bluuebunny/crossref_metadata_2025 huggingface dataset:
A mathematical model of the afferent arteriolar smooth muscle cell
Authors: Layton Harold E , Chen Jing , Moore Leon C , Layton Anita T
Published: June 2021

We have developed a mathematical model of a rat afferent arteriolar smooth muscle cell. The model is based on a model for small vessels by Gonzalez‐Fernandez & Ermentrout (Math Biosci 119:127, 1994). The model incorporates ion transport, cell membrane potential, cellular contraction, and wall mechanics. Model results suggest that interacting Ca2+ and K+ fluxes, mediated by voltage‐gated and voltage‐calcium‐gated channels, give rise to periodic oscillations in cytoplasmic calcium concentration, myosin light chain phosphorylation, and crossbridge formation with attending muscle stress that mediates vasomotion. A parameter set, based on rat afferent arteriole dimensions was identified; with that set, model predictions agree well with findings in the experimental literature. The model's representation of the myogenic response is based on the hypothesis that the voltage dependence of calcium channel openings responds to transmural pressure so that vessel diameter decreases with increasing pressure. The model will be used as an essential component in models of integrated renal hemodynamic regulation. This research was supported in part by NIH grant DK‐42091 and NSF grant DMS‐0715021.
The functional correlates of jaw‐muscle fiber architecture in primates
Authors: Taylor Andrea B. , Vinyard Christopher J.
Published: June 2021

The jaw muscles are responsible for generating jaw forces and movements yet surprisingly little is known about the architecture of these muscles in primates. To better understand the relationship among jaw‐muscle fiber architecture, feeding behavior, and diet, we evaluated and compared masseter and temporalis fiber architecture in marmosets (Callithrix jacchus, Cebuella pygmaea), tamarins (Saguinus oedipus), capuchins (Cebus sp), and large‐bodied hominoids. Muscles were removed en masse from the skull, weighed, and whole muscle and fiber lengths measured. We computed pinnation angles and physiologic cross‐sectional areas (PCSAs). In some cases, fiber architecture differences can be predictably linked to functional and adaptive differences in feeding behavior. Thus, the relatively longer, less pinnate‐fibered muscles in tree‐gouging marmosets compared to nongouging tamarins can be functionally linked to the production of relatively wide jaw gapes during tree gouging. Likewise, C. apella displays relatively larger PCSAs compared to other capuchins, consistent with their occasional exploitation of exceptionally resistant foods. Fiber architecture, however, fails to predict differences in feeding behavior and diet among hominoids. We discuss the implications for reconstructing feeding behavior and diet in extinct taxa. Grant sponsor: NSF; Grant numbers: BCS‐0452160, BCS‐0094666, BCS‐0412153; Grant sponsor: NIH; Grant number: R24 HD050837; Grant sponsor: Duke Undergraduate Research Support and the Biology Research Forum of Trinity College.
High Fat Diet Induces Expression of Cell Cycle Control Gene p16 In the Liver of Obesity Prone Rats Through Epigenetic Modifications
Authors: Zhang Xiyuan , Pan Yuan‐Xiang
Published: June 2021

Objectives

Proliferation of cells is important in the repair and growth of tissues. Our bodies' ability to regenerate tissues decreases as we age. Because of ongoing epidemics of obesity and type 2 diabetes, it's important to find out if high fat diet affects liver regenerate function in obese population.

Methods

Obesity Prone (OP) and Obesity Resistant (OR) rats were fed with high fat diet containing 45 kcal% fat, 35 kcal% carbohydrate and 20 kcal% protein for 3 months. To investigate whether cell cycle control genes were affected by high fat diet, livers from both OP and OR rats were collected and used to isolate RNA, protein, and chromatin. Quantitative real‐time PCR, Western blot and chromatin immunoprecipitation were performed for the analysis of the cell cycle genes.

Results

Our analysis demonstrates for the first time that high fat diet causes a five‐fold increase of p16 (Ink4a) mRNA (p=0.03) in the liver of male OP rats. Acetylation of histone H4, but not H3, at the promoter region is also significantly increased in male OP rats (p=0.003), which may reveal the molecular mechanism of p16 up‐regulation.

Conclusions

Expression of p16 (Ink4a) increases with age in tissues. Significant increase of p16 expression level in the liver of OP rats by high fat diet suggests that high fat diet may lead to an decline in the regenerative capabilities of the liver maintained by p16 (Ink4a).

Grant Funding Source: USDA
The Collaboration for Health, Activity, and Nutrition in Children's Environments (CHANCE): A Program Integrating Parenting and Nutrition Behavioral Education Improves Food, Active Play, and Parenting Practices in Low‐income Families
Authors: Dickin Katherine Lynn , Hill Tisa Fontaine , Dollahite Jamie S
Published: June 2021

CHANCE is designed to help low‐income parents prevent childhood obesity by using effective parenting skills to shape home environments and promote healthy eating and activity. A new dialogue‐based curriculum integrates nutrition, active play, and parenting education in hands‐on workshops for small groups of parents and caregivers of 3–11 year‐old children. In a 30‐month pilot under usual program conditions, 523 eligible participants enrolled and 268 attended at least 6 of 8 sessions. Parents responded positively to workshops and discussed multiple changes they made at home. To assess change in key behaviors, a targeted evaluation tool was validated and data were collected pre‐ and post‐intervention (n=210). Improvements were reported in adult and child intake of fruit, vegetables, low‐fat dairy, soda, and fast food; child active play and screen time; and parenting practices related to family meals and home food environments (paired t‐tests, p<0.001). Race, sex, and urban residence predicted changes in behavior (p<0.05). CHANCE is a feasible, effective way to reach low‐income parents to improve parenting practices and family lifestyle behaviors relevant to obesity prevention. Supported by USDA.
Vitamin D homeostasis is disrupted in a type II diabetic rat model due to aberrant reuptake in kidney
Authors: Anderson Rachel Lynn , Rowling Matthew J
Published: June 2021

Alterations of serum levels of the major circulating form of vitamin D (25‐hydroxycholecalciferol, 25D3) and its active hormone derivative (1,25‐dihydroxycholecalciferol, 1,25D3) have been associated with type II diabetes, but a mechanism for this occurrence has not been fully elucidated. In this study, we used the Zucker Diabetic Fatty (ZDF) rat model to characterize the role of renal reabsorption in vitamin D homeostasis in diabetic and non‐diabetic rats fed vitamin D sufficient (1000 IUs/kg), deficient (0 IUs/kg), and supplemented diets (10000 IUs/kg). Using ELISA, we analyzed serum and urinary 25D3, 1,25D3 and vitamin D binding protein (DBP) levels in 16 wk old ZDF and lean rats. Serum levels of both 25D3 and 1,25D3 were significantly lower in diabetic animals than their non‐diabetic controls fed either vitamin D deficient or sufficient diets. Furthermore, diabetic rats excreted greater proportions of DBP and 25D3 in urine than their respective controls. Vitamin D supplementation markedly increased serum 25D3 in diabetic rats, but had no effect on serum 1,25D3 or urinary excretion ratios. Taken together, these data suggest that compromised renal reabsorption of 25D3 and DBP may be a significant contributing mechanism leading to reduced vitamin D status in diabetes. Supported by USDA (Iowa State University Nutrition and Wellness Center SRPG to MR) and the Iowa State University Experiment Station.
Spectroscopic and structural studies of the mechanism of bicarbonate‐induced allostery in Haemophilus influenzae carbonic anhydrase
Authors: Rowlett Roger S , Samardzic Dejan , Failing Hannah , Williams Jared , Mysliwiec Margaret , Stanford Ian
Published: June 2021

X‐ray crystallographic studies have previously revealed a non‐catalytic, allosteric bicarbonate binding site in H. influenzae β‐carbonic anhydrase (HICA). We have structurally and kinetically characterized several allosteric site variants. We have also structurally, kinetically, and spectroscopically characterized Co(II)‐substituted HICA. In several allosteric site HICA variants there is a “bicarbonate escort site” which appears to be an intermediate binding site for bicarbonate on a pathway leading from bulk solution to the non‐catalytic, allosteric site. Visible spectroscopy of Co(II)‐substituted HICA and heterogeneously substituted Co(II)‐Zn(II) HICA suggest that metal ion sites in an allosteric dimer can communicate with one another, and that bicarbonate can trigger a conformational change in the protein. These results provide direct spectroscopic evidence of intersubunit communication and cooperativity in HICA, and provide compelling structural evidence for the ingress/egress route of bicarbonate ion to/from the allosteric site. This work is supported by NSF grants CHE‐0819686 and MCB‐0741396.
Feeding Omega‐3 Fatty Acids from Different Sources on Tissue Fatty Acid Composition and Oxidative Stability in Rats
Authors: Cordonier Elizabeth , Altman Stephanie Nicole , Gigliotti Joey , Tou Janet
Published: June 2021

Omega‐3 polyunsaturated fatty acid (n‐3 PUFA) sources providing high bioavailability and tissue accretion with the least peroxidation favor maximal health benefits. The study objective was to determine the tissue accretion and stability of n‐3 PUFAs from different sources. Young (28 d) female Sprague‐Dawley rats (n=10/group) were pair‐fed high fat (12% lipid/wt) diets containing either corn oil (CO) or n‐3 PUFA rich oils derived from flaxseed (FO), krill (KO), or menhaden (MO). Rats were individually housed in metabolic cages to determine apparent lipid digestibility: [(lipid intake – fecal lipid)/(lipid intake)] x 100. After 8 wks, liver and brain tissue were collected. Tissue fatty acid content (FA) was determined by gas chromatography and lipid oxidation by enzyme immunoassay for Thiobarbituric Acid Reactive Substances (TBARS). Lipid digestibility was lowest (p<0.05) in KO fed rats. In the brain, docosahexaenoic acid (DHA) was highest (p<0.02) in rats fed KO. In the liver, alpha‐linolenic acid was highest (p<0.001) in rats fed FO; eicosapentaenoic acid was higher (p<0.001) in rats fed KO, MO, and FO compared to CO. DHA was highest (p<0.001) in rats fed KO and MO. TBARs were similar among diet groups. Different sources of n‐3 PUFAs had different effects. Despite low digestibility, KO increased tissue DHA content without increasing tissue oxidation. Funding NRI #1004489 USDA NIFA grant.
Negative design at the carboxy‐terminal beta‐edge of truncated hemolysin A
Authors: Mahoney Jacob , Weaver Todd
Published: June 2021

Hemolysin A (HpmA) from Proteus mirabilis is secreted via the Two‐Partner Secretion pathway, which is utilized by Gram‐negative bacteria as an energy‐independent mechanism for secretion of large virulence proteins. A truncated form of HpmA (HpmA265) has been shown to activate full‐length HpmA in a template‐assisted and thermally stable fashion. HpmA265 was recently analyzed by X‐ray crystallography, revealing a right‐handed β‐helical structure and a dry dimeric interface between exposed C‐terminal β‐edges of HpmA265 subunits. These characteristics are typical of disease‐state proteins of beta‐amyloid diseases such as Alzheimer's and CJD. In order to characterize the possible role of exposed β‐edge interactions in the template‐assisted hemolytic activity, negative design principles were applied by replacing the C‐terminal hydrophobic residues F241 or M245 with positively charged lysine residues. The mutants, F241K and M245K, were then characterized via template‐assisted hemolytic assays, temperature stability studies, and circular dichroism (CD). F241K displayed faster template‐assisted activation and greater heat stability, as well as more organized β‐structure. However, M245K was less active, less heat stable, and showed decreased β‐structure. These results suggest that the incorporation of positive charge in the exposed β‐edge effectively destabilizes the helix, inhibits template‐assisted activation, and correlates with loss of beta signal from CD. This research was supported by NSF grant (MCB0744754) to TW and the UW‐La Crosse Dean's Distinguished Undergraduate Research Fellowship to JM.
The DNA damage inducible protein UmuD inhibits replication
Authors: Beuning Penny J , Sefcikova Jana , Fang Jing , Engen John R , Silva Michelle C
Published: June 2021

All organisms experience DNA damage from a myriad of sources. When bacterial cells experience DNA damage, the SOS response is induced, leading to upregulation of at least 57 genes in E. coli. The SOS regulated genes include those involved in DNA repair and cell cycle regulation. Also induced as part of the SOS response are Y family DNA polymerases, which have the specialized ability to copy damaged DNA and tend to be error‐prone when replicating undamaged DNA. UmuD, a small manager protein, and its cleaved form, UmuD', directly interact with both Y family polymerases as well as the beta processivity clamp and the replicative DNA polymerase. We find that UmuD, but not UmuD′, inhibits primer extension by the DNA polymerase III alpha subunit. Thermal shift experiments show that UmuD undergoes two melting transitions, one likely due to the dissociation of the N‐terminal arms and the other due to unfolding of the globular domain. We used hydrogen‐deuterium exchange mass spectrometry (HXMS) to probe the conformations of UmuD and UmuD'. Our HXMS results reveal that the N‐terminal arm of UmuD is highly dynamic. Our observations are consistent with the proposed model of UmuD and the finding that UmuD is relatively unstructured. Research supported by NSF Career Award MCB‐0845033.
Cinnamon increases liver glycogen, improves insulin sensitivity, and regulates glycogen synthesis related gene expression in an animal model of the metabolic syndrome
Authors: Qin Bolin , Couturier Karine , Batandier Cecile , Awada Manar , Hininger‐Favier Isabelle , Canini Frederic , Roussel Anne‐Marie , Anderson Richard A
Published: June 2021

We have shown that cinnamon (CN), and polyphenols in the aqueous extracts of CN, counteract insulin resistance in in vitro, experimental animal, and in human studies. The objective of this study was to determine the effects of CN on glycogen synthesis in the muscle and liver using an animal model of the metabolic syndrome, the high fat/high fructose diet (HF/HFr) fed rat. Four groups of male Wistar rats were fed for 12 wks with (i) HF/HFr known to induce insulin resistance, (ii) HF/HFr containing 20g CN/kg of diet, (iii) control diet, and (iv) control diet containing 20g of CN/kg of diet. Cinnamon added to the diet led to highly significant increases in liver but not muscle glycogen, especially in animals fed the HF/HFr diet. Cinnamon also improved the decreased Ir, Irs1, Irs2, Glut1, Glut2, Gsk3b, and glycogen synthesis1 in the liver. In muscle, HF/HFr diet decreased expression of Ir, Irs1, Irs2, Glut1, Glut4 and glycogen synthesis1 and CN also reversed these effects. In addition, CN decreased the over‐expression Gsk3b mRNA levels in the muscle of HF/HFr fed rats. In summary, these results suggest that CN increases liver glycogen and improves insulin sensitivity in rats with diet‐induced insulin resistance via regulating glycogen synthesis related gene expression. (Funded by USDA/ARS/USA and French PNRA 007)
Comparison of sodium and potassium intake and excretion: Results from the AMPM Validation Study
Authors: Rhodes Donna G , Clemens John C , Murayi Theophile , Moshfegh Alanna J
Published: June 2021

Due to their relationship to hypertension, reliable assessments for sodium and potassium intake in populations are important. Although a 24hr urinary excretion for each nutrient is believed to provide the best estimate of intake, few studies have used these biomarkers to evaluate the accuracy of a dietary instrument. The purpose of this research is to compare self‐reported dietary intake of sodium and potassium and their corresponding 24hr urinary excretion in the US Department of Agriculture Automated Multiple‐Pass Method (AMPM) Validation Study. The AMPM, used to collect 24hr recalls in What We Eat in America, NHANES, was validated in a large doubly labeled water study and shown to reduce bias in the collection of energy intakes in a study population that included 524 men and women, aged 30–69 years. Using data from subjects (n=465) who completed at least one 24hr dietary recall plus collected a complete 24hr urine during the same period, the validity of sodium and potassium intake is assessed. Reporting accuracy, calculated as the ratio of reported intake to that estimated from the corresponding urinary biomarker, is 0.88 for sodium and 0.95 for potassium. For sodium only, there is a decrease (p<0.5) in reporting accuracy between normal weight and obese individuals; 0.99 vs. 0.75, respectively. Estimations of sodium intake are adjusted for discretionary use of salt in cooking but do not quantify salt added at the table.

Funding source for this study: ARS, USDA
Effects of chronic blockade of cannabinoid 1 receptors in the NTS on blood pressure in Sprague Dawley rats
Authors: Hopp Francis A , Dean Caron , Hillard Cecilia J , Seagard Jeanne L
Published: June 2021

Previously, we have shown that endocannabinoid activation of cannabinoid 1 receptors (CB1Rs) in the nucleus tractus solitarius (NTS) prolongs baroreflex inhibition of renal sympathetic nerve activity (RSNA) via modulation of GABAergic transmission in normotensive Wistar Kyoto (WKY) and Sprague Dawley (SD) rats, but not spontaneously hypertensive rats (SHR). These results suggest that CB1R modulation of GABAergic transmission in SHRs is reduced and could contribute to elevated blood pressure (BP) and sympathetic tone characteristic of this model. This study examined the effects of chronic blockade of CB1Rs, mimicking the decrease in CB1R regulation in SHRs, on BP regulation in SD rats. To block CB1Rs, microinjections of the CB1R antagonist SR141716 (70nl; 10 or 20 micromolar) through indwelling guide cannulae into the barosensitive region of the NTS were performed daily for 7 days in rats instrumented with transmitters for telemetry recording of BP. The higher dose of SR141716 produced a sustained increase in BP by day 3, with only transient increases in BP seen in response to microinjection of low‐dose SR141716. The results suggest that loss of endocannabinoid signaling in the NTS can lead to sustained increases in BP in normotensive rats. VA Medical Research Funds (JLS), American Heart Grant‐in‐Aid 0750010Z (CD), NSF Award IOS 0751613 (CD) and NIDA Award DA09155 (CJH).
Mapping of peptides and proteins in Leech (Hirudo medicinalis) by Mass Spectrometry Imaging
Authors: Sistla Srinivas , Bruand Jocelyne , Ghassemian Majid , Bafna Vineet , Gaasterland Terry , Salzet Michel , Macagno Eduardo
Published: June 2021

Mass Spectrometry imaging (MSI) can be used to obtain the expression profiles of proteins and peptides in various biological tissues under different experimental conditions. In this study, we employed MSI to map the temporal and spatial distributions of many individual molecules in the range of m/z from 1kD to ~10kD in the embryo of the medicinal leech (Hirudo medicinalis). Distributions with specific characteristics such as selective expression in the central nervous system or localized expression in certain areas of the body wall were selected for further analysis. For sequence identification, leech protein/peptide embryo extracts were prepared using different conditions and run on LC ESI QTOF and on LC MALDI instruments. Peaks corresponding to masses associated with selected distributions were sequenced using database dependent and denovo methods. An interesting example of peptides identified in this manner are proteolytic fragments from histones, protease inhibitors like cystatins and other proteins that have a role in the innate immune system of lower invertebrates.

First and second author contributed equally Supported by NSF Grant DBI‐0852081
Effects of allosteric ligands on the folded‐unfolded transitions in GDH: Combining experimental and mathematical modeling approaches
Authors: Lekova Virzhiniya , Bell Ellis
Published: June 2021

Bovine glutamate dehydrogenase catalyzes the oxidative deamination of L‐Glutamate using NAD(P). The enzyme is extensively regulated by a variety of ligands, including the purine nucleotides ADP and GTP. The current study investigates the effect of cofactor substrates and allosteric regulators on the cooperative unfolding of GDH using both empirical and mathematical modeling approaches. Denaturation using varying concentrations of guanidine hydrochloride was monitored using the fluorescence of the three tryptophan residues per subunit. In addition to these equilibrium measurements, kinetics of the fluorescence changes were used to resolve the detectable unfolding intermediates. The kinetic data at intermediate guanidine hydrochloride concentrations was shown to fit to a 3 exponential decay consistent with an unfolding pathway involving intermediates. The shift in wavelength of maximum emission in the equilibrium experiments, which is due to unfolding, was modeled with nonlinear fit of a sigmoidal curve. The developed mathematical model is a useful tool to quantify the effect of ligands on the cooperativity of unfolding and stability of the protein. The results demonstrate that both cofactors increase the cooperativity of unfolding, with negligible effects on stability.

This work is supported by NSF Grant MCB 0448905 to EB
Monoclonal antibodies against diverse xyloglucan structures as probes for plant cell wall chemistry and ultrastructure
Authors: Tuomivaara Sami , Avci Utku , O'Neill Malcolm , Hahn Michael G. , York William S.
Published: June 2021

The plant cell wall is a complex extracytoplasmic organelle whose structure and dynamics are poorly understood at the molecular scale. Traditional biochemical methods have documented plasticity in the cell wall properties during development and in response to stress, but are limited to characterizing bulk features. Alternatively, the structural discriminating power of monoclonal antibodies can be utilized to simultaneously probe the abundances and locations of molecular structures in muro at sub‐organellar resolution, and to examine cell wall components in vitro. We have generated more than 30 distinct monoclonal antibodies with a diverse range of specificities against xyloglucan, an abundant and structurally heterogenous class of carbohydrate polymer found in the plant cell walls. The binding specificities of these antibodies have been exhaustively characterized by ELISA‐based methods. The knowledge of the binding specificities allows structural interpretation of the immunolocalization patterns generated using these antibodies. Moreover, we have demonstrated the utility of these antibodies in structural classification of xyloglucans as well as in probing the biophysical properties of partially reconstituted plant cell walls, thus sheding light on the structure‐function relationships of this polysaccharide. This work is funded by NSF (DBI‐0421683) and DOE (DE‐FG02‐96ER20220).
Trace metal dyshomeostasis is associated with loss of TRPML1 ion channel function
Authors: Eichelsdoerfer Jonathan Lee , Evans Jeffrey , Sorasaenee Karn , Morrison Chevaun , Linder Maria , Slaugenhaupt Susan , Cuajungco Math P
Published: June 2021

Mucolipidosis type IV (ML‐IV) is a human lysosomal storage disorder caused by the dysfunction of TRPML1 ion channel. TRPML1 is believed to play a role in endosomal‐lysosomal biogenesis. Metal accumulation and lysosomal dysfunction have been implicated in neurodegenerative diseases like ML‐IV and Alzheimer's disease. Interestingly, recent evidence suggests that TRPML1 is permeable to divalent cations. We hypothesize that the TRPML1 ion channel function influences metal homeostasis in lysosomes. Inductively‐coupled mass spectrometry analysis of brain tissues from TRPML1 knockout (TRPML1‐KO) mice and wild‐type (WT) littermates showed that isotopic levels of abundant metals sodium, magnesium, and calcium, and levels of trace metals iron, copper, chromium, aluminum, cadmium and manganese, did not vary significantly. The isotopic levels of zinc (Zn‐66) in brain tissues of TRPML1‐KO mice were significantly higher when compared with WT mice. RNA interference of endogenous TRPML1 in cultured human embryonic kidney‐293 cells also showed marked changes in trace metals. In conclusion, trace metal dyshomeostasis in lysosomes appears to be directly related to the loss or reduction of function of the TRPML1 ion channel. Future therapeutic strategy targeting the TRPML1 protein, or trace metal accumulation could be developed to alleviate conditions associated with lysosome‐associated neurodegenerative diseases. This project was funded in part by funds from HHMI and NSF.
Relationships between iron status, serum hepcidin and inflammation in female Soldiers during military training
Authors: Karl J Philip , Lieberman Harris R , Cable Sonya J , Williams Kelly W , Young Andrew J , McClung James P
Published: June 2021

Expression of hepcidin, a key hormone regulating iron homeostasis, may be affected by factors including iron stores and inflammation, but these relationships are not well characterized in humans. This study examined relationships between serum hepcidin concentration, iron status and inflammation in female Soldiers during basic combat training (BCT). Iron status indicators, serum hepcidin, and markers of inflammation were measured in fasted female Soldiers (n=40) during the initial (Pre) and final (Post) weeks of a 9‐wk BCT course. Iron status and inflammation declined during BCT; red cell distribution width and soluble transferrin receptor (sTfR) increased while ferritin, interleukin‐6 (IL‐6) and C‐reactive protein decreased (p≤0.01, all such). Hepcidin concentrations were lower in iron‐deficient compared to iron‐normal Soldiers (p<0.05), and were similar pre‐ versus post‐BCT. Hepcidin was positively correlated with ferritin, transferrin saturation (TS) and IL‐6 pre‐BCT (p<0.05, all such). Post‐BCT, hepcidin was positively correlated with ferritin, hemoglobin and TS, and negatively with sTfR (p<0.05, all such). These data demonstrate relationships between hepcidin, iron status and inflammation, and suggest declining iron status in female Soldiers during BCT may be regulated, in part, by both changes in serum hepcidin and inflammation. Funded by MRMC.
The influence of copper (Cu) deficiency in a cardiomyocyte cell model (HL‐1 cell) of ischemia/reperfusion injury
Authors: Johnson William Thomas , Briske‐Anderson Mary , Curtis Sheena A. , Asher Elizabeth T.
Published: June 2021

Mitochondria are important mediators of cell death and this study examines whether mitochondrial dysfunction caused by Cu deprivation promotes cell death in a cell culture model for ischemia/reperfusion injury in cardiomyocytes. HL‐1 cells (donated by Dr. William C. Claycomb, LSU Health Sciences Center) were cultured for 3 days in the absence or presence of 80 μM TET, a Cu chelator. TET‐treated cells in comparison to untreated cells had lower cytochrome c oxidase activity (32.5±0.4 vs 65.0±4.3 nmol cyt c oxidized·min−1·mg−1) and lower Cu content (0.95±0.06 vs 2.07±0.08 ng Cu/106 cells). After 3 days of TET treatment, ischemia/reperfusion was simulated by replacing medium with nutrient‐free buffer and incubating in a hypoxic atmosphere for two hours followed by incubation in complete medium in a normoxic atmosphere for 90 min. Ischemic cells compared to control cells had reduced mitochondrial membrane potential (ΔΨm) (53±1 vs 42±1% depolarized cells) and increased apoptosis (16±1 vs 11±1% apoptotic cells) but TET treatment had no effect. However, following reperfusion, TET‐ treated compared to untreated cells exhibited lower ΔΨm (55±1 vs 46±1% depolarized cells) and increased apoptosis (9.0±0.2 vs 6.9±0.2% apoptotic cells). These findings indicate that Cu status of cardiomyocytes is a determinant of reperfusion injury through a mechanism that likely involves mitochondrial dysfunction. Support ‐ USDA/ARS
Dietary immunoglobulins affect colon cytokines in mouse model of inflammatory bowel disease
Authors: Jiang Huanyi , Becker Chad W , Pryzbyszewski Joseph , MacDonald Ruth S
Published: June 2021

The incidence of inflammatory bowel diseases (IBD) is increasing in the US. Dietary factors impact the symptoms of IBD, although the food matrix and milieu of the gastrointestinal tract create challenges for therapy. Based on previously documented success in managing necrotizing enteropathy in neonatal pigs using a concentrated form of blood‐derived immunoglobulins, we explored the role of this product in alleviating the symptoms of IBD in a mouse model. ImmunoLin® (Proliant Health Ingredients, Ankeny IA) contains a mixture of mainly IgG with IgA, IgM, IgE, and IgD. We hypothesized that the immunoglobulins in this product would reduce colon inflammation. We treated C57BlackJ mice with 2% dextran sodium sulfate (DSS) for 4 days and then fed AIN‐93G diets containing 0, 11.25, 22.5 or 45 g/kg ImmunoLin® for 7 or 14 days. Colon tissue was snap frozen and 23 cytokines were analyzed by Luminex immunoassay. DSS resulted in elevated expression of most cytokines in the mouse colon. After 14 days the lower doses of ImmunoLin® reduced IL‐1α, IL‐4, IL‐6, IL‐10, MCP‐1 and KC, but the highest dose was ineffective. In a parallel study, feeding the 45g dose for 14 days prior to DSS treatment had no protective effect on these cytokines. Thus, low doses of ImmunoLin® reduced cytokine induction in response to DSS in mice and may have value in human therapy. Funded by USDA special grant to the ISU Center for Designing Foods to Improve Nutrition.
Glutamine: precursor or nitrogen donor for the synthesis of citrulline?
Authors: Marini Juan C , Didelija Inka Cajo , Castillo Leticia , Lee Brendan
Published: June 2021

Glutamine (Gln) is considered the main precursor for citrulline (Cit) synthesis, but no attempts have been made to differentiate the contribution of Gln carbon (Gln‐C) skeleton vs. the nonspecific contribution through NH3 and CO2. To study the contribution of dietary Gln‐N to the synthesis of Cit, the incorporation of 2‐15N and 5‐15N Gln, and 15N ammonium into the 3 N groups of Cit was determined in conscious mice. D5 and U‐13C5 Gln were used to determine the incorporation of Gln‐C. The contribution of dietary arginine (Arg) and proline (Pro) were also determined by the infusion of U‐13Cn tracers. The 2‐15N Gln tracer labeled to a similar extent the 3 N positions of Cit, while 5‐15N Gln and 15N ammonium tracers mainly labeled the ureido group. The total incorporation of 2‐15N and 5‐15N exceeded 25‐fold the incorporation of the D5 tracer. Dietary Arg was the main precursor for Cit synthesis (40% of RaCit), followed by Pro and Gln (3.4 and 0.4% of RaCit, respectively). The incorporation of a single 13C into Cit from Gln and Pro, but not from Arg, was detected. This represents the oxidation of Gln and Pro in enterocytes, with the subsequent utilization of 13CO2 for the synthesis of carbamylphosphate. In conclusion, Gln is not a carbon skeleton precursor for Cit synthesis, but donates nonspecific N and C for its synthesis. Dietary Arg is the main carbon skeleton precursor for Cit synthesis. Support: USDA 2533771314 and NIH KO1 RR24173.
Precursors for the synthesis of citrulline in mice fed arginine free diets
Authors: Marini Juan C , Didelija Inka Cajo , Castillo Leticia , Lee Brendan
Published: June 2021

Dietary arginine (Arg) is the main dietary precursor for citrulline (Cit) synthesis. To test the hypothesis that the contribution of dietary proline (Pro) and glutamine (Gln) increases during the feeding of an Arg free diet, rates of appearance (Ra) and precursor‐intermediate‐product relationships were established by the infusion of tracers in conscious mice. There was no difference in RaCit or RaArg due to the lack of Arg in the diet (152 and 564 μmol•kg−1• h−1, resp.); however, Ra ornithine (Orn) was greater in the Arg sufficient diet (332 vs. 180 μmol•kg−1• h−1). There was an increase in the utilization of plasma Orn for the synthesis of Cit (47 vs. 34 μmol•kg−1• h−1) in the Arg free diet. There was no difference in the utilization of plasma Arg between the two diets for Cit synthesis, either through plasma Orn (~26 μmol•kg−1• h−1) or at the site of citrulline synthesis (~13 μmol•kg−1• h−1). The contribution of dietary Pro to the synthesis of Cit was mainly at the site of Cit production rather than through plasma Orn (16.7 and 4.6 μmol•kg−1• h−1, resp.). Dietary Gln was utilized only at the site of Cit synthesis (4 μmol•kg−1• h−1). Dietary Gln and Pro increased their contribution to the synthesis of Cit during Arg free feeding, but still remained a minor source, despite the absence of Arg in the diet. Endogenous Arg and Orn are able to support Cit synthesis during Arg free feeding. Support: USDA 2533771314 and NIH KO1 RR24173.
Food and nutrient intakes of Native Hawaiian, Filipino and White children and their caregivers
Authors: Novotny Rachel , Vijayadeva Vinutha , Grove John , Gittelsohn Joel , Murphy Suzanne
Published: June 2021

This study examined ethnic differences in food and nutrient intake of children and their caregivers in Hawaii. Caregiver‐child pairs were randomly selected from four low income communities with a high proportion of Native Hawaiian children. Socio‐demographic data and one 24 hour food recall (using a modified USDA five‐step multiple pass method) were collected from each pair (n = 156 pairs: 18 White, 28 Filipino, 110 Native Hawaiian). SAS Proc GLM, ANCOVA, CANCORR and M test were used for data analysis. All models adjusted for season. Community, material style of living and caregiver education were not significant in models, and were dropped from final models. Significant ethnic differences in intakes were (always White higher than either Filipino or Native Hawaiian): 1. children: yogurt food group, 2. caregiver's: whole grain, fruit, meat and cheese food group; calcium, potassium, folate and vitamin C nutrient; and dairy and total fat Healthy Eating Index (HEI). Only caregiver nutrient intake differences remained after adjusting for dairy food group intake. Three multivariate caregiver models testing of the effect of ethnic group on food groups combined, nutrient intakes combined, and HEI component scores combined was significant only for nutrient intakes (p=0.004) and HEI component scores (p=0.01). Funding: USDA 2004‐35215‐14252, Hawaii Department of Health 436851.

Grant Funding Source: MARC
Healthy Vending Snacks on 10 College Campuses: A Comparison of Nutrient Density Scores
Authors: Horacek Tanya , Byrd‐Bredbenner Carol , Colby Sarah E , Hoerr Sharon , Johnson Michelle , Kidd Tandalayo , Phillips Beatrice , Walsh Jennifer
Published: June 2021

This study assessed the energy and nutrient density of snacks sold in vending machines on 10 college campuses using 5 different nutrient density scoring methods. Percent Daily Values were used to calculate nutrient density scores for 450 vending snacks using 4 existing methods: Smart Choices Program, Nutrition Rich‐6, Nutrition Rich‐9, and Limited Nutrient Score. A fifth method, Smart Nutrient Score, was created for this study. Results indicate <5% of vending items were rated as healthful using existing scoring methods. Only one snack, Oats ‘N Honey granola bar (Nature Valley), qualified for the Smart Choices label. Using the Nutrient Rich‐6 and Rich‐9 methods, 3.5% of snacks had a mean of >=10% DV nutrients to encourage (vitamins A & C, fiber) and snacks had a mean of 16.4%DV+/−8.9SD of nutrients to limit (sodium, saturated fats, added sugars), ranging from 0 to 61.4%DV. The Smart Nutrient Score (1 pt for each encouraged nutrient >=10% minus 1 pt for each nutrient to limit >10%) is a summative effect of nutrients to encourage and limit. Fifty‐five foods (12.2%) received a positive Smart Nutrient Score. The system could be a useful way to evaluate and market snacks. Supported by NIFA, USDA.
Blast‐induced traumatic brain injury reduces rotarod performance in Sprague‐Dawley male rats
Authors: Awwad Hibah Omar , Gonzalez Larry P , Lerner Megan R. , Tompkins Paul , Brackett Daniel J , Standifer Kelly M
Published: June 2021

Blast‐induced traumatic brain injury (bTBI) is the most common injury of modern warfare. In 2008, nearly 25,000 U.S. soldiers were diagnosed with TBI, with neuronal and behavioral defects detected up to one year post‐blast. Research is only beginning to reveal the mechanisms of bTBI. Our objective was to determine the effect of our bTBI model on motor function in rats using the rotarod. Non‐blast TBI studies assure the high sensitivity of the rotarod as a vestibulomotor test, where deficits are detected even after recovery from beam balance/walk. Sprague‐Dawley male rats pretrained on the rotarod apparatus prior to blast injury were tested on days 1–4, 7 and 8 post‐blast. bTBI was induced by a blast‐pressure wave generator, with exposures at ~60 and ~80 psi. Rotarod performance on days 1, 2, 7 and 8 post‐blast in 80 psi blast‐injured rats was significantly reduced compared to sham rats, whereas 60 psi blast did not reduce performance. Cerebellar immunoreactivity of the glial fibrillary acidic protein, a reactive astrocyte marker, was significantly increased on day 9 post‐blast in rats exposed to 80 psi blast compared to sham rats. This indicates a direct link between bTBI and increased reactive astrocytes in the cerebellum. This is the first study, to our knowledge, to show a pressure‐dependent effect on rotarod performance as a measure of vestibulomotor function in bTBI. Supported by Department of Defense (CDMRP) – DR080343
Phenotypic and functional genomic analysis of heat and cold stress in transcription factor deletion strains of Saccharomyces cerevisiae
Authors: Kubeck Lauren Nicole , Rodriguez Kenny R , Entzminger Kevin C , Kuelbs Stephanie D , Hubbard Kristine B , Dahlquist Kam D
Published: June 2021

Budding yeast, Saccharomyces cerevisiae, respond to temperature changes by either activating or repressing the transcription of certain genes. The response of budding yeast to increased temperatures has been well studied, but little is known about which transcription factors regulate the response to cold. Therefore, transcription factors previously suggested to be involved in environmental stress responses were studied. Twenty‐nine strains, each deleted for a particular transcription factor were genotyped by PCR and tested for growth impairments at 15, 20, 30, and 37°C on rich media. Strains deleted for the genes INO2, GLN3, SWI4, and ZAP1 were impaired at cold temperatures. A strain deleted for the gene AFT1 was impaired for growth in both heat and cold. Bioinformatic analysis was performed to determine whether the target genes of these transcription factors were induced or repressed due to cold shock in a DNA microarray experiment performed on the wild type parental strain. For example, targets of the Zap1p transcription factor were induced during cold shock, corroborating the results of the temperature‐dependent growth experiment. This work was supported by NSF awards 0634613 (K.C.E., S.D.K., K.R.R., K.D.D.) and 0921038 (L.N.K. and K.D.D.).
Characterization of the Thermotoga maritima ADP‐glucose pyrophosphorylase: Both glgC and glgD Subunits are Required for Optimal Activity and Allosteric regulation
Authors: Matsui Mikiko , Guandique Erick , Tran Margaret , Nisperos Sean , Orry Andrew , Meyer Christopher
Published: June 2021

The T. maritima ADP‐glucose pyrophosphorylase (ADPG PPase) is comprised of both glgC and glgD subunits. Analytical ultracentrifugation, size‐exclusion chromatography, and native‐PAGE analyses all indicate that glgC and glgD exist as monomers that oligomerize to form a glgD/C heterotetramer. Functional assays revealed negligible glgD activity alone; however, glgC showed ~20‐fold stimulation when combined with glgD. The kinetic parameters for glgC alone and the glgD/C complex were determined. glgC was insensitive to a screen of 15 known ADPG PPase effector molecules, but glgD/C exhibited 2.2‐fold activation by FBP. The glgD/C S0.5 values for ATP, G1P, and Mg2+ showed increased apparent affinities than glgC alone and the specific activity was 3.2‐fold higher than glgC at 37°C. The glgD/C S0.5 values for ATP, G1P, and Mg2+ also showed increased apparent affinities and a 2.7‐fold higher specific activity than glgC alone at 75°C. Both glgC K26A and D244N studies resulted in significantly decreased glgC activity, indicating that these two residues are important for catalysis. Supported in part by NSF Grant 0448676.
The Role of Methionine Residues in Activity and Subunit Communication in Malate Dehydrogenase
Authors: Billue Farren , Bell Ellis
Published: June 2021

Malate Dehydrogenase is a dimeric enzyme that catalyzes the oxidation of malate to oxaloacetate in a reaction involving both a hydride transfer [to NAD] and a proton abstraction. The enzyme demonstrates kinetic evidence for the involvement of subunit interactions in the catalytic activity and regulation [by citrate] but little is know concerning the mechanism of communication between the two active sites in the dimer. The subunit interface contains three conserved methionine residues [M62, M64 & M91] that are in close proximity to residues from the opposing subunit [P59, L63, Y273 & I88 as well as between M66 on one subunit and M62 & M91 on the opposing subunit]. These residues together with a Methionine close to the active site [M128] have been mutated using QuikChange mutagenesis to a variety of different amino acids to test their role in subunit communication and catalytic activity. The his‐tagged proteins are expressed, purified using Ni‐NTA affinity chromatography and characterized by MALDI‐tof and Circular Dichroism. The strength of subunit interactions is assessed using guanidine hydrochloride dissociation followed by dynamic light scattering. Initial rate kinetic studies demonstrate changes in both Vmax and Km of various mutants as well as indicating changes in citrate regulation of the protein.

This work is supported by NSF Grant MCB 0448905 to EB and an HHMI summer stipend to FB
The Role of G51 and I140 in Glyoxasomal Malate Dehydrogenase
Authors: Jones Rachel , Gruner Rachel , Bell Ellis
Published: June 2021

Glyoxysomal Malate Dehydrogenase is an enzyme that is crucial to the Krebs Cycle. Clustal analysis determined that G51 and I140 are completely conserved residues in gMDH. G51 is close to P119, and we hypothesized that mutatation to alanine or serine would displace the proline impacting the active site loop. I140 appears to interact with non polar residues around it. To assess the role of these hydrophobic interactions, I140 was mutated to glutamine. Mutants were created using Stratagene® QuickChange protocol. Expressed protein was purified using Ni‐NTA chromatography and kinetic constants were obtained by fitting the data to a Michaelis Menten Equation to give Km and Vmax. G51mutants had higher activity than that of the native gMDH, however the serine mutant affected OAA and Citrate binding, but had no effect on NADH. G51A impacted only NADH binding. The I140Q mutant had a much lower activity and exhibited weaker NADH binding but tighter OAAbinding. This research is being incorporated into multiple lessons appropriate for grades 6–8 in a public school setting to illustrate experimental design and how to properly follow the scientific method, and illustrate the structure and reactivity of molecules.

This work is supported by NSF Grant MCB 0448905 to EB

We would also like to thank ASBMB for funding through its k12 teacher program.
An Evolutionary and Functional Analysis of Vibrio Pathogenicity Island 2
Authors: Napolitano Michael Gregory , Almagro‐Moreno Salvador , Boyd Fidelma
Published: June 2021

The evolution and emergence of pathogenic bacteria is tightly linked to the horizontal transfer of pathogenicity islands (PAIs) and bacteriophages encoding a range of virulence factors. We studied the evolutionary relationships between integrases and excisionases from a group of islands, and used VPI‐2 as a model for pathogenicity island excision. We determined the functional role of the VPI‐2‐encoded integrase (intVPI‐2) and two putative excisionases (vefA and vefB) in excision. We found negligible levels of excision as measured by attB levels in the ΔintVPI‐2 strain whereas overexpression of intVPI‐2showed an increase in excision. In the ΔvefA excision is severely decreased. Overexpression of vefA (VC1785) in the wildtype causes complete excision of the island, while complimenting vefA in either of the deleted strains ΔvefA and ΔvefB leads to only a minor increase in excision. Overexpression of vefA also causes excision of a second island, VPI, critical for pathogenicity. For the first time, we have demonstrated that a factor encoded on one island, vefA, causes the excision of a second. This work was funded by a NSF career grant to EFB and a Beckman Scholars grant to MGN.
Common plant foods consumed in Korea are rich source of phytochemicals
Authors: Yoon G‐A , Cho Y‐S , Yeum K‐J , Chen C‐YO , Tang G , Blumberg J B , Russell R M , Yoon S , Lee‐Kim YC
Published: June 2021

Commonly consumed plant foods (n=95) in Korea were selected and stratified according to botanical family names. Carotenoids and tocopherols were measured by reverse‐phase HPLC, total phenols by the Folin‐Ciocalteu reaction, and antioxidant capacity by the Oxygen Radical Absorbance Capacity assay. β‐Carotene and lutein content were highest in the Compositae, Umbelliferae, and Goosefoot families at 3.10–4.13 and 5.66–7.69 mg/100 g FW, respectively. Tocopherols were identified mainly in the Legunimosae family. Total phenols were highest in Umbelliferae followed by Leguminosae and Compositae at 149.3–199.3 mg gallic acid equivalents/100 g FW. Antioxidant activity was also high in Umbelliferae, Leguminosae and Compositae families. The top 20 plant foods containing high levels of β‐carotene, lutein and total phenols as well as showing high antioxidant activities were, in general, traditional Korean green leafy vegetables such as Myeong Il Yeop, red pepper leaves, Perilla leaves, Mugwort, whereas only parsley, spinach, lettuce, and carrot were in the list of top 20 foods among common western plant foods. Thus, leafy green vegetables from the Compositae and Umbelliferae families, especially those commonly consumed in Korea, are rich in phytochemicals with high antioxidant capacity in vitro. [Supported by USDA ARS 1950‐51000‐065‐08S, USA and BioGreen 21 #20070301034009 RDA, Korea]
An integrated high resolution mass spectrometric and bioinformatic approach for the rapid identification of flavonoids in plant extract
Authors: Aldini G , Regazzoni L , Scevaroli M , Carini M , Chun HK , Cho SM , Park KM , Yeum K‐J
Published: June 2021

An integrated and fully automated high resolution mass spectrometric and bioinformatic approach was set‐up for the rapid and unequivocal identification of flavonoids and other phenol compounds in plant extract. The approach consists of the following steps: 1) direct infusion of the extract into an Orbitrap mass analyzer (resolution 100,000; negative ion‐mode, ESI source); 2) automated identification of the monoisotopic masses and of the corresponding isotopic patterns by a Boolean logic algorithm; 3) searching the experimental monoisotopic masses (tolerance 5 ppm) in a database of flavonoids (http://www.phenol‐explorer.eu/compounds; http://www.metabolome.jp/download/flavonoid/) implemented with phenol compounds (i.e. chalcons). Final identification is achieved by matching the isotopic pattern and by MS/MS fragmentation studies. In particular, experimental MS/MS fragments are matched with those retrieved by the data base http://www.massbank.jp and by the predicted ions obtained by the software ACD/MS‐fragmenter. The method was firstly validated by mixtures of standard polyphenols and was then applied for the identification of phenols and flavonoids contained in an EtOH extract of Angelica keiskei. The method can also be used for semiquantitative analysis using Trolox as internal standard. [Supported by USDA ARS #58‐1950‐7‐707, USA and BioGreen 21 #20070301034009 RDA, Korea]