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SUMMARY Rapid neurotransmitter release depends on the Ca2+-sensor Synaptotagmin-1 and the SNARE complex formed by synaptobrevin, syntaxin-1 and SNAP-25. How Synaptotagmin-1 triggers release remains unclear, in part because elucidating high-resolution structures of Synaptotagmin-1-SNARE complexes has been challenging. An NMR approach based on lanthanide-induced pseudocontact shifts now reveals a dynamic binding mode where basic residues in the concave side of the Synaptotagmin-1 C2B domain β-sandwich interact with a polyacidic region of the SNARE complex formed by syntaxin-1 and SNAP-25. The physiological relevance of this dynamic structural model is supported by mutations in basic residues of Synaptotagmin-1 that markedly impair SNARE-complex binding in vitro and Synaptotagmin-1 function in neurons. Mutations with milder effects on binding have correspondingly milder effects on Synaptotagmin-1 function. Our results support a model whereby their dynamic interaction facilitates cooperation between synaptotagmin-1 and the SNAREs in inducing membrane fusion.

Refereed/Peer-reviewed The taste-masking of bitter-tasting active pharmaceutical ingredients is key to ensuring patient compliance when producing oral pharmaceutical formulations. This is generally achieved via the incorporation of pH-responsive, reverse enteric polymers, that prevent the dissolution of the formulation in the oral environment, but rapidly mediate it within the gastric environment. Reverse enteric polymers are commonly applied as coatings on oral dosage forms via spray atomisation (e.g., fluidised-bed spray coating), and generally exhibit the most efficient taste-masking. However, currently used reverse enteric coatings require high mass gains (% w/w) during coating to mediate taste-masking, and thereby exhibit delayed release within the gastric environment. Therefore, there remains a need for the development of new reverse enteric coatings, that can efficiently taste-mask at low mass gains and maintain rapid release characteristics within the gastric environment. Herein we report the synthesis and evaluation of a series of addition copolymers of 2-vinylpyridine and butyl methacrylate, methyl methacrylate and isobornyl methacrylate. The thermal, solubility, and water absorption properties of the copolymers were effectively tuned by altering the mol% fraction of the constitutive monomers. Based on their physical properties, selected copolymers were preliminarily evaluated for their compatibility with fluidised-bed spray coating, and effectiveness as taste-masking reverse enteric coatings. The copolymers poly[(2-vinylpyridine)-co-(butyl methacrylate)] (mol% ratio 40:60) and poly[(2-vinylpyridine)-co-(butyl methacrylate)-co-(methyl methacrylate)] (mol% ratio 40:50:10) were found to exhibit excellent taste-masking properties following fluidised-bed spray coating onto Suglets® sugar spheres. Suglets® bearing a film coating of either copolymer (5.2–6.5% w/w mass gain) were found to effectively impede the release of a model drug formulation for up to 72 h in a simulated salivary environment, and rapidly release it (

Efforts to mitigate the effects of feral cats through the management of remnant or reintroduced populations of threatened species, are often unsuccessful due to predation by control-averse feral cats, or 'problem individuals'. In order to target these animals, we have developed the Population Protecting Implant (PPI). PPIs are designed to be implanted subcutaneously in a native animal. If the animal is preyed upon, and the implant ingested by a feral cat, release of a toxic payload is triggered in the acidic stomach environment and the problem individual is eliminated. We introduce the first toxic implant incorporating the poison sodium fluoroacetate. Manufactured via fluidised-bed spray coating, toxic implants exhibited uniform reverse enteric coatings and low intra-batch variation. Toxic implants were found to exhibit favourable stability at subcutaneous pH in vitro, and rapidly release their toxic payload in vitro at gastric pH. However, limited stability was demonstrated in rats in vivo (similar to 39-230 d), due to the use of a filament scaffold to enable coating and was likely exacerbated by metachromatic interactions caused by 1080. This work highlights that future development of the PPIs should primarily focus on removal of the filament scaffold, to afford implants with increased in vivo stability. Refereed/Peer-reviewed

Refereed/Peer-reviewed Globally, invasive mammalian predators are responsible for the depredation and extinction of threatened species. The development of innovative control methods that target the most impactful of these predators is crucial to ensure the successful conservation of threatened species. Feral cats (Felis catus) are one of the most damaging invasive mammalian predators, particularly in Australia, where they are estimated to kill more than 1.5 billion mammals, birds, and reptiles annually. Mammals are disproportionally depredated by feral cats, and efforts to reintroduce or conserve remnant populations of threatened mammal species are hampered by shortfalls in current control methods. To address this challenge, we have prototyped the population-protecting implant (PPI), a subcutaneous implant that is designed to target the most dangerous and efficient predators, so called ‘problem individuals’. Conceptually, the implant has a reverse enteric coating that is stable in the subcutaneous environment of a prey animal but dissolves rapidly in the gastric environment of a predator, resulting in the release of a toxic payload and death of the predator. In this work we report a novel approach for the manufacture of PPIs, that combines hot-melt extrusion and fluidised-bed spray coating, to respectively afford implants with accurate drug loading and a uniform reverse-enteric coating. Most of the implants bearing a 300 μm coating were found to effectively contain their payload for more than 280 d in vitro, in a simulated subcutaneous environment (pH 7.4, 37 °C) but release it within 2.5 h in vitro under simulated gastric conditions (pH 1.5, 37 °C). However, two failures were observed at 64 d and 200 d, likely due to the increased osmotic pressure caused by the soluble core. This work highlights a revised implant design that may improve implant stability in vivo and facilitate the translation of the PPI to field use, for the conservation of threatened species. We also introduce a novel and facile approach to determine the loading of non-chromophoric payloads in hot-melt extruded systems that is highly relevant for the determination of active pharmaceutical ingredients in drug-delivery formulations.

Refereed/Peer-reviewed Introduced predators are a significant threat to global biodiversity and are responsible for most of all modern bird, reptile, and mammal extinctions. In Australia, the introduced feral cat (Felis catus) kills 459 million mammals annually and leaves many species facing extinction. Attempted reintroductions of threatened mammal species often fail due to the persistence of intractable feral cats termed "problem individuals" and the swift depredation of the reintroduced population. Biomaterial implants could hold the key to targeting problem individuals. Herein, we report the development of the population-protecting implant, a subcutaneous implant for native mammals. The implant is intended to be inert within the subcutaneous environment for the life of the native mammal and to release a toxic payload in the gastric environment of a feral cat when ingested during a predation event. By toxifying and causing the death of the feral cat, the problem individual is eliminated, and the remaining population of native mammals is protected from further predation. To achieve this, an innovative reverse enteric coating was developed for use as a biomaterial, which exhibited a previously unreported level of pH selectivity for solubility at gastric pH. Large batches of implants were manufactured via fluidized-bed spray coating with a uniform reverse enteric coating and low intrabatch variability. Implants with a 300 μm coating afforded significant stability and retention of the payload at subcutaneous pH in vitro, with rapid release of the payload observed at gastric pH. In addition, implants exhibited favorable stability in vivo in rats, with no observed difference in biocompatibility compared to conventional radio-frequency identification microchips. This work demonstrates a proof of concept of an innovative type of implant and serves as the basis for its future development and translation into the field.

Refereed/Peer-reviewed Predation of threatened fauna by native and introduced predators can drive extinction and prevent population recovery. Most predator management involves exclusion or culling. Evidence suggests that exclusion may have detrimental effects on a prey species' predator awareness. At the same time, culling can cause selection of control-resistant predators. There is increasing interest in harnessing evolutionary processes to drive adaptation of threatened fauna to cope, but there is limited attention on trying this from the predator direction. We need to shift the survival advantage away from predators that avoid lethal control, and go on to kill, towards those that demonstrate behaviors that reduce impact on threatened fauna. Instead of driving undesirable predator selection, could we select through management actions desirable traits to make them "less lethal" to threatened fauna? We draw on experimental research on predator aversion that suggests there may be an alternative way to mitigate the impacts of predators, while maintaining the learning opportunities of prey species. Using the case study of the invasive red fox in Australia, we propose a conceptual framework within which future research and management could occur to select for these desirable traits in predators and develop practical regimes for predator impact mitigation.