bluuebunny/crossref_metadata_2025 huggingface dataset:
Buprenorphine Analogs to Treat Relapse
Authors: Olson Keith M. , Hillhouse Todd E. , Sears Bryan F. , Rysztak Lauren , Husband Stephen M. , Jutkiewicz Emily M. , Traynor John R.
Published: April 2020

Substance use disorders (SUDs) – the overuse and dependence on at least one substance – have a lifetime prevalence of ~10% in the U.S. While buprenorphine (BUP) treatment reduces relapse rates, it is only marginally effective, as 70% of SUD patients relapse within a year. Due to BUP’s marginal effectiveness and abuse potential, there is a need to improve SUD therapies that prevent relapse to multiple drugs of abuse after exposure to stress, drug‐associated context and drug priming. Here we evaluate BUP and two analogs for their ability to inhibit cocaine‐ and stress‐induced relapse using a conditioned place preference (CPP) paradigm. Pharmacologically, BUP is a potent (sub nM) partial agonist at the m‐opioid receptor (MOR) with potent antagonist activity at the k‐(KOR), and d‐opioid receptors (DOR). In vitro BUP can act as a weak (mM) partial agonist at the nociceptin/OrphaninFQ (NOP) receptor with uncertain in vivo consequences. We measured in vitro potency and efficacy using [35S]GTPgS functional assays in CHO cells separately expressing DOR, KOR, MOR or NOP. Since NOP receptor agonists reduce relapse in animal models, we compared BU10119 with NOP agonist activity (EC50 = 120 nM ± 20; EMAX = 44% ± 5%) or BU12004 with NOP antagonist activity (KB = 190 nM ±30) to BUP (EC50 = 1300 nM ± 320; EMAX = 39% ± 7%). Furthermore, BU12004 and BU10119 were MOR antagonists (KB = 0.42 nM ± 0.08 and KB = 1.1 nM ± 0.2, respectively) with less abuse liability than the MOR partial agonist BUP (EC50 = 0.79 nM ± 0.31; EMAX = 34% ± 6%). BUP, BU10119, and BU12004 were all potent antagonists at DOR and KOR. BU10119 (1.0 mg/kg) attenuated cocaine‐primed reinstatement in CPP, but 10 mg/kg BU12004 did not. BUP trended toward reduced drug‐primed reinstatement. The in vivo rank order of BU10119 >BUP >BU12004 matches the rank order for NOP agonism in vitro. Additionally, 1.0 mg/kg of either BU10119 or BU12004 blocked swim stress‐primed reinstatement, ostensibly due to their KOR antagonist activity. Taken together, BU10119 provides a promising lead to treat SUDs with better effectiveness and reduced abuse potential compared to BUP.

Support or Funding Information

Supported by NIDA R01 DA07315 and 035316.
Protein Kinase A isoform dynamics and disease.
Authors: Aoto Phillip , Baker Blaine , Taylor Susan
Published: April 2020

A number of disease mutations in the alpha and beta isoforms of Protein Kinase A have recently been identified. We model these mutations and investigate changes in conformational and protein/ligand interaction dynamics. Changes in partner protein and substrate interactions due to mutation is confirmed by fluorescence polarization binding and competition assays. Computational and biochemical approaches reveal a potential common mechanism among the mutations leading to kinase dysfunction.
Rare Bilateral Variation of the Extensor Indicis Tendon in two Female Cadavers
Authors: Pfennig Mitchell , Porcaro Skyler , Khatchadourian Nayiri , Bughrara Moneb , Burkett Erica , Barremkala Malli
Published: April 2020

In this case study, we discuss the observed anatomical variant of bilateral extensor indicis medii communis (EIMC) muscle. During a routine anatomical dissection of 48 upper limbs from 24 cadavers, two female cadavers (75 and 79 years of age) demonstrated bilateral EIMC (8.3%), while one male cadaver (86 years of age) demonstrated unilateral EIMC (4.2%). The noted EIMC shared a muscle belly with the extensor indicis, but had a distal attachment in the extensor hood of the third digit. This is a rare instance of a bilateral presentation of the extensor indicis medii communis muscle. There has been a limited number of cases in which a bilateral EIMC was discovered; however, such findings have been limited to one cadaver and at a lower frequency. This finding may demonstrate that bilateral EIMC may not be as rare as previously thought. Ultimately due to its infrequency, awareness of this variation may help physicians avoid improper muscle recognition when visualizing the area during procedures regarding the dorsum of the hand, as well as providing a possible site of tendon harvesting.
Comparing Transcriptome Profiles of Silver, Cadmium Selenide/Zinc Sulfide, Indium Phosphide/Zinc Sulfide, and Palladium Quantum Dots in Saccharomyces cerevisiae
Authors: Horstmann Cullen Michael , Kim Daniel , Kim Kyoungtae
Published: April 2020

The primary focus of our research is to obtain global gene expression data in baker’s yeast exposed to sub‐lethal doses of specific nanomaterials, such as nanoparticles (NPs) and quantum dots (QDs), to reveal novel insights on their mysterious and undetermined mechanisms of toxicity. We have investigated the negative effects of several nanomaterials including Silver nanoparticles (AgNPs) and yellow emitting Cadmium Selenide/Zinc Sulfide (CdSe/ZnS) QDs, and are continuing our investigation on other nano‐materials like green emitting CdSe/ZnS QDs, Indium Phosphide/Zinc Sulfide (InP/ZnS) QDs, and Palladium nanoparticles (PdNPs). Despite their promising and ever‐growing list of applications (fluorescent probes, drug delivery systems, etc.) their potential toxicity and long lasting effects on the environment are not well understood. Using high‐throughput technology, such as RNA‐sequencing, we obtained gene expression profiles and the identities of specific differentially expressed genes (DEGs) in yeast exposed to our wide variety of nanomaterials. We have found hundreds of DEGs in cells treated with AgNPs involved in rRNA processing, ribosome biogenesis, cell wall formation, cell membrane integrity, and mitochondrial functions. Additionally, our cell wall stability experiment revealed cells treated with AgNPs were extremely susceptible to cell wall damage. Further, we found hundreds more DEGs in yellow emitting CdSe/ZnS treated cells than in Ag treated cells leading us to conclude that both Ag and CdSe/ZnS treated cells induced a mild cytotoxic effect in yeast. Due to the versatile nature of nanoscale materials, their mechanisms of toxicity can differ vastly based on their physical characteristics. Currently, we are analyzing gene expression profiles of a similar green emitting CdSe/ZnS QD, a “safer” InP/ZnS QD, and PdNPs to determine a better understanding of how these materials induce toxicity. Interestingly, we have noted differences in DEGs between yellow and green emitting CdSe/ZnS QDs, such as fewer DEGs involved in RNA processing and ribosome biogenesis, indicating the two similar QDs may have different mechanisms of toxicity. To our knowledge, we are the first to investigate and compare DEGs induced by these nanomaterials in yeast using RNA‐sequencing. Additionally, we expect and anticipate that our findings will help provide useful data on the toxicity of certain nanomaterials and will expedite change in the EPA’s current regulations and standards on the use of such nanomaterials.
Nutrient modulation of DNA repair in lung cancer
Authors: Eroglu Abdulkerim , Balbuena Emilio
Published: April 2020

Noncommunicable diseases associated with aging and inflammation is rising in recent decades, contributing global burden of disease globally. Among them are cardiovascular diseases, diabetes, and cancer. Our objective is to illustrate the molecular mechanisms behind the chemoprotective effects of major dietary carotenoids. We hypothesize that carotenoids play a role in modulating genome stability. Telomeres are essential for the stability of our genome, and oxidative stress caused by the reactive oxygen species poses a risk for telomere shortening, thereby contributing to the pathogenesis of chronic diseases, including lung cancer. Human telomeres are composed of 10–15 kilobases of TTAGGG repeats. Guanine is the most readily oxidized of the natural bases, and TTAGG repeats are preferred sites for conversion of Guanine to 8‐oxoG, which repaired by base excision repair through an enzyme called OGG1. In the current study, we investigated mRNA and protein expression of OGG1 and retinoic acid receptors (RARs) in human alveolar epithelial cells exposed to cigarette smoke. Cells pre‐treated with various doses of β‐carotene for 24 hours, and the following day exposed to smoke using a smoking chamber. We found out that while β‐carotene increased OGG1 expression at lower doses, it led to a dramatic decrease at higher concentrations suggesting that at higher doses β‐carotene exerted a pro‐oxidant effect. Previously it was reported that the expression of the RARβ, putative tumor suppressor gene, is reduced in lung cancer. In parallel to OGG1 inhibition, we observed decreased levels of RARβ. We are in the process of quantifying 8‐oxoG by HPLC‐MS/MS. We believe that these novel findings will shed light on the mechanism of action of β‐carotene at the molecular level.

Support or Funding Information

USDA‐NIFA Hatch Project
The Effects of Histidine Tags on the Energy Landscape on Acyl Co‐A Binding Protein
Authors: Van Dyck Papa Kobina , Guinn Emily
Published: April 2020

Protein epitope tags play an important role in protein characterization, allowing for easy detection, tracking and isolation in cellular and biochemical experiments. A polyhistidine tag is an amino acid motif that consists of at least six histidine (His) residues, often located at the N‐ or C‐terminus of the protein. Affinity tags, like His tags, are routinely employed as convenient means of purifying recombinantly expressed proteins. However there have been increasing reports suggesting that His tags interfere with the structure and function of proteins. We hypothesize that His tags affect the energy landscape of proteins. Acyl Co‐A Binding Protein (ACBP) is a single‐domain protein that is thought to fold in a two‐state process To test our hypothesis we studied the stability and folding kinetics of ACBP with differentially located His tags in the presence of buffers with varying pHs with a fluorimeter and a stopped‐flow spectrometer. First, we found that buffer pH differentially affected the charge of the His tag: At a pH of 5.3 Histidine is protonated and at 7 Histidine is deprotonated. Second, we observed a significant difference in the kinetics and thermodynamics of the C‐Terminus His tagged ACBP relative to the taglesss ACBP. In a pH of 5.3, the C‐Term His tagged ACBP was less stable whereas in a pH of 7 it was more stable. On the other hand, there was no significant difference in the thermodynamics or the kinetics of the N‐term ACBP to the tagless ACBP. Taken together our studies suggest that the location of the His tag (and ionic environment) markedly affected the energy landscape of ACBP.

Support or Funding Information

DePauw University‐PDF
Sighs Originating in the PreBötC are Driven by Neuroglial Interactions
Authors: Severs Liza , Baertsch Nathan A. , Ramirez Jan-Marino
Published: April 2020

Sighs are large amplitude inspirations that occur periodically during normal breathing and are physiologically important for preventing alveolar collapse. Normal or “eupneic” inspirations and sighs originate from the same neural network, the preBötzinger Complex (preBötC), in the ventral medulla. Given that both sighs and eupnea arise from the same network, it has been challenging to differentiate the underlying mechanisms that concurrently generate both rhythmic behaviors. Using the in vitro brainstem slice preparation, we have recently demonstrated that this is possible through neuroglial coupling. We found that modulating purinergic signaling through P2Y1 receptors strongly alters sigh frequency without altering eupnea. Then, we used optogenetics in combination with extracellular population recordings to drive ChR2 expression under control of the astrocytic promoter, Aldh1l1. Light activation of Aldh1l1 expressing preBötC astrocytes evoked both eupnea and sigh bursts. Evoked sighs and eupnea had the same characteristics of spontaneous bursts, including comparable amplitudes, burst durations, and post‐burst intervals. Furthermore, optogenetic stimulations showed that sighs are tightly coupled to the eupneic frequency, as they occurred with a significantly higher probability when stimulated during the inspiratory cycle. Next, we demonstrated that light‐evoked sighs could be inhibited by applying P2Y1 blockade and low concentrations of the Ca2+ channel blocker cadmium (4μM, to selectively target P/Q‐type receptors). Sighs under control conditions and with application of the muscarinic receptor agonist oxotremorine were elicited within the limits defined by the refractory period. Utilizing calcium imaging with Fura‐2, we next identified a population of brainstem astrocytes that displayed an increase in intracellular calcium corresponding to both sigh and eupnea activity. We hypothesize that this increase in calcium facilitates neuroglial coupling through release of ATP, as preliminary data indicates that release of ATP from astrocytes and conversion to ADP contributes to this calcium change. Taken together, our experiments suggest that the timing characteristics underlying sigh generation are a coordinated action between glial calcium oscillations and the recruitment of preBötC neurons along the inspiratory column through purinergic signaling.

Support or Funding Information

NIH Ruth L. Kirschstein Predoctoral Individual National Research Service Award

NIH R01 HL126523
Characterizing the Thermodynamic Stability of the Holliday Junction Through Spectroscopic Techniques
Authors: Taylor Nicholas Brown , Mukerji Ishita , Savage Rachel , McDonald Julie Lynn , Qiu Rong
Published: April 2020

The Holliday junction is a four‐way DNA structure formed during the process of double strand break repair and DNA recombination in meiosis. Branch migration of the junction is a vital molecular process for the transfer of genetic information from parents to offspring. Understanding this process relative to base pair composition can provide a better picture of the overall structure of the junction and its connection to short inverted repeats that have been linked to chromosome instability. We used a fluorescent nucleoside analog, 4‐amino‐6methyl‐8‐(2‐deoxy‐beta‐d‐ribofuranosyl)‐7(8H)‐pteridone (6‐MAP), to obtain site‐specific information about the melting process, through incorporation of the probe at distinct sites throughout a 34 bp immobile junction. Under our conditions, the junction primarily adopts a conformation where the arms stack co‐axially on each other. We have examined the stability of the junction arms by incorporating the probe at equivalent positions on each arm. From these measurements we have found that the junction follows a 'pseudo‐duplex' model of denaturation, in which one of the coaxially stacked pair of arms melts at a lower temperature relative to the other. Our results also point to a sequential melting pattern in which positions at the end of the arms and the center destabilize before the interior of the arms. The relative proximity to the junction center appears to be one of the factors affecting stability. Our current research investigates how sequence homology influences the stability of the junction, specifically whether the thermodynamic characteristics of an immobile junction are predictive for a mobile one. We have developed a mobile junction with 10‐bp homology in the central region. Previous research by (Voth A.R., Hays F.A., Ho P.S. (2007) Directing macromolecular conformation through halogen bonds. Proc. Natl. Acad. Sci. USA.; 104:6188–6193. doi: 10.1073/pnas.0610531104) has suggested that macromolecular conformations can be directed through the use of halogen bonds. Specific incorporation of brominated uracils in the sequence of two DNA strands of a mobile Holliday junction was shown to bias the structure to a single conformation in crystallographic experiments. Combined spectroscopic data from both the immobile and mobile junction will inform coarse‐grained modeling of the Holliday junction. Our experimental data combined with computational modeling furthers our current understanding of this DNA structure as it relates to branch migration and exchange of genetic information.
Investigating the Functions of Endogenous Neuraminidases Neu1 and Neu3 in Blood Cell and Protein Homeostasis
Authors: Westman Julia S. , Yang Won Ho , Marth Jamey D.
Published: April 2020

Neuraminidases cleave terminal sialic acid linkages on the glycans of glycoproteins and glycolipids. In some cases this exposes underlying galactose linkages that can be recognized by lectins including the endocytic hepatic Ashwell‐Morell receptor that thereby modulate protein function through endocytic clearance. Neuraminidases are widely distributed in vertebrates and microorganisms, although the neuraminidases of mammalian origin seems to differ from the microbial variants in genetic sequences and enzymatic properties. Four mammalian neuraminidases, encoded by NEU1‐4, have been identified and characterized. Mice deficient in the lysosomal resident Neu1 have been reported with molecular features resembling sialidosis, a severe human lysosomal storage disorder, whilst Neu3 deficiency does not result in an obvious physiological phenotype. Neu1 is found in a complex with b‐galactosidase and protective protein/cathepsin A in the lysosome. The subcellular localization of Neu3 has been suggested to be the plasma membrane and endosomal compartments. Interestingly, the membrane attachment mechanism and topology is yet to be fully clarified, however Neu3 is commonly reported to be membrane‐associated. A variety of mammalian cell types express neuraminidases, including endothelial cells as well as hematopoietic lineages spanning platelets, monocytes, and the erythrocyte surface. Both Neu1 and Neu3 have been found in the bloodstream and in extracellular compartments, by one or more secretion mechanisms that are not fully defined. We have previously noted that circulating neuraminidase activity is measurable in normal mammalian blood at physiological pH, and de‐sialylate plasma proteins with increasing molecular age, and this was further demonstrated to result in lectin‐mediated clearance in the determinations of glycoprotein half‐lives. To further investigate the roles of each of these mammalian neuraminidases, we are using multiple approaches in Neu1‐ and Neu3‐deficient mice, including measurements with novel lectins that detect different types of sialic acid linkages and their modifications, among the endothelium and the surfaces of hematopoietic cells in parallel with comparative blood proteomics, flow cytometry, and other molecular identification strategies. These studies will be presented with our current findings that may provide further information regarding mammalian Neu activation and function in glycoprotein homeostasis.

Support or Funding Information

NIH grant HL131474.

Post Doctoral Fellowship: Swedish Research Council dnr 2017‐00192.
Design and Testing of RNA Therapeutics to Block VEGFR2 and EGFR Expression in Human Glioblastoma
Authors: Gawargi Flobater , Hicks Martin Joseph
Published: April 2020

Glioblastoma multiforme (GBM), is the most common and aggressive malignant primary brain tumor with a median survival of 15 months with standard care. Current therapies are limited by the blood brain barrier. Tumor blood vessel formation depends on vascular endothelial growth factor receptor 2 (VEGFR2), while tumor cell proliferation is stimulated by epidermal growth factor receptor (EGFR). Both are important for tumor cell survival. Our novel approach delivers directly to the tumor cells, genes encoding RNA therapeutics. Our strategy is to alter the splicing mechanism of the VEGFR2 and EGFR genes to block their activation, thus stop tumor cell angiogenesis and growth Our current RNA therapeutics demonstrate significant reductions in EGFR and VEGFR2 expression. To increase the efficacy of our RNA therapeutics, we are fine‐tune targeting regions of EGFR and VEGFR2, as well as recruiting splicing silencers and enhancers to the pre‐mRNA transcripts. Can we generate therapy vectors that encode antisense RNA therapeutics that either block or activate splicing motifs and alter the expression of functional VEGFR2 and EGFR in GBM significantly reducing tumor growth? Forty‐five antisense sequences were designed to target the EGFR gene and nine antisense sequences for VEGFR2, to potentially block their activation. The antisense sequences were cloned into our pAAV‐U7‐smOPT therapy vector. In addition, multiple strategies were used to clone the exonic splicing silencer 4G‐quadruplex and five distinct exonic splicing enhancer motifs into the RNA anti‐sense therapy vector, which recruit serine‐arginine (SR) proteins, directing the spliceosome to a desired splice site or intronic poly‐adenylation site. Moreover, another aspect of this research is to isolate the mRNA of multiple tyrosine kinase receptors from GBM cancer cells, clone the cDNA into a T7 expression vector to transcribe control RNA to use in our high throughput sequencing experiments. Multiple GBM cell lines, including U87 and SKMG3 were transfected with our novel therapies. Total mRNA was collected, analyzed, and compared to the same cell lines without treatment, showing a significant reduction in target expression. The collected data allow analysis to measure efficacy of current anti‐VEGFR2 and anti‐EGFR therapeutic strategies which will be tested in a mouse model using adeno‐associated virus (AAV) vector.
Desmopressin inhibit the proliferation and cellular migration in the OV‐90 ovarian cancer cell line
Authors: Calvillo-Robledo Argelia , González-Sánchez Zaira Samantha , Valdez-Urias Fernando , Gómora-Herrera María José , Quintanar-Stephano Andrés
Published: April 2020

Epithelial ovarian cancer (EOC) is the most common and lethal of the ovarian neoplasms, frequently it is detected at advanced stages of the disease resulting in 60–70% of the cases in recurrence and less responsive to chemotherapy. Currently the role played by the neurohypophyseal hormone arginine vasopressin (AVP) and its receptors in the development of EOC is unknown. The purpose of this study was to evaluate the action of desmopressin (DDAVP), an AVP V2 receptor agonist on the cell proliferation and cell migration in cells from the OV‐90 cell line. Cell proliferation was evaluated with the proliferation marker Ki67, whereas the cells migration was evaluated by the wound assay. The AVP V1a and V2 receptors expression were assessed by immunofluorescence. Cell cultures were stimulated during 12, 24, 36 and 48 hours with DDAVP (500 to 1500 mM). In order to discard the cytotoxicity of DDAVP on the OV‐90 cell cultures in control and stimulated cultures the Cytotox 96 kit was used. The experiments were performed three times by duplicate. The results showed that the expression of AVP V1a and V2 receptors are present in both DDAVP stimulated and‐non stimulated cultures. On the other hand in comparison with the control cultures, doses of 1000mM of DDAVP significantly (p<0.05) inhibit both cell proliferation and cellular migration. Present results also suggests a possible inhibitory role of DDAVP on proliferation and cellular migration in the OV‐90 cell lines of epithelial ovarian cancer.

Support or Funding Information

Universidad Autónoma de Aguascalientes PIFF14‐1 and CONACYT 221262. México
THE CONTRIBUTION OF “In Vitro” SKELETAL MUSCLE FIBER BIOCHEMICAL TIMING ANALYSIS IN THE UNDERSTANDING OF ELECTROMYOGRAPHIC SPECTRUM
Authors: Acero-Mondragon Edward Javier , Urdaneta-Paredes Daniel Antonio , Chaustre-Nieto Luis Carlos , Gallego John Jairo , Leon-Ariza Henry , Botero-Rosas Daniel , Gutierrez-Arenaza Gerardo
Published: April 2020

Introduction

“In vivo” electromyography usually reports a frequency domain muscle activity in a broad Hertz spectrum that ranges from 1 Hz to 500 Hz. However, when the contiguous nicotinic biochemical events in rhabdomyocytes type Ia, IIa and IIb are timed “in vitro”, and then converted to Hertz. A reduced electromyographic frequency domain is obtained.

Materials and Methods

Google Scholar databases and National Library of Medicine, were searched for articles published from 1990 to 2019 containing times of kynetic cell signalig in raddomiocytes type Ia IIa and IIB join terms like 1) “Title (TI)= ‘Time Acetylcholine exocytosis’ AND ‘Time Activation nicotinic receptor’ AND ‘Time Depolarization of sarcolemma’ AND ‘Excitation–contraction coupling’ AND ‘Time depolarization T‐tubule” AND ‘Cross‐bridge cycle’. The inclusion criteria were that each study involving a types of striated muscle fiber “In vitro” as an objective measure interaction Acetylcholine‐Nicotinic Recepptor and cascade cell signaling to evaluating speed of despolarizatig and every physiological contraction subphases of contraction. Once every time of physiological event of contraction was found, the time was added and then the mathematical inverse of the period in seconds was converted to frequency in Hertz.

Results

“In vitro” the total time events of contraction summed to striated muscle fiber Type IA were 3,33 ms; Fibers Type IIA were 6,81 ms; and Fibers Type IIB were 7,41 ms. Times that converted to Hertz show fiber type Ia, IIa IIb with 3,03 Hz, 1,46 Hz, 1,34 Hz

Conclusions

The electromiographic Hertz domain is made of a series of “noise” such as the different extracellular events that occur between inferior motor neurons and sarcolemmas. The present work aims to rationalize the obtained “in vitro” frequency spectrum values of 3,03 Hz, 1,46 Hz, 1,34 Hz for type Ia, IIa and IIb respectively.

Support or Funding Information

UNIVERSIDAD DE LA SABANA
A Physical Model for Exploring Drug Design to Treat Break‐bone Fever
Authors: Jordan Sarah , Martindale Aidan W. , Heuss Aaron , Miller Dylan , Tamse Joshua , Hall Bonnie L.
Published: April 2020

Annually, dengue fever affects an estimated 390 million people worldwide (more than the population of the entire United States). Symptoms include high fever, headache, vomiting, and muscle and joint pain so extreme it is also known as “break‐bone fever”. Severe infection can lead to dengue hemorrhagic fever, dengue shock syndrome, and can be fatal. The disease is transmitted by mosquitos in tropical areas, and is caused by the single‐stranded RNA dengue virus (DENV). Currently the only treatment for patients infected with DENV is rest and hydration. The Schiffer lab has focused on the DENV NS2/NS3 serine protease as a potential drug target, because it is essential for DENV maturation. The serine protease inhibitor aprotinin (CP1) was the starting point for design of cyclic peptide (CP) inhibitors targeting DENV protease. A series of CP‐inhibitors were designed and tested, each with varying chemical properties. We designed a physical model of the DENV protease active site that displays the interactions with six different CP‐inhibitors. The model demonstrates how CP‐inhibitor binding is impacted by drug size, flexibility, polarity, and ability to form intermolecular interactions (Lipinski’s Rule of Five). Our model also highlights how drug structure drives drug function, and connects meaningfully to undergraduate studies of biology and chemistry at many levels. The DENV protease active site model, with the set of six CP‐inhibitors, is a useful classroom tool to show how basic biological and chemical principals are relevant to modern drug design.

Support or Funding Information

The CREST Program is funded through DUE‐IUSE #1725940 awarded to Milwaukee School of Engineering Center for BioMolecular Modeling.
Cloning and Expression of Palmitoyl Protein Thioesterases and Acyl Protein Thioesterases for Use in Depalmitoylation Reactions
Authors: Esoe Dave-Preston I. , Erickson Jeffery A. , Hamann Michael J.
Published: April 2020

Palmitoylation is a dynamic process for regulating protein function and localization. It involves the attachment of palmitate to protein cysteine residues by palmitoyl transferases and its removal by palmitoyl protein thioesterase (PPTs) or acyl protein thioesterases (APTs). Techniques to study palmitoylation are difficult to perform and use harsh depalmitoylating reagents like hydroxylamine (HAM), often leading to destruction of the protein being studied. Using recombinant or over‐expressed PPT/APTs could be used as an alternative to HAM for studying protein palmitoylation/depalmitoylation dynamics, as well as investigating PPT/APT substate specificities. We have cloned human APT and PPT cDNA for the production of GST or MBP‐tagged fusion proteins to be used in acyl‐biotin exchange assays. We have also produced mCherry‐tagged versions for co‐expression with a target protein of interest to evaluate their activities. In these experiments, we will focus on the Rho GTPase TCL/RhoJ, which was recently shown to associate with the plasma membrane through palmitoylation. Cherry‐tagged APT/PPTs will be co‐transfected with YFP‐tagged TCL to determine if TCL membrane association diminishes. Overall, the goal of these experiments is to improve techniques for studying protein palmitoylation/depalmitoylation.

Support or Funding Information

This work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).
Letrozole modulates tyrosine phosphorylation in liver and kidney of polycystic ovarian syndrome rats
Authors: Chaiyamoon Arada , Bunsueb Sudtida , Reabroi Somrudee , Iamsaard Sitthichai
Published: April 2020

Letrozole (Letro), a non‐steroidal aromatase inhibitor, is commonly used to reduce estrogen level for breast cancer treatment and delay cancer cell metastasis. Recently, Letro is used to induce the polycystic ovarian syndrome (PCOS) in rat model. Protein phosphorylation on tyrosine residues is a posttranslational modification known to regulate biological processes and physiological functions including reproduction. Although the adverse effects of Letro have been reported, the alterations of tyrosine phosphorylation responsible for liver and kidney functions are not demonstrated. In present investigation, PCOS in rats was induced by daily oral administration of Letro (1 mg/kg BW) for consecutive 21 days. After induction, the effects of Letro on serum biochemical components, liver, and kidney functions were examined. In addition, both tissues were subjected for expression of protein tyrosine phosphorylation using Western blotting analysis. Histopathology of liver and kidney was performed by Masson’s trichrome staining. The results showed that Letro did not affect histological features but markedly increased the serum levels of urea nitrogen, cholesterol, triglyceride, high‐density lipoprotein (HDL), low‐density lipoprotein (LDL), alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP). Interestingly, expressions of tyrosine phosphorylation of 32 and 27 kDa in liver and of 55 and 43 kDa in kidney were increased in PCOS group. In contrast, such expression of a 26 kDa in PCOS kidney was decreased as compared to those of control. Our findings suggest that alterations of liver and kidney parameters in the Letro‐induced PCOS rat model may associate with modulation of the tyrosine phosphorylation.

Support or Funding Information

Khon Kaen University
Modifying an Enzyme to Improve PET Plastic Recycling
Authors: Fenton Robin L. , Duplan Amanda , Hall Bonnie L.
Published: April 2020

Polyethylene terephthalate (PET) is widely used in plastic products that pollute our environment due to improper disposal methods, a cause for global concern. PET plastics are resistant to degradation and thus persist in our environment for lengthy periods of time. The bacterium Ideonella sakaiensis is capable of utilizing PET as a carbon source for growth, using the enzyme PETase to break it down at a very slow rate. We are using enzyme engineering to improve the efficiency of PETase, to make it a viable option in PET recycling. The PETase protein has been successfully over‐expressed and purified, and an HPLC assay developed to quantify PET breakdown. Multiple engineered PETase mutants are being characterized for improved efficiency of PET breakdown and enzymatic stability.
Isolation and Characterization of the Pseudomonas aeruginosa Bacteriophage Vanarele
Authors: Rasmussen Reid J. , LaFerriere Holly J.
Published: April 2020

Antibiotic resistant bacteria have become a major problem in the United States, making treatment of patients a nightmare for hospitals. Phage therapy has reemerged as a possible solution to this ever increasing problem. A novel bacteriophage that infects a biosafety level 2 bacterium, Pseudomonas aeruginosa 15692, was isolated from wastewater in Bemidji, MN and named Vanarele. Antibiotic resistant strains of this bacterium are a major cause of disease in immunocompromised patients. Infection from this bacterium is commonly found in patients with cystic fibrosis and burn wound victims. This bacterium commonly forms biofilms that can be problematic in patient airways and catheters. Characterization of the bacteriophage Vanarele that infects strains of Pseudomonas aeruginosa could provide insight and tools that may be utilized in the treatment of antibiotic resistant infections. The phage was isolated utilizing an enrichment procedure and six rounds of plaque purification. Plaque morphology was examined for the isolated phage and was found to have a slight turbid, halo appearance with an average diameter of 3 mm. Once the bacteriophage had been completely isolated a high titer lysate was harvested. Transmission electron microscopy was then performed to visualize phage size. A capsid size of 40 nm was observed. Currently, the effectiveness of the bacteriophage to break down biofilms formed by multiple strains of Pseudomonas aeruginosa is being tested. The characterization of this phage will add to our knowledge of bacteriophages and may aid in development of new treatments for antibiotic resistant infections.

Support or Funding Information

This work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science.
Determining protein structure with tyrosine bioconjugation
Authors: Moinpour Mahta , Schwartz Jacob
Published: April 2020

A hallmark of many neurodegenerative diseases like Alzheimer’s disease (AD), Amyotrophic Lateral Sclerosis (ALS) and Frontal Temporal Lobar Dementia (FTLD) is the formation of misfolded proteins into aggregates in the neuronal cells. These proteins often have intrinsically disordered regions which are enriched in a low complexity of amino acids such as Serine, Glycine, Glutamic acid and Tyrosine. Disordered proteins are challenging to study with common techniques such as x‐ray crystallography and NMR. Furthermore, common biorthogonal chemical reactions which mostly target lysine and cysteine residues cannot be used to determine the protein fold due to the low variety of amino acid composition in these proteins. However, a unique characteristic of several disordered proteins involved in neurodegenerative disease is their enrichment for tyrosines. Here we have developed an approach to assess tyrosine accessibility as a measure of levels in native vs misfolded forms of the proteins.

To measure tyrosine accessibility we employed the ability of 4‐Phenyl‐1,2,4‐triazoline‐3,5‐dione (PTAD) to conjugate to tyrosine side‐chains. We assessed the targeted labeling of tyrosine’s for peptides, proteins, and cells. We have used this chemical approach to observe the transition of Bovine Serum Albumin (BSA), a well‐characterized and folded protein, to an unfolded state. We found that we could assess the local changes to protein structure by using LC‐MS/MS to quantify levels at each tyrosine position. We could confirm changes using fluorescent detection through click chemistry. Our long‐term goal is combining this approach and proteomics to quantify changes in cells to protein folding in comparable cellular states, such as disease and non‐diseased tissues.

Support or Funding Information

National Institute of Health, American Cancer Society
RNA Therapy Recruitment of hnRNP H to Alter the pre‐mRNA Splicing of the Epidermal Growth Factor Receptor Transcript in Human Glioblastoma
Authors: Montero Reina , Hicks Martin Joseph
Published: April 2020

Glioblastoma Multiforme (GBM) is a prominent form of brain cancer that encompasses the formation of gliomata in astrocytes and oligodendrocytes of the central nervous system. GBM has a mortality rate of 15 months due to resistance to current standard care. Tyrosine Kinase Receptors (TKRs) are key players in the proliferation and development of GBM. Examples of TKRs include, vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR). VEGFR is responsible for the formation of new blood vessels while EGFR signals for the increase in tumor size. DNA transcription occurs within the nucleus to produce pre‐messenger RNA (pre‐mRNA) that is modified by capping, splicing and polyadenylation to form mature mRNA, which is translated into protein by the ribosomal complex. Our strategy aims to deliver a gene therapy that will be targeted to the nucleus of intra‐tumoral cells where the therapy will be transcribed to take on the RNA function to alter pre‐mRNA splicing of TKR genes. The focus of this project involves alternative splicing of EGFR exons 15 to 16, disrupting the expression of the transmembrane domain of exon 17. Without its transmembrane domain, EGFR will bind to its ligand without activating the EGFR pathway. This dominant negative receptor is termed soluble decoy due to its ability to sequester ligands without causing physiological changes within the cell. To ensure that our therapy interacts with hetero‐ribonucleoprotein H to further block splice site recognition, EMSA experiments are performed to confirm RNA‐protein binding affinity and the formation of RNA‐protein complexes. Mammalian HEK 293T cell cultures are grown and transfected with a DNA plasmid to produce hnRNP H. Cell lysis is followed by protein isolation and dialysis. The RNA therapy was generated by T7 transcription of cDNA, followed by RNA extraction and purification via RNA 4% polyacrylamide gels. Varying concentrations of therapeutic RNA were incubated with protein H for 30 minutes at 37 degrees Celsius to evaluate optimal binding concentration. Binding reactions were run through a non‐denaturing gel to separate molecules and verify the formation of RNA‐protein complexes. Results of gel‐shift assays show that the RNA therapy binds the hnRNP H protein forming a complex. This demonstrates RNA therapy recruitment of the splicing silencer to the pre‐mRNA target.
Effects of Testosterone and 17β‐estradiol on Physical Activity Patterns in Mice
Authors: Weeks Kayla A. , Bowen Robert S
Published: April 2020

Physical inactivity causes an increased risk of chronic diseases and can lead to an early death. Notably, sex steroids have been shown to affect physical activity patterns in both humans and rodents. The purpose of this study was to evaluate the effects 17β‐estradiol and testosterone have on wheel‐running behavior in female and male C57BL/6J mice. Initially, testosterone‐deficiency and estrogen‐deficiency were induced by bilateral orchidectomy (n = 4) and ovariectomy (n = 4) surgeries. Control mice (n = 10; 5 males and 5 females) received sham gonadectomies. Wheel running data were collected for seven days following the surgical procedures. After completion of the first experimental period, surgically manipulated mice received implants containing either testosterone (males) or 17β‐estradiol (females) to elevate sex steroid levels back to physiological levels. Wheel running data were collected for a second week. To accumulate a daily turn‐by‐turn record, each wheel revolution was counted and time‐stamped. Turn‐by‐turn data were used to generate seven‐day averages for speed (m·min−1), distance (km), duration (min), onset of wheel running (minutes before (−) or after (+) lights off), completion of wheel running (minutes before (−) or after (+) lights on) and wheel running bouts (number of contiguous blocks of wheel running). Statistical analyses (three‐way ANOVAs) were run for each variable to determine the effects that sex (male vs. female) and sex steroid levels (deficient vs. reintroduced) had on each wheel running parameter between control and experimentally manipulated animals. Follow‐up post‐hoc testing was completed to determine statistically different pair‐wise comparisons. After gonadectomy, distance [F (1,14) = 10.9, p = 0.005], duration [F (1,14) = 5.5, p = 0.035] and bouts [F (1,14) = 18.769, p = 0.001] dropped significantly due to loss of endogenous steroids for both males and females. Speed, the onset of wheel running, and the completion of wheel running was largely unaffected. Females ran less under low estrogen conditions and exhibited reinvigoration following re‐exposure to 17β‐estradiol. Males ran less under low testosterone levels and exhibited reinvigoration following re‐exposure to the steroid. Interestingly, after reintroduction of 17β‐estradiol, female mice approached the daily wheel running task with a more fragmented pattern (4 or more bouts per night), while males exhibited a more fragmented wheel running pattern under low testosterone conditions (5 or more bouts per night) that resolved back to a normal pattern following reintroduction of testosterone. This study suggests that intact mice run almost constantly overnight in longer bouts, while the compromised mice accumulate their wheel running in much smaller bouts sporadically through the night. The continuous pattern approach to wheel running might be associated with the improved resistance to hypokinetic disease progression associated with physically active individuals.
Effects of SCH‐23390 and Alpha‐Methyl‐Tyrosine on the Discriminative Stimulus Effects of Mephedrone in Male Rats
Authors: Friend Ashton Jeremy , Erwin Laura Lynn , Smith William L. , Cozzi Nicholas V. , Winsauer Peter J.
Published: April 2020

The illicit synthetic cathinone mephedrone (4‐methylmethcathinone) often termed a “bath salt” produces discriminative stimulus effects that are similar, but not identical, to those for central nervous system stimulants such as cocaine and methamphetamine. This may be due to its capacity to bind atypically at both dopamine and serotonin transporters compared to those drugs. The purpose of the present study was to further elucidate the mechanism of action underlying mephedrone’s discriminative stimulus effects by administering it with the dopamine type‐1 receptor antagonist SCH‐23390 (n=11) or the catecholamine synthesis inhibitor alpha‐methyl‐tyrosine (alpha‐MT; n=8). Male Long‐Evans rats were trained to discriminate intraperitoneal injections of mephedrone (3.2 mg/kg) from saline under a fixed‐ratio 20 (FR‐20) schedule of food presentation. Mephedrone dose‐effect curves (0.32 – 10 mg/kg) were then established by administering increasing cumulative doses of mephedrone on test days, and comparing those effects with multiple (4–5) injections of saline. The interaction of SCH‐23390 (0.032 – 0.1 mg/kg) and alpha‐MT (200 mg/kg) with mephedrone was examined by administering an acute injection of these drugs prior to increasing cumulative doses of mephedrone. As shown previously by our laboratory, saline administration produced almost exclusively saline‐lever responding while increasing cumulative doses of mephedrone dose‐dependently increased mephedrone‐lever responding. When SCH‐23390 preceded cumulative doses of mephedrone, neither dose alone produced mephedrone‐lever responding and only the larger of the two doses tested (0.1 mg/kg) shifted the mephedrone dose‐effect curve rightward; however, this shift was still less than 2‐fold and produced rate‐decreasing effects when administered alone. Acute administration of alpha‐MT alone also did not produce mephedronelever responding nor decrease rate, and in combination did not produce a marked rightward shift in the mephedrone dose‐effect curve. Taken together, these data indicate that neither SCH‐23390 nor alpha‐MT effectively antagonize the discriminative stimulus effects of mephedrone, and the alpha‐MT data in particular suggest that mephedrone’s discriminative stimulus effects do not depend on newly synthesized catecholamines.
Microvascular Responses Following 5‐Fluoruracil Chemotherapy Administration
Authors: Hammond Stephen T. , Parr Shannon K. , Hilgenfeld Emma G. , Turpin Vanessa-Rose G. , Ade Carl J.
Published: April 2020

BACKGROUND

The pyrimidine antimetabolite 5‐Fluorouracil (5‐FU) is implicated with the second highest rate of cardiotoxicity among cancer chemotherapies, manifesting as vascular vasospasms. Despite this, the pathophysiological mechanisms in which 5‐FU‐induced vasospasms occur is not well understood, but alterations in microvascular endothelial‐dependent and ‐independent pathways have been implicated. This is critical given that decrements in microvascular function are known to occur early in the development of overt cardiovascular disease and like measurements have previously been used to asses pathophysiological mechanisms in numerous patient populations. However, to date, no study has investigated the microvascular function in cancer patients actively receiving 5‐FU chemotherapy. Therefore, we hypothesized that cancer patients treated with 5‐FU would exhibit lower measurements of cutaneous blood flow (CBF) and cutaneous vascular conductance (CVC) following iontophoresis of ACh to that of healthy non‐treated controls.

METHODS

9 5‐FU treated cancer patients (7 M, 2 F; age 62.8 ± 11.1 yr) and 9 healthy controls (6 M, 3 F; age 56.62 ± 9.9 yr) were recruited for this study. Red blood cell flux was measured non‐invasively in the right forearm via Laser Doppler Flowmetry (LDF) and used as an index of CBF. Endothelial‐dependent vasodilation was elicited via iontophoresis of acetylcholine (ACh). Baseline to peak changes CBF and CVC, derived from CBF and mean arterial pressure were calculated.

RESULTS

No significant differences for CBF [5‐FU: 792.78 ± 476.3%, control: 973 ± 682.77%, p = .26] or CVC [5‐FU: 796.76 ± 472.99 %, control: 918.33 ± 638.01.3%, p = .32] were found between groups.

CONCLUSION

To date, our data suggests 5‐FU does not alter Ach‐mediated endothelial‐dependent vasodilation within the cutaneous microcirculation. These findings suggest that endothelial‐independent mechanisms may be linked to 5‐FU cardiotoxicity. Future directions will include a focus on smooth muscle function and vasoconstrictor responses of patients undergoing 5‐FU chemotherapy.