bluuebunny/crossref_metadata_2025_split huggingface dataset:
Curcuminoid analogs, a novel class of drugs for the treatment of Glioblastoma multiforme
Authors: Shivachar Amruthesh C. , Selvam Chelliah , Bhaskaran Mahendran , Hosseini- Zare Maryam Sadat , Ranganna Kasturi 
Published: April 2020

Glioblastoma multiforme is one of the glial‐derived malignant primary brain tumors in adults with a poor prognosis. The average survival period still remains between 14–16 months despite various treatment modalities involving combination of surgery, radiotherapy and chemotherapy with temozolomide, an imidazotetrazine derivative. Curcumin (#1) is a plant derived dietary constituent, and has been shown to play a role in preventing many cancers. We investigated for the first time the potencies and the molecular mechanisms of action of #1 and its three synthetic analogs (#7, #20, and #32) that were prepared through an acid catalyzed condensation with various hydrazine derivatives. Cultures of grade IV glioblastoma multiforme (GBM) MG118 cells (5,000/cm2) were treated with various concentrations (0.01–100μmole) of #1, #7, #20 or #32 for 24 hr. along with temozolomide (0.1–300μmole) as positive control. Cell viability/toxicity was assessed by Cell Titer Glo 2.0 Assay, and by the fluorescent dyes Hoechst 33342/Propidium iodide‐based live‐dead cell assay. Apoptosis was directly assessed by TACS Annexin V‐FITC Apoptosis detection kit. Concurrently, we measured the effects of these analogs on the expression of pro‐angiogenic HIF1α, VEGF, and chemokine receptor 4 (CXCR4) by western blotting analyses and quantitated using GAPDH loading controls. Additionally, we formulated curcuminoid‐loaded nanoparticles & optimized it using Design Expert (DoE) software and then evaluated the prepared formulation for determining the cytotoxic effects in GBM. Our results show that the tested analogs dose‐dependently caused apoptosis, decreased cell viability and inhibited cell proliferation in the order #32> #20> #7> #1>>temozolamide, suggesting that synthetic cucuminoids 32 and 20 are more potent than their parent compound and the current drug temozolomide. Additionally, we noted that these two analogs down‐regulated the expression of VEGF, and anti‐hypoxic HIF1α, and the chemokine receptor CXCR4 a known mediator of cancer cell proliferation and invasion. Thus our preclinical results demonstrate that compounds 32 and 20 may be two novel class of drugs for the treatment of GBM by down‐regulation of key targets that are critical for GBM survival and metastasis.Support or Funding InformationPartial support from an Institutional grant (ACS), and a grant from NIMHD/NIH #2G12MD007605 (KR).
Exploring the Interaction of Msh2‐Msh6 with the Holliday Junction Through ATPase Activity
Authors: Joshi Meera , Li Yan , Lombardo Zane , Hingorani Manju , Mukerji Ishita 
Published: April 2020

DNA mismatch repair systems (MMR) are important in reducing post replicative errors and preventing recombination between homologous sequences. Mismatch repair is highly conserved in most organisms and is initiated by the MutS homodimer in prokaryotes. In eukaryotes, small insertion/deletion loops (IDL), and single base pair mismatches are recognized by the MutS homolog Msh2‐Msh6. Prior research in our lab and by others (Marsischky G. T., Lee S., Griffith J., and Kolodner R. D. (1999) J.Biol.Chem. 274: 7200–7206 doi:10.1074/jbc.274.11.7200) determined the binding affinity of the junction with Msh2‐Msh6 with a Kd = 12.9 ± 1.3 nM, which is similar to the binding affinity for a G:T duplex, suggesting that the Msh2‐Msh6 Holliday Junction interaction is specific. Our investigation aims to understand the Msh2‐Msh6‐junction binding interaction by studying nucleotide utilization upon binding and determining any conformational changes associated with complex formation and ATP hydrolysis. We use the well‐characterized J3 Holliday junction with 17 base pair arms to characterize binding. Malachite green assays measuring inorganic phosphate were performed to obtain the rate of ATP hydrolysis in the presence of Msh2‐Msh6, J3 junction, and other DNA substrates. Msh2‐Msh6 alone has a Kcat = 0.27s−1 in the presence of 100mM NaCl, with the Kcat weakly increasing with a G:T mismatch 34‐mer duplex and the J3 junction to 0.47s−1and 0.6s−1 respectively. We will also report on junction conformational changes associated with protein binding and how protein affinity for the junction is modulated by nucleotide binding.Support or Funding InformationASBMB Undergraduate Research Grant.
Link between Nutritional Status and Gene Expression in Regulatory Associated Protein of Mammalian Target or Rapamycin mRNA (RPTOR) and Rapamycin‐insensitive Companion of Mammalian Target of Rapamycin (RICTR) messenger RNA
Authors: Kostner Danica LeAnn , Spainhour Rebekah , Wertz Blaine LeAnn , Schmidtberger Abigail , Dougherty Megan , Abernathy Oaklee LeAnn , Ball Jenna , Kobayashi Yass 
Published: April 2020

RPTOR and RICTR form a complex with Mammalian Target of Rapamycin (mTOR) called mTORC1 and 2, respectively and activates the transcription of genes associated with different metabolic pathways, including autophagy. Previous studies showed the expression of mTOR mRNA in muscle was increased in response to reduced food intake in channel catfish. However, whether RPTOR and RICTR mRNA expression is influenced by changes in food intake in catfish is unclear. The objective of the current study was to explore the relationship of RPTOR and RICTR mRNA expression and food intake in the brain, liver, and muscle. For the first study, channel catfish were fed daily (control) or fasted for 28 days (fasted). Fish in the third treatment were fasted for the first 14 days then fed daily for 14 days (refed). For the second study, catfish were fed once every 12 (overfed), 24 (control), or 48 hours (underfed, n=4 tanks per treatment) for 28 days. Brain, liver, and muscle expression of RPTOR and RICTR mRNA was measured from the samples collected on day 28. Muscle expression of RPTOR and RICTR mRNA was unaffected by changes in food intake. In contrast, fasting increased brain expression of RICTR mRNA, and increased feeding frequency decreased RPTOR mRNA in the liver. Our results showed that changes in the expression of RPTOR and RICTR mRNA did not coincide with changes in the expression of mTOR mRNA. The exact relationship between food intake and expression of these mRNA species needs further investigation.Support or Funding InformationK‐INBRE P20GM103418
Kinetic control of MERS‐CoV 3CLpro is mediated by non‐conserved residues distal to the active site
Authors: Anson Brandon James , Daczkowski Courtney Marie , Mesecar Andrew David 
Published: April 2020

Middle‐East Respiratory Syndrome (MERS) Coronavirus is a re‐emergent zoonotic pathogen with a 30% fatality rate in humans. The MERS positive‐sense single‐stranded RNA genome is translated into two polyproteins that must be processed by two viral cysteine proteases (3CLpro and PLpro) into 16 non‐structural proteins (nsp) before viral replication can occur. Attempts to drug either of these cysteine proteases have proven difficult. MERS 3CLpro functions as dimer and is unique among all of the coronavirus 3CLpro enzymes studied to date because competitive inhibitors activate the wild‐type MERS 3CLpro enzyme at low inhibitor concentrations and inhibit at higher concentrations. This mechanism of substrate/inhibitor‐induced dimerization seems to be mediated by non‐conserved residues in structural regions distal to the catalytic site. We have identified unique residues that are involved in the inhibitor‐induced dimerization mechanism and we show that these ‘competitive’ inhibitors activate and inhibit in a dose‐dependent manner. In an attempt to provide a structural basis for this mechanism, we crystallized the apo and inhibitor‐bound forms of this enzyme and diffracted them to 1.5 – 2.2 Å. We also show that residues in distinct structural regions synergize to modulate the intrinsic kinetic parameters of this family of enzymes. These parameters include increased turnover (kcat) and a lower dimer dissociation constant (KD). Additionally, a rapid‐equilibrium kinetic mechanism was developed to model the kinetic response of MERS 3CLpro to inhibitors that act via substrate/inhibitor induced activation/inhibition mechanisms.Support or Funding InformationThis project has been funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Department of Health and Human Services, under Contracts No. HHSN272200700058C, HHSN272201200026C, and HHSN272201700060C. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357.Use of the Lilly Research Laboratories Collaborative Access Team (LRL‐CAT) beamline at Sector 31 of the Advanced Photon Source was provided by Eli Lilly Company, which operates the facility.
Inhibition of JNK Signaling During Mast Cell Differentiation Promotes Altered Cytokine Secretion and Degranulation Profiles
Authors: Hicks Natalie J. , Crozier Robert W.E. , Den Hartogh Danja J. , MacNeil Lori A. , MacNeil Adam J. 
Published: April 2020

Mast cells are large granulated leukocytes active in both innate and acquired immunity. These cells differentiate from hematopoietic stem cells, found in the medullary cavity of long trabecular bones. Increased inflammatory activity and numbers of mast cells contribute to allergic disease, which is of increasing concern worldwide. C‐jun N‐terminal kinase (JNK) is a major signaling node in the map kinase pathway that has been shown to contribute to IL‐3 signaling, known to promote murine mast cell differentiation. The aim of this research is to determine the role of JNK signaling in mast cell differentiation. Primary bone marrow‐derived mast cell (BMMC) cultures were established from C57BL/6 mice and differentiated for 7 weeks in the presence or absence of a JNK inhibitor (JNK‐IN‐8). Following removal of the inhibitor for 5 days, phenotypic and functional characteristics of BMMCs were probed using flow cytometry, ELISA, and β‐hexominidase release assays. Flow cytometric analysis of surface expression of the mast cell marker c‐kit, and levels of IgE bound to FcɛRI, a measure of mast cell sensitization, were unaffected by impaired JNK signaling compared to cells grown in the absence of inhibitor over mast cell differentiation, suggesting a normal mast cell phenotype with the potential to be activated via the allergen‐bound IgE‐dependent pathway. Cells differentiated under JNK inhibition showed a significant impairment in the secretion of IL‐6 (p<0.05) and IL‐13 (p<0.05) and a non‐significant impairment in TNF and CCL1 secretion at 24 hrs post‐activation compared to cells grown in the absence of inhibitor. Degranulation was also significantly decreased (by 25%; p<0.05) in BMMCs grown under JNK inhibition. To probe an epigenetic mechanistic role for histone acetylation changes in JNK‐impaired differentiation, expression of HDAC and HAT enzymes were considered by qPCR and western blotting, but were not modulated by JNK. These results suggest that JNK plays an important role in the development of a fully functional mast cell in the context of IgE‐mediated allergic inflammation and may warrant consideration as a strategy to regulate mast cell development in associated pathologies.Support or Funding InformationSupported by the Natural Sciences and Engineering Research Council of Canada (NSERC); Canada Foundation for Innovation (CFI); Government of Ontario and Brock University.
Effect of 7‐Day Whey Protein Supplementation on Steroid Hormones and SHBG in Women with Polycystic Ovary Syndrome (PCOS)
Authors: Zumbro Emily L. , Rao Manisha , Balcom-Luker Shenavia , Broughton K. Shane , LeMieux Monique J. 
Published: April 2020

Polycystic ovary syndrome (PCOS) is the most prevalent endocrinopathy in women of child‐bearing age and is associated with obesity, infertility, insulin resistance, and hyperandrogenemia. Sex‐hormone binding globulin (SHBG) is suppressed as a result of hyperinsulinemia leading to an increase in free testosterone levels commonly observed in PCOS women. High levels of free testosterone contributes to hirsutism, acne, and irregular menstrual cycles in this population resulting in the need for interventions aimed at increasing SHBG concentration in PCOS women. Subsequently, estradiol and progesterone levels suffer in this population resulting in irregular menstrual cycles and infertility. Current treatments for PCOS target symptoms of the disease but not the underlying mechanisms, and typically are associated with adverse side effects. Nutritional interventions, therefore, that can ameliorate PCOS‐related issues are warranted. Whey protein has recently been shown to have improved glycemic homeostasis, reduced chronic inflammation and enhanced protein anabolism effects in people with or without insulin resistance. The purpose of this study was to examine changes in SHBG and free testosterone concentrations following 7 days of whey protein supplementation and to examine differences in baseline measurements of progesterone and estradiol between PCOS women and healthy controls (CON). Women with PCOS and CON consumed 35g of whey protein for 7 days. Baseline and Day 7 concentrations were measured via plasma and serum collection. Progesterone and estradiol concentrations were measured at baseline only whereas free testosterone and SHBG concentrations were measured at baseline and Day 7. Protocols used in this study were approved by the TWU Institutional Review Broad (IRB). Initial analyses indicate progesterone levels were lower in the PCOS group compared to CON. Currently, no other statistical differences are reported at baseline nor over time in any other variable. Additional analyses are currently being conducted. Seven days whey protein supplementation is not a viable intervention to increase fertility in PCOS women by enabling changes in steroid hormone and globulin binding concentrations. Future studies will include a prolonged intervention period to establish potential changes in the aforementioned hormones following chronic whey protein supplementation.Support or Funding InformationGlanbia Nutritionals, Inc., TWU Research Enhancement Program, TWU Human Nutrition Research funds
Cryo‐EM Structure of CtBP2 Confirms Tetrameric Architecture
Authors: Jecrois Anne M. , Bellesis Andrew , Schiffer Celia , Royer William 
Published: April 2020

C‐terminal binding proteins 1 and 2 (CtBP1 and CtBP2) are transcriptional regulators that regulate gene expression. CtBP can act as both an activator and repressor of transcription by interacting with different transcription factors and chromatin modifying enzymes. CtBP is activated under conditions of hypoxia when the NADH level is elevated in the cell, which has direct implication in various forms of cancer. The transcriptional activity of CtBP can confer resistance to apoptosis and/or promotes oncogenesis and metastasis depending on its interacting partners. Due to its role in tumorigenesis and correlation with poor patient prognosis, CtBP is a potential drug target.NADH exerts its transcriptional effect through promoting CtBP oligomerization. The activated oligomer can then associate with other co‐factors to form the CtBP‐mediated repression complex. Although previous studies have proposed dimeric CtBP as the relevant oligomeric state, our studies with multi‐angle light scattering have shown that the primary effect of NADH binding is to promote the assembly of two CtBP dimers into tetramers. Here, we present the 4.5Å CryoEM structure of CtBP2 corroborating that the native state of NADH bound CtBP2 is indeed tetrameric. Together, these results highlight the tetramer as the functional oligomeric form of CtBP proteins.Support or Funding Information• 1 F31 GM129988‐01• R01 GM119014
5‐Aminoethylbenzimdazole Suppresses Lipopolysaccharide (LPS)/Interferon Gamma (IFNγ)‐Induced Inflammatory Responses in Macrophages
Authors: Chen Xin , Smith Amanda , Plummer Christopher , Lei Wei 
Published: April 2020

Tec family non‐receptor tyrosine kinases, including TEC, BTK, ITK/EMT/TSK, BMX and TXK/RLK, are expressed in a variety of immune cells. Previous studies have demonstrated that Tec kinases play pivotal roles in inflammatory disorders such as allergic asthma and atopic dermatitis. Several 5‐aminomethylbenzimdazoles (5‐AMBD) have been reported as highly potent inhibitors of ITK as well as other TEC kinases and showed strong activities on reducing IL‐2 and IL‐4 production. However, the impact of 5‐AMBD on macrophage‐mediated inflammatory responses remains unclear. The goal of this study was to determine the effect of 5‐AMBD on the inflammatory responses in macrophages stimulated with lipopolysaccharide (LPS) and interferon‐gamma (IFNγ). RAW 264.7 cells (a murine macrophage cell line) were stimulated with 10 ng/mL LPS and 1 ng/mL IFNγ for 10 min, then treated with various concentrations of 5‐AMBD. The cell viability and production of nitric oxide (NO) were measured 20 hr after treatment. The total RNA was extracted from cells treated for 4 hr, and then the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNFα), and interleukin 1 beta (IL‐1β) were evaluated using quantitative real‐time PCR. The impact of 5‐AMBD on the activation of nuclear factor kappa B (NF‐κB) was investigated using the RAW 264.7 cells stably expressing the NF‐kB luciferase reporter. 5‐AMBD dose‐dependently reduced the NO production with no significant toxicity to the macrophages. Treatment of 100 nM and 1 μM 5‐AMBD significantly inhibited the expression of iNOS by 50% and 95%, respectively. Meanwhile, 5‐AMBD (1 μM) also reduced the expression of IL‐1β and TNFα. Furthermore, we found that 5‐AMBD dose‐dependently diminished the activation of NF‐κB at 4 hr and 20 hr after treatment. These findings demonstrate that 5‐AMBD has a strong anti‐inflammatory activity in macrophages, and suggest that TEC kinases could be potential drug targets for inflammatory diseases.Support or Funding InformationWork supported by Small Pharmacy Awards for Research and Collaboration from the Presbyterian College School of Pharmacy.
Zebrafish‐inspired strategies for spinal cord repair.
Authors: Mokalled Mayssa H. , Zhou Lili , Mcadow Ryan , Burris Brooke 
Published: April 2020

Spinal cord injury (SCI) causes irreversible loss of sensory and motor functions in mammals. In contrast to mammals, adult zebrafish are capable of efficient, spontaneous recovery after SCI. Whereas glial scarring and neuron loss accentuate spinal cord (SC) damage and inhibit regeneration in mammals, zebrafish are capable of pro‐regenerative glial bridging and effective adult neurogenesis post‐injury. Emerging evidence suggests that in mammals these pro‐regenerative processes are either inefficient or overshadowed by anti‐regenerative effects. We propose that developing efficient SCI treatments will require a deeper understanding of endogenous SC regenerative capacity, and that adult zebrafish present a valuable model to uncover fundamental, evolutionarily concealed mechanisms of mammalian SC regeneration.Although glial bridging and neurogenesis are hallmarks of the robust SC repair observed in zebrafish, the mechanisms that direct these processes are underexplored. Using expression profiling and genetic loss‐of‐function, we screened for and identified a suite of molecular factors and cell populations that are activated after injury and required for SC regeneration. These studies revealed that discrete lineage‐restricted niches of progenitor cells differentially contribute to glial bridging and neurogenesis after SCI in zebrafish. Using a battery of genetic and molecular tools, we identified yap‐ctgfa‐twist1a as a central glial bridging signaling axis, generated the first transcriptional profiles of zebrafish bridging glia, and elucidated glial cell injury responses at the single cell level in zebrafish. We are currently using our understanding of the bridging glial cell fate in zebrafish to perform molecular comparisons with mammalian glial cells, and to reprogram mammalian glia into scarless, pro‐regenerative scaffolds that promote SC regrowth. Taken together, our studies elucidate fundamental mechanisms that direct innate SC regeneration in zebrafish, and use natural SC regeneration as a platform to enhance mammalian SC repair at the molecular and cellular levels.Support or Funding InformationWe acknowledge support from NIH (R01‐NS113915), the Missouri Spinal Cord Injury/Disease Research Program, the McDonnell Center for Cellular and Molecular Neuroscience, the Institute of Clinical and Translational Sciences, and from Washington University School of Medicine to M.H.M.
Intracellular Localization Patterns of Cancer‐associated Thyroid Hormone Receptor Mutants
Authors: Silva Carolina Lopez , Allison Lizabeth A. 
Published: April 2020

The thyroid hormone receptor (TR) is a nuclear receptor that plays a critical role in human development and metabolism, regulating thyroid hormone interactions. TR rapidly shuttles between the nucleus and cytoplasm but is primarily localized to the nucleus at steady state, where it acts as a hormone‐dependent transcription factor. This process of nuclear import and export is mediated by two nuclear localization signals (NLSs) and multiple nuclear export signals (NESs). Previous studies have shown that TR may act as a tumor suppressor by regulating key genes in cell growth and proliferation in response to thyroid hormone. However, if TR shuttling is misregulated, it will not be able to perform its transcriptional regulatory functions. In this project, we analyzed the impact of NLS and NES amino acid substitutions on the intracellular localization patterns of TRα1 mutants previously associated with hepatocellular carcinoma (G24E, M256V, E343A, P269L) and thyroid cancer (K29T, C97A). We transfected mammalian cells with expression plasmids for green fluorescent protein (GFP)‐tagged and mCherry‐tagged TRα1 mutants and examined localization patterns through quantitative fluorescence microscopy. Here, we show that both TRα1 mutations exhibit increased cytosolic distributions. Further, mutant TRα1 (K29T, C97A) had an increased tendency to form nuclear and cytosolic protein aggregates. In co‐transfection experiments, TRα1 (K29T, C97A) failed to co‐localize with LC3, a well‐characterized autophagy marker, but showed significant co‐localization with GFP‐170, a marker for aggresome formation. These data suggest that aggregates formed by TRα1 (K29T, C97A) are actively recruited to cellular aggresomes as a cellular stress response. Overall, our study provides evidence that even minor changes in TR’s primary structure can have a significant impact on its intracellular localization and folding patterns.Support or Funding InformationThis project was funded by the National Institutes of Health. Grant information: 2018–2021, P.I., “Thyroid Hormone Receptor Mislocalization and Pathogenesis,” National Institutes of Health (NIH‐NIDDK) Research Grant # 2 R15 DK058028‐05: $405,273