bluuebunny/crossref_metadata_2025 huggingface dataset:
Retinoic Acid and Retinal Ganglion Cell Survival after Optic Nerve Injury
Authors: Duprey‐Diaz Mildred , Blagburn Jonathan , Blanco Rosa
Published: June 2021

Retinoic acid (RA) is a potent transcriptional activator that may be involved in neuronal plasticity and regeneration in the adult brain. We have shown that the enzyme that synthesizes RA, and the RA receptors, are present in frog retinal ganglion cells (RGCs) and are upregulated after axotomy. The focus of this work is to understand how RA signaling affects RGC survival and its involvement in the activation of intracellular signaling pathways after optic nerve injury. The optic nerve was cut and animals were followed from 1‐6 weeks. Surviving RGCs were retrogradely labeled and counted. Western blot analysis of experimental and control retinas was carried out to measure levels of pro‐survival and pro‐apoptotic proteins, and of activated signaling pathway components, in non‐treated retinas and retinas treated with RA signaling agonists and antagonists immediately after axotomy. Inhibition of endogenous RA signaling significantly decreased the number of RGCs surviving after axotomy; conversely, RA agonist treatment improved RGC survival. RA signaling antagonists abolished axotomy‐induced activation of mitogen‐activated protein kinase (MAPK),phosphoinositide 3‐kinase (PI3K) and signal transducer and activator of transcription 3 (STAT3) in the retina. Application of specific RA receptor subtype agonists into the eye caused an additional increase in the activation of STAT3, but not of the PI3K pathway. We conclude that RA signaling is important for RGC survival after nerve injury, and that further understanding of the role of RA is important for its potential future therapeutic use.

Supported by NIH‐GM 093869 and RCMI‐G12RR03051.
Characterization of DNA dioxygenase TET1 promoter structure and activity
Authors: Juan Li‐Jung
Published: June 2021

Tumor suppressor gene silencing by cytosine methylation contributes to cancer formation. Whether DNA demethylation enzymes counteract this oncogenic effect is just to be revealed. We have demonstrated that the dioxygenase TET1, a DNA demethylation enzyme which converts 5‐methylcytosine to 5‐hydroxymethylcytosine, 5‐formylcytosine and 5‐carboxylcytosine, is an important suppressor of tumor malignancy (Best 5 Article of Cell Reports, 2012). TET1 mRNA expression is significantly low in cancer tissues and its low abundance correlates with poor breast cancer patient survival. Understanding the upstream regulatory mechanism of TET1 expression might be important to control cancer progression. Here we constructed serial deletion mutants of TET1 promoter and identified a specific region to account for the high promoter activity in normal breast cells but low in malignant cancer cell lines.
Evaluation of Bias Associated with High Multiplex Target‐Specific Pre‐Amplification
Authors: Petro Kimberly , Okino Steven , Kong Michelle , Wang Yan
Published: June 2021

We developed a novel PCR‐based pre‐amplification (PreAmp) technology that can increase the abundance of over 350 target genes one million‐fold. To assess bias introduced by PreAmp we utilized ERCC RNA reference standards, a model system that quantifies measurement error in RNA analysis workflows. We assessed three types of bias: amplification bias, dynamic range bias and fold‐change bias. We show that PreAmp does not introduce significant amplification and fold‐change bias, even under high multiplex and high amplification conditions. We do detect dynamic range bias if a target gene is highly abundant and PreAmp occurred for 16 or more PCR cycles; however, this type of bias is easily corrected. To validate PreAmp performance in a gene expression profiling experiment, we analyzed a panel of genes that are regulated during differentiation using the NTera2 stem cell model system. We find that results generated using PreAmp are statistically equivalent to results obtained using standard qPCR without the pre‐amplification step. Importantly, PreAmp maintains patterns of gene expression changes across samples; the same biological insights would be derived from a PreAmp experiment and a standard gene expression profiling experiment. Our PreAmp technology can thus facilitate accurate analysis of extremely limited samples in gene expression profiling experiments.
Investigating and Quantitating RNA Binding to Nanoparticles for Potential Use in Gene Therapy
Authors: Pescador Jorge , Rodriguez Blanca , Maeder Corina , Beall Gary , Lewis Kevin
Published: June 2021

Nanoparticles are defined as molecules that are 1‐100 nm in size in at least one dimension, which contributes to their large surface area to volume ratio. Hydrotalcite, montmorillonite, and halloysite are nanoparticles that have garnered attention as potential drug delivery carriers in recent years. The low toxicity levels of these nanoparticles as well as their ability to interact with biomolecules, such as proteins and nucleic acids, is a key feature for gene therapy applications using nanotechnology. Previous studies have shown that single‐stranded and double‐stranded oligonucleotides and plasmid DNAs bind well to these nanoparticles. Binding of RNA, however, has not been extensively studied. With RNAi becoming an important potential gene therapy tool, finding suitable drug delivery vehicles for siRNAs is of crucial importance. In our studies, we have evaluated the binding efficiency of different types of RNAs (ssRNA, dsRNA, and complex RNA structures) to different nanoparticles and compared them to DNA binding studies with the same nanoparticles. In initial studies focusing on the layered double hydroxide hydrotalcite, a combination of physical and molecular biological assays was employed to assess binding to DNAs and RNAs with different structures. In addition, competition and displacement studies were performed using anionic salts such as carbonate, sulfate, and phosphate to further study the exchange capacity and the binding affinity of one over the other of these anions with the RNAs of interest. In the future, we also aim to characterize the mode of binding using X‐ray diffraction and transmission electron microscopy.
Scorpion Toxin BeKm‐1 Blocks Low K+‐induced Endocytosis of Cell‐surface Human Ether‐a‐go‐go Related Gene (hERG) Channels
Authors: Han Xi , Guo Jun , Li Wentao , Zhang Shetuan
Published: June 2021

The human ether‐a‐go‐go related gene (hERG)‐encoded potassium channel conducts an important current for the repolarization of cardiac action potentials. hERG channel dysfunction leads to abnormal cardiac electrical activities characterized by a prolonged QT interval. We have previously shown that exposure of hERG‐expressing HEK cells to a 0 mM K+medium induces vigorous internalization of hERG but not ether‐a‐go‐go (EAG) channels from the plasma membrane. To investigate the mechanisms of 0 mM K+‐induced hERG endocytosis, we used a chimerical channel model constructed by combining hERG and EAG, as well as drugs that bind to either the internal pore mouth of the hERG channel (E‐4031) or the extracellular edge of the pore entrance (BeKm‐1). Using Western blot analysis and whole‐cell patch clamp, our data show that replacing the S5‐S6 linker of hERG with that of bovine EAG prevented 0 mM K+‐induced hERG endocytosis. Moreover, BeKm‐1 prevented 0 mM K+‐induced hERG internalization. Under the whole‐cell voltage clamp configuration, acute exposure of hERG‐expressing HEK cells to 0 mM K+ solution eliminated hERG conductance within 5 minutes, and the hERG conductance recovered slowly upon re‐exposure to 5 mM K+ solution. The 0 mM K+ exposure‐induced conductance loss was completely prevented by the application of BeKm‐1 (1 µM). We conclude that a conformational change in the outer pore‐mouth of hERG occurs upon removal of extracellular K+, which triggers hERG internalization. BeKm‐1 prevents such conformational change and the subsequent internalization of hERG channels under 0 mM K+ culture conditions.

(Supported by the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Ontario)​
Cholesterol Trafficking for Steroid Biosynthesis in MA‐10 Mouse Tumor Leydig Cells
Authors: Jagannathan Sathvika , Chebbi Seimia , Hullin‐Matsuda Francoise , Kobayashi Toshihide , Papadopoulos Vassilios
Published: June 2021

The hormone‐sensitive and rate‐limiting step in steroid biosynthesis is the movement of cholesterol from intracellular sources to the inner mitochondrial membrane (IMM). Despite the numerous studies on cholesterol trafficking in steroidogenesis, the exact source and mechanism by which cholesterol is transported to IMM remains to be elucidated. D4 is the fourth domain of perfringolysin O protein, which has the ability to bind membranes containing greater than 30 mol% cholesterol of total lipid concentration with high affinity. mCherry‐tagged D4 was used to visualize cholesterol trafficking in MA‐10 cells. Confocal microscopy showed D4‐mCherry localized at the plasma membrane, but upon 45 minutes treatment with dibutyryl‐cAMP (dbcAMP) a significant reduction in plasma membrane labeling was observed. Functional inhibitors of proteins involved in cholesterol import into mitochondria and metabolism, blocked steroid formation and slowed down the movement of D4‐mCherry from the plasma membrane. Recombinant D4‐GFP protein readily bound the outer leaflet of the plasma membrane even after 120 minutes of dbcAMP stimulation, indicating that cholesterol was trafficked from the inner leaflet of the plasma membrane. D4‐mCherry also localized the late endosomes upon dbcAMP stimulation suggesting a route for the cholesterol from plasma membrane to mitochondria. These data suggest that the bulk of the steroidogenic pool of cholesterol, mobilized by cAMP for acute steroidogenesis, likely originates from the inner leaflet of the plasma membrane.

Source: CIHR
Effects of Small Red Bean Powder on Lipid Metabolism and Antioxidant Capacity in High Cholesterol and High Fat Diet Induced Nonalcoholic Fatty Liver Disease (NAFLD) Mice
Authors: Kim Sera , Kim Hyunsook
Published: June 2021

NAFLD is most frequently occurred liver disease caused by high cholesterol and high fat diet. NAFLD is highly related to metabolic disease like obesity, type 2 diabetes and dyslipidemia. In this study, we investigated the effects of small red bean powder on lipid metabolism and antioxidant capacity in NAFLD mice. Mice were randomly divided into 4 groups then fed different diets: normal diets(ND), high cholesterol and high fat diets(HD); and HD diets with 10%(10RB‐HD) and 20%(20RB‐HD) small red bean powder. Body weight gain for 4 groups was ND(5.55±1.27g), HD(15.11±2.00g), 10RB‐HD(8.90±1.17g) and 20RB‐HD(9.54±0.83g). Liver weight in HD(1.63±0.19g) was significantly higher than ND(1.02±0.04g) and decreased in 10RB‐HD(1.23±0.07g) and 20RB‐HD(1.31±0.12g). Abdominal and epididymis adipose tissue weights in all experimental groups were lower than that of HD(0.67±0.13g and 2.29±0.25g). Blood glucose level in 10RB‐HD and 20RB‐HD(156.7±33.78mg/dl, 127.57±31.45mg/dl) was significant lower than HD(274.57±44.76mg/dl). Serum triglyceride and total cholesterol concentration significantly decreased in 10RB‐HD(TG; 118.10±3.38mg/dl, TC; 124.57±8.21mg/dl) and 20RB‐HD(TG; 111.47±2.23mg/dl, TC; 116.51±8.97mg/dl) than HD(TG; 127.88±3.07mg/dl, TC; 201.83±26.19mg/dl). Liver glutathione peroxidase activity in all experimental groups (5.55±0.2104U/ml and 6.34±0.6104U/ml) significantly increased than HD(3.75±0.04U/ml). These results suggest that small red bean powder could improve lipid metabolism and antioxidant capacity, and be meaningful because there are not enough studies for small red bean related to NAFLD. Further lipid and antioxidant indexes are under analysis therefore, it could be more discussed.
Interactions of Human CYP2D6 Polymorphisms with the Mechanism‐Based Inactivator SCH 66712
Authors: Glass Sarah , Osorio Victoria , Fujiwara Rina , Glista Michael , de Waal Parker , Furge Laura
Published: June 2021

Cytochrome P450 enzymes (CYPs) are a heme‐containing enzyme superfamily that have a major role in the metabolism of drugs in humans. CYP2D6 is highly polymorphic with over 100 different allelic variants and is responsible for approximately 15% of CYP‐mediated drug metabolism. Molecular modeling simulations of the allelic variants CYP2D6*1, *17‐2, *17‐3, *34, and *53 with and without docked SCH 66712 ‐ a ligand that is a mechanism‐based inactivator, suggest that SCH 66712 should inactivate these variants. Studies further suggested that varied flexibility and substrate binding‐pocket accessibility within the allelic variants may alter interaction abilities between the enzyme and the ligand. In the current study, CYP2D6 allelic variants were expressed and purified to further characterize these interactions with inactivators. Physical and kinetic studies with the variants will be compared to molecular dynamics models and be used to broaden the understanding of inter‐individual interactions with mechanism‐based inactivators (Support: NIH 1R15‐GM086767‐02).
Specification of Novel Compound E121's Mechanism of Action
Authors: Drummond Shelika , Manzoor Mohammed , Cramer Amanda , Edafiogho Ivan , Soldato Doreen
Published: June 2021

Prior research has shown E121, a novel enaminone compound has immunosuppressive ability and potential as a novel therapeutic drug for inflammatory diseases. E121 has shown significant reduction of TNF‐α and IL‐6 and based on its chemical structure, is suggested to act as a glucocorticoid. The aim of this research project is to further elucidate the biochemical signaling pathway through which E121 conducts its effects. By blocking the E121's possible glucocorticoid receptor with RU‐486, it is hypothesized that E121's immunosuppressive ability will be inhibited. This response was compared with that of dexamethasone (a common glucocorticoid) as a control. In this study, lipopolysaccharide (LPS) stimulated mouse macrophages were treated in different conditions with E121, RU‐486 and dexamethasone. From previous research efforts, E121 has exerted an anti‐inflammatory effect by inducing SOCS1 (Suppressor of Cytokine Signaling). Western Blot of LPS stimulated macrophages in the presence and absence of E121 revealed that higher levels of SOCS1 protein were detected in samples containing increasing amounts of E121. Therefore, E121 appears to induce SOCS1. Preliminary research using RU‐486 as a glucocorticoid receptor antagonist also shows that E121 may require the glucocorticoid receptor. Based on its structure, it may resemble the four ring structure of a glucocorticoid and present a novel therapy for autoimmune diseases as an alternative to steroids.
Structural Dynamics of the NSD Family Histone Methyltransferases
Authors: Teichert Kristian , Iacob Roxana , St. Pierre Roodolph , Ishoey Mette , Chen Sixun , Paulk Joshiawa , Engen John , Bradner James
Published: June 2021

The nuclear SET domain containing protein (NSD) family of histone methyltransferases is an oncogenic family of proteins, whose overexpression is directly involved in cancers, such as acute lymphoblastic leukemia (ALL) and breast cancer. The NSD family consists of three proteins NSD1, NSD2 and NSD3. These proteins are directly responsible for the methylation of multiple lysine residues of histones, a post‐translational modification implicated in transcription‐activating cell survival mechanisms and DNA damage responses. NSD2 in particular is a high‐profile target for inhibition, because it is overexpressed in 20% of multiple myeloma cases. Both NSD2 and NSD3 have evaded crystallization efforts, and only the SET domain of NSD1 has been crystallized. In the absence of a crystal structure for NSD2 and NSD3, we employed homology modeling of the SET domains of NSD2 and NSD3 and hydrogen‐deuterium exchange mass spectrometry (HDX MS) in order to obtain information about the conformation and dynamics of the SET domain in NSD family proteins. HDX MS was also used to monitor and localize binding of drug candidates directed against these proteins. Our results can aid the development of specific inhibitors for members of the NSD family. Support: R21 CA179159‐01A1, Multiple Myeloma Research Foundation Collaborative PPG
Mechanism of Nicotinamide Riboside as an Aid to Weight Loss
Authors: Brenner Charles , Weidemann Benjamin , Trammell Samuel , Coon Joshua , Yu Liping , Migaud Marie , Grobe Justin
Published: June 2021

Caged mice represent an attractive model system with which to dissect the mechanistic basis for dietary and activity interventions because one can control for food amounts, dietary compositions, supplementation, and exercise in addition to genotype. Here we report that oral nicotinamide riboside (NR) supplementation increases weight loss with respect to nonsupplemented C57BL/6 mice. NR‐dependent weight loss was much more effective than resistance to weight gain on high fat diet (HFD), which has been previously reported. To assess weight loss, mice were first fattened on HFD and then assigned to normal chow plus or minus NR. Supplemented and nonsupplemented mice were also randomized to 5 days/week forced exercise or not. NR and exercise promoted weight loss individually and in an additive fashion. To assess the mechanism by which NR promotes weight loss, we measured calorie intake, activity, calorie waisted, resting metabolic rate, body composition, glucose tolerance, insulin sensitivity, and a variety of biochemical and metabolomic parameters related to liver mitochondrial function. The data are consistent with a novel malabsorptive mechanism for weight loss that can be tracked by excreted biomarkers. NR analogs and proteomic analysis of mitochondrial post‐translational modifications are being used to further characterize the mechanistic basis for NR activity in weight loss.
Hypoxia Affects Cell Migration and Wound Healing by Altering Basal Lamina Proteins and Focal Adhesion Turnover
Authors: Onochie Obianamma , Trinkaus‐Randall Vickery
Published: June 2021

Corneal disease and injury remain a major cause of blindness according to the World Health Organization. Certain conditions such as diabetes, corneal infection, and corneal ulceration may cause the cornea to be exposed to a hypoxic state for extended periods of time. Over 95% of the new cases of diabetes are Type II. In these patients, a simple wound can lead to complications which are compounded by the reduced ability to recover from hypoxic stress. An ex vivo corneal organ culture model was employed to examine cell migration. After a 24 hour exposure to hypoxia healing is delayed by 6 hours compared to control. In addition, there is a morphological change at the leading edge of cells. We hypothesize that hypoxia affects cell migration and wound healing by altering the regulation of basal lamina proteins and the turnover of focal adhesions. We determined that wounded rat corneas exposed to a hypoxic environment display decreased epithelial expression of syndecan‐1 and fibronectin. The localization of fibronectin throughout the epithelium and stroma is altered in the hypoxic cornea. Additional studies examined the alterations in expression and co‐localization of focal adhesion proteins. Hypoxia caused a change in the localization of vinculin and paxillin along the basal lamina and within the epithelium. Furthermore, there was a change in localization along the basal lamina of Y118 paxillin in the hypoxic cornea. By determining the changes in basal lamina composition and characterizing the changes in regulation and dynamics of focal adhesion proteins, we aim to improve the treatment of delayed wound healing associated with corneal hypoxia.
Matricellular Protein Cyr61 Is a Key Mediator of Platelet‐derived Growth Factor‐induced Cell Migration
Authors: Cui Mei‐Zhen , Zhang Fuqiang , Hao Feng , An Dong , Zeng Linlin , Xu Xuemin
Published: June 2021

PDGF, a potent chemoattractant, induces cell migration via MAPK and PI3K/Akt pathways. However, the downstream mediators are still elusive. In particular, the role of extracellular mediators is largely unknown. In this study, we identified that the matricellular protein Cyr61, which is de novo synthesized in response to PDGF stimulation, is the key downstream mediator of the ERK and JNK pathways, independent of p38 MAPK and AKT pathways, and thereby mediates PDGF‐induced smooth muscle cell (SMC) migration, but not proliferation. Our results revealed that when Cyr61 was newly synthesized by PDGF, it was promptly translocated to the extracellular matrix and physically interacted with plasma membrane integrins α6β1 and αvβ3. We further demonstrate that Cyr61 and integrins are integral components of the PDGF signaling pathway, via an “outside‐in” signaling route to activate intracellular focal adhesion kinase (FAK), leading to cell migration. Thus, this study provides the first evidence that PDGF‐induced endogenous extracellular matrix component Cyr61 is a key mediator in modulating cell migration by connecting intracellular PDGF‐ERK and JNK signals with integrin/FAK signaling. Therefore, extracellular Cyr61 convergence with growth factor signaling and integrin/FAK signaling is a new concept of growth factor‐induced cell migration. The discovered signaling pathway may represent an important therapeutic target in growth factor‐mediated cell migration/invasion‐related vascular diseases and tumorigenesis.
Biological Evaluation of Simplified Analogs of Protein Kinase C Inhibitor Staurosporine
Authors: Rolph Carly , Pelkey Erin , Mowery Patricia
Published: June 2021

PKC, or protein kinase C, is an enzyme in the family of serine/threonine kinases that is involved in a variety of cell signaling pathways throughout the body. It plays a role in a multitude of diseases and disorders such as diabetes, heart failure, Parkinson's disease, Alzheimer's disease, bipolar disorder, dementia and cancer. PKC is a promising therapeutic target using small molecule inhibitors. One of the best characterized inhibitors is staurosporine, an indolocarbazole derivative, that binds the ATP pocket of PKC and limits its phosphorylation activity. Numerous simplified derivatives of staurosporine have been synthesized, which have promising structural features for inhibiting PKC. An initial screening using an MTT assay has shown that planar analogs have different IC50 values than non‐planar ones, but testing will need to be continued to further determine cytotoxicity.
Evaluation of the Polyketide GEX1A as a Potential Lead for Niemann‐Pick Type C Disease
Authors: Granatosky Eve , Pickering Jarred , Stevens D. , Taylor Richard
Published: June 2021

Niemann‐Pick Type C (NPC) disease is a rare neurodegenerative disorder in which unesterified cholesterol accumulates in the lysosomes as a result of a mutation in either the NPC1 or NPC2 gene. Our efforts to identify a novel treatment for NPC disease are focused on the polyketide natural product GEX1A, derived from Streptomyces chromofuscus. We have observed that GEX1A is capable of improving cholesterol trafficking in NPC cells similar to other small molecules currently under investigation for the treatment of NPC disease, however our recent work suggests that GEX1A may exert its activity through a unique mode of action. Based on these findings, we aim to study the therapeutic potential and biological target of GEX1A. Here we highlight our multidisciplinary approach to accessing GEX1A and analogues and our studies of GEX1A's biological activity in the context of NPC disease.
Targeting the AKT pathway in glioma by using bioinformatics tools
Authors: Bardhi Kostandina , Bardhi Keti , Craig Paul , Bernstein Herbert
Published: June 2021

The aim of this project is to study enzymes involved in glioma for the purpose of identifying better inhibitors. The PI3K/ AKT/mTor pathway is one of the most important pathways related to different cancers, including glioma. Faulty signaling in the PI3K/AKT pathway can lead to resistance to cancer therapies. This is a tyrosine kinase pathway that controls cell growth, proliferation and survival. AKT is a member of the protein kinase b family and it's over phosphorylation can lead to uncontrolled cell growth. Therefore, inhibition of AKT is a good target for glioma therapies. In the PDB there are 88 structure hits for the protein kinase b family and we decided to focus specifically on glycogen synthase kinase 3 (GSK3; PDB entry 4B7T). We employed structural bioinformatics tools to search for inhibitors of AKT. We first used ProMol, a plug‐in for Pymol, to look at the structural homologs of the GSK3 catalytic site. The best alignment was with protein kinase c (1ZRZ). A subsequent BLAST alignment of the two structures revealed 29% identity for 56% sequence coverage. We then used Autodock Vina to screen for ligand‐receptor docking. Several ligands were docked with our target protein, 4B7T. According to the binding affinity scores, two ligands displayed promising binding affinity for the active site of the target protein: 9 ‐ nitro ‐ 5,12 ‐ dihydro ‐ 7H ‐ benzo[2,3] azepino[4,5 ‐ B]indol ‐ 6 ‐ one and pyrimido [4,5‐c]isoquinoline‐1,3,7(2H,4H,8H)‐trione, 9,10‐dihydro‐2,4,9,9‐tetramethyl. These two ligands and others will be explored further.
Inhibition of Human Acetylcholinesterase by Enantiomers of V‐Type Chemical Warfare Nerve Agents and Analogs
Authors: Boeri Michael , Kasten Shane , Cerasoli Douglas
Published: June 2021

Chemical warfare nerve agents are extremely toxic organophosphorus compounds that contain a chiral phosphorus center. Analytical scale methods using a supercritical fluid chromatography (SFC) system in tandem with a mass spectrometer were developed for the separation and detection of individual stereoisomers of VX, VR, and VS, and the analogs V1 thru V4. Separation was evaluated with respect to the chromatography parameter resolution (Rs) with a value of 蠅1.7 indicating complete separation. The ChiralCel OD‐H column yielded Rs values >1.7 for each V‐type compound with mobile phase modifier concentrations 蠄2%. Methods that provided the highest Rs values were scaled up to the semi‐preparative level, allowing for isolation and analysis of individual stereoisomers at >99% purity. Ellipticity (Θ) values of each enantiomer were determined by circular dichroism (CD) spectroscopy. VX, V1, V2, and V3 all had a ΘMAX at 221nm, while VR, VS, and V4 had a ΘMAX at 226nm. For all agents, the first eluting isomer had a negative value at ΘMAX [(‐)Θ221‐226], while the second eluting isomers all had positive values at ΘMAX [(+)Θ221‐226]. Individual enantiomers and racemic mixtures of each agent were then incubated with recombinant human acetylcholinesterase to determine their inhibition rate constants (ki). The (+)Θ221‐226 enantiomer was found to be an average of 2‐3 orders of magnitude more inhibitory than the opposite enantiomer of the same agent.

The views expressed in this poster are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government.

This research was supported by the Defense Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division.
An Optimized Viral Delivery System to Expand the Genetic Code in Mammalian Cells
Authors: Zheng Yunan , Chatterjee Abhishek
Published: June 2021

Co‐translational, site‐specific incorporation of unnatural amino acids (UAAs) by nonsense suppression has proven to be a powerful tool for investigating and engineering protein structure and functions. This methodology utilizes an engineered aminoacyl‐tRNA synthetase/tRNA (aaRS/tRNA) pair, orthogonal to the host cell, to deliver the UAA of interest in response to a nonsense codon. The ability to use this toolbox to investigate and manipulate protein function in human and other mammalian cells can be highly beneficial. The primary challenge is to efficiently co‐deliver all the genetic components, and a lack of understanding regarding optimal expression levels of these components. Viral vectors are ideal vehicles for delivery, potentially enabling application in a variety of mammalian cell lines, and even live tissues and animals.

Using a unique viral delivery vector, and optimal expression of the genetic components, we have substantially enhanced UAA mutagenesis efficiency in mammalian cells. We identified the tRNA as the limiting component, while the traditional aaRS expression level was shown to be unfavorably high. The large and complex cargo could be efficiently packaged within our unique baculoviral delivery vector‐ showcasing the prowfess of this platform.
Sequential Phosphorylation of SIKE by TBK1
Authors: Bell J Ellis , Kerckhove Tanner , Kim Seung‐Hwan , Bell Jessica
Published: June 2021

Our recent work (Marion et al. J. Biol. Chem. (2013) 288:18612) has demonstrated that SIKE

can be multiply phosphorylated by the protein kinase, TBK1, and that phospho‐mimetic mutants representing differently phosphorylated SIKE forms inhibit TBK1 utilization of its other substrate, IRF3, with significantly different inhibition constants. To investigate both the effects and time dependence of site‐specific SIKE phosphorylation we have combined computational (homology modeling using I‐TASSER and docking with TBK1 studies using the ClusPro server ) and experimental approaches ( CD, Thermal melts, Chemical denaturation and Multiwavelength Collisional Quenching studies) of WT SIKE and several phospho‐knockout or phosphor‐mimetic forms of SIKE. In time‐dependent phosphorylation experiments, SIKE with different levels of total phosphorylation was isolated by ion exchange chromatography and unique phosphorylation sites of the separated isoforms identified by fragmentation and MALDI‐TOF or tandem mass spectrometry as appropriate. To investigate the impact of phosphorylation on the structure and stability of SIKE we have used a combination of circular dichroism spectroscopy and multiwavelength collisional quenching to investigate the conformation and flexibility of SIKE and the effects of phosphorylation.. This work is supported NIH Grant: R21AI107447
Acid Gradient Across Plasma Membrane can Drive Phosphate‐Bond Synthesis in Cancer Cells: Acidic Tumor Milieu can Act as a Potential Energy Source
Authors: Dhar Gautam , Sen Suvajit , Chaudhuri Gautam
Published: June 2021

Aggressive cancers exhibit high glucose to lactate conversion accompanied by acid secretion, a phenomenon popularly known as the Warburg effect. The acidic microenvironment and the alkaline cytosol create a proton‐gradient (acid gradient) across the plasma membrane. In this work we demonstrate that cancer cells can utilize proton‐motive force across plasma membrane to drive robust synthesis of intracellular phosphate‐bonds, at a pH range (6.0 to 7.2) seen in tumor milieu (6.15 to 7.4). This was demonstrated in absence of glycolysis and mitochondrial ATP synthesis, and is unique to cancer cells. Assays with viable cancer cells and plasma membrane vesicles utilizing radioactive phosphate confirmed phosphate‐bond synthesis from phosphate, as well as localization of this activity to the plasma membrane. In addition to ATP, predominant formation of pyrophosphate (PPi) from phosphate (Pi) was observed with the plasma membrane vesicles subjected to acid gradient. This was observed in purifed right‐side out vesicles (ROV) that were prepacked with substrates and also with inside‐out vesicles (IOV) but with reversed acid gradient. Cancer cytosols were found capable of converting PPi to ATP, and also stimulate ATP synthesis from Pi from the vesicles in the IOV assay. Possible mechanistic steps were worked out. Acid gradient created by glucose metabolism, as observed in tumors, was found critical for phosphate‐bond synthesis. The findings suggest a role of acidic tumor milieu as a potential energy source in cancer. The robust activity implies that the plasma membrane phosphate‐bond synthesizing machinery and its associative cytosolic counterparts could be potential therapeutic targets against cancers.
Targeting 6‐Phosphofructo‐2‐kinase/Fructose‐2,6‐Bisphosphatase‐4 (PFKFB4) in Cancer
Authors: Telang S. , Trent J. , Chesney J. , Clark J.
Published: June 2021

High glycolytic flux, even in the presence of oxygen (i.e. the Warburg effect), is a central neoplastic hallmark. Oncogenic activation and HIF‐1α over‐expression stimulate glycolysis by activating the 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatases (PFKFB1‐4) which synthesize fructose‐2,6‐bisphosphate (F2,6BP), a potent allosteric activator of the metabolic sensor, 6‐phosphofructo‐1‐kinase (PFK‐1). We recently found that PFKFB4 is highly expressed in multiple cancer cell types with disparate driver oncogenes, is strongly induced by hypoxia via HIF‐1α upregulation and that its suppression leads to decreased F2,6BP, glycolytic flux to lactate and the TCA cycle, increased apoptosis and inhibition of lung tumor growth in vivo. Importantly, compared with normoxia, silencing PFKFB4 in hypoxia significantly decreases survival and proliferation, indicating that PFKFB4 may be preferentially required for hypoxic survival and growth. Taken together, these data provide rationale for the development of small molecule inhibitors targeting PFKFB4.

Using molecular modeling and receptor‐based virtual screening, we have identified a small molecule inhibitor of PFKFB4, termed 5MPN, that selectively inhibits recombinant PFKFB4 activity. We find that 5MPN: (i) decreases the F2,6BP, glucose uptake and proliferation of multiple cancer cells; (ii) selectively suppresses Ras‐transformed bronchial epithelial cell growth without affecting normal bronchial epithelial cells; and (iii) markedly reduces xenograft growth in vivo (IV and PO). Our data indicate that targeting PFKFB4 may be a viable therapeutic strategy against cancer and future studies will be aimed at the examination of 5MPN in combination with PFKFB3 inhibitors and other targeted anticancer agents.
Impaired Mitochondrial Protein Synthesis in Head and Neck Squamous Cell Carcinoma
Authors: Koc Emine , Haciosmanoglu Ebru , Wolf Allison , Claudio Pier Paolo , Koc Hasan
Published: June 2021

Human Head and Neck Squamous Cell Carcinoma (HNSCC) ranks among the sixth most common cancer types worldwide possibly due to the significant role of alcohol and tobacco use in the development of HNSCC. Interestingly, mutations in mitochondrial DNA (mtDNA) have been found to be associated with human head and neck cancers. Here, we investigated the changes in oxidative phosphorylation (OXPHOS) complexes and contribution of mitochondrial translation components in HNSCC. Immunoblotting analyses of human HNSCC tumors have shown that the expression of OXPHOS was impaired at the protein level. Specifically, an overall reduction in one of the mitochondrially encoded subunits of the OXPHOS complexes accentuated a possible defect in mitochondrial translation machinery. Evidence provided in this study suggests that the mitochondrial translation defect(s) could be due to a decrease in expression of one of the essential mitochondrial ribosomal proteins, MRPL11, in head and neck tumor biopsies. We also observed an acquired mitochondrial translation deficiency in the HN8 cell linederived from a lymph node metastasis but not in the HN22 cells derived from the primary tumor of the same patient. Our observations suggest that the mitochondrial translation machinery deserves further investigation for accurate molecular assessment and treatment of HNSCC.
Resveratrol Fails to Protect from Lipid Peroxidation and Cell Viability Loss in a Chronologically Aged GPX2 Mutant of S. cerevisiae.
Authors: Flores‐Ledesma Claudia Guadalupe , Canizal‐Garcia Melina , Moreno‐Jimenez Mario , Morales‐Solorio Maria , Montoya‐Perez Rocio , Madrigal‐Perez Luis Alberto , Saavedra‐Molina Alfredo , Cortes‐Rojo Christian
Published: June 2021

The lifespan of several animal species has been inversely correlated with the unsaturation degree of the fatty acids constituting membranes. In this regard, it has been described that resveratrol may extend the lifespan of several organisms, as S. cerevisiae. This yeast is suitable for carrying out studies exploring the role of fatty unsaturation degree on the rate of aging because the easy of manipulation of its membrane fatty acid composition. In order to explore whether resveratrol is able to extend the lifespan of yeast in relation with a high degree of fatty acid unsaturation and enhanced lipid peroxidation, we tested it effects on chronological aging and lipid peroxidation sensitivity in a phospholipid‐ hydroperoxide glutathione peroxidase 2 (GPX2) mutant growth in the presence of arachidonic acid (C20:4). The GPX2 mutant exhibited lower cell viability at 5 or 10 days of chronological aging in the presence of C20:4 and this phenotype was partially rescued by the addition of resveratrol or the lipophilic antioxidant BHT. However, only BHT protected from cell viability loss at 15 or 20 days of aging. The GPX2 mutant exhibited a phenotype of hypersensitivity to lipid peroxidation at 15 days of aging, which was prevented by BHT but not by resveratrol. These results suggest that resveratrol may not extend the lifespan of yeast by increasing membrane unsaturation degree due to its inability to prevent lipid peroxidation.
Elderly rat heart shows long‐lasting impairment of oxidative balance after forced exercise at early age.
Authors: Braz Glauber Ruda , Pedroza Anderson , Sousa Shirley , Lopes de Morais Andréia , Mendes‐da‐Silva Rosângela , Costa Laís , Batista‐de‐Oliveira‐Hornsby Manuella , Lagranha Claudia
Published: June 2021

It is well known that aging process alters cardiac physiology and the energy metabolism.Reactive oxygen species (ROS) are considered a key factor in the heart aging process. It has been demonstrated that activity of antioxidant systems are modified in aging and consequent weak resistance to ROS accumulation. Studies suggest that exercises ameliorate the process induced by aging. Therefore, the aim of the present study was to evaluate the long‐lasting effects of physical training during the period of critical brain development (i.e 15 ‐ 45 days of age) on oxidative balance in elderly hearts (585 days of age). The treadmill exercise of the rats was performed as previously described (Batista‐de‐Oliveira et al.Experim. Gerontol. 47:452–457). Malondialdehyde‐MDA formation was measured as described by Buege & Aust (Methods Enzymol 52, 302‐310), Carbonyl content as described by Levine et al. (Methods Enzymol 233:346‐57), GSH levels by Hissin & Hilf (Analytical Biochemistry 74:214‐216), SOD activity by Misra & Fridovich (JBC 247(10): 3170‐3175), Catalase‐CAT measured as described by Aebi (Methods Enzymol 105, 121‐126; 1984), and GST by Nascimento et al. (APNM 39:1‐8). Exercise increases MDA formation (75%); increases activity of SOD (24%) and it has no effects in additional enzymes. Our data suggest that submitting young rats to this protocol of moderate exercise does lead to long‐lasting impairment of oxidative balance in the hearts of aged rats.
Rin GTPase deficiency promotes neuroprotection following traumatic brain injury
Authors: Pannell Megan , Cai Weikang , Brelsfoard Jennifer , Carlson Shaun , Littlejohn Erica , Stewart Travis , Saatman Kathryn , Andres Douglas
Published: June 2021

Traumatic brain injury (TBI) affects approximately 1.4 million people in the USA annually and remains a leading cause of death and disability. In addition to the cell death caused by the initial insult, TBI also leads to the activation of secondary cascades including excitotoxicity, free radical production, and glial activation. These mechanisms initiate detrimental consequences for months following injury, obstructing cognition and motor functioning.

Ras family proteins act as GDP/GTP regulated molecular switches governing diverse cellular processes by allowing for the relay of extracellular stimuli into cellular signaling pathway. Rin was identified as a small G‐protein with expression limited to the central nervous system.

Through the generation of a Rin knockout mouse, our lab has elucidated a model in which loss of Rin signaling aids in neuroprotection following TBI. While targeted disruption of Rin does not alter neural development, its loss promotes enhanced adult neurogenesis. Phospho‐specific immunoblotting suggests that Rin deficiency protects primary neuronal cultures from the effects of reactive oxygen species by increasing AKT signaling and blunting p38 MAPK activation. Furthermore, we have shown that the absence of Rin is neuroprotective against TBI, decreasing the amount of hippocampal neurodegeneration and improving cognitive performance after injury. Taken together, these data suggest that endogenous Rin regulates a neuronal pro‐apoptotic cascade and that inhibition of Rin signaling may have broad therapeutic potential by promoting neuronal survival and limiting post‐injury cognitive dysfunction.
Calcyon Regulates Axonal Transport of PI4K11a Vesicles in Cultured Rat Sensory Neurons
Authors: Roth Zak , Shi Liang , Smith Deanna
Published: June 2021

Cytoplasmic dynein is a motor protein representing an essential component in directed microtubule trafficking of membrane bound vesicles. Dynein motors are primarily responsible for retrograde, or minus‐end directed axon transport. Recently, we have shown that Calcyon (Caly, NSG3), a type 2 transmembrane protein important for the targeting of Clathrin Adaptor Protein‐3 (AP3) cargos, interacts with dynein (Muthusamy et al, in preparation). In order to explore Caly's viability as an efficient regulator of dynein activity, we have used time‐lapse microscopy and kymographs to analyze axonal trafficking of PI4K11a organelles in cultured adult rat sensory neurons. PI4K11a is a known Calcyon‐interacting protein involved in endosomal trafficking. The motility of organelles enriched in PI4K11a was shown to be altered by changes in Calcyon expression. In addition, previous studies indicate that the loss of PI4K11a in mice results in an adult onset neurodegeneration phenotype (Simons et al, PNAS, 2009). Based on our studies, we hypothesize that the Caly/PI4K11a interaction is an important regulator of dynein‐dependent axon transport, and any significant disruption of this interaction has the potential to cause axon degeneration.
Colocalization of CHL1 and ROBO1 During Embryonic Development of Mouse Brain
Authors: Wright Amanda , Sohail Saher , Hackel Dana
Published: June 2021

Axons are an integral part of a neuron responsible for transmitting electrical signals within the brain. It is important to understand the mechanics of axon growth, including: how growth cones function and which proteins are involved in axon migration and how they co‐exist with other proteins. This hierarchy of information is essential to combat neurological disorders such as Alzheimer's disease. We currently know of several families of proteins that play major roles in regulating axon guidance. Two such families are neural cell adhesion molecules (CAMs) of the immunoglobulin superfamily (Maness and Schachner, 2007) and the family of SLIT proteins (Andrews, et al., 2007). Close Homolog of L1 (CHL1), a CAM, is part of the mammalian L1 family, which is known to play a role in axon growth and migration of developing neurons (Wright, et al., 2007). Roundabout1 (ROBO1), also a member of the immunoglobulin super family, plays a role in axon guidance in the developing forebrain by serving as a receptor for the guidance cue, SLIT1 (Andrews, et al., 2007). Our objective in this study is to investigate a potential interaction between CHL1 and ROBO1. We hypothesize that these two proteins function together to regulate SLIT‐mediated axon growth in developing neurons. The first step in our investigation involved the use of co‐immunofluorescence staining to visualize CHL1 and ROBO1 colocalization. We demonstrate here that CHL1 and ROBO1 do colocalize in the intermediate zone of the cerebral cortex during axonal outgrowth (E15) and axonal targeting (E16). This colocalization suggests an interaction between these two proteins that may help promote axon growth and guidance. This work was supported by Marymount University's DISCOVER Program and the Clare Boothe Luce Foundation.
AMPKα1 Deficiency in Fibroblasts Promotes Angiogenesis in Athymic Nude Mice
Authors: Song Ping , Zhou Yanhong , Xu Hairong , Ding Ye , Zou Ming‐Hui
Published: June 2021

Angiogenesis is required and essential for tumor development. Increasing evidence suggests that adenosine monophosphate‐activated protein kinase (AMPK), an energy sensor and modulator of redox system, is associated with cancer development. However, the effect of AMPK on tumor development is controversial. Mouse embryo fibroblasts (MEF) were isolated from AMPKα1 knock out (AMPKα1‐KO), AMPKα2‐KO, and wild type (WT) C57BL/6J mouse embryos, and immortalized by using standard 3T3 protocol to investigate the mechanisms of fibroblast‐mediated angiogenesis. Xenograft model data revealed that knock out of AMPKα1, but not AMPKα2, stimulated the cellular proliferation of immortalized MEFs, angiogenesis, and tumor development in athymic nude mice in vivo. Consistent with this finding, the migration of human microvascular endothelial cells (hmvEC) was enhanced by co‐culturing with AMPKα1‐KO MEFs when compared to either WT or AMPKα2‐KO MEFs. Moreover, the network formation of hmvEC was stimulated by conditioned medium from AMPKα1‐KO MEFs, but not from WT or AMPKα2‐KO MEFs. Mechanistically, we found that the protein level of noncanonical nuclear factor kappa B2 (NF‐ĸB2)/p52 was elevated in AMPKα1‐KO MEFs and was responsible for induction of erythropoietin (Epo) expression. Finally, the most conclusive evidence for AMPKα1‐dependent inhibition of EC migration and consequent angiogenesis as well as tumor progression was that the xenograft growth of the inoculated AMPKα1‐KO MEFs was ameliorated by treatment with Epo neutralization antibody. This compelling evidence indicates that AMPKα1, rather than AMPKα2 deletion in fibroblasts, instigates EC migration, angiogenesis, and the resultant tumor development.
Composition Analysis of Hexane Extract Seed Oils, and Study on the Effect of Seed Oil on 3T3‐L1 Adipocytes
Authors: Kim Tae Woo , Kim Kyoung Kon , Kang Yun Hwan , Kim Dae Jung , Choe Myeon
Published: June 2021

In this study, we evaluated the fatty acid composition and physiological activities of 8 seed oils extracted from pepper (Capsicum annuumL.) seed, green tea (Camellia sinensisL.) seed, perilla (Perilla frutescensvar. japonicaHara) seed, peanut (Arachis hypogaeaL.), cotton (Gossypium indicumLAM.) seed, sesame (Sesamum indicumL.) seed, walnuts (Juglans regia L.), and safflower (Carthamus tinctoriusL.) seed. The composition and quality measurements showed that the oils were suitable for consumption as per Food Code standards. The composition analysis showed that the oils were composed of unsaturated fatty acids of majority including linoleic acid and oleic acid. Green pepper, perilla, and peanut seed oils inhibited lipid accumulation. Also, green pepper, perilla, peanut, sesame, walnuts, and safflower seed oils induced leptin secretion. These results show that the effect of seed oils on inhibition of lipid accumulation and induction of leptin secretion may be useful for weight management edible oil.