bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification and Characterization of Modified DNA Aptamers Targeting ENOX2
Authors: Fetter Lisa , Bonham Andrew J. 
Published: June 2021

Human Ecto‐NOX Disulfide‐Thiol Exchanger 2 (ENOX2) is a cell‐surface metalocatalyst which is expressed solely on malignancies of all major human cancer types. ENOX2, along with its ubiquitous parent protein ENOX1, serve as terminal oxidases in plasma membrane electron transport chains, suggesting that such proteins are energetically essential for normal and malignant cell growth. Since ENOX2 is shed from malignancies into the sera and demonstrates site‐specific transcript variants, it presents an attractive target for both diagnostic blood testing and anti‐cancer therapeutic treatments. To better facilitate detection and targeting of ENOX2 in patient sera, a handful of next‐generation aptamers were identified and characterized to determine their specificity and affinity for human ENOX2. These so‐called X‐Aptamers include DNA oligonucleotides that have been chemically modified to further enhance target affinity while greatly diversifying oligonucleotide functionality. Ultimately, these novel ENOX2‐specific X‐Aptamers will be incorporated into an electrode‐based electrochemical biosensor platform to create a rapid, dose‐responsive readout for the presence of ENOX2.Support or Funding InformationThis work is supported by internal grant funding from the Metropolitan State University of Denver.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolite Concentrations Are Unstable during Cell Sorting
Authors: Llufrio Elizabeth M. , Wang Lingjue , Naser Fuad , Patti Gary J. 
Published: June 2021

There is increasing interest in the application of metabolomics to animal models. When performing metabolomics with animal models, however, often researchers are focused on only one specific cell lineage. A common approach for purifying one cell type from a multicellular sample is fluorescent‐activated cell sorting (FACS). Cells can be sorted according to morphology or fluorescent markers, such as GFP. Although necessary to purify cell populations, FACS is a timely process that could potentially influence metabolism. In this study, we examined whether the process of cell sorting causes changes in metabolism, which may complicate interpretation in metabolic studies employing FACS. We used a simplified model in which astrocytes grown in a mono‐culture were either metabolically quenched with methanol immediately after harvesting or metabolically quenched after cell sorting. Samples were analyzed using tandem liquid chromatography/mass spectrometry (LC/MS) with both targeted and untargeted methods. We found that many metabolite concentrations, including central carbon metabolites, amino acids, and nucleotides, were altered after cell sorting. Some structural lipid pool sizes were unchanged. We conclude that cell sorting is not compatible with measuring metabolite concentrations by conventional metabolomics, and we explore several alternative metabolomics strategies that may provide more robust results.Support or Funding InformationGJP received financial support for this work from the National Institutes of Health Grants R21 CA191097 and R35 ES028365, the Alfred P. Sloan Foundation, the Pew Scholars Program in the Biomedical Sciences, the Camille Dreyfus Foundation, and the Edward Mallinckrodt, Jr Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increases in transmembrane glycoprotein NMB (GPNMB), phospho‐ERK1/2, and matrix metallopeptidase (MMP)‐9 follow decline in arylsulfatase B in cystic fibrosis
Authors: Tobacman Joanne Kramer , Bhattacharyya Sumit , Feferman Leo , Sharma Girish 
Published: June 2021

In previous reports, decline in the enzyme arylsulfatase B (ARSB; N‐acetylgalactosamine‐ 4‐sulfatase) was identified in cystic fibrosis (CF) cells in culture and in circulating leukocytes from CF patients. Recently, the transmembrane glycoprotein NMB (GPNMB) was shown to be increased in a CF gene array and in Arylsulfatase B (ARSB; N‐acetylgalactosamine‐4‐sulfatase)‐null mice. These findings are consistent with previous reports that ARSB is reduced in cystic fibrosis (CF). A mechanism by which decline in ARSB, and the resultant increase in chondroitin 4‐sulfate, increased GPNMB expression has been reported. Subsequently, experiments were designed to assess the potential role of GPNMB in CF and the mechanisms by which GPNMB might contribute to CF pathophysiology. In serum and circulating leukocytes from CF and control patients, GPNMB levels were determined by ELISA. GPNMB was also measured in cells and spent media of CFTR‐corrected, CFTR‐uncorrected, and normal human bronchial epithelial cells (BEC) following ARSB siRNA or control siRNA. The GPNMB which co‐immunoprecipitated with β‐1 integrin was determined by ELISA. Matrix metalloproteinase (MMP)‐9 was measured, and the correlations between GPNMB and MMP‐9 in serum and spent media were determined. Phospho‐ERK1/2 in the circulating leukocytes and in the cultured cells was measured. The impact of excess exogenous RGD peptide and GPNMB siRNA on phospho‐ERK1/2 content was determined, and the impact of the RGD peptide, GPNMB siRNA, and ERK phosphorylation inhibitor on MMP‐9 mRNA expression was determined. GPNMB was markedly increased in cells and serum of CF patients compared to controls (p<0.0001, unpaired t‐test, two‐tailed). In uncorrected CF cells and in BEC following ARSB silencing, the GPNMB was significantly increased (p<0.0001; p<0.001). Silencing GPNMB or treatment with excess RGD peptide significantly inhibited the ARSB‐silencing induced increase in phospho‐ERK1/2 in the BEC. Silencing GPNMB, treatment with excess RGD peptide, or treatment with ERK phosphorylation inhibitor blocked the ARSB silencing‐induced increases in MMP‐9 expression in the normal BEC. Findings suggest that decline in ARSB activity caused by decline in CFTR function leads to increased GPNMB, phospho‐ERK1/2, and MMP‐9 expression, which may contribute to disruption of cell signaling and to organ dysfunction in CF.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding Gαq/11 localization and trafficking in uveal melanoma
Authors: Randolph Clinita , Wedegaertner Philip 
Published: June 2021

Constitutively activating mutations in Gαq and Gα11 (Gαq/11) have been reported in up to 93% of uveal melanomas. Although constitutively active Gαq/11 promote uveal melanoma tumorigenesis through activation of multiple downstream pathways, no therapies inhibit constitutively active Gαq/11. Studies suggest that upon activation of some Gα subunits increased palmitate turnover and increased cytoplasmic localization occur. The purpose of our studies is to understand the role of palmitoylation in trafficking and signaling of constitutively active Gαq/11 in uveal melanoma. Using live cell imaging and cellular fractionation of HEK 293 cells and uveal melanoma cells, constitutively active GαqQ209L and GαqQ209P show decreased localization at membranes compared to wild type Gαq, suggesting increased turnover of attached palmitate in constitutively active mutants. Moreover, immunofluorescence microscopy revealed that a palmitoylation‐deficient constitutively active mutant displays a complete loss of plasma membrane localization and an inability to signal as measured by Rho‐ and Rac‐dependent YAP translocation into the nucleus, TEAD‐dependent luciferase activity, and ERK phosphorylation. These studies demonstrate that palmitoylation of mutationally activated Gαq/11 is required for its signaling functions. Although live cell imaging and cellular fractionation experiments reveal decreased membrane localization of constitutively active Gαq compared to wild‐type Gαq, our studies suggest that complete loss of membrane localization abolishes constitutively active Gαqdependent signaling. Disruption of constitutively active Gαq/11 palmitoylation and localization may be an effective strategy for inhibiting constitutively active Gαq/11 oncogenic signaling in uveal melanoma.Support or Funding InformationThis research was funded by NIH grants R01GM056444‐16, R01GM056444‐16S1 (Research Supplements to Promote Diversity in Health‐Related Research) and T32 GM100836.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Trans‐phosphorylation Activates The Yeast Protein Kinase Kin2, An Ortholog Of Human Microtubule Affinity Regulating Kinase
Authors: Ghosh Chandrima , Sathe Leena , Dey Madhusudan 
Published: June 2021

Yeast protein kinases Kin1 and its isoform Kin2 are orthologs of the human microtubule affinity‐regulating kinase (MARK), which are known to regulate multiple cellular processes, including cellular exocytosis and polarity. Recently, we show that Kin kinases regulate cellular protein homeostasis or proteostasis by an unknown mechanism. To decipher the role of Kin kinases in proteostasis network, we focused our studies on the Kin2 kinase. We observed that the N‐terminal half of Kin2 protein (residues 94–510, referred to here as the Kin2mini) fully complemented the Kin2 protein null strain. These data suggest that Kin2mini plays a primary role in cellular processes. The subsequent mass spectrometric analysis of the Kin2mini protein identified three phosphorylated peptides with five phospho‐acceptor residues. Mutations of only two phospho‐acceptor residues Y275 and T281 by non‐phosphorylatable alanines in a single protein (i.e., Kin2mini‐Y275A, T281A) completely abolished the kinase activity both in vivo and in vitro. These results suggest that phosphorylation of the residues Y275 and T281 are important for its kinase function. We also observed that substitution of the residue T281 by a phosphomimetic glutamate (i.e., Kin2mini‐Y275A, T281E) restored the Kin2 kinase function. These data suggest that T281 is the key phosphorylation residue for the Kin2 kinase function. Furthermore, we found that the kinase‐dead Kin2mini‐D248A mutant (D of the conserved catalytic HRD motif) was phosphorylated on the residue T281 in vivo and the recombinant glutathione S‐transferase (GST)‐fused Kin2‐T281E mutant protein, but not the GST‐Kin2 wild type protein, was an active kinase in vitro. Collectively, our data demonstrate that Kin2 kinase is activated by transphosphorylation on the residue T281 by an upstream kinase. Currently we are studying how an active Kin2 kinase initiates a signaling cascade to fine‐tune the proteostasis network. Characterization of the Kin‐kinase signaling pathway in yeast, we believe, will illuminate the inner workings of the MARK signaling pathway in human cells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Authentic research in the teaching laboratory at a large university: comparative studies of malate dehydrogenase isoforms in trypanosomes
Authors: Springer Amy 
Published: June 2021

Offering authentic research experiences to all Biochemistry and Molecular Biology (BMB) majors at a large university can be challenging. BMB at UMass Amherst has developed a series of Course‐Based Undergraduate Research experiences (CUREs), required for all majors. One of these focuses on studying Malate dehydrogenase (MDH) isoforms in Trypanosoma brucei, a protozoan parasite that causes African sleeping sickness. This organism utilizes oxidative phosphorylation of amino acids during growth in its Tsetse fly host, but is strictly glycolytic during growth in the mammalian bloodstream, thus understanding metabolic regulation in T. brucei is important to elucidating how the parasite proliferates in the host. T. brucei has three functional MDH isoforms localized to mitochondria, cytoplasm or glycosomes, respectively. MDH is an important enzyme that catalyzes the oxidation of oxaloacetate to malate, concomitant with the reduction of NAD+ to NADH. It highly conserved and important not only as part of the citric acid cycle reactions but also as part of gluconeogenesis and of the malate aspartate shuttle used to maintain redox balance. The wealth of structural and functional information about MDH from model organisms is used to inform studies of the orthologs from a range of evolutionarily distinct organisms, currently being conducted by a consortium of collaborating faculty from various colleges and universities out as part of the protein‐centric MDH CURE community (MCC). This collaboration gives faculty and students access to community beyond the students and instructors in the course. We have generated clones for recombinant expression of all three trypanosome MDHs and have begun kinetic analyses on the cytoplasmic and glycosomal isoforms, and are optimizing expression levels for the mitochondrial form. Students can design a projects de novo or choose to continue a project from students in a previous semester. During the term students give a presentation and have many writing assignments, with opportunities for peer review and revision, all teams present their results in a department‐wide poster session at the end of the semester. Projects a have included comparisons between recombinant forms with C‐ or N‐terminal tags and between different isoforms, or orthologs of different organisms, function at different temperature, pH or ionic strength. These studies provide tools for further analysis of biochemical and kinetic properties of the T. brucei enzymes and to perform comparative analyses with well characterized mammalian and plant MDH enzymes.Support or Funding InformationNSF IUSE “An Interdisciplinary Faculty Community Using a Protein‐focused Course Based Undergraduate Research Experience (CURE) to Improve Student Learning,”This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
3D Model of Human O‐N‐Acetylglucosamine Hydrolase
Authors: Hawkins Darrius C. , Tasie Ihuoma , Gayle Ashley , Billings Ryan , Harris Aliah , Jones Candace , Van Stry Melanie 
Published: June 2021

The post‐translational modification (PTM) of proteins enable cells to react promptly to internal and external signals through direct and progressive control of protein function. Addition of O‐linked‐n‐acetylglucosamine (O‐GlcNAc) to serine and threonine residues of cytoplasmic, nuclear and mitochondrial proteins is a nourishment and stress response PTM. O‐GlcNAcylation has been linked to several diseases such as diabetes and cancer. For example, increased O‐GlcNAcylation is directly linked to insulin resistance and to hyperglycemia‐induced glucose toxicity, two characteristics of diabetes and diabetic complications. O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA) are enzymes that control the dynamic cycling of this PTM. Here, we have constructed a 3D model of human OGA bound to the transition state analog Thiamet‐G using Jmol and the PDB file 5M7S, a truncated version of the enzyme called Split1 (Roth et al. Nat. Chem. Biol. 2017). This protein is a functional homodimer that is stabilized by a helical bundle formed by helices from each subunit. Thiamet‐G fits in the substrate binding pocket formed by Cys215, Tyr298, and Trp278 and interacts with Gly67, Lys98, Asn280, Glu295, and Asn313 to form hydrogen bonds. Thiamet‐G also interacts with the catalytic residues Glu174 and Glu175. The focus of this project was to explore the protein structure of OGA and design and build a physical model that illustrated key functional features of the protein.Support or Funding InformationFunded in part by NSF‐DUE 1725940 for the CREST Project.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Epigallocatechin‐3‐gallate Suppressed Methylglyoxal‐Induced Apoptosis in SH‐SY5Y Cells
Authors: Cheng An‐Chin , Lee Ming‐Fen , Huang Tzu‐Li , Liou Yi‐Siou , Pan Min‐Hsiung 
Published: June 2021

Glycation is an endogenous process that generates a class of compounds known as advanced glycation end products (AGEs). AGEs are found in age‐related intracellular protein deposits of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Green tea polyphenols have a role in the reversal of age‐related loss of neuronal plasticity and recovery after neuronal lesions. There are several polyphenolic catechins in green tea, where epigallocatechin‐3‐gallate (EGCG) is both the most abundant and most biologically active form. EGCG could exhibit protective effects against AGEs‐induced injury in neuronal cells through its antioxidative properties, suggesting a beneficial role for this tea catechin against neurodegenerative diseases. In the current study, efforts were made to investigate the effects of EGCG on MG‐induced apoptosis. SH‐SY5Y cell were treated with different concentrations of EGCG followed by treatment with MG. Our data exhibited that both the cleaved PARP and cleaved‐caspase‐3 were downregulated by EGCG, and the MG‐induced reduction of Bcl‐2 expression were reversed by EGCG treatment. Moreover, EGCG could protect cells from death via reverse MG‐induced promoting of ERK/JNK pathway, and associated with reduced p53 accumulation. Our results implicate that EGCG could suppress MG‐induced apoptosis.Support or Funding InformationThis study was supported by NSC 100‐2313‐B‐309‐003 to A‐C ChengThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Induction of immunogenic cell death of cancer cell by using chemical detergent treatment and its activation of immune system
Authors: Kimura Tsuyoshi , Nakamra Naoko , Hashimoto Yoshihide , Kishida Akio 
Published: June 2021

Allogenic whole cancer cell vaccines have been proposed for cancer immunotherapy. Immunogenic cell death (ICD) is one of programed cell death and the ICD induced cells show high immunogenicity and can activate immune system effectively. So, ICD induced cancer cell is expected as an autologous cell vaccine. As ICD induction method, chemical and physical methods have been proposed. Among them, high hydrostatic pressure (HHP) treatment can induce ICD of cancer cell. The HHP‐ICD induced cancer cells show higher immune activation than other methods. However, the ICD induction efficiency is still low. Therefore, in order to develop simple and effective ICD induction method, we investigated ICD induction method by using various detergents, such as SDS, Triton X‐100 and Tween 20. HeLa cells were treated with detergents at various concentrations. For the Tween 20 treated HeLa cell, the calreticulin and HMGB1 translocations were confirmed by confocal laser microscopic observation (Fig. 1). This result indicates that immunogenic cell death of HeLa cells was induced by the detergent treatment. For SDS and Triton X‐100, although the cell death was induced, the calreticulin and HMGB1 translocations were not confirmed. The ICD induced HeLa cells were effectively phagocytosed by macrophage‐like cells and the effective secretion of TNF‐alpha from the macrophage‐like cells also confirmed by ELIZA assay (Fig. 2). From these results, it was suggested that the ICD inducted cancer cells by using Tween 20 could activate immune system.Support or Funding InformationJSPS Grant‐in‐Aid for Scientific Research (B)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation of Phospho Tensin Homolog (PTEN) in Cancer Cells and Lipid Peroxides in Peripheral Blood Mononuclear Cells (PBMCs) Following Exposure to Flavonoids
Authors: Garcia Alekzander Sky , Myles Elbert , Boadi William Y. 
Published: June 2021

Plant flavonoids have been shown to offer more protective health benefits against oxidative deoxyribonucleic acid (DNA) damages caused by γ‐ray radiation, UV irradiation, chemicals, and endogenous oxidative stress. The above compounds have been shown to be capable of modulating the activity of enzymes, and affect the behavior of many cell systems. While these may account for the anti‐mutagenic activities of flavonoids in experimental systems, relatively little is known about the mechanisms of the modulation of PTEN activity in cancer cells and the levels of lipid peroxides in peripheral blood mononuclear cells (PMBCs). The objectives of this study was to investigate the effects of three flavonoids: quercetin, kaempferol and genistein on the expression levels of both phospho‐ and total‐PTEN in breast cancer (BT‐549), lung cancer (A‐549) and human preadipocytes (3T3‐L1 which was used as controls). Furthermore, the effects of the flavonoids on lipid peroxides in PMBCs were investigated. Cells were seeded and were exposed to each of the flavonoid at concentrations of 0, 5, 10, 15, 20 and 25 μM and the phosphorylation of PTEN at Ser380, total PTEN levels, and lipid peroxides (i.e., malonaldehyde (MDA before and after the oxidative stress)) levels were measured. The results indicate the flavonoids increased both phospho‐PTEN‐ and total‐PTEN levels increased in a dose‐dependent manner. The effect of quercetin was more pronounced followed by genistein and kaempferol. Exposure of PMBCs cells to the various flavonoids at the different concentrations lowered the MDA levels compared to their respective controls. Levels of MDA in quercetin treated samples were lower compared to kaempferol and genistein. The findings suggest that the flavonoids play an important role in controlling oxidative stress in cells.Support or Funding InformationNational Institutes of Health: 2T34GM007663This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.