bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cell death by gold nanoparticles in MDA‐MB‐231 cells involves different epigenetic alterations: role of surface charge
Authors: Surapaneni Sunil Kumar , Bashir Shafiya , Tikoo Kulbhushan 
Published: June 2021

Triple negative breast cancer represents an important clinical challenge, as these tumours do not respond to endocrinal therapy. Nanotechnology offers a solution to overcome these clinical challenges. Gold nanoparticles (AuNPs) have been used in multiple ways for the prevention of breast cancer due to their unique physicochemical properties, therapeutic payload efficiency of drugs, biological compatibility, theranostic applications and radiation sensitizer effects. Here, we elucidate the molecular mechanism by which AuNPs selectively cause death in triple negative breast cancer (MDA‐MB‐231) cells. Both citrate capped (negatively charged) and cysteamine capped (positively charged) AuNPs significantly decreased the proliferation of MDA‐MB‐231 cells in a dose‐dependent manner and were less toxic to MCF‐10A cells. For the first time, we provide evidence that AuNPs can be used to prevent the progression of triple negative breast cancer by altering Wnt/β‐catenin signalling. AuNPs significantly decreased the protein expressions of β‐catenin, GSK‐3β, Cyclin D1, p53 and increased the protein expressions of phospho‐GSK‐3β, acetyl‐p53 and p‐p38. HR‐TEM was used to study the localisation of AuNPs. It is interesting to report that both AuNPs caused cell death but (−ve charged) AuNPs significantly increased the expression of MKP‐1, dephosphorylated and deacetylated histone H3 at Ser10 and K9/K14 residues respectively. However, (+ ve charged) AuNPs decreased the expression of MKP‐1, phosphorylated and acetylated histone H3 at Ser10 and K9/K14 residues respectively in MDA‐MB‐231 cells. Although both the nanoparticles have almost similar size (between 25–40 nm), the surface charge of AuNPs might contribute to the different patterns of histone modifications. Phosphorylation of histone H3 at ser 10 has been shown to be involved in chromatin condensation during mitosis and increase in phosphorylation of histone H3 suggests that cells might be undergoing mitotic catastrophy with (+ ve charged) AuNPs. This was reflected in lower IC50 values (655 μg/mL) with (+ ve charged) AuNPs in comparison to (−ve charged) AuNPs (720 μg/mL). To the best of our knowledge, we report for the first time that AuNPs potentiates the cell death of MDA‐MB‐231 cells to 5‐fluorouracil (5‐FU). Studies are in progress to gain insights on the sensitization effect of AuNPs on MDA‐MB‐231 cells to 5‐Fluorouracil. This study further warrants the usage of AuNPs either alone or in combination in cancer therapeutics.Support or Funding InformationThe authors are thankful to NIPER, SAS NAGAR, MOHALI, PUNJAB for funding the current experimental workThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Evf2 enhancer long noncoding RNA regulates enhancer interactions across megabase distances
Authors: Kohtz Jhumku , Cajigas Ivelisse , Chakraborty Abhijit , Bastidas Monique , Swyter Kelsey S. , Kohtz Sara J. , Ay Ferhat 
Published: June 2021

A major question in genome biology is how genes are selectively targeted and regulated over megabase (Mb) distances in the genome. In this work, we show that Evf2, a Dlx5/6 ultraconserved enhancer (Dlx5/6UCE) long non‐coding RNA, regulates a small group of 6 genes that are asymmetrically‐positioned across 27 Mb in relation to the Evf2 transcription start site. Evf2 RNA forms large intranuclear clouds that localize to both activated (Umad1, ~1.6 Mb distant) and repressed (Akr1b8, ~27 Mb distant) target genes in mouse developing brain. Measurements between Dlx5/6UCE‐Umad1 and Dlx5/6UCE‐Akr1b8 support that Evf2 controls distances between Dlx5/6UCE and transcriptional targets in subpopulations of neuronal progenitors. Chromosome conformation capture (4Cseq) using the Dlx5/6UCE as bait identifies Evf2 regulated and Evf2 independent Dlx5/6UCE interactions throughout chromosome 6 (~150 Mb). However, gene expression analysis suggests that transcriptional changes occur at only a subset of Evf2‐regulated Dlx5/6UCE interacting sites, supporting a developmental regulation of enhancer promoter contacts prior to gene regulation. These studies support that an autosomal, cloud‐forming enhancer lncRNA regulates genes through both antisense and chromosome topological mechanisms, and controls 3‐D architecture across multi‐megabase regions of a chromosome.Support or Funding InformationThis work is funded by NIMH R01MH094653 and R01MH111267 to J.D.KThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Membrane Allostery and Hydrophobic Binding Sites Control Substrate Specificity of Lipolytic Enzymes
Authors: Mouchlis Varnavas D. , McCammon J. Andrew , Dennis Edward A. 
Published: June 2021

Connecting molecular structure with cellular function is fundamental to understanding the mechanism of action of water‐soluble enzymes acting on cell membranes (PNAS 2015, 112, E516–E525). Cytosolic (cPLA2), calcium‐independent (iPLA2) and lipoprotein‐associated (Lp‐PLA2) provide an ideal system for studying protein‐lipid molecular recognition and membrane functioning. cPLA2 is the main arachidonic acid provider for the eicosanoid pathway, iPLA2 is involved in membrane phospholipid remodeling and Lp‐PLA2 was found to associate with LDL and HDL in human plasma to hydrolyze phospholipids containing short‐chain and oxidized fatty acids. A lipidomics mass spectrometric assay allowed us to define unique specificity of PLA2s towards a wide variety of phospholipids that was not feasible with existing assays. Computational techniques such as molecular dynamics simulations and docking guided by hydrogen/deuterium (H/D) exchange experimental data showed that membrane phospholipids bind to allosteric sites located on the interfacial surface of PLA2s, shifting their conformation from the “closed” to the “open” state. This process enables PLA2s to extract and bind a phospholipid molecule in the active site where the hydrolysis occurs. This is a novel study addressing recognition between water‐soluble enzymes and membranes as well as substrate specificity, binding and interactions using computational techniques guided by experimental data. PLA2s are implicated in chronic inflammatory diseases and understanding their association with membranes as well as their interactions with substrates will allow us to identify potent and selective inhibitors that can be further developed as anti‐inflammatory agents (J. Med. Chem. 2016, 59, 4403–4414).Support or Funding InformationNIH grant GM20501‐41This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Utilization of Artificial Intelligence to Develop a QSAR Model for Predicting P450 Mediated Metabolic Stability
Authors: Gonzalez Eric , Shah Pranav , Zakharov Alexey , Nguyen Dac‐Trung , Torimoto‐Katori Nao , Sakamuru Srilatha , Xia Menghang , Zhao Tongan , Obach R. Scott , Hop Cornelis , Simeonov Anton , Xu Xin 
Published: June 2021

CytochromeP450 (P450) mediated drug metabolism continues to be a key obstacle in thediscovery and development of small molecule therapeutics. Greater understandingof the relationship between chemical structure and P450 metabolism will facilitatestructure optimization that results in increased metabolic stability. NCATS, incollaboration with IQC DMLG members, has endeavored to develop quantitativestructure‐activity relationship (QSAR) models that predict the P450‐mediatedmetabolic stability. It is anticipated that these in silico tools will enhance drug discovery by expediting structureoptimization and lead selection, ultimately accelerating drug development forunmet medical needs. Moreover, the models, and complete datasets from whichthey were developed, will be made publicly available for the benefit ofscientists worldwide. Using a high‐throughput, semi‐automated in vitro assay, the metabolic clearance(half‐life) of more than 4,000 compounds was assayed with recombinant P450 3A4,2C9, and 2D6. The QSAR models were developed using an open‐sourcecheminformatics software, RDkit, with the available Morgan, Avalon, andAtomPair fingerprints in conjunction with a multitask deep learning approach. Multi‐layer feedforward neural networks were implemented in Keras using theTheano backend. The ADAM algorithm was used for minimization of the lossfunction. Hyperparameter optimization was avoided using a pyramidal neuronalarchitecture (including hidden layers) and the ReLu activation function. Themodels were evaluated by fivefold cross‐validation and the balanced accuracy(BA) was calculated for each P450 model. The initial model showed reasonableaccuracy of prediction for all activities, with BA ranging between 0.65 and0.75. To further strengthen the model, the results from a previous NCATSpublication, consisting of 17,000 compounds assayed with the P450‐Glo™ competition assay, were incorporated into the deep learning model to performtransfer of knowledge. As a result, the accuracy of prediction wassignificantly improved. The finalized model will be incorporated in thepublicly available NCATS web services (https://predictor.ncats.io).Support or Funding InformationThis work was supported by the Intramural Research Program of the National Center for Advancing Translational Sciences/National Institutes of Health.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of host‐microbiota signaling molecules with High‐Resolution Metabolomics
Authors: Liu Ken , Saeedi Bejan , Darby Trevor , Ganesh Thota , Jones Rheinallt , Neish Andrew , Jones Dean P. 
Published: June 2021

Human metabolism evolved in the presence of the microbiota. Products of bacterial metabolism can be used directly to support host metabolism (succinate, amino acids) or act as signaling molecules (butyrate, n‐acyl amides). Thus, identifying bacterial metabolites and their role as metabolic intermediates or signaling molecules could lead to new microbiome therapeutic strategies. Using mass spectrometry based High‐Resolution Metabolomics (HRM), we analyzed samples of mouse stool, liver, serum, and brain tissues collected from germ‐free, conventionalized, and altered Schaedler's flora (ASF) mice to identify metabolites enriched in conventional mice. Differentially expressed metabolites belonged to a number of amino acid and citric acid cycle pathways, indicating that gut bacteria alter peripheral tissue metabolic profiles. Furthermore, we identified a previously uncharacterized bacterial metabolite (VB005), whose biological activity is currently unknown. Computational tools for target prediction (SEA) and pathway enrichment analysis (mummichog) suggest that this molecule may be involved in host‐receptor signaling processes and participate in fatty acid metabolism. While these predictions are currently being evaluated, further HRM studies will continue to illuminate the dynamic nature of host‐microbiota signaling.Support or Funding InformationNIEHS P30ES019776, NIEHS R01ES023485, NIH T32‐GM008602This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Improved RNA‐modification mapping through employment of novel ribonucleases and LC‐MS
Authors: Thakur Priti , Limbach Patrick Limbach A. , Addepalli Balasubrahmanyam 
Published: June 2021

More than 150 different chemical modifications have been documented in various types of ribonucleic acids (RNA) [1]. They play key roles in translation, cellular fate, and stabilization of RNA structure [2]. While majority of these modifications are catalyzed by cellular enzymes, others are generated by xenobiotic stress exposure. RNA modifications have been linked to human diseases [3] such as neurological disorders and cancer.Understanding the role of these modifications in human disorders requires precise identification and location of the site in the overall RNA sequence. Nucleobase specific ribonuclease (RNase) enzymes are useful in mapping the location of chemical modification. Here the RNA is digested with a nucleobase‐specific enzyme and the resulting digestion products are subjected to liquid chromatography coupled with mass spectrometry (LC‐MS) ‐based sequencing, a method widely referred to as RNA modification mapping [4].Commercially available RNase T1 (G‐specific) and RNase A (pyrimidine‐specific) have been widely used in RNA modification mapping procedures. To augment the limitations of these enzymes, additional nucleobase‐specific enzymes, RNase MC1 (U‐specific) [5] and cusativin (C‐specific) [6] have been developed that could complement the existing enzymes. Thus, the digestion products generated by multiple enzymes can complement each other thereby improving the sequence coverage and generation of overlapping digestion products of substrate RNA. In the current investigation, we will present reconstruction of modified tRNA sequences from the digestion products generated by digestion of Escherichia coli tRNA by multiple nucleobase‐specific enzymes. We will also demonstrate the utility of this technology to map the stress‐induced damage in the ribosomal RNA. The advantage of such a multi‐pronged approach as opposed to the single enzyme usage in reconstruction of modified RNA sequences will be discussed.Support or Funding InformationFinancial support for this work was provided by the National Institutes of Health (GM058843) and the University of Cincinnati.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Necroptosis in Aging and Age‐Associated Inflammation
Authors: Sathyaseelan Deepa , Richardson Arlan 
Published: June 2021

Aging is characterized by low‐level chronic inflammation, termed sterile inflammation or ‘inflammaging’. Inflammaging is an important risk factor for most age‐related diseases as well as both morbidity and mortality in older people. Inflammaging is characterized by high levels of circulating proinflammatory cytokines. Although the exact cause of inflammaging is not known, several pathways have been suggested to cause chronic inflammation with age, e.g., immunosenescence, cell senescence, and increased levels of damage associated molecular patterns (DAMPs), which are a strong inducer of inflammation. Necroptosis is a newly identified non‐apoptotic form of cell death that plays a major role in inflammation through generation of DAMPs. We hypothesized that necroptosis plays a role in aging and age‐associated increase in inflammation. In support of our hypothesis, we found that levels of phosphorylated mixed lineage kinase domain like (P‐MLKL), a marker of necroptosis, is elevated by 2.5‐fold in epididymal white adipose tissue (eWAT) of 25‐month‐old wild‐type (old‐WT) mice, compared to 9‐month‐old WT (young‐WT) mice. Interestingly, only eWAT, not subcutaneous WAT (sWAT), showed an age‐dependent increase in necroptosis. Dietary restriction (DR), an intervention known to reduce inflammation and increase lifespan, reduced necroptosis in eWAT of old‐DR mice to the level found in young‐WT mice. We also measured the level of necroptosis in several tissues of Sod1−/− (Sod1KO) mice. Sod1KO mice show a 20–30% decrease in lifespan and increased inflammation (e.g., circulating levels of pro‐inflammatory cytokines and pathological markers of inflammation). P‐MLKL is increased 3.7‐fold and 0.4‐fold in eWAT and liver of 9‐month‐old Sod1KO mice. Furthermore, markers of M2 macrophages (anti‐inflammatory) are reduced and M1 macrophages (pro‐inflammatory) are increased in eWAT of Old‐WT and young‐Sod1KO mice. Consistent with the shift of M2 to M1 macrophages, we observed that transcript levels of pro‐inflammatory cytokines TNFa, IL‐6 and IL‐1b are elevated in eWAT of old‐WT and young‐Sod1KO, compared to young‐WT mice. In summary, we show for the first time that necroptosis increases with age, is elevated in an accelerated aging model and is reduced by DR suggesting a role of necroptosis in age‐associated inflammation and possibly aging. Currently, we are testing the effect of inhibition of necroptosis on inflammaging.Support or Funding InformationSupported in part by NIH grants P01‐AG051442 and R01 AG045693This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quantification of Propionate
Authors: Doan Mary , Trefely Sophie , Xu Jimmy , Polanco Juan , Snyder Nathaniel 
Published: June 2021

The metabolism of propionate, a 3‐carbon short chain fatty acid, via propionyl‐CoA to 2‐methyl‐2‐pentenoic acid (2M2PE), a 6‐carbon intermediate, was recently and unexpectedly reported to occur in humans. The enzymology and further metabolism of this pathway remains unclear, but investigations into the metabolism of 2M2PE are an untested avenue in anabolic and catabolic handlings of propionate. To elucidate and quantify this pathway, we employed both targeted and untargeted liquid chromatography‐mass spectrometry. Kinetic and dose response studies in human hepatocellular carcinoma HepG2 cells indicated that 2‐methyl‐2‐pentenoic acid was generated in a propionate‐dependent manner and underwent further metabolism over time. Fibroblasts cells from apparently healthy controls, as well as propionic acidemia and methylmalonic acidemia patients demonstrated similar results confirming this pathway is not dependent on entrance of carbon into the Krebs cycle. In these experiments, surprisingly, simultaneous untargeted metabolomics did not detect any propionate derived metabolites larger than 2M2PE. To extend this work to study human metabolism, we developed a method to quantify propionate and 2M2PE from media, plasma, serum and urine.Support or Funding InformationThis work was supported by NIH grants R03HD092630 and K22ES026235This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cytoplasmic PFK‐2 Activity Affects Mitochondrial PDK4 Levels in the Heart
Authors: Newhardt Maria Fernanda , Kinter Michael , Humphries Kenneth Michael 
Published: June 2021

The healthy heart has a dynamic capacity to respond and adapt to changes in nutrient availability. Disruption of this metabolic flexibility, such as with diabetes and insulin resistance, promotes cardiomyopathy through mechanisms that are not completely understood. A principal regulator of glycolysis in cardiomyocytes is the bifunctional enzyme 6‐phosphofructo‐2‐kinase/fructose 2,6‐bisphosphatase (PFK‐2, gene product of pfkfb2). PFK‐2 controls the levels of fructose 2,6‐bisphosphate (Fru‐2,6‐P2), an allosteric activator of phosphofructokinase‐1. We have recently reported that insulin signaling regulates cardiac PFK‐2 levels and that this central metabolic regulator is constitutively decreased with diabetes and high fat diet feeding. We hypothesize that the sustained loss of PFK‐2 has significant consequences on cardiac metabolic flexibility. In this study, we examined how the heart coordinates changes in metabolic flexibility as it responds to decreases in insulin signaling and identified a role Fru‐2,6‐P2 has in this process. Wild type mice, mice expressing a cardiac‐specific kinase‐deficient PFK2 transgene (GlycoLo mice), and mice expressing a phosphatase‐deficient PFK2 (GlycoHi mice) were fed either a low fat or high fat diet for 7 days. A targeted proteomics approach was then applied to quantify changes in over 150 proteins that are involved in glycolysis, fatty acid oxidation, and cellular antioxidants. Wild type animals placed on a high fat diet displayed a coordinated increase in fatty acid oxidation enzymes but this response was absent in GlycoLo mice and blunted in GlycoHi mice. In particular, pyruvate dehydrogenase kinase 4 (PDK4), which regulates mitochondrial pyruvate dehydrogenase activity, was strikingly increased in GlycoHi mice on the high fat diet (4 fold increase) as compared to wild types (2 fold increase). These results demonstrate a previously undocumented mechanism whereby the levels of Fru‐2,6‐P2 regulate mitochondrial PDK4 levels and suggests a new coordinated means of cardiac metabolic flexibility that is disrupted with diabetes.Support or Funding InformationThis study was supported by the NHLBI, National Institutes of Health (R01HL125625).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A novel role of sphingolipids and mitochondria in glutamate‐induced programmed necrosis in oligodendrocytes
Authors: Novgorodov Sergei A. , Voltin Joshua A. , Li Li , Gooz Monika A. , Lemasters John J. , Gudz Tatyana I. 
Published: June 2021

Oligodendrocytes play a prominent role in axon myelination and provide trophic factors and substrates for neurons. Under pathological conditions (e.g., ischemia/reperfusion, traumatic brain injury and stroke), the increase in extracellular glutamate levels evokes oligodendrocyte cell death, thus contributing to neuronal dysfunction. In contrast to the activation of ionotropic glutamate receptors in neurons, glutamate induces cell death in oligodendrocytes by depleting cells of cystine through the reversal of glutamate/cystine antiport mediated by system xc−. Our experiment revealed ferroptosis as a major contributing factor to glutamate‐initiated primary oligodendrocytes (OLs) death, while the contribution of necroptosis and apoptosis was excluded both on biochemical and pharmacological ground. However, in contrast to general view that disintegration of plasma membrane as a result of uncontrolled lipid peroxidation is the final step in execution of ferroptotic program, we demonstrate that inhibitors of the mitochondrial permeability transition pore, (MPTP) cyclosporine A and NIM811, attenuate glutamate‐induced cell death without affecting lipid peroxidation. This suggests bioenergetic failure as the ultimate cause of cell demise. We also identified a novel controlling factor of ferroptosis‐like cell death in OLs, sphingosine, a sphingolipid whose level in the mitochondrial compartment is increased in response to glutamate. This leads to suppression of the respiratory chain and enhanced production of reactive oxygen species, which contributes to MPTP opening. Experiments indicate that the enhanced sphingosine level originates from activation of acid sphingomyelinase (ASM) in the lysosomal compartment, which produces ceramide with its subsequent hydrolysis to sphingosine by acid ceramidase. The results of these studies highlight a novel mechanism of ASM involvement in governing mitochondrial ROS production and integrity, and suggest an important role of ASM in system xc−‐ dependent ferroptosis in the brain.Support or Funding InformationSupported by NIH grants NS083544 (TIG) and RO1 DK073336 (JJL).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.