bluuebunny/crossref_metadata_2025 huggingface dataset:
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 Information

This 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 Information

NIH R01HL119439, NIH R01GM117177
Exploration of the Role of the CCR4‐NOT Deadenylase Complex Subunit Regena/NOT2 in MicroRNA‐mediated Gene Silencing
Authors: Knox James , Rees Katie , Zbornik Tika , Morin Michael , Bowden Sean
Published: November 2023

Our goal is to determine the cellular mechanisms that regulate microRNA (miRNA)‐mediated gene silencing. MiRNAs are non‐coding RNA molecules that interact with target‐gene messenger RNA transcripts via complementary base pairing, and silence target gene expression via interaction with the miRNA‐induced silencing complex (miRISC). Silencing is accomplished through translation block, mRNA degradation, or a combination of these mechanisms. A forward genetic screen was carried out to identify requirements for miRNA‐mediated silencing in Drosophila melanogaster. miRNA function was assayed using Green Fluorescent Protein (GFP)‐based reporters of gene silencing. The GFP transgenes contain miRNA binding sites, and are regulated by endogenous miRNAs. We have genetic evidence demonstrating roles for both Regena (NOT2), a subunit of the CCR4‐NOT deadenylase complex, and the protein kinase dAkt1, in miRNA‐mediated gene silencing. We are investigating the necessity of this gene in miRNA‐mediated gene silencing at various stages of Drosophila development by determining differences in gene expression in mutant larva lacking functional NOT2. NOT2‐mediated reporter silencing has been shown to be dependent upon the activity of miRNAs as demonstrated by the use of an additional GFP‐based reporter of gene silencing. Reporter gene expression in larval fat body tissues is being assessed to determine whether NOT2 is required for silencing at different developmental time points and in different tissues of Drosophila.. Investigation of the composition, abundance, and localization of known components of the silencing machinery is revealing distinct mechanisms for the regulation of silencing.
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 Information

SUNY 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 Information

NIH R01 HL119439, NIH R01 GM117177
Using Proteomics and Biochemical Approaches to Evaluate the Function of SAKS1 in p97‐associated Process
Authors: Li Shan , Chou Tsui‐Fen
Published: November 2023

p97 is an AAA (ATPase associated with a variety of activities) ATPase that consists of an N‐domain and two ATPase domains (D1 and D2). A group of p97 cofactors, called the Ubiquitin Regulatory X (UBX) domain‐containing cofactors, direct the function of p97 in many different cellular pathways. Among them, the cofactor SAKS1 is able to bind to both p97 and polyubiquitinated proteins through its UBX and UBA (ubiquitin‐associated) domains. SAKS1 enhances the ATPase activity of WT p97 by 2.4‐fold and of the D2‐active mutant by 1.3‐fold, whereas it has no effect on D1‐active mutant. The steady‐state kinetic study demonstrates that SAKS1 increases the catalytic efficiency (Kcat/Km) of both WT and the D2‐active mutant by more than 4‐fold, but does not affect the D1‐active mutant. Thus, SAKS1 regulates mainly the D2 ATPase activity of WT p97. We used co‐immunoprecipitation (IP) and LC‐MS/MS quantitation approaches to analyze interacting proteins of p97 and SAKS1. Analysis of SAKS1‐Flag IP from transient transfection with 293T and U2OS cells demonstrated that SAKS1 likely binds with four other p97 cofactors (p47, Ubxd1, NPL4 and Ufd1). This indicates that SAKS1 not only interacts with p97 directly but also forms mixed p97/cofactor complexes to mediate p97‐associated processes. Besides, we inhibited p97 activity using ATP‐competitive inhibitor (ML240) or noncompetitive inhibitor (NMS‐873) in SAKS1 expressing cells and carried out the IP and LC‐MS/MS analysis, which reveal significant changes in 76 SAKS1‐associated proteins and we are examining their functional role in p97‐associated processes.
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 Information

This 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 Information

This 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 Information

This 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 Information

This 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 Information

This work was supported by NIH grant R21AI107447 and University of San Diego SURE program.
Genomic Characterization of Novel Genes in the Novel Mycobacterium Phage Butters
Authors: Juarez Michelle C. , Grajales Melanie , Mageeney Catherine M. , Kenna Margaret A. , Ware Vassie C.
Published: November 2023

The SEA‐PHAGES program at Lehigh University is a collaborative undergraduate research enterprise focused on isolating and characterizing phages that infect Mycobacterium smegmatis to gain a better understanding of phage genome structure, gene function, and phage biology in general. One specific long term objective of the research program is to characterize phage gene products that function in lytic infection and to evaluate their potential as biocontrol agents to target pathogenic bacteria. Among the phages isolated at Lehigh is Mycobacterium phage Butters – a temperate N cluster phage with one of the smallest known mycobacteriophage genomes (41,491bp) among the >6000 mycobacteriophages isolated and characterized thus far (www.phagesdb.org). Among 66 annotated and primarily uncharacterized genes in Butters, two (gp30, gp31) are “orphams” with no known mycobacteriophage family members and one is N‐specific (gp57). To test the hypothesis that specific genes are essential for phage infection, gene deletions were made using a Bacterial Recombineering Electroporated DNA (BRED) technique to determine if mutant phage could be recovered after gene targeting. Here we focus on gp30 and gp57. Initial identification of mutant plaques was based on notable changes in plaque size phenotype and the presence of deletion‐sized PCR products. Mutant plaques retained the characteristic turbidity of wildtype plaques, indicating little, if any, perturbation of the lysogenic cycle for this temperate phage and a likely requirement for gp30 and gp57 in lytic growth. In most instances, mutant phage plaques (recovered after mixed BRED infection with wildtype phage DNA) could not be individually propagated after multiple plating rounds, suggesting that an essential component (required for bacterial infection) is missing in the population of mutant phages. We report progress in characterizing mutant phages by: 1) analysis of lysogens recovered from mutant plaques that harbor lytic‐incompetent phages, 2) cloning and sequencing of deletion‐sized PCR products recovered from mutant prophage genomes, and 3) plating mixed phage populations on gene complementation strains to recover mutant phages.

Support or Funding Information

Lehigh University, Department of Biological Sciences; Howard Hughes Medical Institute Science Education Alliance
Transcriptomic analysis of host response to the Karp strain of Orientia tsutsugamushi in inbred mice blood
Authors: Miller Stacy‐Ann , Chao Chien‐Chung , Yang Ruoting , Chan Teik‐Chye , Zhang Zhiwen , Chen Hua‐Wei , Ching Wei‐Mei , Hammamieh Rasha , Jett‐Tilton Marti
Published: November 2023

Scrub typhus is caused by the obligate intracellular Gram negative bacterium, Orientia tsutsugamushi (OT). Previous studies have shown mRNA expression profile was altered in PBMC upon in vitro infection by OT. A combined expression profile based on a few selected mRNAs appeared unique to OT infection. To better understand the host response upon infection, we infected C3H mice and monitored gene expression profile in blood at different time post infection.

C3H mice were infected with sham (i.e. PBS only) or Karp strain of OT (250 mLD50) by an artificial route. Mice were sacrificed at different time post infection. RNA was isolated from blood using the automated PAXgene blood miRNA kit on the Qiacube. Agilent SurePrint G3 Mouse GE 8×60K Microarray slides were used. The experimental and universal reference RNA were labeled with CY5 and CY3, respectively using Agilent Low Input Quick Amp Labeling Kit and Two‐Color Spike‐Mix, following by fragmentation and hybridization using the Agilent Gene Expression Hybridization Kit. Arrays were scanned using the Agilent Microarray G4900DA scanner system. Microarray data was extracted using Feature Extraction v 11.0.1.1. Further analysis was performed using functional analysis clustering toolbox (FACT) and customized R code.

Using FACT, we were able to dissect the most important gene expression patterns. Lots of changes (1667 probes) occurred only at 6 h, 12 h (796 probes), then 2 days (828 probes) post infection. Multiple pathways with highly up‐regulated expression occurred at 8 – 12 h. Pathway‐specific qPCR is being performed to confirm these altered mRNAs.

Using a mouse model with an artificial route of infection, we are able to show various genes involved in pathways responded to OT infection. While further validation of these altered genes is needed, the results demonstrated a massive change of gene expression early during the infection progression.

Research was conducted in compliance with the Animal Welfare Act, and all other Federal requirements. The views expressed are those of the author(s) and do not constitute endorsement by the U.S. Army.
The molecular model of PKC life cycle explained through Systems Biology Approach
Authors: Aslam Naveed , Alvi Farah
Published: November 2023

Protein kinase C (PKC) is a family of kinases which is poised to transduce signals emanating from the cell surface. Membranes of most cells provide the regulatory platform for supporting the life cycle and function of PKC enzymes. Maturation, activation and termination are three key regulatory steps of PKC life cycle. These regulatory steps precisely control the level of signaling competent PKC enzyme in the cells and any dysfunction in this regulation results in pathophysiological disease state such as cancer, where PKC levels are observed to be altered. Here, we describe a molecular model of PKC life cycle based on computational approach. Our model explains the fate of PKC during denovo synthesis and lipid mediated activation cycle. Through our systems biology approach we show that life cycle of PKC is controlled by multiple phosphorylation and dephosphorylation events. PKC processing events can be divided into two types 1: Maturation processing of newly synthesized enzyme; 2. Activation of enzyme. Newly synthesized PKC enzyme is constitutively processed through three ordered phosphorylations and is stored in cytosol as a signaling competent inactive molecule. First step in this maturation sequence of PKC is PDK1 mediated phosphorylation at activation loop (Thr500 in PKCβII). Once phosphorylated at activation loop PKC molecule binds with mTORC2 complex and undergoes constitutive auto‐phosphorylations at turn and hydrophobic motifs (Thr641 and Ser660 in PKCβII). Upon stimulation by extra‐cellular stimuli the generation of diacylglycerol (DAG) and Ca+2 at membrane engages the PKC molecule through binding at C2 and C1 domains respectively. This activates the enzyme. Our model explains all these steps in PKC lifecycle through a quantitative approach. Our model also shows that once active PKC molecule is prone to de‐phosphorylation (PHLPP‐mediated) and degradation. This model also explains the stabilizing role of HSP‐60 in rescuing the de‐phosphorylated molecule and assisting its re‐phosphorylation, thus re‐building the pool of signaling competent enzyme after activation cycle. This model shows that for small to moderate activation stimuli the pool of PKC molecule is effectively unchanged however, larger stimuli can down‐regulate the enzyme through degradation pathways. Our results also show that blocking the PHLPP de‐phosphorylation pathway could recover the PKC pool in a dose dependent manner even after a large amplitude stimulus has been applied. Our results also points toward a possible role of HSP‐60 overexpression in recovering the PKC signaling competent pool after large amplitude stimuli has been applied. The results presented in this communication unravel some interesting aspects of PKC life cycle through a computational approach. Enhancing the understanding of PKC life cycle could be instrumental in translating new drugs for this important transducing molecule for diseases like breast cancer.

Support or Funding Information

BioSystOmics
High Throughput Single Cell Gene Expression Profiling by Multiplex qPCR
Authors: Okino Steven , Kong Michelle , Ma Jason , Fenrich Joshua , Jouvenot Yann , Wang Yan
Published: November 2023

Single cell gene expression analysis is a powerful technique that provides a unique and insightful perspective on biological pathways and processes. Here we present a robust workflow that enables fast and accurate analysis of up to 100 genes in isolated single cells. Our workflow is highly sensitive, by assessing RNA reference standards we find that a single RNA transcript is detected with about 80% efficiency. We used this workflow to study differentiation in cultured NTera2 cells (NT2), a human embryonic stem cell model system. We analyzed untreated NT2 cells, and NT2 cells treated with low and high doses of retinoic acid (RA) for 10 days to initiate differentiation to a neuronal lineage. The expression levels of 16 genes were quantified in 164 single cells by multiplex real‐time qPCR with two technical replicates. The entire experiment, from cultured cells to results, can be completed in 2–3 days and requires four 384‐well qPCR plates for gene expression quantification. We find that control cells and cells treated with a high dose of RA (10 uM) are relatively homogeneous in the expression levels of the targeted genes. However cells treated with a low dose of RA (0.25 uM) exhibit significant heterogeneity with respect to gene expression; about half of the cells are similar to the high‐dose RA cells, the other cells exhibit a wide range of partial differentiation. Interestingly, we find that LEFTY2 expression is almost exclusive to the low dose RA cells and strongly correlates with partial differentiation. A time‐course study analyzing cell populations reveals that LEFTY2 is only transiently expressed in differentiating NT2 cells with peak expression at 3 days of high dose RA treatment. These findings imply that, in NT2 cells, LEFTY2 is a potential biomarker of early differentiation. In summary, we present an accurate, sensitive and robust single cell analysis procedure that uses standard reagents and platforms. We envision that this workflow will enable researchers to investigate cell heterogeneity in biological pathways in a cost‐effective way.
Quantifying HbF Using a Modified Kleihauer‐Betke Assay
Authors: Singh Rhena , Randolph Tim R
Published: November 2023

Sickle cell anemia is one of the most commonly inherited diseases, annually affecting approximately 275,000 births worldwide. The disease is most prevalent in African countries and countries in which a large proportion of the population is of African descent, such as the Caribbean nation of Haiti. Hydroxyurea is the only FDA approved drug that is used to mitigate sickle cell symptoms by reducing the percentage of HbS and increasing HbF. Since hydroxyurea is also mildly carcinogenic, HbF levels must be monitored during treatment to determine the minimum effective dosage or to stop the drug in non‐responders. The objective of this study is to develop and validate an inexpensive and simple method of measuring HbF levels in sickle cell patients undergoing Hydroyurea treatment. This method uses a modified Kleihauer‐Betke (KB) principle to elute HbA and HbS in a weak citric acid solution while retaining HbF. Normal blood samples (source of HbA) were collected from the research team, de‐identified cord blood samples (source of HbF) were provided by the St. Louis Cord Blood Bank at Cardinal Glennon's Children's Hospital and de‐identified sickle cell blood samples (source of HbS) were provided by Cardinal Glennon Children's Hospital. The procedure involves the mixing of 20 uL of type specific cord blood/normal blood, or cord blood/sickle blood mixtures that were standardized to 35% hematocrit to 4 mL of citrate solution (0.07 M sodium citrate in a 0.06 M sodium phosphate buffer solution). Samples were incubated at 370C for 6 minutes, centrifuged for 30 seconds and the supernatant read spectrophotometrically at 395 nm. As HbA or HbS levels increase (decreasing levels of HbF), the absorbance also increases showing a strong positive linear correlation (r2 = 0.99, N=5). This simple and cost‐effective method shows promise to be used in underdeveloped countries for HbF quantification.

Support or Funding Information

RS is supported by the DeNardo Education and Research Foundation.
Phenotypic studies of PHO13Δ in Saccharomyces cerevisiae
Authors: Kellogg Courtney , Mills Kimbria J , O'Handley Suzanne , Gehret Austin U.
Published: November 2023

PHO13 in Saccharomyces cerevisiae (bakers yeast) is a para‐nitrophenylphosphatase (pNPPase) family of the haloacid dehalogenase (HAD) superfamily. The natural substrate for PHO13 was previously unknown and a knockout phenotype has only been observed in strains engineered to grow on xylose; however, the purified enzyme has been identified in our lab to specifically hydrolyze 2‐phosphoglycolate, classifying it as a phosphoglycolate phosphatase (PGPase). In photosynthetic organisms, 2‐phosphoglycolate is generated by the oxygenation reaction of RuBisCO in the Calvin Cycle. To be recycled back into general metabolism, photosynthetic PGPase converts 2‐phosphoglycolate (2‐PG) to glycolate. In non‐photosynthetic yeast, 2‐PG must be generated by some other mechanism, possibly by oxidative damage to DNA. Regardless of its origin, 2‐PG must be catabolized due to its ability to inhibit triose phosphate isomerase (TPI). Yeast cells lacking functional Pho13p (pho13Δ) are being investigated for growth a phenotype(s) that supports TPI inhibition including the inability to grow in the absence of exogenous inositol. To increase intracellular levels of 2‐PG, cells are being treated with hydrogen peroxide. While many conditions are currently being explored, no phenotype has been apparent under normal growth conditions or in the presence of hydrogen peroxide in complete medium.

Support or Funding Information

Funded by NIH AREA Grant GM066715‐01
Flavin‐N5‐oxide: A new redox state in flavin enzymology
Authors: Sapra Aparna , Palfey Bruce
Published: November 2023

Flavoproteins comprise of one of the most studied family of enzymes and catalyze a number of redox reactions. The flavin prosthetic group can exist either in oxidized or one or two electron reduced form during the redox chemistry. All previously studied flavin‐dependent monooxygenase have been shown to catalyze oxygenation by a flavin‐C4a‐hydroperoxide, which transfer a single oxygen atom to its organic substrate. However, recent study have shown an unprecedented oxygenating species in the flavoprotein EncM that is involved in the biosynthetic pathway of the antibiotic enterocin [1]. EncM catalyzes the oxygenation of a polyketide substrate using a stable flavin‐N5 oxide species. This leads to a rare Favoriskii‐type oxidative rearrangement of the carbon skeleton chain of its substrate. The enzyme gets reduced during substrate turnover, followed by its reaction with molecular O2 to converts it back to the flavin N5‐oxide species via an unknown pathway. The N5‐oxide represents a new redox state for flavin chemistry. It can transfer an oxygen atom obtained from the reduction of O2. While the flavin‐C4a‐peroxyflavin species is highly unstable, the flavin‐N5 oxide is a stable redox intermediate, and its identification has been confirmed by absorbance spectroscopy, mass spectroscopy, isotope labelling[2]. EncM exits as a dimer with distinct flavin and substrate binding domains and a a8‐histidyl‐FAD covalent linkage with positively charges residues surrounding it: a structure common in many oxidases. This study sheds light on the mechanism, potential substrates and the structural basis that leads to this unique flavin redox state.
Identification of Mechanistic Targets for Optimization of a Cyanobacterial Hydrocarbon Production Bioprocess
Authors: Beardsley Ellis , Rajakovich Lauren , Pandelia Maria‐Eirini , Krebs Carsten , Bolinger Martin , Booker Squire J.
Published: November 2023

Cyanobacteria utilize sunlight to fix carbon dioxide to form biomolecules such as fatty acids. These microbes can convert fatty acids into diesel fuel hydrocarbons via a two‐enzyme pathway, which has been targeted for application in renewable fuel bioprocesses. The first enzyme, acyl‐acyl carrier protein reductase (AAR), catalyzes a two‐electron, NADPH‐dependent reduction of its Cn‐fatty acyl‐ACP substrate to yield a Cn‐fatty aldehyde. This reaction provides the substrate for the second enzyme, aldehyde‐deformylating oxygenase (ADO), which catalyzes the oxygenative conversion of the fatty aldehyde into the corresponding Cn‐1‐alkane and formate. To gain insight into how to improve product turnover of the ADA enzyme, kinetic and spectroscopic techniques were used. This revealed an O2‐based unproductive pathway that compromises alkane yields. Time dependent traces showed that under high O2 conditions a delay of only 30s was necessary for maximum intermediate formation, while for the low O2 conditions it took the intermediate 50s to generate. Mix Freeze‐Quench experiments were performed under analogous conditions for the stopped‐flow experiment, but once the intermediate was formed, it was reacted with reduced ferredoxin and frozen in a cryosolvent. Electron Paramagnetic Resonance spectra showed formation of the peroxyl radical under high and low O2 conditions. Under high O2 conditions more peroxyl radical was formed and less alkane product, but the alkane product can be recovered under low O2 conditions. This suggests that ADO requires O2, however under conditions of excessive O2 alkane formation is inhibited. Future efforts in bioprocesses should focus on avoiding this unproductive pathway to maximize alkane production. Through investigation of ADO catalysis, opportunities for optimization of this pathway have been identified to make it a viable option for the production of renewable biofuels.
New function of enzyme involved in the formation of a carbon‐nitrogen triple bond
Authors: Kobayashi Michihiko
Published: November 2023

We have studied microbial metabolism of nitrile at the protein and gene levels. We discovered a nitrile‐synthesizing enzyme linked with nitrile hydratase (which is an industrial enzyme used for the production of amide from nitrile) in the nitrile metabolism. The nitrile‐synthesizing enzyme, which is involved in the synthesis of a carbon‐nitrogen triple bond, is a unique enzyme, which catalyzes the dehydration of aldoxime [R‐CH=N‐OH] into nitrile [R‐CN] even in an aqueous solution. This enzyme contains a heme as the prosthetic group. Unlike the utilization of H2O2 or O2 as a mediator of the catalysis by other heme‐containing enzymes, this enzyme is notable for the direct binding of a substrate to the heme ion. We previously reported that ferrous enzyme, containing a five‐coordinated high‐spin heme and a histidine residue as its proximal ligand, is the reactive form of the enzyme, and that another histidine in the distal heme pocket plays a crucial role in the catalysis. We determined the crystal structure of the enzyme and clarified the detailed reaction mechanism. Here, we for the first time found catalase activity of the enzyme, when 10 mM H2O2 was used as a substrate. We also determined kinetic parameters of this reaction. Based upon the three‐dimensional structure of this enzyme, we have tried to construct a mutated enzyme, which shows higher activity than the wild type enzyme.
Entamoeba spp. as Models for the Effects of Environmental Stress in Protists
Authors: Hackey Meagan , Leitao Joshua , Espinosa Avelina
Published: November 2023

The Entamoeba lineage includes protists residing in the gut of organisms including komodo dragons, turtles, snakes, and humans. Some Entamoeba varieties exhibit pathogenicity. In vitro assays were performed to test environmental conditions such as temperature and pH for impact in growth and survival of Entamoeba. Trophozoites incubated at high temperatures and extreme pH showed significantly reduced cell surface area, indicating stress. Slightly increased temperature and decreased pH values resulted in little change or slightly improved cell health, based on cell shape, confluency, cell density, aggregation, and accumulation of waste. We suggest that one consequence of climate change could be the increase of pathogenic protists such as E. histolytica, E. invadens VK‐1: NZ, and E. invadens IP‐1, which pose a significant threat to human and animal health worldwide. The secretion of small molecular weight proteins in cultures incubated above 37°C was detected. Protein gel electrophoresis and mass spectrometry analysis identified heat shock (HSP70) and chaperonin proteins similar to those identified in aggregation studies. These proteins have been reported in E. invadens, E. histolytica, Giardia lamblia and Trichomonas vaginalis as down regulated under stressful conditions. We are currently exploring how Entamoeba varieties respond to changes in salt concentration. The identification of cell‐signaling proteins that decrease Entamoeba aggregation in response to extracellular conditions could aid in finding alternatives to managing pathogenic Entamoeba spp. in the changing environment.

Support or Funding Information

Supported by the Rhode Island National Science Foundation EPSCoR Summer Undergraduate Research Fellowship.
Antiproliferative effect of Resveratrol and Oxaliplatin on colon cancer cells
Authors: Daniel Nalini P , Ezekiel Uthayashanker
Published: November 2023

Current cancer treatments include radiation, chemotherapy and surgery. Treatment of primary tumors with radiotherapy and/or chemotherapy has been observed to increase the rate of metastasis. While these treatments contribute to overall well‐being, they are a double‐edged sword and may also cause unintended and painful side effects. Consumption of a diet rich in phytochemicals is efficacious in inhibiting colon cancer. Phytochemicals are plant‐based compounds that are usually nontoxic and have significant effects on cancer cells. They also have been used in a synergistic manner with chemotherapy drugs. This study assessed different plant‐based compounds for effect on colon cancer cells and resveratrol in combination with the known chemotherapy drug, oxaliplatin, displayed interesting results. Resveratrol is found in the skin of red grapes and red wine and has shown promise as an anti‐tumor agent. Our studies focused on assessment of varying concentrations of resveratrol or oxaliplatin used as single compounds and oxaliplatin in combination with resveratrol with resveratrol held constant at the 50uM (IC50) concentration. Results indicated that resveratrol or oxalipatin treatment alone elicited anti‐proliferative activity. Oxaliplatin exhibited greater antiproliferative activity than resveratrol alone. In the double compound treatment analysis, resveratrol (held constant at 50uM) plus oxaliplatin showed significant inhibition at lower concentration (1.56uM) than that of either single compound treatment alone. At higher concentrations, no significant differences were found between oxaliplatin and double‐compound treated cells. Preliminary results suggested that resveratrol plus the chemotherapeutic agent oxaliplatin may inhibit at lower concentrations due to synergistic activities. Studies have shown that both compounds reduce expression of the inhibitor of apoptosis protein known as survivin. Therefore, future experiments will be focused on expression level of micro RNA‐16 and micro RNA‐34a that modulates the survivin gene expression by post transcriptional regulation.
Glycome Sequencing; Are We There Yet?
Authors: REINHOLD VERNON NYE
Published: November 2023

Sequence challenges provided by the uncoded glycomes of proteins and lipids are, in principle, comparable to the glycosaminoglycans and proteoglycans. But, in neither case, are there protocols or technologies comparable to those of genes and proteins.

But the demands of personalized medicine and the early successes of carbohydrate antibodies and vaccines strongly suggest this is a research limitation that must be addressed soon. Described in this brief presentation is an overview of a “bottoms‐up” sequencing approach that extends from the glycans of proteins and lipids to the glycosaminoglycans. After sample preparation all sequencing is dependent on ion fragmentation and spatial resolution in the ion trap (MSn). Here two fundamental fragment ions are basic, the common B‐ion and the more elusive A‐ion. This latter ion is time dependent and is generated by combinations of electron rearrangement and group elimination which importantly varies with linkage point. Such detailed accounting provides a comprehensive spatially acquired sequence.

Support or Funding Information

PEG; Common Fund