Chemistry, Department of

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Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: INTRACELLULAR β-O-LINKED N-GLYCOLYLGLUCOSAMINE (GlcNGc), UDP-GlcNGc, AND THE BIOCHEMICAL AND STRUCTURAL RATIONALE FOR THE SUBSTRATE TOLERANCE OF β-O-LINKED β-N-ACETYLGLUCOSAMINIDASE

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2012-08-17
Abstract: 

TheO-GlcNAcmodificationinvolvestheattachmentofsingle-O-linkedN-acetylglucosamine residues to serine and threo-nine residues of nucleocytoplasmic proteins. Interestingly, pre-vious biochemical and structural studies have shown thatO-GlcNAcase (OGA), the enzyme that removesO-GlcNAc fromproteins, has an active site pocket that tolerates variousN-acylgroups in addition to theN-acetyl group of GlcNAc. Theremarkable sequence and structural conservation of residuescomprising this pocket suggest functional importance. Wehypothesized this pocket enables processing of metabolic vari-ants ofO-GlcNAc that could be formed due to inaccuracy withinthe metabolic machinery of the hexosamine biosynthetic path-way. In the accompanying paper (Bergfeld, A. K., Pearce, O. M.,Diaz, S. L.,Pham, T., and Varki, A. (2012)J. Biol. Chem.287,28865–28881),N-glycolylglucosamine (GlcNGc) wasshown to be acatabolite of NeuNGc. Here, we show that the hexosamine sal-vage pathway can convert GlcNGc to UDP-GlcNGc, which isthen used to modify proteins withO-GlcNGc. The kinetics of incorporation and removal ofO-GlcNGc in cells occur in adynamic manner on a time frame similar to that ofO-GlcNAc.Enzymatic activity ofO-GlcNAcase (OGA) toward a GlcNGcglycoside reveals OGA can process glycolyl-containing sub-strates fairly efficiently. A bacterial homolog (BtGH84) of OGA,from a human gut symbiont, also processesO-GlcNGc sub-strates, and the structure of this enzyme bound to a GlcNGc-derived species reveals the molecular basis for tolerance andbinding of GlcNGc. Together, these results demonstrate thatanalogs of GlcNAc, such as GlcNGc, are metabolically viablespecies and that the conserved active site pocket of OGA likelyevolved to enable processing of mis-incorporated analogs ofO-GlcNAc and thereby prevent their accumulation. Such plas-ticity in carbohydrate processing enzymes may be a generalfeature arising from inaccuracy in hexosamine metabolicpathways.

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Insights into O-Linked N-Acetylglucosamine ([0-9]O-GlcNAc) Processing and Dynamics through Kinetic Analysis of O-GlcNAc Transferase and O-GlcNAcase Activity on Protein Substrates*

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2012-05-01
Abstract: 

Cellular O-linked N-acetylglucosamine (O-GlcNAc) levels are modulated by two enzymes: uridine diphosphate-N-acetyl-D-glucosamine:polypeptidyltransferase (OGT) and O-GlcNAcase (OGA). To quantitatively address the activity of these enzymes on protein substrates, we generated five structurally diverse proteins in both unmodified and O-GlcNAc-modified states. We found a remarkably invariant upper limit for k(cat)/K(m) values for human OGA (hOGA)-catalyzed processing of these modified proteins, which suggests that hOGA processing is driven by the GlcNAc moiety and is independent of the protein. Human OGT (hOGT) activity ranged more widely, by up to 15-fold, suggesting that hOGT is the senior partner in fine tuning protein O-GlcNAc levels. This was supported by the observation that K(m,app) values for UDP-GlcNAc varied considerably (from 1 μM to over 20 μM), depending on the protein substrate, suggesting that some OGT substrates will be nutrient-responsive, whereas others are constitutively modified. The ratios of k(cat)/K(m) values obtained from hOGT and hOGA kinetic studies enable a prediction of the dynamic equilibrium position of O-GlcNAc levels that can be recapitulated in vitro and suggest the relative O-GlcNAc stoichiometries of target proteins in the absence of other factors. We show that changes in the specific activities of hOGT and hOGA measured in vitro on calcium/calmodulin-dependent kinase IV (CaMKIV) and its pseudophosphorylated form can account for previously reported changes in CaMKIV O-GlcNAc levels observed in cells. These studies provide kinetic evidence for the interplay between O-GlcNAc and phosphorylation on proteins and indicate that these effects can be mediated by changes in hOGT and hOGA kinetic activity.

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Supervisor(s): 
Canadian Institutes of Health Research (CIHR)
Natural Sciences and Engineering Research Council of Canada (NSERC)

Limits on Performance and Survival of Juvenile Sockeye Salmon (Oncorhynchus Nerka) During Food Deprivation: A Laboratory-Based Study

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2021-03-24
Abstract: 

Long-distance migrations can be energetically demanding and can represent phases of high mortality. Understanding relationships between body condition and migratory performance can help illuminate the challenges and vulnerabilities of migratory species. Juvenile anadromous sockeye salmon (Oncorhynchus nerka) may migrate over 1000 km from their freshwater nursery habitats to estuary and ocean feeding grounds. During the period corresponding to the seaward migration of sockeye salmon, we held smolts in the laboratory to ask the following: (i) Does non-feeding migration duration influence prolonged swim performance and survival? (ii) What are the relationships between individual body condition and swim performance and survival? Wild sockeye salmon were intercepted during their migration and held without food for up to 61 days to represent the non-feeding freshwater migration and the extremes of poor estuary habitat. We conducted 40 sets of prolonged swim trials on 319 fish from 3 treatment groups that represented entrance to the marine environment on (i) an average,(ii) a delayed and (iii) a severely delayed migration schedule. Experimentally controlled freshwater migration duration did not impact swim performance or survival. Swim performance decreased concomitant with condition factor, where smolts with a Fulton’s condition factor of <0.69 were less likely (<50% probability) to complete the swim test (90 min swim test, at ~0.50 m/s). Survival of salmon smolts in the laboratory was less likely at energy densities of less than 3.47 MJ/kg. Swim performance decreased much sooner than survival, suggesting that swim performance, and therefore condition factor, may be a good indicator of survival of migratory smolts, as fish with reduced swim performance will likely be predated. These two relationships, one more ecologically relevant and one more clinical, help reveal the limits of long-distance migration for juvenile salmon and can be used to determine population-specific starvation risk associated with various freshwater and marine habitat conditions.

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Design, Synthesis, Pharmacokinetics, and Biodistribution of a Series of Bone-Targeting EP4 Receptor Agonist Prodrugs for Treatment of Osteoporosis and Other Bone Conditions

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2021-03-10
Abstract: 

A series of bone-targeting EP4 receptor agonist conjugate pro-drugs were prepared wherein a potent EP4 receptor agonist was bound to a biologically inactive, bisphosphonate-based bone-targeting moiety. Single and double radiolabeled conjugates were synthesized and were shown to be stable in blood, to be rapidly eliminated from the bloodstream and to be effectively taken up into bone in vivo after intravenous dosing. From these preliminary studies a preferred conjugate 4 (also known as C3 and Mes-1007) was selected for follow up bio-distribution and elimination studies. Double radiolabeled conjugate 4 was found to partition largely to liver and bones and both labels were eliminated from liver at the same rate indicating the conjugate was eliminated intact. Quantification of the labels in bones indicated that free EP4 agonist (EP4a)(2a) was released from the bone-bound 4 with a half-time of about 7 days. When dosed orally, the radiolabeled 4 was not absorbed and passed through the gastrointestinal tract essentially unchanged and only traces of radiolabel were found in liver, blood or bones. 4 was found to bind rapidly and completely to powdered bone mineral or to various forms of calcium phosphate to form a stable matrix suitable for implant and that could made into powders or solid forms and be sterilized without decomposition or release of 4. Basic hydrolysis released free EP4 agonist 2a quantitatively from the material.

Document type: 
Article

Functional and Versatile Superhydrophobic Coatings via Stoichiometric Silanization

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2021-02-12
Abstract: 

Superhydrophobic coatings have tremendous potential for applications in different fields and have been achieved commonly by increasing nanoscale roughness and lowering surface tension. Limited by the availability of either ideal nano-structural templates or simple fabrication procedures, the search of superhydrophobic coatings that are easy to manufacture and are robust in real-life applications remains challenging for both academia and industry. Herein, we report an unconventional protocol based on a single-step, stoichiometrically controlled reaction of long-chain organosilanes with water, which creates micro- to nano-scale hierarchical siloxane aggregates dispersible in industrial solvents (as the coating mixture). Excellent superhydrophobicity (ultrahigh water contact angle >170° and ultralow sliding angle <1°) has been attained on solid materials of various compositions and dimensions, by simply dipping into or spraying with the coating mixture. It has been demonstrated that these complete waterproof coatings hold excellent properties in terms of cost, scalability, robustness, and particularly the capability of encapsulating other functional materials (e.g. luminescent dyes).

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Arrays of Microscale Linear Ridges with Self-Cleaning Functionality for the Oxygen Evolution Reaction

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2021-01-06
Abstract: 

Gas management during electrocatalytic water splitting is vital for improving the efficiency of clean hydrogen production. The accumulation of gas bubbles on electrode surfaces prevents electrolyte access and passivates the electrochemically active surface area. Electrode morphologies are sought to assist in the removal of gas from surfaces to achieve higher reaction rates at operational voltages. Herein, regular arrays of linear ridges with specific microscale separations were systematically studied and correlated to the performance of the oxygen evolution reaction (OER). The dimensions of the linear ridges were proportional to the size of the oxygen bubbles, and the mass transfer processes associated with gas evolution at these ridges were monitored using a high speed camera. Characterization of the adhered bubbles prior to detachment enabled the use of empirical methods to determine the volumetric flux of product gas and the bubble residence times. The linear ridges promoted a self-cleaning effect as one bubble would induce neighboring bubbles to simultaneously release from the electrode surfaces. The linear ridges also provided preferential bubble growth sites, which expedited the detachment of bubbles with similar diameters and shorter residence times. The linear ridges enhanced the OER in comparison to planar electrodes prepared by electrodeposition from the same high purity nickel (Ni). Linear ridges with a separation distance of 200 µm achieved nearly a two-fold increase in current density relative to the planar electrode at an operating voltage of 1.8 V (vs Hg/HgO). The electrodes with linear ridges having a separation distance of 200 µm also had the highest sustained current densities over a range of operating conditions for the OER. Self-cleaning surface morphologies could benefit a variety of electrocatalytic gas evolving reactions by improving the efficiency of these processes.

Document type: 
Article

Elucidating the Role of Precursors in Synthesizing Single Crystalline Lithium Niobate Nanomaterials: A Study of Effects of Lithium Precursors on Nanoparticle Quality

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2021-01-25
Abstract: 

A number of solution-based procedures have been realized for the synthesis of lithium niobate (LiNbO3) nanoparticles (NPs). Relatively little is, however, known about the influences of the selection of lithium (Li) precursors on the resulting dimensions, shapes, crystallinity, and purity of the products. A comparative study is provided herein on the role of different Li precursors during the synthesis of LiNbO3 NPs. To the best of our knowledge, this study provides the first systematic comparison of the effects of various Li reagents on the preparation of LiNbO3 NPs through solvothermal processes. This solution-phase approach was tuned by the inclusion of Li precursors that either lacked carbon based anions (e.g., F−, Cl−, Br−, I−, OH−, NO3−, or SO42−) or contained carbon-based anions (e.g., C2H5O−, C2H3OO−, C5H7OO−, or CO32−). All other variables were held constant during the synthesis, such as reaction temperature, solvent, niobium precursor, and surfactants. The results of these studies suggest that the type of Li precursor selected plays an important role in nanoparticle formation, such as through controlling the uniformity, crystallinity, and aggregation of LiNbO3 NPs. The average diameter of the resulting NPs can also vary from ∼30 to ∼830 nm as a function of the Li reagent used in the synthesis. The selection of Li precursors also influences the phase purity of the products. This comparative study on the preparation of crystalline LiNbO3 NPs represents a critical step forward to understand the influences and roles of precursors in the design of synthetic processes for the preparation of a variety of alkali metal niobates (e.g., including NaNbO3 and KNbO3) and crystalline metal oxide-based NPs containing other transition metals (e.g., titanium, tantalum).

Document type: 
Article

Presence of an EML4-ALK Gene Fusion Detected by Microfluidic Chip DNA Hybridization

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2021-01-28
Abstract: 

Non-small cell lung cancer (NSCLC) accounts for ∼80-85% of all lung cancer cases, and the EML4-ALK fusion oncogene is a well-known contributor to NSCLC cases. Expensive methods such as FISH, IHC, and NGS have been used to detect the EML4-ALK fusion oncogene. Here, a cost-effective and facile method of detecting and differentiating an EML4-ALK fusion oncogene from the wild-type gene has been accomplished by DNA hybridization using the microfluidic biochip. First, oligonucleotide probes were confirmed for successful detection of immobilized sense strands. Second, capture of the sense PCR product strands (fusion and WT) and their subsequent detection and differentiation were accomplished. Our proof-of-concept study shows the ability to detect 1% fusion products, among WT ones.

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Production, Purification, and Radiolabeling of the 203Pb/212Pb Theranostic Pair

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2021-02-01
Abstract: 

Background

Lead-212 (212Pb, t1/2 = 10.6 h) and lead-203 (203Pb, t1/2 = 51.9 h) are an element-equivalent, or a matched theranostic radioisotope pair that show great potential for application in targeted radionuclide therapy (TRT) and single-photon emission computed tomography (SPECT), respectively. At TRIUMF we have produced both 203Pb and 212Pb using TRIUMF’s TR13 (13 MeV) and 500 MeV cyclotrons, and subsequently purified and evaluated both radioisotopes using a series of pyridine-modified DOTA analogues in comparison to the commercially available chelates DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and TCMC (1,4,7,10-tetraaza-1,4,7,10-tetra(2-carbamoylmethyl)cyclododecane).

Results

Proton irradiation (12.8 MeV) of natural and enriched thallium-203 (203Tl) targets gave 203Pb saturation yields of 134 ± 25 and 483 ± 3 MBq/μA, respectively. Thorium-228 (228Th, t1/2 = 1.9 y), a by-product of 232Th proton spallation on TRIUMF’s main 500 MeV beamline (beamline 1A, BL1A), was recovered to build a 228Th/212Pb generator with the ability to deliver up to 9–10 MBq of 212Pb daily. Both lead isotopes were purified via solid phase extraction chromatography (Pb resin), and isolated in an acetate form ([203/212Pb]Pb(OAc)2) suitable for direct radiolabeling of chelators and bioconjugates. A series of cyclen-based chelators (herein referred to as DOTA-1Py, -2Py, and -3Py) along with established chelates DOTA and TCMC were evaluated for their ability to complex both 203Pb and 212Pb. All chelates incorporated 212Pb/203Pb efficiently, with higher radiolabeling yields observed for the 212Pb-complexes.

Conclusion

The production of 203Pb and 212Pb was established using TRIUMF 13 MeV and 500 MeV cyclotrons, respectively. Both production methods provided radiometals suitable for subsequent radiolabeling reactions using known and novel chelates. Furthermore, the novel chelate DOTA-3Py may be a good candidate for biomolecule conjugation and further theranostic 212Pb/203Pb studies.

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Free Radicals Formed by H Atom Addition to Allenes as Determined by Muon Spin Spectroscopy

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2020-12-04
Abstract: 

Allyl and vinyl radicals are important intermediates in diverse areas of chemistry, ranging from combustion to syn-thesis. However, questions remain about the competitive formation of these radicals from allenes. Here we present a study of proto-typical allyl and vinyl radicals formed by H atom addition to allenes. They were studied by forming the analogous muonium ad-ducts, since muonium (Mu) behaves as a light isotope of hydrogen, and muoniated species can be characterized by muon spin spec-troscopy. Two techniques were employed: Transverse-Field Muon Spin Resonance (TF-μSR), and Muon Level Crossing Reso-nance (μLCR), which allow for the measurement of muon hyperfine constants (hfcs) and other nuclear hfcs, respectively, and thus aid identification of the formed radicals. TF-μSR has already been used to determine that two radicals are formed by Mu addition to 1,1-dimethylallene, but μLCR techniques were undeveloped at the time of that study, so assignments were based on ESR data of similar allyl and vinyl radicals. We report here the muon spin spectroscopy of multiple radicals detected from positive muon irradi-ation of 1,1-dimethylallene and 1-methoxyallene in solution. The radicals were identified by comparison of muon and proton hfcs with ESR data and the results of DFT calculations. The conclusion is that muonium (and by extension, the H atom) can add to all three carbons of the allene system, albeit with preference for the central carbon.

Document type: 
Article