bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Effect of Phosphodiesterase 4 Inhibition on Gene Expression Changes Produced by Long Acting β2‐adrenoceptor Agonists in BEAS‐2B Airway Epithelial Cells
Authors: Joshi Radhika , Yan Dong , Hamed Omar , Giembycz Mark A 
Published: June 2021

Phosphodiesterase 4 (PDE4) inhibitors are prescribed as add‐on therapy to a subset of COPD patients that suffer from exacerbations despite treatment with a combination of long‐acting β2‐adrenoceptor agonists (LABA), long‐acting muscarinic receptor antagonist and inhaled corticosteroids. The molecular mechanisms of their beneficial effects in COPD are ill‐defined. We have previously shown that LABAs, through their effect on the canonical Gs‐cAMP pathway, promote genomic effects in airway epithelial cells. Herein, we hypothesized that PDE4 inhibitors, by inhibiting cAMP degradation, promote similar genomic effects, and further potentiate the effects of LABAs in a human bronchial epithelial cell line. Additionally, we investigated whether this interaction between PDE4 inhibitors and LABAs occurred in an additive or a synergistic manner.Gene expression changes were determined by population‐based RNA‐sequencing in BEAS‐2B cells treated with the PDE4 inhibitor, roflumilast N‐oxide (RNO), alone and in combination with LABA, salmeterol. RNO showed a weak stimulation of gene expression by itself but showed a strong rank‐order correlation with salmeterol‐induced gene expression changes, suggesting a common mechanism of action. RNO augmented the expression of all salmeterol‐regulated genes, but the magnitude of augmentation varied between genes. This variation can be explained by an estimated 7.5‐fold difference in sensitivities of genes to salmeterol. Deming regression analysis of the gene expression changes produced by the combination of salmeterol and RNO versus the sum of their individual effects showed a deviation away from unity slope that may be explained by a synergistic effect of RNO at some DEGs. Consistent with these results, PDE4 inhibitors, GSK 256066, and roflumilast potentiated the effects of LABAs, indacaterol, and formoterol, on a panel of randomly‐selected genes as measured by RT‐PCR.Collectively, these data suggest that PDE4 inhibition may synergistically augment LABA‐induced gene expression changes in airway epithelial cells. This interaction between LABAs and PDE4 inhibitors may contribute to the therapeutic benefits of their combination in COPD.Support or Funding InformationCanadian Institutes for Health Research (PJT 152904)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cellular and Molecular Dynamics of Vertebrate Eye Morphogenesis: How Neural Crest Shapes the Embryonic Eye
Authors: Kwan Kristen M. , Bryan Chase D. , Casey Macaulie A. 
Published: June 2021

Precise and stereotyped 3‐dimensional tissue structure, established during the process of morphogenesis, is essential for proper organ function. In the visual system, aberrant structural development of the embryonic eye can lead to visual impairment and blindness. Despite increasing knowledge of pathways controlling the differentiation of many cell types in the eye, we still lack a basic understanding of the mechanisms controlling its morphogenesis. Using a combination of 4‐dimensional timelapse imaging, computational approaches, and zebrafish genetics, we seek to unravel the cellular and molecular basis, including both intrinsic and extrinsic cues, for the morphogenesis of the initial embryonic eye structure, the optic cup. One particular extrinsic cell population of interest is the neural crest: migratory mesenchymal cells are essential for the development and morphogenesis of many epithelial organs. In mouse, eye defects have been described in a neural crest mutant, but the mechanisms underlying aberrant optic cup development are unknown. Using zebrafish, we asked when and how neural crest contacts the developing eye, and how optic cup defects arise.We find that at the earliest stages of optic vesicle formation, neural crest cells are in contact with the optic vesicle; by the end of optic cup morphogenesis, these neural crest derived cells encapsulate the optic stalk and optic cup, except for its distal, lens‐facing side. To test the role of neural crest, we utilize the zebrafish tfap2ats213;foxd3zdf10 double mutant: in the absence of neural crest, optic cup invagination is defective, and using 4D cell tracking, we pinpointed the cell movements disrupted by loss of neural crest.What molecule is neural crest providing to control optic cup morphogenesis? Strikingly, using electron microscopy, we directly observe loss of basement membrane assembly in the neural crest mutant. Nidogen, a laminin‐collagen crosslinking protein expressed by neural crest and essential for optic cup morphogenesis in embryonic stem cells, appears to be a crucial factor: dominant negative nidogen disrupts wild type optic cup morphogenesis without affecting neural crest migration, and overexpression of wild type nidogen partially rescues optic cup morphogenesis in neural crest mutants. There are four nidogen genes in zebrafish (nid1a, nid1b, nid2a, and nid2b), and we are now generating quadruple mutants to examine loss‐of‐function phenotypes.Our data suggest that neural crest provides crucial extracellular matrix factors to regulate optic cup morphogenesis, and highlight diverse roles mesenchyme can play in epithelial organ development and morphogenesis.Support or Funding InformationNIH/NEI R01EY025378, R01EY025780 to KMK. CDB was supported by the University of Utah T32 Developmental Biology Training Grant (NIH T32HD007491).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The physiological hypertrophic agonist, norepinephrine, is able to induce PLC‐mediated PI4P hydrolysis in cardiac myocytes via a pool of internal β‐adrenergic receptors
Authors: Nash Craig , Wei Wenhui , Smrcka Alan 
Published: June 2021

Previously, we have shown that PLC∑‐mediated phosphatidylinositol‐4‐phosphate (PI4P) can be activated by raising cAMP in cardiac myocytes, however, activation by Isoproterenol was unable to induce PI4P hydrolysis. In the accompanying abstract by Wei et al, we have demonstrated that the cell permeant β‐agonist, Dobutamine is able to induce PI4P hydrolysis. Here, we seek to determine if the physiological agonist, norepinephrine is able to induce PI4P hydrolysis, despite being membrane impermeant. In contrast to Iso, norepinephrine induced sustained PI4P hydrolysis that was blocked by the cell permeant β‐adrenergic antagonist, metoprolol (100μM). The cell impermeant βAR antagonist, sotalol (5mM) could not block norepinephrine effects, suggesting that a pool of internal β‐adrenergic receptors is responsible for NE‐mediated PI4P hydrolysis. To directly determine if internal β1‐ARs in NRVMs can be activated by norepinephrine, we monitored translocation of a YFP‐tagged mini Gs protein in β1AR transfected NRVMs2. Norepinephrine induced robust and rapid recruitment of mini Gs to both the plasma membrane, and the perinuclear region corresponding to the Golgi apparatus. Golgi localization could be reversed by Metoprolol but not Sotalol. Isoproterenol, on the other hand, induced a strong recruitment to the plasma membrane but not to the Golgi. NE‐mediated PI4P hydrolysis is also abrogated by an inhibitor of a cation transporter Oct3, Corticosterone (100μM). Oct3 has been recently shown to be required for epinephrine‐mediated β1‐AR stimulation at the Golgi envelope in HELA cells3. Taken together, these data suggest that norepinephrine can activate β‐adrenergic receptors in internal membranes, potentially at the Golgi apparatus, stimulating PI4P hydrolysis. These internal GPCRs may be responsible for cardiac hypertrophy and heart failure and targeting them may allow for the development of selective therapies which can treat heart failure with little to no effect on other cardiac functions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Validation of a Manual Cell Counting Assay for Assessing the Kinetics of Cell Unclumping Reagents in Drug Discovery
Authors: Zem Gregory , Nahapetyan V , Crocker K , Tobar D , Santos S Delos , Nazarian D , Hovannes J , Beltran G , Dubin R , Reque L , Singh P , Sarkisyan L , Cardona B , Bachinela G , Oppenheimer S B 
Published: June 2021

This laboratory has developed and used a manual (non‐automated) cell counting assay for examining the kinetics of cell interactions in a yeast model (FASEB J 32:113; 31: 539; 30: 44; 30:49; 29: 52; 29: 65; Acta Histochem 116: 1514; 108: 311). Manual cell counting is often considered as a gold standard in some applications because of direct observation of results (Comparison of Cell Counting Methods…Inhal Toxicol 28(9): 410–429). For many years this assay has been used to evaluate the efficacy of reagents to unclump yeast cell clumps, with possible human applications in unclumping cancer cell clumps, unclumping thrombocytic blockages and reducing biofilms. In this study the assay is further validated by not only counting single cells as done in the past, but also counting cell clumps and number of cells per clump. To accomplish this, in over 400 trials by 14 investigators, sodium citrate dihydrate and magnesium sulfate were tested at 1, 2 and 3 mg per ml deionized water for their effects on fixed (Prefer fixative, Anatech Ltd, Battle Creek, MI) yeast (Saccharomyces cerevisae) over 60 min (with agitation every 20 min) on glass microscope slides and plotting all data with standard error and calculating p values comparing experimentals and controls in each category of results. It was generally observed that when there was no significant change in percentage of single cells in experimentals and controls (no reagent) there was no significant change in number of clumps and number of cells per clump. When there was a significant increase in percent single cells (suggesting that the reagent caused cell unclumping), there was a reduction in clump number and number of cells per clump. A specific example of this finding in 60 trials was that 3 mg sodium citrate dihydrate per ml deionized water caused a 35% increase in single cells by 60 min. The number of clumps decreased by 56% and the number of cells per clump decreased by 39%. Trends for magnesium sulfate were generally similar to those for sodium citrate dihydrate. In summary, this manual assay was further validated here by assessing cell clumps as well as the usual single cells. This assay is considered to be a gold standard for precise assessment of cell unclumping reagents in drug discovery.Support or Funding InformationSupported by California State University, Northridge Biology Department and Center for Cancer and Developmental Biology Foundation and Corporation accounts including U.S. Presidential Award contribution.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activation of AMPK‐FoxO3a pathway mediated autophagy inhibits fibroblast‐myofibroblast differentiation in pulmonary fibrosis
Authors: Gao Jian 
Published: June 2021

Aimto investigate the mechanism of AMPK‐FoxO3a pathway mediated autophagy during fibroblast‐myofibroblast differentiation in PF.Methodswe used BLM to induce PF in SD rats, and treatment with an AMPK activator(metformin) in model group. The pathological change and collagen deposition of lung tissue was observed by H&E and Masson staining. Meanwhile, we detected related proteins by immunohistochemistry and western blot. Meanwhile, human lung fibroblast were stimulated with TGF‐β1, and the cells were treated with AMPK activator(Metformin and AICAR), and activator and inhibitor of autophagy. Furthermore, the FoxO3a small RNA was used to interference cells. The related proteins and autophagy were detected by western blot.ResultsIn vivo, AMPK could reduce the destruction of alveolar structure and collagen deposition. In addition, AMPK could increase the expression of FoxO3a and autophagy level. In vitro, the protect effect of AMPK was abolished when FoxO3a was silenced by small RNA. Meanwhile, activation of autophagy by starvation and rapamycin could decrease TGF‐β1 induced fibroblast differentiation. However, when autophagy was inhibited by 3‐MA or chloroquine, the TGF‐β1 induced FMD was aggravated again under the pre‐treatment with metformin.ConclusionAMPK‐FoxO3a pathway could attenuate FMD by increasing autophagy in PFSupport or Funding InformationNational Natural Science Foundation of China No. 81473267, 81274172.
imageThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Maternal Electronic Cigarette Use Can Enhance Offspring Susceptibility to Hypoxic‐Ischemic Brain Injury
Authors: Sifat Ali Ehsan , Nozohouri Saeideh , Villalba Heidi , Vaidya Bhuvaneshwar , Abbruscato Thomas 
Published: June 2021

Prenatal exposure to tobacco smoke and nicotine is believed to interfere with fetal brain development predisposing offspring to different neurobehavioral and neuropsychological disorders. Included in this is increased neonatal vulnerability to hypoxic‐ischemic encephalopathy (HIE) which is a major cause of neonatal death and child disability in the US. These effects could be, in part, mediated by fetal nicotine exposure. Use of electronic cigarettes (e‐Cigs), commonly known as vaping, has been rapidly increasing in recent times in the general population, including women of reproductive age. E‐Cig use during pregnancy is also increasing because of the addictive properties of nicotine along with the perception of the relative safety of e‐Cigs. In this study, we aimed to investigate the effects of maternal e‐Cig use on neonatal brain development and HIE utilizing a combination of in vitro and in vivo models. Pregnant CD1 mice were exposed to e‐Cig vapor (2.4% nicotine). Primary cortical neurons were isolated from e‐Cig exposed fetus and cultured for seven days with subsequent exposure to oxygen‐glucose deprivation followed by reoxygenation (OGD/R) to mimic ischemia‐reperfusion injury. Hypoxic‐ischemic brain injury was induced in 8–9 days old mouse pups by a combination of left common carotid artery ligation and 15 minutes exposure to 8% oxygen. We found that e‐Cig exposed cortical neurons demonstrated decreased cell viability and increased caspase‐3 expression in OGD/R condition. These effects were accompanied by a decrease in neurite formation, mitochondrial dysfunctions, and decreased glucose uptake & glucose transporter expression in OGD/R condition. Our preliminary data also indicate increased sensitivity to HI brain injury in prenatally e‐Cig exposed mouse pups. Additionally, in utero e‐Cig exposed mice offspring displayed a significantly increased level of hyperactivity at postnatal day 45 in open field test. These results indicate that maternal e‐Cig exposure could lead to offspring behavioral abnormalities and enhance HI brain injury. Further studies are needed to demonstrate the long‐term anatomical and behavioral effects of HI brain injury in e‐Cig exposed offspring pups. This study is instrumental in elucidating the possible deleterious effects of maternal e‐Cig use in the general population.Support or Funding InformationR01 DA029121This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Carbamate Insecticides Modulate G Protein‐Dependent Signaling in Cells Expressing Melatonin Receptors
Authors: Jones Anthony Jordan , Mastrandrea Lucy D. , Rajnarayanan Rajendram R. , Dubocovich Margarita L. 
Published: June 2021

Melatonin targets MT1 and MT2 melatonin receptors that are expressed in brain as well as peripheral tissues in order to signal “time‐of‐day” messages for the synchronization of circadian rhythms. In mammals, the homeostatic balance of glucose metabolism and insulin release is modulated by Gαi protein‐dependent melatonin receptor (MTR) signaling in pancreatic β‐cells (Kemp et al. 2002). In‐silico screening paired with competition binding has demonstrated that carbamate insecticides with similar pharmacophores to melatonin have affinity for the mammalian MT1 and MT2 melatonin receptors (Jones et al. 2015 EB Abs.; Popovska‐Gorevski et al. 2017; Glatfelter et al. 2017 EB Abs.). Therefore we hypothesize that exposure to these environmental chemicals can disrupt melatonin receptor signaling. The objective of this study was to examine the effect of carbamate insecticides on G protein dependent‐signaling pathways on human MT1 and MT2 melatonin receptors stably expressed in CHO cells and in rat INS‐1 β‐cells expressing MT1. The apparent efficacy of carbamates (ie., carbaryl, fenobucarb, bendiocarb, and carbofuran) assessed by affinity ratios derived from competition with 2‐[125I]‐iodomelatonin binding, with or without G protein inactivation by GTP, suggested antagonist efficacy on hMT1 receptors (no affinity shift with GTP; KiGTP/KiControl:0.59 – 2.3) and agonist efficacy on hMT2 receptors (rightward affinity shift with GTP; KiGTP/KiControl: 4.8 – 11). The same insecticides tested at 10 μM did not modify forskolin‐stimulated cAMP levels in CHO‐hMT1 cells but inhibited stimulated cAMP formation in CHO‐hMT2 cells (47%–72% of melatonin effect on stimulation). Carbaryl (10 pM – 100 μM) acted as a partial agonist at both hMT1 (log IC50= −5.8 ± 0.3 M; Emax= 55% melatonin effect) and hMT2 (log IC50 = −10.0 ± 0.4 M; Emax= 67% melatonin effect) receptors with over 10,000 times higher potency for hMT2 compared to hMT1. Next, carbaryl at 10 μM, but not at 1 μM, inhibited forskolin‐stimulated insulin release from INS‐1 pancreatic β‐cells by 25% (96% melatonin effect) as determined by radioimmunoassay. These data suggest that carbamate insecticides with similar structures to melatonin can alter G protein‐dependent signaling in tissues that express melatonin receptors. Exposure to these chemicals could potentially contribute to the development of diabetes or other metabolic disorders as disruption of β‐cell melatonin receptor signaling and insulin release are linked to increased risk of disease (Tuomi et al. 2016).Support or Funding InformationSupported by ES 023684 to MLD and RVR.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pharmacokinetics and pharmacodynamics of curcumin in regulating antioxidant and epigenetic gene expression in healthy human volunteers
Authors: Cheng David , Li Wenji , Wang Lujing , Brunetti Luigi , Kong Ah‐Ng 
Published: June 2021

Curcumin is a major component of the spice, turmeric (Curcuma longa) often used in food or as a dietary supplement. Many preclinical studies on curcumin suggests benefit in many diseases due to its antioxidant and epigenetic effects. The few human studies and curcumin's unfavorable pharmacokinetics (PK) have limited its potential, leading researchers to study and develop formulations to improve its PK. The purpose of this clinical study is to describe the pharmacokinetics and pharmacodynamics (PK/PD) of commercially marketed curcumin in normal, healthy human volunteers. Twelve volunteers received 4 g of curcumin capsules with standard breakfast. Plasma samples were collected at specified timepoints and analyzed for curcumin levels. RNA was extracted and analyzed for expression of select antioxidant and epigenetic histone deacetylase (HDAC) genes. Plasma levels of parent curcumin were not detected by HPLC‐ITMS/MS/MS. However, curcumin glucuronide, a major metabolite of curcumin, was detected as soon as 30 min. These observations of little to no curcumin and low levels of metabolite are in line with previous studies. Antioxidant genes Nrf2, HO‐1, NQO1, and HDAC1, HDAC2, HDAC3, and HDAC4 were quantified by qPCR. Curcumin glucuronide pharmacokinetics are well‐described by a one‐compartment model and the PK/PD of curcumin glucuronide and its effect on antioxidant and epigenetic gene expression are explained by indirect response model (IDR). Increasing AUC or exposure to curcumin glucuronide was correlated with overall PD response. Physiologically based pharmacokinetic modeling (PBPK) and simulation using Simcyp correlated well with our observed data in the human volunteers. In vitro experiments on curcumin glucuronide in HepG2C8 cells also show that Nrf2‐ARE luciferase activity increases after 24‐hour treatment. These results show that poor bioavailability of curcumin remains a challenge but that oral administration of curcumin delivers detectable levels of curcumin glucuronide and may mediate the antioxidant and epigenetic effects of curcumin.Support or Funding InformationGrant: This work was supported in part by institutional funds and R01AT007065 from the National Center for Complementary and Alternative Medicines (NCCAM) and the Office of Dietary Supplements (ODS).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.