Background: Cdc14 phosphatases help control mitosis by dephosphorylating sites (Ser/Thr-Pro) targeted by cyclin-dependent kinases (Cdks). Results: Cdc14 family phosphatases strongly prefer phosphoserine over phosphothreonine at Cdk sites. Conclusion: By discriminating among Cdk sites, Cdc14 may participate in setting the order and timing of Cdk substrate dephosphorylation. Significance: Mechanisms governing the timing of Cdk site dephosphorylation are crucial for proper coordination of late mitotic events.

Lipid droplets (LDs) provide a reservoir for triacylglycerol storage and are a central hub for fatty acid trafficking and signaling in cells. Lipolysis promotes mitochondrial biogenesis and oxidative metabolism via a SIRT1/PGC-1a/PPARa-dependent pathway through an unknown mechanism. Herein, we identify that monounsaturated fatty acids (MUFAs) allosterically activate SIRT1 toward select peptide-substrates such as PGC-1a. MUFAs enhance PGC-1a/ PPARa signaling and promote oxidative metabolism in cells and animal models in a SIRT1-dependent manner. Moreover, we characterize the LD protein perilipin 5 (PLIN5), which is known to enhance mitochondrial biogenesis and function, to be a fattyacid-binding protein that preferentially binds LDderived monounsaturated fatty acids and traffics them to the nucleus following cAMP/PKA-mediated lipolytic stimulation. Thus, these studies identify the first-known endogenous allosteric modulators of SIRT1 and characterize a LD-nuclear signaling axis that underlies the known metabolic benefits of MUFAs and PLIN5.

Managing student needs for effective learning in a large-enrollment, introductory organic chemistry course can be a challenging task. Because instructor time is at a premium, it is imperative to find resources that engage the students in active learning and provide them with feedback about their understanding of course content. Appropriately designed online homework systems can provide this level of engagement. Here we describe our experience with using the Sapling Learning online homework system in an organic chemistry course for prepharmacy majors. Based on the literature describing the effectiveness of online homework in general chemistry and other courses, we hypothesized that student engagement in the online homework would be associated with students’ performance in the course, but that studying with the textbook problems would provide a greater benefit because the material was more sophisticated. We found that engagement in using the system was positively correlated with course grade, and that student perceptions of the system were overwhelmingly positive. Surprisingly, we also found that spending more time studying with the problems in the textbook was not associated with significantly better performance than using the online homework system alone. We speculate that this is because the immediate feedback given by the online system more effectively reinforces the topics. Furthermore, we found that despite the perceived utility of the system and its relationship to final course grades, students still seemed to require an extra-credit incentive to incorporate the use of the system into their study habits. This case study suggests that learning reinforcement through real-time feedback and extra credit (or other points-based) incentives to motivate participation are important components of achieving student benefits from an online homework system.

Background The venoms of predators such as spiders, scorpions, cone snails, sea anemones, and snakes, have been an excellent source of pharmacological diversity for drug discovery and as pharmacological tools for elucidating the structure, function, and physiological properties of ion channels. Here we describe the first known peptide antagonist of the nociceptor ion channel transient receptor potential ankyrin 1 (TRPA1). Results We constructed a recombinant cDNA library encoding ∼100 diverse GPI-anchored peptide toxins (t-toxins) derived from spider venoms and screened this library by co-expression in Xenopus oocytes with TRPA1. This screen resulted in identification of protoxin-I (ProTx-I), a 35-residue peptide from the venom of the Peruvian green-velvet tarantula, Thrixopelma pruriens, as the first known high-affinity peptide TRPA1 antagonist. Interestingly, ProTx-I was previously identified as an antagonist of voltage-gated sodium (NaV) channels. To identify the surfaces of ProTx-I by which it binds to these distinct ion channel types, we constructed a t-toxin library of ProTx-I alanine-scanning mutants and screened this library against NaV1.2 and TRPA1. This revealed distinct partially overlapping surfaces of ProTx-I by which it binds to these two ion channels, and whose specific chemical features explain its higher affinity for NaV1.2 than for TRPA1. Importantly, this mutagenesis yielded two novel ProTx-I variants that are only active against either TRPA1or NaV1.2, but not both. By testing its activity against chimeric channels, we identified the extracellular loops of the TRPA1 S1-S4 gating domain as the ProTx-I binding site. Conclusions These studies establish screening of t-toxin libraries of native and mutated toxins, which we term “toxineering”, as a generally applicable method for isolation of novel ion channel modifiers and for design of ion channel modifiers with altered target selectivity. They also suggest that ProTx-I will be a valuable pharmacological reagent for addressing the biophysical mechanisms of TRPA1 gating, the physiology and pathophysiology of TRPA1 function in nociceptors, and for potential clinical application in the context of pain and inflammation.