bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phosphorylation of GPCR Kinase 2 in Intact Cells: A Proteomic Approach
Authors: Sterne‐Marr Rachel , Vijay Jesus , Keyoskey Joshua , Shareef Afzaal 
Published: June 2021

G protein‐coupled receptor (GPCR) kinases (GRKs) were first discovered by their ability to phosphorylate GPCRs in an agonist‐dependent manner. GRKs belong to the AGC kinase family of kinases. Activation of AGC kinases is often accompanied by phosphorylation of activation loop residues as well as other conformational changes in catalytically important residues. Crystal structures of GRK2 suggest that the activation segment region is in a relatively “active” conformation in the absence of phosphorylation. Nonetheless, a phosphoproteomic study (Oppermann et al., 2009) indicated that GRK2 Ser350 and Thr353 can be phosphorylated in intact cancer cells. Ser350 and Thr353 reside in the activation loop and P+1 site of the peptide‐binding site, respectively. To test the role of Ser350 and Thr353 phosphorylation in GRK2 function, we substituted alanine and aspartate at these two positions and tested the ability to phosphorylate the beta‐adrenergic receptor. We found that Thr353 is particularly important for phosphorylation of beta‐adrenergic receptor by GRK2 in COS‐7 cells, since conversion to Ala or Asp prevented substrate phosphorylation. In cells transfected with both beta‐adrenergic receptor and GRK2, multiple forms of GRK2 were detected in cells treated with agonist. The multiple forms are consistent with various phosphorylation states. Our goal is to assess Ser350/Thr353 phosphorylation in intact cells using phosphoproteomic approaches. To this end, we engineered a hexahistidine tag on GRK2 expressed from a mammalian cell vector to facilitate its purification and have demonstrated that the tagged kinase is fully functional in agonist‐dependent receptor phosphorylation. We are further developing methods to assess GRK2 phosphorylation by isoelectric focusing and mass spectrometry.Support or Funding InformationThis work was funded by the National Science Foundation MCB0744739 and Siena College Center for Undergraduate Research and Creative Activity (CURCA).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deciphering DYRK1A Signaling using Proteomics and Transcriptomics
Authors: Poss Zachary , Ebmeier Christopher , Simpson Helen , Pagratis Maria , McClure‐Begley Tristan , Old William 
Published: June 2021

Trisomy 21 is the most common genetic manifestation of intellectual disability, with deleterious brain phenotypes thought to be caused by increased dosage of critical genes on chromosome 21, including the kinase DYRK1A. Additionally, individuals with Down syndrome are at an increased risk of developing Alzheimer's disease, immune dysfunction and certain cancers, and DYRK1A has been implicated in each of these conditions.Although it is clear that DYRK1A is critical for neural development, little is known about the substrates it modifies and how its dosage and activity contribute to trisomy 21 phenotypes. We employed phosphoproteomic and transcriptomic approaches in a 3D neuronal model of the developing trisomy 21 brain and found that DYRK1A phosphorylates and regulates key factors involved in splicing and transcription regulation. As both of these processes are central to neural differentiation, these data suggest that DYRK1A activity may be critical for the transition from progenitor to differentiated neurons. Additionally, dysregulation of this transition may contribute to phenotypic brain hallmarks exhibited within the trisomy 21 population, which express an extra copy of DYRK1A.Support or Funding InformationLinda Crnic Institute for Down Syndrome, Anna and John J. Sie Foundation, DARPAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Use of a research‐based framework to guide instructional design and assessment related to noncovalent interactions in a biochemical context
Authors: Loertscher Jennifer , Feola Stephanie , Lemons Paula , Lewis Jennifer , Mercer Allison , Minderhout Vicky 
Published: June 2021

Noncovalent interactions are difficult for students to conceptualize throughout the undergraduate chemistry sequence, which results in misconceptions that can make mastering complex biochemistry concepts difficult. In an effort to elevate the quality of teaching and learning in this area, a construct map was developed that outlines a common learning trajectory of the physical basis of interactions. Utilizing this guideline, a series of activities and assessments were created and implemented by a biochemistry educator who participated in the iterative workshops that developed the construct map. We have analyzed students' responses to newly‐developed instructional materials and exam questions and have characterized themes related to students' understanding of noncovalent interactions. We ultimately hope to gain insights that will result in further changes in teaching practice to better support student learning. This work highlights the interplay of research and practice in undergraduate level biochemistry curriculum.Support or Funding InformationNational Science Foundation DUE‐1224868This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Design and construction of a structural model of the bifunctional GlmU protein in complex with N‐acetyl‐D‐glucosamine‐1‐phosphate and uridine‐diphosphate‐N‐acetylglucosamine
Authors: Tollar M. Roarke , Edwards Titus D. , Nguyen Nhan , Novak Walter R.P. 
Published: June 2021

The focus of this project was to explore the structure of the GlmU protein from E. coli (PDB ID: 2OI7) and to design and build a physical model that illustrates the key functional features of the protein. GlmU is a bifunctional bacterial protein that synthesizes uridine‐diphosphate‐N‐acetylglucosamine (UDP‐GlcNAc) from D‐glucosamine 1‐phosphate, acetyl‐CoA, and UTP. The structure described here is in complex with the products desulfo‐coenzyme A, N‐acetyl‐D‐glucosamine‐1‐phosphate and UDP‐GlcNAc. The N‐terminal uridyltransferase domain is composed of two beta‐hairpins and a seven‐stranded beta‐sheet surrounded by alpha‐helices. The C‐terminal acetyltransferase domain is a left‐handed parallel beta‐helix. GlmU functions biologically as a homotrimer. The three N‐terminal uridyltransferase active sites are independently formed by each monomer; however, each of the three acetyltransferase active sites is comprised of residues from the beta‐helix domain of all three monomers. This site is formed by one face of the beta‐helix and a loop region of the first monomer, a different face of the second monomer beta‐helix, and the C‐terminal tail of the third monomer.Support or Funding InformationThis work was funded in part by NSF‐DUE 1725940 for the CREST Project and the Wabash College Haines Biochemistry 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.
Mutant huntingtin affects p53 function in a context‐dependent manner in a cell model of Huntington's disease
Authors: Ellison Genevieve A. , Garcia Loren S. , Cornett Jonathan C. 
Published: June 2021

Huntington's disease (HD) is an adult‐onset neurodegenerative disease caused by expansion of a polyglutamine (polyQ) repeat in the N‐terminal region of the huntingtin (htt) protein. In HD, multiple N‐terminal mutant htt fragments, smaller than the first 500 amino acids, have been found to accumulate in disease tissue. These N‐terminal htt fragments associate abnormally with many cellular proteins, disturbing their function. The p53 tumor suppressor protein has been shown to abnormally interact with mutant htt and is associated with cellular dysfunction in HD. However, it is unknown whether different htt fragments affect p53 function differentially. Here, we investigated the affect of different length N‐terminal mutant htt fragments on p53 localization and function in a human cell model of HD. We found that shorter htt fragments cause more accumulation of p53 protein in the nucleus which is associated with decreased cell viability. Our results demonstrate the importance of mutant htt protein context in HD. While the p53 pathway is implicated as a potential target to ameliorate Huntington disease progression, the therapeutic benefit may be limited by the relative abundance of shorter N‐terminal htt fragments in target cells.Support or Funding InformationThis work was supported by the National Science Foundation under Grant No. 1458212. JC was supported in part by a faculty fellowship from the Appalachian College Association.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mucus‐inspired low‐fouling barriers based on self‐assembled glycopeptide nanofibers
Authors: Hudalla Gregory , Restuccia Antonietta 
Published: June 2021

Mucus is a glycoprotein‐rich gel that protects epithelial tissue surfaces from non‐specific interactions with micro‐organisms and macromolecules. Synthetic mucus‐like barriers could enable new opportunities to confer implantable materials with non‐fouling properties that are essential for biocompatibility and long‐term efficacy. However, mucus glycoproteins (i.e., “mucins”) derived from natural sources are highly variable and recombinant mucin production is impractical, which has led to increasing interest in synthetic mucin analogs. Here we report synthetic carbohydrate‐modified peptides (i.e., “glycopeptides”) that self‐assemble into beta‐sheet nanofibers in water. The dense carbohydrate coating on the surface of glycopeptide nanofibers is similar to that of naturally derived mucins. Carbohydrates decorating the surface of glycopeptide nanofibers are specifically recognized by their cognate lectins. Additionally, the type of carbohydrate appended onto the nanofiber can be efficiently varied via glycosyltransferase enzymes. Using this approach, we synthesized nanofibers decorated with n‐acetylglucosamine (GlcNAc) and n‐acetyllactosamine (LacNAc) that selectively bind to wheat germ agglutinin (WGA) and galectins, respectively. At millimolar concentrations in water, glycopeptide nanofibers entangled into viscoelastic hydrated gels (i.e., “hydrogels”) (Figure 1a–b). GlcNAc‐decorated glycopeptide hydrogels bound and retained WGA, but were permeable to various non‐lectin proteins, including albumin and green fluorescent protein, as well as the mannose‐binding lectin, concanavalin A. GlcNAc‐decorated glycopeptide hydrogels coated onto a glass surface reduced E. coli adhesion by ~90%, whereas non‐glycosylated peptide hydrogels only reduced bacterial adhesion by 50%. Additionally, glycopeptide hydrogels coated on glass robustly inhibited adhesion of macrophages (Figure 1c) and fibroblasts (Figure 1d) in the presence of serum, whereas non‐glycosylated peptide hydrogels were permissive to non‐specific cell adhesion. Together, these data demonstrate that self‐assembling glycopeptides can provide synthetic barriers with selective macromolecule permeability and resistance to non‐specific cell interactions similar to natural mucinous gels. Moving forward, we anticipate that glycopeptide hydrogels will be broadly useful as mucus‐inspired surface coatings to improve the biocompatibility and efficacy of implantable devices, drug delivery vehicles, and biosensors.Support or Funding InformationNSF Career award, DMR‐1455201This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comprehensive High Throughput One‐Pot Analysis of Glycoproteins through Tool for Rapid Analysis of Glycopeptide by Permethylation (TRAP) Method.
Authors: Shajahan Asif , Supekar Nitin T. , Heiss Christian , Azadi Parastoo 
Published: June 2021

Glycoproteins plays key roles in various cellular functions such as protein folding, protein targeting, receptor function, cell–cell recognition, protein stability, signaling, and other cellular functions. Currently a comprehensive and universal tool for the automated analysis of glycopeptides through their tandem mass spectrometry and data processing is a tedious process. We recently developed a method, Tool for Rapid Analysis of glycopeptide by Permethylation (TRAP), that simplifies glycoprotein analysis by integrating glycan sequencing and glycopeptide analysis in a single experiment. We employed this approach for the detailed analysis of glycosidic linkages, composition, glycan sequencing, glycosylation site mapping and sequencing of peptide moiety of glycopeptides of glycoproteins from both prokaryotes and eukaryotes. Here we are presenting an easy to use, robust, suitable for large as well as small sample sets and also cost‐effective glycoprotein analysis tool through TRAP approach. A permethylation protocol which can be automated and suitable for reliable permethylation of glycopeptides from the protease digest of glycoproteins was developed. Initial studies on glycopeptides from a model glycoprotein bovine RNAse B indicates that improved sensitivity and throughput of permethylation reactions can be achieved in conducting the reaction in microscale. This approach would enable convenient, rapid, and reliable high‐throughput screening of protein glycosylation and can be employed as a cost‐effective kit for glycosylated biomarker discoveries. In order to improvise the comprehensive glycosylation analysis and throughput, we have also developed an LC‐MS/MS platform for the analysis of glycopeptides processed through TRAP. We have conducted the analysis of standard glycoproteins such as bovine RNase B, human transferrin, and bovine fetuin and our preliminary results indicate that TRAP hyphened with LC‐MS/MS enables characterization of isomers of each glycoform at each glycosylation sites of complex glycoprotein mixtures.Support or Funding InformationThis research was supported by the National Institutes of Health (NIH) funded research, Grant R21GM122633; resource for biomedical glycomics, Grant P41GM103490; instrument grant, Grant S10OD018530; and U.S. Department of Energy grant, Grant DE‐SC0015662, to the Complex Carbohydrate Research Center.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Concentration‐Dependent Actions of Piperidine Alkaloids on the Inhibition of Fetal Movement in Day 40 Pregnant Goats and Comparison to Cell‐Based Models.
Authors: Green Benedict , Welch Kevin D. , Lee Steve T. , Kem William R. 
Published: June 2021

Anabasine and anabaseine are potent and effective agonists at nicotinic acetylcholine receptors (nAChR). Anabasine in livestock species is teratogenic and has been shown to cause developmental defects that include arthrogyrposis, kyposis, lordosis, scoliosis, and torticollis. We have postulated that these fetal defects are due to the complete and sustained inhibition of fetal movement. The purpose of this research was to determine if cell‐based predictions of teratogenicity from TE‐671 cell experiments translate to inhibition of fetal movement in a day 40 pregnant goat model. We hypothesized that anabasine, anabaseine, epibatidine, and DMPP which are potent and effective agonists atfetal muscle‐type nAChR expressed by TE‐671 cells, would be effective at completely inhibiting fetal movement in a goat model the putative mechanism of teratogenicity in livestock species. Pregnant goats were I.V. dosed with anabasine, anabaseine, epibatidine, DMPP, or saline control on gestation day 40 and the number of fetal movements were measured by ultrasound periodically for 8 hours. The experiment was designed to only briefly inhibit fetal movement(< 3 h) and as a result, no terata were observed in any of the kids from the pregnant does dosed with any of the compounds in this study. Anabasine, at a dose of 0.8 mg/kg, completely inhibited fetal movement for 1.5 hours after dosing, and its actions were dose‐dependent with an IC50 value of 0.1 mg/kg. Anabaseine, epibatidine, and DMPP did not completely inhibit fetal movement in day 40 pregnant goats. The results from this experiment suggest that while experiments with TE‐671 cells provide valuable information and predictions of the actions of plant alkaloids on fetal movement, in vivo experiments are still required in order to determine the ability of an alkaloid to inhibit fetal movement in livestock species and to predict teratogenic potential.Support or Funding InformationFunding provided by USDA ARSThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Buprenophine functions as a weak dopamine releaser in the absence of heroin but blocks the dopamine releasing effects of heroin in its presence
Authors: Issacs Dominic , Krystinziak Greg , Schelp Scott , Pultorak Katherine , Oleson Erik 
Published: June 2021

With the rise in opioid use and opioid‐related death in modern society, it is imperative to understand how these drugs affect neural circuits, particularly those implicated in drug‐seeking and motivated behavior. Generally, all drugs of abuse are thought to increase mesolimbic dopamine concentrations through various mechanisms, and it is widely believed these fluctuations in dopamine concentrations (and subsequent hypodopaminergic states) are implicated in the acquisition, persistence, and maintenance of addiction. Here we sought to identify increases in phasic dopamine in the nucleus accumbens of rats after intravenous diacetylmorphine (heroin) administration. Then, we sought to identify the effects on phasic dopamine after buprenorphine administration, followed by subsequent heroin administration. Utilizing fast‐scan cyclic voltammetry in awake behaving animals, we administered cumulative animal‐equivalent doses based on clinical investigations that correlated subjective measures of the drug's effect to dosage in human subjects. Either heroin or buprenorphine administered alone causes large increases in dopamine transient amplitude and frequency, whereas heroin administration after buprenorphine causes a decrease in the amplitude and frequency of transients. These data suggest a modulatory effect of buprenorphine on opioid receptors in the presence of exogenous opiates.Support or Funding InformationFunding for the project was provided by NSF grant IOS‐1557755, NIH grant R03DA038734, Boettcher Young Investigator Award and NARSAD Young Investigator Award to EBO and an institutional UROP to KJP.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Correlation between Toluene Exposure and Toxicity Effects of Toluene on Drosophila Melanogaster Fecundity and Offspring Survivability
Authors: MacGregor Michael Charles , Trivino Vanessa , Luu Brenda , Moreno Angela , Kovar Erin , Arredondo Dalia , Rosell Rosemarie , Ledesma Elmer 
Published: June 2021

Toluene is a volatile organic compound that is a common indoor contaminant. According to the U.S. Department of Health and Human Services, chronic toluene exposure negatively affects organ systems, such as nervous and respiratory, and acts as a fetotoxic agent in the development of humans. Toluene exposure causes damage to human myelin sheaths, thus we propose that damage to the fly nervous system will be similar to that seen in humans exposed to toluene. Our ultimate goal is to determine the effects of toluene exposure on developmental and neuronal processes in D. melanogaster. In this first phase, we hypothesize that toluene‐exposed adults, at the interpolated Lethal Exposure (LE50), will demonstrate decreased fecundity and that offspring from toluene‐exposed females will have decreased survivability from the egg to adult stage. We developed a continuous flow system that delivers known concentrations of gaseous toluene to populations of D. melanogaster. Ten to twenty adult female flies per trial were exposed for 24 hours to known concentrations of toluene. Surviving females were collected, counted and mated to non‐exposed adult males. The toluene‐exposed females were placed on a colored agar plate to lay eggs for 24 hours. Eggs were counted to determine fecundity (number of eggs produced by a single female fly). Survivability of eggs to later life stages, was measured by counting number of larvae, pupae, and adults divided by number eggs, larvae, and pupae respectively. The interpolated LE50 of gaseous toluene was between 1000 and 1500 ppm after 24‐hour exposure. Fecundity of toluene‐exposed females decreased by approximately 50% when compared to airexposed females. The survivability of offspring of air‐exposed flies from egg to larval stages was 84% higher than published studies. We observed that larval eclosure from eggs laid by toluene‐exposed females was 35–50% higher on days 1 and 2 when compared to larval eclosure from eggs of air‐exposed exposed females. On days 3–6 the eclosure rate was relatively similar. These data showed toluene's effects on fecundity and survivability were measurable, however, additional replicates are in progress to fully assess the life table characteristics of offspring of toluene‐exposed female flies. Further studies will focus on toluene's effect on nervous and reproductive organs of fruit flies, along with a study on the mutagenic effects of toluene on fly DNA.Support or Funding InformationU.S. DOE, Hispanic‐Serving Institutions, STEM Articulation Grant P031C110128This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.