Isolation and characterisation of two epithelial-like cell lines from the gills of Chrysophrys auratus (Australasian snapper) and Oncorhynchus tshawytscha (Chinook salmon) and their use in aquatic toxicology.

In vitro gill models are becoming increasingly important in aquatic toxicology, yet the fish gill invitrome is underrepresented, encompassing approximately 0.1% of extant species. Here, we describe the establishment and characterisation of two gill-derived, epithelial-like cell lines isolated from fish species of significant importance to New Zealand: Chrysophrys auratus (Australasian snapper) and Oncorhynchus tshawytscha (Chinook salmon). Designated CAgill1PFR (Chrysophrys auratus, gill 1, Plant & Food Research) and OTgill1PFR (Oncorhynchus tshawytscha, gill 1, Plant & Food Research), these cell lines have each been passaged greater than each 70 times over several years and are considered spontaneously immortalised. Both cell lines required serum for growth and exhibited differential responses to basal media formulations. CAgill1PFR was sensitive to low temperatures (4 °C) but replicated at high temperatures (30 °C), whereas OTgill1PFR was sensitive to high temperatures but remained viable at low temperatures, mirroring the natural environment of their host species. Immunostaining revealed expression of epithelial cell markers cytokeratin and E-cadherin, alongside positivity for the mesenchymal cell marker, vimentin. CAgill1PFR was more sensitive to the environmental toxin 3,4 dichloroaniline than OTgill1PFR through measurements of metabolic activity, membrane integrity, and lysosomal function. Furthermore, CAgill1PFR produced less CYP1A activity, indicative of ongoing biotransformation processes, in response to beta-naphthoflavone than OTgill1PFR. These cell lines expand the toolbox of resources and emphasise the need for species-specific aquatic toxicology research.

Selection and characterization of DNA aptamers for the rat major urinary protein 13 (MUP13) as selective biorecognition elements for sensitive detection of rat pests.

Among invasive mammalian predators, rats represent a major threat, endangering ecosystem functioning worldwide. After rat-control operations, detecting their continued presence or reinvasion requires more sensitive and lower cost detection technologies. Here, we develop a new sensing paradigm by using a specific rat urine biomarker (MUP13) to unambiguously signal the presence of rats. As the first step towards a new remote surveillance technology, aptamers were selected to MUP13 using the Flu-Mag SELEX method. Six aptamer candidates were initially screened by dot blot and two of them (Apt-2.5 and Apt-1.4) exhibited high affinity and specificity. Both aptamers were further characterized by bead-based assay to confirm affinity and selectivity. The lead aptamer candidates were then applied to fluorescence anisotropy (FA) and surface plasmon resonance (SPR)-based biosensor platforms, showing dissociation constants in the nanomolar range and high specificity towards their target. The SPR biosensor had limits of detection of 13.8 and 7.5 nM for Apt-2.5 and Apt-1.4, respectively, which are more than three orders of magnitude lower than the physiological concentrations found in rat urine. Selectivity of the aptamers, when comparing with other major urinary proteins, was excellent, indicating strong efficacy in specific detection of rats. In order to validate the aptamer Apt-2.5 for use with real world samples a FA-based assay was performed on a rat urine sample. The assay showed that the aptamer could detect recombinant MUP13 spiked in filtered urine and the natural MUP13 in unfiltered urine, as a first step into translation to real world application. These are the first known assays to detect and quantify a MUP biomarker of rats.

Expression, purification and characterisation of the recombinant possum lipocalin vulpeculin.

BACKGROUND: Lipocalins are a large family of proteins, which possess a highly conserved eight-stranded antiparallel beta-barrel structure as distinctive trait. This family includes Major Urinary Proteins (MUPs) from rats and mouse, studied for their role in urinary protein-mediated chemosignalling. Vulpeculin has been identified as the most abundant protein in the urine of the common brushtail possum, Trichosurus vulpecula. On the basis of high similarity with other MUPS, we hypothesised that vulpeculin might have a role in possum chemosignalling and investigated its stability and binding ability. METHODS: We expressed and purified vulpeculin using an E.coli-based system and confirmed correct folding by circular dichroism (CD) spectroscopy. Thermal stability was studied by CD and binding properties were investigated using two optical probes N-phenyl-naphthylamine (NPN) and 8-anilino-1-naphthalene sulphonic acid (ANS). RESULTS: CD revealed a secondary structure typical of a predominantly ß-sheet protein, consistent with the beta barrel structure of the lipocalin family. Vulpeculin showed a high level of thermostability, as assessed by CD, exhibiting a small shift in the secondary structure even at 95 °C. Binding assays indicated that vulpeculin cannot accommodate the NPN ligand but can bind ANS. CONCLUSION: The urinary secretion, high degree of sequence similarity with other lipocalins, its beta sheet structure assessed by CD and potential to bind hydrophobic ligands in the hydrophobic cavity or an external hydrophobic pocket, suggest vulpeculin may be involved in possum chemosignalling. GENERAL SIGNIFICANCE: This work represents a first step towards the further investigation of the newly discovered lipocalin and its role in possum chemosignalling.

An ultrasensitive electrochemical impedance-based biosensor using insect odorant receptors to detect odorants.

Herein, we present that insect odorant receptors reconstituted into the lipid bilayers of liposomes can be successfully immobilized onto a gold surface and selectively and sensitively detect odorant molecules. The odorant receptors (OrXs) Or10a, Or22a, and Or71a from the common fruit fly, Drosophila melanogaster, were recombinantly expressed, purified and integrated into nano-liposomes (100-200 nm). These liposomes were covalently attached to the self-assembled monolayers (SAMs) of a 6-mercaptohexanoic acid (MHA)-modified gold surface. X-ray Photo Electron Spectroscopy (XPS) and Quartz Crystal Microbalance with Dissipation (QCM-D) measurements confirmed the successful modification of the gold surface and immobilization of liposomes. Atomic Force Microscopy (AFM) revealed that the liposomes were covalently attached to the surface without any disruption of vesicles. The liposomes tethered to the gold sensor surface were then treated with a range of known ligands of various concentrations. We demonstrated by Electrochemical Impedance Spectroscopy (EIS) that an OrX/liposome EIS sensor can sensitively and selectively detect its known ligand to femtomolar concentrations by detecting a change in electrical signal upon binding. Our study is the first step towards using purified insect odorant receptors alone in biosensors to enable the development of novel ultrasensitive volatile sensors for medical diagnostic, air quality, food safety and border security applications.

Biosensing with Insect Odorant Receptor Nanodiscs and Carbon Nanotube Field-Effect Transistors.

Insect odorant receptors have been reconstituted into lipid nanodiscs and tethered to carbon nanotube field-effect transistors to function as a biosensor. Here, four different insect odorant receptors (ORs) from Drosophila melanogaster (DmelOR10a, DmelOR22a, DmelOR35a, and DmelOR71a) were expressed in Sf9 cells, purified, and reconstituted into lipid nanodiscs. We have demonstrated that each of these ORs produce a selective and highly sensitive electrical response to their respective positive ligands, methyl salicylate, methyl hexanoate, trans-2-hexen-1-al, and 4-ethylguaiacol, with limits of detection in the low femtomolar range. No detection was observed for each OR against control ligands, and empty nanodiscs showed no specific sensor signal for any of the odorant molecules. Our results are the first evidence that insect ORs can be integrated into lipid nanodiscs and used as primary sensing elements for bioelectronic nose technologies.

Data on preparation and characterization of an insect odorant receptor based biosensor.

Insect Odorant receptors (OrXs) can be used as the recognition element in a biosensor as they demonstrate high levels of sensitivity and selectivity towards volatile organic compounds. Herein, we describe a method to express and purify insect odorant receptors and reconstitute them into artificial lipid bilayers (liposomes). These OrX/liposomes were covalently attached to a gold surface and characterized using quartz crystal microbalance with dissipation monitoring (QCM-D). The interaction of OrX/liposomes immobilized on a gold surface to positive and negative odorants were studied by means of electrochemical impedance spectroscopy (EIS) and QCM-D. The data presented in this article are related to the research article titled "An ultrasensitive electrochemical impedance-based biosensor using insect odorant receptors to detect odorants" [1].

Rapid Identification of Novel Inhibitors of the Human Aquaporin-1 Water Channel.

Aquaporins (AQPs) are a family of membrane proteins that function as channels facilitating water transport in response to osmotic gradients. These play critical roles in several normal physiological and pathological states and are targets for drug discovery. Selective inhibition of the AQP1 water channel may provide a new approach for the treatment of several disorders including ocular hypertension/glaucoma, congestive heart failure, brain swelling associated with a stroke, corneal and macular edema, pulmonary edema, and otic disorders such as hearing loss and vertigo. We developed a high-throughput assay to screen a library of compounds as potential AQP1 modulators by monitoring the fluorescence dequenching of entrapped calcein in a confluent layer of AQP1-overexpressing CHO cells that were exposed to a hypotonic shock. Promising candidates were tested in a Xenopus oocyte-swelling assay, which confirmed the identification of two lead classes of compounds belonging to aromatic sulfonamides and dihydrobenzofurans with IC50 s in the low micromolar range. These selected compounds directly inhibited water transport in AQP1-enriched stripped erythrocyte ghosts and in proteoliposomes reconstituted with purified AQP1. Validation of these lead compounds, by the three independent assays, establishes a set of attractive AQP1 blockers for developing novel, small-molecule functional modulators of human AQP1.