A strategy was developed to extend the lifetime of an peptide-based substrate for Abl kinase in the cytosolic environment. Small β-turn structures were added to the peptide’s N-terminus to block entry into peptidase catalytic sites. The influence of the size of the β-turn and two covalent cross-linking strategies on the rate of hydrolysis was assessed. The most peptidase-resistant substrate was degraded at a rate of 0.6 pmol mg−1 s−1 and possessed a half-life of 20.3 ± 1.7 min in a Baf/BCR-ABL cytosolic lysate, representing 16- and 40-fold improvements, respectively, over that of a control peptide lacking the β-turn structure. Furthermore, the kcat/KM value of this peptide was 432 μM−1 min−1, a 1.25X increase over the unmodified control, verifying that the added β-turn did not hinder the substrate properties of the peptide. This improved peptide was microinjected into single Baf/BCR-ABL cells and substrate phosphorylation measured. Zero to forty percent of the peptide was phosphorylated in the single cells. In contrast, when the control peptide without a β-turn was loaded into cells, the peptide was too rapidly degraded to detect phosphorylation. This work demonstrates that small β-turn structures can render peptides more resistant to hydrolysis while retaining substrate efficacy and shows that these stabilized peptides have the potential to be of high utility in single-cell enzyme assays.

Regulation of the ubiquitin–proteasome system (UPS) to treat select types of cancer has become a popular area of drug discovery research. The FDA approval of proteasome inhibitors Bortezomib and Carfilzomib in the treatment of multiple myeloma has led to an increased need for chemical reporters capable of detecting and quantifying protein ubiquitination and the activity of members of the UPS including E3 ubiquitin ligases and the proteasome in the tumor cells of the patients. One limitation of peptide-based reporters is their rapid degradation in the cellular environment by cytosolic peptidases. Conversely, β-hairpin “protectides” exhibit a pronounced secondary structure that significantly increases their lifetime under cellular conditions. The goal of this work was to develop a family of novel, ornithine-rich protectides that could act as primary degrons serving as substrates for in vitro ubiquitination. The fluorescent peptide-based reporters were demonstrated to be highly resistant to degradation in multiple myeloma cell lysates. The most stable β-hairpin primary degron, containing a single ornithine residue at the N-terminus, OWRWR [Ac-OWVRVpGO(FAM)WIRQ-NH2], demonstrated rapid ubiquitination kinetics and a 20-fold increase in stability when compared with an unstructured primary degron. A screen of E1 and E3 enzyme inhibitors in cell lysates showed that ubiquitination of OWRWR was significantly impaired by inhibitors of the SCF family of E3 ligases. Furthermore, this is the first report demonstrating the use of an ornithine residue on a primary degron as a ubiquitination site. This study serves as a strong foundation for the development of stable, fluorescent, peptide-based reporters capable of quantifying protein ubiquitination and the enzymatic activity of members of the UPS.

Problem: Opportunities for self-directed learning were missing from our medical school curriculum in general and on our radiology electives specifically. Our objective was to explore the feasibility and benefits of using medical students in the development of our studentcreated teaching files. Approach: In 2018, a website was developed at our institution to support medical student radiology education and create a repository for the online publication of student-developed teaching cases. Students participating in radiology clerkships at our institution had an opportunity to submit case presentations for publication to our online teaching file following peer review. The medical students participated in the peer review process facilitated by the faculty director of radiology undergraduate medical education. The faculty member oversaw the training of new student editors and the development of a peer review guide. Outcomes: The peer review guide included goals of the teaching file project and direction regarding the peer review process. Student editors were trained using the peer review guide in conjunction with individual meetings with the faculty mentor. At twenty-four months, 82 student-developed cases had been published to the online teaching file following medical student peer review. The teaching file had garnered 3884 page views. Next Steps: The medical student-led peer review process meets core competencies in selfdirected learning. The authors plan to explore the application of peer-assisted learning theories to the editing and peer-review process.

Functional magnetic resonance imaging (fMRI) is widely used by researchers to noninvasively monitor brain-wide activity. The traditional assumption of a uniform relationship between neuronal and hemodynamic activity throughout the brain has been increasingly challenged. This relationship is now believed to be impacted by heterogeneously distributed cell types and neurochemical signaling. To date, most cell-type- and neurotransmitter-specific influences on hemodynamics have been examined within the cortex and hippocampus of rodent models, where glutamatergic signaling is prominent. However, neurochemical influences on hemodynamics are relatively unknown in largely GABAergic brain regions such as the rodent caudate putamen (CPu). Given the extensive contribution of CPu function and dysfunction to behavior, and the increasing focus on this region in fMRI studies, improved understanding of CPu hemodynamics could have broad impacts. Here we discuss existing findings on neurochemical contributions to hemodynamics as they may relate to the CPu with special consideration for how these contributions could originate from various cell types and circuits. We hope this review can help inform the direction of future studies as well as interpretation of fMRI findings in the CPu.