bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Effects of Copper and Zinc on PSN gene expression in a Drosophila Alzheimer's Model
Authors: DeGouveia Kelsey , Rogers Ryan Patricia 
Published: June 2021

Based on the current trajectory of Alzheimer's disease, the number of Americans affected are said to increase by 13.5 million people, approximately 16% of the elderly population, by the year 2050. [1] Alzheimer's disease is characterized by the presence of amyloid‐β plaques and neurofibrillary tangles resulting in loss of cognition, memory, behavior, and locomotion.[2] Amyloid‐β plaques are proteolytic fragments of the amyloid precursor protein (APP), which is a resultant of processing through the amyloidogenic pathway. [2,3,4] It is believed that elevated consumption of copper or zinc will increase or decrease the toxicity of amyloid‐β in the brain respectively due to its interactions with presenilin, a component of the γ‐secretase complex responsible for the cleavage of APP.[2,3,4] In order to begin mapping out the amyloidogenic pathway, levels of the PSN gene, the Drosophila melanogaster ortholog responsible for coding for the presenilin protein. In order to begin mapping out the amyloidogenic pathway, levels of the PSN gene will be quantified over various time points and under different dietary conditions. It is hypothesized that increased copper consumption will upregulate the expression of PSN, promoting the amyloidogenic cleavage of APP, therefore speeding up the progression of the disease, while zinc will have an opposite effect and potentially break down amyloid‐β aggregates. The proposed experiments will ultimately develop a more efficient model of Alzheimer's disease in Drosophila melanogaster by monitoring physical and genetic effects of dietary changes.Support or Funding InformationThis work was supported by funds from the Department of Sciences at Wentworth Institute of Technology.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Relating Collapse to Sequence and Behavior in Glutamine‐Rich Domains
Authors: Garelli Colton J. , Scott Adam S. , Ellis Jamie P. 
Published: June 2021

Intrinsically disordered proteins, or IDPs, are ubiquitous throughout eukaryotic proteomes with particularly important roles in biosignalling and transcription regulation. Within this category, glutamine‐rich (Q‐rich) domains appear commonly within transcription activators. Q‐rich domains are known to collapse rather than fold upon binding and have a high tendency to aggregate. Their behavior appears to be mediated by solvent interaction with some evidence that water is trapped within the collapsed domains. These traits appear important in gene activation, yet effective pathways to evaluate the behavior of Q‐rich domains remain elusive. In this work, we develop fluorescence methods to experimentally observe the solvent exchange between bulk media and trapped water within collapsed Q‐rich domains. In conjunction with experimental observation, we have evaluated constructs for known parameters including disorder, solubility, and aggregation propensity to directly align experimental results with available prediction systems. We composed a library of disordered constructs from known transcription activators and repressors. Specifically, we selected peptides from within the Auxin response factor (ARF) family of proteins in Arabidopsis thaliana to utilize a biologically characterized genome with natural sequence variation including percent glutamine content. The control used was κ‐casein, an inexpensive protein that has been previously shown to collapse and trap water. We optimized a general expression, purification, and labeling protocol to isolate the library of constructs with a solvent‐sensitive fluorophore. Our methods allow us to directly observe both solvent exposure and exchange on constructs of varying glutamine content. This work provides important new tools to measure and compare Q‐rich domains. These tools can be applied to clarify the relationship between sequence and behavior in Q‐rich domains, specifically the biophysical implications of peptide‐solvent interactions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Engineering the PETase Enzyme to More Efficiently Break Down PET Plastics
Authors: Duplan Amanda , Hall Bonnie L. 
Published: June 2021

Polyethylene terephthalate (PET), a thermoplastic polyester resin containing a high ratio of aromatic components, is widely used in plastic products. PET is unreactive and difficult to degrade, and thus overproduction and improper disposal methods have caused the world's oceans and ecosystems to be polluted with plastic waste. In 2016, Yoshida et al. discovered Ideonella sakaiensis, a bacterium able to use PET as a carbon source for growth. The enzyme PETase was found to break down PET plastics, but at a slow rate. Our goal is to engineer a PETase with a higher efficiency of PETase hydrolysis. The first step in the process is overexpression and purification of the PETase protein. Colorimetric and liquid chromatography assays will be optimized for determining the rate and efficiency of PET breakdown. PETase mutants will then be engineered to improve enzyme activity under the desired conditions. An optimized PETase enzyme capable of degrading plastics could be the first step to repairing our polluted ecosystems.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biophysical Characterization of Aptenodytes forsteri Cytochrome P450 19 (Aromatase)
Authors: Zarate‐Perez Francisco , Velázquez‐Fernández Jesús B. , Jennings Gareth K. , Shock Lisa S. , Lyons Charles E. , Hackett John C. 
Published: June 2021

Cytochrome P450 19 (aromatase) catalyzes the conversion of androgens to estrogens in a sequence of three reactions that each depend on NADPH and O2. As the terminal enzyme in estrogen biosynthesis, it is critical for the maintenance of myriad tissues and has emerged as a successful target for the treatment of estrogen‐dependent breast cancer. Aromatase is a phylogenetically‐ancient enzyme and its breadth of expression in other species has highlighted distinct physiological functions. In songbirds, estrogen production is required for programming of neural circuits controlling song. Aromatase also plays a role in sex determination of fish, reptiles, and amphibians. Biophysical characterization of the human enzyme has been limited by low yields from recombinant expression systems. Accordingly, we sought to identify an aromatase homolog with enhanced expression properties in the absence of stabilizing ligands. This work describes the expression, purification, and biophysical characterization of Aptenodytes forsteri (Emperor penguin) aromatase (afCYP19A1). Using human cytochrome P450 reductase as a redox partner, the enzyme kinetic parameters for turnover of androstenedione were like the human enzyme and mass spectrometry analysis conformed that afCYP19A1 likewise catalyzes the transformation through 19‐hydroxy‐ and 19‐oxoandrostenedione intermediates. Equilibrium binding studies revealed that androstenedione and anastrozole had the highest affinity for the enzyme and were closely followed by 19‐hydroxy‐androstenedione and testosterone. The affinity of 19‐oxoandrostenedione was an order‐of‐magnitude lower. Ligand‐binding kinetics were determined by stopped‐flow UV‐visible spectroscopy. Both androstenedione and testosterone where determined to bind to the enzyme in a step‐wise fashion with a spectroscopically‐detectable intermediate. In summary, these studies describe the first biophysical characterization of a non‐human aromatase that displays strikingly similar enzyme kinetics and ligand binding properties to the human enzyme despite bearing 75% sequence identity. In addition, they provide access to an alternative model to study the catalytic mechanism of the enzyme using methods requiring substantial amounts of ligand‐free enzyme.Support or Funding InformationNational Institutes of Health National Institute of General Medical Sciences [Grant R01GM114168]This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery of Tumor Necrosis Factor Receptor Binders using Yeast Surface Display
Authors: Vunnam Nagamani , Szymonski Sophia , Sachs Jonathan N. , Hackel Benjamin 
Published: June 2021

Tumor Necrosis Factor Receptors (TNFRs) are essential regulators of the immune system. Dysregulation of TNF receptors play a role in the pathology of many autoimmune diseases. Available treatments for TNF‐related diseases cause adverse side effects by unintentionally inhibiting ligand binding to off‐target receptors. In our previous work we showed an alternative therapeutic strategy for inhibition of the pro‐apoptotic activities of TNF receptors by specifically targeting receptor‐receptor interactions, without interrupting ligand binding. In the current study, we employed two small protein scaffold libraries, T7 phage gene 2 protein (Gp2) and affibody, to discover binders that specifically modulate the signaling activities of death receptor 5 (DR5) and TNFR1. Using yeast display and directed evolution, we will identify Gp2 and affibody variants with high binding affinity, stability and specificity to DR5 and TNFR1. These small protein scaffolds can be used as therapeutic agents in TNFR related diseases.Support or Funding InformationNIH 1R01 EB023339‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Construction of Chimera Prolyl Endopeptidases to Determine Role of Domains in Substrate Size Specificity
Authors: Alakija Femi , Clack Beatrice 
Published: June 2021

Eurygaster integriceps Puton (commonly known as the Sunn pest) is a member of the family Scutelleridae. It is a major pest of wheat and is commonly found in Middle East, northern Africa, Russia, and Eastern Europe (Critchley, 1998). The Sunn pest secretes a proteolytic enzyme, a prolyl endopeptidase (spPEP) into the wheat grains with subsequent degradation of gluten (Darkoh et al., 2010), the major protein in wheat. The spPEP is a unique prolyl endopeptidase in the family of S9 serine proteases because it recognizes large proteins (gluten) as substrates ranging from 30kDa to greater than 140kDa. All other prolyl endopeptidases are purported to bind only peptides shorter than 30 amino acids in length. PEPs consist of two conserved domains, a hydrolase domain which includes the catalytic triad (Ser554, Asp641, and His680) and a seven bladed β‐propeller domain (Juhász et al., 2006). To understand the role with which the two domains have on substrate specificity, chimeras were constructed in which domains were swapped between the sunn pest prolyl endopeptidase (spPEP), mammalian prolyl endopeptidase (hPEP), and the bacteria prolyl endopeptidase (bPEP). Forward and reverse primers identical within the hinge region of either spPEP, hPEP, or bPEP connecting the two domains were used in polymerase chain reaction (PCR) to amplify the vector and the respective domain of each PEP. Ligation independent cloning (LIC) was used to clone the insert DNA into the LIC vector (pNYCOMPS‐LIC‐ccdB‐FH10T+(N‐term) expression vector). The product chimeras include spPEP hydrolase/hPEP β‐propeller, spPEP hydrolase/bPEP β‐propeller, hPEP hydrolase/spPEP β‐propeller, hPEP hydrolase/bPEP β‐propeller, bPEP hydrolase/spPEP β‐propeller, and bPEP hydrolase/hPEP β‐propeller. Each of the chimeras were expressed in BL21 (DE3) pTFe. The cell lysates were used to determine the enzyme activity for each of the chimera using Gly‐Pro‐p‐nitroanalide (GPpNA) as substrate.Support or Funding InformationOffice of Research and Sponsored Programs, Stephen F. Austin State University, and Ed & Gowen Cole, Nacogdoches. Texas.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rational Design of pH Sensitive MS2 Virus‐Like Particles for Drug Delivery Applications
Authors: Alvarez‐Benedicto Ester , Hartman Emily , Francis Matthew B. 
Published: June 2021

Virus‐like particles (VLPs) have emerged as promising drug delivery and imaging scaffolds. VLPs made from the MS2 Bacteriophage Coat Protein (CP) enter cells via receptor‐mediated endocytosis and are believed to accumulate in the endosomal pathway. This pathway involves a decrease in pH levels that can potentially trigger the disassembly of acid sensitive VLPs, leading to cargo release. However, MS2 CPWT VLPs are highly stable in the endosomal pH range (4.5–5.5), which could slow endosomal release of cargo within the cell. Here, we set out to identify an acid‐sensitive variant of the MS2 CP that could lead to faster cargo release in the acidic environment of the endosome. We identified a previously‐unknown double mutant, CPT71H/E76C, that exhibits significant acid sensitivity in vitro at the endosomal pH range, in contrast to MS2 CPWT. The population of CPT71H/E76C VLPs was measured after a 4 h incubation at 37°C in a pH range from 1.0 to 7.3 with High Performance Liquid Chromatography using a Size Exclusion Chromatography column (HPLC‐SEC). Acid screens showed CPT71H/E76C to have 50% less well‐formed VLP at pH 4.9. These mutations alter a key hydrogen bond interaction, which could be the reason for increased acid sensitivity. To explore this hypothesis, capsids were alkylated with N‐ethylmaleimide, a moiety that could restore the hydrogen bond between the residues, and screen for acid sensitivity. Results showed modified capsids had an acid sensitivity similar to CPT71H, suggesting the hydrogen bond is involved in this property. Acid sensitive CPT71H/E76C could potentially improve the delivery of drugs to cancer cells.Support or Funding InformationNIH‐ MARC (5T34GM007821‐38) and the Amgen Scholars FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Utilization of multiple lipid sources in the production of biodiesel by using lipase nanoparticles from Candida rugosa in a nonaqueous system
Authors: Rivera Hector A. , O'Neill Crystal A. , Sharma Rohit K. , Thapa Bibek , Bovea Vanessa B. , Griebenow Kai 
Published: June 2021

One of the major challenges for the sustainable fatty acid methyl or ethyl ester (FAME/EE, a.k.a biodiesel) production is the smart utilization of low‐cost feedstocks, e.g., waste cooking oil (WCO), brown grease, or algal lipids. Although traditional catalyst systems could be used to transform such feedstocks into biodiesel, some well‐established drawbacks are associated with their use. Alternatively, lipases can convert the FFA‐rich feedstocks into FAME/EE in a manner with less environmental impact. However, low enzyme activity in organic solvents and high cost remain challenging for the industrial application of lipase nanoparticles. In this study, we are addressing these drawbacks of lipase by developing lipase nanoparticles (LNP), cross‐linked lipase nanoparticles (CLNP), and immobilized LNP on iron oxide nanoparticles (IONP). Lipase from Candida rugosa was selected as an economic widely available source and purified to ca. >90%. Different formulations of nanoparticles were formed, and detailed surface and morphological characterizations were performed by using the various microscopy techniques, e.g., scanning and transmission electron microscopy, and atomic force microscopy, dynamic‐light‐scattering, and magnetic properties by the vibrating‐sample magnetometer. For structural characterization, Fourier‐transform infrared spectroscopy and X‐ray diffraction were used. Identification of fatty acid esters was done by gas chromatography coupled to a mass spectrometer. The activity of the LNP, CLNP, and LNP‐IONP formulations increased 10‐, 20‐, and 32‐fold in n‐hexane, 18‐, 42‐, and 54‐fold in 1,4 dioxane, and 13‐, 29‐, and 43‐fold in THF, respectively, compared to free lipase. Detailed analysis of fatty acid ester formation was performed using the different combinations of substrates (WCO and brown grease, methanol and ethanol), lipase formulations (free lipase, LNP, CLNP, and LNP‐IONP), and solvent systems (hexane, 1,4 dioxane, THF, and t‐butanol). The nanoparticulate lipase formulations converted a high percentage of fatty acid esters into biodiesel in less time and the overall operational stability of the system improved considerably.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential modulation of microtubule stability by inhibitor‐bound Kinesin‐5
Authors: Kim Catherine , Kim Elizabeth , Liu Liqiong , Wojcik Edward 
Published: June 2021

Kinesin‐5 forms a homotetramer that mediates the assembly of the bipolar mitotic spindle apparatus in many eukaryotes. Thought primarily to crosslink and slide antiparallel microtubules during spindle morphogenesis, this motor protein has been the target for the development of small molecule anti‐mitotic agents as potential cancer therapeutics. Most of the discovered effectors bind to a single site formed by loop‐5 wherein they mediate allosteric inhibition of the motor ATPase activity and motility. All of the loop‐5 inhibitors block ADP release and are therefore argued to all inhibit the motor in a cognate manner. Furthermore, at the single molecule level, the inhibitors have been shown to trap the monomeric motor domain in a weakly microtubule‐bound state that exhibits one‐dimensional diffusive movements along microtubules in vitro. Herein we show that, despite these similarities, different loop‐5 pocket inhibitors can elicit remarkably distinct impacts on the motor‐bound microtubule dynamics. We observe that monastrol‐bound human Kinesin‐5 motor domains can drive the net depolymerization of microtubules in vitro, while the STLC‐bound motors do not. Furthermore, while monastrol and STLC both induce metaphase arrest with mono‐polar spindles in cultured cells, monastrol also causes a significant decrease in microtubule density in these spindles. We conclude that the monastrol‐inhibited motor appears to drive microtubule depolymerization in vitro and in vivo. Therefore, loop‐5 pocket inhibitors of human Kinesin‐5 are not all synonymous, and can modulate a range of microtubule interaction which likely will impact their clinical efficacy.Support or Funding InformationThis work was funded by grants from the National Institutes of Health (R01GM097350) and the School of Graduate Studies at LSU Health Sciences Center – New Orleans.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of Ramachandran Outlier Prevalence for X‐ray Crystallographic Model Structure Validation
Authors: Vavrinak David A. , Weiss Charles 
Published: June 2021

There are numerous errors in published crystal structures, and the prevalence of Ramachandran outliers can be a valuable tool in identifying these errors. However, it is necessary to know which outliers are valid in a PDB file and which ones result from errors in the X‐ray crystallographic model structure. To address this problem, the Python language and NumPy library were used to compile data on approximately eight thousand high‐quality X‐ray crystal structures of proteins published in a public database to extract relevant data to provide insight concerning the probability and validity of a Ramachandran outlier assignments. Ramachandran analyses were performed by Phenix using the Phenix/Python application programming interface. The data collected include each residue's identity, Ramachandran outlier evaluations, phi and psi angles, outlier score and type, and information regarding the location of the residue in the protein. The above information along with metadata about the protein crystal structures are stored and maintained in an HDF5 file. The results of the data analysis using machine learning and other statistical methods will be presented.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nano‐biomaterial and functional genomics research
Authors: Mydin Rabiatul Basria S. M. N. 
Published: June 2021

Nanotechnology has become a foremost research interest, which provides an opportunity to explore functional devices at nanometer regime and make it possible to design biomaterials compatible with nanoscale topography. The nanometric scaled topography of biomaterial plays a critical role in cellular stability and cell survival. This nanostructure surface has been shown to modulate diverse cellular responses of cell adhesion, migration, proliferation and differentiation. As for medical implant surface technology, this nano architecture structures may be beneficial for nano‐mechanical‐adaptation response which activates gene regulation involved in cytoskeleton remodelling of extracellular matrix alterations, plasma membrane modulations, locomotory behaviour, intracellular signalling response for cell metabolism in mechanosensitivity activities. On the other hand, for therapeutic research the present cell‐nano‐biomaterial study has provided profound understanding in regulation of cellular survival and cell growth signals, activation of DNA damage and DNA repair mechanisms and activation of redox regulator pathways. More research should be conducted to explore nanomaterial–nanomolecular interactions by performing SNP and DNA methylation studies as this knowledge is important in explaining the gene regulation and self–renewal potential of nano‐biomaterial interaction in the cell systems. Furthermore, comprehensive study using confocal laser scanning microscopy coupled with specific markers are needed for the understanding of cell migration on nano‐biomaterial surface especially on the cytoplasmic spreading behaviour involving filopodia and lamellipodia. Likewise, the metabolomics and bio‐kinetics of nanomaterial studies are also examples of important research areas to be explored. The valuable antioxidant properties of some nano‐biomaterials have created research gaps in product life cycle, circulatory system, half‐life of the antioxidant properties and other possible chemical reactions from the nanomaterials. Knowledge of these aspects will create opportunities for better understanding which may help researchers to develop better nano‐biomaterials products to be used in medicine and health‐line services.Support or Funding InformationThe author are thankful to the Ministry of Education (MOE) Malaysia for funding this work under Transdisciplinary Research Grant Scheme (TRGS) grant no. 6769003 and Universiti Sains Malaysia (USM) for USM‐Short Term Research Grant (304/CIPPT/6315073).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.