bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mice Expressing Fluorescent Protease‐Activated Receptor‐2 as a Novel Tool to Visualize Intracellular Receptor Trafficking
Authors: Teng Shavonne , Latorre Rocco , Jensen Dane , Schmidt Brian L. , Bunnett Nigel 
Published: April 2020

Protease‐activated receptor‐2 (PAR2) is a G‐protein coupled receptor (GPCR) that plays a critical role in mediating the proinflammatory and pronociceptive actions of serine and cysteine proteases in many tissues. In common with a growing number of GPCRs, PAR2 can signal from endosomes as well as the plasma membrane, and sustained endosomal signaling underlies disease‐relevant processes. Our knowledge of the role of PAR2 in health and disease states is hampered by an inadequate understanding of its cellular and subcellular localization. Since antibodies to GPCRs often lack sensitivity and specificity, and are unsuitable for studying receptor trafficking in living cells, we used a knockin approach to generate mice expressing PAR2 fused to monomeric ultrastable GFP (muGFP). To investigate whether mouse PAR2 fused at the C‐terminus to muGFP was functional, we compared agonist‐evoked signaling and trafficking of PAR2‐muGFP and PAR2 with a C‐terminal HA11 epitope expressed in KNRK cells. Trypsin and the PAR2 selective peptide agonist 2‐Furoyl‐LIGRLO‐NH2 similarly stimulated calcium signals and PAR2 endocytosis. Thus, muGFP does not disrupt PAR2 signaling and trafficking. We then generated knockin mice expressing mouse PAR2 fused at the C‐terminus to muGFP. RT‐PCR revealed similar levels of expression of PAR2 in PAR2‐muGFP and wild‐type mice. Western blotting of colonic extracts using a GFP antibody revealed expression of PAR2‐muGFP at the protein level. We localized PAR2‐muGFP by immunofluorescence and confocal microscopy using the GFP antibody. PAR2‐muGFP was highly expressed in epithelial cells in the digestive tract and skin, and was detected at lower levels in the enteric nervous system and pancreatic islets. In the unstimulated state, PAR2‐muGFP was principally localized to the plasma membrane of colonocytes. Exposure to trypsin or 2‐Furoyl‐LIGRLO‐NH2 induced redistribution of PAR2‐muGFP from the plasma membrane to endosomes. PAR2‐muGFP mice provide a valuable tool to study the localization, regulation, and trafficking of PAR2 in intact animals in healthy and diseased conditions.Support or Funding InformationNIH
Valproic Acid Alters Activities of Key Enzymes of Glucose Oxidative Metabolism, Citric Acid Cycle and Neurotransmitter Synthesis in Embryonic Rat Brain
Authors: Triyasakorn Korawin , Lai James C.K. , Awale Prabha 
Published: April 2020

Although studies have investigated the role of gamma‐aminobutyric acid (GABA)ergic signaling in rodent neural development and behaviors relevant to autism, the energetics that underlie neural developmental changes are poorly understood. Here, we employed a valproic acid (VPA) rat model of autism to investigate the effects of VPA treatment on key enzymes of glucose oxidative metabolism and citric acid cycle (namely lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and glutamate dehydrogenase (GDH)) in rat brain. Pregnant Wistar rats were administered 600 mg/kg of VPA by intraperitoneal injection on day 12.5 of gestation and embryonic (E) day 15 and E17 rat brains were assayed for the activities of the enzymes. Both E15 and E17 male and female rat models of VPA‐induced autism showed significantly changed whole brain activities of the enzymes studied compared to the corresponding values in control rats. The dysregulated GDH activity could alter the excitatory‐inhibitory balance in the brain of embryonic rats during development. Thus, our findings may have pathophysiological/pathogenic implications in autism and may ultimately lead to the discovery of new therapeutic target(s) for autism.Support or Funding InformationSeed Grant Idaho State University College of Pharmacy
Respiratory Dysfunction in a Mouse Model of Spinocerebellar Ataxia 7
Authors: Fusco Anna F. , Pucci Logan , McCall Angela L. , Dhindsa Justin , Kahn Amanda , Switonski Pawel , Strickland Laura , Spada Albert La , ElMallah Mai K. 
Published: April 2020

Spinocerebellar ataxia type 7 (SCA‐7) is a neurodegenerative polyglutamine disease within the family of spinocerebellar ataxias. Classically SCA7 is a disease of ataxia and vision loss. However, patients with SCA also have difficulty breathing, swallowing, coughing and maintaining a stable open airway which often result in fatal respiratory failure. While respiratory measures have been reported in other SCA mouse models, ventilation in a SCA‐7 mouse has not yet been studied. Using the SCA‐7 mouse model (Atxn7266Q/+) we sought to characterize the respiratory pathology of SCA‐7.Whole body plethysmography (WBP) was used to assess respiratory dysfunction in the Atxn7266Q/+ mouse both at baseline breathing (FiO2: 0.21; nitrogen balance) and during a hypercapnic and hypoxic challenge (Fi:CO2: 0.07, FiO2: 0.10; nitrogen balance). Neurobehavioral testing and strength was assessed using the inverted screen test and the grip strength test. Post mortem histological analysis was performed on the phrenic and hypoglossal motor neurons and nerves.At 4 weeks of age, the SCA‐7 mice are indistinguishable from their unaffected littermates. However, by 8 weeks of age, SCA‐7 mice perform poorly on both neurobehavioral and strength tests as compared to their WT littermates. 50% of the SCA‐7 mice died by 9 weeks, while littermate controls thrived. SCA‐7 mice have elevated respiratory rate at baseline, as well as reduced tidal volume, minute ventilation, peak inspiratory flow, peak expiratory flow, and inspiratory time during the respiratory challenge. These results indicate that the SCA‐7 mice have respiratory dysfunction. To understand the respiratory deficits, ongoing studies are histologically analyzing the diaphragm neuromuscular junction, phrenic and hypoglossal nerves, as well as phrenic and hypoglossal motor nuclei. Preliminary nerve analysis of the hypoglossal and phrenic nerves reveals significant axonal pathology. In conclusion, WBP analysis and preliminary histological nerve data reveals respiratory dysfunction in the Atxn7266Q/+ mouse model.Support or Funding Information
Funding: R01 HD099486‐01; 1R21NS098131‐01
Interrogating the Activity of Environmental Contaminants Against the Druggable GPCR‐ome
Authors: Wilkinson Joshua , Lehmler Hans-Joachim , Roman David 
Published: April 2020

Exposure to environmental contaminants can result in a myriad of conditions including developmental disorders, pulmonary conditions, and neurological pathologies. Literature precedent and new, emerging data, demonstrates specific GPCRs can be activated by some classes of environmental contaminants, including PCBs, pesticides, chloropropanols, and others. When interrogating GPCR’s with small‐molecule based screens, the traditional paradigm is to test hundreds of thousands of compounds against a single target. In recent literature, an open‐source method “PRESTO‐TANGO” (Parallel Receptor‐ome Expression and Screening via Transcriptional Output – TANGO) has been described as a feasible approach to simultaneously screen the entire druggable human GPCR‐ome against a smaller collection of compounds via a G protein‐independent β‐arrestin recruitment assay. In this work, we have adopted a similar approach to the PRESTO‐TANGO methodology, enabling us to screen activity of a number of environmental contaminates against the known druggable GPCR‐ome of 314 GPCRs. From our initial screen, we identified several potential ligand‐receptor interactions. Secondary dose response experiments uncovered what we believe to be a novel ligand‐receptor relationship between PCBs and the Sphingosine‐1‐phoshpate receptor (S1PR) family. Experiments to validate the relationship between the PCBs and S1PR family, as well as investigate the relevant signaling cascades impacted are currently underway. Ultimately, this work utilizes a modified form of the PRESTO‐TANGO assay as a novel screening platform to elucidate bioactivity of environmental contaminants.Support or Funding InformationThis work was supported by the University of Iowa Environmental Health Science Research Center (NIEHS P30 ES05605 D.L.R)
Inflammation and the Cardiovascular Responses to Exercise in Patients with Peripheral Artery Disease
Authors: Craig Jesse C. , Hart Corey R. , Layec Gwenael C. , Kwon Oh Sung C. , Richardson Russell S. , Trinity Joel D. 
Published: April 2020

BackgroundPatients with peripheral artery disease (PAD) demonstrate an exaggerated exercise pressor response (EPR). Heightened inflammation critically contributes to this exaggerated EPR in animal models of PAD, however, this relationship remains largely unexplored in humans. Therefore, we tested the hypotheses that the blood pressure response to exercise would be exaggerated in patients with PAD compared to controls; and that markers of inflammation would be positively related to this response.MethodsEight patients with PAD (ABI: 0.67±0.11) and eight healthy controls (HC; ABI: 1.16±0.07) matched for age, sex, physical activity, and physical characteristics completed plantar flexion exercise at both absolute (2.7 W) and relative (40% max; PAD: 2.1±0.2 vs HC: 4.3±0.4 W, p<0.001) intensities. Popliteal artery blood flow (Flow) was measured via Doppler ultrasound and systemic mean arterial pressure (MAP) via Finapres. Resting systemic and intramuscular markers of inflammation and oxidative stress were quantified from plasma and muscle biopsies.ResultsThe exercise‐induced ΔMAP was greater for PAD at both the absolute (PAD: 20±3 vs HC: 9±2 mmHg, p<0.01) and relative (PAD: 23±5 vs HC: 11±2 mmHg, p=0.02) exercise intensities. The ΔFlow was lower for PAD compared to HC during both absolute (PAD: 251±53 vs HC: 545±66 ml/min, p<0.01) and relative intensities (PAD: 242±61 vs HC: 595±89 ml/min, p<0.01). Inflammatory markers including plasma tumor necrosis factor alpha (TNFα: ↑134%), interleukin‐6 (IL6: ↑92%), and intramuscular IL6 (↑77%) were elevated in PAD compared to HC (p<0.05). Additionally, markers of oxidative stress including intramuscular protein carbonyls (PC: ↑57%) and 4‐hydroxynonenal (HNE: ↑80%) were elevated in PAD (p<0.05). TNFα, plasma IL6, and PC were positively correlated with ΔMAP (r: 0.57–0.82, all p<0.05) while plasma and intramuscular IL6, PC, and HNE were negatively correlated with ΔFlow (r: 0.64–0.78, all p<0.05).ConclusionsThe present findings indicate an association between markers of inflammation with the cardiovascular responses to exercise in humans with PAD. Specifically, heightened inflammation and oxidative stress appear to contribute to the exaggeration the exercise pressor reflex and attenuated blood flow response during lower limb exercise in PAD. Therapeutically, targeting systemic and local inflammation may be a viable option to improve the blood pressure and blood flow responses to exercise in patients with PAD.
HIV‐1 Matrix Protein Interactions with Cellular tRNAs
Authors: Ejimogu Jason , Parker Connor , Bauerle Mahlet , Palm Cheyenne , Brown Janae Baptiste , Summers Michael 
Published: April 2020

The human immunodeficiency virus (HIV) is a retrovirus that infects roughly 40 million people globally that attacks CD4+ T‐cells, thus impairing the immune system. Highly active antiretroviral treatment against the virus is available; however, the viral genome frequently mutates, which gives rise to evasion of current therapies and highlights the need for new drugs. Better understanding molecular mechanisms of viral replication may lend to this effort. One process necessary for HIV replication is membrane trafficking of the Gag polyprotein (Gag) to the plasma membrane via Gag’s N‐terminal matrix domain (MA). MA mediates membrane targeting by means of electrostatic interaction and anchoring to the plasma membrane using a covalently linked myristoyl moiety. Recent virology studies demonstrated that MA interacts with cellular tRNAs, including tRNALys3 and tRNAGlyGCC, before binding to the plasma membrane, but the role of tRNA on assembly is poorly understood. The long‐term goal of this work is to study the MA:tRNA complex and elucidate its 3D structure as characterization of this interaction may shed light on the function of tRNA during membrane targeting. To achieve this goal, conditions to stabilize MA and the tRNA independently and in complex must be established. Nuclear magnetic resonance (NMR) and gel electrophoresis were employed to identify conditions that stabilized the tRNA and MA independently. Electrophoretic mobility shift assays (EMSAs) were carried out to probe MA:tRNA interactions under these conditions and showed complex formation upon addition of a stoichiometric ratio of MA to tRNA. This work lays a foundation to study thermodynamics of the MA‐tRNA interaction and determine the three‐dimensional structure of the complex.Support or Funding InformationThis research was sponsored by NIH/NIGMS MARC U*STAR T34 HHS 00026 National Research Service Award to UMBC, HHMI, and NIH grants R01GM042561 and 8R01AI50498.
A Horseshoe Kidney with Convoluted Vasculature: Gross and Histological Anatomy
Authors: Cook Ryan L. , Herrold Christina B. , Auten Matthew W. , Grise Joy , Santos Rosaysela , Zdilla Matthew J. , Lambert H. Wayne 
Published: April 2020

This report documents the incidental finding of a horseshoe kidney identified during routine dissection in a gross anatomy course. The anatomic examination demonstrated malrotated bilateral kidneys, which were fused at the inferior poles. Histologic examination revealed no significant pathologic change; however, the fused portion was shown to have an intact cortex and collecting system. Furthermore, the renal vasculature was anomalous with multiple accessory and convoluted arterial vessels. An aortic aneurysm was also noted just superior to the renal fusion. Operative procedures and diagnostic imaging performed in the region of such an anatomical variation may be complicated by unanticipated vascular patterns. Therefore, special attention should be paid to vasculature in the setting of a horseshoe kidney.Support or Funding InformationT. Walley Williams Summer Anatomy Research Fellowship;WV Research Challenge Fund [HEPC.dsr.17.06]
Crystal structures and Directed Evolution of the α‐L rhamnosidase RHA‐P from Novosphingobium sp. PP1Y
Authors: Terry Brendan , Driza Danicah Vinh , Izzo Vivianna , Sazinsky Matthew Howard 
Published: April 2020

Flavonoid natural products are well‐known for their beneficial antimicrobial, antitumor, and anti‐inflammatory properties. However, some of these natural products often are rhamnosylated, which severely limits their bioavailability. The lack of endogenous rhamnosidases in the human GI tract not only prevents many of these glycosylated compounds from being of value in functional foods but also limits the modification of natural product libraries being tested for drug discovery. RHA‐P is a catalytically efficient, thermostable α‐L‐rhamnosidase from the marine bacterium Novosphingobium sp. PP1Y that selectively hydrolyzes α‐1,6 and α‐1,2 glycosidic linkages between a terminal rhamnose and a flavonoid moiety. Due to its promiscuity, relative thermostability compared to other known rhamnosidases, and catalytic efficiency even in significant concentrations of organic solvents, RHA‐P shows potential for biocatalytic applications. In a recent work, we reported the 2.2. Å resolution crystal structure of RHA‐P and, in part through in silico docking experiments, offered insight into the substrate preference of RHA‐P for glycosylated aryl compounds such as hesperidin, naringin, rutin, and quercitrin. This work reports the 3.0 Å resolution crystal structure of a E506Q RHA‐P mutant soaked with naringin and applies the structural data to‐date to the semi‐rational directed evolution of highly thermostable mutants of RHA‐P with retained activity on flavonoid glycosides.Support or Funding InformationThis research was supported by FARB 2014 ORSA 132082 (VI), the Beckman Scholars program (BT), and the Pomona College Chemistry Department (BT and DD). Use of the Stanford Synchroton Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE‐AC02‐76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393).
Long non‐coding RNAs regulate NF‐κB activation, inflammation, and glucose metabolism
Authors: Mandal Subhrangsu S. , Obaid Monira , Chini Avisankar 
Published: April 2020

Inflammation plays a central role in immune response and the inflammatory response is closely linked with metabolism. Our recent study demonstrates that lncRNA HOTAIR plays key roles in NF‐kB activation, cytokine expression, and inflammation. Here, we investigated if HOTAIR plays any role in regulation of glucose metabolism in immune cells during inflammation. Our study demonstrate that lncRNA HOTAIR regulates the expression of glucose transporter isoform 1 (Glut1) which controls the glucose uptake into macrophages. SiRNA‐mediated knockdown of HOTAIR suppressed the LPS‐induced expression of Glut1 and glucose update in macrophages suggesting key roles of HOTAIR in LPS‐induced Glut1 expression and glucose metabolism. HOTAIR induces NF‐kB activation which in turn increases Glut1 expression and glucose metabolism in response to LPS. Beyond HOTAIR, we also discovered several novel lncRNAs (termed as LinfRNA:
Long‐noncoding
inflammation associated
RNA) which are closely associated with inflammation and metabolism. Overall, our study demonstrated that HOTAIR and LinfRNAs play key roles in NF‐kB activation and inflammation and regulate metabolic programming in immune cells potentially to meet the energy needs at the priming of immune response to pathogenic infection or other stressors.Support or Funding InformationNational Institute of Health grant # 1R15 ES019129‐01
Impaired Bmp signaling in Foxl1+ telocyte influence the maintenance of the intestinal stem cell niche
Authors: Perreault Nathalie , Servant Raphaelle , Pomerleau Veronique , Jones Christine , Boudreau Francois 
Published: April 2020

New components of the intestinal stem cell niche are constantly being discovered. Recently, the subepithelial Foxl1+telocytes (TCFoxl1+) were found to play a key role in maintaining and regulating intestinal crypt homeostasis. TCFoxl1+ produce crucial niche components such as WNT2b, WNT5a and RSPO3 as well as the Bone Morphogenic Proteins (BMP) inhibitors Gremlin 1 and 2. Previous work in our laboratory showed that epithelial BMP signaling is involved in terminal differentiation and maturation of cells from the secretory lineage. Our recent work showed that the loss of mesenchymal BMP signaling leads to the development of a pathological microenvironment inducing colonic and gastric polyposis in older mice, drawing attention to the impact of a pathogenic microenvironment in polyposis.By genetic means, we propose to address how BMP signaling in TCFoxl1+ modulates the intestinal stem cell niche. Using the Cre/loxP system, we generated mice with a deletion of BmpR1a in all gastrointestinal subepithelial TCFoxl1+. Histological analysis and terminal differentiation assessment were performed with cellular staining and immunofluorescence (H&E, Alcian Blue and Lysozyme). Niche components were analysed by RT‐qPCR from crypt epithelium or total ileal RNA extracts. H&E staining on 9 months‐old BmpR1aΔFoxl1+ and control mice ileum demonstrated perturbed architecture in the BmpR1aΔFoxl1+ such as fused and enlarged villi compared to control mice. Alcian Blue staining showed an increase of goblet cells in the mutant mice. Electron microscopy showed that Paneth cells from mutant mice have larger vesicles than controls. The RT‐qPCR analysis showed a decrease in WNT signaling targets in the crypts epithelium cells. Indeed, LGR5 is decreased by 50% and mTERT by 34% in the mutant mice, whereas the stem cell marker BMI1 is not modulated. Maturity of Paneth cells is impaired in the mutant mice as shown by a 41% decrease in SOX9 expression. This lack in Paneth cell maturity is also reflected by a 60% decrease in WNT3 expression in BmpR1aΔFoxl1+ mice, an important stem cell niche factor. Secretory cell determination transcription factor ATOH1 is decreased by 35% in the mutant mice. Analysis from total ileal extract revealed a decrease of WNT2b, a key niche factor from the mesenchymal cells.Thus, our results show that altering Bmp signaling in subepithelial TCFoxl1+ modulates crucial niche factors influencing intestinal stem cells homeostasis and their subsequent determination.Support or Funding InformationCanadian Institutes of Health Research‐Institute of Nutrition, Metabolism and Diabetes
Ezh2 loss is a haploinsufficient mediator of cell lineage in prostate cancer and has divergent effect on androgen receptor expression
Authors: Wadosky Kristine M. , Wang Yanqing , Goodrich David 
Published: April 2020

Patients with advanced, metastatic, and/or treatment‐resistant prostate cancer (PCa) can develop aggressive tumor variants with neuroendocrine (NE) features. These PCa variants are found in 20–30% of patient autopsies relapsing from androgen deprivation therapy (ADT), can lack androgen receptor (AR), and have altered histology and clinical course. A recent comprehensive study of patients with advanced PCa shows that alteration in tumor suppressor gene RB1 is the strongest genomic predictor of survival. We have previously shown that loss of Rb1 promotes NE lineage transformation in genetically engineered mouse models (GEMMs) of PCa. Rb1 deficiency also induced pervasive changes in gene expression characteristic of NE variants in human PCa. These changes included increased expression of histone methyltransferase Ezh2. These data led us to hypothesize that Ezh2 is a driver of NEPC transformation in Rb1‐deficient PCa. To test this hypothesis, we developed new GEMMs of Rb1‐deficient PCa lacking Ezh2. Data show that overall survival of PBCre4:Ptenf/f:Rb1f/f:Ezh2f/+ (DKOE/+) mice with loss of one Ezh2 allele is significantly increased compared to PBCre4:Ptenf/f:Rb1f/f (DKO) (42 weeks vs. 38 weeks). To our surprise, overall survival of PBCre4:Ptenf/f:Rb1f/f:Ezh2f/f (DKOE/E) was decreased compared to DKO (33 weeks vs. 38 weeks). Histological analysis and expression of endstage primary tumors from DKOE/E and DKOE/+ diverge from the original DKO model in regards to AR expression. While DKO tumors are ARlow‐NEpos, DKOE/E tumors are ARneg‐NEhigh and DKOE/+ tumors are ARhigh‐NEhigh, which has been confirmed by RNA sequencing. Interestingly, ARhigh‐NEhigh tumors in DKOE/+ is similar to the “amphicrine” histological PCa variant that has recently been reported in patients. Together, these data suggest that Ezh2’s effect on PCa progression in the absence of Rb1 is haploinsufficient. Future work is required to investigate of the exact mechanism of Ezh2’s contribution to NE lineage, AR expression, and AR activity in advanced PCa.Support or Funding InformationThis work was supported by the National Cancer Institute funding sources R01CA207757, R01CA207757‐03S1, R01CA234162 to D.G. and 3P30CA016056 to Roswell Park.