In situ scanning probe microscopy studies of tetanus toxin-membrane interactions

Despite the considerable information available with regards to the structure of the clostridial neurotoxins, and their inherent threat as biological warfare agents, the mechanisms underpinning their interactions with and translocation through the cell membrane remain poorly understood. We report herein the results of an in situ scanning probe microscopy study of the interaction of tetanus toxin C-fragment (Tet C) with supported planar lipid bilayers containing the ganglioside receptor G(T1b). Our results show that Tet C preferentially binds to the surface of fluid phase domains within biphasic membranes containing G(T1b) and that with an extended incubation period these interactions lead to dramatic changes in the morphology of the lipid bilayer, including the formation of 40-80 nm diameter circular cavities. Combined atomic force microscopy/total internal reflection fluorescence microscopy experiments confirmed the presence of Tet C in the membrane after extended incubation. These morphological changes were found to be dependent upon the presence of G(T1b) and the solution pH.     

Do Male Cook Strait Giant Weta Prudently Allocate Sperm?

Sperm production is costly and so males are expected to prudently allocate sperm to matings in a manner that maximizes their fitness. Sperm competition hypotheses predict that when facing increased sperm competition risk males should increase their investment in ejaculates. In contrast, when facing high future mating opportunities, males are expected to decrease their sperm investment in the current mating. This is because males should keep in reserve an amount of sperm proportional to their expected future mating opportunities. We experimentally tested whether male Cook Strait giant weta (Anostostomatidae: Orthoptera: Deinacrida rugosa) phenotypically adjust their investment in ejaculates in relation to their perceived risk of sperm competition and future mating opportunities. D. rugosa is a large flightless orthopteran insect in which males pass multiple spermatophores to females during a day-long mating bout. Contrary to expectation, we found that low female availability (i.e. increased sperm competition risk) had no effect on male resource allocation to sperm (i.e. number of spermatophores) compared to controls whereas, contrary to expectation, males experiencing high female availability increased their ejaculate investment by transferring significantly more spermatophores to their mates. Our results might be a consequence of males being insensitive to increased presence of rival males, reducing their allocation to sperm under increasingly risky circumstances, or due to females prolonging copulations when their perceived future mating opportunities are low.     

Cyclooxygenase-1 overexpression decreases Basal airway responsiveness but not allergic inflammation

Pharmacological inhibition or genetic disruption of cyclooxygenase (COX)-1 or COX-2 exacerbates the inflammatory and functional responses of the lung to environmentally relevant stimuli. To further examine the contribution of COX-derived eicosanoids to basal lung function and to allergic lung inflammation, transgenic (Tr) mice were generated in which overexpression of human COX-1 was targeted to airway epithelium. Although no differences in basal respiratory or lung mechanical parameters were observed, COX-1 Tr mice had increased bronchoalveolar lavage fluid PGE(2) content compared with wild-type littermates (23.0 +/- 3.6 vs 8.4 +/- 1.4 pg/ml; p < 0.05) and exhibited decreased airway responsiveness to inhaled methacholine. In an OVA-induced allergic airway inflammation model, comparable up-regulation of COX-2 protein was observed in the lungs of allergic wild-type and COX-1 Tr mice. Furthermore, no genotype differences were observed in allergic mice in total cell number, eosinophil content (70 vs 76% of total cells, respectively), and inflammatory cytokine content of bronchoalveolar lavage fluid, or in airway responsiveness to inhaled methacholine (p > 0.05). To eliminate the presumed confounding effects of COX-2 up-regulation, COX-1 Tr mice were bred into a COX-2 null background. In these mice, the presence of the COX-1 transgene did not alter allergen-induced inflammation but significantly attenuated allergen-induced airway hyperresponsiveness, coincident with reduced airway leukotriene levels. Collectively, these data indicate that COX-1 overexpression attenuates airway responsiveness under basal conditions but does not influence allergic airway inflammation.     

Scalable purification of Bacillus anthracis protective antigen from Escherichia coli

The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor that are produced by the Gram-positive bacterium, Bacillus anthracis. Current vaccines against anthrax use PA as their primary component. In this study, we developed a scalable process to produce and purify multi-gram quantities of highly pure, recombinant PA (rPA) from Escherichia coli. The rPA protein was produced in a 50-L fermentor and purified to >99% purity using anion-exchange, hydrophobic interaction, and hydroxyapatite chromatography. The final yield of purified rPA from medium cell density fermentations resulted in approximately 2.7 g of rPA per kg of cell paste (approximately 270 mg/L) of highly pure, biologically active rPA protein. The results presented here exhibit the ability to generate multi-gram quantities of rPA from E. coli that may be used for the development of new anthrax vaccines and anthrax therapeutics.     

8-plex quantitation of changes in cerebrospinal fluid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzheimer's disease

An 8-plex version of an isobaric reagent for the quantitation of proteins using shotgun methods is presented. The 8-plex version of the reagent relies on amine-labeling chemistry of peptides similar to 4-plex reagents. MS/MS reporter ions at 113, 114, 115, 116, 117, 118, 119, and 121 m/z are used to quantify protein expression. This technology which was first applied to a test mixture consisting of eight proteins and resulted in accurate quantitation, has the potential to increase throughput of analysis for quantitative shotgun proteomics experiments when compared to 2- and 4-plex methods. The technology was subsequently applied to a longitudinal study of cerebrospinal fluid (CSF) proteins from subjects undergoing intravenous Ig treatment for Alzheimer's disease. Results from this study identify a number of protein expression changes that occur in CSF after 3 and 6 months of treatment compared to a baseline and compared to a drug washout period. A visualization tool was developed for this dataset and is presented. The tool can aid in the identification of key peptides and measurements. One conclusion aided by the visualization tool is that there are differences in considering peptide-based observations versus protein-based observations from quantitative shotgun proteomics studies.     

A systematic review of adaptive wildlife management for the control of invasive,non-native mammals,and other human–wildlife conflicts

1. We are entering an era where species declines are occurring at their fastest ever rate, and the increased spread of non-native species is among the top causes. High uncertainty in biological processes makes the accurate prediction of the outcomes of management interventions very challenging. Adaptive management (AM) offers solutions to reduce uncertainty and improve predictability so that the outcomes of interventions can continuously improve.
2. We quantitatively assess the extent to which AM is used for managing vertebrates, with a focus on invasive non-native species (INNS). Using the Web of Science, we evaluated 3992 articles returned by the search terms ‘adaptive management’ or ‘adaptive harvest management’ against seven recommended elements of AM (engagement with stakeholders, defining objectives, forecasting and estimating uncertainty, implementing management, monitoring populations, adjusting management in response to monitoring, and improving forecasting and reducing uncertainty in response to monitoring populations).
3. The use of AM for vertebrates was reported in 56 (1%) of the evaluated studies; including four for managing INNS. Of these, ten studies excluding INNS and no studies of INNS management implemented all seven recommended elements of AM. Those elements infrequently implemented were as follows: the use of analysis or models to forecast and represent uncertainty (44%) and the feedback of monitoring data to improve forecasting and reduce uncertainty (25%).
4. Complete active AM has rarely been implemented and reported for managing INNS, despite the significant advantages it offers. Among studies purporting to have implemented AM, most did not use analyses or models to forecast and represent uncertainty, while most defined objectives, implemented management, and monitored populations.
5. Improvements to ongoing control programmes and much broader adoption of the AM approach are required to increase the efficiency and success of INNS management campaigns and reduce their negative impacts on native species.

     

Precopulatory choice for cues of material benefits in tree crickets

The relative importance of direct and indirect benefits modelsof mate choice is a central question in sexual selection, butseparating the two models is very difficult because high qualitymales often provide both better direct benefits and better genes.In tree crickets, Oecanthus nigricornis, females have the opportunityto gain both directly and indirectly from mate choice. Femaletree crickets exercise premating choice for large males, butthe model underlying this choice is unknown. In this study weexamine the proximate cues used by female tree crickets to rejectmales, and show that the ability of males to provide food isa central cue. In contrast, we find no evidence that the relativesize of mates is important in mate rejection. The fact thatthe major phenotypic cues of male quality are related to foodprovisioning suggests that the role of genetic benefits in shapingfemale preferences is limited by the extent to which food provisioningsignals genetic quality.