bluuebunny/crossref_metadata_2025 huggingface dataset:
Exploring a potential link between cell cycle regulation and Cancer Testis gene misregulation
Authors: Alhewati Sabha Salem , Goetsch Paul
Published: June 2021

Oncogenesis and misexpression of germline genes like Cancer Testis (CT) genes, called the soma‐to‐germline transformation, are commonly linked, but the mechanisms that drive the phenomenon remain underexplored. In the model system Caenorhabditis elegans, a soma‐to‐germline transformation occurs when the function of the highly conserved DREAM complex (for Dp, Retinoblastoma(Rb)‐like, E2F, and MuvB) is attenuated. In mammalian cells, DREAM's activity overlaps with the Retinoblastoma(Rb) protein to mediate establishment of cell cycle exit into quiescence, or G0. We hypothesize that CT gene misexpression in cancer cells occurs following loss of DREAM and Rb activity, similar to the soma‐to‐germline transformations that occur in C. elegans. Therefore, we expect that cancer cell lines that express CT genes will be unable to maintain an effective cell cycle exit. To test our hypothesis, cell cycle distributions of 8 cancer cell lines were analyzed using propidium iodide (PI) staining and flow cytometry before and after serum starvation. We observed that only 3 of the cell lines failed to arrest under growth limiting conditions, with the other 5 exhibiting some enrichment in G0/G1, indicating that these cell lines that did not arrest might exhibit upregulated expression of CT genes, and DREAM complex and Rb are deregulated. However, the cell lines that we observe G0arrest may still be dysfunctional in either DREAM complex or Rb activity. Therefore, we will next test expression analysis will test whether CT genes are differentially regulated between these groups and whether DREAM and/or Rb regulation is attenuated. This study is the first step towards understanding whether cell cycle regulation and the soma‐to‐germline transformation are linked in mammalian cells.

Support or Funding Information

The Saudi Arabian Cultural Mission (SACM)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein‐DNA interactomes of cardiac transcription factors GATA4 and NKX2‐5
Authors: Carrasquillo Emmanuel , Rodriguez Jose Arcadio
Published: June 2021

Every year over 100,000 new cases of Congenital Heart Disease (CHD) are reported, being one of the most common disease in neonates. CHDs are characterized by anomalies in the heart and its great vessels during development and physiology. Transcription Factors (TFs) such as GATA4 and NKX2‐5 play a vital role in the regulation of genes involved in human heart development and functionality. Identified point mutations in the DNA‐binding domain (DBD) of GATA4 and NKX2‐5 are among multiple mutations associated to CHD. We hypothesize that non‐synonymous mutations in the DBD of GATA4 and NKX2‐5 will alter their DNA‐binding properties ultimately affecting the whole transcription pathway for a proper heart development. To address this hypothesis, we want to study how the GATA4 and NKX2‐5 DNA‐protein interactomes are affected by the non‐synonymous mutations found in CHD patients. We have successfully cloned the DNA binding domain (DBD) of GATA4 and NKX2‐5 via Gibson‐assembly. Both of which were over‐expressed and purified. Functional analysis protocols using electrophoretic mobility shift assay (EMSA) confirms protein binding to its DNA binding sites and therefore functional. We will measure the comprehensive protein‐DNA interactomes of missense mutants of GATA4 and NKX2‐5 using High‐Throughput Systematic Evolution of Ligands by Exponential Enrichment (HT‐SELEX), and use bioinformatics tools to predict changes in gene targets between wild‐type and mutant TFs.

Support or Funding Information

This research was supported by:

1) Research Initiative for Scientific Enhancement (RISE) Grant #: 5R25GM061151‐17

2) NIH SC1GM127231

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Methylation of RB1 CpG island by SUV39H1/DNMT3A promotes malignant melanoma development
Authors: Kim Garam , Choi Hong Seok
Published: June 2021

Melanoma is among the most aggressive and treatment‐resistant human cancers. Aberrant histone H3 methylation at Lys 9 (H3K9) correlates with carcinogenic gene silencing but the significance of suppressor of variegation 3–9 homolog 1 (SUV39H1), a H3K9‐specific methyltransferase, in melanoma initiation and progression remains unclear. Here we show that SUV39H1‐mediated H3K9 trimethylation (H3K9me3) facilitates retinoblastoma (RB1) promoter CpG island methylation by interacting with DNMT3A and decreasing RB mRNA and protein in melanoma cells. Reduced RB abundance, in turn, impairs E2F1 transcriptional inhibition leading to increased peptidyl‐prolyl cis‐trans isomerase NIMA interacting 1 (PIN1) levels, human keratinocyte neoplastic cell transformation, and melanoma tumorigenesis via enhanced RAF1/MEKs/ERKs signaling pathway activation. In a synergistic model with B16‐F1 murine melanoma cells, SUV39H1 and PIN1 overexpression increased melanoma growth, which was abrogated by their inhibition in SUV39H1‐overexpressing B16‐F1 mice. SUV39H1 also positively correlated with PIN1 expression in human melanoma. Our studies establish SUV39H1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.

Support or Funding Information

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number : HI18C1083).

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 Role of αvβ3 Integrin Proteins in Glaucoma
Authors: Fassler Amy
Published: June 2021

Glaucoma, the leading cause of vision loss worldwide, occurs when elevated intraocular pressure damages the optic nerve. Intraocular pressure increases when there is a blockage of the trabecular meshwork, a drainage system at the base of the cornea. This process is dictated by a bidirectional signalling protein composed of a specific combination non‐covalently bonded α and β integrin subunits called the αvβ3 integrin. In an inactive state, the integrin is is positioned as a switchblade and its cytoplasmic tails are held together by a salt bridge. To become activated, the integrin must extend; the salt bridge must be broken, and the cytoplasmic tails spread apart. During outside‐in signalling, a ligand binds to the αvβ3 integrin which forces it to re‐conform to the active state and results in the transmission of mechanical and chemical cues from the extracellular matrix into the cell by means of a protein cascade. This leads to the trabecular meshwork becoming more dense and interwoven. A possible hypothesis to explain the connection between integrins and glaucoma is that the trabecular meshwork can not turn off the αvβ3 integrin which results in a reduced outflow of the aqueous humor and thus, increased intraocular pressure. Understanding the modulation of intraocular pressure by integrins can lead to therapeutic targets to treat glaucoma. The Marshfield High School MSOE Center for BioMolecular Modelling for SMART Team used 3D Modelling and printing technology to examine structure‐ function relationships of the αvβ3 integrin protein.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Carbonic Anhydrase Expression in Experimental Colitis: A Possible Mechanistic Link with Na‐H Exchanger
Authors: Khan Islam , Al‐Shamali Amna
Published: June 2021

In this study we investigated a role and regulation of carbonic anhydrase (CA) and a possible mechanistic link with the NHE expression in experimental colitis. Intra‐rectal instillation of trinitrobenzenesulphonic acid was used to induce colitis in rats. On day 6 post‐TNBS, colon and ileal segments were taken out and used to measure expression levels of the CA‐I, ‐II and ‐IV isoforms by ECL western blot analysis. These isoforms together with NHE‐1 were significantly suppressed in the inflamed colon. Immunoprecipitation and confocal immunofluorescence demonstrated co‐localization of CA‐I and CA‐II, but not the CA‐IV isoform with NHE‐1 in the colon. However, the binding of CA isoforms with NHE‐1 was more suppressed as compared to their expression level in inflamed colon. Only CA‐I was found to interact with an 83 aa long NHE‐1 C‐terminal peptide. Ex‐vivo studies demonstrated suppression of CA‐I and CA‐II isoforms by TNF‐alpha treatment of control colon segments. Inflammation in the colon was confirmed by measuring body weight, myeloperoxidase activity, colon hypertrophy, mucus staining and inflammatory cells infiltration. These findings demonstrate inflammation‐induced reduction in the expression of CA isoforms which is regulated by TNF‐alpha. Reduction in the binding of CA isoforms with NHE‐1 C‐terminal peptide suggest inflammation‐induced uncoupling of the two transporters. Interaction of CA with the NHE‐1 is important to ensure availability of H+ for NHE‐1 activity. In conclusion both expression and coupling of CA and NHE‐1 are altered and may account for the suppression of NHE‐1 activity in inflamed colon.

Support or Funding Information

Financial support through a grant (MB 03/15) by the Research Sector, Kuwait 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.
Crystal structure of a dimerization domain of Drosophila Caprin protein
Authors: Zhou Xia , Zhu Jiang , Du Zhihua , Huang Xiaolan
Published: June 2021

The Drosophila Melanogaster Caprin protein (dCaprin), as well as the human Caprin‐1 and Caprin‐2 proteins, are prototypic members of the caprin (cytoplasmic activation/proliferation‐associated protein) protein family. Caprin proteins contain two highly conserved homologous regions (HR1 and HR2) and C‐terminal RGG motifs characteristic of RNA‐binding proteins. The detailed molecular mechanisms of caprin protein functions remain largely unknown. We had previously determined the crystal structure of a ~120‐residue fragment of Caprin‐1 and Caprin‐2 within the HR1 region. Here, we report the determination of the crystal structure of a corresponding fragment in dCaprin. Our structural studies show that all three structures of dCaprin, Caprin‐1, and Caprin‐2 have a similar novel all α‐helical fold that self‐associates to form a homodimer. Structural comparison reveals that the molecular interactions mediating homodimerization of the three proteins are largely conserved in the three systems. Presence of a dimerization domain within HR1 may represent an evolutionarily conserved feature of the caprin protein family. Interestingly, while the caprin proteins adopt similar overall dimeric structures, the structures have quite different molecular surface properties. The different surface properties of the caprin dimeric structures may dictate their intermolecular interaction with specific protein partners.

Support or Funding Information

SIUC Seed 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.
DNA‐binding specificity of TBX5 and its coding variants associated with Holt‐Oram Syndrome
Authors: Cesar Brenda I , Carrasquillo Emmanuel , Rodriguez Jose A
Published: June 2021

Transcription factors (TFs) are important sequence‐specific DNA binding proteins that regulate gene expression and control cellular state. Evaluation of the DNA‐binding properties of transcription factors is fundamental for understanding their possible roles in controlling gene regulatory networks. TBX5 is a transcription factor that has been identified as an important regulator in organ development. TBX5 mutations have been associated to Holt‐Oram Syndrome, a genetic disorder that affects upper limb formation and often the heart. We aim to determine the specificity of DNA‐binding of TBX5 and its T‐Box DNA‐binding domain and how it changes when the protein mutates. We have cloned TBX5 and its T‐Box domain that contains a His‐ and Strep‐Tag affinity tags that we plan to use for purifying after we overexpress it in E. coli cells. We will then analyze the DNA‐binding specificity of TBX5 and the changes it conveys when it mutates using Cognate Site Identification by High‐Throughput Systematic Evolution of Ligands by Exponential enrichment (HT‐SELEX), as this will allow us to better understand the impact of TBX5 mutations on its 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.
Isolation and characterization of lipase inhibitory activity from Solanum tuberosum L
Authors: Katimbwa Dorsilla Anono , Lim Jinkyu
Published: June 2021

Previous studies using 14 different cultivars of Solanum tuberosum L.showed that patatins, lipid acyl enzymes, have varied activities and are regulated by lipase inhibitory compound. In this study we isolated the lipase inhibitory compound by serial solvent extraction and using several chromatographic methods. The inhibitory compound showed high solubility in water and weak solubility in butanol and ethyl acetate. This solubility property suggests that the molecule may have both hydrophilic and hydrophobic moieties. Inhibition kinetics study showed that the compound may have mixed type inhibition as it may interact with the substrate and is markedly influenced by the structure of ester bond or lipid emulsion. The compound also exhibited high potency with an estimated IC50 of 216 ng/mL in the impure extract. The potency of the lipase inhibition of the compound is comparable to the commercially available anti‐obesity drug, Orlistat, whose LC50 was 0.4 ng/mL. Taken together with stability of the inhibitory compound during storage, high temperature and low pH stability, it presents many possibilities in developing applications in anti‐obesity remedies with less likelihood of side‐effects related with hydrophilic lipase inhibitors currently in use in the global market.

Support or Funding Information

Korea National Research Fund

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Electrochemical Investigation of R2lox, a Novel Class of Heterobimetallic Mn/Fe Protein
Authors: Schneider Camille R. , Miller Effie K. , Smith Zachary R. , Shafaat Hannah S.
Published: June 2021

In order to sustain life, nature accomplishes a diverse array of chemical reactions; one such way this diversity is achieved is through the use of metalloenzymes. In Mycobacterium tuberculosis, a unique Mn/Fe heterobimetallic cofactor has recently been discovered in the R2‐like ligand binding oxidase metalloprotein, R2lox.1 While the function of this protein remains elusive, the cofactor is similar to that of the ribonucleotide reductase Ic subclass, which holds the one‐electron oxidizing equivalent necessary for enzymatic function in the form of a Mn(IV)/Fe(III) metal cofactor.2 R2lox, following oxygen activation, forms an unprecedented tyrosine‐valine ether crosslink in its active site, indicative of two‐electron chemistry, ultimately giving a stable Mn(III)/Fe(III) resting state.1–3 However, the means by which these virtually identical metal cofactors accomplish these different types of chemistry is unknown. In addition, while R2lox can also incorporate a Fe/Fe cofactor when incubated with exclusively Fe(II), the Mn/Fe cofactor is proposed to be the biologically relevant cofactor, despite going against the well‐established Irving‐Williams series.4 To begin to probe potential reactivity in this system, R2lox was investigated using electrochemistry techniques. Both the Fe/Fe and Mn/Fe cofactors were studied in three different R2lox variants. For all three variants tested, only the Mn/Fe cofactor displayed redox activity under the conditions tested with an oxidation process at biologically accessible potentials. Following this initial characterization, a pH dependence was conducted to investigate potential proposed states.5 This study represents the first electrochemical characterization of R2lox and illuminates the potential reactivity of this novel cofactor. As electrons are a form of chemical currency, the reduction potentials reported here provide a handle on the accessible energy available for catalysis.

Support or Funding Information

Ohio State University Graduate School Presidential Fellowship; 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.
Protein‐Polymer Bioconjugation Effects on Activity and Stability of Cellulase (FnCel5a) with Varying Molecular Weights of Polymer Chains
Authors: Ram Natasha
Published: June 2021

An alternative method for energy production which decreases dependency on nonrenewable resources and utilizes a greater amount of the available plant biomass is cellulosic ethanol production. However, there is a need for a more cost effective and energy efficient method to produce a stable form of cellulase that can withstand the harsh chemical and physical industrial processes. One method to enhance protein stability has been demonstrated through protein‐polymer bioconjugation. Thermophilic cellulase, FnCel5a, has been shown to have increased activity when conjugated to polymers via amidation reactions onto lysine residues of the enzyme. However, due to numerous lysine residues there was a lack of site‐specific binding of the polymers. Therefore, a novel site‐specific approach was taken in this study to conjugate polymers onto cellulase via a maleimide method using thiolene click chemistry onto a cysteine residue. In addition, polymer chain length was manipulated to investigate its effects on enzymatic stability and activity using the amidation method. Polymerization methods of RAFT and ATRP were utilized to synthesize N, N‐dimethylacrylamide (DMAm) polymers and OEOA polymers of varying degrees of polymerization. These polymers were conjugated using a “graft to” method onto lysine residues. SDS‐PAGE electrophoresis confirmed conjugation of the polymers. And an activity assay using carboxymethylcellulose substrate indicated the presence of a maximum percent enzymatic activity with increased polymer chain length. For site‐specific conjugation, a mutagenesis at C296S and C102S was done for selective polymer binding. SDS‐PAGE electrophoresis confirmed DMam and OEOA polymer binding at the C296 position of FnCel5a. Overall this study confirmed the effective use of click chemistry method as a technique for site‐specific polymer binding of RAFT and ATRP at a cysteine residue. This method can also be utilized to better investigate how polymer chain length can affect the stability and activity of FnCel5a with controlled site‐specific reactions.

Support or Funding Information

REU Site: Summer Research in Chemistry and Biochemistry at Miami University at Miami University, Award No. 1460862

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 DEXTRIN CAPPED CADMIUM SULFIDE QUANTUM DOTS WITH BLOOD COMPONENTS: COAGULATION AND HEMOLYSIS STUDIES
Authors: Rodríguez‐López Anahí , Rodríguez‐Fragoso Patricia , De la Cruz Gerardo González , Rodriguez‐Fragoso Lourdes , Reyes‐Esparza Jorge
Published: June 2021

Evaluate at interaction of dextrin capped cadmium sulfide quantum dots with blood components. The intravenous administration of quantum dots (QDs) has been widely reported as a promising alternative for delivery of drugs to specific cells. Therefore, previous studies have pointed out the importance of studying of, at least, factors like coagulation effect; and their hemolytic character when nanoparticles are intended to be administered for intravenous injection. The hemolytic activity and platelets aggregation of the nanoparticles was tested on isolated rat erythrocytes and platelets. Atomic force microscopy (AFM) facilitates the real‐time studies of blood cells and the morphological, structural, optical properties of the CdS‐dex QDs. In this work a methodology for erythrocytes and platelets study by platelet aggregation, hemolysis and AFM was developed. The results show that CdS‐Dex/QDs caused platelet aggregation at concentration‐dependent manner, this effect being greater at 1000 ug/mL, where the increase was up to 57% (p<0.05). The AFM measurements establish a more direct correlation between the surface topography of platelets with the surface nanostructure and reveal that modified the morphology of the platelets from the concentration of 0.01 ug/mL. The CdS‐Dex/QDs do not cause lysis of erythrocytes significantly, although they modified their morphology, suggesting that they can cause eriptosis. The CdS‐Dex/QDs, caused effects in oval and biconcave morphology of the erythrocytes, making it round and of edges not defined, semi‐flat and with cracks in its surface and lacking central depression in a concentration‐dependent manner and observed in its surface some artifacts that may correspond to agglomerated NPs. We found that CdS‐dex/QDs caused aggregation of rats platelets in vitro; however; there were both quantitative and qualitative differences depends to various concentrations.

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 assembly and function of poxvirus proteins
Authors: Jafari Nafiseh Jandaghi , Szymczyna Blair
Published: June 2021

Proteins play essential roles in biological processes, and atomic details of their structure and function provide an insight into their biological roles and how they can be manipulated for therapeutic purposes. One class of proteins that are still poorly characterized at the structural level are those involved in pox virus replication. This work describes the structural and functional studies of several proteins involved in Tanapox virus replication replication. The Tanapox virus is a poxvirus that is related to the medially important vaccinia, variola and Monkeypox viruses. The variola virus is the causative agent of smallpox, which is estimated to have caused 300–500 million deaths in the 19th century alone and is a potential bioterrorism threat. Structural and functional insights into the proteins and biomolecular complexes that regulate the lifecycle of the virus will aid in the development of therapeutic agents against poxviruses and the development of poxviruses as therapeutic tools against cancer. The main goals of the study were to assess the structure and function of Tanapox proteins using biochemical and biophysical methods. Many Tanapox virus protein construct samples were generated and studied. Nuclear magnetic resonance (NMR) spectroscopy was performed on several samples to assess their structural and conformationally dynamic properties. Circular dichroism spectroscopy, size exclusion chromatography and electrophoretic mobility shift assays were used to determine the roles of these proteins molecular function. The results reveal that Tanapox virus proteins are generally insoluble when expressed alone. While biophysical studies of insoluble proteins are difficult, the samples generated are ideal for generating antibodies for in vivo functional studies.

Support or Funding Information

Thank you to Dr. Karim Essani for providing Tanapox genome.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Long‐Term Consumption of High Protein Disrupts Dog Gut Microbiome and Metabolites
Authors: Gebreselassie Eden Ephraim Ephraim , Jewell Dennis E.
Published: June 2021

There are controversial views on the optimum level of protein required by adult dogs. According to the guidelines by the Association of American Feed Control Officials, adult maintenance dog foods must contain a minimum of 18% protein based on dry matter. However, there are varying levels of protein in commercial dog foods ranging from 18% to over 60%. We conducted a study to evaluate the impact of a long‐term consumption of low (18.99%), medium (25.34%) and high (45.77%) protein foods on the body composition, metabolites and gut microbial composition of 30 adult dogs. All dogs were maintained on a prefeed food for 14 days and were randomly assigned to one of the 6 different groups of 5 dogs. Each group consumed the low, medium and high protein foods, each for 90 days, in a distinct sequence following the William's Latin Square Design. Body composition and levels of metabolites and microbial composition were assessed at the end of each 90 days feeding period. The body composition of the dogs was not affected by the varying levels of protein consumption. However, dogs had increased circulating levels of urea and indole sulfates after the consumption of the high protein food compared to the lower protein levels. High protein consumption also led to increased fecal levels of indole and branched‐chain fatty acids and reduced levels of the short‐chain fatty acids butyrate, propionate and lactic acid. Further, the consumption of high protein food increased the proportions of the genera Clostridium and Streptococcus that are implicated to have negative health associations. This was accompanied by a reduction in the beneficial bacteria belonging to the genera Prevotella and Bifidobacterium. The results suggest that long‐term consumption of high protein foods is associated with negative microbial and metabolic profiles.

Support or Funding Information

The study was funded by Hill's Pet Nutrition, Inc. Topeka, Kansas, United States of America

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the CRISPR‐Cas13b systems in Porphyromonas gingivalis
Authors: Zhou Xia , Zhu Jiang , Du Zhihua , Huang Xiaolan
Published: June 2021

CRISPR‐based technologies have revolutionized biochemical and biomedical research. In the past few years, many CRISPR systems have been discovered and characterized. These CRISPR systems greatly expand the CRISPR toolkit. Cas13b is the most recently identified CRISPR system. The usefulness of the Cas13b system as a gene editing tool has been demonstrated in human cells. Cas13b system can be exploited to achieve not only RNA knockdown, but also highly specific and efficient RNA editing. The RNA editing ability of the Cas13b‐based technology has been used for the correction of human disease‐relevant mutations. Despite the rapid progress of Cas13b research, little is known about the structures and catalytic mechanisms of this important family of CRISPR enzymes. A cross‐disciplinary research approach is employed in our study to characterize the two CRISPR‐Cas13b systems in Porphyromonas gingivalis (Pgi5 and Pgi8 Cas13b systems). Both Cas13b enzymes possess dual RNase activities for pre‐crRNA processing (to produce the mature crRNAs) and target RNA cleavage. The systems are capable of mRNA knockdown in human cells.

Support or Funding Information

1. SIUC Seed Grant

2. NIH Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Celecoxib Inhibits Proliferation, Mitochondrial Respiratory Rate, and Membrane Potential in Myoblasts
Authors: Kolb Alexander , Reynoso Marinaliz , Geddis Alyssa , Matheny Ronald
Published: June 2021

Nonsteroidal anti‐inflammatory drugs (NSAIDs) are commonly prescribed for their analgesic and anti‐inflammatory properties, and their use is widespread in the general, athletic, and military populations. It is relatively well‐documented that long‐term consumption of NSAIDs is associated with increased risk of heart attack, stroke, gastrointestinal bleeding, and kidney disease; however, much less is known regarding the positive or negative effects of NSAIDs in skeletal muscle. Given this, we performed a series of experiments to determine the degree and mechanisms through which the NSAID celecoxib perturbs cultured skeletal muscle cell proliferation, intracellular signaling, and mitochondrial function. Myoblasts were incubated with celecoxib or NS‐398 (a selective COX2 inhibitor) for 48‐h. Cell viability was tested using 3‐(4,5‐Dimethylthiazol‐2‐Yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) assay, anabolic signaling pathways were evaluated by Western blot, mitochondrial metabolism was determined via cytochrome c oxidase activity assay, and mitochondrial membrane potential was determined by membrane‐sensitive JC‐1 dye. First, cells were treated with celecoxib at concentrations ranging from 200 μM to 0.1 μM. Cell viability was significantly depressed (100% to 19%; P<0.01) by celecoxib in a dose‐dependent fashion at concentrations from 200 μM to 3.125 μM. Myoblasts treated with NS‐398 showed no decrease in viability. Phosphorylation of ribosomal protein S6 (S240/244) was decreased by 77% in myoblasts treated with 50 μM celecoxib (P<0.05), whereas treatment with NS‐398 had no effect on S6 phosphorylation. Celecoxib inhibited the mitochondrial respiratory rate of the following Kreb's cycle intermediates (percent reduction in parenthesis): cis‐aconitic acid (25%; P<0.01), alpha‐keto‐glutaric acid (50%; P<0.05), succinic acid (45%; P<0.05), and L‐malic acid (37%; P<0.05). Fifty and 25 μM celecoxib reduced mitochondrial membrane potential by 40% and 25%, respectively (P<0.001 for both) Together, these data demonstrate that decreased myoblast viability following celecoxib treatment was associated with reductions in proliferative signaling, mitochondrial enzyme activity, and membrane potential. The mechanism(s) and degree to which celexoxib‐induced perturbations of mitochondrial enzyme activity influence myoblast viability remain to be determined.

The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. This abstract has been approved for public release with unlimited distribution.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of MAP kinase docking specificity with yeast genetic screens
Authors: Shi Guangda , Turk Benjamin
Published: June 2021

Mitogen‐activated protein kinase (MAPK) pathways play pivotal roles in making vital cellular decisions. In order to carry out their unique functions, each MAPK recognizes its own repertoire of substrates. Aside from a rather degenerate phosphosite motif (S/T‐P), MAPKs utilize a non‐catalytic site docking interaction to specifically recognize their substrates as well as their regulators. Docking interactions occur between a linear docking site peptide (D‐site) and a conserved docking groove on the MAPK. While a general D‐sites sequence motif has been described, unique features of sequences that specifically target individual MAPKs is not completely understood. Investigations into these interactions is difficult due to the weak and transient nature of the interaction.

Here I present screening methods using S.cerevisiae to identify functional docking site sequences that direct to specific MAPKs. I found that co‐expressing a mammalian MAPK and its MKK activator in yeast leads to growth suppression. I have used this phenomenon as the basis of a pooled yeast genetic screen, where cells expressing MKK variants compete in one pooled culture. The change in representation of every single D‐site in the culture over time is monitored by next‐generation sequencing. Variants having more functional D‐sites activate the pathway better, grow slower, and are depleted from the population. I generated and screened a library of ~12,000 candidate D‐sites found in the human proteome against the MAPKs p38α and JNK1.

These screens revealed sequence motifs that allow discrimination between the two MAPKs, and also defined a D‐site dependent MAPK interactome which contains many new targets that could broaden our understanding of MAPK biology. Our screens serve as guides to more completely understand weak interactions that play an important factor in the dynamic nature of MAPK signaling networks.

Support or Funding Information

This research was supported by National Institutes of Health grant R01 GM104047.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein phosphatase 2A‐B56 controls mitosis by interacting with LS/TPI/V motif containing protein interactors
Authors: Chaudhuri Sibapriya , Moorhead Gregory
Published: June 2021

Mitosis, like many other cellular processes, is tightly regulated by reversible protein phosphorylation which is achieved by coordinated activities of mitotic protein kinases and mitotic protein phosphatases. PP2A‐B56 is an important mitotic protein phosphatase which contributes to the timely execution of several mitotic events. Recent proteomic studies show that PP2A‐B56 recognizes a short linear motif, LXXIXE, on its interactors. We hypothesize that LS/TPI/V is a consensus motif for the recruitment of PP2A‐B56 in an isoform‐dependent manner, particularly during mitosis. We also speculate that this motif is phosphorylated during mitosis and dephosphorylated at mitotic exit. Recruitment of PP2A‐B56 is regulated by the phosphorylation status of this motif and this, in turn, plays an important role in controlling the progression of the cell cycle. Our findings show that LS/TPI/V motif is present in more than 100 mitotic regulators and several of them interact with PP2A‐B56 in a phosphorylation‐dependent and isoform‐dependent manner. Further understanding of how interactions between the target proteins and PP2A‐B56 affect mitotic progression and/or mitotic exit will provide valuable insight into the mechanisms of specific recruitment and mitotic functions of PP2A‐B56.

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 MAL/SRF Pathway Regulates Desmosome Protein Expression and Localization in Cancer Cells
Authors: Dubash Adi D , Eldredge Luke
Published: June 2021

The desmosome (DSM) is a cell‐cell adhesion complex required for the mechanical stability of tissues and regulation of other biological processes, such as proliferation and migration. Proteins from three families make up the complex: Transmembrane cadherins connect adjacent cells, plaque proteins stabilize attachment on the intracellular face, and the plakin protein desmoplakin (DSP) anchors the DSM complex to the intermediate filament cytoskeleton. Many studies have reported diverse mechanisms by which cancer progression alters DSM gene expression, but the signaling pathways that govern the expression and localization of DSM constituent proteins remain elusive. Recent work has identified the transcription factor serum response factor (SRF) as a regulator of mRNA levels and localization of DSM proteins such as the cadherin Desmoglein‐1. Here, we treated several cancer cell lines with siRNA and a pharmacological inhibitor (CCG‐1423) of SRF and its cofactor, MAL, to broadly investigate the role of these transcription factors in regulating DSM gene expression and protein localization. Our results demonstrate that abrogation of MAL/SRF signaling through both siRNA and pharmacological inhibition results in a decrease in mRNA levels of DSP. In addition, localization of DSP to borders is also reduced upon inhibition of MAL/SRF signaling. We observed similar changes for the plaque proteins Plakophilin‐2 and Plakoglobin, but not for desmosomal cadherins. As DSP is required to maintain the strength of DSM attachments, we also investigated the role of MAL/SRF signaling on the adhesion strength between cells. Our data highlight a novel link between MAL/SRF signaling and DSP expression, and contribute to a growing understanding of the variety of signaling pathways involved in mediating DSM gene expression.

Support or Funding Information

This project was funded by an NIH South Carolina IDeA Networks of Biomedical Research Excellence (SC INBRE) grant #5P20GM103499‐17 which provided a Developmental Research Project (DRP) sub‐award #22050‐ZA14 to AD.

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 Phylogenetics of Rhipicephalus sanguineus and its Role as a Vector of Rocky Mountain Spotted Fever
Authors: Schaefer Charles , Allen Jonathan , Yao Tony , Owen Haley , Lisowski Sasha , VandenBrooks John
Published: June 2021

Rocky Mountain Spotted Fever (RMSF) is a tick‐borne disease caused by the intracellular bacteria Rickettsia rickettsii. The traditionally accepted vectors for this disease include ticks from the genus Dermacentor, however this genus is not endemic to the site of a recent outbreak in Arizona. Rhipicephalus sanguineus, also known as the Brown Dog Tick, is an ectoparasite that can be found throughout the United States. It has been hypothesized that this species exists as several distinct groups that form what can be more accurately described as a species complex. The Brown Dog Tick has been linked to this recent outbreak of RMSF in Arizona, despite no previous evidence of this species acting as a vector elsewhere in the US. Given the widespread habitat of the Brown Dog Tick, its potential as a vector for RMSF is a pressing public health risk. In this study, we sought to determine if differences associated with the genetic diversity of the Brown Dog Tick are responsible for its role as a vector. Ticks identified as R. sanguineous (n=241) were sorted into lineages based on phylogenetic analysis of a concatenated sequence of the 16S, 12S and COX1 genes. We used “Tropical” and “Temperate” marker sequences from identified samples on genbank (Table 1). Our results demonstrate the presence of multiple clades geographically distributed Arizona beyond just the “Tropical” and “Temperate” delineation. These tick samples were also tested for infection with R. rickettsii using PCR for the R. rickettsii OMPA gene. However, there was no significant difference between the phylogenetic tick clades (Chi‐squared, p=0.21). This suggests the genetic lineage of the vector tick is not the primary factor responsible for the outbreak of RMSF in Arizona and the surrounding regions.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Microbial Co‐occurrence Patterns and Keystone Species in the Gut Microbial Community of Mice in Response to Stress and Chondroitin Sulfate Disaccharide
Authors: Li Robert W. , Liu Fang , Li Xhaojie , Xue Changhu , Tang Qingjuan
Published: June 2021

Detecting microbial interactions is essential to the understanding of the structure and function of the gut microbiome. In this study, microbial co‐occurrence patterns were inferred using a random matrix theory based approach in the gut microbiome of mice in response to chondroitin sulfate disaccharide (CSD) under healthy and stressed conditions. While the overall size and structure of the global networks were similar, the network composition was distinctly different among various experimental conditions. The exercise stress disrupted the network composition and microbial co‐occurrence patterns. 34 Operational Taxonomic Units (OTU) were identified as module hubs and connectors, likely acting as generalists in the microbial community. Mucispirillum schaedleri acted as a connector in the stressed network in response to CSD supplement and may play a key role for bridging intimate interactions between the host and its microbiome. Several modules strongly correlated with physiological parameters were detected. For example, Modules M02 (under stress) and S05 (stress + CSD) were strongly correlated with blood urea nitrogen levels (r = 0.90 and − 0.75, respectively). A positive correlation between node connectivity of the OTUs assigned to Proteobacteria with superoxide dismutase activities under stress (r = 0.57, P <0.05) provided further evidence that Proteobacteria can be developed as a potential pathological marker. Moreover, the results suggest that altered network topologies may have important functional implications. Our findings provided novel insights into microbial interactions in gut microbial communities of the mice in response to stress and CSD supplement and may facilitate future endeavor in microbial community engineering.

Support or Funding Information

National Natural Science Foundation of China (No. U1606403 to CX)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Preliminary Results of Assessing the Impact of a High School Science Outreach Program on the Confidence, Attitudes, and Skills of the Participants
Authors: Calderon Irene , Mina Rina
Published: June 2021

Science outreach has been shown to enhance science education and cultivate students' interest in science. The Summit Country Day School, an independent Cincinnati‐based co‐educational K‐12 school, established a Milwaukee School of Engineering (MSOE) Center for Biomolecular Modeling Modeling a Protein Story (MAPS) team this school year, with the overall goal of increasing the scientific and technical skills of its participants. Through this 22 week program, the participants studied the structure‐function relationship of an assigned protein (Aquaporin) by researching relevant scientific articles and using software (Jmol) for molecular modeling to 3D print the protein structure. Seven underclassmen participated; two science faculty members facilitated and mentored. A Likert survey was developed to assess and compare the confidence, attitudes, and skills of the participants. Specifically, the tool evaluated these learning goals: 1) selecting relevant scientific articles, 2) assessing important points in the selected articles, 3) presenting the summary of the articles and their ideas to the group, 4) writing a scientific abstract, and 5) managing computer software for molecular modeling for 3D printing. The tool was administered at the beginning and halfway through the program, with planned administration at its conclusion. Wilcoxon signed‐rank test was performed to assess statistically significant differences between the survey responses at the two time periods. The survey findings demonstrated no statistically significant differences between the responses at the beginning of the program and at the halfway through the program. However, there were overall trends for improvement in the confidence, attitudes, and skills of the participants, especially regarding understanding scientific articles (Fig. 1). In conclusion, participating in a MAPS team generally increases the confidence, attitudes, and skills of the participants in regard to scientific activities. The lack of statistical significance may be because of the short duration of the program and small sample size.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TmAFP Interaction with an Ice Lattice
Authors: MacGillivray Brendan , Greig James , Bai Kathleen , Fu Celine , Fujikawa Ayaka , Hopkins Graeme , Illianu Richard , Kim Anna , Lalapet Tarun , Miao Crystal , Ramirez Victoria , Vo Minh Thu
Published: June 2021

The mealworm beetle, Tenebrio molitor, has an antifreeze protein (TmAFP), which keeps it from freezing solid in sub‐zero temperatures. TmAFP prevents the growth of ice crystals by using repeating coils of a highly conserved 12‐amino acid loop sequence to bind to the ice surface. This structure creates a regular array of threonine‐residues which form specific hydrogen bonds with the ice lattice. As a small protein (8.4 kDa) with this repeating structure, TmAFP is among the simplest AFP structures to be crystallized. This structural simplicity may make it the perfect candidate to use as a tool in prolonging organs that have been harvested for transplant. A current problem is that organs are only viable for transplant for a day at the most. The best way to keep organs viable for prolonged periods of time would be to store them at freezing temperatures. However, if the organ freezes, ice crystals will kill cells and destroy the tissue. By introducing antifreeze proteins into organs before harvesting, the hope is to extend the lifespan of the organ for transplantation. The Ashbury College MSOE Center for BioMolecular Modeling SMART Team used 3D modeling and printing technology to examine structure‐function relationships between TmAFP and ice formation.

Support or Funding Information

MSOE ‐ Center for Biomolecular Modelling ‐ co‐sponsor Diane Munzenmaier

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Flow Cytometry Analysis of Anastasis in Cultured Mammalian Lymphocytes
Authors: Moran Colleen Joy , Lauzon Robert
Published: June 2021

Apoptosis is a physiological form of programmed cell death characterized by phosphatidylserine externalization, caspase activation, and genomic DNA fragmentation. Recent studies have shown that apoptosis may be reversed even in its late stages, calling the previous notion of a ‘point of no return’ into question. This recovery process has been termed anastasis (‘rising to life’), but its underlying mechanisms are not well understood. Our lab has developed a model of studying anastasis using cultured mouse HT‐2 lymphocytes dependent on the cytokine interleukin 2 (IL2). Our previous findings have shown that HT‐2 cells accumulate in the G1 stage of the cell cycle following 24 hours of IL2 deprivation and can be rescued by adding IL2 back into the culture medium. In this study, we have used quantitative flow cytometry to investigate which apoptotic events are congruent with anastasis. HT2 cells were deprived of IL2 for 24 hours and then rescued for periods of 0, 24, 48, and 72 hours. CD25 surface expression (high‐affinity IL2 receptor) and phosphatidylserine externalization (annexin V staining) were subsequently monitored prior to and following recovery, as markers of T cell activation and early stage apoptosis, respectively. Control HT2 lymphocytes continuously maintained in IL2 comprised a single viable population of CD25HIGHAnnexin−cells. Cells deprived of IL2 for 24 hours were observed to fall into two populations: CD25LOWAnnexin+early apoptotic and CD25LOWAnnexinnon‐apoptotic cells. In both populations, CD25 expression was reduced approximately 10‐fold when compared to IL2‐maintained controls. During anastasis, three distinct viable cell populations emerged: 1) CD25LOWAnnexin+cells that did not progress into the later stages of apoptosis, 2) CD25LOWAnnexin−non‐apoptotic cells, and 3) CD25HIGHAnnexin−non‐apoptotic cells. With advancing recovery time, viable CD25LOWpopulations decreased as CD25HIGHcells increased. By 72 hours post‐recovery, the majority of cells were actively cycling, annexin negative cells. These findings suggest that IL2‐dependent mouse HT‐2 lymphocytes may be a promising model to study the mechanisms of anastasis.

Support or Funding Information

Union College Faculty and Student Research Grants

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