bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutating the Telomere Gene
Authors: Palmero Brett , Sandler Anna , Dejneka Alexandra , Kirk Karen E
Published: June 2021

Telomeres are protein‐nucleotide sequences at the ends of chromosomes that serve to prevent the loss of genetic information during cell replication. A telomerase ribonucleoprotein complex includes telomerase reverse transcriptase (TERT) and an RNA template (TER) and is responsible for adding telomeric DNA. Our lab has been studying the biogenesis of TER in Aspergillus nidulans, a molecule that is in part structurally similar to that of Saccharomyces cerevisiae and partly to that of mammalian cells. Research in S. cerevisiae has shown that the telomerase RNA appears to leave the nucleus and is potentially assembled in the cytoplasm. Whether TER migrates to the cytoplasm is not known in A. nidulans, but its ability to form a multi‐nucleate state known as a heterokaryon can be used to determine the localization of TER. We assay for the presence of two genetically distinct nuclei via the heterokaryon test, a molecular tool that has allowed us to deduce whether the RNA products for TER and TERT leave the nucleus. We knocked out the genes for TER and TERT, analyzed the results of the heterokaryon test, and determined if assembly of the active enzyme occurs in the nucleus or the cytoplasm. Our results suggest assembly in the nucleus, a scenario that is similar to humans but not to yeast. These results further suggest the potential of A. nidulans as a useful model organism to study the localization of telomerase components.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining the association of the TrmB‐like protein OxsR to chromatin binding and oxidative stress in Haloferax volcanii
Authors: Mondragon Paula Giselle , Hwang Sungmin , Schmid Amy , Maupin‐Furlow Julie
Published: June 2021

Archaea are single‐celled microorganisms that are well known for their ability to survive and thrive in harsh environmental conditions; however, the factors that mediate homeostasis during redox and/or oxidative stress are not fully understood this domain of life. OxsR (redox stress responsive regulator, HVO_2970) is a TrmB‐like protein of arCOG002242 that is increased several‐fold in abundance during hypochlorite stress based on quantitative proteomic analysis of the halophilic archaeon Haloferax volcanii. TrmB (transcription regulator of mal operon) family proteins are typically transcription factors that bind sugar/effector molecules and regulate the expression of carbon metabolism and transport genes. OxsR has a winged‐helix DNA binding domain but lacks the sugar/effector binding domain common to members of the TrmB family. Here we find that OxsR has a conserved cysteine residue (C24) at a predicted homodimer interface that may to be intimately associated with redox homeostasis in this archaeon. OxsR is required: i) for tolerance to oxidative stress and ii) for an increase in the level of transcripts associated with DNA repair and oxidative stress response. Current work is focused on determining how OxsR senses redox stress, binds DNA, and regulates transcript levels by use of RNAseq, ChIP‐Seq and ChIP‐PCR methods. To this end, we have prepared an RNA library for RNAseq analysis and integrated a C‐terminal hemagglutinin (HA) tag onto the oxsR gene locus for the ChIP methods. Overall, OxsR is an apparent redox sensor required for elevated transcript levels of DNA repair and oxidative stress response pathways. The distribution of OxsR homologs with conserved cysteine residue(s) in Euryarchaeota and Crenarchaeota phyla suggests this type of redox sensor is widespread in Archaea.

Support or Funding Information

Funds for this project were awarded to JM‐F through the Bilateral NSF/BIO‐BBSRC program (NSF 1642283), the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences, Physical Biosciences Program (DOE DE‐FG02‐05ER15650) and the National Institutes of Health (NIH R01 GM57498).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cysteine‐Mediated Dimerization of the Transmembrane Domain of Mucin 16
Authors: Li Edwin , Butch Nicole , Micua Taylor
Published: June 2021

Mucins are proteins in the epithelial tissue that produce mucus barriers to provide moisture and also prevent harmful, foreign material from entering the cell. These proteins are heavily glycosylated, and can be secreted to the extracellular space or be embedded in the plasma membrane. The overexpression of transmembrane mucins have been correlated with the development of cancerous growths. For example, the transmembrane mucins mucin 1 (MUC1) and mucin 16 (MUC16) have been associated with breast and ovarian cancer, respectively.

The role of MUC1 in tumor progression has been extensively studied. Dimerization of MUC1 leads to its trafficking to the nucleus where it can alter the expression of genes that regulate cell growth. The cysteine‐glutamine‐cysteine (CQC) motif in the cytosolic juxtamembrane domain of MUC1 is the main driving force of this dimerization, primarily through the formation of disulfide bonds. Contrary to MUC1, there is not much information regarding the possible intracellular role of MUC16 in tumor formation and progression. Similar to MUC1, MUC16 also has two cysteine residues at or near the juxtamembrane domain. Furthermore, recent studies have observed the presence of MUC16 in the nucleus. Thus, we hypothesized that there may be a similarity in the mechanism of trafficking of MUC1 with that of MUC16.

We are using the ToxR activity assay to assess the dimerization of the transmembrane domains of MUC1 and MUC16 with and without the juxtamembrane cysteines. Our preliminary results show that, in the presence of the cysteine residues, the transmembrane domain of MUC16 dimerizes as strongly as that of MUC1. Our results also show that the strength of dimerization, in both MUC1 and MUC16 are associated with the formation of disulfide bonds. These results suggest a possible mechanism of MUC16 nuclear trafficking, similar to that of MUC1, involving cysteine‐mediated dimerization.

Support or Funding Information

Nicholas & Susan Nicholaides Research Fellowship, and the SJU Summer Scholars 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.
Superposition of Macromolecular Electron‐Density Maps in X‐ray Solution Scattering (SAXS)
Authors: Nguyen Nhan D , Grant Thomas D
Published: June 2021

Understanding the structure of biological macromolecules is important for determining their functions and cellular activities. With the growth of computational techniques in crystallography, X‐ray Solution Scattering Data (SAXS) becomes more applicable to reconstruct a wide range of 3D biomolecular structures from a low‐resolution profile by its easier use and simpler sample preparation. A novel program called (DENsity from Solution Scattering) resolves these disadvantages of low‐resolution data and retrieves 3D structural information of the complexes in solution. Due to containing the lack of information of 1D scattering data, these SAXS profiles require more modeling procedures and novel iterative algorithm to retrieve desired macromolecular structures. Our lab's previous publication on Nature elucidates the modeling algorithm using the Fast Fourier Transform to calculate and reconstruct 3D electron density maps of each profile. To obtain better 3D maps of macromolecules, we face another challenge of averaging method. The averaging step involves principal axis alignment, enantiomer selection, overlap score optimization among multiple (20) electron density maps. The updated version has been worked out by minimizing interpolation overuse and independently running with new Python codes that no longer require heavy EMAN2 installation as previous for the reconstruction purpose.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evolution of Specificity and Stability in the Folding Trajectory of Caspase
Authors: Shrestha Suman , Clark Clay
Published: June 2021

Proteins must fold properly and efficiently to accomplish cellular functions. Over the course of evolution, the energy landscape must be maintained such that the protein folds in the right conformation, however, how protein's energy landscape is maintained or altered is unclear. To study how a protein's energy landscape changed over time, we characterized the folding trajectories of caspase homologs and their common ancestor. Caspases are an ancient class of cysteinyl proteases that control apoptosis, responsible for cell differentiation, and maintain cellular homeostasis in multicellular organisms. The caspase family is an excellent model to study protein evolution because substrate specificity and conformational selection are retained through hundreds of millions of years of evolution. Some regulatory features are ancient, and therefore common, while other features are modern and cluster‐specific. In this study, we performed enzyme kinetic assay using synthetic tetrapeptides and substrate phage display using peptide library on ancestral reconstructed and modern proteins to study the evolution of substrate specificity. In addition, we examined urea‐induced equilibrium unfolding properties to evaluate thermodynamic stability and folding pathways. We found that common ancestor of effector caspases showed promiscuity over substrates (D/L/I/VXXD) which is specified later on in modern proteins in different lineages; caspase‐3 and ‐7 prefers DXXD whereas, caspase‐6 prefers VXXD. On the other hand, equilibrium unfolding result suggests all the human effector caspases unfolds via four‐state process; the native dimer (N2) undergoes an isomerization to a dimeric intermediate (I2) and to a monomeric intermediate (I) before completely unfolded (U). Overall, the data show that the folding landscape was established in the early ancestor (>650 MYA) and enzyme specificity evolved from the common landscape. A discrepancy in the overall ΔG0conf, in the folding process between extant caspases, may be due to subsequent mutations in the subfamily.

Support or Funding Information

Department of Biology, University of Texas at Arlington

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 Evolution and Mechanism of Enzyme Specificity and Stability of Caspase‐3
Authors: Yao Liqi , Clark Clay
Published: June 2021

The caspase family of proteases is an excellent model when analyzing protein evolution. Caspases are in a large family, and proteins evolved from a common ancestor. Caspases in the apoptotic initiator subfamily form a stable monomer, and their activities result from dimerization. Apoptotic effector caspases, however, can only form stable dimers. Urea folding/unfolding studies showed that human caspase‐3, an effector caspase, folds by a four‐state equilibrium process in which the native dimer (N2), unfolds through a non‐active dimeric intermediate (I2), a non‐native monomeric state (I) and an unfolded monomeric state (U). We compared the folding process of caspases from other species in order to examine whether the folding mechanism is conserved in other species. We show that zebrafish caspase‐3b also folds by the four‐state model. In addition, the human caspase‐3 dimer is stabilized by several salt bridges across the dimer interface, and dimerization is sensitive to pH due to the presence of two histidine residues in the salt bridges. In contrast, zebrafish caspase‐3a and ‐3b do not use histidines in the salt bridges, so the zebrafish dimers appear less sensitive to pH.

Following evolution of the dimer, mutations in effector caspases resulted in changes in substrate specificity. The caspase‐6 subfamily shows preference to valine at the P4 position, while caspase‐3 shows preference to aspartate at the P4 position. However, recent research shows that zebrafish caspase‐3a has relaxed specificity and allows either valine and aspartate at the p4 position. In contrast, the closely related zebrafish caspase‐3b is similar to human caspase‐3 with ~100‐fold preference for D over V at P4. Our data shows that there are two ways to change enzyme specificity. The first one is through improving H‐bonds to the P4 aspartate by decreasing the H‐bond distance between carboxyl group in P4 and asparagine from active site loop 3. A second mechanism that results in selection of D over V is by including a polar cap near the S4 site. This mechanism is observed in caspase‐3b from zebrafish and appears to be a general strategy from the scaffold of the common ancestor. We show that mutations that break the salt bridge in human caspase‐3 results in relaxed specificity compared to wild type protein.

In conclusion, protein conformational ensemble and enzyme specificity are two important ways to regulate caspase activity. Gaining the knowledge with regards to the mechanisms with which these proteins and their related families evolved may assist in uncovering how and why the various functions evolved in these 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.
A Novel Inexpensive Media for the Growth of Escherichia coli Expressing a Recombinant Lipase
Authors: Okpuzor JOY , Bello Adebayo J , Igwo‐Eziekpe Miriam , Toye Voke , Adewole Adeniyi R , Agbabiaje Olayinka
Published: June 2021

There is an ever increasing demand for industrial enzymes for different applications to satisfy the needs of the world's ever‐growing population. Nigeria currently is at a precipice and urgent measures are needed for self‐sustainability in a distressed economy for most of her industrial processes. The progression of synthetic biology techniques has been a continuous driving force for sustainable development on industrial enzymes. Lipases (EC 3.1.1.3) are important biocatalysts due to their utility in various industrial applications. For industrial processes, it is necessary to define the best set of conditions for cell growth, protein induction and recovery. This work therefore investigates the conditions necessary for optimum expression of novel recombinant lipase (sourced originally from marine sponge Haliclona simulans metagenome) in Escherichia coli.

A codon‐optimized gene that codes for lipase Lpc53E1, a member of the group VIII family of lipases from Haliclona simulans was heterologously expressed in E. coli by induction of graded concentrations (0.1 mM, 0.25 mM, 0.5 mM and 1 mM) of Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) when the cell's density (OD600) reached 0.4, 0.5, 0.6 and 0.7 accordingly. Analysis of the SDS‐PAGE revealed the presence of lipase with an approximate molecular weight (MW) of 44 kDa. The recombinant lipase exhibited a higher halo‐tolerance and thermostable properties up to 60°C when the cell density reached 0.7 OD600 after induction with 0.5 mM IPTG. A novel and newly formulated inexpensive media for cell growth of the E. coli culture showed distinct colonies comparable to the LB control. This suggests an alternative and cheaper media for this microbial culture. Thus this inexpensive media from locally sourced materials would reduce the cost of operations in industries requiring lipase in developing countries.

Support or Funding Information

The grant for this research was awarded by the Central Research Committee, University of Lagos, Nigeria (CRC, 2017/01)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification and Characterization Chemical Compounds that Inhibit Lysyl‐tRNA Synthetase from Pseudomonas aeruginosa
Authors: Balboa Samantha , Hu Yanmei , Bullard James M
Published: June 2021

Introduction

Pseudomonas aeruginosa is an opportunistic pathogen and a major cause of nosocomial infections. The enhanced ability of P. aeruginosa to acquire resistance to most antibiotics places it in a special class of pathogens, the so‐called “superbugs”. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis and are validated targets for development of antibacterial agents. The gene encoding P. aeruginosa lysyl‐tRNA synthetase (LysRS) was cloned and the resulting overexpressed protein was purified to 98% homogeneity.

Methods and Results

Aminoacylation assays were used to measure kinetic parameters for interactions of LysRS with tRNA and ATP:PPi assays were used to determine the kinetic parameters for interactions with lysine and ATP. The KM values for interaction with lysine, ATP and tRNALys were determined to be 45.3, 627, and 3.3 μM, respectively. The kcatobs values were calculated to be 13.3, 22.8, and 0.35 sec−1. This resulted in kcatobs/KM values of 0.29, 0.036, 0.11 s−1μM−1. P. aeruginosa LysRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen natural product (800) and synthetic (890) compound libraries to identify compounds with the ability to inhibit the function of the enzyme. Three compounds (BM01D09, BT06F11, and BT08F04) were identified and inhibitory activity was confirmed. These compounds inhibited the ability of LysRS to aminoacylate tRNALys with IC50 values of 35.3, 66.9, and 36.4 μM, respectively. The minimum inhibitory concentration (MIC) was determined for each compound against a panel of ten pathogenic bacteria. Each compound exhibited broad spectrum activity. Time‐kill studies indicated a bacteriostatic mode of inhibition for each compound against cultures of S. aureus, however in cultures of M. catarrhalis, BT06F11 and BT08F04 was observed to be bactericidal. When tested in cultures of human embryonic kidney 293 (HEK‐293) cell lines using MTT cell proliferation assays, BT06F11 was not toxic at any concentration tested. BM01D09 was observed to only inhibit human cell growth at high concentrations (CC50 = 370 μM). However, BT08F04 was toxic at much lower concentrations (CC50 = 61 μM).

Conclusion

LysRS from P. aeruginosa was characterized and developed into a screening platform to identify potential anti‐bacterial compounds. Three compounds were identified as having inhibitory activity against the aminoacylation of LysRS. The compounds were then characterized for inhibition of enzymatic activity, bacterial growth and toxic effects in human cell cultures.

Support or Funding Information

National Institutes of Health (grant number: 1SC3GM098173)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Dihydromotuporamine C Derivatives on Actin Assembly Dynamics
Authors: Heidings James Bernard , Mathin Amirah , Skruber Kristen , Muth Aaron , Phanstiel Otto , Kang Hyeran
Published: June 2021

Dihydromotuporamine C (motu C) is a small molecule isolated from a New Guinea sea sponge that has demonstrated anti‐metastatic and anti‐migratory properties both in vivo and in vitro. Many cancers do not pose a threat to a patient's life until they metastasize. Actin filament assembly dynamics play a critical role in cancer cell metastasis, cancer cell movement, and force generation. In this study we investigate how motu C derivatives modulate actin filament assembly kinetics in vitro, using biophysical and biochemical assays. The effect of motu C on average filament lengths was determined by direct visualization of actin filaments incubated with motu C derivatives, using total internal reflection fluorescence (TIRF) microscopy. We demonstrate that one particular motu C derivative, motuCH233, reduces filament length significantly. Pyrene actin assembly kinetics indicate that motuCH233 also effectively decreases the amount of polymerized actin, which may potentially affect cancer cell migration. Outcomes from this project can be used to further optimize the molecular design of anti‐migratory small molecules.

Support or Funding Information

This study was supported by the UCF start‐up fund and In‐House grant for Hyeran Kang.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural and Dynamics of Glucocorticoid Receptor Ligand Binding Domain in Complex with PGC1α and Synthetic Glucocorticoids
Authors: Liu Xu , Wang Yashuo , Ortlund Eric
Published: June 2021

Synthetic glucocorticoids are important pharmaceutical drugs that are known to bind to the ligand binding domain (LBD) of glucocorticoid receptor (GR), a member of the nuclear receptor superfamily. These binding will induce conformational changes within GR for the subsequent DNA binding and different co‐regulators recruitments, ultimately lead to activate or repress target gene expression. One of the crucial co‐regulators is peroxisome proliferator‐activated gamma coactivator 1‐α (PGC1α), known to regulates energy metabolism by direct interacting with GR to modulate gene expression. However, the mechanisms through which PGC1α changes GR conformation to drive transcription are unknown. Here, an ancestral variant of GR (GR2) was utilized as a tool to produce stable protein for biochemical and structural studies. We report the first high resolution X‐ray crystal structures of GR2 LBD in complexes with PGC1α and dexamethasone and hydrocortisone. These structural analyses reveal how distinct steroid drugs bind to GR with different affinities via unique hydrogen bonding and hydrophobic interactions. Hydrogen‐deuterium exchange mass spectrometry showed different effects on GR2 LBD allosteric communication after glucocorticoids binding. Molecular dynamics simulations and network analysis identified the key residues involved in the intramolecular communication between ligand binding site and activation function surface, which is the PGC1α binding site.

Support or Funding Information

This work was supported by W.M. Keck Foundation Medical Research Grant to E. A. O. X. L. was supported by an American Heart Association postdoctoral fellowship [17POST33660110].

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Branched Amphiphilic Peptide Capsules: Agents for Gene Delivery and Immunomodulation
Authors: Tomich John M. , Sukthankar Pinakin R. , Barros Sheila de M. , Withaker Susan K. , Wilkinson‐Nutsch Kayla M. , Avila L. Adriana
Published: June 2021

Branched Amphiphilic Peptides Capsules (BAPCs™) are a novel class of nano‐carriers constituted from the spontaneous co‐assembly of two unique, engineered peptide sequences. We recently reported on a thermally induced variant of BAPC™ that demonstrates promise as a vehicle for gene delivery and transfection. These BAPCs™ display a uniform size of 20–30 nm; they are easy to synthesize, stable and produce minimal immunogenic and inflammatory responses, in contrast to those commonly observed with viral and cationic lipid based approaches for gene delivery. BAPCs™ act as cationic nucleation centers allowing nucleic to wrap around them in a nucleosome like fashion, generating peptide‐nucleic complexes with sizes ranging from 50 to 250 nm. In this study, we combine the pre‐constituted BAPCs™ with a plasmid DNA construct encoding the green fluorescent protein (eGFP) in order to generate DNA/peptide complexes. We utilize these complexes to successfully transfect mammalian cells in vitro, yielding higher transfections rates than the commercially available lipid based reagent Lipofectin®, with minimal cytotoxicity. We then test the transfection system for the in vivo delivery of a DNA vaccine, previously designed to activate immune responses capable of controlling tumors induced by type 16 human papilloma virus (HPV‐16). We successfully demonstrate the efficacy of the DNA/BAPCs™ complexes in the in vitro activation of murine dendritic cells, and the enhancement of the associated vaccine's anti‐tumor potency, without significant toxic effects in vaccinated mice. Together these results indicate that the interaction of double stranded DNA to the cationic BAPCs™ nanoparticles represents a promising new in vitro and in vivo non‐viral gene delivery system.

Support or Funding Information

Partial support for this project was provided by the Terry Johnson Cancer Center at Kansas State University, The Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD) at Kansas State 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.
Induction of Neoplastic Transformation, AP‐1 Signaling, PDCD4 signaling, and DNA Damage by Metal Oxide Nanoparticles Involves ROS‐Mediated MAPK Pathways in JB6 Cells
Authors: Barber Tabatha L. , Aldinger Joni , Arnold Joshua , Ding Min
Published: June 2021

Occupational exposures to metal oxide nanoparticles have been reported to be harmful to human health, with possible risk of cancer among workers from many sectors. Metal Oxide nanoparticles have not, to our knowledge, been extensively examined for potential carcinogenic or genotoxic effects. To investigate the mechanisms of TiO2, WC‐Co, and CuO‐induced pathogenesis, the effect of metal oxide fine and nanoparticles on AP‐1‐MAPKs and ROS generation were investigated. The results indicated that CuO, WC‐Co, andTiO2 nanoparticles caused a 2‐fold increase or more in AP‐1 activity in JB6 cells. The induction of AP‐1 activity in cultured cell lines was time and dose‐dependent. The signal transduction pathways for AP‐1 activation were also investigated. Western Blot analysis demonstrate that CuO, WC‐Co, and TiO2 nanoparticles stimulate phosphorylation of p38 MAPK and ERKs. CuO, WC‐Co, and TiO2 also generated ROS when incubated with the cells as measured by electron spin resonance (ESR). Nano‐sized CuO, WC‐Co, and TiO2 generated more ROS than the fine sized particles when incubated with the cells. COMET assay suggests that exposure of the cells to CuO resulted in DNA damage. Cells pre‐treated with ROS inhibitors sodium formate and Polyvinylpyridine‐N‐Oxide (PVPNO) before exposure to CuO and WC‐Co nanoparticles demonstrate a significant reduction in p‐38 and ERK activity, suggesting the H2O2 may have a role in the ROS mechanism. Soft agar transformation assays demonstrated that there is a significant increase in colony formation in JB6/AP‐1 cells treated with CuO, WC‐Co, and TiO2 nanoparticles as compared to the control, indicative of neoplastic transformation. Unraveling the complex mechanisms associated with these events may provide insights into the initiation and progression of metal oxide‐induced pathogenesis.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Utilization of a DNA Nanoswitch to Detect Parallel Poladenylic Duplex Formation
Authors: Brylow Karl , Pickard Martha Anne G. , Anecelle Matthew R. , Gleghorn Michael L.
Published: June 2021

Polyadenylic molecules (poly A) are single stranded nucleic acids containing a series of repeating adenine bases. Polyriboadenylic (poly (rA)) strands form parallel duplexed helices in acidic conditions. Our group has generated and utilized a technique to determine if polyadenylic sequences can pair when each sequence differs in composition. We have adapted a DNA nanoswitch developed by others to detect duplex formation where we can control the composition of each strand that is being tested for interaction. This method is superior to traditional UV melting studies, given the low specificity for detection of duplex species in a complex mixture of polyadenylic strands with different compositions. Our results show that nanoswitches can be used to detect poly(rA) duplex formation as a function of pH and we have identified interesting binding between polyadenylic strands that have different compositions. Our methodology not only provides fundamental information regarding parallel polyadenylic duplex formation, but also can help in design of future and potentially pH‐sensing biological nanotechnology.

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 Beacon‐based Fluorescence Magnetic Nanoprobes for Tumor‐related HSP90 mRNA In‐suit Detection and Imaging
Authors: Chen Zhongyuan , Feng Yi , Xie Xiaoxue , Shen Xue , Yang Hong , Wu Chuihui , Liu Yiyao
Published: June 2021

Heat shock proteins (HSPs) are a group of molecular chaperones that help proteins folding to their native conformation, protect cells from stressful environments. However, certain HSPs are overexpressed by malignant cells to survive under various therapies, inhibited cancer cells from senescence, apoptosis, stabilized lysosome membrane and promoted autophagy. Therefore, real‐time detection of the expression level of HSPs in living cells is required for both clinical cancer diagnosis application and oncology research to have a better understanding in anti‐apoptosis mechanism of malignant cells. Recently, delivery of molecular beacons that detect tumor‐related genes into cancer cells via nanocarriers shows promising results in the field of nanomedical diagnostics. In this study, uniformly sized and shaped superparamagnetic iron oxide nanocubes (SPIONCs) were synthesized by thermal decomposition and transferred into the aqueous phase via surface exchange with DMSA. G4 PAMAM dendrimer and Pluronic‐P123 were successively linked on SPIONCs‐DMSA in order to increase positive charge meanwhile reduce cytotoxicity. High specificity and sensitivity molecular beacon that targeting HSP90α mRNA was designed and synthesized and was condensed on PAMAM‐functionalized SPIONCs to form nanoprobes. TEM, XRD, TGA, ξ‐potential, DLS, VSM, fluorescence spectroscopy, agarose gel electrophoresis were carried out to characterize the morphology, composition, and properties of the nanoprobes. The mean diameter of as‐synthesized SPIONCs is 11 nm and increased to 100 nm after forming nanoprobes, which is stable in both water and culture media. Proliferation and hemolysis assays of the nanoprobes show low cytotoxicity and good hemocompatibility. LSCM confirmed that after 24h incubation with cells, the nanoprobes were uptake efficiently and located inside lysosome, and successfully escaped to the cytoplasm after 48h incubation. More importantly, the fluorescence of nanoprobes was observed significantly only in the HSP90 overexpressed MDA‐MB‐231 cells, instead of HSP90 low‐expressed MCF‐10A cells, which literally “highlights” a difference between normal and malignant cells, therefore provides a promising nanoplatform for real‐time gene expression level analysis and imaging in living cells.

Support or Funding Information

We would like to thank the financial supports, in whole or in part, by the National Natural Science Foundation of China (31470959, 81671821, 81471785), the Basic Research Program of Sichuan Science and Technology (2017JY0019, 2017JY0217), and the Fundamental Research Funds for the Central Universities (ZYGX2016Z001) for financial supports.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphorylation of PEA‐15 in HIV‐TAT Mediated Disruption of Blood‐Brain Barrier
Authors: Cabezas Andres , Hassan Sherouk , Crespo‐Flores Sergio , Wei Yufeng
Published: June 2021

Trans‐activator of transcription, TAT, one of the proteins that is expressed by the HIV genome, has been heavily associated with HIV‐associated cognitive morbidities. In vitro experiments have shown that the presence of TAT disrupts blood‐brain barrier (BBB) tight junctions and overall integrity. The mechanisms for TAT associated BBB disruption remain largely unknown.

Phosphoprotein enriched in astrocytes (PEA‐15) is a ubiquitously expressed protein that is best known for its ability to regulate critical cellular pathways via protein‐protein interactions. PEA‐15 gains multiple protein binding affinities depending on the phosphorylation states of its residues Ser104 and Ser116. When PEA‐15 is phosphorylated at both Ser104 and Ser116 it gains high affinity to the Fas‐associated death domain (FADD). When protein‐protein binding with FADD, PEA‐15 blocks FADD from inducing apoptosis by preventing it from activating Caspase‐8.

By blocking FADD effector functions via transfecting a phosphomimetic mutant of PEA‐15, in which both Ser104 and Ser116 are replaced with Aspartic Acid (PEA‐15‐DD), into primary human brain microvascular endothelial cells, we set to explore the mechanisms of HIV‐TAT mediated disruption of BBB tight junctions.

Support or Funding Information

This research is supported by NIH Grant R21DA046223

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Examining lateral development through CXCL14 modulation of CXCL12‐CXCR4 mediated gene expression in Danio rerio
Authors: Calderon‐Zavala Ariana Carolina , Wall Nancy
Published: June 2021

The lateral line is a mechanosensory system used by fish to sense the movement of water. It is evolutionarily related to the inner‐ear in humans. For both organisms, the binding of the CXCL12 (SDF‐1 ligand) to the CXCR4 receptor induces conformational changes needed to activate signal transduction. This signaling results in numerous cellular responses such as cell fate, chemotaxis, and gene transcription. Interestingly, researchers have found that another signaling molecule, CXCL14, can also bind to the CXCR4 receptor with high affinity (Tanegashima et al., 2013). As a result, CXCL14 modulates CXCL12‐mediated chemotaxis, presumably acting as an allosteric regulator. In our lab, we our concerned with the allosteric relationship between CXCL14 and CXCL12 and how those relationships affect gene expression in lateral line development. To examine the effects of CXCL14 on CXCL12‐mediated gene transcription, zebrafish embryos were microinjected with CXCL14 antisense morpholino and incubated for a period of three timepoints: 24 dpf, 36 dpf, and 48 dpf, when lateral line development occurs. Our preliminary results suggest that the absence of CXCL14 affects the gene expression of CXCL14, CXCL12, CXCR4, CXCR7, epcam, claudin, and snail1b during lateral line development, thereby suggesting the allosteric capabilities of CXCL14. Further studies will be conducted to determine the degree to which CXCL14 affects the transcription levels of these genes.

Support or Funding Information

WiscAMP‐NSF #1400815

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An Evaluation of Palladin Overexpression in Pancreatic Cancer Cells
Authors: Bush Jason , Vander Karamjot , Chu‐Bacher Rowena , Ramirez Joel
Published: June 2021

Pancreatic cancer (PC) is the fourth leading cause of cancer in United States with overall survival rates of less than 5% (ACS, 2018). Its aggressive nature and late onset of physical symptoms lead to poor prognoses. Recognition of biomarkers is clearly an imperative undertaking for the disease. To tackle this problem, we evaluated the secreted protein profile of pancreatic cancer cell lines derived from different stages. In total, nine human pancreatic cancer cell lines were successfully adapted into serum‐free environments from which conditioned media was collected by a combination of ultracentrifugation, molecular weight cutoff, protein precipitation, gel‐based separation, and tryptic digestion followed by mass spectrometry protein identification. A small subset of proteins has been identified and validated biochemically including Palladin (PALLD), a pancreatic cancer susceptibility protein shown to be up‐regulated in previous studies. PALLD is a scaffolding protein that supports remodeling of the actin cytoskeleton in association with integrins at focal adhesion sites to coordinate cell migration. We demonstrate a possible interaction between PALLD and the beta5 integrin (ITGB5) indicative of the increased motile phenotype in PC. Taken together, our results suggest that PALLD overexpression is associated with enhanced motility in pancreatic cancer cells and could represent a potential biomarker or therapeutic strategy against pancreatic cancer progression.

Support or Funding Information

This work was funded in part through intramural programs at Fresno State to KV, RCB, JAB, and through NCI P20CA138025 to JAB.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neuronal membrane proteasomes target the nascentome to generate extracellular peptides that modulate neural network activity
Authors: Margolis Seth Shatkin , Ramachandran Kapil , Turker Fulya
Published: June 2021

In mammals, activity‐dependent changes in neuronal function require coordinated regulation of the protein synthesis and protein degradation machinery. However, the biochemical evidence for this balance and coordination is largely lacking. To investigate this we initially used acute metabolic radiolabeling of stimulated primary mouse neurons to follow the fate of polypeptides being newly synthesized. We observed polypeptides being newly translated exclusively during neuronal stimulation were rapidly degraded by the neuronal membrane proteasome (NMP) and not the cytosolic proteasome. This turnover correlated with enhanced production of NMP‐derived peptides into the extracellular space which have the capacity to mediate neuronal signaling in part through NMDA receptors. Using in‐depth, global, and unbiased mass spectrometry, we identified the nascent protein substrates of the NMP. Among these substrates, we found that immediate‐early gene products c‐Fos and Npas4 were targeted to the NMP during ongoing activity‐dependent protein synthesis. Moreover, we found that turnover of nascent polypeptides and not full‐length proteins through the NMP occurred independent of canonical ubiquitylation pathways. We propose that these findings generally define a neuronal activity‐induced protein homeostasis program of coordinated protein synthesis and degradation through the NMP. This generates a new modality of neuronal signaling in the form of extracellular peptides with potential significance for our understanding of nervous system development and function.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Correlation Between Accumulation of Alzheimer's Disease Markers in Brain and Retina of Individuals with Late Stage of the Disease
Authors: Salloum Liza , Bondarenko Vladimir
Published: June 2021

This study was aimed to determine if there is a correlation between accumulation of various Alzheimer's disease (AD) markers in the brain and retina of individuals in the late stages of the disease. Characterizing the distribution and accumulation of these markers in the retina and brain may be important for early diagnosis of neurodegenerative diseases such as AD.

We examined total tau and phosphorylated tau protein accumulations in different areas of the brain and in retinal tissues from aged individuals with AD in order to compare the intensity and sensitivity of these biomarkers.

Tissues from the hippocampus and temporal lobe of four different cadavers were analyzed. Two cadavers had diagnosed Alzheimer's disease, and two cadavers served as a non‐AD control. We utilized cryosectioning to prepare tissue sections for immunostaining. Tissues obtained from individuals without a history of Alzheimer's disease or other types of dementia were used as negative controls. Detection of tau was performed using a primary antibody against human tau proteins and amyloid‐b, followed by a universal secondary antibody.

Although the staining for AD markers was greater than controls for both brain tissues, the density was higher in the hippocampus than in the temporal lobe. The density of the total tau (TT) was greater than that of phosphorylated tau (PT181) in both the hippocampus and temporal lobe. We also found this to be the case in the brain tissues of a control, indicating that total tau may be a more sensitive biomarker than phosphorylated tau.

These data demonstrate that total tau may be a stronger biomarker than phosphorylated tau in quantifying protein accumulations in the brains of Alzheimer's disease cadavers. This raises the intriguing possibility that there may be more sensitive biomarkers that can better predict the course of neurodegenerative diseases such as AD. Thus, future studies will concentrate on the accumulation of tau in the brain and retina of individuals during the earlier stages of Alzheimer's disease, as well as the sensitivity and accuracy of different biomarkers.

Support or Funding Information

Touro University Nevada intramural 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.
Examination of downstream targets of the FKH‐8 transcription factor in dopaminergic neurons
Authors: Anthony Austin , Nelms Brian
Published: June 2021

The transcription factor forkhead‐8 (FKH‐8) is enriched in dopamine (DA) neurons and is required for DA neuron function. Disruption of the fkh‐8 gene mimics the swimming‐induced paralysis (SWIP) phenotype characteristic of loss of the dopamine transporter (DAT). DAT is responsible for the reuptake of excess dopamine from the synaptic cleft after its pre‐synaptic release, and loss of DAT function results in excess dopamine in the synaptic cleft and spillover into extra‐synaptic areas. This spillover of dopamine results in the hyperdopaminergic state seen via the SWIP phenotype.

However, neither the expression nor function of DAT‐1 are altered in fkh‐8 mutants compared to wildtype worms, and FKH‐8 does not seem to impact genes in the dopamine synthesis pathway. While FKH‐8 has been shown to regulate important DA neuron functions through phenotypic assays, downstream genes have yet to be identified. Because FKH‐8 acts independently of the well‐studied DAT‐1 transporter, it is likely that its targets will include a number of novel genes whose impact on dopamine signaling have yet to be revealed.

Cell‐specific RNA sequencing comparing gene expression in dopamine neurons isolated from wildtype vs. mutant organisms will identify what genes are important in dopamine signaling in the model organism Caenorhabditis elegans. More specifically, it will reveal targets and gene expression patterns that are altered by loss of FKH‐8. in dopamine neurons.

We anticipate that using this unbiased approach to identify novel genes critical for these functional outcomes in C. elegans will provide fundamental new biological insights as well as potential novel molecular targets for therapeutic intervention.

Support or Funding Information

NSF CREST BioSS Center Grant HRD15‐47757

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding the Role of Cyclin Dependent Kinase 5 (Cdk5) in Insulin Related Neurodegenerative Disease
Authors: Tran Brandon , Aguanno Ann
Published: June 2021

Cyclin Dependent Kinase 5 (Cdk5) is a member of the cyclin dependent kinase family of proteins that play key roles in the regulation of the cell cycle. The activity of these enzymes is dependent upon association with a cyclin activating partner. Unlike other members of this family, however, Cdk5 appears to play no role in cell cycle regulation. Cdk5 is ubiquitously expressed in all cells. However maximum kinase activity is found in post‐mitotic neurons and glial cells, where Cdk5's cyclin activating partners exhibit high expression. Known substrates of Cdk5 include proteins that modulate cell morphology and motility. Our lab has shown that Cdk5 plays an essential role in the developing nervous system primarily through studies of chemical inhibition of Cdk5 in a neuronal model. Specifically, we employed the well‐known PC12 cell line which can be induced to transdifferentiate into neuronal‐like cells. Inhibition of Cdk5 during this process reduced the neuronal characteristics these cells typically exhibit, mimicking neuronal degradation or degeneration. Multiple published studies support Cdk5's role in the developing mammalian nervous system. Recent investigations have shown an increased correlation between the likelihood of developing neurodegenerative diseases and insulin resistance associated with Type 2 diabetes. Insulin resistance results in the over‐secretion of insulin (hyperinsulinemia) into the blood stream and causes a down regulation of insulin receptors in peripheral tissue. Although acute hyperinsulinemia in the central nervous system (CNS) may have cognitive enhancing effects, chronic hyperinsulinemia appears to be neurotoxic. We are exploring this potential relationship between insulin levels and the neuron by exploiting our neuronal model cell line, PC12. Using this model, we examine the role of normal and aberrant Cdk5 activity in the response of differentiated neuronal‐like PC12 cells to varying concentrations of insulin in culture. Our aim is to test the reports of deregulation of brain insulin signaling as an early marker in AD. Here we show our preliminary results of the transdifferentiation of PC12 cells while Cdk5 activity is manipulated under hyperinsulinemic conditions. Our findings underscore the value of this model as a means to examine the potential role of Cdk5 as a link between AD and Type 2 diabetes. Given the increased Type 2 Diabetes diagnosis and the aging of American society implications for this study are self‐evident.

Support or Funding Information

The Rose M. Badgely Charitable Trust, Joseph Alexander Foundation, Sister Colette Mahoney Science Research Grant and the Marymount Manhattan College Faculty Award.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Measuring T‐cell avidity and enrichment using an acoustic force‐based technology
Authors: Scheper Wouter , Merino‐Rodriguez Elena , Braster Rens , Sitters Gerrit , Oswald Felix , Schumacher Ton , Candelli Andrea
Published: June 2021

The key driver for effective immunotherapies is the overall strength binding of the immune cell and the target cell(e.g. tumor cells). The overall strength is known as ‘avidity’, a parameter reflecting interaction efficiency. One of the main obstacles has been the generation of immunotherapies promoting effective and long lasting immune responses, due to the lack of tools measuring this parameter. Here, we have made used of acoustic forces as a novel method to isolate immune cells based on their avidity to specific targets, such as (tumor) cells. The force that cell target need to experience to be separated is called ‘rupture force’. In this study, we were able to identify the rupture force of tumor specific and non specific T cells and separate these different populations applying controllable acoustic forces. T cells engineered with a melanoma antigen recognizing T cell receptor needed 6 times more relative force than the control, nonspecific T cells, to be separate from their melanoma cells. These findings indicate that the specific melanoma T cells bind with higher efficiency and higher avidity than the nonspecific ones, and that they can be separated with this method. Furthermore, 1.4 to 3.6‐fold enrichment of high avidity T cells from a mixed specific:nonspecific (1:10) T cell population was obtained after their isolation using acoustic forces and confirmed using FACS measurements. Therefore, we demonstrate here that acoustic forces can be used as a tool to measure avidity.

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