bluuebunny/crossref_metadata_2025 huggingface dataset:
Specificity of glycosaminoglycan‐protein interactions: the role of desolvation (1007.5)
Authors: Sarkar Aurijit , Desai Umesh
Published: June 2021

Why do most glycosaminoglycan (GAG)‐protein interactions lack specificity? We hypothesized that desolvation of highly charged groups, which is highly unfavorable, plays an as‐yet‐unrecognized dominant role. We performed molecular dynamics (MD) simulations of water binding to antithrombin (AT) and thrombin (T), two prototypic ‘specific’ and ‘non‐specific’ GAG‐binding proteins respectively, to parse the role of desolvation energy (DE). The DE for Arg/Lys sidechains was a substantial ~4.5 kcal/mol, which counteracts enthalpic gains during hydrogen‐bond formation with GAG sulfates. This DE was irrespective of the location ‐ within the heparin‐binding site (HBS) or elsewhere ‐ on either protein, suggesting that hydrogen‐bonding by Arg/Lys residues with GAG sulfates does not explain the interaction specificity. However, DE for selected uncharged sidechain‐ and/or backbone‐ donors may play a dominant role. Specifically, Asn45 of AT contributes singularly to GAG recognition with a much‐reduced DE, while no such equivalent uncharged residue exists for T. These results find support in mutagenesis and crystallography studies reported in literature. The key finding that GAG specificity for proteins arises from hydrogen‐bonding with a select group of uncharged donors due to lower DE penalty is being extended to the serpin family of proteins.

Grant Funding Source: Supported by NIH grants R01 HL090586 and P01 HL107152 to URD, and NCCR grant S10 RR027411 to VCU.
Hyaluronan rafts in airway epithelial cells (1007.6)
Authors: Abbadi Amina , Lauer Mark , Swaidani Shadi , Hascall Vincent
Published: June 2021

Hyaluronan (HA) is a linear, non‐sulfated glycosaminoglycan that is normally formed as a unmodified structure but uniquely modified during inflammation. This modification involves the transfer of heavy chains (HC) from IαI to form HC‐HA complexes mediated by TSG‐6. These complexes are identified in bronchial secretions from asthmatics that also show an up‐regulation in their pulmonary TSG‐6 expression. HC‐HA matrices are leukocyte adhesive as demonstrated in many other pathological conditions including idiopathic arterial pulmonary hypertension, asthma and cystic fibrosis. In all cell types studied to date, the leukocyte‐adhesive HA cables synthesized by these cells remain attached to the cell surface. However, while airway epithelial cells (AECs) also form HA cables on their cell surface in response to a variety of inflammatory stimuli, we have identified that they can also actively release a majority of their leukocyte‐adhesive HA structures into the extracellular space from their apical surface even when cells are not treated with inflammatory stimuli. These matrices do not resemble the rope‐like HA cables, but occur in distinct clumps or rafts of HA that can capture and embed leukocytes from cell suspensions. Not only were these leukocyte adhesive structures observed in vitro but also in the bronchoalveolar lavage of naïve mice. Furthermore, we have been able to identify the synthesis of heavy chain 3, previously reported to be constitutively expressed by human proximal tubular epithelial cells as urinary trypsin inhibitor (UTI), which may directly contribute to heavy chain modification of the HA matrix in lung inflammation. This provides evidence that AECs can manufacture their own HC‐donor. By characterizing the structure of these cell‐free HA rafts, the mechanism by which they are synthesized and their effect on leukocyte activation and behavior, we will have a better understanding of their role in human health and disease.
Endo F3 glycosynthase mutant enables chemoenzymatic synthesis of core fucosylated triantennary complex type glycopeptides and glycoproteins (1007.7)
Authors: Giddens John , Lomino Joseph , Wang Lai‐Xi
Published: June 2021

Chemoenzymatic synthesis of glycoproteins, which enables the production of glycoproteins with homogenous glycans, is an essential tool for structural and functional studies of glycoproteins. The endo‐β‐N‐acetylglucosaminidase from Elizabethkingia meningosepticum, Endo F3, is an endoglycosidase that can hydrolyze complex type N‐glycans and also possess transglycosylation activity. We have generated glycosynthase mutants by site‐directed mutagenesis and have found that the glycosynthases were capable of efficiently transferring complex type N‐glycans to GlcNAc‐peptide/protein without product hydrolysis. Our experimental data have further shown that the Endo‐F3 glycosynthases are able to use tri‐antennary glycan oxazolines as substrates for transglycosylation, which were unable to achieve by previously reported endoglycosidase mutants derived from Endo‐A, Endo‐M, Endo‐D and Endo‐S. The usefulness of the Endo‐F3 mutants was demonstrated by the synthesis of CD52 antigen and by the efficient Fc glycosylation remodeling of monoclonal antibody rituximab. The binding of these novel glycoforms of rituximab to FcγRIIIa and galectin‐3 is being analyzed. This chemoenzymatic approach permits an efficient synthesis of core fucosylated bi‐ and tri‐antennary glycopeptides and the glycosylation remodeling of intact monoclonal antibodies, making it possible to probe the structural and functional roles of the attached glycans in more details.

Grant Funding Source: Supported by NIH R01 GM080374
Endoplasmic reticulum stress induces alveolar epithelial cell apoptosis through the activation of the unfolded protein response (1010.10)
Authors: Nguyen Hang , Dang MyTrang , Ono Shinji , Morimoto Konosuke , Uhal Bruce
Published: June 2021

STUDY OBJECTIVE: Our prior work showed that ER stress‐induced apoptosis of AECs is regulated by the autocrine angiotensin (ANG)II/ANG1‐7 system. MG132 or surfactant protein C (SP‐C) BRICHOS domain mutation G100S induced apoptosis in human AECs by activating cathepsin D or decreasing ACE‐2. This study tested the hypothesis that ER stress‐induced apoptosis of human AECs might be mediated by the activation of the UPR thus regulates the ANGII/ANG1‐7 system.

METHODS: A549 cells were challenged with proteasome inhibitor MG132 or SP‐C BRICHOS domain mutant G100S. The level of UPR markers and angiotensin components were measured by Western blotting. AEC apoptosis was measured by caspase activation and nuclear fragmentation.

RESULTS: MG132‐ or G100S mutation‐induced ER stress activated the UPR pathways (eIF2α, ATF4, IRE1, XBP1), which led to an increase of cathepsin D or a decrease of ACE‐2. These changes were inhibited by the presence of UPR inhibitors, thus blocking the AEC apoptosis caused by the ER stress.

CONCLUSIONS: These data show that ER stress induces apoptosis in human AECs through UPR‐mediated pathways that in turn regulate the autocrine ANGII/ANG1‐7 system. It is also demonstrated that ER stress‐induced AEC apoptosis can be blocked by inhibition of the UPR pathways.

Grant Funding Source: PHS HL‐45136
Effects of exposure to bisphenol A on hepatocytes and pre‐adipocytes in vitro (1010.7)
Authors: Macis Elias , Kostrominova Tatiana
Published: June 2021

In recent years, environmental pollutants/endocrine disrupting chemicals (EDCs) have received special attention as metabolic disruptors that produce adverse developmental, reproductive, neurological, cardiovascular, metabolic and immune effects. Bisphenol A (BPA) is one of the most abundant EDCs present in the environment. We focused our study on the effects produced by the exposure to BPA on cell viability, apoptosis and DNA fragmentation in FaO hepatocytes and 3T3 pre‐adipocytes in vitro. In particular, we were interested whether there are considerable differences in the sensitivity to cytotoxic effects of BPA in hepatocytes and pre‐adipocytes when evaluated under the same experimental conditions. Exposure to 10 and 100 μM of BPA showed that although morphology of both cell lines was affected after 24 and 48 hours of exposure, the effect on 3T3 pre‐adipocytes was more pronounced. Most probably exposure to BPA attenuated cell growth or caused cell detachment since the number of viable cells attached to the substrate did not show statistically significant differences when evaluated by trypan blue exclusion method. Several methods for apoptotic DNA fragmentation evaluation were compared. Qiagen DNeasy kit for DNA isolation showed the best detection sensitivity. Exposure to 100 μM of BPA for 48 hours showed presence of apoptotic DNA ladder when evaluated by gel electrophoresis.

Support: IUN and IUSM‐NW.

Grant Funding Source: Undergraduate Research Fund IUN
Magnolol affects the expression of polyamine biosynthesis and catabolism‐linked proteins in PC3 human prostate cancer cells in vitro (1011.1)
Authors: McKeown Brendan , Hurta Robert
Published: June 2021

This study examined the effect of magnolol on the expression of proteins involved in polyamine biosynthesis/catabolism in PC3 prostate cancer cells in vitro. Magnolol, 80 μM for 6 hours, decreased cellular viability by ~50%. Magnolol exposure decreased expression of ornithine decarboxylase (ODC), a key protein in polyamine biosynthesis, and antizyme‐inhibitor (AZI) with no apparent change to expression of antizyme (AZ), an ODC inhibitor. Expression of spermidine/spermine N1‐acetyltransferase, a key protein in polyamine catabolism, was also decreased. These magnolol‐mediated changes to ODC & AZI expression suggest that changes in polyamine biosynthesis would occur. An AZI/AZ ratio decrease in response to magnolol favors growth repression of PC3 cells. Cellular signaling proteins were also evaluated, with no apparent change occurring in ERK1, ERK2, p‐ERK1, p‐ERK2, p38, p‐JNK2, PI3Kp110 & AKT expression. Decreased expression of p‐p38, JNK1, JNK2, PI3Kp85, p‐PI3Kp85 & p‐AKT and increased expression of p‐JNK1 were observed. This study demonstrates that magnolol can alter the behavior of human prostate cancer cells in vitro by affecting activities associated with growth regulation. (Supported by the Jeanne and J.‐Louis Levesque Foundation.)

Grant Funding Source: Supported by the Jeanne and J.‐Louis Levesque Foundation
Understanding how the distal pocket environment affects the ligand binding affinity of nitrite to heme proteins (1013.17)
Authors: Garcia Adriana , Gomez Rocio , Wojdyla Sylvia , Lough Lea , Esquerra Raymond
Published: June 2021

Cardiovascular disease is the leading cause of death worldwide. Nitric oxide (NO) plays a fundamental role in cardiovascular health and disruptions in normal NO physiology is associated with the progression of cardiovascular disease. A relatively new mechanism by which heme proteins can support vasodilatation during hypoxia is by converting nitrite (NO2‐) to NO. This NO2‐ reductase activity plays essential roles in a variety of physiological processes. Because there is a large range in NO2‐ affinity and NO2‐ reductase activity in heme proteins with the same active site, this research seeks to better understand how the protein environment controls the binding chemistry of NO2‐ to the heme active site. Our goal is to determine how the distal pocket environment affects the binding affinity of NO2‐ to metmyoglobin mutants. Our hypothesis is that the distal pocket environment can increase the binding affinity of NO2‐ by way of electrostatic and steric interactions. To do this, we will compare the binding affinity of a series of distal pocket mutants that affect hydrogen bonding, polarity, and the size of the distal pocket. We will correlate coordination chemistry and binding affinity in order to establish a clear picture of how the protein environment controls NO2‐ binding affinity in heme proteins. We found that polar distal residues exhibit greater binding affinity to NO2‐ than non‐polar residues due to hydrogen bonding. These results indicate that proteins with polar distal residues will exhibit greater NO2‐ reductase activity. Understanding how the protein environment influences NO2‐ binding in heme proteins helps toward understanding how these proteins generate NO physiologically, and in designing therapeutics based on the NO2‐ reductase activity of heme proteins.
Phosphatidic acid activates mTORC1 by displacing the inhibitor DEPTOR (1013.19)
Authors: Plaisier Christina , Yoon Mee Sup , Wu Cong , Truong Nga , Chen Jie
Published: June 2021

The lipid second messenger, phosphatidic acid (PA), is a critical mediator of mitogenic activation of the mammalian target of rapamycin complex 1 (mTORC1). Previously we showed that PA binds to mTOR and activates mTORC1, but the mechanism of this activation remains incompletely understood. Here we propose a novel mechanism of mTORC1 activation by PA. Using mass spectrometry analysis to identify mTOR interactions regulated by PA, followed by confirmation by co‐immunoprecipitation, we discovered that the interaction between mTOR and its inhibitor DEPTOR was disrupted by PA. Dissociation of DEPTOR by PA coincided with activation of mTORC1. Phospholipase D1 (PLD1) catalyzes the production of PA and is also an established mediator of mitogenic activation of mTORC1. Treatment of cells with phorbol myristate acetate and lysophosphatidic acid, known to activate PLD1, recapitulated the DEPTOR displacement and mTORC1 activation achieved with exogenous PA, and this effect was decreased by PLD1 inhibition. Furthermore, by examining a panel of PA molecules with various fatty acid chains, we found that only the PA species known to be produced by PLD displaced DEPTOR and activated mTORC1 both in vitro and in cells. Taken together, our results strongly suggest that PA produced by PLD1 regulates mTORC1 activity by displacing the inhibitor, DEPTOR.

Grant Funding Source: Supported by NIH GM089771 and AR048914 grants to J. Chen.
Plasma membrane recovery kinetics of a microfluidic intracellular delivery platform (1013.2)
Authors: Poceviciute Roberta , Sharei Armon , Jensen Klavs , Langer Robert
Published: June 2021

Intracellular delivery of macromolecules continues to be a challenge in both research and therapeutic applications. Recently, physical delivery methods, such as electroporation and sonoporation, have emerged that provide direct access to the cytoplasm by disrupting the plasma membrane. For a rapid development of such methods the mechanisms behind their action need to be understood. We use the microfluidic shear‐based CellSqueeze delivery platform to examine the kinetics of plasma membrane recovery after disruption and its dependence on the calcium content in the surrounding buffer. Delivery experiments with HeLa cells show that the pores close ten times faster when calcium is present in the surrounding buffer. If we eliminate calcium from the buffer, we are able to improve delivery efficiency five‐fold without sacrificing cellular viability. Membrane repair characteristics initially observed in HeLa cells are shown to translate to primary naïve murine T cells, suggesting that calcium may have a similar role across many cell types. Subsequent manipulation of other delivery parameters enables the delivery of larger macromolecules, such as antibodies, into naïve t cells. The work provides insight into the membrane repair process in response to mechanical disruption and could potentially enable the development of improved delivery methods.

Grant Funding Source: Supported by National Institutes of Health Grants RC1 EB011187‐02 and R01GM101420‐01A1
Direct PKA‐PDE interactions lead to localized cAMP signaling domains in adult cardiomyocytes (1013.7)
Authors: Boras Britton , Taylor Susan , McCulloch Andrew
Published: June 2021

The β‐adrenergic pathway in cardiomyocytes activates protein kinase A (PKA) to phosphoregulate several Ca2+ handling proteins, including L‐type Ca2+ channels, ryanodine receptors, and sarco/endoplasmic reticulum Ca2+‐ATPases (SERCA) (via phospholamban), resulting in inotropic, lusitropic and chronotropic responses. Based on PKA kinetics measured in vitro, almost all PKA should be activated by basal concentrations of cAMP, limiting its dynamic range in response to a stimulus event. Several studies have postulated a role for local degradation of cAMP by phosphodiesterases (PDE) in maintaining microdomains with lower cAMP concentrations around A kinase anchoring proteins (AKAP), which bind both PKA and PDE. In this study we use 3‐D diffusional modeling around cell geometries from transmission electron microscopy (TEM) images to show that local clustering of PDEs is insufficient to maintain cAMP microdomains necessary for lower PKA basal activation. We instead propose a new model for PKA activation which incorporates direct binding between PDE’s and the regulatory subunit of PKA as a pathway to stimulate PDE activity and inhibit PKA activation. We used in vitro kinetic experiments with 3H tagged cAMP and purified PKA and PDE to quantify this interaction and explore its specificity both for PKA and PDE isoforms.

Grant Funding Source: Supported by: NIH 5 T32 HL 105373‐2
Human p190A RhoGAP binds eIF3A and exists in a complex with the 48S translational pre‐initiation complex (1013.8)
Authors: Parasuraman Prasanna , Walker James , Li Bihua , Mulligan Peter , Haas Wilhelm , Bernards André
Published: June 2021

By regulating the actin cytoskeleton Rho GTPases control biological processes such as cell migration, adhesion and polarity. p190A RhoGAP is an important yet poorly understood Rho GTPase regulator. To shed further light on the functions of p190A RhoGAP, we performed mass spectroscopic analysis of associated proteins. Here we report that human p190A, but not its related paralog p190B, forms a stable complex with multiple subunits of the 48S translational pre‐initiation complex. Complex formation involves a direct interaction between the first FF domain of p190A and the PCI/PAM domain of eIF3A. This interaction does not appear to be regulated by Y308 phosphorylation, previously found to control the p190A directed cytoplasmic sequestration of TFII‐I, but may instead by controlled by phosphorylation of S296 by an as yet unknown protein kinase. Although other reasons for the detected interaction cannot be ruled out, we speculate that p190A RhoGAP may serve as an anchoring protein allowing the localized translation of specific mRNAs.

Grant Funding Source: RO1 GM084220
The problem with low pricing: buffet lunch and regret (1019.15)
Authors: Sigirci Ozge , Wansink Brian
Published: June 2021

Objective: Will the price people pay for an All‐you‐can‐eat buffet influences them to overeat and to feel guilty about how much they eat? This has implications for consumers, restaurants and public health.

Methods: A field experiment was conducted in which diners at an All‐you‐can‐eat (AYCE) restaurant were either charged $4 or $8 for an Italian lunch buffet. The ratings of consumers who eat different number of pieces of pizza were taken for overeating, physical discomfort, and guilt items. Their ratings were analyzed using 2x3 between groups ANOVA.

Results: Consistent with expectation disconfirmation theory, diners who paid $4 for their buffet rated themselves as physically more discomfortable and has eaten more than they should have comparing to the diners who paid $8 for the buffet (p<0.05). Interestingly, even if they ate the exact same number of pieces, diners who paid $4 for their buffet rated the overeating, feeling of guilt and physical discomfort items higher than the diners who paid $8 for the buffet.

Conclusions: Paying less for an AYCE experience has a number of surprising consequences.

Interestingly, low‐ priced diners feel themselves as physically more discomfortable, and guiltier comparing to the low‐priced diners even if they ate exactly the same amount with high‐priced diners.
Intensive use of primary literature in science courses can supplement and prepare undergraduate students for laboratory‐based jobs and graduate programs (1020.7)
Authors: Varamini Behzad
Published: June 2021

Many schools across America, particularly smaller, liberal arts, undergraduate‐intensive schools, may lack the human and capital resources necessary to fully prepare science, pre‐medical, and nutrition undergraduates for the job market or graduate school, as laboratory techniques and critical research skills are coveted by employers and graduate school admissions committees alike. As such, it is the responsibility of the undergraduate department to seek creative avenues of supplementing normal lecture and laboratory classes with opportunities for students to interact with widely employed biological techniques and current topics in medicine and nutrition. As a result of this need, the CREATE strategy (consider, read, elucidate hypotheses, analyze and interpret the data, and think of the next experiment) has proven effective in undergraduate science education. Herein described is, for the first time, the application of the CREATE strategy to upper division courses at Biola University, and measurable outcomes related to student learning, preparedness, critical thinking, and satisfaction. Overall, it is clear that the CREATE strategy can help students become more well‐rounded and better prepared technicians and critical thinkers, particularly in atmospheres where laboratory research opportunities are minimal.
Increasing insulin resistance among U.S. adolescents ages 12‐19: NHANES 2003‐2010 (1024.17)
Authors: Gee David , Davis Erika
Published: June 2021

Type 2 diabetes is increasingly becoming a public concern among adolescents. The aim of this study was to utilize the National Health and Examination Survey (NHANES) to assess the prevalence trends of risk factors for diabetes mellitus in US adolescents. This study observed 2,716 adolescents from ages 12 to 19 in the NHANES survey years 2003‐2006 and 2007‐2010. There was no significant change in the prevalence of diabetes and pre‐diabetes based on fasting plasma glucose values. There was also no significant change in the prevalence of obesity. However, the prevalence of elevated glycohemoglobin (HbA1c) concentrations (5.7% ‐ 6.5%) increased from 1.8% to 4.1% of the population (p<0.02). This increase in elevated HbA1c was seen predominantly in normal weight adolescents (1.3% to 3.6%, P = 0.04). In addition, there was a significant increase in average fasting insulin from 11.1 uU/mL in 2003‐06 to 12.6 uU/mL in 2007‐10 (p < 0.001). A significant increase in fasting insulin was only seen in normal weight adolescents (7.7uU/ml to 9.8% uU/ml, p <0.001). The increase in elevated HbA1c and fasting insulin values suggests that insulin resistance is increasing among US adolescents, particularly those in the normal weight category. Despite no significant change in fasting plasma glucose, the trend of increasing fasting insulin and HbA1c concentrations is a concern for future diabetes risk.
Proportionality of regional fat pattern changes with weight change (1031.14)
Authors: Davidson Robert , Gibson Glenn
Published: June 2021

Objective. There has been some debate in the literature regarding fat pattern changes with weight change, particularly whether fat loss is proportional or if you can ‘spot reduce’. Using published papers reporting changes in regional fat due to various interventions as measured with DEXA we determined the proportionality of fat change in arm, leg, arm and leg, and trunk regions.

Methods. Twenty‐three studies were identified from Pub Med searches that reported regional fat data before and after an intervention resulting in fat change (gain or loss). The initial and final percentage of total body fat occurring in each region (arms, legs, or arms and legs, and trunk) for each intervention was compared. To see if the magnitude of fat change influenced regional fat pattern changes, total body fat change was compared to the ratio of final to initial percentage of body fat in each region.

Results. The average ratio of final percent of total body fat in a region to the initial percent of total body fat in the region was 0.98, 0.99, 0.99, and 1.02 for arm, leg, arm and leg, and trunk regions, respectively. Linear regression of plotted initial versus final percent of total body fat in each region gave slopes of 0.84, 0.88, 1.09, 0.80, and 1.00 for arm, leg, arm and leg, trunk, and all regions combined, respectively.

Conclusions. Regional fat pattern changes are proportional to the initial percentage of body fat in each region.
Effects of mild calorie restriction on hepatic lipid metabolism and inflammation in mice (1034.14)
Authors: Park Chan Yoon , Park Soyoung , Kim Min Soo , Han Sung Nim
Published: June 2021

Calorie restriction (CR) has been reported to improve liver adiposity and inflammation. However, most CR models use 30~60% calorie reduction, which is hard to achieve in human. We investigated the effects of mild CR on hepatic lipid metabolism and inflammation. Seven‐wk‐old C57BL/6 mice were fed control diet containing 10% kcal fat (Control) or high fat diet containing 60% kcal fat (HFD) or reduced amount of control diet to achieve 14% calorie restriction (CR). Hepatic and epididymal IL‐1, IL‐6, Mcp‐1, Tnf‐, and Socs3 mRNA, hepatic Pparg, Ppara, Fasn, Srebf‐1c, Adipor2, and Cpt1 mRNA and hepatic Sirt1 and pStat3/Stat3 protein expression levels were determined. Hepatic expression of genes related to lipogenesis, Pparg and Srebf‐1c, were significantly lower in CR compared with HFD. The mRNA levels of pro‐inflammatory cytokines Mcp‐1, IL‐6, IL‐1 and Tnf‐ in adipocyte were significantly lower in CR compared with HFD. In liver, only Mcp1 mRNA level was lower in CR than Control and HFD (63% and 74% lower). Serum leptin level was significantly lower and hepatic Lepr mRNA level was significantly higher in CR than Control. Hepatic expression of Socs3 was higher in CR than HFD (154% higher) and correlated with Lepr (r = 0.699, P <0.001) and pStat3/Stat3 ratio (r =0.596, P =0.004). In conclusion, mild CR reduced hepatic lipid synthesis, attenuated hepatic Mcp‐1 expression, and improved leptin sensitivity.

Grant Funding Source: Supported by the grant from the National Research Foundation (NRF) of Korea, (NRF‐2010‐0024878).
Effect of n‐3 fatty acids and their derivatives on the expression of inflammatory genes (1034.15)
Authors: Allam‐Ndoul Benedicte , Guenard Frederic , Barbier Olivier , Vohl Marie‐Claude
Published: June 2021

Background: Uncontrolled inflammation participates in the development of chronic inflammatory diseases. Beneficial effects of the consumption of long chain n‐3 polyunsaturated fatty acids (PUFAs), mostly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on inflammation have been reported. Lipid mediators produced from EPA and DHA (resolvins) play a central role in the resolution of inflammation. Objective: To study the effects of EPA, DHA, resolvin D1 (RvD1) and resolvin D2 (RvD2) on the expression of inflammatory genes in cultured THP1 macrophages. Methods: Cells were incubated for 6h and 24h in the presence of n‐3 PUFAs (EPA, DHA; 100, 50, 25, 10 μM and resolvins (RvD1, RvD2; 1μM, 100, 50, 25, 10 nM). Total RNA was isolated and TNF‐alpha, IL‐6 and IL‐10 mRNA levels were analyzed by real‐time PCR. Results: In THP1 cells, EPA, DHA, RvD1 and RvD2 down‐regulated IL‐6 and TNF‐alpha gene expression, and up‐regulated IL‐10 expression in a time‐dependent manner with higher extent observed after 24h. TNF‐alpha, IL‐6 and IL‐10 gene expression demonstrated a dose‐dependent response following treatment with EPA and DHA (10, 25 and 50 μM). No association was found between gene expression and resolvins concentrations in dose‐response experiments. Conclusion: We demonstrate a time‐ and dose‐dependent regulation of TNF‐alpha, IL‐6 and IL‐10 gene expression levels in macrophages under EPA and DHA treatment.

Grant Funding Source: NSERC
Role of Wnt5b in fatty acid synthesis in primary cultured mouse hepatocytes (1037.6)
Authors: Siriwardhana Nalin , Kim Jung Han
Published: June 2021

Wnt proteins are secreted glycoproteins that act in autocrine and paracrine fashions and known to regulate fundamental cellular functions including cell differentiation, cell growth and apoptosis. Emerging evidence indicates that Wnts are also involved in the development of metabolic disorders. Large‐scale gene expression analysis of liver of an obesity mouse model fed high fat diets displayed decreased expression of number of genes including Wnt5b. In this study, we investigated the role of Wnt5b in lipid metabolism using RNA interference in primary cultured mouse hepatocytes. Hepatocytes were isolated from C57BL/6J mice and transiently transfected with siRNA targeting mouse Wnt5b or control siRNA. Cells were then maintained in culture medium for 48 hours before assays. For fatty acid synthesis, cells were incubated with 3H2O (0.5 mCi/ml) for 2 hours, followed by saponification. Fatty acids were extracted and the 3H content of fatty acids determined. For fatty acid oxidation, cells were incubated with 22 M [9,10(n) 3H] palmitate substrate for 1 hour and the degree of incorporation of 3H into H2O determined. In the cells transfected with Wnt5b siRNA, fatty acid synthesis increased 1.9 fold compared with that in the control siRNA transfected cells (p<0.05). There were no differences in beta‐oxidation. These results may suggest that Wnt5b plays a role in hepatic lipogenesis.
The effect of almond consumption on satiety and the postprandial metabolic response in high‐risk pregnant women (1040.5)
Authors: Henderson Mary , Sawrey‐Kubicek Lisa , Mauldin Kasuen , King Janet
Published: June 2021

Over one third of all American pregnant women are obese. Managing weight gain during pregnancy is difficult because hormonal changes induce an anabolic state facilitating tissue gain. The pregnancy hormonal effects on appetite and energy intake are unknown. Almonds are satiating and have a lower net metabolizable energy, which may explain their role in managing body weight and aiding in weight loss. As well, results from a number of clinical trials have shown other health benefits of almond intake including reducing oxidative stress and inflammation and promoting vascular health and glycemic control. A cross‐over, randomized control trial examining the metabolic response to 2 oz of almond consumption versus dairy fat consumption in 2 standardized, isocaloric and equal macronutrient composition test meals, was done in 20 overweight/obese (pre‐pregnancy BMI = 25‐40) Asian and Latina women during 30‐36 weeks of gestation. Satiety was assessed by visual analogue scale questionnaires, circulating ghrelin, leptin, and GLP‐1 concentrations, and ad libitum food intake at a meal following the postprandial study. Our early data suggest that consuming 2 oz of almonds improves satiety, reduces appetite, and may help promote healthy maternal weight gain during pregnancy.

Grant Funding Source: Supported by the Almond Board of California
Role of thioredoxin‐binding protein 2 in cardiac oxidative stress and energy metabolism of rats supplemented with vitamin D (1041.14)
Authors: Santos Priscila , Rafacho Bruna , Gonçalves Andréa , Pires Vanessa , Fernandes Ana Angélica , Minicucci Marcos , Azevedo Paula , Okoshi Marina , Zornoff Leonardo , Paiva Sergio
Published: June 2021

Vitamin D (VD) is important for cardiac development and functioning but, recent studies have shown that VD receptor is undetectable in cardiac muscle. On the other hand, in the heart is present a thioredoxin‐binding protein (TBP‐2) which is up regulated by VD. The aim of this study was to evaluate whether supplementation with different doses of VD results in changes in TBP‐2 protein expression, cardiac oxidative stress and energy metabolism. Methods: 48 male rats allocated into 4 groups: control (C, n=12) received standard chow; VD3 (n=12), VD5 (n=12) and VD10 (n=12) received 3000, 5000 and 10000 IU of cholecalciferol/kg chow for 2 months. Statistical analysis was performed by ANOVA or Kruskal‐Wallis test and Test for Linear Trend. Results: The TBP‐2 protein expression was higher in VD10 group compared to control and VD3 in a dose‐dependent manner (p<0.001). In relation to the oxidative stress, the thioredoxin (Trx) protein expression decreased in dose‐dependent manner (p=0.019) and lipid hydroperoxide (LH) was higher in VD10 compared to other groups. The increase in LH was dose dependent (p<0.001). In relation to cardiac energy metabolism, the peroxisome proliferator‐activated receptor‐γ coactivator‐1 α (PGC‐1α) protein expression was lower in VD10 compared to control in dose‐dependent manner (p=0.002). Activity of the enzymes: 3‐hydroxyacyl coenzyme‐A dehydrogenase and citrate synthase was lower and lactate dehydrogenase was higher in VD10 compared to other groups. Conclusion: The increased expression of TBP‐2 in rats with VD supplementation may be associated with increased oxidative stress and changed cardiac energy metabolism in these animals.

Grant Funding Source: Supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Comparison of tissue concentrations in male and female C57BL/6 mice in response to vitamin K manipulation (1041.4)
Authors: Harshman Stephanie , Smith Donald , Karl James , Shen Xiaohua , Booth Sarah , Fu Xueyan
Published: June 2021

The tissue‐specific response to dietary vitamin K (VK) manipulation has not been well studied in mice. This limits the use of genetically modified mouse models in VK studies. The objective of this study was to determine the sex‐specific effects of dietary VK manipulation on serum, liver and extra‐hepatic tissue VK concentrations in C57BL/6 mice (n=64). Mice were weight‐matched and pair‐fed a control diet (1.4±0.08 mg phylloquinone (PK) /kg) or VK‐deficient diet (31±0.45 µg PK/kg) for 28 days. Liver, kidney, brain, pancreas, adipose tissue, and serum PK and menaquinone‐4 (MK‐4) concentrations were measured by HPLC. Data were log transformed and analyzed by a general linear model, stratified by diet. Male and female mice responded differently to dietary manipulation of VK in a tissue‐dependent manner (for all sex by diet interactions p<0.031). Serum PK concentrations did not differ between males and females within each diet (p>0.95); no serum MK‐4 was detected. Females on the control diet had significantly higher PK and MK‐4 concentrations in adipose tissue, kidney, liver, pancreas, and brain compared to males (p<0.01). Only MK‐4 concentrations in adipose tissue, liver, and pancreas did not differ between males and females on the deficient diet (p>0.44). Sex‐specific differences in response to VK manipulation need to be considered when using animal models of VK metabolism.

Grant Funding Source: Supported by the USDA/ARS Cooperative Agreement No. 58‐1950‐7‐707
Novel pathway for iron deficiency in pediatric non‐alcoholic steatohepatitis (1042.1)
Authors: Moya Diana , Baker Susan , Liu Wensheng , Garrick Michael , Kozielski Rafal , Baker Robert , Zhu Lixin
Published: June 2021

Non‐alcoholic steatohepatitis (NASH) is an obesity related liver disease characterized by steatosis and inflammation. Iron may play a role in NASH pathology as it catalyzes the production of reactive oxygen species. To examine the iron status in pediatric NASH, serum iron, ferritin, and soluble transferrin receptor 1 in NASH patients were compared to those in the National Health and Nutrition Examination Survey database. Serum iron and soluble transferrin receptor 1 were decreased while serum ferritin was increased in NASH patients. No detectable iron was observed in NASH liver by Perls’ Prussian blue staining. Microarray and quantitative real‐time PCR indicated that the mRNA of transferrin, transferrin receptor 2 and catalase were significantly elevated in NASH patients, and that transferrin mRNA was positively correlated with catalase mRNA. To test the hypothesis that oxidative stress induces iron deficiency, HepG2 cells were treated with H2O2, and the mRNA of transferrin and catalase were induced and positively correlated. We conclude that there is a tendency towards iron deficiency in pediatric NASH patients. Increased liver gene expression of transferrin and transferrin receptor 2 supports a status of iron deficiency. Our finding that H2O2 induces the expression of transferrin, and consequently, decreased iron absorption, suggests a novel mechanism for iron deficiency in NASH patients.
Ascorbate‐induced iron transport through ferroportin involves IRP2 and HIF2α (1042.2)
Authors: Scheers Nathalie , Sandberg Ann‐Sofie
Published: June 2021

In previous studies we have shown that ascorbate status of intestinal Caco‐2 cells affects the cellular levels of iron‐absorption related proteins (DMT1, DCYTB, ferritin, and ferroportin). The aim of the present study was to dig deeper into the ascorbate effects to investigate the mechanisms behind. We have measured ferroportin, IRP2, and HIF2α expression in response to different ascorbate concentrations with ELISA and Western blot analyses, which resulted in peaking protein levels at 100 µM followed by a decrease at higher ascorbate concentrations. In conclusion, Iron‐independent regulation of ferroportin by IRP2 seems to be responsible for the ascorbate‐mediated decrease in ferroportin with increasing ascorbate status.

Grant Funding Source: The Swedish Council for Environment, Agricultural Sciences and Spatial Planning funded this study.
Combination of modified formulation and bioprocessing techniques as a way to improve nutritional and health potential of bread (1044.13)
Authors: Garcia Campayo Vicenta , Han Sonia , Gaspard Dan , Aimutis William , Casper Jeffrey
Published: June 2021

Formulation and processing can improve nutritional, functional, and health attributes of foods. In breads, addition of whole wheat (WW) enhances content of dietary fiber, proteins, and non‐nutrients: minerals and antioxidants. Bioaccessibility of these compounds is partial due to cross‐linking to polymers and/or physical entrapment within the complex plant cell wall matrix and can be improved by technological processes. We study the effect of two bioprocessing techniques: A) Enzyme (pre‐ vs in situ treatment), B) dough mixing method (straight vs sponge and dough), in the release of antioxidants (free Ferulic Acid (FA) and Steryl Ferulates), in vitro digestibility, and antioxidant bioaccessibility in WW or Aleurone‐containing WW (AL‐WW) breads. Sponge and‐dough processing and enzyme treatment increases 1.5‐4.0 times the free FA in AL‐WW breads but only in‐situ enzyme treatment enhances free FA (about 1.4 times) in WW breads. Free Steryl Ferulates also increased with both processing methods in AL‐WW breads. Straight dough but not enzyme treatment enhances in vitro digestibility of AL‐WW bread starch fractions while the combination of sponge time and enzyme treatment benefit in vitro digestibility of WW bread. Bioaccessibility of antioxidant compounds (ORAC) is higher in AL‐WW vs WW bread. Thus, nutritional properties and health benefits of bread are influenced by formulation and processing techniques.
Impact of allicin on enzyme activity, cytokine secretion, and gene expression dynamics in oxidatively‐stressed epithelial cells (1045.19)
Authors: Horn Nathan , Ajuwon Kolapo , Miller Guy , Adeola Olayiwola
Published: June 2021

Environmental stress can alter cell permeability by influencing tight junctions and inflammatory mediators. Allicin is a botanical derived from garlic that has anti‐inflammatory and antioxidant properties. The objective of the current experiment was to determine if allicin can mitigate oxidative stress using a Caco‐2 cell model. The experiment was arranged in a 3 x 2 factorial with three concentrations of allicin (0, 40, or 80 µM) and two concentrations of oxidative stressor, hydrogen peroxide (0 or 100 µM). Cells were incubated with allicin or hydrogen peroxide for 24 or 3 h, respectively. There was an increase in reactive oxygen species with hydrogen peroxide incubation, whereas a combination of allicin and hydrogen peroxide increased superoxide dismutase activity. Supplementation with allicin ameliorated the effects of hydrogen peroxide on catalase activity and reactive oxygen species. Gene expression of NFκB tended to increase and occludin expression decreased with hydrogen peroxide treatment, whereas treatment with allicin resulted in decreased expression of claudin‐4 and occludin, and an increase in IL‐8 secretion. Results from this study show that hydrogen peroxide induced oxidative damage through epithelial cell antioxidant enzymes, tight junction gene expression, and inflammatory mediators, but allicin partially mitigated epithelial cell oxidative damage.
Inhibition of digestive enzymes by tea polyphenols: enzymological and in silico studies (1045.34)
Authors: Glisan Shannon , Sae‐Tan Sudathip , Grove Kimberly , Yennawar Neela , Lambert Joshua
Published: June 2021

Tea (Camellia sinensis) has shown anti‐obesity effects in animals. Tea polyphenols can inhibit digestive enzymes such as pancreatic lipase (PL), phospholipase A2 (PLA2), and trypsin in vitro. Our objective was to explore the mechanisms of inhibition by tea polyphenols by enzymology and in silico modeling. Theaflavin‐3,3’‐digallate (TFdiG), theaflavin‐3’‐gallate, theaflavin‐3‐gallate, and theaflavin inhibit PL with IC50 = 1.9, 4.2, 3.0, and 32.9 µM, respectively, indicating that the location of the galloyl ester is essential for inhibitory potency. TFdiG exhibits a mixed mode of inhibition. Theaflavins were found to bind to a pocket adjacent to the active site in PL; galloyl‐containing theaflavins are able to form a hydrogen bond with His264, which forms part of the catalytic triad. Similar interactions are predicted with green tea catechins. Catechins also inhibit PLA2. Modeling studies show that these compounds occupy a tunnel that leads to the active site and are stabilized by van der Waals bonds. This interaction is expected to result in noncompetitive inhibition. We found that (‐)‐epigallocatechin‐3‐gallate (EGCG) inhibited trypsin (IC50 = 193 µM) in a non‐competitive manner. Modeling studies show that EGCG interacts with amino acids around the active site and hinders substrate binding. These findings provide insight into the mechanisms by which tea polyphenols can inhibit key digestive enzymes.

Grant Funding Source: NIH AT004678
Ursolic acid, a promising dietary bioactive compound of anti‐obesity (1045.40)
Authors: Wang Yanwen , He Yonghan
Published: June 2021

Ursolic acid has recently been reported to possess a promising anti‐obesity effect. A number of studies have been conducted to elucidate the underlying mechanisms, which include the inhibition of pancreatic lipase activity, promotion of muscle hypertrophy, increase of brown fat, thermogenesis, energy expenditure, and lipolysis. Our recent study in 3T3‐L1 preadipocytes has demonstrated that ursolic acid inhibits cell differentiation and adipogenesis. This compound modulates the expression or activity of many proteins or enzymes involved in fat cell differentiation, fatty acid synthesis and oxidation. Further studies have shown that ursolic acid increases the phosphorylation and activity of AMPK and the protein expression of Sirt1. The anti‐adipogenic effect of UA can be reversed by AMPK siRNA but not Sirt1 inhibitor. Moreover, when LKB1 is silenced or inhibited, the effect of UA on AMPK activation diminishes. These results demonstrate that ursolic acid inhibits preadipocyte differentiation and adipogenesis through LKB1/AMPK pathway. While focusing on our recent report on the effect and mechanism of action of ursolic acid on fat cell differentiation and adipogenesis, other mechanisms related to energy and fat metabolism and body weight will be briefly discussed.

Grant Funding Source: CIHR
Phytochemical genistein promotes pancreatic beta‐cell survival and exerts anti‐diabetic effect via GPR30‐mediated mechanism (1045.44)
Authors: Wang Aihua , Liu Dongmin , Luo Jing , Suh Kyung‐Shin , Moore Willam , Alkhalidy Hana
Published: June 2021

Loss of pancreatic beta‐cell mass is central to the development of both type 1 and type 2 diabetes (T2D). Chronic hyperglycemia and hyperlipidemia cause beta‐cell apoptosis, thereby contributing to the pathogenesis of T2D. We investigated the effects of phytochemical genistein on apoptosis and function of beta‐cells and further determined the mechanism underlying its actions ex vivo and in vivo. We show that genistein promoted viability, inhibited apoptosis, and reduced caspase‐3 activity of INS1 beta‐cells and human and mouse islets chronically exposed to palmitate (0.5 mM) and high glucose (HG, 20 mM). In addition, exposure of beta‐cells to genistein activated PI3K/Akt signaling and up‐regulated anti‐apoptotic protein Bcl‐2 expression. The anti‐apoptotic effect of genistein is independent of classical estrogen receptor‐mediated signaling machinery, but it may be achieved through the G‐protein‐coupled receptor GPR30‐mediated activation of cAMP signaling. Oral administration of genistein (50 mg/kg BW/day) improved hyperglycemia, glucose tolerance, and blood insulin levels in wide‐type obese diabetic mice, but these effects were abolished in GPR30 null mice. These findings demonstrate that genistein exerts anti‐diabetic effect and promotes beta‐cell survival via GPR30‐mediated signaling pathway.

Grant Funding Source: supported by grants from the NCCAM of NIH (R01AT007077) and the ADA (7‐11‐BS‐84) to DL.
Brassicaceae spp. spout extract inhibits ROS production induced by bisphenol A during the differentiation of 3T3‐L1 preadipocytes (1045.8)
Authors: Lee Jong Seok , Lee Young‐Jun , Kim Jae‐Hwan , Kim Dan‐Bi , Kim Young‐Hyun , Lee Ok‐Hwan , Lim Jeong‐Ho
Published: June 2021

Bisphenol A (BPA), an environmental endocrine disrupting chemical, is contained in polycarbonate plastic products. BPA plays as a mixed agonist‐antagonist with respect to steroid hormones such as estrogens. Recent studies have suggested that BPA affects adipocytes diffenetiation and lipid accumulation by regulation of adipogenic transcription factors. In this study, we evaluated the influence of BPA on oxidative stress during the differentiation of 3T3‐L1 preadipocytes. We also evaluated that inhibitory effects of Brassicaceae spp. spout extracts such as Raphanus sativus L, Brassica oleracea var gongyloides, Brassica rapa var. glabra Regel on BPA‐induced oxidative stress during the differentiation of 3T3‐L1 preadipocytes. Our results showed that BPA treatment significantly increased ROS production in 3T3‐L1 cells compared with control by down‐regulating expression of antioxidant enzyme such as manganese superoxide dismutase (Mn‐SOD) and glutathione reductase (GR). However, Brassicaceae spp. spout extracts significantly inhibited BPA‐induced ROS production. In addition, Brassicaceae spp. spout extracts increased the level of the expression of antioxidant enzyme such as Mn‐SOD and GR. These results indicated that Brassicaceae spp. spout extracts consider as inhibitors that attenuate BPA‐induced adipocytes dysfunction.