bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of a Robust Direct Binding Assay for Phospholipid‐Sensing NR5A Nuclear Receptors
Authors: D'Agostino Emma Hope , Cornelison Jeffery , Mays Suzanne , Flynn Autumn , Jui Nathan , Ortlund Eric 
Published: June 2021

The human NR5A nuclear receptors, steroidogenic factor‐1 (SF‐1, NR5A1) and liver receptor homolog‐1 (LRH‐1, NR5A2), are phospholipid‐sensing regulators of steroidogenesis, development, and metabolism. Their control of such diverse biological processes renders them attractive pharmacological targets for the treatment of several cancers and metabolic diseases such as obesity, type II diabetes, and non‐alcoholic fatty liver disease. However, the evaluation of candidate endogenous ligands and the development of small molecule modulators have been hindered by the lack of a robust direct binding assay. Using a structure‐guided approach, we recently developed a potent NR5A agonist (EC50 of 15 nM) targeting the ligand‐binding pocket. We have leveraged this molecule to create a high‐affinity fluorescent probe that binds SF‐1 and LRH‐1 with affinites of 12 nM and 1 nM, respectively. This probe has enabled the creation of a fluorescence polarization (FP)‐based competition assay suitable for both synthetic small molecules and mammalian phospholipids. FP is solution‐based, allowing proteins to retain their native conformation, and the competition format of the assay ensures that binding will only be detected in the ligand‐binding pocket. We have found that for a set of 25 LRH‐1 agonists derived from the previously‐reported RJW100 scaffold, affinities determined by FP correlate with potencies in a luciferase reporter assay, suggesting that in vitro binding affinity may be predictive of in‐cell activity for this class of molecule. Moreover, binding affinities reported for other NR5A agonists harboring different chemical scaffolds agree with our FP‐based measurements. Finally, we show that the previously‐reported dilauroylphosphatidylcholine agonist binds LRH‐1 with a Ki of 80 nM, suggesting that this assay may be used to evaluate candidate endogenous ligands for these receptors.Support or Funding InformationR01 DK115213 02EHD has additional support from the National Science Foundation Graduate Research Fellowship and the Emory University NIH‐5T32GM008367‐27. SGM has additional support from F31 DK111171 03.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Progress towards development of an ELISA for quantifying avian CBG
Authors: Antunes Ivy , Johnson Elizabeth , Malisch Jessica , Mertz Pamela S. 
Published: June 2021

Corticosterone (CORT) is the avian equivalent to mammalian cortisol, one of the stress hormones expressed as part of the acute stress response. The biochemical response to environmental stressors is highly conserved amongst vertebrates, allowing for use of avian models in mammalian stress research. Corticosterone binding globulin (CBG) is a carrier protein for CORT which binds ~80% of CORT in the bloodstream. Current research indicates that only unbound CORT is active, suggesting that CBG acts as a regulator as well as a carrier of CORT. CBG declines under chronic stress which would correlate with higher levels of free CORT during the acute stress response. There are also studies which indicate that the CBG‐CORT complex has a biological role separate from CORT delivery or inactivation, and studies which indicate that unbound CBG can play a role in cell signaling pathways independent of CORT binding. There is evidence in humans that the CBG‐CORT complex can increase expression of cyclic adenosine monophosphate, but it is unknown if it has the same effect in birds. Routine quantification of CBG could lead to a more clear definition surrounding its role in the stress response and clarify some of its other biological functions. This project reports on the progress of refining an avian CBG enzyme linked immunosorbent assay (ELISA) using a novel CBG antibody against zebra finch (Taeniopygia guttata), and developing a standard for use in the ELISA for quantifying CBG. The current accepted technique for quantifying CBG is a radioactive assay which is not accessible to all researchers, highlighting the necessity of development of an ELISA to be an easier, safer, and more accessible method for quantificationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondrial Oxidative Stress And Adipocyte Protein Carbonylation
Authors: Huang Yimao , Hauck Amy , Chen Yue , Bernlohr David A 
Published: June 2021

At the molecular level, obesity‐linked type 2 diabetes is characterized by chronic low‐grade inflammation driven by resident macrophages resulting in potentiated adipocyte oxidative stress, mitochondrial dysfunction, lipid oxidation and altered gene expression. Among the many mechanisms linking inflammation to metabolic disease, protein carbonylation has been considered a primary event linking oxidative stress to insulin resistance. Protein carbonylation refers to oxidative posttranslational modification of proteins with reactive lipid aldehydes such as 4‐hydroxy trans 2,3 nonenal (4‐HNE), 4‐hydroxy trans 2,3 hexenal (4‐HHE), or 4‐oxo trans 2,3 nonenal (4‐ONE). Lipid aldehydes are formed at high levels under conditions of increased oxidative stress in visceral, but not subcutaneous, fat tissue and has long been considered a biomarker of oxidative stress in adipocytes, neurons, and muscle cells. Since 4‐HNE and 4‐ONE are derived from lipid peroxidation, C11‐BODIPY581/591, a peroxidation‐sensitive sensor, was used to profile which organelle in 3T3‐L1 exhibited the greatest oxidation. Using C11‐BODIPY581/591, the mitochondrion exhibited the greatest probe peroxidation and is therefore the likely source of reactive lipid aldehydes in fat cells. Moreover, carbonylation is increased coincident with lipogenesis suggesting that ROS sources in the mitochondrion drive reactive lipid aldehyde formation. Despite the localization of lipid hydroperoxides to the mitochondrion, carbonylated proteins accumulate in the nucleus of visceral, but not subcutaneous, white adipose tissue. Proteomic analysis of carbonylated nuclear proteins reveals that zinc‐finger proteins (ZFP) including the estrogen related receptors a and g are primary targets and result in loss of DNA binding activity. Mapping of carbonylation sites reveals that the DNA/RNA binding domain of ZFPs are primary targets. These results suggest that oxidative stress and lipid peroxidation in the mitochondrion may lead to retrograde signaling of aldehydes to the nucleus culminating in the carbonylation‐dependent inactivation of nuclear zinc‐finger proteins.Support or Funding InformationSupported by NIH DK053189 to DABThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sterols promote atlastin‐mediated endoplasmic reticulum membrane fusion in Saccharomyces cerevisiae
Authors: Jun Youngsoo , Lee Miriam , Moon Yeojin 
Published: June 2021

Sterols play critical roles in various membrane fusion events, including SNARE‐mediated membrane fusion, mainly by modulating the physical properties of biological membranes; however, it remains unclear whether they also function in atlastin‐mediated endoplasmic reticulum (ER) membrane fusion. Although ergosterol, the major sterol in yeast, is essential for fusion of Sey1p (yeast atlastin)‐containing liposomes with an ER‐mimicking lipid composition, fusion of phosphatidylcholine/phosphatidylserine liposomes does not require sterols. Here, we examined whether sterols are important for Sey1p‐mediated ER fusion in Saccharomyces cerevisiae using an in vitro ER fusion assay with isolated yeast ER microsomes. Ergosterol‐specific ligands inhibited microsome fusion, indicating that ergosterol is critical for ER fusion. However, microsomes isolated from yeast strains lacking genes that encode enzymes involved in synthesis of ergosterol from lanosterol still fused, suggesting that other sterols can replace ergosterol and support Sey1p‐mediated ER fusion. Importantly, disruption of sterol‐binding motifs in the transmembrane regions of Sey1p markedly reduced ER fusion. Furthermore, Sey1p physically interacted with Erg11p, which converts lanosterol into an ergosterol precursor, suggesting that Sey1p recruits Erg11p to fusion sites and thereby enriches ergosterol, which, in turn, may recruit more Sey1p. This positive feedback loop may facilitate ER membrane fusion by concentrating fusion factors at fusion sites.Support or Funding InformationThis work was supported by a grant from the National Research Foundation (NRF) of Korea (2016R1A5A1007318) awarded to the Cell Logistics Research Center and by a grant from the GIST Research Institute at the Gwangju Institute of Science and Technology.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Epac1 is required for cAMP‐mediated endothelial barrier stabilization
Authors: Radeva Mariya Yosifova , Schuster Katharina , Ponce Alexander Garcia , Døskeland Stein‐Ove , Curry FitzRoy E , Reed Rolf K. , Waschke Jens 
Published: June 2021

Regulation of intercellular junctional complexes is critical for the control of endothelial barrier function and dependent on cAMP‐mediated Rac1 activation. Recently, the exchange protein activated by cAMP (Epac1) has been shown to serve as a tonic stabilizer of the endothelial barrier. Here, we further elucidate the role of Epac1 in cAMP and Rac1‐mediated endothelial barrier regulation.Transendothelial‐electrical‐resistance (TER) measurements in newly generated immortalized myocardial endothelial cells derived from Epac1‐knockout (KO) mice revealed significant reduction in the baseline TER, when compared to wild‐type (WT) cells, indicating that Epac1 is required for maintenance of endothelial barrier function. This effect was associated with fragmented VE‐cadherin staining and reduced localization of tight junctional proteins ZO‐1 and occludin along cell borders. In WT‐cells, application of forskolin (F) and rolipram (R) resulted in increased TER associated with linearization and augmentation of VE‐cadherin immunostaining at adherens junctions. In contrast, Epac1‐KO‐cells did not respond to F/R treatment indicating that Epac1 is crucial for cAMP‐mediated endothelial barrier stabilization. In comparison to WT‐cells, Epac1‐KO‐cells revealed significantly increased protein levels of Rac1, whereas Rac1 basal activity was not enhanced, indicating a defect in Rac1 activation. However, in Epac1‐KOcells, cAMP‐mediated Rac1 activation was not abolished and the basal cAMP‐concentration was significantly increased. Additionally, in Epac1‐deficient cells F/R‐mediated increase in cAMP levels was less compared to WT cells. Taken together, our data show that Epac1 is crucial for cAMP‐mediated barrier stabilization by mechanisms which at least in part are independent of Rac1 regulation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dual Specificity Phosphatase and Pro Isomerase Domain Containing 1 (Dupd1) is Upregulated During Neurogenic Skeletal Muscle Atrophy and is Differentially Expressed in MuRF1‐null Mice
Authors: Cooper Lisa , Waddell David 
Published: June 2021

Skeletal muscle atrophy is caused by a decrease in muscle size and strength and results from a range of physiological conditions, including denervation, immobilization, corticosteroid exposure and aging. Dual Specificity Phosphatase and Pro Isomerase Domain Containing 1 (Dupd1) has been identified as a novel atrophy‐induced gene in skeletal muscle and has been found to be differentially regulated in wild‐type mice compared to Muscle RING Finger 1 (MuRF1) knockout mice in response to denervation. Muscle RING Finger 1 (MuRF1), an E3 ubiquitin ligase, has previously been shown to be upregulated under almost all conditions of skeletal muscle wasting. Preliminary data described in this study provides evidence that MuRF1 may play a more complex role in the muscle atrophy cascade than previously believed and appears to be necessary for full activation of a subset of neurogenic atrophy‐induced genes, including Dupd1. A recent study that utilized microarray analysis of skeletal muscle isolated from wild‐type and MuRF1‐null mice revealed that Dupd1 expression is significantly induced at 3 days and 14 days post‐denervation in wild‐type mice, while Dupd1 expression is dramatically blunted in the MuRF1‐null mice at both 3 days and 14 days post‐denervation. qPCR analysis revealed that Dupd1 is significantly upregulated during differentiation of cultured muscle cells. To determine how Dupd1 might be transcriptionally regulated in skeletal muscle, fragments of the promoter region of Dupd1 were cloned, fused to a reporter gene and found to be highly inducible in response to ectopic expression of MyoD and myogenin. Furthermore, site‐directed mutagenesis of conserved E‐box elements in the proximal promoter of Dupd1 rendered the Dupd1 reporter genes incapable of being induced by overexpression of myogenic regulatory factors. The Dupd1 gene codes for a dual specificity phosphatase (DUSP) that can recognize and dephosphorylate tyrosine, serine and threonine amino acids of substrate proteins. Finally, we have found that Dupd1, an atypical DUSP, can modulate the ERK branch of the MAP Kinase signaling in muscle cells and inhibits muscle cell differentiation when ectopically expressed in proliferating muscle cells. Elucidating the role of Dupd1 in muscle will help contribute new insight into the molecular mechanisms for skeletal muscle atrophy.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants and a University of North Florida Foundation Board Grant to D.W.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stem Cell‐Based Modelling of Brown Adipogenesis Using Hypothermal Conditions
Authors: Leija Hilda Anaid Lugo , Velickovic Ksenija , Bloor Ian , Law James , Sacks Harold , Symonds Michael , Sottile Virginie 
Published: June 2021

In vivo cold exposure is the main stimulus to activate the unique mitochondrial uncoupling protein (UCP)1, known for mediating heat production in brown and beige adipocytes. In vitro, UCP1 adipogenic response under hypothermic conditions requires an extended evaluation in order to be comparable to an in vivo cellular function. We have developed a new in vitro culture system to induce adipogenic browning in bone marrow (BM) derived mesenchymal stem cells (MSC), using exposure to reduced temperature during differentiation. Incubation at 32°C instead of 37°C over the course of the culture treatment was seen to promote brown adipogenesis with increased adipocyte response and upregulation of adipogenic and thermogenic factors including UCP1. Adipocytes differentiated under hypothermic conditions displayed enhanced uncoupled respiration and metabolic adaptation, as well as significant durable changes in cellular and molecular features. These results suggest that MSC available from accessible tissue sources can be differentiated in vitro using cold temperature conditions to favor the appearance of functional beige‐like adipocytes. This cell‐based model represents a new valuable tool to screen for pro‐browning factors in vitro and elucidate the mechanism of metabolic adaptation relevant to the browning process.Support or Funding InformationThis work was funded by the EU‐CASCADE fellowship scheme funded by the EU's 7th FP PCOFUND‐GA‐2012‐600181 (K.V.), CONACyT, Mexico (H.A.L.L.), and received support from The Cardiometabolic Disease Research Foundation (Los Angeles, USA) and the Biotechnology and Biological Sciences Research Council (BBSRC, Multidisciplinary Super Resolution Microscopy Facility grant BB/L013827/1).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lysophosphatidic Acid Regulates Erythropoiesis at Different Hematopoietic Hierarchy
Authors: Chiang Jui‐Chung , Lin Kuan‐Hung , Chen Wei‐Min , Lee Hsinyu 
Published: June 2021

Hematopoiesis, a complex developmental process to form blood components and replenish the blood system, involves multiple intracellular and extracellular mechanisms. We previously demonstrated that lysophosphatidic acid (LPA), a lipid growth factor has opposing regulatory effects on erythrocyte differentiation through activating LPA receptor 2 and 3, yet the underlie mechanisms in vivo remained unclear. Examining the LPA receptors expression profile in murine myeloid cells indicates that LPA2 is highly expressed on common myeloid progenitor (CMP), whereas expression of LPA3 increases with later erythrocyte differentiation. Therefore, we hypothesize that the expression of LPA2 and LPA3 determine the hematic homeostasis of mammalian hematopoietic progenitors. In vitro colony forming unit (CFU) assays of murine sorted progenitor cells reveal that LPA2 agonist, GRI977143, reduces the erythroblast differentiation potential of CMP. In contrast, LPA3 agonist,1‐oleoyl‐2‐O‐methyl‐rac‐glycerophosphothionate (OMPT), increases the production of erythrocyte from megakaryocyte erythrocyte progenitor (MEP). In addition, LPA2 agonist treatment reduces erythroid, CMP and MEP populations in mice. These results indicate that activation of LPA2 predominantly inhibits myeloid differentiation at the early stage of myelopoiesis. By contrast, activation of LPA3 increases production of terminally differentiated erythroid cells. We also demonstrate that LPA3 agonist successfully restored phenylhydrazine (PHZ)‐induced acute hemolytic anemia in mice. Our results reveal distinct roles of LPA2 and LPA3 at different hematopoietic hierarchy in vivo, suggesting potential therapeutic strategies for the treatment of anemia.Support or Funding InformationThis research was supported by a grant from Ministry of Science and Technology, Taiwan (MOST 107‐2311‐B‐002‐009‐)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Galectin‐3 Levels in Hepatic Tissue from Lean and Obese Zucker Rats
Authors: Schuster Jane E , Motawe Zeinab Y. , Trujillo Andrea N. , Breslin Jerome W. 
Published: June 2021

Obesity and metabolic syndrome are of major concern for physicians worldwide. Galectin‐3 is a β‐galactoside‐binding protein that has been shown to have regulatory functions in inflammatory responses as well as fibrosis of the liver, lung, heart and blood vessels. Galectin‐3 is widely expressed in multiple types of immune cells as well as epithelial and endothelial cells, and its properties have been explored as a therapeutic target for nonalcoholic fatty liver disease liver fibrosis, heart disease and other obesity‐associated diseases. We previously observed elevation of galectin‐3 in mesenteric fat from male and female 12‐week‐old obese Zucker rats compared to their lean counterparts. We hypothesized that galectin‐3 expression levels would be elevated in the liver in obese animals. Livers were collected from 20 Zucker rats (5 male, 5 female lean rats and 5 male, 5 female obese Zucker rats) for histology and protein analysis. After protein lysates were collected and total protein levels were normalized, galectin‐3 levels were quantified with the Wes Simple Western capillary electrophoresis assay. The results showed that livers of obese rats of both sexes display typical fatty liver pathology and have a significantly higher level of Galectin‐3 expression. The results show that increased galectin‐3 expression accompanies fatty liver in obese rats. Galectin‐3 may represent a future target for preventing tissue dysfunction in obesity and metabolic syndromeSupport or Funding InformationAPS Frontiers in Physiology FellowshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Crystallographic Structural Elucidation of E. coli HU and a Four‐Way Junction
Authors: Khun Charya , Mukerji Ishita 
Published: June 2021

E. coli histone‐like HU protein binds with high affinity to DNA recombination intermediates, such as the Holliday junction, to facilitate prokaryotic DNA transcription, compaction, recombination, and replication. While previous Förster resonance energy transfer (FRET) studies have determined possible structural models of HU‐junction interaction, X‐ray crystallographic data is needed to confirm those models. Studies have shown that crystals can be obtained with a dodecamer junction complex. However, we believe that HU can also be successfully crystallized with J20, that is, a junction with 20 base pairs per strand. We believe that 20 base pairs per arm is sufficient in limiting the flexibility of our junction. Our investigation aims to determine the most favorable conditions for HU‐J20 crystallization by using various solvents, pH, and viscosities. In our study, we show that our HU samples are pure, nuclease‐free, and bind with high affinity to J20. In addition, we show that our J20 samples are at least 90% properly annealed via a non‐denaturing polyacrylamide gel. Moreover, we have observed the formation of small crystals in a few of our wells, primarily around pH 8.5 in 20% polyethene glycol with 0.2 M ammonium chloride, and 0.005 M calcium dichloride. Going forward, we will screen around our successful conditions with higher protein concentrations. We will also use magnesium dichloride solutions in addition to calcium dichloride solutions, as previous studies have shown that magnesium improves the stabilization of DNA. Upon the development of crystals, we hope to solve the structure of HU‐J20 via diffraction.Support or Funding InformationAmerican Society of Biochemistry and Molecular Biology Undergraduate Research Award, Wesleyan Math and Science Scholars Program, Wesleyan UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.