What \u27zyme is it? : A comparison of the effectiveness of various enzyme cleaners on soft contact lenses

A clinical study was conducted to determine the cleaning efficacy of soft contact lens enzymatic cleaners available on the market, including Alcon\u27s new one-drop wonder, SupraClens. Soft contact lenses from each of the four CL groups were coated with a 0.1% artificial lysozyme solution before being enzymatically cleaned by six commonly used protein removers. The treated lenses were dehydrated and analyzed for remaining protein deposits. Allergan\u27 s Ultrazyme was not only able to remove protein from each of the four types of lenses, but was able to dissolve the protein as well. It was also noted the liquid enzyme removed protein as effectively as the tablet forms. The study also evaluated the cost-efficiency of the enzymatic cleaners tested. Alcon\u27s Opti-Zyme enzymatic cleaner and CIBA Vision\u27s Unizyme enzymatic cleaner were found to be the best buys over a years time. Alcon\u27s SupraClens came out to be one of the most expensive, although for contact lens wearers with faster protein build-up times, it might be more beneficial in terms of decreasing discomfort, ocular infections, and allergic reactions

Pharmacokinetic and Biodistribution Assessment of a Near Infrared-Labeled PSMA-Specific Small Molecule in Tumor-Bearing Mice

Prostate cancer is themost frequently diagnosed cancer in men and often requires surgery. Use of near infrared (NIR) technologies to perform image-guided surgery may improve accurate delineation of tumor margins. To facilitate preclinical testing of such outcomes, here we developed and characterized a PSMA-targeted small molecule, YC-27. IRDye 800CW was conjugated to YC-27 or an anti-PSMA antibody used for reference. Human 22Rv1, PC3M-LN4, and/or LNCaP prostate tumor cells were exposed to the labeled compounds. In vivo targeting and clearance properties were determined in tumor-bearing mice. Organs and tumors were excised and imaged to assess probe localization. YC-27 exhibited a dose dependent increase in signal upon binding. Binding specificity and internalization were visualized by microscopy. In vitro and in vivo blocking studies confirmed YC-27 specificity. In vivo, YC-27 showed good tumor delineation and tissue contrast at doses as low as 0.25 nmole. YC-27 was cleared via the kidneys but bound the proximal tubules of the renal cortex and epididymis. Since PSMA is also broadly expressed on the neovasculature of most tumors, we expect YC-27 will have clinical utility for image-guided surgery and tumor resections

Functional Analyses of ABHD17 Enzymes

Post-translational modifications (PTMs) play a crucial role in trafficking proteins for many location-dependent cellular functions. Protein S-acylation describes the addition of long chain fatty acids (predominantly the C16:0 palmitate) to cysteines via a thioester bond. This reversible modification thus allows for controlled regulation of protein membrane tethering during various cellular processes. Indeed, S-palmitoylated proteins include kinases, small GTPases and transmembrane receptors, which function in response to diverse signaling events. It is important to study the enzymes that catalyze this modification since it is involved in many essential pathways. Enzymes such as APT1 and APT2 have thoroughly been studied and shown to depalmitoylate a myriad of S-palmitoylated substrates and the development of selective inhibitors has accelerated the discovery of new substrates. ABHD17 has more recently been shown to depalmitoylate substrates in cell-based studies. However, not much is known about its substrate recognition mechanism and cellular function and my dissertation helps to bridge this gap in knowledge. The first chapter presents a detailed introduction to protein depalmitoylases, their different cellular roles and methods that have been developed to study them. The following chapter focuses on understanding the in vitro activity of ABHD17 and the development of potential inhibitors to study its function. The third chapter outlines a proteomics strategy that can be used to study S-palmitoylation of multiple proteins at a time and was applied to understand how ABHD17 regulates S-palmitoylation in cells. Finally, future experiments are proposed in Chapter 4 to further improve our understanding of how ABHD17 works and what its cellular function is.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/153388/1/mcheusk_1.pd

Ion Mobility and Gas-Phase Covalent Labeling Study of the Structure and Reactivity of Gaseous Ubiquitin Ions Electrosprayed from Aqueous and Denaturing Solutions

Gas-phase ion/ion chemistry was coupled to ion mobility/mass spectrometry analysis to correlate the structure of gaseous ubiquitin to its solution structures with selective covalent structural probes. Collision cross section (CCS) distributions were measured to ensure the ubiquitin ions were not unfolded when they were introduced to the gas phase. Aqueous solutions stabilizing the native state of ubiquitin yielded folded ubiquitin structures with CCS values consistent with previously published literature. Denaturing solutions favored several families of unfolded conformations for most of the charge states evaluated. Gas-phase covalent labeling via ion/ion reactions was followed by collision-induced dissociation of the intact, labeled protein to determine which residues were labeled. Ubiquitin 5+ and 6+ electrosprayed from aqueous conditions were covalently modified preferentially at the lysine 29 and arginine 54 positions, indicating that elements of three-dimensional structure were maintained in the gas phase. On the other hand, most ubiquitin ions produced in denaturing conditions were labeled at various other lysine residues, likely due to the availability of additional sites following methanol- and low-pH-induced unfolding. These data support the conservation of ubiquitin structural elements in the gas phase. The research presented here provides the basis for residue-specific characterization of biomolecules in the gas phase

Galaxy Zoo: the effect of bar-driven fuelling on the presence of an active galactic nucleus in disc galaxies

We study the influence of the presence of a strong bar in disc galaxies which host an active galactic nucleus (AGN). Using data from the Sloan Digital Sky Survey and morphological classifications from the Galaxy Zoo 2 project, we create a volume-limited sample of 19756 disc galaxies at 0.01<z<0.05 which have been visually examined for the presence of a bar. Within this sample, AGN host galaxies have a higher overall percentage of bars (51.8 per cent) than inactive galaxies exhibiting central star formation (37.1 per cent). This difference is primarily due to known effects: that the presence of both AGN and galactic bars is strongly correlated with both the stellar mass and integrated colour of the host galaxy. We control for this effect by examining the difference in AGN fraction between barred and unbarred galaxies in fixed bins of mass and colour. Once this effect is accounted for, there remains a small but statistically significant increase that represents 16 per cent of the average barred AGN fraction. Using the $L_{\rm {[O\,{\small {iii}}]}}$/MBH ratio as a measure of AGN strength, we show that barred AGNs do not exhibit stronger accretion than unbarred AGNs at a fixed mass and colour. The data are consistent with a model in which bar-driven fuelling does contribute to the probability of an actively growing black hole, but in which other dynamical mechanisms must contribute to the direct AGN fuelling via smaller, non-axisymmetric perturbation

Gas-Phase Ion/Ion Chemistry for Structurally Sensitive Probes of Gaseous Protein Ion Structure: Electrostatic and Electrostatic to Covalent Cross-Linking

Intramolecular interactions within a protein are key in maintaining protein tertiary structure and understanding how proteins function. Ion mobility-mass spectrometry (IM-MS) has become a widely used approach in structural biology since it provides rapid measurements of collision cross sections (CCS), which inform on the gas-phase conformation of the biomolecule under study. Gas-phase ion/ion reactions target amino acid residues with specific chemical properties and the modified sites can be identified by MS. In this study, electrostatically reactive, gas-phase ion/ion chemistry and IM-MS are combined to characterize the structural changes between ubiquitin electrosprayed from aqueous and denaturing conditions. The electrostatic attachment of sulfo-NHS acetate to ubiquitin via ion/ion reactions and fragmentation by electron-capture dissociation (ECD) provide the identification of the most accessible protonated sites within ubiquitin as the sulfonate group forms an electrostatic complex with accessible protonated side chains. The protonated sites identified by ECD from the different solution conditions are distinct and, in some cases, reflect the disruption of interactions such as salt bridges that maintain the native protein structure. This agrees with previously published literature demonstrating that a high methanol concentration at low pH causes the structure of ubiquitin to change from a native (N) state to a more elongated A state. Results using gas-phase, electrostatic cross-linking reagents also point to similar structural changes and further confirm the role of methanol and acid in favoring a more unfolded conformation. Since cross-linking reagents have a distance constraint for the two reactive sites, the data is valuable in guiding computational structures generated by molecular dynamics. The research presented here describes a promising strategy that can detect subtle changes in the local environment of targeted amino acid residues to inform on changes in the overall protein structure

Experimental Determination of Activation Energies for Covalent Bond Formation via Ion/Ion Reactions and Competing Processes

The combination of ion/ion chemistry with commercially available ion mobility/mass spectrometry systems has allowed rich structural information to be obtained for gaseous protein ions. Recently, the simple modification of such an instrument with an electrospray reagent source has allowed three-dimensional gas-phase interrogation of protein structures through covalent and noncovalent interactions coupled with collision cross section measurements. However, the energetics of these processes have not yet been studied quantitatively. In this work, previously developed Monte Carlo simulations of ion temperatures inside traveling wave ion guides are used to characterize the energetics of the transition state of activated ubiquitin cation/sulfo-benzoyl-HOAt reagent anion long-lived complexes formed via ion/ion reactions. The ΔH‡ and ΔS‡ of major processes observed from collisional activation of long-lived gas-phase ion/ion complexes, namely collision induced unfolding (CIU), covalent bond formation, or neutral loss of the anionic reagent via intramolecular proton transfer, were determined. Covalent bond formation via ion/ion complexes was found to be significantly lower energy compared to unfolding and bond cleavage. The ΔG‡ values of activation of all three processes lie between 55 and 75 kJ/mol, easily accessible with moderate collisional activation. Bond formation is favored over reagent loss at lower activation energies, whereas reagent loss becomes competitive at higher collision energies. Though the ΔG‡ values between CIU of a precursor ion and covalent bond formation of its ion/ion product complex are comparable, our data suggest covalent bond formation does not require extensive isomerization