A real-time,fluorescence-based assay for Rho-associated protein kinase activity

Inhibitors of Rho-associated protein kinase (ROCK) enzymatic activity have been shown to reduce the invasive phenotype observed in metastatic hepatocellular carcinoma (HCC). We describe the design, synthesis, and evaluation of a direct probe for ROCK activity utilizing a phosphorylation-sensitive sulfonamido-oxine fluorophore, termed Sox. The Sox fluorophore undergoes an increase in fluorescence upon phosphorylation of a proximal amino acid via chelation-enhanced fluorescence (CHEF, ex. = 360 nm and em. = 485 nm), allowing for the direct visualization of the rate of phosphate addition to a peptide substrate over time. Our optimal probe design, ROCK-S1, is capable of sensitively reporting ROCK activity with a limit of detection of 10 pM and a high degree of reproducibility (Z’-factor = 0.6 at 100 pM ROCK2). As a proof-of-principle for high-throughput screening (HTS) we demonstrate the ability to rapidly assess the efficacy of a 78 member, small molecule library against ROCK2 using a robotics platform. We identify two previously unreported ROCK2 inhibitor scaffolds, PHA665752 and IKK16, with IC₅₀ values of 3.6 μM and 247 nM respectively. Lastly, we define conditions for selectively monitoring ROCK activity in the presence of potential off-target enzymes (PKCα, PKA, and PAK) with similar substrate specificities.     

Monitoring real-time curing of epoxies in situ using single-sided NMR

Adhesively bonded joints using epoxy are widely used in aircraft and aerospace structures. Quality control and defect detection during epoxy curing in such applications is critical. We used single-sided nuclear magnetic resonance (NMR) to nondestructively probe and spatially resolve the change in the characteristic NMR relaxation time (T₂) of epoxies during curing on a substrate. Time-dependent T₂ values were fit to a Weibull function to model temporal changes in the NMR measurables. Our results demonstrate that the reduction in molecular mobility of various epoxy/curing agent mixtures occurs more rapidly at the interface than in the bulk. Further use of single-sided NMR to acquire spatially resolved T₂ data will provide a route for elucidatory epoxy curing studies. © 2020 Wiley Periodicals, Inc. J. Polym. Sci. 2020 , 58, 616–623     

Incremental Tuning Up of Fluorous Phenazine Acceptors

In a simple, one‐step direct trifluoromethylation of phenazine with CF₃I we prepared and characterized nine (poly)trifluoromethyl derivatives with up to six CF₃ groups. The electrochemical reduction potentials and gas‐phase electron affinities show a direct, strict linear relation to the number of CF₃ groups, with phenazine(CF₃)₆ reaching a record‐high electron affinity of 3.24 eV among perfluoroalkylated polyaromatics.     

The notion of motion: covariational reasoning and the limit concept

This paper investigates outcomes of building students’ intuitive understanding of a limit as a function's predicted value by examining introductory calculus students’ conceptions of limit both before and after instruction. Students’ responses suggest that while this approach is successful at reducing the common limit equals function value misconception of a limit, new misconceptions emerged in students’ responses. Analysis of students’ reasoning indicates a lack of covariational reasoning that coordinates changes in both x and y may be at the root of the emerging limit reached near x = c misconception. These results suggest that although dynamic interpretations of limit may be intuitive for many students, care must be taken to foster a dynamic conception that is both useful at the introductory calculus level and is in line with the formal notion of limit learned in advanced mathematics. In light of the findings, suggestions for adapting the pedagogical approach used in this study are provided.     

Characterization and Optimization of Injectable In Situ Crosslinked Chitosan-Genipin Hydrogels

In recent years, there has been an increased interest in injectable, in situ crosslinking hydrogels due to their minimally invasive application and ability to conform to their environment. Current in situ crosslinking chitosan hydrogels are either mechanically robust with poor biocompatibility and limited biodegradation due to toxic crosslinking agents or the hydrogels are mechanically weak and undergo biodegradation too rapidly due to insufficient crosslinking. Herein, the authors developed and characterized a thermally-driven, injectable chitosan-genipin hydrogel capable of in situ crosslinking at 37 °C that is mechanically robust, biodegradable, and maintain high biocompatibility. The natural crosslinker genipin is utilized as a thermally-driven, non-toxic crosslinking agent. The chitosan-genipin hydrogel's crosslinking kinetics, injectability, viscoelasticity, swelling and pH response, and biocompatibility against human keratinocyte cells are characterized. The developed chitosan-genipin hydrogels are successfully crosslinked at 37 °C, demonstrating temperature sensitivity. The hydrogels maintained a high percentage of swelling over several weeks before degrading in biologically relevant environments, demonstrating mechanical stability while remaining biodegradable. Long-term cell viability studies demonstrated that chitosan-genipin hydrogels have excellent biocompatibility over 7 days, including during the hydrogel crosslinking phase. Overall, these findings support the development of an injectable, in situ crosslinking chitosan-genipin hydrogel for minimally invasive biomedical applications.