bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of CREG1 as a Master Regular of Liver Function
Authors: Jahan Iffat , Bulla Gary A
Published: June 2021

Mammalian development involves a complex system of regulatory signals and reactions resulting in highly differentiated cell types with specific structure and function. The liver is a major organ that has been studied extensively to understand underlying genetic processes responsible for specification, establishment and maintenance of tissue identity. Hepatoma and hepatoma variant cell lines have been used as a model to understand genetic networks responsible for liver function. Whole genome microarray analysis of hepatocyte cell lines has revealed candidate genes that may serve as regulators or master regulators of liver specificity. In two previous studies, the role of candidate gene Cellular Repressor of E1A Stimulated Gene (CREG1) on regulation of liver‐specific gene expression was determined using transfection studies combined with utilization of quantitative real‐time polymerase chain reaction (qRTPCR). Both studies found strong activation (10–100‐fold) of transcription factor Hnf6 and the downstream gene Serpina1 (a marker gene used to identify liver function). These findings suggested that CREG1 might act through Hnf6 to regulate Serpina1 via a Locus Control Region (LCR). However, while one study showed that of CREG1 overexpression in a hepatoma variant cell line resulted in modest activation of liver‐specific transcription factors Hnf1, Hnf4 and Hnf3, the second study suggested robust activation of these genes as measured by qRT‐PCR. In the current study, a CREG1 expression vector was reintroduced into two variant cell lines and gene activation profiles monitored to establish validity of the previous studies. However, we postulated that CREG‐1 overexpression, validated by qRT‐PCR, can activate expression of hepatocyte transcription factor Hnf6 and the downstream gene Serpina1.

However, analysis of gene silencing of liver and non‐liver cell types using whole genome microarray screening will help to identify, compare and elucidate the effects of various candidate tissue specific genes. Moreover, we are currently interested to see the reintroduction of liver‐enriched factors in hepatoma variants fully reactivates liver gene expression. Thus, understanding the underlying mechanism and role of each gene helps to develop therapeutic treatment targets for treating liver diseases.

Support or Funding Information

Eastern Illinois University, Department of Biological Science

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kub5‐HeraRPRD1B regulates CDK1 expression through the DREAM complex
Authors: YEH I‐JU , Motea Edward A , Boothman David A
Published: June 2021

Triple Negative Breast Cancer (TNBC) is a subtype with more aggressive, less differentiated capacity and enhanced metastatic ability. Although TNBC tumors are more responsive to neoadjuvant chemotherapy compared to receptor‐positive tumors, development of resistance to cytotoxic chemotherapy and early relapse is more common. Due to the lack of effective therapies, understanding the underlying mechanisms of TNBC aggressiveness and poor prognosis is a major focus of research with particular interest in identifying new therapeutic targets.

Kub5‐HeraRPRD1B (K‐H) is a novel factor involved in preserving genomic stability and regulates BRCA1 function by mediating the expression of CDK1, a master regulator of cell cycle, DNA replication, and DNA repair. This regulation has been reported in MDA‐MB‐231 cells, a model of TNBC. However, the underlying mechanism regulated CDK1 expression by K‐H is unknown. Here, we found that the CDK1 promoter is essential for the recruitment of K‐H. We also examined the interaction between K‐H and the complex for the regulation of cell cycle‐dependent gene expression, DREAM complex (dimerization partner, RB‐like, E2F and multi‐vulval class B complex) by immunoprecipitation. The interaction between K‐H and the DREAM complex is further validated using a Proximity ligation assay (PLA). Furthermore, a mutation in K‐H (R106A) results in the loss of an interaction between K‐H and the DREAM complex as well as K‐H and RNAPII, leading to decreased CDK1 levels. These results suggest that CDK1 expression is upregulated by K‐H and its complex formation with the DREAM complex and RNAPII. Thus, we hypothesize that K‐H modulates CDK1 expression through the recruitment of the DREAM complex to the CDK1 promoter region in a sequence‐specific manner. Our studies reveal a novel strategy for developing therapies that target the interaction between K‐H and the DREAM complex, potentially inhibiting the growth of cancer cells.

Support or Funding Information

This work is supported by NIH grant 1 R01 CA201489‐04

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
IN VITRO EVALUATION OF THE CYTOTOXIC POTENTIAL OF Securidacalongepedunculata ON HUMAN BREAST ADENOCARCINOMA (MCF‐7) CELLS
Authors: MINARI JOSEPH BAMIDELE
Published: June 2021

The prevalence of cancer has increased interest in the role of complementary and alternative medicine, employing the use of plant products. Securidacalongepedunculata (SL) serves as a major component of anticancer decoctions in Nigeria. The present study evaluates the cytotoxic potential of the crude extract and fractions of SL root bark against human breast adenocarcinoma (MCF‐7) cell line. The root back of SL was pulverized and extracted with 80% methanol to yield a crude extract which was then submitted to liquid–liquid fractionation with dichloromethane (DCM) and butanol (BUT). The extract and fractions were subjected to Gas Chromatography‐Mass Spectrometry (GC‐MS). MCF‐7 cell line was treated with graded concentrations (9.77 – 5000 μg ml−1) of the aqueous methanol crude extract as well as the DCM and BUT fractions for 24 or 48 hours. Cell viability was thereafter measured by XTT proliferation assay. The activity of the aqueous methanolic crude extract was found to be both dose‐dependent and time independent with IC50 values of 2241μg ml−1 and 1808 μg ml−1 after 24 and 48 hours respectively. However, the DCM and BUT fractions were significantly (p < 0.05) cytotoxic with IC50 values of 86.27 μg ml−1 and 12.08 μg ml−1 respectively after 48 hours of treatment. The study lends credence that BUT fractions of SL may contain lead agent with anticancer properties and that informed its use in alternative medicine.

Support or Funding Information

SELF FUNDING

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterizing the Alternative Splicing Dynamics of Cav1.3, a Calcium Ion Channel Involved in Autism Spectrum Disorder
Authors: Mirkovic Jovan , Sabzanov Samuel , Wang Ruizhi , Hossain Faruk , Ruggiu Matteo
Published: June 2021

Voltage‐gated calcium channels move calcium ions across cellular membranes in response to action potential stimuli, which mediate processes including cellular signaling, neurotransmitter discharge, muscle contraction, and gene expression. They are multimer proteins composed of alpha‐1, beta, alpha‐2/delta, and gamma subunits. Variety in calcium channels is afforded by different subtypes of alpha‐1, including the A, B, C, D, E, and S isoforms.

Cav1.3 is a member of the L‐type calcium channels and is categorized in the high‐voltage activated family. It employs the alpha‐1 subunit encoded by the CACNA1D gene. The alpha‐1 subunit forms four homologous repeat domains, each consisting of six transmembrane helical segments. Cacna1d knock out mice suffer from bradycardia and congenital deafness, testifying to the significance of the alpha‐1 subunit in the sinoatrial node and in auditory hair cells. Naturally occurring missense mutations in alternatively spliced regions of CACNA1D, such as the mutually exclusive exons 8a and 8b encoding part of the sixth transmembrane segment of domain I, have been reported to be gain‐of‐function mutations, which cause aldosterone‐producing adenomas, aldosteronism, seizures, and autism spectrum disorder, highlighting the importance of alternative splicing for Cav1.3 function.

Although the diseases derived from CACNA1D mutations have been characterized, the splicing dynamics of the CACNA1D pre‐mRNA that gives rise to the different alpha‐1 isoforms is not well understood. Here, we describe the alternatively spliced isoforms of Cacna1d and their distribution in mouse tissues.

Support or Funding Information

National Institute of General Medical Sciences Grant 1R15GM119099‐01

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RNA Therapeutic Strategies to Block VEGFR2 Expression and Angiogenesis in Glioblastoma Multiforme
Authors: Muralidharan Koushik , Gawargi Flobater I , Fuoco Kerianne
Published: June 2021

Glioblastoma multiforme (GBM), a grade IV tumor of the central nervous system, is the most common malignant primary brain tumor, and has a median survival of only 14 months. Poor survival is due to a lack of efficacy in current therapies to pass the blood‐brain barrier (BBB). GBM tumors are characterized by angiogenesis, which is essential for tumor growth and survival. Endothelial cells form the walls of new blood vessels, bridging the gap between the growing tumor and the established vasculature. The membrane receptor that activates tumors to recruit endothelial cells to promote neo‐angiogenesis is vascular endothelial growth factor receptor 2 (VEGFR2). Changes in VEGFR2 expression to block its activation would inhibit the development of new blood vessels within the tumor microenvironment. We have designed novel therapies to bypass the BBB and deliver the genetic sequences of anti‐sense RNA molecules to alter the splicing pattern and expression of the VEGFR2 transcript. Critical splice sites, enhancers, silencers and intronic polyadenylation signals within the VEGFR2 pre‐mRNA transcript were analyzed using bioinformatics databases and RNA‐modeling tools. Based on this, nine different antisense sequences were generated to target and block these elements and were cloned into our therapeutic platform vector, pAAV‐U7‐smOPT. This vector directs the antisense RNA therapeutic to the spliceosome machinery. GBM cells were cultured and transfected with the therapeutic vectors, and the total protein and RNA was collected and analyzed. A172 and U87 GBM cells treated with antisense therapy revealed a greater than two‐fold reduction of VEGFR2. Current directions include direct delivery to the central nervous system using an adeno‐associated viral (AAV) gene therapy vector. In addition, we are isolating AAV‐exosomes with the potential to increase tropism toward the tumor microenvironment.

Support or Funding Information

Bristol‐Myers Squibb, Independent College Fund of New Jersey, Johnson and Johnson

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Histone H4 Acetyltransferase NuA4 is Required for Appropriate RNA Splicing in Saccharomyces cerevisiae
Authors: Palfini Lawrence M , Maisner Rose , Gunning Thomas , Lin Mengjia M. , Kress Tracy L
Published: June 2021

Eukaryotes undergo transcription and splicing simultaneously, allowing for coordination and regulation of these two processes. Currently, there are a few known connections between chromatin modification, which regulates transcription, and splicing found in yeast and metazoa1,2, yet there is still much to study. The NuA4 histone acetyltransferase works with Swr1 chromatin remodeling enzyme to promote transcription3–5. First, NuA4 acetylates histone H4, which is required for the recruitment of Swr1. Next, Swr1 inserts the histone variant H2A.Z, which is subsequently acetylated by NuA4. Interestingly, in Saccharomyces cerevisiae, both NuA4 and Swr1 play a key role in the transcription of the ribosomal protein genes7, which make up a large fraction of the intron containing genes. Thus, NuA4, Swr1, and H2A.Z are excellent candidates for coordinating transcription and splicing. A role for H2A.Z in splicing was recently identified6. Interestingly, data from our lab suggest that NuA4 may have functions in RNA splicing that are independent of H2A.Z deposition. Using directed genetic screens we identified positive (suppressive) interactions between splicing factor gene mutations and mutations that catalytically inactivate NuA4. Additionally, using MPE‐seq8 and RT‐qPCR we found that mutations that block the catalytic activity of NuA4 alter the splicing of a subset of RNAs. Our data suggest that NuA4 plays a role in regulating RNA splicing and we are currently performing chromatin immunoprecipitation experiments to determine whether ESA1 catalytic activity impacts the association of splicing proteins with nascent RNAs during transcription. Together, our data support a model in which NuA4 interacts with the splicing machinery to coordinate transcription and splicing.

Support or Funding Information

Research Corporation for the Advancement of Science (Cottrell College Science award no. 20186) National Institutes of Health (R15GM122026).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Basis of Phenotypic Variation in the DNA Damage Response of Acinetobacter baumannii
Authors: DePamphilis Matthew , Ching Carly , Godoy‐Carter Veronica
Published: June 2021

Classified as an “ESKAPE” pathogen, Acinetobacter baumannii is one of the leading causes of hospital‐acquired (nosocomial) infections. This Gram‐negative opportunistic pathogen is able to survive desiccation and rapidly acquire multi‐drug resistances. The bacterium's successful mutagenic and antibiotic resistant capabilities are linked to its unique regulation of the DNA Damage Response (DDR). In similarity to many other bacteria, the DDR in A. baumannii depends on RecA, a highly conserved recombinase. However, in A. baumannii, the DDR is bimodal and promotes phenotypic variation; meaning that a fraction of cells—with the same genetic information— respond very well to DNA damage while others do not. It is predicted that this bimodal gene expression is a bet‐hedging strategy that contributes to the persistent survivability of A. baumannii by providing a mechanism for acquisition of advantageous mutations, such as those for antibiotic resistance.

We have shown that the recA gene transcript contains an extended structured 5′UTR that influences bimodal gene expression. One of the 5′UTR elements, an outer loop, possesses an influential role as a cis‐regulatory element. We have shown that RNA binding proteins (RBPs) bind specifically to the native UTR when DNA damage is induced. We have identified one of these proteins as a chaperonin known for its ability to bind to RNA and stabilize structure. We aim to further investigate these proteins and their sequence specific binding as we hypothesize that the outer loop is a binding site for RBPs, which would loosen and open the UTR to promote translation of the recA transcript. These findings provide insight to recA regulation, which in turn affects the DDR in A. baumannii. Understanding the DDR will permit to devise novel strategies to eliminate A. baumannii from clinical wards.

Support or Funding Information

This work is supported by grants from Northeastern University's Office of Undergraduate Research and Fellowships and Northeastern University's Honors Department.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modifying Mouse Herpesvirus‐Induced Vasculitis and Serpin Anti‐Inflammatory Treatment ‐ Profound Effects of the Gut Microbiota
Authors: Lucas Alexandra , Yaron Jordan , Zhang Liqiang
Published: June 2021

Introduction

Inflammatory vascular syndromes (IVS) are rare but devastating arterial disorders. A lethal IVS model can be induced by mouse gamma herpesviral (MHV68) infection in interferon gamma receptor knockout mice (IFNγR−/−). Previously, a serine protease inhibitor (serpin), virus‐derived Serp‐1 protein and Serp‐1 reactive center loop (RCL) peptide S‐7 (G305TTASSDTAITLIPR319) significantly improved survival and reduced aortic inflammation after MHV68 infection. As the microbiome is reported to have profound effects on immune response, we investigated the role of the gut bacteria in MHV68‐infected IVS mouse models with and without treatment.

Method

IFNγR−/− mice (N=56) were split into control or 10‐day antibiotic groups prior to infection with MHV68. Mice were treated with saline, Serp‐1 or RCL peptides S‐2, S‐7, S‐8 or modified S‐7 peptides MPS‐8 and MPS‐9. Fecal pellets were collected and 16S rRNA was Illumina sequenced from genomic DNA extracts.

Results

Suppression of gut bacteria accelerated mortality and reduced survival from 60 to 20 days (P=0.036) in MHV68 IVS. Survival was reduced from 70% at 150 days to 20% at 30 days with Serp‐1 treatment (P=0.003) and 0% at 30 days with S‐7 treatment (P<0.0001), while S‐2 was inactive and S‐8 trended towards improvement (N=14). In contrast, modified S‐7 RL peptides, MPS‐8 and MPS‐9, improved survival after suppression of gut bacteria (P<0.001, N=10). Both Serp‐1 and S‐7 significantly altered microbiome community structure (beta‐diversity), while S‐7 also altered community richness (alpha‐diversity), in the large intestine versus saline controls. Lactobacillales were significantly reduced and Streptophyta increased in MHV68 infection with saline treatment, while Bacillales were significantly reduced and Lactobacillales increased in Serp‐1 treatments. Treatment with normal gut micorbial gavage improved survival after antibiotic suppression of gut bacteria.

Conclusion

Microbiome richness, structure, or sub‐population composition, play a crucial role in modulating lethal vasculitic syndromes and treatment response. Serp‐1 and S‐7 show significant therapeutic benefit in MHV68 infection‐induced IVS in mice, which is abolished by gut microbiome suppression. MPS‐8 and MPS‐9 recover efficacy in this model. Significant microbiome changes occurred in Serp‐1‐ and S‐7‐treated, gut bacteria‐suppressed mice.

Support or Funding Information

Arizona State University ‐ Start up funding American Heart Association Grant in aid National Institutes of Health RO1

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure of the WFIKKN2 Follistatin Domain and GDF8 Antagonism
Authors: McCoy Jason C , Thompson Tom
Published: June 2021

Growth Differentiation Factor 8 (GDF8), also known as Myostatin, is a member of the TGF‐β superfamily and is a potent negative regulator of skeletal muscle growth. Disruption of GDF8 signaling or injection of GDF8 inhibitors results in muscle hypertrophy. Muscle mass can also be stimulated through the up‐regulation of secreted GDF8 protein antagonists, such as Follistatin. In contrast, transgenic overexpression of GDF8 causes muscle wasting. Thus, in animals the balance of muscle mass is highly regulated by the appropriate levels of active GDF8, both during development and in the adult. Given that GDF8 plays a profound role on muscle biology there has been a strong interest in developing therapeutics that target GDF8 to alleviate muscle wasting in a variety of diseases or to treat cancer cachexia where tumors have been shown to upregulate GDF8. As such, anti‐GDF8 therapeutics are within phase II and III clinical trials that directly target GDF8 or block signaling receptors. GDF8 is inhibited by a number of extracellular antagonists, one such antagonists, WFIKKN2, exhibits exceptional specificity for GDF8 and GDF11 (90% identity). Similar to the less specific Follistatin family of antagonists, WFIKKN2 utilizes a number of small modular domains including at least one follistatin (FSD) domain. Although there is vast knowledge regarding the Follistatin family FSDs and how they contribute to GDF8 antagonism very little is known about how the WFIKKN2 FSD interacts with GDF8. To address this, we isolated the WFIKKN2 FSD and characterized its binding interaction with GDF8 and GDF11. In addition, we solved the crystal structure of the WFIKKN2 FSD to 1.39Å. The structure revealed conserved, surface exposed, hydrophobic residues that when mutated to alanine reduced full‐length WFIKKN2 antagonism of GDF8. Comparison of the WFIKKN2 FSD to follistatin and FSTL3 indicates that WFIKKN2 utilized a different surface of the FSD to interact with GDF8. Further comparison reveals drastic differences in the domain arrangement classified as either an open or closed conformation. Collectively, the results highlight that although FSDs have conserved domain architecture they exhibit unique functions in biology.

Support or Funding Information

NIH RO1: GM114640 American Heart Association: 18PRE33990312

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dimerization of Yeast Ste14 Isoprenylcysteine Carboxyl Methyltransferase
Authors: Ratliff Anna , Bains Sahej , Funk Amy , Griggs Amy , Hrycyna Christine
Published: June 2021

Isoprenylcysteine carboxyl methyltransferase (Icmt) is an integral membrane protein localized to the endoplasmic reticulum that is responsible for the post‐translational α‐carboxyl methylesterification of the C‐terminus in CaaX proteins. CaaX proteins (where C is Cys, a is generally an aliphatic amino acid and X is one of several amino acids) include a variety of regulatory proteins, most notably Ras. The Icmt from S. cerevisiae, Ste14, is comprised of six transmembrane (TM) domains in which TM1 contains a putative dimerization motif, G31XXXG35XXXG39, where G is a glycine amino acid residue and X is a subset of hydrophobic amino acids. Ste14 has been shown biochemically to form functional homodimers or higher order oligomers, yet further studies must be performed to better understand this mechanism. To explore these elements as possible sequence and/or structural determinants for dimerization, we used cysteine‐scanning mutagenesis to generate a library of single cysteine mutants for each residue in TM1 (amino acids 25–42). These TM1 cysteine mutants were characterized with immunoblot analyses to assess protein expression, activity, stability, and the ability to form dimers. We determined that residues S27, Y28, L30, G31, G35, and G39 are critical for both methyltransferase activity and the structural stability of the protein. Interestingly, several of the TM1 cysteine mutants that are proposed to lie on the same face of an α‐helix in a helical wheel diagram formed dimers upon the addition of sulfhydryl specific cross‐linkers. We further validated the homodimerization of Ste14 by size exclusion chromatography, multi‐angle light scattering and small‐angle X‐ray scattering. Together, these data suggest that the active form of the Ste14 Icmt is, in fact, a homodimer and these data will ultimately be useful as we further explore the mechanism of action of Ste14.

Support or Funding Information

1. Purdue Research Foundation

2. National Institutes of Health/NIGMS (Grant #: 5R01GM106082‐05)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Genotype on the Ergogenic Effects of Caffeine in Collegiate Distance Runners
Authors: Kline Gabriel M , Millard Julie T
Published: June 2021

Although caffeine is the world's most popular stimulant, used in both athletic and cognitive contexts, its effects vary widely between individuals. In athletics, caffeine effects depend on dose but may include increases in lipolysis, pain tolerance, and time to exhaustion. While the effects of caffeine on cognitive performance are mixed, caffeine consumption has been linked to increased performance on visual processing tasks and reaction time tasks. Despite the evidence supporting the beneficial effects of caffeine on athletic and cognitive performance, variation in genes related to caffeine metabolism and mechanism of action may moderate its performance‐enhancing effects. For example, a single nucleotide polymorphism (rs762551) of the gene for the liver enzyme CYP1A2 has been reported to influence caffeine outcomes, with “slow” metabolizers having reduced benefits of caffeine during exercise and increased risk of heart attack. Similarly, a single nucleotide polymorphism (rs5751876) of the adenosine receptor gene ADORA2A has been reported to modulate caffeine consumption habits, caffeine‐induced anxiety, and the ergogenic effects of caffeine. In this study, 12 collegiate distance runners were genotyped at CYP1A2 and ADORA2A loci. To assess the effects of caffeine on athletic and cognitive performance, participants underwent two treadmill exhaustion tests and two cognitive battery tests, with one trial being under the influence of low dose caffeine and the other a placebo trial. This work may contribute to a better understanding of the variable effects of caffeine consumption on athletic performance, as well as inform the timing and use of caffeine for optimal performance enhancement.

Support or Funding Information

Supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM0103423.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CYP 2D6* 4 polymorphism in Polycythemia vera patients in Turkish population
Authors: Cifci Kezban Ucar , Okat Zehra
Published: June 2021

Studies have shown the association of susceptibility to several cancers with gene encoding enzymes' polymorphisms which engage in xenobiotics' biotransformation. In this study, our main motivation is to determine the relation between cytochrome P450 (CYP) 2D6* 4 polymorphisms and Polycythemia vera (PV) incidence in Turkish population. When patients and controls were compared in terms of CYP 2D6* 4 poor metabolizer (PM) and heterozygous extensive metabolizer (HEM) genotype frequency, it has been found that the patients have 1.35% PM and 32.43% HEM frequency, and controls have 2.63% PM and 21.05% HEM frequency. The correlation between the results of CYP 2D6 genotype analysis and the risk of disease in patients and controls was examined. We found that PM, HEM and extensive metabolizer (EM) genotypes were unrelated with the risk of PV (OR 0.51, 95% CI 0.04–5.71, OR 1.80, 95% CI 0.86–3.75, χ2 1.93, P: 0.164, OR 0.61, 95% CI 0.30–1.24, χ2 1.40, P: 0.235). Our results suggested that the CYP 2D6* 4 polymorphism, in the studied population, does not play an important role in PV etiology.

Support or Funding Information

The funding for the research came from Marmara University Scientific Research Projects Coordination Unit under the project SAG‐C‐DRP‐130515‐0162. Part of the study was published in Marmara Medical Journal in 2018.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis and Characterization of Esterified Fmoc‐Amino Esters as Potential Butyrylcholinesterase Inhibitors
Authors: Pingul Bianca Ysabel , Ramirez Jennifer , Schwans Jason
Published: June 2021

Neurodegenerative diseases such as Alzheimer's disease (AD) is the sixth leading cause of death in the United States and affects 5.7 million Americans. While cures for this disease have not yet been discovered, several pharmaceuticals are available to alleviate symptoms. These compounds typically target the enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Specifically, for individuals with AD it has been found that while AChE activity is slightly decreased or unaffected, BChE activity is increased. The increased BChE activity leads to a depletion of the neurotransmitter acetylcholine, and this is implicated in the progression of dementia. Thus, inhibitors of BChE are sought in AD treatment. We previously found Fluorenylmethyloxycarbonyl (Fmoc)‐amino acids selectively inhibit BChE, leading to a potential new class of cholinesterase inhibitors. While the role of the amino acid side chain was explored, the effects of modifying the carboxylate group were not investigated. Specifically, the enzyme binds the cationic substrate acetylcholine, but the Fmoc‐amino acids are anionic. We postulated Fmoc‐amino acid esters may be more potent inhibitors, as the ester ablates the negative charge. In addition, the ester allows incorporation of substituents that increase van der Waals interactions that may interact favorably with the enzyme. To test this model, a series of Fmoc‐amino acids were esterified using an alcohol and EEDQ (N‐Ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline), purified by chromatography, and characterized by NMR and HPLC. The solubility and inhibition properties for the Fmoc‐containing esters were evaluated using UV‐Vis spectroscopy. Kinetic studies suggest the Fmoc‐Leu‐esters are more potent inhibitors compared to the Fmoc‐Leu‐O− but are limited by solubility. We are now investigating additional Fmoc‐amino acid esters bearing cationic groups to enhance solubility such as lysine, and our initial results suggest the lysine‐based compounds are potent BChE inhibitors. Overall, the results may guide the design of new Fmoc‐containing compounds that specifically and effectively inhibit BChE.

Support or Funding Information

This project is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers R25GM071638, UL1GM118979, TL4GM118980, RL5GM118978, and T34 GM008074.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting Dxr/IspC to develop drugs against malaria and tuberculosis
Authors: Girma Misgina Belachew , Ball Haley , Dowd Cynthia , Couch Robin
Published: June 2021

The methylerythritol phosphate pathway (the MEP pathway) of isoprenoid biosynthesis is essential for the survival of many pathogenic organisms, including Plasmodium falciparum and Mycobacterium tuberculosis, causative agents of malaria and tuberculosis, respectively. The MEP pathway is absent in humans, making it an important drug target. There are seven enzymes in this pathway. 1‐Deoxy‐D‐xylose 5‐phosphate reductoisomerase (Dxr/IspC) catalyzes the first committed step in the pathway. It converts the substrate 1‐Deoxy‐D‐xylose 5‐phosphate (DXP) into 2‐C‐methyl‐D‐erythritol 4‐phosphate (MEP), which in turn is the substrate for the downstream enzymatic steps. We have expressed and purified IspC of Plasmodium falciparum (PfIspC) and Mycobacterium tuberculosis (MtIspC) using E. coli as an expression system. Finally, we carried out enzyme inhibition assays using novel IspC inhibitors, showing IC50 results ranging from 0.28 – 5.3 μM.

Support or Funding Information

Self‐sponsored.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanism Studies of Thiosemicarbazone Inhibition on Human Topoisomerase IIα
Authors: Lyons Kyle Robert , Toma Maryo , Lisic Edward , Jiang Xiaohua
Published: June 2021

Thiosemicarbazones (TSCs) has been shown as noncompetitive inhibitors of human Topoisomerase IIα (TopoIIα). Metal‐TSC complexes demonstrated higher activities than their ligands. Metal‐TSC was shown to bind on TopoIIα near the ATP binding pocket. Thus in the absence of ATP, metal‐TSC complexes can increase TopoIIα‐induced cleavage complexes, unlike other inhibitors of TopoIIα. The metal ions of TSC play a predominant role in the activity of TSCs. Cu‐TSC complexes are the most active ones compared to their Pd and Pt counterpart. In this study, we study the activity of metal‐TSC effects on TopoIIα in the presence and absence of ATP molecule and try to understand the mechanism how metal‐TSC interact with TopoIIα.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigations of the Mechanism of Action for Lung Cancer Cell Death by a 4′‐Trifluoromethoxy Substituted Chalcone Derivative
Authors: Stantliff Trevor Martin , Krzysiak Amanda J.
Published: June 2021

Chalcones are a diphenyl compound that serves as a natural precursor to flavanones in plants.

Chalcones have been shown to have anticancer and antimicrobial activities. Chemoprevention activity of chalcones are of high interest in medicinal chemistry because of the simple laboratory synthesis and modification via aldol condensation. Previously this lab created and screened a library of synthetic chalcones against A549 lung adenocarcinoma cell line for antiproliferation properties. We identified a strong drug candidate (4′‐Trifluoromethoxy substituted chalcone) for A549 growth inhibition. However, the cause of inhibition by the substituted chalcone remains to be identified. We began to explore the mechanism of action of this drug by looking at physical characteristics of cell death with microscopy. Then, we conducted viability and cytotoxic assays such as MTS and Trypan blue for cell death quantification. Cytotoxicity was determined with a Lactate Dehydrogenase Assay. Western Blots were ran to identify dose dependency on the specific apoptotic proteins PARP and Caspase‐3. Using flow cytometry, we investigated a more accurate quantification of cells undergoing apoptosis versus necrosis. Here we present the results of our investigation into the apoptotic and necrotic cell death of lung cancer A549 cells by a chalcone derivative.

Support or Funding Information

Project Funded by Bellarmine University Department of Chemistry and Physics and Bellarmine University Student Government Association.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Yiqi‐Huoxue Granule reduces pro‐thrombotic factors induced by lipopolysaccharide in HUVECs via upregulating KLF2 and inhibiting NF‐κB
Authors: Wang Zhentao , Wang Xinzhou , Gao Shuibo , WU HONG
Published: June 2021

Yiqi‐Huoxue Granule (YQHX) is a widely used traditional Chinese formula for the treatment of cardiovascular diseases in clinical practice in China. Our present study explored the effects of YQHX on ameliorating inflammatory events triggered by lipopolysaccharide (LPS) in HUVECs. YQHX significantly suppressed the generation of pro‐thrombotic factors such as plasminogen activator inhibitor‐1 (PAI‐1) and tissue factor (TF), whereas increased the protein expression of Kruppel‐like factor 2 (KLF2). The increase of PAI‐1 and TF were reduced by KLF2 knockdown with Lenti‐shKLF2 vector. Furthermore, YQHX remarkably lowered the phosphorylation levels of nuclear factor κB (NF‐κB) p65 and IκB induced by LPS. In addition, YQHX inhibited the production of PAI‐1 and TF in an NF‐κB‐dependent manner, which is manifested by using PDTC, a specific NF‐κB inhibitor. In short, the results demonstrated that YQHX notably exerts the anti‐thrombotic reaction induced by LPS via inhibiting the upregulation of KLF2 expression and the activity of NF‐κB signaling in HUVECs, suggesting that KLF2 and NF‐κB signaling may be promising therapeutic strategies for intervention of inflammation‐associated atherosclerosis.

Support or Funding Information

This work was financially supported by grants from the National Natural Science Foundation of China (No. 81473453, 81673800), the Science & Technology Innovation Talents in Universities of Henan Province (No. 14HASTIT028), and the Henan Science and Technology Project (No. 142102310040).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimization of Shell Grinding Techinques for Purification of Proteins from Zebra Mussel Shells
Authors: Freiermuth Margaret , Grant Jennifer E.
Published: June 2021

Wisconsin and Minnesota lakes are under attack from invasive Zebra Mussels, Dreissena polymorpha, a freshwater mussel species native to Eurasia. The Zebra Mussels were introduced to the Great Lakes around 1985–1986 and have continued to aggressively invade the surrounding areas of Minnesota and Wisconsin. Outcompeting native species, including fish, for food sources, Zebra Mussels are thriving. Oddly, this confluence of attributes, ie fast growth but undesired presence, offers a unique opportunity to capitalize on the economic potential of this invasive species' presence in Wisconsin. Here we examine the protein content found in the Zebra Mussel's shell, and optimization of the protocol for extracting proteins from this matrix, with particular emphasis on techniques for grinding the shells. This will be the first step in determining a possible commercial use in a large pool representing an underutilized potential resource that is also a pest.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Towards Extraction of Proteins from the Shells of Dreissena polymorpha
Authors: Junaidi Nabil , Grant Jennifer E.
Published: June 2021

The zebra mussel, Dreissena polymorpha, is one of the most invasive aquatic species of Midwest United States. Its biogeochemical impact on American waters is regarded as a quintessential example of a difficult and current ecological problem, since there does not exist a working management scheme to control its biofouling and parasitic effects. Yet, the untapped proteomic diversity of aquatic species, especially freshwater mollusks, leave the zebra mussel as an attractive resource for the large‐scale screening of peptides that may confer selective advantages to its life cycle, such as antibiotic resistance. The first step along this pathway is to optimize procedures for extracting functional peptides from zebra mussel shells. Here we discuss basic research aimed at identifying successful extraction of proteins using detergent‐based strategies.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
New tools for autoimmune kidney disease: Design of antigen binding proteins against the thrombospondin type‐1 containing domain 7A
Authors: Stoddard Serena Daniella , Stoddard Shana Victoria
Published: June 2021

Idiopathic membranous nephritis, (IMN) is an autoimmune disease (AD) affecting the kidney. Conventional treatment for AD uses immunosuppressive medicines that suppress the entire immune system, rather than target specific autoimmune disorder antibodies. This causes patients to become immunocompromised, and at risk of not being able to fight off common infections. A more specific therapy to treat AD is needed to prevent the suppression of the entire immune system. Thus the goal of this work was to develop antigen binding proteins (ABP) which could prevent autoantibody binding to the antigen of interest in an AD. ABP targeting the thrombospondin type‐1 containing domain 7A (THSD7A) antigen of IMN were developed. Domain 2 of THSD7A was used as the target site for binding. These designs were carried out through in silico mutagenesis of template monobody candidates. Homology modeling of designed proteins was performed using the Phyre2 server for all ABP. The Rosetta docking2 protocol was used to determine strength of protein‐protein interactions between the ABP and domain 2 of THSD7A. Molecular interactions were evaluated to determine which mutations strengthened binding of the ABP to the THSD7A. Several mutations were shown to improve overall binding compared to the template monobody (3UYO) which was found to have an interface score of −2.511, measured in Rosetta energy units (REU). The most promising binding efficiency scores and placement of an ABP was shown to be the C‐side location of the 3UYO ABP template. Introduction of an electrostatic interaction through the single point mutation S88D, produced an interface score of −5.557 REU. The four point mutation G43W, T72R, S88D, S91D introducing three electrostatic interactions, one hydrophobic interaction, resulted in an interface score of −4.793 REU. Currently protein expression and experimental testing is underway to evaluate binding in vitro. This work will assist in the design of antigen specific therapies for AD, leading to a more targeted therapeutic suppression of the immune system.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of antigen‐specific blocking monobodies against the β2‐glycoprotein I receptor for the inhibition of systemic lupus erythematosus
Authors: Rasasack Itthipoaln , Stoddard Shana Victoria
Published: June 2021

Autoimmune diseases occur when the body's immune system attacks healthy tissues and organs through the use of antibodies. Current treatments suppress the patient's entire immune system through immunosuppressive drug therapies. These drugs decrease the body's effective immune response to genuine pathogens, increasing the patient's susceptibility to having major complications from basic infections. Therefore, treatments that can eliminate disease progression, as well as preserve the healthy function of the patient's immune system, should be considered. Systemic lupus erythematosus (SLE) is a systemic autoimmune disorder affecting a predicted five million individuals worldwide. One autoantigen targeted in SLE is the β2‐glycoprotein I receptor (β2GPI). The work here focuses on the development of protein monobodies capable of inhibiting interactions between the autoantibody and autoantigen that elicit the immune response. Binding between the monobody and autoantigen may prevent antibody binding, decrease autoimmune complexes, and ultimately halt disease progression. Using in silico programs Phyre2 and Chimera, mutations to the monobody templates 3K2M, 3T04, and 5A43 were introduced to improve binding between the monobody and proposed antibody‐binding β2GPI domains 1, 2, and 5. In silico program ROSIE was used to predict protein‐protein binding scores between the monobody candidates and proposed β2GPI domains. Initial binding scores (in Rosetta Energy Units, or REU) between monobody templates 3K2M, 3T04, and 5A43 with β2GPI domain 5 were −3.105 REU, −2.795 REU, and −3.038 REU, respectively. A double mutation to 5A43 (V46R‐E48H) improved 5A43's binding score with β2GPI domain 5 to −4.122 REU. A slight improvement to binding was made to 3K2M with a single mutation (N47H), with a score of −3.446 REU to β2GPI domain 5. More mutations to each monobody template are being evaluated. Enzyme‐linked immunosorbent assays (ELISAs) will be conducted to evaluate antibody inhibition by monobody candidates in vitro. The goal of the research presented here is to develop safer and effective treatments of autoimmune disease.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Computational Approaches for the Analysis of Tandem Affinity and Proximity‐Based Purifications in R
Authors: Velasquez Erick Francisco , Garcia Yenni , Gao Lucy , Whitelegge Julian , Torres Jorge
Published: June 2021

During the past decade, the incorporation of mass spectrometry data to identify protein‐protein interactions and associations has become extremely popular, and presents an exciting opportunity to exploit the protein interactome. However, the experimentation often produces large amounts of proteomic data that can be hard to handle, delaying scientific discovery. It has become clear that when handling the data, cleaning the data of false positives is as important as verifying associations. To address this issue, scientists have developed databases like the Contaminant Repository for Affinity Purification (CRAPome) that provide spectral count data for a collection of controls. However, the data can be hard to interpret without a more rigorous statistical analysis. This work describes an open source proteomic pipeline that incorporates tools to identify false positives and visualize interactions/associations in a user friendly manner. Taking advantage of R, a popular statistical programming language, and Shinny apps we developed a program that aims to standardize analysis of protein interaction/association experiments. The pipeline allows scientists to submit proteomic data and easily apply suggested significance tests to clean their experiments against their own control and the CRAPome if they wish to do so. Often these types of experiments are done through a purification step and so we have extended the platform to include predictive machine learning algorithms that highlight false positives based on intrinsic data that describes a protein that might stick to the column. After analysis, is carried out we couple the platform to protein interaction network visualization tools such as Cytoscape, to create a comprehensive analysis pipeline that is user friendly. In the future we plan on extending this platform by applying neuronal networks to further predict protein‐protein interactions, adding to a fully automated pipeline, for the analysis of protein‐interaction/association experiments.

Support or Funding Information

The work was possible through USPHS National Research Service Award 5T32GM008496 and the Whitcome Pre‐doctoral Training Program and the UCLA MolecularBiology Institute

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An Integrated Framework for Genome Analysis Reveals Numerous Previously Unrecognizable Structural Variants in Leukemia Patient Samples
Authors: Broach James , Xu Jie , Schleicher Emily , Song Fan , Pool Christopher , Hennessy Max , Sheldon Kathryn , Annageldiyev Charyguly , Sharma Arati , Chang Yuanyuan , Hastie Alex , Miller Barbara , Goldenberg David , Claxton David , Moldovan Geroge‐Lucian , Yue Feng
Published: June 2021

We have applied a combination of whole genome sequencing and optical genomic mapping to a number of adult and pediatric leukemia samples, which revealed in each of these samples a large number of structural variants not recognizable by current standard genomic analyses. We developed computational methods to determine which of those variants likely arose as somatic mutations. The method identified 97% of the translocations previously reported by karyotype analysis of these samples and revealed an additional fivefold more such somatic rearrangements. The method identified on average tens of previously unrecognizable inversions and duplications and hundreds of previously unrecognizable insertions and deletions. These structural variants affected a number of leukemia associated genes as well as cancer driver genes not previously associated with leukemia and genes not previously associated with cancer. A number of variants only affected intergenic regions but caused cis‐acting alterations in expression of neighboring genes. Analysis of TCGA data indicates that several of the recurrently mutated genes significantly affect survival of AML patients. Our results suggest that current genomic analysis methods fail to identify a majority of structural variants in leukemia samples and this lacunae may hamper diagnostic and prognostic efforts.

Support or Funding Information

NIH grants R35GM124820, R01HG009906, and U01CA200060; Four Diamonds Fund; George Lafferty Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.