bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibiting epidermal growth factor receptor signal transduction via metabolic oligosaccharide engineering sensitizes pancreatic cancer cells to drugs (788.4)
Authors: Mathew Mohit , Tan Elaine , Saeui Christopher , Bovonratwet Patawut , Bhattacharya, Rahul , Yarema Kevin
Published: June 2021

Metabolic Oligosaccharide Engineering is a versatile technique that can be used to change patterns of glycosylation by altering the availability and abundance of biosynthetic precursors of glycans; for example, 1,3,4‐O‐Bu3ManNAc can be used to increase flux through the sialic acid biosynthetic pathway. Because glycosylation is a non‐template driven process, such an increase in the levels of sialic acid results in the favoring of increasingly sialylated glycoforms. In a previous study we found that 1,3,4‐O‐Bu3ManNAc increased the sialylation of dozens of N‐linked glycoproteins in SW1990 pancreatic cancer cells including Epidermal Growth Factor Receptor (EGFR) by ~2.6 fold. EGFR is known to play important roles in cancer progression but relatively little is known about how alterations to the glycosylation of this protein affect its behavior. Therefore we found it interesting that the 1,3,4‐O‐Bu3ManNAc‐driven increase in EGFR sialylation inhibited signal transduction of this receptor beginning at the level of phosphorylation and extending downstream to the transcriptional level. We reasoned that these cancer cells would have a decreased capacity for EGFR signaling and be more susceptible to drugs such as Erlotonib and Gefitinib that target EGFR phosphorylation. This premise was confirmed experimentally, with combination indexes of ~0.3 showing strong synergy between 1,3,4‐O‐Bu3ManNAc and either drug. Further studies using additional approaches (e.g., non‐natural ManNAc analogs or modulation of glycolytic flux) also show synergy in preliminary experiments, opening the door to multi‐pronged approaches that combine the benefits of glycosylation‐induced EGFR signal suppression and synergistic interactions with existing cancer therapeutics.

Grant Funding Source: Supported by R01CA112314
Complex N‐linked glycans serve as a determinant for exosome/microvesicle cargo recruitment (788.5)
Authors: Liang Yaxuan , Eng William , Colquhoun David , Dinglasan Rhoel , Graham David , Mahal Lara
Published: June 2021

Exosomes, also known as microvesicles (EMVs), are nano‐sized membrane particles secreted from nearly all mammalian cell types. These nanoparticles play critical roles in many physiological processes including cell‐cell signaling, immune activation and suppression, and are associated with disease states such as tumor progression. The biological functions of EMVs are highly dependent on their protein cargo, which can dictate pathogenicity. While some mechanisms have been proposed for the regulation of EMV cargo selection, little attention has been paid to N‐linked glycosylation as a potential sorting signal. Previous work from our laboratory found a conserved glycan signature for EMVs, which differed from the overall cellular membranes, suggesting a potential role for glycosylation in EMV biogenesis. In this study, we explore further the role of glycosylation in EMV cargo selection. We identify specific glycosylated targets and associated proteins and demonstrate alteration of their recruitment as a function of their glycosylation status upon pharmacological manipulation. Further, we show that genetic manipulation of the glycosylation levels of a MV cargo glycoprotein EWI‐2 directly impacts its recruitment as a function of N‐linked glycan sites. Taken together, our data provided strong evidence that N‐linked glycosylation directs glycoprotein sorting into EMVs and serves as a determinant of EMV cargo selection.

Grant Funding Source: Supported by Mizutani Foundation for Glycoscience
O‐GlcNAc occurs in the protozoan parasite Toxoplasma gondii and is part of the histone code (790.4)
Authors: Silmon de Monerri Natalie , Nardelli Sheila , Calloway Myrasol , Nieves Edward , Angeletti Ruth , Weiss Louis , Wu Peng , Kim Kami
Published: June 2021

O‐GlcNAcylation, the addition of N‐acetylglucosamine to Serine or Threonine residues, is an abundant, dynamic posttranslational modification that occurs in many eukaryotes; very little is known about its role in early eukaryotes including protozoa. Here, we describe O‐GlcNAcylation in the protozoan parasite Toxoplasma gondii, which causes one of the most common parasitic infections in humans and warm‐blooded animals, Toxoplasmosis. T. gondii is an obligate intracellular parasite, which has the unique ability of being able to switch between two developmental stages, a phenomenon that is crucial for pathogenesis and is thought to be regulated by sensing of nutrient availability and metabolism. How parasites sense changes in the host cell environment is unclear; since O‐GlcNAc is important for metabolic sensing in other eukaryotes, this led us to hypothesise that O‐GlcNAc is a metabolic sensor in T. gondii, regulating developmental programming. Here, we demonstrate that both the T. gondii homologue of O‐GlcNAc transferase (TgOGT) and O‐GlcNAc are present in nuclear and cytoplasmic compartments in T. gondii parasites. As O‐GlcNAc is an important epigenetic regulator, we focussed on its role on T. gondii histones, on which we show by mass spectrometry and immunoprecipitation that it is also present, indicating that it is a dynamic modification in T. gondii. To identify more O‐GlcNAcylated proteins associated with chromatin, we performed immunoprecipitations and mass spectrometry. Surprisingly, native ChIP‐chip experiments localised O‐GlcNAc to centromeric and telomeric regions of the T. gondii genome, where it colocalises with other epigenetic modifiers, suggesting that it plays a role in mitotic chromatin dynamics. Current efforts include overexpression of dominant negative mutants and conditional knockdown to investigate the function of TgOGT in T. gondii.

Grant Funding Source: R21AI101801‐01A1
Complexation of phospholipase C isozymes regulate cytosolic and nuclear signaling and insulin secretion (796.14)
Authors: Cline Gary
Published: June 2021

Insulin secretion from the pancreatic islet β‐cell is stimulated not only by glucose, but also by binding of acetylcholine to the M3 muscarinic receptor. Acetylcholine binding activates PLCβ2. Postpandrial increases in glucose metabolism by the β‐cell lead to Ca2+ influx and activates PLCδ1. Thus, the β‐cell has redundant mechanisms to increase the provision of PLC‐derived second messengers of insulin secretion. PLCβ2 and PLCδ1 isozymes can exist in an enzymatically‐inactive complexed state that is dissociated by either cholinergic agonists or by elevated glucose. Dissociation of the PLCβ‐δ complex enables PLC enzymatic activity.

Our Co‐IP studies indicate that the PLCβ‐δ complex exists in the INS‐1 β‐cell line, and that treatment with muscarinic agonist, carbachol, led to dissociation of PLCβ2 from PLCδ1. Spatial localization of PLC isozymes was evaluated with indirect immunofluorescence microscopy. At basal conditions, PLCβ2 and PLCδ1 were colocalized throughout the cytosol, but were excluded from the nucleus. Dissociation of the PLCβ‐δ complex occurred with carbachol or high glucose, and notably a pronounced translocation of the PLCδ1 to the nucleus occurred.

These data are the first showing that 1.) PLCβ2 and PLCδ1 exist in a complexed state in β‐cell lines, 2.) PLCβ‐δ decomplexation occurs upon cholinergic or glucose stimulation, and 3.) PLCδ1 translocates to the nucleus. Intriguingly, dissociation and activation of the PLC isozymes has the potential to generate both cytosolic and nuclear signaling intermediates. These studies are timely in light of the recent appreciation of the paracrine role of acetylcholine release from islet α‐cells, and the association between variants in human muscarinic receptor gene and increased risk of type 2 diabetes.
p300 Is a preferred client protein for nuclear chaperones (800.6)
Authors: Byun Jung , Gardner Kevin
Published: June 2021

The KAT3 family of histone acetyl‐transferases, p300 and CBP, are a class of highly versatile epigenetic regulators capable of forming a diverse array of chromatin regulatory complexes that readily adapt to a wide variety of gene promoter contexts to control transcription. These properties require forming multiple conformations through flexible positioning and folding of their various functional domains. Here we show that HSP90 and HSP70 molecular chaperones play a major role in assisting p300/CBP in these molecular functions at gene promoters. Disruption of this chaperone function alters the genome‐wide distribution of p300 and CBP while causing global changes in histone acetylation and gene expression. Major targeted genes include diverse regulators of mammalian protein degradation pathways, thus revealing a previously unrecognized function for molecular chaperones in the epistatic regulation of protein homeostasis through epigenetically regulated transcription of numerous components of protein degradation pathways essential for maintaining protein quality control in the cell.
A role for the transcription factor YY1 in chromosome segregation (801.1)
Authors: Rizkallah Raed , Daraiseh Susan , Tawfiq Reema , Danesh Dorreen , Alexander Karen , Hurt Myra
Published: June 2021

Chromosomal abnormalities, such as polyploidy, aneuploidy, and micronuclei formation, are distinguishing hallmarks of cancer cells. Micronuclei are small extra‐nuclear bodies containing whole, or fragments of, chromosomes originating from defects in the attachment to the microtubule spindle during mitosis. The clinical correlation between increases in the frequency of micronuclei occurrence and advancement of malignancies is widely documented. However, many of the causative factors of these defects during tumorigenesis are not fully understood yet. Due to the significance of this phenotype in risk‐prediction, prognosis, diagnosis, and treatment of cancer there is a critical need to identify all of its direct and indirect causes. The transcription factor YY1 is a multifunctional protein playing key roles in growth and development, cellular proliferation and differentiation, DNA repair, apoptosis, and a wide array of biological processes. Importantly, YY1 is deregulated in many tumor types and proposed to have oncogenic effects. However, the implication of YY1 in chromosome segregation and micronuclei formation has never been reported. Our data show that knockdown of YY1 results in significant increases in kinetochore‐positive micronuclei frequencies in HeLa cells. This is indicative of whole‐chromosome missegregation defects. Interestingly, this effect was also associated with prolongation of the duration of mitosis, indicative of mitotic defects. Our ongoing research aims at identifying the downstream mediators and upstream regulators of YY1 in the regulation of mitotic cell division and chromosome segregation.
Rab11A and Rab11B distinctly regulate protease‐activated receptor‐1 recycling and constitutive degradation (802.10)
Authors: Coronel Luisa , Grimsey Neil , Canto Isabel , Stephens Bryan , Phan Cattien , Handel Tracy , Trejo JoAnn
Published: June 2021

Protease‐activated receptor‐1 (PAR1) is a G protein‐coupled receptor (GPCR) expressed throughout the vasculature. Thrombin irreversibly activates PAR1, therefore receptor signaling and expression is tightly regulated. Upon activation, PAR1 is rapidly internalized and sorted to lysosomes for degradation, a process critical for signal termination. Whereas naïve PAR1 is constitutively internalized, generating an intracellular pool of PAR1 that recycles and repopulates the cell surface with uncleaved PAR1 after thrombin stimulation, a process critical for rapid recovery of thrombin signaling. The mechanisms that mediate recycling of naive PAR1 are not known. Using a siRNA library screen of 140 membrane trafficking proteins, we identified Rab11B as a key regulator of PAR1 trafficking. We confirmed that siRNA depletion of Rab11B, and not Rab11A reduced PAR1 surface expression, but did not affect expression of the thromboxane GPCRs, TPα and TPβ. Rab11B knockdown had no affect on PAR1 constitutive internalization, but blocked PAR1 recycling resulting in loss of surface expression and enhanced receptor degradation that was blocked by lysosomal protease inhibitors. In contrast to Rab11B, knockdown of Rab11A by siRNA disrupted PAR1 sorting from endosomes to lysosomes leading to accumulation, without affecting surface expression, suggesting that Rab11A and Rab11B distinctly regulate PAR1 trafficking. The mechanisms by which Rab11A and Rab11B distinctly regulate intracellular trafficking of PAR1 are not known and currently being investigated. Given that elevated PAR1 expression contributes to malignancy of certain cancers, understanding the mechanisms that control PAR1 surface expression could aid in the development of therapeutic approaches.

Grant Funding Source: Supported by NIH R01 GM090689, Western States Affiliate AHA Postdoctoral Fellowship and the NIGMS In
Imaging kinase: connexin43 interactions using phosphorylation activity biosensors and phospho‐specific antibodies (802.2)
Authors: Cone Angela , Cavin Gabriel , Ambrosi Cinzia , Hakozaki Hiroyuki , Newton Alexandra , Sosinsky Gina
Published: June 2021

Connexin43 (Cx43) is a ubiquitous gap junction protein with widespread tissue expression, serving important functions in cell homeostasis and signaling. The C‐terminus of Cx43 contains multiple phosphorylation sites and protein binding domains that are involved in regulation of connexin trafficking and channel gating. Several kinases phosphorylate Cx43 (Protein Kinase A, Protein Kinase C, Akt (also known as Protein Kinase B), Src, CK1, MAPK) at specific serines or tyrosines. We used kinase biosensors,for dynamic readouts of function, and combined it with image analysis to obtain spatial information. We fused a genetically encoded fluorescence resonance energy transfer‐based reporter for PKC delta activity, delta C kinase activity reporter (dCKAR) to the C‐terminus of Cx43 (Cx43‐dCKAR) and expressed it in Cos7 cells. Similarly, we genetically appended an activity reporter for Protein Kinase A (AKAR‐CR) to Cx43 and expressed it in Cos7 cells. Time lapse imaging of stimulated PKC resulted in phosphorylation of Ser368 Cx43‐dCKAR channels and discrete domains in the center of the gap junction, followed by internalization and degradation. Phosphorylated Cx43‐AKAR‐CR was predominantly found in the Golgi apparatus and to a lesser extent around the plaque edges. Thus, the spatio‐temporal localizations of PKC and PKA phosphorylated Cx43 represent degradative and synthetic pathways, respectively.

Grant Funding Source: Supported by NIH grants GM072881, GM043154, GM103412
Scaffolding of atypical protein kinase C to p62 is disrupted by the pseudosubstrate peptide ZIP (802.4)
Authors: Tsai Li‐Chun , Xie Lei , Xie Li , Bourne Philip , Newton Alexandra
Published: June 2021

Atypical protein kinase C (aPKC) enzymes, aPKCλ and aPKCζ, play key roles in metabolic signaling. A myristoylated peptide based on the autoinhibitory pseudosubstrate fragment of PKCζ, zeta inhibitory peptide (ZIP), has been extensively used to inhibit aPKC activity, however we have recently shown that ZIP does not inhibit the catalytic activity of aPKC isozymes in cells (Wu‐Zhang et al., (2012) JBC 287:12879‐85). Here, we identify a novel interaction between the pseudosubstrate segment of PKCλ and the protein scaffold p62, a known binding partner, that is disrupted by ZIP in cells. First, we used protein‐protein interaction network analysis, structural modeling, and protein‐protein docking to predict that ZIP, a basic peptide, binds an acidic surface on the PB1 domain of p62. This predicted interaction is additional to the known interaction between the PB1 domain of aPKC, which immediately precedes the pseudosubstrate segment, and p62. Second, we used fluorescence resonance energy transfer (FRET) experiments to show that ZIP disrupts the interaction of CFP‐tagged p62 and YFP‐tagged PKCλ in cells via a mechanism that depends on an intact pseudosubstrate segment. In contrast, auranofin, a known disrupter of the PB1:PB1 domain interaction of aPKC and p62, disrupts the interaction of wild‐type PKCλ, a construct lacking the pseudosubstrate, but not a construct in which the PB1 domain is deleted. Our data are consistent with ZIP altering cellular function by displacing binding partners from the acidic face of the PB1 domain of p62, a region which we identify as an interaction surface for the pseudosubstrate segment of aPKCs.

Grant Funding Source: This work was supported by 5T32DK007494 and P01 DK054441.
Dietary diversity and food insecurity in Mexican children 2 to 4 years old (805.5)
Authors: Mundo Rosas Verónica , De la Cruz Vanessa , Jimenez Alejandra , Shamah Teresa
Published: June 2021

Objective: Assess dietary diversity (DD) and adequacies of energy and nutrients intake of Mexican children two to four years old according to the level of food insecurity (FI) in your home. Methods: We included information of 955 children and their homes from the ENSANUT‐2012. Dietary data was obtained with a food frequency questionnaire of food. An index of DD and adequacies of energy and nutrients were built. Multinomial models were used to calculate adjusted prevalences of DD according to the classification of IA at home using the Latin American and Caribbean Scale Food Safety. Results: The highest proportion of children with low DD (53.6%) live in severe insecurity (IS). Children with moderate and severe insecurity not cover the recommended intake of energy. Conclusion: DD, energy and nutrients consumption are related to the IA at home. It is important to improve access to foods that provide micronutrients.
Trend in caffeine intake and food and beverage sources of caffeine in U.S. children over the last decade: analysis of What We Eat In America/National Health and Nutrition Examination Survey (NHANES) 2001‐2010 (806.17)
Authors: Fulgoni Victor , Schorin Marilyn
Published: June 2021

WWEIA data and the food groupings within were used to assess caffeine intake and food and beverages sources of caffeine in the diets of children and adolescents 6‐18 years of age (N=13,131) over the last decade. Regression analyses were conducted to determine a p‐for‐trend across the NHANES data releases (2001‐2002, 2003‐2004, 2005‐2006, 2007‐2008, and 2009‐2010). For the total population, total caffeine intake remained stable across the five NHANES releases (beta coefficient: ‐0.5 mg/release; p= 0.6191). Major contributors of caffeine (mean ± SE, mg/d) in the diet across the decade were regular soda (42±2); tea (10±1), coffee (6±1), diet soda (2±0.3), and energy drinks (1±0.3). There was a significant p‐for‐trend of ‐3 mg/release for regular sodas (p<0.0001) and 1.1 mg/release for coffee (p<0.01). Energy drinks increased 0.3 mg/release (p<0.05), but their contribution was only 1 mg/d. Among caffeine consumers (N=9,400), total caffeine intake remained stable across the five NHANES releases (beta coefficient: ‐0.2 mg/release; p= 0.8581). Also among caffeine consumers, caffeine contributed by each beverage category evaluated remained stable across the decade. For the total population, total caffeine intake has remained stable among children over the decade with a decrease in contribution from soft drinks and a slight increase from coffee.

Grant Funding Source: American Beverage Association
Nutritional implications of marijuana use in an HIV infected population in the Miami Adult Studies on HIV cohort in Miami (810.7)
Authors: Kienzle Cinda , Campa Adriana , Kurtz Joy , Li Yinghui , Greer Pedro , Martinez Sabrina , Stewart Tiffanie , Boyer Jacqueline , Baum Marianna
Published: June 2021

OBJECTIVE: To determine the relationship of nutritional status and MJ use in HIV+ population .

METHODS: Dietary intake, food insecurity, plasma levels of nutrients, anthropometrics, CD4 count, viral load and ART use were determined with consent in 201 HIV+ participants. Multivariate and logistic regressions were used.

RESULTS: Mean age was 45.2 years, mostly males (64%) and Blacks (72.5%); 27% used MJ. MJ users were younger (mean age 42 vs. 46, P<0.001) and used alcohol and tobacco more frequently than non‐users (94% vs. 87%, P<0.001). MJ use was associated with better ART adherence (β=0.99, P=0.043), higher CD4 cell count (β=112.87, P=0.03) and lower homocysteine (β=‐1.05, P=0.051). MJ users trended toward increased BMI (β=1.47, P=0.08) and %fat mass (β=3.02, P=0.08). In contrast, MJ users were more food insecure (52% vs. 33%, P=0.015) and had lower % body cell mass (β=‐3.3, P=0.018) and serum iron (β=‐13.16, P=0.018). Caloric intake did not differ between MJ users and non‐users.

CONCLUSION: MJ use is associated with some positive effects on HIV disease progression, but the effect on body composition seems to be from fat accumulation. Studies are needed to determine the effects of MJ on health in HIV infection.

Grant Funding Source: NIDA and NIAAA
Body image and weight control in youth: 9‐year international trends from 24 countries (811.5)
Authors: Quick Virginia , Nansel Tonja , Liu Danping , Lipsky Leah , Due Pernille , Iannotti Ronald
Published: June 2021

This study’s purpose was to examine 9‐year trends in misperceptions of body size and dieting for weight loss among adolescents from 24 countries, and explore the influence of country‐level overweight prevalence. Body size perception and dieting for weight loss were assessed in the Health Behaviour in School‐aged Children survey conducted in 24 countries cross‐sectionally at 3 time points (2001/02, 2005/06, 2009/10). Logistic regression models examined change over time in over‐ and under‐estimation of body size and dieting for weight loss. Compared to Time 1, overweight adolescents had greater odds of body size underestimation at Time 3 (OR=1.68 for girls, OR=1.10 for boys), while non‐overweight adolescents had lower odds of body size overestimation at Time 3 (OR=0.87 for girls, OR=0.89 for boys). Controlling for country‐level overweight prevalence attenuated these relationships. Compared to Time 1, overweight and non‐overweight boys were 10% more likely to diet at Time 3, while overweight and non‐overweight girls were 19% and 16%, respectively, less likely to diet at Time 3. Controlling for country‐level overweight prevalence did not impact trends in dieting for weight loss. Findings suggest body size perceptions among adolescents may have changed over time concurrent with shifts in country‐level body weight.

Grant Funding Source: Supported by Maternal and Child Health Bureau of the Health Resources and Services Administration
Fruit juice, fruit drinks and atole are associated with obesity and lipid profile in school‐aged children from rural México (811.8)
Authors: Garcia Olga , Zavala Gerardo , Caamaño María , Ronquillo Dolores , Martínez Guadalupe , Camacho Mariela , Rosado Jorge
Published: June 2021

Beverages such as fruit drinks (natural and artificially flavored), soft drinks and 100% fruit juices are regularly consumed by children as part of their diet. 198 children (8 ± 1.5 years) participated in a cross‐sectional study to evaluate the association of beverage intake with obesity, lipid profile and glucose concentration. Body fat (DXA), height, weight and waist circumference were measured in all children. Diet was evaluated with a food frequency questionnaire and three 24hr recalls. A fasting blood sample was taken to analyze lipid profile and glucose. 17% of the daily energy intake of these children came from beverages. Linear regressions adjusted for socio‐demographic variables showed some significant associations (p<0.05). High intake of 100% fruit juices was associated with high body and abdominal fat, waist‐to‐height ratio, and high triglyceride concentration. Fruit drinks were positively associated with higher BMI z‐score and triglycerides. Intake of atole (maize based beverage with added sugar) was associated with high triglycerides levels. Milk intake was associated with lower LDL concentration. Soda intake was not associated with any of the variables studied. In conclusion, high intake of 100% fruit juices, fruit drinks and atole in school‐aged children from rural México, are associated with obesity and alterations in lipids profile.

Grant Funding Source: Supported by CONACYT
Evaluation of protein‐phytochemical interactions in a hypoallergenic edible peanut matrix (813.10)
Authors: Plundrich Nathalie , White Brittney , Davis Jack , Lila Mary Ann
Published: June 2021

Allergenic peanut proteins (PP) are stable to digestion, remain immunoreactive and trigger IgE‐mediated allergic reactions in PP sensitive individuals. In this study, a hypoallergenic peanut protein‐phytochemical matrix (PPPM), produced by complexing cranberry juice (CB) with light roast peanut flour, was evaluated to determine the nature of protein‐phytochemical interactions and the stability of the binding post‐ingestion. PPPM or unmodified peanut flour were subjected to a simulated gastric fluid digestion. Aliquots of the enzymatic mixture were taken at different time points and inhibited with either sodium bicarbonate (pH ~7‐8) or heat (pH ~2) to stop the digestive process. Digested PP fragments and undigested PP were examined on SDS‐PAGE and screened for their peanut‐specific IgE binding capacity using Western blotting, respectively. SDS‐PAGE revealed that PP were more soluble at a low pH than at a basic pH. PP treated with CB had reduced solubility at both acidic and basic pH, compared to untreated PP. However digestion of both PPPM and unmodified peanut flour resulted in formation of highly soluble small molecular weight PP fragments at either pH. Examination of the Western blots revealed that digested PP fragments or undigested PP from PPPM exhibited altered IgE binding, compared to proteins from unmodified peanut flour. Modification of PP with CB phytochemicals led to the formation of insoluble complexes with altered IgE binding to the protein epitopes, suggesting altered PP digestion and IgE binding capacity.

Grant Funding Source: Supported by Everett W. Byrd Endowment and NC State appropriations for NCSU at Kannapolis
Role of pH and emulsification agents in the solubility and sensory properties of branched chain amino acids (813.3)
Authors: Quintanilla Andrew , Silva Luciana , Kristen Espinosa , Dunsmore Kristin , Joy Jordan , Wells Shawn , Wildman Robert
Published: June 2021

The Branched Chain Amino Acids (BCAAs), leucine, isoleucine, and valine, are essential amino acids with unique roles in muscle protein synthesis making them popular among athletes and physically active individuals. However, BCAAs are also among the most hydrophobic amino acids and are not palatable in an isolated form. This creates challenges for commercialization in ready‐to‐drink and beverage powders. Instantization with lecithin can improve solubility as can adjusting the pH level of the BCAAs recipe with acids. In addition, total acidity and specific acid types also provide desirable taste characteristics, especially for fruit‐flavored products. Thus, the purpose of this study was to characterize the solubility of instantized BCAAs based on pH as well as to identify the optimal pH range at which solubility and taste are both optimized for a proprietary BCAA ingredient (CHAIN‐SOL, Dymatize Nutrition). Based on orange flavored, sweetened and acidified (citric acid) recipe, CHAIN‐SOL underwent a series of pH challenges (4.5 to 2.5) to determine optimal dissolvability as well as sensory scoring by a trained panel. It was determined that a pH range of 3.2‐3.6 was the optimal pH overlap range for desirable dissolving of amino acids and consumer palatability. It is concluded that both instantization and pH targeting are critical considerations in the formulation of BCAA and likely other amino acid based beverages and powders.
Effect of weight loss on lipid metabolism in the mature horse (814.4)
Authors: Bush Morgan , Zambito Jennie , Spooner Holly , Barnes Kimberly
Published: June 2021

Obesity currently affects over half of the nation’s horses, and causes concern as it poses increased disease risks and impairs various metabolic functions. Weight loss is considered a viable option to correct these maladies. Our objective was to determine if weight loss affected markers of lipid metabolism. Eight, mixed sex horses (5‐19 yr) were kept at an obese body condition score (BCS, 7‐8) for two weeks, then entered a six week period of 30% caloric restriction and light exercise to reach a moderate BCS (5). Blood samples collected every other week were analyzed for non‐esterified fatty acid (NEFA) and triglyceride concentration. Neck fat biopsies were also collected for ex vivo lipolysis analysis and media were analyzed for NEFA and free glycerol release. Plasma triglyceride and serum NEFA did not differ due to change in BCS or percent weight loss. Lipolysis, as indicated by release of NEFA, was decreased by change in BCS (p=0.01) and tended to be decreased by percent weight loss (p=0.07). Glycerol release was correlated (r=0.47, p=0.001) with NEFA release, but not significantly altered by change in BCS or percent weight loss. As an indicator of fatty acid oxidation, acyl‐CoA dehydrogenase mRNA expression was measured in skeletal muscle, but was not significantly affected. Therefore, as these horses transitioned to a leaner phenotype they exhibited only minor changes in lipid metabolism, indicating that while overweight they were not unhealthy.
Altered expressions of multiple gene pahways with aging in a mammal model and reversal of aging‐related changes by Cordycep sinensis Cs‐4 (818.2)
Authors: Tan Ning‐Zhi , Qiao Nan , Barger Jamie L. , Ferguson Scott B , Prolla Tomas A. , Bartlett Mark , Weindruch Richard , Han Jing‐Dong , Zhu Jia‐Shi
Published: June 2021

Aging is a complex process, involving multiple biological systems and function pathways. An anti‐aging fermented product Cordyceps sinensis Cs‐4 extends lifespan in natural aging mice. It improves antioxidant capacity, glucose‐lipid‐energy metabolisms, immune functions, physical endurance, etc. This study was to profile the multifactorial aging‐induced changes in gene pathway expressions (GE), and to explore molecular mechanisms of Cs‐4’s anti‐aging effect in aged mice. GE were examined on gastrocnemius from young (5 mo), old (25 mo) and old Cs‐4 treated (0.3 g/kg) mice (n=5 each). Differentially expressed GO and KEGG pathways were identified with using GSEA with a setting of FDR q<0.01. Compared to young controls, 288 gene clusters were up/down regulated in the old muscle: e.g., telomere related, stem cell signaling related, apoptosis, cell structural integrity, DNA repair, transcription‐translation, inflammatory‐immune responses, carcinogenesis, mitochondria related, metabolisms, cell communications, etc. Cs‐4 opposed these aging related changes in GE. A remarkable negative correlation was noted between aging related changes in GE and Cs‐4’s effects (PCC = ‐0.80). In conclusion, aging is a multifactorial, complex process, although there are many single gene hypotheses for aging. Cs‐4 reverses the aging related changes in GE, as the mechanisms of its lifespan extending effects.
Branched‐chain amino acid supplementation does not exacerbate disease progression or decrease survival time in a murine model of metastatic cancer (824.2)
Authors: Ward Nathan , Poff Angela , Sherwood Jacob , Seyfried Thomas , D'Agostino Dominic
Published: June 2021

Cancer cachexia is the loss of muscle mass associated with advanced‐stage cancer. Dietary supplementation of branched‐chain amino acids (BCAAs) is a proposed nutritional strategy to preserve muscle mass in cachexic patients. BCAAs, especially leucine, activate the mammalian target of rapamycin complex 1 (mTORC1), triggering protein biosynthesis and promoting cell motility. Thus it is proposed that increased levels of BCAAs, specifically leucine, will enhance tumor invasiveness through activation of mTORC1. To determine if BCAA supplementation exacerbates metastatic disease, we tested the effect of supplementing a standard rodent diet (SD) and ketogenic diet (KD) with a leucine‐rich dietary supplement of BCAAs (10% by weight) on cancer progression and survival in a murine model of metastatic cancer. The KD has been shown to significantly slow tumor progression and increase survival time in this metastatic model. Supplementation of the SD did not significantly enhance tumor growth or impact survival time. In contrast, we observed a trend of increased mean survival time with BCAA supplementation (19% increase; NS). Supplementation of the KD significantly slowed tumor growth and increased mean survival time by 46% (p<.05). Our results do not support the hypothesis that BCAA supplementation will exacerbate metastatic disease and support further study of its efficacy in mitigating cancer cachexia.
A 14‐day in‐house dietary modification of a ‘Western’ diet to an ‘African’ diet changes the microbiota, its metabolome, and biomarkers of colon cancer risk (825.5)
Authors: Vipperla Kishore , Ou Junhai , Wahl Elaine , Ruder Elizabeth , O'Keefe Stephen
Published: June 2021

Methods Twenty age and sex matched middle aged healthy African Americans were studied on their usual diet and then again after dietary modification to a low meat and fat, high fiber ‘traditional African’ diet, prepared and delivered individually in‐house under close supervision for 2 weeks. Phutu‐ corn meal, Samp‐ dried corn kernels, sugar beans, fruits and vegetables provided 30‐35 Kcal/Kg of energy, 14% protein, 17% fat, 72% carbohydrate, 40 g dietary fiber, and 32 grams resistant starch. The diet was well tolerated overall and their body weights remained stable. Pre‐ and post‐dietary modification fecal and colonic evacuate samples were collected to measure the microbiota and their metabolites and colonoscopies were performed to assess mucosal health and to obtain biopsies for the measurement of inflammatory and proliferative biomarkers (Ki67‐ identified by immunohistochemical staining).

Results Following dietary change, significant increases were measured in the expression of functional microbial genes encoding butyrate (BcoA gene, 1.1E+10±1.8E+10 vs. 2.5E+10±9.3E+09 copies/g feces, p= 0.04) and methane synthesis (mcrA gene, 6.5E+5±1.2E+5 vs. 1.6E+6±3.9E+5 copies/g feces, p= 0.01); fecal short chain fatty acids, e.g. butyrate concentrations (3.56±0.96 vs. 8.61± 2.10 µmol/gm feces, p=0.034); expired breath methane levels (12.8±3 vs. 20.3±8.3 ppm, p=0.02); Finally, mucosal Ki67 expression was significantly reduced (p<0.0001).

Conclusion In this study, short term dietary change in high colon cancer risk African Americans to a low meat and fat, and high fiber “African” diet promoted saccharolytic fermentation and the production of anti‐neoplastic metabolites such as butyrate in association with a reduction in biomarkers of colon cancer risk. These results support the recommendations for reducing meat and fat and increasing fiber to suppress colon cancer in high‐risk populations.

Grant Funding Source: Supported by National Institutes of Health, R01 CA135379 (O’Keefe) and UL1 RR024153 and UL1TR000005
Cellular pyridoxal phosphate restriction in cultured HepG2 cells decreases formation and flux of metabolites in the transsulfuration pathway including production of hydrogen sulfide biomarkers (827.6)
Authors: DeRatt Barbara , Ralat Maria , Kabil Omer , Banerjee Ruma , Gregory Jesse
Published: June 2021

Cystathionine beta‐synthase (CBS) and cystathionine gamma‐lyase (CSE) are pyridoxal phosphate‐dependent transsulfuration enzymes responsible for the majority of endogenously produced hydrogen sulfide (H2S) and concurrently formed biomarkers, lanthionine (lan) and homolanthionine (hlan). H2S modulates physiological functions in the cardiovascular and central nervous systems. We investigated the effects of restricted vitamin B6 supply on cultured HepG2 cell metabolism by analyzing transsulfuration pathway kinetics. Cell culture media contained pyridoxal (PL) concentrations at severely deficient (10 nM), marginal (50 nM), adequate (200 nM) and supraphysiological (2000 nM) levels. Metabolic flux studies using [U‐13C5] L‐methionine and (3,3 D2) L‐cysteine indicated lower fractional synthesis rates (FSR) and steady state concentrations of lan and hlan at 10 and 50 nM PL (P<0.027). The FSR of cystathionine was higher in cells cultured in 50 nM PL than other PL concentrations (P<0.001), consistent with the greater sensitivity of CSE than CBS to B6 deficiency. In‐vitro H2S production capacity in assays with cell lysates and these results confirmed that H2S production decreased in B6 deficiency. These findings support a hypothesis that impaired H2S production may be involved in the association between vitamin B6 inadequacy and cardiovascular disease.

Grant Funding Source: Supported by NIH Grant DK072398
Effect of vitamin D on ROS, ICAM‐1, and monocyte adhesion in human umbilical vein endothelial cells treated with high glucose and acetoacetate (828.10)
Authors: Kanikarla Marie Preeti , Jain Sushil
Published: June 2021

Monocyte adhesion to the endothelium plays a central role in the progression of endothelial dysfunction and cardiovascular disease (CVD). This study examines whether VD supplementation is beneficial in preventing endothelial dysfunction in diabetes. HUVEC were treated with 1, 25‐(OH)2‐D3 (0‐25nM, 24h) and later exposed to the ketone body acetoacetate (AA, 4mM) or high glucose (HG, 25mM) for 24h. There was an increase in ROS, ICAM‐1 expression, and monocyte adhesion in HUVEC treated with AA or HG. VD (25nM) supplementation significantly lowered ROS (41%, p<0.05), ICAM‐1 (73%, p=0.05) and p‐NFκB expression (50%, p<0.05), and IL‐8 (16%, p=0.05) and MCP‐1 (41.2%, p<0.001) secretion along with monocyte adhesion (37.5%, p=0.05) in AA treated HUVEC. VD had a similar beneficial effect on HG treated HUVEC. Interestingly, we also saw an upregulation in GSH and GCLC with VD. We hypothesize that the beneficial effect of VD is mediated by the upregulation of GCLC and GSH, a potential antioxidant. To explore this idea, we knocked down GCLC using siRNA. Results show that beneficial effect of VD was abolished on ketone‐ or HG‐induced ICAM‐1 in GCLC knockdown cells. This suggests that VD mediates its beneficial effects via the upregulation of GCLC. This study provides the biochemical mechanism through which VD can lower ROS, ICAM‐1 expression, monocyte‐endothelial adhesion, and thereby lower the risk of CVD in diabetes.

Grant Funding Source: Supported by NIH and Malcolm Feist Chair in Diabetes
Anti‐hyperglycemic effect of selected sugar alcohols (829.32)
Authors: Kang Yu‐Ri , Jo Sung‐Hoon , Yoo Jong‐Il , Cho Jae‐Bong , Kim Eui‐Joong , Apostolidis Emmanouil , Kwon Young‐In
Published: June 2021

In this study, we estimated the anti‐hyperglycemic effect of the most widely used seven sugar alcohols including xylitol, erythritol, maltitol, isomalt, mannitol, sorbitol, and inositol. Sugar alcohols, also known as polyols, are considered as sugar substitutes because they are contain low calories and some sugar alcohols do not raise blood glucose. However, there are few studies on the anti‐hyperglycemic activity of sugar alcohols. Therefore, we investigated inhibitory activity of sugar alcohols against rat intestinal α‐glucosidase, porcine pancreatic α‐amylase, and sucrase using in‐vitro. We also examined phenolic‐linked antioxidant activity, oxygen radical absorbance capacity (ORAC) to evaluate antioxidant activity of sugar alcohols. Among seven sugar alcohols xylitol had the highest α‐glucosidase inhibitory activity and maltitol had the highest sucrase inhibitory activity. These result indicated that some sugar alcohols exhibit considerable anti‐hyperglycemic activity with inhibitory activity about carbohydrate absorption‐linked enzymes such as α‐glucosidase and sucrase.
Black carrots fermented with Aspergillus oryzae improves cognitive function and insulin resistance in β‐amyloid‐infused diabetic rats (829.4)
Authors: Park Sunmin , Kim Da Sol , Kang Suna , Shin Bae Keun
Published: June 2021

We investigated whether the extracts of black carrot (BC) and BC fermented with Lactobacillus plantarum (BCLP) and Aspergillus oryzae (BCAO) can protect against cognitive dysfunction and glucose dysregulation in rats with Alzheimer’s disease and type 2 diabetes. Partial pancreatectomy and ICV β‐amyloid (25‐35) infusion into the CA1 region were fed either control diet (CON), 3% BC, BCAO, or BCLP in a high fat diet for 8 weeks. BCAO increased the contents of total anthocyanins compared to the BC. BC mainly contained cyanidin glycosides whereas after fermentation, the contents of peonidin derivatives increased in the ascending order of BCLP<BCAO. BCAO markedly decreased β‐amyloid deposition and improved hippocampal insulin signaling (pAkt◊pGSK◊pTau) in AD rats. Cognitive function was markedly impaired in AD rats and the cognitive dysfunction was prevented in an ascending order of CON<BC and BCLP<BCAO. BCAO blocked the decrease of whole body glucose infusion rates and the increase of hepatic glucose output at hyperinsulinemic state in AD rats. Interestingly, second phase insulin secretion during hyperglycemic clamp was rampantly increased in AD rats with remarkable decreased β‐cell mass. The BCAO treatment corrected the impaired insulin secretion and decreased β‐cell mass in AD rats. In conclusions, BCAO prevented their impairment of cognitive and glucose homoeostasis in AD diabetic rats as much as Non‐AD rats.
Anti‐inflammatory effects of catechol and ferulic acid derivatives through NF‐κB activation in Raw264.7 cells (830.21)
Authors: Kim Jin‐Kyoung , Kwon Young‐In , Jang Hae‐Dong
Published: June 2021

Catechol and ferulic acid derivatives are naturally occurring substances found in fruits, vegetables and flowers and are consumed as dietary phenolic compounds. Several previous studies reported that catechol and ferulic acid derivatives have potentials for various biological activities. The present study was designed to investigate the anti‐inflammatory effects of catechol and ferulic acid derivatives in Raw 264.7 cells. The anti‐inflammatory activities of catechol and ferulic acid derivatives investigated by analyzing NO and PGE2 production, inducible nitric oxide synthase(iNOS), and cyclooxygenase‐2 (COX‐2)expression as a biomarker in Raw 264.7 macrophage cell demonstrated that caffeic acid phenethyl ester, carnosic acid, ferulic acid ethyl ester, 6‐shogaol significantly attenuated the production of NO and PGE2 and the expression of iNOS and COX‐2, indicating that the reduction in NO and PGE2 formation may be attributed to suppression of iNOS and COX‐2 expression resulting from the control of NF‐κB activation with IKK modification. These results propose that these compounds could be used as a potential natural source for the treatment of oxidative stress and related diseases.
Antioxidant properties of Rubus fruit anthocyanins in hydrogen peroxide‐treated RAW264.7 cells (830.28)
Authors: Jung Hana , Kwak Ho‐Kyung , Hwang Keum Taek
Published: June 2021

Antioxidant properties of ethanol extracts (EE) of Rubus fruits (Blackberry; BB, Korean raspberry; KR, and black raspberry; BR) and their anthocyanin fractions (AF) were examined employing H2O2‐stimulated RAW264.7 macrophage cells. H2O2‐induced reductions of cell viabilities were significantly prevented by all the tested AF (20 µg/mL) and BBE (200 µg/mL). When cells were treated with EE (100 µg/mL) and AF (10 µg/mL) from KR and BR for 4 h, activities of superoxide dismutase and glutathione peroxidase significantly increased. Protein expressions of nuclear factor‐erythroid 2p45‐related factor 2 were increased in the cells pre‐treated with all the tested AF (20 µg/mL). Significant dose‐dependent reductions of intracellular reactive oxygen species (ROS) were observed in all the AF (5‐20 µg/mL) treated cells. In addition, all the AF treated cells showed a significant increase in cellular ferric reducing antioxidant power. Moreover, H2O2‐induced DNA fragmentation and oxidation were markedly reduced as measured by Hoechst staining and comet assay. Rubus fruit anthocyanins may have cellular protective effect by up‐regulating antioxidant enzymes and preventing DNA oxidation.
Grape pomace bioactivity from Brazilian wine industry: toxicity and antioxidant properties in human hepatocarcinoma cells (830.31)
Authors: El Bacha Tatiana , Sales Nathalia , Minuzzo Daniela , Cruz Ana Paula , Cabral Lourdes , Torres Alexandre
Published: June 2021

Wine industry produces large quantities of by‐products rich in bioactive compounds from grapes, which could be utilized for nutraceutical purposes. We aim to evaluate antioxidant activity (AC) and bioactivity of grape pomace extract from Brazilian wine industry in human hepatocarcinoma cells (HepG2). The content of bioactive compounds and AC of extracts were determined by ORAC, TEAC (ABTS•‐) and TRAP assays. Bioactivity was assessed on HepG2 and on normal human fibroblasts (BEAS). Cellular viability was accessed by MTT reduction and cellular antioxidant capacity (CAC) by dichlorofluorescein oxidation, under short‐, medium‐, and long‐term incubation periods. Total phenolics, flavonoids and anthocyanins of extracts where, respectively, (mean±SD) 473.8±52.4 mg GAE/100g, 147.5±2.77 mg CE/100g and 105.5±7.27 mg cyanidin3‐glycoside/100g. Extract AC was (mean±SD; µmol TE/g) 27.1±0.33, 9.33±6.74, 122.2±2.40. HepG2 viability reduced in a time‐ and dose‐dependent manner. Short‐term incubation had no effect whereas medium‐ and long‐term incubation induced, respectively, a 37% and a 75% reduction in viability. BEAS viability was not affected by the extract, regardless of time and concentration used. Interestingly, short‐term incubation promoted a dose‐response increase in HepG2 CAC, suggesting that decreased oxidative stress signals to decrease HepG2 proliferation. Taken together, wine industry by‐products present important antioxidant activity and potential selective anticancer and antiproliferative effects on HepG2 cells.

Grant Funding Source: Supported by FAPERJ, CNPq, CAPES, EMBRAPA (Brazil)
AGEs‐induced pro‐atherosclerosis pathway: endothelium are regulated by the AGEs‐RAGE axis (832.12)
Authors: Nam Mi‐Hyun , Hong Chung‐Oui , Koo Yun‐Chang , Lee Kwang‐Won
Published: June 2021

During hyperglycemia, reducing sugars react with the amino groups of proteins non‐enzymatically to form Schiff base and Amadori products, which undergo formation of advanced glycation end‐products (AGEs). Binding AGEs and receptor for AGEs (RAGE) generated oxidative stress and several vascular cells triggered secretion of inflammatory cytokine and abnormal signal transduction but the underlying mechanism remains unclear. We evaluate the hypothesis that the AGEs‐RAGE axis might mediate combined signals contributed to the local regulations in co‐cultures of human monocytes (THP‐1), endothelial cells (HUVEC) and aortic vascular smooth muscle cell (SMC). The results revealed that production of reactive oxygen species (ROS) and adhesion of monocytes to endothelium induced AGEs were decreased by RAGE knockdown HUVEC. We further discovered that TNF‐α and IL‐1β as pro‐inflammatory cytokines were markedly suppressed in AGEs‐stimulated HUVECs and THP‐1 simultaneously knockdown of RAGE. Besides, SMC were down‐regulated inflammatory cytokines and decreased proliferation in co‐culture system. In conclusion, AGEs stimulation of vascular cells causes binding of RAGE, enhances the production of ROS, adhesion of monocytes, and expression of cytokines eventually contributes to the progression of atherosclerosis.