bluuebunny/crossref_metadata_2025 huggingface dataset:
High‐ throughput Carbonic Anhydrase Activity and Inhibitor Screening Assays
Authors: Hazra Suratna , Tchaga Grigoriy , Yan Gordon July
Published: June 2021

Carbonic Anhydrase (CA) is a zinc based metalloprotease present in at least 14 different isoforms in mammals. It catalyzes the reversible hydration of carbon dioxide to form bicarbonate and proton, thus playing an important role in pH and CO2 homeostasis. Changes in CA activity are associated with various diseases such as glaucoma, type II diabetes mellitus, lung and liver diseases etc. As CA is also an important target for cancer therapy developing high‐throughput carbonic anhydrase activity and inhibitor screening assays is very important.

Carbonic anhydrase activity or carbon dioxide hydration rate is widely measured by using potentiometer or a stopped‐flow instrument as reflected by the change in pH over time. However, this method is not suitable for high‐throughput assay. Consequently, in BioVision, Inc. we have launched the first high throughput Carbonic anhydrase activity assay kit, detecting the esterase activity of this enzyme. This kit utilizes the zinc‐hydroxide mechanism for carbon dioxide hydration and hydrolysis of an ester substrate resulting in a release of a chromophore signal that is proportional to the enzyme activity.

Support or Funding Information

Biovision Inc.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification and Characterization of Bioactive Cyclotides in Viola odorata using PepSAVI‐MS
Authors: Parsley Nicole C. , Kirkpatrick Christine L. , Hicks Leslie M.
Published: June 2021

Antimicrobial resistance (AMR) is present in every country worldwide and has a direct impact on global health, agriculture and economy. Treatment of common bacterial, viral and fungal infections is becoming increasingly challenging as new mechanisms for AMR develop, accelerated by the misuse and overuse of antimicrobial drugs. Botanically‐derived bioactive peptides are an attractive solution to the ongoing deficit of new antimicrobial compounds, with the ability to be highly selective, potent, and inexpensive to produce on a large scale. Our lab has developed the PepSAVI‐MS (Statistically‐guided bioActive Peptides prioritized VIa Mass Spectrometry) pipeline, an adaptable method for the analysis of natural product libraries to rapidly identify bioactive peptides. Using PepSAVI‐MS to screen the cyclotide‐rich botanical species Viola odorata against a variety of human and plant pathogens, we have identified several putative, uncharacterized cyclotides. Cyclotides are a class of cyclic, disulfide‐rich, plant‐derived peptides that boast a diverse range of potent antimicrobial bioactivities, indicating a highly stable, evolvable scaffold. Additionally, we have identified novel bioactivities of a known nematocidal V. odorata cyclotide, Cycloviolacin O8 (cyO8), including anticancer activity against PC3 prostate, MDA‐MB‐231 breast, and OVCAR ovarian cancer cell lines, and for the first time in literature, cyclotide antifungal activity against the common fungal pathogen Fusarium graminearum. Furthermore, to address the identity of the putative, traditionally difficult‐tosequence V. odorata cyclotide species, we demonstrate a proof‐of‐principle 193 nm ultraviolet photodissociation (UVPD) mass spectrometry approach using cyO8 to elucidate the fragmentation efficiency of this information‐rich technique.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of the nuclease resistance of DNA four‐way junctions.
Authors: Moorhead Gaston , Huang Raymond , Kebede Yonatan , Smith Andrew , Estevez Luis , Troisi Maria , Bell Anthony Jerome
Published: June 2021

Oligodeoxynucleotides (ODN) offer great promise as therapeutics due to their high binding specificity, low toxicity and ease of synthesis. Hence, it is not surprising that ODNs are being developed as antigene and antisense reagents. ODNs can also control gene expression by targeting proteins. One popular approach uses double stranded DNA as transcription factor decoy sequences that are directed against transcription factors such as NF‐kB and specificity protein 1 (Sp1). More recently, researchers have used a similar approach to target proteins in the extracellular matrix. In these studies, nucleic acids are directed against the damage‐associated molecular pattern (DAMP) molecule, High Mobility Group B1 (HMGB1). We hypothesize that ODNs based on DNA four‐way junctions (4WJs) can be used to target HMGB1. To enhance the ex vivo stability of 4WJs, the helical termini of a DNA junction are modified to generate the ODN, i‐J1. The objective of this study is to investigate the nuclease resistance of i‐J1, a control 4WJ (J1) and the intramolecular 4WJ, J4 against three nucleases. The three nucleases are: DNase I, Exonuclease III (Exo III) and bacteriophage T5 Exonuclease (T5 Exo). DNase I cleaves dsDNA and ssDNA in a largely non‐specific manner to generate mono‐ and oligodeoxyribonucleotides. Exo III has expanded functionally; the enzyme acts as a 3′®5′ exonuclease, 3′‐repair diesterase, 3′‐phosphomoesterase and ribonuclease. Here, we focus on the 3′®5′ exonuclease activity of Exo III. T5 Exo hydrolyzes dsDNA and ssDNA in a 5′®3′ direction to release mono‐, di‐, tri‐nucleotides and oligonucleotides. The nuclease protection data shows that i‐J1 and J4 have significantly higher nuclease than J1 against DNase I and Exo III. We suspect the increase in nuclease resistance in linked to changes in the minor groove dimensions of i‐J1 and J4. A binding model is presented to explain the increase in nuclease resistance of i‐J1 and J4.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cleaving Glutathione S‐Transferase from a B‐cell Receptor Protein to Improve Interactions as seen in EMSAS
Authors: Egbuchulam Angela , Lares Monica
Published: June 2021

The purpose of my project is understanding the specific amino acid interactions for the B‐cell Receptor, BAFF‐R, protein interacting with a RNA aptamer. A RNA aptamer is a peptide that binds to a molecule of interest. We had to express our protein with a GST tag. We used different lab skills, such as gel shift assay, to investigate the interaction between the BAFF‐R and its RNA aptamer. However, the results were inconclusive as we saw an unexpected binding pattern. We hypothesize that cleaving the Glutathione S‐Transferase (GST) tag using thrombin will provide for our expected binding pattern in an EMSA.

Support or Funding Information

Funded by Office of Undergraduate Research in Sonoma State University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Searching Protease Inhibitors by a Phage Display Kunitz‐Type Library.
Authors: da Silva Gustavo Felippe , de Lourdes Borba Magalhães Maria , de Moraes Julio Cesar , Echeverri Lina Maria Salazar
Published: June 2021

Phage Display is a powerful tool for identification of protein ligands against a myriad of targets. In our study, we selected a small protein member from the Kunitz family to serve as a scaffold in the MIII 13 phage coat protein. The protein, originally a scorpion toxin that inhibits a potassium‐dependent voltage channel, has 33 residues composed of two beta sheets and an alpha helix stabilized by disulfide bonds. After cloning the toxin gene into an M13 phagemid, we used Kunkel's mutagenesis, an additional uracil‐DNA glycosylase treatment, and rolling circle amplification (RCA) procedures to produce a synthetic Kunitz‐type phage display library. We drove the mutations in specific residues within two specific loops as well as within the alpha helix of the scaffold protein, keeping the cysteines intact. We were able to produce a library of over a million different proteins. To validate the ability of this library to bind specific targets, trypsin was used as target. Since other studies have proved Kunitz's natural proteins as protease inhibitors, we expect our displayed Phage protein library to generate new potent and specific protease inhibitors for medical as well as biotechnological purposes.

Support or Funding Information

This work was supported by “State University of Santa Catarina” (UDESC) and Fundação de Amparo a Pesquisa e Inovação do Estado de Santa Catarina (FAPESC).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pentachloropseudilin inhibits TGF‐β signaling by promoting TGF‐β type II receptor degradation via non‐raft internalisation
Authors: Chung Chih‐Ling
Published: June 2021

Transforming growth factor beta 1 (TGF‐β) is a multifunctional cytokine that regulate several cellular processes including cell proliferation, differentiation and apoptosis. In addition to normal physiological function, TGF‐β has been reported that involved in many diseases such as tumor progression and fibrosis. Therefore, studying inhibitor of TGF‐β signaling pathway may provide new therapeutic strategies of those diseases. Pentachloropseudilin (PClP), a chlorinated phenylpyrrole compound isolated from Actinoplanes sp., and found in marine origin, was identified as an allosteric inhibitor of myosin family. However, how PClP influence TGF‐β signaling remains unknown. In this study, we found that PClP is a potent inhibitor of transforming growth factor‐beta signaling. PClP suppressed TGF‐β‐induced PAI‐1 reporter activation and Smad2/3 phosphorylation with a low IC50. PClP also inhibited TGF‐β‐induced epithelial–mesenchymal transition (EMT) and cell migration. Results from cell surface labeling and immunoblot analysis indicated that PClP suppressed TGF‐β response by promoting degradation of TGF‐β type II receptor (TβR‐II). The proposed mechanism is that PClP directs TβR‐II translocation to non‐raft fraction leading to accelerated internalisation and rapid degradation of TβR‐II.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Screening of in silico identified inhibitors of breast cancer resistance protein (BCRP) transporter in BCRP‐overexpressing MCF‐7 M100 cancer cells
Authors: Bequeaith Beau A. , Nanayakkara Amila K. , Vogel Pia D. , Wise John G.
Published: June 2021

The overexpression of breast cancer resistance protein (BCRP) is associated with multidrug resistance (MDR) in specific cancers. BCRP is an ATP‐binding cassette (ABC) transporter that is also functionally important in the blood brain barrier. The ability of BCRP to export cancer chemotherapeutics to sub‐therapeutic concentrations renders chemotherapy treatment ineffective in cancers that overexpress BCRP. BCRP can also be problematic in its ability to inhibit the delivery of drugs to the brain. There are currently no clinically approved inhibitors of BCRP. The identification of inhibitors of BCRP that have the potential for clinical use would therefore greatly improve drug delivery systems to both cancers and the brain.

Using computational approaches, we have identified a number of small, drug‐like molecules that have been predicted to inhibit BCRP. We report here the results of testing these small molecules in vitro in a BCRP‐overexpressing breast cancer cell line (MCF‐7 M100). The compound, Hoechst 33342, is known to be a transport substrate of BCRP, and was used here in assays that attempted to identify BCRP inhibition. Since Hoechst 33342 can be pumped out of the cells by BCRP, inhibition of BCRP leads to its intracellular accumulation. The BCRP activity assay used here takes advantage of the fact that accumulated Hoechst 33342 binds to DNA in the nucleus where it becomes highly fluorescent. The fluorescence of cells treated with putative inhibitors was compared with vehicle treated cells as an indication of BCRP inhibition. Nearly 100 experimental compounds were assayed at 10 μM in this study and compounds that exhibited three‐fold or greater accumulation over the vehicle treated control group were tested for inhibition of BCRP activity at lower concentrations. Five compounds have been identified so far that have demonstrated effective BCRP inhibition at nM concentrations. These compounds are currently being tested for the ability to reverse MDR in the BCRP‐overexpressing MCF‐7 M100 cell line.

Support or Funding Information

This work was supported by NIH NIGMS [R15GM09477102] to John G. Wise, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, the Hamilton Undergraduate Research Scholars Program, and a private gift from Ms. Suzy Ruff of Dallas, Texas.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure‐based drug design targeting the malty sweet Mycobacterium tuberculosis GlgE
Authors: Petit Cecile , Kapil Sunayana , Veleti Sri Kumar , Lindenberger Jared , Sucheck Steven J. , Ronning Donald R.
Published: June 2021

In 2016, 8.1 % of new cases (601,000 people) and 20 % of previously treated cases of Tuberculosis (TB) were diagnosed as Multiply Drug Resistant Tuberculosis (MDR‐TB) cases, amongst which 10 % are Totally Drug Resistant Tuberculosis (TDR‐TB) cases worldwide. In America, MDR‐TB new cases make 1.5 % of the cases among which none of the TDR‐TB cases were successfully treated in 2016. In addition to TB and HIV treatment being contraindicated, the rise of MDR‐TB and TDR‐TB emphasize the need for new drugs and drug targets. Identified in 2010, the maltosyltransferase GlgE is an interesting drug target in that it has no homologues in humans and its inhibition results in the death of the microorganism in 14 days. Based on the structure of GlgE's natural ligand, maltose‐1‐phosphate, and the known enzymatic mechanism, substrate and transition‐state analogs were designed as potential inhibitors of the enzyme and co‐crystallized with an MtbGlgE homolog. To improve the resolution of our X‐ray structures to more accurately analyze the interactions between the inhibitors and the residues in the active site, ScoGlgEI has been used as a surrogate. Sharing 53 % identity with MtbGlgE, we created a ScoGlgEI‐V279S variant as to obtain 100 % identity between the active sites of the two homologues. Based on the information obtained from X‐ray structures of ScoGlgEI‐V279S in complex with our initial inhibitor, 2,5‐dideoxy‐3‐O‐α‐D‐glucopyranosyl‐2,5‐imino‐D‐mannitol (DDGIM), a 2‐fold inhibition improvement has been made on our second‐generation inhibitors bringing the IC50 from 102 ± 8 μM to 45 ± 4 μM. Recently published, the crystal structures of ScoGlgEI‐V279S in complex with these early‐ and late‐dissociative transition‐state‐like inhibitors were solved at 2.5 Å and 3.2 Å showing that improvement was obtained through the addition of a phosphonate to our DDGIM inhibitor, resulting in an increased number of hydrogen bonded interactions between the enzyme and the inhibitors. To increase potency of our next generation inhibitor, additional interactions with the enzyme were sought outside the active site. The release of a structure of ScoGlgEI‐V279S in complex with its substrate brought more information to increase the number of interactions between ScoGlgEI‐V279S and the next generation of inhibitors. The details of the crystal structure of ScoGlgEI‐V279S in complex with our third‐generation inhibitor was obtained at 3.35 Å and the determined inhibitory activity will be discussed.

Support or Funding Information

NIH grant AI105084

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Disease‐causing mutations of Lamin A lead to loss of calcium homeostasis in the endo/sarcoplasmic reticulum
Authors: Chi Ya‐Hui , Lin Wen‐Hsin , Wang Wan‐Ping , Kao Cheng‐Heng , Wang Jing‐Ya , Teng Yuan‐Chi , Tsai Ting‐Fen
Published: June 2021

Mutations in nuclear lamin A are responsible for several rare human diseases, including the Emery‐Dreifuss muscular dystrophy (AD‐EDMD) and the Hutchinson‐Gilford progeria syndrome (HGPS). Here we show that structure of the endo/sarcoplasmic reticulum (ER/SR) is disrupted in HGPS cells and skeletal muscle of Lmna mutant mice. However, only a slight increase in ER stress‐associated genes was observed. Microarray analysis revealed that expression of several calcium signaling‐associated genes were disturbed in the skeletal muscle of Lmna mutant mice compare to the wild type cohorts. Sacrolipin (Sln), a SERCA activity modulator, is highly expressed and serves as a protective factor for the lifespan of Lmna−/− mice. In a myoblast cellular system inducibly expressing progerin to model HGPS, we found increased store operated Ca2+ entry (SOCE) and colocalization of Stim1 and Orai1. These findings suggest that the nuclear lamin A is structurally and functionally connected to the robustness of ER/SR for calcium homeostasis.

Support or Funding Information

This work was supported by grants from the Ministry of Science and Technology, Taiwan (MOST 105‐2633‐B‐400‐001 and MOST 105‐2320‐B‐400‐020).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein‐Protein Interaction Prediction Yields WNK‐SPAK/OSR1 Pathway as Regulator of Kir2.1 and Kir2.3 K+ Channels
Authors: Taylor Clinton Albert , An Sung‐Wan , Kankanamalage Sachith Gallolu , Huang Chou‐Long , Cobb Melanie Houston
Published: June 2021

The WNK‐SPAK/OSR1 pathway is a key regulator of cellular ion transport and homeostasis, and has a role in physiological processes such as neurotransmission and blood pressure regulation. WNK kinases respond to changes in intracellular Cl−, osmolarity, and cell volume by activating the closely related kinases, SPAK and OSR1. The pathway mediates ion flux through Na+, K+, and Cl‐− cotransporters and ion channels via SPAK and OSR1‐dependent and independent mechanisms. SPAK and OSR1 use a protein interaction domain to bind linear R‐F‐x‐V/I sequence motifs in WNKs, cotransporters, and other proteins. Our goal was to identify novel SPAK and OSR1 interacting proteins by further defining the specificity determinants of the interaction, and predicting new interactions using the refined specificity profile. We used peptide arrays and biophysical measurements to define specificity, and used this information to search and rank motifs in the Uniprot database. All motifs not conserved in mice, not solvent exposed, and not present in the cytosol or nucleus were excluded. Nearly all of the previously validated interactions clustered within the top ten percent of our rankings and none were excluded, thus providing strong evidence that our ranking methodology was successful. Members of the inward rectifier K+ channel family were predicted to have a high probability of interacting with SPAK and OSR1 based on our rankings. We determined that OSR1 kinase activity activates two of the channels, Kir2.1 and Kir2.3, and activation is dependent on the presence of the binding motif. SiRNA knockdown of OSR1 and chemical inhibition of WNK kinases disrupted NaCl‐induced plasma membrane localization of Kir2.3. Our results suggest that activation of the WNK‐SPAK/OSR1 pathway enhances Kir2.1 and Kir2.3 channel activity by increasing plasma membrane localization of the channels. Our interaction predictions imply that additional inward rectifier K+ channel family members may be regulated in a similar manner. On a broader scale, our interaction predictions may help identify additional novel regulatory functions of the WNK‐SPAK/OSR1 pathway.

Support or Funding Information

This work was supported by National Institutes of Health GM53032 and Welch Foundation I‐1243 for M.H.C. Additional funding support to C.L.H.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PGC‐1α, a Novel Substrate for Protein Arginine Methyltransferase 7?
Authors: Cuala Janielle , Osuji Immaculeta , Zurita‐Lopez Cecilia
Published: June 2021

Peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α) is a protein that plays an important role in the regulation of energy metabolism. It participates in the regulation of both carbohydrate and lipid metabolism and is activated by insulin. For these reasons, it has been implicated in obesity, aging, diabetes and cardiomyopathy. Despite many studies on the role of this master regulator, the precise way it becomes regulated remains unknown. A recent study found that PGC‐1α becomes phosphorylated at Ser570 by kinase B (Akt/PKB) and this stops it from promoting gluconeogenesis and fatty acid oxidation. PGC‐1α is also modified by protein arginine methyltransferase 1 (PRMT1) at several arginine residues, which enhance its regulation. In this study, we set out to determine whether PGC‐1α is modified by PRMT7, another member of the PRMT family. We hypothesize that PRMT7 methylates PGC‐1α because it contains many RXR motifs, a known PRMT7 motif. In addition, they both are active at similar temperatures. To test this hypothesis, truncated constructs corresponding to PGC‐1α and full‐length PRMT7 were expressed and purified in bacterial cells. In vitro methylation reactions using tritiated AdoMet were carried out. Through fluorography of the methylation reactions, it was determined that PRMT7 methylates PGC‐1α within amino acid residues 532–640. Upon confirmation of the methylated arginine residue(s) through immunoblotting and mass spectrometry, further studies will be carried out to investigate the function of arginine methylation by PRMT7. These results will enhance our understanding of PGC‐1α with the broader goal of understanding its regulation in the context of obesity and diabetes.

Support or Funding Information

CSULA Minority Access to Research Careers‐Undergraduate Student Training for Academic Research (MARC‐U*STAR), Grant: T34 GM008228 and Louis Stokes Alliance for Minority Participation (LSAMP), Grant: HRD‐1302873

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Contribution of Sinorhizobium meliloti Flagella on Biofilm Formation and Symbiosis During Host Colonization
Authors: Amir Zhainib Adel , Chen Joseph C.
Published: June 2021

Many bacteria transition from a motile to a sessile, aggregate state, known as a biofilm, ensuring bacterial survival in harsh environments. Biofilms pose serious threats to public health due to their association with bacterial resistance to antimicrobial treatments against infections. Sinorhizobium meliloti, a nitrogen‐fixing bacterium, forms biofilms on abiotic surfaces and on roots of leguminous plants. Our laboratory is currently investigating the contribution of biofilm to symbiosis between Sinorhizobium meliloti and the model host legume Medicago truncatula. Recently our laboratory detected a nonsense mutation in the podJ gene of the commonly used S. meliloti laboratory strain, Rm1021. By correcting this mutation, the new strain, podJ+, showed an ability to produce a polar adhesive called holdfast, and to build more robust biofilms. PodJ regulates flagellar motility and chemotaxis in S. meliloti, and the full‐length protein appears to be required for holdfast production, which in turn contributes to surface adhesion and biofilm formation. However, previous studies showed that holdfast alone is insufficient for biofilm formation; for example, holdfast and flagella together facilitate the surface attachment of cells in the related bacterium Caulobacter crescentus. The goal of this project is to investigate whether the flagella of S. meliloti contribute to holdfast‐dependent biofilm formation in S. meliloti. Additionally, I would like to determine whether the presence of flagella improves symbiosis of S. meliloti with the legume host. To achieve this goal, the presence of flagella was examined in strains with mutations in flagellar genes, in both the podJ+ background (JOE3759) and the original, uncorrected background (Rm1021). First, soft‐agar plates were used to assess the flagellar motility of the strains: this test indicates which strains synthesize or lack flagella. To confirm the presence and absence of flagella and to assess the formation of adhesive clusters in these strains, scanning electron microscopy and scanning transmission electron microscopy were utilized to examine the cells. Next, competitive infection assays were performed to assess the effect of flagella on symbiosis efficiency. This was performed by inoculating the Medicago host simultaneously with podJ+ strains that lack or produce flagella, thereby, determining which strain colonizes more root nodules. These results will help uncover the molecular pathways by which rhizobia form biofilms, thereby providing the basis for understanding and controlling biofilm formation in related pathogens.

Support or Funding Information

NIH RISE program

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Genomics Education Partnership: Course‐based Research Experiences for Undergraduates
Authors: Van Stry Melanie , Saville Ken , Leung Wilson , Lopatto David , Elgin Sarah C.R.
Published: June 2021

The Genomics Education Partnership (GEP) is a collaboration between faculty from over 100 diverse institutions with the goal of providing authentic course‐based undergraduate research experiences (CUREs) in bioinformatics and genomics for undergraduate students. The GEP faculty have developed curricula for undergraduates at all levels that allow faculty to implement bioinformatics instruction and research within their courses. The research projects have primarily focused on the comparative genomics of the Muller F element (dot chromosome) in several Drosophila species. This chromosome is unusual in that it is largely heterochromatic, but contains ca. 80 genes that are expressed at levels similar to those found in euchromatin. Our students have focused on sequence improvement (finishing), and the annotation of protein‐coding genes and transcription start sites. Through these evolutionary comparisons, we are gaining insights into the regulation of gene expression within this heterochromatic environment. Our most recent paper on the expansion of the Drosophila ananassae F element includes 239 undergraduate students and 31 faculty co‐authors. Our faculty and student surveys, pre/post‐quiz results, and focus groups suggest that students gain the most from CUREs when faculty treat them like members of a research team. They also learn through formative failures — when challenges arise that must be resolved. In addition to research projects, we are developing new curricula, such as the “Understanding Eukaryotic Genes” modules, designed to familiarize beginning students with genome browsers while teaching eukaryotic gene structure, and units on Hidden Markov Models and Dynamic Programming for more advanced students. The GEP is collaborating with Galaxy to create G‐OnRamp (http://gonramp.org), a workflow that constructs UCSC Assembly Hubs and JBrowse genome browsers for newly sequenced eukaryotic genomes. G‐OnRamp enables faculty to engage students in novel annotation projects from a variety of organisms (e.g., Puerto Rican parrot, parasitoid wasps). If interested in participating in a grant‐supported workshop either June 12–15 or July 16–19, 2018 to learn to use G‐OnRamp, contact Sarah Elgin (selgin@wustl.edu).

Support or Funding Information

Supported by NSF IUSE #1431407 and NIH R25GM119157 to SCRE.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The role of the ESCRT pathway in prion disease
Authors: Lawrence Jessica
Published: June 2021

Prion diseases are infectious neurodegenerative disorders caused by PrPSc, a misfolded and aggregated isoform of the cellular prion protein, PrPC. During late disease stages, PrPSc spreads through the CNS, causing neurodegeneration, gliosis, and ultimately death, however the mechanisms of prion cell to cell transport remain poorly defined. Here we investigated the role of exosomes in the intercellular spread of prions. Exosomes are extracellular nanovesicles that originate from the endosomal pathway and are important for intercellular communication, transporting proteins and mRNA to nearby cells. Exosomes harbor infectious prions isolated immortalized cell lines and mice, and these exosomes efficiently cause prion disease in mice. Exosomes are produced by the ESCRT pathway, an endosomal pathway consisting of four protein complexes functioning sequentially. Previous research has shown that knocking down components of this pathway reduce prion conversion and release in vitro. We hypothesize that prions exploit the exosome pathway as the primary mechanism of prion spread within the CNS. We have manipulated the ESCRT pathway in neuronal cells to understand its role in both prion conversion and spread in vitro. We have discovered that inhibiting the initial stages of prion entry into endosomes severely impacts both prion generation as well as autophagic degradation pathways.

Support or Funding Information

NIH F31 NS103588

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterizing Native American mitochondrial DNA Haplogroups in Puerto Ricans by Next‐Generation Sequencing (NGS)
Authors: Matos Paola M. , Martínez‐Cruzado Juan C.
Published: June 2021

Puerto Ricans are an admixed people with three different parental populations: European, African, and Native Americans. When Puerto Rico became a United States colony in 1898, the results of the census reported no trace of Native Americans (e.g. Taínos) on the island. Therefore, the percentage of Native American ancestry in Puerto Ricans has been questioned. Using mtDNA and restriction length polymorphism analysis (RFLP), my lab identified and quantified the frequency of four different Native American haplogroups (e.g A, B, C, and D). However, the genetic diversity within these haplogroups across the entire island needs further characterization. The experimental design consists of three phases: 1) genotype individuals using Real Time‐PCR, 2) library preparation for next‐generation sequencing, 3) mapping and sequencing of the mtDNA genome. Over 320 human saliva DNA samples have been screened. So far, 102 individuals have been identified for haplogroup A and 26 for haplogroup C. Further work will include the continuation of genotyping of individuals, and preparation of samples for sequencing and mapping of the mtDNA genome for the four haplogroups. This project will help discover the continental origins of the different lineages within each haplogroup and their time of arrival to the island.

Support or Funding Information

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institute of Health under Award Number: T34GM008419

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ultrasensitive Immunoassay for Ex‐Vivo Human Soluble Epoxide Hydrolase Detection
Authors: Li Dongyang , Cui Yongliang , Morisseau Christophe , Bellien Jérémy , Hammock Bruce
Published: June 2021

Soluble epoxide hydrolase (sEH) is a potential pharmacological target for the treatment of hypertension, inflammation, cancer, pain and multiple cardiovascular related diseases and a marker of inflammation. Detecting and quantifying sEH appears an important diagnostic tool for numerous pathologies. However, current methods rely on expensive technologies such as radioactive markers or mass spectrometry that are available by the bench side. Here, we report the development of an ultrasensitive nanobody based immunoassay for human sEH ex vivo. Nanobodies are the variable domain of the heavy chain antibody, which permit high stability, ease of genetic manipulation, bacterial expression, and ability for continuous manufacture, thus providing a constant high quality biochemical reagent. Llama nanobodies against non‐denatured human sEH were used as the detection antibody in a sandwich ELISA format with polyclonal anti‐sEH as the capture antibody. To enhance the reporting signal, the conventional HRP labeled anti‐HA tag tracer was replaced with polymeric horseradish peroxidase (PolyHRP) reporter system. This yields a 141‐fold increase in the sensitivity and 57‐fold decrease in limit of detection. Systematic comparison of three different tracers in four ELISA formats demonstrated the overwhelming advantage of PolyHRP as a label for nanobody based immunoassay. The enhanced sEH immunoassay was further evaluated in terms of selectivity against other epoxide hydrolases and detection of the target protein in human tissue homogenate samples. Comparison with a radioactive enzyme activity based assay and a Western‐blot for sEH detection reveals good correlation with the immunoassay. Moreover, the enhanced ELISA was used successfully to detect the sEH level in peripheral blood mononuclear cells isolated from 90 patients with different disease status, which have in general very low abundance of sEH hard to detect by classic methods. The work demonstrates the promise of nanobody based ELISA as a diagnostic tool for detection of and other pharmacologically and toxicologically important proteins.

Support or Funding Information

National Institute of Health (Superfund P42ES04699 and R01 ES002710) and National Natural Science Foundation of China (81402722)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pharmacological inhibition or genetic ablation of soluble epoxide hydrolase attenuates obesity‐induced nonalcoholic fatty liver disease
Authors: Zhang Jianan , Yang Jun , Wang Weicang , Yang Haixia , Sanidad Katherine z. , Yang Szu‐Hao , Sukamtoh Elvira , Zhang Guodong
Published: June 2021

The prevalence of nonalcoholic fatty liver disease (NAFLD) in western countries is 20–30%; it is of critical importance to identify new therapeutic target, in order to reduce the risks of NAFLD and associated diseases. Here, we report that the expression of soluble epoxide hydrolase (sEH) is increased in liver tissues of a high‐fat diet‐induced NAFLD model; in addition, pharmacological inhibition or genetic ablation of sEH attenuates high‐fat diet‐induced NAFLD, suggesting that sEH could be a novel therapeutic target of NAFLD. We find that after 8‐week dietary feeding of high‐fat diet (60%cal), the expression of sEH is dramatically increased in liver. To explore the roles of sEH in obesity‐induced NAFLD, we test whether pharmacological inhibition or genetic ablation of sEH affects the development of NAFLD. We find that oral administration of TPPU and t‐TUCB, which are two different sEH inhibitors, attenuates high‐fat diet‐induced fat accumulation in liver (assess by liver triglyceride analysis, oil red o staining and H&E histology staining), and reduces expressions of pro‐inflammatory genes in liver (Mcp‐1 and Tnf‐α). Similar results are observed in sEH knockout mice. Regarding the mechanisms, we find that pharmacological inhibition or genetic ablation of sEH suppresses the expression of Srebf1, Ppar‐γ, Cd36 and Dgat2, which play critical roles in lipid synthesis; and increases the expression of Ppar‐α, regulating fatty acid β‐oxidation. Together, these results support that sEH could be a promising therapeutic target for NAFLD, suppressing fat accumulation and inflammation in liver. This could lead to rapid human translations, because the pharmacological inhibitors of sEH are being evaluated in human clinical trials targeting multiple disorders.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the In Vivo Catalytic Efficiency of CYP2C9 M1L, a Novel and Common Variant in the Yup'ik Alaska Native Population
Authors: Henderson Lindsay M. , Hopkins Scarlett , Boyer Bert B. , Thummel Kenneth E.
Published: June 2021

The Yup'ik Alaska Native people are under‐represented in genetic research, but have unique pharmacogene variation that may critically impact their response to drug therapy. Full gene resequencing of CYP2C9 in the Yup'ik population identified novel, relatively common CYP2C9 variants, including CYP2C9M1L (M1L), a coding region single nucleotide polymorphism hypothesized to confer a poor metabolism activity phenotype by disrupting the start codon. M1L is present at a minor allele frequency of 7.1% in the Yup'ik, placing the population at risk for adverse drug interactions with narrow therapeutic index CYP2C9 substrates such as (S)‐warfarin, phenytoin, and tolbutamide. This study's objective was to characterize the catalytic efficiency of MIL in vivo by evaluating the pharmacokinetic behavior of naproxen, a probe for CYP2C9 activity, in genotyped Yup'ik participants. Targeted genotyping was conducted to identify heterozygous and homozygous M1L individuals from 906 representative members of the Yup'ik population. CYP2C9 mediates the O‐demethylation of naproxen to its 6‐O‐desmethyl metabolite, which is glucuronidated or sulfonated to secondary metabolites, all of which are excreted into the urine. Thus, a change in the urinary metabolite to parent ratio is indicative of a change in CYP2C9 intrinsic formation clearance to 6‐O‐desmethyl naproxen. We developed and validated a novel LC/MS method for simultaneous quantification of (S)‐naproxen, (S)‐6‐O‐desmethyl naproxen, and (S)‐naproxen acyl glucuronide in human urine. Preliminary analysis of one‐half of the final study population revealed an average ratio of 6‐O‐desmethyl naproxen to unchanged naproxen of 18.0 ± 7.7 (n = 7), 10.1 ± 4.5 (n = 7), and 15.6 (n = 2) for the M1L reference, heterozygote, and homozygote variant groups, respectively. ANOVA analysis was borderline significant (p = 0.06) and pairwise comparison of the homozygous reference and heterozygous groups (p = 0.06) suggested reduced activity for the M1L variant. The effect of the novel M1L variant and its potential to alter the pharmacokinetics of drugs metabolized by CYP2C9 have clinical implications, including phenotypic misclassification from currently available pharmacogenetic tests, in the Alaska Native population.

Support or Funding Information

P01 GM116691, T32 GM 007750

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of glutathione‐S‐transferases in the metabolism of the anti‐cancer agent and aromatase inhibitor, exemestane
Authors: Teslenko Irina L. , Chen Gang , Xia Zuping , Lazarus Philip
Published: June 2021

Breast cancer is the most commonly diagnosed cancer in women, with approximately 1 in 8 women in the U.S. developing breast cancer over their lifetime. The majority of breast cancers diagnosed each year are estrogen receptor positive (ER+). Drugs targeting the ER pathway are a major treatment modality for ER+ patients and are additionally prescribed for the prevention of breast cancer. Exemestane (EXE) is an inhibitor which blocks the enzyme aromatase in the final step of estrogen biosynthesis. Studies examining the long‐term use of EXE as a chemopreventative agent demonstrated a reduction in breast cancer incidence by more than 65%. However, response and toxicity varied widely among individuals, which may be attributed to genetic polymorphisms in enzymes that metabolize EXE. Data from our lab demonstrate that a cysteine conjugates of EXE and its active metabolite 17β‐dihydro‐EXE (17β‐DHE) make up 77% of the total metabolite in urine. Cysteine conjugates are formed from metabolism of glutathione (GSH) conjugates in a reaction catalyzed by the glutathione‐S‐transferases (GST) family of the enzymes. The goal of the present study was to identify hepatic GSTs active in the metabolism of EXE and 17β‐DHE and to determine kinetic parameters for the identified enzymes. In a screening of the Protein Atlas, a total of 12 cytosolic and 3 microsomal GSTs were found to be hepatically expressed. EXE‐GSH conjugation activity in human liver samples was screened by incubating GSH with EXE or 17β‐DHE (0.125–1.0 mM) in cytosol (HLC) or microsomes (HLM) prepared from human livers. GSH conjugates were formed for both cellular fractions, indicating that both cytosolic and microsomal GST enzymes are active against EXE and 17β‐DHE. GSH conjugates were detected by UPLC‐MS and quantified using synthesized EXE‐GS and 17β‐DHE‐GS standards. The purity and identity of both standards and internal standards were confirmed using NMR and UPLC‐MS and found to be > 95% pure. Individual recombinant human GSTs were then screened to determine which enzyme is primarily responsible for the metabolism of EXE. Commercially available cytosolic GSTs were purchased, and their activity was verified with a common GST substrate, 1‐chloro‐2.4‐dinitrobenzene. Results from the individual EXE‐GSH conjugation assays indicate the cytosolic enzymes GSTA1, GSTM3 and GSTP1 are active against EXE and 17β‐DHE; studies of microsomal GSTs are on‐going. These data suggest that several GSTs, both cytosolic and microsomal, may be important in the phase II conjugation of EXE and its major active metabolite, 17β‐DHE.

Support or Funding Information

This study is supported by National Institutes of Health (NIH) (Grant R01‐ES025460 and R01‐CA164366) and the Health Sciences and Services Authority of Spokane

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Tumor Conditioned Media from Prostate Cancer Cells on Fibroblast Cell Morphology, Viability and Gene Expression
Authors: Kaur Sukhneeraj Pal , Cummings Brian S.
Published: June 2021

Cancer is a heterogeneous collection of neoplastic cells, surrounded by non‐malignant, stromal cells, and extracellular matrix (ECM), among other constituents. Cross‐talk between tumor and stromal cells forms a dynamic tumor microenvironment (TME) that mediates cell growth, proliferation, and metastasis. The TME can act to inhibit cancer growth and proliferation during the initial stages of carcinogenesis. However, cancer cells are able to evade these inhibitory signals, and subsequently reshape the TME, making it more amenable to their own growth. This gives rise to numerous cancer‐associated stromal cells, including cancer‐associated fibroblasts (CAFs). CAFs further modulate the TME by regulating the release of growth signals, cell death, ECM remodeling, angiogenesis, and epithelial‐mesenchymal transition. To study the ability of prostate cancer to alter the TME in vitro, we exposed human bone fibroblasts (HS‐5 cells) to tumor conditioned media isolated from a human neuroendocrine prostate cancer cell line (DU145) and assessed changes in cell morphology, viability (using MTT assay) and the expression of Group IIA secretory phospholipase A2 (PLA2G2A), as well as its regulatory proteins, glypican‐1 (GPC‐1) and Group IVA phospholipase A2 (PLA2G4A), using qPCR. Exposure of HS‐5 cells to TCM from DU145 cells altered cellular morphology to a more mesenchymal phenotype typically seen in active myofibroblasts. TCM also decreased MTT staining in a time‐dependent manner that was independent of the presence of serum in the TCM. Changes in cell morphology and viability correlated to increases in the expression of PLA2G2A, GPC‐1 and PLA2G4A. The effect of TCM was time‐ and serum‐dependent, and was differential for each gene studied. Alteration in cellular morphology and increases in PLA2G2A, GPC‐1 and PLA2G4A in HS‐5 cells correlated to increases in the expression of fibroblast activation protein‐α, which is a CAF marker. To elucidate the mechanisms mediating changes in HS‐5 cells exposed to TCM, we investigated the expression of GLi1, a transcription factor involved in the activation of the sonic hedgehog signaling pathway. We found that TCM increased GLi1 mRNA levels in HS‐5 cells, with greater increases being observed using TCM containing serum. Collectively, these data demonstrate that media from DU145 cells can induce morphological and genotypic changes in human fibroblasts similar to those observed in CAFs. As such, this model holds promise for identifying molecular mechanisms by which prostate cancer cells alter the TME and induce prostate cancer cell growth.

Support or Funding Information

National Institute of Biomedical Imaging and Bioengineering NIBIB (EB0160100)Department of Defense Prostate Cancer Research Program Idea Development Award (PC150431 GRANT11996600)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FOXO1 Couples Reactive Oxygen Species in Diabetic Atherosclerosis
Authors: LIU Jingjin , Yan Dan , Xie Xiang , XIA Zhengyuan
Published: June 2021

Diabetic atherosclerosis, distinct from other type of atherosclerosis, is progressing rapidly which is the leading cause of morbidity and mortality in the entire world, and no effective therapy has been established so far. Thus, an intensive search for new therapeutic targets in diabetic atherosclerosis is needed. A growing evidences are that post translational modifications of FOXO1 triggered by hyperglycemia‐induced reactive oxygen species (ROS) which is most likely primarily responsible for hyperglycemia, plays an important role in diabetic atherosclerosis progression.

Here, we investigated whether the inhibition of FOXO1 alters diabetes‐associated atherosclerosis. Our study found that pharmacologic inhibition of FOXO1 markedly suppressed the vascular remodeling in DM though attenuating overproduction of ROS, increasing antioxidant function in conjunction with enhancing antioxidant activity, and reducing ROS induced inflammation and fibrosis in carotid artery. Moreover, in T1DM rats, FOXO1 translocate into the tunica intima of carotid artery after treatment of pharmacologic inhibition of FOXO1, which coupled with the activation of PI3K/Akt/mTOR pathway. In addition, inactivation of PI3K/Akt/mTOR pathway in diabetic carotid artery was restored by pharmacologic inhibition of FOXO1 and the anti‐ROS effect of pharmacologic inhibition of FOXO1 was partially inhibited by a PI3K inhibitor (LY294002) and also enhanced p‐Akt/Akt protein ratio. Therefore, we hypothesis that pharmacologic inhibition of FOXO1 attenuate hyperglycemia‐induced ROS generation associated with activation of PI3K/Akt/mTOR pathway signaling, which may be a promising target for treating diabetic atherosclerosis and subsequent cardiovascular diseases.

Support or Funding Information

RGC General Research Fund: GRF 17124614 (09/2014‐08/2017), GRF 17123915M (09/2015‐08/2018)

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