bluuebunny/crossref_metadata_2025_split huggingface dataset:
In silico exploration of the mechanism of translesion synthesis by DinB
Authors: Perez Elevit , Benitez Benedetta Sampoli 
Published: November 2023

In prokaryotic organisms, environmental stress elicits the up‐regulation of a variety of genes involved in the so‐called SOS response. One major aspect of this response is the increase of mutation frequency. Some of the mutations may confer unique characteristic to the organism that will be beneficial for adapting to the environment, therefore accelerating evolution during stress. Y‐family DNA polymerases are a class of error‐prone polymerases able to perform DNA translesion synthesis. E. coli DNA Polymerase IV (also called DinB from the name of the gene) is a Y‐family DNA polymerase implicated in stress‐induced mutagenesis. As other DNA polymerases, DinB has the general architecture of a right hand, with palm, thumb and fingers subdomains. In addition, Y‐family polymerases have an extra domain, namely the “little fingers” and have a more solvent accessible active‐site than their high‐fidelity replicative counterparts. Kinetics studies have shown that DinB is particularly efficient at bypassing damaged guanosines, such as the N2‐furfuryl‐dG, and even more efficient in lesion bypass on damaged DNA than in replication of undamaged DNA. Recent hydrogen exchange experiments have suggested that the protein undergoes a conformational change in the presence of certain DNA damages on the templating strand, but not others, implying a possible mechanism of bypass discrimination. However, the exact nature of such movement is not well characterized. Using molecular dynamics (MD) simulations we explored different DNA/protein/incoming nucleotides combinations to dissect the protein conformational change. We were also able to identify the important residues implicated in this process. All systems were prepared starting with the available crystal structure, solvated and equilibrated using CHARMM‐GUI. MD simulations were run for 10 ns with the NAMD simulation package and multiple runs were done for the same system to ensure reproducibility of the data.Support or Funding InformationThis work was partly funded by the Rose M. Badgeley Residuary Charitable Trust.
Eukaryotic translation initiation factor eIF4G utilizes different domains to coordinate with eIF4A, eIF4B and eIF4E in binding 3′ Cap‐independent translation element of Barley Yellow Dwarf Virus
Authors: Zhao Pei , Goss Dixie 
Published: November 2023

Barley Yellow Dwarf Virus (BYDV) lacks a 5′ (7‐methyl guanosine) cap as well as a 3′poly A tail. Like many plant viruses, BYDV contains a cap independent translation element (CITE) in the 3′ untranslated region of the viral mRNA. BTE (Barley Yellow Dwarf Virus like cap‐independent element) is one of the well characterized CITEs. BTE mediated translation primarily depends on eukaryotic initiation factor eIF4G. BTE binds to eIF4G; however, the details of BTE initiated translation are still unclear. Three eIF4G deletion mutants with different domain organization were used to investigate BTE interaction with eIF4G: eIF4G601‐1196 is the eIF4G truncated fragment containing amino acids from 601 to 1196, including eIF4E, central eIF4A, eIF4B binding domains and the possible BTE binding region; eIF4G601‐1488 is a longer fragment with one additional C‐terminal eIF4A binding domain; eIF4G742‐1196 is a shorter deletion mutant lacking the eIF4E binding sequence. eIF4G601‐1196 binds BTE as efficiently as wild type eIF4G and supports translation. Translation initiation factor eIF4A and eIF4B with ATP (helicase complex) stimulate eIF4G601‐1196 binding with BTE but not eIF4G601‐1488, suggesting that the helicase complex function relies on the eIF4G central eIF4A binding domain, not the C‐terminal eIF4A binding domain. eIF4E, upon binding with eIF4G mutants which have eIF4E binding region, significantly increases the binding to BTE. These data suggest eIF4E has a significant role in eIF4G‐RNA binding even in the absence of the cap structure.Support or Funding InformationNSF MCB 1513737
CHARACTERIZATION OF THE HIV‐1 5′UTR DIMERIZATION MECHANISM
Authors: Santos Justin Leonel Cruz , Frank Heather , McCowin Sayo , Sciandra Carly , Keane Sarah C , Summers Michael F 
Published: November 2023

Human Immunodeficiency Virus Type‐1 (HIV‐1) is responsible for a pandemic that affects roughly 35 million people worldwide. To date, there is no cure, and the array of antiretroviral medications serves only to diminish the progression of the disease and prevent a more rapid deterioration of the immune system. Many antiretrovirals target four main stages of the viral life cycle: entry, reverse transcription, integration, and maturation. Genome selection for packaging, however, is relatively less clearly understood, thus no current therapies that target this process have been developed. HIV‐1 genome packaging has been shown to be a highly selective process in which only dimeric, unspliced, genomes are recognized by the nucleocapsid (NC) domain of the Gag polyprotein for encapsidation. The nature of dimerization, which is mediated by elements within the 5′ untranslated region (5′‐UTR) of the genome, is hypothesized to be either a “kissing” interaction involving the six nucleotide palindromic (GCGCGC) dimerization initiation site (DIS) sequence, or an extended dimer conformation featuring a more extensive intermolecular interface. Our studies aim to elucidate the dimerization mechanism using NMR spectroscopy. Previous studies in the Summers laboratory identified that the HIV‐1 5′‐UTR exists as an extended dimer at high concentrations and after long incubation times at 37 °C. The U5:AUG region, which is distal to the DIS, has been identified as one of the sites with an intermolecular interface. We hypothesize that dimer formation is initiated by kissing interactions and transitions to an extended conformation over time. We utilized NMR spectroscopy to test this hypothesis using a mutagenesis strategy know as long‐range Adenosine Interaction Detection (lr‐AID). 1D NMR data collected on small U5:AUG control RNAs containing either UUA/UAA or UUG/UAA lr‐AID signals revealed isolated chemical shifts at 6.5 ppm, and 6.7 ppm, respectively. We incorporated the UUA/UAA and the UUG/UAA sequences within the U5:AUG region of the 5′‐UTR and observed the appearance of a chemical shift at 6.5 ppm in a biologically relevant time frame. Currently, we are conducting time‐dependent dimerization studies using native gel electrophoresis techniques in order to further characterize this transition.Support or Funding InformationThis research was funded by NIH/NIGMS grant 1P50GM103297 and sponsored by NIH/NIGMS MARC U*STAR T34 08663 National Research Service award to UMBC. It was conducted at the Howard Hughes Medical Institute at UMBC, with support from the Summer Biomedical Training Program (SBTP). Supported in part by the Howard Hughes Medical Institute's Precollege and Undergraduate Science Education Program.
The JAK‐STAT pathway is regulated by miRNA mediated Ribosomal Frameshifting
Authors: Dinman Jonathan D. , Khan Yousuf A. , Advani Vivek M. , Flickenger Zachary R 
Published: November 2023

Programmed −1 ribosomal frameshifting (‐1 PRF) is a translational recoding mechanism whereby a translating ribosome is induced to slip one nucleotide in the 5′ direction by a complex mRNA cis‐acting element (1). In the context of cellular mRNAs, such events overwhelmingly direct ribosome to premature termination codons (PTC), rendering these mRNAs substrates for the nonsense mediated mRNA decay (NMD) pathway (2). Approximately 10% of all human genes have at least one predicted significant −1 PRF motif, suggesting that −1 PRF is extensively used to regulate gene expression in human cells (3). Here we have characterized the −1 PRF signals in the mRNAs encoding human Janus Kinase 2 (JAK2) and Signal Transducer and Activator 1 (STAT1), both of which are involved in JAK/STAT intracellular signaling. Efficient −1 PRF by these sequence elements was confirmed both genetically and biochemically. Preliminary data suggests that JAK2‐mediated −1 PRF is inhibited by two miRNAs in a sequence specific manner. Furthermore, Jak2‐mediated −1 PRF appears to be involved in a regulatory feedback loop with cytokine signaling. Studies are underway to characterize the role of −1 PRF and the miRNAs in regulating the JAK‐STAT pathway. This study will serve to understand the role of translational recoding in immune signaling.Support or Funding InformationNIH R01HL119439, NIH R01GM117177
Effect of 5‐Azacytidine on Micro‐RNA Levels in the HL‐60 Cell Line
Authors: Gillard Mitchell , Stafford Nicholas , Militello Kevin , O'Donnell Robert W. 
Published: November 2023

The abnormal expression of four micro‐RNAs in patients with acute myeloid leukemia correlates with poor clinical outcomes for these patients. Working with the HL‐60 acute promyelocytic leukemia cell line, an AML subtype, the first goal of this project was to determine if the HL‐60 cell line expressed micro‐RNAs associated with myeloid leukemogenesis. Using a micro‐RNA isolation kit and qRT‐PCR, we were able to detect the presence of miR‐223, one of the four major micro‐RNAs associated with myeloid leukemogenesis. The three other micro‐RNAs known to be involved in myeloid leukemogenesis, miR‐155, miR‐29b‐1 and miR‐196b, were near the limit of detection of the assay. After determining that miR‐223 was stably expressed in the HL‐60 cell line, the next goal was to test whether 5‐azacytidine, a DNA methylation inhibitor and effective chemotherapeutic agent for AML, could alter the expression of miR‐223. We found that increasing concentrations of 5‐azacytidine affected the levels of miR‐223 expression. Using a derivative of 5‐azacytidine, 5‐aza‐2′‐deoxycytidine, the final and ongoing goal of this experiment is to determine whether the effects of 5‐azacytidine on micro‐RNA expression in the HL‐60 cell line are the result of blocking DNA methylation, blocking RNA methylation, or by a novel mechanism. We believe that these series of experiments will provide greater insight into the role of micro‐RNAs in cancer and help determine whether their inhibition with 5‐azacytidine is clinically relevant.Support or Funding InformationSUNY Geneseo Department of Biology
Translational recoding in human disease
Authors: Advani Vivek M , Kobylarz Ryan C , Dinman Jonathan D. 
Published: November 2023

Translational recoding is emerging as an important post‐transcriptional regulatory mechanism. Prior studies from our laboratory have explored how Programmed −1 Ribosomal Frameshifting (−1 PRF) fits within this paradigm. Computational analysis predicts ~10% of the genes in humans have at least one functional −1 PRF signal which translates to 1,943 high probability candidates in the human genome. Analysis of predicted −1 PRF signals across 20 genomes suggests that it is a universal molecular mechanism1, and demonstrate that >99% of such events function as mRNA destabilizing elements through at least two pathways2,3. In published work we have explored the biological significance of the connection between −1 PRF and NMD on telomere maintenance in yeast4 and the immune response in humans5. Here, we are exploring how abnormalities in ribosome function are implicated in both congenital and acquired syndromes, broadly classified as ‘ribosomopathies’, contribute to disease pathogenesis. Preliminary findings using yeast and human cell‐based models for X‐linked Dyskeratosis Congenita (X‐DC), Spinocerebellar ataxia 26 family (SCA26) and Diamond‐Blackfan anemia (DBA) as models suggest that these genetically inherited defects are caused by translational fidelity defects, with attendant effects on mRNA abundance and gene expression. The observations that global dysregulation of −1 PRF has deleterious effects on gene expression leads us to hypothesize that −1 PRF plays an important role in regulating cellular gene expression. Through the First‐year Innovative and Research Experiences (FIRE) ‐ Found in Translation (FIT) Undergraduate Research Program at the University of Maryland, we have validated functional −1 PRF signals in over 40 human genes, involved in processes including immune signaling, translation initiation, cancer, anemias and other diseases. This study will increase our understanding of how defects in the interplay between mRNA and ribosomes contribute to human disease.Support or Funding InformationNIH R01 HL119439, NIH R01 GM117177
A Novel Lysosomal GPCR Sorting Pathway Regulated by Alpha‐arrestins and ALIX
Authors: Dores Michael Robert , Lin Huilan , Trejo JoAnn 
Published: November 2023

Endocytic sorting and lysosomal degradation is critical for the turnover of transmembrane receptors and the attenuation of receptor signaling. However, little is known about the diversity of molecular mechanisms that regulate receptor lysosomal sorting. Here, we describe a novel pathway that mediates the endosomal trafficking of G protein‐coupled receptors (GPCRs), the largest family of signaling receptors expressed in mammalian cells. Many GPCRs are ubiquitinated following activation and internalized to the early endosome where they interact with ubiquitin‐binding adaptor proteins that facilitate receptor packaging into intraluminal vesicles of multivesicular endosomes. In contrast, we have found that ubiquitination and ubiquitin‐binding adaptor proteins are dispensable for the lysosomal sorting of multiple GPCRs. Since defects in GPCR lysosomal sorting lead to persistent signaling and contribute to cancer progression, it is critical to define the components of this novel endosomal trafficking pathway. We have discovered that a subset of GPCRs are bound directly by the adaptor protein ALIX, which facilitates receptor interaction with the ESCRT‐III (endosomal sorting complex required for transport) complex and sorting into intraluminal vesicles. Furthermore, we have used gene silencing and quantitative confocal microscopy to demonstrate a novel function for the alpha‐arrestin ARRDC3 in regulating ALIX‐mediated trafficking of GPCRs. Importantly, ARRDC3 is a tumor suppressor that is downregulated in metastatic and highly invasive breast cancers. We have showed that ARRDC3 recruits the E3 ubiquitin ligase WWP2 to mediate ubiquitination of ALIX in response to GPCR activation. ALIX ubiquitination is critical for GPCR degradation, as depletion of ARRDC3 or WWP2 blocks receptor lysosomal sorting. These findings also indicate that GPCR signaling regulates ALIX function through ARRDC3 and WWP2, suggesting that GPCRs may control other ALIX‐mediated processes like cytokinesis and the biogenesis of exosomes. The ALIX lysosomal sorting pathway demonstrates the diversity of molecular mechanisms that regulate receptor trafficking at the late endosome, and represents a novel mechanism for attenuating the signaling of specific GPCRs through lysosomal degradation.Support or Funding InformationThis work was supported by NIH/NIGMS R01 GM090689 (to J. Trejo). M.R.D. is a San Diego IRACDA Fellow supported by NIH K12 GM06852.
Phenotypic Characterization of LRS1‐2 Knockout Mutants in Arabidopsis thaliana
Authors: Lozzi Brittney , Keuls Rachel , Erickson Patti , Erickson F. Les 
Published: November 2023

Ubiquitination is an important step in post‐translational protein modification and is tightly regulated within the cell. The Arabidopsis thaliana lateral root stimulator 1 (LRS1) gene encodes a Damaged DNA Binding WD40‐repeat (DWD) protein that may play a role in ubiquination regulation. The DWD motif suggests an interaction with E3 ubiquitin ligases, but recent findings indicate that LRS1 may regulate deubiquitinases (DUBs), which reverse ubiquitination via cleavage of the bond between ubiquitin and its target protein. LRS1 is highly conserved among eukaryotic species, including humans, worms, and yeast, where it is involved in DNA repair, glutamate receptor abundance, and biosynthesis of mitochondrial subunits necessary for respiration. To determine its biological function in plants, T‐DNA insertion mutants of LRS1 have been identified. Initial observations of the lrs1‐1 allele revealed reduced lateral root growth with otherwise normal development. A second allele, lrs1‐2, has been identified and genotyped using PCR. Homozygous lrs1‐2 mutants and their sibling, wild‐type segregants have been isolated after multiple rounds of back‐crossing mutants to the parental wild type Columbia (Col‐0). Both mutant lines are being analyzed for altered developmental and biological phenotypes, including germination rates, sensitivity to auxin and its transport, and reproductive capacity. lrs1 mutants are also being grown on media containing phenanthroline, isopropyl (N‐3‐chloro‐phenyl)‐carbamate, and nordihydroguaiaretic acid because Saccharomyces cerevisiae mutants lacking the LRS1 homolog, DUB‐associated factor 1 (DUF1), are sensitive to these compounds. By exposing the mutant plant lines to different chemical and environmental stress conditions, a phenotype may be revealed that provides a better understanding of the cellular activities of LRS1 and its function in plants.Support or Funding InformationThis project is supported by an NSF Major Research Instrumentation grant and Salisbury University's Henson Undergraduate Research Fund.
Engineered Mannose‐Binding Lectin (FcMBL) Decreases Macrophage Phagocytosis
Authors: Chen Angeline , Waterhouse Anna , Rodas Melissa , Cuneo Garry , Watters Alexander , Super Michael , Ingber Donald 
Published: November 2023

Mannose‐binding lectin (MBL) is an innate immune system opsonin that binds a large variety of pathogen‐associated molecular patterns (PAMPs) on bacteria, fungi, viruses, parasites and toxins, leading to the activation of the lectin compliment pathway, coagulation, and opsonization of pathogens for phagocytosis. Due to its opsonizing abilities, MBL can be used to diagnose and treat blood‐borne infections, including sepsis. We have engineered a recombinant protein (FcMBL) by fusing the Fc domain of IgG1 to the pathogen‐binding domains of MBL. FcMBL preserves the capabilities of wild‐type MBL to recognize and bind pathogen, while avoiding the subsequent immune responses normally produced by wild‐type MBL, such as complement activation, coagulation, and inflammation. In the present study, we have differentiated THP‐1 acute monocytic leukemia cells into monocyte‐derived macrophages (MDM) using 12‐O‐tetradecanoylphorbol‐13‐acetate (PMA) and used these to test MBL‐dependent phagocytosis. Phagocytosis levels were determined using flow cytometry by measuring the fluorescent signal of pHrodo® Red E. coli BioParticles®, which fluoresce upon macrophage ingestion due to a change in pH from the extracellular environment (pH 7.4) to the inside of a phagosome (pH 5). Characterizing FcMBL as an effective opsonin capable of recognizing and binding to a diverse range of pathogens but without activating an immune response typical of wild‐type MBL will provide useful information about the use of FcMBL as a therapeutic protein.Support or Funding InformationThis work was supported by Defense Advance Research Projects Agency (DARPA) Grant N66001‐11‐1‐4180 and Contract HR0011‐13‐C‐0025 and the Wyss Institute for Biologically Inspired Engineering at Harvard University.
Generation of chimeric high‐affinity monoclonal antibody against DYKDDDDK peptide (FLAG)
Authors: Ikeda Koki , Sasaki Fumiyuki , Saeki Kazuko , Okuno Toshiaki , Koga Tomoaki , Yokomizo Takehiko 
Published: November 2023

DYKDDDDK peptide (FLAG) is a useful tool to investigate the function and localization of proteins whose antibodies are not available. Although several monoclonal antibodies (mAbs) for FLAG are commercially available, they sometimes fails to detect specific signals, rather detects a plenty of nonspecific signals upon the assays. We recently established a hybridoma that produces a high‐affinity mAb for FLAG (clone 2H8, Sasaki F, Anal Biochem. 2012). 2H8 antibody is sensitive enough to detect FLAG‐fused proteins by flowcytometry and immunoprecipitation. However, we observed nonspecific signals with 2H8 mAb in immunohistochemistry, because 2H8 mAb is mouse origin. In the present study, we tried to generate chimeric 2H8 mAb with Fc fragments derived from human immunoglobulin in order to reduce the nonspecific signals. We first cloned 5′ terminus cDNA (Fab region) of heavy and light chains of 2H8 mAb by 5′‐Rapid Amplification of cDNA End (5′‐RACE), and fused them to 3′ terminus cDNA (Fc region) of heavy and light chains of human IgG1. We next transiently transfected both chimeric plasmids into human embryonic kidney HEK293 cells with polyethyleneimine (PEI), and then medium was collected and subjected to flowcytometry. Human chimeric 2H8 mAb detected FLAG‐tagged protein. Taken together, we succeeded to generate human chimeric high‐affinity mAb against FLAG. We will further determine whether chimeric 2H8 decreases background signals with mouse tissues in the future.Support or Funding InformationThis work was supported by Grants‐in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) Nos. 22116001, 22116002, 15H05901, 15H05904, and 15H04708.
Functional analysis of Suppressor of IKKɛ (SIKE) in migration and phagocytosis
Authors: Fahey Sean M , Slykas Frank A , DiChristofano Nick D , Dawood Mariam K , Bell Jessica K 
Published: November 2023

Innate immunity is a critical, non‐specific early defense system that allows the body to respond to pathogens prior to the recruitment of specific antibody defenses. This process involves the recruitment and subsequent migration of nearby immune cells to the site of infection, allowing the pathogen to be consumed via phagocytosis to prevent widespread infection. Migration and phagocytosis require the rapid disassembly, rearrangement, and assembly of a network of structural proteins known as the cytoskeleton. The link between cytoskeletal rearrangements in these processes and detection of pathogen, like viral patterns by Toll‐like receptor 3, are not well defined. TLR3 initiates proinflammatory and type I interferon responses via the TANK binding kinase 1 (TBK1) pathway. Suppressor of IKKe (SIKE) was originally proposed as a suppressor of IKKe and TBK1, playing an important inhibitory role in interferon β production. However, more recent studies have indicated SIKE acts instead as a preferred substrate of TBK1. Mass spectrometry and confocal microscopy further indicate that SIKE interacts with the cytoskeletal proteins actin, tubulin, and a‐actinin. This revised understanding of SIKE suggests that SIKE acts as a bridge between the viral stimulation of TBK1 activity and the resulting cytoskeletal rearrangements. To examine the role of SIKE in cytoskeletal rearrangement, SIKE function in cellular migration and phagocytosis were assessed. A CRISPR/Cas 9 SIKE knockout was developed in the KMB7‐derived HAP1 cells. The HAP1 SIKE−/− showed no detectable SIKE expression compared to the parental HAP1 cells. To determine the role of SIKE in migration, parental and HAP1 SIKE−/− were used in cell migration assays +/− dsRNA to simulate viral infection conditions. A lentiviral‐mediated shRNA SIKE knockdown was completed in the mouse macrophage RAW264.7 cell line. Using SL1344 Salmonella typhimurium strain, gentamicin protection assays were completed to determine SIKE's role in phagocytosis. Introduction of SL1344 Salmonella typhimurium at MOI ≤ 100 into RAW 264.7 showed successful infection of cells by confocal microscopy and gentamicin protection assay.Support or Funding InformationThis work was supported by NIH grant R21AI107447 and University of San Diego SURE program.