bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two Distinct Regulatory Mechanisms of Transcriptional Initiation in Response to Nutrient Signaling
Authors: Barman Priyanka , Ferdoush Jannatul , Sen Rwik , Kaja Amala , Bhaumik Sukesh R. 
Published: June 2021

SAGA (Spt‐Ada‐Gcn5‐Acetyltransferase) and TFIID (transcription factor IID) promote the formation of the PIC (pre‐initiation complex) at the promoters of two distinct sets of genes for transcriptional initiation. However, it is unknown whether SAGA and TFIID are involved in the PIC formation at the same gene. Interestingly, we find that TFIID and SAGA differentially participate in the stimulation of PIC formation (and hence transcriptional initiation) at the promoter of PHO84, a gene for the high‐affinity inorganic phosphate (Pi) transporter for crucial cellular functions, in response to nutrient signaling. Thus, a gene can be regulated by both TFIID and SAGA. We show that transcriptional initiation of PHO84 occurs predominantly in a TFIID‐dependent manner in the absence of Pi in the growth medium. Such TFIID dependency is mediated via the NuA4 (nucleosome acetyltransferase of H4) histone acetyltransferase (HAT). Intriguingly, transcriptional initiation of PHO84 also occurs in the presence of Pi in the growth medium, predominantly via the SAGA complex, but independently of NuA4 HAT. Thus, Pi in the growth medium switches transcriptional initiation of PHO84 from NuA4‐TFIID to SAGA dependency. Further, we find that both NuA4‐TFIID‐ and SAGA‐dependent transcriptional initiations of PHO84 are facilitated by the 19S proteasome subcomplex or regulatory particle (RP) via enhanced recruitment of the coactivators SAGA and NuA4 HAT, which promote TFIID‐independent and ‐dependent PIC formation for transcriptional initiation, respectively. NuA4 HAT does not regulate activator binding to PHO84, but rather facilitates PIC formation for transcriptional initiation in the absence of Pi in the growth medium. On the other hand, SAGA promotes activator recruitment to PHO84 for transcriptional initiation in the growth medium containing Pi. Collectively, our results demonstrate two distinct stimulatory pathways for PIC formation (and hence transcriptional initiation) at PHO84 by TFIID, SAGA, NuA4, and 19S RP in the presence and absence of an essential nutrient, Pi, in the growth media, thus providing new regulatory mechanisms of transcriptional initiation in response to nutrient signaling. Further, we find that proteolytic function of the proteasome is also involved in transcriptional regulation of PHO84.Support or Funding InformationNIH 2R15GM088798‐01, NIH 2R15GM088798‐02 and NIH 2R15GM088798‐03This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating Conditional Mutations in MTR4: an RNA Helicase Associated with the Nuclear Exosome
Authors: Isenhour Anthony , Fulop Hannah , Pingali Hema , Hilliker Angie 
Published: June 2021

The nuclear exosome is a complex of proteins in the nucleus that processes and degrades various RNAs. In doing so, it associates with various other proteins, including Mtr4, an RNA helicase that processes and degrades RNA. MTR4 is an essential gene conserved from yeast to humans. Mtr4 associates with various complexes, such as TRAMP and the nuclear exosome, to degrade aberrant RNAs and process RNAs such as the 5.8s rRNA and the U4 snRNA (1, 2). The ATPase region of Mtr4 has been shown to be essential, but the N‐ and C‐termini are not fully characterized (3). Current questions about MTR4 focus on how Mtr4 chooses which RNAs to process and degrade, as well as what regions of the protein affect said functions.Through random mutagenesis of the N‐ and C‐ termini of Mtr4, we selected for conditional mutants that are phenotypically defective only at high temperature. Because Mtr4 is essential, conditional mutants serve as in vivo tools to investigate how minor alterations to MTR4's protein sequence affect the cell. We have generated five C‐terminal conditional mutants, three N‐terminal conditional mutants, and a large library of lethal mutants. Through chemiluminescent northern blotting we have been able to confirm that our C‐terminal mutants are not defective in 5.8s rRNA processing. N‐terminal mutants testing is still underway, as well as U4 processing tests. Understanding the integral residues of Mtr4 will allow us to greater understand how it processes some RNAs and degrades others.Support or Funding InformationNIH R15 GM128068‐01; Arts & Sciences (UR)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Towards the Elucidation of Novel Viral Factors that Hijack Host Ubiquitination Pathways
Authors: Lippa Geoffrey Michael , Muehl Madison , Lapham Joshua 
Published: June 2021

Viruses, like human immunodeficiency virus (HIV), have the ability to hijack host pathways to promote disease pathogenesis. The process of ubiquitination is used by the cell for regulating signal transduction, DNA replication, cellular development, transcription, and the cell cycle. Ubiquitination and the proteasome work together to degrade a variety of cellular target proteins involved in the processes described. The covalent attachment of a ubiquitin protein to a cellular substrate requires a multi‐protein complex consisting of a scaffold protein, adaptor module, substrate receptor, RING protein, and E2 protein. In HIV, the Cullin‐RING E3 ligase complex utilizing Cullin5, Rbx2, E2, Elongin B, Elongin C, and Vif (viral infectivity factor), amongst other accessory proteins results in the targeted degradation of antiretroviral factor APOBEC3G. Other viruses have followed suit countering these natural processes by expressing viral proteins capable of sequestering or enhancing host ubiquitination conducted by CRL complexes. Research efforts have been made in discovering both host proteins targeted in this fashion and those viral proteins responsible for this aberrant activity. Through bioinformatics and structural analysis new potential viral proteins involved in the above CRL complex have been discovered. These targets are predicted to play a role in viral fitness for Hepatitis C virus (NS5A), West Nile Virus (NS1), and Lassa virus (Z‐protein) due to their similarities with the host substrate receptor SOCS2. We are currently in the process of co‐expressing parts of the CRL complex with these viral targets in E. coli with the goal of evaluating their interactions. Utilizing in vitro pull‐down assays and thermodynamic techniques we hope to verify the viral proteins involved in the CRL complex and their mechanisms of interaction laying the groundwork for new avenues of anti‐viral therapeutics.Support or Funding InformationFunding was achieved through Alfred Research Grants for Undergraduate Research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Detection of Protein Palmitoylation Via Click Chemistry and Flow Cytometry
Authors: Sime Drake , Wilander Benjamin , Hamann Michael J 
Published: June 2021

Palmitoylation is the process of attaching palmitate groups to protein cysteine residues. Many proteins have dynamic palmitoylation modifications that are added and removed to regulate protein function. Techniques to study protein palmitoylation, however, are slow and difficult to perform. We are in the process of developing a rapid technique for measuring protein palmitoylation using the palmitate analogue17‐ODYA and click chemistry followed by flow cytometry. To work out assay conditions, the Rho family GTPase TC10 is being used as a positive palmitoylation control, as it is known to be palmitoylated on its C‐terminal tail. To perform the analysis, 17‐ODYA is added to cells transfected with YFP‐tagged versions of TC10, and lysates from the cells are treated with click chemistry reagents to selectively conjugate the terminal alkyne group of 17‐ODYA with Alexa‐647 azide. The protein is isolated with magnetic anti‐FLAG beads which then are analyzed for YFP and Alexa‐647 fluorescence using flow cytometry. The current results indicate the method may be effectively detecting palmitoylation of TC10; however, the results will need to be validated using Western blotting as well as TC10 mutants that are incapable of being palmitoylated. Overall, the method will significantly enhance the time required to detect protein palmitoylation compared to traditional blot‐based techniques.Support or Funding InformationThis work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Insight into the recognition mechanism of EcoRV
Authors: Zahran Mai , Imhof Petra 
Published: June 2021

EcoRV, a restriction enzyme in Escherichia coli, destroys invading foreign DNA by cleaving it at the center step of a GATATC sequence. In the EcoRV–cognate DNA crystallographic complex, a sharp kink of 50° has been found at the center base‐pair step (TA). In a previous research, we have examined the interplay between the intrinsic propensity of the cognate sequence to kink and the induction by the enzyme, and have understood the mechanism of action of the enzyme and the intrinsically behavior of the cognate sequence to undergo a kink allowing the proper placement of the atoms for cleavage. We have also demonstrated that the enzyme requires 2 magnesium ions at the catalytic site to perform the cleavage.In the present study, we perform multiple independent molecular dynamics simulations to investigate the behavior of a mutant EcoRV (E45A), in the presence of one or two magnesium ions and complexed to three DNA sequences: the cognate sequence, GATATC (TA); the non‐cognate sequence, GAATTC (AT); and with the cognate sequence methylated on the first adenine GACH3TATC (TA‐CH3). The results give further insight into the recognition and cleavage mechanism of EcoRV.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Amyloid‐Beta Mediated Pro‐Oxidative Effects in Human Retinal Cells Involve the Small GTPase Rac‐1: Potential Therapeutic Role of FTI277 for Age‐Related Macular Degeneration
Authors: Owens‐Walton Jeunice , Thounaojam Menaka , Gutsaeva Diana , Bartoli Manuela 
Published: June 2021

There is a need for new therapies for age‐related macular degeneration (AMD), the leading cause of blindness in elderly. Fibrils of amyloid beta (Aβ), a causative factor of Alzheimer's disease (AD), have been identified among the constituents of “drusen”, yellowish deposits located in the retinal pigment epithelium (RPE) of AMD patients and considered diagnostic hallmarks of the disease. Increased production of reactive oxygen species in RPE is in important pathogenic step for AMD and involves NAD(P)H oxidases such as NOX2 and NOX4. Here we have conducted a study analyzing the effects of oligomers of Aβ (1–42) in inducing oxidative stress in human retinal pigment epithelial cells (HRPE) and evaluated the role of the small GTPase Rac‐1, required for NOX2 and 4 activation, in this process. Using a Cell‐ROX assay we found that Aβ (1–42) (5mM for 12hours) stimulated the formation of reactive oxygen species (ROS) in HRPE. Aβ (1–42)‐promoted ROS production and contemporaneous activation of Rac‐1, measured by translocation of the protein to the plasma membrane (cell fractionation and Western blotting) and by increased ratio of the GTP bound versus the GDP bound form of the protein (assessed by immunoprecipitation). Same doses of the inverse peptide Aβ (42‐1), used as negative control, had no effects. Activation of Rac‐1 is preceded and dependent on farnesylation. Treatment of the cells with the farnesyl transferase inhibitor FTI‐277 (25mM) blocked Aβ (1–42)‐induced activation of Rac‐1 and consequent ROS formation in HRPE. In summary, our results suggest a role for Aβ in promoting oxidative stress and dysfunction of the RPE, two effects seen during AMD. In addition, we have identified FTI‐277 as a potential new therapy for AMD because on its inhibitory effects towards Rac‐1.Support or Funding InformationSupported by the Medical Scholars Program, Medical College of Georgia, Augusta, GA.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of the nuclease resistance of intramolecular DNA four‐way junction composed of phosphorothioate bonds
Authors: Klein Mitchell , Parker Elliott , Troisi Maria , Smith Samara , Wade Elizabeth , Moorehead Gaston , Bell Anthony Jerome 
Published: June 2021

Oligodeoxynucleotides (ODN) offer great promise as therapeutics due to their high binding specificity, low toxicity and ease of synthesis. Hence, it is not surprising that ODNs are being developed as antigene and antisense reagents. ODNs can also target proteins to control gene expression. One strategy uses double stranded DNA as “decoy” sequences that are directed against transcription factors such as NF‐kB and specificity protein 1. More recently, researchers have used a similar approach to target proteins in the extracellular matrix. In these studies, cruciform and bent DNA are directed against the damage‐associated molecular pattern molecule, High Mobility Group B1 (HMGB1). We hypothesize that immobilized DNA four‐way junctions (4WJs) can target HMGB1. To enhance the stability of immobilized 4WJs for ex vivo studies, the helical termini are modified to generate the intramolecular constructs. Phosphorothioate (PS) bonds are inserted into the 4WJ lattice to enhance nuclease resistance. In this study, the nuclease resistance of five intramolecular 4WJs and the DNA control, J1 are evaluated. J1 is a classic immobilized 4WJ that is composed of four asymmetric strands of DNA. Three nucleases are used: DNase I, Exonuclease III (Exo III) and bacteriophage T5 Exonuclease (T5 Exo). DNase I is an endonuclease that cleaves dsDNA and ssDNA in a largely non‐specific manner. Exo III has expanded functionally. Here, we focus on the 3′®5′ exonuclease activity of the enzyme. T5 Exo hydrolyzes dsDNA and ssDNA in a 5′®3′ direction. The nuclease protection data shows that each intramolecular 4WJ has significantly higher nuclease resistance values than J1 against DNase I and Exo III. Circular dichroism studies are being developed to investigate the minor groove binding properties of J1 vs. the intramolecular 4WJs. We hypothesize that perturbations within the minor groove of intramolecular 4WJs may enhance the nuclease resistance of these constructs vs. J1.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure‐Activity Relationships of Chalcone Derivatives as Inhibitors of Breast Cancer Cell Growth
Authors: Neal Hadley E. , Stantliff Trevor M. , Harrod Tighe , Hill Amber , Barrios Francis , Krzysiak Amanda 
Published: June 2021

Chalcones are polyphenols composed of two aromatic rings linked by an aliphatic three carbon chain. Chalcones and their derivatives have been shown to have numerous types of biological activity including anti‐inflammatory, antioxidant, anti‐infective, antiviral, anticancer and antitumor. Previous work in our lab discovered that anti‐cancer activity benefitted from electron donating substitutions to the B ring. Here, we are exploring the flexibility of the bridge structure using tetralone and indanone to generate a more rigid link between the aromatic rings. Using tetralone and indanone combined with benzaldehydes bearing carious electron donating groups a diverse library of chalcones was generated. These different compounds were first tested in noncancerous HEK 293 cells and have continued being tested in breast cancer cells (MCF7). An initial screening assay is performed at 50 μM. Compounds showing fifty percent or greater inhibition are taken on for more detailed analysis to determine an IC50. In both cases, cells are counted and seeded at specific densities, followed by drug treatment forty‐eight hours later, then cell viability is measured using an MTS assay on a visible spectrophotometer forty‐eight hours after drug treatment. Our results indicate the flexibility is an important feature for biological activity.Support or Funding InformationBellarmine University Chemistry and Physics DepartmentThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In silico Prediction of Immunogenic Sites on PMN Antigen and Design of Epitope Blocking Caps
Authors: Welsh Colin L , Hayes Candace A , May Xavier A , Stoddard Shana V 
Published: June 2021

Autoimmune disorders (AD) are a type of disease in which an individual's immune system attacks the cells of its own body. Currently, AD are the 2nd leading cause of chronic long term illness. The primary method of treating AD is the use of non‐specific immunosuppressant drugs, suppressing the entire immune system and leaving the patient vulnerable to infections. Antigen specific therapies have been proposed in order to replace treatment with immunosuppressant drugs for AD. The work here focuses on developing a new approach to AD therapy which targets the epitope site (ES) on the antigen, with an emphasis on the kidney specific AD primary membranous nephropathy, which affects 10–12 million people worldwide. Using in silico mutagenesis the ES on the phospholipase A2 receptor (PLA2R) were predicted and epitope binding proteins (EBP) were developed to target these regions. Using Epitopia and EPCES two regions on the CTLD1 domain of PLA2R were identified as potential ES. These ES are characterized in terms of the number of residues, their electrostatic potential, and their hydrophobicity. In silico prediction of immunogenicity showed the single nucleotide polymorphisms M292V, H300D, and G1106S do not directly contribute to immunogenic sites on PLA2R. In silico mutagenesis of two protein templates (5IMK and 4JE4) was performed to design EBP structures. The Rosetta protein‐protein docking server was used to evaluate the interface score of designed EBP to the PLA2R antigen, and disruption of binding to their original binding partner via molecular mechanics calculations. In silico mutations to the 5IMK monobody increased the binding interface score from −4.259 rosetta energy units (REU) to −8.903 REU. Similarly, in silico mutations to the 4JE4 monobdy increased the interface score from −3.964 REU to −5.867 REU. Data shows the introduction of charged residues to the monobody contributes to the most significant gains in interface score. Further in silico mutations were introduced to disrupt the binding preference of 5IMK and 4JE4 to their original targets. Binding potency was reduced from −7.851 REU to −3.87 REU for 5IMK, and from −12.263 REU to −3.693 REU for 4JE4. This work could be used to develop a new antigen specific therapy approach for PMN and AD in general.Support or Funding InformationWilliam Randolph Hearst FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparative transcriptome profiling of genes and pathways related to acquired and intrinsic resistance to BRAF inhibitor
Authors: Lee Michael , Hwang Sung‐Hee 
Published: June 2021

Melanoma cells have been shown to respond to BRAF inhibitors; however, intrinsic and acquired resistance limits their clinical application. In this study, we performed RNA‐Seq analysis with BRAF inhibitor‐sensitive (A375P) and ‐resistant (A375P/Mdr with acquired resistance and SK‐MEL‐2 with intrinsic resistance) melanoma cell lines, to reveal the genes and pathways potentially involved in intrinsic and acquired resistance to BRAF inhibitors. A total of 546 differentially expressed genes (DEGs), including 239 up‐regulated and 307 down‐regulated genes, were identified in both intrinsic and acquired resistant cells. Gene ontology (GO) analysis revealed that the top 10 biological processes associated with these genes included angiogenesis, immune response, cell adhesion, antigen processing and presentation, extracellular matrix organization, osteoblast differentiation, collagen catabolic process, viral entry into host cell, cell migration, and positive regulation of protein kinase B signaling. In addition, using the PANTHER GO classification system, we showed that the highest enriched GOs targeted by the 546 DEGs were responses to cellular processes (ontology: biological process), binding (ontology: molecular function), and cell subcellular localization (ontology: cellular component). Ingenuity pathway analysis (IPA) network analysis showed a network that was common to two BRAF inhibitor‐resistant cells. Taken together, the present study may provide a useful platform to further reveal biological processes associated with BRAF inhibitor resistance, and present areas for therapeutic tool development to overcome BRAF inhibitor resistance.Support or Funding InformationSupported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2016R1D1A1B03930193)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Black Crappie Sarcoma: Searching for a Piece of the Puzzle
Authors: BOYD KAYLA 
Published: June 2021

Black Crappie Sarcoma (BCS) is a disease primarily affecting fresh water lake systems in western Wisconsin. It manifests in the form of a tumor in front of the pelvic fin on black crappies, Pomoxis nigromaculatus. A defining characteristic are lesions on the body that are also found on the pectoral, anal, and caudal fins. The cause of the sarcoma and its complete effects on the body of the infected fish is unknown. Past efforts in revealing any information about BCS have yielded little to no results, in part because of the complexity of the pathogen. Lack of information is causing a rise in concern for lake residents as the disease appears to be spreading, although the route of transmission is unclear. The primary goal of the study is to investigate differential protein expression in sick fish tissue including muscle, heart, and spleen. These tissues were selected because damage had been reported in those areas on infected fish in past investigations. This study also explores techniques for protein extraction from fish organ tissue. The findings in this study will provide evidence of a molecular change in infected black crappies and will provide future ichthyologists the means to perform proteome studies more efficiently and effectively when examining disease.Support or Funding InformationSupported by donations from Bone Lake Management District, Polk County Association of Lakes and Rivers, Lake Wapogasset/Bear Trap Improvement Association, Pipe & North Pipe Lake Rehabilitation and Protection District, and private donationsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.