Sixty years from discovery to solution: crystal structure of bovine liver catalase form III

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86968/1/S0907444911024486.pd

Polymers as Heterogeneous Growth Promoters for Protein Crystallization.

Obtaining suitable single crystals for X-ray diffraction remains a major bottleneck in the structural characterization of new compounds. Nowhere is this more apparent than in structural biology where the challenges of crystal growth are considerable. In cases where traditional growth methods fail to yield suitable protein crystals, the target is often reconsidered, rather than the crystallization approach. In the studies described in this thesis, polymer-induced heteronucleation (PIHn), a powerful technique well-established in the realm of small molecule crystallization, is tailored to meet the challenges inherent to protein crystallization. Based on the premise that polymers designed for small molecule crystallization may not be best suited to facilitating biomolecule crystallization in PIHn, the heteronucleant composition in PIHn was modified by introducing less hydrophobic crosslinking agents, which led to increased crystal size and new form access. The polymers were additionally redeployed in a variety of formats suitable for sitting and hanging-drop vapor diffusion crystallizations. The power and utility of these advances was demonstrated through the application of PIHn to bovine liver catalase (BLC) and concanavalin A (conA). For BLC, this approach led to increased crystal size and ultimately the first X-ray crystal structure of a crystal form of BLC that was previously too small for structural characterization. Additionally, in the presence of the heteronucleants two novel forms of conA were discovered and analyzed using single crystal X-ray diffraction. Forming single crystals does not ensure structural characterization. Crystal harvesting often leads to crystal damage for delicate protein crystals, which can be amplified when using PIHn due to the propensity for crystals to adhere to the heteronucleants. This led to a growth strategy to allow nucleation on crystal mounts coated with polymer heteronuclei. In order to achieve selective control of nucleation, the surface chemistry of the crystal mount was modified using different heteronuclei. This method successfully controlled crystal growth for conA and BLC. A conA crystal grown directly on the crystal mount was characterized using X-ray diffraction, illustrating that the quality of the crystal was not negatively impacted by the presence of the functionalized crystal mount.Ph.D.BiochemistryPolymer chemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/127205/2/3519586.pd

Structural Modifications of Polyethylenimine to Control Drug Loading and Release Characteristics of Amorphous Solid Dispersions

Crystalline drugs with low solubility have the potential to benefit from delivery in the amorphous form. The polymers used in amorphous solid dispersions (ASDs) influence their maximum drug loading, solubility, dissolution rate, and physical stability. Herein, the influence of hydrophobicity of crosslinked polyethylenimine (PEI) is investigated for the delivery of the BCS class II nonsteroidal anti-inflammatory drug flufenamic acid (ffa). Several synthetic variables for crosslinking PEI with terephthaloyl chloride were manipulated: solvent, crosslinking density, reactant concentration, solution viscosity, reaction temperature, and molecular weight of the hyperbranched polymer. Benzoyl chloride was employed to cap amine groups to increase the hydrophobicity of the crosslinked materials. Amorphous deprotonated ffa was present in all ASDs; however, the increased hydrophobicity and reduced basicity from benzoyl functionalization led to a combination of amorphous deprotonated ffa and amorphous neutral ffa in the materials at high drug loadings (50 and 60 wt %). All ASDs demonstrated enhanced drug delivery in acidic media compared to crystalline ffa. Physical stability testing showed no evidence of crystallization after 29 weeks under various relative humidity conditions. These findings motivate the broadening of polymer classes employed in ASD formation to include polymers with very high functional group concentrations to enable loadings not readily achieved with existing polymers

Safety and outcomes of intravenous thrombolytic therapy in ischemic stroke patients with COVID-19: CASCADE initiative

BACKGROUND: There is little information regarding the safety of intravenous tissue plasminogen activator (IV-tPA) in patients with stroke and COVID-19. METHODS: This multicenter study included consecutive stroke patients with and without COVID-19 treated with IV-tPA between February 18, 2019, to December 31, 2020, at 9 centers participating in the CASCADE initiative. Clinical outcomes included modified Rankin Scale (mRS) at hospital discharge, in-hospital mortality, the rate of hemorrhagic transformation. Using Bayesian multiple regression and after adjusting for variables with significant value in univariable analysis, we reported the posterior adjusted odds ratio (OR, with 95% Credible Intervals [CrI]) of the main outcomes. RESULTS: A total of 545 stroke patients, including 101 patients with COVID-19 were evaluated. Patients with COVID-19 had a more severe stroke at admission. In the study cohort, 85 (15.9%) patients had a hemorrhagic transformation, and 72 (13.1%) died in the hospital. After adjustment for confounding variables, discharge mRS score ≥2 (OR: 0.73, 95% CrI: 0.16, 3.05), in-hospital mortality (OR: 2.06, 95% CrI: 0.76, 5.53), and hemorrhagic transformation (OR: 1.514, 95% CrI: 0.66, 3.31) were similar in COVID-19 and non COVID-19 patients. High-sensitivity C reactive protein level was a predictor of hemorrhagic transformation in all cases (OR:1.01, 95%CI: 1.0026, 1.018), including those with COVID-19 (OR:1.024, 95%CI:1.002, 1.054). CONCLUSION: IV-tPA treatment in patients with acute ischemic stroke and COVID-19 was not associated with an increased risk of disability, mortality, and hemorrhagic transformation compared to those without COVID-19. IV-tPA should continue to be considered as the standard of care in patients with hyper acute stroke and COVID-19