In this investigation, venom from the giant Neotropical hunting ant Dinoponera australis (Order: Hymenoptera) has been harvested and subjected to chromatographic separations for the purpose of elucidating possible peptides that display neuroactivity by bioassay guided venom fractionation (BGVF). The venom of this arcane solitary predator paralyzes small invertebrate prey and causes highly exaggerated pain in large vertebrates. The hypothesis that the venom has a peptide component highly effective in modulating neuronal conduction by depolarization of cellular membranes has been tested and subsequent biochemical characterization has been performed to elucidate the primary structure. The data suggests that the modulation of neuronal conduction appears to result from the formation of a de novo pore that allows non-selective ion movements in a concentration dependent manner. The venom contains a variety of proteinaceous candidates and one particular peptide from the venom, -Dinoponeratoxin Da-1837, has been observed to cause very fast, large and sustained depolarization in two types of normally quiescent peripheral neurons (primary cultures of trigeminal and dorsal root ganglia) in whole cell patch clamp recordings. The profound depolarization is due to non-selective cationic flux which is irreversible at high concentrations. Preliminary studies suggest that the peptide also has a minor inhibitory effect on voltage-gated sodium channels, which does not contribute to the depolarization. Membrane assays with microsomes, fluorescent probes and lipid bilayers confirmed peptide-induced non-selective and concentration dependent permeabilization of the membrane. The primary structure of the peptide was determined by iterations of product ion scans in multiple configurations utilizing high resolution tandem mass spectrometry, commonly referred to as MS-MS data dependent acquisition. -DpTx Da-1837 is an eighteen residue peptide that is highly hydrophobic, positively charged at physiological pH and has one atypical post translational modification, i.e. C-terminal peptidyl-lysine. The authentication of the toxin was confirmed by the successful solid phase synthesis of an analog that showed neither biochemical nor physiological variation from the properties of the peptide isolated from Dinoponera australis. The conclusion of this study was the creation of derivative analogs that provide the platform for the first fundamental step in drug discovery: establishing the structure-function relationship. Although the purpose of these cytolytic peptides in venom may be to capture prey or discourage predation, the discoveries of new molecules that affect cell viability by interactions with the cellular envelope provide the genesis for studies of targeted cell death. As a novel anti-microbial agent or as a potent tumor suppressor, the development of peptide derivatives could also help direct the development of new therapeutic interventions.

Previous reports have shown that killed cells of S. aureus potentiate the lethal effect of crude staphylococcal alpha toxin for mice. In the present study this synergistic effect has been demonstrated with highly purified preparations of alpha toxin. Time studies indicate that the active principle does not appear to be released until approximately two hours following administration of the whole cells.

Yes

Yes

Yes

Yes

Yes / Julian P. Perry Robinson discusses the implementation of the General Purpose Criterion in the Chemical Weapons Convention.

Yes / Negotiation of the BTWC in the late 1960s resulted at entry into force in a much weaker treaty regime than had been envisaged in the original proposal put forward by the British. In this video Nicholas A. Sims describes the ways in which specific parts of the original proposal were weakened.

Yes / Nicholas Sims, Reader in International Relations, London School of Economics, 'Evaluation of Proposals: Resumed Session of Biological and Toxin Weapons Convention Fifth Review Conference', November 2002.

Almost all bacteria contain genes that may lead to their growth stasis and death.Normally, these toxins are believed to be neutralized with their cognate antitoxinsfrom a toxin-antitoxin (TA) operon. These modules are also abundant in pathogenic bacteria suggesting a role for them both in normal bacterial physiology and pathogenicity. Their functions have been subject to intense debates. Due to the cell killing capability of the toxin and the gene silencing capability of the antitoxin, they have been utilized for basic research, biotechnology and medical applications. However, further advancements of these applications have been impeded by our limited knowledge of the biology of TAs. Among these TA systems is the Ibs/Sib (A-E) family. Here, we discuss our efforts in characterizing these systems, with a focus on the IbsC/SibC member. Studying them has shown to not be straightforward due to the complexity of their underlying mechanisms and the current approaches being laborious and lacking sensitivity to be applied to these low abundant molecules. We have developed fluorescence-based platforms to take advantage of sensitive and high throughput and resolution techniques such as Fluorescence Assisted Cell Sorting (FACS) to study these molecules instead of relying on traditional culturing methods. While developing these platforms, we gained insights about the biology and regulation of these molecules. To expand this knowledge, we actively pursued investigating the regulation of these molecules at the transcriptional and post-transcriptional levels, both in their native context and in artificial systems. The rest of this thesis summarizes our efforts in solving one of the biggest pieces of the Ibs/Sib puzzle, namely their physiological expressions. With the strategies we have optimized for specific detection of these low abundance molecules, and the knowledge of their biology and regulation presented, we are now at an exciting phase to interrupt the long pause in the study of functions by these molecules and advancement of TA-based applications. / Thesis / Doctor of Philosophy (PhD) / Almost all bacteria contain genes that may lead to their growth stasis or death. Normally, these toxins are believed to be neutralized with their cognate antitoxins. In spite of the efforts to understand these toxin-antitoxin (TA) systems, their physiological roles are subject to intense debate. These systems are hard to study mainly because 1) they are only activated under specific conditions and 2) they are low in abundance. Current approaches are not high throughput and sensitive enough. In this thesis, we developed DNA constructs, bacterial strains and methodologies to facilitate the study of these molecules, particularly the Ibs-Sib family. We next employed these tools to gain a fundamental knowledge of their expression under different conditions, which revealed surprising information about the function of these molecules. We believe that future studies can greatly benefit from the tools offered here to tremendously enhance our understanding of these systems and lead to useful applications.