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Cone snails use separately evolved venoms for prey capture and defence. While most use a harpoon for prey capture, the Gastridium clade that includes the well-studied Conus geographus and Conus tulipa, have developed a net hunting strategy to catch fish. This unique feeding behaviour requires secretion of "nirvana cabal" peptides to dampen the escape response of targeted fish allowing for their capture directly by mouth. However, the active components of the nirvana cabal remain poorly defined. In this study, we evaluated the behavioural effects of likely nirvana cabal peptides on the teleost model, Danio rerio (zebrafish). Surprisingly, the conantokins (NMDA receptor antagonists) and/or conopressins (vasopressin receptor agonists and antagonists) found in C. geographus and C. tulipa venom failed to produce a nirvana cabal-like effect in zebrafish. In contrast, low concentrations of the non-competitive adrenoceptor antagonist ρ-TIA found in C. tulipa venom (EC50 = 190 nM) dramatically reduced the escape response of zebrafish larvae when added directly to aquarium water. ρ-TIA inhibited the zebrafish α1-adrenoceptor, confirming ρ-TIA has the potential to reverse the known stimulating effects of norepinephrine on fish behaviour. ρ-TIA may act alone and not as part of a cabal, since it did not synergise with conopressins and/or conantokins. This study highlights the importance of using ecologically relevant animal behaviour models to decipher the complex neurobiology underlying the prey capture and defensive strategies of cone snails. [ABSTRACT FROM AUTHOR]

Theconventional setup of in vitrobioassays in microplatesdoes not prevent the loss of volatile compounds, which hampers thetoxicological characterization of waterborne volatile disinfectionby-products (DBPs). To minimize the loss of volatile test chemicals,we adapted four in vitrobioassays to a headspace-freesetup using eight volatile organic compounds (four trihalomethanes,1,1-dichloroethene, bromoethane, and two haloacetonitriles) that covera wide range of air–water partition coefficients. The nominaleffect concentrations of the test chemicals decreased by up to three ordersof magnitude when the conventional setup was changed to a headspace-freesetup for the bacterial cytotoxicity assay using bioluminescence inhibitionof Vibrio fischeri. The increase of apparent sensitivitycorrelated significantly with the air–water partition coefficient.Purge and trap GC/MS analysis revealed a reduced loss of dosed volatilecompounds in the headspace free setup (78–130% of nominal concentration)compared to a substantial loss in the conventional set up (2–13%of the nominal concentration). The experimental effect concentrationsconverged with the headspace-free setup to the effect concentrationspredicted by a QSAR model, confirming the suitability of the headspace-freeapproach to minimize the loss of volatile test chemicals. The analogueheadspace-free design of the bacterial bioassays for genotoxicity(umuC assay) and mutagenicity (Ames fluctuation assay) increased thenumber of compounds detected as genotoxic or mutagenic from one tofour and zero to two, respectively. In a bioassay with a mammaliancell line applied for detecting the induction of the Nrf-2-mediatedoxidative stress response (AREc32 assay), the headspace-free setupimproved the apparent sensitivity by less than one order of magnitude,presumably due to the retaining effect of the serum components inthe medium, which is also reflected in the reduced aqueous concentrationsof compounds. This study highlights the importance of adapting bioanalyticaltest setups when volatile/semivolatile compounds are present in thesample to avoid the loss of chemicals and thus to avoid underestimatingthe toxicity of mixtures and complex environmental samples. [ABSTRACT FROM AUTHOR]

Purpose: This study aimed to identify serum glycoprotein biomarkers for early detection of high-grade serous ovarian cancer (HGSOC), the most common and aggressive histotype of ovarian cancer., Experimental Design: The glycoproteomics pipeline lectin magnetic bead array (LeMBA)-mass spectrometry (MS) was used in age-matched case-control serum samples. Clinical samples collected at diagnosis were divided into discovery (n = 30) and validation (n = 98) sets. We also analysed a set of preclinical sera (n = 30) collected prior to HGSOC diagnosis in the UK Collaborative Trial of Ovarian Cancer Screening., Results: A 7-lectin LeMBA-MS/MS discovery screen shortlisted 59 candidate proteins and three lectins. Validation analysis using 3-lectin LeMBA-multiple reaction monitoring (MRM) confirmed elevated A1AT, AACT, CO9, HPT and ITIH3 and reduced A2MG, ALS, IBP3 and PON1 glycoforms in HGSOC. The best performing multimarker signature had 87.7% area under the receiver operating curve, 90.7% specificity and 70.4% sensitivity for distinguishing HGSOC from benign and healthy groups. In the preclinical set, CO9, ITIH3 and A2MG glycoforms were altered in samples collected 11.1 ± 5.1 months prior to HGSOC diagnosis, suggesting potential for early detection., Conclusions and Clinical Relevance: Our findings provide evidence of candidate early HGSOC serum glycoprotein biomarkers, laying the foundation for further study in larger cohorts., (© 2023 The Authors. Proteomics - Clinical Applications published by Wiley-VCH GmbH.)

Aberrant protein glycosylation is a characteristic of diverse diseases which has been explored as biomarkers. To support translational serum glycoprotein biomarker discovery and validation, we developed a semi-automated workflow using individual lectin-coupled magnetic beads to conduct lectin pulldowns in a high-throughput format. Lectins are naturally occurring glycoprotein binding proteins widely used in glycobiology. While lectin-affinity isolation has been coupled to mass spectrometry-based proteomics, the lectin magnetic bead array (LeMBA) platform allows technically robust screening and measurement of clinical cohorts. This chapter describes detailed lectin-magnetic bead coupling, serum denaturation, lectin magnetic bead pulldown, and on-bead trypsin digest. The resulting tryptic peptides are analyzed by untargeted or targeted liquid chromatography-mass spectrometry (LC-MS), for biomarker discovery, or qualification/validation, respectively. LeMBA-MS generates quantitative data for glycoforms based on lectin affinity of the glycoprotein coupled with MS measurement of one or more prototypic peptides and has successfully been used to discover and validate novel serum cancer glycoprotein biomarkers. This chapter includes detailed protocols for two different liquid handlers, along with recommendations on quality control measures for clinical biomarker studies., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)