bluuebunny/crossref_metadata_2025_split huggingface dataset:
Understanding the Thermodynamic and Kinetic Properties of the Thermophilic Oxoglutarate Carboxylase BC domain to Increase Activity for use in Biofuel Crops
Authors: Dillard Lucas , Rose Robert 
Published: June 2021

For many biofuel crops, carbon fixation is limiting –making the production of these alternative fuels economically unviable. The first green revolution has led to significant advances in crop yields in the past half century. Crop yield is now mostly limited by plant biology. To overcome this limitation, a condensed reverse TCA (crTCA) cycle has been developed in the workhorse species camelina that rapidly fixes atmospheric carbon. The biotin‐dependent carboxylase Oxoglutarate Carboxylase (OGC) represents the slow step of the crTCA cycle engineered in camelina. OGC –a thermophile –is 500‐fold less active at mesophilic temperatures than at its optimal 70 °C. Engineering OGC to preserve high activity at mesophilic temperatures presents an opportunity to further improve the rate of carbon fixation in the crTCA pathway, and possibly increase yields in oil crops –reducing the cost of biofuels.Biotin‐dependent carboxylases such as OGC are a ubiquitously found family of proteins that consist of three domains: The Biotin Carboxylase (BC) domain, Biotin Carboxyl Carrier Protein (BCCP), and the Carboxy Transferase (CT) domain. We choose to analyze the BC domain as previous studies suggest the rate limiting step of the enzyme to result from 1) conformational changes associated with lid closure in the domain or 2) deprotonation of a species within the active site. We report kinetic and thermodynamic ATPase activity measurements of the thermophilic OGC BC domain measured via discontinuous assay coupled with direct quantitation of ADP production via HPLC. ATP concentration dependent activity was observed at temperatures ranging from 48 °C to 76°C, with kcat at 76 °C being 93 min−1. A subsequent Eyring plot was generated to measure the free energy of the transition state of OGC BC. Along with this, kinetic deuterium isotope effect experiments are being performed to measure energetics of deprotonation in the active site. Lid motions are also currently being measured with FRET. These studies will aid in the identification of mutations that may increase the activity of the BC domain at mesophilic temperatures and facilitate the use of OGC to increase biofuel crop yields.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Detection of Celiac Disease Autoantibodies with a Rapid and Noninvasive Diagnostic Biosensor
Authors: Maldonado Marcos , Nguyen Anna , Bonham Andrew J 
Published: June 2021

Celiac Disease (also known as gluten‐sensitive enteropathy and nontropical sprue) is estimated to affect between 0.5–1.0% of the general population of the United States (1.6 million to 3.3 million during 2017) with many of these cases being under‐diagnosed. Celiac Disease (CD) is the genetic hypersensitivity to ingested exogenous gluten. Subsequent exposure to gluten related proteins lead to the development of an autoimmune response, causing damage to the lining of the gastrointestinal tract. Due to increased inflammation, symptomatic individuals present with abdominal pain, diarrhea, nausea, and many other gastrointestinal complications. However, some individuals affected by CD are asymptomatic creating increased diagnostic error. An accurate diagnosis of CD remains challenging; current diagnostic procedures involve multi‐day serological screening, followed by invasive gastrointestinal biopsies of suspected lesions. Baring this in mind, there is a pressing need for a rapid, non‐invasive, and accurate detection method to diagnose individuals suffering from this disease. Our method is based on the known autoimmune interactions related to CD. Specifically, we observe the formation of a structured complex between a unique epitope—a chimera of tissue transglutaminase 2 and truncated gluten peptide—and the autoantibodies produced in response to the presence of gluten related proteins. Conducted within our research, we demonstrate a CD‐related diagnostic tool for early, accurate, and rapid disease identification of affected individuals. This tool is a electrochemical DNA‐based biosensor (E‐DNA) that relies on peptide epitopes acting as capture probes to detect levels of disease‐specific antibodies. This commonly used biosensor is utilized to detect small changes in electrical conductivity to determine whether or not a target molecule has been bound. In principle, this sensor design can be utilized for unprocessed finger‐lancet blood samples to provide a rapid digital readout, allowing for efficient and accurate diagnoses of CD affected patients.Support or Funding InformationThis work is supported by internal grant funding from the Metropolitan State University of Denver.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Design and Application of a Mutational Sensor for Characterizing AID Activity
Authors: Woods Allison Taylor , Zelazowska Monika , Plummer Joshua , Mu Yunxiang , McBride Kevin 
Published: June 2021

Activation‐induced cytidine deaminase (AID) is a mutator enzyme that introduces uracil:guanine mismatches in Immunoglobulin (Igh) genes during the antibody diversification processes of class switch recombination and somatic hypermutation in B lymphocytes. When this happens, one of three pathways is possible. Firstly, the DNA can be directly replicated with the mutation, causing a transition (C → T) mutation. Secondly, during genomic mutation, the repair enzyme Uracil N‐Glycosylase (UNG) can act to base excise the mutation in either an error free fashion, replacing the original cytidine in the mutated spot, or thirdly, an error prone fashion, in which a transversion mutation (C → G/A) will then be introduced. Certain “hotspots” in the DNA sequence increase mutational frequency: cytosine residues are strongly favored when they are part of an RGYW sequence, or its reverse complement, sequence WRCY (where W=A/T, R=A/G, and Y=C/T). However, AID can also produce off target damage to genes, including oncogenes, that result in lymphomas. Therefore, tight regulation of AID mutator activity is necessary for genomic stability. In this study, we designed a cellular mutation reporter assay to determine the role of AID in regulating mutational activity. Mutations introduced by AID were measured with the use of constructs containing a green fluorescent protein (GFP) with premature STOP codons which‐ upon mutation‐ will “turn on” GFP and can be screened by flow cytometry. The use of this construct allows for mutational screening to be done in any cell type in which AID is naturally occurring, such as lymphomas and leukemias, rather than having one cell type with the reporter gene built into the cell line (current method). Once the constructs were successfully created via PCR SOEing reactions, we verified their ability to serve as mutational sensors. To achieve this, cells were co‐transfected with constructs and AID into BOSC cells and analyzed for rates of nucleotide reversion via flow cytometry. AID is shown to reproducibly revert two constructs with premature stop codons in contextual hotspots from GFP− to GFP+, as seen by the increased presence of GFP when analyzed via flow cytometry. The data show that the GFP mutational sensor works and serves as a functional system for determining mutational analysis among BOSC cells via flow cytometry analysis.Support or Funding Information1. Allison Woods is an undergraduate scholar supported in part by the MARC Program at St. Mary's University and the Summer Program in Cancer Research at MD Anderson Cancer Center support by National Cancer Institute (R25CA181004).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Detection of Mycoplasma secreted protein P48 via Electrochemical DNA‐based Biosensor
Authors: James Anika , Bonham Andrew J 
Published: June 2021

Mycoplasma bacteria are highly infectious agents of human disease and laboratory contamination. For example, Mycoplasma pneumonia infects almost two million people every year with contagious upper respiratory infections also known as atypical or ‘walking pneumonia’. In addition to human disease, Mycoplasma is a major source of contamination of laboratory human cell cultures. However, current methods of detection for Mycoplasma strains, such as molecular‐based assays, PCR and serological analysis, are time consuming, expensive, and less suitable for working under stringent conditions such as extreme temperature or pH. To provide a rapid and accurate alternative, we have focused on detecting the presence of Mycoplasma with a diagnostic electrochemical biosensor that detects lipoprotein P48, which is shed from the surface of several strains into the surrounding blood serum or growth media. This biosensor is based on a previously identified DNA aptamer that binds to the secreted P48 protein with high affinity. We have expressed P48 recombinantly in E. coli to serve as a positive control, and our results show rapid and sensitive binding of the target, as well as convenient electrochemical signaling from these binding events. This approach will allow us to potentially improve both prevention and diagnosis of Mycoplasma in cell culture platforms as well as patients who present a proposed infection.Support or Funding InformationThis work is supported by internal grant funding from the Metropolitan State University of Denver.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biodegradation of Surgical Polypropylene Meshes
Authors: Fiedler Anna K 
Published: June 2021

Surgical meshes of a variety of materials, such as polypropylene, are commonly used in many procedures to support tissues. From our previous studies on polypropylene meshes, samples of mesh removed from patients often demonstrated biodegradation proportional to the amount of time that had passed since implantation. To expand this field of study, we tested the mesh sample in E. coli media to observe if degradation would occur in a controlled, single‐organism environment. Mesh samples were immersed in either an E. coli media or a control solution; the biodegradation was measured at one‐, two‐ and three‐month intervals. At this point the samples were harvested from both E. coli media and the control media for analysis, and results were then compared. A scanning electron microscope was used to identify surface defects and capture images of the material deterioration. Additionally, infrared spectroscopy was used to qualitatively identify oxygen present in the samples, while energy dispersive X‐ray spectroscopy quantifiably determined oxygen levels and degradation. Results from this study demonstrate that bioerosion and increased oxygen content can be observed in the mesh structure due to E. coli activity. Future plans include the synthesis of a monomer with collagen as a functional group to improve interactions with living tissues while maintaining structural integrity.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 diversity of Baeg‐nyong cave and characterization of the antibiotics extracted from Streptomyces exfoliatus
Authors: Park Shinae , Cho Yong‐Joon , Jung Da‐yea , Jo Kyung‐nam , Lee Jung‐Shin 
Published: June 2021

Baeg‐nyong (BN) cave is a one of the natural cave and critical zone observatory (CZO) in the Republic of Korea, but the role of microbial species in BN cave is still not well‐known. To analyze the microbial diversity of the cave, we collected a moonmilk, which is a white deposit of limestone cave. Because moonmilk has different textures depending the moisture contents, we performed metagenomics for two moonmilks (Dry and wet moonmilk) by next generation sequencing (NGS). The results of metagenomics revealed that moonmilks have very different microbial community and dominant species to each other. Specifically, Chromatiales, Pseudomonas, and Steroidobacter groups are present in high percentages in dry moonmilk, and Xanthomonadales and Arthrobacter group are dominant bacteria in wet moonmilk. Specifically, Streptomyces species of Actinobacteria are only present in dry moonmilk, and surface analysis showed that only dry moonmilk contained round‐shaped calcite structures, which might be affected by Streptomyces. Therefore, we isolated nine Streptomyces spp. from dry moonmilk and characterized their morphological, cultural, physiological, and biochemical properties. Among the nine Streptomyces species, only one species, as known as Streptomyces exfoliatus, showed the antimicrobial activity against to some bacteria including E. coli and B. subtilis. In addition, we extracted the antimicrobial compound from S. exfoliatus and analyzed HPLC and various assays to establish the antibiotics of S. exfoliatus isolated from BN cave.Support or Funding InformationThis research is supported by NRF‐2015R1D1A1A02061743 and NRF‐2015R1A4A1041105.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Palmitic Acid Stimulates Apoptosis and Extracellular Vesicle Release in Renal Proximal Tubule Epithelial Cells
Authors: Cobbs Alyssa A , Zhang Yuanyuan , Chen Xiaoming , George Jasmine , Huang Ming Bo , Bond Vincent , Zhao Xueying 
Published: June 2021

Abnormal levels of lipids in the blood are associated with a myriad of chronic diseases. Lipotoxicity contributes to kidney deterioration through cellular dysfunction and apoptosis in renal epithelial cells. A predominant saturated fatty acid, palmitic acid, stimulates the release of extracellular vesicles (EVs) in hepatocytes and induces an inflammatory phenotype in macrophages. However, the role of EVs in the progression of chronic kidney disease remains vaguely explored. This study was aimed to investigate whether palmitic acid increases the release of EVs in renal tubular epithelial cells. Rat proximal tubular epithelial cell line, NRK‐52E, was incubated with 1% bovine serum albumin (BSA), BSA‐conjugated palmitic acid (PA), and BSA‐conjugated oleic acid (OA) for 24–48 hours. The EVs released into conditioned media were isolated by ultracentrifugation and quantified by nanoparticle‐tracking analysis (NTA). According to NTA, the size distribution of EVs was 30–50 nm with similar mode sizes in all experimental groups. Moreover, BSA‐induced EV release was significantly enhanced in the presence of PA, whereas EV release was not altered by the addition of OA. PA‐enhanced EV release was associated with an induction of cell apoptosis reflected by an increase in cleaved caspase‐3 protein levels and Annexin V positive cells analyzed by flow cytometry. Additionally, confocal microscopy confirmed the uptake of lipid‐induced EVs by recipient renal proximal tubular cells. Collectively, our results indicate that PA stimulates EV release from cultured proximal tubular epithelial cells. Thus, extended characterization of lipid‐induced EVs may constitute new signaling paradigms contributing to chronic kidney disease pathology.Support or Funding InformationThis work was supported by NIH/T32 (5T32HL103104‐07), NIH (SC1DK112151), NIH/NCRR/RCMI (8G12MD007602) and (8U54MD007588)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Studying the Role of Zinc (Zn2+) in Cellular Transitions Under Acidic Conditions
Authors: Hu Yuli , Case Maddie , Lickfelt Megan , Li Yang V. 
Published: June 2021

The maintenance of ionic homeostasis is critical for cell metabolism, cell growth, and cell survival. It has been shown that pH and zinc dyshomeostases are linked to pathophysiological conditions such as cancerous mutations, urinary diseases, gastric infections, and ischemia. In particular, we observed that there were concentration increases of intracellular free zinc after acidic treatments. We further hypothesize that zinc may be a mediator for the acidic treatment‐induced cellular changes. A spindle shape or fibroblast‐like shape has been used as an indicator of decreased cell‐to‐cell adhesion, increased cellular motility, and an increased possibility of metastasis in cancer. Our preliminary experiments showed that the accumulation of spindle cells occurred after acidic treatments and that zinc chelation reduced the accumulation, indicating the role of Zn2+ in the accumulation of spindle cells. Spindle cell accumulation is a sign for cancer cell synchronizations at a particular phase in cell cycle, which provide windows for potential cancer therapeutics that target cell cycles. Our preliminary experiments showed that acidic treatments enhanced the expression of vimentin, which was attenuated by an application of zinc chelator, suggesting that zinc plays an important role in cellular transitions under acidic conditions. The significance of this research is to provide a novel mechanism that zinc may be a mediator in acidic treatment‐induced cellular changes. Acidic environment is seen in physiological condition such as gastric ulcer, and in abnormal conditions such as ischemia and cancer. It will be interesting to see how zinc is involved in these processes.Support or Funding InformationThanks to the Osteopathic Heritage Foundations, Graduate Assistantship Program at Ohio University Heritage College of Osteopathic Medicine for supporting graduate student Yuli Hu.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
LY294002‐induced growth inhibition of cancerous cells is inked to downregulation of MEK‐ERK pathway and adhesion molecules
Authors: Kavege Llona , Baquier Jocelyn , Duff Angela , Hu Tang 
Published: June 2021

Phosphatidylinositol‐4,5‐bisphosphate 3‐kinases (PI3Ks) regulate several important cellular and subcellular processes including cell survival, proliferation, and differentiation. PI3K‐Akt is one of most commonly activated signal pathways in human cancer. Thus, targets PI3K provides a great therapeutic opportunity in cancer treatment. LY294002 was the first synthetic small molecule inhibitor that can reversibly target PI3K family members and several other lipid kinases. However, its effect on cellular adhesion molecules (CAMs) and its link to MEK/ERK pathway are largely unknown. In this study, we present evidence that LY294002 significantly inhibited proliferation of human myeloid leukemia (TF‐1a and MV4‐11) and epithelia cancer (Hep‐G2 and MCF‐7) cells in suspension and adherent cell culture, respectively, measured by MTT and XTT assays. LY294002 also dramatically inhibited the colony‐forming cells derived from TF‐1a, MV4‐11, and Hep‐G2 cell lines, measured by semi‐solid agar culture. The adherent culture of Hep‐G2 and MCF7 showed substantial cell detachment from the culture substrate and formation of aggregates suspended in the culture medium, leading to disruption of the cell monolayer. Similar but smaller aggregates were also found in suspension culture of TF‐1a and MV4‐11 cells. As expected that LY294002 downregulated the phosphorylation of PI3Kp85 and Akt analyzed by Western blot. Unexpectedly, the expression of MEK and ERK, two components of Ras‐Raf‐MAPK pathway and adhesion molecule, integrin‐linked kinase (ILK) and focal adhesion molecule (FAK) were also significantly downregulated in MV4‐11 cells in response to LY294002. In contrast, GM‐CSF and PMA stimulated the expression of MEK and ERK in MV4‐11 and TF‐1a cells, respectively. Addition of LY294002 and PD‐98059 blocked GM‐CSF‐and PMA‐induced MEK and ERK. Lastly, ERK immunoprecipitants from GM‐CSF‐treated cells stimulates the phosphorylation of transcription factor Elk‐1, whereas the immunoprecipitants from LY294002‐treated cells downregulates the phosphorylation of Elk‐1. Briefly, our data suggest that LY294002 inhibited growth of human cancerous cells partially due to its disruption of the adhesion molecules, which is linked to downregulation of MEK‐ERK pathway.Support or Funding InformationBarry University Faculty Stimulus Grant and NIH‐NIGMS RISE R25 GM059244‐17This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Is Less Actually More? Investigating the Synergistic Relationship between Chemotherapy Agents and NHE1 Inhibitors in Ovarian Cancer Cells
Authors: Larson Allie C , Wallert Mark A. 
Published: June 2021

Chemotherapeutic regimens continue to be the conventional therapy utilized in the treatment of ovarian cancer. These treatment plans often include the use of cytostatic medications such as paclitaxel, rucaparib, olaparib, carboplatin, and cisplatin. The mechanisms of action for each of these oncogenic therapeutics differs substantially. Paclitaxel prevents the polymerization of microtubules in an attempt to hinder mitotic completion. Rucaparib and olaparib both inhibit PARP, an enzyme associated with repairing damaged DNA. Carboplatin and cisplatin also initiate apoptosis through the inhibition of DNA repair. However, they do so by crosslinking the purine bases on DNA. Although the use of chemotherapy agents has been instrumental in reducing cancer‐related deaths, the approbation of these drugs has largely been mitigated due to the adverse side effects they procure. Furthermore, the capacity of chemotherapy drugs to repeatedly eradicate cancer is often limited because of developed resistance. It has thus become of interest to investigate methodologies for reducing not only the required chemotherapy drug dosage, but also the threat of disease recurrence. Such a synergistic effect is hypothesized to occur through the coupling of chemotherapy agents with direct inhibitors of NHE1. NHE1, or the Na+‐H+ Exchanger Isoform 1, is a transport protein that is often hyperactivated in cancer cells and contributes, through maintenance of a pH inversion, to cancer cell proliferation. Inhibition of this transport protein results in intracellular acidification, an environmental requirement for the initiation of apoptosis. Applying this inhibition with an accompanying chemotherapeutic has thus interested researchers as a possible therapeutic approach towards mitigating neoplastic development. The potential of these drug‐inhibitor pairs has been evaluated through a reduction in proliferation rates and quantified via XTT assays. The larger the growth inhibition, the more powerful the combination therapy. Ultimately, the drug‐inhibitor pairs will be applied in a spheroid assay to better assess the potential of the therapy in a three‐dimensional environment.Support or Funding InformationLueken Family Cancer FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.