bluuebunny/crossref_metadata_2025 huggingface dataset:
Sub‐diaphragmatic vagal nerve stimulation alleviates rodent hypertension associated with gut dysbiosis and reduced serotonergic vagal afferent signaling
Authors: Yang Tao , Ayaz Basak Donertas , Araujo Alan , Dirr Elliott W. , Baekey David M. , Otto Kevin J. , de Lartigue Guillaume , Zubcevic Jasenka
Published: April 2020

Introduction

The gut‐brain vagal axis, of which serotonin (5HT) is an important mediator, can be altered by the gut microbiota. Low vagal tone and autonomic imbalance is present in conditions marked by gut dysbiosis including in hypertension (HTN); however, the effect of gut dysbiosis on vagal sensory/afferent function in HTN is unknown. We hypothesized that HTN‐associated gut dysbiosis will decrease serotonergic vagal afferent signaling from the gut and cause autonomic imbalance leading to increased blood pressure (BP). Moreover, we propose that restoring the vagal gut‐brain axis by chronic vagal stimulation will alleviate HTN.

Methods

Fecal matter transplant (FMT) was performed from either the spontaneously hypertensive (SHR) or Wistar‐Kyoto rats (WKY) to control WKY. BP and autonomic variables were recorded using radiotelemetry. Colonic 5HT, expression of 5HT3a receptors (5HT3aR) along the vagal gut‐brain axis, and cardiovascular responses to systemic 5HT3aR agonist injection were determined in the FMT rats, WKY and SHR. In vivo Ca2+ imaging was performed in gCAMP‐labeled nodose ganglia (NG) neurons in response to 5HT3aR agonist in WKY and SHR. Lastly, SHR were implanted with cuff electrodes on the sub‐diaphragmatic ventral vagal trunk for chronic vagal stimulation (sVNS) while measuring BP and autonomic variables using radiotelemetry. Endpoint bacterial 16s sequencing determined the effects of sVNS on SHR gut dysbiosis.

Results

FMT from SHR but not WKY significantly increased BP (P<0.05 vs baseline) and resulted in autonomic imbalance (P<0.05 vs baseline) in control WKY. This was associated with reduced colonic 5HT levels (P<0.01 vs FMT from WKY), as well as reduced 5HT3aR gene expression in vagal sensory (NG) neurons (p<0.05 vs FMT from WKY) and the brainstem region receiving vagal sensory projections (P<0.01 vs FMT from WKY). Furthermore, NG neuronal firing in response to 5HT3aR agonist corresponded with a time‐locked reduction in the HR in WKY, but both 5HT3aR agonist‐induced NG firing rates and HR responses were significantly blunted in the SHR (P<0.05). In addition, 5HT3aR agonist‐dependent HR responses were also reduced in the WKY following FMT from SHR (P<0.05 vs FMT from WKY). Crucially, chronic sVNS alleviated SHR HTN over eight weeks (P<0.05 vs SHAM SHR), without reversing gut dysbiosis, suggesting that the observed BP effect may primarily be due to restoration of the vagal afferent gut‐brain signaling.

Conclusions

HTN‐associated gut dysbiosis reduces serotonergic gut‐brain vagal signaling and increases BP. Restoring vagal afferent tone is effective in alleviating HTN in the SHR.
SPARC: The Structure and Location of Vagal Nociceptive Nerve Fibers in the Mouse Trachea
Authors: Patil Mayur , Kim Seol-Hee , Taylor-Clark Thomas E. , Kollarik Marian
Published: April 2020

Vagal afferent innervation of the airways and lungs comprises of afferent fibers from vagal nodose neurons (developmentally derived from epibranchial placodes) and vagal jugular neurons (derived from neural crest). Here we evaluated the structure and the location in tracheal wall of afferent nerve fibers innervating the trachea focusing on the nociceptive TRPV1‐positive fibers (putative C‐fibers). We used genetic neuronal tracing in several mutant mice, including two new strains we generated. The wholemount preparations of the trachea were immunostained for fluorescent proteins and imaged by spinning‐disk confocal microscopy. In the TRPV1‐Flp/Tac1‐Cre/RC:FLTG mice (N=3) in which GFP is selectively expressed in cells positive for both TRPV1 and Tac1 (in the vagal system the marker of TRPV1‐positive fibers derived from neural crest) we observed dense innervation of the trachea. In the anterolateral tracheal wall the TRPV1+Tac1+ fibers were located beneath the epithelium in the mucosa above tracheal ligaments (which connect tracheal rings) running transversally (i.e. in parallel with tracheal rings). These fibers ramified richly and repetitively. In the dorsal (membranous) tracheal wall the TRPV1+Tac1+ fibers were also located beneath the epithelium in the mucosa above tracheal smooth muscle, running craniocaudally and often branching. These fibers occasionally penetrated through the tracheal smooth muscle. Some TRPV1+Tac1+ fibers were also observed in the epithelium in the sublaryngeal region of the trachea. In the TRPV1‐Cre mice unilaterally injected with AAV9‐FLEX‐EGFP or AAV9‐FLEX‐tdTomato virus vector into vagal ganglia (N=4), in which only the vagal TRPV1‐positive fibers express GFP or tdTomato, the vagal TRPV1+ fibers had the same pattern as TRPV1+/Tac1+ fibers, but with expected lower density and patchy distribution. Similarly, in the Tac1‐Cre mice unilaterally injected with AAV9‐FLEX‐EGFP vector into vagal ganglia (N=3) in which only the vagal Tac1‐positive fibers express GFP had the same pattern. In contrast, in the TRPV1‐Flp/P2X2‐Cre/RC:FLTG mice (N=3) in which GFP is selectively expressed in cells positive for both TRPV1 and P2X2 (the marker of vagal nociceptors derived from placodes) the TRPV1+/P2X2+ fibers were almost absent in the trachea. However, in the P2X2‐Cre mice unilaterally injected with AAV9‐FLEXEGFP vector into vagal ganglia (N=2) in which GFP is selectively expressed in vagal P2X2− positive fibers, the P2X2+ fibers had distinct distribution from TRPV1+Tac1+ and often located in the tracheal smooth muscle and tracheal epithelium. Our data indicate that vagal afferent nociceptive (TRPV1+) nerve fibers (putative C‐fibers) innervating the mouse trachea originate almost exclusively from jugular ganglion neurons. In contrast, vagal nodose ganglia project only TRPV1‐fibers into the trachea. Our results are in agreement with electrophysiological studies in other species.

Support or Funding Information

NIH SPARC
Copper Modulation and Reactive Oxygen Species Production Across Yeast Lifespan
Authors: Sherlock Zachary , Bauer Sofia , Bestwick Megan L.
Published: April 2020

The mitochondrial electron transport chain (ETC) machinery is the primary site of superoxide formation, a reactive oxygen species (ROS) associated with premature cell aging. A primary defense against ROS in yeast (Saccharomyces cerevisiae) is the enzyme superoxide dismutase (Sod1p). This enzyme requires copper and zinc as metal cofactors in the conversion of superoxide to hydrogen peroxide, a less harmful ROS. How mitochondrial function contributes to yeast chronological lifespan (CLS) is particularly of interest, specifically how essential metals for mitochondrial function and their role in the production of ROS drive the aging process of yeast cells. Our aim is to determine the effect exogenous copper treatment on the CLS of yeast strains lacking Sod1p and its copper chaperone, Lys7p. Our results indicate that low levels of exogenous copper (0.25 mM copper sulfate or less in restricted nutrient media) extend yeast chronological lifespan, especially in cells lacking Lys7p. All cell types see a dramatic reduction in lifespan when exogenous copper levels are increased to 2.0 mM copper sulfate or higher. This indicates that a small increase in the amount of copper in the media is beneficial for yeast in these restricted media conditions. Our current studies are to assess protein levels by western blot and gene expression by qRT‐PCR of various components the yeast ETC machinery, specifically Cox2 and Cox4, to mechanistically understand how exogenous copper in a small dose is contributing to lifespan extension. ROS levels are also being assessed via fluorescent staining. By using biochemical dyes such as Dihydroethidium (DHE) for cytosolic ROS, and MitoSox and mitochondria superoxide production, we are able to monitor ROS production and show localization of these metabolic byproducts with both fluorescent microscopy and FACS at various points of the chronological lifespan yeast.
Contribution of Mitochondrial Reactive Oxygen Species to Chronic Hypoxia‐Induced Pulmonary Hypertension
Authors: Yan Simin , Walker Benjimen R. , Jernigan Nikki L. , Resta Thomas C.
Published: April 2020

Preliminary data from our laboratory support a major role for mitochondria‐derived reactive oxygen species (mitoROS) in mediating elevated basal pulmonary arterial tone following chronic hypoxia (CH) in rats. However, neither the effect of CH on mitoROS production in pulmonary arterial smooth muscle cells (PASMCs), nor the contribution of mitoROS to pulmonary hypertension (pHTN) in this setting are understood. We hypothesized that CH increases PASMC mitoROS production, and that mitoROS contribute to the progression of CH‐induced pHTN. To test these hypotheses, we measured mitoROS production by fluorescence detection of the mitochondrial O2− indicator MitoSOX in primary cultures of PASMCs from control and CH (4 wk, 0.5 atm) rats. Experiments were conducted in the presence of the mitochondria‐targeted antioxidants MitoQ, MitoTEMPO or vehicle. We additionally measured indices of pHTN in rats treated with the mitochondria‐targeted antioxidant MitoQ (500 μM) or vehicle in their drinking water for the entire 4 wk duration of normoxic or hypoxic exposure. Groups were compared statistically by two‐way ANOVA. Values of n indicate numbers of animals. We found that MitoSOX fluorescence was significantly (p < 0.05) greater in PASMCs from CH rats compared to normoxic controls, and this response was prevented by MitoQ and MitoTEMPO (n=6–7). Furthermore, MitoQ attenuated CH‐induced increases in right ventricular (RV) systolic pressure in isoflurane‐anesthetized rats, as well as RV hypertrophy as assessed by Fulton’s index (n=4, p < 0.05). We conclude that CH increases mitoROS production in PASMCs, and that mitoROS provide an important contribution to the development of pHTN following CH.

Support or Funding Information

This work was supported by NIH grant R01 HL132883 (to T.C. Resta).
SREBPs in the control of intestinal homeostasis and tumorigenesis
Authors: Engelking Luke , Xu Yanchao
Published: April 2020

Lipids are key regulators of the growth and differentiation of intestinal epithelial cells, including intestinal stem cells (ISC). High‐fat diets and obesity are serious risk factors for gastrointestinal cancers in humans. Sterol regulatory element‐binding proteins (SREBPs) are transcription factors that control lipid synthesis and uptake in the intestine. Our recent studies have shown that SREBPs and their major products, sterols (from SREBP‐2) and unsaturated fatty acids (from SREBP‐1) have a critical role in sustaining growth of the intestinal epithelia: (1) The loss of Scap, which controls intracellular cleavage events required for SREBP activation, reduces lipid synthesis and causes a lethal intestinal injury in mice characterized by a loss of intestinal crypts, which house ISCs. (2) When SREBP‐2 is inactivated in intestinal epithelium, hyperplasia of intestinal crypts and hypertrophy of the intestine results. In SREBP‐2 knockouts, sterol synthesis falls while unsaturated fatty acid synthesis does not, owing to an increase in SREBP‐1. (3) Despite this overgrowth phenotype, ablation of SREBP‐2 in intestinal epithelia of mice blocks the formation of adenomas caused by mutant APC. These findings indicate that SREBPs govern intestinal epithelial homeostasis in complex ways. We propose that SREBPs maintain homeostasis of intestinal epithelia by producing specific, yet‐to‐be identified lipid metabolites that sustain the proliferation of ISCs. These pathways control intestinal organ size and malignant potential, which bears on human diseases of under‐ or over‐proliferation of the intestine, such as short gut syndrome and colorectal cancer, the 3rd most common cause of cancer death in the U.S.

Support or Funding Information

NIH‐R01DK121842, NIH‐K08DK102652
Characterization of the Tyrosyl‐S and Tyrosyl‐Z tRNA Synthetases from Pseudomonas aeruginosa and Discovery of Inhibitors
Authors: Bullard James M. , Hughes Casey A. , Escamilla Yaritza
Published: April 2020

Introduction

Pseudomonas aeruginosa, a Gram‐negative opportunistic pathogen, is a leading cause of nosocomial infections. The ability of this bacteria to form resilient biofilms on implanted medical devices results in high mortality rates. Patients with cystic fibrosis (CF) are especially at risk as P. aeruginosa colonizes the lungs of these patients forming biofilms which leads to chronic infections and is a leading cause of death. Aminoacyl‐tRNA synthetases (aaRSs) catalyze the covalent attachment of amino acids to their cognate tRNAs. These enzymes are essential for protein synthesis in bacteria and serve as validated targets for the development of new anti‐infectives. Unlike in other bacteria, the P. aeruginosa genome contains two genes (tyrS and tyrZ) which encode two distinct TyrRS enzymes, which are very different at the amino acid level containing only 27% conserved amino acid residues.

Methods and Results

The kinetic parameters for the interaction of these enzymes with their three substrates (tRNA, ATP, tyrosine) were determined using timed aminoacylation and ATP:PPi exchange assays. The KM for interaction with tRNA, ATP, and tyrosine were 1.5, 204, 172 μM and 2.1, 496, 29 μM for TyrRS‐S and TyrRS‐Z, respectively. The kcat values for interaction with tRNA, ATP, tyrosine were 0.19, 1.0, 3.8 s−1 and 1.9, 3.8, 3.1 s−1 for TyrRS‐S and TyrRS‐Z, respectively. Both forms of TyrRS were developed into screening platforms using scintillation proximity assay (SPA) technology. Using this assay, a synthetic compound library from ChemDiv, Inc. containing over 2000 distinct compounds was screened to detect compounds with the ability to inhibit the function of TyrRS. Four compounds (BCD37H06, BCD38C11, BCD49D09, and BCD54B04) were confirmed as hit compounds. All four compounds inhibited the activity of TyrRS‐S and one of the compounds inhibited TyrRS‐Z. The compounds exhibited IC50 values in the low μM range. These compounds were found to have moderate broad spectrum inhibitory activity (MICs) against a panel of ten pathogenic bacteria. The compounds were all shown to be bacteriostatic in time‐kill kinetic assays. In competition assays, none of the compounds competed with tyrosine for binding, however BCD38C11 and BCE49D09 were competitive with ATP for binding. None of the compounds inhibited growth of human cell cultures.

Conclusion

TyrRS‐S and TyrRS‐Z from P. aeruginosa were characterized and used for development of screening platforms to identify compounds that have the potential for development as antibacterial agents against drug resistant pathogenic organisms.

Support or Funding Information

The authors are grateful for the financial support provided by the National Institutes of Health (grant number: 1SC3GM098173).
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 Information

WesternU 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 Information

This 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 Information

Supported 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 Information

FAPESP: 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 Information

This 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 Information

Research 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 Information

NIH 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 Information

NIH 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 Information

Support 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.
Adiposity of the B6 Mouse Lung
Authors: Meyerholz David K. , Beck Amanda P.
Published: April 2020

In biomedical research, mice are studied using a variety of techniques that require a baseline knowledge of microscopic anatomy, these include histochemistry, immunohistochemistry and immunofluorescence. Many biomedical personnel lack this background in their training. The lung is primarily composed of two zones: the conducting zone (airways) and the respiratory zone (alveoli). Morphologic differentials for alveolar structures are few in the lung, but in our experience adipose tissues in the lung can be a source of confusion and misinterpretation. In this study, we examined lungs from adult B6 mice (>3 months, n=16, female) that were obtained from de‐identified archival control tissues (UI Comparative Pathology Laboratory) to define lung adiposity. White adipose tissues (WAT) were detected in all lung samples (100%, 16/16). WAT was often seen as small aggregates of 5–15+ adipocytes embedded in loose connective tissue adjacent to large airways/vessels. In sites of smaller airway branching, WAT was rarely detected and usually only as a few solitary adipocytes. WAT adipocytes were distinguished from alveolar structures by their location, significantly reduced cellularity, and residence often in a collagenous matrix. Brown adipose tissues (BAT) were not observed in this survey. We have characterized the localization and appearance of adipose tissue in the lungs of female B6 mice. Awareness that WAT is present in lung tissues along with knowledge of its distinguishing features versus alveoli can help investigators make more clear and accurate interpretations of the lung data.

Support or Funding Information

NIH
Cosolvent Molecular Dynamics Simulation‐Based Discovery of Potential Allosteric Sites on Regulator of G Protein Signaling 4
Authors: Chan Wallace , Carlson Heather , Traynor John
Published: April 2020

Regulator of G Protein Signaling 4 (RGS4) is member of a family of intracellular proteins that are characterized by the presence of a 125 amino‐acid RGS homology domain (RH) which binds to the Ga subunit of heterotrimeric G proteins. By doing so RGS4 acts as a GTPase‐accelerating protein or GAP to expedite the conversion of the GTP‐bound active form of Gα subunit of heterotrimeric G proteins to the GDP‐bound inactive form, thereby terminating signaling downstream of G protein coupled receptors (GPCRs). RGS4 is expressed widely in both the periphery and central nervous system and has been implicated in pain, schizophrenia, and the control of cardiac contractility. Inhibitors of RGS4 have been developed, but these all bind covalently to a cysteine (Cys95) in the RH domain. Therefore, we sought to identify alternative druggable sites on this protein using cosolvent molecular dynamics simulations. This technique employs a low concentration of organic probes (pyrimidine, acetonitrile, isopropyl alcohol) to find druggable “hot spots” on a protein based on the binding preference of the probes. This methodology has the advantage of utilizing protein dynamics to find druggable pockets that would be otherwise inaccessible with a single snapshot crystal structure. The top predicted sites were located in all areas of the RH domain of RGS4. Subsequently, statistical coupling analysis, a technique that identifies co‐evolving residues from analysis of multiple sequence alignments, was used to map out a functionally‐significant sector on the surface of RGS4 which overlapped with the identified hot spots, allowing for prioritization. The first site identified was expectedly the Gα subunit binding site. The second was located in a cleft in the proximity of lysine residues thought to be the binding site for phosphatidylinositol (3,4,5)‐trisphosphate (PIP3) binding. Since PIP3 is a known inhibitor of the GAP activity of RGS4, future directions entail the investigation of PIP3‐bound conformations of RGS4 by molecular dynamics simulation to better understand the mechanism of deactivation. Of other predicted sites, one is in close proximity to the purported allosteric site that contains Cys95, while the others are putative novel sites for allosteric control of RGS4.

Support or Funding Information

Supported by R01 DA‐035316.
Attenuation of Scopolamine‐Induced Learning and Memory Impairment by Ceriporia lacerata Mycelial Culture in C57BL/6 Mouse Model
Authors: Lee Sujin , Yun Hyun Seok , Oh Jisun , Kim Jong-Sang
Published: April 2020

Ceriporia lacerata (provided from FUGENBIO Co., Ltd., Seoul, S. Korea) is one of white‐rot fungi. The submerged culture and its extract of the fungal mycelia have been reported to contain various bioactive substances, including extracellular polysaccharide and β‐glucan, and to exert the anti‐diabetic effect in mice. Our in vitro study showed that the ethanol extract of C. lacerata mycelial culture powder protected HT22 mouse hippocampal neuronal cells from glutamate‐induced cytotoxicity. The present study aimed at examining whether oral supplementation of the cultured C. lacerata mycelia assists the restoration of learning and memory capability diminished by scopolamine injection in C57BL/6 mice. Results exhibited that the treatment with C. lacerata mycelial culture powder (□) improved spatial learning and memory impairment in scopolamine‐injected mice as tested by behavioral tests (Y‐maze task, Passive avoidance task, and Morris water maze task), (□) reduced histological damage in the hippocampal CA1 region, (□) increased the expression level of cytoplasmic heme oxygenase‐1 (HO‐1) in hippocampal tissue homogenates, and (□) decreased the levels of oxidative stress markers in the plasma, in comparison to mice treated with scopolamine alone. These findings suggest that oral supplementation of C. lacerata mycelial culture powder could prevent scopolamine‐induced learning and memory deficit possibly by protecting hippocampal damage from oxidative stress.

Support or Funding Information

This work was supported by the “Food Functionality Evaluation Program” with the Ministry of Agriculture, Food, and Rural Affairs (2019).
Using NMR Derived Structural Information Complemented by Molecular Docking and MM/GBSA Rescoring to Mitigate CYP3A4 Time‐Dependent Inhibition
Authors: Wilderman Ross , Halpert James
Published: April 2020

Time‐dependent inhibition (TDI) of human CYP3A4 is a significant challenge in drug development and is the cause of multiple clinically relevant drug‐drug interactions. The vasodilator diltiazem causes TDI when metabolized by CYP3A4. We hypothesized that introduction of steric bulk into the CYP3A4 active site by site‐directed mutagenesis will disrupt active site contacts, alter diltiazem orientation in the CYP3A4 active site, and decrease TDI, as previously seen with midazolam. CYP3A4‐ligand interactions were monitored using spectral binding titrations. In parallel, we performed molecular docking of diltiazem and the N‐demethylation products into CYP3A4 models of previously tested mutants that introduce steric bulk into the enzyme active site, followed by molecular mechanics generalized Born surface area (MM‐GBSA) rescoring. An NMR‐derived model of diltiazem in the CYP3A4 active site revealed that the dimethylamino group that undergoes CYP3A4‐mediated N‐demethylation is oriented toward the heme iron. Diltiazem caused a Type II spin shift upon binding to CYP3A4, indicating interaction of the amine with the heme iron. Docking of diltiazem, N‐desmethyldiltiazem, or N, N‐didesmethyldiltiazem into wild‐type CYP3A4 resulted in the amino group of each compound oriented toward the heme iron. In contrast, docking these three compounds into CYP3A4 I120W resulted in the amino group orienting away from the heme. Furthermore, T309Y docking results showed reorientation of diltiazem, which would prevent the initial N‐demethylation event, and I301W and A370F resulted in N‐desmethyldiltiazem reorientation. MM‐GBSA rescoring of docking experiments confirmed that all compounds used orient with the amino group toward the heme in wild‐type CYP3A4, all amino groups away from the heme in I120W, and reorientation of N‐desmethyldiltiazem in I301W and A370F. Furthermore, in rescoring experiments, N‐desmethyldiltiazem was reoriented in the Y307W active site, and N, N‐didesmethyldiltiazem was reoriented in the T309Y active site in addition to reorientation of diltiazem. Substitution of I120 and T309 with a larger residue decreased CYP3A4 TDI. In summary, a hybrid NMR‐computational approach was used to rapidly generate a model of diltiazem in complex with CYP3A4. These results indicate that NMR‐derived models of protein‐ligand interactions provide a structure‐based approach for decreasing metabolic liabilities of CYP enzyme substrates.

Support or Funding Information

This work was supported by internal funding from the University of Connecticut to James Halpert.