bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of Heterologously Expressed Putative Cytolysins from “Brachyspira hampsonii “
Authors: Keith Brandon A , Harding John C , Hill Janet E , Loewen Matthew E 
Published: November 2023

Cytolysins produced by Brachyspira hyodysenteriae and “Brachyspira hampsonii” strain 30446 are believed to be important virulence factors in the pathology of swine dysentery. To date four putative cytolysin genes termed tlyA, tlyB, tlyC, and hlyA have been identified, all of which are present in both Brachyspira hyodysenteriae and “Brachyspira hampsonii”. While the contributions of tlyB, tlyC, and hlyA to the pathogenesis of swine dysentery are not yet known, tlyA knockout strains of Brachyspira hyodysenteriae have shown decreased virulence in mouse models. In addition, homologues of tlyA are believed to be important virulence factors for human pathogens such as Helicobacter pylori and Mycobacterium tuberculosis, functioning as both cytolysins and contributing to antibiotic resistance through ribosomal RNA methylation. While active hlyA protein has been purified from Brachyspira hyodysenteriae broth cultures, studies of tlyA, tlyB, and tlyC to date have focused on their activity when introduced into Escherichia coli via plasmid. These results were cast into doubt several years later upon the discovery that many lab strains of Escherichia coli exhibit a hemolytic phenotype when foreign genes were introduced. To examine the effects of these putative cytolysins the tlyA and tlyC genes were amplified from “Brachyspira hampsonii” genomic DNA and ligated into expression vectors. The resulting protein products were overexpressed in Escherichia coli and purified by Ni2+ and amylose affinity chromatography. Cytolytic activity of purified recombinant tlyA and tlyC on a sheep erythrocyte suspension was examined, and no cytolysis greater than a phosphate buffered saline control was observed from tlyA at concentrations ranging from 0 to 88 μg/mL or from tlyC at concentrations ranging from 0–34 μg/mL. At the time of writing, attempts are being made to express and purify the α‐toxin from Staphylococcus aureus utilizing the same methodology in order to rule out inactivation of tlyA and tlyC by the purification process. While it is also possible that higher concentrations of tlyA and tlyC are required for hemolysis or that full activity of these proteins require unidentified cofactors or post‐translational modifications not possible in our current Escherichia coli based expression system, the possibility that tlyA and tlyC have been misannotated as cytolysins cannot be ruled out.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC) 371364 /2010 to MEL & Alberta Livestock and Meat Association project 2013R054R to JCH, MEL & JEH
Understanding the Phylogenetic Relationship of Bacterial Arginine Kinases
Authors: Stock Katie , Aryal Manish , Fraga Dean 
Published: November 2023

Arginine kinases (AKs) are transferase enzymes that are part of the phosphagen kinase family. These enzymes catalyze the transfer of phosphate from ATP to L‐arginine and are important in the energy buffering mechanisms in invertebrates and unicellular organisms. Previous studies have attempted to understand the relationship of the bacterial AKs to other phosphagen ianses. Atleast eight bacterial AKs have been discovered through examining bacterial genome sequences available on‐line. Using phylogenetic analyses it was found previously that the bacterial AKs can be separated into two clusters, cluster A and cluster B. This work will describe a more complete characterization of the bacterial AKs, both biochemically and phylogenetically to better understand their relationships to each other and to other PKs. Our initial results support the observation that there are two ‘forms’ of the bacterial AKs, a kinetically slow form and a kinetically faster form.Support or Funding InformationCollege of Wooster
Temporal Dynamics of Apoptotic Activity in Jurkat Cells
Authors: Morris Sean Kyle , Reif Randall 
Published: November 2023

Apoptosis, a process in which a cell systematically triggers its own death in response to DNA damage or external stimuli, is widely utilized in the body. Malfunction of the apoptosis process can lead to serious health problems such as cancer. There are several known pathways that execute apoptosis utilizing a family of enzymes called Caspases. The (intrinsic) pathway is the focus of this research, and initiates caspase activity through mitochondrial depolarization.The goal of this research project was to find suitable initiators of apoptosis in Jurkat T‐Lymphocytes and elucidate their temporal dynamics with respect to caspase activity. Microfluidic devices were fabricated to capture cells and view this process. To identify suitable inducers of apoptosis to use, cells were exposed to several compounds and monitored over six hour time periods using Fluorescence Microscopy. Caspase activation was confirmed with the use of a caspase‐specific fluorogenic probe, L‐bisaspartic acid rhodamine 110.Anti‐CD95, staurosporine, and hydrogen peroxide were used as inducers of apoptosis resulting in induction rates of 8.88%, 1.66%, and 1.24% with un‐induced rates of 0.59%, 1.88%, and 0.24% respectively. On a microfluidic device, cells were induced with Hydrogen Peroxide and their fluorescence intensity was measured over time. Apoptotic cells with active caspase enzymes consistently reached peak intensity two hours after caspase activation began. Using these results, mitochondrial depolarization will be tracked simultaneously with caspase activity utilizing MitoTracker Red.Support or Funding InformationThis research would not be possible without support from the Department of Chemistry at the University of Mary Washington, as well as a grant from the University of Mary Washington Summer Science Institute.
Mechanism of Transport of Ferric Enterobactin through FepA in Gram Negative Bacteria
Authors: Majumdar Aritri , Trinh Vy , Moore Kyle J. , Smallwood Chuck R. , Newton Salete M.C. , Klebba Phillip E. 
Published: November 2023

BACKGROUND AND PURPOSELigand‐gated porin (LGP) receptors of Gram‐negative bacterial outer membrane (OM), typified by FepA, FhuA, BtuB, possess a complex β‐barrel transmembrane channel occluded by a globular N‐terminal domain. The structure poses immediate questions on the mechanism of ligand transport through the receptor protein. Our previous results with in‐vivo fluorescent labeling of engineered Cysteine residues on FepA indicated a ‘Ball and Chain’ mechanism of transport in which the N‐domain is expelled into the inter‐membrane periplasmic space during Ferric enterobactin (FeEnt) ligand passage. This study further investigates whether the N‐domain exits the barrel as a unit or unfolds into the periplasm or forms a ‘transient pore’ within the barrel allowing ligand passage.METHODWe constructed a series of double‐cysteine mutants located i) in the N‐domain such that disulphide bonds should not interfere with transport if it exits the channel as a unit ii) in the N‐domain and the β‐barrel such that the cysteines can come close and form double bonds only if the N‐domain exists the channel during ligand uptake. We tested these mutants for ligand uptake capabilities in nutrition tests and conventional radiochemical assays in the presence and absence of a reductant (β‐mercaptoethanol) of disulphide bonds.RESULTS AND CONCLUSIONOur results suggest that three double‐cysteine mutations G27C/R126C, A33C/E120C and L125C/V141C prevent transport of FeEnt in the absence of β‐mercaptoethanol. The location of these residues on the N‐domain point to a mechanism in which the globular N‐domain undergoes conformational change to allow tight ligand passage.Support or Funding Information1R21AI1157187 granted to PEK
Isomerized aspartate residues in rodent eye lens protein act as a marker of biological aging
Authors: Warmack Rebeccah Austin 
Published: November 2023

Proteins in biological systems are known to accumulate spontaneous conformational alterations before breakdown and recycling. The racemization and isomerization of aspartic acid in proteins is one such modification that can result in loss of structure and function. Aspartic acid racemization specifically has been studied as a potential measure of chronological aging within low protein turnover mammalian tissues. However, very few of these studies have been optimized for shorter‐lived animals such as rodents, which are increasingly used as genetic and metabolic models of aging. In this study, we have developed a new sensitive approach for the measurement of biological age by taking advantage of the formation of isomerized aspartyl residues of the rodent eye lens. Using the human protein L‐isoaspartate/D‐aspartate methyltransferase (PCMT1) as an analytical reagent, we are able to rapidly and conveniently quantify the levels of L‐isoaspartate within lens protein extracts. We found the levels of isomerized aspartic acid to be about 35 times higher in the extracts of 18 month‐old rats versus two month‐old rats. Importantly, it was observed that the accumulation appeared to plateau in rats of 18 months and older, suggesting the presence of potentially novel mechanisms for the removal of altered proteins that may develop with age.Support or Funding InformationRuth L. Kirschstein National Research Service Award GM007185
Role of Janus‐Kinase 3 in Lung Inflammation
Authors: MISHRA JAYSHREE , VERMA RAJ K , YANG XIUDONG , KUMAR NARENDRA 
Published: November 2023

ObjectiveMucin hyper production is common in inflammatory and allergic lung disease such as bronchial asthma, chronic obstructive pulmonary disease, bronchiectasis and cystic fibrosis. Excessive mucin production in the lung leads to obstruction of airways. Hence, it is critical to understand the mechanisms that regulate mucin production under normal physiological conditions and the reason for it's over production during inflammatory lung diseases. Janus kinase 3 (Jak3) is a member of the JAK family of intracellular non‐receptor tyrosine kinase that interacts with receptors for IL‐2, IL‐5, IL‐7, IL‐9, and IL‐15 through the common γ‐chain.2 Though previously Jak3 was thought to be expressed only in hematopoietic tissues, however recent studies have shown that Jak3 is expressed in many organ tissues including lungs of human and mice. Previously, we demonstrated that Jak3 plays an essential role in cytoskeletal remodeling, wound repair, and mucosal homeostasis in intestinal epithelial cells however the role of Jak3 in lung inflammation is not known.Materials and MethodsFreshly dissected lung tissue from control and Jak3 knockout mice were immunostained for Jak3 ((Invitrogen) as well as different transcription factors like Muc1, Muc2, Hif‐1, Hif‐2 (Novus Biologicals), p(142) β‐catenin (Abcam), p(654) β‐catenin (Santa Cruz Biotechnology), STAT3, pSTAT3, p‐STAT6 (Cell Signaling Technology). Phosphorylation status of these transcription factors in both WT and Jak3 KO mice were assessed through western analysis. Level of pro inflammatory cytokines in the lung tissue as well as the serum sample was measured using Multianylate cytokine assay kit from Qiagen.Summary of ResultsOur results show that lung epithelial cells of mice express Jak3 and knock out of Jak3 resulted in substantial decrease in expression of mucin (Muc1 and Muc2). Absence of Jak3 decreases the expression of transcription factors HIF‐1, HIF‐2, and STAT3. Absence of Jak3 also decreased the tyrosine phosphorylation level of transcription factors such as STAT3, STAT6, and β‐catenin as well as increased expression of pro‐inflammatory cytokines such as IL‐6 and IL‐17A in the lung tissue lysate.ConclusionOur data suggest that Jak3 plays an important role in lung inflammation through regulating mucin expression. Further, we will determine how Jak3 regulates different lung epithelial functions such as epithelial barrier function, ion and fluid transport function and the underlying molecular mechanism of it.Support or Funding InformationCrohn's & Colitis Foundation of America (CCFA Ref # 2188) and NIH (DK081661) to NKNIH (sub awarded) 1R43GM109528‐01 to NK and JM
Using Anti‐Fungal Bacteria to Contest Snake Fungal Disease
Authors: Malone Katherine 
Published: November 2023

Often referred to as indicator species, amphibians and reptiles are abundant and vital in ecosystems across the world. However, across the globe there is a decrease in this abundance due to various factors. Many species are already critically endangered or on the brink of extinction. One of these factors is the spread of various fungal diseases.Ophidiomvces ophiodiicola, also known as Snake Fungal Disease (SFD), is a new fungi spreading and residing on the skin of snakes. Although SFD does not immediately harm the infected reptile, in combination with other disturbances or stressors SFD could pose a serious threat to the reptile populations infected. The geographic range and the quantity of SFD infections are relatively unknown. Through this research we strive to isolate a residential bacteria from either reptiles’ skin or gut that posses antifungal properties along with estimate the extent of SFD infections. The first phase of this project was to collect skin and fecal samples from 18 species of snakes and lizards from the southeastern and southwestern US. We will then perform DNA extraction and real time PCR to determine if Ophidiomvces ophiodiicola was present on the skin or in the gut of each animal. We will also isolate resident bacterial species from the skin and gut and perform in vitro challenge assays against the snake fungal pathogen to determine the probiotic antifungal effects of resident skin bacteria. Antifungal bacterial species could be used for future probiotic field based treatments of reptiles at risk of infection from Ophidiomvces ophiodiicola.
Novel Brain‐specific Isoforms of Regulator of G protein Signaling 6 (RGS6)
Authors: Ahlers Katelin E , Stewart Adele , Yang Jianqi , Luo Zili , Fisher Rory A 
Published: November 2023

RGS proteins act as gatekeepers for cellular responses induced by G protein coupled receptors (GPCRs) by facilitating inactivation of heterotrimeric G protein signaling. By acting as GTPase activating proteins for Gα subunits, an activity bestowed by the semiconserved RGS domain present in all RGS members, RGS proteins determine the magnitude and duration of G protein signaling initiated by GPCRs. RGS6 is a member of the R7 subfamily distinguished by two additional unique domains, DEP/DHEX and GGL, which are important for membrane targeting and protein stability of these members, respectively. RGS6 specific modulation of Gαi/o protein activity has been implicated in regulation of several disease states, particularly in the CNS, including: alcoholism, anxiety/depression, and Parkinson's disease. In addition, RGS6 is unique in that it remains the only RGS7 family member that is known to regulate G protein‐independent pathways promoting pro‐apoptotic and growth suppressive actions in cancer. Potentially key to RGS6's G protein‐independent signaling as well as its modulation of G protein signaling are previously unidentified domains that arise via alternative mRNA splicing. Indeed, when our laboratory first cloned RGS6 from human brain we identified 36 distinct RGS6 isoforms. Recently, using western blot, we identified at least two additional RGS6 isoforms in the brain that are larger (~61 and 69kDa) than the ubiquitously expressed 56kDa form of the protein. The functions for these RGS6 variants and how they arise (either through protein modification or additional RNA splicing) is unknown. Here, we demonstrate that these brain specific isoforms, along with the 56kDa form, are robustly down‐regulated in human glioma, indicating they may be important for the prevention of tumorigenesis in the brain. In addition, utilizing RGS6 specific primers to perform PCR amplification of RGS6 from a human brain cDNA library (Clontech), we have discovered novel RGS6 species not identified in the initial cloning effort. These transcripts include novel exons, some of which appear to encode early stop codons and others which, when included in the final transcript, are predicted to produce proteins around the size of the brain specific isoforms previously identified by western blot. Interestingly, several of the identified exons appear to be shared primarily between humans and other primates. Together, this research will lay the groundwork for future experiments to elucidate the functional significance of RGS6 alternative mRNA splicing in normal brain function as well as pathology.Support or Funding InformationNIH CA161882
GRK2 Constitutively Downregulates Delta Opioid Receptor Activity in the Periphery
Authors: Doyle Allison P , Gomez Ruben , Henry Michael A , Jeske Nathaniel A 
Published: November 2023

Opioids remain the gold standard for prescribed analgesic care. However, systemic opioid receptor agonists carry systemic negative side effects that can contraindicate long‐term administration. In contrast, peripherally restricted opioids would produce significant analgesia while avoiding centralized side effects. Unfortunately, peripheral opioid receptors are analgesically incompetent through an unknown mechanism. Here we report that G Protein‐Coupled Receptor Kinase 2 (GRK2) naively associates with plasma membrane delta opioid receptor (DOR) in peripheral sensory neurons to limit receptor responsiveness. Importantly, bradykinin receptor activation in sensory neurons stimulated GRK2 movement away from plasma membrane DOR and onto the cytosolic scaffolding protein Raf Kinase Inhibitory Protein (RKIP). Protein kinase C (PKC)‐dependent phosphorylation of RKIP dimerized the scaffolding protein to sequester GRK2, restoring full DOR functionality in sensory neurons. Additional results indicate that naive GRK2 association with plasma membrane DOR prevents optimal Gβ subunit association with the receptor, thereby reducing DOR‐mediated inhibition of voltage‐gated Ca+2 channels in sensory neurons responsible for peripheral analgesic competence. Together, these results identify a novel, non‐internalizing role for GRK2 in maintaining peripherally‐expressed DOR in an analgesically incompetent state, thereby providing a mechanism that may be targeted pharmaceutically to increase peripheral opioid analgesic efficacy.Support or Funding InformationNINDS/RO1 NS082746 (N.A.J.), NIDCR/T32 DE14318 (A.P.D.), and NIDCR/F31 DE025551 (A.P.D.) from NIH funded this work.
Bitter Taste Receptor T2R4 Mediated Inactivation of Rac1 GTPase in Response to Quinine
Authors: Sidhu Crystal , Jaggupilli Appalaraju , Chelikani Prashen , Bhullar Rajinder P 
Published: November 2023

Rac1 is a member of the Rho family of low molecular mass GTP binding proteins (GTPases). It regulates the dynamics of actin cytoskeleton by causing membrane ruffling, chemotaxis, and lamellipodia formation. Rac1 has been recently implicated in cancer metastasis and progression. As is the case with other GTPases, Rac1 cycles between the active GTP‐bound form and the inactive GDP‐bound form. This step is facilitated by a variety of Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). T2R4 is a bitter taste receptor that belongs to the GPCR (G protein‐coupled receptor) family of membrane proteins. In addition to mediating bitter taste sensations from the tongue, T2R4 and other T2Rs have been recently found in tissues other than the oral cavity e.g. nasal epithelium, airways, brain, gastrointestinal tract and male reproductive system suggesting a much broader physiological function for these receptors. T2Rs have been implicated in bronchodilation and in vasoconstriction. Quinine is an antimalarial drug and is one of the most bitter tasting compounds known. It is a natural alkaloid with a quinoline ring and a bicyclic quinuclidine. Quinine is a known agonist for T2Rs whereas BCML (Nα,Nα‐Bis(carboxymethyl)‐L‐lysine) acts as an inverse agonist. Since Rac1 is activated via various receptors like tyrosine kinase (RTKs), integrins and GPCRs; we have investigated the potential role of T2R4 in regulating Rac1 activity. In this study, HEK293T cells stably expressing T2R4/Gα16/44 were transiently transfected with HA‐Rac1 followed by treatment with Quinine or Quinine plus BCML for 15 min. After incubation, active Rac1 was pulled‐down from cell lysate using GST‐PAK1 and anti‐Rac1 monoclonal antibody was used in Western blots to quantify amount of active Rac1. The results demonstrated that Quinine treatment resulted in significant (p<0.001) reduction in the amount of active Rac1 whereas in the presence of BCML, Quinine failed to cause any significant change in the amount of active Rac1 when compared to untreated cells. This study is the first to show inhibitory downstream action of a T2R agonist on Rac1 function and further investigation will help in better understanding the extra‐oral physiological roles of T2Rs.Support or Funding InformationThis work is supported by a grant from Research Manitoba and the College of Dentistry.
The Coordination of Notch and Integrin Signaling by Resveratrol
Authors: LaFoya Bryce , Albig Allan 
Published: November 2023

Integrins are transmembrane proteins which form for cell‐cell and cell‐extracellular matrix connections, allowing environmental signals to be relayed into a cell through their signal transduction capabilities. The Notch signaling pathway has a role in many cellular processes such as differentiation and proliferation. The regulation of this pathway can provide a cell with molecular cues which are used to make cell‐fate decisions. Both αVβ3 integrin and Notch signaling have been implicated in many normal processes of vascular biology such as angiogenesis and arteriovenous development as well as pathological processes such as the development of atherosclerosis and tumor vascularization. Studies on trans‐resveratrol, a stilbene produced in several plant species, have demonstrated beneficial effects on vascular health. Resveratrol is known to regulate two important signaling pathways in endothelial biology: Notch and αVβ3 integrin signaling. Through luciferase reporter assays and western blot analysis we have demonstrated that trans‐resveratrol promotes Notch1 induced transcriptional activation, but does not show the same effect when siRNA is used to knockdown β3 integrin. This data suggests, that resveratrol is able to induce transcription of Notch target genes via an αVβ3 dependent mechanism. Using co‐immunoprecipitation and in vitro kinase assays, we have identified an interaction between Notch1 and SRC kinase, a principle intermediary of integrin signaling. Additionally, using site directed mutagenesis we have identified tyrosine residues within the transcriptionally active portion of Notch1 which are important for Notch activation. Having established these results, we turn our attention towards the importance of the Notch1‐αVβ3 integrin signaling axis in the major cellular hallmarks of atherosclerosis.Support or Funding InformationBoise State Biomolecular Sciences Ph.D. ProgramNIH #1R15GM102852‐01