Search

Predation by invasive mammalian species is one of the key drivers of native species' population declines and extinctions. Current management of invasive species focuses on their removal from the landscape. However, total removal can be difficult, costly and even impossible. If eradication is not achieved, reductions in predator numbers are often temporary. New tactics are needed to target predators in situ, to reduce their negative impacts. We test the efficacy of conditioned taste aversion (CTA), a tactic that could reduce the impact of predation on target prey species. By associating nausea with a specific food source, it may be possible to condition an aversion to a target bait, and ultimately to live animals in the wild. To assess if wild invasive red foxes (Vulpes vulpes) can be conditioned to avoid a specific food source, we used baits (fried deboned chicken) containing encapsulated levamisole, an anthelmintic agent known to induce nausea leading to emesis and/or diarrhea at high dosages with no long‐term side effects. We buried baits at 30 stations across an open landscape. After treatment, reductions in control baits taken (at least 30%) were observed for 68 days, indicating the use of CTA had successfully reduced bait consumption by red foxes in a wild context. To our knowledge, this study represents the first successful test of CTA to a meat bait in a wild red fox population. Our results suggest that CTA shows promise as a tool to reduce the predation of vulnerable animals providing an alternative tactic to manage the impacts of invasive mammalian predators where eradication is currently impossible. [ABSTRACT FROM AUTHOR]

The use of pH-responsive polymeric micelles is a promising approach to afford the targeted, pH-mediated delivery of hydrophobic drugs within the low-pH tumour milieu and intracellular organelles of cancer cells. However, even for a common pH-responsive polymeric micelle system—e.g., those utilising poly(ethylene glycol)-b-poly(2-vinylpyridine) (PEG-b-PVP) diblock copolymers—there is a lack of available data describing the compatibility of hydrophobic drugs, as well as the relationships between copolymer microstructure and drug compatibility. Furthermore, synthesis of the constituent pH-responsive copolymers generally requires complex temperature control or degassing procedures that limit their accessibility. Herein we report the facile synthesis of a series of diblock copolymers via visible-light-mediated photocontrolled reversible addition-fragmentation chain-transfer polymerisation, with a constant PEG block length (90 repeat units (RUs)) and varying PVP block lengths (46–235 RUs). All copolymers exhibited narrow dispersity values (Đ ≤ 1.23) and formed polymeric micelles with low polydispersity index (PDI) values (typically <0.20) at physiological pH (7.4), within a suitable size range for passive tumour targeting (<130 nm). The encapsulation and release of three hydrophobic drugs (cyclin-dependent kinase inhibitor (CDKI)-73, gossypol, and doxorubicin) were investigated in vitro at pH 7.4–4.5 to simulate drug release within the tumour milieu and cancer cell endosome. Marked differences in drug encapsulation and release were observed when the PVP block length was increased from 86 to 235 RUs. With a PVP block length of 235 RUs, the micelles exhibited differing encapsulation and release properties for each drug. Minimal release was observed for doxorubicin (10%, pH 4.5) and CDKI-73 exhibited moderate release (77%, pH 4.5), whereas gossypol exhibited the best combination of encapsulation efficiency (83%) and release (91% pH 4.5) overall. These data demonstrate the drug selectivity of the PVP core, where both the block molecular weight and hydrophobicity of the core (and accordingly the hydrophobicity of the drug) have a significant effect on drug encapsulation and release. These systems remain a promising means of achieving targeted, pH-responsive drug delivery—albeit for select, compatible hydrophobic drugs—which warrants their further investigation to develop and evaluate clinically relevant micelle systems. [ABSTRACT FROM AUTHOR]

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 (~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. [ABSTRACT FROM AUTHOR]

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 (<10 min) within a simulated gastric environment. The results demonstrated the potential of poly[(2-vinylpyridine)-co-(butyl methacrylate)] copolymers to form effectively taste-masked, reverse enteric dosage forms, and suggested that these copolymers may provide improved performance compared to currently available polymers. [ABSTRACT FROM AUTHOR]

Characterizing interactions of Synaptotagmin‐1 with the SNARE complex is crucial to understand the mechanism of neurotransmitter release. X‐ray crystallography revealed how the Synaptotagmin‐1 C2B domain binds to the SNARE complex through a so‐called primary interface and to a complexin‐1‐SNARE complex through a so‐called tripartite interface. Mutagenesis and electrophysiology supported the functional relevance of both interfaces, and extensive additional data validated the primary interface. However, ITC evidence suggesting that binding via the tripartite interface occurs in solution was called into question by subsequent NMR data. Here, we describe joint efforts to address this apparent contradiction. Using the same ITC approach with the same C2B domain mutant used previously (C2BKA‐Q) but including ion exchange chromatography to purify it, which is crucial to remove polyacidic contaminants, we were unable to observe the substantial endothermic ITC signal that was previously attributed to binding of this mutant to the complexin‐1‐SNARE complex through the tripartite interface. We were also unable to detect substantial populations of the tripartite interface in NMR analyses of the ITC samples or in measurements of paramagnetic relaxation effects, despite the high sensitivity of this method to detect weak protein complexes. However, these experiments do not rule out the possibility of very low affinity (KD > 1 mm) binding through this interface. These results emphasize the need to develop methods to characterize the structure of synaptotagmin‐1‐SNARE complexes between two membranes and to perform further structure–function analyses to establish the physiological relevance of the tripartite interface. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Ca2+-triggered neurotransmitter release depends on the formation of SNARE complexes that bring the synaptic vesicle and plasma membranes together, on the Ca2+ sensor synaptotagmin-1 and on complexins, which play active and inhibitory roles. Release of the complexin inhibitory activity by binding of synaptotagmin-1 to the SNARE complex, causing complexin displacement, was proposed to trigger exocytosis. However, the validity of this model was questioned based on the observation of simultaneous binding of complexin-I and a fragment containing the synaptotagmin-1 C2 domains (C2AB) to membrane-anchored SNARE complex. Using diverse biophysical techniques, here we show that C2AB and complexin-I do not bind to each other but can indeed bind simultaneously to the SNARE complex in solution. Hence, the SNARE complex contains separate binding sites for both proteins. However, total internal reflection fluorescence microscopy experiments show that C2AB can displace a complexin-I fragment containing its central SNARE-binding helix and an inhibitory helix (Cpx26-83) from membrane-anchored SNARE complex under equilibrium conditions. Interestingly, full-length complexin-I binds more tightly to membrane-anchored SNARE complex than Cpx26-83, and it is not displaced by C2AB. These results show that interactions of N- and/or C-terminal sequences of complexin-I with the SNARE complex and/or phospholipids increase the affinity of complexin-I for the SNARE complex, hindering dissociation induced by C2AB. We propose a model whereby binding of synaptotagmin-1 to the SNARE complex directly or indirectly causes a rearrangement of the complexin-I inhibitory helix without inducing complexin-I dissociation, thus relieving the inhibitory activity and enabling cooperation between synaptotagmin-1 and complexin-I in triggering release. [Copyright &y& Elsevier]

Neurotransmitter release depends critically on the Ca2+ sensor synaptotagmin-1 and the SNARE proteins syntaxin-1, synaptobrevin, and SNAP-25, which mediate membrane fusion by forming tight SNARE complexes that bridge the synaptic vesicle and plasma membranes. Interactions between the SNARE complex and the two C2 domains of synaptotagmin-1 (the C2A and C2B domains) are believed to play a key role in coupling Ca2+ sensing to membrane fusion, but the nature of these interactions is unclear, in part because of a paucity of data obtained by quantitative biophysical methods. Here we have analyzed synaptotagmin-1/SNARE complex interactions by monitoring the decrease in the intensities of one-dimensional 13C-edited 1H NMR spectra of 13C-labeled fragments of synaptotagmin-1 upon binding to unlabeled SNARE complex. Our results indicate that there is a primary binding mode between synaptotagmin-1 and the SNARE complex that involves a polybasic region in the C2B domain and has a sub-micromolar affinity. Our NMR data, combined with precipitation assays, show that there are additional SNARE complex/synaptotagmin-1 interactions that lead to aggregation and that involve in part two arginines at the bottom of the C2B domain. Overall, this study shows the importance of disentangling the contributions of different types of interactions to SNARE complex/synaptotagmin-1 binding and illustrates the usefulness of one-dimensional NMR methods to analyze intricate protein interactions. [ABSTRACT FROM AUTHOR]

Injectable, thermoresponsive hydrogels are promising candidates for the delivery, maintenance and controlled release of adoptive cell therapies. Therefore, there is significant interest in the development of cytocompatible and biodegradable thermoresponsive hydrogels with appropriate gelling characteristics. Towards this end, a series of thermoresponsive copolymers consisting of poly(caprolactone) (PCL), poly(ethylene glycol) (PEG) and poly(propylene glycol) (PPG) segments, with various PEG:PPG ratios, were synthesised via ring-opening polymerisation (ROP) of ε-caprolactone and epoxy-functionalised PEG and PPG derivatives. The resultant PCL–PEG–PPG copolymers were characterised via proton nuclear magnetic resonance (1H NMR) spectroscopy, gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). The thermoresponsive characteristics of the aqueous copolymer solutions at various concentrations was investigated using the inversion method. Whilst all of the copolymers displayed thermoresponsive properties, the copolymer with a ratio of 1:2 PEG:PPG exhibited an appropriate sol–gel transition (28 °C) at a relatively low concentration (10 wt%), and remained a gel at 37 °C. Furthermore, the copolymers were shown to be enzymatically degradable in the presence of lipases and could be used for the encapsulation of CD4+ T-cell lymphocytes. These results demonstrate that the thermoresponsive PCL–PEG–PPG hydrogels may be suitable for use as an adoptive cell therapy (ACT) delivery vehicle. [ABSTRACT FROM AUTHOR]

Rapid neurotransmitter release depends on the Ca2+ sensor Synaptotagmin-1 (Syt1) and the SNARE complex formed by synaptobrevin, syntaxin-1 and SNAP-25. How Syt1 triggers release has been unclear, partly because elucidating high-resolution structures of Syt1-SNARE complexes has been challenging. An NMR approach based on lanthanide-induced pseudocontact shifts now reveals a dynamic binding mode in which basic residues in the concave side of the Syt1 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 Syt1 that markedly impair SNARE-complex binding in vitro and Syt1 function in neurons. Mutations with milder effects on binding have correspondingly milder effects on Syt1 function. Our results support a model whereby dynamic interaction facilitates cooperation between Syt1 and the SNAREs in inducing membrane fusion.