bluuebunny/crossref_metadata_2025_split huggingface dataset:
Activity and Kinetic Characterization of Four Human CYP2D6 Polymorphisms using the Substrates Bufuralol and Dextromethorphan
Authors: Glass Sarah , Furge Laura Lowe 
Published: November 2023

Human cytochrome P450 enzymes (CYPs) are a heme‐containing enzyme superfamily that have a major role in the metabolism of drugs along with other endogenous and exogenous chemicals. Human CYP2D6 is responsible for approximately 12% of CYP‐mediated drug metabolism. There are over 100 different allelic variants of CYP2D6 with metabolic profiles ranging from poor to ultra‐rapid. Four CYP2D6 allelic variants, three with a small series of distal mutations (*34, *17‐2, *17‐3) and one possible ultra‐metabolizer (*53), were expressed and purified in E. coli to further characterize their interactions with the substrates bufuralol and dextromethorphan using HPLC with UV detection. Km and vmax values were measured for comparison between variants and the reference *1. Previous molecular dynamics simulations of these allelic variants with and without a ligand bound suggested that varied flexibility and active site accessibility altered interaction with substrates. The current metabolic and kinetic studies are compared to these molecular dynamics models to broaden the understanding of substrate interactions for CYP2D6 variants with local and distal mutations relative to the active site.Support or Funding InformationNIH 1R15‐GM086767‐02
Investigating the Mechanism of Wnt‐Induced Chromosomal Instability
Authors: Stiers Aubrey , Willert Karl 
Published: November 2023

Wnt signaling is essential for early embryonic development, the maintenance of stem cell pluripotency, determination of cell polarity, and cellular differentiation. Alterations in Wnt signaling activity have dramatic effects, resulting in developmental defects and contributing to the development of cancer. We recently found that perturbations in Wnt signaling lead to chromosomal instability (CIN), which results in aneuploidy, a hallmark of many cancer cells. However, we currently do not understand the mechanism by which Wnt promotes this CIN. We hypothesize that the protein p53 (gene name = TP53), often described as “the guardian of the genome”, acts downstream of Wnt to influence the fidelity of chromosome segregation. The goals of my research are to further study the effect of Wnt signaling on CIN and to explore the potential involvement of p53 in this process. To this end, I am utilizing DNA fluorescence in situ hybridization (FISH) as a means to detect and quantify the frequency of chromosome mis‐segregation across a population of cells. This method will allow me to detect changes in chromosome numbers in response to Wnt stimulation over time. In addition, I am creating a human embryonic stem cell line (H1/WA01) that will express fusion proteins composed of a fluorescent protein and either a histone (H2B) or tubulin. These transgene expressing H1 cells will be used to visualize mitosis and chromosome mis‐segregation. To address the involvement of p53, I will monitor both changes in gene expression of TP53 and changes in p53 protein abundance and modification, including phosphorylation and acetylation. Any observed changes in TP53 gene expression or p53 protein in response to Wnt signaling may contribute to CIN and hence to the occurrence of aneuploidy. These studies into the mechanism of Wnt‐induced CIN and the role of p53 will provide valuable insights into the development of cancer and may help identify a novel target of cancer therapeutics.Support or Funding InformationThis work is supported by a grant from the CIRM (RB1‐01406, to K.W.) and was made possible in part by the California Institute for Regenerative Medicine (CIRM) Major Facilities Grant (FA1‐00607) to the Sanford Consortium for Regenerative Medicine.
Transcriptional Changes in Aging Photoreceptor Cells of Drosophila melanogaster
Authors: Brennan Kaelan Joseph , Hall Hana , Weake Vikki Marie 
Published: November 2023

As the human population ages, there is an increased risk for ocular diseases such as age‐related macular degeneration (AMD), glaucoma, and diabetic retinopathy. Specifically, AMD is characterized by deterioration of the macula of the eye and is the leading cause of the loss of central vision in those individuals over 60. Twin studies have previously shown that non‐genetic, environmental factors contribute more than 50% of the overall risk for developing age‐related ocular diseases such as AMD. For this reason, epigenetic mechanisms have been thought to play a major role in the environmental‐based risks associated with AMD. The epigenetic factors that contribute to vision loss in the aging eye have yet to be identified, but are thought to play a pivotal role in maintaining proper ocular ability, as misregulation of these factors could lead to transcriptional changes in gene expression. We hypothesize that as photoreceptor cells age, there are changes in gene expression that contribute to the loss of vision seen later in life. Because age‐related changes in gene expression are cell autonomous, Drosophila melanogaster photoreceptor neurons were chosen as a model system due to the likelihood that these post‐mitotic cells are more sensitive to changes in gene expression. Photoreceptor cells are also likely to be at an increased vulnerability to the effects of aging because they must constantly express high levels of proteins involved in phototransduction in order to maintain their function throughout the adult lifespan. Using these Drosophila photoreceptors, we are conducting a study on the effects of aging on gene expression. Experiments to globally characterize the transcriptome of isolated nuclei from the R1‐R6 photoreceptor cells of male Drosophila were performed using RNA‐sequencing technology. We then used cluster analysis on this sequencing data to identify genes that exhibited statistically significant changes in gene expression at specific fly‐age time points. Using this cluster analysis data, we will prioritize genes from each cluster to generate subsets of genes to test through qRT‐PCR experiments on both dissected eye and isolated nuclear samples. This qRT‐PCR will allow us to validate and quantify the age‐related changes in gene expression observed by RNA‐sequencing in photoreceptor nuclei. From this, we can begin to ask questions about the role of epigenetic factors in regulating these changes in gene expression and how these factors are affected by aging.Support or Funding InformationNational Institutes of Health
Regulation of Protein Kinase R (PKR) activation during cell stress by TRBP phosphorylation
Authors: Chukwurah Evelyn Ekene , Patel Rekha C 
Published: November 2023

A crucial component of cellular stress response is the attenuationof protein synthesis by phosphorylation of the α‐subunit of the eukaryotictranslation initiation factor, eIF2. This allows the cell to mount anappropriate response to the stressful conditions, while minimizing energyexpended on protein translation. However, a prolonged eIF2α phosphorylation dueto sustained stressful conditions results in the induction of apoptosis.PKR is one of four well‐characterized serine‐threoninekinases responsible for mediating translation inhibition in response to viralinfection, endoplasmic reticulum (ER) stress, oxidative stress, and serumstarvation. During these conditions, PKR is activated via interaction either withdouble stranded RNA (dsRNA) generated during viral infections or with itscellular activator PACT (Protein Activator of PKR), resulting in itsautophosphorylation, activation of its catalytic function, and subsequent eIF2αphosphorylation.PKR's kinase activity is inhibited by the double stranded RNA binding protein TRBP (TAR RNA Binding Protein) by a direct interaction, as well as by sequestration of PKR's dsRNA activators and PACT, there by preventing PKR autophosphorylation and downstream protein translation inhibition. Various studies have linked dysregulation of PKR activation with various neuro degenerative and neuromuscular disorders.Recent findings indicate that TRBP also inhibits PKR inother cellular contexts (i.e. mitosis, obesity), but suggest that its abilityto inhibit PKR is dependent on various post‐translational modifications that significantly alter its binding affinity for PKR and ultimately affect PKRactivation in those contexts.We investigated changes in both PKR‐TRBP interactions and TRBP‐mediated PKR inhibition during cell stress. We used various biochemical methods to study TRBP phosphorylation, its interaction with PKR, and effect on PKR's kinase activity. Various phospho‐mimetic and phospho‐defective mutants of TRBP were used to examine the role of phosphorylation in mediating PKRinhibition. Our results indicate a functional role of TRBP phosphorylation inregulating PKR activation in response to cellular stress, and elucidate animportant mechanism by which the deleterious sustained response to stress is down regulated.
EXAMINING APOPTOSIS IN A SEPSIS CELL CULTURE MODEL
Authors: Davis Bethany , Del Gaizo Moore Victoria 
Published: November 2023

Sepsis, a systemic inflammatory response to infection of the blood by pathogenic organisms, impacts millions of people around the world every year. Pro‐inflammatory cytokines are released to fight the infection but many times lead to organ damage such as Acute Kidney Injury (AKI). AKI, a disorder characterized by the abrupt deterioration in kidney function, has a broad spectrum of consequences ranging from mild kidney injury to permanent loss in renal function and kidney failure. Common amongst critically ill patients, the development of AKI is believed to be due, at lease in part, to apoptosis. Apoptosis, or programmed cell death, is controlled by a multitude of cellular mechanisms. Cytokines can initiate the extrinsic pathway of apoptosis by binding to death receptors on the cell surface. While it is definitively known that apoptosis plays a large role in immune cell decline under septic conditions, its influence in AKI is not entirely understood. The objective of this study is to determine an efficient means of examining the role of cytokines in inducing apoptosis in renal cells in order to develop greater insight into the pathogenesis of sepsis‐associated AKI. Human immune THP1 cells were treated with a synthetic LPS to stimulate cytokine secretions. Cultured human embryonic kidney (HEK) cells were treated with either these naturally secreted or recombinant cytokines. Then flow cytometry of the HEK cells stained with Annexin V‐FITC determined the amount of apoptosis in the culture sample. Flow cytometry data demonstrates that recombinant cytokines allow for quicker analysis than naturally secreted cytokines. Apoptosis amongst HEK cells treated with recombinant cytokines for 48 hours was comparable to treatment with naturally secreted cytokines for a duration of 120 hours. Treatment with recombinant cytokines results in much quicker analysis because it excludes the time necessary for THP1 stimulation and cytokine recovery as well as significantly reduces the duration of treatment. Recombinant cytokines also have the benefit of easily picking precise concentrations of TNF‐alpha and other cytokines. With the greater understanding of the apoptotic processes associated with sepsis, the biochemical mechanism at the foundation of AKI can be further elucidated and the creation of more effective treatments for this disease may be possible.Support or Funding InformationElon University Undergraduate Research Program (BD), Elon University College Fellows Program (BD), FR&D (VM)
Site Specific Oxidation of Calmodulin by Methionine Sulfoxide Reductase A in Drosophila
Authors: TARAFDAR SREYA , Rusan Nasser M. , Levine Rodney L. 
Published: November 2023

Methionine sulfoxide reductase A stereospecifically catalyzes the reduction of S‐methionine sulfoxide to methionine and is important in defense against oxidative stress. Recently, we reported that methionine sulfoxide reductase A is also a stereospecific methionine oxidase, capable of oxidizing free and peptide‐bound methionine to S‐methionine sulfoxide. The control mechanism that prevents futile cycling is hypothesized to be through interaction with a postulated regulatory protein. Thus, cyclic oxidation and reduction of methionines in proteins could function as a redox‐based mechanism of cellular regulation. We also recently reported that methionine sulfoxide reductase A stereospecifically and selectively oxidizes Met77 in calcium‐bound calmodulin and can fully reduce it as well. Taking advantage of the powerful genetic tools of Drosophila, we seek to assess the physiological significance of methionine sulfoxide reductase A‐mediated reversible oxidation of calmodulin Met77. To accomplish this, we used site directed mutagenesis to generate transgenic animals carrying calmodulin mutated at Met77. Calmodulin Met77 was mutated to glutamine, alanine, leucine, or valine. Glutamine was included because it mimics methionine sulfoxide, thus creating a constitutively oxidized methionine sulfoxide state. We are currently analyzing the effect of these mutations on life span, fertility, sensation, and viability in a calmodulin null background.Support or Funding InformationThis research was supported by the Intramural Research Program of the NIH, NHLBI.
Lysophospholipid Receptors, As Novel Danger Associated Molecular Pattern Receptors And Homeostasis Associated Molecular Pattern Receptors Modulate Inflammation – Novel Paradigm And Therapeutic Potential
Authors: Wang Xin , Li Ya‐Feng , Fu Hangfei , Johnson Candice , Shao Ying , Cueto Ramon , Wang Hong , Yang Xiao‐Feng 
Published: November 2023

To determine whether lysophospholipids (LPLs) serve as danger‐associated molecular pattern molecules (DAMPs) in initiating inflammations, we analyzed the tissue expressions of all 26 LPL receptors in 23 human tissues and 21 mouse tissues. We also examined the expression changes of the 26 LPL receptors in five prototypic chronic sterile inflammations including rheumatoid arthritis (RA), metabolic inflammatory diseases such as coronary artery disease (CAD), metabolic syndrome, type 2 diabetes and type 1 diabetes. We made the following important findings: 1) Three LPLs including lysophosphatidylinositol (LPI), lysophosphatidylserine (LysoPS) and lysophosphatidylethanolamine (LPE) bind to two specific G protein‐coupled LPLs receptors (LPL‐GPCRs) while other four LPLs bind to more than five LPL‐GPCRs, respectively; 2) Lysophosphatidic acid (LPA), lysophingolipid sphingosine 1‐phosphate (S1P) and lysoyphosphatidylcholine (LPC) have ubiquitous physiological signaling functions among tissues whereas other LPLs, especially LPI, may have tissue‐specific functions; 3) The expressions of certain LPL‐GPCRs are significantly upregulated in human and mouse RA, CAD, and other metabolic inflammatory diseases; 4) Mechanistically, pro‐inflammatory cytokine signaling regulates the expressions of LPL receptors; 5) Toll‐like receptor (TLR), presumably bound by other DAMPs, regulates the expressions of LPL receptors; and 6) In cross‐talking, LPL receptor signaling regulates the expressions of TLRs; 7) We propose for the first time that, in parallel to the well‐accepted danger associated molecular pattern receptor model, novel homeostasis‐associated molecular pattern molecules (HAMPs) receptors (HAMPRs) are the receptors for binding to signals that are generated from endogenous metabolic processes and can initiate anti‐inflammatory/homeostatic signaling and promote inflammation resolution. From this point of view, we propose a new paradigm of that LPLs are either pro‐inflammatory DAMPs or anti‐inflammatory HAMPs for regulating the onset of inflammation, inhibition of inflammation, and resolution of inflammation. This new paradigm and new concept of HAMP receptors would significantly improve our understanding on the roles of LPLs and other endogenous metabolites in regulating the pathogenesis of inflammations and eventually lead to the future development of novel therapeutics for rheumatoid diseases, metabolic inflammatory diseases, cardiovascular diseases, and inflammatory cancers.Support or Funding InformationNIH RO1 HL 108910‐01
FTY720 Induces Necroptosis by Regulating Ceramide Signaling at the Plasma Membrane
Authors: Ndeto Rose , Ogretmen Besim 
Published: November 2023

Sphingolipids, important signaling molecules in cells, have recently been explored as cancer therapy targets. FTY720 (Fingolimod, Gilenya) is an FDA approved sphingosine analogue drug used for the treatment of multiple sclerosis (MS). FTY720 is phosphorylated by sphingosine kinase 2 (SK2), to generate P‐FTY720 to exert its immunosuppressive properties through binding to sphingosine 1‐phosphate receptors (S1PRs). FTY720 also exhibits anti‐cancer properties. Our previous studies indicated that one of the mechanism by which FTY720 induces cell death is through necroptosis. FTY720, similar to ceramide, directly binds to I2PP2A/SET (Inhibitor 2 of PP2A), consequently activating the tumor suppressor protein phosphatase 2A (PP2A). The activated PP2A then induces cell death by stimulating the activity of Receptor‐Interacting Protein Kinase‐1 (RIPK1), involved in necroptosis signaling. It is known that FTY720 can modulate sphingolipids metabolism in cells. However, little is known about the roles of FTY720 in ceramide signaling and regulation of necroptosis in lung cancer. We hereby seek to investigate the mechanisms of FTY720 in inducing necroptosis with regard to ceramide signaling. Preliminary cell viability data indicate that inhibitors of ceramide generation partially protect cells against FTY720‐induced cell death. Interestingly, using confocal microscopy, we show that FTY720 and its non‐phosphorylatable analogues lead to the formation of specific ceramide‐multi‐protein complexes at the plasma membrane involved in plasma membrane disruption for induction of necroptosis. Future mechanistic studies will help us understand the details of how these ceramide‐dependent lipid‐protein complexes are formed at the plasma membrane and how they are involved in the regulation of necroptosis in response to cellular stress evoked by FTY720 and other therapeutic agents, leading to tumor suppression.Support or Funding InformationRO1CA088932, RO1CA173687, P30GM103339
The lipid transfer protein STARD3 modulates intracellular cholesterol localization
Authors: TOMASETTO Catherine Laure , WILHELM lea , DRIN Guillaume , ALPY Fabien 
Published: November 2023

STARD3 [(StAR)‐related lipid transfer domain protein 3] belongs to the START domain protein family, a group of protein having lipid transfer properties. STARD3 (also known as MLN64 or CAB1), is overexpressed in ~25% of human breast cancer. On cancer tissue sections, we noted that STARD3 labelled large vesicles often at a perinuclear position. Consistently, we found that STARD3 is a resident protein from late‐endosomes (LE). Structurally, STARD3 is a multifunctional protein composed several conserved regions. The N‐terminal moiety called the MENTAL domain is anchored in the late endosome (LE) membrane and mediates oligomerization and cholesterol binding. At the C‐terminal end of this domain, STARD3 contains a FFAT‐like motif interacting with VAP proteins. The C‐terminal half of the protein bears a cytoplasmic START domain involved in cholesterol transfer. By interacting with the endoplasmic reticulum resident proteins VAPs, STARD3 creates membrane contact sites between endosomes and the endoplasmic reticulum, and remarkably remodels the subcellular architecture. We recently addressed how STARD3 expression modulates cholesterol localization within the cell. Using cholesterol‐specific fluorescent probes, we noted the presence of cholesterol‐enriched endosomes at the expense of other cellular compartments in STARD3 positive cells. Of interest, this STARD3‐dependent intracellular cholesterol redistribution relies both on the activity of the START domain and on the presence of membrane contact sites.Support or Funding InformationThe Institut National du Cancer ‐INCA (France) and The Ligue Nationale Française contre le Cancer
Integration of Undergraduate Research into the Biomolecular Science Curriculum Using Thematically Linked Laboratory Courses in Biochemistry, Chemical Biology, and Neurobiology
Authors: Johnson R. Jeremy , Hoops Geoffrey , Kowalski Jennifer 
Published: November 2023

Participation in undergraduate research increases student interest and retention in science, recruits students into scientific careers, and instills greater self‐confidence in research skills. With these measurable benefits, incorporating greater authentic, research‐based learning into undergraduate science education has become a major thrust of undergraduate laboratory education. In this presentation, we will describe the introduction of three thematically connected, research‐based laboratories into the molecular science curriculum at Butler University. In these courses, students first synthesize and characterize novel fluorescent imaging agents, use these imaging agents to catalogue the substrate specificity of serine hydrolases from Mycobacterium tuberculosis, and then perform parallel methodologies to measure neural signaling in Caenorhabitis elegans. These three linked courses expose undergraduate students to a diverse set of modern methodologies, increase the level of student inquiry throughout the semester, and culminate in student led undergraduate research projects. The introduction of open‐ended, hypothesis driven research has increased undergraduate student exposure to and participation in scientific research, challenged students to integrate multiple scientific disciplines within the context of a larger scientific investigation, and benefited faculty scholarship output. We will provide an overview of the experimental design for each course, present students’ experimental results, describe measurable student learning gains, and provide examples of how student‐led independent projects have been transitioned into publishable scientific research.Support or Funding InformationFunding provided by an NSF TUES Award (DUE‐1140526).