bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chemoproteomic discovery of ligand binding hotspots in the lipid kinome
Authors: Hsu Ku‐Lung 
Published: June 2021

Diacylglycerol kinases (DGKs) are integral components of signal transduction cascades that regulate cell biology through phosphorylation of the ubiquitous secondary messenger diacylglycerol. Methods for direct evaluation of DGK activity in native biological systems are lacking and needed to develop chemical probes for studying isoform‐specific functions. Here, I will discuss efforts from my group to utilize ATP acyl phosphate activity‐based probes and quantitative mass spectrometry to map previously undefined ATP‐ and small molecule‐binding sites of representative members from all subtypes of the DGK family. We use our chemoproteomics strategy to discover an unusual binding mode for the DGK‐alpha (DGKA) inhibitor ritanserin, including interactions at a novel binding site remote from the ATP binding pocket. I will also describe our efforts towards discovery of fragment leads for future development of highly potent and selective DGKA inhibitors. Collectively, our studies illustrate the power of chemoproteomics to site‐specifically profile protein‐small molecule interactions and reveal key ligand binding sites for selective inactivation of the DGK family of lipid kinases.Support or Funding InformationThis work was supported by the University of Virginia, the LaunchPad for Diabetes Program funded by the Manning Family Foundation at the University of Virginia, National Institutes of Health Grants (DA035864 and DA043571) and U.S. Department of Defense (W81XWH‐17‐1‐0487 to K.‐L.H.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modified HPLC method for detection of hydroxyoctadecadienoic acid with greater sensitivity
Authors: Chesmore Nathan J. , Zhang Wenjing , Richards Mark P. , Shanmuganayagam Dhanansayan 
Published: June 2021

Hydroxyoctadecadienoic acid (HODE) is an oxidized fatty acid derivative of linoleic acid that is also a product of lipoxygenase‐mediated reactions. It has been implicated in the development of cardiovascular disease, differentiation of adipocytes, and several pro‐ and anti‐inflammatory mechanisms. HPLC, GC/MS, and LC/MS methods have been developed for the assay of HODE(s) from simple assay schemes to lipidomic approaches in fatty acid signaling, metabolism, and regulation research. Current HPLC methods used to quantify unmodified HODE(s) in fatty acid matrixes lack the required sensitivity necessary to detect levels of HODE found in tissue and serum. Sensitizing agents that bind HODE(s) or all sample fatty acids may be used to increase the signal of analytes by post‐column detectors. Here we describe the use of 2‐(2,3‐Naphthalimino)ethyl trifluoromethanesulfonate (NT) as a sensitizing fluorophore for the detection of 13(S)‐HODE in fatty acid matrixes using a c18 column for separation. NT fluorophore detection increases the sensitivity by more than 15‐fold compared to unmodified HODE conjugated diene absorbance at 234nm without modifications for fluorophore‐HODE conjugate stability. Pre‐column labeling of HODE with NT fluorophore in fatty acid matrixes also markedly increases the c18 column separation efficiency of the HODE‐fluorophore conjugate, reducing the amount of solvent and analysis time needed if shorter columns are used.Support or Funding InformationThe research was supported by the Biomedical & Genomic Research Group Discretionary Fund (University of Wisconsin‐Madison) and by USDA Formula Project Grant (WIS01972) National Institute of Food and Agriculture, USDA).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Personalized Diagnosis for Lafora Disease, a Fatal Epilepsy
Authors: Wayne Jeremiah L. , Brewer M. Kathryn , Gentry Matthew S. 
Published: June 2021

Lafora disease (LD) is a fatal, genetic disorder characterized by progressive neurodegeneration, myoclonus (i.e. uncontrolled muscle spasms), and epilepsy. LD patients present with seizures in adolescence that become increasingly severe and frequent, suffer rapid cognitive decline, and typically die within ten years of onset. Abnormal carbohydrate deposits known as Lafora bodies are found in the brains of LD patients and have been shown to drive disease progression.Approximately 50% of LD cases are caused by mutations in the Epilepsy progressive myoclonus 2A (EPM2A) gene that encodes a protein called laforin. Laforin is the only protein in humans that can release phosphate from carbohydrates. In LD patients, mutations in laforin lead to excess phosphate and abnormal branching in cellular carbohydrate stores, causing carbohydrate accumulation and toxicity. There are >50 different laforin mutations that have been described in LD patients, and some patients have milder forms of the disease. We have shown that not all laforin mutations are the same and that many mutations may have milder effects on protein function. Our biochemical analysis of disease mutations would allow us to predict disease outcome based on a patient's individual genetics.Recently an unusual case of late‐onset LD was described in a patient who lived to the age of 59. This patient contained a novel laforin mutation, where a specific amino acid at position 321 was changed from a phenylalanine to a cysteine (F321C). The objective of this study was to determine the effect of the F321C mutation on laforin function. We generated and purified recombinant laforin F321C, tested its biochemical effects using activity, binding, and stability assays, and found that this mutation and a previously described LD patient mutation, F321S, only mildly alter the function of laforin, providing a biochemical explanation for the very mild clinical phenotype. Our studies also establish a biochemical avenue for rapid, personalized diagnoses of LD patients, enabling doctors to predict patient progression and design treatment schemes that are specific to patients.Support or Funding InformationThis study was supported by NIH Grants F31NS093892 (M.K.B), R01NS070899 and P01NS097197 (M.S.G.), and NSF Grant IIA‐1355438 (M.S.G.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cultural Analysis and Demographics of Caprine Consumption among Texas Residents
Authors: Dimgba Chibuikem Samuel , Copeland Beverly , Ojumu Oluwagbemiga 
Published: June 2021

BackgroundAccording to report, nearly 1.5 million heads of goat are consumed annually within the US. The popularity of goat meat is increasing due to the increase in consumption from the immigrant population according to D'Rozario and Gaung, (2015), with this increase in demand for goat meat, the purpose of this research was to study the current and projected consumption patterns of Caprine products in Texas and identify target populations for the distribution of value‐added caprine products.MethodsA survey was developed, validated and piloted to measure food acceptability, cultural influences, demographics, generational acceptance, and geographic influences. Communities were selected by stratified sampling. Individuals were selected to participate through community outreach activities. Data were collected by trained research personnel.Data AnalysisThe data were analyzed using the statistical package for the Social Sciences (SPSS)ResultsThe sample size consisted of 79 study participants, with the majority (48%) being African Americans. When combined with participants who identified as African or Caribbean, blacks accounted for 58% of the total participants. This was followed by whites with 24% and Hispanics with 13% of the sample. Of the 79 participants who participated in the study, 44 (56%) reported having tasted goat meat before and 35 (44%) said they had never tasted it. Of the 44 who reported having tasted goat meat, only 15 (19%) reported actually eating it. The majority of the participants, 69, reported not seeing goat meat in their local grocery stores. Only 7 (9%) reported having seen goat meat in the grocery store.Support or Funding InformationEvans Allen Capacity Funds, NIFAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nicotinic Receptor Dysfunction Underlies Abnormal Responses to Muscarinic Receptor Antagonist Treatment in DYT1 Dystonia
Authors: Downs Anthony M. , Fan Xueliang , Hess Ellen J. 
Published: June 2021

DYT1 dystonia is an inherited dystonia caused by a mutation in the TOR1A gene, which encodes the torsinA protein. Trihexyphenidyl (THP) a nonselective muscarinic receptor antagonist is the small molecule drug of choice for the treatment of DYT1 dystonia, but it is poorly tolerated due to significant side effects. A better understanding of both the underlying pathology of DYT1 dystonia and the mechanism of action of THP is needed for the development of better treatments. Abnormalities in dopamine (DA) transmission have been implicated in DYT1 and other forms of dystonia. Previous studies have shown that Dyt1 knockin mice have reduced extracellular striatal DA in vivo and reduced striatal DA release ex vivo. Acetylcholine (ACh) is known to play a key role in regulating striatal DA release, and muscarinic receptor antagonists are used to treat dystonia which suggests the hypothesis that ACh signaling mediates the DA release deficiency in DYT1 dystonia. To test this hypothesis, we used ex vivo fast scan cyclic voltammetry (FSCV) and in vivo microdialysis to investigate ACh regulation of striatal DA release in Dyt1 knockin mice. We found that THP increased striatal DA release as assessed by ex vivo FSCV and in vivo microdialysis. However, THP was less effective at enhancing DA release in DYT1 mice relative to WT mice in the ex vivo FSCV experiment (mean increase WT: 65% vs DYT1: 35%). In striatum, blocking mACh autoreceptors on cholinergic interneurons increases ACh release. ACh then activates nicotinic acetylcholine receptors (nAChRs) located on DA terminals to enhance DA release. To determine if abnormal nAChR responses could account for the attenuated response to THP in Dyt1 knockin mice, we performed a dose response experiment with the α4 nAChR subunit selective antagonist dihydro‐β‐erythroidine. We found that Dyt1 mice are more sensitive to nAChR antagonism, which reduces DA release (IC50: WT= 29.46nM, DYT1= 12.26nM). These data suggest that nAChRs mediate the differential effects of THP in Dyt1 mice. These data also suggest that nAChRs may be suitable therapeutic targets for DYT1 dystonia. Future studies will evaluate nAChR positive allosteric modulators for DA‐enhancing effects.Support or Funding InformationSupported by: DoD W81XWH‐15‐1‐0545 and NIH T32‐GM008602This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of key regions mediating human melatonin type 1 receptor biased signaling revealed by natural variants
Authors: HEGRON Alan , PLOUFFE Bianca , BONNEFOND Amelie , GAO Wenwen , FROGUEL Philippe , LE GOUILL Christian , JOCKERS Ralf , BOUVIER Michel 
Published: June 2021

BackgroundMelatonin is a circulating neurohormone mainly released from the pineal gland in a circadian manner regulating many physiological functions, such as glycemia homeostasis. A misregulation of the glycemia could lead to Type 2 Diabetes (T2D). Recently, a genetic variant (rs2119882) found on melatonin type 1 receptor (MT1) promoter has been associated to higher risks to develop T2D.HypothesisVariants on the MT1 coding region could modify receptor signalling and consequently modulate incidence to develop T2D.Methodswe performed a genome‐wide association studies with both normoglycemic individuals and T2D patients and 32 non synonymous polymorphisms of MT1 were identified. Our primary aim was to functionally characterize these variants to further verify if there is an association between specific impaired signaling pathways and higher risks to develop T2D. Affinity for melatonin (Kd) and cell surface expression of variants were measured using 125I‐melatonin binding assays and enzyme‐linked immunosorbent assay (ELISA) respectively to be sure that all receptors were expressed at the cell surface. Melatonin dose‐response curves were performed at equivalent cell surface expression to determine MT1s efficacy and potency to activate each G protein using a bioluminescence resonance energy transfer (BRET) assay monitoring proximity between G alpha and G gamma proteins as activation indicator. Finally, b‐arrestin 2 recruitment to cytoplasmic membrane upon MT1s stimulated with melatonin was measured by monitoring BRET between RlucII‐fused b‐arrestin and the cytoplasmic membrane targeted sensor (rGFP‐fused to CAAX box of KRas).ResultsResults obtained by the functional characterization indicate several types of signaling signatures. Indeed, some mutations show a biased signaling between G protein activation and b‐arrestin recruitment, while others show G protein isoform selectivity.ConclusionAssociation studies between patients having MT1 mutations and these signaling signatures will help us to understand in a better way the relationship between melatonin and insulin and represent an opportunity to identify new potential genetic markers of higher risks to develop T2D.Support or Funding InformationThis work was supported by a grant from CIHR to MB, from FRM, ANR and Université Paris 5 Descartes to RJ and from NIH, NSF and DARPA to OL. PhD student fellowship from Université de Montréal to AH, postdoctoral fellowships from CIHR and from Diabetes Canada to BP and from FRM to AK also funded this work.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Can Inhibition of Soluble Epoxide Hydrolase Ameliorate Increased Susceptibility to DSS Induced Colitis in Soybean Oil Fed Mice?
Authors: Deol Sonia Poonamjot , Yang Jun , Yu Jacqueline , Trivedi Amol , Morisseau Christophe , Hammock Bruce D. , Sladek Frances M. 
Published: June 2021

The incidence of inflammatory bowel disease (IBD) in the U.S. has increased significantly since 1980, preceded by a remarkable increase in the prevalence of soybean oil (SO) in the American diet. Although the role of SO in the etiology of IBD is ambiguous, one of its major components, linoleic acid (LA), is associated with an increased risk of ulcerative colitis (UC). LA is metabolized into oxylipins (also referred to as leukotoxins) via a two‐step reaction involving cytochrome P450 enzymes and the enzyme soluble epoxide hydrolase (sEH). We have recently shown that a diet high in SO (and thus LA) increases the accumulation of oxylipins generated by the cytochrome P450 (CYP)/soluble epoxide hydrolase (sEH) pathway in the liver (PMID: 28970503). Our lab has previously shown that LA decreases the protein levels of the transcription factor hepatocyte nuclear factor 4 alpha (HNF4α) in wild type mouse livers (PMID: 19440305) and that a proper balance of HNF4α isoforms is required for the development and proper functioning of the gut (PMID: 27166517). To investigate the effect of dietary soybean oil on intestinal health, mice were fed either a low‐fat, high fiber vivarium chow diet (Viv) or a high soybean oil plus fiber diet (SO+f) with 19 kcal% LA and the same amount of fiber as Viv. Both male and female C57BL/6N (WT) mice and HNF4α exon‐swap mice (genetically engineered to express just the P1‐form of HNF4α so that in tissues, such as intestine, where both P1 and P2 promoters are active, only HNF4α1/2 is expressed instead of both HNF4α1/2 and HNF4α7/8 (PMID: 16498401)) were put on the diets at weaning. Colitis was induced by Dextran Sodium Sulfate (DSS) treatment eight to nine weeks after weaning. Increased weight loss was observed in both male and female HNF4α (A1HMZ) transgenic mice compared to WT mice, regardless of diet. Differences in other parameters, such as spleen weight, colonic and small intestinal length, that could be attributed to differences in diet and/or HNF4α isoform were also observed. Experiments with an inhibitor of soluble epoxide hydrolase are underway to determine whether soybean oil affects the balance between the HNF4α isoforms via LA diols and whether blocking the production of these oxylipins will make the mice more resistant to DSS induced colitis.Support or Funding InformationSupported by CCFA CDA 454808 (P.D.), USDA NIFA Hatch project CA‐R‐NEU‐5680 (F.M.S.), NIEHS grant R01‐ES002710 (B.D.H.), Superfund Research Program P42 ES04699 (B.D.H.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting the Right Ventricle as a Treatment Strategy for Pulmonary Arterial Hypertension
Authors: Gorr Matthew W. , Liang Wenjing , Muthusamy Abinaya , Sriram Krishna , Insel Paul A. 
Published: June 2021

Pulmonary arterial hypertension (PAH) is characterized by remodeling of the pulmonary artery within the lung that leads to increased pulmonary vascular resistance (PVR). This increase in PVR can result in progressive remodeling of the right ventricle (RV) and eventual right heart failure. Current therapy aims to decrease PVR via vasodilation of the PA, but this strategy does not work for some patients, and is not a cure for the disease. This study aims to uncover novel therapeutic targets by assessing the RV for expression of G protein‐coupled receptors (GPCRs) during PAH. GPCRs are the target of more than 30% of approved drugs and regulate multiple cellular processes by activation on the cell membrane. We utilized a rat model of PAH in which Sprague Dawley rats were placed in hypoxia (10% O2) for two weeks, or normoxia as a control (n=3–4/treatment). Placement in hypoxia resulted in RV remodeling evident by an increase in the RV weight/left ventricle (LV) + septum weight (hypoxia‐treatment: 0.458 ± 0.036 vs. control rats: 0.210 ± 0.020; mean ± standard deviation, Student's t‐test p<0.0001). We then utilized RNA sequencing (RNA‐seq) of the RV and the left ventricle (LV) tissue coupled with differential expression analysis to identify GPCRs expressed with significantly (false discovery rate <0.1) altered expression in the RV of hypoxia‐treated rats compared to controls. In the RV, 37 GPCRs were altered in expression in hypoxia‐treated rats vs. control rats compared to 7 GPCRs altered in the LV. To examine the cell‐specific expression of these targets, cardiac myocytes and fibroblasts were isolated from a mouse model of PAH (10% O2 for 3 weeks with weekly injection of SU5416). qPCR of the target GPCRs revealed that most of the differentially expressed GPCRs were altered in the cardiac myocytes but not in the fibroblasts. These results indicate that a subset of genes is differentially expressed in the RV compared to the LV both in normoxia and hypoxia, particularly in the cardiac myocytes. This raises the possibility that treatments for PAH could be directed at GPCRs selectively expressed in RV remodeling, thus providing a novel means of treating an unmet need for patients with RV hypertrophy and failure.Support or Funding InformationSupported by NIH Grant: T32HL007444‐34 and DoD Grant: W81XWH‐14‐1‐0372This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endothelial Cell Caveolin‐1 Depletion Modulates TGF and Notch Signaling Associated with Pulmonary Arterial Hypertension
Authors: Oliveira Suellen DArc Santos , Chen Jiwang , Castellon Maricela , Chernaya Olga , Colamonici Oscar , Bonini Marcelo G. , Machado Roberto , Minshall Richard 
Published: June 2021

Reduced expression of caveolin‐1 (Cav1) in endothelial cells (ECs) is associated with pulmonary vascular remodeling. Pulmonary vascular pathology in global Cav1−/− mice is prevented when eNOS is also deleted. Thus, we hypothesized that eNOS‐derived oxidants in absence of Cav‐1 result in dysfunctional ECs and pathological vascular remodeling. Hypoxia exposure following VEGFRII inhibition in Sprague‐Dawley rats produced a significant reduction in pulmonary Cav‐1 expression. Moreover, C57BL6 WT (Tie2.Cre−;Cav1lox/lox) mice exposed for 1 month to hypoxia exhibited an increase in the right ventricular systolic pressure (RVSP) from 25.33 to 31.68 ± 1.75 mmHg which was even higher in EC‐Cav1−/− (Tie2.Cre+;Cav1lox/lox) mice (27.95 to *38.48 ± 1.76 mmHg) when compared to normoxia. The increase in pulmonary arterial pressure induced by hypoxia was associated mild cardiac hypertrophy, increased plasma TGF‐β levels, and increased microvascular area/thickness indicative of vascular remodeling. Interestingly, under normoxic conditions, we observed reduced VEGF level in the lungs of EC‐Cav1−/− mice (8998 ± 1062 and 4927 ± 505.4 pg/mL, n=3–7 mice) suggesting Cav‐1 expression may be required to maintain normal VEGF signaling. Since VEGFRII inhibition may promote EC injury, we hypothesized that in the absence of endothelial Cav‐1, a defect in VEGF‐dependent vascular repair after injury may be the mechanism underlying pathological vascular remodeling. Then, mouse lung ECs (MLECs) isolated from WT, Cav1−/−, and Cav1−/−;eNOS−/− mice were used to assess gene and protein expression, and to quantify indices of angiogenesis. We have observed a defect in tube formation in Cav1−/− MLECs which was abo‐lished with genetic ablation of eNOS−/−. Moreover, in support of these findings, we observed increased Notch‐2 and ‐3 gene expression, and reduced Notch‐1 cleavage in Cav1−/− but not Cav1−/−;eNOS−/− MLECs. Finally, our data suggest the absence of endothelial Cav‐1 may promote pathological vascular remodeling by modulating TGF‐β and Notch signaling.Support or Funding InformationCNPq Fellowship‐CsF/Brazil; HL125356; DOD W911NF‐15‐R‐002.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.