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Conformationally strained trans- cyclooctenes (TCOs) engage in bioorthogonal reactions with tetrazines with second order rate constants that can exceed 10 6 M -1 s -1 . The goal of this study was to provide insight into the stability of TCO reagents and to develop methods for stabilizing TCO reagents for long-term storage. The radical inhibitor Trolox suppresses TCO isomerization under high thiol concentrations and TCO shelf-life can be greatly extended by protecting them as stable Ag(I) metal complexes. 1 H NMR studies show that Ag-complexation is thermodynamically favorable but the kinetics of dissociation are very rapid, and TCO•AgNO 3 complexes are immediately dissociated upon addition of NaCl which is present in high concentration in cell media. The AgNO 3 complex of a highly reactive s-TCO-TAMRA conjugate was shown to label a protein-tetrazine conjugate in live cells with faster kinetics and similar labeling yield relative to a 'traditional' TCO-TAMRA conjugate.

The nematode Caenorhabditis elegans uses simple building blocks from primary metabolism and a strategy of modular assembly to build a great diversity of signaling molecules, the ascarosides, which function as a chemical language in this model organism. In the ascarosides, the dideoxysugar ascarylose serves as a scaffold to which diverse moieties from lipid, amino acid, neurotransmitter, and nucleoside metabolism are attached. However, the mechanisms that underlie the highly specific assembly of ascarosides are not understood. We show that the acyl-CoA synthetase ACS-7, which localizes to lysosome-related organelles, is specifically required for the attachment of different building blocks to the 4'-position of ascr#9. We further show that mutants lacking lysosome-related organelles are defective in the production of all 4'-modified ascarosides, thus identifying the waste disposal system of the cell as a hotspot for ascaroside biosynthesis., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Environmental conditions experienced during animal development are thought to have sustained impact on maturation and adult lifespan. Here we show that in the model organism C. elegans developmental rate and adult lifespan depend on larval population density, and that this effect is mediated by excreted small molecules. By using the time point of first egg laying as a marker for full maturity, we found that wildtype hermaphrodites raised under high density conditions developed significantly faster than animals raised in isolation. Population density-dependent acceleration of development (Pdda) was dramatically enhanced in fatty acid β-oxidation mutants that are defective in the biosynthesis of ascarosides, small-molecule signals that induce developmental diapause. In contrast, Pdda is abolished by synthetic ascarosides and steroidal ligands of the nuclear hormone receptor DAF-12. We show that neither ascarosides nor any known steroid hormones are required for Pdda and that another chemical signal mediates this phenotype, in part via the nuclear hormone receptor NHR-8. Our results demonstrate that C. elegans development is regulated by a push-pull mechanism, based on two antagonistic chemical signals: chemosensation of ascarosides slows down development, whereas population-density dependent accumulation of a different chemical signal accelerates development. We further show that the effects of high larval population density persist through adulthood, as C. elegans larvae raised at high densities exhibit significantly reduced adult lifespan and respond differently to exogenous chemical signals compared to larvae raised at low densities, independent of density during adulthood. Our results demonstrate how inter-organismal signaling during development regulates reproductive maturation and longevity.

Inhibition of β-secretase BACE1 is considered one of the most promising approaches for treating Alzheimer's disease. Several structurally distinct BACE1 inhibitors have been withdrawn from development after inducing ocular toxicity in animal models, but the target mediating this toxicity has not been identified. Here we use a clickable photoaffinity probe to identify cathepsin D (CatD) as a principal off-target of BACE1 inhibitors in human cells. We find that several BACE1 inhibitors blocked CatD activity in cells with much greater potency than that displayed in cell-free assays with purified protein. Through a series of exploratory toxicology studies, we show that quantifying CatD target engagement in cells with the probe is predictive of ocular toxicity in vivo. Taken together, our findings designate off-target inhibition of CatD as a principal driver of ocular toxicity for BACE1 inhibitors and more generally underscore the power of chemical proteomics for discerning mechanisms of drug action.

Bioorthogonal reactions, including the strain-promoted azide-alkyne cycloaddition (SPAAC) and inverse electron demand Diels-Alder (iEDDA) reactions, have become increasingly popular for live-cell imaging applications. However, the stability and reactivity of reagents has never been systematically explored in the context of a living cell. Here we report a universal, organelle-targetable system based on HaloTag protein technology for directly comparing bioorthogonal reagent reactivity, specificity, and stability using clickable HaloTag ligands in various subcellular compartments. This system enabled a detailed comparison of the bioorthogonal reactions in live cells and informed the selection of optimal reagents and conditions for live-cell imaging studies. We found that the reaction of sTCO with monosubstituted tetrazines is the fastest reaction in cells; however, both reagents have stability issues. To address this, we introduced a new variant of sTCO, Ag-sTCO, which has much improved stability and can be used directly in cells for rapid bioorthogonal reactions with tetrazines. Utilization of Ag complexes of conformationally strained trans-cyclooctenes should greatly expand their usefulness especially when paired with less reactive, more stable tetrazines.

The development and lifespan of C. elegans are controlled by the nuclear hormone receptor DAF-12, an important model for the vertebrate vitamin D and liver X receptors. As with its mammalian homologues, DAF-12 function is regulated by bile acid-like steroidal ligands; however, tools for investigating their biosynthesis and function in vivo are lacking. A flexible synthesis for DAF-12 ligands and masked ligand derivatives that enable precise temporal control of DAF-12 function was developed. For ligand masking, photocleavable amides of 5-methoxy-N-methyl-2-nitroaniline (MMNA) were introduced. MMNA-masked ligands are bioavailable and after incorporation into the worm, brief UV irradiation can be used to trigger the expression of DAF-12 target genes and initiate development from dauer larvae into adults. The in vivo release of DAF-12 ligands and other small-molecule signals by using photocleavable MMNA-masked ligands will enable functional studies with precise spatial and temporal resolution., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Small-molecule ligands of nuclear hormone receptors (NHRs) govern the transcriptional regulation of metazoan development, cell differentiation, and metabolism. However, the physiological ligands of many NHRs remain poorly characterized, primarily due to lack of robust analytical techniques. Using comparative metabolomics, we identified endogenous steroids that act as ligands of the C. elegans NHR, DAF-12, a vitamin D and liver X receptor homolog regulating larval development, fat metabolism, and lifespan. The identified molecules feature unexpected chemical modifications and include only one of two DAF-12 ligands reported earlier, necessitating a revision of previously proposed ligand biosynthetic pathways. We further show that ligand profiles are regulated by a complex enzymatic network, including the Rieske oxygenase DAF-36, the short-chain dehydrogenase DHS-16, and the hydroxysteroid dehydrogenase HSD-1. Our results demonstrate the advantages of comparative metabolomics over traditional candidate-based approaches and provide a blueprint for the identification of ligands for other C. elegans and mammalian NHRs., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In the model organism Caenorhabditis elegans, a family of endogenous small molecules, the ascarosides function as key regulators of developmental timing and behavior that act upstream of conserved signaling pathways. The ascarosides are based on the dideoxysugar ascarylose, which is linked to fatty-acid-like side chains of varying lengths derived from peroxisomal β-oxidation. Despite the importance of ascarosides for many aspects of C. elegans biology, knowledge of their structures, biosynthesis, and homeostasis remains incomplete. We used an MS/MS-based screen to profile ascarosides in C. elegans wild-type and mutant metabolomes, which revealed a much greater structural diversity of ascaroside derivatives than previously reported. Comparison of the metabolomes from wild-type and a series of peroxisomal β-oxidation mutants showed that the enoyl CoA-hydratase MAOC-1 serves an important role in ascaroside biosynthesis and clarified the functions of two other enzymes, ACOX-1 and DHS-28. We show that, following peroxisomal β-oxidation, the ascarosides are selectively derivatized with moieties of varied biogenetic origin and that such modifications can dramatically affect biological activity, producing signaling molecules active at low femtomolar concentrations. Based on these results, the ascarosides appear as a modular library of small-molecule signals, integrating building blocks from three major metabolic pathways: carbohydrate metabolism, peroxisomal β-oxidation of fatty acids, and amino acid catabolism. Our screen further demonstrates that ascaroside biosynthesis is directly affected by nutritional status and that excretion of the final products is highly selective., (© 2012 American Chemical Society)

Objective: To verify the role of haptoglobin, a major acute-phase reactant protein in cattle, as a marker to identify health/disease status in cattle and further assess its potential in improving food safety., Sample Population: Serum samples from various cattle groups: clinically normal cattle comprising steers (n = 157) and culled dairy cows (n = 92) before death (antemortem [AM]); retained carcasses (n = 57) railed off the line during postmortem (PM) inspection; and apparently AM normal culled dairy cows (n = 57)., Procedure: Efficacy of the simplified monoclonal antibody-based enzyme immunoassay was established by comparing results of haptoglobin tests performed independently on aliquots of serum samples by 3 laboratories., Results: Haptoglobin concentration was significantly (P< or = 0.0001) different between the PM retained carcass group (n = 57) and the AM steer (n = 157) and culled dairy cow (n = 92) groups. In addition, haptoglobin concentration in AM steers (n = 157) and culled dairy cows (n = 92) was significantly (P < or = 0.0012) different, possibly reflecting a higher percentage of underlying pathologic or inflammatory conditions in animals of the latter group. Evaluation in 3 laboratories of sera from a group of culled dairy cows (n = 57), each laboratory performing a different test procedure, indicated that correlation of haptoglobin concentrations was good between the reported test procedure and the unmodified test and the classical hemoglobin-binding assay that measures peroxidase activity., Conclusion: Haptoglobin determination is effective in identifying diseased and healthy cattle. It may be a potentially important tool for application at the farm and slaughterhouse as an aid in improving food safety.

Improved enzyme immunoassay (EIA) procedures achieved by incorporating sodium azide during predilution of serum samples in a solid-phase EIA for the detection of anti-Toxoplasma antibody in swine using a peroxidase conjugate and in all washes of a bovine brucellosis rapid card test EIA using alkaline phosphatase conjugate are reported. Without this modification, substantial background interference was encountered that showed direct correlation with the degree of hemolysis of the serum samples. Anti-Toxoplasma gondii antibody-negative samples, separated by subjective groupings based on degree of hemolysis, into "clear", "slight", and "gross/total" samples, had a mean +/- standard deviation of 0.150 +/- 0.072, 0.187 +/- 0.105, and 0.232 +/- 0.108, respectively. The incorporation of sodium azide during the initial step of serum dilution dramatically eliminated the background, giving a mean +/- standard deviation of 0.079 +/- 0.029, 0.076 +/- 0.022, and 0.081 +/- 0.029, respectively. The level of endogenous peroxidase activity, a possible factor for this nonspecific interference, was considerably elevated in some of the swine sera. The clear, slight, and gross/total categories had relative levels of 1%, 2%, and 51% peroxidase activity compared to the conjugate peroxidase activity of 100%. Whereas sodium azide could be used only in sample predilution in the swine toxoplasmosis peroxidase-conjugate test, in the bovine brucellosis alkaline phosphatase-conjugate card test it could be used in all wash cycles. Many brucellosis card test results were visually uninterpretable because of significant background color when the manufacturer's wash reagent was used. The substitution of a wash reagent containing sodium azide eliminated background color, giving a visually unambiguous test.