bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of Notch signalling using fucose analogs
Authors: Hao Huilin , Liu Zilei , Wu Peng , Haltiwanger Robert 
Published: April 2020

Notch is an evolutionarily conserved cell surface receptor for short range cell‐cell communication. Notch signalling has critical roles in many cell‐fate related events, such as cell differentiation, proliferation and survival. Dysfunction of Notch signalling is associated with various congenital disorders and a wide range of human malignancies. Notch is an important drug target for cancer therapy. However, the initial efforts to inhibit Notch activity with γ‐secretase inhibitors (GSIs) have failed because of severe gastrointestinal toxicity. Novel Notch‐targeting strategies are urgently needed. One unique characteristic of the Notch receptor is the heavy O‐glycosylation of its extracellular domain (ECD). The O‐linked glycans, especially O‐fucose added by POFUT1 (Protein O‐Fucosyltransferase 1), are necessary for proper Notch function, such as protein stability, trafficking and interaction with ligands. We hypothesize that disrupting Notch O‐fucosylation can be a potential approach for Notch inhibition. We have demonstrated that L‐fucose analogs with carbon 6 modifications are potent Notch inhibitors, especially 6‐alkynyl and 6‐alkenyl fucose. The analogs are efficiently incorporated to Notch EGF repeats by POFUT1, and inhibit Notch signalling at sub‐micromolar concentrations. Interestingly, these analogs inhibit Delta but not Jagged‐mediated Notch activity, suggesting they are the first small molecule, ligand‐specific Notch inhibitors, and can be a powerful tool for Notch study. Notch controls the early differentiation of T cells in thymus. Blocking Notch signalling with the analogs showed a complete inhibition of T cell differentiation with Delta ligands in an in vitro OP9 cell culture system. We have preliminary results showing the analogs also showed inhibitory effects in vivo using fetal thymus organ culture (FTOC). Many cancers have mutations in Notch pathway components that make Notch hyperactive. We found the analogs strongly inhibit the constitutively active signalling of NOTCH1 mutants identified from acute lymphoblastic leukemia (T‐ALL) patients. We are now examining the anti‐tumor ability of the analogs in various cancer cell lines where Notch or Notch pathway players contribute to tumorigenesis. The 6‐alkynyl fucose analog can also act as a probe for EGF repeat O‐fucosylation, and help to discover novel POFUT1 substrates. Additional fucose analogs are being synthesized and evaluated to find more effective inhibitors or analogs with distinct activities (e.g. Notch activation, Jagged‐specific or ligand‐nonspecific inhibition).Support or Funding InformationThis work was supported by NIH grant GM061126.
Acute Systemic Inhibition of Inflammation Augments Endothelium‐dependent Dilation in Women with a History of Preeclamptic Pregnancy
Authors: Stanhewicz Anna E. , Dillon Gabrielle A. , Serviente Corinna F. , Alexander Lacy M. 
Published: April 2020

Women who have had preeclampsia demonstrate microvascular endothelial‐dysfunction, mediated in part by reduced nitric oxide (NO)‐dependent dilation. Preeclamptic pregnancies are associated with elevated inflammation, and inhibition of inflammation attenuates endothelial damage in animal models of preeclampsia. However, it is unclear if systemic inhibition of inflammation improves endothelial function in women after a preeclamptic pregnancy. Using the cutaneous microcirculation as a model, we hypothesized that acute systemic inhibition of inflammation (oral salsalate; 1500mg/twice daily/4 days) would improve endothelium‐ and NO‐dependent vasodilation in women with a history of preeclampsia (PrEC) but not in women with a history of uncomplicated pregnancy (HC). Eleven HC (30±1yrs, 9±2 months post‐partum) and 8 PrEC (29±3yrs, 6±1 months post‐partum) participated in a double‐blind placebo‐controlled study. Following each treatment, 2 intradermal microdialysis fibers were placed in the skin of the ventral forearm for graded infusion of acetylcholine (ACh, 10−7–102 mM) or ACh+15mM L‐NAME (NO synthase antagonist). Red cell flux was measured over each site by laser‐Doppler flowmetry (LDF). Cutaneous vascular conductance was calculated (CVC=LDF/mean arterial pressure) and normalized to maximum (%CVCmax; 28mM SNP + local heat 43°C). ACh‐induced (75±3 vs. 92±4%CVCmax; p=0.002) and NO‐dependent (18±8 vs. 32±4%; p=0.04) vasodilation were attenuated in PrEC compared to HC following placebo. Salsalate augmented ACh‐induced (92±3%CVCmax; p=0.001) and NO‐dependent (38±4%; p=0.04) dilation in PrEC compared to placebo but had no effect in HC (all p>0.05). Women who have had preeclampsia demonstrate attenuated microvascular endothelial function compared to women with a history of uncomplicated pregnancy. Salsalate treatment augmented endothelium‐dependent vasodilation via NO‐mediated pathways in these women, suggesting that inflammatory signaling plays a role in mediating persistent endothelial‐dysfunction in women with a history of preeclampsia. ClinicalTrials.gov registration # NCT03482440Support or Funding InformationNIH K99 HL138133 (Stanhewicz); NIH R01 HL093238 (Alexander); NIH T32 AG049676 (Serviente)
Neuroprotective Effects of Estrogen on Beta‐Amyloid Induced Neurotoxicity in Murine Hypothalamic Cells
Authors: Persaud Nikita Sudarshana , Fletcher Jada , Hindieh Jennifer , Chakraborty Sanjoy , Chakraborty Tandra R. 
Published: April 2020

Alzheimer’s disease (AD) is a neurodegenerative affliction marked by significant cognitive decline and eventual dementia. The two most notable pathological hallmarks of AD include beta‐amyloid plaques, formed from the extracellular aggregation and deposition of beta‐amyloid fibrils, and the intracellular accumulation of hyperphosphorylated tau as neurofibrillary tangles in the brain. Previous research has suggested a possible neuroprotective property of estrogen, the female sex hormone, in AD. Despite the implications of a hypothalamic role in the early pathophysiology of AD, such as disturbances in sleep and emotional affect, scant literature exists outside of cortical and hippocampal AD research. This study is designed to replicate AD models using a control cell line, SH‐SY5Y human neuroblastoma cells, and an experimental cell line, N38 murine hypothalamic cells in culture, by incubating these cells with oligomerized human beta‐amyloid 42 (Aβ42). Each cell line is then treated with estrogen in order to observe the hormone’s efficacy as a neuroprotective agent. Cell viability, cytotoxicity and proliferation were measured in order to determine estrogen’s neuroprotective potential, using a variety of bioassays such as MTT and LDH. Preliminary results show lower mitochondrial activity and higher rates of cell lysis when treated with physiologically relevant concentrations of estrogen and low beta‐amyloid concentrations in the SH‐SY5Y cell line. In addition, the low concentration of Aβ42 (10μmol) appeared to have neuroproliferative effects on the SH‐SY5Y cells as opposed to its expected neurotoxicity. Conversely, N38 cells treated with estrogen at dosages higher than physiologically relevant concentrations, were found to have higher mitochondrial activity than cells treated with only Aβ42 after 24 hours, indicating a possible neuroprotective effect of estrogen at high concentrations on the hypothalamic cell line. Cell lysis and cytotoxicity of Aβ42 in N38 cells will be tested in order to further elucidate the pathology of the hypothalamus in AD, as well as estrogen’s potential neuroprotective effect on this unexplored brain region.Support or Funding InformationBiology Department, Adelphi University, Garden City, New York. Department of Biological Sciences, New York City College of Technology/CUNY, New York, NY.
Deciphering the Mechanisms of Action of Altertoxin II with Genome‐Wide CRISPR/Cas9 Knockout Screening
Authors: Robles Andrew J. , Dai Wentao , Vaseva Angelina , Risinger April L. , Cichewicz Robert H. , Mooberry Susan L. , Houghton Peter J. 
Published: April 2020

Ewing sarcomas (ES) are bone and soft tissue tumors that affect children and adolescents. These cancers are characterized by chromosomal translocations that result in expression of an oncogenic fusion protein (typically EWSR1‐FLI1). Despite advances in cure rates for many childhood malignancies, advanced, metastatic or relapsed ES remain a challenge. We identified the fungal metabolite altertoxin II (ATXII) as having highly selective activity against six ES cell lines compared to a panel of pediatric and adult cancer cells. On average, ATXII is 94‐fold (range 16‐ to 400‐fold) more potent against ES cells compared to rhabdomyosarcoma (RMS) cells, indicating high selectivity for ES. Mechanism of action studies indicated that ATXII selectively induces DNA damage in ES cells. However, the high degree of selectivity for ES suggests other mechanisms may be involved. To determine if ES sensitivity to ATXII is a dominant or recessive phenotype, we hybridized sensitive EW8 cells (ES) with resistant Rh30 cells (RMS) and determined the sensitivity of these hybrids to ATXII. Five single‐cell subclones derived from these hybrids were resistant to ATXII, suggesting that sensitivity to ATXII is a recessive phenotype in ES cells. Based on these data, we further investigated the mechanism of action of ATXII to identify which cellular pathways confer resistance. A genome‐wide CRISPR‐Cas9 knockout screen was conducted in resistant Rh30 cells using the GeCKOv2 library. After selection with puromycin, the transduced cells were treated for three weeks with ATXII or vehicle and genomic DNA was isolated every 4 days. These samples were subsequently prepared for next‐generation sequencing to identify sgRNAs that are depleted in the ATXII‐treated samples compared to vehicle‐treated control samples. Sequencing of these samples has recently been completed and initial bioinformatic analysis was conducted. Preliminary results have identified multiple differentially expressed sgRNAs in ATII‐treated Rh30 cells compared to vehicle‐treated cells, suggesting loss of these genes influences growth in the presence of ATXII. Overall, these studies have the potential to identify new molecular targets for the development of ES therapies.Support or Funding InformationFunded by P01CA165995 (PJH), R01GM107490 (RHC, SLM), T32CA148724 (Cancer Biology Training Program) and RP170345 (CPRIT Training Program)
Further Optimization of Heterologous Glucoraphanin Biosynthesis Through Co‐Expression Analysis
Authors: Pignolet Roxanna , Endelman Benjamin , Barnum Collin , Shih Patrick M. 
Published: April 2020

It is estimated that 38.4% of adults in the United States will be diagnosed with cancer (National Cancer Institute). With this high rate of incidence, the exploration of new options for cancer prevention and treatment is of high importance. Plant secondary metabolites are strong candidates for study due to their potential to be used in anti‐cancer dietary supplements. Isothiocyanates are products of the glucoraphanin biosynthesis pathway that have been demonstrated to display potent anti‐cancer and anti‐inflammatory properties. Previous studies have identified the genes in the core glucoraphanin biosynthesis pathway (CGBP); however, heterologous expression of the CGBP in tobacco has produced glucoraphanin at only 0.35 μmol g‐1 dry weight (Mikkelsen et al. 2010). In order to increase our titer, we used co‐expression analysis to identify a total of forty‐two gene candidates for optimizing the CGBP. We tested these candidates for their effect on glucoraphanin biosynthesis by transiently expressing the CGBP with candidate genes in tobacco. LC‐MS analysis of the transformed leaf tissue for glucoraphanin content revealed that six of the forty‐two genes increased glucoraphanin production. We also found several genes that produced significantly less glucoraphanin per gram dry weight when combined with the CGBP than the CGBP alone. The increases in heterologous glucoraphanin production implicate the six genes as important factors in the glucoraphanin biosynthetic pathway with the potential to raise concentrations closer to recommended supplement quantities.Support or Funding InformationThis project is funded by NIH Grant R00AT009573
Role of myeloid cell CYP19A1 in lung airway tissue repair following naphthalene induced epithelial injury in a mouse model
Authors: Han Weiguo , kovalchuk Nataliia , Ding Liang , Liu Pengfei , Yang Weizhu , Zhang Qing-Yu , Van Winkle Laura , Ding Xinxin 
Published: April 2020

CYP19A1 (the aromatase) is a key enzyme in the biosynthesis of estrogens, which have important regulatory functions, including roles as a major mitogenic stimulus for cellular proliferation. In this study, we are testing the hypothesis that macrophage CYP19A1 can increase estrogen production and/or regulate the function of macrophages at sites of tissue injury, thereby influencing the balance between normal tissue repair and pathogenic outcomes related to abnormal epithelial repair. A myeloid cell (including macrophage) specific Cyp19a1‐null mouse model was generated and characterized. Wild‐type and Cyp19a1‐null mice were treated with naphthalene (200 mg/kg, IP) to induce acute lung injury, or with corn oil as a vehicle control. Mouse lung was examined at 24 hour, 72 hours, or 14 days after the treatment, for extent of acute airway injury, post‐injury cellular proliferation, and tissue repair, respectively. At 24 hours, a genotype difference in the extent of naphthalene‐induced airway injury was not detected. At 72 hours, the fraction of airway epithelial cells that are positive for the cell proliferation marker Ki67 was higher in naphthalene‐treated wild‐type mice (40%–70%) than in naphthalene‐treated Cyp19a1‐null mice (20–30%); whereas the fraction was much lower in corn oil treated control mice (3–5%). At 14 days, the bronchiolar epithelium was completely recovered in wild‐type mice, but only partially (<30%) in the Cyp19a1‐null mice, as indicated by the abundance of Club cells (positive for Clara cell secretary protein) in the airway epithelium. Thus, the loss of macrophage CYP19A1 did not impact the degree of naphthalene‐induced lung injury; but it impeded the post‐injury cellular proliferation and tissue repair in the airways.Support or Funding InformationSupported in part by NIH grant ES020867
Epigenetic regulation of phenylethanolamine N‐methyltransferase: implications for adrenaline biosynthesis
Authors: Khurana Sandhya , Tharmalingam Sujeenthar , Murray Alyssa , Tai T.C. 
Published: April 2020

Adrenaline, a neurohormone and neurotransmitter, plays an extensive role in the physiological response to stress, and the sympathetic regulation of blood pressure. Phenylethanolamine N‐methyltransferase (PNMT), the terminal enzyme in the catecholamine biosynthetic pathway, directly responsible for adrenaline synthesis, is elevated in hypertensive animals; genetic linkage studies have associated the PNMT gene to the development of hypertension. Epigenetic changes are heritable changes in gene expression that are a consequence of the modification of the DNA (methylation at CpG sites) or histones (e.g. by methylation, acetylation) that facilitate packaging the DNA into nucleosomes. Changes in the epigenome have been associated with incidences of cancer, metabolic disorders and cardiovascular pathologies. In this study, alteration in PNMT expression modifiable by epigenetic regulation was examined using the rat adrenal pheochromocytoma derived PC12 cells. In vitromethylation of a PNMT promoter driven luciferase construct, using CpG methylases, consequently lead to a radical decrease in promoter activation, even in presence of dexamethasone (Dex) or Forskolin (Fsk), otherwise potent activators of PNMT expression. Further, the influence of a DNA methylation inhibitor 5‐aza‐2′‐deoxycytidine (5aza2DC), and histone deacetylase inhibitor valproic acid (VPA) was examined. Transcript analysis of endogenous PNMT, and PNMT promoter driven luciferase assays, both revealed that PNMT transcription is elevated in presence of these epigenetic modifiers, and these compounds can interact with the activation by Dex or Fsk to modulate PNMT expression. The extent of CpG methylation at the promoter of PNMT using bisulphite‐sequencing, and transcription factor binding sites that are sensitive to epigenetic modification using site directed mutagenesis, are currently being examined. The data suggests that PNMT regulation is sensitive to epigenetic modification which can have repercussions for adrenaline biosynthesis, and consequently on its role as a neurotransmitter.Support or Funding InformationCIHR
Spectroscopic and Biochemical Studies on Metallo‐β‐lactamase IMP‐1
Authors: Zhang Huan 
Published: April 2020

β‐Lactamases are a primarily cause for clinical resistance to β‐lactam antibiotics, such as penicillins, cephamycins, carbapenems, and cephalosporins. Therefore, there are considerable efforts to discover inhibitors of β‐lactamases, which can be given in combination with existing β‐lactams as a treatment for antibiotic resistant bacterial infections. According to previous studies, several types of metallo‐β‐lactamases inhibitors have been discovered by organic chemists: metal‐ion binding inhibitors, covalent inhibitors, allosteric inhibitors, and the inhibitors with uncharacterized mechanisms. The blaIMP gene was initially found in clinical imipenem‐resistant Serratia marcescens and Pseudomonas aeruginosa isolates in Japan. The IMP enzymes deliver broad substrate specificities, and comparing to penicillins they have greater binding affinities for carbapenems and cephalosporins. IMP‐1, the first variant identified, is the most studied IMP variant, and there have been many reported inhibitors of IMP‐1. However, most of these studies reported IC50 and/or MIC values, and most of the inhibitors have not been characterized for mechanism of inhibition. This lack of understanding of mechanism of inhibition could potentially be reducing our efforts to find effective inhibitors of IMP variants and other metallo‐β‐lactamases. We used spectroscopic and biochemical studies to probe the mechanism of inhibition for several known metallo‐β‐lactamase inhibitors with IMP‐1. Equilibrium dialysis was used to incubate IMP‐1 with different inhibitors, and metal analyses were used to determine the zinc content in the resulting samples. This technique allows for us to determine whether an inhibitor strips zinc from IMP‐1. Native mass spectrometry (Orbitrap) of IMP‐1 samples containing inhibitors were used to confirm the equilibrium dialysis results and also to determine whether an IMP‐1/zinc/inhibitor ternary complex forms. Native mass spectrometry can be used to determine whether inhibitor binds to the zinc‐containing form of IMP‐1, but this technique cannot determine whether the inhibitor(s) bind to the metal ions in the active site. Therefore, we utilize UV‐VIS spectrophotometry with cobalt‐substituted analogs of IMP‐1. This technique can be used to probe whether the inhibitor binds to one or both of the metal ions. We also probe inhibitor binding to the cobalt‐containing enzyme by using Electron Paramagnetic Resonance (EPR) spectroscopy. We have discovered that EPR can be used to determine whether inhibitors bind to both metal ions. By using these techniques, we hope to identify inhibitors that form ternary complexes, rather than those that strip metal ions from the metallo‐β‐lactamases. We believe that the inhibitors that form ternary complexes are the best candidates for rational re‐design efforts.Support or Funding InformationNational Institutes of Health (NIH) and Miami University
The Development of 3D Printable Stretch Bioreactors
Authors: Howell Andrew P. , Fischer Kristin 
Published: April 2020

IntroductionCyclic stretch bioreactors for research and medical field applications have proven beneficial in enhancing muscle tissue growth when compared to static cultures. However, the cost for many commercially available cyclic stretch bioreactors carry large price tags and have limited customizability. Three‐dimensional (3D) modeling combined with 3D printing allows for highly customizable cyclic stretch bioreactors to be designed and printed at a lower cost while potentially maintaining the high degree of precision seen in commercially available bioreactors. The importance of this work is to reduce the cost of cyclic stretch bioreactors to increase availability for use in research and medical fields.Materials and MethodsThe 3D modeling software, TinkerCAD, and a Flashforge Creator Pro 3D printer with FlashPrint operating software were used for this project. Poly(‐lactic acid) (PLA) filament purchased from Flashforge was used to print several ready‐for‐assembly pieces. O‐rings and glass were purchased from McMaster‐Carr. For lid fabrication, an O‐ring was placed in a molded channel in the body of the bioreactor and a glass cover was affixed. The chamber was sealed and subjected to an air tight test as well as a water tight test. The chamber was submerged for an hour in water first and then dried prior to filling the chamber with water, inverting the chamber for one hour, and checking for leaks.Results and DiscussionA one‐chamber‐four‐well system was chosen as it allowed for multiple scaffolds to be stretched at once, minimized the potential sealing failure points, and maximized the utility of the chamber’s small footprint. Models for a one‐chamber‐four‐well system were successfully printed using PLA filament. A notable feature of the model is an integrated channel for an O‐Ring in conjunction with a glass lid to allow for sealing of the chamber to ensure an air and water tight internal environment. The chamber successfully maintained all seals for the air tight test, but during the water tight test some water seeped into the chamber frame. This seepage into the frame could lead to contamination and loss of samples.ConclusionsConstruction for a cyclic stretch bioreactor, using 3D printing, is an attractive option for lowering cost while increasing customizability. The production of a water and air tight one‐chamber‐four‐well system can allow for multiple replicates per study and can allow for easy replication of the chamber itself by printing the number of chambers required for the study. Further investigation into the development of 3D printable cyclic stretch bioreactors is a worthwhile effort with future work for this bioreactor including addressing sealing or friction issues, the creation of an adaptable computer control unit with accompanying motor system, and cellularized scaffold testing.Support or Funding Information‐The Hampden‐Sydney Honors Research Council and The Virginia Academy of Science