bluuebunny/crossref_metadata_2025_split huggingface dataset:
Endoplasmic Reticulum Stress Induction of Yeast Bax Inhibitor (BXI1) Gene Expression Does Not Entirely Require IRE1
Authors: Finn John Patterson , Parker Wesley , Cardona Andrew , Pineda Edy , Austriaco Nicanor O.P. 
Published: April 2020

Bax Inhibitor (BI‐1/TMBIM6) is a regulator protein within the Unfolded Protein Response (UPR) that influences rates of apoptosis within cells. Studies have shown that BI‐1 is upregulated in human cancer cells like prostate and breast, therefore it is important to study the factors that control BI‐1 within a cell. In this paper, the Saccharomyces cerevisiae homolog of BI‐1, BXI1, is studied to better understand the signaling pathway that influences concentrations of BXI1, specifically the Inositol Requiring Enzyme 1 (IRE1) pathway. Ire1p is an Endoplasmic Reticulum (ER) bound transmembrane ligand receptor which detects the UPR and activates downstream targets which play further roles in regaining homeostasis. Here, our lab used two genetically modified S. cerevisiae strains, both with Green Fluorescent Protein (GFP) bound to BXI1 (Bxi1p‐GFP) and one lacking genetic information to code for IRE1 (ΔIre1) to test the effect of the IRE1 pathway on the expression of BXI1. When cells were cultured in media containing either tunicamycin or DDT, we observed an increase in the expression of BXI1 that was not entirely dependent on the IRE1 pathway. This suggests that there may be other signaling pathways that influence the expression of BXI1.Support or Funding InformationOur laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.
Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by the Nuclear Bile Acid Receptor, Farnesoid X Receptor
Authors: Smyth Jessica S. , Bussayajirapong Anchalin , Lajczak-McGinley Natalia K. , Foulke-Abel Jennifer , Donowitz Mark , Keely Stephen J. 
Published: April 2020

Introduction and AimsThe intestinal epithelium functions to transport nutrients, fluid and electrolytes, while at the same time acting as a barrier to the entry of harmful substances. CFTR is a transmembrane Cl− channel important in regulating intestinal fluid transport and is implicated in the pathogenesis of a number of intestinal diseases. Bile acids, classically known for their roles in lipid digestion, are now also recognised as important enteric hormones that regulate many aspects of epithelial function. Indeed, it is thought that bile acid metabolism is a primary mechanism by which the microbiota communicates with its human host. Here, we set out to investigate the role of the nuclear bile acid receptor, farnesoid x receptor (FXR), in regulating epithelial CFTR expression.MethodsT84 human colonic epithelial cells were cultured as polarised monolayers on permeable supports and treated bilaterally with the FXR agonist, GW4064 (5 μM), over a range of times. Levels of FGF19, an index of FXR activation, CFTR, FXR, FOXA1, and miR‐494 were measured by qRT‐PCR, western blotting, or ELISA.ResultsTreatment of the cells with GW4064 significantly increased FGF19 mRNA expression by 1235 ± 237.3 fold (n = 8; p < 0.05) and protein by 719.6 ± 92.0 pg/mL (n = 5; p < 0.01) after 48 hrs. Moreover, GW4064 downregulated CFTR mRNA to 0.47 ± 0.1 fold after 12 hrs (n = 8; p < 0.01) and protein levels to 0.36 ± 0.1 fold after 48 hrs, compared to vehicle‐treated controls (n = 4; p < 0.05). Transcriptomic analysis confirmed FXR‐induced downregulation of CFTR in primary human colonic enteroids. Studies in Ussing chambers showed that GW4064 treatment for 48 hrs inhibited Cl− secretory responses to the Ca2+‐dependent agonist carbachol (CCh; 100 μM) and the cAMP‐dependent agonist, forskolin (10 μM) by 79.9 ± 7.5 % (n=4; p <0.01) and 74.2 ± 8.9 % (n=4; p < 0.01), respectively. Expression of miR‐494, which is known to target CFTR, was not increased by GW4064 treatment in T84 cells. However, mRNA expression of FOXA1, a transcription factor that regulates CFTR, was inhibited by 33.2 ± 5.2% after 3 hrs (n = 4; p <0.05).ConclusionThe nuclear bile acid receptor, FXR, regulates colonic epithelial CFTR expression. Such actions are likely to be important in the setting of microbial regulation of host intestinal physiology in health and disease. As such, FXR represents an excellent target for development of new drugs to treat intestinal diseases associated with dysregulated CFTR function.Support or Funding InformationThis work was funded by a Science Foundation Ireland Principal Investigator Award to SJK
ALTERATION OF MITOCHONDRIAL MEMBRANE POTENTIAL (ΔΨM): POTENTIAL ROLE IN THE MECHANISM OF MEHP‐INDUCED INHIBITION OF STEROID BIOSYNTHESIS IN MA‐10 LEYDIG CELLS
Authors: Traore Kassim , Li Yunbo , Papadopoulos Vasilios , Zirkin Barry 
Published: April 2020

INTRODUCTION AND OBJECTIVESMono‐(2‐ethylhexyl) phthalate (MEHP) is the active metabolite of di‐(2‐ethylhexyl) phthalate (DEHP), a plasticizer with endocrine disruptor activity that is widely used in the manufacturing industry. MEHP inhibits luteinizing hormone (LH)‐stimulated steroid biosynthesis in Leydig cells by molecular mechanisms that remain unclear. In the present study, using MA‐10 mouse tumor Leydig cells, we examined changes in mitochondrial function caused by MEHP exposures in relationship to possible link to alteration of progesterone biosynthesis.METHODSThe effect of MEHP on LH‐stimulated progesterone production was assessed after treating MA‐10 cells with (0–300 μM) MEHP for 24 hrs. Progesterone production was quantified by enzyme‐linked immunosorbent assay (ELISA). To measure oxygen consumption, cells were treated with MEHP (0–300 μM) for 24 hrs, and oxygen consumption was monitored using a Clarke‐type oxygen electrode. Mitochondrial membrane potential (ΔΨm) was assessed using the JC‐1 dye mitochondrial membrane potential indicator. Mitochondria‐derived superoxide was measured using the MitoSOX™ red mitochondrial superoxide indicator.RESULTSExposures of MA‐10 cells to 0–300 μM MEHP caused inhibition LH‐stimulated progesterone biosynthesis in a dose‐dependent manner. The decrease in the Leydig ability to produce progesterone correlated with a loss in the Leydig cell mitochondrial membrane potential (ΔΨm), and was linked to perturbation of the capacity to energize the mitochondrial membrane. MEHP exposure was suggested to cause a disruption in the electron flow through the electron transport chain, evidenced by a decrease in the ability to consumption molecular oxygen and an increase in the generation of superoxide in the mitochondria.CONCLUSIONSTogether, these observations suggest that MEHP inhibits steroid biosynthesis in MA‐10 cells via a mechanism involving a loss of the mitochondrial membrane potential (ΔΨm) and an increase in the oxidative stress level, two crucial changes known to affect mitochondrial function and cause inhibition of STAR‐mediated cholesterol transport and steroid biosynthesis.Support or Funding InformationThis material is based upon work supported by NIH grant SC2GM099578 and by the North Carolina Biotechnology Center, NCBC grant #2019‐IDG‐1017.
Cytosolic glutathione‐S‐transferase A1 (GSTA1) plays a primary role in the metabolic clearance of the aromatase inhibitor, Exemestane
Authors: Teslenko Irina L. , Chen Gang , Watson Christy , Lazarus Philip 
Published: April 2020

Exemestane (EXE) is used to treat estrogen receptor positive (ER+) breast cancer in postmenopausal women by inhibiting the enzyme aromatase in the final step of estrogen biosynthesis. Variability exists in both overall patient response and adverse events, which may be attributed to inter‐individual variation in EXE metabolism. EXE is metabolized primarily to the active metabolite, 17β‐dihydro‐EXE (17β‐DHE), and preliminary studies have demonstrated that cysteine conjugates of EXE and 17β‐DHE comprise 77% of total EXE metabolites in the urine of patients. Cysteine conjugation is a multi‐step process, the first of which is glutathione (GSH) conjugation, catalyzed by the glutathione‐S‐transferase (GST) family of the enzymes. The goal of the present study was to identify the hepatic GSTs active in the metabolism of EXE and 17β‐DHE. In a screening of the Human Protein Atlas, 12 cytosolic GSTs were found to be hepatically expressed and were initially screened by incubating 0.5 μg of pure recombinant GST enzyme with EXE or 17β‐DHE (125 μM) and reduced glutathione (GSH). The resulting GS‐conjugates of EXE and 17β‐DHE were detected and quantified using UPLC‐MS/MS. Results from the conjugation assays indicated that the cytosolic enzyme GSTA1 is highly active against both EXE and 17β‐DHE, while GSTM3 and GSTM1 are only moderately active against EXE. No other recombinant cytosolic GST enzymes tested (GSTA2, GSTA4, GSTK1, GSTM2, GSTM4, GSTO1, GSTP1, GSTZ1 and GSTT1) exhibited activity against either EXE or 17β‐DHE. KM values for human liver cytosol (HLC) and purified recombinant GSTA1 were similar for both EXE (45 μM vs. 77 μM, respectively) and 17β‐DHE (54 μM and 30 μM, respectively) as substrate, suggesting that GSTA1 may be the main driver for EXE and 17β‐DHE glutathione conjugation in human liver cytosol. Using EXE as a substrate, Vmax value for GSTA1 was >7‐fold higher than that observed for GSTM3 (9.8 nmol/min/mg vs. 1.3 nmol/min/mg, respectively). The EXE intrinsic clearance (ClINT) for GSTA1 (130 nl/min/mg) was >10‐fold higher than that observed for GSTM3 (11 nl/min/mg). These data suggest that GSTA1 may be an important component in the metabolic pathway contributing to the elimination of EXE in breast cancer patients.Support or Funding InformationThis work is supported by grants from NIH, National Cancer Institute (grant R01‐CA164366; to P. Lazarus) and the Health Sciences and Services Authority of Spokane, Washington (grant WSU002292 to College of Pharmacy and Pharmaceutical Sciences, Washington State University)
S‐Nitrosoglutathione Reductase Inhibition Attenuates Long Term Effects of Neonatal Hyperoxia on Airway Hyperreactivity
Authors: Sopi Ramadan B. , MacFarlane Peter M. , Gaston Benjamin , Martin Richard J. , Raffay Thomas M. 
Published: April 2020

Bronchopulmonary dysplasia survivors display long‐term obstructive lung disease with airway hyperreactivity (AHR). While often treated with asthma medications, patient responses are variable. We have previously shown that the endogenous smooth muscle relaxant molecule, S‐nitrosoglutathione (GSNO), is degraded in neonatal hyperoxia by upregulated GSNO reductase (GSNOR) in the murine lung (Raffay, et al. Molecular Pharmacology 2016; 90(4):418‐26). We hypothesize that inhibition of GSNOR is a novel approach to attenuate neonatal hyperoxia‐induced AHR in room air recovered mice. Newborn C57BL/6 mice were randomized on the first day of life and assigned to room air (21% O2) or hyperoxic (60% O2) groups for three weeks to induce bronchopulmonary dysplasia AHR. Animals were then recovered in room air until six weeks of age. AHR was assessed in vitro using precision‐cut living lung slice preparations in response to increasing doses of bath‐applied methacholine (MCh, 0.25–64 μM). Lung slices were pre‐incubated with or without a GSNOR inhibitor (100 μM N6022) or a β2 adrenergic receptor agonist (10 or 100 μM levalbuterol). AHR is reported as percent change in airway lumen area from baseline (± SEM). 2–3 airways were imaged and averaged per animal per condition. Neonatal hyperoxia significantly increased airway contractile responses to MCh in room air recovered mice with a mean maximal effect (Emax) of 71.9 ± 2.7% change from baseline compared to the room air control Emax of 39.8 ± 5.2% (p<0.01). GSNOR inhibition significantly attenuated hyperoxic effects on AHR (32.2 ± 2.0%, p<0.001). Pre‐incubation of hyperoxic slices with either a GSNOR inhibitor or a β2 adrenergic receptor agonist (100 μM) also attenuated the response to the maximal dose of MCh (64 μM) compared to vehicle (both p<0.01). These studies show that neonatal hyperoxia‐exposed mice display longer‐term in vitro AHR to MCh and that inhibition of GSNOR reverses hyperoxia‐induced AHR comparable to a β2 adrenergic receptor agonist. We speculate that GSNO‐based therapies may serve as novel treatments for AHR in bronchopulmonary dysplasia survivors that do not respond to β2 adrenergic receptor agonists.Support or Funding InformationSupported by NIH 5K08HL133459‐03 and ATS Research Foundation Unrestricted Critical Care Grant
Evaluation of Lysis and Recovery of Genomic DNA from Microbiota to Maximize Efficiency for DNA Sequencing
Authors: Proctor Caleb McKinley , Okparanta Akelachi , Ryan Gabriella , Easparro Brandon , Nash Rodney 
Published: April 2020

Microbes, specifically microbes found in mixed populations in guts, soil, feces, and other tissues, have been the object of much research for commensal relationships for decades. Many of these microbes are ubiquitous across many species or biomes but are present very in low quantities due to the inherent challenges of microorganism survival in environmental conditions. While, in some cases, only present in low quantity, soil microorganisms are a critical component of our ecosystem. Soil microbiome studies aim to characterize the microbial repertoire and to correlate the microbial population with soil and ecological functions. Given the time involved and complexity of culture‐based enrichments, along with the inability to grow some species in culture, many modern studies involve direct cell lysis and DNA recovery from soil followed by PCR or genetic sequencing for target enrichment, detection, and analysis. In order to evaluate the organisms, present in such low quantities, methods must be developed to lyse all organisms present in soil of various sizes and toughness’. In this study we evaluate the lysis and recovery of a microbiological standard using commercially available kits and genetic sequencing to show an ideal bead milling matrix for microbiota in soil.Support or Funding InformationFunding for this project was provided by Omni International
Bacterial Expression of Chimeric Escherichia coli and Trypanosoma brucei DNA Methyltransferases
Authors: Taber Cassandra C. , Militello Kevin T. 
Published: April 2020

Our laboratory is interested in DNA and RNA methylation in E. coli and T. brucei as little is known about this form of epigenetic regulation in microorganisms. One methyltransferase being studied at this time is a putative DNA methyltransferase (TbDmt) from Trypanosoma brucei. The exact function of TbDmt is unknown but the protein strongly resembles bacterial DNA methyltransferases such as DNA cytosine methyltransferase (EcDcm) from E. coli. To test our hypothesis that TbDmt is a DNA methyltransferase, we expressed TbDmt in bacteria and created two chimeric protein sequences switching the DNA binding domain and enzymatic domain of EcDcm and TbDmt. Exchanging the DNA binding domain and enzymatic domain of TbDmt with a known methyltransferase may help us discover the function of the enzyme and, if it is a methyltransferase, what DNA sequence is targeted for methylation. Plasmids were made containing the sequences for EcDcm, TbDmt, and both chimeric proteins where the genes are adjacent to the lac operator. E. coli were transformed with the plasmids and expression was induced with IPTG. All four proteins were produced at 20°C, but the proteins with the TbDmt enzymatic domain were less soluble than those with the EcDcm enzymatic domain. The plasmids were re‐isolated after three hours of growth in the presence of IPTG. The plasmids were then digested with eight restriction enzymes blocked by methylation. Each digestion was run on an agarose gel with DNA from uninduced cells and unmethylated phage lambda DNA as controls. EcDcm methylated at its expected site, 5′CCWGG3′, as determined by resistance to PspGI. In addition, there is partial resistance to HpaII in cells containing EcDcm indicating methylation at 5′CCGG3′ or related sequences. The proteins were denatured using guanidium HCl, isolated and analyzed by SDS‐PAGE with Coomassie blue staining. EcDcm and the chimeric protein with the EcDcm enzymatic domain and TbDmt DNA binding domain were successfully purified and were then purified using partial denaturing conditions. These proteins will be tested for methyltransferase activity. TbDMT and the chimera with the TbDMT enzymatic domain were not able to be purified using the guanidium HCl denaturing method so stronger denaturants will be tested in attempt to purify the proteins. Further work will be done in purifying the proteins using both denaturing and nondenaturing conditions to produce functional enzymes for use in methylation assays. This work will contribute to the limited knowledge of methyltransferases in bacteria and protists.Support or Funding InformationNIH grant 1R15AI133428‐01 to KTM
Assessment of the Lateral Ventricle as a Means to Identify Pre‐Dementia
Authors: Ritz Brianna Kathryn , Zdilla Matthew 
Published: April 2020

Dementia is a component of several neurological diseases including Alzheimer Disease. Unfortunately, predictive factors regarding dementia are elusive and early diagnosis remains challenging; yet, early diagnosis is a key factor in decreasing the severity of the disease. The authors hypothesized that ventricular expansion characteristic of hydrocephalus ex vacuo may be reflective of dementia diagnosis. Therefore, this study assessed the anatomy of the lateral ventricles via MRI in an unremarkable population (n=733), a pre‐dementia population (i.e., a population who was considered unremarkable at the time of MRI but subsequently developed dementia) (n=82), and a population of individuals with diagnosed dementia (n=235). Significant differences in total left‐ and right‐sided lateral ventricle volumes were found between unremarkable and pre‐dementia populations (LEFT: t=10.332, Mean difference=10,627mm3, 95% CI of Mean = (8,607.7mm3, 12,645mm3; p<0.0001. RIGHT: t=9.89, Mean difference=9,700mm3, 95% CI of Mean = 7775mm3, 11,625mm3; p<0.0001.). Also, significant differences in left‐sided and right‐sided inferolateral (i.e., inferior horn) ventricle volumes were found between unremarkable and pre‐dementia populations (LEFT: t=16.966, Mean difference=798mm3, 95% CI of Mean = 706mm3, 891mm3; p<0.0001. RIGHT: t=17.552, Mean difference=778mm3, 95% CI of Mean = 691mm3, 864mm3; p<0.0001). Moreover, significant differences were identified in ventricular volumes between the unremarkable population and the dementia population; however, no significant differences were found in the ventricular volumes between the pre‐dementia and dementia population. Therefore, an anatomical assessment of the lateral ventricles and their inferior horns may aid in the early identification of individuals who are at risk of having diagnosed dementia.Support or Funding InformationFunding: WV Research Challenge Fund [HEPC.dsr.17.06] and NASA West Virginia Space Grant Consortium, Training Grant #NNX15AI01H
GATA‐4 interacts with the UCS domain of the Myosin Chaperone, Striated Muscle UNC‐45
Authors: Odunuga Odutayo , Anderson Morgan , Duncan Drake 
Published: April 2020

Striated muscle UNC‐45 (SM UNC‐45 or UNC‐45B) protein acts as a chaperone for cardiac and skeletal muscle myosins; regulating their folding, assembly into thick filaments, interaction with other sarcomeric proteins, and degradation. GATA‐4 is a transcription factor that regulates the expression of several cardiac muscle genes, including myosin. A previous study by Chen et al (2012) demonstrated for the first time that SM UNC‐45 (UNC‐45B) physically interacted with GATA‐4 and promoted its transcriptional activity in an overexpression experiment. The aim of this study was to identify the specific domain in UNC‐45B that interacted with GATA‐4. Pull‐down assays showed that GATA‐4 bound to the UCS domain of UNC‐45B. The major revelation from these studies is that UNC‐45B has the potential to exert both short‐term (protein level) and long‐term (gene level) controls over myosin and therefore muscle structure and function. Experiments are ongoing to determine whether UNC‐45B acts as a chaperone on GATA‐4 protein.Support or Funding InformationRobert A. Welch Foundation Grant #AN‐0008
Determining the Kinetics of Desferroxiamine D from Streptomyces coelicolor using Isothermal Titration Calorimetry
Authors: Goncuian Eliana , Hoffmann Katherine 
Published: April 2020

The long‐term objective for this research is to create a new class of antibiotics that targets the non‐ribosomal peptide synthetase independent siderophore (NIS) pathway in bacteria. The focus of my current research is to quantify the first and second order kinetic constants and obtain the kinetics curve of our model system, Desferroxiamine D (DesD), from Streptomyces coelicolor. DesD reacts with ATP as a cofactor, and either the small (hydroxysuccinylcadaverine, HSC), medium or large (desferrioxamine G, or dfoG) substrates in an iterative manner. We have so far collected activity curves with ATP and dfoG limiting, and are pursuing HSC curves. To do this we used Isothermal Titration Calorimetry (ITC) to collect data and GraphPad Prism 8.2 along with Malvern MicroCal Peaq ITC software to fit the kinetics curves and determine the kinetics constants. We successfully gathered and analyzed ITC data for both the catalytic (wild type, Wt) and non‐catalytic (mutant, R306Q) forms of DesD and determined the optimal buffer conditions for solubility. We also gathered data regarding the activity of our Wt DesD enzyme in order to determine the lifespan and the time of optimal catalytic activity. We have found that our enzyme is still active within 96 hours where it was previously thought to have a 36–48 hour catalytic lifespan.In conducting these experiments, we observed that an industry standard amount of magnesium chloride (MgCl2) in our solution caused an oscillating signal during equilibration of the ITC prior to enzyme injection. We hypothesized that the data reflects metal chelation of the Mg by our substrate (dfoG). We plan on conducting a binding experiment using the ITC in order to quantify the substrate (dfoG) and MgCl2 interaction, and ultimately recommend a new standard amount of MgCl2 to the field for future experiments. Other future directions include determining the kinetic constants of Wt DesD interacting with its small substrate HSC (which must be synthesized and purified,) and determining the catalytic role of other residues in the active site by site‐directed mutagenesis and ITC. This experimental assay and data will be the foundation for future drug design targeting DesD.Support or Funding InformationThis research was supported in part by the National Science Foundation (NSF‐RUI grant 1716986 to K.M.H.), and by the John Stauffer Charitable Trust.