bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular GPS: Receptor and G‐protein dynamics that drive selectivity in GPCRs
Authors: Sandhu Manbir , Dysthe Matthew , Sivaramakrishnan Sivaraj , Vaidehi Nagarajan 
Published: June 2021

Upon binding to agonists, G‐protein coupled receptors (GPCRs) couple selectively to specific G‐proteins (such as Gs, Gi, and Gq) or β‐arrestins to activate specific signaling pathways. Some GPCR agonists exhibit differential responses to signaling pathways, a phenomenon called “functional selectivity”. The mechanism by which the GPCR‐agonist pairing leads to G‐protein selectivity is relatively obscure. Structure based design of G‐protein selective ligands remains a challenge due to the paucity of information on the structural elements of the ligand‐GPCR‐G‐protein complexes that lead to G‐protein selectivity. We have used a combination of a multi‐resolution Molecular Dynamics (MD) method for enhanced conformational sampling of protein complexes, called the Generalized Newton‐Euler Inverse Mass Operator (GNEIMO) method and a genetically encoded FRET biosensor technique called Systematic Protein Affinity Strength Modulation (SPASM), to measure G‐protein specificity in live cells. The combination of GNEIMO and SPASM form a powerful, complementary and scalable toolset for studying the structural basis of G‐protein selectivity in several GPCR‐G‐protein pairings. SPASM sensors measure the interaction (through FRET) of two proteins tethered by a flexible α‐helical ER/K_linker; this tool permits measurement of non‐cognate GPCR‐G‐protein interactions which cannot be measured by traditional biochemical assays used for cell signaling. This allows for an unprecedented comparison of relative intrinsic affinities of different G‐proteins to a specific GPCR. In this study, we have generated the conformational ensemble for 7 class A GPCRs bound to a full agonist and a C‐terminal peptide from Gαs‐, Gαi‐, and Gαq‐proteins. We predicted the putative hotspot residues on the G‐protein that leads to selectivity and tested these mutations using SPASM for switching specificity between Gs, Gi and Gq receptors. We observe Gs peptide binding to a different location in the intracellular surface of GPCRs compared to Gi and Gq peptides. The homologous, anionic residues, E355 (Gq) and D350 (Gi), orient the G protein C‐terminus away from the Gs‐specific cavity in the GPCR interface. Gi binding is further distinguished from Gq at the conserved position of G352 (Gi) and N357 (Gq). The smaller G352 residue functions as a size‐filter for the smaller cavity of Gi‐signaling GPCRs. This work provides insight to molecular mechanisms governing G‐protein selectivity, and is imperative to the development of functionally selective ligands.Support or Funding InformationNIH R01 GM117923‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Oxycholesterol protects against LXR‐mediated steatosis through Epstein‐Barr virus‐induced GPCR 2 (EBI2) in human and mouse hepatocytes
Authors: Huang Jin , Kang Saeromi , Park Soo‐Jin , Im Dong‐Soon 
Published: June 2021

BackgroundOxycholesterols are metabolites of cholesterol and several of them can act on the G protein‐coupled receptor, Epstein‐Barr virus‐induced G‐protein coupled receptor 2 (EBI2). Pivotal functions of oxycholesterols in immune regulation have been proven. However, the function of oxycholesterols in hepatocytes has not been fully investigated.METHODSAn in vitro model of liver X receptor (LXR)‐mediated hepatocellular steatosis was used to assess the role of oxycholesterols and EBI2 in hepatocytes.RESULTST0901317, a specific LXR activator, induced lipid accumulation in Hep3B human hepatoma cells. This lipid accumulation was inhibited by 7α,25‐dihydroxycholesterol (DHC), the most potent agonist of GPR183. The protective effects of DHC were inhibited by NIBR189, a specific EBI2 antagonist. T0901317 treatment induced expression of SREBP‐1c and ChREBP, key transcription factors for lipid synthesis. DHC inhibited the induction of SREBP‐1c and ChREBP proteins in a EBI2‐dependent manner. Using specific inhibitors of cellular signaling components, DHC‐induced inhibition of lipid accumulation was found to be mediated through Gi/o proteins, p38 MAPKs, PI3K, and AMPK. Furthermore, the protective effect of DHC was confirmed in HepG2 cells and mouse primary hepatocytes.CONCLUSIONTherefore, the present data suggest that DHC‐EBI2 signaling in hepatocytes may protect against lipid accumulation in the liver.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Targeting and Inactivation of Glutaminase and CDK4/6 in Mantle Cell Lymphoma
Authors: Gibbs Bettine E. , Masamha Chioniso P. 
Published: June 2021

Mantle Cell Lymphoma (MCL) is an aggressive B‐cell non‐Hodgkin's lymphoma (NHL) subtype that accounts for 7% of all NHL cases. Upon relapse, standard salvage regimens are ineffective and the disease is considered clinically incurable. Hence, novel therapeutic strategies that effectively target deregulated pathways that contribute to MCL pathogenesis are warranted. The hallmark of MCL is a chromosomal translocation t (11:14) event that results in the constitutive expression of the G1‐phase cell cycle regulatory oncogene, cyclin D1 (CCND1), resulting in uncontrolled cell proliferation. Cyclin D1 binds to and activates cyclin dependent kinases 4 and 6 (CDK4/6) and the complex phosphorylates the tumor suppressor gene product retinoblastoma (RB), a negative regulator of the cell cycle. MCL patients whose tumors express cyclin D1 have reduced survival. Uncontrolled cell proliferation results in metabolic alterations of tumor cells to support the energy and biosynthetic needs of unregulated cell growth. Adaptations by malignant cells involve changes in the expression of genes involved in metabolism. This results in both increased glucose and glutamine uptake and metabolism in tumor cells. Recent studies in metabolically reprogrammed tumor cells have shown increased levels of glutaminase, the enzyme involved in the first steps of glutamine metabolism. We hypothesize that molecular targeting of proliferation and glutamine metabolism will induce synergistic tumor cell death. In vitro studies with a MCL cell line were completed using Palbociclib, a potent and selective inhibitor of CDK4/6 which was recently approved by the FDA for the treatment of HR+/HER2− metastatic breast cancer. MTT assays were also carried out using BPTES which inhibits enzymatic activity by binding to the glutaminase tetramer, stabilizing it in an inactive conformation. Cell viability assays show that treatment of MCL cells with Palbociclib resulted in a dose dependent reduction in cell viability. Treatment with 5μM BPTES alone resulted in a slight reduction in cell viability. However, treatment with 5μM BPTES sensitizes cells to treatment with different concentrations of Palbociclib. Studies are ongoing using different concentrations of BPTES with a series of concentrations of Palbociclib to determine the synergistic effects of the combination treatment. As studies continue, quantification of synergy will be relayed using the Chou‐Talalay method with computerized simulation. Preliminary cell cycle assays using flow cytometry show that treatment of cells with Palbociclib induce G1‐phase cell cycle arrest. Establishing a treatment regimen that achieves a synergistic therapeutic effect may allow for integration of a novel molecular targeted approach to treating MCL.Support or Funding InformationButler University Holcomb research award, AFPE Gateway to Research awardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Long‐term Somatic Hypoalgesia as a Marker of Pathological Conditions in Gastrointestinal Tract
Authors: Filaretova Ludmila , Punin Yuriy , Sudalina Maria , Yarushkina Natalia 
Published: June 2021

Pain is traditionally considered as an evolutionarily developed defensive response to noxious stimulus. Short‐term pain suppression in response to stressful factors (stress‐induced analgesia) is an important adaptive reaction that is required for survival if further injury or death were threatened. In contrast, long‐term pain suppression may be considered as a sign of pain abnormality induced by disturbance of endogenous pain mechanisms under circumstances of pathology. Indomethacin (IM) is commonly used in clinic; however, it has a side ulcerogenic effect on the gastrointestinal tract. Previously we have shown that the formation IM‐induced injury in gastrointestinal tract is accompanied by a decrease in somatic pain sensitivity lasting, a least, for 72 h after IM injection (long‐term somatic hypoalgesia). Here we tested hypothesis that suppression of pathological process in gastrointestinal tract may prevent somatic hypoalgesia and normalize pain regulation in conscious rats. For this we studied effects of “stress preconditioning” on the gastrointestinal tract and somatic pain sensitivity under circumstances of ulcerogenic action of IM (35 mg/kg, sc). Preliminary (24 h) fasted rats were subjected one of four exposures (“stress preconditioning”) before IM administration: a) cold restraint (30 min, t=10 degree C, 1 h before IM); b) CRF (1.25 mkg/kg, ip, 30 min before IM) administration; c) capsaicin (0.5/kg, sc, 1 h before IM) administration, d) forced treadmill running (30 min, 1 h before IM). Pain sensitivity was assessed by tail flick latencies (tail flick test) or number of pain responses induced by acetic acid (“writhing test”). IM administration caused the formation of gastric erosion (2–4 h after injection) and small intestine injury (24, 48, 72 h after injection) that was accompanied by an increase in tail flick latencies (long‐term somatic hypoalgesia). Somatic hypoalgesia 48 h after IM injection was also supported by a decrease in number of pain responses induced by acetic acid. IM‐induced pathological process in the gastrointestinal tract was followed by changes in plasma corticosterone levels, cardiovascular (heart rate, blood pressure) and somatic (weights of thymus and adrenals, body weight) parameters. Each preconditioning exposure attenuated IM‐induced gastric mucosa injury and prevented somatic hypoalgesia. The results obtained suggest that long‐term somatic hypoalgesia may be one of characteristics of pathological process in the gastrointestinal tract induced by ulcerogenic action of IM.Support or Funding InformationThe study was supported by grant of Russian Science Foundation (RSF) №14‐15‐00790.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Circadian Disruption Accelerates Atherosclerosis in ApolipoproteinE‐deficient Mice
Authors: Pendergast Julie S. , Chalfant Jeffrey M. , Howatt Deborah A. , McGann Eric G. 
Published: June 2021

The circadian system is a network of molecular clocks located throughout the body. These clocks coordinate daily rhythms of behavior and physiology with environmental cycles. Shift work, which chronically disrupts circadian rhythms, increases the risk of developing cardiovascular diseases. The mechanisms by which this happens, however, are largely unknown. Our long‐term goal is to determine how disruption of circadian rhythms causes cardiovascular diseases. Atherosclerosis is the build‐up of plaques in arteries and can lead to myocardial infarction and stroke. In this study, we sought to investigate whether circadian disruption accelerates atherosclerosis in mice. Wild‐type mice do not develop atherosclerotic lesions. Therefore, we studied C57BL/6J ApolipoproteinE‐deficient (ApoE−/−) mice because they spontaneously develop atherosclerotic lesions. We first characterized circadian rhythms in ApoE−/− mice. We found that circadian behavioral rhythms, including free‐running periods of activity in constant darkness and constant light, phase angles of entrainment, and phase shifts to light pulses, were similar between wild‐type and ApoE−/− mice. These data show that ApoE−/− mice had no deficits in their circadian behavior rhythms or light responsiveness. Next, we determined if molecular circadian rhythms in tissues were altered in ApoE−/− mice. We analyzed the expression of the circadian timekeeping protein, PERIOD2, in central and peripheral tissues using a luciferase reporter. We found that PERIOD2::LUCIFERASE rhythms in tissues were similar in wild‐type and ApoE−/− mice. We next sought to determine the effect of circadian disruption on atherosclerosis in ApoE−/− mice. Housing mice in constant light profoundly disrupted locomotor activity rhythms in ApoE−/− mice. We also found that atherosclerotic lesion areas were increased in ApoE−/− mice housed in constant light for 3 months compared to those in control 12h light‐12h dark. Together, these data demonstrate that ApoE−/− mice have normal circadian rhythms and chronic circadian disruption accelerates atherosclerosis in ApoE−/− mice.Support or Funding InformationThis study was funded by National Institutes of Health grant P20GM103527, the Gertude F. Ribble Trust, and the University of Kentucky.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Impact of Primary Myoblast Transplantation on Functional Vasodilation Following Arteriogenesis in Mice with Diet‐Induced Obesity
Authors: Tran Christopher Minh , Hamzeinejad Vahid , Cardinal Trevor Ryan 
Published: June 2021

Peripheral arterial occlusive disease (PAOD) is characterized by reduced blood flow to the lower limbs, causing claudication and tissue loss. Proximal collateral arteries are natural bypasses to atherosclerotic occlusions in major arteries. A variety of cell‐based therapies have been tested clinically to enhance collateral arteriogenesis. Unfortunately, their efficacy has been poor, despite promising results from early clinical and preclinical studies, possibly because most preclinical studies are performed with mice that lack co‐morbidities present in most patients that are known to impair arteriogenesis. Therefore, additional cell‐types must be explored in animal models that more closely recapitulate the clinical phenotype to determine if pro‐regenerative therapies are a viable treatment for PAOD. Myogenic cells are an attractive candidate, given their co‐localization with the skeletal muscle vasculature niche and their ability to enhance angiogenesis. Therefore, we tested the hypothesis that primary myoblasts would rescue functional vasodilation in the superficial gracilis collateral following arteriogenesis in mice with diet‐induced obesity (DIO).In these studies, C57Bl/6 mice were fed a high‐fat diet from 6‐weeks of age; experiments were performed at about 4‐months of age. First, we performed a pilot study to evaluate functional vasodilation (vasodilation in response to electrically‐stimulated gracilis muscle contraction) in unoperated animals. As expected, vasodilation was impaired in the right (42±4 vs. 58±4 μm) and left (40±5 vs. 52±5 μm), as compared to lean controls.Having established impaired vasodilation in mice with DIO, we ligated the femoral artery in mice to model obesity and the chronic ischemia aspect of PAOD and measured functional vasodilation at day‐7 following surgery. Pilot data suggests that functional vasodilation is reduced in mice with DIO at day‐7 following surgery (resting: 80±10 μm, dilated: 82±8 μm) and that cell transplantation appears to enhance arteriogenesis and improve functional vasodilation (resting: 93±8 μm, dilated: 108±8 μm). Additional experiments are necessary to confirm these findings, but the data suggest that myoblasts strengthen the arteriogenic response. Future studies may involve additional time points to investigate the time course and duration of myoblast‐rescued arteriogenesis and functional vasodilation following arterial occlusion.Support or Funding InformationResearch was funded by the California Institute of Regenerative Medicine.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effect of docosahexaenoic acid on macrophage polarization
Authors: Ding Shih‐Torng , Peng Yu‐Ju , Chen Yu‐Shan 
Published: June 2021

Macrophages are one of the most important immune cells in the innate immune system. The native monocytes (M0) differentiate into different forms of activated macrophage depending on the cytokines in the microenvironment. The activated macrophages include classic/M1 macrophages and alternative/M2 or regulatory macrophages (Mreg). Docosahexaenoic acid (DHA), a long chain n‐3 polyunsaturated fatty acid is a common dietary supplement which against numerous diseases. In vitro study, we pre‐treated native THP‐1 cells with DHA and induced polarization by PMA. In THP‐1‐derived macrophages, pre‐treatment of DHA reduced the expression of M1 and increased M2/Mreg markers. In vivo study, oral administration of DHA decreased M1 and increased M2‐like macrophages in mice. Moreover, DHA decreased M1‐stimulating cytokines (IL‐12 and IFN‐γ) and increased M2/Mreg‐stimulating cytokines (IL‐4 and IL‐10) in blood. In conclusion, our results showed that DHA inhibited M1 and induced M2 macrophage polarization in THP‐1 cells and mice.Support or Funding InformationThe researcht was supported by Ministry of Science and Technology, Taiwan.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Human Placental Stem Cell Therapy in Stroke: Endothelial/Smooth Muscle Mechanisms Underlying Protection
Authors: Barzegar Mansoureh , Minagar Alireza , Wang Yuping , Yun Winny Jungmi , Boyer Christen , Cananzi Sergio Giuseppe , Alexander Steven Jonathan 
Published: June 2021

Following thrombolytic therapy in stroke, ischemia/reperfusion (I/R) mediated inflammation impairs maintenance of blood brain barrier (BBB), enhance expression of endothelial and leukocyte adhesion molecules and perturbs normal blood flow regulation. Currently, benefits of stem cell therapy (SCT) in stroke are thought to reflect secretion of protective and trophic factors which limit acute brain I/R injury. We studied human placenta mesenchymal stem cells (hPMSC) as a potent, high‐yield and inexpensive tool in SCT for human stroke therapy. We found that acute intravenous hPMSC administration significantly reduced infarct size (p<0.05) at 24h in our acute (1h) model of middle cerebral artery occlusion (MCAO) in rats. Our hypothesis was that acute benefits of hPMSCs in stroke might reflect suppression of post‐I/R immune, inflammatory and BBB disturbances which limit the onset and progression of stroke injury following restoration of blood flow. We first examined hPMSC support of BBB via mobilization of junctional proteins in human brain endothelial cells (hCMEC‐D3) in conjunction with FITC‐Dextran and FITC‐biotin‐gelatin avidin permeability assays plus western blotting and immunolocalization of junctional proteins. We also assessed the ability of hPMSC to suppress inflammation driven hCMEC‐D3 binding of macrophages (RAW246.7). Lastly we studied the influence of hPMSC on contractility of human brain vascular smooth muscle cells (hBVSMC) in collagen gel contraction assays.hPMSC significantly (p<0.001) enhanced barrier integrity at 24h in oxygen/glucose deprived/reperfused (OGDR) brain endothelial monolayers (in both FITC‐dextran and FITC‐biotin assays. We found that hCMEC‐D3 which were contact‐‐independently co‐cultured with hPMSCs expressed significantly higher levels of VE‐cadherin and α‐catenin under OGDR conditions (p<0.001); surprisingly, we did not see significant increases in occludin or claudin‐5 expression. Additionally, the spatial localization of VE‐cadherin and α‐catenin at hCMEC‐D3 junctions was apparently increased under OGDR, but not control conditions after hPMSC treatment. We also saw that hPMSC significantly (p<0.0001) reduced binding of macrophages to activated brain endothelial monolayers. Lastly, we found that hBVSMC contraction was significantly increased by hPMSC (p=0.0001), an effect which was reversed by Angiotensin II Type 1 receptor inhibition (Losartan, 10 uM) (p=0.004), but not by thromboxane A2 or endothelin‐1 blockade.We conclude that hPMSC release stable factors which beneficially and acutely influence endothelial barrier integrity, interactions with immune cells and vasoregulation to improve recovery in stroke.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lymphatic Wall Remodeling with Systemic and Tissue‐Associated Inflammation in Obese Zucker Rats
Authors: Trujillo Andrea N. , Breslin Jerome W. 
Published: June 2021

Lymphatic vessels have an intimate relationship with adipose tissue, and a growing number of reports tie dysfunction of lymphatics to obesity. A large gap currently exists in the knowledge of the pathophysiological mechanisms involved in obesity‐induced lymphatic dysfunction. The goal of this study was to investigate the extent to which obesity gives rise to inflammation and remodeling of the wall of rat mesenteric collecting lymphatic vessels. ZUCKER‐Leprafa (obese) and ZUCKER‐Leprafa/+ (lean) male and/or female rats, in conformance with the FASEB Statement of Principles for the use of Animals in Research and Education, were used for this study. Age groups included 8, 12 and 26–32 wks. Collecting lymphatic vessels were isolated from the mesentery and were mounted onto resistance‐matched glass micropipettes to determine passive wall mechanics. These experiments were performed in a 37°C albumin physiological salt solution (APSS without Ca2+) bath with luminal pressure stepped from 1–5 cm H2O. In addition, mesenteric arcades with adipose tissue and collecting lymphatics were dissected, fixed in 4% paraformaldehyde, processed and embedded in paraffin for sectioning and immunostaining with antibodies to rat‐specific CD68 (macrophage marker) and caveolin‐1 (adipocyte marker) for the quantification of crown‐like structures (CLSs). Blood (serum) and peri‐lymphatic adipose tissue (PLAT) were also collected and analyzed with the currently available panel of 67 inflammatory biomarkers through the Rat Biomarker Testing Service by RayBiotech. The results show that lymphatic wall thickness is significantly greater in obese Zucker rats compared to age‐matched counterparts. Wall:lumin ratio (X100) and medial cross‐sectional area were also significantly greater in obese versus lean male rats at all ages. Many CLSs were observed surrounding mesenteric collecting lymphatics isolated from obese male 12 and 26–32 wk rats. Additionally, CLSs significantly increased with age. In an analyses of serum biomarkers obtained from 12‐wk old lean and obese, male and female rats, we observed that many of the markers in PLAT (IL‐6, IL‐13, adiponectin) and in serum (GM‐CSF, IL‐4, Gas1) were differentially regulated by obesity and sex. The data support the concept that obesity causes an inflammatory environment in the mesentery that is associated with lymphatic vessel wall remodeling and changes to passive wall mechanics. The data also show significant differences in the profiles of inflammatory mediators increased by obesity in male and female Zucker rats, highlighting the importance of studying both sexes.Support or Funding InformationSupported by NIH L40HL097863This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.