bluuebunny/crossref_metadata_2025 huggingface dataset:
Exploiting Non‐Canonical Amino Acids to Alter Enzyme Substrate Specificity
Authors: Windle Claire , Nelson Adam , Pearson Arwen , Berry Alan
Published: June 2021

Enzymes are constructed from the 20 proteogenic amino acids, but Nature has expanded the chemistries available in proteins by recruiting cofactors or inserting non‐canonical amino acids (ncAAs). Until recently, protein engineering was restricted to using the 20 proteogenic amino acids. Using a chemical method, we are able to produce proteins containing ncAAs quickly and in high yields. This method works by first converting a cysteine into a dehydroalanine intermediate, before reacting with a thiol to produce the non‐canonical side chain. Initial studies focussed on replacing the catalytic lysine in the aldolase N‐acetyl‐neuraminic acid lyase (NAL) with a γ‐thialysine, this enzyme has been characterised both kinetically and crystallographically.

Using this methodology, we have explored the effect of an increased amino acid repertoire on enzyme substrate specificity by inserting multiple ncAAs (~16) at various positions (~11) throughout the active site of NAL, and screening for altered activity. This has produced an ncAA‐containing aldolase with a 10 fold increased kcat, with a shortened substrate, over that of the wild‐type. Saturation mutagenesis has also revealed that this increase in activity is not achievable with any of the proteogenic amino acids. Crystallographic and computational studies have been used to elucidate the mechanism by which this ncAA increases activity.

This work highlights that activities previously thought inaccessible by use of the 20 proteogenic amino acids may now be achievable by use of ncAAs.
Ginnalins and Maplexins from Maple Species Inhibit the Formation of Advanced Glycation Endproducts
Authors: Ma Hang , Liu Weixi , Frost Leslie , Niesen Daniel , Yuan Tao , Dain Joel , Seeram Navindra
Published: June 2021

Glycation is a non‐enzymatic process leading to the formation of Advanced Glycation Endproducts (AGEs). The accumulation of AGEs in vivo plays an important role in the pathology of complications in diabetes. Inhibitors of AGEs, especially polyphenols from plant sources, have shown immense potential for diabetes mellitus management. In this study we evaluated the anti‐glycative effects of a series of maple gallotannins, namely ginnalins A‐C and maplexins F and J, at individual stages of protein glycation. A combination of analytical methods were used to determine AGE formation in various in vitro model systems. In the BSA‐fructose assay, maplexin F was the most potent inhibitor of AGEs formation with an IC50 value of 15.8 µM. MALDI‐TOF analysis showed that all maple gallotannins prevented the production of Amadori products, an intermediate formed in the early stage of glycation. Moreover, maple gallotannins were effective in preventing the formation of protein cross‐linking structures. Lastly, the CD spectroscopy demonstrated that the conformational changes of globular protein were reduced in the presence of maple gallotannins, suggesting that these compounds exhibited protective effect on protein's secondary structure. In conclusion, our study demonstrated that gallotannins from maple species display inhibitory activities against the formation of AGEs. Further in vivo studies are needed to evaluate the anti‐AGEs properties of maple gallotannins for their potential applications for the treatment of diabetes and related complications.
A Comparative Study of Different Protein Immobilization Methods for the Construction of an Efficient Nano‐structured Lactate Oxidase‐CNT‐Biosensor
Authors: Pagan Miraida , Suazo Damaris , del Toro Nicole , Griebenow Kai
Published: June 2021

The development of portable and robust biosensors that exhibit a superior sensitive response and reasonable long‐term stability has gained significant attention especially for monitoring human health and physical conditions. Since lactate levels in blood are a long established indicator for the physical fitness in humans, our long term goal is to develop a stable enzyme‐based biosensor for monitoring human health. The model protein for this sensor is lactate oxidase (Lox). In order to construct the biosensor, the protein was attached to a carbon nanotube (CNT) electrode. The use of CNT electrodes will help not only to warrant proper immobilization of the biomolecule to the sensor but also to provide the highest sensitivity during sensing. The central hypothesis of this work is that the use of a covalent immobilization method to attach the protein onto the CNT sensor will positively affect the long‐term stability of the sensor. To address this aim, lactate oxidase (Lox) was covalently attached to the CNT electrode via EDC‐carbodiimine coupling to provide a durable attachment. This will avoid the loss of protein via leaching when it is in contact with the analyte solution during analysis thus enhancing the long‐term stability of the sensor. Cyclic voltammetry and amperometric experiments were conducted to study the biosensor response, the sensitivity, and the detection limits. XPS demonstrated the covalent attachment of Lox onto the CNT electrode. Temperature experiments were conducted in order to study the stability of the sensor. Our results, demonstrate that the covalently immobilization of the protein onto the CNT sensor increased significantly the long‐term stability of the biosensor.
Characterization of a Small‐molecule Modulator of IRE1α Activity
Authors: Salami‐Oyenuga Jemilat , Raina Kanak , Crews Craig
Published: June 2021

Endoplasmic Reticulum (ER) stress due to the accumulation of unfolded/misfolded proteins in the ER lumen is resolved by a series of cellular processes, known collectively as the Unfolded Protein Response (UPR). UPR signaling is responsible for maintaining cell health under ER stress conditions and its misregulation has been implicated in the pathogenesis of several diseases. To remediate misregulated UPR, new ways to probe/modulate UPR signaling have to be developed. Along these lines, our lab has synthesized a novel small molecule (NA14) that interacts with and modulates the downstream activity of Inositol Requiring Enzyme 1 (IRE1α), a central player in one of the three signaling branches responsible for ER stress response. We have characterized the interaction between NA14 and recombinant human IRE1α protein using thermal shift assays and studied its effects on IRE1α kinase and ribonuclease enzymatic activities. We have also tested the toxicity of NA14 to HEK293T human kidney cells and treated these cells with NA14 to study its effects on downstream IRE1α and UPR signaling.

We show that NA14 binds to recombinant human IRE1α and also inhibits its kinase and ribonuclease enzymatic activities. We also show that NA14 is not cytotoxic to HEK293T cells. We are currently working on understanding the effects of NA14 on downstream human IRE1α and UPR signaling. Till date, we have used a luciferase reporter assay to show that NA14 downregulates the splicing of X‐Box Binding Protein 1 (XBP1) mRNA, one of the key consequence of IRE1α activation.

Our aim is to have a clear understanding of the mechanism of regulation of IRE1α activity by NA14. This would enable its use as a chemical probe for studying ER stress pathways in normal and disease contexts.
Myocardial afadin prevents cardiac dysfunction in chronic pressure‐overloaded mice
Authors: Ogita Hisakazu , Zankov Dimitar
Published: June 2021

Afadin, an adaptor protein of cell adhesion molecules nectins, plays a key role in embryonic morphogenesis and tissue organization together with or independent of nectins. However, little is understood about the contribution of myocardial afadin to the heart pathophysiology. In cardiomyocytes, afadin was found to reside in the intercalated disks. We then generated conditional knockout mice lacking myocardial afadin (afa‐/‐ mice) by the Cre/loxP system to evaluate the significance of loss of afadin. Afa‐/‐ mice were born at expected ratio and their phenotype was indistinguishable from littermate controls. Heart rate, blood pressure, and cardiac dimension and contractility showed similar results. In parallel, gross heart morphology, heart weight, histology, and intercalated disks structure in afa‐/‐ mice did not show detectable changes compared to control mice. We next used a model of chronic pressure overload (8 weeks) created by transverse aortic constriction operation. Control mice developed concentric cardiac hypertrophy, but no deterioration of cardiac function was found. Afa‐/‐ mice developed eccentric hypertrophy, and cardiac function was impaired. Interstitial fibrosis was increased, probably due to the disorganized cardiac remodeling by loss‐of‐function of afadin. Our data suggest essential participation of myocardial afadin in preventing cardiac dysfunction through the blockade of fibrosis in responses to chronic pressure overload.
Structure and function of ADCK3, an ancient mitochondrial protein involved in coenzyme Q biosynthesis
Authors: Johnson Isabel , Stefely Jonathan , Reidenbach Andrew , Pagliarini David
Published: June 2021

Coenzyme Q (Q) is an essential lipid best known for its role as an electron carrier in the electron transport chain. Although required for human health, Q biosynthesis is not completely understood and the functions of many Q biosynthetic proteins remain unclear. The atypical mitochondrial kinase, ADCK3, is a Q biosynthetic protein of unknown function belonging to the UbiB protein kinase‐like family. Here, we determine a crystal structure of ADCK3, the first crystal structure of a UbiB protein. We characterize the unique alanine‐rich nucleotide binding loop that dictates an unusual binding preference for ADP over ATP. We find that this preference can be reversed with a single alanine to glycine mutation, which also enables autophosphorylation while inhibiting Q biosynthesis in vivo. Furthermore, we identify an N‐terminal domain that blocks the substrate binding pocket—another inhibitory feature. This work serves as a foundation for further structure‐function analyses aimed at defining the role of ADCK3 in Q biosynthesis as part of a larger effort to treat human Q deficiency.
Defining the Role of Arylsulfatase B (N‐Acetylgalactosamine 4‐Sulfatase) in Cellular Metabolism
Authors: Bhattacharyya Sumit , Feferman Leo , Tobacman Joanne
Published: June 2021

Arylsulfatase B (ARSB; N‐acetylgalactosamine 4‐sulfatase) is the lysosomal and membrane enzyme that removes 4‐sulfate groups from the non‐reducing end of chondroitin 4‐sulfate and dermatan sulfate, and thereby regulates their degradation. Studies have demonstrated decline in ARSB in patients with cystic fibrosis and in malignant human mammary, prostate, and colonic epithelial tissues. Several molecules, including Interleukin‐8, high molecular weight kininogen, BMP4, and galectin‐3, bind preferentially to more or less sulfated C4S, affecting cell‐cell and cell‐extracellular matrix interactions. Previously, we reported that hypoxia reduced ARSB activity and that decline in ARSB replicated the effects of hypoxia on the expression of 84 genes in a hypoxia gene array. Both hypoxia and silencing ARSB decreased the ratios of reduced glutathione to GSSG and of cellular sulfhydryls to sulfides. Recent experiments in ARSB‐deficient mice and human epithelial cells following ARSB silencing have shown: increased serum lactate, increased 2,3‐DPG, increased extracellular acidfication, reduced oxygen consumption rate, reduced mitochondrial membrane potential, disruption of inner and outer mitochondrial membranes by electron microscopy, and increased NADPH to NADP and NADH to NAD ratios. These findings suggest profound effects of decline in ARSB on cellular metabolism and oxidation‐reduction reactions. Sulfate reduction is utilized in protists, plants, and algae for energy production. Impairment of sulfate reduction due to unavailability of sulfate due to ARSB deficiency may help to explain the occurrence of aerobic glycolysis in human cells.
Differential Effects of FAK and FGFR Inhibitors on Motility and Proliferation of L1‐Positive versus L1‐Negative Glioblastoma Cells
Authors: Anderson Hannah , Galileo Deni
Published: June 2021

Glioblastoma multiforme (GBM), a highly invasive astrocytoma, is the deadliest form of brain cancer. Glioblastoma cells express the L1CAM cell adhesion protein (L1), which is cleaved at the cell surface and binds to both integrins and fibroblast growth factor receptors (FGFRs) in an autocrine/paracrine manner. Focal adhesion kinase (FAK), which is activated by L1‐integrin interactions, and FGFR are tyrosine kinases that initiate signaling cascades stimulating GBM motility and proliferation. We hypothesized that inhibitors of FGFR and FAK would have differential effects on the motility and proliferation of T98G glioblastoma cells expressing L1 compared to those with attenuated L1 expression. Short hairpin RNA delivered by a lentiviral vector was used to block L1 expression. Cell motility was measured through quantitative time‐lapse microscopy, and proliferation was measured by cell cycle analysis and quantified as the population of cells in S phase. FAK inhibitor Y15 had a greater effect in L1‐positive versus L1‐negative cells on motility (‐45.3% vs. ‐5.6%) and S phase population (‐6.5% vs. ‐3.5%). FAK inhibitor PF431396 also produced a greater reduction in motility (‐57.2% vs. ‐15.4%) and S phase population (‐11.7% vs. ‐6.2%) in L1‐positive versus L1‐negative cells. An inhibitor of FGFR, PD173074, reduced motility (‐37.8% vs. +2.3%) and S phase population (‐8.6% vs. ‐0.4%) in L1‐positive cells only. We conclude that inhibitors of FAK and FGFR drastically decrease L1‐stimulated motility and proliferation in glioblastoma cells, indicating that they have chemotherapeutic potential for GBM tumors expressing L1. Research support was provided by the Delaware Governor's Bioscience Fellowship and the Center for Advanced Technology.
Investigating the Role of Antioxidants in Protecting Saccharomyces cerevisiae from Oxidative Damage
Authors: Thompson‐Van Hook Nichole , Mordan‐McCombs Sarah
Published: June 2021

The free radical theory of aging suggests that cellular aging is due to the buildup of oxygen‐derived free radicals which damage biological molecules. These free radicals, known as reactive oxygen species (ROS), are produced as a secondary product of oxidative phosphorylation in the mitochondria. Cells maintain existing mechanisms to neutralize ROS, via both enzymatic and non‐enzymatic means. The thiol antioxidants, such as glutathione, form disulfide bonds to reduce the oxidative potential of ROS. Multiple studies have implicated alterations of the glutathione‐glutaredoxin system in tissue degeneration and cellular aging, and genetic mutations in antioxidant enzymes have been correlated with altered antioxidant capacity and lifespan in Drosophila, PC‐12 neuronal cells, and yeast. In this study, GSH2, glutathione synthetase, was deleted from S. cerevisiae to investigate the effect of altering the endogenous antioxidant balance. We hoped to mimic the changes that are believed to occur in the earliest stages of neurodegenerative diseases, such as Alzheimer's and Parkinson's. ΔGSH2 deletion mutants were pre‐treated with the commercially available thiol antioxidant cysteamine to determine its ability to protect the cells from oxidative damage caused by hydrogen peroxide. We hypothesized that, due to the structural similarities between cysteamine and glutathione, cysteamine would act as a substitute for glutathione when GSH2 was lost. Here we show alterations in gene and protein expression of relevant glutathione‐glutaredoxin system proteins as a result of the loss of GSH2 function.

This work is supported by the Biology Department at Franklin College and by a grant from the Indiana Academy of Science.
Characterization of Myelin P2 and Slitrk Echinoderm Genes and Their Expression During Enteric Nervous System Regeneration
Authors: Monahan Vargas Ernest , Otero James , García Arrarás José
Published: June 2021

Holuthuria glaberrima is an echinoderm known for its ability to regenerate its internal organs including its enteric nervous system (ENS). Unfortunately, there is a limited amount of nervous component markers and many of the molecular events that occur during the regeneration of this system in the sea cucumber are still unknown. New insights into the regeneration of the holothurian ENS may be provided by determining the genes involved and differentially expressed in this process. Using a database generated by Next Generation Sequencing (NGS), we identified two genes that might be associated with regeneration: Slitrk and the gene that encodes Myelin P2 protein. Slitrk proteins have been shown to modulate neurite outgrowth in vertebrate animals. Myelin P2 protein is one of the components of the myelin sheath in vertebrate glial cells, and holothurian glia has been shown to play an important role in the regeneration of the holothurian CNS. We have shown that these genes are present and expressed in normal and regenerating sea cucumber by using various bioinformatic tools, sqPCR and immunohistochemistry. We established a gene expression profile of various tissues and at different stages of intestinal regeneration. To view the spatial localization of Myelin P2 protein, we developed an antibody and localized its expression to the ceolomic epithelium of the intestine and muscle. Finally, our work also explores the evolution of myelin‐related proteins, an interesting development from our findings, since myelin is not found in invertebrate nervous systems. In conclusion, we believe that understanding the role of these genes may aid in the elucidation of possible patterns of ENS regeneration of H. glaberrima.
Dimerization of EphA2 in Plasma Membranes
Authors: Hristova Kalina , Singh Deo , King Christopher , Salotto Matt , Gupta Nisha
Published: June 2021

EphA2 is a single pass transmembrane protein and a member of the largest Receptor Tyrosine Kinase (RTK) family. EphA2 regulates cell‐cell interactions during embryonic development in humans and is known as an oncoprotein as well as a tumor suppressor. The Eph receptors differ from other RTKs since they form oligomers when they bind their ligands. However, the interactions between the Eph‐A2 receptors in the absence of ligand have not been investigated. We used spectral FRET in conjunction with two photon microscopy to probe unliganded EphA2 dimerization in HEK 293 cells. We measured the FRET efficiency with high precision over a concentration range that spans three orders of magnitude and includes the physiological range of receptor expressions. We show that EphA2 forms dimers in the absence of ligand. In addition, mutagenesis studies reveal that contacts between the EphA2 receptors that are important for ligand‐mediated clustering are also important for unliganded dimerization. We therefore propose that unliganded EphA2 dimers are an important intermediate in EphA2 signal transduction.
Pumpkin Seed Oil Composition Analysis and Research on the Prevention Effects of Vascular Disease
Authors: Kim Kyoung Kon , Kang Yun Hwan , Kim Dae Jung , Kim Tae Woo , Lee Jeong Il , Choe Myeon
Published: June 2021

Pumpkin seed oil (PSO) was investigated for parasite elimination activity and therapy of disorders of the prostate gland and the urinary bladder. However, analysis of components and efficacies of PSO were not enough. In this study, we confirmed the composition and identified that may be a help to vessels. Gas chromatography coupled with mass spectrometric (GC‐MS) analysis system was used for PSO composition assay. Cytotoxicity and cell proliferation were confirmed by Cell Counting Kit‐8 (CCK‐8) assay. Nitric oxide (NO) production was measured with Griess reagent, and intercellular adhesion molecule (ICAM)‐1 and vascular cell adhesion molecule (VCAM)‐1 mRNA expression were measured by reverse transcription polymerase chain reaction (RT‐PCR). As result, PSO revealed the presence of several components like linoleic acid, oleic acid, palmitic acid and stearic acid. The cytotoxic effect of PSO was not observed and PSO increased nitric oxide production on human umbilical vein endothelial cell (HUVEC). Additionally, TNF‐α induced cell proliferation and ICAM‐1 of HUVEC were inhibited by PSO treatment, but VCAM‐1 was not significantly decreased. Taken together, these results show that PSO is worthy of study as candidate food material for improvement of vascular disease.
Comparative Energy Flow Analysis on Dairy Farms in North Carolina and Malawi
Authors: Chizonda Shalimbala , Dos Passos Adsos , Sun Yawang , Allen Jonathan
Published: June 2021

Milk is a major nutrient source in many Western diets, but in developing countries like Malawi, milk intake is low; increasing its availability from local sources could significantly improve nutrient intake in the population. The objective of this study was to compare energetic inputs, outputs, and efficiencies of dairy farms in Malawi (Southeast Africa) and North Carolina to assess sustainable milk production. Feed consumption and milk production data were collected from Katete Dairy herd in Lilongwe, Malawi, consisting of 408 cattle with 108 lactating cows, and the NCSU Dairy Research and Teaching Farm, housing 245 Holstein and 55 Jersey cows and 170 lactating cows. Both are teaching farms that employ extensive record keeping. Milk production averaged 34 and 19.5 kg/cow/day at NCSU and Katete, respectively. Feed energy intake was 85.8 mcal/cow/d at NCSU and 135 mcal/cow/d at Katete. Thus, return of feed energy as milk energy was 24.7% efficient at NCSU and 11.3% efficient in the lower producing cows at Katete. Manure is recycled for crop production at Katete, and is processed with a solids separation and lagoon system at NCSU. Additional factors that impact the overall farm efficiencies include inputs of petroleum fuels and electricity, and the number of calves, heifers and dry cows fed. The project identifies factors in dairy production that should be considered to improve efficiency, sustainability and milk supply. A greater supply of locally produced milk in Malawi could improve essential nutrient intakes.
Educator Interactions at Head Start Lunches: A Context for Nutrition Education
Authors: Swindle Taren , Whiteside‐Mansell Leanne
Published: June 2021

Early childhood educators in center‐based care share lunch with preschool children over 150 times in a school year. Unfortunately, research shows that, in terms of nutrition education, lunch is a missed opportunity (Larson et al., 2011). Worse, educator behaviors were often negative in a landmark 2001 study of Head Start (Gable & Lutz, 2001). The current study examined Head Start educators lunchtime interactions with children with the goal of (a) updating the state of mealtimes in the Head Start setting and (b) expanding observations to a greater number of classrooms (n =21) across different centers (n= 7).

Lunch observations were conducted by observers who were trained to code phrases by lead and assistant teachers into either 4 positive or 4 negative categories. A broad category related to behavior management was also recorded when firm directives, not appropriate transitions, were used. Interrater reliability of 85% or greater was obtained for all observers.

Table 1 presents the mean number of times each target interaction was observed. Lead teachers were more positive than assistant teachers in their comments and generally more vocal. On average, 54% of lead teachers and 61% of assistant teachers' comments were not supportive of a healthy, nurturing food environment.

Findings illustrate that a high proportion of interactions at Head Start meals between educator and child are either negative or focused on behavioral control (57% of total comments). Children were commanded to eat 4 times more frequently than they were directed to internal hunger cues. This descriptive, naturalistic, observational study highlights the continued need for improved policy and training for educators acting as role models for at‐risk children.

Table 1. Frequency of Educator Interactions by Teacher Role

LEAD
ASSISTANT
TOTAL

Positive Language

Positive comments about food served (e.g., I am really enjoying the carrots today. Yummy!)
4.64
3.29
7.93

Hunger cues (e.g., Are you full? How does your belly feel?)
1.50
1.00
2.50

Encourage trying in a positive way (e.g., Would you like to try the peas? Peas are good for our bodies.)
4.38
2.25
6.63

Exploring foods (e.g., What does it smell like? How does it feel in your mouth?)
3.39
2.15
5.54

Negative Language

Pressure to eat (e.g., Eat your food. Take a bite. Clean your plate.)
6.22
4.17
10.39

Threats to encourage eating (e.g., If you don't eat, you'll be over here by yourself.)
0.45
0.39
0.84

Discourage manipulating food (e.g., Use your manners. Eat, don't play. That's sticky and nasty.)
1.81
2.26
4.07

Hurry to finish eating (e.g., We're waiting on you. Let's hurry so we can go to recess.)
1.10
1.02
2.12

Behavior Language

Focus on behavioral control (e.g., Turn around. Sit up straight. Hands in your lap.)
6.89
5.75
12.64
Using Random Forest Method to Select Most Valued Items on a Healthy Weight [Obesity Risk] Survey for Young Children
Authors: Aden Christopher , Drake Christiana , Shilts Mical , Townsend Marilyn
Published: June 2021

Random forest is an alternative statistical method to generalized linear models. The objective was to use and compare the two statistical methods that identify the most predictive questions for various outcomes including predicting overweight status at a later time point. Head Start and WIC parents and their young children provided data: parent self‐administered 43‐item Healthy Kids (HK) at 4 time points, nine 24‐hour diet + activity recall logs, 3 blood samples and child anthropometrics collected at 3 time points. An importance plot determined the relative importance of each question by computing the average error when the particular question is omitted from a tree. Of the 43 behavioral items tested, 16 are selected for the final model. The chosen model has a similar classification rate as the stepwise regression procedure in looking at overweight status; it also provides the most important variables in this prediction task, along with weights. The advantage of random forest is that it selected models reasonably supported by nutritional knowledge, and avoided problems faced by stepwise regression in fitting large models. Random forest has potential for use in nutritional studies.

Supported by National Research Initiative #2009‐55215‐05019 & Agriculture and Food Research Initiative #2010‐85215‐20658 from USDA National Institute of Food and Agriculture, Human Nutrition & Obesity 93330.
Effects of Consuming Dairy Fat vs Plant‐based Fat with and without Milk Fat Globule Membrane on Postprandial Inflammatory Markers in Overweight and Obese Adults
Authors: Demmer Elieke , Van Loan Marta , Rivera Nancy , Zivkovic Angela , Smilowitz Jennifer , German J , Gertz Erik , Rogers Tara
Published: June 2021

Not all foods rich in SFA are equivalent and may vary in their effect on postprandial inflammation. Additionally, milk fat globule membrane (MFGM) is suggested to have anti‐inflammatory properties. To evaluate the effects of SFA from dairy (WC, whipping cream) vs. a plant source (P, palm oil) on postprandial inflammation markers with and without MFGM a randomized controlled 4‐way cross‐over trial was conducted. Overweight adults with 2 metabolic syndrome (MetS) traits or obese individuals consumed 4 isocaloric high‐fat meals separated by a 1‐2wk washout period. Serum was collected at 0, 1, 3, and 6hrs postprandially and analyzed for inflammatory markers (IL‐6, IL‐8, IL‐10, IL‐18, TNFα, CRP, SAA, sICAM, sVCAM) and metabolic markers (glucose, insulin, triglycerides, HDL‐C, and LDL‐C). Following all high‐fat mixed meals, postprandial TG concentrations rose steadily and peaked at 3hrs (p<0.005). There was a time effect for all markers, yet, no treatment effect was observed. Using incremental area under the curve sICAM, there was a treatment effect for P vs. P+MFGM (p=0.039). Using 3+ MetS traits as a covariate, insulin concentrations were lower at 1hr in response to WC+MFGM vs. P and WC (p=0.05 and p=0.01 respectively) and for P+MFGM vs. WC (p=0.04). In individuals with MetS the addition of MGFM dampened the postprandial insulin response. SFA from dairy does not trigger a greater inflammatory response than a plant SFA source.

Funding:

National Dairy Council
Effect of Dairy Foods on Insulin Resistance
Authors: Hilpert Kirsten , West Sheila , Harrington Emily , Kris‐Etherton Penny
Published: June 2021

While studies have shown an inverse relationship between dairy consumption and metabolic abnormalities such as hypertension, obesity, diabetes, metabolic syndrome and insulin resistance, the mechanisms have not been fully elucidated. Some evidence exists that dairy‐rich diets produce an ideal ionic cellular environment which may improve insulin resistance. Adults with untreated hypertension (n=19) were fed 3 experimental diets (5 wk each) in a randomized crossover study design. Treatments included a high dairy, high fruits and vegetables diet (30% fat, 7% saturated fat [SFA], 3.4 servings [svg]/d dairy, 9.6 svg/d fruits and vegetables ( F&V)), a low dairy, high fruits and vegetables diet (30% fat, 7% SFA, 0.4 svg/d dairy, 9.6 svg/d F&V), and an average Western diet (36% fat, 15% SFA, 0.4 svg/d dairy, 3.6 svg/d F&V). Blood levels of insulin and glucose were measured at baseline and after each diet period. Insulin resistance was determined using the homeostatic model assessment (HOMA) equation and the quantitative insulin‐sensitivity check index (QUICKI). There were no significant differences between treatments for any endpoint. The addition of 3 servings of dairy did not impact markers of insulin resistance in this small study of hypertensive, non‐diabetic participants.

Funding:

National Dairy Council, GCRC of Penn State University NIH M01 RR10732
Effects of low dose dried plum (50 g) on bone mineral density and bone biomarkers in older postmenopausal women.
Authors: Metti Dina , Shamloufard Pouneh , Cravinho Amanda , Delgado Cuenca Paulina , Kern Mark , Arjmandi Bahram , Hooshmand Shirin
Published: June 2021

Our previous findings in osteopenic postmenopausal women indicated that daily consumption of 100 g dried plum for one year is highly effective in increasing bone mineral density (BMD), as well as improving indices of bone turnover. The objective of our current study was to examine whether 50 g dried plum would be as effective as 100 g dried plum in reversing bone loss in osteopenic older postmenopausal women. Forty Eight osteopenic women (65‐79 years old) were randomly assigned into one of three treatment groups: 1) 50 g dried plum; 2) 100 g dried plum; and 3) control (0 g dried plum) with forty two subjects completing the study. All groups received 500 mg calcium and 400 IU vitamin D as a daily supplement. Blood samples were collected at baseline, three and six months to assess biomarkers of bone turnover. Physical activity recall and three‐day food records were obtained at baseline, three and six months to examine physical activity and dietary confounders as potential covariates. Both doses of dried plum significantly increased total body BMD. Tartrate resistant acid phosphatase‐5b (TRAP‐5b, a marker of bone resorption) decreased at three months and six months in both dried plum groups. These results confirm the ability of dried plum in improving BMD in older postmenopausal women and suggest that lower doses of dried plum (i.e. 50 g) may be as effective as 100 g dried plum in reversing bone loss in older, osteopenic postmenopausal women. Hence, our findings suggest that the consumption of a reasonable amount of dried plum is beneficial for older, osteopenic women.
The Center for Nutrition, Learning, & Memory: Together Addressing a Grand Challenge
Authors: Moulton Chris , Wee Emily , Kuchan Matthew , Garleb Keith , Cohen Neal
Published: June 2021

In 2011, Abbott Nutrition and the University of Illinois at Urbana‐Champaign (UIUC) embarked upon a Grand Challenge in the creation of the nation's first interdisciplinary nutrition and cognition research center, now known as the Center for Nutrition, Learning, and Memory (CNLM). The Center's objectives are to advance the breadth and depth of scientific knowledge at the intersection of nutrition and neuroscience, and to translate that knowledge into meaningful benefits for cognitive health across the lifespan. To fulfill this mission, the CNLM annually solicits a research competition and funds interdisciplinary, team‐based projects in basic research, method development, and translational/clinical research. Key areas of focus include promotion of neuronal development, enhancement of learning and memory, and mitigation of age‐related cognitive decline. Funded projects are selected by an Executive Committee equally comprised of Abbott & UIUC representatives. Since its inauguration, the CNLM has funded 30 interdisciplinary research projects involving 15 UIUC departments at a rate of over $25 million. Through these projects, the CNLM collaborative network has expanded to include 15 national & international partner institutions along with Abbott scientists across the globe.
Dietary dairy proteins improve lipoprotein profiles in C57/Bl6 mice
Authors: Price Tara , Wu Xiuzhi , Baskaran Sangeetha , Walzem Rosemary
Published: June 2021

A third of HDL forms in the gut; HDL's Apo A1 also acts in innate immune function. Thus microbiota may alter HDL biology. Gut microbiota differed in C57/Bl6 mice fed from weaning to 13wks with diets providing 30%EN fat, 50%EN carbohydrate and 20%EN protein as either isolated soy protein (ISP), dried whole milk powder (DWMP), milk protein concentrate (MPC) or milk fat globule membrane (MFGM). Growth rates, feed intakes and final body weights were similar. Overall, dairy proteins had similar effects on lipoprotein classes. Relative to ISP, dairy proteins increased triacylglycerol‐rich lipoproteins (2.8% v.1.4%) and HDL (75.6% v. 70.0%) and decreased LDL (21.6% v. 28.5%, p<0.05) as determined by AUC for fluorescently labeled lipoproteins imaged after isopycnic separation. Small dense LDL were reduced 70% in dairy protein‐fed mice (p<0.005). HDL2a increased ~12% in mice fed MPC and MFGM (p<0.05). Cytokines and inflammatory marker genes showed a non‐inflammatory state for all groups. Gut permeability and plasma LPS concentrations were low for all groups; permeability was least (p<0.05) in DWMP fed mice. Overall gut motility was similar. Small intestinal and hepatic ApoA1 gene expression was similar among diet groups indicating that neither gene expression nor inflammation led to differences in HDL. Consumption of dairy proteins appears to improve lipoprotein profiles in C57/Bl6 mice when consumed within the context of a US Dietary Guidelines macronutrient distribution. Funding provided by National Dairy Council project #1120 administered by Dairy Research Institute.
The Serine Protease PARK13 Degrading Misfolded Parkinson's Disease Proteins And Confers Cellular Protection
Authors: Basak Indranil , Patil Ketan , Larsen Jan Petter , Møller Simon
Published: June 2021

Mutations in HTRA2/Omi/PARK13 have been associated with Parkinson's disease (PD) and PARK13 show neuroprotective properties in mice. However, how PARK13 confers neuroprotection is largely unknown. We have developed Arabidopsis thaliana as a new multicellular and complementary plant model for studying PD‐associated proteins. We have shown that Arabidopsis AtPARK13, similar to human PARK13 (hPARK13), is a mitochondrial protease that confers thermotolerance. At elevated temperatures protein unfolding is more prevalent and we have demonstrated that unfolded casein acts as a better substrate for AtPARK13 as compared to native casein. Following these findings we showed that AtPARK13 is capable of degrading the PD proteins α‐synuclein (SNCA) and DJ‐1 with higher activity in response to their unfolded states. We also demonstrated that hPARK13 can degrade SNCA and clinical DJ‐1 mutated variants without autophagy involvement. We further investigated this phenomenon in neuroblastoma cells showing that monomeric SNCA is degraded by hPARK13. Interestingly, we further showed that higher order SNCA states are targets of cytosolic, membrane‐bound and nuclear hPARK13 suggesting that hPARK13 acts on different SNCA states depending on the subcellular localization. Our study not only suggests a new mechanism for PARK13 but also sheds light on its possible involvement in neurodegenerative diseases.
Gene Expression Profiling of Zebrafish (Danio rerio) Exposed to the Steroidogenesis Inhibitor Ketoconazole
Authors: See Mary Jean , Bencic David , Wang Rong‐Lin , Villeneuve Daniel , Ankley Gerald , Biales Adam
Published: June 2021

Ketoconazole (KTC) has been shown to alter the reproductive function of small fish through inhibition of enzymes in the steroid biosynthesis pathway. The purpose of this research was to examine the transcriptomic response of adult zebrafish (Danio rerio) to KTC exposure. Zebrafish were treated up to 96 h with 400 µg/L KTC in a flow‐through system. Total RNA was isolated from brain, liver and gonad and gene expression changes were identified using a two color, high density zebrafish microarray. Differentially‐expressed genes were identified (p<0.05; FDR<30%) then were analyzed by gene ontology term clustering, gene set enrichment analysis (GSEA) and Ingenuity Pathway Analysis (IPA). Results in liver showed a possible disruption of cholesterol and steroid biosynthesis and activation of cytokine signaling pathways. Analyses of brain gene expression demonstrated oxidative stress, down regulation of an oxioreductase and increase in estrogen receptor 2b (esr2b). In ovary, oxidative stress response, oxidative phosphorylation and hormone receptor regulated cholesterol metabolism were enriched along with an increase in esr2b expression. Pathways known to be effected by inhibition via KTC; cholesterol biosynthesis, steroid biosynthesis and xenobiotic metabolism, are finely controlled at multiple levels of regulation. The results presented here provide insight into the transcriptional regulation of these broad and interactive processes at an isolated time point.
Structural Basis for Bacterial Quorum Sensing‐mediated Oxalogenesis
Authors: Oh Juntaek , Goo Eunhye , Hwang Ingyu , Rhee Sangkee
Published: June 2021

Bacterial quorum sensing (QS), a cell‐to‐cell communication process in many Proteobacteria, controls the gene expression for bacterial population‐wide characteristics, including bioluminescence, motility, and virulence‐related factors. Recently, QS has been recognized to provide further benefits at the population level by regulating the production of public goods, the function of which could be beneficial to all members of the group. In Burkholderia species, oxalic acid was recently identified as an excreted public good for the QS‐dependent growth. In these species, QS‐mediated oxalogenesis via the oxalate biosynthetic component (obc) is a cellular event indispensable for the survival of bacteria in the stationary phase. Specifically, the acidity of the oxalic acid produced regulates the pH of the environment, avoiding a possible sudden collapse of the bacterial population caused by an alkaline pH in the stationary phase of their growth. In B. glumae, obc consists of two genes encoding ObcA and ObcB for coordinating the production of oxalic acid, as well as acetoacetate and CoA, by using oxaloacetate and acetyl‐CoA as substrates. In contrast, the two substrates are also utilized by citrate synthase in the TCA cycle. To investigate any structural and functional differences between ObcA and citrate synthase, we are now carrying out structural and functional analysis of ObcA. Here, we will report our progresses on crystallization and structure determination of ObcA from B. glumae. Our analyses will provide structural insights into the first step in oxalogenesis and the mechanistic features of ObcA. This work was supported by Next Generation BioGreen 21 program of Rural Development Administration (Plant Molecular Breeding Center) of Republic of KOREA.
Structure of a Bacterial Ketoisomerase/N‐acetyltransferase
Authors: Chantigian Daniel , Thoden James , Holden Hazel
Published: June 2021

3‐acetamido‐3,6‐dideoxy‐a‐D‐galactose, Fuc3NAc, is an unusual dideoxysugar found in the O‐antigens of various Gram‐negative bacteria and in the S‐layer glycans of some Gram‐positive bacteria. The biosynthetic pathway for its production in Aneurinibacillus thermoaerophilus L420‐91T was elucidated in 2003. In the case of A. thermoaerophilus, all of the enzymes required for the production of dTDP‐Fuc3NAc exist on separate polypeptide chains. However, this is not the case for other organisms. In Shewanella denitrificans, a Gram‐negative bacterium, a gene (fdtD) has been identified that putatively encodes for a bifunctional protein with both 3,4‐ketoisomerase and N‐acetyltransferase activities (the third and fifth steps in the pathway). The quaternary structure of this hypothetical protein is intriguing since the sugar isomerases and the N‐acetyltransferases are typically dimers and trimers, respectively. Curious as to its molecular architecture, we initiated a biochemical and structural analysis on this protein. From our investigation, it is now known that the quaternary structure of the enzyme is hexameric with 322‐symmetry. Each polypeptide chain of the hexamer folds into two specific domains connected by a flexible loop. The N‐terminal ß‐helix motif harbors the active site responsible for the N‐acetylation reaction. The C‐terminal region folds into an antiparallel flattened ß‐barrel and contains the active site responsible for isomerization. Biochemical assays verified the two proposed catalytic activities of the enzyme and revealed that the 3,4‐keto isomerization event leads to inversion of configuration about the hexose C‐4' carbon. This work represents the first molecular characterization of a bifunctional protein involved in the biosynthesis of Fuc3NAc.
Cyclic AMP/PKA‐Mediated Regulation of Mitochondria and Branched‐Chain Amino Acid Metabolism in S49 Lymphoma Cells
Authors: Wilderman Andrea , Guo Yurong , Divakaruni Ajit , Perkins Guy , Zhang Lingzhi , Murphy Anne , Taylor Susan , Insel Paul
Published: June 2021

The role of mitochondria in responses mediated by the second messenger cAMP acting via protein kinase A (PKA) is poorly understood. To assess such a role, we used quantitative proteomic analysis of mitochondria‐enriched fractions and performed functional and morphological studies of wild‐type (WT) and kin‐ (PKA‐null) murine S49 T lymphoma cells. We found increased abundance of 110 proteins, including 8 involved in branched‐chain amino acid (BCAA) metabolism, in WT, but not kin‐, S49 cells incubated for 16 hr with the cell‐permeable cAMP analog 8‐CPT‐cAMP (CPT). CPT also increased mRNA expression of genes involved in BCAA metabolism in WT, but not kin‐, S49 cells. In addition, CPT enhanced survival of WT cells (but not kin‐ cells or WT cells in the presence of the PKA inhibitor H89) grown in media lacking glutamine. Studies using a Seahorse XF analyzer revealed that CPT enhanced the maximal rate of respiration and the capacity of WT cells to oxidize BCAA, while diminishing glycolytic flux (indicated by extracellular acidification). Basally, mitochondria of WT and kin‐ S49 cells have similar structural features but 16 hr incubation of WT (but not kin‐) cells with CPT produced changes of apoptosis (e.g. decreased mitochondrial density and size, and loss of cristae). Together, these findings show that cAMP, acting via PKA, regulates BCAA metabolism, mitochondrial function and structure. Regulation of mitochondria thus likely contributes to cAMP/PKA‐mediated cellular responses. Support: HHMI (S.S.T.), NIH DK54441 (S.S.T.) and GM066232 (P.A.I.)
Putative Role of Glutathione S‐transferases as Mediators of Cell Growth, Development, and Differentiation in the Eukaryotic Model Organism, Dictyostelium discoideum
Authors: Garige Mamatha , Walters Eric
Published: June 2021

In addition to their role in detoxification, Glutathione S‐transferase (GST) enzymes influence signal transduction, development, and differentiation in many eukaryotic cells. Transcriptome analysis of the cellular slime mold, Dictyostelium discoideum (strain AX4) expresses five GST (α1‐α5 class) transcripts whose function(s) remain unknown. This study characterized the activity and expression of GST isozymes in D. discoideum at various stages of growth and development. A GSH‐agarose pull down assay of the lysates from vegetative amoebae identified two protein species of ~24kDa; mass spectrophotometric analysis identified GSTα3> GSTα1>GSTα2 as predominant species. Measurements of GST activity in amoeba using the substrate 1‐chloro‐2‐4‐dinitrobenzene (CDNB) revealed functional enzyme activity, which was significantly reduced by as much as 50% in the presence of 500 nM ethacrynic acid (EA, a GSTs inhibitor). Ethacrynic acid also delayed the formation of aggregates and multicellular development. Levels of GSTs activity differed significantly between vegetative amoebae and multicellular stages of development (aggregate, slug, culmination, fruiting body), indicating the importance of GSTs to influence cell signaling and gene expression during morphogenesis and differentiation. Current studies aim to determine: a) the levels of each isozyme and their substrate specificities; b) the impact of GST null mutants on growth and development of the organism. These studies will contribute to elucidating the expanding the role and function of GST enzymes in eukaryotic signal transduction that regulates cell growth, chemotaxis, and differentiation.
A Systematic Review of the Conceptualization and Measurement of Sustainable Diets
Authors: Jones Andrew , Hoey Lesli , Blesh Jennifer , Green Ashley , Miller Laura , Shapiro Lilly
Published: June 2021

There is currently no clear operational conceptualization or metric of a “sustainable diet”. We aimed to identify the range of conceptualizations of and approaches to measurement of sustainable diets through a comprehensive, systematic review of the scientific and grey literatures. Two independent reviewers screened the titles and abstracts of 719 articles that were identified through the systematic searching of 15 databases using a uniform set of search terms focused on use of the term “sustainable diet”. In total, 95 empirical and conceptual articles were included in the final review. Few empirical studies have attempted to directly measure the sustainability of diets. We identified only 24 such studies, nearly all (n=23) centered on high‐income countries. All but five of the identified studies assessed sustainability through measurement of greenhouse gas emissions (GHGE) associated with the production, processing, and transport of a limited variety of foods. Six studies considered the nutritional quality of diets in addition to GHGE and these primarily emphasized differences between meat‐based and vegetarian diets. Five studies examined the economic costs of diets alongside environmental considerations. Conceptualizations of the sustainability of diets spanned a range of environmental, social, economic, and health‐related domains. Empirical data measuring the sustainability of diets are limited, particularly in low‐ and middle‐income countries, and emphasize only one of several principal domains of sustainability.