bluuebunny/crossref_metadata_2025 huggingface dataset:
Stabilization of intrinsic cardiac neuronal processing mitigates neurally induced atrial arrhythmias
Authors: Richer Louis Philippe , Ardell Jeffrey L. , Vinet Alain , Kus Teresa , Cardinal René , Vermeulen Michel , Armour John Andrew
Published: June 2021

Our objective was to determine whether neuronally‐induced atrial arrhythmias can be suppressed by stabilizing the intrinsic cardiac nervous system. In anaesthetized dogs, trains of 5 electrical stimuli (1 mA; 1 ms) were delivered immediately after the P wave of the ECG to mediastinal nerves associated with the superior vena cava. Regional atrial electrical events were monitored with 191 right and left atrial epicardial unipolar electrodes. Mediastinal nerve sites that reproducibly initiated atrial arrhythmias were identified. These sites were re‐stimulated following hexamethonium (1 mg/kg, iv) blockade or pre‐emptive spinal cord stimulation (SCS: the dorsal aspect of the T1–T3 spinal cord was stimulated electrically at 50 Hz; 0.2 ms; 90% of motor threshold) with parameters sufficient to reduce intrinsic cardiac neuronal activity by 70% for up to 1 hour. Stimulating select mediastinal nerves sites consistently elicited atrial tachyarrhythmias (85% of which progressed to atrial fibrillation, AF) with dispersion of epicardial breakthrough sites. Either hexamethonium or pre‐emptive SCS reduced the incidence of mediastinal nerve‐induced AF (to 11% and 40%, respectively) while increasing pacemaker stability. We conclude that excessive and heterogeneous activation of the intrinsic cardiac nervous system results in atrial arrhythmias. The anti‐arrhythmic effects of stabilizing intrinsic cardiac neuronal processing, via either pharmacological or electrical neuromodulation, indicates a potential novel therapeutic approach to atrial arrhythmia suppression. Supported by the CIHR, the Québec Heart & Stroke Foundation and the NIH.
Structural and functional cardiac cholinergic deficits in adult neurturin knockout mice
Authors: Mabe Abigail M. , Hoover Donald B.
Published: June 2021

Neurturin (NRTN) is a neurotrophic factor required during development for normal cholinergic innervation of the mouse heart. This study used NRTN knockout (KO) and wild type (WT) mice to determine the impact of NRTN deletion on adult intracardiac cholinergic neurons and nerve fibers and on cardiac cholinergic function. Immunohistochemical analysis of atrial tissue stained for vesicular ACh transporter revealed that sinoatrial node cholinergic nerve density was reduced by more than 40% in 8 week KO hearts. Additionally, the number of cholinergic neurons in KO hearts was 80% less than in WT, and KO neurons were 22% smaller in size. Atrial ACh levels were significantly lower in 16 week KO mice compared to WT (1.8±0.3 vs 3.7±0.8 pmol/mg wt; P<0.02), as expected from cholinergic neuron and nerve fiber deficits. Maximum heart rate (HR) responses to right vagal nerve stimulation (VNS) were decreased in 16 week KO (38±6 vs 81±3% decrease at 20Hz; P<0.0001), with no difference in baseline HR. KO animals took longer to reach maximum bradycardia during VNS and longer to recover to baseline HR upon stimulus termination. Data from isolated atrial preparations showed no change in postjunctional cholinergic sensitivity. These results demonstrate that NRTN KO leads to structural and functional cholinergic deficits in the heart but residual cholinergic nerve fibers retain ability to modify cardiac function. Supported by the AHA SE Affiliate.
Insufficient GABA inhibition underlies periodic and irregular breathing in methyl‐CpG‐binding protein 2 (Mecp2) deficient mice
Authors: Bissonnette John , Knopp Sharon
Published: June 2021

Mutations in the MECP2 gene cause Rett syndrome (RTT) whose phenotype includes periodic (PB) and irregular breathing. Using telemetry to record pleural pressure we have found that heterozygous Mecp2 deficient female mice have frequent episodes of PB. The amount of PB was augmented by breathing 40 % oxygen and decreased in 12% oxygen, indicating that peripheral chemoreception is not responsible for the increased controller gain (J Appl Physiol in Press). Medullary respiratory neurons (Brain Research 710:150,1996; J Neurophysiol 80:2368,1998) and phrenic motoneuons (J Neurosci 19:2368,1999) receive concurrent inhibition with excitation during inspiration. Mecp2+/− are relatively deficient in the β3 subunit of GABAA receptors (Hum Mol Genet 14:483,2005). We hypothesized that insufficient GABA inhibition underlies the increased controller gain in Mecp2 deficient animals. NO‐711 a GABA transport inhibitor reduced the number of PB episodes from 18.1±4.9 to 3.5±1.8/hr. and extended the longest period without PB from 26.5±6 to 74.5±23.7 min. Similarly the GABA receptor modulator diazepam reduced the incidence to 3.2±0.7/hr and extended PB free to 96.7±35.9 min. NO‐711 reduced the coefficient of variation for TTOT from 35±3.4 to 22.6±3.8%. These data indicate that augmenting GABA reduces PB and respiratory irregularity seen in Mecp2 deficient mice to the levels observed in wild type mice. MOD 6‐FY06‐314
The elevation of serum tumor necrosis factor (TNF)‐α and hypothalamic prostaglandin (PG) D2 levels in lipopolysaccharide (LPS)‐induced hypothermia in rats: Possible role of cyclooxygenase (COX)‐1
Authors: Akarsu Eyup S. , Usanmaz Suzan E. , Mamuk Soner
Published: June 2021

LPS‐induced hypothermia is an adaptive thermoregulatory strategy against immunological challenge in rats. It appears that a selective proinflammatory cytokine (TNF‐α) elevation occurs in the peripheral circulation at the initial phase. Then, an increased PG synthesis may take place in brain, since COX inhibitors abolish the hypothermia. We measured hypothalamic PGD2 and PGE2 levels at this phase. The effects of COX‐1 selective inhibitors (valeryl salicylate [VS] or SC‐560) were also evaluated. LPS (E. coli O111:B4, 250 μg/kg, ip) produced monophasic hypothermia in biotelemetered adult male Wistar rats. The serum TNF‐α elevation was temporally related with the response. The LPS‐injected rats were sacrificed by microwave irradiation. The 4‐Bromomethyl‐7‐methoxycoumarine derivatives of PGD2 and PGE2 were measured by HPLC in hypothalamic homogenates. The PGD2 increased from 58±5 pg/mg (basal) to 274±18 pg/mg wet tissue weight. The PGE2 levels did not increase. SC‐560 (1 mg/kg, sc) abolished the hypothermia and TNF‐α elevation. But, VS (20 mg/kg, sc) inhibited hypothermia and hypothalamic PGD2 increase, without any effect on serum TNF‐α levels. Data suggest that peripheral induction of TNF‐α may also trigger the PGD2 synthesis in hypothalamus, possibly through COX‐1 dependent mechanisms in LPS‐induced hypothermia in rats.

Supported by TUBITAK (SBAG‐AYD‐381) and Ankara University ()
Interaction of Magnolol and exercise training on blood oxidative stress and aortic adhesion molecules in hyperlipidemic rabbits
Authors: Chang Weng‐Cheng , Yu Ya‐Mei
Published: June 2021

This study was aimed to investigate the interaction of Magnolia officinalis and exercise training on attenuating the process of atherosclerosis of the cholesterol‐fed rabbits. The results showed an addictive effect of Magnolol supplement and exercise training on preventing the aortas of the hyperlipidemic rabbits from atherosclerotic lesion. On the biochemical level, serum low‐density lipoprotein (LDL) and total cholesterol (TC), blood malondialdehyde (MDA) levels, and P‐selectin levels of the aortic arches were synergistically reduced in the hyperlipidemic rabbits by combination of exercise training and Magnolol supplement. Nonetheless, blood free radical counts, aortic SOD‐1 and VCAM‐1 levels were not addictively reduced in the hyperlipidemic rabbits co‐treated with exercise training and Magnolol supplement. On morphological study, lipid lesions of the aorta resulted from high‐fat diet were fully prevented in the combined treatment of exercise training and Magnolia extract. This study indicates that co‐treatment of exercise training and Magnolol supplement exert synergistically and could be a promising alternative therapy for atherosclerosis.
Modulation of apoptotic signaling by hexokinase binding to mitochondria in myeloma cells
Authors: Jefferson Andrew , McGrath Ryan , Landowski Terry , Lynch Ronald M.
Published: June 2021

Mitochondria (MT) synthsize ATP and act as a central mediator of apoptosis. Sustained Ca2+ uptake into MT results in the release of cytochrome c and activation of downstream mediators of apoptosis. The proteosome inhibitor bortezomib (BZ) activates apoptosis via ER stress, and this response is ameliorated by blocking MT Ca2+ uptake with ruthenium red. To evaluate MT Ca2+, we loaded MT with a Ca2+‐sensitive fluorophore Rhod‐2. Activation of apoptosis via sustained elevation in intracellular Ca2+ caused an increase in MT Ca2+ and RR blocks this response, as predicted. Binding of the enzyme hexokinase (HK) to the outer mitochondrial membrane (OMM) is suggested to modulate MT Ca2+ uptake and apoptosis. The N‐terminus of HK is required for MT binding. To compete native HK from the OMM, we prepared a HK N‐term‐GFP (ntHK‐GFP) construct. Following tranfection into myeloma cells, the ntHK‐GFP bound to MT, however analysis of native HK using immunochemistry indicated that expression of HK itself was increased with a significant proportion remaining MT bound. To avoid the effects of upregulation of native HK, we developed a ntHK‐GTP construct that includes a TAT sequence for acute uptake into cells. The construct is produced in and isolate from bacteria. Current studies are investigating the effect of modulating MT‐HK binding on MT Ca2+ signaling, the induction of apoptosis by BZ, and Ca2+ store release.
Effect of cadmium on cellular protein and glutathione synthesis and expression on stress proteins in eastern oysters, Crassostrea virginica Gmelin
Authors: Ivanina Anna , Cherkasov Anton , Sokolova Inna
Published: June 2021

Cadmium (Cd) is an important toxicant in estuarine and coastal environments which can strongly affect energy balance of aquatic organisms by increasing the organism's basal energy demand and reducing its aerobic capacity. Mechanisms of Cd‐induced increase in basal metabolic costs are not well understood and may involve elevated detoxification costs due to the synthesis of cellular protective proteins and glutathione (GSH). We studied the effects of Cd exposure on protein and GSH synthesis and expression of stress protein (heat shock proteins HSP60, HSP70 and HSP90) and metallothioneins in isolated gill and hepatopancreas (HP) cells of the eastern oysters Crassostrea virginica. Our study showed that exposure to Cd (10–2000 ìM for 4 h) resulted in dose‐dependent increase in the rate of the protein synthesis in oyster cells. GSH synthesis was inhibited by the highest Cd concentration (2 mM) especially in HP cells. Elevated protein synthesis was associated with the increased expression of metallothioneins and heat shock proteins. Stress protein response considerably differed between gill and HP cells. In HP, there was an increase in expression of MT mRNA, but no change in HSP protein level in response to Cd exposure. In gill cells no significant increase in MT expression was found and level of HSP60 and 70 significantly increased in Cd‐exposed cells.
Selenoproteins regulate contractility during hyperthyroidism‐induced cardiac hypertrophy
Authors: Hoffmann Peter R , Hoffmann FuKun , Schoendorf David , Cheng Yu , Berry Marla J , Shohet Ralph V
Published: June 2021

We assessed the hypothesis that selenoproteins are important for the heart during hyperthyroidism‐induced myocardial hypertrophy. Mice were injected with 1mg/kg triiodothyronine (T3) or vehicle control for 8 days. Expression levels of several selenoproteins were found to increase in heart tissue with T3‐treatment, with some of the most elevated mRNAs including selenoprotein R (Sel R), thioredoxin reductase−1, and both glutathione peroxidase (GPX)−1 and −3. T3‐treatment induced increases in thioredoxin reductase activity as well as GPX−4 and Sel R, but not GPX−1, proteins. We next utilized a mouse model (trspfl/fl X αMHC‐Cre), which involves reduced expression of Sec‐tRNA in the heart. The mice expressing reduced levels of Sec‐tRNA in the heart (Cre+ mice) developed similar levels of myocardial hypertrophy upon T3 injection compared to negative controls (Cre− mice) as determined by heart mass:body mass. As expected, T3‐treatment of Cre− mice resulted in altered cardiac parameters including increased fractional shortening, decreased systolic left ventricular inner diameter, and increased intraventricular septal thickness. In contrast, these cardiac parameters were not altered in Cre+ mice. These results suggest that restricting the elevation of selenoprotein synthesis by reducing Sec‐tRNA levels may lead to a loss of contractility needed for maintaining cardiac function during hypertrophy.
Establishing a tool to assess product health claims and improve regulation of bioactive compounds as functional ingredients in foods
Authors: Black Lucinda Jane , Eriksen Folmer , Gry Jorn , Kiely Mairead , Kroon Paul , Pilegaard Kirsten , Plumb Jenny , Sheehan Darina , Finglas Paul
Published: June 2021

Non‐nutrient bioactive compounds inherent in plant foods are widely associated with beneficial health effects. As a result, bioactive compounds are becoming increasingly important as functional ingredients in food products with proposed health benefits, such as reduction of disease risk, improvement in growth and development, and optimisation of health. Authorisation of health claims within the current marketplace is imperative to ensure valid representation of food products. In response to the need for access to bioactive compound data, a single online resource has been established which collates international data on the composition and biological effects of plant‐based bioactive compounds. The database derives information from peer‐reviewed published literature and covers 16 major compound classes from approximately 600 commonly consumed plants. All data is quality assessed using a defined rating system and reporting is user‐driven to support the specific requirements of the user. The database is likely to serve as an invaluable tool for product health claim assessment, regulation of bioactive compounds as functional ingredients, estimation of exposure to bioactive compounds and for supporting diet and health research. Support: This project is part of the European Food Information Resource Network (EuroFIR), which is funded by the EU under the 6th Framework Programme. Project contract number: FP6‐513944.
Effects of a traditional Asian medicine (TAM) on Profile of Mood States (POMS) in Healthy Stressed Subjects
Authors: Talbott Shawn , Talbott Julie
Published: June 2021

We evaluated the effects of a dietary supplement containing a traditional Asian medicine (TAM) on perceptions of mood, energy, tension, and fatigue in healthy men and women experiencing moderate levels of psychological stress. TAM contains 16 herbal/fruit extracts with historical uses in traditional Asian medicine for improving energy levels and mood (longan, lycium (goji), astragalus, jujube date, gac, cili, hawthorn, seabuckthorn, coriolus, rhodiola, schisandra, tangerine peel, ginger, mangosteen, litchi (lychee) and durian). Previous work has found TAM to reduce fatigue in active subjects following physical stress. We hypothesized that psychologically stressed subjects may benefit from TAM with reduced tension and fatigue. Subjects (N=100, 32 men, 68 women, age = 39 + 11 years) were pre‐screened for moderate levels of psychological stress and randomly assigned to TAM (3oz daily, N=50) or Control (N=50) for 4 weeks. Each subject completed a Profile of Mood States (POMS) survey at baseline and 4‐weeks. There were no significant differences between groups at baseline. Following supplementation, TAM had significantly higher scores for Global Mood State (+19%) and Vigor (+47%), and lower scores for Tension (−24%) and Fatigue (−42%) compared to control. These results suggest that TAM consumption is associated with improvements in subjective perceptions of mood, energy, tension, and fatigue.
Selenium Deficiency, but Not Vitamin E Deficiency, Activates the Mouse Liver Nrf2‐ARE Pathway
Authors: Burk Raymond F , Hill Kristina E , Nakayama Akihiro , Mostert Volker , Levander Ximena A , Motley Amy K , Freeman Michael L , Austin Lori M
Published: June 2021

Selenium (Se) and vitamin E are essential micronutrients with antioxidant properties. Se functions through selenoproteins and vitamin E reacts with oxidizing molecules in membranes. The relationship of these micronutrients with the Nrf2‐antioxidant response element (ARE) pathway was investigated using ARE‐reporter mice and Nrf2−/− mice. Weanling male mice were fed Se‐deficient (0 Se), vitamin E‐deficient (0 E), or control diet for 16 or 22 weeks. The ARE‐reporter was elevated 450 fold in 0 Se liver but was not elevated in 0 E liver. Antioxidant enzymes induced by Nrf2‐ARE—glutathione S‐transferase (GST), NAD(P)H quinone oxidoreductase (NQOR), and heme oxygenase‐1 (HO‐1)—were elevated in 0 Se livers but not in 0 E livers. Deletion of Nrf2 had varying effects on the inductions, with GST induction being abolished by it but induction of NQOR and HO‐1 still occurring. Thus, Se deficiency, but not vitamin E deficiency, induces a number of enzymes that protect against oxidative stress and modify xenobiotic metabolism through Nrf2‐ARE as well as other stress‐response pathways. We conclude that Se deficiency causes cytosolic oxidative stress but that vitamin E deficiency does not. It follows that the oxidant defense mechanisms in which these antioxidant nutrients function are autonomous. Supported by NIH grant ES02497.
Polymorphonuclear leukocyte (PMN) migration across vascular endothelial cells (EC) in the absence or presence of IL‐1β differentially regulate expression of EC adhesion molecules ICAM‐1 and E‐selectin
Authors: Stephen Jancy , Katada Kazuhiro , Cukiernik Mark , Bihari Aurelia , Potter Richard F , Cepinskas Gediminas
Published: June 2021

Our findings indicate that PMN migration across activated EC results in down‐regulation of EC nuclear transcription factor, NFκB. However, the subsequent functional consequences are largely unknown.

In this study we assessed the effects of PMN transendothelial migration on NFκB‐dependent expression of EC adhesion molecules, ICAM‐1 and E‐selectin during inflammation.

To this end human endothelial cells (HUVEC) were grown in Traswell inserts and stimulated with IL‐1β (1ng/ml; 4 hrs). Subsequently, PMN were added and allowed to migrate across HUVEC in response to fMLP (10−8M) chemotactic gradient in the absence (i.e. across washed HUVEC) or presence of IL‐1β. ICAM‐1 and E‐selectin gene expression in EC was assessed by RT‐PCR after 4 or 24 hrs following PMN migration.

The obtained results indicate that migration of PMN across IL‐1β treated and subsequently washed HUVEC results in a marked down‐regulation of IL‐1 β‐induced ICAM‐1, and to a lesser degree, E‐selectin gene expression as assessed 4 and 24 hrs following PMN migration. On the contrary, migration of PMN across activated HUVEC in the presence of IL‐1β results in a further increase in both, ICAM‐1 and E‐selectin expression.

Taken together these findings indicate that migrating PMN differentially modulate expression of EC adhesion molecules during inflammation and can offer EC with both, anti‐ and pro‐inflammatory stimuli (HSFO‐NA5580/NA6171)
Comparing student centered and traditional teaching techniques in an undergraduate human anatomy laboratory section
Authors: Notebaert Andrew , Fagenbaum Ray , Hand Brian
Published: June 2021

We sought to determine if students in a student centered laboratory section of an undergraduate anatomy course would perform better on classroom examinations compared to students taught in a traditional section from the same lecture course. Fifty four students from one undergraduate human anatomy class volunteered for the study. Grouping was based on enrollment in laboratory sections (37 control, 17 experiment). The control group was taught in a traditional manner using lecture and focusing on facts while the experimental group received instruction using class discussion focusing on concepts. Demographic data and scores from three assignments focusing on factual recall were compared using independent t‐tests at a significance of p<.05. There were no differences between groups based on age or perceived difficulty of course. The control group had a significantly lower grade point average (3.21±.3 versus 3.41±.4, t50=2.063, p=.044). For the graded assignments, there were no significant differences between control and experimental scores for the first quiz (20 points, 16.16±2.6 versus 15.76±3.50), laboratory test (28 points, 21.03±3.6 versus 22.35±4.4), and lecture test (60 points, 44.65±7.3 versus 47.06±7.7). Students taught in a student centered laboratory section performed as well as students in traditional laboratory sections on graded assignments focusing on fact recall. This project was not funded.
Mouse models to study the role of CD34 in allergy and inflammatory diseases
Authors: Blanchet Marie‐Renee , Maltby Steven , Bennett Jami , McNagny Kelly
Published: June 2021

The CD34 antigen is widely used as a stem cell marker. However, the exact role of CD34 expression is still misunderstood. Our lab is currently using mouse models of allergy and inflammation induced in CD34‐null or chimeric mice in order to elucidate the role of CD34 expression in inflammation.

Asthma, hypersentitivity pneumonitis (HP), experimental animal encephalomyetis (EAE) or arthritis was induced in CD34‐null and wild type mice. These pathologies are characterized by activation of various CD34‐expressing cells. Disease progression and cell recruitment and function were compared between wild type and CD34‐deficient mice.

In lung inflammation, CD34‐null mice are protected against development of disease and show various cell trafficking defects. In EAE, mast cells accumulate in the CNS of CD34‐null mice, possibly through an emigration defect. Finally, in arthritis, CD34‐null mice show higher susceptibility to disease due to lack of CD34 on vascular endothelia.

These results suggest a role for CD34 in hematopoietic cell trafficking, either through CD34 expression on inflammatory cells or vascular endothelia. Also, CD34 expression on vascular endothelia is involved in progression of arthritis. The use of these mouse models in combination with CD34‐null mice has allowed us to better understand the role of CD34 expression in development of inflammation and allergy.
Correlating Costal Curvature and Lung Volume in the Genus Homo
Authors: Eaves‐Johnson K. Lindsay
Published: June 2021

Due to a general, though undocumented, sense that little diagnostic information can be gleaned from them, ribs and overall thoracic morphology have been comparatively understudied relative to other anatomical regions in human paleontology. This pilot study tests the influence of skeletal thoracic shape on respiratory variables (e.g., total lung capacity, functional residual capacity, etc.) using computed tomography (CT), to expand our understanding of modern and Neandertal thoracic patterning. The mixed‐sex CT sample consists of 25 respiratorily normal subjects and 10 subjects with chronic obstructive pulmonary disorder (COPD), all between the ages of 20 and 60. This homogenous, living CT sample is compared with a regionally heterogeneous, mixed‐sex skeletal sample from the extremes of human climatic distribution (n=48), as well as the Levantine Neandertal specimens Kebara 2 (original) and Tabun C1 (cast). Preliminary results suggest that, while gross spirometric measures, such as total lung capacity, agree well with the predicted model, further study is needed to delineate the skeletal parameters most amenable to comparison for such variables as functional residual capacity. Additionally, comparisons of the Levantine Neandertals with the modern sample fail to demonstrate any significant temporal excursion from the range of modern variability with respect to these parameters.
A Primer for Understanding Neuro‐Immune Interactions”
Authors: Sanders Virginia
Published: June 2021

One of the most exciting discoveries in neuroscience has been that CD4+ T lymphocytes possess selective effector functions that are involved in mediating nerve injury, survival, and/or repair. The predominant CD4+ T cell subtypes are the T‐helper type 1 (Th1), Th2, Th17, inducible T‐regulatory‐1 (Tr1), and natural Foxp3+ T‐regulatory (Foxp3+Treg) cell. These CD4+ T cell subtypes express distinct cytokine profiles, regulate each other's development and function, and decide the nature and intensity of most immune responses. Th1 and Th17 cells are considered to be proinflammatory subtypes, while Th2, Tr1, and Foxp3+Treg cells are considered antiinflammatory. Although human CD4+ effector T cells do not polarize their cytokine profiles as clearly as murine cells, their functional polarization is clearly similar. The role played by each effector CD4+ T cell subtype in mediating neurodestruction versus neuroprotection, as well as the mechanisms responsible for each effect, will be discussed.
The Ddc1 checkpoint clamp subunit activates Mec1 kinase during the DNA damage checkpoint
Authors: Burgers Peter M , Navadgi Vasundhara M , Majka Jerzy
Published: June 2021

The DNA damage checkpoint pathway operates to prevent cell cycle progression in response to various genotoxic agents. In Saccharomyces cerevisiae, Mec1‐Ddc2 (human ATR‐ATRIP) is the kinase that initiates most checkpoint pathways. The checkpoint clamp is a heterotrimeric ring, Rad17‐Mec3‐Ddc1 (human 9‐1‐1), and shows structural similarity to the replication clamp PCNA. It is recruited to gapped DNA by the Rad24‐RFC clamp loader and is required for activation of the effector kinase Rad53 (human Chk1 or Chk2) in response to DNA damage.

In vitro, the checkpoint clamp is loaded in an ATP‐dependent step onto 5’‐junctions of partial duplex DNA by the Rad24‐RFC clamp loader. The loaded clamp serves to activate Mec1 kinase activity. Activated Mec1 phosphorylates the Ddc1 and Mec3 subunits of the clamp, the Rad24 subunit of the loader, the Rpa1 and Rpa2 subunits of RPA, and the downstream kinase Rad53. We have used this activation system to probe the nature and functionality of interactions between Ddc1 and Mec1.
Visualization of Viruses and Embryos
Authors: Lansford Rusty
Published: June 2021

Our research aims to dynamically visualize and characterize embryonic development at sub‐cellular resolution. Instead of labeling and following small numbers of cells at a time, we am have developed the means to optically record all or nearly all the cells within a fluorescent protein (FP)‐expressing transgenic quail embryo simultaneously. Cell and tissue movements in the developing embryos are then recorded using multispectral, time‐lapse fluorescent microscopy in 3D.

We are tracking the motion of single virus particles and of single viral genomes in living cells using real‐time fluorescence imaging methods. We aim to produce “molecular movies” that will provide mechanistic insight into the biological processes involved in viral mediated gene therapy.
Stress‐induced dysphoria is encoded by the Dynorphin‐Kappa Opioid system
Authors: Chavkin Charles
Published: June 2021

Sustained stressful experience can induce despair and increase the risk of clinical depression and drug abuse. We found that repeated exposure of mice to stress (forced swim or social‐defeat) activated the dynorphin‐kappa opioid receptor system to produce analgesia, immobility, sensitization of the reward system to cocaine, and reinstatement of extinguished cocaine‐place preference. The cellular and molecular basis of these responses were described in a series of studies demonstrating that chronic stress evoked CRF/Urocortin release and subsequent CRF‐R2 receptor mediated dynorphin release. Surprisingly, aversion caused by stress and CRF were blocked by disruption of the dynorphin kappa opioid system. Dynorphin‐induced dysphoria was found to be mediated by p38 MAPK activated through a GRK3/arrestin dependent mechanism. Stress‐induced release of dynorphin activates kappa receptors broadly in brain. In particular, repeated stress caused activation of both KOR and p38 MAPK co‐expressed in GABAergic neurons. The p38 inhibitor SB203580 selectively blocked dynorphin‐induced aversion. The convergence of stress‐induced aversion on the dynorphin system was unexpected and implicates dynorphin activation of p38 MAPK signaling as a key component mediating the aversive properties of stress.
Chemical Genetic Approaches for Studying DNA Replication
Authors: McHenry Charles S , Dallmann Garry
Published: June 2021

DNA replication of bacterial chromosomes is dependent upon approximately 20 essential proteins. We are taking a chemical biology approach to discover inhibitors of each stage of the replicative reaction. These inhibitors should be of general utility to the scientific community, both as specific blocks for replication reaction stages that can be used in cellular experiments and as specific mechanistic and structural probes for studies with purified proteins. The use of fully reconstituted reactions provides a particularly productive approach. Not only are the essential enzymatic activities targets, but also the various protein‐protein interactions and conformers associated with discrete kinetic steps. Inhibitor identification is accomplished by a global robotic screen for all targets in a single well of a microtiter plate coupled with a deconvolution strategy that guides us to the specific target. The use of reconstituted replicases from model Gram‐negative and Gram‐positive organisms permits immediate identification of compounds with broad spectrum potential. A battery of specificity assays permit weeding out compounds that act by non‐specific mechanisms.
Riboswitches as new antibiotics targets
Authors: Blount Ken F , Breaker Ronald R
Published: June 2021

New cellular targets are needed to reinvigorate antibacterial drug discovery. This need could potentially be filled by riboswitches—messenger RNA (mRNA) structures that regulate gene expression in bacteria. Riboswitches are unique from other RNAs that serve as drug targets in that they have evolved to form structured and highly selective receptors for small drug‐like metabolites. In most cases, metabolite binding to the receptor represses the expression of the gene or genes coded by the mRNA. If a new metabolite analog were designed that binds to the receptor, the gene(s) regulated by that riboswitch could be repressed, with a potentially lethal effect to the bacteria. Recent work suggests that certain antibacterial compounds discovered decades ago function at least in part by targeting riboswitches. Herein we will summarize the experiments validating riboswitches as drug targets, describe the existing technology for riboswitch drug discovery, and discuss the challenges that may face riboswitch drug discoverers.
Vitamin D‐induced changes in the gene expression profile of the RWPE1 human prostate epithelial cell (PEC) line relevant to cancer prevention
Authors: Kovalenko Pavel L , Zhang Zhentao , Clinton Steven K , Fleet James C
Published: June 2021

1 alpha, 25 dihydroxyvitamin D3 (1,25 D) has hypothesized anti‐prostate cancer effects but the potential molecular mechanisms are unknown. We examined the effect of 1,25 D (+/â 100 nM, 6, 24, 48 h) on transcript profiles of proliferating RWPE1 cells, an immortalized PEC line with a normal phenotype that is growth arrested by 1,25 D (Affymetrix U133 Plus 2.0, n=4/treatment per time). Statistical analysis revealed many transcript level changes at a 5% false detection rate; 6 h, 1571 (61% up), 24 h, 1827 (60 %), 48 h, 3570 (38 %). Only 262 transcripts were regulated similarly at all time points (182 up, 80 down); later responses were likely secondary expression patterns associated with growth arrest. We confirmed expression of 10 transcripts previously reported as 1,25 D regulated in primary PEC and validated changes by RT‐PCR. 503 of the 6 h transcripts were previously reported as 1,25 D regulated in skin cancer cells (80% up); 327 of these promoters contained putative 1,25 D response elements (78% up) revealing potential new 1,25D targets that may modulate prostate carincinogenesis. Gene Set Analysis revealed that 6 h treatment suppressed genes sets of WNT target genes, genes upregulated by NF‐kB, and transcription factors associated with cancer progression. Our data suggest an early response to 1,25 D for prostate cancer prevention that acts via multiple pathways.
Folate intake at RDA levels is inadequate for Mexican American men with the methylenetetrahydrofolate reductase 677TT genotype
Authors: Solis Claudia , Veenema Kristin , Ivanov Alexandre , Tran Sally , Li Rui , Wang Wei , Moriarty David , Maletz Charles , Caudill Marie A
Published: June 2021

This controlled feeding study investigated the adequacy of the folate RDA, 400 mcg/d as dietary folate equivalents (DFE), for Mexican American men with the MTHFR 677CC or TT genotype. Because of the interdependency between folate and choline, the influence of choline intake on folate status was also assessed. Mexican American men (n=60; 18‐55 y) with the MTHFR 677CC (n=31) or TT (n=29) genotype consumed 438 mcg DFE/d and total choline intakes of 300, 550 (choline AI), 1100 or 2200 mg/d for 12 wk. Folate status response was assessed via serum folate (SF), RBC folate, plasma total homocysteine (tHcy) and urinary folate. SF decreased (P<0.001) 66% to 7.9 +/− 0.7 nmol/L (mean +/− SEM) in men with the 677TT genotype and 62% to 11.3 +/− 0.9 nmol/L in the 677CC genotype. Plasma tHcy increased (P<0.0001) 170% to 31 +/−3 umol/L in men with the 677TT genotype and 18% to 11.6 +/− 0.3 umol/L in the 677CC genotype. At the end of the study, 34% (677TT) and 16% (677CC) had SF concentrations <6.8 nmol/L and 79% (677TT) and 7% (677CC) had tHcy concentrations >14 umol/L. Choline intake did not influence the response of the measured variables. These data show that the folate RDA is not adequate for men of Mexican descent particularly for those with the MTHFR 677TT genotype. Supported by NIH Grant #S06GM053933 and the California Agricultural Research Initiative.
Nonalcoholic steatohepatitis is associated with increased hepatocyte apoptosis, JNK activation and Bax protein
Authors: Wang Yan , Ausman Lynne M. , Russell Robert M. , Greenberg Andrew S. , Wand Xiang‐Dong
Published: June 2021

High‐fat diet enriched in n‐6 polyunsaturated fatty acids (PUFA) induces non‐alcoholic steatohepatitis (NASH). The potential mechanisms involved in this process have not been defined. Male Sprague‐Dawley rats were fed ad libitum Lieber‐DeCarli diet at either 35% energy from fat (control) or 71% energy from fat (high‐fat) enriched in n‐6 PUFA for 6 weeks. Histopathological examination showed key features of NASH (steatosis, inflammatory cell infiltration, ballooning degeneration) by the high‐fat diet, which were accompanied with induction of tumor necrosis factor‐a mRNA, high lipid peroxidation products (4‐hydroxynonenal and malondialdehyde), and cytochrome p4502E1. The high‐fat diet caused significantly increased apoptotic cells (3.3 ± 0.4 [Mean ± SD] vs.1.8 ± 0.1/field as assessed by the Terminal dUTP Nick‐End Labeling assay) and also activated caspase‐3 and poly ADP‐ribose polymerase in liver. This increased apoptosis was associated with higher levels of phosphorylated c‐Jun‐NH2‐kinase (JNK) and pro‐apoptotic Bax protein but not anti‐apoptotic proteins Bcl‐2 and Bcl‐xl. This study indicates that the increased oxidative stress, high JNK activation and imbalance of pro‐ and anti‐apoptotic proteins in Bcl‐2 family all contribute to the high fat diet‐induced apoptosis that may play a significant role in the pathogenesis of NASH.
HFE inhibits non transferrin‐bound iron uptake via downregulating Zip14 in HepG2 cells
Authors: Gao Junwei , Zhao Ningning , Knutson Mitchell D. , Enns Caroline A.
Published: June 2021

Mutations in the Hereditary Hemochromatosis protein, HFE, cause iron overload. Hepatocytes are particularly affected. We investigated how HFE regulates both transferrin (Tf)‐bound and non Tf‐bound iron uptake in HepG2 cells, a human hepatoma cell line. Expression of HFE in this cell line decreased both Tf‐bound (50%) and non Tf‐bound iron uptake (30%). To determine which iron transporter might be involved in iron uptake in HepG2 cells, we measured mRNA levels of two known iron transporters. The mRNA level of Zip14 was five times that of DMT1 (divalent metal transporter 1). Expression of HFE resulted in a decrease in the levels of Zip14 with no change in the mRNA level. Knockdown of Zip14 with siRNA abolished the effect of HFE on non Tf‐bound iron uptake. HeLa cells have no detectable Zip14. When HeLa cells expressing HFE under the tet‐repressible promoter were transfected with Zip14, non‐Tf‐bound iron uptake was inhibited by 40% with the induction of HFE and the levels of Zip14 decreased. These results suggest that HFE controls the stability of Zip14 and therefore the iron loading of hepatocytes.

Supported by NIH grants DK054466 (CAE) and DK0655964 (MK)
Modulation of transferrin‐bound iron uptake by Zip14
Authors: Zhao Ningning , Gao Juwei , Enns Caroline A , Knutson Mitchell D
Published: June 2021

All cells acquire iron through internalization of iron‐loaded transferrin (Tf) via the Tf receptor. The aim of the present study was to examine the effect of Zip14, a transmembrane metal ion transporter, on the uptake of iron from Tf. HEK293T cells were transfected with mouse Zip14 cDNA or empty vector and incubated for 2, 6, 8, 10, 16 h with 59Fe‐Tf, and the radioactivity incorporated into the cells was measured. At all time points, Zip14‐transfected cells took up more 59Fe from Tf than did control cells. At 8 h, Zip14‐overexpressing HEK cells took up 37 ± 6% more 59Fe than did controls. The effect of Zip14 on Tf‐bound iron uptake was also studied in HepG2 cells, which endogenously express abundant Zip14. Treatment of HepG2 cells with Zip14 siRNA decreased Zip14 mRNA and protein levels and markedly reduced Tf‐mediated iron uptake. These data strongly implicate a role for Zip14 in the uptake of iron from Tf, suggesting that Zip14 plays a role in normal iron metabolism. Funded by NIH grants DK065064 (MDK) and DK054488 (CAE).
Skeletal muscle protein synthesis in the elderly: Age, gender, and androgen supplementation
Authors: Henderson Gregory C , Dhatariya Ketan , Ford G. Charles , Klaus Katherine A , Mujeeb Mehrukh , Nair K. Sreekumaran
Published: June 2021

Skeletal muscle protein synthesis rates decline with age.

PURPOSE:

We sought to determine if DHEA supplementation in elderly men (EM) and elderly women (EW) and low dose testosterone (T) supplementation in EM would raise protein synthesis rates.

METHODS:

87 EM (with low bioavailable T and DHEAS) and 57 EW (with low DHEAS) were studied before and after 1 year in which EM received either T (5 mg/d), DHEA (75 mg/d), or placebo (P) and EW received either DHEA (50 mg/d) or P. To assess age effects, 30 young men and 32 young women were studied on a single occasion as controls. [15N]phenylalanine and [2H4]tyrosine tracers were infused and isotopic enrichment was measured in plasma and v. lateralis muscle. Whole body amino acid kinetics were normalized to fat free mass, and mixed muscle protein fractional synthesis rate (FSR) was measured using plasma phenylalanine as the precursor.

RESULTS:

Despite induction of elevated androgen levels, no significant differences were seen between treated groups and the P group in either EM or EW. Whole body protein synthesis and muscle FSR were lower in elderly than in young (P < 0.05) and were higher in women than men (P < 0.05), with no age‐by‐gender interaction.

CONCLUSIONS:

Neither DHEA nor T supplementation for one year had any significant impact on age‐related decline in protein synthesis, while both age and gender do impact protein synthesis rates. NIH grants P01 AG14283 and UL1‐RR‐024150‐01.
Nutritional status and susceptibility to malaria in Senegalese 1–5‐year‐old children
Authors: FilloL Florie , Cisse Badara , Boulanger Denis , Sokhna Cheikh , Simondon Kirsten
Published: June 2021

In rural sub‐Saharan Africa, malaria and malnutrition are two major causes of morbidity and mortality of children under 5. To explore whether nutritional status at baseline has an impact on the susceptibility to malaria morbidity, a cohort of 874 rural Senegalese children aged 12–70 months was followed during one malaria season from July to December. Data on nutritional status and density of P. falciparum parasite were collected at the start of rainy season. Malaria morbidity was monitored each week through home visits by field workers. In multivariate models, both underweight and wasted children were at lower risk of experiencing at least one subsequent clinical malaria attack (OR=0.61 (95%CI: 0.40, 0.94), P=0.03 and OR=0.34 (0.15, 0.76), P<0.01, respectively). While stunting did not induce significantly higher susceptibility to clinical malaria, it increased the risk of presenting a high parasitemia at the onset of the rainy season (OR=2.42 (1.12, 5.24) P=0.03). Geometric mean density parasitemia of the first clinical malaria attack was lower in stunted children as compared to their non‐stunted counterparts (P<0.05), and the same was true for anaemic children (P=0.003). Our findings suggest that stunting and wasting may act differently on susceptibility to malaria, which might be due to different mechanisms involved in specific immune response, related to qualitative differences in nutrient deficits. Funding: Gates Foundation and IRD.
The impact of three supplements with identical micronutrient content on morbidity in Mexican children
Authors: Leroy Jef L , García‐Guerra Armando , Neufeld Lynnette
Published: June 2021

The objective of this analysis was to assess the impact of three types of nutrition supplements with identical micronutrient content on morbidity (>3 stools per day, dysentery, fever, severe cough, cough with mucus and/or vomiting) in children.

Communities (18 per supplement) were randomly assigned to receive a fortified food (FF), syrup (SY) or Sprinkles (SK). Children 6 to 12 m of age, beneficiaries of the Oportunidades program in Mexico were eligible to participate. Supplements were delivered daily and compliance and morbidity were recorded. Appropriate statistical analyses for cluster randomized trials were used. Data on 480 children (6 to 12 mo at baseline) in the first 6 months of supplementation were analyzed (FF n=137; SY n=159; SK n=184).

No statistically significant differences between groups were found at baseline. Children were ill 16 d on average. In unadjusted analysis, there were no statistically significant differences between supplementation groups in morbidity. When adjusting for season, age, sex and height‐for‐age Z‐score at baseline, lagged compliance was associated with lower morbidity (P<0.05). Ensuring adequate compliance with supplementation programs may lower child morbidity.

The Oportunidades program funded this study and holds the rights to the data presented.
Treatment with 1,25 dihydroxyvitamin D3 stimulates adipocytic differentiation of porcine mesenchymal stem cells
Authors: Mahajan Avanika , Stahl Chad H.
Published: June 2021

Depending on the species and culture conditions, 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to have pleiotropic effects on the differentiation of mesenchymal stem cells (MSC). In this study, we characterized the effects of 1,25(OH)2D3 on the differentiation of porcine MSC. The MSC were isolated from bone marrow of young pigs and grown in DMEM + 10% fetal bovine serum and antibiotics (BM). Cells received either BM, BM + 10−8 M 1,25(OH)2D3 or BM + 10−7 M 1,25(OH)2D3 with complete media changes every 3d for a total of 12d of culture. On d 3, 6, 9, and 12 cell proliferation was measured using a MTS‐based assay, and samples were collected for gene expression analysis. Regardless of treatment, there was a linear increase in cell numbers over time up to 6d (P < 0.05), after which the cell numbers plateaued. Proliferation was reduced by treatment with 1,25(OH)2D3 in a dose dependent manner (P < 0.05). The concentrations of mRNAs encoding peroxisome proliferator‐activated receptor gamma (PPARG), CCAAT enhancing binding protein (CEBP), lipoprotein lipase (LPL) and adipocyte‐binding protein 2 (AP2) were increased (P < 0.05) by treatment with 1,25(OH)2D3 in a dose‐dependent manner. These changes seen with 1,25(OH)2D3 treatment are suggestive of an increase in adipocytic differentiation.

This work was supported in part by the BRDC (Peoria, IL) and the North Carolina Agriculture Research Service.
Long‐term adherence to the 2005 Dietary Guidelines for Americans is associated with lower risk of diabetes: 20‐year findings from the Coronary Artery Risk Development in Young Adults (CARDIA) study
Authors: Zamora Daisy , Gordon‐Larsen Penny , Jacobs David R. , Loria Catherine , Popkin Barry M.
Published: June 2021

BACKGROUND: Little is known about the long‐term benefits of adherence to current dietary recommendations.

OBJECTIVE: To study 20‐year diet quality (DQ) change and 20‐year diabetes mellitus (DM) incidence in black and white adults.

METHODS: Data were obtained from the 1985–86 and 2005–6 examinations of the CARDIA study. Participants were 18–30 years old at baseline and were recruited from 4 US cities (n=1838). We used the UNC Diet Quality Index (updated to reflect the 2005 Dietary Guidelines for Americans) to calculate DQ scores (possible range 0–100) at years 0 and 20. Participants were divided into quartiles of 20‐year DQ change. We used logistic regression to calculate odds ratios (OR) for DM (155 cases) adjusted for baseline body mass index (BMI), age, sex, education, race, clinic, and 20‐year changes in physical activity, BMI, smoking, and energy intake.

RESULTS: Mean change in DQ score for each quartile was −12 (1st), 4 (2nd), 16 (3rd), and 31 (4th). Compared to people in the 1st quartile, the OR of DM incidence were 0.70 (95% CI: 0.40, 1.20) for those in the 2nd, 0.53 (94% CI: 0.30, 0.95) for those in the 3rd, and 0.47 (95% CI: 0.25, 0.86) for those in the 4th quartile.

CONCLUSION: We found a trend of decreased DM risk as DQ improved over 20 years, independently of baseline BMI and its change. Our results are consistent with the idea that adherence to current dietary recommendations may help in the prevention of DM.
Activation of TLR5 Protects Against Radiation, Acute Colitis, and Food‐Borne Pathogens
Authors: Vijay‐Kumar Matam , Aitken Jesse , Neish Andrew , Rojas Mauricio , Gewirtz Andrew
Published: June 2021

We investigated the safety and efficacy of systemically‐administered bacterial flagellin to protect mice against a variety of challenges. Compared to LPS, mice given flagellin experienced less weight loss, exhibited transient hypoglycemia and suffered no detectable lung inflammation. In addition, flagellin was a weak inducer of death in D‐galactosamine sensitized mice. Furthermore, flagellin was highly effective as a radioprotectant offering up to 100% protection against the mortality that uniformly occurred in PBS‐treated mice. Flagellin offered significant protection at doses from 1 to 100 ug and required both TLR5 and MyD88. Optimal protection resulted from administration 2 h prior to exposure to radiation but significant (about 60%) protection was observed when flagellin was administered 4 h post‐irradiation. A single 50 ug dose also conferred significant protection against acute DSS colitis, rotavirus infection, and Salmonella‐induced mortality. In conclusion, flagellin is less toxic in vivo than LPS and protects mice against a broad range of challenges and, as such, may have practical value in a scenario in which an at‐risk population requires rapidly administered non‐specific protection in anticipation of, or in response to, an array of disparate threats.