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Motivation Cell-type-specific surface proteins can be exploited as valuable markers for a range of applications including immunophenotyping live cells, targeted drug delivery and in vivo imaging. Despite their utility and relevance, the unique combination of molecules present at the cell surface are not yet described for most cell types. A significant challenge in analyzing ‘omic’ discovery datasets is the selection of candidate markers that are most applicable for downstream applications. Results Here, we developed GenieScore, a prioritization metric that integrates a consensus-based prediction of cell surface localization with user-input data to rank-order candidate cell-type-specific surface markers. In this report, we demonstrate the utility of GenieScore for analyzing human and rodent data from proteomic and transcriptomic experiments in the areas of cancer, stem cell and islet biology. We also demonstrate that permutations of GenieScore, termed IsoGenieScore and OmniGenieScore, can efficiently prioritize co-expressed and intracellular cell-type-specific markers, respectively. Availability and implementation Calculation of GenieScores and lookup of SPC scores is made freely accessible via the SurfaceGenie web application: www.cellsurfer.net/surfacegenie. Contact Rebekah.gundry@unmc.edu Supplementary information Supplementary data are available at Bioinformatics online.

Autoimmune diabetes is characterized by chronic inflammation of pancreatic islets which ultimately destroys insulin-producing β-cells. In the islet, elevated proinflammatory cytokines lead to increased transcription of inducible nitric oxide synthase (iNOS) and its downstream product, nitric oxide (NO). Elevated levels of NO for prolonged periods lead to β-cell apoptosis. The exact causes of autoimmune diabetes are not well defined and, although genetic predisposition is a factor, low concordance rates of autoimmune diabetes among monozygotic twins indicates a role for epigenetic factors. Recent studies suggest that the bromodomain and extraterminal domain (BET) family of epigenetic regulatory proteins contribute to the onset and progression of autoimmune diabetes. BET proteins are epigenetic readers of sites of lysine acetylation found commonly on histones and have emerged as promising drug targets for a wide variety of diseases. We hypothesize that BET proteins increase proinflammatory cytokine-induced transcription in β-cells leading to β-cell damage and that selective inhibition of BET bromodomains with small molecules will prevent or reverse the progression of autoimmune diabetes. Here, we show that BET bromodomain inhibitors decrease the β-cell response to proinflammatory cytokines in an insulinoma β-cell line (INS832/13) and primary rat islets. BET inhibitors attenuated cytokine-induced transcription of inflammatory mediators, including iNOS. This decreased transcription is mediated through inhibition of the NF-κB signaling pathway by BET inhibitors. Using knockdown of individual BET proteins in a β-cell line, we identified the importance of individual BET family members in the altered transcriptional profile characteristic of autoimmune diabetes. This work uncovers important mechanisms of disease onset and progression of autoimmune diabetes, laying the groundwork for more targeted treatments with drug-like small molecules. Disclosure J. Nord: None. S. Wynia-smith: None. B. C. Smith: None. R. Jones lipinski: None. Funding American Diabetes Association (1-18-IBS-068 to B.C.S.); National Institutes of Health (R01DK119359, T32HL134643)

Peptide cleanup is essential for the removal of contaminating substances that may be introduced during sample preparation steps in bottom-up proteomic workflows. Recent studies have described benefits of carboxylate-modified paramagnetic particles over traditional reversed-phase methods for detergent and polymer removal, but challenges with reproducibility have limited the widespread implementation of this approach among laboratories. To overcome these challenges, the current study systematically evaluated key experimental parameters regarding the use of carboxylate-modified paramagnetic particles and determined those that are critical for maximum performance and peptide recovery and those for which the protocol is tolerant to deviation. These results supported the development of a detailed, easy-to-use standard operating protocol, termed SP2, which can be applied to remove detergents and polymers from peptide samples while concentrating the sample in solvent that is directly compatible with typical LC-MS workflows. We demonstrate that SP2 can be applied to phosphopeptides and glycopeptides, and that the approach is compatible with robotic liquid handling for automated sample processing. Altogether, the results of this study and accompanying detailed operating protocols for both manual and automated processing are expected to facilitate reproducible implementation of SP2 for various proteomics applications and will especially benefit core or shared resource facilities where unknown or unexpected contaminants may be particularly problematic.

Non-steroidal anti-inflammatory drugs (NSAIDs) are a powerful class of inhibitors targeting two isoforms of the family of cyclooxygenase enzymes (COX-1 and COX-2). While NSAIDs are widely used in the management of pain, in particular as a treatment for osteo- and rheumatoid arthritis, their long-term use has been associated with numerous on- and off-target effects. As the carboxylic acid moiety present in common NSAIDs is responsible for some of their adverse effects, but is not required for their anti-inflammatory activity, we sought to mask this group through direct coupling to glucosamine, which is thought to prevent cartilage degradation. We report herein the conjugation of commonly prescribed NSAIDs to glucosamine hydrochloride and the use of molecular docking to show that addition of the carbohydrate moiety to the parent NSAID can enhance binding in the active site of COX-2. In a preliminary, in vitro screening assay, the diclofenac-glucosamine bioconjugate exhibited 10-fold greater activity toward COX-2, making it an ideal candidate for future in vivo studies. Furthermore, in an intriguing result, we observed that the mefenamic acid-glucosamine bioconjugate displayed enhanced activity toward COX-1 rather than COX-2.

The mammalian genome contains approximately 200 phosphatases that are responsible for catalytically removing phosphate groups from proteins. In this review, we discuss dual specificity phosphatase 5 (DUSP5). DUSP5 belongs to the dual specificity phosphatase (DUSP) family, so named after the family members' abilities to remove phosphate groups from serine/threonine and tyrosine residues. We provide a comparison of DUSP5's structure to other DUSPs and, using molecular modeling studies, provide an explanation for DUSP5's mechanistic interaction and specificity toward phospho-extracellular regulated kinase, its only known substrate. We also discuss new insights from molecular modeling studies that will influence our current thinking of mitogen-activated protein kinase signaling. Finally, we discuss the lessons learned from identifying small molecules that target DUSP5, which might benefit targeting efforts for other phosphatases. © 2017 American Physiological Society. Compr Physiol 7:1449-1461, 2017.

Caenorhabditis elegans produces a complex mixture of ascaroside pheromones to control its development and behavior. Acyl-CoA oxidases, which participate in β-oxidation cycles that shorten the side chains of the ascarosides, regulate the mixture of pheromones produced. Here, we use CRISPR-Cas9 to make specific nonsense and missense mutations in acox genes and determine the effect of these mutations on ascaroside production in vivo. Ascaroside production in acox-1.1 deletion and nonsense strains, as well as a strain with a missense mutation in a catalytic residue, confirms the central importance of ACOX-1.1 in ascaroside biosynthesis and suggests that ACOX-1.1 functions in part by facilitating the activity of other acyl-CoA oxidases. Ascaroside production in an acox-1.1 strain with a missense mutation in an ATP-binding site at the ACOX-1.1 dimer interface suggests that ATP binding is important for the enzyme to function in ascaroside biosynthesis in vivo. Ascaroside production in strains with deletion, nonsense, and missense mutations in other acox genes demonstrates that ACOX-1.1 works with ACOX-1.3 in processing ascarosides with 7-carbon side chains, ACOX-1.4 in processing ascarosides with 9- and 11-carbon side chains, and ACOX-3 in processing ascarosides with 13- and 15-carbon side chains. It also shows that ACOX-1.2, but not ACOX-1.1, processes ascarosides with 5-carbon ω-side chains. By modeling the ACOX structures, we uncover characteristics of the enzyme active sites that govern substrate preferences. Our work demonstrates the role of specific acyl-CoA oxidases in controlling the length of ascaroside side chains and thus in determining the mixture of pheromones produced by C. elegans.

Caenorhabditis elegans produces ascaroside pheromones to control its development and behavior. Even minor structural differences in the ascarosides have dramatic consequences for their biological activities. Here, we identify a mechanism that enables C. elegans to dynamically tailor the fatty-acid side chains of the indole-3-carbonyl (IC)-modified ascarosides it has produced. In response to starvation, C. elegans uses the peroxisomal acyl-CoA synthetase ACS-7 to activate the side chains of medium-chain IC-ascarosides for β-oxidation involving the acyl-CoA oxidases ACOX-1.1 and ACOX-3. This pathway rapidly converts a favorable ascaroside pheromone that induces aggregation to an unfavorable one that induces the stress-resistant dauer larval stage. Thus, the pathway allows the worm to respond to changing environmental conditions and alter its chemical message without having to synthesize new ascarosides de novo. We establish a new model for biosynthesis of the IC-ascarosides in which side-chain β-oxidation is critical for controlling the type of IC-ascarosides produced., eLife digest Small roundworms such as Caenorhabditis elegans release chemical signals called ascarosides in order to communicate with other worms of the same species. Using the ascarosides, the worm can tell its friends, for example, how crowded the neighborhood is and whether there is enough food. The ascarosides thus help the worms in the population decide whether the neighborhood is good – meaning they should hang around, eat, and make babies – or whether the neighborhood is bad. If so, the worms should develop into a larval stage specialized for dispersal that will allow them to find a better neighborhood. Roundworms make the ascarosides by attaching a long chemical ‘side chain’ to an ascarylose sugar. Further chemical modifications allow the worms to produce different signals. In general, to signal a good neighborhood, worms attach a structure called an indole group to the ascarosides. To signal a bad neighborhood, worms make the side chain very short. But how does a worm control which ascarosides it makes? Zhou, Wang et al. now show that C. elegans can change the meaning of its chemical message by modifying the ascarosides that it has already produced instead of making new ones from scratch. Specifically, as their neighborhood runs out of food, C. elegans can use an enzyme called ACS-7 to initiate the shortening of the side chains of indole-ascarosides. The worm can thus change a favorable ascaroside signal that causes the worms to group together into an unfavorable ascaroside signal that causes the worms to enter their dispersal stage. Although Zhou, Wang et al. have focused on chemical communication in C. elegans, the findings could easily apply to the many other species of roundworm that produce ascarosides. Knowing how worms communicate will help us to understand how worms respond to their environment. This knowledge could potentially be used to interfere with the lifecycles and survival of parasitic worm species that harm health and crops.