bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kinetic control of signaling: role of RGS proteins and GRKs
Authors: Sharma Garima , Bastepe Murat , Jackson Edwin K. , Masuho Ikuo , Martemyanov Kirill A. , Andresen Bradley T. 
Published: April 2020

G protein‐coupled receptors interact with the GDP‐bound heterotrimeric G‐protein, G protein‐coupled receptor kinases (GRKs), and arrestins through their cytosolic surface. Previous studies suggest that the interaction of GRKs and the heterotrimer with the receptor occur in the same space; therefore, there is likely steric hindrance allowing only one pathway to operate at a time. Supporting this claim, experimentally induced competition for the intracellular heterotrimeric G‐protein binding site results in a reduction in GRK and Gα function. We created a Gαs chimera, called sis, containing most of the helical domain of Gαi1. In initial studies, sis prevented GRK2, 4, 5, and 6‐mediated reductions in isoproterenol‐mediated cAMP accumulation. Therefore, we tested the hypothesis that sis disrupts GRK/arrestin function due to an alteration in the turnover of the sis protein. To exclude any background effects of endogenous Gαs, we used GnasE2−/E2− MEFs re‐expressing Gαs (control) or sis (experimental) as well as the β2‐adrenergic receptor. Experimentally, cAMP accumulation in the presence and absence of receptor and GRK inhibitors, receptor‐Gs kinetics, and receptor internalization were measured. The introduction of the Gαi1 helical domain into Gαs did not significantly affect the biochemistry of the chimeric sis protein; however, GRK function and cAMP production altered. GRK2 failed to attenuate isoproterenol‐mediated cAMP accumulation in sis‐expressing cells, but attenuated cAMP accumulation in Gs‐expressing cells. Accordingly, the rate of endocytosis decreased in sis‐expressing cells compared to Gαs‐expressing cells at 10 μM, but not 0.1 and 1 μM, isoproterenol (0.77 ± 0.10 versus 1.00 ± 0.14 min−1). The kinetics of 10 μM isoproterenol‐mediated cAMP production demonstrates that the rate of accumulation of cAMP is similar, yet sis‐expressing cells have an increase in maximal response, and the rate of degradation of cAMP fits two different models. Degradation of cAMP in Gαs‐expressing cells is fit to a classical first‐order equation with a rate constant of 0.068 ± 0.012 luminescence/min, whereas, in sis‐expressing cells, the degradation of cAMP fit a linear zero‐order model with a slope of −0.012 ± 0.0012 luminescence/min. As there is 10 μM of isoproterenol present, we hypothesized that the receptor is nearly always active, and sis has altered kinetics and GRK activity due to rapid cycling between the cyclase and receptor. To test this second hypothesis, exploratory experiments are being conducted to determine if the alterations observed can be explained through a chimerically‐derived interaction with RGS proteins. The collected preliminary data suggest that altered kinetics of signaling alter GRK function and receptor internalization, and consequently, RGS proteins may indirectly inhibit GRK function.Support or Funding InformationWesternU MSPS program funds and NIH grants DK073911 (MB), DK079307 (EKJ), and HL109002 (EKJ) supported this work.
The Effects of Ocean Acidification in the California sea hare (Aplysia californica)
Authors: Zlatkin Rebecca L. , Grosell Martin , Heuer Rachael M. 
Published: April 2020

Fish and some invertebrates have demonstrated to be strong acid‐base regulators following exposure to ocean acidification‐relevant carbon dioxide (CO2) levels through the accumulation of bicarbonate (HCO3−) in extracellular and intracellular fluids. This allows for pH compensation, however, internal pCO2 and HCO3− remain elevated. This compensation has been hypothesized to cause negative behavioral impairments, by altering ion gradients (HCO3−/Cl−) along neuronal cell membranes that affect the function of the GABAA receptor, an important inhibitory receptor in the central nervous system of vertebrates and invertebrates. Aplysia californica, known for their simple and well‐mapped nervous system, have the potential to be strong model organisms to examine the relationship between CO2 compensation and impaired behavior in invertebrates. We hypothesized that CO2‐exposed animals would compensate for an acidosis by accumulating HCO3−. In the second portion of the study, we hypothesized that two important behaviors, the righting reflex and tail‐withdrawal reflex, would be impaired following CO2 exposure, since this has been noted in many other species. Aplysia were exposed for 4–11 days to either control (400 μatm), 1,200 μatm (close to end of century predictions), or 3,000 μatm CO2. Hemolymph pH was measured using a custom gas‐tight chamber with a fiber‐optic pH microsensor, and HCO3− and pCO2 were calculated using pH and measurements of total CO2 using the Henderson‐Hasselbach equation. The amount of time it took the animal to right following release from the water column (righting reflex), and the amount of time it took the animal to relax its tail to 50% of the original length (tail‐withdrawal reflex) following a mild tail depression were recorded. We observed that animals were able to fully compensate pH at 1,200 μatm CO2, but experienced a significant but mild acidosis at the 3,000 μatm level. Hemolymph HCO3− and pCO2 increased at both CO2 levels, and were statistically significant when compared to controls. Increased CO2 exposure did not significantly affect the righting reflex, but tail‐withdrawal reflex demonstrated a significant 36–37% decrease in relaxation time at both CO2 levels, suggesting increased boldness and altered behavior at projected future CO2 levels. Given these findings, Aplysia are promising models to study CO2‐induced behavioral impairments since they are capable of regulating HCO3− and have a CO2‐impaired behavior linked to simple neural networks amenable to electrophysiological testing. Further research using this model may help illuminate physiological mechanisms underlying CO2‐induced behavioral impairments noted in many marine species that could threaten ecosystems in future oceans.Support or Funding InformationThis research was supported by the National Institute of Health Bridge to Baccalaureate Program (Award R25GM050083).
Epithelial Repair in the Rabbit Cornea Following Aspects of Epithelial Injury and Repair after Mustard Exposure
Authors: Gordon Marion K. , Gerecke Donald R. , Zhou Peihong , Hahn Rita , Joseph Laurie B. , Chang Yokechen 
Published: April 2020

Sulfur mustard (SM; bis (2‐chloroethyl) sulfide) is a potent vesicating agent that has been used in chemical warfare. Exposure of the eyes to SM can cause corneal erosions, epithelial defects, neovascularization, microblistering, and/or dry eye disease, all injuries that can lead to corneal epithelium sloughing and ultimately blindness. In the present studies, the effects of ocular exposure to SM vapor (0.420 mg/L for 8 min) on the corneas of male New Zealand white rabbits was characterized at 1, 3, 7, 14, 21 and 28 days post‐exposure. During this time, corneal thickness increased from ~400 microns to 600–900 microns. Unexposed control corneas were clear throughout the time course. However, after SM exposure increased corneal thickness was associated with decreased corneal opacity and neovascularization. Histologically, SM exposure induced epithelial‐stromal separation at 3 days, by which time 16% of the epithelia had separated from the stroma, and by 28 days detachment had decreased to 9%. Also observed was post‐SM epithelial thinning, increased stromal disorganization, and edema. Sporadic keratocytes were also evident in the edematous stroma by day 14. At 28 days post‐SM, an inflammatory cell infiltrate was observed in the stroma. In control corneas, keratin 17, a marker of wound repair, was constitutively expressed in the limbus and bulbar conjunctiva. By 14 days post‐SM, keratin 17 was expressed in the peripheral corneal epithelium, and by 28 days it was expressed in the central cornea. PCNA, a proliferation marker, was constitutively expressed in the nuclei of control corneal epithelial cells. Three days post‐SM, PCNA expression was decreased along the damaged basal layer. Twenty eight days post‐SM, PCNA was upregulated in the nuclei of hyperplastic cells of the central cornea and cells of the limbus, indicating wound repair. These data suggest that limbal and /or conjunctival epithelial cells play a role in the repair of SM‐induced corneal injury. A better understanding of the mechanisms of SM‐induced corneal damage will lead to the development of new therapeutics for the treatment of these debilitating ocular injuries. Supported by NIH grant AR055073.Support or Funding InformationSupported by NIH grant AR055073.
TIME COURSE CARDIOVASCULAR AND NEUROIMMUNE DYSFUNCTIONS IN A METABOLIC SYNDROME MODEL: IMPACT OF AEROBIC TRAINING
Authors: Bernardes Nathalia , da Silva Dias Danielle , Stoyell-Conti Filipe Fernandes , Freitas Sarah Cristina , Paixão Camila Cristina , Llesuy Susana Francisca , Irigoyen Maria-Cláudia , De Angelis Kátia 
Published: April 2020

This study analyzed the effects of aerobic exercise training (ET) in the time course of the dysfunctions observed in metabolic syndrome model (SHR + fructose overload). Animals were randomly distributed into two groups: hypertensive + fructose (HF) and hypertensive + fructose + ET (HFT). The fructose overload (100g/l) and the ET (treadmill 1h/d, 5d/wk) was initiated at 30 days of life, and the animals (n=6/group/time) were evaluated after 7, 15, 30 and 60 days. The ET (HFT group) induced an increase in baroreflex sensitivity for bradycardic response at 7 (100%), 15 (86%), 30 (76%) and 60 (74%) days of protocol and for tachycardia response (TR) in 7 (34%), 15 (31%) and 30 (49%) days of protocol as compared to sedentary group (HF). However, the ET did not attenuate the impairment in TR at 60 days of protocol. There was a reduction in the relation of TNF‐a/IL‐10 in fat tissue (40%) and IL‐1β in the spleen (30%) in the HFT group when compared to the HF group in 15 days of protocol. The nitrites in plasma were increased in HFT group as compared to HF group at 7 and 15 days of protocol. Regarding oxidative stress in plasma at 60 days of protocol, there was an increase in SOD activity (74%) and a decrease in lipid peroxidation (46%) and in protein oxidation (60%) in the HFT group when compared to the HF group. The ET reduced blood glucose (15%) and increased insulin sensitivity (31%) at the end of the protocol, although it did not alter blood triglycerides or blood pressure. Fructose consumption increased NFkB and p47 phox protein expression at day 60. However, aerobic exercise training was able to reduce NFkB at day 30 and 60, and p47 phox at day 60. In conclusion, the hemodynamic, autonomic, inflammatory and oxidative stress disorders seem to be time dependent in SHR with fructose overload. On the other hand, aerobic ET is effective in attenuate disorders in this model of metabolic syndrome.Support or Funding InformationFAPESP: 2018/17183‐4
Hyperosmolality Induces Nuclear Translocation of Osmotic Stress Transcription Factor 1 in Oreochromis Mossambicus Cells
Authors: MacNiven Leah Marie , Hamar Jens Carlton , Kültz Dietmar 
Published: April 2020

Euryhaline fish tolerate a wide range of environmental salinity by employing molecular mechanisms for coping with the associated osmotic stress. We have previously shown that osmotic stress transcription factor 1 (OSTF1) is part of these mechanisms. OSTF1 is transiently and rapidly upregulated in gill epithelial cells of tilapia (Oreochromis mossambicus) exposed to hyperosmolality. Hyperosmotic induction of OSTF1 was shown in tilapia gills, a response that was reproduced in the tilapia OmB cell brain fibroblast cell line (OmB). OSTF1 shares the signature sequence of the TSC‐22 family predicting that it is a transcription factor. If in fact OSTF1 is a transcription factor, we hypothesize it will localize to the nucleus during hyperosmotic stress. Using molecular cloning, OSTF1 was tagged with enhanced green fluorescent protein (EGFP) at either the C‐ or N‐terminus. Using fluorescent microscopy we showed that the fusion proteins were retained in the cytosol under iso‐osmotic conditions. To evaluate potential nuclear translocation of OSTF1 during hyperosmotic stress, we subjected OmB cells expressing the OSTF1:EGFP fusion protein to hyperosmotic media and imaged at time intervals from 5 minutes to 4 hours using a Leica Dmi8 microscope. At four hours and 650 mOsmol/kg, subcellular localization quantified by LASX image analysis (Leica) demonstrated that OSTF1:EGFP was mostly localized to the nucleus. This result supports our hypothesis that OSTF1 is indeed an osmotically inducible transcription factor. Current work evaluates the persistency of nuclear localization of OSTF1 during sustained hyperosmotic stress and isosmotic recovery.Support or Funding InformationThis study was funded by a grant from NSF (IOS‐1656371).
Omega‐3 Fatty Acid and Glucocorticoid Additively Suppress Pro‐inflammatory/ Interferon‐Regulated Gene Responses and Inflammasome Activation in Novel Alveolar Macrophage Model
Authors: Heine Lauren K. , Wierenga Kathryn A. , Lewandowski Ryan , Chauhan Preeti S. , Harkema Jack R. , Pestka James J. 
Published: April 2020

Glucocorticoids (GCs, a.k.a. steroids) are a widely used treatment in chronic inflammatory and autoimmune diseases, but have many injurious side effects. Dietary omega‐3 fatty acids (O3FA) are effective anti‐inflammatory agents with potentially distinct and overlapping mechanisms of action with GCs. However, the potential for combining these anti‐inflammatory agents as a steroid‐sparing strategy is understudied. The goal of this study was to assess the interactive effects of the O3FA docosahexaenoic acid (DHA) and GC dexamethasone (DEX) in modulating inflammasome activation and interferon‐regulated gene (IRG) response in Max Planck Institute (MPI) cells, a novel murine alveolar macrophage model derived from fetal liver. MPI cells were pre‐treated with 0, 5, 10 or 20 μM DHA‐BSA conjugate for 24 hr, followed by 0, 0.01, 0.1, or 1 μM DEX for 1 hr. This was followed by stimulation with 20 ng/mL lipopolysaccharide (LPS) for 2 or 4 hr. At this point, cells were either i) directly harvested and analyzed for pro‐inflammatory and IRG gene expression by qRT‐PCR, or ii) incubated with 5 μM nigericin for 45 min to achieve inflammasome activation, as measured by IL‐1β in the supernatant. LPS treatment significantly induced both pro‐inflammatory and IRG responses in this cell line, and was also required for nigericin‐induced IL‐1β release. Singular treatment with either DHA or DEX alone both exhibited concentration‐dependent reduced inflammatory gene expression and inflammasome activation. When combined, DHA and DEX suppressed these responses in an additive manner. Taken together, this model offers a tool to identify interrelated mechanisms of action of O3FAs and GCs with the long‐term goal of implementing this combination in new steroid‐sparing therapies against inflammatory and autoimmune diseases.Support or Funding InformationResearch funded in part by the Dr. Robert and Carol Deibel Family Endowment fund
SPARC: Atomistic Modeling of Autonomic Stimulation for a Multi‐Scale Neurocardiovascular Simulator.
Authors: DeMarco Kevin R. , Dawson John R. D. , Bekker Slava , Yarov-Yarovoy Vladimir , Clancy Colleen E. , Vorobyov Igor 
Published: April 2020

Heart rhythm and vascular tone are modulated by stimulation of the autonomic nervous system, and dysregulation of sympathetic or parasympathetic stimuli is associated with multiple cardiovascular disorders such as arrhythmias and hypertension. At the molecular level, such neuromodulation is established by stimulating myocyte G protein‐coupled receptor (GPCR) mediated signal transduction pathways with neurotransmitters like norepinephrine or acetylcholine. In this study, funded by NIH Common Fund program “Stimulating Peripheral Activity to Relieve Conditions” (SPARC), we used atomistic modeling to identify structural, energetic, and kinetic determinants of several molecular processes crucial for cardiovascular neuromodulation such as neurotransmitter interactions with GPCR / G protein complexes at the neuroeffector junction, as well as G protein interactions with adenylyl cyclase (AC), a key enzyme for downstream subcellular signaling. To determine these parameters, we developed atomistic models of human β1‐ and β2‐adrenergic receptors (β1AR and β2AR) as well as muscarinic acetylcholine receptor M2 (M2R) in different conformational states with and without bound Gs or Gi protein using available structures and Rosetta structural modeling. These models were validated via molecular docking of βAR agonists norepinephrine and isoproterenol and MR agonists acetylcholine and carbachol to compare with experimental crystal structures. Model structural stabilities were assessed via microsecond‐long all‐atom molecular dynamics (MD) simulations with and without bound ligand. Enhanced sampling atomistic MD simulations were used to estimate state‐dependent receptor ‐ ligand binding affinities and association / dissociation rates, which were compared with available experimental values. Also, coarse‐grained and Brownian dynamics implicit‐solvent techniques were utilized to assess G protein interactions with AC and GPCRs. This information will be used to inform functional kinetic models of autonomic control of myocyte subcellular signaling, a crucial component of our predictive multi‐scale neurocardiovascular simulator.Support or Funding InformationNIH SPARC 1OT2OD026580 (C.E.C., I.V.), UC Davis Pharmacology T32GM099608 (J.R.D.D.), AHA career development award 19CDA34770101 (I.V.), NIH NHLBI 5R01HL128537 (C.E.C., V.Y.Y.) and 5U01HL126273 (C.E.C., V.Y.Y.). Computer time allocations: Anton 2 PSCA18077P (I.V., C.E.C., K.R.D.), XSEDE MCB170095 (I.V., K.R.D.), NCSA Blue Waters Broadening Participation Allocation (C.E.C., I.V., K.R.D.).
Glycoprotein Aging with Increased Mannose Exposure Linked to Cardiovascular Disease through the Macrophage Mannose Receptor (Mrc1)
Authors: Restagno Damien , Pimienta Genaro , Yang Won Ho , Aziz Peter V. , Haslund-Gourley Benjamin S. , Smith Jeffrey W. , Marth Jamey D. 
Published: April 2020

N‐glycosylation is common to the vast majority of secretory proteins that are found at the cell surface or secreted. We have previously discovered that as secreted glycoproteins age in the blood circulation their glycan linkages are progressively hydrolyzed from the terminal position inward in what appears to be primarily a stepwise exo‐glycosidic process starting with neuraminidases and followed by a series of glycosidases that results in the exposure of different potential lectin ligands. We have also published that this mechanism includes cell surface glycoproteins of the intestinal tract. This intrinsic mechanism of glycoprotein aging is common to all plasma glycoproteins and appears to determine the half‐lives of glycoproteins in circulation in the limited cases where it has been studied ‐ specifically two circulating alkaline‐phosphatase enzymes. The presence of lectins that recognize further aged glycoproteins includes the mannose receptors, which would detect mannose ligands exposed following loss of sialic acid, galactose, and N‐acetylglucosamine linkages. Herein we have investigated the identities of glycoproteins modulated by mannose exposure with increasing age, and those are further regulated in their clearance and function by the endogenous macrophage mannose receptor (Mrc1). Mrc1 is found on macrophages and specialized cells in most organs as well as the vasculature and has a broad spectrum of both glycan and protein ligands due to its complex domain‐repeat structure composed of three distinct binding sites. A cysteine rich domain and a fibronectin like domain allow the binding of sulfated glycoproteins and collagens respectively. The lectin domain of Mrc1 can bind and endocytose mannosylated particles ranging from pathogens and allergens to mannosylated glycoproteins. Our studies reveal that the Mrc1 receptor has a lectin function that controls the half‐lives of specific glycoproteins in blood circulation, including those that have a regulatory role in cardiovascular and inflammatory processes.Support or Funding InformationNIH grant HL131474.
Bioinformatic Prediction of Neuropathic Pain Signaling Pathways in Rheumatoid Arthritis after high throughput miRNA analysis
Authors: Francois John Anthony , Delgado-Cruzata Lissette Anthony , Alvarez Milena Rodriguez , Almodovar Nickolas 
Published: April 2020

microRNAs (miRNAs) are a special subgroup of RNAs which work to prevent the expression of genes at the post‐transcriptional level by “silencing” them. That is, gene expression is prevented. This ultimately affects the pathways that they are a part of and the products produced by these pathways. Due to the ability of miRNA to bind to any base pair complementary to its own thus inhibiting expression of the sequence it is bound to, they are often involved in multiple cellular pathways. The relation between a specific clinical outcome and an exact pathway that a miRNA is involved is hard to establish because of the sheer number of potential pathways one miRNA could be involved in. Bioinformatic predictions are useful because they allow us to identify the most probable pathway. That is, through the careful analysis of miRNA’s targets and the prediction of the targets’ interactions in biological mechanisms. In this project, we predicted the pathways related to deregulation of miR‐223‐3p and miR‐16‐5p. Both miRNAs were correlated with neuropathy‐related clinical measures (neuropathic pain (ID Pain) and the Toronto Clinical Neuropathy Score (TCNS) respectively (Pearson Coeff.ID Pain=0.556, p=0.014) and (Pearson Coeff.TCNS= −0.5, p=0.02)). To predict possible neuropathic pain signalling pathways in people afflicted with rheumatoid arthritis, we used the miRDB database. We identified 1,485 targets and selected targets with scores of 60+ to carry out a gene ontology analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG). Conducted through DAVID Software from the Laboratory of Human Retrovirology and Immunoinformatics, KEGG identified 72 pathways as the most likely affected by deregulation of miR‐223‐3p and miR‐16‐5p. Several pathways on this list including the FoxO, neurotrophin and sphingolipid signalling pathways are related to pain and neuropathic outcomes. Future studies will have to determine whether both miRNAs affect these pathways directly and in relationship to RA.Support or Funding InformationSupport for student stipends, supplies, and/or equipment used in this research was supplied by the Program for Research Initiatives in Science and Math (PRISM) at John Jay College. PRISM is funded by the Title V program within the U.S. Department of Education; the PAESMEM program through the National Science Foundation; and New York State’s Graduate Research and Technology Initiative and NYS Education Department CSTEP program.