201 results on '"Isaac J"'
Search Results
2. Increasing Biodiversity and Land-Use Efficiency Through Pea (Pisum aestivum)-Canola (Brassica napus) Intercropping (Peaola)
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Isaac J. Madsen, Janice M. Parks, Maren L. Friesen, and Robert E. Clark
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intercrop ,peaola ,canola ,pea (field) ,soil microbiome ,Chemistry ,QD1-999 ,Engineering geology. Rock mechanics. Soil mechanics. Underground construction ,TA703-712 - Abstract
Intercropping is an ancient agricultural management practice quickly re-gaining interest in mechanized agricultural systems. Mechanized management practices have led to a decreased biodiversity at the macro- and micro-fauna levels. These agricultural practices have also resulted in the degradation of soil and long-term inefficiencies in land, water, and nutrients. The inland Pacific Northwest (iPNW) of the United States of America is a wheat-dominated cropping system. The integration of winter and spring legumes and oilseeds has improved the biodiversity and nutrient-use efficiency of the cropping systems. This article examines the feasibility of pea-canola (peaola) intercropping in dryland production systems of the iPNW. In two site years, small plot peaola trials were established near Davenport, WA. Overall, the land equivalence ratio (LER) of peaola was found to be 1.46, showing an increase in efficiency of the system. Increasing the N fertilizer application rates did not affect peaola yield, indicating that peaola has low demand for N inputs. The effects of peaola on insects and bacterial diversity were examined on replicated large scale strip trials. Peaola was found to have significantly greater numbers of beneficial insects than the monoculture controls. There were no significant differences between the diversity of the soil bacterial communities found in peaola vs. pea and canola monocultures. However, we found that the strict core soil bacterial microbiome of peaola was larger than the monocultures and included core members from both the canola and pea soil microbiomes. In conclusion, the widespread adoption of peaola would likely increase the biodiversity and increase the land use efficiency of dryland production systems in the iPNW.
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- 2022
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3. An Isochroman Analog of CD3254 and Allyl-, Isochroman-Analogs of NEt-TMN Prove to Be More Potent Retinoid-X-Receptor (RXR) Selective Agonists Than Bexarotene
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Peter W. Jurutka, Orsola di Martino, Sabeeha Reshi, Sanchita Mallick, Michael A. Sausedo, Grant A. Moen, Isaac J. Lee, Dominic J. Ivan, Tyler D. Krall, Samuel J. Peoples, Anthony Perez, Lucas Tromba, Anh Le, Iraj Khadka, Ryan Petros, Brianna M. Savage, Eleine Salama, Jakline Salama, Joseph W. Ziller, Youngbin Noh, Ming-Yue Lee, Wei Liu, John S. Welch, Pamela A. Marshall, and Carl E. Wagner
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retinoid-x-receptor ,retinoid ,rexinoid ,leukemia ,small molecule therapeutic ,structure-activity-relationship ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Bexarotene is an FDA-approved drug for the treatment of cutaneous T-cell lymphoma (CTCL); however, its use provokes or disrupts other retinoid-X-receptor (RXR)-dependent nuclear receptor pathways and thereby incites side effects including hypothyroidism and raised triglycerides. Two novel bexarotene analogs, as well as three unique CD3254 analogs and thirteen novel NEt-TMN analogs, were synthesized and characterized for their ability to induce RXR agonism in comparison to bexarotene (1). Several analogs in all three groups possessed an isochroman ring substitution for the bexarotene aliphatic group. Analogs were modeled for RXR binding affinity, and EC50 as well as IC50 values were established for all analogs in a KMT2A-MLLT3 leukemia cell line. All analogs were assessed for liver-X-receptor (LXR) activity in an LXRE system to gauge the potential for the compounds to provoke raised triglycerides by increasing LXR activity, as well as to drive LXRE-mediated transcription of brain ApoE expression as a marker for potential therapeutic use in neurodegenerative disorders. Preliminary results suggest these compounds display a broad spectrum of off-target activities. However, many of the novel compounds were observed to be more potent than 1. While some RXR agonists cross-signal the retinoic acid receptor (RAR), many of the rexinoids in this work displayed reduced RAR activity. The isochroman group did not appear to substantially reduce RXR activity on its own. The results of this study reveal that modifying potent, selective rexinoids like bexarotene, CD3254, and NEt-TMN can provide rexinoids with increased RXR selectivity, decreased potential for cross-signaling, and improved anti-proliferative characteristics in leukemia models compared to 1.
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- 2022
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4. Oligomannose Glycopeptide Conjugates Elicit Antibodies Targeting the Glycan Core Rather than Its Extremities
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Dung N. Nguyen, Bokai Xu, Robyn L. Stanfield, Jennifer K. Bailey, Satoru Horiya, J. Sebastian Temme, Deborah R. Leon, Celia C. LaBranche, David C. Montefiori, Catherine E. Costello, Ian A. Wilson, and Isaac J. Krauss
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Chemistry ,QD1-999 - Published
- 2019
- Full Text
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5. Efecto de la temperatura de calcinación sobre la concentración de grupos silanoles en superficies de SiO2 (SBA–15)
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Reyna Ojeda-López, Isaac J. Pérez-Hermosillo, J. Marcos Esparza-Schulz, and Armando Domínguez-Ortiz
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Activation ,Adsorption ,Silanols Groups ,SBA-15 ,Calcination Temperature ,Chemistry ,QD1-999 - Abstract
The preparation of SBA-15 materials involves both, calcination and activation processes. The effects of the temperatures of these stages on the silanol superficial concentration are studied experimentally. The characterization techniques employed were: nitrogen adsorption, infrared spectroscopy, nuclear magnetic resonance, and X-ray diffraction. To improve the concentration of silanols groups on the solid surface, the obtained results indicate that the best temperatures of calcination and activation are 623K and 373K, respectively.
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- 2014
6. Incorporating Biological Mass Spectrometry into Undergraduate Teaching Labs, Part 2: Peptide Identification via Molecular Mass Determination
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Arnquist, Isaac J. and Beussman, Douglas J.
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Mass spectrometry has become a routine analytical tool in the undergraduate curriculum in the form of GC-MS. While relatively few undergraduate programs have incorporated biological mass spectrometry into their programs, the importance of these techniques, as demonstrated by their recognition with the 2002 Nobel Prize, will hopefully lead to increased opportunities for undergraduate students to experience the power of these techniques. We present a laboratory experiment that allows students to determine the molecular mass of a peptide based on the spectrum obtained from analysis using electrospray ionization mass spectrometry (ESI-MS). Since ESI-MS often produces multiply charged ions, students must use isotopic patterns to first determine the charge state on the ion signals prior to calculating the molecular mass and thus the identity of the peptide. If the charge state determination is not done correctly, large mass errors larger can occur, while students routinely observe mass errors of approximately 0.01% when the analysis is done correctly. (Contains 1 table and 2 figures.) [For Part 1, see EJ820900.]
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- 2009
7. Incorporating Biological Mass Spectrometry into Undergraduate Teaching Labs, Part 1: Identifying Proteins Based on Molecular Mass
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Arnquist, Isaac J. and Beussman, Douglas J.
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Biological mass spectrometry is an important analytical technique in drug discovery, proteomics, and research at the biology-chemistry interface. Currently, few hands-on opportunities exist for undergraduate students to learn about this technique. With the 2002 Nobel Prize being awarded, in part, for the development of biological mass spectrometry, more academic institutions will hopefully incorporate these techniques into their curricula. We present the first in a series of experiments designed to introduce students to the analysis of biological molecules using mass spectrometry. In this experiment, students analyze a medium-sized protein using electrospray ionization and identify it based on molecular weight. Since electrospray ionization produces multiply charged ions and since isotopic distributions cannot be easily observed for highly charged ions, a mathematical treatment of sequentially charged ions is used to determine the charge state on the ion signals and thus calculate the molecular weight. Averages of several differently-charged ion signals from the same analyte are used to obtain an average molecular weight. We have included several representative protein spectra, suitable for classroom use, in the Supplemental Material. (Contains 1 table, 1 figure, and 2 notes.)
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- 2007
8. Prolonged Reactive Oxygen Species Production following Septic Insult
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Vladimir P. Badovinac, Garry R. Buettner, Isaac J. Jensen, Roger R. Berton, Patrick W. McGonagill, Thomas S. Griffith, Elvia E. Silva, and Brett A. Wagner
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Adult ,Male ,Antioxidant ,Adolescent ,medicine.medical_treatment ,Lymphocyte ,Immunology ,Ascorbic Acid ,Severity of Illness Index ,Antioxidants ,Article ,Immature Monocyte ,Sepsis ,Mice ,Young Adult ,Immune system ,Disease severity ,Animals ,Humans ,Immunology and Allergy ,Medicine ,Aged ,Aged, 80 and over ,chemistry.chemical_classification ,Reactive oxygen species ,Vitamin C ,business.industry ,General Medicine ,Middle Aged ,medicine.disease ,Healthy Volunteers ,Disease Models, Animal ,medicine.anatomical_structure ,chemistry ,Case-Control Studies ,Leukocytes, Mononuclear ,Female ,Reactive Oxygen Species ,business - Abstract
The dysregulated host response and organ damage following systemic infection that characterizes a septic event predisposes individuals to a chronic immunoparalysis state associated with severe transient lymphopenia and diminished lymphocyte function, thereby reducing long-term patient survival and quality of life. Recently, we observed lasting production of reactive oxygen species (ROS) in mice that survive sepsis. ROS production is a potent mechanism for targeting infection, but excessive ROS production can prove maladaptive by causing organ damage, impairing lymphocyte function, and promoting inflammaging, concepts paralleling sepsis-induced immunoparalysis. Notably, we observed an increased frequency of ROS-producing immature monocytes in septic hosts that was sustained for greater than 100 days postsurgery. Recent clinical trials have explored the use of vitamin C, a potent antioxidant, for treating septic patients. We observed that therapeutic vitamin C administration for sepsis limited ROS production by monocytes and reduced disease severity. Importantly, we also observed increased ROS production by immature monocytes in septic patients both at admission and ∼28 days later, suggesting a durable and conserved feature that may influence the host immune response. Thus, lasting ROS production by immature monocytes is present in septic patients, and early intervention strategies to reduce it may improve host outcomes, potentially reducing sepsis-induced immunoparalysis.
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- 2021
9. Synthesis of Mannosidase-Stable Man3 and Man4 Glycans Containing S-linked Manα1→2Man Termini
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Isaac J. Krauss, Mahesh Neralkar, Richard L Redman, and Leiming Tian
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Mannosidase ,Glycan ,Glycosylation ,biology ,010405 organic chemistry ,Chemistry ,Organic Chemistry ,Thio ,010402 general chemistry ,01 natural sciences ,Biochemistry ,0104 chemical sciences ,chemistry.chemical_compound ,In vivo ,biology.protein ,Stereoselectivity ,Physical and Theoretical Chemistry ,HIV vaccine ,Antibody - Abstract
Oligomannose glycans are of interest as HIV vaccine components, but they are subject to mannosidase degradation in vivo. Herein, we report the synthesis of oligosaccharides containing a thio linkage at the nonreducing end. A thio-linked dimannose donor participates in highly stereoselective glycosylations to afford trimannose and tetramannose fragments. Saturation transfer difference nuclear magnetic resonance (STD NMR) studies show that these glycans are recognized by HIV antibody 2G12, and we confirm that the reducing terminal S-linkage confers complete stability against x. manihotis mannosidase.
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- 2021
10. Functional and structural characterization of allosteric activation of phospholipase Cε by Rap1A
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Angeline M. Lyon, Elisabeth E. Garland-Kuntz, Monita Sieng, Andrea T. Marti, Arielle F. Selvia, Isaac J. Fisher, and Jesse B. Hopkins
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0301 basic medicine ,Ras-related protein 1 (Rap1) ,Models, Molecular ,G protein ,Allosteric regulation ,small-angle X-ray scattering (SAXS) ,GTPase ,Biochemistry ,Receptor tyrosine kinase ,GTP Phosphohydrolases ,03 medical and health sciences ,conformational change ,Phosphoinositide Phospholipase C ,Allosteric Regulation ,Protein Domains ,X-Ray Diffraction ,cardiovascular disease ,Scattering, Small Angle ,cell signaling ,structural biology ,Humans ,Binding site ,phospholipase C ,Molecular Biology ,030102 biochemistry & molecular biology ,Phospholipase C ,biology ,diacylglycerol ,Chemistry ,calcium intracellular release ,protein kinase C (PKC) ,rap1 GTP-Binding Proteins ,Pleckstrin Homology Domains ,Cell Biology ,Recombinant Proteins ,Cell biology ,Protein Structure, Tertiary ,Pleckstrin homology domain ,Protein Transport ,030104 developmental biology ,Structural biology ,biology.protein ,Mutagenesis, Site-Directed ,phosphatidylinositol signaling ,membrane enzyme ,Signal Transduction ,Protein Binding - Abstract
Phospholipase Ce (PLCe) is activated downstream of G protein–coupled receptors and receptor tyrosine kinases through direct interactions with small GTPases, including Rap1A and Ras. Although Ras has been reported to allosterically activate the lipase, it is not known whether Rap1A has the same ability or what its molecular mechanism might be. Rap1A activates PLCe in response to the stimulation of β-adrenergic receptors, translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLCe was proposed to the primary binding site for Rap1A, we first confirmed using purified proteins that the RA2 domain is indeed essential for activation by Rap1A. However, we also showed that the PLCe pleckstrin homology (PH) domain and first two EF hands (EF1/2) are required for Rap1A activation and identified hydrophobic residues on the surface of the RA2 domain that are also necessary. Small-angle X-ray scattering showed that Rap1A binding induces and stabilizes discrete conformational states in PLCe variants that can be activated by the GTPase. These data, together with the recent structure of a catalytically active fragment of PLCe, provide the first evidence that Rap1A, and by extension Ras, allosterically activate the lipase by promoting and stabilizing interactions between the RA2 domain and the PLCe core.
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- 2021
11. Multilevel effects of alcohol among early adolescents in an urban school district
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Chao Liu, Isaac J. Washburn, Julie M. Croff, Ronald B. Cox, and Clinton Broadbent
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chemistry.chemical_compound ,chemistry ,Environmental health ,education ,Early adolescents ,Alcohol ,School district ,Psychology - Abstract
Aims: To examine how interpersonal interactions within and between the social networks formed by teachers, parents, students, and others shape the unique culture of the school, which in turn, reciprocally exerts a determining influence on each individual in the network. Design: Cross-sectional study exploring whether factors associated with alcohol use at the individual level also exert influence on the culture of a school. Setting: Twelve middle schools within an urban school district in the Midwestern United States. Participants: Seventh grade students (N = 1,620). Measures: Lifetime alcohol use behaviors; mothers’ and fathers’ involvement in their child’s education; and peer deviance. Findings: The findings of this study suggest that as parents’ involvement in education increases, and as peer deviance decreases, there are direct benefits to the child, and a protective effect for other children within the school when, in the aggregate, parental involvement increases and peer deviance decreases. Conclusions: The effect of interventions to improve parent involvement and reduce peer deviance, with examinations at the individual-level and school-level, warrant future study.
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- 2020
12. Enrichment of Charged Monomers Explains Non-monotonic Polymer Volume Fraction Profiles of Multi-stimulus Responsive Copolymer Brushes
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Edwin C. Johnson, Joshua D. Willott, Timothy J. Murdoch, Stuart W. Prescott, Ben A. Humphreys, Isaac J. Gresham, Grant B. Webber, Andrew Nelson, Wiebe M. de Vos, Erica J. Wanless, MESA+ Institute, and Membrane Science & Technology
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chemistry.chemical_classification ,Materials science ,Atom-transfer radical-polymerization ,22/2 OA procedure ,Substrate (chemistry) ,02 engineering and technology ,Surfaces and Interfaces ,Polymer ,010402 general chemistry ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,Methacrylate ,01 natural sciences ,0104 chemical sciences ,chemistry.chemical_compound ,Monomer ,chemistry ,Polymerization ,Chemical engineering ,Electrochemistry ,Copolymer ,General Materials Science ,Neutron reflectometry ,0210 nano-technology ,Spectroscopy - Abstract
Multi-stimulus responsive poly(2-(2-methoxyethoxy)ethyl methacrylate-co-2-(diethylamino)ethyl methacrylate) [P(MEO2MA-co-DEA)] 80:20 mol % copolymer brushes were synthesized on planar silica substrates via surface-initiated activators continuously regenerated via electron transfer atom transfer radical polymerization. Brush thickness was sensitive to changes in pH and temperature as monitored with ellipsometry. At low pH, the brush is charged and swollen, while at high pH, the brush is uncharged and more collapsed. Clear thermoresponsive behavior is also observed with the brush more swollen at low temperatures compared to high temperatures at both high and low pH. Neutron reflectometry was used to determine the polymer volume fraction profiles (VFPs) at various pH values and temperatures. A region of lower polymer content, or a depletion region, near the substrate is present in all of the experimental polymer VFPs, and it is more pronounced at low pH (high charge) and less so at high pH (low charge). Polymer VFPs calculated through numerical self-consistent field theory suggest that enrichment of DEA monomers near the substrate results in the experimentally observed non-monotonic VFPs. Adsorption of DEA monomers to the substrate prior to initiation of polymerization could give rise to DEA segment-enriched region proximal to the substrate.
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- 2020
13. Coupling lipid nanoparticle structure and automated single particle composition analysis to design phospholipase responsive nanocarriers
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Hanna M. G. Barriga, Isaac J. Pence, Margaret N. Holme, James J. Doutch, Jelle Penders, Valeria Nele, Michael R. Thomas, Marta Carroni, Molly M. Stevens, Commission of the European Communities, Engineering & Physical Science Research Council (E, Engineering & Physical Science Research Council (EPSRC), and Royal Academy Of Engineering
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Technology ,label-free dynamic monitoring ,Chemistry, Multidisciplinary ,Materials Science ,Materials Science, Multidisciplinary ,lipid nanoparticles ,PRESSURE ,09 Engineering ,Physics, Applied ,DELIVERY ,X-Ray Diffraction ,Scattering, Small Angle ,phospholipase D ,SCATTERING ,General Materials Science ,Nanoscience & Nanotechnology ,RNA, Small Interfering ,Science & Technology ,02 Physical Sciences ,Chemistry, Physical ,Mechanical Engineering ,Physics ,CANCER ,Lipids ,Chemistry ,Physics, Condensed Matter ,Mechanics of Materials ,Phospholipases ,Physical Sciences ,Liposomes ,X-RAY ,Science & Technology - Other Topics ,Nanoparticles ,enzyme-responsive systems ,03 Chemical Sciences - Abstract
Lipid nanoparticles (LNPs) are versatile structures with tunable physicochemical properties that are ideally suited as a platform for vaccine delivery and RNA therapeutics. A key barrier to LNP rational design is the inability to relate composition and structure to intracellular processing and function. Here Single Particle Automated Raman Trapping Analysis (SPARTA) is combined with small-angle X-ray and neutron scattering (SAXS/SANS) techniques to link LNP composition with internal structure and morphology and to monitor dynamic LNP-phospholipase D (PLD) interactions. This analysis demonstrates that PLD, a key intracellular trafficking mediator, can access the entire LNP lipid membrane to generate stable, anionic LNPs. PLD activity on vesicles with matched amounts of enzyme substrate is an order of magnitude lower, indicating that the LNP lipid membrane structure can be used to control enzyme interactions. This represents an opportunity to design enzyme-responsive LNP solutions for stimuli-responsive delivery and diseases where PLD is dysregulated.
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- 2022
14. Probing conformational transitions towards mutagenic Watson–Crick-like G·T mismatches using off-resonance sugar carbon R1ρ relaxation dispersion
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Atul Rangadurai, Eric S. Szymanski, Honglue Shi, Hashim M. Al-Hashimi, and Isaac J. Kimsey
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0301 basic medicine ,Speed wobble ,Base pair ,Chemistry ,Relaxation (NMR) ,Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Tautomer ,0104 chemical sciences ,03 medical and health sciences ,Molecular dynamics ,Crystallography ,030104 developmental biology ,Kinetic isotope effect ,Spectroscopy ,Dynamic equilibrium - Abstract
NMR off-resonance R1ρ relaxation dispersion measurements on base carbon and nitrogen nuclei have revealed that wobble G·T/U mismatches in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-abundance, and mutagenic Watson–Crick-like conformations. As Watson–Crick-like G·T mismatches have base pairing geometries similar to Watson–Crick base pairs, we hypothesized that they would mimic Watson–Crick base pairs with respect to the sugar-backbone conformation as well. Using off-resonance R1ρ measurements targeting the sugar C3′ and C4′ nuclei, a structure survey, and molecular dynamics simulations, we show that wobble G·T mismatches adopt sugar-backbone conformations that deviate from the canonical Watson–Crick conformation and that transitions toward tautomeric and anionic Watson–Crick-like G·T mismatches restore the canonical Watson–Crick sugar-backbone. These measurements also reveal kinetic isotope effects for tautomerization in D2O versus H2O, which provide experimental evidence in support of a transition state involving proton transfer. The results provide additional evidence in support of mutagenic Watson–Crick-like G·T mismatches, help rule out alternative inverted wobble conformations in the case of anionic G·T−, and also establish sugar carbons as new non-exchangeable probes of this exchange process.
- Published
- 2020
15. Pharmacological Properties of δ-Opioid Receptor–Mediated Behaviors: Agonist Efficacy and Receptor Reserve
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Kathryn E. Livingston, Emily M. Jutkiewicz, Alexander Disney, Stephen M. Husbands, Ruizhuo Chen, John R. Traynor, Kenner C. Rice, Amanda M. Shafer, and Isaac J. Dripps
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0301 basic medicine ,Agonist ,medicine.drug_class ,Pharmacology ,Partial agonist ,Piperazines ,Drug Discovery and Translational Medicine ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Opioid receptor ,Naltrindole ,Receptors, Opioid, delta ,Convulsion ,medicine ,Animals ,Potency ,Receptor ,Behavior, Animal ,Chemistry ,Naltrexone ,Buprenorphine ,Drug Partial Agonism ,030104 developmental biology ,Opioid ,Benzamides ,Molecular Medicine ,medicine.symptom ,030217 neurology & neurosurgery ,medicine.drug - Abstract
δ-Opioid receptor (δ-receptor) agonists produce antihyperalgesia, antidepressant-like effects, and convulsions in animals. However, the role of agonist efficacy in generating different δ-receptor–mediated behaviors has not been thoroughly investigated. To this end, efficacy requirements for δ-receptor–mediated antihyperalgesia, antidepressant-like effects, and convulsions were evaluated by comparing the effects of the partial agonist BU48 and the full agonist SNC80 and changes in the potency of SNC80 after δ-receptor elimination. Antihyperalgesia was measured in a nitroglycerin-induced thermal hyperalgesia assay. An antidepressant-like effect was evaluated in the forced swim test. Mice were observed for convulsions after treatment with SNC80 or the δ-opioid receptor partial agonist BU48. Ligand-induced G protein activation was measured by [(35)S]guanosine 5′-O-[γ-thio]triphosphate binding in mouse forebrain tissue, and δ-receptor number was measured by [(3)H]D-Pen(2,5)-enkephalin saturation binding. BU48 produced antidepressant-like effects and convulsions but antagonized SNC80-induced antihyperalgesia and G protein activation. The potency of SNC80 was shifted to the right in δ-receptor heterozygous knockout mice and naltrindole-5′-isothiocyanate–treated mice, and the magnitude of potency shift differed across assays, with the largest shift occurring in the thermal hyperalgesia assay, followed by the forced swim test and then convulsion observation. Naltrindole antagonized these SNC80-induced behaviors with similar potencies, suggesting that these effects are mediated by the same type of δ-receptor. These data suggest that δ-receptor–mediated behaviors display a rank order of efficacy requirement, with antihyperalgesia having the highest requirement, followed by antidepressant-like effects and then convulsions. These findings further our understanding of the pharmacological mechanisms mediating the in vivo effects of δ-opioid receptor agonists. SIGNIFICANCE STATEMENT: δ-Opioid receptor (δ-receptor) agonists produce antihyperalgesia, antidepressant-like effects, and convulsions in animal models. This study evaluates pharmacological properties, specifically the role of agonist efficacy and receptor reserve, underlying these δ-receptor–mediated behaviors. These data suggest that δ-receptor–mediated behaviors display a rank order of efficacy requirement, with antihyperalgesia having the highest requirement, followed by antidepressant-like effects and then convulsions.
- Published
- 2020
16. Feasibility and reliability of continuously monitoring alcohol use among female adolescents and young adults
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Erica K. Crockett, Isaac J. Washburn, Julie M. Croff, Micah Hartwell, and Ashleigh L. Chiaf
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Adult ,medicine.medical_specialty ,Health (social science) ,Adolescent ,Alcohol Drinking ,030508 substance abuse ,Medicine (miscellaneous) ,Poison control ,Alcohol ,Article ,Occupational safety and health ,Young Adult ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Injury prevention ,Humans ,Medicine ,Prospective Studies ,030212 general & internal medicine ,Young adult ,Prospective cohort study ,Transdermal ,business.industry ,Reproducibility of Results ,Repeated measures design ,chemistry ,Physical therapy ,Feasibility Studies ,Blood Alcohol Content ,Female ,0305 other medical science ,business - Abstract
INTRODUCTION AND AIMS. Transdermal alcohol sensors allow objective, continuous monitoring and have potential to expand current research on adolescent and young adult alcohol use. The purpose of this manuscript is to evaluate the feasibility and reliability of transdermal alcohol sensor use among female adolescents as compared to female young adults. DESIGN AND METHODS. This trial included 59 female adolescents and young adults aged 14–24 years who reported drinking during the previous month. All participants were asked to wear a Giner Wrist Transdermal Alcohol Sensor (WrisTAS)-7 over a 1 month prospective study. Participants came to the research lab weekly to complete a detailed self-report of behaviours, including day of drinking events, amounts and types of alcohol use and length of drinking events. Estimates of blood alcohol concentration (eBAC) were computed from self-report data using the Matthew and Miller, NHTSA and Zhang equations. Daily transdermal alcohol concentration (TAC) peaks and calculated eBAC peak data were analysed with paired-samples t-tests and repeated measures correlations for validity comparisons. RESULTS. All participants (100%, n = 59) completed the trial, however, two participants were removed due to greater than 50% of missing transdermal alcohol sensor data. Of the 57 participants, the data included 1,722 days of continuous alcohol monitoring. Missing data was recorded more frequently among female adolescents at about (11.78%) as compared to female young adults (8.59%; χ(2) = −18.40, P < 0.001). Participant self-report of drinking occurred with greater frequency (374 events) than detected by the WrisTAS transdermal alcohol sensors (243 events). On days when self-report and sensor data indicated a drinking event, participants’ eBAC was moderately correlated with TAC, after accounting for repeated measures. DISCUSSION AND CONCLUSIONS. This study finds that transdermal alcohol sensors are moderately reliable when sensor data is paired with self-report. This objective data collection method may improve the ability to collect alcohol curves among adolescents.
- Published
- 2020
17. Distinct trajectories of fruits and vegetables, dietary fat, and alcohol intake following a breast cancer diagnosis: the Pathways Study
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Lawrence H. Kushi, Andrew Rundle, Heather Greenlee, Zaixing Shi, Isaac J. Ergas, Jeanine M. Genkinger, Ying Kuen Cheung, Janise M. Roh, and Marilyn L. Kwan
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Adult ,Dietary Fiber ,0301 basic medicine ,Cancer Research ,Calorie ,Alcohol Drinking ,Health Behavior ,Breast Neoplasms ,Alcohol ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Social support ,0302 clinical medicine ,Breast cancer ,Vegetables ,Humans ,Medicine ,Dietary fat ,Aged ,Aged, 80 and over ,business.industry ,Middle Aged ,medicine.disease ,Dietary Fats ,030104 developmental biology ,Oncology ,chemistry ,Fruit ,030220 oncology & carcinogenesis ,Fruits and vegetables ,Female ,Alcohol intake ,business ,Psychosocial ,Demography - Abstract
PURPOSE: To identify distinct diet trajectories after breast cancer (BC) diagnosis, and to examine characteristics associated with diet trajectories. METHODS: We analyzed 2,865 Pathways Study participants who completed ≥2 food frequency questionnaires at the time of BC diagnosis (baseline), and 6 and 24 months after baseline. Trajectory groups of fruit and vegetable (F/V) intake, % calories from dietary fat, and alcohol intake over 24 months were identified using group-based trajectory modeling. Associations between diet trajectories and sociodemographic, psychosocial, and clinical factors were analyzed using multinomial logistic regression. RESULTS: Analyses identified 3 F/V trajectory groups, 4 dietary fat groups, and 3 alcohol groups. All 3 F/V trajectory groups reported slightly increased F/V intake post-diagnosis (mean increase=0.2–0.5 serving/day), while 2 groups (48% of participants) persistently consumed 40% of calories from fat). While most survivors consumed
- Published
- 2019
18. Revealing A-T and G-C Hoogsteen base pairs in stressed protein-bound duplex DNA
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Hsuan-Fu Liu, Uyen Pham, Isaac J. Kimsey, Hashim M. Al-Hashimi, Maria A. Schumacher, Honglue Shi, and Stephanie Gu
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Models, Molecular ,Dna duplex ,Stereochemistry ,Base pair ,AcademicSubjects/SCI00010 ,Biology ,urologic and male genital diseases ,Crystallography, X-Ray ,Genome ,chemistry.chemical_compound ,Protein Domains ,Structural Biology ,Databases, Genetic ,Genetics ,Base Pairing ,Binding Sites ,Base Sequence ,Computational Biology ,Hydrogen Bonding ,DNA ,Dna double helix ,DNA-Binding Proteins ,chemistry ,Mutation ,Damage repair ,Nucleic Acid Conformation ,Thermodynamics ,hormones, hormone substitutes, and hormone antagonists ,Protein Binding - Abstract
Watson–Crick base pairs (bps) are the fundamental unit of genetic information and the building blocks of the DNA double helix. However, A-T and G-C can also form alternative ‘Hoogsteen’ bps, expanding the functional complexity of DNA. We developed ‘Hoog-finder’, which uses structural fingerprints to rapidly screen Hoogsteen bps, which may have been mismodeled as Watson–Crick in crystal structures of protein–DNA complexes. We uncovered 17 Hoogsteen bps, 7 of which were in complex with 6 proteins never before shown to bind Hoogsteen bps. The Hoogsteen bps occur near mismatches, nicks and lesions and some appear to participate in recognition and damage repair. Our results suggest a potentially broad role for Hoogsteen bps in stressed regions of the genome and call for a community-wide effort to identify these bps in current and future crystal structures of DNA and its complexes.
- Published
- 2021
19. A Highly Ordered, Nanostructured Fluorinated CaP-Coated Melt Electrowritten Scaffold for Periodontal Tissue Regeneration
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Marco C. Bottino, Arwa Daghrery, Isaac J. de Souza Araújo, Brian H. Clarkson, Jos Malda, Jessica A. Ferreira, Sarit B. Bhaduri, George J. Eckert, Equine Musculoskeletal Biology, and dES RMSC
- Subjects
Periodontium ,Scaffold ,bones ,melt electrowriting ,Periodontal ligament stem cells ,Polyesters ,Biomedical Engineering ,Pharmaceutical Science ,engineering.material ,Article ,Biomaterials ,Coating ,Osteogenesis ,In vivo ,periodontal regeneration ,medicine ,Animals ,Progenitor cell ,periodontitis ,Periodontitis ,Wound Healing ,Tissue Engineering ,Tissue Scaffolds ,Chemistry ,Regeneration (biology) ,Adhesion ,3D printing ,medicine.disease ,Rats ,scaffolds ,engineering ,Biophysics - Abstract
Periodontitis is a chronic inflammatory, bacteria-triggered disorder affecting nearly half of American adults. Although some level of tissue regeneration is realized, its low success in complex cases demands superior strategies to amplify regenerative capacity. Herein, highly ordered scaffolds are engineered via Melt ElectroWriting (MEW), and the effects of strand spacing, as well as the presence of a nanostructured fluorinated calcium phosphate (F/CaP) coating on the adhesion/proliferation, and osteogenic differentiation of human-derived periodontal ligament stem cells, are investigated. Upon initial cell-scaffold interaction screening aimed at defining the most suitable design, MEW poly(𝝐-caprolactone) scaffolds with 500 µm strand spacing are chosen. Following an alkali treatment, scaffolds are immersed in a pre-established solution to allow for coating formation. The presence of a nanostructured F/CaP coating leads to a marked upregulation of osteogenic genes and attenuated bacterial growth. In vivo findings confirm that the F/CaP-coated scaffolds are biocompatible and lead to periodontal regeneration when implanted in a rat mandibular periodontal fenestration defect model. In aggregate, it is considered that this work can contribute to the development of personalized scaffolds capable of enabling tissue-specific differentiation of progenitor cells, and thus guide simultaneous and coordinated regeneration of soft and hard periodontal tissues, while providing antimicrobial protection.
- Published
- 2021
20. Translational biophotonics with Raman imaging: clinical applications and beyond
- Author
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Conor L. Evans and Isaac J. Pence
- Subjects
medicine.medical_specialty ,Disease progression ,Raman imaging ,Spectrum Analysis, Raman ,Biochemistry ,Analytical Chemistry ,Therapeutic monitoring ,Biophotonics ,symbols.namesake ,Chemistry ,Electrochemistry ,medicine ,symbols ,Environmental Chemistry ,Medical physics ,Spectroscopy ,Raman scattering - Abstract
Clinical medicine continues to seek novel rapid non-invasive tools capable of providing greater insight into disease progression and management. Raman scattering based technologies constitute a set of tools under continuing development to address outstanding challenges spanning diagnostic medicine, surgical guidance, therapeutic monitoring, and histopathology. Here we review the mechanisms and clinical applications of Raman scattering, specifically focusing on high-speed imaging methods that can provide spatial context for translational biomedical applications., The technological developments of high-speed spontaneous and coherent Raman scattering based imaging for translational applications including surgical guidance, histopathology, and pathophysiological monitoring.
- Published
- 2021
21. Directed Evolution of 2'-Fluoro-Modified, RNA-Supported Carbohydrate Clusters That Bind Tightly to HIV Antibody 2G12
- Author
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Isaac J. Krauss and Richard L Redman
- Subjects
Glycan ,biology ,Chemistry ,Carbohydrates ,RNA ,Sequence (biology) ,General Chemistry ,Carbohydrate ,HIV Antibodies ,010402 general chemistry ,Directed evolution ,01 natural sciences ,Biochemistry ,Affinities ,Catalysis ,Article ,0104 chemical sciences ,Colloid and Surface Chemistry ,biology.protein ,Cluster (physics) ,Antibody - Abstract
Carbohydrate binding proteins (CBPs) are attractive targets in medicine and biology. Multivalency, with several glycans binding to several binding pockets in the CBP, is important for high-affinity interactions. Herein, we describe a novel platform for design of multivalent carbohydrate cluster ligands by directed evolution, in which serum-stable 2′-fluoro modified RNA (F-RNA) backbones evolve to present the glycan in optimal clusters. We have validated this method by the selection of oligomannose (Man(9)) glycan clusters from a sequence pool of ~10(13) that bind to broadly neutralizing HIV antibody 2G12 with 13 to 36 nM affinities.
- Published
- 2021
22. Assessment of Relative Potencies of Nitrogen Sources on Seedling Root Systems
- Author
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William L. Pan and Isaac J. Madsen
- Subjects
Agronomy ,chemistry ,biology ,Seedling ,chemistry.chemical_element ,Root system ,biology.organism_classification ,Agronomy and Crop Science ,Nitrogen - Published
- 2019
23. Characterizing micro-to-millisecond chemical exchange in nucleic acids using off-resonance R1ρ relaxation dispersion
- Author
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Eric S. Szymaski, Isaac J. Kimsey, Hashim M. Al-Hashimi, Atul Rangadurai, and Honglue Shi
- Subjects
Nuclear and High Energy Physics ,Materials science ,Field (physics) ,Population ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,030218 nuclear medicine & medical imaging ,Analytical Chemistry ,03 medical and health sciences ,0302 clinical medicine ,Dispersion (optics) ,education ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy ,chemistry.chemical_classification ,Quantitative Biology::Biomolecules ,education.field_of_study ,Spins ,Chemical shift ,Biomolecule ,Relaxation (NMR) ,DNA ,Magnetostatics ,0104 chemical sciences ,Models, Chemical ,chemistry ,Chemical physics - Abstract
This review describes off-resonance R(1ρ) relaxation dispersion NMR methods for characterizing microsecond-to-millisecond chemical exchange in uniformly (13)C/(15)N labeled nucleic acids in solution. The review opens with a historical account of key developments that formed the basis for modern R(1ρ) techniques used to study chemical exchange in biomolecules. A vector model is then used to describe the R(1ρ) relaxation dispersion experiment, and how the exchange contribution to relaxation varies with the amplitude and frequency offset of an applied spin locking field, as well as the population, exchange rate, and differences in chemical shifts of two exchanging species. Mathematical treatment of chemical exchange based on the Bloch-McConnell equations is then presented and used to examine relaxation dispersion profiles for more complex exchange scenarios including three-state exchange. Pulse sequences that employ selective Hartmann-Hahn cross-polarization transfers to excite individual (13)C or (15)N spins are then described for measuring off-resonance R(1ρ)((13)C) and R(1ρ)((15)N) in uniformly (13)C/(15)N labeled DNA and RNA samples prepared using commercially available (13)C/(15)N labeled nucleotide triphosphates. Approaches for analyzing R(1ρ) data measured at a single static magnetic field to extract a full set of exchange parameters are then presented that rely on numerical integration of the Bloch-McConnell equations or the use of algebraic expressions. Methods for determining structures of nucleic acid excited states are then reviewed that rely on mutations and chemical modifications to bias conformational equilibria, as well as structurebased approaches to calculate chemical shifts. Applications of the methodology to the study of DNA and RNA conformational dynamics are reviewed and the biological significance of the exchange processes is briefly discussed.
- Published
- 2019
24. Accurate and efficient representation of intramolecular energy in ab initio generation of crystal structures. II. Smoothed intramolecular potentials
- Author
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Isaac J. Sugden, Claire S. Adjiman, Constantinos C. Pantelides, and Engineering & Physical Science Research Council (EPSRC)
- Subjects
Chemistry, Multidisciplinary ,Ab initio ,Crystal structure ,FORMS ,Classification of discontinuities ,crystal structure prediction ,Materials Chemistry ,Statistical physics ,0912 Materials Engineering ,Representation (mathematics) ,0306 Physical Chemistry (incl. Structural) ,Lattice energy ,Science & Technology ,Crystallography ,Chemistry ,Metals and Alloys ,computational chemistry ,POLYMORPHISM ,Atomic and Molecular Physics, and Optics ,Electronic, Optical and Magnetic Materials ,Crystal structure prediction ,Intramolecular force ,Physical Sciences ,Inorganic & Nuclear Chemistry ,STRUCTURE PREDICTION ,LANDSCAPES ,0301 Analytical Chemistry ,Energy (signal processing) - Abstract
The application of crystal structure prediction (CSP) to industrially relevant molecules requires the handling of increasingly large and flexible compounds. A revised model for the effect of molecular flexibility on the lattice energy that removes the discontinuities and non-differentiabilities present in earlier models (Sugden et al., 2016), with a view to improving the performance of CSP is presented. The approach is based on the concept of computing a weighted average of local models, and has been implemented within the CrystalPredictor code. Through the comparative investigation of several compounds studied in earlier literature, it is shown that this new model results in large reductions in computational effort (of up to 65%) and in significant increases in reliability. The approach is further applied to investigate, for the first time, the computational polymorphic landscape of flufenamic acid for Z′ = 1 structures, resulting in the successful identification of all three experimentally resolved polymorphs within reasonable computational time.
- Published
- 2019
25. Oligomannose Glycopeptide Conjugates Elicit Antibodies Targeting the Glycan Core Rather than Its Extremities
- Author
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Nguyen, Dung N., Xu, Bokai, Stanfield, Robyn L., Bailey, Jennifer K., Horiya, Satoru, Temme, J. Sebastian, Leon, Deborah R., LaBranche, Celia C., Montefiori, David C., Costello, Catherine E., Wilson, Ian A., and Krauss, Isaac J.
- Subjects
Chemistry ,QD1-999 ,Research Article - Abstract
Up to ∼20% of HIV-infected individuals eventually develop broadly neutralizing antibodies (bnAbs), and many of these antibodies (∼40%) target a region of dense high-mannose glycosylation on gp120 of the HIV envelope protein, known as the “high-mannose patch” (HMP). Thus, there have been numerous attempts to develop glycoconjugate vaccine immunogens that structurally mimic the HMP and might elicit bnAbs targeting this conserved neutralization epitope. Herein, we report on the immunogenicity of glycopeptides, designed by in vitro selection, that bind tightly to anti-HMP antibody 2G12. By analyzing the fine carbohydrate specificity of rabbit antibodies elicited by these immunogens, we found that they differ from some natural human bnAbs, such as 2G12 and PGT128, in that they bind primarily to the core structures within the glycan, rather than to the Manα1 → 2Man termini (2G12) or to the whole glycan (PGT128). Antibody specificity for the glycan core may result from extensive serum mannosidase trimming of the immunogen in the vaccinated animals. This finding has broad implications for vaccine design aiming to target glycan-dependent HIV neutralizing antibodies., Vaccines that elicit antibodies against oligomannose carbohydrates may protect against HIV, but carbohydrate trimming in serum is a critical factor that may have thwarted successful vaccine attempts.
- Published
- 2019
26. Impact of building block structure on ion transport in cyclopropenium-based polymerized ionic liquids
- Author
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Karen I. Winey, Benjamin Paren, Luis M. Campos, Isaac J. Knudson, Jessica L. Freyer, and Raghavachary Raghunathan
- Subjects
chemistry.chemical_classification ,Materials science ,Polymers and Plastics ,Organic Chemistry ,Bioengineering ,02 engineering and technology ,Polymer ,Conductivity ,010402 general chemistry ,021001 nanoscience & nanotechnology ,01 natural sciences ,Biochemistry ,0104 chemical sciences ,Ion ,chemistry.chemical_compound ,chemistry ,Chemical engineering ,Ionic liquid ,Ionic conductivity ,Molecule ,Counterion ,0210 nano-technology ,Glass transition - Abstract
Ion transport and morphology are studied in cationic polymers based on tris(dialkyl)aminocyclopropenium ions tethered onto polystyrene (PS-TAC) and Cl− counterions. To investigate how molecular structure impacts single-ion transport and physical properties, the substituents on TAC were varied. Modifying the polarity, along with the size and flexibility of the functional groups on TAC significantly changes the glass transition temperature. Ionic conductivity of the PS-TAC with branched functional groups is 1–2 orders of magnitude lower than more compact and bulkier moieties on polymers at their glass transition temperature, Tg. Decreasing the size of the substituents correlates with increasing ionic conductivity in the polymers with non-polar functional groups. However, it is the geometry of the functional groups on the cation (isopropyl, ring, or linear) in PS-TAC that has a much larger effect on conductivity than the size of the group itself. Mimicking the transport properties of ionic liquids using polymers imbued with mechanical stability is essential for the development of robust, non-volatile electrolytes for batteries and fuel cells. Changing a number of variables in this tunable PS-TAC system is a step towards developing soft materials design rules for selecting solid ion supports.
- Published
- 2019
27. Polymorphism in p-aminobenzoic acid
- Author
-
Martin R. Ward, Iain D. H. Oswald, Isaac J. Sugden, Roger J. Davey, and Aurora J. Cruz-Cabeza
- Subjects
Chemistry ,Energy landscape ,02 engineering and technology ,General Chemistry ,010402 general chemistry ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,01 natural sciences ,0104 chemical sciences ,Crystal structure prediction ,law.invention ,Drug compound ,Crystallography ,Polymorphism (materials science) ,law ,P-Aminobenzoic acid ,High pressure ,QD ,General Materials Science ,Crystallization ,0210 nano-technology - Abstract
We review the polymorphism of p-aminobenzoic acid (pABA), a model drugcompound whose crystallisation and polymorphic behaviour has been extensively studied in recent years. Beyond the well-known and characterised α and β forms, pABA also crystallises as a γ polymorph, which is structurally similar to the α form. In addition we also compare the newly reported δ form, obtained by high pressure crystallisation and through compression of the α-form. A structural analysis and comparison of all of the forms is presented, the conditions by which each of them is obtained summarised. Crystal structure prediction calculations have also been carried out in order to probe the solid form energy landscape of this compound. The overall picture of the polymorphism of pABA, reveals, surprisingly, the rarity of the β form.
- Published
- 2019
28. The Neuropeptides of Ocular Immune Privilege, α-MSH and NPY, Suppress Phagosome Maturation in Macrophages
- Author
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Isaac J. Benque, Pu Xia, Robert Shannon, Andrew W. Taylor, and Tat Fong Ng
- Subjects
0303 health sciences ,Chemistry ,Phagocytosis ,medicine.medical_treatment ,T cell ,Immunology ,General Medicine ,Phagolysosome ,Article ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,Cytokine ,medicine.anatomical_structure ,Immune privilege ,Phagosome maturation ,medicine ,Immunology and Allergy ,Macrophage ,030217 neurology & neurosurgery ,030304 developmental biology ,Phagosome - Abstract
The ocular microenvironment has evolutionarily adapted several mechanisms of immunosuppression to minimize the induction of inflammation. Neuropeptides produced by the retinal pigment epithelial cells regulate macrophage activity. Two neuropeptides, α-melanocyte–stimulating hormone (α-MSH) and neuropeptide Y (NPY), are constitutively expressed by the retinal pigment epithelial cells. Together these two neuropeptides induce anti-inflammatory cytokine production in endotoxin-stimulated macrophages and suppress phagocytosis of unopsonized bioparticles. These neuropeptides do not suppress the phagocytosis of opsonized bioparticles; however, they do suppress phagolysosome activation or formation. In this report, we studied the possibility that α-MSH with NPY suppress phagosome maturation within macrophages using opsonized OVA-coated magnetic beads to isolate and analyze the phagosomes. The magnetic bead–containing intercellular vesicles were isolated and assayed for Rab5, Rab7, LAMP1, Iad, and OVA. The macrophages cotreated with α-MSH and NPY were suppressed in Rab7 recruitment to the phagosome with suppression in LAMP1 expression but not in Iad expression. The results demonstrated that the α-MSH/NPY cotreatment suppressed phagosome maturation. In addition, the α-MSH/NPY–cotreated macrophages were suppressed in their ability to Ag stimulate CD4+ T cell proliferation. These results imply a potential mechanism of ocular immune privilege to divert Ag processing to prevent autoreactive effector T cells from binding their target cognate Ag within the ocular microenvironment.
- Published
- 2018
29. Stereoselective Homocrotylation of Aldehydes: Enantioselective Synthesis of Allylic-Substituted Z/E-Alkenes
- Author
-
Isaac J. Krauss and Leiming Tian
- Subjects
Allylic rearrangement ,Chemistry ,Reagent ,Intramolecular force ,Yield (chemistry) ,Organic Chemistry ,Enantioselective synthesis ,Regioselectivity ,Stereoselectivity ,Physical and Theoretical Chemistry ,Selectivity ,Biochemistry ,Combinatorial chemistry - Abstract
Cyclopropanated allyl- and crotylboron reagents participate in homoallylation and homocrotylation reactions that enable enantioselective access to motifs that otherwise require many steps to synthesize. In this study, we investigated the effect of substituents α- to boron, predicted either to counteract or reinforce the 1,3- selectivity of the parent reagents. We then investigated the transformation of the substituted homocrotylation products in intramolecular photocycloadditions to produce stereochemically complex natural-product-like scaffolds, finding that flow conditions enhanced the regioselectivity and yield.
- Published
- 2018
30. delta-Melt: Nucleic acid conformational penalties from melting experiments
- Author
-
Isaac J. Kimsey, Yu Xu, Hashim M. Al-Hashimi, Honglue Shi, Hala Abou Assi, Huiqing Zhou, Atul Rangadurai, and Bei Liu
- Subjects
chemistry.chemical_classification ,Chemistry ,Chemical physics ,Biomolecule ,Nucleic acid ,Sequence (biology) - Abstract
Thermodynamic propensities of biomolecules to adopt non-native conformations are crucial for understanding how they function, but prove difficult to measure experimentally. Combining optical melting experiments with chemical modifications and mutations, we developed delta-Melt for measuring the energetic penalties associated with nucleic acid conformational rearrangements and how they vary with sequence and physiological conditions. delta-Melt is fast, simple, cost effective, and can characterize conformational penalties inaccessible to conventional biophysical methods.
- Published
- 2021
31. Measuring thermodynamic preferences to form non-native conformations in nucleic acids using melting experiments reveals a rich sequence-specific DNA conformational landscape
- Author
-
Bei Liu, Hala Abou Assi, Atul Rangadurai, Yu Xu, Hashim M. Al-Hashimi, Isaac J. Kimsey, Huiqing Zhou, Honglue Shi, and John D. Boom
- Subjects
education.field_of_study ,chemistry.chemical_compound ,Computational chemistry ,Chemistry ,Population ,Nucleic acid ,Sequence (biology) ,education ,Function (biology) ,DNA - Abstract
Thermodynamic preferences to form non-native conformations are crucial for understanding how nucleic acids fold and function. However, they are difficult to measure experimentally because this requires accurately determining the population of minor low-abundance (+ and A-T Hoogsteen and A-T base open states for nearly all sixteen trinucleotide sequence contexts and found distinct sequence-specific variations on the order of 2-3 kcal/mol. This rich landscape of sequence-specific non-native minor conformations in the DNA double helix may help shape the sequence-specificity of DNA biochemistry. Thus, melting experiments can now be used to access thermodynamic information regarding regions of the free energy landscape of biomolecules beyond the native folded and unfolded conformations.Significance StatementThermodynamic preferences of nucleic acids to adopt non-native conformations are crucial for understanding how they function but prove difficult to measure experimentally. As a result, little is known about how these thermodynamic preferences vary with sequence and structural contexts, physiological conditions, and chemical modifications. Here, we show that modifications stabilizing non-native conformations and rendering them the major state, in conjunction with melting experiments, enable facile measurements of thermodynamic preferences to form various non-native conformations in DNA and RNA. delta-melt provided rare insights into the cooperativity of forming tandem Hoogsteen base pairs and revealed large and distinct sequence-specific preferences to form G-C+ and A-T Hoogsteen and A-T base open conformations in DNA, which may contribute to sequence-specific DNA biochemistry.
- Published
- 2020
32. Nanoscale Molecular Quantification of Stem Cell−Hydrogel Interactions
- Author
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Stacey C. Skaalure, Stephanie A. Maynard, Isaac J. Pence, Charlotte Lee-Reeves, Amy Gelmi, Thomas E. Whittaker, Molly M. Stevens, Julia E. Sero, Commission of the European Communities, Medical Research Council (MRC), Wellcome Trust, Engineering & Physical Science Research Council (EPSRC), and Engineering and Physical Sciences Research Council
- Subjects
integrin alpha 5 beta 1 ,Technology ,Chemistry, Multidisciplinary ,General Physics and Astronomy ,02 engineering and technology ,ADHESION ,01 natural sciences ,Regenerative medicine ,ACTIVATION ,General Materials Science ,SPECIFICITY ,RGD ,biology ,Chemistry ,Chemistry, Physical ,General Engineering ,Adhesion ,021001 nanoscience & nanotechnology ,3. Good health ,INTEGRIN ALPHA(5)BETA(1) ,DIFFERENTIATION ,Self-healing hydrogels ,Physical Sciences ,single cell force spectroscopy ,Science & Technology - Other Topics ,Stem cell ,AFM ,integrin α5β1 ,0210 nano-technology ,PEG hydrogel ,Integrin ,Materials Science ,FIBRONECTIN ,BETA ,Materials Science, Multidisciplinary ,010402 general chemistry ,Article ,FORCE ,TISSUE REGENERATION ,SDG 3 - Good Health and Well-being ,dSTORM ,Nanoscience & Nanotechnology ,MODULATION ,Cell adhesion ,Science & Technology ,Force spectroscopy ,0104 chemical sciences ,Fibronectin ,biology.protein ,Biophysics - Abstract
A common approach to tailoring synthetic hydrogels for regenerative medicine applications involves incorporating RGD cell adhesion peptides, yet assessing the cellular response to engineered microenvironments at the nanoscale remains challenging. To date, no study has demonstrated how RGD concentration in hydrogels affects the presentation of individual cell surface receptors. Here we studied the interaction between human mesenchymal stem cells (hMSCs) and RGD-functionalized poly(ethylene glycol) hydrogels, by correlating macro- and nanoscale single-cell interfacial quantification techniques. We quantified RGD unbinding forces on a synthetic hydrogel using single cell atomic force spectroscopy, revealing that short-term binding of hMSCs was sensitive to RGD concentration. We also performed direct stochastic optical reconstruction microscopy (dSTORM) to quantify the molecular interactions between integrin α5β1 and a biomaterial, unexpectedly revealing that increased integrin clustering at the hydrogel-cell interface correlated with fewer available RGD binding sites. Our complementary, quantitative approach uncovered mechanistic insights into specific stem cell-hydrogel interactions, where dSTORM provides nanoscale sensitivity to RGD-dependent differences in cell surface localization of integrin α5β1. Our findings reveal that it is possible to precisely determine how peptide-functionalized hydrogels interact with cells at the molecular scale, thus providing a basis to fine-tune the spatial presentation of bioactive ligands.
- Published
- 2020
33. Structure and Hydration of Asymmetric Polyelectrolyte Multilayers as Studied by Neutron Reflectometry: Connecting Multilayer Structure to Superior Membrane Performance
- Author
-
Wiebe M. de Vos, Andrew Nelson, Isaac J. Gresham, Joshua D. Willott, Stuart W. Prescott, Dennis M. Reurink, Membrane Science & Technology, and MESA+ Institute
- Subjects
Materials science ,Polymers and Plastics ,Organic Chemistry ,UT-Hybrid-D ,Permeance ,Polyelectrolyte ,Article ,Styrene ,Inorganic Chemistry ,chemistry.chemical_compound ,Membrane ,Sulfonate ,chemistry ,Chemical engineering ,Nafion ,Materials Chemistry ,Neutron reflectometry ,Acrylic acid - Abstract
Porous membranes coated with so-called asymmetric polyelectrolyte multilayers (PEMs) have recently been shown to outperform commercial membranes for micropollutant removal. They consist of open support layers of poly(styrene sulfonate) (PSS)/poly(allylamine) (PAH) capped by denser and more selective layers of either PAH/poly(acrylic acid) (PAA) or PAH/Nafion. Unfortunately, the structure of these asymmetric PEMs, and thus their superior membrane performance, is poorly understood. In this work, neutron reflectometry (NR) is employed to elucidate the multilayered structure and hydration of these asymmetric PEMs. NR reveals that the multilayers are indeed asymmetric in structure, with distinct bottom and top multilayers when air-dried and when solvated. The low hydration of the top [PAH/Nafion] multilayer, together with the low water permeance of comparable [PAH/Nafion]-capped PEM membranes, demonstrate that it is a reduction in hydration that makes these separation layers denser and more selective. In contrast, the [PAH/PAA] capping multilayers are more hydrated than the support [PSS/PAH] layers, signifying that, here, densification of the separation layer occurs through a decrease in the mesh size (or effective pore size) of the top layer due to the higher charge density of the PAH/PAA couple compared to the PSS/PAH couple. The [PAH/PAA] and [PAH/Nafion] separation layers are extremely thin (∼4.5 and ∼7 nm, respectively), confirming that these asymmetric PEM membranes have some of the thinnest separation layers ever achieved.
- Published
- 2020
34. Multiplexing physical stimulation on single human induced pluripotent stem cell-derived cardiomyocytes for phenotype modulation
- Author
-
Amy Gelmi, Brian Wang, Molly M. Stevens, Worrapong Kit-Anan, Sahana Gopal, Michele Becce, Cesare M. Terracciano, Sian E. Harding, Manuel Mazo, Vincent Leonardo, Ciro Chiappini, Isaac J. Pence, Anika Nagelkerke, British Heart Foundation, and Commission of the European Communities
- Subjects
Cell type ,Technology ,0206 medical engineering ,Induced Pluripotent Stem Cells ,Materials Science ,Biomedical Engineering ,Bioengineering ,cardiomyocyte ,02 engineering and technology ,Biochemistry ,Article ,cell shape ,Biomaterials ,Cell membrane ,Engineering ,physical interaction ,0903 Biomedical Engineering ,Physical Stimulation ,medicine ,Humans ,Myocytes, Cardiac ,Induced pluripotent stem cell ,Engineering, Biomedical ,microfabrication ,Materials Science, Biomaterials ,Science & Technology ,Chemistry ,1004 Medical Biotechnology ,1099 Other Technology ,technology, industry, and agriculture ,Stiffness ,Cell Differentiation ,General Medicine ,Adhesion ,Muscle stiffness ,021001 nanoscience & nanotechnology ,020601 biomedical engineering ,Phenotype ,human induced pluripotent stem cells ,medicine.anatomical_structure ,calcium handling ,Biophysics ,medicine.symptom ,0210 nano-technology ,Biotechnology ,Biofabrication - Abstract
Traditional in vitro bioengineering approaches whereby only individual biophysical cues are manipulated at any one time are highly inefficient, falling short when recapitulating the complexity of the cardiac environment. Multiple biophysical cues are present in the native myocardial niche and are essential during development, as well as in maintenance of adult cardiomyocyte (CM) phenotype in both health and disease. This study establishes a novel biofabrication workflow to study and manipulate hiPSC-CMs and to understand how these cells respond to a multiplexed biophysical environment, namely microscopic topography (3D shape resembling that of adult CM) and substrate stiffness, at a single cell level. Silicon masters were fabricated and developed to generate pillars of the desired 3D shapes, which would be used to mould the designed microwell arrays into a hydrogel. Polyacrylamide was modified with the incorporation of acrylic acid to provide a carboxylic group conjugation site for adhesion motifs, without comprising its capacity to modulate the stiffness. In this manner, individual parameters can be finely tuned independently within the hydrogel: the dimension of 3D shaped microwell and its stiffness. The design allows the platform to isolate single hiPSC-CMs to study solely biophysical cues in an absence of cell-cell physical interaction. Under physiologic-like physical conditions (3D shape resembling that of adult CM and 9.83 kPa substrate stiffness), isolated single hiPSC-CMs exhibit increased Cx-43 density, cell Peer reviewed version of the manuscript published in final form at Biofabrication (2020). membrane stiffness and calcium transient amplitude; co-expression of the subpopulation-related MYL2- MYL7 proteins; while displaying higher anisotropism in comparison to pathologic-like conditions (flat surface and 112 kPa substrate stiffness). This demonstrates that supplying a physiological or pathological microenvironment to an isolated single hiPSC-CM in absence of any physical cell-to-cell communication in this biofabricated platform leads to a significantly different set of cellular features, thus presenting a differential phenotype. Importantly, this demonstrates the high plasticity of hiPSC-CMs even in isolation. The ability of multiple biophysical cues to significantly influence isolated single hiPSC-CM phenotype and functionality highlights the importance of fine-tuning such cues for specific applications. This has the potential to produce more fit-for purpose hiPSC-CMs. Further understanding of human cardiac development is enabled by the robust, versatile and reproducible biofabrication techniques applied here. We envision this system to be easily applied to other tissues and cell types where the influence of stiffness and cellular shape plays also an important role in its physiology. Keywords: microfabrication, cardiomyocyte, physical interaction, cell shape, calcium handling, human induced pluripotent stem cells
- Published
- 2020
35. Hypothalamic-pituitary-adrenal axis regulation and organization in urban and rural song sparrows
- Author
-
Scott Davies, Elizabeth R. Gilbert, Samuel J. Lane, Kendra B. Sewall, Michelle L. Beck, Michael G. Emmerson, Catherine Hucul, and Isaac J. VanDiest
- Subjects
Male ,medicine.medical_specialty ,Hypothalamo-Hypophyseal System ,Hippocampus ,Pituitary-Adrenal System ,030209 endocrinology & metabolism ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Endocrinology ,Mineralocorticoid receptor ,Glucocorticoid receptor ,Receptors, Glucocorticoid ,Corticosterone ,Internal medicine ,medicine ,Animals ,030304 developmental biology ,0303 health sciences ,biology ,biology.organism_classification ,medicine.anatomical_structure ,chemistry ,Hypothalamus ,Animal Science and Zoology ,Melospiza ,hormones, hormone substitutes, and hormone antagonists ,Hypothalamic–pituitary–adrenal axis ,Glucocorticoid ,Sparrows ,medicine.drug - Abstract
Urban habitats present animals with persistent disturbances and acute stressors not present in rural habitats or present at significantly lower levels. Differences in the glucocorticoid stress response could underlie colonization of these novel habitats. Despite urban habitats characterization as more stressful, previous comparisons of urban and rural birds have failed to find consistent differences in baseline and stress induced glucocorticoid levels. Another aspect of glucocorticoid regulation that could underlie an animal’s ability to inhabit novel habitats, but has yet to be well examined, is more efficient termination of the glucocorticoid stress response which would allow birds in urban habitats to recover more quickly after a disturbance. The glucocorticoid stress response is terminated by negative feedback achieved primarily through their binding of receptors in the hippocampus and hypothalamus and subsequent decreased synthesis and release from the adrenals. We investigated if male song sparrows (Melospiza melodia) in urban habitats show more efficient termination of the glucocorticoid stress response than their rural counterparts using two approaches. First, we measured glucocorticoid receptor, mineralocorticoid receptor and 11β-HSD2 (an enzyme that inactivates corticosterone) mRNA expression in negative feedback targets of the brain (the hippocampus and hypothalamus) as a proxy measure of sensitivity to negative feedback. Second, we measured plasma corticosterone levels after standardized restraint and again following a challenge with the synthetic glucocorticoid, dexamethasone, as a means of assessing how quickly birds decreased glucocorticoid synthesis and release. Though there were no differences in the hypothalamus of urban and rural song sparrows, urban birds had lower glucocorticoid receptor and 11β-HSD2 mRNA expression in the hippocampus. Further, urban and rural birds had similar reductions in corticosterone following the dexamethasone challenge, suggesting that they do not differ in how quickly they decrease glucocorticoid synthesis and release. Thus, urban and rural song sparrows display similar termination of the glucocorticoid stress response even though urban birds have decreased hippocampal glucocorticoid receptor and 11β-HSD2 abundance.
- Published
- 2020
36. Potential chemopreventive, anticancer and anti-inflammatory properties of a refined artocarpin-rich wood extract of Artocarpus heterophyllus Lam
- Author
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Jianan Zhang, Rupika Delgoda, Guodong Zhang, JeAnn Murray, Roy Porter, Isaac J. Morrison, Nicholas J. Sadgrove, James Barker, Moses K. Langat, and Jingwen Lin
- Subjects
Male ,medicine.drug_class ,Science ,Anti-Inflammatory Agents ,Azoxymethane ,alliedhealth ,Antineoplastic Agents ,Diseases ,Pharmacology ,chemistry ,Biochemistry ,Anti-inflammatory ,Article ,Proinflammatory cytokine ,Artocarpus ,chemistry.chemical_compound ,Mice ,medicine ,Animals ,Humans ,Cytotoxicity ,IC50 ,Cytochrome P-450 CYP2C9 ,Cancer ,chemistry.chemical_classification ,Multidisciplinary ,biology ,Molecular medicine ,Chemistry ,Plant Extracts ,biology.organism_classification ,cs_r ,Colitis ,HCT116 Cells ,Wood ,In vitro ,Mice, Inbred C57BL ,Enzyme ,Mannose-Binding Lectins ,Medicine ,Plant Lectins ,Colorectal Neoplasms ,Plant sciences - Abstract
Colorectal cancer (CRC) represents the third leading cause of death among cancer patients below the age of 50, necessitating improved treatment and prevention initiatives. A crude methanol extract from the wood pulp of Artocarpus heterophyllus was found to be the most bioactive among multiple others, and an enriched extract containing 84% (w/v) artocarpin (determined by HPLC–MS–DAD) was prepared. The enriched extract irreversibly inhibited the activity of human cytochrome P450 CYP2C9, an enzyme previously shown to be overexpressed in CRC models. In vitro evaluations on heterologously expressed microsomes, revealed irreversible inhibitory kinetics with an IC50 value of 0.46 µg/mL. Time- and concentration-dependent cytotoxicity was observed on human cancerous HCT116 cells with an IC50 value of 4.23 mg/L in 72 h. We then employed the azoxymethane (AOM)/dextran sodium sulfate (DSS) colitis-induced model in C57BL/6 mice, which revealed that the enriched extract suppressed tumor multiplicity, reduced the protein expression of proliferating cell nuclear antigen, and attenuated the gene expression of proinflammatory cytokines (Il-6 and Ifn-γ) and protumorigenic markers (Pcna, Axin2, Vegf, and Myc). The extract significantly (p = 0.03) attenuated (threefold) the gene expression of murine Cyp2c37, an enzyme homologous to the human CYP2C9 enzyme. These promising chemopreventive, cytotoxic, anticancer and anti-inflammatory responses, combined with an absence of toxicity, validate further evaluation of A. heterophyllus extract as a therapeutic agent.
- Published
- 2020
37. Severity of Sepsis Determines the Degree of Impairment Observed in Circulatory and Tissue-Resident Memory CD8 T Cell Populations
- Author
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Isaac J. Jensen, Vladimir P. Badovinac, Derek B. Danahy, John T. Harty, Thomas S. Griffith, Frances V. Sjaastad, Scott M. Anthony, Stephanie van de Wall, and Steven J Moioffer
- Subjects
Immunology ,Inflammation ,Vascular permeability ,Mice, Transgenic ,CD8-Positive T-Lymphocytes ,Lymphocytic Choriomeningitis ,Article ,Sepsis ,chemistry.chemical_compound ,Mice ,T-Lymphocyte Subsets ,medicine ,Immunology and Allergy ,Cytotoxic T cell ,Animals ,Humans ,Lymphocytic choriomeningitis virus ,Cells, Cultured ,business.industry ,medicine.disease ,Mice, Inbred C57BL ,chemistry ,Apoptosis ,Organ Specificity ,Circulatory system ,Blood Circulation ,Disease Progression ,medicine.symptom ,Vaccinia ,business ,Immunologic Memory ,CD8 - Abstract
Sepsis reduces the number and function of memory CD8 T cells within the host, contributing to the long-lasting state of immunoparalysis. Interestingly, the relative susceptibility of memory CD8 T cell subsets to quantitative/qualitative changes differ after cecal ligation and puncture (CLP)–induced sepsis. Compared with circulatory memory CD8 T cells (TCIRCM), moderate sepsis (0–10% mortality) does not result in numerical decline of CD8 tissue-resident memory T cells (TRM), which retain their “sensing and alarm” IFN-γ–mediated effector function. To interrogate this biologically important dichotomy, vaccinia virus–immune C57BL/6 (B6) mice containing CD8 TCIRCM and skin TRM underwent moderate or severe (∼50% mortality) sepsis. Severe sepsis led to increased morbidity and mortality characterized by increased inflammation compared with moderate CLP or sham controls. Severe CLP mice also displayed increased vascular permeability in the ears. Interestingly, skin CD103+ CD8 TRM, detected by i.v. exclusion or two-photon microscopy, underwent apoptosis and subsequent numerical loss following severe sepsis, which was not observed in mice that experienced moderate CLP or sham surgeries. Consequently, severe septic mice showed diminished CD8 T cell–mediated protection to localized skin reinfection. Finally, the relationship between severity of sepsis and demise in circulatory versus tissue-embedded memory CD8 T cell populations was confirmed by examining tumor-infiltrating and nonspecific CD8 T cells in B16 melanoma tumors. Thus, sepsis can differentially affect the presence and function of Ag-specific CD8 T cells that reside inside tissues/tumors depending on the severity of the insult, a notion with direct relevance to sepsis survivors and their ability to mount protective memory CD8 T cell–dependent responses to localized Ag re-encounter.
- Published
- 2020
38. Inducing Experimental Polymicrobial Sepsis by Cecal Ligation and Puncture
- Author
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Vladimir P. Badovinac, Frances V. Sjaastad, Isaac J. Jensen, Roger R. Berton, and Thomas S. Griffith
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0301 basic medicine ,Lipopolysaccharide ,medicine.drug_class ,Immunology ,Peritonitis ,Wounds, Penetrating ,Monoclonal antibody ,Article ,Immunophenotyping ,Sepsis ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Immune system ,medicine ,Animals ,Humans ,Cecum ,Ligation ,business.industry ,Septic shock ,Coinfection ,Zymosan ,Immunity ,medicine.disease ,bacterial infections and mycoses ,Pathophysiology ,030104 developmental biology ,chemistry ,Models, Animal ,business ,030215 immunology - Abstract
Numerous models are available for the preclinical study of sepsis, and they fall into one of three general categories: (1) administration of exogenous toxins (e.g., lipopolysaccharide, zymosan), (2) virulent bacterial or viral challenge, and (3) host barrier disruption, e.g., cecal ligation and puncture (CLP) or colon ascendens stent peritonitis (CASP). Of the murine models used to study the pathophysiology of sepsis, CLP combines tissue necrosis and polymicrobial sepsis secondary to autologous fecal leakage, as well as hemodynamic and biochemical responses similar to those seen in septic humans. Further, a transient numerical reduction of multiple immune cell types, followed by development of prolonged immunoparalysis, occurs in CLP-induced sepsis just as in humans. Use of the CLP model has led to a vast expansion in knowledge regarding the intricate physiological and cellular changes that occur during and after a septic event. This updated article details the steps necessary to perform this survival surgical technique, as well as some of the obstacles that may arise when evaluating the sepsis-induced changes within the immune system. It also provides representative monoclonal antibody (mAb) panels for multiparameter flow cytometric analysis of the murine immune system in the septic host. © 2020 Wiley Periodicals LLC. Basic Protocol: Cecal ligation and puncture in the mouse.
- Published
- 2020
39. Synergistic effects of frothers, collector and salt on bubble stability
- Author
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Yueyi Pan, Ghislain Bournival, Stuart W. Prescott, Seher Ata, and Isaac J. Gresham
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Surface tension ,Coalescence (physics) ,chemistry.chemical_compound ,Adsorption ,Polypropylene glycol ,Chemical engineering ,Chemistry ,General Chemical Engineering ,Sodium ,Bubble ,chemistry.chemical_element ,Xanthate ,Propylene glycol methyl ether - Abstract
This work investigates synergistic and antagonistic effects of flotation reagents and salt on the air–liquid interface and stability of air bubbles under quasi-static conditions. Frothers of different families (aliphatic alcohol, polypropylene glycol and propylene glycol methyl ether), potassium amyl xanthate (PAX), a common collector used in the flotation of sulphide minerals, and sodium chloride (NaCl) were employed in the study. Adsorption of frother and collector molecules from individual and mixed solutions was studied through surface tension and Quartz Crystal Microbalance with Dissipation (QCM–D) measurements, while bubble stability was examined by the coalescence time of the bubble pairs in these solutions. The results show that NaCl enhanced the adsorption of frothers, while PAX's impact on the adsorption of the frother was found to be rather insignificant compared to the salt. The bubble coalescence time measurements, however, pointed to a significant synergistic effect between PAX and two frothers i.e., Methyl Iso Butyl Carbinol (MIBC) and Di Propylene Glycol (DPG) as reflected in the improvement of bubble stability. In the ternary solution systems (frother/NaCl/PAX), the concentration of salt in the MIBC mixture solution was found to have an opposite impact on the bubble lifetime in the DPG and DPM solutions. For instance, bubble lifetime increased with higher NaCl concentrations added to the DPG and DPM solutions, whereas it decreased in the MIBC solutions.
- Published
- 2022
40. Competitive specific ion effects in mixed salt solutions on a thermoresponsive polymer brush
- Author
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Stuart W. Prescott, Edwin C. Johnson, Isaac J. Gresham, Erica J. Wanless, Hayden Robertson, Grant B. Webber, and Andrew Nelson
- Subjects
chemistry.chemical_classification ,Hofmeister series ,Iodide ,Salt (chemistry) ,02 engineering and technology ,Electrolyte ,010402 general chemistry ,021001 nanoscience & nanotechnology ,Polymer brush ,01 natural sciences ,0104 chemical sciences ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Biomaterials ,Solvent ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,chemistry ,Chemical engineering ,Molecule ,0210 nano-technology ,Ethylene glycol - Abstract
Hypothesis Grafted poly(ethylene glycol) methyl ether methacrylate (POEGMA) copolymer brushes change conformation in response to temperature ('thermoresponse'). In the presence of different ions the thermoresponse of these coatings is dramatically altered. These effects are complex and poorly understood with no all-inclusive predictive theory of specific ion effects. As natural environments are composed of mixed electrolytes, it is imperative we understand the interplay of different ions for future applications. We hypothesise anion mixtures from the same end of the Hofmeister series (same-type anions) will exhibit non-additive and competitive behaviour. Experiments The behaviour of POEGMA brushes, synthesised via surface-initiated ARGET-ATRP, in both single and mixed aqueous electrolyte solutions was characterised with ellipsometry and neutron reflectometry as a function of temperature. Findings In mixed fluoride and chloride aqueous electrolytes (salting-out ions), or mixed thiocyanate and iodide aqueous electrolytes (salting-in ions), a non-monotonic concentration-dependent influence of the two anions on the thermoresponse of the brush was observed. A new term, δ , has been defined to quantitively describe synergistic or antagonistic behaviour. This study determined the specific ion effects imparted by salting-out ions are dependent on available solvent molecules, whereas the influence of salting-in ions is dependent on the interactions of the anions and polymer chains.
- Published
- 2020
41. Plumbagin induces testicular damage via mitochondrial-dependent cell death
- Author
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Isaac J. Bello, Todiimu O. Omodara, John Oludele Olanlokun, Olufunso O. Olorunsogo, and Olubukola T. Oyebode
- Subjects
Male ,medicine.medical_specialty ,Apoptosis ,Mitochondrion ,Toxicology ,Lipid peroxidation ,chemistry.chemical_compound ,Internal medicine ,Testis ,medicine ,Animals ,Rats, Wistar ,Receptor ,Adenosine Triphosphatases ,biology ,Cell Death ,Chemistry ,Cytochrome c ,General Medicine ,Plumbagin ,Sperm ,Spermatozoa ,Caspase 9 ,Mitochondria ,Rats ,Endocrinology ,Mitochondrial permeability transition pore ,Proto-Oncogene Proteins c-bcl-2 ,biology.protein ,Lipid Peroxidation ,Tumor Suppressor Protein p53 ,Naphthoquinones ,Signal Transduction - Abstract
Different aspects of reproductive functions are regulated by mitochondrial-controlled events. This study investigated the effect of plumbagin (PL) on testicular mitochondria with a view to unravelling the mechanism of the antifertility potential of plumbagin in testis of healthy rats. Thirty-two male Wistar strain albino rats were randomly allocated into four groups of eight animals each. The control or healthy group received orally 0.1 % DMSO while animals in the remaining three groups received 2.5 mg PL/kg bdwt, 5.0 mg PL/kg bdwt and 10 mg PL/kg bdwt, respectively, for 14 days. In study two, twenty-four male Wistar rats were randomly divided into three (3) groups and were orally administered 0.1% DMSO (control), 30 and 100 mg/kg PL, respectively once daily for 72 h. Rat testis mitochondria were isolated using differential centrifugation. The mitochondrial Permeability Transition (mPT) pore, mitochondrial ATPase (mATPase) activity and mitochondrial lipid peroxidation were assessed spectrophotometrically. Expression of apoptotic proteins (p53, Bax, Bcl-2) and the release of cytochrome c were determined by immunochemical technique. Reproductive receptors (FSH, PR), the expression of aromatase, Testis Specific Kinase-1 {TESK-1} were quantified by RT-PCR. The various doses of plumbagin (2.5, 5.0 and 10 mg/kg bdwt) induced opening of the testicular mPT pore by 2, 5 and 8 folds, respectively, after 14 days of oral administration. These doses of plumbagin also caused enhancement of mATPase activity, elevated generation of mLPO as well as increases in the concentrations of caspases 9 and 3. Sperm analysis revealed that these doses of PL also caused significant decreases in sperm count and motility and increased sperm abnormalities compared to control. Interestingly, these effects were accompanied by dose-dependent expressions of the Bak, p53 and cytochrome c release. Conversely, the abundance of anti‐apoptotic Bcl-2 protein decreased relative to control. The levels of transcripts of FSH and progesterone receptors as well as TESK-1 and aromatase decreased significantly relative to control. Furthermore, PL strongly inhibited p53-MDM2 compared to control. Altogether, these findings show that plumbagin damages testicular cells through the activation of mitochondrial pathway involving the p53 protein network.
- Published
- 2020
42. Functional and structural characterization of allosteric activation of Phospholipase Cε by Rap1A
- Author
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Elisabeth E. Garland-Kuntz, Monita Sieng, Arielle F. Selvia, Jesse B. Hopkins, Isaac J. Fisher, Angeline M. Lyon, and Andrea T. Marti
- Subjects
Pleckstrin homology domain ,biology ,Chemistry ,Allosteric regulation ,biology.protein ,GTPase ,Phospholipase ,Binding site ,Receptor ,Receptor tyrosine kinase ,G protein-coupled receptor ,Cell biology - Abstract
Phospholipase Cε (PLCε) is activated downstream of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) through direct interactions with small GTPases, including Rap1A and Ras. While Ras has been reported to allosterically activate the lipase, it is not known whether Rap1A has the same ability, or what its molecular mechanism might be. Rap1A activates PLCε in response to the stimulation of β-adrenergic receptors (β-ARs), translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLCε was proposed to the primary binding site for Rap1A, we first confirmed using purified proteins that the RA2 domain is indeed essential for activation by Rap1A. However, we also showed that the PLCε pleckstrin homology (PH) domain and first two EF hands (EF1/2) are required for Rap1A activation, and identified hydrophobic residues on the surface of the RA2 domain that are also necessary for activation by the GTPase. Finally, small angle X-ray scattering (SAXS) showed that Rap1A binding induces and stabilizes discrete conformational states in PLCε variants that can be activated by the GTPase. This data, together with the recent structure of a catalytically active fragment of PLCε, provide the first evidence that Rap1A, and by extension Ras, allosterically activate the lipase by promoting and stabilizing interactions between the RA2 domain and the PLC core.
- Published
- 2020
43. Mechanism Underlying Anti-Markovnikov Addition in the Reaction of Pentalenene Synthase
- Author
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Ramasamy P. Kumar, Daniel D. Oprian, Jason O. Matos, Alison C. Ma, MacKenzie Patterson, and Isaac J. Krauss
- Subjects
Models, Molecular ,Protein Conformation ,Stereochemistry ,Markovnikov's rule ,Cyclopentanes ,Carbocation ,Hyperconjugation ,Crystallography, X-Ray ,Biochemistry ,Catalysis ,Article ,Carbenium ion ,chemistry.chemical_compound ,Catalytic Domain ,Side chain ,Pentalenene synthase ,Intramolecular Lyases ,Addition reaction ,Alkyl and Aryl Transferases ,biology ,Chemistry ,Active site ,Regioselectivity ,Streptomyces ,Cyclization ,biology.protein - Abstract
Most terpene synthase reactions follow Markovnikov rules for formation of high energy carbenium ion intermediates. However, there are notable exceptions. For example, pentalenene synthase (PS) undergoes an initial anti-Markovnikov cyclization reaction followed by a 1,2-hydride shift to form an intermediate humulyl cation with positive charge on the secondary carbon C9 of the farnesyl diphosphate substrate. The mechanism by which these enzymes stabilize and guide regioselectivity of secondary carbocations has not heretofore been elucidated. In an effort to better understand these reactions, we grew crystals of apo-PS, soaked them with the non-reactive substrate analog 12,13-difluorofarnesyl diphosphate, and solved the x-ray structure of the resulting complex at 2.2 Å resolution. The most striking feature of the active site structure is that C9 is positioned 3.5 Å above the center of the side chain benzene ring of residue F76, perfectly poised for stabilization of the charge through a cation-π interaction. In addition, the main chain carbonyl of I177 and neighboring intramolecular C6,C7-double bond are positioned to stabilize the carbocation by interaction with the face opposite that of F76. Mutagenesis experiments also support a role for residue 76 in cation-π interactions. Most interesting is the F76W mutant which gives a mixture of products that likely result from stabilizing a positive charge on the adjacent secondary carbon C10 in addition to C9 as in the wild-type enzyme. The crystal structure of the F76W mutant clearly shows carbons C9 and C10 centered above the fused benzene and pyrrole rings of the indole side chain, respectively, such that a carbocation at either position could be stabilized in this complex, and two anti-Markovnikov products, pentalenene and humulene, are formed. Finally, we show that there is a rough correlation (although not absolute) of an aromatic side chain (F or Y) at position 76 in related terpene synthases from Streptomyces that catalyze similar anti-Markovnikov addition reactions.
- Published
- 2020
44. Computational Screening of Organic Semiconductors: Exploring Side-Group Functionalisation and Assembly to Optimise Charge Transport in Chiral Molecules
- Author
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Jenny Nelson, Joseph A. Weatherby, Erin R. Johnson, Isaac J. Sugden, Alejandro Santana-Bonilla, Julia A. Schmidt, Francesco Salerno, Kim E. Jelfs, Matthew J. Fuchter, The Royal Society, Commission of the European Communities, and Engineering & Physical Science Research Council (EPSRC)
- Subjects
Technology ,Materials science ,Chemistry, Multidisciplinary ,Materials Science ,Materials Science, Multidisciplinary ,ENERGETICS ,FLUORINATION ,ENERGY ,chemistry.chemical_compound ,MOLECULES ,0302 Inorganic Chemistry ,Molecule ,0912 Materials Engineering ,Organic electronics ,0306 Physical Chemistry (incl. Structural) ,Science & Technology ,Crystallography ,Intermolecular force ,Rational design ,HELICENES ,Material Design ,PERFORMANCE ,HARTREE-FOCK ,Crystal structure prediction ,Organic semiconductor ,Chemistry ,Helicene ,chemistry ,Chemical physics ,MOBILITY ,Physical Sciences ,CRYSTAL-STRUCTURE PREDICTION ,Inorganic & Nuclear Chemistry ,LANDSCAPES - Abstract
Molecular materials are challenging to design as their packing arrangement and hence properties are subject to subtle variations in the interplay of soft intermolecular interactions that are difficult to predict. The rational design of new molecular materials with tailored properties is currently hampered by the lack of knowledge of how a candidate molecule will pack in space and how we can control the polymorphs we can experimentally obtain. Here, we develop a simplified approach to aid the material design process, by the development of a screening process that is used to test 1344 helicene molecules that have potential as organic electronic materials. Our approach bridges the gap between single molecule design, molecular assembly, and the resulting charge-carrier mobilities. We find that fluorination significantly improves electron transport in the molecular material by up to 200%; the reference [6]helicene packing showed a mobility of 0.30 cm2 V-1 s-1, fluorination increased the mobility to up to 0.96 and 0.97 (13-fluoro[6]H and 4,13-difluoro[6]H), assuming an outer reorganisation energy of 0.30 eV. Side groups containing triple bonds largely lead to improved transfer integrals. We validate our screening approach through the use of crystal structure prediction to confirm the presence of favourable packing motifs to maximize charge mobility.
- Published
- 2020
45. Incorporating biological mass spectrometry into undergraduate teaching labs, part I: identifying proteins based on molecular mass
- Author
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Arnquist, Isaac J. and Beussman, Douglas J.
- Subjects
Mass spectrometry -- Analysis ,College students -- Education ,Proteomics -- Study and teaching ,Science experiments ,Chemistry ,Education ,Science and technology - Abstract
Students are given an unknown protein and are asked to analyze and identify the molecular mass of medium -sized proteins using ESI-MS or Electrospray ionization-Mass spectrometer.
- Published
- 2007
46. Efficient screening for ternary molecular ionic cocrystals using a complementary mechanosynthesis and computational structure prediction approach
- Author
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Sharmarke Mohamed, Abeer F. Shunnar, David H. Bowskill, Bhausaheb Dhokale, Hector H. Hernandez, Isaac J. Sugden, Panče Naumov, and Durga Prasad Karothu
- Subjects
Chemistry, Multidisciplinary ,Ionic bonding ,SOLID-STATE LANDSCAPE ,Crystal structure ,010402 general chemistry ,Crystal engineering ,01 natural sciences ,POTENTIAL-FUNCTION MODELS ,Catalysis ,crystal structure prediction ,law.invention ,ENERGY ,Computational chemistry ,law ,Crystallization ,mechanosynthesis ,CO-CRYSTALS ,SUPRAMOLECULAR SYNTHONS ,Science & Technology ,Full Paper ,STABILITY ,010405 organic chemistry ,Chemistry ,green chemistry ,Organic Chemistry ,SALT ,DISTRIBUTED MULTIPOLE ANALYSIS ,General Chemistry ,Full Papers ,PHARMACEUTICAL COCRYSTALS ,0104 chemical sciences ,Crystal structure prediction ,X-ray diffraction ,crystal engineering ,X-ray crystallography ,Physical Sciences ,CRYSTAL-STRUCTURE PREDICTION ,Mechanosynthesis ,molecular ionic cocrystals ,Ternary operation ,03 Chemical Sciences - Abstract
The discovery of molecular ionic cocrystals (ICCs) of active pharmaceutical ingredients (APIs) widens the opportunities for optimizing the physicochemical properties of APIs whilst facilitating the delivery of multiple therapeutic agents. However, ICCs are often observed serendipitously in crystallization screens and the factors dictating their crystallization are poorly understood. We demonstrate here that mechanochemical ball milling is a versatile technique for the reproducible synthesis of ternary molecular ICCs in less than 30 min of grinding with or without solvent. Computational crystal structure prediction (CSP) calculations have been performed on ternary molecular ICCs for the first time and the observed crystal structures of all the ICCs were correctly predicted. Periodic dispersion‐corrected DFT calculations revealed that all the ICCs are thermodynamically stable (mean stabilization energy=−2 kJ mol−1) relative to the crystallization of a physical mixture of the binary salt and acid. The results suggest that a combined mechanosynthesis and CSP approach could be used to target the synthesis of higher‐order molecular ICCs with functional properties., Spot on predictions! The mechanosynthesis of ternary molecular ionic cocrystals (ICCs) with significantly different physicochemical properties has been achieved in less than 30 min of grinding. The crystal structures of the ICCs were successfully predicted by using computational methods. The results pave the way for the efficient screening of higher‐order multicomponent crystal forms with functional properties (see figure).
- Published
- 2019
47. Infrared-Driven Charge-Transfer in Transition Metal-Containing B12X122– (X = H, F) Clusters
- Author
-
Patrick J. J. Carr, Rick A. Marta, W. Scott Hopkins, Michael J. Lecours, Isaac J. S. De Vlugt, Eric Fillion, Ahdia Anwar, and Vincent Steinmetz
- Subjects
010405 organic chemistry ,Infrared ,Hydride ,Chemistry ,Dodecaborate ,010402 general chemistry ,7. Clean energy ,01 natural sciences ,Dissociation (chemistry) ,0104 chemical sciences ,Crystallography ,Transition metal ,Density functional theory ,Infrared multiphoton dissociation ,Physical and Theoretical Chemistry ,Spectroscopy - Abstract
Density functional theory (DFT) calculations and infrared multiple photon dissociation (IRMPD) spectroscopy are employed to probe [TM·(B12H12)]− and [TM·(B12H12)2]2– clusters [TM = Ag(I), Cu(I), Co(II), Ni(II), Zn(II), Cd(II)]. A comparison is made between the charge-transfer properties of the clusters containing the hydrogenated dodecaborate dianions, B12H122–, and the fluorinated analogues, B12F122–, for clusters containing Cd(II), Co(II), Ni(II), and Zn(II). IRMPD of the [TM·(B12H12)]− and [TM·(B12H12)2]2– species yields B12H11– via hydride abstraction and B12H12– in all cases. To further explore the IR-induced charge-transfer properties of the B12X122– (X = H, F) cages, mixed-cage [TM(B12H12)(B12F12)]2– [TM = Co(II), Ni(II), Zn(II), Cd(II)] clusters were investigated. IRMPD of the mixed-cage species yielded appreciable amounts of B12F12– and B12H12– in most cases, indicating that charge-transfer to the central TM cation is a favorable process; formation of B12F12– is the dominant process for the Co(II...
- Published
- 2018
48. Tolerance to high‐internalizing δ opioid receptor agonist is critically mediated by arrestin 2
- Author
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Areeb Akbari, Amynah A. Pradhan, Ana Vicente-Sánchez, Alycia F Tipton, Isaac J. Dripps, Heba Akbari, and Emily M. Jutkiewicz
- Subjects
Male ,0301 basic medicine ,G protein ,media_common.quotation_subject ,Analgesic ,Pain ,Pharmacology ,Piperazines ,03 medical and health sciences ,0302 clinical medicine ,Seizures ,Receptors, Opioid, delta ,Arrestin ,medicine ,Animals ,Potency ,Receptor ,Internalization ,media_common ,Mice, Knockout ,Analgesics ,Chemistry ,Chronic pain ,Brain ,Drug Tolerance ,medicine.disease ,Research Papers ,beta-Arrestin 1 ,030104 developmental biology ,Opioid ,Guanosine 5'-O-(3-Thiotriphosphate) ,Benzamides ,Female ,030217 neurology & neurosurgery ,medicine.drug - Abstract
Background and purpose Opioid δ receptor agonists are potent antihyperalgesics in chronic pain models, but tolerance develops after prolonged use. Previous evidence indicates that distinct forms of tolerance occur depending on the internalization properties of δ receptor agonists. As arrestins are important in receptor internalization, we investigated the role of arrestin 2 (β-arrestin 1) in mediating the development of tolerance induced by high- and low-internalizing δ receptor agonists. Experimental approach We evaluated the effect of two δ receptor agonists with similar analgesic potencies, but either high-(SNC80) or low-(ARM390) internalization properties in wild-type (WT) and arrestin 2 knockout (KO) mice. We compared tolerance to the antihyperalgesic effects of these compounds in a model of inflammatory pain. We also examined tolerance to the convulsant effect of SNC80. Furthermore, effect of chronic treatment with SNC80 on δ agonist-stimulated [35 S]-GTPγS binding was determined in WT and KO mice. Key results Arrestin 2 KO resulted in increased drug potency, duration of action and decreased acute tolerance to the antihyperalgesic effects of SNC80. In contrast, ARM390 produced similar effects in both WT and KO animals. Following chronic treatment, we found a marked decrease in the extent of tolerance to SNC80-induced antihyperalgesia and convulsions in arrestin 2 KO mice. Accordingly, δ receptors remained functionally coupled to G proteins in arrestin 2 KO mice chronically treated with SNC80. Conclusions and implications Overall, these results suggest that δ receptor agonists interact with arrestins in a ligand-specific manner, and tolerance to high- but not low-internalizing agonists are preferentially regulated by arrestin 2.
- Published
- 2018
49. Role of signalling molecules in behaviours mediated by the δ opioid receptor agonist SNC80
- Author
-
Kenner C. Rice, John R. Traynor, Emily M. Jutkiewicz, Isaac J. Dripps, Brett T. Boyer, and Richard R. Neubig
- Subjects
0301 basic medicine ,Pharmacology ,Agonist ,Cell signaling ,biology ,Chemistry ,medicine.drug_class ,G protein ,Cell biology ,δ-opioid receptor ,RGS4 ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Regulator of G protein signaling ,Knockout mouse ,Arrestin ,medicine ,biology.protein ,030217 neurology & neurosurgery - Abstract
Background and Purpose G protein-coupled receptors exist in multiple conformations that can engage distinct signaling mechanisms which in turn may lead to diverse behavioral outputs. In rodent models, activation of the delta opioid receptor (δ-receptor) has been shown to elicit antihyperalgesia, antidepressant-like effects, and convulsions. We recently showed that these δ-receptor-mediated behaviors are differentially regulated by the GTPase-activating protein regulator of G protein signaling 4 (RGS4), which facilitates termination of G protein signaling. To further evaluate the signaling mechanisms underlying δ-receptor-mediated antihyperalgesia, antidepressant-like effects, and convulsions, we observed how changes in Gαo or arrestin proteins in vivo affected behaviors elicited by the δ-receptor agonist SNC80 in mice. Experimental Approach Transgenic mice with altered expression of various signaling molecules were used in the current studies. Antihyperalgesia was measured in a nitroglycerin-induced thermal hyperalgesia assay. Antidepressant-like effects were evaluated in the forced swim test. Mice were also observed for convulsive activity following SNC80 treatment. Key Results In Gαo RGS-insensitive heterozygous knock-in mice, the potency of SNC80 to produce antihyperalgesia and antidepressant-like effects was enhanced with no change in SNC80-induced convulsions. Conversely, in Gαo heterozygous knockout mice, SNC80-induced antihyperalgesia was abolished while antidepressant-like effects and convulsions were unaltered. No changes in SNC80-induced behaviors were observed in arrestin 3 knockout mice. SNC80-induced convulsions were potentiated in arrestin 2 knockout mice. Conclusions and Implications Taken together, these findings suggest that different signaling molecules may underlie the convulsive effects of the δ-receptor relative to the antihyperalgesic and antidepressant-like effects.
- Published
- 2018
50. A ratiometric iron probe enables investigation of iron distribution within tumour spheroids
- Author
-
Jacek L. Kolanowski, Benjamin Chekroun, Angela G Torrisi, Isaac J. Carney, Trevor W. Hambley, Zelong Lim, and Elizabeth J. New
- Subjects
0301 basic medicine ,Iron ,Cell ,Biophysics ,Iron Chelating Agents ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Biomaterials ,03 medical and health sciences ,Iron homeostasis ,Coumarins ,Spheroids, Cellular ,Tumour spheroid ,Tumor Cells, Cultured ,medicine ,Humans ,Distribution (pharmacology) ,Calcein AM ,Fluorescent Dyes ,Chemistry ,Metals and Alloys ,Fluoresceins ,0104 chemical sciences ,3. Good health ,030104 developmental biology ,medicine.anatomical_structure ,Chemistry (miscellaneous) ,sense organs ,Colorectal Neoplasms - Abstract
Iron dysregulation is implicated in numerous diseases, and iron homeostasis is profoundly influenced by the labile iron pool (LIP). Tools to easily observe changes in the LIP are limited, with calcein AM-based assays most widely used. We describe here FlCFe1, a ratiometric analogue of calcein AM, which also provides the capacity for imaging iron in 3D cell models.
- Published
- 2018
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