bluuebunny/crossref_metadata_2025 huggingface dataset:
An interactive method for teaching anatomy of the human eye for ophthalmology rotations using suid eyes
Authors: Kivell Tracy L , Doyle Sara K. , Madden Richard H , Mitchell Terry , Sims Ershela
Published: June 2021

Much research has shown the benefits of additional anatomical learning and dissection beyond the first year of human gross anatomy during postgraduate medical training. We have developed an interactive method for teaching second‐year medical students in their ophthalmology rotation at Duke University School of Medicine. We provide review lectures on the detailed anatomy of the adult human eye and orbit as well as the development of the eye. This is followed by an anatomical demonstration of the human orbit and eye on a prosected human cadaver, projecting the dissection of the detailed anatomy onto a computer screen. Each student then dissects a fresh suid eye under a regular‐strength microscope, identifying the external musculature and focusing on the detailed anatomy of the internal eyeball. The anatomy and size of the suid eye is comparable to that of the human and thus this method provides students a valuable learning opportunity to dissect anatomy that is most often not preserved or unidentifiable in the human cadaver.

Grant Funding Source

NSF
Single‐Stranded, Primer‐Template, and Blunt‐End DNA Selectivity by Klenow and Klentaq Polymerases
Authors: Wowor Andy J. , Datta Kausiki , Thompson Greg , LiCata Vince J.
Published: June 2021

Understanding substrate selection by DNA Polymerase I is important for characterizing the balance between DNA replication and repair for this enzyme in vivo. Due to their sequence and structural similarities, Klenow and Klentaq, the "large fragments" of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus, are considered functional homologues. We have examined the DNA binding thermodynamics of Klenow and Klentaq to different DNA structures: single‐stranded DNA (ss‐DNA), primer‐template DNA (pt‐DNA), and double‐stranded DNA (ds‐DNA). The DNA binding affinity trend for Klenow from weakest to tightest binding is ds‐DNA < pt‐DNA < ss‐DNA. This is in contrast to Klentaq's DNA binding trend: ss‐DNA < pt‐DNA ≈ ds‐DNA. Both Klenow and Klentaq release more ions and have larger heat capacity changes when binding to pt‐DNA and ds‐DNA than when binding to ss‐DNA in KCl buffer. It is also found that Mg2+ significantly shifts the ds‐DNA binding affinity of Klenow, but not Klentaq. The differences in DNA structural selectivity of the two polymerases suggest that the in vivo functions of these two supposedly homologous polymerases are different, and that Taq polymerase is more likely to be involved in ds‐break repair and end‐preservation in vivo. Funded by the NSF and the LBRN.
Protein splicing of the Pyrococcus abyssi lon protease intein
Authors: Schufreider Ann K. , O'Brien Kathryn M. , McGill Melissa A. , Reitter Julie N. , Mills Kenneth V.
Published: June 2021

The lon‐b protease of the extreme thermophile Pyrococcus abyssi is interrupted by an intein. Inteins are intervening polypeptides that catalyze their own excision from, and the concurrent ligation of, the flanking polypeptides, called exteins. We have shown that the P. abyssi lon‐b protease intein can facilitate protein splicing when over‐expressed in E. coli as a fusion protein with affinity domains serving as exteins. We have generated inteins with site directed mutations of the nucleophilic residues at the splice junctions, and these inteins cannot facilitate splicing. In other inteins, such mutations lead to cleavage at the splice junctions, but these side reactions are not as significant with the P. abyssi lon intein. We have also made mutations to study the influence of the penultimate Lys on splicing and/or C‐terminal side reactions. This Lys replaces a highly conserved His. We are also studying the influence of Gly residues in the extein that flank the intein insertion site. This material is based upon work supported by the National Science Foundation under Grant No. 0320824 and CAREER grant No. 0447647.
Role of a flexible loop in the mechanism of desulfonation by alkanesulfonate monooxygenase
Authors: Xiong Jingyuan , Ellis Holly R
Published: June 2021

The alkanesulfonate monooxygenase (SsuD) catalyzes the oxidation of alkanesulfonate to aldehyde and sulfite in the presence of O2 and FMNH2 provided by an FMN reductase (SsuE). Studies have shown that SsuD activity is completely abolished with amino acid substitutions of Arg297 located on a flexible loop. The flexible loop is proposed to close over the active site when substrates bind. In the presence of substrates, wild‐type SsuD is less susceptible to rapid proteolysis by trypsin, whereas SsuD variants of Arg297 are less protected. These results suggest that Arg297 may facilitate loop closure over the active site with the binding of substrates.

Investigations were performed to evaluate loop movement and the role of Arg297 in facilitating loop closure. Phe261 located on the loop was mutated to Trp to generate a fluorescent probe to monitor loop movement. F261W SsuD variant showed a higher fluorescence intensity than wild‐type, supporting the use of this variant as a fluorescent probe. Fluorescence stopped‐flow experiments and substrate titrations were performed with wild‐type and F261W SsuD in order to assess the rate of loop closure. Similar experiments were performed in the presence of SsuE to determine the effect of SsuE and SsuD interactions on the loop movement. The results of these studies provide insight into the role of loop dynamics in the mechanism of desulfonation.

Work was supported by NSF Grant MCB0545048.
The Effects of Serine on Local Motions in 3‐Phosphoglycerate Dehydrogenase
Authors: Falk Bradley , Bell Ellis
Published: June 2021

3phosphoglycerate dehydrogenase catalyzes the first committed step in serine biosynthesis and the tetrameric enzyme is allosterically inhibited by serine binding to an regulatory domain some 60A from the active site. To investigate the role that ligand induced changes in local motion may play in serine inhibition, local flexibility has been studied using limited proteolysis approaches of both the native protein and a D264N mutation that we have previously shown lacks serine inhibition but binds serine normally. These studies are complemented by an analysis of temperature factors from the crystal structures of native and serine inhibited enzyme and an analysis of the effects of serine on the strength of subunit interactions by dynamic light scattering in the presence and absence of guanidine hydrochloride. Together these approaches shown serine induced changes in local flexibility of the protein rather than simple rotation of rigid domains.

This research is supported by a Grant from the National Science Foundation: MCB 0448905 to EB
Probing the Role of Aspartate‐378 in the Regulation of ADPGlucose Pyrophosphorylase from Rhodobacter sphaeroides
Authors: Fernandez Paola , Matsui Mikiko , Orry Andrew , Meyer Christopher R
Published: June 2021

ADP‐glucose pyrophosphorylase (ADPG PPase) catalyzes the rate‐limiting step in glucan synthesis. The R. sphaeroides ADPG PPase is activated by F6P, pyruvate, and FBP and inhibited by PEP and phosphate. The structure of the similar A. tumefaciens ADPG PPase (2008 Biochemistry 47, 4439‐4451) indicates that a salt‐bridge connecting R33 and D378 is involved in formation of an allosteric site. Mutations of R33 resulted in desensitization to effectors. The D378A,N, and E enzymes were generated and characterized to probe the role of size, shape and charge. Without effectors, all of the enzymes had a lower apparent affinity for ATP while the D378A and D378E enzymes exhibited a ten fold lower and two‐fold higher Vmax compared to wild‐type, respectively. In contrast to wild‐type and D378N, E enzymes, the D378A did not exhibit K‐type activation. All of the altered enzymes exhibited both lower fold activation by F6P, FBP, and pyruvate and a higher apparent affinity for activators, consistent with a change in the allosteric site resulting in an altered enzyme form which allows for tighter ligand binding to the exposed R33. In silico ligand docking studies are in progress. Supported in part by NSF Grant 0448676.
Differential MAPK and cytokine responses to human rhinovirus by human macrophages is a function of receptor‐mediated signal transduction
Authors: Schuler Bryce , Raugi Dana , Wilson Megan , Hall David J
Published: June 2021

In both children and adults, asthma exacerbations are caused primarily by viral respiratory infections, with human rhinovirus (HRV) being the most common infectious agent detected. Major and minor group rhinoviruses enter cells by binding to the cell surface molecules ICAM‐1 and LDL‐R respectively that are present on both macrophages and epithelial cells. Although epithelial cells are the site of productive HRV infection, previous studies have implicated macrophages in establishing the cytokine dysregulation relevant to asthma exacerbations in response to rhinovirus challenge. Even though major and minor group rhinoviruses are nearly genetically identical, differences in cytokine release and MAPK activation were observed indicating a differential virus response. Major and minor group HRV elicited differential production of the asthma relevant cytokines CCL20, MCP‐1 and IL‐10. Furthermore, differential activation of the MAPKs p38, JNK and ERK5 was observed between the major and minor group HRV treatments. This is the first report of genetically similar viruses with different receptors eliciting dissimilar cytokine release and MAPK activation from macrophages suggesting that receptor dependence plays a role in establishing the inflammatory microenvironment initiated in the human airway upon exposure to rhinovirus. This work was supported by the McNair program, NIH R15 AI065505‐01A1 and NSF 0521112.
Truncated trafficking protein TRAPPC6A(del29‐42) counteracts tumor suppressor WOX1‐mediated apoptosis
Authors: Chang Jean‐Yun , Chang NanShan
Published: June 2021

Whether apoptosis occurs during abnormal protein trafficking is largely unknown. Here, we isolated a TGF‐beta1‐induced truncated trafficking protein particle complex 6A, TRAPPC6A(del29‐42), which bound tumor suppressor WOX1 (also named WWOX or FOR). The wild type TRAPPC6A is a component of transport protein particle (TRAPP) complex in vesicle trafficking to the Golgi complex. Transient overexpression of TRAPPC6A(del29‐42) induced apoptosis of oral SCC‐15 and many other types of cells. Ectopic TRAPPC6A(del29‐42) is localized in the cytoplasm and nucleus. By co‐immunoprecipitation, endogenous WOX1 interacted with TRAPPC6A. Also, by FRET analysis, TRAPPC6A(del29‐42) physically bound to the C‐terminal SDR domain of WOX1. TRAPPC6A(del29‐42) counteracts with WOX1 in inducing apoptosis probably due to direct binding. We determined the presence of WOX1/TRAPPC6A aggregates in the hippocampus of patients with Alzheimer's disease (AD). Importantly, TGF‐beta1 induced aggregation of WOX1 and TRAPPC6A in vitro, indicating a novel mechanism for the pathogenesis of AD. [Supported by NSC 96‐2628‐B‐006‐045‐MY3, 96‐2628‐B‐006‐041‐MY3, NHRI EX97‐9705BI, Taiwan, and DoD BC075692, USA].
Using Research in a First Year Biology Class to Promote Critical Thinking
Authors: Bell Ellis
Published: June 2021

In their first semester students interested in majoring in the life sciences take an "Introduction to Biological Thinking" class to expose them to critical thinking and research questions and skills as well as some basic chemistry and biology. In the early part of the semester students are also introduced to the primary literature. Using National Science Foundation funded research on Malate Dehydrogenase, students learn how to analyze clustal results, develop a hypothesis and design a mutant to test the hypothesis. The students write a proposal and conduct the research before giving a presentation of the outcomes of the work. In the early part of the course and at the end of the course students are assessed for their understanding of what it takes to develop a hypothesis and design appropriate experiments to test the hypothesis and on their ability to use the primary literature. Significant gains in these skills are found. A number of the students go on to start independent research projects with faculty, and over 90% of the students indicate that they have a much better appreciation for what it takes to do science.

This research is supported by a Grant from the National Science Foundation: MCB 0448905 to EB
Metabolizable energy value of resistant maltodextrin
Authors: Baer David J , Mai Volker , Okuma Kazuhiro , Tagami Hiroyuki , Kanahori Sumiko , Henderson Theresa , Stote Kim S , Paul David R , Gordon Dennis T , Rumpler William V
Published: June 2021

Resistant maltodextrin (RM) is a soluble dietary fiber with desirable functional properties that is resistant to digestion by human enzymes but fermentable. To determine the metabolizable energy (ME) value of RM, a randomized, controlled dose‐response trial was conducted. Each subject (n=14 healthy males) was assigned to a treatment sequence that consisted of 3 treatments with controlled diets: a control diet (0 g/d RM + 50 g/d maltodextrin [MD]) and 2 levels of dietary RM (25 g/d RM + 25 g/d MD and 50 g/d RM + 0 g/d MD). Daily, subjects received their treatment divided into 2 doses, 1 at breakfast and 1 at dinner. After a 14‐d adaptation to each treatment, excreta were collected for a 7‐d period. Fecal amount of dry mass, energy, nitrogen, RM, and total carbohydrate increased with dose of RM (P<0.0001) and total fecal bacteria number also increased. Fat excretion did not differ among treatments. The energy value of RM based on the replacement of 25 g/d MD for RM ranged from 0.1 kcal/g to 4.1 kcal/g with a mean value of 2.0 kcal/g, and the energy value of RM based on the replacement of 50 g/d of MD for RM ranged from 1.8 to 3.9 kcal/g with a mean value of 2.5 kcal/g. Thus, at the higher intake of resistant MD, the ME value of RM is higher than at the lower intake. The variability of the ME value is lower at the higher intake of RM compared to the lower intake (SD of 0.7 vs. 1.2, respectively). Supported by USDA and Matsutani Chemical Industry Co., Ltd.
Cholecystokinin and Ghrelin are blunted in overweight women compared to normal weight women after protein preloads.
Authors: Randolph Jody , Cheema Mandeep , Edirisinghe Indika , Burton‐Freeman Britt
Published: June 2021

CCK and ghrelin have opposing roles in appetite regulation and are influenced by incoming nutrients. Protein induces satiety, although its potency varies by source. Whether protein provides equivalent satiety independent of body fatness has not been well characterized bio‐behaviorally. In a randomized crossover design, 10 normal weight (NW, BMI 22.06 ± 1.56) and 10 overweight (OW, BMI 31.82 ± 2.97) unrestrained women (23.65y ± 6) consumed 1 of 4 test beverage preloads (2MJ) containing 40g dairy protein isolates; milk protein (MPI), whey (WPI), and casein (CPI), or no protein control (NPC). Cholecystokinin (CCK), ghrelin, insulin, glucose and subjective satiety (SS; by VAS) were assessed over 5 h at defined intervals post preload. Preload (p<0.006) and time (p<0.0001) affected CCK, insulin, glucose, and SS (n=16 of 20). Preload by BMI interactions were observed for CCK (p=0.02), ghrelin, (p<0.004), glucose (p= 0.01), hunger (p=0.0001) and fullness (p=0.005). The CCK response to preloads was blunted in OW vs NW. Ghrelin was blunted in OW vs NW after CPI and MPI. Insulin was higher in OW vs NW (p=0.04); and as a group (n=16), WPI and CPI induced higher insulin than NPC or MPI. Glucose was higher after NPC vs protein preloads (p<0.001). Hunger and fullness corresponded with CCK and ghrelin responses to preloads suggesting OW people are receiving inadequate signaling to regulate energy intake. Funding provided by USDA NRI.
Snacking is associated with reduced risk for overweight and reduced abdominal obesity in adolescents aged 12‐18 years: NHANES, 1999‐2004
Authors: Keast Debra Rose , O'Neil Carol E. , Nicklas Theresa A.
Published: June 2021

The aim of this study was to examine associations of snacking with weight status and abdominal obesity in adolescents aged 12‐18 years (n = 5,811). Snacks/drinks were combined when eating occasions were named in the 24‐h recall, but analysis separated snacks from snacks/drinks that were only drinks. Adolescents were classified by frequency of snack consumption (0, 1, 2, 3, 4+ snacks per day), and by % of daily energy intake obtained from snacks (0, <10, 10‐19, 20‐29, 30‐39, 40% or more). CDC Growth Charts were used to define overweight (BMI =85th percentile), and abdominal obesity was waist circumference =90th percentile for age and gender. Covariate‐adjusted prevalence of overweight and abdominal obesity, and odds ratios were determined using PROC REGRESS and PROC LOGISTIC of SUDAAN. Compared to non‐snackers, prevalence of overweight and abdominal obesity was 22‐35% and 45‐49% lower, respectively, for those who ate 3‐4+ snacks per day, and 31‐32% and 49‐50% lower, respectively, for those who obtained 30‐40% or more calories from snacks. Odds ratios for overweight and abdominal obesity ranged from 0.70 (0.52, 0.94) to 0.50 (0.37, 0.69), and from 0.66 (0.47, 0.94) to 0.42 (0.24, 0.74), respectively, for 2‐4+ snacks per day; and from 0.54 (0.40, 0.75) to 0.56 (0.41, 0.76), and from 0.43 (0.27, 0.68) to 0.44 (0.28, 0.68), respectively, for 30‐40%+ calories from snacks. Adolescents who eat snacks are less likely than non‐snackers to be overweight or obese. Supported by Frito Lay and USDA
The impact of dairy and sweetened beverage consumption on diet and weight of a multi‐ethnic population of Head Start mothers
Authors: O'Neil Carol E. , Nicklas Theresa A. , Liu Yan , Franklin Frank A.
Published: June 2021

The objective of this study was to assess the association of milk and sweetened beverage (SwB) consumption with nutrient intake, dietary adequacy, and weight of Head Start mothers. Using a cross sectional, secondary analysis, Black (43%), Hispanic‐American (33%), and White (24%) women (n=609) were divided into four beverage consumption groups: high milk/low SwB, high milk/high SwB, low milk/low SwB, and low milk/high SwB. 24 hr diet recalls from 3 non‐consecutive days were averaged to determine nutrient intake. Dietary adequacy was determined with the Mean Adequacy Ratio (MAR). Mean body mass index (BMI) for the beverage consumption groups was compared; there were no differences among the groups (mean BMI 30.8+0.3 [SE]). Women in the high milk/low SwB group had higher intakes of vitamins A, D, and B6; riboflavin; thiamin; folate; phosphorus; calcium; iron; magnesium; and potassium (p<0.0125 for all) when compared with the other groups. MAR was highest in the high milk/low SwB (71.8+0.8) and lowest in the low milk/high SwB (58.4 + 0.8) groups (p<0.0125). Women in the high milk/low SwB group consumed more nutrient dense foods. Overall consumption of milk was low. Consumption of high milk/low SwB was associated with improved nutrient intake, including the short fall nutrients: calcium, potassium, magnesium, and vitamin A. Supported by Dairy Management, Inc. and USDA.
The effect of whole grain consumption (WG) on diet quality and Healthy Eating Index (HEI) scores in children aged 1 to 18 years: Results from NHANES 1999‐2004
Authors: Cho Susan S. , Zanovec Michael T. , O'Neil Carol E. , Nicklas Theresa A.
Published: June 2021

The objective of this study was to examine the effect of WG consumption on diet quality & nutrient intake in children: 2‐5 years [y] (n=2,278), 6‐12 y (n=3,868), & 13‐18 y (n=4,931) using a secondary analysis of NHANES 1999‐2004 data. Age groups were divided into four WG consumption groups: >0 to <0.6, >0.6 to <1.5, >1.5 to <3.0, & >3.0 servings/day. Nutrient intakes & HEI scores were calculated. The mean number of servings of WG consumed was 0.45, 0.59, & 0.63 for children 2‐5 y, 6‐12 y, & 13‐18 y, respectively. In 2‐5 y, HEI, & intake of energy, fiber, vitamins A, C, B1, B6, folate, magnesium, phosphorus, & iron increased with increasing consumption of WG; whereas, protein, added sugars, total fat, saturated fat (SFA), monounsaturated fat (MUFA), & cholesterol decreased. In 6‐12 y, HEI, & intake of energy, fiber, vitamins A, C, B6, folate, magnesium, potassium, phosphorus, iron, & sodium increased with increasing consumption of WG; intake of protein, SFA, MUFA, polyunsaturated fat, & cholesterol decreased. In 13‐18 y, HEI & intake of energy, fiber, vitamins A, E, B1, B2, B6, folate, magnesium, phosphorus, & iron increased with increasing consumption of WG; intake of protein, total fat, SFA, MUFA, & cholesterol decreased. Consumption of WG was low; diet quality & nutrient intake in children significantly improved with increasing consumption of WG. Supported by USDA and Kellogg's Corporate Citizenship Fund.
Development of a web‐based module about eating competence for low‐income audiences
Authors: Lohse Barbara A , Stotts Jodi L , Patterson Jill , Horacek Tanya , Do Miyoung , White Adrienne , Greene Geoffrey
Published: June 2021

"About Eating" (AE) is a 4‐lesson web‐based module (adapted from Project WebHealth, a program for college students) addressing constructs of the Satter Eating Competence Model. Following cognitive interviews with 25 low‐income men and women for the 4 modules, revisions were made in wording, graphics, lesson length and layout. Finalized lessons were assessed online by 19 reviewers with links to evaluation tools (ie., food security screener, Satter Eating Competence Inventory for Low Income (ecSI/LI). Reviewers were mostly female, white, non‐college educated, overweight or obese, food stamp participants, with marginal to very low food security and not eating competent (mean ecSI/LI 26.2 ± 10.6; possible range 0 ‐ 48). Each lesson was assessed independently for usability, navigation, design, reading level, and interest. All 4 lessons were reviewed favorably. More than 75% of reviewers denoted lessons as useful; 84% or more identified lessons as interesting. Navigation issues were identified by 7% of the reviewers for 2 of the lessons and at least 6% felt the lessons were difficult to read. Length and design were reviewed positively for all 4 lessons. Findings informed additional revisions prior to use with a larger low‐income audience. Availability of tested web‐based lessons on eating competence facilitates education about eating competence and impact testing in low‐income audiences. Funded by USDA CSREES NRI, USDA FNS SNAP‐Ed and Penn State University.
Dried Plum Suppresses the OVX‐Induced Increase in Bone Turnover in Adult Osteopenic Ovariectomized Rats
Authors: Lim Yin F , Wang Yan , Rendina Elizabeth , Bu So Young , Cullen Diane M. , Marlow Denver , Clarke Stephen L. , Lucas Edralin A. , Smith Brenda J.
Published: June 2021

Dietary supplementation with dried plum (DP) has been shown to reverse bone loss in osteopenic animals, but our understanding as to how bone metabolism is altered remains unclear. To address this issue, 6‐month‐old female Sprague Dawley rats (n=84) were either sham‐operated or ovariectomized (OVX) and assigned to: Sham‐ control diet (AIN‐93M), OVX‐control diet, OVX‐5% (w/w) DP, OVX‐15% DP, OVX‐25% DP or the positive control OVX+PTH (80 μg/kg/d x 5 days/wk). Post‐operatively, rats were maintained on control diet (6 wks) until osteopenia was confirmed, followed by 6 wks of treatment. DP (15% and 25%) improved vertebral bone density (p<0.05) compared to the OVX group, but did not restore bone density to that of the sham group. μCT analyses of the vertebral body demonstrated that trabecular bone volume, number, thickness were decreased (p<0.05) by OVX, and DP enhanced these structural properties. Finite element analysis revealed partial restoration of bone strength and stiffness by DP. All doses of DP decreased the OVX‐induced increase in bone turnover based on serum PINP and the 15% and 25% reduced bone resorption. In contrast, PTH maintained the increase in bone turnover and resorption associated with OVX. These data suggest that DP's effects on bone are mediated by slowing bone turnover and further studies should investigate how these effects are mediated. (Supported by USDA Grant #2006‐35200‐17383)
Holocarboxylase synthetase physically interacts with histone H3 to mediate biotinylation of K9 and K18
Authors: Bao Baolong , Pestinger Valerie , Zempleni Janos
Published: June 2021

Holocarboxylase synthetase (HCS) mediates the covalent binding of biotin to lysine (K) residues in histones H2A, H3, and H4; HCS knockdown disturbs gene regulation and causes distinct phenotypes in eukaryotes. HCS binds to chromosomes despite lacking a strong DNA‐binding domain. Here we tested the hypothesis that binding of HCS to chromosomes is mediated by chromatin proteins. HCS‐associated proteins were precipitated by using an antibody to HCS. Histone H3 was among the most abundant HCS‐bound proteins, and its identity was confirmed by mass spectrometry. Next, we produced recombinant GST‐HCS fusion protein and its substrate p67. The purified HCS fusion protein was bioactive, as evidenced by biotinylation of p67 in vitro. Finally, we synthesized H3‐based peptides containing the biotinylation targets K9 and K18. These peptides were incubated with recombinant HCS and cofactors.HCS catalyzed biotinylation of both K9 and K18 in peptides, as judged by probing with streptavidin, anti‐H3K9bio, and anti‐H3K18bio. In conclusion, these studies provide evidence that HCS physically interacts with histone H3, and that HCS mediates biotinylation of K9 and K18 in histone H3. (Supported by NE ARD Hatch Act funds, NIH DK063945, DK077816 and ES015206, USDA CSREES 2006‐35200‐17138, and NSF EPSCoR EPS‐0701892.)
Enrichment of H3K4bio, H3K9bio, H3K18bio, and H4K8bio in distinct genomic loci
Authors: Pestinger Valerie , Wijeratne Subhashinee Samudra Kumari , Zempleni Janos
Published: June 2021

Covalent histone modifications play crucial roles in chromatin structure and gene expression. We previously reported a novel modification of histones H3 and H4: biotinylation of lysine (K) residues by holocarboxylase synthetase (HCS). Biotinylation sites include H3K4bio, H3K9bio, H3K18bio, H4K8bio, and H4K12bio. We demonstrated that H4K12bio represses genes and is enriched in repeat elements such as pericentromeric alpha satellite repeats, telomeres, and long terminal repeats. Biological functions of biotinylated histones other than H4K12bio are unknown. Here we tested the hypothesis that H3K4bio, H3K9bio, H3K18bio, and H4K8bio are enriched at distinct genomic loci in humans and other eukaryotes. As a first step towards testing our hypothesis we validated polyclonal antibodies against H3K4bio, H3K9bio, H3K18bio, and H4K8bio. Antibodies against H3K9bio, H3K18bio, and H4K8bio met stringent specificity requirements and are currently being used to map novel histone marks in the human genome by ChIP‐to‐PCR, ChIP‐to‐chip, and ChIP‐to Seq assays. We anticipate these studies providing insights in the biological functions of H3K9bio, H3K18bio, and H4K8bio (Supported by UNL ARD Hatch Act funds, NIH DK063945, DK077816 and ES015206, USDA CSREES 2006‐35200‐17138, and NSF EPSCoR EPS‐0701892.)
Glucoraphanin is hydrolyzed by lactobacilli in vitro and rat cecal microbiota in vitro and in situ.
Authors: Lai Ren‐Hau , Miller Michael J , Jeffery Elizabeth H
Published: June 2021

Glucoraphanin (GP) is a major glucosinolate in broccoli. To become active as an anticarcinogen, GP needs hydrolysis to the isothiocyanate sulforaphane (SF), either by the endogenous plant enzyme myrosinase or possibly by microbiota in the GI tract. Five Lactobacillus species (L. gasseri, L. acidophilus, L. casei, and two of L. plantarum) and rat cecal digesta (containing live microbiota) were evaluated for GP metabolism. The degradation of GP by the tested Lactobacillus spp. ranged from 36 to 49% after 24 h incubation, but the major hydrolytic product was a nitrile, which has no bioactivity, and no SF was found. The cecal digesta from naïve and GP pretreated rats showed 40 and 56% degradation of GP, respectively, after 24 h incubation in MRS, a medium that supports growth of lactobacilli. Again, the major hydrolytic product was a nitrile, although one sample produced SF (36% of the GP substrate). Also, when GP was injected directly into the cecum of rats, isothiocyanate metabolites were found in the portal blood stream. This is the first study to show directly that GP can be metabolized by cecal microbiota to SF in situ, and then absorbed at the cecum. Supported by USDA/NRI 05‐02622 and a University of Illinois Toxicology Fellowship to RHL.
Change in plasma carotenoids in response to a 3‐week carrot juice intervention among breast cancer survivors
Authors: Butalla Amy Christine , Crane Tracy E , Patil Bhimu , Cussler Ellen C , Carino‐Thompson Patricia A , Thomson Cynthia A
Published: June 2021

Evidence suggests that breast cancer recurrence may be reduced among women with higher plasma carotenoid levels post‐treatment (Rock 2005). Convenient dietary approaches to increase plasma carotenoids to protective levels are needed. Breast cancer survivors (n=33) participated in a randomized, controlled trial in which they consumed 8 fl.oz of fresh carrot juice daily for 3 weeks. Subjects were randomized to purple (n=23) or orange (n=10) carrots (Daucus carota) to test the hypothesis that daily juice intake would be associated with a significant rise in plasma total carotenoids and a‐ and β‐carotene. Mean baseline carotenoid levels were 1.24±0.73 umol/L for total carotenoids and 0.11±0.12 umol/L and 0.39±0.38 umol/L for a‐ and β‐carotene, respectively. Post‐feeding, levels increased significantly (p<0.001) to 4.80±1.91 umol/L, 1.52±0.75 umol/L, and 2.03±1.05 umol/L, for total carotenoids, α‐ and β‐carotene. Analysis by carrot type showed that mean change in carotenoids was not significantly different between the two carrot types (p=0.78). Mean total plasma carotenoids post‐feeding reached levels previously shown to be protective against breast cancer recurrence (4.19 umol/L). Daily consumption of 8 fluid ounces of carrot juice may represent an effective and simple dietary strategy to enhance survival among breast cancer survivors. Funded by: USDA‐CSREES, TAES 06‐118409, Texas A&M‐VFIC.
Associations between body fat and vitamin K status in older women
Authors: Shea M. Kyla , Saltzman Edward , Gundberg Caren , Waddell Catherine , Bennett Grace , Booth Sarah L
Published: June 2021

Fat soluble nutrients are stored in fat tissue. Yet, the association between body fat and vitamin K status is not clear. We examined associations between % body fat (%BF) and 3 circulating measures of vitamin K status [plasma phylloquinone (plasma K1), uncarboxylated prothrombin (PIVKA), uncarboxylated osteocalcin (%ucOC)] cross‐sectionally in 262 women (60‐80 yrs) using analysis of covariance adjusted for age, vitamin K intake, triglycerides, and smoking. Higher %BF was associated with lower plasma K1 (p‐trend<0.01) and higher PIVKA (p‐trend<0.01), both indicative of lower vitamin K status. There was a non‐significant trend, but %ucOC did not differ across %BF tertiles (p=0.08). Linear regression was then used to assess the association between plasma K1 and the functional measures of PIVKA and %ucOC (as outcomes) in the separate %BF tertiles. The strength of the inverse association between plasma K1 and PIVKA increased across tertiles: unstd βT1:‐0.33(p=0.08), T2:‐0.45 (p=0.02), and T3:‐0.51(p=0.02), while the association between plasma K1 and %ucOC was weaker and non‐significant among women in the highest %BF tertile: unstd βT1:‐9.9(p=0.01), T2:‐18.9(p<0.01), and T3:‐3.0(p=0.50). Further studies are needed to elucidate the meaning of the associations between body fat and the measures of vitamin K status. Research support: NIA A914759 & USDA ARS (cooperative agreement 58‐1950‐7‐707)
The Hsp70.3 isoform of Hsp70, but not Hsp70.1, contributes to NF‐kappaB‐dependent cardioprotection of late ischemic preconditioning
Authors: Tranter Michael , Ren Xiaoping , Wilhide Michael , Jones W. Keith
Published: June 2021

It has been shown by our lab and others that the transcription factor NF‐κB is necessary for the development of the late window of protection after ischemic preconditioning (IPC) in the heart. However, the specific mechanisms and downstream gene expression profiles that underlie the cardioprotection of NF‐κB in late IPC remain unknown. To investigate NF‐κB‐dependent gene expression in IPC, we conducted a directed microarray analysis to compare the gene expression patterns of wild‐type and NF‐κB dominant negative mice 3.5 hrs after an IPC stimulus. Among the potential cardioprotective candidates identified by gene array analysis as being regulated in an NF‐κB‐dependent manner after IPC were both isoforms of heat shock protein 70 (Hsp70.1 and Hsp70.3). An in vivo mouse model of IPC followed 24 hours later by a 30 minute ischemia and reperfusion was used to assess the impact of Hsp70 isoform expression on late IPC induced cardioprotection. As previously shown by others, an Hsp70.1/70.3 double knockout mouse model failed to exhibit a cardioprotective phenotype during the late window of IPC. However, the Hsp70.1 single isoform knockout exhibited late IPC cardioprotection similar to that of wild‐type mice. In conclusion, this work demonstrates that while both Hsp70.1 and Hsp70.3 expression are induced by NF‐κB after an IPC stimulus, only the Hsp70.3 isoform contributes to cardioprotection in the late phase of IPC. This work was supported by NIH grant R01 HL63034 (WKJ) and NSF award number 0333377.
Thromboxane A2 and ADP play synergistic roles in platelet activation by von Willebrand factor
Authors: Kermode John C , Patil Chetan , Majumdar Suvankar , Zheng Qi
Published: June 2021

High shear stress in a stenotic artery triggers interaction of von Willebrand factor (VWF) with its platelet receptor, glycoprotein Ib‐IX‐V (GpIb‐IX‐V). This leads to platelet activation by a poorly defined mechanism. We have examined the signaling mechanism on ristocetin‐mediated binding of VWF to human platelets. Intracellular calcium was monitored with Fura‐PE3 or Oregon Green 488 BAPTA‐1 and Fura Red. Platelet aggregation was assayed simultaneously from light transmittance, and activation through secretion of serotonin. Treatment of platelets with the cyclo‐oxygenase inhibitor aspirin or the thromboxane A2 receptor antagonist SQ 29,548 abolished the VWF‐induced calcium transient and platelet activation. Selective antagonists of each purinoceptor subtype were also examined. Antagonism of P2Y12 receptors with MRS 2395 suppressed both the calcium transient and platelet activation by VWF. In contrast, antagonism of the P2Y1 or P2X1 receptors had minimal effect. Blockade of either the thromboxane or ADP pathway eliminated the platelet responses, implying that they contribute synergistically to VWF signaling. This intrinsically weak mechanism and strict requirement for both secondary agonists may preclude inadvertent platelet activation when a little VWF binds to GpIb‐IX‐V during passage through a region of modestly elevated shear stress. [Supported by NSF and AHA (Greater Southeast).]
Some like it hot: temperature profiles of diving elephant seals
Authors: Meir Jessica Ulrika , Ponganis Paul J
Published: June 2021

To account for the repetitive, long duration dives of the northern elephant seal, Mirounga angustirostris, it has been hypothesized that this species must either become "hypometabolic", or, contrary to current concepts of aerobic dive limits, must resort to glycolysis and lactate accumulation during dives. As recent studies have revealed reductions in regional temperature during diving, it is possible that hypothermia may serve as a mechanism of reducing oxygen consumption and extending aerobic dive time. To document core body temperature during diving in this species, a thermistor was inserted into extradural vein, hepatic sinus, or aortic sites and a backpack recorder attached to translocated, juvenile elephant seals. Venous temperature during routine dives remained near 37°C. Arterial temperature profiles, however, often revealed increases during diving (similar to that of exercising mammals), even to greater than 40°C, with subsequent decreases during each surface interval. Decreases in venous and arterial temperature to as low as 31‐34°C did occur in long (>30 min) dives, though these drops were transient and re‐warming occurred prior to the end of dive. These data suggest that hypothermia‐induced hypometabolism is an unlikely contributor to metabolic suppression while diving. Funding: NSF grant IOS‐0641801 & SIO Halliday Student Field Research Award
Glycosylation of aquaporins in the freeze tolerant tree frog, Hyla chrysoscelis
Authors: Alnajjar Khadijeh , Pandey Ram Naresh , Frisbie James , Goldstein David L.
Published: June 2021

HC‐1, ‐2, and ‐3 are aquaporins expressed in tissues of the freeze‐tolerant treefrog Hyla chrysoscelis. We hypothesize that the aquaporins are involved in osmoregulation during cold acclimation and freezing. Western blots reveal distinct protein bands at the nominal MW of the proteins (29 ‐ 31 kDa), and also at higher MW. We hypothesized that the high MW "smear" represents glycosylated forms of aquaporin involved in proper protein folding and localization to the membrane. The purpose of the present project was to investigate the magnitude of this glycosylation and its importance in quantifying aquaporin expression. We used Peptide N‐glycosidase (PNGase F), an enzyme that cleaves the oligosaccharides attached to extracellular asparagine residue, to examine N‐linked glycosylation of HC‐1, 2, and 3. In all tissues examined (skin, muscle, stomach, liver, blood, kidney, small and large intestine), addition of PNGase F decreased the intensity of signal at high MW and increased the intensity of the 29 ‐ 31 kDa band. We conclude that variation in aquaporin protein expression in treefrogs experiencing cold or freezing is associated with changing patterns of glycosylation. However, even with PNGase F, we continue to observe high MW bands in some tissues and conditions; further studies are exploring whether this represents an alternative form, O‐linked, glycosylation. Supported by NSF IOB‐0517301 and APS UGSRF.
Exercise training and low dose radiation protect skeletal muscle from high dose radiation
Authors: De Lisio Michael , Phan Nghi , Kaczor Jan J , Tarnopolsky Mark A , Boreham Doug , Parise Gianni
Published: June 2021

Radiation is a lethal, carcinogenic, mutagenic and oxidative stress‐inducing stimulus at high doses (HDR). In low doses (LDR), however, radiation is a hormetic stimulus that protects against oxidative stress by activating cellular defence mechanisms. Exercise training (EX) activates many of the same defence systems, suggesting that EX may be a potent radioprotectant. To examine whether EX could protect skeletal muscle against HDR, we divided mice into four groups (n=16/group): no intervention (SED), EX, LDR, and a group receiving both interventions (ExLDR). A sample of mice from each group received a high dose of radiation (HDR). Markers of oxidative damage, antioxidant enzyme activity and mitochondrial enzyme activity were measured. EX enhanced antioxidant capacity as exhibited by an increase in total, and MnSOD activity which was further increased in response to HDR. LDR did not alter basal antioxidant capacity, however, total, Mn‐, CuZnSOD, and catalase activity all increased in response to HDR. The increase in catalase activity was likely a response to the decreased GPx activity observed in LDR. The mitochondrial enzymes, cytochrome c oxidase and citrate synthase increased activity in EX and LDR following the HDR. Here, we demonstrate the ability of two hormetic stimuli, EX and LDR, to protect skeletal muscle from exposure to a HDR. Funding provided by US DOE (DE‐FG02‐07ER64343) and NSERC.
Muscle Regeneration utilizing ECM, MSCs and mechanical loading
Authors: Farrar Roger P. , Song Tae J , Hammers David W , Merritt Edward K , Walters Thomas J
Published: June 2021

Previously we, (Merritt et al, 2008), reported that an implantation of a decellularized extracellular cellular matrix (ECM) into a skeletal muscle defect of 1 x 1 cm of the lateral gastrocnemius (GAST) of Lewis rats, would stimulate partial recovery of muscle function. With an ECM repair, the maximal force of the lateral GAST with a defect was 80.7% ± 8.4, 77.4% ± 4.2, and 84.0% ± 3.6 of the non‐operated limb at 7,14 and 28 days. These data suggest that after 28 days of recovery from a defect injury, the lateral GAST does not recover full functionality. In the current study we infused bone marrow derived mesenchymal cells (MSCs) into the defect area with an inserted ECM and mechanically loaded the muscle utilizing the ladder climb training regime described by (Lee et al, 2004). These rats underwent resistance training every third day for 6 weeks. The resistance‐trained GAST with the ECM and infused MSCs demonstrated total recovery of mass and function with values of 1220 ±30mg and tetanic specific tension, 21.1±1.1 (N/cm2), X±SEM, respectively. These values were not different than the contralateral lateral head of the GAST of the rats that underwent resistance training. They are, however, significantly greater than defect muscles of sedentary rats with ECM and infused MSCs (Merritt et al 2009).

Funded in part by a DOD grant, DAMD W81XWH‐06‐1‐0540, to RPF
Renal proximal tubule secretion of urate is regulated by a stress‐induced pathway
Authors: Bataille Amy M , Renfro J. Larry
Published: June 2021

Urate is an important antioxidant at normal plasma concentrations (~ 300 µM) in both birds and humans, and is the major nitrogenous waste in birds. Plasma urate concentrations are primarily regulated by urinary excretion, and ~70% of renal urate excretion is due to tubular secretion. The steps in tubular secretion include basolateral entry via Oat1/3‐like transport and apical membrane exit through Mrp4 transport. Factors that regulate this pathway remain uncertain. Cellular stress has been shown to affect transport properties in other systems by altering both stress protein levels and transporter expression or activity. Here, the effect of zinc‐induced cell stress on active transepithelial urate secretion by chicken renal proximal tubule epithelial cell monolayers was determined. Direct measure of net secretion following a six‐hour conditioning stress with 250 µM ZnCl2, then 1.5 hour zinc‐free recovery, inhibited urate transport with no change in Mrp4 expression, as shown by fluorescence immunohistochemistry and real‐time PCR, and no change in glucose transport or transepithelial resistance. Acute exposure to zinc had no effect suggesting involvement of a cellular adaptation to stress. Preliminary data show an AMP kinase activator (AICAR, 0.1 mM) strongly inhibited urate secretion. We tentatively conclude that cellular stress may inhibit active renal urate secretion. Supported by NSF.
Serotonin‐mediated Ca2+ signaling in pulmonary arterial myocytes and the combined influence of maturation and high‐altitude exposure
Authors: Dawson Antoinette , Osman Noah , Pearce William J , Longo Lawrence D , Wilson Sean M
Published: June 2021

The pulmonary vasculature regulates lung blood flow in order to maintain blood oxygenation. However, chronic hypoxia (CH) such as that induced by high‐altitude exposure leads to changes and dysfunctions in the pulmonary vasculature. Serotonin (5‐HT) is an inflammatory mediator that triggers cytosolic Ca2+ increases and pulmonary arterial smooth muscle cell (PASMC) contractility. We have recently shown maturation and CH‐stress alters Ca2+ ‐dependent contractility in pulmonary arteries from sheep. The present study extends these findings by testing two hypotheses, these being that maturation enhances while CH reduces 5‐HT ‐generated Ca2+ signaling in sheep PASMCs. These hypotheses were assessed by performing confocal fluorescence microscopy of fluo‐4 in PASMCs in‐situ from fetal and adult sheep that were housed under normoxic conditions or at 12,470 feet (CH) for ~ 110 days. Basal Ca2+ activity was greater in adult than fetus and in CH‐adults as compared to their normoxic counterparts. In the presence of 10 μM 5‐HT, adult PASMCs from CH and normoxic sheep had similar firing rates. However, 5‐HT‐mediated cell firing was blunted in PASMCs from CH fetus. Maturation of PASMCs from normoxic sheep resulted in increased basal and 5‐HT‐elicited Ca2+ ‐ reactivity. These findings provide the first evidence that maturation and CH interact to alter 5‐HT‐dependent Ca2+ signaling. Support from NSF, NIH, UM, and LLUMC.
A novel method to quantify contribution of electrical restitution to alternans of repolarization in cardiac myocytes: A simulation study
Authors: Traxel Stuart Joseph , Patwardhan Abhijit
Published: June 2021

There is evidence that alternans of repolarization, i.e. a beat to beat alternation in the action potential duration (APD), is a precursor to ventricular fibrillation. Electrical restitution is considered important in predicting the onset of alternans in cardiac myocytes. Restitution predicts action potential duration (APD) based on the preceding diastolic interval (DI). Determining how much restitution contributes to alternans is not possible with existing methods. We used mathematical models and control of DI using a novel pacing protocol to quantify DI dependent contribution to alternans. Using the canine ventricular model (CVM), we paced at one end of the simulated tissue and found the DI dependent contribution as a rate of increase in the amplitude of alternans as a function of tissue length. Through manipulation of different ionic currents, we compared the new method with standard and dynamic restitution to quantify the contribution of different ionic currents towards DI independent and dependent components. When the L‐type calcium current was altered, we found non‐conformal changes between standard restitution and our method at a controlled DI value of 30 msec. These results suggest that the new method allows for unmasking of features of mechanisms of alternans not seen with current methods.

Supported by grant CBET 0730450 from the National Science Foundation.
Elucidation of the role of aquaporins and glyceroporins in freeze tolerance of Hyla chrysoscelis
Authors: Wysong Robert , Smith Aubrey , Goldstein David
Published: June 2021

Cope's gray tree frog (Hyla chrysoscelis) can tolerate freezing of its extracellular water. While the exact mechanism of freeze tolerance is unknown, aquaporins may play a role by allowing water and solutes like glycerol to move between compartments prior to and during freezing. Previously, two aquaporins (HC1, HC2) and one glyceroporin (HC3) were identified in Hyla chrysoscelis in a tissue and temperature dependent manner. To further elucidate the role of aquaporins in freeze tolerance, three putative aquaporins (HC7, HC8 and HC0) have been identified. Degenerate PCR primers were developed using conserved amino acid regions from Xenopus tropicalis and compared to other species with high sequence homology. For the putative aquaporins, mRNA was isolated from the fat, large intestines, and the eye for HC7, HC8 and HC0 respectively. The templates were amplified using RT‐PCR and products (326 bp, 377 bp and 400 bp respectively) were examined by electrophoresis. Further experiments are underway to confirm these putative aquaporins, and to examine their tissue and temperature dependence. Supported by NSF IOB‐0517301.