bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of Cancer‐Associated Mutations in SF3B1 on Yeast pre‐mRNA Splicing
Authors: Groubert Brent Joseph , Kaur Harpreet , Paulson Joshua C , Hoskins Aaron
Published: June 2021

In eukaryotes, a mRNA maturation step called splicing removes non‐coding intronic regions from pre‐mRNAs. Splicing is conducted by an enzyme called the spliceosome which uses a variety of protein and snRNA components in order to perform its catalytic function. One of these highly conserved components is the U2 snRNP protein SF3B1. In humans, mutation of SF3B1 can lead to improper regulation of alternative splicing. Many human diseases such as myelodysplastic syndromes (MDS), endometrial cancer (UCEC), bladder carcinoma (BLCA), and acute myeloid leukemia (LAML) have been linked to SF3B1 mutation and dysregulation of alternative splicing.

The goal of this study is to introduce SF3B1 mutations observed in UCEC, BLCA, or LAML cancer patients into the homologous yeast protein (HSH155) to gain further insight into the consequences of these mutations on splicing. I have used site‐directed mutagenesis to incorporate these mutations into the HSH155 gene on plasmids and then shuffled these plasmids into a budding yeast strain in which the HSH155 gene was deleted from the genome. I then used temperature‐dependent growth assays and an in vivo splicing reporter to measure phenotypic responses due to these point mutations. These experiments will identify how cancer‐linked mutations change conserved, fundamental steps carried out by the spliceosome and how perturbation of these steps can led to changes in alternative splicing.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Structure and Function of Estrogen Receptor β: Essential Roles in Gene Regulation
Authors: Dai Eddie , Liu Samuel , Kholi Khushi
Published: June 2021

The Olathe North MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine the structure‐function relationships and regulatory roles of ERβ. Estrogens are steroid hormones synthesized from cholesterol in cells of the ovary and other tissues expressing the enzyme aromatase. These hormones are secreted into the bloodstream and modulate numerous physiological functions through gene regulation. To achieve this, estrogens pass through cell and nuclear membranes, facilitated by their lipophilic and miniscule nature. In target cells, estrogens bind to nuclear transcription factors called estrogen receptors (ERs), the two primary types being estrogen receptor alpha (ERα) and beta (ERβ). ERs typically form either homodimers with an identical molecule or heterodimers of ERα with ERβ. Estrogen binding occurs at ER's ligand binding domain (LBD), activating the ER's DNA binding domain (DBD). The ER dimer then binds to DNA at estrogen response elements (EREs) located in the promoter regions of estrogen‐responsive genes, allowing the ER to regulate the transcription of target genes. However, this process is only one of four mechanisms by which ERs function. In addition to this genomic mechanism, ERs can also function through tethered, nongenomic, and ligand‐independent mechanisms. In all these processes, ERs' role as a gene regulator is determined by the physical change in the structure of various ER domains, making it essential to analyze the ERs' protein structure. In this review, we focus on the structure and function of ERβ because of its largely unexplored physiological significance and clinical potential. ERβ is most highly expressed in the ovaries, mammary glands, prostate, testes, gastrointestinal tract, and immune system. Transcriptional regulation by ERβ is essential for ovarian follicle maturation and ovulation. Additionally, ERβ is involved in neurogenesis, vascular function modulation, cardiac myocyte maintenance, and metabolism regulation, among other physiological functions. In a pathological context, recent studies suggest ERβ is tumor suppressing while ERα increases tumor growth. Specifically, ERβ can inhibit proliferation while inducing differentiation and apoptosis in tumor cells. Thus, activation of ERβ by synthetic agonists, known as selective estrogen receptor modulators (SERM), has clinical significance concerning the treatment of breast, prostate, colon, and ovarian cancers. Here, we present a review of the latest research on ERβ in conjunction with its physical model, emphasizing ERβ's regulatory roles in human health.

Support or Funding Information

This is a SMART Team project supported through the contributions of Dr. M.A. Karim Rumi of the University of Kansas Medical Center, the Milwaukee School of Engineering, and the Olathe Medical Professions 21st Century Academy.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterizing the Role of Metal Ion Interactions in the Elasticity and Strength of Slug Glue: Steps Toward a Novel Medical Adhesive
Authors: Lazo Christopher Gallego , Smith Andrew
Published: June 2021

Stitches are often cumbersome for patients and pose a risk of infection. Thus, there is a great interest in developing medical glues that could serve as an alternative, but current adhesives are limited. Analysis of the tough, sticky defensive secretion of the slug Arion subfuscus has guided the development of a novel medical glue, and further work characterizing the slug glue should lead to deeper insights into how to make a hydrogel glue. The glue of A. subfuscus consists of a double network of carbohydrates and proteins and is held together by sacrificial bonds. These sacrificial bonds are central to the high performance of the glue, but they have yet to be identified definitively. Calcium and magnesium seem to be most likely to form intermolecular cross‐links in the glue due to their high abundance, and the abundance of calcium‐binding proteins in the glue. The role of calcium and magnesium was tested by manipulating their interactions within the glue to observe how this impacts its elasticity. Metal ion interactions were controlled by soaking glue samples in different pH buffers and determining the resulting elasticity. The glue's elasticity was measured in cut glue samples in a tensometer. The association between metal ions and glue when under different pH buffers was measured by using an atomic absorption spectrometer to determine whether the metals stayed bound in the glue or diffused out at different pH values. We found that glue elasticity varied with pH, showing a steady decline as pH dropped below 5.5. Between 5.5 and 7.5 no significant change in elasticity was apparent. This change in stiffness was not due to dissociation of calcium and magnesium, however, as they diffused completely out of the glue within sixty minutes regardless of pH. Thus, there was no correlation between the change in elasticity with pH and the loss of these metal ions, and their hypothesized role as sacrificial bonds was not supported. Further investigation aims to look into the relationship between zinc ions or imine bonds and glue elasticity and strength.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Illuminating Protein Interactions within a Repressive Auxin Response Factor
Authors: Johnston Shelby R , Garner Reid M , Ellis Jamie P
Published: June 2021

Auxin response factors (ARFs) are transcription factors that activate or repress gene expression in most plant species. While almost all ARFs contain three domains, the small structured C‐terminal Phox and Bem1 (PB1) domain shows the most potential for defined regulatory interactions. Each ARF PB1 domain interacts through opposing cationic and anionic surfaces to other PB1 domains, including ARFs and Aux/IAA proteins. This interaction provides a necessary pathway for hormonal regulation of transcription. From Arabidopsis thaliana, the PB1 domains of both activators ARF5 and 7 show the potential to form homo‐ and hetero‐oligomers from studies of model mutant and wild‐type proteins. To date, no similar characterization of ARF repressor PB1 domains has been performed. Our work combines genetic analysis with experimental characterization of the PB1 domain of repressor ARF1 to test for unique biophysical traits. The PB1 domains carry significant sequence diversity for potential behavioral differences. Within A. thaliana, the 20 distinct ARF PB1 domains are only 8.5% identical. ARF repressor PB1 domains differ significantly from their activating counterparts as well as show significant diversity. The repressing ARFs form clades that are distinct from each other including some within the same subclade as the activators. Analysis of wild type ARF1 has shown that ARF1 PB1 forms a mixture of trimers, tetramers, as well as the larger oligomers common to ARF5 and 7. The conserved charged residues appear to be the primary mechanism with which they form oligomers. We used the natural fluorescence of tryptophan to observe structural changes during binding. Two tryptophans are present in the intradomain interface of the beta‐grasp fold common to PB1 domains. Through fluorescence intensity and collisional quenching experiments, we quantified the relative protection of the tryptophans from their environment. Comparing ARF1 with ARF5 and 7 as well as under conditions of homo‐ and hetero‐oligomerization provided another insight to commonalities and distinctions between these regulatory proteins. The distinct traits found between an ARF repressor and homologous ARF activators may illuminate the role of sequence diversity and the evolved retention of multiple variants of ARF proteins.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interaction of PKR and E3 protein present in vaccinia virus
Authors: Patnaik Sikta
Published: June 2021

E3 protein from vaccinia virus is a key host‐range protein, which suppresses the innate antiviral immune response of the infected cells. In response to the viral infection, Protein Kinase R (PKR) of the host cell initiates the immune response via a cascade of reactions. E3 attacks PKR to suppress the immune response but the mechanism of PKR inhibition by E3 is unknown. Interaction studies of PKR and E3 suggested a physical interaction between the kinase domain of PKR and E3 that has biochemical relevance. E3 follows two different pathways when inhibiting inactivated PKR and activated PKR respectively. Further analysis of the interaction revealed that the inhibition of PKR activation by the E3 protein does not correlate with the dsRNA binding ability of PKR or E3.

Support or Funding Information

National Institute of Health (NIH)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tamm‐Horsfall Protein and Properdin Biochemical and Functional Interactions
Authors: Rhodes Diana CJ
Published: June 2021

Tamm‐Horsfall protein (THP), also known as uromodulin, is an abundant protein in normal mammalian urine that interacts with numerous cells and molecules of the immune system, including various complement proteins such as C1q and factor H. Properdin (factor P), which potentiates complement activation by stabilizing the alternative pathway C3 convertase, previously has been shown to bind to THP. In the present study, an enzyme‐linked immunosorbent assay (ELISA) was used to examine the effect of ionic strength, pH, and heparin on the binding between these two oppositely charged proteins. Additionally, since THP has been shown to act as a cofactor, like factor H, in factor I‐mediated C3b degradation, we investigated whether properdin altered this cofactor activity of THP. All ELISA buffers contained 1 mM MgCl2 and 1 mM CaCl2 because earlier studies showed a requirement for divalent cations in this reaction. As measured by ELISA, binding between THP and properdin was similar (K~ 10−8 M) in 20 mM, 60 mM, and 90 mM NaCl solutions, but no significant binding was detected in 154 mM NaCl buffers. There was a reduction in binding capacity, but not overall affinity in 120 mM NaCl buffers. Binding of properdin to immobilized THP in 60 mM NaCl was similar at pH 6.0, 7.5, and 8.5. The THP/properdin interaction was very sensitive to heparin, with 0.008 units of heparin/ml producing a 50% inhibition of binding of 2 μg/ml properdin to immobilized THP. In vitro cofactor assays analyzed on silver‐stained SDS‐PAGE gels demonstrated that while 300 ng of properdin inhibited by more than 50% the ability of 1 ng factor H to serve as a cofactor in factor‐I mediated C3b degradation, properdin appeared to increase by about 25% the ability of THP to serve as a cofactor in factor I‐mediated C3b degradation. Properdin, by itself, was not able to act as a cofactor in factor I‐mediated C3b degradation. In conclusion, THP and properdin bind more readily in lower ionic strength buffers than in higher ionic strength solutions, but these interactions are stable over a range of pH conditions typically found in urine. Heparin is very effective at inhibiting the binding between properdin to THP. On a functional level, properdin may augment THP's ability to act as a cofactor in C3b degradation, while it inhibits factor H's cofactor activity.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Does the MS2 L protein work with E. coli host factors?
Authors: Streff Kimberly J , Piefer Andrew J
Published: June 2021

MS2 is a small single stranded RNA bacteriophage that escapes its infected Escherichia coli host through an L protein‐driven lysis mechanism. The L protein consists of 75 amino acids, with a hydrophilic N‐terminal that contains mainly basic residues and a hydrophobic C‐terminal end. Prior studies have shown that the L protein is necessary for the cell to lyse, however it is not known if L alone is sufficient for cell lysis. In our laboratory we have shown that expression of the L protein in the absence of other viral proteins causes E. coli lysis within 40 minutes of expression. Further, the actual mechanism for lysis is not completely understood and potentially requires a protein‐protein interaction with E. Coli host factors. Recent studies by Chamakura et. al [Journal of Bacteriology, 12, 199 (2017)] using a genetic screen identified DnaJ, a chaperone protein, as a host factor involved in the lysis mechanism. However, this study did not describe a direct protein‐protein interaction between L and DnaJ. Thus, the question remains whether DnaJ along with MS2 L is necessary and sufficient for lysis, or are there additional host proteins involved? In order to answer this question and investigate host protein interactions using a biochemical approach, a hemagglutinin epitope tag (HA) will be inserted into a plasmid containing the MS2 L gene and an arabinose‐inducible promoter. Once transformed into E. coli, the HA tagged L protein can be detected using a Western Blot and purified by immunoprecipitation. Using these methods, it is expected that any host protein that interacts with the L protein will be co‐immunoprecipitated. Future studies will use mass spectrometry to identify these co‐immunoprecipitated proteins.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Mechanisms of Isocitrate Dehydrogenase 1 Mutants in Driving the Oncogenic Neomorphic Reaction
Authors: Quichocho Giovanni Garrido , Xu Ruth , Luna Lucas , Matteo Diego Avellaneda , Scott Michelle , Fonbon Jeon Erik , Sohl Christal D.
Published: June 2021

Brain tumors result in a mortality rate > 60% within 5 years of diagnosis. Mutations in cytosolic isocitrate dehydrogenase 1 (IDH1) drive over 70% of low grade gliomas and secondary glioblastomas. IDH1 catalyzes the NADP+‐dependent formation of α‐ketoglutarate (α‐KG) from isocitrate via oxidative decarboxylation. Mutation of IDH1 results in a neomorphic activity, the formation of the oncometabolite, D‐2‐hydroxyglutarate (D2HG) from α‐KG in the presence of NADPH, via reduction. The most common mutations occur at residue 132, where arginine (R132) is typically replaced by histidine (R132H). We have shown previously that replacing arginine with other amino acids resulted in a trend in catalytic efficiency, with larger, more polar residues at the 132‐position promoting the normal reaction, and smaller, more hydrophobic residues driving the neomorphic reaction. Based on previous data with R132H and R132L, we hypothesize that R132Y and R132I, having similar structures respectively, will promote similar rates of catalytic efficiency in the neomorphic reaction, and R132P will have little to no catalytic activity due to likely disruption of local secondary structure. To test this hypothesis, we generated cDNA constructs and heterologously expressed and purified R132I, R132P, and R132Y‐IDH1 from E. coli to determine how these residues affect to the normal or neomorphic reaction. Kinetic assays were performed to determine the rates of the neomorphic reaction of the IDH1 mutant in the presence of NADPH and αKG. We found that R132Y‐IDH1 was the most efficient in catalyzing the neomorphic reaction followed by R132I, and R132P‐IDH1, having the lowest catalytic activity for the neomorphic reaction. By replacing the R132 residue with a wide range of amino acids, and studying catalytic efficiency of each mutation, we will be able to discover the molecular mechanisms that facilitate this neomorphic activity and help inform drug design for selective mutant IDH1 inhibitors that are effective against a wide range of tumor‐relevant mutants.

Support or Funding Information

This work was funded by the National Institutes of Health R00 CA187594 (C.D.S.), U54CA132384 (SDSU) & U54CA132379 (UC San Diego), MARC 5T34GM008303 (SDSU), and IMSD 5R25GM058906 (SDSU), as well as the California Metabolic Research Foundation (SDSU) and SDSU startup funds (C.D.S). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Function of active site residues in the dUTPase from Epstein‐Barr Virus
Authors: Robbins Alexander
Published: June 2021

Epstein‐Barr Virus, which causes infectious mononucleosis, contributes to many diseases such as Burkitt lymphoma and other cancers. The viral genome encodes a dUTPase, an enzyme that catalyzes the reaction of dUTP + H2O to dUMP + PPi + H+. This is an intermediate reaction involved in the formation of the thymidine nucleotide. The dUTPase found in humans is a trimeric enzyme containing three active sites while the viral enzyme is monomeric. Due to variation in the structures of the human and viral proteins, there may be differences in each enzyme's catalytic activity. Our results demonstrate the importance of a metal cofactor for activity of the viral enzyme. The role of active site residues in substrate binding and the catalysis of dUTP to dUMP is being examined by site‐directed mutagenesis. Characterization of the EBV dUTPase may facilitate the discovery of inhibitors. Inhibition of dUTPase decreases the formation of thymidine nucleotides creating a high dUTP/dTTP ratio. Uracil becomes incorporated into DNA in place of thymine which causes mutations that may be detrimental to cell survival. Selectively inhibiting the dUTPase from Epstein‐Barr Virus provides a mechanism to target cells infected with the virus without damaging uninfected cells.

Support or Funding Information

UWL Undergraduate Research and Creativity Grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kinetic survey of evolutionary diverse isoforms of malate dehydrogenase
Authors: Sampson Grant , Chang Nathan , Greene Erika , Provost Joseph
Published: June 2021

Malate dehydrogenase (MDH) catalyzes the redox formation of malate to oxaloacetate (OAA). Several isoforms exist. Mitochondrial MDH plays a well‐known role in the last step of the Krebs cycle while cytosolic MDH is involved in the NADH shuttle and other metabolic pathways. Plants, fungi, and protists express MDH in the glyoxisomes, where a variant of the Krebs cycle takes place. To better understand the unique enzymatic differences between evolutionarily distinct MDH isoforms, we have synthesized C‐terminal His tagged with a protease cleavage site and codon optimized 13 different isoforms of MDH for expression in E. coli. These include MDH from Chlorobaculum parvum, a green sulfur photosynthetic bacterium which uses sulfide, sulfur or thiosulfate as electronic donors; Streptomyces avermitilis, a soil bacterium with unique behaviors including insecticidal properties; saline tolerant Haloarcula marismortui, a Halophilic red Archaeon found in the Dead sea; Aquaspirillium arcticum bacterium which is cold tolerant living under the snow and ice of the pacific northwest; as well as all forms of yeast and human MDH. The purpose of this study is to evaluate structural and functional differences between each isoform of MDH, purified using affinity chromatography. Enzyme assays were performed to determine Km/Vmax of each isoform under varying OAA and NADH concentrations and to assess the inhibitory effects of citrate. Thermal stability assays were performed to assess resilience to heat, cold, and determine optimal storage conditions. A final cladogram was constructed to represent the evolutionary diversity between MDH isoforms. This work will help uncover the shifts in primary and structural evolutionary changes with functional kinetics of each MDH isoform and support use of these enzymes in the classroom.

Support or Funding Information

Funded in part by NSF IUSE DUE 1726932.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimization of Cell Free Protein Synthesis: Development of a Restriction Endonuclease Fitness Strategy
Authors: Tovar Sofia Aguilera , Hanlan Nicklaus , Ong Shi Qing , Wagner Jacob , Eckdahl Anthony , Edman Elise , Roland Kyla , Travieso Tatianna , Willis Caroline , Campbell A. Malcolm , Heyer Laurie J , Poet Jeffrey L , Eckdahl Todd T
Published: June 2021

Our undergraduate researchers are interested in advancing the field of metabolic engineering for the production of useful metabolites that serve as biofuels, pharmaceuticals, or industrial chemical feedstocks. We published an approach to the metabolic engineering of bacteria called programmed evolution, which introduces variation in regulatory elements for the production of enzymes that control orthogonal metabolic pathways. Programmed evolution uses in vivo fitness modules that enable selection by employing riboswitches to transduce the production of desired metabolites into antibiotic resistance. Because of limitations imposed by in vivo programmed evolution such as crosstalk between orthogonal and native bacterial metabolism, the toxicity of some metabolites, and poor representation of large variation spaces, we want to reinvent programmed evolution as an in vitro strategy. This preliminary report describes our first steps of comparing lysates from two different strains of E. coli and using them to carry out cell‐free protein synthesis (CFPS). We also designed, constructed, and have begun testing a fitness module based on the expression of the restriction enzyme SmaI. We are working toward a proof‐of‐concept system in which combinations of DNA regulatory elements that enhance the production of caffeine demethylase are selected for their ability to produce theophylline. Theophylline production will turn on a riboswitch that leads to a burst of SmaI expression. Localized SmaI activity is expected to preferentially cleave successful control elements over unsuccessful ones, allowing purification and identification of previously unknown requirements for CFPS‐controlled metabolism. The successful development of in vitro programmed evolution would enable researchers to exploit the power of selection to discover combinations of control elements for the optimal yield of metabolites that are not easily produced in vivo.

Support or Funding Information

NSF RUI grant MCB‐1613203 to Davidson College and MCB‐1613281 to Missouri Western State University, Davidson College Martin Genomics Program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Multiplex Imaging of Activity‐Dependent Changes in Neuronal Redox Dynamics Using Compartment‐Specific Redox Probes
Authors: Radhakrishnan Saranya , Norley Jake , Leroy Nathan , Tantama Mathew
Published: June 2021

Reactive oxygen species (ROS) are integral components of redox signaling in cells and mediate physiologically essential signaling across compartments. Under pathological conditions, excessive presence of ROS can lead to oxidative stress that damages cellular components and leads to cellular dysfunction and death. To understand dynamic redox changes and identify the mechanisms that disrupt redox homeostasis, we need high‐precision tools that can be used to define redox processes in live cells under physiological and pathophysiological conditions.

Genetically encoded fluorescent sensors that measure cellular redox status can be targeted to specific subcellular compartments to study the redox changes in intracellular compartments during live‐cell imaging. Reduction‐oxidation‐sensitive green fluorescent proteins (roGFPs) are redox sensors designed by modifying a green fluorescent protein to encode two cysteines engineered proximal to the chromophore, and a change in green fluorescence is observed upon reduction of the disulfide bond formed by the cysteines. However, it has remained technically challenging to quantitatively correlate spatially distinct redox dynamics with subcellular resolution.

To address this challenge, we developed genetically‐encoded sensors that can be used along with roGFP to visualize redox changes in multiple compartments within the same cell. Our approach utilized Förster‐type resonance energy transfer (FRET) in a spectral relay strategy that extends the fluorescence emission of the redox‐sensitive green fluorescent protein (roGFP) into red emission wavelengths. We targeted the sensors to mitochondria and cytosol in primary hippocampal neurons and monitored sensor response to oxidizing and reducing agents. Upon neuronal stimulation of the neurons with glutamate, we observed an increase in mitochondrial oxidation accompanied by a cytosolic reduction. Pre‐treatment with antioxidants like MitoTEMPO and N‐Acetyl Cysteine attenuated both the neuron‐activity dependent cytosolic reduction and mitochondrial oxidation implying that these redox changes were coupled. In a mixed culture system, astrocytes are known to have a neuroprotective effect from oxidative insults and glutamate excitotoxicity. We targeted the redox sensors in a neuron‐astrocyte co‐culture system to observe the neuroprotective effect of astrocytes on neuronal redox dynamics. Using this sensor in combination with the roGFP, we can conduct live‐cell imaging with spatio‐temporal accuracy to delineate sub‐cellular changes in redox dynamics that result in pathological oxidative stress.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increasing Efficiency of Inducing Pluripotency with a Novel Cell Penetrating Peptide‐Adaptor System
Authors: Oja Laura‐Maria , Clearman Kelsey R. , Hudson Martin L. , McMurry Jonathan L.
Published: June 2021

Somatic stem cells have been used to treat a number of disorders for decades but are limited in their ability to differentiate and self‐renew. Induced pluripotent stem cells (iPSCs) not only have the ability to generate cells of all three germ layers, but bypass the ethical concerns related to embryonic stem cells. They also hold the prospect of utilizing a patient's own cells, thus avoiding host‐vs‐graft issues. In 2006, Takahashi and Yamanaka discovered that expression of only four genes were required to induce pluripotency: Oct4, Sox2, Klf4 and c‐Myc (OSKM). Since then, pluripotency has been induced most effectively by transfection or transduction of these genes. However, efficiencies are low, expression is difficult to control, and off‐target effects are a concern. Inducing pluripotency by exogenous delivery of OSKM proteins using cell‐penetrating peptides (CPPs), while promising, is also inefficient due to of poor penetration and entrapment in endosomes. Our novel CPP‐adaptor system overcomes entrapment by relying on the calcium flux during endosomal trafficking. The adaptor, TAT‐CaM, is a recombinant protein consisting of commonly used CPP, TAT, fused to calmodulin. The protein of interest (the ‘cargo’) has an engineered calmodulin binding site (CBS). TAT‐CaM binds purified CBS‐cargo outside the cell where the Ca2+ concentration is relatively high, but after CPP‐mediated endocytosis, cargo dissociates from TAT‐CaM inside endosomes as calcium concentrations drop, leaving the CPP‐adaptor trapped but cargo free to escape to the cytoplasm and beyond. We have used our adaptor system to deliver submicromolar doses of CBS‐Oct4 protein, inducing pluripotency as measured by expression of stem state markers and alkaline phosphatase staining. CBS‐Sox2 and CBS‐Klf4 have also been delivered and while insufficient to induce pluripotency, play an efficiency enhancing role. While not directly comparable to published results, CPP‐mediated efficiency as measured expression of stem‐state markers is higher than current transfection‐ and transduction‐mediated methods. Parameters such as concentration and timing of doses and molar ratios of OSK to each other and TAT‐CaM affect efficiency. iPSCs created via our CPP‐adaptor system can be cultured, expanded over multiple passages and differentiated into other cell types such as cardiomyocytes. Success in this work will lead to a virus‐ and nucleic acid‐free, nontoxic, tunable method for inducing pluripotency.

Support or Funding Information

This work was supported by NIH grant R15GM120691 and R15NS100632

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Size of protein is a major factor that affects retention on preparative IMAC columns
Authors: ODUNUGA ODUTAYO O , TOVAR CARMEL N
Published: June 2021

Immobilized metal affinity chromatography (IMAC) is a specific high‐capacity technique used in large‐scale purification of proteins. IMAC exploits the ability of immobilized metal ions to form coordination bonds with atoms in the side chains of certain amino acids. The technique is generally robust. However, several factors still affect column binding capacity, retention, yield and purity of proteins during IMAC. It was observed that the recovery of 6 × histidine, (His)6‐tagged proteins from metal affinity columns differ significantly depending on the size of the protein. To test this observation, we determined the effect of protein size, flow‐rate, number and position of (His)6 tag on the retention of highly expressing proteins on commercial Ni2+ and Co2+ IMAC columns. All experiments were performed in phosphate buffer to eliminate interference of amine‐containing buffers with the binding of the (His)6 tag to the columns. Column retention was determined as the ratio of protein of interest in the supernatant (input) to flow‐through (output). Data obtained suggest that regardless of the flow‐rate, (His)6 tag position and number, the size of protein is a major factor affecting column retention and therefore recovery during column IMAC purification. Small and medium‐sized proteins (~50 kDa) have higher column retention than bigger proteins, resulting in higher recovery. These outcomes provide important information to consider when performing IMAC.

Support or Funding Information

Welch Foundation Grant # AN‐0008 SFASU CoSM SURE 2018

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Single Neuron and Microglia Gene Expression Networks Demonstrate Cellular Subphenotype Shifts and Altered Glial‐Neuronal Signaling in Solitary Nucleus during Alcohol Withdrawal: A Time Series
Authors: O'Sullivan Sean J , Park James , Mcintosh‐Clark Damani , Schwaber James S
Published: June 2021

Alcohol use disorder remains a major societal and healthcare challenge motivating investigation into novel mechanisms of alcohol addiction. Negative‐reinforcement models of addiction postulate that avoidance of negative physical and emotional symptoms during withdrawal drive drug‐seeking. Alcohol withdrawal syndrome is characterized by autonomic dysregulation and negative emotion implicating the strongly connected amygdala and solitary nucleus (NTS) involved in autonomic inputs, outputs, and emotional states in the pathophysiology of alcohol withdrawal. Our previous work demonstrates a neuroinflammatory state in the amygdala during alcohol withdrawal, which we speculate contributes to the negative physical and emotional withdrawal symptoms and consequently drug‐seeking. Here, we investigated the effect of alcohol withdrawal on gene expression in single glucagon‐like peptide 1 (GLP‐1) neurons, adrenergic neurons, and microglia in the NTS. GLP‐1 neurons in this region receive autonomic inputs via vagal afferents and signal to amygdala neurons co‐expressing GLP‐1 receptors and corticotropin‐releasing factor. NTS adrenergic neurons respond to higher‐order inputs to maintain cardiovascular homeostasis. Control, ethanol‐dependent, and rats undergoing ethanol withdrawal at 8, 32, and 176 hour time points were sampled. Single cells were harvested using using laser‐capture microdissection, and microfluidic RT‐qPCR was employed to generate a high‐dimensional dataset. Cellular subphenotypes were characterized and local glial‐neuronal signaling networks were described based on gene networks that defined these subphenotypes. We found an increase in inflammatory subphentoypes in the 8 and 32 hour withdrawal condition with the 176 hour withdrawal condition demonstrating decreased GABA receptor expression. Preproglucagon (Gcg), the GLP‐1 precursor transcript, was found to be upregulated only in alcohol withdrawal conditions. The levels of this transcript opposed levels of the norepinephrine precursor tyrosine hydroxylase transcript suggesting these two signaling ligands have oppositional regulation in alcohol withdrawal.

Support or Funding Information

U01HL133360‐01

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Simulating protein and nucleic acid sequence co‐evolution
Authors: Camenares Devin
Published: June 2021

Protein structures and interactions can be predicted by identifying co‐evolution of residues, both within the same protein or across different proteins and nucleic acids. While conserved residues might represent invariant features of a protein's structure, co‐evolving residue pairs can suggest a sequence‐specific and consequential interaction. Different computational approaches have been used to detect co‐evolution in sequence alignments, but biological data is often used as a benchmark. Such evaluation is important due in part to the confounding presence of indirect co‐evolution. However, using biological data as a benchmark is problematic, because not all the interactions and evolutionary constraints can be known a priori. Instead, the ideal benchmark would be a set of sequences generated with constraints such that they simulate co‐evolution. This was achieved using new cross‐platform, user friendly software tools, which were then used to benchmark and evaluate existing detection methods. This new software can generate sequences in which intramolecular, intermolecular, and indirect co‐evolution can be simulated. Systematic tuning of these evolutionary constraints shed new light on how they drive co‐evolution between residues. Better understanding how to detect co‐evolution and the residue interactions they predict can lead to a wide range of insights important for synthetic biologists interested in engineering new, orthogonal interactions between two proteins or nucleic acids.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Automating the design of structure‐switching aptamer biosensors
Authors: Haider Austin , Bulow Aviva , Bonham Andrew J
Published: June 2021

For both personalized medicine and academic research, there is pressing need for tools to enable the rapid interrogation of an array of biological targets associated with diseased states. Electrochemical, DNA‐based (E‐DNA) biosensors utilize the dynamic nature of nucleotide polymers to quantify the concentration of a specific peptide, substrate, or heavy‐metal species contained in a biological sample. A critical limitation of E‐DNA biosensors is the complexity associated with designing a nucleotide sequence that maximizes sensitivity while maintaining specificity. This dilemma is further complicated by the vast number of conformations that can exist for a given sequence. While great strides have been made in computationally predicting the folded structures that DNA and RNA‐based biosensors may assume, interpreting whether such predicted structures would be effective biosensors and using that knowledge to design functional E‐DNA biosensors ab initio remains challenging, suggesting the utility of an in silico approach to E‐DNA sensor design. We have created a program called Fealden, which automates the process of aptamer‐based biosensor generation. Fealden utilizes several programmatic paradigms to build an ideal E‐DNA biosensor sequence; the validity of this sequence is then assessed by assigning thermodynamic values and conformational distance metrics to each possible fold. Using these metrics, Fealden can provide the user with several candidates for an E‐DNA aptamer biosensor that are sensitive to the target of interest. Fealden has already been applied to optimize the detection of the antibiotic tobramycin with minimal human interaction. This work provides insights into applying an algorithmic approach to utilize the wide pool of existing aptamers as E‐DNA biosensors in practical applications spanning such diverse fields as food safety, environmental monitoring, and clinical diagnostics.

Support or Funding Information

This work is supported by internal grant funding from the Metropolitan State University of Denver.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Curcumin Induces Apoptosis by regulating the cell cycle proteins in MDA‐MB‐231 Breast CancerCells
Authors: Ali Mohammed Showkat , Smiley Rebecca
Published: June 2021

Background

Curcumin, a natural polyphenolic compound found in turmeric, has potential cancertreatment efficacy, including apoptosis‐induction, in many cancer cell types. Cancer cells arecharacteristically anti‐apoptotic, highly proliferative andcontinue to live indefinitely. Controlof cell cycle progression and the initiation of apoptosis in breast cancercells is considered to be a potentially effective strategy for the prevention/treatment of the disease. The triple‐negative breast cancer cell line MDA‐MB‐231 does not express progesterone, estrogen, or human epidermal growth factor receptors, is highly aggressive and is difficult to treat. This study aims to elucidate the mechanism of curcumin‐induced apoptosis and its effects on cell cycle proteins in the MDA‐MB‐213 breast cancer cell line.

Methods

Apoptosis was measured by flow cytometric analysis of AnnexinV/Propidium Iodide expression. Cell cycle arrest was measured by Western Blot analyses of p21Waf‐1, p53 and retinoblastoma protein pRb, as well as the cyclin dependent kinases Cyclin B1, and Cyclin D1.

Results

Flow cytometry data suggest that curcumin induces both early and late stage apoptosis in MDA‐MB‐231 cells. Cell cycle inhibitory protein p21Waf‐1 was significantly increased in curcumin‐treated cells compared to untreated cells. Tumor suppressor protein p53 and Cyclin D1 were significantly down‐regulated (p=.0.0001 and p=0.014, respectively), pRb, another tumors uppressor protein, was unchanged, and Cyclin B1 was upregulated (p=0.0006).

Conclusion

Given that upregulation of p21Waf‐1 can lead to apoptosis and can disrupt CDKs/cyclin complexes necessary for cell cycle progression, and that down‐regulation of Cyclin D1, which is required for progression through the G1/S phase and whose over expression is associated with most breast cancers, can lead to cell‐cycle arrest at G1, curcumin appears to be effective in both apoptosis induction, demonstrated also in the flow cytometric results, and in cell‐cycle arrest at G1. The upregulation of cyclinB1, which is involved in the G2/M phase transition, and the down‐regulation ofp53 by curcumin, indicate that additional mechanisms are involved with curcumin exposure. Our findings will aid in the development of future therapeutic strategies, including curcumin as a novel treatment supplement, to effectively induce both cell‐cycle arrest and apoptosis in breast tumors.

Disclaimer: The views expressed in this manuscript are those of the author and do not reflect the official policy of the Department of Army, Department of Defense, or U.S. Government.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cell Growth Inhibition and Transglutaminase 2 Expression in Human Erythroleukemia Cells (K562) Following Exposure to Retinoic Acid and Sodium Butyrate
Authors: Spencer Gabi Rose , Birckbichler Paul J
Published: June 2021

Transglutaminase (TG‐2) is a calcium‐dependent enzyme that has been linked to numerous biological functions including cell adhesion, extracellular matrix, stabilization, wound healing, receptor signaling, cellular proliferation, and cellular motility. This enzyme is also thought to play a role in cancer, as TG‐2 expression is elevated in several types of cancer cells. Some studies have also shown that patients with neurodegenerative diseases like Parkinson's, and Alzheimer's have exhibited elevated levels of TG‐2 which may be linked to the formation of protein aggregates that cause these diseases. The goal of this study was to explore the biological responses of human erythroleukemia cells (K562) to retinoic acid and sodium butyrate. The cells were treated with DMSO (20 uL), sodium butyrate (100 uM), retinoic acid (20 uM), or a combination of sodium butyrate (100 uM) and retinoic acid (20 uM). Preliminary data has shown that TG‐2 is expressed more in cells treated with sodium butyrate, while retinoic acid does not have an effect on TG‐2 expression. The control cells grew continually over the 72‐hour observation period, while the growth of cells treated with sodium butyrate and the mix of both chemicals were slowed to approximately 50% of the growth of control cells. Immunofluorescent photo‐microscopy was used to measure the expression of TG‐2. The cells were also tested for apoptosis using fluorescent microscopy to differentiate between viable, necrotic, and apoptotic cells. Future research will attempt to localize the actual location of the TG‐2 when treated with both chemicals, and if it promotes or suppresses the viability of the cancer cells. Studies are also underway to determine the amount of TG‐2 that is present in each of the samples.

Support or Funding Information

Funding provided by Department of Chemistry, Slippery Rock University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Benzimidazole scaffolds as potential anticancer agents: Synthesis and Biological evaluation
Authors: VEMANA HARI PRIYA , Barasa Leonard , Surubhotla Nirupama , Kong Jing , Ha Sin Sil , Palaguachi Christian , Croft John L. , Yoganathan Sabesan , Dukhande Vikas V.
Published: June 2021

Cancer continues to be one of the leading causes of mortality worldwide. Current anti‐cancer agents have a major limitation of drug resistance. Continued search for newer and safer anticancer drugs is critical and this led us to synthesize a small library of structurally unique benzimidazoles with anticancer potential. Various analogs of these compounds were derived from indole‐2‐carboxylic acid, fatty acids and alpha amino acids, providing a panel of diverse structures. Synthesized compounds were tested in three different cancer cell lines‐HepG2 (human hepatocellular carcinoma), HeLa (human cervical cancer), A549 (human lung carcinoma) and one transformed cell line HEK293 (human embryonic kidney). Many of the compounds tested induced cytotoxicity in a concentration‐dependent manner. Cell viability studies using MTT assay showed that indole and fatty acid based benzimidazoles were most potent followed by amino acid derivatives. Presence of protective functional groups decreased activity of the compounds. In order to investigate the mechanism of the cytotoxicity of the compounds, cleaved caspase 3 expression was measured and a fluorescence‐based apoptosis‐necrosis assay was performed. These results confirmed the presence of apoptosis process in cell death induced by the benzimidazole compounds. Furthermore, cell cycle alterations accompanied the cell death. In addition to anticancer activity, several of these compounds also showed promising antibacterial activity against S. aureus and S. epidermidis. Taken together, our findings demonstrate that the novel benzimidazole compounds could serve as potential leads for the further development of new anticancer agents.

Support or Funding Information

Funding for this research was provided by College of Pharmacy and Health Sciences, St John's University, Queens, NY

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