bluuebunny/crossref_metadata_2025_split huggingface dataset:
tRNA Methylation Controls Bacterial Multi‐Drug Resistance
Authors: Masuda Isao , Matsubara Ryuma , Christian Thomas , Rojas Enrique R. , Yadavalli Srujana S. , Goulian Mark , Huang Kerwyn Casey , Hou Ya‐Ming 
Published: June 2021

Bacterial multi‐drug resistance is a burgeoning crisis whose origin remains mechanistically opaque. Through altered permeability or efflux activity, the membrane prevents many antibiotics from reaching high enough intracellular concentrations to exert a therapeutic effect. We show here that protein synthesis of membrane‐associated genes involves translation of proline CC[C/U] codons, which require N1‐methylation of guanosine 37 (m1G37) on the 3′‐side of the tRNA anticodon. TrmD is the bacterial methyl transferase that catalyzes m1G37‐methylation. Removal of m1G37 by trmD inactivation reduces biosynthesis of membrane proteins, impairs membrane structure and mechanics, and sensitizes Gram‐negative bacteria to multiple classes of antibiotics and suppresses development of resistance and persistence. Codon engineering in the major efflux gene tolC removes the dependence on trmD for biosynthesis of membrane proteins. TrmD activity requires the conserved G37 in tRNAPro; replacement of G37 with C37 increases membrane permeability and accelerates cell death. These findings demonstrate the potential to diminish multi‐drug resistance by inaction of TrmD and its m1G37‐tRNA product.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sensing Changes in Cellular Iron Metabolism: Regulation of IRP1 by FBXL5 and Cytosolic Iron‐Sulfur Cluster Assembly
Authors: Johnson Nathan B. 
Published: June 2021

Iron is a micronutrient essential for the viability of nearly all cells, but in excess, iron can overwhelm antioxidant defense mechanisms resulting in damage of cellular structures and cell death. Therefore, cellular iron levels must be maintained within narrow limits to prevent iron deficiency and iron toxicity. In order to maintain optimal iron levels, mammalian cells utilize Iron Regulatory Proteins (IRP). IRP1 and IRP2 are iron‐regulated RNA binding proteins that control the fate of mRNAs encoding proteins important for maintaining cellular iron homeostasis. For example, in iron replete cells IRP RNA binding is inactivated resulting in decreased iron uptake by transferrin receptor 1 and increased iron storage by ferritin. IRP1 RNA binding is inactivated primarily by the insertion of a [4Fe–4S] cluster by the cytosolic iron‐sulfur cluster assembly (CIA) system converting it into the cytosolic isoform of aconitase (c‐acon). IRP1 can also be inactivated by the iron‐dependent E3 ligase FBXL5 that also targets IRP2. Since, human diseases associated with impaired Fe‐S cluster assembly can disrupt cellular iron metabolism, we sought to understand the mechanism by which CIA and FBXL5 regulate IRP1. I will discuss a model for how CIA and FBXL5 collaborate to control the level of IRP1 RNA binding activity and optimize iron metabolism to meet cellular needs for proliferation.Support or Funding InformationThis work was supported by National Institutes of Health Grant RO1 DK66600, United States Department of Agriculture Hatch Project WIS01667, and the Iron Metabolism Research Fund.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Asymmetric Configurations in a Reengineered Homodimer Reveal Multiple Subunit Communication Pathways in Protein Allostery
Authors: Lanfranco Maria Fe , Garate Fernanda , Engdahl Ashton , Maillard Rodrigo 
Published: June 2021

Many allosteric proteins form homo‐oligomeric complexes to regulate a biological function. In homo‐oligomers, subunits establish communication pathways that are modulated by external stimuli like ligand binding. A challenge for dissecting the communication mechanisms in homo‐oligomers is identifying intermediate liganded states, which are typically transiently populated. However, their identities provide the most mechanistic information on how ligand‐induced signals propagate from bound to empty subunits. Here, we dissected the directionality and magnitude of subunit communication in a reengineered, single‐chain version of the homodimeric transcription factor cAMP Receptor Protein (CRPSC). By combining wild‐type and mutant subunits in various asymmetric configurations, we revealed a linear relationship between the magnitude of cooperative effects and the number of mutant subunits. We found that a single mutation is sufficient to change the global allosteric behavior of the dimer even when one subunit remains wild‐type. Dimers harboring two mutations with opposite cooperative effects had different allosteric properties depending on the arrangement of the mutations. When the two mutations were placed in the same subunit, the resulting cooperativity was neutral. In contrast, when placed in different subunits, the observed cooperativity was dominated by the mutation with strongest effects over cAMP affinity relative to wild‐type. These results highlight the distinct roles of intrasubunit interactions and intersubunit communication in allostery. Finally, dimers bound to either one or two cAMP molecules had similar DNA affinities, indicating that both asymmetric and symmetric liganded states activate DNA interactions. These studies have revealed the multiple communication pathways that homo‐oligomers employ to transduce signals.Support or Funding InformationThis work was supported with startup funds from Georgetown University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Natural product bioactive peptide discovery using PepSAVI‐MS
Authors: Hicks Leslie 
Published: June 2021

As current methods for antibiotic drug discovery are being outpaced by the rise of antimicrobial resistance, new methods and innovative technologies are crucial to replenish our dwindling arsenal of antimicrobial therapeutics. While natural products are a well‐studied source of biologically active small molecules, peptidyl factors contributing to their medicinal properties remain largely unexplored. To this end, we have developed the PepSAVI‐MS (Statistically‐guided bioactive peptides prioritized via mass spectrometry) pipeline1 to identify bioactive peptide targets from complex biological samples. MS/MS techniques such as CID, ETD, UVPD are implemented for de novo characterization. To validate this pipeline, we have demonstrated successful detection and identification of a known antimicrobial peptide, cycloviolacin O2 (cyO2), from the botanical species Viola odorata. Additionally, we have widened the known antimicrobial spectrum for V. odorata cyclotides, including antibacterial activity of cyO2 against A. baumannii and novel anticancer activities for cycloviolacins by their cytotoxicity against ovarian, breast and prostate cancer cell lines. The developed platform is highly versatile as it is adaptable to any natural product source of peptides and can test against diverse physiological targets, including bacteria, fungi, viruses, protozoans, and cancer cells for which there is a developed bioassay. As such, we demonstrate extension of this pipeline to fungal and bacterially‐sourced AMPs through the identification of the killer toxin KP4 from Ustilago maydis2 and the bacteriocin bac‐21 from Enterococcus faecalis harboring pPD13. Bac‐21 is identical in nucleotide sequence to another enterococcal bacteriocin, AS‐484, but herein we have experimentally validated the protein sequence of bac‐21 for the first time. Additionally, we begin to probe the vast array of botanical natural product sources to prioritize highly active species for downstream analysis.Support or Funding InformationUniversity of North Carolina Chapel HillThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Small Molecule Differentiation of RNA Structures Using Pattern Recognition
Authors: Hargrove Amanda E. 
Published: June 2021

The critical importance of RNA elements in gene regulation and the potential of targeting RNA with small molecules are increasingly appreciated, yet many questions remain unanswered in small molecule:RNA recognition. These questions include which RNA structures can best be differentiated by small molecules and how small molecules can be used as tools to probe RNA structure and function. We developed a method termed Pattern Recognition of RNA with Small Molecules (PRRSM) that evaluates the ability of small molecules to classify RNA structures under a variety of conditions and in turn uses these small molecule “receptors” to identify site‐specific RNA structural changes. Insights gained from the assay, the current scope of the method, and future directions will be discussed.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optical Imaging Tools for Elucidating the Roles of Anions in Cellular Signaling
Authors: Dodani Sheel 
Published: June 2021

Living systems have evolved finely tuned mechanisms to coordinate transporters, chaperones, and non‐proteinaceous ligands to spatially and temporally position ions for normal physiology. In this context, freely diffusing and protein‐bound anions link to signal transduction, regulation of intracellular pH, and even detoxification. However, a breakdown in cellular homeostasis can lead to anion dysregulation in a wide range of diseases including neuropathic pain, cancer, and cystic fibrosis suggesting that anions could be signals of cellular status. Despite this biological significance, chemical biology approaches to identify the cellular stores, protein targets, and signaling roles of biologically relevant anions, are still underdeveloped, but could provide new insights for the role of anions in human health and disease. To this end, in this presentation I will describe the development and applications of turn‐on fluorescent biosensors for the real‐time imaging of anions in biological systems.Support or Funding InformationAcknowledgment is made to the Welch Foundation (Grant No. AT‐1918‐20170325).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Promotion Of Muscle Satellite Cell Proliferation By cAMP‐Induced Transcription
Authors: Berdeaux Rebecca , Akhmedov Dmitry 
Published: June 2021

The cAMP‐activated transcription factor CREB stimulates proliferation of myogenic progenitor cells during embryonic development, and genetic activation of CREB stimulates proliferation during muscle regeneration. Proliferation of muscle progenitor cells, known as satellite cells, declines during aging. We hypothesized that pharmacologic activation of the cAMP‐CREB pathway would promote satellite cell proliferation after injury and that cAMP‐regulated CREB co‐activators (CRTCs) contribute to CREB‐regulated target gene expression during satellite cell proliferation. To test this, we created ROSA26‐GsDREADD knock‐in mice for tissue‐specific expression of a Gs‐coupled DREADD (Designer Receptor Exclusively Activated by Designer Drugs, “GsD”). After tamoxifen‐induced expression of GsD in muscle satellite cells in young adult mice, activation of the receptor by the GsD ligand CNO (clozapine N‐oxide) increased proliferation after muscle injury. Consistent with our hypothesis, myoblasts over‐expressing constitutively active CRTC2 had increased cell proliferation in vitro, and Crtc2 knockout mice had reduced proliferation after muscle injury. Our results indicate that cAMP signaling enhances satellite cell proliferation in vivo and cAMP regulated CREB coactivator 2 (CRTC2) contributes to the proliferative response after muscle injury. ROSA26‐GsDREADD mice can therefore be used to stimulate cAMP signaling in specific cell populations in vivo to study the effects of pharmacologic activation of cAMP signaling without using ligands that have pleiotropic effects in other cell types.Support or Funding InformationResearch reported in this abstract was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01‐AR059847. DA was supported by a Postdoctoral Fellowship from the American Heart Association (15POST25090134). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kynurenine, a tryptophan metabolite that increases with age, induces skeletal muscle atrophy and reactive oxygen species.
Authors: Kaiser Helen , Yu Kanglun , Mendhe Bharati , Fulzele Sadanand , Johnson Maribeth , McGee‐Lawrence Meghan , Hamrick Mark W. 
Published: June 2021

Loss of muscle mass and power with age in the form of sarcopenia is associated with a loss of independence and a substantial decrease in quality of life due to an increase in falls and fractures. The cellular and molecular mechanisms underlying sarcopenia are not well‐understood; however, heterochronic parabiosis experiments have revealed that circulating factors are likely to play a role, and reactive oxygen species (ROS) are also known to increase in skeletal muscle with aging. This study investigated the relationship between kynurenine, a circulating tryptophan metabolite which increases with age, and markers of muscle oxidative stress. C2C12 myoblasts were treated with kynurenine in a dose‐dependent manner (0, 1, 10 μM) and ROS was measured using an Amplex red assay. Female C57BL/6 mice 6 months of age were treated with kynurenine (10 mg/kg BW) or with saline (vehicle control) for 4 weeks. Muscle mass and fiber size were measured from the quadriceps femoris ex vivo, and ROS was measured from paraffin‐embedded muscle sections using immunostaining for 4HNE. ROS was increased 2‐fold in C2C12 myoblasts treated with kynurenine at both low and high concentrations (P<.01). Quadriceps weight relative to body weight was reduced (10%) and muscle fiber size was significantly lower (P<.01) in young mice treated with kynurenine compared to controls. ROS was increased by 20% (P<.05) in young mice treated with kynurenine compared to control based on 4HNE staining. Our data reveal that the circulating tryptophan metabolite kynurenine can induce muscle wasting and increase reactive oxygen species in skeletal muscle, suggesting that pharmacological approaches to inhibit kynurenine production may provide a therapeutic strategy for the prevention of sarcopenia.Support or Funding InformationFunding for this research was provided by the National Institute on Aging (P01 AG036675).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Age‐induced Hepatic Steatosis and Inflammation of Murine Livers is Influenced by MCP‐1
Authors: Stahl Elizabeth , LoPresti Samuel , Delgado Evan , Alencastro Frances , Wilkinson Patrick , Duncan Andrew W. , Brown Bryan N. 
Published: June 2021

Aging is linked to the onset and severity of several chronic liver diseases, including non‐alcoholic fatty liver disease, alcoholic liver disease, and hepatitis, which correlate with high levels of macrophage infiltration, inflammation, and immunosenescence. Recent findings suggest that liver macrophages can be divided into two unique subsets: CD68+ embryonically‐derived Kupffer cells and CD11b+ bone marrow derived cells, which exhibit specific cell functions. It is currently unclear how the macrophage populations residing in the liver are phenotypically and functionally impacted by the natural aging process, which may have implications for the onset and progression of inflammatory disease and liver pathology in elderly populations.The aim of this study is to characterize the liver macrophage compartment from young (8–10 week) and aged (18–20 month) C57BL/6 wild‐type mice. First, we identified a significant increase in the total number of F4/80+ macrophages residing in aged livers. Preliminary in vitro studies showed that aged macrophages were primed towards inflammatory reaction, including increased phagocytosis and nitric oxide production, in response to LPS and interferon‐gamma. In addition, the aged macrophage population contained a greater proportion of CD11b+ cells, which are most likely derived from circulating monocytes. To confirm this, we determined that aged hepatocytes secrete enhanced levels of monocyte chemoattractant protein‐1 (MCP‐1), an important signaling molecule for monocyte recruitment following injury. Although aged livers do not show signs of injury or damage, we determined that hepatocytes from aged livers have increased triglyceride content and a shifted fatty acid profile. Of note, the aged mice received the same diet as young controls, and do not show signs of glucose intolerance. Future studies will examine how inhibition of MCP‐1 signaling will impact macrophage recruitment, inflammation, and steatosis in aged livers towards a better understanding of age‐related liver pathology.Support or Funding InformationNIH‐NIBIB Training Grant (T32‐EB001026); NIH‐NIDDK NRSA Fellowship (F31‐DK112633‐01A1); Pittsburgh Liver Research Center Pilot & Feasibility GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Postnatal Organogenesis by Transdifferentiation
Authors: Willenbring Holger 
Published: June 2021

Transdifferentiation is a physiological backup mechanism and alternative to stem‐cell‐mediated organ regeneration. In adult mammals, findings of transdifferentiation have been limited to replenishing lost cells in preexisting structures, i.e., in the presence of a fully developed scaffold and niche. Here we show that hepatocytes in the adult liver can transdifferentiate to build a structure that failed to form in development––the biliary system in mice that mimic the hepatic phenotype of human Alagille syndrome. The bile ducts forming the new biliary system are fully functional as evidenced by reversal of cholestasis, liver injury and liver fibrosis. Our results reveal the potential of mammalian transdifferentiation and may inform efforts to build organs containing a complex tubular system.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phospholipid Regulation of Inflammatory Processes and Viral Infection
Authors: Voelker Dennis R. 
Published: June 2021

Gas exchange between the external environment and erythrocyte hemoglobin takes place within the alveolar compartment of the lung, which contains an extracellular, phospholipid‐rich complex known as pulmonary surfactant. The major phospholipid classes of pulmonary surfactant consist of phosphatidylcholine (~70%), phosphatidylglycerol (PG) (~10%) and phosphatidylinositol (PI) (~2%). Dipalmitoyl‐phosphatidylcholine plays an essential role in reducing surface tension within the alveoli during respiration, but the roles played by phosphatidylglycerol (PG) and phosphatidylinositol (PI) remained enigmatic for decades. PG and PI are uniquely secreted by alveolar epithelial cells, and their concentrations in this extracellular compartment greatly exceed that of any other tissue. Unexpectedly, studies of the innate immune Toll‐like receptors (TLRs) have revealed that PG and PI are potent antagonists of the activation of multiple TLRs including TLRs 1,2,3,4 and 6. The lipids bind to the receptors and prevent their activation by cognate ligands, and consequently prevent downstream activation of inflammatory signaling pathways, and release of inflammatory mediators such as interleukins 6 and 8, interferon‐lambda, eicosanoids and TNF‐alpha. In addition to suppressing activation of TLRs, the lipids also inhibit infection by major respiratory viruses including respiratory syncytial virus (RSV) and influenza A virus (IAV). Both PG and PI prevent the viral infections in cultured cells, and in vivo in mice. PG also completely abrogates lethal challenge of mice with pandemic H1N1‐IAV. Additional studies with ferrets further demonstrate protection against pandemic H1N1‐IAV. The lipids act as decoy receptors for both RSV and IAV, and block virus attachment to epithelial cell surfaces. We have constructed a library of 25 analogs of POPG and PI, which vary in both the structure of the polar headgroup and their hydrophobic moieties. Some of these analogs exhibit improved pharmacological properties compared with PG and PI. These studies demonstrate regulation of innate immune processes by pulmonary surfactant phospholipids, and also reveal their potential use for suppressing inflammatory processes in the lung and inhibiting establishment and spread of respiratory viral infections.Support or Funding InformationSupported by NIH Grants HL 094629, U19‐AI 125357, GM 118819This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.