanan, A., Wiley, M. W., Glasser, S. T., Wong, C. P., Johnson, G. S., Chang, J. H., Löhr, C. V., Williams, D., Dashwood, R. H., Hendrix, D. A., Ho, E. (2017) Long noncoding RNAs and sulforaphane: a target for chemoprevention and suppression of prostate cancer. The Journal of nutritional biochemistry 42: 72-83.

Beaver, L. M., Nkrumah-Elie, Y. M., Truong, L., Barton, C. L., Knecht, A. L., Gonnerman, G. D., Wong, C. P., Tanguay, R., Ho, E. (2017) Adverse effects of parental zinc deficiency on metal homeostasis and embryonic development in a zebrafish model. The Journal of nutritional biochemistry 43: 78-87.

Blumberg JB, Frei B, Fulgoni III, VL, Weaver CM, Zeisel SH.  Contribution of dietary supplements to nutritional adequacy by socioeconomic subgroups in adults of the United States.  Nutrients  10:  2018  doi:10.3390/nu10010004

Blumberg JB, Frei BB, Fulgoni VL, Weaver CM, Zeisel SH.  Contribution of dietary supplements to nutritional adequacy in race/ethnic population subgroups in the United States.  Nutrients  9:1295-1304, 2017.

Blumberg JB, Frei BB, Fulgoni VL, Weaver CM, Zeisel SH.  Impact of Frequency of Multi-Vitamin/Multi-Mineral Supplement Intake on Nutritional Adequacy and Nutrient Deficiencies in U.S. Adults.  Nutrients  9:849-863, 2017.

Bobe G, TJ Cobb, SW Leonard, S Aponso, CB Bahro, D Koley, E Mah, RS Bruno, MG Traber (2017). Increased static and decreased capacity oxidation-reduction potentials in plasma are predictive of metabolic syndrome. Redox Biol, 12:121-128.

Camara Teixeira D, Cordonier EL, Wijeratne SSK, Huebbe P, Jamin A, Jarecke S, Wiebe M, Zempleni J. A cell death assay for assessing the mitochondrial targeting of proteins. J Nutr Biochem (in press)

Chen, Y. S., Wang, R., Dashwood, W. M., Löhr, C. V., Williams, D., Ho, E., Mertens-Talcott, S., Dashwood, R. H. (2017) A miRNA signature for an environmental heterocyclic amine defined by a multi-organ carcinogenicity bioassay in the rat. Archives of toxicology.

Coborn JE, Deporter DP, Mavanji, V, Sinton CM, Billington CJ, Kotz CM, Teske, JA. Role of orexin A in the ventrolateral preoptic area on components of total energy expenditure. 2017.International Journal of Obesity. 41(8):1256-1262. Epub 2017 April 10.

Coborn JE, Houser MA, Perez-Leighton CE, Teske, JA Role of sex and the environment in moderating weight gain due to inadequate sleep. 2017. Current Obesity Reports. 6(4):397-404. Epub 2017 Nov. 28.

Collins FL, Kim SM, McCabe LR, Weaver CM.  Ch. 14  Intestinal Microbiota and Bone Health: The Role of Prebiotics, Probiotics, and Diet  In:  Molecular and Integrative Toxicology – Bone Toxicology.  Smith S, Varela A, Samadfam R (eds)  pg 417-443, 2017.

Conley MN, Wong CP, Duyck KM, Hord N, Ho E, Sharpton TJ. Aging and serum MCP-1 are associated with gut microbiome composition in a murine model.  (2016) PeerJ. Mar 31;4:e1854. doi: 10.7717/peerj.1854. eCollection 2016.

Conley, MN, Maccha, A, Roberts, C, Sharpton, TJ, Urszula T. Iwaniec, UT, Hord NG. (2016) Increasing dietary nitrate has no effect on bone loss or gut microbiome in ovariectomized rats.  Mol Nutr Food Res. 2017 May;61(5). doi: 10.1002/mnfr.201600372. Epub 2017 Mar 30.

Cotton BM, SA Diamond, T Banh, Y-H Hsiao, RM Cole, J Li, CT Simons, RS Bruno, MA Belury,Y Vodovotz. (2017). Raspberry ketone fails to reduce adiposity beyond decreasing food intake in C57BL/6 mice fed a high-fat diet. Food Funct, 8(4):1512-1518.

Cristian Del Bo, Valeria Deon, Jonica Campolo, Marisa Porrini, Dorothy Klimis-Zacas and Patrizia Riso. A serving of blueberry (V. corymbosum) reverses endothelial dysfunction in young smokers and non-smokers: a randomized, controlled, crossover study, Food and Function, DOI: 10.1039/c7fo00861a, 2017.

Dallas, D. C., German, J. B. (2017) Enzymes in human milk. In: Isolauri E, Sherman P, Walker W, editors. Intestinal Microbiome: Functional Aspects in Health and Disease. Basel, Switzerland: Karger Publishers; p. 129-36.

Datta A., Grün IU, Kwasniewski, MT, and Fernando, LN. 2017. Comparison of Two Adsorbent Based de-Bittering Procedures for Neem (Azadirachta indica A. Juss) Tea- Effect on Polyphenols, Anti-Oxidant Capacity, Color and Volatile Profile. Plant Foods for Human Nutrition 72(1):88-95; DOI : 10.1007/s11130-016-0595-9; Impact Factor 2.368.

Datta, A., Fernando, L.N. and Gruen, I.U. 2015. Effects of two de-bittering strategies on flavonols, phenolic compounds and anti-oxidant activity of Neem tea. Institute of Food Technologists Annual Meeting, Chicago, IL, July 11-14, Pres. #098-058.

Datta, A., Fernando, L.N., and Gruen, I.U. 2014. Investigation of Phytosterols in Leaves and Bark of Neem using High Performance Liquid Chromatography. Institute of Food Technologists Annual Meeting, New Orleans, LA, June 21-24, Pres. # 206-143.

Delimont NM, Fiorentino NM, Kimmel KA, Haub MD, Rosenkranz SK, Lindshield BL. Long-term, multiple daily-condensed tannin supplementation in increasing concentrations does not affect iron status or bioavailability: results from the Tannin-dose response trial. Curr Dev Nutr. 1(10): 2017.

Delimont NM, Rosenkranz SR, Haub M, Lindshield BL. Salivary Proline-Rich Proteins May Reduce Tannin-Iron Chelation: A Systematic Narrative Review. Nutr Metab. 14(47): 2017. https://doi.org/10.1186/s12986-017-0197-z.

Demers-Mathieu, V., Nielsen, S. D., Underwood, M. A., Borghese, R., Dallas, D. C. (2017) Changes in proteases, antiproteases and bioactive proteins from mother's breast milk to the premature infant stomach. Journal of Pediatric Gastroenterology and Nutrition 66(2): 318-324.

Demers-Mathieu, V., Nielsen, S. D., Underwood, M. A., Borghese, R., Dallas, D. C. (2017) Analysis of milk from mothers who delivered prematurely revealed few changes in proteases and protease inhibitors across gestational age at birth and infant postnatal age. Journal of Nutrition 147(6): 1152-1159.

Demers-Mathieu, V., Qu, Y., Underwood, M. A., Borghese, R., Dallas, D. C. (2017) Premature infants have lower gastric digestion capacity for human milk proteins than term infants. Journal of Pediatric Gastroenterology & Nutrition.

DePorter DP, Coborn JE, Teske, JA. Partial sleep deprivation reduces the efficacy of orexin-A in the ventrolateral preoptic area to stimulate physical activity and energy expenditure. 2017. Obesity (Silver Spring). 25(10):1716-1722. Epub 2017 August 17.

Keller, RM, Beaver, L, Prater, MC, Hord, NG. (2017) Dietary Nitrate and Nitrite Concentrations in Food Patterns and Dietary Supplements. Nutrition Today, December 27, 2017; doi: 10.1097/NT.0000000000000253.

Duca FA, Bauer, PV, Waise TM, Rasmussen BA, Abraham MA, Dranse HJ, Puri A, O’Brien CA, Lam TK. Metformin alters upper small intestinal microbiota that impact a glucose-SGLT1 sensing glucoregulatory pathway. Cell Metab 2017 (epub).

Ellison D., Morris C.E., Locatelli B., Sheil D., Cohen J., Murdiyarso D., Gutierrez V., van Noordwijk M., Creed I. F. Pokorny J., Gaveau D., Spracklen D., Bargués Tobella A., Ilstedt U., Teuling R., Gebrehiwot S. G., Sands D.C., Muys B., Verbist B., Springgay E., Sugandi Y., Sullivan C. 2017. Trees, forests and water: cool insights for a hot world. Global Environmental Change (in press)

Farruggia C., M. Kim, M. Bae, Y. Lee, T. X. Pham, Y. Yang, M. J. Han, Y. Park, J. Lee. Astaxanthin exerts anti- inflammatory and antioxidant properties in macrophages in NRF2-dependent and independent manners. J Nutr Biochem: In review.

Fratantonio D,* Shu J,* Howard K, Baier SR, Giraud D, Cui J, Zempleni.MicroRNAs from chicken eggs are bioavailable and alter gene expression in peripheral blood mononuclear cells in humans (submitted). *These authors contributed equally

Gaur S, Kuhlenschmidt, TB, Kuhlenschmidt, MS, Andrade, JE. Effect of Oregano Essential Oil and Carvacrol on Cryptosporidium parvum infectivity in HCT-8 Cells. Parasitology International. 67(2):170-175 (2017).

Giudici K, Weaver CM.  Calcium:  Physiology and Metabolic Aspects.  In:  Calcium and Vitamin D Physiology, Nutrition and Associated Diseases.  Ed  Martini LA  Editora Manole  2017.

Hord, NG and Conley M. (2016)  Regulation of dietary nitrate and nitrite: balancing essential physiological roles with potential health risks, In: Nitrates and Nitrites in Human Health and Disease, Editors: Joseph Loscalzo, M.D., Ph.D. (Harvard University) and Nathan S. Bryan, PhD (Texas), New York, NY, Spring Science and Busines Media, Second Edition, In Press (book chapter).

Housley L, Magana AA, Hsu A, Beaver LM, Wong CP, Stevens JF, Choi J, Jiang Y, Bella D, Williams DE, Maier CS, Shannon J, Dashwood RH, and Ho E. (2018) Untargeted Metabolomic Screen Reveals Changes in Human Plasma Metabolite Profiles Following Consumption of Fresh Broccoli Sprouts.  Mol Nutr Food Res. 2018 Jan 28. doi: 10.1002/mnfr.201700665. [Epub ahead of print]

Johnson, G. S., Li, J., Beaver, L. M., Dashwood, W. M., Sun, D., Rajendran, P., Williams, D., Ho, E., Dashwood, R. H. (2017) A functional pseudogene, NMRAL2P, is regulated by Nrf2 and serves as a coactivator of NQO1 in sulforaphane-treated colon cancer cells. Molecular Nutrition & Food Research 61(4):

Juraschek SP, Miller ER, III, Weaver CM, Appel, LJ.  Effect of sodium reduction and the DASH diet by level of baseline blood pressure:  Pronounced benefits among adults with higher blood pressure.  J Am Col Card 2017. (In Press)

Kim B., C. Farruggia, C. S. Ku, T. X. Pham, Y. Yang, M. Bae, Casey J. Wegner, N. J. Farrell, E. Harness, Y. Park, Sung I. Koo, J. Lee. Astaxanthin inhibited inflammation and fibrosis in the liver and adipose tissue of mouse models of diet- induced obesity and nonalcoholic steatohepatitis. J Nutr Biochem 2017; 43:27-35.

Kim B., M. Bae, Y. Park, H. Ma, T. Yuan, N. Seeram, J. Lee. Blackcurrant anthocyanins stimulated cholesterol transport via post-transcriptional induction of LDL receptor in Caco-2 cells. Eur J Nutr 2017; Epub ahead of print on July 17, 2017.

Kim, B. G., Y. Liu, and H. H. Stein. 2017. Effects of ileal digesta collection time on standardized ileal digestibility of amino acids in corn, soybean meal, and distiller’s dried grains with solubles fed to growing pigs. J. Anim. Sci. 95:789-798.

Kindler JM, Pollock NK, Laing EM, Oshri A, Jenkins NT, Isales CM, Hamrick MW, Ding KH, Hausman DB, McCabe GP, Martin BR, Hill Gallant KM, Warden SJ, Weaver CM, Peacock M, Lewis RD.  Insulin resistance and the IGF-I-cortical bone relationship in children ages 9 to 13 years.  J Bone Min Res 32:1537-1545, 2017.

Kotz CM, Perez-Leighton CE, Teske JA, Billington CJ. Spontaneous Physical Activity Defends Against Obesity. 2017. Current Obesity Reports. 6(4):362-370. Epub 2017 Nov. 3

Lashley J, Cummings-Saul R, Bennett A, Lindshield BL. Cultivating Textbook Alternatives From the Ground Up: One Public University’s Sustainable Model for Open and Alternative Educational Resource Proliferation. Int Rev Res Open Distributed Learn. 18(4): 2017. http://dx.doi.org/10.19173/irrodl.v18i4.3010.

Lee J., Y. Yue, Y. Park, and S.-H. Lee (2017) 3,3'-Diindolylmethane Suppresses Adipogenesis via AMPK-Dependent Mechanism in 3T3-L1 Adipocytes and Caenorhabditis elegans, J. Med. Food 20: 646-652.

Lee Y-K, Hyun T, Lyu E-S, Oh S-Y, Park H, Ro H-K, Heo Y-R, Kim M-H, Weaver CM Choi M-K.  Serum calcium is associated with dyslipidemia in Korean adults:  a cross-sectional study.  Trace Elements Eletrolytes  34:159-165, 2017.

Leiferman A, Shu J, Grove R, Cui J, Adamec J, Zempleni J. A diet defined by its content of bovine milk exosomes and their RNA cargos has moderate effects on gene expression, amino acid profiles and grip strength in skeletal muscle in C57BL/6 mice (submitted)

Li J, TN Sapper, E Mah, MV Moller, JB Kim, C Chitchumroonchokchai, JD McDonald, RS Bruno. (2017). Green tea extract treatment reduces NFκB activation in mice with diet-induced nonalcoholic steatohepatitis by lowering TNFR1 and TLR4 expression and ligand availability. J Nutr Biochem, 41:34-41.

Lillehoj, H., Y. Liu, S. Calsamiglia, M. E. F. Miyakawa, F. Chi, R. L. Craven, and G. Cyril. 2018. Phytochemicals as potential antibiotic alternatives to promote growth and enhance host health: A report from the Second International Symposium on Alternative to Antibiotics. Vet. Res. (In press)

Liu, Y., C. D. Espinosa, J. J. Abelilla, G. A. Casas, L. Vanessa Lagos, S. A. Lee, W. B. Kwon, J. K. Mathai, D. M. D. L. Navarro, N. W. Jaworski, and H. H. Stein. 2018. Non-antibiotic feed additives in diets for pigs. Anim. Nutr. (In press)

Márquez Loza, A., Elias, V., Wong, C. P., Ho, E., Bermudez, M., Magnusson, K. R. (2017) Effects of ibuprofen on cognition and NMDA receptor subunit expression across aging. Neuroscience 344: 276-292.

Mathai, J. K., Y. Liu, and H. H. Stein. 2017. Values for digestible indispensable amino acid scores (DIAAS) for some dairy and plant proteins may better describe protein quality than values calculated using the concept for protein digestibility-corrected amino acid scores (PDCAAS). Br. J. Nutr. 117-490-499.

Mella R, Schmidt C, Romagnoli P, Teske JA, Perez-Leighton CE. The food environment, preference and experience modulate the effects of Exendin-4 on food intake and reward. 2017. Obesity. 25(11):1844-1851. Epub 2017 Nov 1.

Moisa, SJ, P Ji, JK Drackley, SL Rodriguez-Zas, and JJ Loor. 2017. Transcriptional changes in mesenteric and subcutaneous adipose tissue from Holstein cows in response to plane of dietary energy. J. Anim. Sci. Biotech. doi: 10.1186/s40104-017-0215-z

Mónica Fernández-Aparicio, Alexandre Bernard, Laurent Falchetto, Pascal Margert, Bruno Chauvel, Christian Steinberg, Cindy E. Morris, Stephanie Gibot-Leclerc, Angela Boari, Maurizio Vurro, David A. Bohan, David C. Sands and Xavier Reboud. (2017 Investigation of amino acids as herbicides for control of Orobanche minor parasitism in red clover. (2017). Frontiers in Plant Science vol. 8, article 842.

Morris C.E., Sands D.C. 2017. Impacts of microbial aerosols on natural and agro-ecosystems: immigration, invasions and their consequences. IN : Delort A.M., Amato, P. (eds.) Microbiology of Bioaerosols. Wiley, (in press).

Morris C.E., Soubeyrand S., Bigg E.K., Creamean J.M., Sands D.C. 2016. A framework to assess the contribution of bioaerosols to the outcome of meteorological contexts favorable for rainfall. bioRxiv archives http://biorxiv.org/content/early/2016/08/21/070532.  (NOTE THAT THIS IS NOT A

Murray, N. M., O'Riordan, D., Jacquier, J.-C., O'Sullivan, M., Cohen, J., Heymann, H., Barile, D., Dallas, D. C. (2017) Validation of a paper-disk approach to facilitate the sensory evaluation of bitterness in dairy protein hydrolysates from a newly developed food-grade fractionation system. Journal of Sensory Studies 32(3): e12266.

Nielsen, S. D., Beverly, R. L., Dallas, D. C. (2017) Peptides released from foremilk and hindmilk proteins by breast milk proteases are highly similar. Frontiers in Nutrition, Nutrition Methodology 4(54).

Nielsen, S. D., Beverly, R. L., Qu, Y., Dallas, D. C. (2017) Milk bioactive peptide database: A comprehensive database of milk protein-derived bioactive peptides and novel visualization. Food Chemistry 232: 673-682.

Opoku-Acheampong AB, Henningson JN, Lindshield BL. 5α-reductase 1 mRNA levels are positively correlated with TRAMP mouse prostate most severe lesion scores. PLOS ONE. 12(5): e0175874, 2017 https://doi.org/10.1371/journal.pone.0175874.

Opoku-Acheampong AB, Henningson JN, Lindshield BL. The Impact of Finasteride and Dutasteride Treatments on Proliferation, Androgen Receptor, Apoptosis, 5α-Reductase 1 and 5α-reductase 2 in TRAMP Mouse Prostates. Heliyon. 3(7): 2017. https://doi.org/10.1016/j.heliyon.2017.e00360.

Palomera-Sanchez, Z., Watson, G. W., Wong, C. P., Beaver, L. M., Williams, D., Dashwood, R. H., Ho, E. (2017) The phytochemical 3,3'-diindolylmethane decreases expression of AR-controlled DNA damage repair genes through repressive chromatin modifications and is associated with DNA damage in prostate cancer cells. The Journal of nutritional biochemistry 47: 113-119.

Panagiotis Tsakiroglou, Sharon Ashworth, James Webber, Cristian DelBo and Dorothy Klimis-Zacas, Anthocyanins and Phenolic acids extracted from wild blueberries (V. angustifolium) differentially modulate endothelial cell migration through RhoA and RAC1 (in preparation)

Panagiotis Tsakiroglou, VandenAkker, N., Del Bo’, C., Riso, P., and Klimis-Zacas, D., The Role of Berry Bioactives on Angiogenesis. Invited Review, Food and Function (in preparation)

Panagiotis Tsakiroglou, Weber, J.A., Ashworth, SL. and Klimis-Zacas, D., Angiogenesis in endothelial cells is differentially modulated by ACNs and PAs from wild blueberries (V. angustifolium) through AKT/eNOS pathway (in preparation)

Parrish JB and Teske JA. Acute partial sleep deprivation due to environmental noise increases weight gain by reducing energy expenditure in rodents. 2017. Obesity (Silver Spring) Jan;25(1):141-146. Epub 2016 November 29.

Pham T. X., M. Bae, Y. Lee, Y. Park. J. Lee. Transcriptional and post-transcriptional repression of histone deacetylases by docosahexaenoic acid in macrophages. J Nutr Biochem: In revision. 

Pham T. X., Y. Park, M. Bae, J. Lee. The potential role of an endotoxin-like mechanism for the anti-inflammatory effect of Spirulina platensis in macrophages: Insight into energy phenotype. J Med Food 2017; 20: 201-210.

Pham T.X., J. Lee. Epigenetic regulation of adipokines. Int J Mol Sci 2017; 18:1740; doi:10.3390/ijms18081740.

Picariello, G., Addeo, F., Ferranti, P., Nocerino, R., Paparo, L., Passariello, A., Robinson, R. C., Barile, D., Dallas, D. C., Berni Canani, R. (2016) Antibody-independent identification of bovine milk-derived peptides in breast-milk. Food & Function 7(8): 3402-3409.

Shams-White MM, Chung M, Du M, Fu Z, Insogna KL, Karlsen MC, LeBoff MS, Shapses SA, Sackey J, Wallace TC, Weaver CM.  Dietary protein and bone health:  a systematic view and meta-analysis from the National Osteoporosis Foundation.  2017  10.3945/ajcn.116.145110.

She, Y., Y. Liu, and H. H. Stein. 2017. Effects of graded levels of microbial phytase on apparent total tract digestibility of calcium and phosphorus and standardized total tract digestibility of phosphorus in four sources of canola meal and in soybean meal fed to growing pigs. J. Anim. Sci. 95:2061-2070.

Shen P., Y. Yue, K.-H. Kim, and Y. Park (2017) Piceatannol Reduces Fat Accumulation in Caenorhabditis elegans, J. Med. Food 20: 887-894.

Shen P., Y. Yue, Q. Sun, N. Kasireddy, K.-H. Kim, and Y. Park (2017) Piceatannol Extends the Lifespan of Caenorhabditis elegans Via DAF-16, Biofactors, 43: 379-387.

Sloffer, EM, Gaur, S, Engeseth, NJ, Andrade JE. Evaluation of accelerated shelf life testing in the development of a shea butter-containing lipid nutrition supplement for Sub-Saharan Africa.  Foods. 6(11), 97 (2017).

Taylor K. Bloedon, Rock E. Braithwaite, Imogene A. Carson, Dorothy J. Klimis-Zacas, Impact of whole berry fruit consumption on exercise-induced oxidative stress and inflammation: a systematic review and meta-analysis, Nutrition Reviews, 2018 (under review)

Traber MG, E Mah, SW Leonard, G Bobe, RS Bruno. (2017). Metabolic syndrome increases dietary α-tocopherol requirements as assessed using urinary and plasma vitamin E catabolites: a double-blind, crossover clinical trial. Amer J Clin Nutr, 105(3):571-579.

Velkova, A., Diaz, J. E. L., Pangilinan, F., Molloy, A. M., Mills, J. L., Shane, B., Sanchez, E., Cunningham, C., McNulty, H., Cropp, C. D., Bailey-Wilson, J. E., Wilson, A. F. and Brody, L. C. (2017) The FUT2 secretor variant p.Trp154Ter influences serum vitamin B12 concentration via holo-haptocorrin, but not holo-transcobalamin, and is associated with haptocorrin glycosylation. Hum. Mol. Genet. 26: 4975-4988.

Vogel KA, Martin BR, McCabe LD, Peacock M, Warden SJ, McCabe GP, Weaver CM.  The effect of dairy intake on bone mass and body composition in early pubertal girls and boys:  A randomized controlled trial.  AJCN 105:1214-1229, 2017.

Vorland CJ, Martin BR, Weaver CM, Peacock M, Hill Gallant KM.  Phosphorus balance in adolescent girls and the effect of supplemental dietary calcium.  JBMR Plus  70:2841-2848, 2017.

Wang L, Sadri M, Giraud D, Zempleni J. RNase H2-dependent PCR and elimination of confounders in sample collection, storage and analysis strengthen evidence that microRNAs in bovine milk are bioavailable in humans. J Nutr (in press)

Wang, R., Kang, Y., Löhr, C. V., Fischer, K. A., Bradford, C. S., Johnson, G., Dashwood, W. M., Williams, D., Ho, E., Dashwood, R. H. (2016) Reciprocal regulation of BMF and BIRC5 (Survivin) linked to Eomes overexpression in colorectal cancer. Cancer letters 381(2): 341-8.

Wang, R., Yu, Z., Sunchu, B., Shoaf, J., Dang, I., Zhao, S., Caples, K., Bradley, L., Beaver, L. M., Ho, E., Löhr, C. V., Perez, V. I. (2017) Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2-independent mechanism. Aging cell 16(3): 564-574.

Weaver CM, Hill Gallant KM  Ch. 44 Osteoporosis:  The Early Years.  In:  Nutrition in the Prevention and Treatment of Disease  2017, 4^th Ed.  Coulston, AM, Boushey, CJ, Ferruzzi MG, DeLahanty LM, eds.  Elsevier, Inc.  Pp 969-989.

Weaver CM, Miller JW.  Challenges in conducting clinical nutrition research.  Nutr Rev  doi:  10.1093/nutrit/nux026

Wikoff D, Welsh BT, Henderson R, Brorby GP, Britt J, Myers E, Goldberger J, Lieberman HR, O’Brien C, Peck J, Tenebein M, Weaver C, Harvey S, Urban J, Doepker C.  Systematic review of the potential adverse effects of caffeine consumption in healthy adults, pregnant women, adolescents and children. Food Chem Tox 1-64, 2017.

Wu X., M. Song, P. Qiu, K. Rakariyatham, F. Li, Z. Gao, X. Cai, M. Wang, F. Xu, J. Zheng, and H. Xiao (2017) Synergistic chemopreventive effects of nobiletin and atorvastatin on colon carcinogenesis, Carcinogenesis, 38: 455-464.

Wu X., M. Song, X. Gao, Y. Sun, M. Wang, F. Li, J. Zheng, and H. Xiao (2017) Nobiletin and its colonic metabolites suppress colitis-associated colon carcinogenesis by down-regulating iNOS, inducing antioxidative enzymes and arresting cell cycle progression, J. Nutr. Biochem, 42L 17-25.

Xia Z., D. J. McClements, and H. Xiao (2017) Influence of Lipid Content in a Corn Oil Preparation on the Bioaccessibility of β-Carotene: A Comparison of Low-Fat and High-Fat Samples, J. Food Science, 82: 373-379.

Yang Y., M. Bae, Y. Park, Y. Lee, T. X. Pham, S. Rudraiah, J. Manautou, S. I. Koo, J. Lee. Histone deacetylase 9 plays a role in the anti-fibrogenic effect of astaxanthin in hepatic stellate cells. J Nutr Biochem 2017; 40: 172-177.

Yildiz G, Andrade, JE, Engeseth, NJ, Feng, H. Functionalizing soy protein nano-aggregates with pH-shifting and mano-thermo-sonication. Journal of Colloid Interface Sciences. 2017 505(1): 836-846.

Yuan, D., T. Hussain, B. Tan, Y. Liu, P. Ji, Y. Yin. 2017. The Evaluation of Antioxidant and Anti-Inflammatory Effects of Eucommia ulmoides Flavones Using Diquat-Challenged Piglet Models. Oxid. Med. Cell Longev. doi: 10.1155/2017/8140962.

Zempleni J. Milk exosomes in infant nutrition. Shelley McGuire, ed. (book chapter, invited contribution, in preparation)

Zempleni J. Milk exosomes. Annual Review of Animal Biosciences (review article, invited contribution, in preparation)

Zempleni J. Milk exosomes: beyond dietary microRNAs. Nutr Genes (in press)

Zhong F, M Xu, RS Bruno, KD Ballard, J Zhu. (2017). Targeted high performance liquid chromatography tandem mass spectrometry-based metabolomics differentiates metabolic syndrome from obesity. Exp Biol Med, 242(7):773-780.

Zhou F, Paz HA, Shu J, Sadri M, Cui J, Fernando SC, Zempleni J. Dietary bovine milk exosomes and their RNA cargos elicit changes in the composition of the intestinal microbiome and the hepatic transcriptome in C57BL/6 mice (submitted)

Zou Y, Zou X, Li X, Guo G, Ji P, Wang Y, Li S, Wang Y, Cao Z. 2017. Substituting oat hay or maize silage for portion of alfalfa hay affects growth performance, ruminal fermentation, and nutrient digestibility of weaned calves. Asian-Austr. J. Anim. Sci. doi: 10.5713/ajas.17.0210.