bluuebunny/crossref_metadata_2025 huggingface dataset:
Tapping the Power of Plasma Lipidome to Improve Hypertriglyceridemic Waist as a Predictor of Future Type 2 Diabetes
Authors: Mamtani Manju , Meikle Peter , Kulkarni Hemant , Wong Gerard , Weir Jacquelyn , Barlow Christopher , Dyer Thomas , Almasy Laura , Mahaney Michael , Comuzzie Anthony , Duggirala Ravindranath , Blangero John , Curran Joanne
Published: June 2021

Hypertriglyceridemic waist is considered a specific predictor of T2D because it combines the predictive value of waist circumference (WC) and serum triglycerides (TG). Our objective was to investigate if the addition of specific triacylglycerol (TAG) lipid species to WC performs better than the combination of WC and TG to predict future T2D in initially diabetes‐free individuals (n=771, 9197 person‐years follow‐up). The participants were from the San Antonio Family Heart Study and the plasma lipidome was quantified using high‐performance liquid chromatography and mass spectrometry. A forward addition approach to optimize the integrated discrimination index (IDI) identified five independent and significant TAG species out of 43 species investigated.

Selection of TAG species using stepwise forward added IDI
Association of TAG species with T2D

Step
TAG Retained
IDI
p
Scale
Coefficient
p

1
TAG 16:0/16:0/18:2
0.0278
0.0031
10‐4
0.6915
0.0004

2
TAG 14:1/18:1/18:1
0.0264
8.9x10‐5
10‐3
‐0.4125
0.0003

3
TAG 18:0/18:2/18:2
0.0025
0.0391
10‐3
‐0.2426
0.0278

4
TAG 16:0/18:1/18:1
0.0075
0.0224
10‐5
1.1917
0.0105

5
TAG 14:0/18:2/18:2
0.0070
0.0209
10‐3
0.5095
0.0308

A summary TAG score was constructed from these five TAG species based on logistic regression model as shown in Table 1. Comparison of the predictive performance of the TAG score with that of TG concentration demonstrated the additive value of the TAG score but not of TG to basic clinical predictors of T2D including prediabetes.

Statistic
Base Model Estimate (p)
Base Model + Prediabetes Estimate (p)

ΔAROC TAG Score
0.0374 (0.0063)
0.0274 (0.0014)

IDI TAG Score
0.0726 (5.3x10‐7)
0.0585 (9.9x10‐6)

NRI TAG Score
0.6008 (5.7x10‐10)
0.5937 (8.9x10‐10)

ΔAROC TG
0.0035 (0.4472)
0.0014 (0.6156)

IDI TG
0.0059 (0.0407)
0.0011 (0.2827)

NRI TG
0.3500 (0.0002)
0.1553 (0.0578)

Our results provide two interesting implications: first, not all TAG species are similar (either in strength or direction of association) with regards their predictive value for future T2D; and second, few TAG species from the plasma lipidome can be combined with WC to significantly improve the prediction of future T2D.

This work was supported in part by NIH grants, AT&T Foundation anad facilities supported by NIH grants.
Exogenous Arachidonic Acid Mediates Permeability of Human Brain Microvessel Endothelial Cells through Prostaglandin E2 Activation of EP3 and EP4 Receptors
Authors: Dalvi Siddhartha , Nguyen Hieu , On Ngoc , Mitchell Ryan , Aukema Harold , Miller Donald , Hatch Grant
Published: June 2021

The blood‐brain barrier, formed by microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. Arachidonic acid (ARA; 5,8,11,14‐cis‐eicosatetraenoic acid) is a conditionally essential polyunsaturated fatty acid [20:4(n‐6)] and is a major constituent of brain lipids. The current study examined the transport processes for ARA in confluent monolayers of human brain microvessel endothelial cells (HBMEC). Addition of radioactive ARA to the apical compartment of HBMEC cultured on Transwell® inserts resulted in rapid incorporation of radioactivity into the basolateral medium. Knock down of fatty acid transport proteins did not alter ARA passage into the basolateral medium due to the rapid generation of prostaglandin E2 (PGE2), an eicosanoid known to facilitate opening of the blood‐brain barrier. Permeability following ARA or PGE2 exposure was confirmed by an increased movement of fluorescein‐labeled dextran from apical to basolateral medium. ARA‐mediated permeability was attenuated by specific cyclooxygenase‐2 inhibitors. EP3 and EP4 receptor antagonists attenuated the ARA mediated permeability of HBMEC. The results indicate that ARA increases permeability of HBMEC monolayers likely via increased production of PGE2 which acts upon EP3 and EP4 receptors to mediate permeability. These observations may explain the rapid influx of ARA into the brain previously observed upon plasma infusion with ARA.
Absorption of Molecular Forms of Dietary Docosahexaenoic acid (DHA) and Their Potential to Provide DHA to the Brain
Authors: Subbaiah Papasani , Dammanahalli Karigowda. , Yang Peng , Malhotra Pooja , Bi Jian , O'Donnell J , Alrefai Waddah
Published: June 2021

In this study, we tested the hypothesis that the presence of dietary DHA in the sn‐1 position of phosphatidylcholine (PC) results in greater incorporation of absorbed DHA into the phospholipids of plasma lipoproteins, compared to its presence in triacylglycerol (TG) or in the sn‐2 position of PC, because of the positional specificities of the pancreatic enzymes. Micelle containing the DHA either in lyso PC (LPC) form (surrogate for the dietary sn‐1 DHA PC) or as free fatty acid (surrogate for dietary DHA TG and sn‐2 DHA PC) were infused intraduodenally into lymph fistula rats, and the concentration of DHA in PC and TG of lymph chylomicrons and HDL was determined by GC and LC/MS. The total amount of DHA absorbed was comparable with the two preparations. However the ratio of DHA in PC/TG in lymph was much higher (0.4) after LPC‐DHA infusion, compared to free DHA infusion (0.1). Also, more DHA appeared in HDL fraction of lymph after LPC‐DHA infusion, and more of the HDL DHA was in PC than in TG. Similar results were obtained with CACO‐2 cells grown on micropore membranes in transwell plates. The two types of DHA micelle were added to the apical medium and the lipoproteins in the basolateral medium were analyzed after 24 h. The results showed a 4‐fold higher incorporation of DHA into PC in presence of LPC DHA compared to free DHA. These data indicate that the dietary DHA in the sn‐1 position of PC is potentially more available than the DHA in sn‐2 position of PC (krill oil) or in TG (fish oil) for uptake by the brain since the phospholipid form of DHA, especially from HDL, is transported more efficiently into the brain. Supported by NIH R21 AT008457.
The G Protein‐Coupled Receptor TGR5 Plays a Protective Role Against the Development of Non‐Alcoholic Fatty Liver Disease
Authors: Jadhav Kavita , Zhang Yanqiao
Published: June 2021

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of conditions which ranges from simple steatosis to non‐alcoholic steayohepatitis (NASH) and liver cirrhosis. Although only a small percentage of people with steatosis develop serious liver disease, NAFLD is often associated with obesity and insulin resistance. The aim of this study is to establish that activation of the G‐protein coupled receptor TGR5 by its synthetic ligand INT‐777 leads to a reduction in hepatic lipid accumulation. TGR5+/+ mice were treated with either vehicle or INT‐777 for 7 days and then sacrificed. Hepatic lipid analysis revealed that administration of INT‐777 led to a significant reduction in liver triglycerides and cholesterol. In addition, TGR5+/+ LDLr‐/‐ and TGR5‐/‐ LDLr‐/‐ mice were fed a methionine choline‐deficient diet for induction of fibrosis. Hepatic lipid analysis of these mice showed that liver and plasma triglycerides were significantly higher in TGR5‐/‐ LDLr‐/‐ mice. Furthermore, hematoxylin and eosin (H and E) of liver sections from these mice displayed higher lipid droplet deposition compared to TGR5+/+ LDLr‐/‐ mice. Also, qPCR analysis of liver gene expression revealed a marginal yet significant increase in some fibrosis genes in the TGR5‐/‐ LDLr‐/‐ mice indicating that TGR5 may have a protective role in liver fibrosis. These results point to the possibility that TGR5 could be a potential therapeutic target for the treatment of NAFLD, even in its advanced stages.
Inositol Depletion Perturbs the Vacuolar‐ATPase: A Novel Mechanism of Action of Valproate
Authors: Deranieh Rania , Shi Yihui , Tarsio Mauraeen , Kane Patricia , Greenberg Miriam
Published: June 2021

Depletion of inositol has profound effects on cell function and has been implicated in the therapeutic effects of drugs used to treat bipolar disorder. We have previously shown that one such drug, valproate (VPA), depletes inositol by inhibiting myo‐inositol 3‐phosphate synthase, the enzyme that catalyzes the first and rate‐limiting step of inositol biosynthesis. To elucidate the cellular consequences of inositol depletion, we screened the yeast deletion collection for VPA‐sensitive mutants and identified mutants in vacuolar sorting and the V‐ATPase.

Inositol depletion caused by VPA or by starvation of ino1Δ cells perturbed the vacuolar structure, decreased V‐ATPase activity and proton pumping in isolated vacuolar vesicles. VPA compromised the levels of PI3,5P2, which is necessary for stabilization of the V‐ATPase complex, and resulted in reduced V‐ATPase activity. Osmotic stress, known to increase PI3,5P2 levels, did not restore the compromised PI3,5P2 levels, nor did it induce vacuolar fragmentation in VPA‐treated cells, suggesting that perturbation of the V‐ATPase is a consequence of altered PI3,5P2 homeostasis under inositol‐limiting conditions. The findings of this study are the first to demonstrate that inositol depletion caused by starvation of an inositol synthesis mutant or by the inositol‐depleting drug VPA leads to perturbation of the V‐ATPase.
Protein Arginine Methylation of Tubulin Beta Decreases Binding of Taxol in Neuro2a Cells
Authors: Piller Sabine , Jwad Noor , Hejazi Leila , Gamsjaeger Roland , Sucher Nikolaus
Published: June 2021

Tubulin α and β heterodimers are the building blocks of microtubules, which are part of the cytoskeleton in eukaryotic cells. Microtubules play critical roles in both development and synaptic function in neurons and are the target of various anti‐cancer drugs. Tubulins undergo multiple post‐translational modifications which influence microtubule stability, motor protein guidance and contribute to the “microtubule code” that selectively and specifically regulates the functions of microtubules. Here we report that tubulin β is methylated on two novel arginine (R) residues. Using mass spectrometry, R62 and R282 were identified to be methylated in Neuro2a cells. The location of these methylated residues suggests a role in microtubule stability. Furthermore, R282 is involved in binding to microtubule stabilizing drugs such as taxol and epothilone derivatives and mutants of R282 are associated with anti‐cancer drug resistance. Computer modelling using protein‐docking simulations on taxol binding to tubulin β with and without methylation of R282, suggested that methylation on R282 reduces taxol binding. This prediction was supported experimentally by cell cycle analysis of Neuro2a cells using flow cytometry. Inhibition of methylation by AdOx caused an increased effect of taxol with more cells being arrested in the G2 phase. Hence our findings add a new player to the multitude of post‐translational modifications of the “microtubule code” and have potentially important implications for anti‐cancer drug design, brain development and synaptic function.
Function of KpsS, KpsC, and NeuE in Escherichia coli K1 Polysialic Acid Biosynthesis
Authors: Thon Vireak , Peterson Dwight , Vionnet Justine , Vann Willie
Published: June 2021

Pathogenic Escherichia coli are often encapsulated with acidic polysaccharides such as polysialic acid. We have shown that four genes, kpsS, kpsC, neuE and neuS, in the E. coli K1 gene cluster are essential for initiation and elongation in E. coli K1 polysialic acid biosynthesis. NeuS is a polysialyltransferase. A previous report demonstrated that homologues of kpsC and kpsS gene products in N. meningitidis are cytidine 5′‐monophosphate‐2‐keto‐3‐deoxyoctulosonate (CMP‐KDO) transferases. These enzymes are involved in the biosynthesis of the putative glycolipid terminus of terminus of polysialic acid. Our objective in this report is to determine the function of KpsC, KpsS, and NeuE of E. coli in initiation of polysialic acid synthesis. We have successfully expressed in a soluble form and purified KpsC, KpsS, and NeuE of E. coli K1 to near homogeneity. This is the first time these full length membrane proteins have been expressed in a soluble form. We used HPLC and TLC methods to demonstrate that E. coli K1 KpsS and KpsC can transfer KDO to a fluorescence glycolipid acceptor substrate. The fluorescent products of KpsS and KpsC were isolated and used to investigate the function of the NeuE in polysialic acid biosynthesis.
RNA‐Seq Reveals Differential hnRNP‐RNA Interactions During VSV Infection
Authors: Davidson Adam , Lyles Douglas , Alexander Rebecca
Published: June 2021

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a class of RNA‐binding proteins that carry out numerous cellular functions. hnRNPs serve as RNA shuttling proteins in healthy cells, transporting nascent RNA from the nucleus to the cytoplasm. Recent work by others has shown that hnRNP A1, hnRNP K and hnRNP C1/C2, among others, are relocalized to the cytoplasm during infection by vesicular stomatitis virus (VSV), the purpose of which is not fully understood.

Vesicular Stomatitis Virus (VSV) is a non‐segmented negative strand virus of the Rhabdoviridae family. Genomic VSV is transcribed by virally encoded RNA‐dependent RNA polymerase to generate the mRNA responsible for protein synthesis in the host cell. As a weak human pathogen, it is a model organism for the more infectious viral agents influenza and rabies. VSV is an emerging player in oncolytic viriotherapy, as it is capable of tumor suppression through a variety of mechanisms including cellular hypoxia, inflammatory cytokine release and direct cell lysis.

In the context of innate immunity, the interaction between hnRNPs and both viral and cellular mRNA is explored through RNA‐Seq, a deep‐sequencing approach. Each hnRNP displays a differential affinity for cellular and viral transcripts between mock‐infected and VSV‐infected states, indicating the occurrence of RNA exchange and a directed purpose for this host‐pathogen interaction.
Fibroblast‐Macrophage Interactions: Regulation by Modification of Extracellular Matrix
Authors: Mazur Anna , Kelly Thomas , Post Steven
Published: June 2021

Inflammation is associated with modifications of the extracellular matrix (ECM) that are recognized by receptors expressed by a variety of inflammatory cells. During inflammation, fibroblasts are activated and increase their expression of type I collagen and Fibroblast Activation Protein (FAP). FAP is a member of the post‐prolyl peptidase family and is primarily expressed by activated fibroblasts. Although the biological role of FAP is not clear, deletion of FAP in mice is associated with decreasing the immune response in cancer. To understand the mechanism underlying the immunomodulatory role of FAP, we examined the relative ability of FAP to cleave collagen and regulate macrophage adhesion. Using quenched fluorescent substrates, we show that FAP, but not the related post‐prolyl proteases DPPIV and POP, selectively cleaves type I collagen. When used as an adhesion substrate, FAP‐cleaved type I collagen increased the adhesion of primary macrophages in comparison to native collagen. Chelating divalent cations did not inhibit macrophage adhesion to FAP‐cleaved collagen indicating that this effect is independent of integrins. In contrast, FAP‐mediated cleavage of collagen did not increase the adhesion of macrophages isolated from mice lacking the Class A Scavenger Receptor (SR‐A). Together, our results identify an unknown biological role for FAP in modifying the ECM and increasing macrophage adhesion. Taken further, our study describes a novel way of communication between activated fibroblasts and macrophages, which relies on modifications of ECM.
Altering Caspase Specificity Using an Intracellular Directed Evolution Approach
Authors: MacPherson Derek , Hill Maureen , Wu Peng , Hardy Jeanne
Published: June 2021

Caspases are among the most specific of proteases, making them ideal targets for engineering new specificity and developing new protease‐based biotherapeutics. However, conventional high‐throughput methods for protein evolution are not amenable to caspases as they are multi‐chain, and intracellular. Increasing the complexity, active sites of caspases are highly flexible to allow for accommodation of various substrates, making a rational design approach difficult To overcome these challenges, we developed a method for evolving function in cytosolic proteases, based on our novel genetically‐encoded caspase activatable‐GFP reporter (CA‐GFP), which is activated from a dark to a fluorescent state by proteolysis.

Our first application of this technology, we engineered caspase‐7, which cleaves the amino acid sequence DEVD to recognize and cleave the new sequence VEID, recognized by caspase‐6. Saturation mutagenesis at key residues within the active site of the enzyme allowed us to sort for variants with altered specificity by flow cytometry. Variants able to cleave the target sequence were purified, then characterized using traditional Michaelis‐Menten kinetics, protein substrate assays and x‐ray crystallography. We identified several variants that displayed kinetic activity and cleavage patterns similar to the wild type caspase‐6. X‐ray crystal structures of the evolved variants bound to the casp‐6 and ‐7 cognate substrates VEID, and DEVD reveal how the mutations affect the activity of the enzyme, which could not be rationally predicted, emphasizing the strength of this approach. Utilizing this method we are able to reengineer cytosolic proteases to cleave novel targets and pave the way for biotherapeutic applications.

This work is supported in part by NIH GM080532.
An ApnAase / mRNA Decapping Nudix Hydrolase from Mycobacterium leprae
Authors: Zhu Peipei , DiDonato Tessa , O'Handley Suzanne
Published: June 2021

The diadenosine polyphosphatases (ApnAases) / mRNA decapping enzymes are a family of enzymes within the Nudix hydrolase superfamily. The diadenosine polyphosphatases from Legionella pneumophilia and Bartonella bacilliformis have been found to be important in each pathogen's ability to invade its host cells. An ApnAases / mRNA decapping enzyme from Mycobacterium tuberculosis has been characterized in our laboratory and a homolog to this enzyme has been uncovered in Mycobacterium leprae. We have cloned and expressed the M. leprae homolog and have determined that it is an ApnAase. While the enzyme has good expression, it is rather insoluble. We have lowered the expression temperature, lowered the concentration of IPTG, incorporated GroESL, and used a Rosetta cell line to try to increase solubility, but without much effect. We are still working to increase solubility, before we purify and further characterize the enzyme. If these Mycobacteria ApnAases / mRNA decapping enzymes are found to be involved in invasiveness and thus in virulence, then these enzymes could be potential novel antibiotic targets in M. tuberculosis and M. leprae. This research was supported by a NIH AREA Grant and a RIT Graduate Student Grant.
Pharmacological Properties of Anthocleista vogelii against CCl4 Induced Toxicity
Authors: IROANYA ONYEKACHI , ODUOLA TAIWO , AKAGHA MILDRED , OLADUNJOYE ENITAN
Published: June 2021

The Hematopoietic, Hepatoprotective Lipid Peroxidative, Enzymic & Non Enzymic properties of 70% aqueous EtOH extract of Anthocleista vogelii leaves (AEEAV) was ascertained using Wistar albino rats.

The animals (30) were divided into 6 groups. Groups 1 & 2 received distilled water (10 ml/kg p.o.) for 7 days & served as normal & toxin controls respectively. Groups 3, 4 & 5 were treated with 200, 400 & 800 mg/kg of AEEAV (p.o.) respectively while Group 6 received Silymarin (300 mg/kg p.o.) for 7 days. On the 7th day, group 1 recieved 1 ml/kg of liquid paraffin (i.p.) while Groups 2, 3, 4, 5 & 6 received 1 ml/kg of 50 % (v/v) CCl4 in liquid paraffin (i.p.) 6 hrs after the last dose. After 24 hrs the animals were sacrificed.

In serum liver and kidney, AEEAV dose dependently showed significant (p<0.05) increase in Catalase Glutathione Peroxidase Superoxide Dismutase Reduced Glutathione & Glutathione S Transferase levels with decrease in the level of MDA compared to the normal & toxin control groups. On administration of 800 mg/kg of AEEAV, ALP, AST and ALT levels were significantly low (p<0.05) compared to 200 & 400 mg/kg but almost the same as silymarin. AEEAV showed significant (p<0.05) increase in Albumin and Total Protein levels but decreased the concentrations of Triglyceride Cholesterol Urea Bilirubin &Creatinine compared to toxin control group. There was significant (p<0.05) increase in PCV, Hb, platelet & MCH levels compared to the toxin control group.

The toxic effect of CCl4 was significantly attenuated (p<0.05) on treatment with AEEAV by restoring the liver function to near normal indicating the protection of hepatic cells, increasing the rate of erythropoiesis and antioxidant activity.
An α‐Acetoxy‐Tirucallic Acid Isomer Inhibits Akt/mTOR Signaling and Induces Oxidative Stress in Prostate Cancer Cells
Authors: El Gafaary Menna , Büchele Berthold , Syrovets Tatiana , Schmidt Christoph , Simmet Thomas
Published: June 2021

Here we provide evidence that 3α‐acetyloxy‐tir‐8,24‐dien‐21‐oic acid (αATA(8,24)) inhibits the Akt/mTOR signaling. αATA(8,24) and other tirucallic acids were isolated from the acetylated extract of the oleo gum resin of Boswellia serrata to chemical homogeneity. Compared to related tirucallic acids, αATA(8,24) was the most potent inhibitor of the proliferation of androgen‐insensitive prostate cancer cells in vitro and in vivo, in prostate cancer xenografted onto chick chorioallantoic membranes. αATA(8,24) induced loss of cell membrane asymmetry, caspase 3 activation, and DNA fragmentation in vitro and in vivo. These effects were selective for cancer cells, because αATA(8,24) did not exert overt toxic effects either on peripheral blood mononuclear cells or on the chick embryo. At the molecular level, αATA(8,24) inhibited the Akt1 kinase activity. Prior to all biochemical signs of cellular dysfunction, αATA(8,24) induced inhibition of the Akt downstream target mTOR as indicated by dephosphorylation of S6K1. This event was followed by decreased expression of cell cycle regulators, such as cyclins, cyclin‐dependent kinases, and phospho retinoblastoma protein, which led to inhibition of the cell cycle progression. In agreement with mTOR inhibition, αATA(8,24) and rapamycin increased the volume of acidic vesicular organelles. In contrast to rapamycin, αATA(8,24) destabilized lysosomal and mitochondrial membranes, and induced ROS production in cancer cells. The ability of αATA(8,24) to inhibit Akt/mTOR signaling and to induce simultaneously oxidative stress could be exploited for the development of novel antitumor therapeutics with a lower profile of toxic side effects.
Inhibition of Neisseria meningitidis Group X N‐Acetylglucosamine‐1‐Phosphotransferase by Donor Substrate Analogues
Authors: Cottman‐Thomas Ebony , Ming Shonoi , Tedaldi Lauren , Wagner Gerd , Chen Yi , Chen Xi , Cai Cao , Linhardt Robert , Vann Willie
Published: June 2021

Neisseria meningitidis is a major cause of bacterial meningitis in the meningitis belt of Sub‐Saharan Africa. N. meningitidis serogroup X is increasing prevalence in the meningitis belt. The capsular polysaccharide is a homopolymer of (α1‐4)‐linked N‐acetyl‐D‐glucosamine (GlcNAc)‐1‐phosphate. We have shown that the enzyme responsible for formation of this polymer is a GlcNAc‐1‐phosphotransferase that catalyzes the transfer of a GlcNAc‐1‐phosphate from UDP‐GlcNAc to the 4‐hydroxyl of a terminal GlcNAc‐1‐phosphate on the growing polysaccharide chain.

In this investigation we explore its interaction with substrates analogs of the sugar nucleotide donor, UDP‐GlcNAc. Recently a sugar nucleotide inhibitor was described for glycosyltransferases based on a proposed conformation change induced by donor binding. We demonstrated that several of these novel analogs of UDP‐GlcNAc are indeed inhibitors. The best inhibitors contained a bulky thien‐2‐yl group at the 5‐positon of uracil.

UDP‐GalNAc, which differs from the UDP‐GlcNAc only by the orientation of the sugar 4‐hydroxyl group, and UDP‐Glc, which differs by the absence of an acetamido group were neither substrates nor inhibitors suggesting that the 4‐hydroxyl and the 2‐acetamido may be important for interaction of the sugar moiety. Both UDP‐trifluoroacetylglucosamine and UDP‐azidoacetylglucosamine were good inhibitors of GlcNAc‐1‐phosphoryltransferase, confirming the importance of the acetamido group.
Internal Abstraction of Dynemicin A: A QM/MM Approach
Authors: Beane Angus , Jaini Arjun , Miller Bill , Parish Carol
Published: June 2021

Enediynes exhibit promise as anti‐tumor drugs because of their ability to induce cell apoptosis. After undergoing Bergman cyclization, the enediyne is converted into para‐benzyne, a diradical. These radicals are responsible for abstracting hydrogen from DNA, which causes the DNA to unravel, leading to cell death. Past research has shown that dynemicin A, an enediyne, easily binds to and abstracts hydrogen from DNA. However, the molecular mechanism through which this abstraction occurs is unknown. Following Bergman cyclization of the enediyne to form the diradical, we hypothesize two possible mechanisms for the subsequent reaction: 1.) the diradical directly abstracts hydrogen from DNA or 2.) the diradical moiety first abstracts a hydrogen from a proximate, intramolecular methyl group, and then the resulting methyl radical abstracts a hydrogen from the DNA. The Quantum Mechanics/Molecular Mechanics (QM/MM) package ONIOM available within Gaussian09 was used to investigate the energetics of these reactions.
The Effect of a Sulforaphane and Maitake Mushroom Extract Combination on the Expression of NAD(P)H:Quinone Oxidoreductase 1 (NQO1) and Glutathione S‐Transferase theta‐1 (GSTT1)
Authors: Erwin Sharis , Ownby Stacy , Slider Laxmi , Cornblatt Grace
Published: June 2021

Humans are exposed to numerous environmental threats on a daily basis. Our bodies are able to naturally eliminate these toxins in a series of reactions catalyzed by Phase 1 and Phase 2 enzymes, which occur primarily in the liver. Phase 1 enzymes convert toxins into reactive intermediates which can diffuse through cell membranes and negatively interact with DNA, proteins, or lipids. These reactive intermediates are further modified by Phase 2 enzymes which solubilize the reactive intermediates allowing for excretion from the body. Here we examine a novel blend of sulforaphane and maitake mushroom extract and their effect on the detoxifying enzymes. Previously, our laboratory has shown that the combination of sulforaphane and maitake mushroom extract can induce such detoxifying enzymes as UDP glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1). In this study, we focus on the effect of the combination of sulforaphane and maitake mushroom extract on Phase 2 enzymes in cultured liver cells over a series of time points using qRT‐PCR, Western blot analysis, and immunofluorescence. Elevated levels of NQO1 and GSTT1 were observed over a course of 72 hours. This demonstrates that the combination of sulforaphane and maitake mushroom extract can induce the production of the phase 2 enzymes needed to promote clearance of potentially reactive intermediates.

Funding: Nutramax Laboratories, Consumer Care, Inc.
Combinatorial In Silico Drug‐Lead Optimization for Targeted Inhibitors of P‐Glycoprotein
Authors: Olengue Ketetha , McCormick James , Vogel Pia , Wise John
Published: June 2021

P‐glycoprotein (P‐gp) is an ATP‐powered efflux pump of the plasma membrane that is responsible for removing toxins from cells. While this is beneficial in normal cells, P‐gp overexpression is often responsible for the failure of chemotherapies in cancer and HIV treatments. Because P‐gp transports drugs that encompass a wide variety of structures, multidrug resistances (MDR) to many unrelated therapeutics is often a consequence of P‐gp overexpression. Attempts to identify inhibitors of P‐gp that could be used to reverse MDR have often failed because they are also pumped out of the cell. We recently identified three inhibitors that target the nucleotide binding domains of P‐gp and avoid the drug binding domains. These compounds reverse MDR in cancer cell lines. A big problem in optimizing any drug‐lead is that the number of possible chemical variations of the lead always out‐numbers those that can be synthesized and tested. We present here a novel computational approach that generates many thousands of compounds related to a drug‐lead structure, analyzes them for interaction with the target protein, and rates them for improvements in target binding potential. We have applied this approach to inhibitors of P‐gp that reverse multidrug resistances in cancer and report on the application of these variants to high performance computational drug docking experiments with Pgp. These methods aimed at expediting the optimization of drug leads should be generalizable to many different systems. Supported by NIH NIGMS [Grant 1R15‐GM094771‐01A1] to PDV and JGW, and SMU Undergraduate Research Assistantship, SMU Engaged Learning, and Hamilton Undergraduate Research Scholarships to KO.
Biochemical Reactivities of 1,2‐Dehydro‐N‐β‐Alanyldopamine And The Mode of Action of Bioactive 1,2‐Dehydrodopyl Peptides
Authors: Barek Hanine , Evans Jason , Sugumaran Manickam
Published: June 2021

The objective of the present study is to investigate the biochemical transformations of 1,2‐dehydro‐N‐β‐alanyldopamine (dehydro NBAD), which belongs to the class of bioactive 1,2‐dehydrodopa containing compounds possessing crosslinking and cementing potentials, antioxidant activities, and antibiotic activities. These activities can be ascribed the reactivity of 1,2‐dehydrodopyl unit. To examine their mode of action, we conducted biochemical studies with dehydro NBAD. Tyrosinase oxidized dehydro NBAD to a transient quinone methide imine amide rather than the conventional quinone. This quinone methide rapidly reacted with the starting material forming oligomeric products. Liquid chromatography mass spectrometry analysis of the reaction confirmed the production of dimers and other oligomers. Laccase also oxidized dehydro NBAD to its dimeric products, without producing quinone methide, probably by free radical coupling. Dehydro NBAD is sensitive to alkaline pH and exhibited rapid nonenzymatic oxidation generating oligomers. During this nonenzymatic reaction, facile production of superoxide anions could be observed. Thus dehydro NBAD seems to exhibit its reactivities through the formation of highly reactive quinone methide, and free radicals. These reactions account for the crosslinking as well as antioxidant and antibiotic potentials of dehydrodopa containing units found in a myriad of organisms.
Antibacterial Evaluation of 2‐oxo‐4‐(phenylamino)‐2H‐chromene‐3‐carbaldehyde
Authors: Govori Sevdije , Spahiu Sabrie , Haziri Arben
Published: June 2021

This study presents the synthesis, spectral analysis and antibacterial evaluation of 2‐oxo‐4‐(phenylamino)‐2H‐chromene‐3‐carbaldehyde (3). Compound (3) was synthesized by action of aniline (a) on 4‐chloro‐2‐oxo‐2H‐chromene‐3‐carbaldehyde (2). The synthesized compound was screened for in vitro test of its antibacterial activity against three bacterial cultures: Hafnia alvei, Pseudomonas aeruginosa and Enterobacter cloacae. The antibacterial activity of synthesized compound was compared to antibacterial activity of novobiocin as standard drug. The biological evaluation of compound (3) generally showed moderate activity against Hafnia alvei, Pseudomonas aeruginosa and Enterobacter cloacae compared with novobiocin.
Phosphonylated Acetylcholinesterase: Probing the Mechanism of a Small Molecule Reactivator
Authors: Cadieux C Linn , Wang Haoyu , Zhang Yuchen , Koenig Jeffrey , Shih Tsung‐Ming , McDonough John , Koh John , Cerasoli Douglas
Published: June 2021

Currently fielded treatments for nerve agent intoxication include atropine, an acetylcholine receptor antagonist, and pralidoxime (2PAM), a small molecule reactivator of acetylcholinesterase (AChE). 2PAM reactivates nerve agent‐inhibited (NA‐)AChE via direct nucleophilic attack by the oxime moiety on the phosphorus center of the bound nerve agent. Due to a permanently charged pyridinium motif, 2PAM is not thought to cross the blood brain barrier and therefore cannot act directly in the neuronal junctions of the brain. In this study, a non‐permanently charged, non‐oxime molecule initially identified using pesticide‐inhibited AChE was tested for reactivation potential against NA‐AChE. The inhibitory and reactivation potentials of the molecule were determined with native AChE and AChE inhibited with tabun, sarin, soman, cyclosarin, VX, or VR and then compared to those of 2PAM. Then, several structural analogues of the candidate were used to probe the reactivation mechanism of the molecule. Additionally, the experimental condition under which reactivation was performed was altered in an attempt to determine the active form of the molecule. Finally, guinea pigs were used to examine the protective efficacy of the compound after exposure to sarin. Together, the results of both in vitro and in vivo testing will inform the design of future NA‐AChE small molecule reactivators.
This research was supported by the Defense Threat Reduction Agency ‐Joint Science Technology Office, Medical S&T Division.
Restoration of enzymatic activity of energy related proteins in traumatic brain injured rats following the administration of gamma‐glutamylcysteine ethyl ester
Authors: Rice Brittany , Reed Tanea , Sowell Rena , King Christina
Published: June 2021

The brain serves as the control center for the body. Its function is primarily dependent upon sufficient supply of glucose for energy metabolism. The glycolytic pathway and Kreb's cycle are important in producing ATP for the brain. Brain dysfunction resulting from an external force is known as traumatic brain injury (TBI). Symptoms range from physical to psychological and effects can be mild, moderate, or severe depending on the extent of the injury. TBI is associated with oxidative damage resulting in reduced energy metabolism. Antioxidants are substances responsible for the inhibition of oxidation. Gamma‐glutamylcysteine ethyl ester (GCEE) is an ethyl ester moiety of gamma‐glutamylcysteine that exhibits antioxidant activity by increasing glutathione production. This study investigates the enzymatic activity of two energy related enzymes, malate dehydrogenase and glyceraldehyde‐3‐phosphate dehydrogenase that have been identified as being nitrated in moderate TBI. To test the hypothesis that the administration of GCEE will normalize enzymatic activity post‐TBI, adult male Wistar rats were divided equally into three groups: Sham, Saline, and GCEE treatment (150 mg/kg). Upon sacrifice, enzymatic activity was indirectly measured spectrophotometrically by assessing the absorbance of reduced β‐nicotinamide adenine dinucleotide at 340nm. Preliminary data demonstrates that the administration of GCEE post‐TBI robustly increases enzymatic activity bolstering the hypothesis. These results are promising and indicate potential therapeutic strategies to restore enzymatic activity in the brain post‐TBI.
Structure and Activity of Inteins from Pyrococcus abyssi and Pyrococcus horikoshii
Authors: Jaramillo Mario , Williams Jennie , Reitter Julie , Mills Kenneth
Published: June 2021

Protein splicing is the self‐catalyzed excision of an intervening polypeptide (intein) from flanking polypeptides (exteins), concomitant with the ligation of the exteins. Similar inteins found in two thermophilic archaebacteria, Pyrococcus abyssi (Pab) and Pyrococcus horikoshii (Pho), interrupt the DNA Polymerase II. Comparison of the Pab PolII NMR structure and a Pho PolII homology model, along with 75% sequence identity between the inteins, reveals a common fold with the exception of a longer, disorganized loop in the Pab intein. The longer loop may provide a less rigid structure that allows splicing to occur at lower temperatures. Both splicing and uncoupled cleavage at the intein junctions occur to a greater extent at lower temperatures for the Pab intein than with the Pho intein. The relative rigidity of the inteins was compared by treatment of each with the thermostable protease thermolysin, which cleaves proteins in flexible regions. Thermolysin cleavage was more efficient at lower temperatures for the Pab intein. We are currently examining the relative strength of a disulfide bond that links the catalytic Cys residues as a measure of the flexibility of both inteins' active sites by titration with DTT.
Protein Function Prediction Using ProMOL and PyMOL
Authors: Hart Kaitlin , McKay Talia , Tedla‐Boyd Weinishet , Mills Jeffrey , Bernstein Herbert , Craig Paul
Published: June 2021

We use ProMOL and PyMOL to predict the function of structures that lack functional annotation. There are currently over 3700 proteins in the Protein Data Bank that do not have an assigned function. ProMOL, a plugin for the molecular graphics system PyMol, was developed for the purpose of characterizing protein structures of unknown function. The program was created, and is constantly being revised, by members of the RIT and Dowling College communities. ProMOL compares query protein structures against a library of active site motif templates. ProMOL calculates Levenshtein distances and RMSD values, and generates a 3D residue alignment visual between the motif active site residues and corresponding query residues. The protein queries which generate the best results are then submitted to NCBI's BLAST algorithm, the University of Helsinki's Dali alignment tool, and Sanger Institute's Pfam database. If the data produced by these four bioinformatics programs lead to a preliminary conclusion concerning the function of an uncharacterized structure, the behavior of the protein is then tested in vitro. To date, there have been over 90 significant “hits” for over 70 uncharacterized protein structures using ProMOL's motif library. These structures are currently being analyzed via BLAST, Dali, and Pfam. While computational data analysis in an ongoing process, the most promising alignments to date include PDB entries 2HRZ, 4EZI, 3L1W, and 2O14. This work has been supported in part by National Institute of General Medical Sciences grants 2R15GM078077‐02, 3R15GM078077‐02S1, and 3R15GM078077‐02S2.
Molecular characterization and phosphorylation site mapping of the Arabidopsis CRINKLY4 receptor‐like kinase (ACR4)
Authors: Shah Shweta , Meyer Matthew , Rao A
Published: June 2021

ACR4 is required for overall growth and development of the plant. ACR4 architecture possesses an extracellular ligand binding domain, a transmembrane (TM) helix, and an intracellular domain (ICD). Previously, we have characterized the biochemical and biophysical properties of the ICD independent of the TM domain (Meyer et al., Biochemistry. 2011, 50:2170). However, the molecular association of the TM domain with plasma membrane and its effect on the functioning of ICD domain is still poorly understood. The main objective of this study is to characterize ACR4 ICD in association with the TM domain. We have recombinantly expressed soluble protein with a N‐terminal 6H‐SUMO tag and the TM domain followed by ICD (SUMO‐TM‐ICD). Gel filtration experiments suggest that the protein is a monomer. The far UV‐CD spectrum shows minima at 208 nm and 222 nm, characteristic of alpha helical structures, indicating that the protein is properly folded. We have also carried out a comprehensive mapping of the TM‐ICD autophosphorylation sites. A total of 20 phosphorylation sites were identified. Significantly, among the 20 phosphorylation sites, two occurred at tyrosine residues, suggesting that in vitro the kinase activity of ACR4 may not be restricted to Ser/Thr residues. Furthermore, in order to mimic the natural membrane bound environment, we have prepared SUMO‐TM‐ICD Nanodiscs. The SUMO‐TM‐ICD Nanodiscs were characterized by TEM and were found to be ~20 nm in size. SUMO‐TM‐ICD Nanodisc remains functionally active, affording great potential for further characterization of SUMO‐TM‐ICD in its near‐native environment.
c‐JUN N‐terminal kinase (JNKα1) binds and phosphorylates endothelial nitric oxide synthase at S116, differentiating it from p38 and ERK‐mediated regulation
Authors: Caldara Amber , Chrestensen Carol , Salerno John , McMurry Jonathan
Published: June 2021

Recent studies identified direct submicromolar affinity interactions of mitogen activated protein kinases (MAPK) p38 and ERK with endothelial nitric oxide synthase (eNOS) via a pentabasic sequence in the autoinhibitory insertion of eNOS that resembles a MAPK binding motif. The neuronal isoform, which lacks the pentabasic motif, did not bind MAPK. In the present study, eNOS binding by another MAPK, c‐Jun N‐terminal kinase (JNKα1), was examined using optical biosensing. Similar to p38 and ERK, high affinity binding was measured with a 31 nM KD as determined by fit to a one‐state global model. Rate constants were determined. kon was 4125 M‐1s‐1 and koff was 1.3 x 10‐4 s‐1. Immunoblotting identified phosphorylation at S116, contrasting with ERK, which phosphorylated S602, and p38, which phosphorylated both. Phosphorylation did not inhibit NO production nor alter the conformational manifold. The results underscore the importance the newly discovered noncanonical MAPK binding site on eNOS as a regulatory element bound by an array of MAPK. Further, they contribute to the emerging paradigm of eNOS as a junction of multiple signaling pathways
Ketosis and hyperbaric oxygen metabolic therapy elicits potent anti‐cancer effects in vitro and in vivo
Authors: Poff Angela , Ward Nathan , Seyfried Thomas , D'Agostino Dominic
Published: June 2021

The Warburg effect and tumor hypoxia underlie a unique cancer metabolism characterized by glucose dependency and abnormal mitochondrial function. We have previously shown that this phenotype can be exploited therapeutically by glucose restriction with the ketogenic diet, dietary ketone supplementation, or hyperbaric oxygen (HBOT). We hypothesized that combining these therapies would be an effective non‐toxic treatment regimen, so we tested this combination therapy in the VM‐M3 mouse model of metastatic cancer. Treated mice exhibited decreased tumor growth rate, tumor vascularization, and metastatic spread, and lived twice as long as controls. To further characterize the effects of these therapies, we measured proliferation, viability, ROS production, and expression of key signaling molecules in VM‐M3 cells exposed to the following conditions: high glucose (control), low glucose (LG), ketone supplementation (βHB), hyperbaric oxygen (HBOT), or combination therapy (LG+βHB+HBOT). Proliferation and viability of VM‐M3 cells were significantly decreased in LG, βHB, HBOT, and combination‐treated cells at all‐time points tested. ROS (superoxide) production was increased in HBOT and combination‐treated cells compared to controls. Treatment with βHB significantly decreased expression of HIF‐1α and pAkt in VM‐M3 cells while HBOT inhibited HIF‐1α expression but increased pAkt expression. This data strongly supports further investigation into mechanisms of action and the use of these metabolic therapies as a potential cancer treatment.
Unsaturated Fatty Acids Stimulate Tumor Growth through Stabilization of β‐Catenin
Authors: Kim Hyeonwoo , Rodriguez‐Navas Carlos , Kollipara Rahul , Kapur Payal , McDonald Jeff , Brugarolas James , Kittler Ralf , Ye Jin
Published: June 2021

Cancer cells alter their metabolism to adapt for their uncontrolled proliferation. One of such alterations leads to accumulation of free fatty acids (FAs), which facilitate proliferation of some cancer cells through a mechanism yet to be identified. Here we report that excess unsaturated FAs block degradation of β‐catenin, the aberrant accumulation of which triggers development of various cancers. Interestingly, unsaturated FAs stabilize β‐catenin through a mechanism independent from that mediated by Wnt, a protein known to block degradation of β‐catenin. Unlike Wnt, unsaturated FAs do not affect ubiquitination of β‐catenin. Instead, the lipids block degradation of ubiquitinated β‐catenin through Fas‐associated factor 1 (FAF1), a protein known to facilitate degradation of β‐catenin through its interaction with the protein. We now show that FAF1 directly binds to unsaturated FAs. This interaction triggers polymerization of FAF1, causing dissociation of the protein from β‐catenin and consequently stabilization of β‐catenin. This finding is clinically relevant, as excess unsaturated FAs accumulated in clear cell renal cell carcinoma promote growth of the tumor through stabilization of β‐catenin. Our study indicates that overaccumulation of unsaturated FAs is a novel mechanism for cancer cells to acquire excess β‐catenin.

This study is supported by grants from NIH (HL‐20948), Welch foundation (I‐1832), and Clayton Foundation for Research.
Impact of Calcineurin Homologous Protein and NHE1 on Lung Cancer Cell Adaptation to Hypoxia and Serum Deprivation
Authors: Cottle Wayne , Wallert Mark , Provost Joseph
Published: June 2021

The Sodium Hydrogen Exchanger Isoform 1 (NHE1) is vital in maintaining pH homeostasis and is regulated by several proteins including both isoforms of Calcineurin Homologous Protein 1 and 2 (CHP1 and CHP2). The metabolic rate of cancer cells is elevated and in hypoxic and serum deprived developing tumors intracellular pH (pHi) is significantly decreased. Cells must adopt survival mechanisms to escape these conditions. Earlier work suggests that CHP1 is required for basic NHE1 proton transport while CHP2 increases the proton transport kinetics. CHP2 expression may activate NHE1 allowing for pHi homeostasis. Therefore, we investigated CHP binding to NHE1 as a means for adapting to hypoxic and serum deprivation conditions. Using Quartz Crystal Microbalance (QCMd) we measured protein‐protein interactions between NHE1 and CHP1 or CHP2. We also cultured H1299 cells with chemically induced hypoxia using dimethyloxaloylglycine (DMOG) and with serum deprivation in H1299 lung cancer cells. Cell exposed to hypoxia and serum deprivation showed a decreased pHi 0.2‐0.4 pH units. When NHE1 is inhibited by 5‐(N‐Ethyl‐N‐isopropyl) amiloride (EIPA) the decrease in pHi was significantly lower than non EIPA treated cells, indicating NHE1 supports cell viability. The impact of hypoxia and serum deprivation on intracellular pH was also investigated in H1299 cells with reduced NHE1 or CHP2 expression (shRNA knockdown). Together this work suggests that CHP2 plays an important role in cell survival in lung tumor development.