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Immobilization of proteins and enzymes on solid supports has been utilized in a variety of applications, from improved protein stability on supported catalysts in industrial processes to fabrication of biosensors, biochips, and microdevices. A critical requirement for these applications is facile yet stable covalent conjugation between the immobilized and fully active protein and the solid support to produce stable, highly bio-active conjugates. Here, we report functionalization of solid surfaces (gold nanoparticles and magnetic beads) with bio-active proteins using site-specific and biorthogonal labeling and azide-alkyne cycloaddition, a click chemistry. Specifically, we recombinantly express and selectively label calcium-dependent proteins, calmodulin and calcineurin, and cAMP-dependent protein kinase A (PKA) with N-terminal azide-tags for efficient conjugation to nanoparticles and magnetic beads. We successfully immobilized the proteins on to the solid supports directly from the cell lysate with click chemistry, forgoing the step of purification. This approach is optimized to yield low particle aggregation and high levels of protein activity post-conjugation. The entire process enables streamlined workflows for bioconjugation and highly active conjugated proteins.

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Implantable, bioresorbable drug delivery systems offer an alternative to current drug administration techniques; allowing for patient‐tailored drug dosage, while also increasing patient compliance. Mechanistic mathematical modeling allows for the acceleration of the design of the release systems, and for prediction of physical anomalies that are not intuitive and may otherwise elude discovery. This study investigates short‐term drug release as a function of water‐mediated polymer phase inversion into a solid depot within hours to days, as well as long‐term hydrolysis‐mediated degradation and erosion of the implant over the next few weeks. Finite difference methods are used to model spatial and temporal changes in polymer phase inversion, solidification, and hydrolysis. Modeling reveals the impact of non‐uniform drug distribution, production and transport of H⁺ions, and localized polymer degradation on the diffusion of water, drug, and hydrolyzed polymer byproducts. Compared to experimental data, the computational model accurately predicts the drug release during the solidification of implants over days and drug release profiles over weeks from microspheres and implants. This work offers new insight into the impact of various parameters on drug release profiles, and is a new tool to accelerate the design process for release systems to meet a patient specific clinical need.

 

Background: Identification and quantitation of newly synthesized proteins (NSPs) are critical to understanding protein dynamics in development and disease. Probing the nascent proteome can be achieved using non-canonical amino acids (ncAAs) to selectively label the NSPs utilizing endogenous translation machinery, which can then be quantitated with mass spectrometry. We have previously demonstrated that labeling the in vivo murine proteome is feasible via injection of azidohomoalanine (Aha), an ncAA and methionine (Met) analog, without the need for Met depletion. Aha labeling can address biological questions wherein temporal protein dynamics are significant. However, accessing this temporal resolution requires a more complete understanding of Aha distribution kinetics in tissues. Results: To address these gaps, we created a deterministic, compartmental model of the kinetic transport and incorporation of Aha in mice. Model results demonstrate the ability to predict Aha distribution and protein labeling in a variety of tissues and dosing paradigms. To establish the suitability of the method for in vivo studies, we investigated the impact of Aha administration on normal physiology by analyzing plasma and liver metabolomes following various Aha dosing regimens. We show that Aha administration induces minimal metabolic alterations in mice. Conclusions: Our results demonstrate that we can reproducibly predict protein labeling and that the administration of this analog does not significantly alter in vivo physiology over the course of our experimental study. We expect this model to be a useful tool to guide future experiments utilizing this technique to study proteomic responses to stimuli. 

Introduction: Inquiry-based learning is vital to the engineering design process, and most crucially in the laboratory and hands-on settings. Through the model of inquiry-based design, student teams are able to formulate critical inputs to the design process and develop a stronger and more relevant understanding of theoretical principles and their applications. In the junior-level Biotransport laboratory course at Purdue University’s Weldon School of BME, the curriculum utilizes the engineering design process to guide students through three (3) different modules covering different Biotransport phenomena (diffusivity, mass transport, and heat transfer). Students are required to research, conceptualize, and generate hypotheses around a module prompt. Students design, execute, and analyze their own experimental setups to test the hypotheses within an autodidactic peer-learning structure. Methods: A multi-year study was completed spanning from 2014 to 2016, assessing students’ end of course evaluations. With an integration of the flipped lecture into the lab being first implemented in 2015 (prior to 2015, the flipped lecture was a stand-alone course offered outside of the lab sections), the data presented here offers a comparison of student evaluations between these two course structures. Per the student response rates, the sample size for each year was: n=81 (2016); n=60 (2015); n=48 (2014). The surveys were anonymous and a host of questions related to overall course satisfaction, structure, and content were posed. Results: Analysis of the data showed a consistent increase in overall student satisfaction with the course following the implementation of the new structure. The percent of students giving a satisfactory rating or higher for the 2014, 2015 and 2016 course offerings was 79%, 89%, 92%, respectively. This shows a significant difference between 2014 and 2016. Conclusion: The integration of a flipped lecture into the lab successfully improved student satisfaction and self-perceived understanding of course material. This format also improved the delivery of content to students as assessed by maintaining pertinence to the lab topics and clear understanding of learning concepts.