bluuebunny/crossref_metadata_2025 huggingface dataset:
Hair Cell Development and Regeneration in Zebrafish Lacking the Cannabinoid Receptor2
Authors: Deida Tiffany Tossas , Colon Luis , Rodríguez Roberto , Santana Alexis , Behra Martine
Published: June 2021

Hair cells (HCs) are mechanosensory receptors found in sensory epithelia in the vertebrate inner ear that detect sound and head movements. HCs are fragile and prone to damage from various causes like disease, aging, and noise pollution. Unlike mammals, lower vertebrates including birds, amphibians and fish can regenerate HCs. To understand the underlying molecular mechanisms of HCs regeneration, we study the lateral line (LL) in a powerful genetic and regenerative animal model: the zebrafish. The LL is a skin‐deep fish sensory organ that is evolutionarily linked to the vertebrate sensory epithelia of the inner ear. It is composed of stereotypical distributed sensory patches called neuromasts (NMs) which contain HCs that are highly similar in function, cellular structure and gene expression to HCs of the inner ear. LL‐HCs are deflected and activate by surrounding water movements. Using CRISPR‐Cas9 genome editing technology, our laboratory has previously generated a null allele of the cnr2 gene (cnr2upr1) which is encoding the cannabinoid receptor 2 (CB2). We found that cnr2 mRNA is highly expressed in LL‐HCs. So, we hypothesized that CB2 has a role in the development and the regeneration of LL‐HCs. We counted LL‐HCs at different developmental stages in wild type and homozygote mutant larvae (cnr2upr1/upr1) to see if their number is affected in the absence of a functional CB2 protein. Next, to trigger vigorous HCs regeneration, we synchronously ablated LL‐HCs by copper treatment of 5‐day post‐fertilization (dpf) wild type and homozygote animals. We counted regenerating LL‐HCs for 3‐day post‐treatment. We will present our preliminary results that will ultimately allow us to conclude on the involvement of CB2 in development and in regeneration of the LL and on a potential link between endocannabinoid signaling and sensory epithelia development and regeneration.

Support or Funding Information

CREST

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Use of NMR to probe changes in the flexibility of acid‐stress chaperone HdeB from its inactive to active state
Authors: Abasi Lannah , Crowhurst Karin
Published: June 2021

HdeB is an intrinsically disordered periplasmic chaperone essential to the acid resistance of numerous pathogenic bacteria. It, along with its homolog, HdeA, binds to vulnerable periplasmic proteins at low pH and prevents them from irreversibly aggregating in the acidic stomach. The activity of these two proteins is responsible for the survivability of pathogenic bacteria and resulting proliferation of dysentery. At physiological pH, HdeB is an inactive folded homodimer and remains a structurally unchanged, fully folded dimer in its active state (at pH 4–5), suggesting that the mechanism of chaperone activation is a result of changes in its dynamics (internal motions). The long‐term goal of this study is to gain insight into HdeB's unique chaperoning mechanism by characterizing its pH dependent changes in structure and dynamics. In order to achieve atomic level specificity and detect subtle conformational changes, we are utilizing Nuclear Magnetic Resonance (NMR) spectroscopy to compare HdeB's flexibility and structure in its inactive and active states at pH 6.0 and 4.5, respectively. Isotopically labeled HdeB was recombinantly expressed and purified. Subsequently, protein backbone and side chain dynamics were examined at pH 6 and pH 4.5 utilizing R2 and R1 relaxation experiments, which identify fast (ps–ns) time scale motions within the structure; this indicates local motions, such as methyl rotations. Additionally, intermediate (μs‐ms) time scale motions, which reflect global conformational changes and flexibility, were identified using CPMG relaxation dispersion experiments. Notable changes in dynamics were observed with activation. At pH 6.0, most residues with μs‐ms motions are clustered at the dimer binding interface (BC loop and helix B). With the transition to pH 4.5, μs‐ms motions expand within the dimer binding interface (W27, T33, G37, T40, K53) and extend beyond it (L16δ, I55δ). Furthermore, at pH 4.5, Helix B decreases in solvent protection, suggesting decreased burial and a loosening of the dimer binding interface. Since this helix is extremely hydrophobic, loosening exposes residues that are well suited to interacting with the exposed hydrophobic regions of an unfolding client protein, likely enabling interactions crucial to substrate binding. Interestingly, the largest changes with activation were observed near multiple titratable residues, making us suspect a protonation change. Continuing research will focus on experimentally determining acid dissociation constants to confirm this.

Support or Funding Information

We gratefully acknowledge the support of the NIH for research funds (SC3‐GM116745) and BUILD PODER for research support to L.A., as well as the NSF for funding the purchase of our NMR spectrometer (CHE‐1040134).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Arabidopsis Histone Deacetylase 5 is Required for Pseudomonas‐Induced Deacetylation of Histone H3 at Lysine 9
Authors: van Dijk Karin , Kim Panya , Hubbard Troy , Nguyen Linda , Guo Ming , Alfano Jim
Published: June 2021

The gram‐negative plant pathogen Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) depends on a type III secretion system (T3SS) to colonize plant tissue, and cause disease. The T3SS facilitates injection of virulence proteins called effector proteins (T3Es) directly into host cells. The T3Es have been shown to target components of plant immunity and are essential for Pto to cause disease. We have been interested in determining the extent that Pto T3Es are involved in modulating host chromatin to favor pathogenesis. Through Immunoblot and Chromatin Immunoprecipitation analyses we found a rapid deacetylation of host histone H3 lysine 9 (H3K9) along innate immunity related genes in response to Pto. This deacetylation was dependent on both T3Es and the host histone deacetylase HDA5. Moreover, Arabidopsis plants lacking HDA5 had an enhanced resistance to Pto infections and produced more callose in response to Pathogen Associated Molecular Pattern exposure. Moreover, through analyses of specific T3E Pto mutants and transient and transgenic expression of specific T3Es, we found that only a small subset of the T3Es are needed for the HDA5‐triggered deacetylation. Collectively our data show a Pto T3E‐induced deacetylation by HDA5 along immunity related genes in Arabidopsis.

Support or Funding Information

This work was supported by NSF award #1557417, NSF award # 0940177 and NIGMS award # 8P20GM103427

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Use of directed peptide libraries for discovery of substrates of Arabidopsis CDPKs
Authors: Harmon Alice C. , Curran Amy C. , Harper Jeffrey F.
Published: June 2021

CDPKs are a family of 34 calcium‐dependent protein kinases in Arabidopsis. Because of functional redundancy, reverse genetic methods have not been successful in revealing functions of the majority of the family members. We have used directed peptide libraries to discover substrates of these kinases and to compare substrate specificity. Over 650 synthetic peptides, biotinylated at their N‐termini, were tested as substrates of four representative CDPKs in SPA (GE Healthcare) assays. The peptide sequences were based on known Arabidopsis phosphoproteins, proteins common to both Arabidopsis and rice that contain the CDPK phosphorylation motif R/K‐X‐X‐S/T, and peptides used to assay activities of animal protein kinases. The CDPKs phosphorylated 188 peptides. Phosphorylation of 20 of the parent proteins has been corroborated in vivo. CPK34 phosphorylated 162 peptides (23 unique); CPK 1 phosphorylated 129 peptides (4 unique); CPK10 phosphorylated 133 peptides (20 unique); and CPK 16 phosphorylated only five peptides (none unique). These data demonstrate both the substrate overlap and specificity of the CDPKs and reveal new potential in vivo substrates. This work was supported by a grant from the National Science Foundation (MCB‐0114769).
Teenagers consume far more discretionary calories and far less fruits and vegetables than that recommended in the Dietary Guidelines for Americans, 2005
Authors: Bowman Shanthy A. , Moshfegh Alanna J. , Friday James E. , Martin Carrie L.
Published: June 2021

The Dietary Guidelines for Americans (DGA), 2005, recommend that Americans reduce their discretionary calorie intakes, the excess of which may lead to obesity, and increase intake of fruits and vegetables. The objectives of this study are to (1) determine the discretionary calorie, fruit, and vegetable intakes of 14 to 19 year old males (N=831) and females (N=787) in What We Eat In America, National Health and Nutrition Examination Survey 2003–2004, who had complete dietary data on day 1 of the survey, and (2) evaluate their diets in comparison to the DGA recommendations. The estimated mean intakes of males and females were compared using t‐tests at alpha =0.05 level of significance. Males consumed significantly more total energy per day than females (2756±77 vs. 2019±28 kcal), discretionary calories from fat (744±27 vs. 573±14 kcal), and added sugars (502±18 vs. 381±15 kcal). However, both males and females consumed on average 46% of their total energy as discretionary calories (19% from added sugars and 27% from fat). Both males and females ate only 1 cup equivalent of fruits and 1.3 cup equivalents of vegetables, far below the recommended levels of 2.5 and 2 cup equivalents of fruits and 3.5 and 2.5 cup equivalents of vegetables for males and females, respectively. The study showed that teenagers should reduce discretionary calorie intakes and increase fruit and vegetable intakes. Funding source for this study is USDA.
The effects of clofibrate on malonyl‐CoA inhibition of carnitine pamitoyltransferase I (CPT I) and fatty acid oxidation in liver of neonatal pigs
Authors: Shim Kwanseob , Xi Lin , Herfel Tina , Odle Jack
Published: June 2021

Previous studies demonstrated that clofibrate increased CPT I activity and fatty acid oxidation in piglets, but the role of malonyl‐CoA, a physiological inhibitor of CPT I, has not been clearly elucidated. Thirty colostrum‐deprived newborn pigs were fed milk replacer and orally gavaged with either vehicle (2 % Tween 80) or clofibrate (75 mg /kg body weight) +/− etomoxir (5 mg/ kg body weight) once daily for up to 7 days, and the sensitivity of liver CPT I to malonyl‐CoA inhibition and fatty acid oxidation were determined using 1‐14C‐octanoic, oleic and erucic acids. Administration of clofibrate significantly increased liver CPT I activity (60%) and malonyl‐CoA IC50 values (56%). Addition of etomoxir potently inhibited CPT I activity, but malonyl‐CoA IC50 remained unchanged. Consistent with CPT I activity, clofibrate increased mitochondrial fatty acid oxidation by 53%, but did not affect the rate of peroxisomal beta‐oxidation. Etomoxir did not alter the rate of fatty acid oxidation despite a 30% reduction in CPT I activity. These results indicate that the increase in fatty acid oxidation induced by clofibrate is due to both an increase in CPT I activity and a decrease in sensitivity of CPT I to malonyl‐CoA inhibition, suggesting that malonyl‐CoA plays an important role in the induction of fatty acid oxidation by clofibrate. Supported by CSREES, USDA NRI program award 2007‐35206‐1 7897.
Somatotropin Enhanced Muscle Protein Synthesis in Growing Pigs Is Not Modulated by Insulin
Authors: Wilson Fiona A , Nguyen Hanh V , Suryawan Agus , Orellana Renan A , Jeyapalan Asumthia S , Davis Teresa A
Published: June 2021

Chronic, 7‐day treatment of growing pigs with porcine somatotropin (ST) promotes protein synthesis and doubles postprandial levels of insulin, a hormone that enhances translation initiation. This study aimed to determine whether the ST induced increase in skeletal muscle protein synthesis was mediated through an insulin‐induced stimulation of translation initiation. Pancreatic glucose‐amino acid clamps were performed in overnight fasted pigs after 7–10 days of ST (150 μg/kg/d) or vehicle treatment, to reproduce 1) fasted (5 μU/ml), 2) fed (25 μU/ml), and 3) fed‐ST treated (50 μU/ml) insulin levels, while glucose and amino acids were maintained at baseline fasting levels. Fractional protein synthesis rates were determined and translational control mechanisms were examined. Effectiveness of ST treatment was confirmed by reduced urea nitrogen (P<0.001) and elevated IGF‐1 levels (P < 0.01) in plasma. Skeletal muscle protein synthesis was independently increased by both insulin (P < 0.07) and ST (P < 0.05). Insulin enhanced eIF4G·eIF4E complex formation as well as PKB and eIF4G phosphorylation, but ST‐treatment did not alter translation initiation factor activation. We conclude that the ST‐induced stimulation of skeletal muscle protein synthesis in growing pigs is independent of the insulin‐associated activation of translation initiation. (Supported by USDA NRI 2005‐35206‐15273)
Non‐viral delivery of therapeutic nucleic acids to investigate the role of transcriptional networks in the ischemic heart
Authors: Tranter Michael , Wilhide Michael E , Liu Yemin , Ren Xiaoping , Reineke Theresa , Jones W Keith
Published: June 2021

Nuclear factor‐kappaB (NF‐κB) is a key player in the physiological outcomes of myocardial ischemic events including ischemia‐reperfusion (I/R), permanent occlusion (PO), and the cardioprotection of ischemic preconditioning (IPC). Our lab has demonstrated that NF‐κB plays a protective role in late IPC and after PO, yet has an injurious role after I/R. Such results suggest the transcriptional activation of differential gene sets by each ischemic event. This was confirmed in our lab by a gene microarray analysis comparing I/R, PO, and IPC. Our microarray results also suggested potential cross‐talk of NF‐κB with other transcriptional networks, such as those regulated by HIF‐1α, AP‐1, and HSF‐1. Our aim is to deliver transcription factor decoys and interfering RNAs to the heart in vivo and begin to elucidate the relative role of these gene networks in myocardial ischemia. We have demonstrated nucleic acid delivery to the heart via novel polyglycoamidoamine (PGAA) glycopolymers that act through formation of nanoscale “polyplexes” and enhance cellular uptake via endocytosis resulting in nearly transmural transfection of the myocardium without eliciting cytotoxicity. Our work provides a high‐throughput in vivo approach to the study of the regulation of interlaced gene networks during myocardial ischemic events and will augment the identification of new clinical targets for the treatment of myocardial ischemia. This work was supported by NIH grant R01 HL63034 (WKJ) and NSF award number 0333377.
EFFECTS OF ISOVOLEMIC HEMODILUTION ON TISSUE OXYGEN CONSUMPTION USING A HEMOGLOBIN‐BASED OXYGEN CARRIER AND HUMAN SERUM ALBUMIN
Authors: Song Bjorn Kyungsuck , Pittman Roland N.
Published: June 2021

This microcirculatory study compared the effects on oxygen transport of two hemodilution fluids: HBOC‐201 (Biopure Corp., Cambridge, MA) a Hemoglobin‐Based Oxygen Carrier (HBOC), and 5.9% Human Serum Albumin (HSA) an iso‐oncotic non‐oxygen carrying colloid solution. Measurements using intravital microscopy were made on the spinotrapezius muscle of Sprague‐Dawley rats. Interstitial PO2 was measured using phosphorescence quenching microscopy, and recorded before and after isovolemic hemodilutions (HD) at hematocrits of 40% (baseline), 30% (moderate HD) and 15% (severe HD). Oxygen consumption (VO2) of the spinotrapezius muscle was derived from PO2 recordings following the rapid inflation of a Saran bag placed around the objective. When the bag was inflated, blood flow in the muscle was arrested and PO2 rapidly fell over several seconds; the rate of decline of PO2 was proportional to VO2. For moderate HD with HBOC‐201 interstitial PO2 did not change from baseline conditions (Hct~40%), while HD with HSA showed a decrease. For severe HD (Hct~15%) both PO2 and VO2 were significantly lower for the HSA group than for the HBOC‐201 group. These findings indicate that HBOC‐201 maintains both a higher PO2 and VO2 during hemodiluted states compared with a non‐oxygen carrying colloid solution (HSA).

(Supported by Naval Medical Research Center, DOD and NIH grant HL18292)
Effects of maturation on intracellular Ca2+ homeostasis in ovine pulmonary arterial smooth muscle cells
Authors: Goyal Ravi , Creel Kara D , Chavis Erica , Smith Gregory D , Longo Lawrence D , Wilson Sean M
Published: June 2021

Cytosolic Ca2+ is central to vascular reactivity and alterations are involved in pulmonary hypertension (PH). Apart from altered vascular reactivity, maladaptations in 5‐HT signaling are important to the development of PH in newborn and adult, where there may be distinctive differences in 5‐HT signaling. Using fluorescence imaging of Ovine pulmonary arterial smooth muscle cells (PASMCs) loaded with Fura‐2 the hypothesis that Ca2+ transport and release pathways are under‐developed in fetus was tested. The data show that 5‐HT (10 μM) stimulation elicited Ca2+ elevations in very few fetal PASMCs as compared to adult. However, other parameters of Ca2+ signaling, which might influence 5‐HT induced Ca2+ elevations were unchanged with maturation; these included basal Ca2+, sarcoplasmic reticulum (SR) Ca2+ storage, plasma membrane and SR Ca2+ transport, and capacitative Ca2+ entry. Moreover, 5‐HT mediates Ca2+ release through IP3 signaling, but this pathway appears intact since 10 μM phenylephrine‐ and 1 μM angiotensin II‐mediated Ca2+ elevations were unchanged by maturation. Interestingly, in cells with 5‐HT responses the magnitude of the Ca2+ elevation was unchanged with age. Overall, this work suggests that 5‐HT mediated Ca2+ signaling adapts with maturation, possibly through changes in receptor expression or post‐receptor coupling. (Support from UM, Sigma Xi, NSF and NIH)
Slow rate of neuromuscular activation contributes to impaired movement acceleration and peak power in mobility‐limited older adults
Authors: Clark David J , Patten Carolynn , Reid Kieran F , Carabello Robert , Phillips Edward M , Fielding Roger A
Published: June 2021

Peak muscle power is a robust predictor of functional motor performance in older adults. A critical constituent of peak power is effective neuromuscular activation. This study investigated the relationship between pre‐movement activation rate, acceleration (Acc), and peak power (Ppeak) for a leg press task in healthy and mobility‐limited older adults.

METHODS 19 middle aged healthy (MH, 40–55y, 9 male), 19 older healthy (OH, 70–85y, 14 male), and 14 older mobility‐limited (OML, 70–85y, 7 male, SPPB ≤ 9) adults participated. EMG was acquired from the quadriceps during 5 max effort leg press trials at 40% of 1‐rep max. EMG was rectified, smoothed, and normalized to peak from a maximal voluntary contraction (MVC). EMG from each trial was aligned to movement onset, then averaged and fit using linear regression to yield activation rate (slope).

RESULTS OML demonstrated markedly lower activation rate (.13 %MVC/ms, p<.02) than MH (.24) and OH (.26). Similarly, Acc (4.4 m/s2, p<.01) and Ppeak (536W, p<.01) were impaired in OML relative to MH (6.5, 1057) and OH (5.8, 980). Across groups, activation rate was strongly related to Acc (R2=.66, p<.0001) and Ppeak (R2=.55, p<.001).

CONCLUSION Attenuated rate of neuromuscular activation is associated with reduced movement acceleration and power production in mobility‐limited older adults.

Supported by USDA 58‐1950‐7‐707and NIA RO1‐AG‐18844
E‐NTPDase1 and alkaline phosphatase control chemotaxis of human neutrophils by generating adenosine from released ATP
Authors: Corriden Ross , Insel Paul A. , Chen Yu , Junger Wolfgang G.
Published: June 2021

Human polymorphonuclear neutrophils (PMN) release ATP in response to stimulation by chemoattractants. Released ATP and its product adenosine regulate chemotaxis by activating P2 and P1 receptors, respectively. Here we investigated the ecto‐ATPases that generate adenosine from released ATP. We find that ecto‐nucleoside‐triphosphate‐diphosphohydrolases 1 (E‐NTPDase1, CD39), which hydrolyses ATP to AMP, and alkaline phosphatase (ALP), which converts AMP to adenosine, are highly expressed in human PMN and that E‐NTPDase1 and ALP are recruited to the leading edge upon polarization of PMN in a chemotactic gradient. While E‐NTPDase1 is tightly associated with the leading edge, ALP remains more broadly distributed across the cell membrane. Inhibitors of E‐NTPDase1 reduced ATP hydrolysis by 60%, while inhibitors of ALP completely blocked the generation of adenosine. Inhibition of both enzymes significantly reduced the migration speed of PMN towards chemoattractant in a gradient field. Thus, E‐NTPDase1 and ALP play important roles in the control of PMN chemotaxis by generating extracellular adenosine from released ATP. (Supported by grants from NIGMS and DOD)
Dexamethasone allows sugar‐induced trafficking of the glucocorticoid receptor in rat small intestine
Authors: Douard V. , Elshenawy S. , Lagunoff D. , Ferraris R.
Published: June 2021

Fructose (F) consumption has increased dramatically and is linked to childhood obesity but mechanisms regulating neonatal F absorption are unknown. The intestinal F transporter GLUT5 cannot be enhanced by luminal F in rats < 14 d old unless the gut is primed with dexamethasone (D). The effect of D requires transcription and is mediated by the glucocorticoid receptor (GR). We tested the hypothesis that in 10 d old pups, D‐ and F‐induced GLUT5 upregulation involves trafficking of the GR from cytoplasm to nucleus. Intestines of 10 d pups (+ D), were perfused in vivo with F or glucose. GR mRNA levels were similar in all 4 groups but GR levels in the nucleus increased markedly only in D‐primed, F‐perfused pups. Regulation of KPNA2, a nuclear importin whose mRNA levels are age‐sensitive and increase with F perfusion, seems linked with that of GLUT5. KPNA2 is also transcriptionaly upregulated and levels are high in nuclei of F‐perfused, D‐primed 10d pups, indicating that KPNA2 could be the importin carrying GR to the GRE in the GLUT5 promoter. Ser‐211 is phosphorylated when GR is active. Using a specific Ser211‐GRp antibody, we failed to observe GRp in the nucleus, indicating that phosphorylation is not necessary in D‐allowed, F‐induced GR trafficking into the nucleus. In 10 d pups, regulation of GLUT5 involves KPNA2‐mediated trafficking of the GR to the nucleus. Support: NIH RDK075617A, NSF IOS‐0722365
Studies of Ion Transport at Extreme Temperature
Authors: Cuppoletti John , Birn Sarah S , Tewari Kirti P , Chakrabarti Jayati , Malinowska Danuta H
Published: June 2021

Biological transport proteins have potential for use in many biotechnology applications. However, high temperatures or harsh chemical environments could limit their use. This study examines the stability of ion channels at high temperature. Preliminary studies using gramicidin showed H+ transport at unexpectedly high temperature. In the present study a ClC‐like channel from the archeobacter hyperthermophile, Methanococcus jannaschii (mj) which grows at a temperature optimum of 85°–90°C, was cloned from mj genomic DNA. It was ligated into pcDNA3.1/V5‐His TOPO and stably expressed in HEK293 cells. Cl− currents were studied by whole cell patch clamp electrophysiology. ClCmj‐expressing HEK293 cells (but not mock transfected cells) exhibited voltage activated Cl− currents which were inhibited by 500 μM CdCl2. When membranes were isolated from ClCmj‐expressing HEK293 cells and fused to planar lipid bilayers, single Cl−channels were evident at room temperature (25°C) which persisted at elevated temperatures. No channels were evident with membranes from mock transfected HEK293 cells. These studies demonstrate that ion channels from these organisms will be functional at elevated temperatures as required for applications in biotechnology. Supported by AFOSR.
Soy‐based Formula Does Not Adversely Affect Immune System Development in Neonatal Piglets
Authors: Horton Chris , Burris Ramona L. , Badger Thomas M. , Nagarajan Shanmugam
Published: June 2021

Breast‐fed infants are less susceptible to infections and allergic reactions than those fed formula, suggesting that diet may influence neonatal immune system development. The goal of the present study was two fold: 1. to determine the effect of formula feeding relative to breast‐feeding on neonatal immunity; and 2) to determine whether soy‐based formula has any adverse effects on neonatal immune system development. Piglets were breast‐fed by the sow or weaned at 2 days to a milk‐ or soy‐based formula. At PND 21, splenocytes from the sow‐fed group showed 3 to 4 fold higher T lymphocyte proliferation compared to the formula‐fed groups. The increase in T‐lymphocyte proliferation was also reflected in circulating levels of IgG and IgM in sow‐reared piglets. Notably, both formula diets showed greater NK cell cytotoxicity than the sow‐fed piglets. Our findings suggest that the neonatal innate immune system in sow‐reared piglets is functionally better developed than that in formula‐fed piglets. Comparison between soy‐ and milk‐based formulas did not show any differences in NK cell cytotoxicity, total circulating antibody levels or lymphocyte proliferation, suggesting that soy formula does not have adverse effects on neonatal immune system development. Supported by ARS CRIS#6251‐51000‐005‐03S and in part by INBRE program funded by NIH.
Commonly reported US adult multivitamin/mineral (MVM) products: analysis of labeled vs. analytical values for 19 vitamins and minerals
Authors: Andrews Karen , Roseland Janet , Zhao Cuiwei , Schweitzer Amy , Holden Joanne M , Perry Charles , Dwyer Johanna , Picciano Mary Frances , Fisher Kenneth , Saldanha Leila , Yetley Elizabeth , Douglass Larry
Published: June 2021

Adult MVM products, representing over 55% of national market share, were analyzed for nutrient content. Nationally representative samples of each product were obtained in 6 retail locations. Products were sent to independent laboratories for analysis, along with quality control materials. For each nutrient, mean percent differences from label (%DL) and among‐lot variability were calculated. To identify patterns of variance, regression analyses were conducted for %DL vs. labeled values for the 35 products. For 12 nutrients (calcium, copper, iron, magnesium, manganese, niacin, phosphorus, potassium, riboflavin, vitamin C, alpha‐tocopherol, and zinc), mean %DLs for all products were <12%. For 7 nutrients (selenium, vitamin B6, chromium, thiamin, vitamin B12, folic acid, iodine), mean %DL values ranged from 14% to 46%. Future study will include the regression analysis of data for MVM products that garner a lower market share to determine if variance patterns can be used to predict %DL for products to be included in the Dietary Supplement Ingredient Database. This research was funded by USDA and the Office of Dietary Supplements at the National Institutes of Health.
Resolution of Genetic Structure in White‐Tailed Deer: A tool for investigating an outbreak of CWD in Illinois
Authors: Kelly Amy Catherine , Mateus‐Pinilla Nohra , Diffendorfer Jay , Beissel Tom , Shelton Paul , Killefer John , Ruiz Marilyn , Brown William , Novakofski Jan
Published: June 2021

Microsatellite markers have been developed and tested in a multitude of organisms, and are useful because of their potential to efficiently resolve genetic relationships between individuals in large populations. The objective of this study was to use a panel of microsatellite markers with high‐throughput analysis to obtain genetic information in white‐tailed deer from Illinois. Chronic Wasting Disease (CWD), a cervid transmissible spongiform encephalopathy, was identified in this area in 2002. Inferring structure and movement from genetic information may clarify the relative contribution of animal and environmental components of CWD transmission. Using fourteen microsatellites, multi‐locus genotypes were compiled for over 700 deer sampled as part of ongoing CWD surveillance in Illinois. Markers were tested for inbreeding patterns and deviations from Hardy‐Weinburg equilibrium, indicators of potential bias related to microsatellites. Of the 14 markers, 11 were in Hardy‐Weinburg equilibrium and three were associated with inbreeding with in sub‐populations. Genotypes were evaluated for genetic isolation by distance and used to infer trends of gene flow and migration, factors that could aid in the prediction of CWD spread across the landscape. Research funded by USGS and IDNR.
Long‐Chain Omega‐3 fatty acids associated with better cognitive function and less depressive symptoms in a population of Puerto Rican adults in Boston, MA
Authors: Arsenault Lisa Nicole , Scott Tammy M , Tucker Katherine L
Published: June 2021

The omega‐3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) found in fatty fish are increasingly recommended for promoting brain health with aging. Studies have reported protective associations between dietary DHA/EPA or fatty fish and incident dementia, but few have reported specific measures of cognitive functioning or focused on non‐white populations. Utilizing baseline data from the ongoing Boston Puerto Rican Heath Study, we examined the associations between dietary DHA/EPA and cognitive performance and depressive symptoms in Puerto Rican adults aged 45+. Diet was assessed with a food frequency questionnaire designed for the population. Cognitive function was assessed with a neuropsychological battery of tests and depression by the Center for Epidemiologic Depression Scale (CES‐D). Adjusting for age, sex, education, total energy, saturated fat, BMI, multivitamin use, drinking, acculturation, and household income; dietary DHA/EPA (log transformed) was significantly associated with global cognition, learning, and executive functioning. MMSE score (β=0.35, p=0.007), word list immediate (β = 1.21, p=0.007) and long‐term recall (β=0.34, p=0.03), letter fluency (β=1.71, p=0.0001), and Stroop Task (β=0.94, p=0.038). CES‐D score was also significantly associated with DHA/EPA intake (β=−1.58, p=0.01). Research supported by NIA AG023395 and USDA ARS 58‐1950‐7‐707.
DNA Structure Selectivity of Escherichia coli versus Thermus aquaticus DNA Polymerase I
Authors: Budiman Wowor Andy James , 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), primed‐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 released more ions when binding to pt‐DNA and ds‐DNA than when binding to ss‐DNA in KCl buffer. ΔCp is the temperature dependence of the enthalpy of a reaction. Both of these non‐sequence specific binding proteins exhibit relatively large heat capacity changes (ΔCp) upon DNA binding. Klenow binding to pt‐DNA and Klentaq binding to pt‐DNA and ds‐DNA show the highest ΔCp values. The thermodynamic differences among the different DNA structural preferences for the two polymerases suggest that the in vivo functions of these two supposedly homologous polymerases are different. This work was supported by NSF and Louisiana Biomedical Research Network.
The dependence of three P. abyssi inteins on extein sequence for efficient protein splicing
Authors: Kerrigan Adam M. , Powers Taryn L. , Dorval Deirdre M. , Reitter Julie N. , Mills Kenneth V.
Published: June 2021

Protein splicing is catalyzed by an intervening polypeptide, called an intein. Inteins facilitate their own excision from the flanking polypeptides, called exteins, as well as the ligation of the exteins. Three inteins interrupt the ribonucleotide reductase of the extreme thermophile Pyrococcus abyssi. We have separately over‐expressed each intein in E. coli as a fusion protein with flanking affinity domains. By site directed mutagenesis, we have altered the sequences flanking each intein to determine the flexibility of the intein insertion site. We have also made mutations to residues suspected of catalyzing the splicing steps in each intein, and plan to study the influence of these mutations on the rate and extent of splicing.

This material is based upon work supported by the National Science Foundation under Grant No. 0320824 and CAREER grant No. 0447647.
Molecular dynamics simulation of the NikR protein: Equilibrium conformational fluctuations reveal inter‐domain allosteric communication pathways
Authors: Bradley Michael John , Chivers Peter , Baker Nathan
Published: June 2021

The Ni2+ and DNA binding protein NikR is involved in nickel regulation in Escherichia coli through transcriptional repression of the NikABCDE nickel permease. NikR is a homotetramer and each chain contains both a DNA binding ribbon‐helix‐helix (RHH) domain and a Ni2+ binding regulatory ACT (aspartokinase, chorismate mutase, TyrA) fold. In this study we have utilized an equilibrium molecular dynamics (MD) simulation in order to explore the conformational dynamics of the NikR tetramer and determine important residue interactions within and between the RHH and ACT domains. Using two different correlation measures based on fluctuations in atomic position and non‐covalent bonding, we have defined a series of residue interactions that describe an allosteric communication pathway between the Ni2+ and DNA binding sites. Several of the residues identified by our analyses have been shown experimentally to be important for NikR function. An additional subset of the selected residues structurally connects experimentally important residues and may help coordinate allosteric communication between the ACT and RHH domains. Site‐directed mutagenesis of E. coli NikR and subsequent biophysical characterization will reveal the role of these residues in allosteric activation of this regulatory protein.

Support: NSF grant MCB‐0520877, NSF Graduate Research Fellowship to MJB
Food Safety Interventions are Effective in Increasing Knowledge within Different Ethnic Groups
Authors: Dixon Zisca , Huffman Fatma
Published: June 2021

The purpose of the study was to evaluate the most effective method to deliver food safety information to consumers. Culturally appropriate food safety tools were used to train a convenience sample (n=576). Group 1 (n=264) received food safety training using lectures and videotapes. Group 2 (n=130) received the same training as Group 1, with a hands‐on demonstration component. The Control group (n=182) received nutrition education. Knowledge was measured using a pre‐/post‐/follow up test design. Most of the participants from Group 1 were older, female, Hispanic, skilled with a 12th grade/GED education. For Group 2, participants were young, female, Black, unskilled and had 12th grade/GED education. The Control group were young, female, Hispanic, unskilled with a 12th grade/GED education. Means and SEM for pre‐/post‐/follow up test for Group 1 were 56.9±1.0, 71.2±64.0 and 64.0±1.1, respectively. For Group 2 the results were 51.3±1.4, 75.0±1.5 and 63.9±1.5, respectively. Control group results were 56.9±1.2, 58.3±1.2 and 62.0±1.3, respectively. Food safety knowledge was not significantly different between groups at pre‐test. However at post‐test, there were significant (p<0.001) differences in knowledge with Group 2 showing the greatest increase. Group 2, with mostly Black participants and similar educational background as the other groups, demonstrated greater knowledge with the hands‐on demonstrations.

This study was supported by funding from the National Integrated Food Safety Initiative of the Cooperative State Research, Education, and Extension Service, of the United States Department of Agriculture No. 2003‐04270.
Histone biotinylation represses retrotransposons in whole organisms, decreasing production of viral particles and retrotranspositions
Authors: Chew Yap Ching , West John T. , Kratzer Stephanie J. , Ilvarsonn Anne M. , Eissenberg Joel C. , Zempleni Janos
Published: June 2021

Repeat elements such as LTR retrotransposons constitute ~35% of the human genome. Transposition of these elements leads to genomic instability and may cause cancer. Previously, we have discovered an epigenetic mechanism that mediates LTR silencing: binding of biotin to lysine‐12 in histone H4 (K12BioH4), mediated by HCS. Here we provide novel evidence for a role of biotin in LTR silencing and genomic instability. First, we discovered that K9BioH2A is enriched at LTRs in Jurkat cells. The enrichment of K9BioH2A and K12BioH4 depended on biotin supply and was abolished by HCS knockdown. Decreased abundance of K9BioH2A and K12BioH4 in biotin‐deficient Jurkat cells increased LTR transcripts, increasing the risk for retrotranspositions. Second, we supplemented adults with 600 μg/d biotin for 4 wk. Biotin supplementation increased K12BioH4 at LTRs and decreased LTR transcripts in lymphocytes. Third, the production of MMTV particles increased in biotin‐deficient murine breast carcinoma cells compared with biotin‐normal controls. Fourth, the increased incidence of retrotranspositions was greater in HCS knockdown Drosophila compared with wild‐type controls. Our finding suggested that histone biotinylation plays a meaningful role in decreasing retrotranspositions and cancer risk. Support: University of Nebraska ARD, NIH DK063945 and ES015206, USDA 2006‐35200‐17138, NSF EPSCoR EPS‐0701892, and NSF MCB 0615831.
Moderate iron deficiency and physical training affect body fat accretion in growing male rats
Authors: McClung James P. , Andersen Nancy E. , Tarr Tyson N. , Stahl Chad H. , Young Andrew J.
Published: June 2021

Recent human studies demonstrate a relationship between iron deficiency and obesity, but it is not known whether physical training (PT) modulates the effects of iron deficiency on body composition and fat accretion. The objective of this study was to determine whether the effects of moderate iron deficiency on body fat accretion would be modulated by PT in growing rats. Male Sprague‐Dawley rats were fed iron adequate (IA, 45 mg/kg) or moderately iron deficient (ID, 10 mg/kg) diets ad libitum for 12 wks. Rats were assigned to four treatment groups (n = 10 per group): IA sedentary (IAS), IA physically trained (IAPT), ID sedentary (IDS), or ID physically trained (IDPT). PT involved running on motorized running wheels at 4 M/min for 1 h/d for 5 d/wk. After 12 wks, ID rats were not anemic, but iron status was diminished, as indicated by increased (P < 0.05) red cell distribution width ([mean ± SD] IAS = 13.8 ± 0.9, IAPT = 13.8 ± 1.1, IDS = 15.1 ± 0.5, IDPT = 14.1 ± 0.8 %). Body weight, lean body mass, and bone mineral content did not differ between groups. In contrast, the IDS rats had greater (P < 0.05) body fat as compared to all other groups (IAS = 31.8 ± 2.9, IAPT = 31.8 ± 2.0, IDS = 38.6 ± 6.7, IDPT = 32.8 ± 4.5 %). Thus, moderate iron deficiency may increase body fat accretion in growing animals, and PT appears to attenuate that effect. Research supported by MRMC.
Differential analysis of the serum proteome from bull terrier pups with lethal acrodermatitis
Authors: Grider Arthur , Casal Margret L
Published: June 2021

The biochemical lesion in bull terriers that causes lethal acrodermatitis (LAD) has not been identified, though the phenotype is similar to the systemic zinc deficiency syndrome in humans, acrodermatitis enteropathica. Currently, there is no biomarker for the disease. The goal of this research is to determine whether differential analysis of control and affected dog serum proteomes will identify proteins that might be used as biomarkers for the disease. Serum was obtained from two affected pups (15.6 wks of age) and one control (17.1 wks of age). Cibachron blue spin columns were used to remove albumin. The samples (100 g protein) were prepared for two‐dimensional gel electrophoresis. Following staining with colloidal Coomassie Blue, the gel images were analyzed for differential protein expression. Five proteins were increased in the serum from the unaffected bull terrier pup and three proteins exhibited increased expression in the serum from the affected bull terrier pups. These proteins may serve as biomarkers to identify LAD heterozygotes. The identifications of the differentially expressed proteins will be determined using peptide mass fingerprinting following mass spectrometry and database analysis. Funded in part by NCRR 2P40RR002512‐19 and USDA Hatch GEO00513.
Adenosine contributes to complement‐activation‐related paradoxical bradycardia
Authors: Bedocs Peter , Baranyi Lajos , Szebeni Janos , Bunger Rolf
Published: June 2021

Intravenous injection of complement (C)‐activating drugs in pigs can cause immediate cardiac anaphylaxis associated with myocardial ischemia and relative bradycardia during severe hypotension. We tested whether this paradoxical bradycardia could be mediated by myocardial adenosine. Pigs were injected i.v. with adenosine or various C‐activating drugs with or without pretreatment with nonselective adenosine receptor (A) blocker theophylline, or selective adenosine A1/A2 receptor antagonists cyclopentyl‐xanthine (CPX) and ZM24135, respectively. ECG and hemodynamic parameters were recorded.

Results:

Injection of 20–40 mg/kg adenosine caused a sharp hypotension with transient bradycardia, which mimicked the effect of C‐activating drugs. Both of these bradycardias were significantly inhibited by theophylline and CPX, but not by ZM24135, providing first pharmacological evidence that the phenomenon was mediated by the cardiac A1 receptor. In presence of CPX C‐activation related paradoxical bradycardia was converted to the physiological tachycardia.

Conclusion:

Complement activation related pseudoallergy (CARPA), severe bradyarrhythmia is mediated in part by cardiac adenosine, activating the A1 receptor subtype. Blockade of the A1 receptor using CPX could prevent potentially lethal paradoxical bradyarrhythmias due to liposomal drugs or hemorrhagic shock. Support: USUHS and Bay‐Nano
Identification of Na‐driven anion exchanger (NDAE) splice variants from Malpighian (renal) tubules of the adult yellow‐fever mosquito
Authors: Yamahiro Atsuko , Piermarini Peter M. , Beyenbach Klaus W.
Published: June 2021

To date, only one member of the bicarbonate transporter (SLC4) superfamily has been cloned from an insect—i.e., the Na‐driven anion exchanger (NDAE) of Drosophila (Romero et al., 2000, J Biol Chem 275: 24552). The goal of this study was to identify cDNAs encoding NDAE in Malpighian tubules of the yellow‐fever mosquito, Aedes aegypti. Using the recently completed Aedes genome, we designed gene‐specific primers to highly conserved regions of NDAE for conducting 5′ and 3′ RACE on Malpighian tubule cDNA. The 3′ RACE indicated that splicing does not occur at the end of the open‐reading frame (ORF) or in the 3′ ‐untranslated region (UTR). However, the 5′ RACE demonstrated extensive splicing in the 5′ ‐UTR immediately before the predicted start codon, resulting in transcripts with unique 5′ UTRs that we designate as NDAE‐A, NDAE‐B, and NDAE‐C. The putative functions of these unique 5′ UTRs are not known, but it is possible that they play a role in the translational efficiency or stability of a particular transcript within the Malpighian tubule. One splice variant (NDAE‐A’ ) contained a deletion of 162 nucleotides within the ORF, which would encode an NDAE with a cytosolic N‐terminal domain missing 54 amino acids. In conclusion, Aedes Malpighian tubules express a diversity of NDAE transcripts. Functional properties remain to be elucidated. Supported by NSF Grant IOB‐0542797 to KWB.
DNA and Metal Binding of the E. coli Transcription Factor RcnR
Authors: Iwig Jeffrey Scott , Leitch Sharon , Maroney Michael , Chivers Peter
Published: June 2021

Escherichia coli RcnR is a founding member of a new family of bacterial ligand‐responsive transcription factors. RcnR controls the expression of the nickel and cobalt efflux protein RcnA. To better understand the functions of this family, we have investigated the DNA and metal binding properties of RcnR. We find that RcnR binds a pair of inverted repeats in the rcnA promoter. DNA binding is negatively regulated by the direct interaction of RcnR with Ni(II) and Co(II) in vitro and in vivo. X‐ray absorption spectroscopy and mutagenesis experiments reveal a 6‐coordinate metal site with a His3(N/O)2S ligand set for Ni(II) and Co(II), where the strength of the thiolate interaction allows for discrimination of the two metals. Structural and functional comparisons with the regulatory sites of NikR, another nickel responsive regulator in E. coli, allow for a better understanding of how different nickel levels can be sensed within the same cell. Residues at the N‐terminus of this protein fold are important for recognition of metals that require higher coordination numbers, while the conserved cysteine aids in recognition of metals with the same preferred coordination number. Sequence analysis of other related proteins reveals a four amino acid fingerprint that enables predictions for ligand specificities, leading to an emerging picture of the diversity of this protein family. This work is supported by NSF Grant MCB‐0520877.
The COG complex interacts with Syntaxin5a to stabilize intra‐Golgi SNARE complex and to direct trafficking of recycling intra‐Golgi vesicles
Authors: Lupashin Vladimir , Smith Richard D , Shestakova Anna M
Published: June 2021

The COG complex is an eight subunit evolutionary conserved peripheral Golgi membrane hetero‐oligomeric protein complex. In humans, mutations in COG1, COG7 or COG8 subunits cause type II Congenital Disorders of Glycosylation. Knockdown of COG3 or COG4 induces accumulation of intra‐Golgi transport vesicles and Golgi ribbon breakdown. Life microscopy of HeLa cells stably expressing CFP‐COG6 and YFP‐COG3 reveals significant diversity in molecular dynamic of COG complex subunits.

The COG complex interacts with intra‐Golgi SNARE molecules. In yeast cells the COG complex preferentially binds to the SNARE complexes containing Golgi t‐SNARE Sed5p. In HeLa cells hCog4p and hCog6p interact with Syntaxin5a, the mammalian homolog of Sed5p. FRET reveals an in vivo interaction between Syntaxin5a and the COG complex. Knockdown of the mammalian COG complex decreases Golgi SNARE mobility, produces an accumulation of free Syntaxin5, and decreases the steady‐state levels of intra‐Golgi SNARE complex. Finally, overexpression of the hCog4p N‐terminal Syntaxin5a‐binding domain destabilizes intra‐Golgi SNARE complexes, disrupting the Golgi. These data suggest that the COG complex orchestrates vesicular trafficking similarly in yeast and mammalian cells by binding to the t‐SNARE Syntaxin5a/Sed5p and enhancing the stability of intra‐Golgi SNARE complexes.

Supported by grant from the NSF (MCB‐0645163)