bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hybrid Rat Diversity Program (HRDP): A rat resource for mapping complex traits
Authors: Dwinell Melinda R , Takizawa Akiko , Lazcares Lynn , Schilling Rebecca , Hoffman Matthew , Geurts Aron , Shimoyama Mary 
Published: April 2020

Rats are a major model for studying complex disease mechanisms, behavioral phenotypes, environmental factors, and for drug development and discovery. Inbred rat strains control for genetic background and allow for repeated, reproducible molecular, cellular, and whole animal phenotyping. Genetic susceptibility to disease, sensitivity to environmental elements, and pharmacogenomics are critical components of the concepts of precision medicine. A resource that combines the power of genetic stability within strains, power for genomic mapping strategies, and strength in an animal model that mirrors many human disease traits is the goal of this project. Through the development of the Hybrid Rat Diversity Program (HRDP), a powerful panel of inbred rats with genomic, physiologic, and behavioral phenotype data is beginning to serve as a catalyst for research in many fields. When complete, the HRDP will consist of 96 inbred rat strains to maximize power to detect specific genetic loci associated with complex traits while maximizing the genetic diversity among strains. The 96 strain panel consists of 33 genetically diverse inbred strains and two panels of recombinant inbred panels: FEXL/LEXF (33 strains, Japan) and HXB/BXH (30 strains, Czech Republic). Embryo resuscitation and breeding is well underway at the Medical College of Wisconsin (MCW). Whole genome sequencing for 32 of the 96 strains is complete, having been generated on am Illumina NovaSeq S4 to achieve 30X coverage. Liver and brain transcriptome analysis is underway through PhenoGen (
http://phenogen.ucdenver.edu) at the University of Colorado. Genomic, phenotype, and strain information will be made available through the Hybrid Rat Diversity Panel portal at the Rat Genome Database (
http://rgd.mcw.edu). The HRDP will provide a genetically stable population of rat strains with fully sequenced genomes, transcriptomes for brain and liver, and general phenotypic characterization to be used for systems genetic studies and fine mapping of complex traits.Support or Funding InformationNational Institutes of Health, Office of the Director, R24OD024617
Assessing CMPI potentiation of (α4)3(β2)2 nicotinic acetylcholine receptor using the Monod‐Wyman‐Changeux allosteric model.
Authors: Deba Farah , Akk Gustav , Hamouda Ayman K. 
Published: April 2020

Positive allosteric modulators (PAMs) of neuronal nicotinic acetylcholine receptors (nAChRs) enhance ACh potency and/or efficacy by binding at site(s) distinct from the ACh, i.e., orthosteric binding sites. Previous work has established CMPI (3‐(2‐chlorophenyl)‐5‐(5‐methyl‐1‐(piperidin‐4‐yl)‐1H‐pyrrazol‐4‐yl)isoxazole as a nAChR PAM that selectively potentiates (α4)3(β2)2 nAChR. In (α4)3(β2)2 nAChR, co‐application of CMPI maximally potentiated responses to ACh EC10 by ~400% with potentiation EC50 of ~0.3 μM. CMPI produced a left‐shift of the ACh concentration‐response curve of (α4)3(β2)2 nAChR enhancing ACh potency at (α4)3(β2)2 nAChRs by ~100 fold with no significant effect on the ACh maximal response. Mutational and computational analyses have identified a CMPI binding site that partially overlaps the ACh binding site (ABS) at the α4:α4 subunit extracellular interface which exists only in the (α4)3(β2)2 nAChR. Here, we are characterizing CMPI potentiation of 4 classes of (α4)3(β2)2 nAChR agonists: 1) a full agonist binding at α4:α4 ABS (ACh); 2) a full agonist not binding at the α4:α4 ABS (5I‐A85380); 3) partial agonists binding at the α4:α4 ABS such as cytisine; and 4) partial agonists not binding at the α4:α4 ABS such as TC‐2559. CMPI potentiation of nicotine, cytisine, and 5‐A855380 was similar to that for ACh (potentiated responses to low but not saturating agonist concentrations enhancing the potency but not the efficacy of these agonists). In contrast, CMPI potentiated responses to low and high concentration (up to 30 μM) of TC‐25559 enhancing its efficacy. However, in (α4)3(β2)2 nAChR containing alanine substitution at α4H116 which allows TC‐2559 to bind at α4:α4 ABS, CMPI enhanced potency but not the efficacy of TC‐2559‐induced currents. Analysis of the findings in the framework of the Monod‐Wyman‐Changeux model indicates that CMPI acts at the α‐α interface with a gating efficacy that is equivalent to that of ACh but higher than the gating efficacy of cytisine at this site. At the α4H116A mutated α‐α interface, TC‐2559 has a higher efficacy than ACh or CMPI. The precise nature of the modulatory effect is thus multifactorial, dependent on the concentrations and nature of the combined agonists.Support or Funding InformationNational Institutes of Health/National Institute of Neurological Disorders and Stroke [Grant NS‐093590] (A.K.H).
Defining the Molecular Basis of KIR Channel Mechanosensitivity in Vascular Smooth Muscle
Authors: Fletcher Jacob , Welsh Donald G. 
Published: April 2020

In cerebral arteries, the inwardly rectifying potassium channel (KIR) contributes to the setting of membrane potential and development of myogenic tone. KIR channels have been recently identified as a target of mechanical regulation in vascular smooth muscle, by which their activity is suppressed by pressure. However, the mechanism underlying this process remains unknown. The actin cytoskeleton and caveolae are often part of signaling domains implicated in established models of mechanosensitivity. Consequently, this study investigated whether these distinct structural components enable KIR pressure sensing in rat cerebral arterial smooth muscle. Whole‐cell patch clamp electrophysiology was used to monitor KIR activity and initial experiments confirmed that this current was suppressed by mechanical stimulation. Pre‐treatment of cells with actin‐disrupting agents, Latrunculin A and Cytochalasin D, prevented the suppression of KIR, indicative of the cytoskeleton’s participation. As KIR channels reside in caveolae, we next assessed the involvement of caveolin proteins in mechanoregulation. As expected, inhibition of caveolin‐1 signaling with blocking peptides diminished the ability of KIR channels to respond to pressure. The current data highlights that the actin cytoskeleton and caveolae form key parts of a signal transduction structure that confers mechanosensitivity to KIR. Ongoing work seeks to identify other KIR‐protein interactions in cerebral arterial smooth muscle using pull‐down and proximity ligation assay techniques. The functional impact of signaling complex disruption will also be assessed with vessel myography.Support or Funding InformationResearch supported by the Canadian Institutes of Health Research.
Regulation of presynaptic CB1 receptors in persistent inflammation
Authors: Bouchet Courtney A. , Suchland Katherine L. , Ingram Susan L. 
Published: April 2020

Opioids are the golden‐standard for pain treatment and therapeutics targeting the cannabinoid system are on the rise; yet the molecular mechanisms by which these systems are regulated is not fully understood. Opioids and cannabinoids can elicit analgesia through activation of mu‐opioid receptors (MOR) or cannabinoid 1 (CB1) receptors, respectively, in the ventrolateral region of the periaqueductal gray (vlPAG). Both receptors suppress GABA release from the presynaptic terminal under basal conditions. Our studies examine how persistent inflammation affects presynaptic MOR and CB1 receptor signaling in the vlPAG. Adult male and female Sprague Dawley rats (postnatal day 30–60) are naïve or injected with Complete Freund’s Adjuvent (CFA) into one hindpaw to induce inflammation for 5–7 days. Using electrophysiology to measure miniature inhibitory postsynaptic currents (mIPSCs), we find that CB1‐mediated inhibition of GABA mIPSC frequency is significantly reduced in inflamed rats compared to naïve rats. MOR‐mediated inhibition of mIPSC frequency is not altered. This effect is consistent in both males and females. There are no effects of inflammation on mIPSC amplitude. Previous work has shown that neuropathic pain, chronic THC exposure, or persistent inflammation reduces CB1 receptor protein levels while mRNA levels are unchanged suggesting that CB1 receptors may be regulated via local protein synthesis. We find that protein synthesis inhibitors, anisomycin (20 μM) or cyclohexamide (80 μM) reduce presynaptic CB1 receptor‐mediated inhibitionof GABA release in the vlPAG but do not affect presynaptic MOR function. These results provide evidence that presynaptic CB1 and MOR are regulated differentially under basal conditions and in persistent inflammation. These data have important implications for the future treatment of pain with pharmaceuticals targeted at either CB1 or MOR receptors.Support or Funding InformationNIDA T32 Training Grant; NIH NIDA R01DA042565
HSP27 Mediates PAR1‐stimulated Pro‐inflammatory Vascular Responses
Authors: Rada Cara 
Published: April 2020

Endothelial cells line the lumen of blood vessels and create a semi‐permeable barrier that is disrupted during vascular inflammation resulting in increased permeability, tissue edema, and subsequent organ failure. Endothelial dysfunction also results in cytokine production and leukocyte recruitment. However, the molecular mechanisms responsible for the pro‐inflammatory state remains to be elucidated. Protease‐activated receptor‐1 (PAR1) is a G protein‐coupled receptor for the coagulant protease thrombin and mediates hemostasis, thrombosis, and inflammatory response to vascular injury. However, the distinct mechanism of how endothelial PAR1 promotes vascular pro‐inflammatory responses remains poorly understood. We recently discovered that thrombin activation of PAR1 stimulates p38 activation through a non‐canonical pathway mediated by TAB1 to promote endothelial permeability. PAR1‐induced p38 activation has been linked to vascular barrier disruption; however, the mechanism by which p38 signaling contributes to pro‐inflammatory responses is not known. We found through a phosphoproteomics screen that thrombin‐stimulated p38 signaling relies on phosphorylation of heat shock protein 27 (HSP27). Moreover, we found that this pathway is physiologically relevant by showing PAR1‐activated HSP27 modulates the closing of the endothelial barrier in vitro and vascular leakage in vivo. We show HSP27 activity is both p38‐ and PAR1‐dependent. Finally, we show PAR1‐induced p38 signals to HSP27 through two p38 downstream kinases, MAPKAPK2 (MK2) and MAPKAPK3 (MK3), to directly phosphorylate HSP27 on specific serines. Together these studies reveal a novel pathway in which thrombin‐induced p38 signals through MK2 and MK3 to regulate HSP27 activity leading to recovery of the endothelial barrier and promoting resolution of pro‐inflammatory responses.Support or Funding InformationT32 Pharmacological Sciences Training Grant (NIGMS T32 GM007752) Tobacco Related Disease Research Program (TRDRP) Pre‐doctoral fellowship (27DT‐0009)
Yerba mate increases mitochondrial uncoupling in adipose tissue
Authors: Walton Chase M. , Saito Erin , Jacobsen Samuel , Hansen Jason M. , Bikman Benjamin T. 
Published: April 2020

Yerba mate is commonly consumed in cultures throughout Central and South America with decades of research revealing numerous effects, including improvements in cardiometabolic outcomes. Among these benefits is an increased resistance to weight gain, as well as altered energy expenditure. However, additional research is needed to elucidate the effects of yerba mate on mitochondrial parameters. Through cell culture and rodent models, we tested mitochondrial respiration and ATP production in adipose to determine the direct effects of yerba mate on mitochondrial coupling status. We found that adipocytes experienced a substantial increase in oxygen flux, without a comparable increase in ATP generation, indicating increased mitochondrial uncoupling. Similar findings were observed in subcutaneous adipose tissue in rodents fed yerba mate chronically. These observations in subcutaneous adipose, which is unique in its capacity to “beige” or undergo mitochondrial biogenesis and uncoupling. Altogether, these results suggest that a portion of the metabolic benefit of regular consumption of yerba mate may be a result of increased adipose mitochondrial uncoupling.Support or Funding InformationNone.
Genome‐wide Analysis of Heparan Sulfate Biosynthesis
Authors: Liu Qing , Weiss Ryan , Spahn Philipp , Chiang Austin , Li Jing , Hamill Kristina , Tor Yitzhak , Lewis Nathan , Esko Jeffrey 
Published: April 2020

Heparan sulfate proteoglycans (HSPGs) are expressed on virtually all animal cells and are involved in many important biological processes. Each HSPG consists of a core protein with one or more covalently attached linear heparan sulfate (HS) chains composed of alternating glucosamine and uronic acids that are heterogeneously N‐ and O‐sulfated. The arrangement and orientation of the sulfated sugar residues of HS specify the location of distinct ligand binding sites on the cell surface, and these modifications can vary temporally during development and spatially across tissues. While most of the enzymes involved in HS biosynthesis have been studied extensively, much less information exists regarding the specific mechanisms that give rise to the variable composition and binding properties of HS. A genome‐wide CRISPR/Cas9 screen was developed to uncover novel genes other than those encoding known HS biosynthetic enzymes. A lentiviral single guide RNA (sgRNA) library was utilized to knock down gene expression across the entire genome in a human malignant melanoma cell line. Subsequently, high‐throughput screens were adapted to identify sgRNAs that induce resistance to cytotoxins whose action depends on HSPGs and decrease binding of HS‐dependent ligands to the cell surface. Among the resulting targets, we identified well‐known HS biosynthesis enzymes as well as candidate genes with a previously unrecognized link to HS biosynthesis. The top overlapping hits from the screens were then characterized based on gene function and validated to regulate HS assembly. Overall, these studies demonstrate novel approaches to uncovering regulatory factors and provide a better understanding of the genetic regulation of HS biosynthesis. Furthermore, these studies lead us to new methods to manipulate heparan sulfate and its activities in other cellular processes that go awry in human diseases.Support or Funding InformationR01GM33063 (to J.E.), R21CA199292 (to J.E. and N.L.), R35GM119850 (to N.L.), and K12HL141956 (to R.W.)
Keratan sulfate disaccharide: specific targeting to langerin and possible applications to COPD
Authors: Taniguchi Naoyuki , Ohkawa Yuki , Fujinawa Reiko , Kanto Noriko , Harada Yoichiro , Kizuka Yasuhiko 
Published: April 2020

The C‐type lectin receptor, langerin, is expressed in Langerhans cells (LCs) that are located in the dermis along with other dendritic cells (DCs). Although there have been several reports on the sugar‐binding properties of langerin, the physiological and pathological output upon the recognition of each sugar ligand by langerin are still under investigation. We propose here that the keratan sulfate (KS) disaccharide L4 and oligomeric derivatives thereof, triangle‐L4 and pendant‐L4, are functional ligands of langerin that, on binding, evoke anti‐inflammatory responses. ELISA assays revealed that triangle‐L4 and pendant‐L4 showed stronger binding to recombinant langerin than L4, and we here found that these L4 and derivatives thereof suppressed the expression of inflammatory cytokines in langerin‐expressing primary bone marrow‐derived dendritic cells (BMDCs). Furthermore, in an elastase‐induced mouse model of chronic obstructive pulmonary disease (COPD), the administration of L4 mitigated the extent of emphysema in the lung, suggesting that KS‐related langerin ligands might serve as new drug candidates for the treatment of COPD and other inflammatory diseases. We also discuss several unsolved questions that need to be tackled for our understanding of the functions of langerin and the development of langerin‐targeted therapy by using glycomimetics.Support or Funding InformationThis study was supported for Naoyuki Taniguchi by Japan Agency for Medical Research and Development (AMED, 18ae0101070h0003) and Grants‐in‐Aid for Scientific Research (KAKENHI KIBAN B, 15H04700 and Challenging Exploratory Research, 15K14481).
Abundance of Ssh1p modulates Sec61p stability and topogenesis
Authors: Kim Sungmin , Kim Hyun Ah 
Published: April 2020

Protein translocation across the endoplasmic reticulum (ER) membrane is mediated by translocons. Two translocons in the ER of Saccharomyces cerevisiae are the Sec61 and the Ssh1 (
Sec
sixty‐one
homolog
1) complexes. They contain Sec61p and Ssh1p as a pore‐forming core subunit, respectively, and share Sss1p as a complex stabilizing factor. Ssh1p is not essential for cell growth in yeast, and it remains unclear why yeast cells keep this non‐essential translocon component.Upon overexpression of Ssh1p, we observed a severe growth defect and rapid destabilization of Sec61p at high temperature. We reasoned that overexpressed Ssh1p, which is scarce in steady state might recruit Sss1p from the Sec61 complex, impairing its stability. To test this idea, co‐overexpression of Sss1p with Ssh1p was carried out and data show that it partially rescues a growth defect and Sec61 stability. Furthermore, overexpression of the Sss1p‐interaction‐defective mutant of ssh1p had no effect on growth or Sec61p stability. We also assessed synthesis of single‐spanning membrane proteins in SSH1 deletion and overexpression strains and found that Ssh1p is involved in biased topogenesis of single‐spanning membrane proteins.Our data characterized cellular roles of Ssh1p that it might contribute for □) adjusting Sec61p stability and □) mediating selective topologenesis of membrane proteins.
A Randomized, Placebo‐Controlled Trial Evaluating Effects of Lebrikizumab on Airway Eosinophilic Inflammation and Remodeling in Uncontrolled Asthma (CLAVIER)
Authors: Austin Cary D. , Edick Melissa Gonzalez , Ferrando Ronald E. , Solon Margaret , Baca Miriam , Mesh Kathryn , Bradding Peter , Gauvreau Gail M. , Sumino Kaharu , Fitzgerald Mark , Israel Eliott , Bjermer Leif , Bourdin Arnaud , Arron Joseph R. , Choy David F. , Olsson Julie K. , Abreu Francis D. , Howard Monet D. , Wong Kit D. , Cai Fang D. , Peng Kun D. , Putman Wendy S. , Holweg Cécile T. J. , Matthews John G. , Kraft Monica , Woodruff Prescott G. 
Published: April 2020

BackgroundThe anti–interleukin 13 (IL‐13) monoclonal antibody lebrikizumab improves lung function in patients with moderate‐severe uncontrolled asthma, but its effects on airway inflammation and remodeling are unknown. CLAVIER was designed to assess lebrikizumab’s effect on eosinophilic inflammation and remodeling.MethodsWe performed bronchoscopy on patients with uncontrolled asthma before and after 12 weeks of randomised double‐masked treatment with lebrikizumab (n=31) or placebo (n=33). The primary endpoint was relative change in airway subepithelial eosinophils per mm2 of basement membrane (cells/mm2). Pre‐specified secondary and exploratory outcomes included change in IL‐13–associated biomarkers and measures of airway remodeling.FindingsThere was a baseline imbalance in tissue eosinophils and high variability between treatment groups. There was no discernible change in adjusted mean subepithelial eosinophils/mm2 in response to lebrikizumab (95% CI, −82·5%, 97·5%). As previously observed, FEV1 increased after lebrikizumab treatment. Moreover, subepithelial collagen thickness decreased 21·5% after lebrikizumab treatment (95% CI, −32·9%, −10·2%), while fractional exhaled nitric oxide, CCL26, and SERPINB2 mRNA expression in bronchial tissues also diminished. Lebrikizumab was well tolerated, with a safety profile consistent with other lebrikizumab asthma studies.InterpretationWe did not observe reduced tissue eosinophil numbers in association with lebrikizumab treatment. However, in pre‐specified secondary analyses, lebrikizumab treatment was associated with diminished degree of subepithelial fibrosis, a feature of airway remodeling, as well as improved lung function and reduced key pharmacodynamic biomarkers in bronchial tissues. These results reinforce the importance of IL‐13 in airway pathobiology and suggest that neutralization of IL‐13 may reduce asthmatic airway remodeling.Clinical trial registrationNCT02099656Support or Funding InformationThe study was sponsored by F. Hoffmann‐La Roche Ltd.
Histidine Reactive Chemicals Modify the Thermus thermophilus CuA Redox Center
Authors: Acevedo Zachary , Hunsicker-Wang Laura 
Published: April 2020

The electron transport chain is a series of redox protein complexes that couple the shuttling of electrons with the pumping of protons, in order to create the electrochemical gradient that drives ATP synthesis. The first redox center of the terminal complex from the Thermus thermophilus bacteria has been isolated as TtCuA and has a binuclear copper center bridged by two cysteines and ligated by two histidines and two weak axial ligands. H157 of TtCuA, a solvent exposed ligating histidine, has demonstrated modification by diethyl pyrocarbonate (DEPC), an exogenous chemical modifier, that is facilitated by the residue’s fast proton exchange with the solvent. The H157‐DEPC adduct is reversible over 48 hours at room temperature, as supported by mass spectrometry, electrochemistry, and visible CD spectroscopy. This is one of two modifications, out of 12, that becomes removed. Due to the lability of the H157‐DEPC bond, it is possible that the H157 Nɛ is primed for proton pumping in the electron transport chain.In order to model a possible in vivo modification, TtCuA was exposed to 4‐hydroxynonenal (HNE), an endogenous lipid peroxidation product associated with multiple metabolic and neurodegenerative diseases. Exposure of TtCuA to 20 equivalents of HNE appears to reduce the protein over 48 hours and four modifications are observed via mass spectrometry. Exposure of the Pseudomonas aeruginosa azurin protein, a mononuclear blue‐copper denitrification protein with similar ligand groups to TtCuA, is not reduced by HNE, despite modification. Azurin also lacks a ligating histidine that can be modified by DEPC. In conclusion, the binuclear copper center of TtCuA has hyperreactivity in comparison to a similarly ligated mononuclear copper center.Support or Funding InformationMurchison Undergraduate Research Fellowship, Trinity University Chemistry Department, Mach Research Fellowship, Welch Foundation