bluuebunny/crossref_metadata_2025_split huggingface dataset:
Administration of a novel β‐hydroxybutyrate ester after radiation exposure suppresses in vitro lethality and chromosome damage, attenuates bone marrow suppression in vivo
Authors: Kemper Martin F , Miller Alexandra , Pawlosky Robert J , Veech Richard 
Published: November 2023

Cellular mechanisms to protect against and repair radiation damage depend on reducing equivalents from oxidative metabolism. Two recent studies explored compounds (ex. Ex‐Rad and TP‐508) with efficacy in preventing injury or reducing its severity but neither takes advantage of cellular metabolism to this end. We report for the first time that a novel ketone body ester (KE) protects against injury and death in cells and animals exposed to several types of radiation.The unique metabolism of ketone bodies shifts biochemical set points, particularly of the NADP/H system which is essential to cellular antioxidant function and oxidative damage repair. Additionally, the demonstrated activity of the ketone bodies as histone deacetylase inhibitors and enhancers of DNA repair may make these compounds uniquely suited to combat radiation injury.We show that addition of KE to culture media provides significant protection to cultured osteoblasts against γ‐radiation lethality when administered before or after radiation. KE significantly attenuated gamma radiation induced chromosomal injury in an escalating dose model (60–70% reduction). Ketone ester supplementation also significantly reduced proton radiation induced γH2Ax foci, a marker of double stranded DNA breaks, and overall proton radiation lethality. KE protection extends to other radiation types, with an average dose reducing factor of 30%.In vivo confirmation of these findings is ongoing, but preliminary data indicates that many bone marrow chromosomal damage markers were attenuated by 50% when KE was administered by gavage 24 hours after γ‐radiation exposure. Importantly, the reticulocyte/erythrocyte ratio in bone marrow, a marker of erythrogenesis and an important indicator for survival at the radiation exposure level studied was significantly rescued by KE.Support or Funding InformationFunding was provided by the NIH/NIAAA, Department of Defense and Defense Medical Research and Development Program
Novel Method for the Detection of Glucose Uptake: Direct Measurement of Glucose Levels in Cultured Cells
Authors: Yan Kevin , Li Shunan , Lin Yuan , Saddar Sonika , Tchaga Grigoriy , Yan Gordon 
Published: November 2023

Glucose uptake is the transport process of glucose from the outside of cells into cells across the cell membrane. It is one of the key regulation steps of glucose metabolism. Study of glucose uptake provides important information for understanding glucose metabolism and its regulation in normal and disease development. Here we report the development of a novel glucose uptake detection method based on direct measurement of glucose levels in cultured cells. In this method, we utilized the inhibitor, 3‐bromopyruvate (3BP), to temporarily inhibit hexokinases, the first enzyme that metabolizes glucose in cells. Thus, glucose metabolism was blocked, which leads to glucose accumulation rather than its rapid metabolism inside the cells. Glucose uptake was then detected by direct measurement of the glucose levels inside of the cells using Glucose Oxidase/HRP/OxiRedTM enzyme based method. In comparison with current widely used methods, this direct glucose uptake detection method has the following advantages: i) No radioisotope materials are used; ii) It directly detects glucose without using structurally modified glucose derivatives; iii) Glucose from culture medium is transported inside of cells, and then detected with minimum medium changes and washing processes. This simple, sensitive and direct glucose uptake detection method provides a powerful tool for studying the process of glucose uptake and its regulation, as well as for screening and characterization of drugs that regulate glucose uptake during normal and disease development.Support or Funding InformationThis project was supported by the New Product Development Funds from R&D Division of BioVision Inc.
Characterizing the Structural Requirements of the Human Antiviral Protein Apobec3G
Authors: Casazza Rebecca , Sheehy Ann 
Published: November 2023

Apobec3G (A3G) is an endogenous, human protein that restricts Human Immunodeficiency Virus Type 1 (HIV‐1) infection. A3G exhibits cytidine deaminase activity and when packaged into virus particles, has been shown to hypermutate the viral genome during the critical process of reverse transcription in the next round of target cells. HIV‐1 combats this cellular defense with Viral Infectivity Factor (Vif), a viral protein that binds to A3G resulting in its polyubiquitination and degradation in the 26S proteasome. Research has shown an uncoupling of cytidine deamination and antiviral activity, indicating that A3G may restrict infection via multiple mechanisms. It has been proposed that, in addition to its well‐characterized cytidine deamination activity, A3G may also sterically hinder the reverse transcription process by blocking reverse transcriptase from accessing the viral RNA. A3G has characterized RNA‐binding activity although the regions of the protein involved in this interaction have not been fully delineated.To investigate the A3G structural requirements for this proposed alternate mechanism, an A3G mutant library was generated and examined for antiviral and deaminase activity. When tested for functional activity, several mutants identified in an N‐terminal domain appeared to demarcate a region that, when mutated, exhibited decreased antiviral activity, but retained wildtype levels of deaminase activity. This phenotype suggests that this putative domain may be implicated in A3G's alternate antiviral mechanism. Computational analysis of this region of A3G indicate that it is characterized by a highly‐positive surface charge. The region that the novel domain falls within has previously been implicated in nucleic acid binding. We hypothesize that this novel domain has a role in facilitating A3G binding to the negatively‐charged viral RNA backbone. This surface charge is primarily conferred by three specific residues within the domain of interest. Here, the importance of this charge in A3G's alternate mechanism is investigated through site‐directed mutagenesis of the three positively charged residues, and subsequent assessment for antiviral and deaminase activity.
Analysis of immune molecule expression in mouse adipose tissue and deep fat
Authors: Juresic Sanny , Ochrietor Judith D 
Published: November 2023

Many recent studies indicate that adipose tissue plays a role in innate immunity and inflammation. This lab has been studying the expression of Basigin gene products and the innate immune receptor TLR4 with respect to neural immune functions. Basigin gene products are members of the immunoglobulin superfamily. In the neural retina, Basigin forms a transport metabolon with monocarboxylate transporter 1 (MCT1), which is vital for the function of photoreceptor neurons in mice. Basigin is also expressed on monocytes and epithelial cells throughout the body, but has been less studied in connective tissues like adipose. Recent studies by this laboratory and others suggest that Basigin interacts with the innate immune receptor TLR4, which has the ability to initiate expression of pro‐inflammatory cytokines. The purpose of the present study was to examine the expression of Basigin gene products and TLR4 in mouse adipose and deep visceral fat tissues in an effort to determine whether they participate in immune functions of those tissues. Detergent‐solublized protein lysates and RNA were isolated from mouse adipose and deep visceral fat and used in ELISA and quantitative reverse transcriptase polymerase chain reaction (q‐RT‐PCR), respectively specific for Basigin gene products and TLR4. It was determined that the ubiquitously expressed Basigin is expressed within adipose tissue and deep fat, but the photoreceptor‐specific Basigin‐2 was not. In addition, TLR4 was observed in adipose tissue, but not in the visceral deep fat tissue. The results of this study are consistent with the literature and suggest that Basigin may play a role in obesity‐induced inflammation.Support or Funding InformationUniversity of North Florida Office of Undergraduate Studies and the Office of Academic Affairs
The Roles of GPD1 in Maintaining Blood Glucose Levels under Fasting Conditions
Authors: Sato Tomoki , Morita Akihito , Mori Nobuko , Miura Shinji 
Published: November 2023

Glucose is used as one of the energy sources in many organs. Under feeding conditions, glucose can be obtained from dietary carbohydrate. On the other hand, when external supply of energy is interrupted, such as fasting, carbohydrate preserved in liver and glycogenic precursor derived from other organs are used to maintain blood glucose levels. Glycerol and glycogenic amino acid derived from adipocyte and skeletal muscle, respectively, are utilized as glycogenic precursor. But, it hasn't become clear which glycogenic precursor is more important in gluconeogenesis. The recent study revealed that Metformin, therapeutics for treating type 2 diabetes, depressed gluconeogenesis from glycerol by inhibition of glycerol‐3‐phosphate dehydrogenase (GPD) 2. This result suggests that glycerol have a potent impact on gluconeogenesis. In this study, we focused attention on GPD1, which is the enzyme that related gluconeogenesis using glycerol, and examined the roles of GPD1 in gluconeogenesis and importance of glycerol as glycogenic precursor. Using GPD1 null mutant model BALB/cHeA mice (HeA mice), we measured the change of blood glucose levels under fasting conditions and gene expressions related to gluconeogenesis in liver. BALB/cBy mice (By mice) were used as a control. In HeA mice, blood glucose levels at 1 to 4 hour after fasting were significantly higher than the By mice. The hepatic gene expressions involved in gluconeogenesis and aminotransferase such as glucose‐6‐phosphatase and alanine amino transferase were also higher than the By mice. Moreover, we examined availability of the glycogenic amino acid by alanine tolerance test. At 30 min after alanine administration, blood glucose level was significantly higher in the HeA mice. In short, these data suggests that lacking of GPD1 maintained blood glucose levels under fasting state by enhancing gluconeogenesis from glycogenic amino acid and release of glucose into the blood stream.Support or Funding InformationThis study was supported by the Council for Science, Technology and Innovation (CSTI), Cross‐ministerial Strategic Innovation Promotion Program (SIP, No.14533567), and “Technologies for creating next‐generation agriculture, forestry and fisheries” (funding agency: Bio‐oriented Technology Research Advancement Institution, NARO), Grants‐in‐Aid for Scientific Research (KAKENHI, No. 15J10165, 15K00827) from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT, Tokyo), and University of Shizuoka Grant for Scientific and Educational Research.
Metabolomics reveals the Glycerophosphodiester Phosphodiesterase GDPD5 to regulate an invasive phenotype in lung cancer cells and potentially constitute a novel drug target
Authors: Nordstrom Anders , Broom Oliver , Witek Barbara 
Published: November 2023

Study ObjectiveEpithelial to Mesenchymal Transition (EMT) is a process by which epithelial cells lose their cell polarity and cell‐cell adhesion, and gain migratory and invasive properties required for invasion and metastasis. The discovery of new targets affecting this process opens up for novel cancer therapies.MethodsWe explored the metabolic profiles of A549 lung cancer cells undergoing EMT induced by TGF‐β in a time series using LC‐MS based metabolomics. Follow up on the gene and protein leads suggested by changes in the metabolic profiles included gene expression measurement with qPCR, gene knockdown (lentiviral siRNA), gene over expression (V5 vector), protein analysis using FACS and western blotting. Phenotypic evaluation was performed by transwell invasion assay and zymography. The publically available gene expression database Kaplan‐Meier Plotter was queried for survival analysis correlating with GDPD5 gene expression.ResultsIntracellular glycerophosphocholine (GPC) levels were increased 3‐fold within 24 hours after TGF‐β application. GPC is an intermediate in the phosphocholine (PC) metabolism and is the substrate for a family of phosphodiesterase's. Administration of TGF‐β together with the phosphodiesterase inhibitor dipyridamole increased the GPC levels but not N‐cadherin, a marker for EMT, suggesting that GPC itself is no driver of EMT. Genes in the Glycerophosphodiesterase family were downregulated when treated with TGF‐β, supporting accumulation of GPC. One member, Glycerophosphodiester phosphodiesterase 2, GDE2 (GDPD5) displayed a pattern of increased mRNA and protein expression upon TGF‐β administration. Knockdown of GDE2 using siRNA decreased the invasive phenotype and the amount of protease excreted into the media whereas stable overproduction of GDE2 increased the invasive phenotype and protease excretion. Interestingly the phosphodiesterase inhibitor dipyridamole, an approved drug for blood vessel dilation and inhibition of blood clot formation, also reduced protease excretion in GDE2 over producing cells. Finally, querying a public gene expression database gave that high expression of GDE2 is associated with decreased overall survival in lung, breast and gastric cancers.ConclusionsThese data suggests a central role of phosphodiesterase's and GDE2 in particular for the invasion and metastasis process and identifies a previously unrecognized EMT signaling mechanism which potentially could constitute a therapeutic target.
Effect of Cinobufotalin on Three Different Ovarian Cancer Cell Lines
Authors: Afroze Syeda H , Tobin Richard , Peddaboina Chander , McCormick Timothy C , Newell‐Rogers Martha K , Zawieja David C , Kuehl Thomas J , Uddin Mohammad N 
Published: November 2023

ObjectiveCinobufotalin (CINO), a cardiotonic steroid (CTS) or bufadienolide, is extracted from the skin secretions of the traditional Chinese medicine giant toads (Chan su). CINO has been used as a cardiotonic, diuretic and hemostatic agent. Previously we have shown that CINO inhibits cytotrophoblast cell function. Recently others have shown that CINO inhibits function of a lung cancer cell line. In this study, we assessed the effect of CINO on three different ovarian cancer cell lines; SK‐OV‐3, CRL‐1978 and CRL‐11731 to assess whether the effect of CINO is cell specific.Study DesignWe evaluated the effect of CINO on three ovarian cancer cells by treating cultures with 0.1, 1, 5, and 10 μM concentrations of CINO. Cell proliferation, migration and invasion were measured by using a CellTiter Assay (Promega), Cytoselect Assay (Cell Biolabs) and by using a FluoroBlock Assay (BD) respectively. Proliferating Cell Nuclear Antigen (PCNA) was evaluated in cell lysates of CINO treated cells by western blot analysis. Cell cycle arrest and cell viability were determined by fluorescence‐activated cell sorting (FACS) analysis. We performed Annexin V staining by immunofluorescence to evaluate the pro‐apoptotic protein expression. In addition mitochondrial membrane potential was measured using MMP kit and by FACS analysis.ResultsConcentrations of CINO at 0.5 μM inhibit SK‐OV‐3, CRL‐1978, and CRL‐11731 ovarian cancer cells proliferation, migration and invasion (p < 0.05) without cell death and loss of cell viability, but cell viability differed among cell lines. Each cell line differed in response to CINO doses for PCNA expression (p < 0.05) as well as Annexin V pro‐apoptotic protein expression. CINO decreases mitochondrial membrane potential (p < 0.05) for SK‐OV‐3. However, in CRL‐1978 and CRL‐11731, mitochondrial membrane potential (p < 0.05) is increased in response to CINO treatment.ConclusionCINO is cell specific, as each cancer cell line responds differently. These data demonstrate that the mode of action of CINO is different on these three types of ovarian cancer cells.
Src Family Kinases as Proteasome Regulators and Therapeutic Targets for Treatment of Diffuse Large B‐cell Lymphoma
Authors: Cann Marissa , Priestman Melanie , Wang Qunzhao , Lawrence David 
Published: November 2023

Diffuse large B‐cell lymphoma (DLBCL) patients can be categorized into two clinically relevant subtypes: activated B‐cell (ABC) and germinal center B‐cell (GCB). Since patients with ABC DLBCL tend to have a much worse prognosis than their GCB DLBCL counterparts, there is great interest in identifying effective drug targets for the treatment of this subtype. Chronic, overactive signaling through the B‐cell receptor and the anti‐apoptotic NF‐κB pathway are hallmarks of the ABC, but not the GCB DLBCL subtype. Interestingly, the Src family kinases (SFKs) are known to play important roles in both of these signaling pathways. Additionally, since the proteasome is also important for maintaining NF‐κB activity and is a target of interest for the treatment of ABC DLBCL, we hypothesized that the SFKs play a role in the regulation of the proteasome. In this study we explored this role and its potential as a therapeutic target for the treatment of DLBCL. We treated a panel of ABC and GCB DLBCL cell lines with the SFK inhibitor dasatinib and found that the ABC DLBCL cell lines are significantly more sensitive to dasatinib than the GCB DLBCL cell lines. To determine if this sensitivity to dasatinib was linked to regulation of the proteasome by SFKs, a set of novel peptide sensors were used to monitor the proteasome's chymotrypsin‐like, trypsin‐like, and caspase‐like activities. Using these sensors, we demonstrate that treatment of DLBCL cell lines with the SFK inhibitor dasatinib results in a marked decrease in chymotrypsin‐like proteasome activity for the ABC DLBCL cell lines, whereas nominal changes are observed in the GCB DLBCL cell lines. Biochemical analyses demonstrate that while there is minimal change in expression of the β5 proteasome subunit, which is responsible for the proteasome's chymotrypsin‐like activity, dasatinib treatment induces a decrease in expression of the proteasome 19S regulatory particle in isolated proteasomes. However, 19S expression remains unchanged in whole lysates, indicating that the SFKs might regulate the proteasome through a chaperone protein that helps assemble or stabilize proteasome complexes. Taken together, our data suggest that inhibition of the SFKs with dasatinib affects proteasome assembly in addition to B‐cell receptor signaling, thus making ABC DLBCL cell lines particularly sensitive to dasatinib treatment.Support or Funding InformationThis project was supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Award Number TL1TR001110. The project is currently supported by the National Cancer Institute of the National Institutes of Health under Award Number F31CA196153. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
mTORC2 Regulation of the Hexosamine Biosynthetic Pathway (HBP) in Response to Nutrients
Authors: Vega‐Cotto Nicole Marie , Moloughney Joseph , Kim Peter , Wu Chang‐Chih , Lynch Thomas , Zhang Sisi , Adlam Matthew , Guntaka Sai , Chou Po‐Chien , Rabinowitz Joshua D. , Werlen Guy , Jacinto Estela 
Published: November 2023

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that forms two distinct protein complexes, mTORC1 and mTORC2. mTORC1 has emerged as a central controller of cell metabolism. The role of mTORC2 in cell metabolism is less clear, but so far it has been shown to be involved in the regulation of actin cytoskeleton organization, cell growth and proliferation, and cell survival. The extracellular signals that regulate mTORC2 and its downstream targets remain to be elucidated. In a screen to identify mTORC2 targets or effectors, our lab identified glutamine:fructose‐6‐phosphate amidotransferase (GFAT1) as a physical interactor of mTOR. GFAT1 is the rate limiting enzyme of the hexosamine biosynthetic pathway (HBP). The HBP utilizes glucose metabolites and glutamine as substrates to produce UDP‐GlcNAc, which is used for protein glycosylation. We found that mTORC2 is involved in regulating GFAT1 expression and the HBP. Metabolomics analysis of mTORC2‐disrupted cells also revealed defects in metabolites of the HBP. Our preliminary findings reveal that mTORC2 could regulate GFAT1 in response to the levels of nutrient metabolites. Our studies provide insights on how mTORC2 regulates a biosynthetic pathway in response to nutrients.Support or Funding InformationNCI
Experimental Demonstration of Localized Excess Protons at a Water‐Membrane Interface and the Effect of other Cations on Their Stability
Authors: Saeed Haitham A. , Lee James W. 
Published: November 2023

The electrostatic localization of excess protons at a water‐membrane interface has now been clearly demonstrated experimentally, which corrects the Mitchellian proton delocalization view. To demonstrate the fundamental behavior of localized excess protons in a pure water‐membrane‐water system in relation to the newly derived pmf equation, excess protons and excess hydroxyl anions were generated by utilizing an “open‐circuit” water‐electrolysis system and their distributions were tested using a proton‐sensing aluminum membrane. The proton‐sensing film placed at the membrane‐water interface displayed localized proton activity while that placed into the bulk water phase showed no excess proton activity during the entire experiment. These observations clearly match with the prediction from the proton‐electrostatics localization hypothesis that excess protons do not stay in water bulk phase; they localize at the water‐membrane interface in a manner similar to the behavior of excess electrons in a conductor. Proton‐electrostatics localization hypothesis predicts that high concentration of other cations such as Mg++, K+, or Na+ could partially delocalize the electrostatically localized protons. Therefore, determination of the cation exchange equilibrium constant is another significant way to test this hypothesis. Since protons are the smallest cations and can exist as part of the water molecules, the predicted equilibrium constant for protons to electrostatically occupy the cation sites at the water‐membrane interface is likely to be much larger than one. Results indicated that the equilibrium constant for protons (H+) to electrostatically occupy the cation sites at the water‐membrane interface was about 107 as predicted. Therefore, electrostatically localized protons at the water‐surface interface are quite stable. These findings have significance not only in the science of bioenergetics but also in the fundamental understanding for the importance of water to life in serving as a proton conductor for energy transduction.Support or Funding InformationThis research was supported by Dr. Lee's start‐up research funds provided by the Department of Chemistry and Biochemistry, the College of Sciences, the Office of Research at Old Dominion University, and the ODU Research Foundation.