bluuebunny/crossref_metadata_2025_split huggingface dataset:
New analogs of 3‐pyridyl ether as potential new ligands with high affinity and efficacy at selective nAChRs (1059.10)
Authors: Ogunjirin Adebowale , Fortunak Joseph , Brown LaVerne , Xiao Yingxian , Davila‐Garcia Martha 
Published: June 2021

The development of subtype‐selective ligands for nAChRs holds high potential for better, more targeted therapeutic management of conditions where nicotinic acetylcholine receptors are implicated, including brain and peripheral neuropathies. Here it is proposed that subtype selectivity for a particular nAChR may be achieved by small structural modifications of a ligand, including a change in the distance between the electronegative elements of the compound containing the nicotinic pharmacophore. In order to test this hypothesis, analogs of A‐84543 (compound 1) were designed and synthesized as potentially new nAChR subtype‐selective ligands. A‐84543 and four analogues (compounds 2‐5) were characterized in rat brain tissues. All synthesized analogs of A‐84543 were partially characterized and their binding affinities and subtype‐selectivity and function were determined using stably transfected cell lines expressing different nAChR subtypes. Of all the analogs, only compound 1 and 2 were shown to have higher selectivity for β2‐containing, compared to β4‐containing nAChRs, with a selectivity ratio between α3β4 and α4β2 of 1,350 and 530 fold, respectively. Finally, A‐84543 was shown to be a partial agonist at α3β4 nAChRs (EC50 = 160 ± 40 nM, Emax of 45%), while compound 2 was a full agonist and had higher potency (EC50 = 24 ± 1 nM, Emax of 100%), compared to 100 µM nicotine. Based on its higher efficacy and good selectivity ratio, compound 2 can be useful as a pharmacological tool for β2‐containing as well as α3β4*‐containing nAChRs receptors if selective pharmacological tools are used to protect or mask the other receptors. From our results we conclude that compound 2, has good potential as a lead compound to produce more selective ligands.Grant Funding Source: This research was supported in part by NIH grant R24MH06762701 and 5U54NS039407 to MDG.
Nitric oxide attenuates the enhanced expression of Giα proteins in vascular smooth muscle cells from spontaneously hypertensive rats: molecular mechanisms (1065.15)
Authors: Sarkar Oli , Paul Ravinder , Anand‐Srivastava Madhu B. 
Published: June 2021

We have previously shown that the nitric oxide donor, SNAP, decreased the expression of Giα proteins and associated functions in A10 vascular smooth muscle cells. The present study was undertaken to investigate if SNAP can also decrease the expression of Giα proteins and associated signaling in aortic vascular smooth muscle cells (VSMC) from 12‐week‐old Wistar‐Kyoto (WKY) and Spontaneously Hypertensive rats (SHR) and to further explore the underlying mechanism(s). Treatment of VSMC with SNAP (100 µM) for 24h decreased the expression of Giα‐2, ‐3 proteins. This decrease was subverted by inhibiting peroxynitrite with wither Mn (III) tetrakis (4‐benzoic acid) porphyrin (MnTBAP) or uric acid, and by inhibiting MEK with PD98059. However, inhibition of guanylate cyclase by 1H‐(1, 2, 4) oxadiazolo (4, 3‐a) quinoxalin‐1‐one (ODQ) was unable to restore the SNAP‐induced decrease in Giα‐2, ‐3. In addition, the enhanced activity of NADPH oxidase and protein levels of its subunits (Nox‐4, p47phox and p22phox) were decreased by SNAP. The decreased expression of NADPH oxidase subunits was restored towards control levels by MnTBAP. Furthermore, SNAP treatment decreased the enhanced levels of O2‐, TBARS and protein carbonyl in SHR to control levels. SNAP also attenuated the increased phospholyration of PDGFR, EGFR and IGF‐1R and c‐Src. These results suggest that SNAP decreased the enhanced expression of Giα proteins in VSMC from SHR by attenuating the increased oxidative stress, growth factor receptor activation and ensuing ERK1/2 signaling and not by a cGMP‐dependent mechanism.Grant Funding Source: CIHR
Phospholipase C‐epsilon links G‐protein coupled receptor activation to inflammatory responses in astrocytes (1066.5)
Authors: Dusaban Stephanie , Purcell Nicole , Kunkel Maya , Smrcka Alan , Heller Brown Joan 
Published: June 2021

Neuroinflammation underlies many diseases of the central nervous system (CNS) and the role of astrocytes in this process is increasingly recognized. Thrombin and the lysophospholipids, LPA and S1P are generated during injury and can activate G‐protein coupled receptors (GPCRs) on astrocytes. We hypothesized that GPCRs that couple to RhoA induce inflammatory gene expression in astrocytes through phospholipase C‐epsilon (PLCε). Using wild‐type (WT) and PLCε (KO) astrocytes, we recently demonstrated that thrombin, LPA, and S1P signal through PLCε to mediate sustained PKD activation and subsequently activation of NFκB to induce expression of inflammatory genes including COX‐2, IL‐1β, and IL‐6 (Dusaban, SS et al. PNAS, 2013). We are currently investigating the molecular mechanism by which PLCε mediates sustained PKD activation and downstream inflammatory responses. Preliminary data using Golgi targeted PKD FRET reporters suggests that PLCε mediates sustained activation of PKD at the Golgi. We also find that disrupting the Golgi with brefeldin A inhibits both PKD activation and COX‐2 expression. We further determined that COX‐2 gene expression was induced through PLCε following in vitro wounding and in vivo cortical stab wound injury. In light of growing evidence for a role of glial cells and S1P receptors in neuroinflammation, we investigated the S1P receptor subtypes coupled to PLCε and its downstream targets. Taking advantage of receptor KO astrocytes and receptor knockdown, we determined that coupling occurs primarily through the S1P3 receptor. Future work is aimed at identifying other GPCRs that signal through PLCε to mediate neuroinflammatory responses.Grant Funding Source: GM 36927
Role for DNA repair signaling in coronary artery stenosis (1071.9)
Authors: Meloche Jolyane , Pflieger Aude , Provencher Steeve , Bonnet Sébastien 
Published: June 2021

Coronary artery stenosis is a vascular disease characterized by sustained inflammation and oxidative stress, leading to DNA damage. Despite these detrimental conditions, coronary artery smooth muscle cells (CoASMC) show increased proliferation and suppressed apoptosis leading to luminal narrowing. PARP‐1 is a critical enzyme acting as DNA damage sensor by promoting either DNA repair or apoptosis depending on the amount of damage. Recent studies demonstrated the implication of the glycogen synthase 3 (GSK3) enzyme, which when inhibited, favors DNA repair (promoting cell survival and proliferation) and resistance to apoptosis (by promoting mitochondrial hyperpolarization). Thus, we hypothesized that increased DNA damage in coronary artery of patients with stenosis promotes PARP‐1 activation and GSK3 inhibition. In freshly isolated human CoASMC issued from control or stenosed arteries, we measured DNA damage, PARP‐1 expression and phosphorylated GSK3 protein levels. We demonstrated that CoASMC from stenosed arteries exhibit increased DNA damage, enhanced PARP‐1 expression and GSK3 inhibition. These cells also present mitochondrial membrane hyperpolarization and consequently show increased proliferation and suppressed apoptosis. Our study suggests an important role of DNA damage signaling and metabolism dysfunction in coronary stenosis and opens the door to new avenues of investigation and treatment.Grant Funding Source: Supported by CIHR grants
Interaction between the Cl‐ conductance associated proteins bestrophins and TMEM16A? (1077.2)
Authors: Aalkjaer Christian , Dam Vibeke , Boedtkjer Donna , Matchkov Vladimir 
Published: June 2021

We have previously demonstrated two Ca2+ activated Cl‐ conductances in smooth muscle cells (SMC). One which is cGMP dependent and Zn2+ sensitive (IClcGMP) and one which is not (ICl). We have also shown that IClcGMP, but not ICl, disappears after knock‐down of bestrophin3. We here tested the role of TMEM16A for the two currents. In vivo downregulation of TMEM16A in segments of rat mesenteric small arteries with siRNA resulted in disappearance of both IClcGMP and ICl. Downregulation of TMEM16A also led to decreased expression of bestrophin1, 2 and 3. Immunoprecipitation of TMEM16A revealed bestrophin3 in the precipitate, while immunopreciptation of bestrophin3 revealed TMEM16A in the precipitate. Close apposition of TMEM16A and bestrophin3 in SMC was confirmed with proximity ligation assay. We suggest that TMEM16A is the poreforming unit in SMC and that bestrophin(s) may be subunit(s) which modifies the biophysical and pharmacological characteristics of the Cl‐ conductance of TMEM16A, although the data does not rule out the possibility that bestrophins are poreforming subunits idependently from TMEM16A.Grant Funding Source: Supported by The Danish Research Council
The effect of substrate elasticity on the adhesion and elasticity of vascular smooth muscle cells (1077.8)
Authors: Hong Zhongkui , Jin Minshan , Bunyak Filiz , Ersoy Ilker , Sun Zhe , Li Zhaohui , Palaniappan Kannappan , Meininger Gerald 
Published: June 2021

In previous studies, we found vasoactive agonists induce coordinated changes in elasticity and integrin‐mediated adhesion to extracellular matrix (ECM) in primary vascular smooth muscle cells (VSMC) that were cultured on plastic/glass dishes. In this study, we hypothesized that the mechanical properties/stiffness of the substrate may play an important role in determining cellular elasticity and adhesion in VSMC. To simulate the in vivo mechanical environment of the ECM for VSMCs, a flexible polyacrylamide (PA) gel 2D substrate with tunable elasticity was used for cell attachment. A comparison of two substrate elasticities (7 and 30 kPa) were tested and collagen‐I was coated on the surface of PA gel to provide a biologically functional substrate to promote adhesion of VSMCs. An atomic force microscope (AFM) was used to measure VSMC elasticity and VSMC adhesion to FN coated AFM probes. Quantitative characterization of changes in cell morphology, adhesion, and elasticity were determined by custom‐developed software. Initial results showed a significant increase in cell elasticity/stiffness (~ +120%), adhesion force (~ +30%), and adhesion probability (~ +100%) of the VSMCs cultured on the stiffer (30 kPa) substrates compared with the cells cultured on softer substrates (7 kPa). These findings support the hypothesis that elasticity of substrate exerts an effect on the mechanical and adhesive properties of VSMCs.Grant Funding Source: NIH P01HL095486
The role of the VEGF/eNOS signaling pathway in cardiovascular development: a novel target to advance cardiovascular regeneration (1078.11)
Authors: Gentile Carmine , dos Remedios Cristobal , Drake Christopher , Davies Michael 
Published: June 2021

In vivo and in vitro models of murine and human endothelial cells (ECs) and cardiomyocytes (CMs) have been used to study the emerging roles played by the vascular endothelial growth factor (VEGF)/endothelial nitric oxide synthase (eNOS) signaling pathway in cardiovascular development. In vivo studies using mouse embryos showed that eNOS is first expressed by Flk‐1 (VEGF receptor 2)‐positive ECs of the endocardium earlier than in CMs. In vivo studies also showed that this occurs as early as at 8 days post‐coitum in mice, a developmental stage when both ECs and CMs are proliferating. Our in vitro studies using human umbilical vein endothelial cells (HUVECs) identified cyclin B1 as a novel molecular target of the VEGF/eNOS signaling pathway controlling EC proliferation. VEGF/eNOS‐mediated S‐nitrosylation of cyclin B1 in HUVECs occurs following formation of the “signalosome”, the multimolecular complex responsible for the S‐nitrosylation of several target proteins via HSP90. Pharmacological inhibition of HSP90 using 17‐AAG reduced the VEGF/eNOS‐mediated proliferative effects on human ECs. Taken together, our studies showed that the VEGF/eNOS signaling pathway via S‐nitrosylation of cyclin B1 represents a novel molecular target for cardiovascular regeneration in patients with cardiovascular disease. Current studies are focused on establishing the role of the VEGF/eNOS signaling pathway in CM development.Grant Funding Source: Supported by Marcus Blackmores Fellowship and the Translational‐in‐Aid‐Grant (Bosch Institute)
Prevention of ischemic vascular injury: targeting cellular stress in the endothelium with 5‐aminoimidazole‐4‐carboxamide 1‐β‐D‐ribofuranoside (1084.12)
Authors: Johnson Sarah , Banek Christopher , Gillham Haley , Needham Karen , Gilbert Jeffrey 
Published: June 2021

A variety of stressors induce endothelial cells (ECs) to produce excessive oxygen radicals, triggering vascular inflammation, cellular stress and apoptotic activation which may have deleterious effects on vascular function and contribute to the pathophysiology of hypertension during preeclampsia. Although the benefits of AICAR on ischemic‐reperfusion (I/R) injury in the myocardium are well established, the role of AICAR on specifically on vascular ECs is much less clear. Thus, we hypothesized AICAR would promote cytoprotective pathways in ECs, reducing stress and attenuating apoptotic activity under hypoxic stress. To model vascular ischemia as might be found in the preeclamptic placenta, human umbilical vascular endothelial cells (HUVECs) were cultured at physiologic normoxic (8% O2) or hypoxic (1% O2) conditions and treated with AICAR (0.2, and 2mM). Cell protein and media were collected for further analysis. AICAR increased (p<0.05) pAMPKα/AMPKα in hypoxic conditions, but not in normoxia. AICAR pretreatment (2mM, 1hr) and subsequent exposure to hypoxia (24hr) showed decreased (p<0.05) BiP expression and cell‐adhesion molecule ICAM‐1 compared to normoxic control. AICAR reduced (p<0.05) cellular metabolic activity measured by MTT assay in both 8 and 1% O2. Further, ET‐1 secretion was reduced (p<0.05) following AICAR treatment (434 ±14 vs. *179 ±1pg/mL). Endothelial permeability was increased (p<0.05) with AICAR at 12 (869%) and 24 hours (315%) compared to the control. HUVEC adhesion was reduced (p<0.05) following AICAR treatment (‐91.57%), and CC blocked this effect. These data suggest by slowing cellular metabolic activity, reducing cell stress and apoptotic activity, AICAR could be a novel cytoprotective factor for improvement of vascular endothelial function in preeclampsia as well as other types of cardiovascular disease.Grant Funding Source: Supported by National Institute of Health RO1HL114096 and AHA 10SDG2600040
Src glutathionylation and its role in oxidant and TGFβ1‐mediated Src activation (1095.13)
Authors: Zhang Hongqiao , Forman Henry 
Published: June 2021

Src is a critical enzyme involved in epithelial‐mesenchymal transition that contributes to metastasis and possibly fibrosis. Previous studies have demonstrated that oxidants are involved in Src activation by various stimuli. As glutathionylation of critical cysteine residues is an important mechanism of redox signaling, we studied the glutathionylation of Src and its role in Src activation in response to TGF‐β1, hydrogen peroxide (H2O2) and α,β‐unsaturated aldehydes. Human H358 lung carcinoma cells were transfected with FLAG‐tagged human c‐Src constructs, exposed to agents or control media. Then, FLAG‐Src was immunoprecipitated with anti‐FLAG antibody. Src protein was glutathionylated upon exposure to TGF‐β1 and H2O2, but not to aldehydes. Roles of individual cysteines in c‐Src activation were examined by mutating the cysteine (to alanine), transfecting cells with mutant c‐Src, and then examining oxidant‐stimulated auto‐phosphorylation of c‐Src at Tyr418. Mutation at C248 or C501 abolished Src activation by oxidants and TGF‐β1 and glutathionylation was absent in these mutants. The data suggests that glutathionylation plays a critical role in c‐Src activation by both TGF‐β1 and oxidative stimuli.Grant Funding Source: ES020942
Bioinformatic analysis of differential protein expression in calu‐3 cells exposed to carbon nanotubes (1095.7)
Authors: Li Pin , Lai Xianyin , Witzmann Frank , Blazer‐Yost Bonnie 
Published: June 2021

Carbon nanotubes (CNT) are widely used in industry, medicine and scientific research. To examine potential impact on human health, the human airway epithelial cell line, Calu‐3, was exposed to a physiologically relevant (100 ng/mL) concentration of either single‐ and multi‐wall CNTs for 24 h. Label‐free quantitative mass spectrometry showed differential protein expression of 281‐282 proteins of which 231 were common to both nanotube exposures. The majority of the proteins showed decreases suggesting a general stress response to protect cells. Ingenuity Pathway Analysis was used for bioinformatic analysis. The STRING database was used to analyze functional protein networks. Common proteins changes occurred within the cellular functions of cell death and survival, cell‐to‐cell signaling and interaction, cellular assembly and organization, cellular growth and proliferation, infectious disease, molecular transport and protein synthesis. Some proteins like cadherin 1, STAT1, plakoglobin, and PYCARD, appear in several functional categories and tend to be in the center of the protein networks, suggesting important roles in multiple cellular functions and activities altered in response to CNT exposure.Grant Funding Source: Supported by Grants from the National Institutes of Health (R01GM085218 and RC2ES018025)
Control of thin filament lengths by sarcomeric tropomodulin isoforms: insights from mouse models (1102.2)
Authors: Gokhin David , Fowler Velia 
Published: June 2021

The uniform lengths of skeletal muscle thin filaments are controlled by actin subunit exchange at pointed ends (P‐ends). Each P‐end is capped by two sarcomeric tropomodulin (Tmod) molecules, with Tmod1 and Tmod4 isoforms present in a 1:9 ratio. In Tmod1‐null sarcomeres, P‐ends are capped by Tmod3 (normally a sarcoplasmic reticulum‐associated Tmod isoform) and Tmod4, preserving normal thin filament lengths. In Tmod4‐null sarcomeres, P‐ends are capped solely by Tmod1, also preserving normal lengths. The mild phenotypes of Tmod1‐null and Tmod4‐null muscle highlight skeletal muscle’s ability to deploy compensatory mechanisms to specify correct thin filament lengths in the absence of one sarcomeric Tmod isoform during myofibril assembly and muscle development. To discern the effect of sarcomeric Tmod depletion from mature muscle, we measured thin filament lengths in the mdx and mdx/mTR mouse models of Duchenne muscular dystrophy, in which Tmods are calpain‐proteolyzed in a muscle use‐dependent manner. Proteolysis of Tmod1 and/or Tmod4 in mdx and mdx/mTR muscles results in 10‐12% increases in thin filament lengths, due to addition of actin subunits onto P‐ends. Collectively, these results show that Tmod1 and Tmod4 are individually dispensable for initial specification of muscle‐specific thin filament lengths, but important for maintenance of correct thin filament lengths in mature contracting myofibrils.Grant Funding Source: Supported by NIH and MDA.
Phosphodiesterase inhibition increases PGC‐1α in muscle cells (1102.32)
Authors: Rahnert Jill , Zheng Bin , Woodworth‐Hobbs Myra , Hudson Matthew , Price S 
Published: June 2021

Muscle wasting associated with chronic diseases, such as type I diabetes (DM), can be countered by restoring the reduced expression of the transcriptional coactivator PGC‐1α. Previous reports indicate that PGC‐1α transcription can be regulated by CREB in a process that requires nuclear localization of CRTC (CREB‐regulated transcription coactivator). We recently found that CRTC protein is reduced in skeletal muscle of diabetic rats whereas CREB is highly phosphorylated (i.e., activated). Nuclear translocation of CTRC is promoted by dephosphorylation via calcineurin (CnA) whereas nuclear export occurs by phosphorylation via the salt‐inducible kinases (SIK). Inhibition of phosphodiesterases (PDE), which raise cyclic AMP (cAMP) levels, results in increased PGC‐1α mRNA expression. cAMP also inhibits SIK suggesting this pathway may contribute to CRTC localization and thus PGC‐1α activity. Our experiments tested the hypothesis that inhibition of PDE increases the amount of nuclear CRTC and PGC‐1α transcription. Myotubes were transfected with luciferase reporter plasmids (Luc) and treated with the general PDE inhibitor IBMX (250uM) for 6 hours. IBMX increased PGC‐1α‐Luc promoter activity as well as the transcriptional activity of CREB. Deletion of the CRE site in PGC‐1α‐Luc prevents the increase in luciferase activity suggesting IBMX increases PGC‐1α activity via CREB. In addition, protein analysis indicates IBMX increases the level of nuclear CRTC. Together, these data imply that raising cAMP levels by inhibiting PDEs results in increased PGC‐1α transcriptional activity by a mechanism involving CREB and its coactivator, CRTC.Grant Funding Source: Supported by NIH RO1 DK95610, NIH T32 DK007656 and VA Merit X01BX001456
Effect of meal composition on postprandial blood glucose levels in middle aged and young adults (1107.6)
Authors: Koch Carynn , Koch Jennifer , Hopper Mari 
Published: June 2021

Effect of meal composition on postprandial blood glucose levels was determined in two cohorts, one middle aged (MA) (35‐55 years) and one young adult (YA) (19‐24 years). Subjects first completed an OGTT following an overnight fast. Over the next two weeks, and in random order, subjects consumed two breakfast meals varying in glycemic load: 1) high glycemic load (HGL); 2) low glycemic load (LGL). An Accucheck® glucometer determine fasted, 30, 60, and 120 minute postprandial blood glucose levels.Although fasted blood glucose levels were normal for all individuals, the mean value for the MA group was significantly higher than YA (87.1±0.70 vs. 82.9±0.67 mg/dl; p=0.0001). Mean blood glucose values following OGTT were normal and similar for the two groups. Following HGL and MGL meal consumption, blood glucose values for MA were higher than YA at 30 and 60 min and AUC’s were also significantly higher (13724±388 vs. 12780±354; p=0.042 and 12896±335 vs. 11703±212; p=0.003).The MA individuals with high waist circumference (WC>88cm) demonstrated significantly higher OGTT values than MA with normal waist circumference at 30 and 60 min (149.3±4.8 vs. 128.0±8.1; p=0.0273 and 147.2±7.6 vs. 118.6±11.6; p=0.0373). Following meal consumption high waist circumference individuals had higher blood glucose values than normal waist circumference at all time points with significant differences following MGL at 30 and 60 minutes.Grant Funding Source: Support received from the University of Southern Indiana Early Undergraduate Research Program