bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assessing the Effects of Inorganic Arsenic on IL‐1β and TNFɑ Secretion, Gene Expression, and DNA Methylation in Murine Macrophages to Gauge Immunotoxic Effects of Heavy Metals
Authors: DeLong Lauren Nicole , Barbosa Jeremie Rose , Nyland Jennifer 
Published: June 2021

Trivalent inorganic arsenic, arsenite, is a natural component of the earth and is often found in drinking water in areas where the composition is high. Some studies have suggested that, especially at moderate to high exposure levels, arsenite is toxic to individuals who are chronically exposed through drinking water, characterized by an increase in inflammatory cytokine release, specifically interleukin‐1β (IL‐1β) and tumor necrosis factor‐ɑ (TNFɑ). Other epidemiological studies at low to moderate exposures did not find the same correlation with inflammation. We aim to characterize and quantify the effects of arsenite exposure to macrophages to gauge any potential inflammatory response.M1 macrophages are key players in inflammatory response and secrete IL‐1β and TNFɑ in response to Danger Associated Molecular Patterns (DAMPs) released by neighboring cells undergoing necrosis, in coordination with Pathogen Associated Molecular Patterns (PAMPs), molecules associated with pathogens, such as lipopolysaccharide, or LPS. We obtained a murine macrophage cell line to study the effects of arsenic on inflammatory cytokine secretion in vitro. A dose response curve was developed including varying concentrations of arsenite with and without 50 ng/mL LPS, using the same treatments without arsenite as controls. Cytolethality was determined by differential staining and counting. Secretion of cytokines after 24 hours of exposure was quantified using ELISA. Results were statistically analyzed using Student's t‐test and One‐Way ANOVA. IL‐1β was not differentially secreted for any concentration of arsenite, but cytolethality and TNFɑ secretion were significantly increased for 200 nM arsenite with LPS as opposed to 200 nM arsenite without LPS, possibly indicating that some inflammatory augmentation occurs in the presence of both arsenite exposure and a PAMP.Future steps include analyzing alterations in expression of genes responsible for proteins involved in the secretory pathways of these cytokines. For example, the genes involved in manufacturing the pieces of the NLRP3 inflammasome, key in production of IL‐1β, or TNFɑ Converting Enzyme, involved in cleavage of the TNFɑ precursor, may be potential targets. Additionally, we will explore differential DNA methylation using methylation sensitive and insensitive restriction enzyme digestion to examine epigenetic control of cytokine secretion in response to arsenite. These results will be reported to appraise the immunotoxic effects of arsenic exposure.Support or Funding InformationThis research is supported by the Salisbury University Department of Biological Sciences and the Salisbury University Honors College.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fas‐associated factor 1 (FAF1) binds to mineralocorticoid Receptor (MR) through its SUMO‐interacting motifs and negatively modulates MR transcriptional activity
Authors: Lin Ding‐Yen 
Published: June 2021

Fas‐associated factor 1 (FAF1) was originally isolated as a Fas(CD95)‐associated protein factor and was subsequently found to interact with numerous other proteins that are involved in various cellular events including Fas‐mediated apoptosis, NF‐kB, Wnt/β‐catenin pathways, mineralocorticoid receptor (MR) mediated transactivation and ubiquitin‐dependent processes. Here, we define two Small Ubiquitin‐like Modifier (SUMO)‐interacting motifs (SIMs) within FAF1 and show it to be crucial for transcriptional modulation of MR. Our study demonstrated that the SIMs of FAF1 do not play a significant role in the regulation of its subcellular localization, Fas‐mediated apoptosis, NF‐kB and Wnt/β‐catenin pathways. Remarkably, FAF1 interacts sumoylated MR and represses aldosterone‐activated MR transactivation in a SIMs‐dependent manner. Moreover, silencing of endogenous FAF1 in stably MR‐expressing cells resulted in an increase in the induction of MR target genes by aldosterone, indicating that FAF1 functions as a MR corepressor. We further provide evidence to suggest that the mechanism of FAF1/SIMs‐mediated MR transrepression involves inhibition of MR N/C interaction. Sumoylation has been linked to impart repressive properties on several transcription factors and cofactors. Our findings therefore provide the mechanistic insight underlying SUMO‐dependent transcriptional repression of the MR.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Insight into Parkinson's Disease from Yeasts: Combined impact of covalent modifications & familial mutations on α‐synuclein
Authors: Ganev Yoan Petrov , Thomas Rosemary , Roman Alexandra , Mwale Chisomo , Mountain Joseph , Balaram Ariane , Marshall Morgan , DebBurman Shubhik 
Published: June 2021

Parkinson's Disease (PD) is a neurodegenerative disorder linked to the loss of dopaminergic neurons in the midbrain. A key pathological marker of PD is the presence of Lewy bodies, which are mainly composed of misfolded α‐synuclein protein. α‐synuclein is a highly post‐translationally modified protein. While phosphorylation and nitration of α‐synuclein are well studied in the context of PD pathology, less is known about sumoylation, which is proposed to be neuroprotective based on limited studies. The majority of sumoylation takes place on the lysine‐96 and lysine‐102 sites of α‐synuclein, and it increases the protein's solubility. The goal of this research was to better understand the role of sumoylation in regulating α‐synuclein toxicity, and we performed four studies towards it. First, we evaluated the effects of blocking sumoylation on α‐synuclein in the well‐established budding yeast model for PD and found that α‐synuclein becomes more aggregated, gains toxicity, and loses localization at the plasma membrane. Second, we evaluated the effects of altering sumoylation pathways by using yeast strains with reduced (ulp1ts) or excessive sumoylation (smt3ts), and found that α‐synuclein aggregates more with reduced sumoylation, but becomes less toxic with increased sumoylation. Third, we asked how altering phosphorylation of α‐synuclein would alter sumoylation's protective role and found that blocking phosphorylation (in α‐synuclein already blocking sumoylation) reduced the protein's toxicity. Finally, we began evaluating whether blocking sumoylation and altering phosphorylation on familial PD mutant versions of α‐synuclein would exacerbate its toxicity. We found preliminary evidence that the toxicity of the A53T mutation increases when sumoylation is blocked. In the future, we will conduct further studies to understand how sumoylation affects other variants and modifications of α‐synuclein.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quantifying the Aggregation Propensity of IAPP from Diabetic and Nondiabetic Species
Authors: Moffet David , Palato Larry , Pilcher Shannon , Rinauro Dillon , Menefee Kate , Tun Angela , Jauregui Betssy , Shapiro Sarah , Nossiff Olivia , Nguyen Viviane 
Published: June 2021

The aggregation of the 37‐amino acid polypeptide human Islet Amyloid Polypeptide (hIAPP, amylin), as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of pancreatic β‐islet cells in type 2 diabetes. It is known that several organisms express non‐amyloidogenic variants of IAPP (such as rat and mouse) and are not known to develop diabetes naturally. Conversely, several organisms express highly amyloidogenic variants of IAPP (such as human, cat and primates) and are known to develop diabetes. Despite the significant amount of genetic information available, no comprehensive study has been conducted to directly correlate IAPP aggregation potential to the propensity to develop diabetes within the animal kingdom. In this work, we will compare the aggregation potential of naturally occurring IAPP variants from organisms known to, or known not to, develop diabetes. IAPP peptide variants were chemically synthesized. Each variant was tested for its amyloid aggregation potential using Circular Dichroism and Atomic Force Microscopy.Support or Funding InformationNational Institute of Health (Grant#: 1R15DK112172)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Defining the fumarase water binding site
Authors: Weaver Todd Michael 
Published: June 2021

Fumarase C, a class II family member, catalyzes the nearly catalytically perfect reversible conversion of fumarate to S‐malate as part of the Krebs Cycle. Despite decades of research and high quality structural information the exact mechanism for diffusion limited catalysis remains elusive. In order to more fully understand the contribution of various active site residues to the fumarase‐catalyzed reaction, a series of crystal structures and kinetic parameters for a series of active site fumarase variants have been determined. The first set of variants targeted the water 26 (W26) binding site formed via Ser98, Thr100, Asn141 and His188. Alterations at the water‐binding site, termed the W26 cluster, affected the Km and kcat. The structural data for each W26 cluster replacement notes only slight shifts in this structural water location with maximal movement of 4.9 Å in the H188A active site leading to an overall 3500‐fold reduction in activity. The second set of variants replaced the highly conserved glutamic acid at position 331 with either an aspartic acid or a glutamine residue. Glu331, contained within the fumarase signature sequence, forms a shortened hydrogen bond to stabilize His188 just behind the W26 cluster. The Glu331 to aspartic acid or glutamine substitution create significant reductions in kcat and limited effect on Km values even though there are few structural changes in the position of W26. Based upon the structural and kinetic data we propose the following (1) W26 provides a model for both the S‐malate 2‐[OH] binding site during the S‐malate dehydration reaction and the water binding site during the fumarate hydration reaction, (2) Asn141 and Glu331 facilitate Nδ1‐H to Nɛ2‐H tautomeric interconversion of His188 and (3) His188 acts as the catalytic acid to protonate the S‐malate 2‐[OH] during the S‐malate à fumarate dehydration.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kinetic Characterization and Chemotherapeutic Relevant Inhibition of Human Malate Dehydrogenase 1 and 2.
Authors: Wardenfelt Stacey , Dwyer Tim 
Published: June 2021

Docetaxel and doxorubicin are chemotherapeutic agents used to fight cancers ranging from prostate cancer to uterine cancer to leukemia to soft tissue sarcomas. However, patients can often develop resistance to docetaxel, and doxorubicin can be cardiotoxic which limits its usefulness. Recent data suggest that both of these problems are in part caused by overexpression of the mitochondrial malate dehydrogenase (MDH2) protein in tumor cells and that inhibitors of this enzyme, such as mebendazole and visnagin, could greatly increase the efficacy of docetaxel and doxorubicin. Studies have yet to address the how these inhibitors affect the cytoplasmic isoform of malate dehydrogenase (MDH1) and the possible side effects the inhibition of this non‐target enzyme could have. One of the functions of MDH1 is to help in gluconeogenesis. Malignant cells are often more dependent on glycolysis for energy generation than normal cells. Since gluconeogenesis is antithetic to glycolysis, inhibiting an enzyme that helps in gluconeogenesis (such as MDH1) could promote glycolysis and, in essence, enhance the growth of tumor cells. The current study has utilized the pMAL expression system (New England Biolabs) to express human MDH1 and MDH2. The expressed enzymes have been kinetically characterized in the absence and presence of known and new inhibitors with the idea of finding an inhibitor that is specific for MDH2.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimization of the Enzymatic Synthesis of UDP‐Xylose and Characterization of Enzymes Involved
Authors: Cook Matthew , Culbertson Alan , Zabotina Olga 
Published: June 2021

The plant cell wall is of interest for agriculture and biofuel production; understanding the biosynthesis of plant cell walls is critical for improving the viability of biorenewable energy as a replacement for traditional energy sources. Xyloglucan is an important cell wall constituent and has high degrees of xylosylation. However, studies of xylosylation of hemicelluloses and other biomolecules are hampered, partially due to the low supply of the primary substrate for xylosylation, UDP‐xylose (UDPX). The goal of this project has been to optimize the known biological pathway for UDPX synthesis for laboratory use. The two‐enzyme UDPX biosynthesis pathway produces UDPX from UDP‐glucose (UDPG), a more readily available substrate chemical. The project also characterized enzymatic properties of the enzymes UDP‐glucose dehydrogenase (Ugd) and UDP‐xylose synthase (Uxs), which perform the catalytic activities in the pathway of interest. These enzyme homologs have not been characterized previously.Ugd and Uxs were expressed and purified using the plasmids prepared in Dr. Adam Barb's lab. While homologs of Ugd and Uxs have been partially characterized before, neither has been studied to the degree required for this project and the specific homologs used herein have never been reported. Enzymatic assays were used to determine the optimum conditions for each enzyme and spectrophotometry and high‐performance liquid chromatography were used to measure the products of reactions. Biologically relevant conditions including pH, salt content, reducing agent content, presence of metal ions, et cetera were optimized. Subsequently, methods of combining the reaction systems have been attempted. Due to the significantly different pH optimums of the two enzymes and the generation of inhibitory compounds, the synthesis of UDPX cannot be performed as a one‐pot reaction and will require two independent, consecutive reactions. Additionally, kinetics data collected has provided interesting insights into the enzymatic properties of Ugd and Uxs. Thus, it was shown that Ugd experiences substrate inhibition, but this substrate inhibition can be relieved by the addition of ATP, despite ATP not being involved in the reaction.The primary goal of the project has been the efficient synthesis of UDPX from UDPG. To that end, the individual assay conditions have been improved to over 90% conversion efficiency for both enzymes, individually. The conversion of UDPG to UDPX is now being performed in a consecutive reaction series and the efficiency of this reaction series is being improved.Support or Funding InformationThis work was supported by NSF‐MCB grant #1121163This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting DXP Synthase using TPP Mimics
Authors: Alsalahi Amera , Girardi Peter , Callahan Kevin , Sridhar Anand 
Published: June 2021

A global emergence of antibiotic‐resistant bacteria, largely due to the misuse and over use of antibiotics, has led to an increase in demand for the development of new antibacterial agents. In order to develop new antibiotics, researchers need to identify new targets that have high specificity for certain bacterial pathways. The enzyme D‐xylulose 5‐phosphate (DXP) synthase catalyzes the first step in the MEP (methylerythritolphosphate) pathway in human pathogens, which produces the biological molecules necessary to regulate numerous cellular functions, including central metabolism. Inhibition of DXP synthase would significantly reduce the rate of DXP production and the MEP pathway and thus is an ideal candidate for antibiotic development. DXP synthase requires the cofactor, thiamin pyrophosphate (TPP) for its activity. Preventing TPP from complexing with DXP synthase will inhibit the activity of the enzyme and consequently inhibit the MEP pathway. To test the efficacy of DXP synthase inhibitors both DXP synthase and the sequential enzyme of the MEP pathway, DXP reductoisomerase (IspC), were purified from E.coli using nickel affinity chromatography. Conversion from DXP to 2‐C‐Methyl‐D‐erythritol 4‐phosphate (MEP) by IspC coincides with NADPH oxidation that can be measured using spectroscopic techniques. The establishment of this in vitro system will allow us to test the efficacy novel DXP synthase inhibitors.Support or Funding InformationThis project was funded by SJFC internal funds.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‐Targeted Kinase Inhibitor Treatment of KRAS Mutant Lung Cancer
Authors: Sridharan Ramya , Shah Janki , Shamay Yosef , Heller Daniel 
Published: June 2021

Lung cancer is the leading cause of mortality in the US, affecting 160,000 patients annually with a five year survival of 17%. The majority of lung cancers harbor KRAS mutations that over‐activate the RAS kinase in the RAS‐MAPK signaling pathway, RAS‐RAF‐MEK‐ERK. This leads to increased pathway activity and increased cell division and tumor growth. Kinase inhibitors (KIs), which target key effectors of cancer signaling pathways, constitute a major strategy to treat KRAS‐driven lung cancer. Although RAS itself is difficult to drug with KIs due to the lack of binding pockets, KIs inhibiting downstream MEK kinases of the RAS‐MAPK pathway have shown to be most effective in vitro. However, MEK inhibitors suppress signaling in non‐cancer cells, so their dosing is limited by toxicity and results in a narrow therapeutic index. Additionally, the anti‐tumor effects of MEK inhibitors in KRAS‐driven lung cancer are further diminished by the activation of compensatory pathways, resulting in drug resistance and tumor resurgence. Although the co‐administration of a second KI can mitigate this resistance, many such combinations of KIs are highly toxic.We recently developed a new nanomedicine platform to package kinase inhibitors with high drug loading. These indocyanine nanoparticles (INPs) alter the systemic biodistribution of the KI to improve the drug's therapeutic index via tumor targeting. The INPs target caveolin receptors overexpressed on cancer endothelium to increase the local concentration of kinase inhibitor at the tumor site and reduce the systemic exposure of non‐cancerous tissues. Thus, the selective targeting decreases the toxicity of systemic KI exposure to concomitantly improve the therapeutic index.Support or Funding InformationThis work was supported in part by the NIH New Innovator Award (DP2‐HD075698), the Cancer Center Support Grant (P30 CA008748), the Pershing Square Sohn Cancer Research Alliance, the Honorable Tina Brozman Foundation for Ovarian Cancer Research, the Expect Miracles Foundation ‐ Financial Services Against Cancer, the Anna Fuller Fund, the Louis V. Gerstner Jr. Young Investigator's Fund, the Frank A. Howard Scholars Program, Cycle for Survival, the Alan and Sandra Gerry Metastasis Research Initiative, Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research, the Experimental Therapeutics Center, the Imaging & Radiation Sciences Program, and the Center for Molecular Imaging and Nanotechnology of Memorial Sloan Kettering Cancer.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Imaging Exosomes using Super Resolution Microscopy
Authors: Chen Chen , Zong Shenfei , Wang Zhuyuan , Cui Yiping 
Published: June 2021

Exosomes are small membrane vesicles originated from late endosomal compartments of cells with a diameter ranging from 30–100 nm. Studies have demonstrated that exosomal miRNAs can influence the biological processes of recipient cells. Therefore, direct imaging and tracking of exosomes and exosomal miRNAs in recipient cells are essential for exosome‐related functional analysis. However, the moderate spatial resolution of conventional fluorescence microscope limits the precise localization of exosomes considering their relatively small size (<100 nm). Here, we took advantage of single molecule localization microscopy (SMLM) to realize the visualization and intracellular functional analysis of exosomes and exosomal miRNAs in recipient cells.First, the multi‐color SMLM imaging of exosomal membrane receptors (CD63 and HER2) and exosomal miRNAs (mir‐21 and mir‐31) were accomplished with more reliable structural information and localization accuracy. Exosomes taken up by normal cells can be more precisely visualized through SMLM. In our research, the motion of exosomes within the intercellular filamentous structures was observed successfully, indicating one kind of the intercellular communication pathways. According to the dual‐color SMLM imaging of exosomes and lysosomes, we confirmed that exosomes can be transported into lysosomes for further degradation after binding to recipient cells. Moreover, as one kind of the natural carriers of the cellular cargoes, exosomes can encapsulate mir‐21 inside to prevent enzyme degradation during transferring and then release them into recipient cells. Specifically, these released exosomal mir‐21 were able to target the 3'UTR of TPM1 mRNA, which can repress the expression of the target gene.These findings can provide new insights into the pathway of intercellular communications and affirm that SMLM is a powerful technique for the exploration of exosome‐mediated cancer metastasis, as well as the role of exosomal miRNAs in gene regulation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.