bluuebunny/crossref_metadata_2025 huggingface dataset:
Galectin‐9 is a Novel Modulator of Epithelial Restitution
Authors: Robinson Brian Scott , Saeedi Bejan , Naudin Crystal , Arthur Connie , Darby Trevor , Jones Rheinallt , Stowell Sean , Neish Andrew
Published: June 2021

Galectins represent a diverse class of secreted lectins with high‐affinity for □‐galactoside residues present on glycoprotein and glycolipid glycans. Through regulation of protein stabilization at the cell surface, membrane localization, receptor activation, and interactions with the ECM, galectins can affect a diverse set of cellular activities required for epithelial tissue homeostasis and response to injury. To date, five mucosal‐associated galectins (Galectin‐1, 3, 4, 6, and 9) have been identified, each with different glycan binding properties and expression patterns, yet the roles of these galectins in epithelial restitution remains poorly understood. In order to investigate the role of individual galectins in these processes, we have begun to characterize the phenotypes of individual galectin‐deficient animals. We demonstrate that Galectin‐9 (Gal‐9) deficient mice show increased susceptibility to multiple models of epithelial damage and restitution, including radiation‐induced gastrointestinal syndrome and DSS‐induced colitis. Given its localization to intestinal crypts, we next tested the ability of Gal‐9 deficient crypts to grow in ex vivo cultures. We observe that organoid cultures obtained from Gal‐9 deficient animals have marked growth defects and reduced complexity compared to their wild‐type counterparts. Finally, on a molecular level, we see alterations in several signaling pathways required for epithelial restitution and cellular differentiation. In summary, these studies highlight the requirement of galectins in regulating epithelial function, and demonstrate a specific role for Gal‐9 in mediating these effects.

Support or Funding Information

This work was supported by NIH R01 AI064462. BR was supported by the Research Training in Translational Gastroenterology and Hepatology Training grant (NIH T32 DK108735).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chemical‐proteomic strategies to investigate reactive cysteines
Authors: Weerapana Eranthie
Published: June 2021

Cysteine residues regulate the activity of diverse proteins via post translational modification by endogenous electrophiles and reactive oxygen species. These modified cysteine residues do not conform to a conserved sequence or structural motif, rendering the global identification of regulatory cysteines a considerable challenge. To identify regulatory cysteines in human proteomes, we have developed chemical‐proteomic tools to monitor cysteine reactivity both in vitro as well as directly in living cells. These global profiling methods have been applied to monitor the susceptibility of cysteines to S‐nitrosation and to identify redox‐active disulfides induced by epidermal‐growth factor (EGF) stimulation. In order to identify cysteine residues hypersensitive to S‐nitrosation, we developed a chemical‐proteomic platform that ranks cysteines in the human proteome by sensitivity to a variety of transnitrosating agents. Our proteomic studies identified several previously unannotated cysteines, which we proceeded to demonstrate were important regulators of protein activity. To identify cysteine oxidation events triggered by EGF stimulation, we developed a caged cysteine‐reactive probe to enable profiling of cysteine reactivity directly in living cells. We identified several redox‐active disulfides that were formed upon EGF stimulation for future functional characterization. Overall, our studies aim to unearth novel functional cysteine residues in the proteome, with the long‐term goal of identifying new modes of protein regulation through reactive cysteines.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Shrinking the Metabolome for Systems Biology Reveals a New Metabolic Function of an Old Protein
Authors: Patti Gary
Published: June 2021

When using liquid chromatography/mass spectrometry to perform metabolomics, it is common to detect thousands of signals from biological samples. A substantial number of these signals, however, cannot be biochemically identified. This has complicated the use of metabolomics for discovery profiling and created uncertainty about the number of “unknown metabolites” in a data set. Here we describe a suite of informatic and experimental approaches that were applied to completely annotate one metabolomic data set. The analysis revealed that >90% of our signals corresponded to artifacts, contaminants, and redundancies. A workflow will be presented for removing such signals to simplify discovery analysis by metabolomics. As an example, a study will be described in which we applied metabolomics to discover a new function of the protein carnitine palmitoyltransferase I (CPT1) that is unrelated to fatty acid oxidation.

Support or Funding Information

This work was supported by the National Institutes of Health Grant R35 ES028365, the Alfred P. Sloan Foundation, the Pew Scholars Program in the Biomedical sciences, 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.
Subclavius Posticus Muscle: Accessory Muscle with Clinical Relevance to Thoracic Outlet Syndrome
Authors: Arrick Denise Mary , Stevens Kelsey , Inglis Stuart , Das Manas
Published: June 2021

During a routine anatomical dissection, an aberrant muscle was discovered in the left neck region in a cadaver of an 80‐yr‐old male. The accessory muscle originated from the superior angle of the scapula and inserted to the superior margin of the first rib cartilage. The muscle ran anterior to the trunks of the brachial plexus and subclavian vessels. Due to its origin and insertion, the aberrant muscle was considered to be the subclavius posticus muscle. The subclavius posticus muscle has been reported to play a role in thoracic outlet syndrome, which occurs by neurovascular structures being compressed in the thoracic outlet region. The variants of subclavius posticus muscle, its development and its clinical significance to thoracic outlet syndrome will be discussed.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sexual Dimorphism of the Fetal, Neonatal, and Infantile Supraorbital Rim between Races
Authors: Koons Aaron W. , Zdilla Matthew J.
Published: June 2021

The adult orbital rim is known to exhibit sexual dimorphism. However, the development of the orbital sexual dimorphism is poorly understood. Moreover, there has been little study of sexual dimorphism in the supraorbital rim in the early development of fontal bones. Therefore, this study assessed 188 supraorbital rims among black and white females and males of fetal, neonatal, and infantile age. The population consisted of 47 black female, 36 white female, 54 black male, and 51 white male supraorbital rims. Canonical variate analysis revealed statistically significant contour differences among all groups (p<0.0001). Further, all groups separated by Mahalanobis distances greater than 11 except for black females and white males (Mahalanobis distance = 6.84). The results of this study suggest that sexual dimorphism of the supraorbital rim exists within and between races, even among fetuses.

Support or Funding Information

WV Research Challenge Fund [HEPC.dsr.17.06] and [HEPC.dsr.14.13]

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tyr1 phosphorylation of RNA polymerase II CTD directs and expands the CTD code.
Authors: Mayfield Joshua E. , Mehaffey M. Rachel , Irani Seema , Walker Levi , Robinson Michelle , Burkholder Nathaniel Tate , Prescott Nicholas , Kathuria Karan , Brodbelt Jennifer S. , Zhang Yan Jessie
Published: June 2021

The C‐terminal domain of RNA polymerase II (CTD) acts as a platform to recruit factors that facilitate gene expression, RNA processing, and integrate transcription with the essential cellular processes of DNA repair and histone modification. This domain is composed of a species‐specific number of repeats of the consensus amino acid heptad YSPTSPS, classically numbered as Tyr1‐Ser2‐Pro3‐Thr4‐Ser5‐Pro6‐Ser7. The targeted recruitment of these myriad factors is accomplished by post‐translational modification (PTM) of these heptads, specifically phosphorylation of Tyr1, Ser2, Ser5, and Ser7. Although the contribution of these marks in isolation is extensively studied, the ability of these marks to co‐exist or influence one another has not been rigorously investigated.

We take the first steps to characterize “cross‐talk” between phosphorylation marks and reveal a previously unknown role for Tyr1 phosphorylation in diversifying and directing CTD modification events. Tyr1 phosphorylation has been previously implicated in RNA polymerase II stability, antisense and enhancer transcription, anti‐termination, and as an indicator of DNA damage. However, the mechanism through which Tyr1 influences this diverse array of functions is unknown. Using a combination of mass spectrometry, molecular biology, and targeted cellular assays we find that Tyr1 phosphorylation generates a chemically diverse CTD landscape and can influence the specificity of downstream modification enzymes, resulting in novel and uncharacterized marks in the CTD code. We provide direct evidence for combinatorial CTD phosphorylation events, expand the lexicon of CTD coding marks, and demonstrate unexplored mechanisms to precisely control eukaryotic transcription and transcription coupled‐processes.

Support or Funding Information

This work is supported by grants from the National Institutes of Health (R01 GM104896 to Y.J.Z. and R21EB018391 to J.S.B.) and Welch Foundation (F‐1778 to Y.J.Z. and F‐1155 to J.S.B.). Voyager DE‐PROT MALDI MS data was collected in the University of Texas at Austin Proteomics Facility. Funding from the UT System for support of the UT System Proteomics Core Facility Network is gratefully acknowledged.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FIH is an oxygen sensor for G9a/GLP‐driven epigenetic regulation of metastasis‐related genes in ovarian cancer
Authors: KANG JENGMIN , Park Jong‐Wan
Published: June 2021

The dioxygenases PHD1‐3 and FIH are the oxygen sensors for hypoxia‐inducible factor (HIF)‐driven transcription of hypoxia‐induced genes. However, little is known about the oxygen sensors responsible for oxygen‐dependent epigenetic regulation. We here show that FIH plays additional roles as an oxygen sensor in G9a‐ and GLP‐driven epigenetic regulation. FIH hydroxylates and inhibits G9a and GLP under normoxia. When the FIH reaction is limited under hypoxia, G9a and GLP are activated and repress the metastasis suppressor genes, thereby triggering cancer cell migration and peritoneal dissemination of ovarian cancer xenografts. FIH and G9a expressions are reciprocally associated with clinical outcomes of ovarian cancer. Mutations of FIH‐target motifs in G9a and GLP were identified in human cancers, and they exhibit excessive H3K9 methylation and facilitate cell invasion. This study provides insight into a new function of FIH as an upstream regulator of oxygen‐dependent chromatin remodeling. It also implies that the FIH‐G9a/GLP pathway could be a potential target for inhibiting hypoxia‐induced cancer metastasis.

Support or Funding Information

This work was supported by a grant from the National Research Foundation of Korea (2017R1A4A1015015 and 2016R1A2A1A05005082 to J.W.P). Kang J received a scholarship from the BK21‐plus education program provided by the National Research Foundation of Korea.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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 Information

The authors are thankful to NIPER, SAS NAGAR, MOHALI, PUNJAB for funding the current experimental work

This 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 Information

This work is funded by NIMH R01MH094653 and R01MH111267 to J.D.K

This 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 Information

NIH grant GM20501‐41

This 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 Information

This 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 Information

NIEHS P30ES019776, NIEHS R01ES023485, NIH T32‐GM008602

This 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 Information

Financial 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 Information

Supported in part by NIH grants P01‐AG051442 and R01 AG045693

This 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 Information

This work was supported by NIH grants R03HD092630 and K22ES026235

This 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 Information

This 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 Information

Supported 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.
Molecular basis for the maintenance of lipid asymmetry in the outer membrane of Escherichia coli
Authors: Chng Shu‐Sin , Yeow Jiang , Tan Kang Wei , Holdbrook Daniel , Chong Zhi‐Soon , Marzinek Jan , Bond Peter
Published: June 2021

A distinctive feature of the Gram‐negative bacterial cell envelope is the presence of an asymmetric outer membrane (OM), where lipopolysaccharides (LPS) and phospholipids (PLs) reside in the outer and inner leaflets, respectively. This unique lipid asymmetry renders the OM impermeable to external insults, thus allowing survival of bacteria in harsh environments. In Escherichia coli, the OmpC‐Mla system is responsible for maintenance of OM lipid asymmetry. Osmoporin OmpC and the OM lipoprotein MlaA form a complex proposed to remove PLs from the outer leaflet of the OM. How this complex is organized in the OM to perform this function is not known. In this report, we define the molecular architecture of the OmpC‐MlaA complex to gain insights into its function in PL transport. We show that MlaA sits entirely within the OM lipid bilayer, and interacts with the OmpC trimer at its dimeric interfaces. Molecular dynamics simulations reveal a membrane‐spanning hydrophilic channel within MlaA, suggesting a path for PL translocation across the OM. We demonstrate that a hydrophobic hairpin loop adjacent to this putative channel is critical for modulating the activity of MlaA; restricting flexibility of this structure significantly perturbs the function of the complex. Finally, we establish that OmpC plays an active role in maintaining OM lipid asymmetry together with MlaA. Our work provides a glimpse into the molecular mechanism of how the OmpC‐MlaA complex may extract PLs from the outer leaflet of the OM, and highlights key features that could be exploited in the development of future antimicrobial drugs.

Support or Funding Information

National University of Singapore Graduate School for Integrative Sciences and Engineering scholarship (to J.Y.).

Singapore Ministry of Education Academic Research Fund Tier 3 grant (MOE2012‐T3‐1‐008) grant to (P.J.B.).

Singapore Ministry of Education Academic Research Fund Tier 1 and Tier 2 (MOE2013‐T2‐1‐148) grants (to S.‐S.C.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Unfolded Protein Response Regulator, ATF6, Promotes Mesodermal Differentiation
Authors: Kroeger Heike , Grimsey Neil , Paxman Ryan J. , Chiang Wei‐Chieh , Plate Lars , Jones Ying , Shaw Peter X. , Trejo JoAnn , Tsang Stephen H. , Powers Evan , Kelly Jeffery W. , Wiseman R. Luke , Lin Jonathan H.
Published: June 2021

ATF6 encodes a transcription factor that is activated during the Unfolded Protein Response to protect cells from ER stress. Loss of ATF6α and its paralog ATF6β, results in embryonic lethality, notochord dysgenesis, and in people, loss of ATF6α specifically, results in malformed neuroretina and congenital vision loss. These phenotypes implicate an essential role for ATF6 during vertebrate development. We investigated the function of ATF6 in development using human stem cells undergoing differentiation into multipotent germ layers, nascent tissues, and organs. We artificially activated ATF6 in stem cells with a recently identified small molecule ATF6 agonist, and we inhibited ATF6 using iPSCs from patients harboring ATF6 mutations. We discovered that ATF6 suppresses pluripotency, enhances differentiation, and surprisingly, guides stem cells toward mesodermal cell fates. Our findings reveal a novel role for ATF6 during differentiation and identify a new strategy to robustly create mesodermal tissues through modulation of the ATF6 arm of the UPR.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential effect of OCRL1 patient mutations on protein localization, and sensitivity to FDA‐approved drug suppression of cellular phenotypes associated with Lowe syndrome
Authors: Ramadesikan Swetha , Madhivanan Kayalvizhi , Aguilar R. Claudio
Published: June 2021

Lowe Syndrome (LS) is a lethal X‐linked genetic disorder caused by mutations in the OCRL1 gene, which encodes the lipid phosphatase Ocrl1. LS children present with congenital cataracts, low molecular weight proteinuria and mental retardation as characteristic clinical symptoms. Progressive renal dysfunction is the main cause of death. Ocrl1 localizes to the trans Golgi network (TGN), early endosomes and the plasma membrane and has been shown to participate in vesicle trafficking. We established that LS patient cells are defective in fluid phase uptake and ciliogenesis, which are critical processes required for kidney function. Over 100 OCRL1 mutations have been identified in LS, but their specific impact on cellular processes is unknown. However, our results indicate that different mutations have diverse effect on Ocrl1 localization and on triggering cellular phenotypes.

We expressed GFP‐Ocrl1 (WT and several patient mutants) in human kidney proximal tubule (HK2) cells and LS fibroblasts lacking endogenous Ocrl1. The co‐localization of WT/mutant Ocrl1 with various organelle markers was quantitatively analyzed and different cellular processes/phenotypes were monitored. Our data indicates that in general, N‐terminal PH domain mutations resulted in redistribution of Ocrl1 to the cytoplasm, ASH‐RhoGAP domain mutations prevented localization of Ocrl1 to TGN and mutations in the phosphatase domain resulted in Golgi apparatus (GA) morphological abnormalities.

Importantly, using a drug repurposing approach we identified two FDA‐approved compounds able to differentially suppress LS cellular phenotypes; the ability of these drugs to alleviate defects associated with specific Ocrl1 patient mutants was also analyzed. Our data indicate that while some patient OCRL1 mutations require treatment with both drugs, others only necessitate one for LS cellular phenotype suppression.

This study determined for the first time the effect of different patient mutation on the Ocrl1 intracellular localization and on various LS‐associated cellular phenotypes. Further, it explored how different OCRL1 mutations determined the sensitivity of cells to the newly identified FDA‐approved drugs with ameliorating effects on LS phenotypes. It also contributes to expand our understanding of the molecular mechanism of LS pathogenesis and provide the basis for the development of a LS‐specific therapeutic.

Support or Funding Information

Lowe Syndrome TrustNational Institutes of Health (NIH)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Altered Metabolism and Mitochondrial Lipid Content Drive the Evasion of Apoptosis
Authors: Ross Kyle A. , Wyeth Alexandra J. , Dittenhafer‐Reed Kristin E.
Published: June 2021

Mitochondria are the metabolic centers of the cell and critical players in initiating programmed cell death. Mitochondrial dysfunction and dysregulated apoptosis contribute to a number of disease states, including inherited mitochondrial syndromes, neurological disorders, and cancer. Critical to both mitochondrial function and apoptosis is the architecture of mitochondrial membranes. A mitochondrion is comprised of an inner and outer membrane composed of numerous phospholipids, including cardiolipin, a phospholipid found exclusively in the mitochondria. Cardiolipin binds to cytochrome c in the inner mitochondrial membrane, preventing its release and the initiation of intrinsic apoptosis. Defects in genes required for the maintenance of mitochondrial membrane structure and cardiolipin composition have been identified, and represent an emerging class of mitochondrial disorders. However, a detailed molecular understanding of the synthesis and regulation of mitochondrial membrane phospholipids is lacking. The overall objective is to investigate mechanisms of increased cardiolipin biosynthesis and to study the role of cardiolipin content and composition in regulating the initiation of intrinsic apoptosis. Cancer cells are often characterized by an increased dependence on glucose. These cancers are often resistant to chemotherapies, representing a significant clinical problem. Current research employing cellular phospholipid quantitation has identified that altered cellular glucose metabolism regulates cardiolipin synthesis. Further, the effects of altered cardiolipin levels on cell viability and cytochrome c release upon RNA mediated knockdown of cardiolipin biosynthesis enzymes were studied. Data acquired from cell viability and immunofluorescence microscopy assays suggest that knockdown of cardiolipin synthase (CRLS1), resulting in decreased cardiolipin levels, decreased cell viability after drug treatment, and increased cytochrome c release. Alternatively, loss of lysophosphatidic acid phosphatase type 6 (ACP6), hypothesized to increase cardiolipin levels, resulted in decreased cytochrome c release, which increased cell viability. These results will allow for an increased fundamental understanding of cardiolipin physiology in order to uncover the therapeutic potential of targeting new mitochondrial pathways to treat human disease like cancer.

Support or Funding Information

Dow Scholars ProgramHope College Division of Natural and Applied Sciences

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protease OMA1 modulates mitochondrial metabolism and cristae structure through interaction with MICOS complex
Authors: Levytskyy Roman Myroslavovych , Viana Martonio Ponte , Khalimonchuk Oleh
Published: June 2021

Mitochondria are highly dynamic organelles, whose metabolic outputs are known to correlate with changes in the organelle's network and ultrastructure. Defects in these activities appear to underlie a variety of human diseases, however many molecular aspects of mitochondrial involvement in these pathologies are far from clear. Mitochondrial inner membrane protease OMA1 is a critical regulator of mitochondrial dynamics in higher eukaryotes. It is best known to modulate mitochondrial morphology through proteolytic processing of the dynamin‐like GTPase OPA1, thereby promoting rapid partitioning of mitochondrial network in response to various homeostatic challenges. OMA1 is also known to perform protein quality control functions, and to maintain the stability of respiratory supercomplexes, thus influencing mitochondrial energy metabolism. Mechanistic aspects, as well as functional connection of these important activities remain largely unaddressed.

Here we demonstrate that in addition to its role in mitochondrial fragmentation, OMA1 is required to maintain mitochondrial ultrastructure through an interaction with mitochondrial contact site and cristae organizing system (MICOS) ‐ a protein complex critical for stability of mitochondrial architecture. This specific interaction is mediated by MIC60 MICOS subunit, and – under conditions of cellular stress – influences the recruitment of MICOS proteins MIC10 and MIC27 without affecting mitochondrial fission and fusion machinery proteins. We further show that OMA1‐MICOS association is important for metabolic fine‐tuning of mitochondria in response to homeostatic insults such as mitochondrial depolarization. Moreover, in the absence of OMA1, these activities can be partially compensated for by expression of an artificial tether protein that bridges outer and inner mitochondrial membranes, thereby emulating tunable MICOS complex.

Our findings demonstrate that OMA1‐mediated support of mitochondrial ultrastructure is important for modulation of mitochondrial bioenergetic outputs in response to homeostatic challenges and highlight previously unanticipated role for this protease in mitochondrial physiology.

Support or Funding Information

National Institutes of Health grants R01GM108975 (to O.K.) and P30 GM103335 (to Redox Biology Center at UNL)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.