Discovery and characterization of a linear cyclotide from Viola odorata: Implications for the processing of circular proteins

Cyclotides are mini-proteins of 28-37 amino acid residues that have the unusual feature of a head-to-tail cyclic backbone surrounding a cystine knot. This molecular architecture gives the cyclotides heightened resistance to thermal, chemical and enzymatic degradation and has prompted investigations into their use as scaffolds in peptide therapeutics. There are now more than 80 reported cyclotide sequences from plants in the families Rubiaceae, Violaceae and Cucurbitaceae, with a wide variety of biological activities observed. However, potentially limiting the development of cyclotide-based therapeutics is a lack of understanding of the mechanism by which these peptides are cyclized in vivo. Until now, no linear versions of cyclotides have been reported, limiting our understanding of the cyclization mechanism. This study reports the discovery of a naturally occurring linear cyclotide, violacin A, from the plant Viola odorata and discusses the implications for in vivo cyclization of peptides. The elucidation of the cDNA clone of violacin A revealed a point mutation that introduces a stop codon, which inhibits the translation of a key Asn residue that is thought to be required for cyclization. The three-dimensional solution structure of violacin A was determined and found to adopt the cystine knot fold of native cyclotides. Enzymatic stability assays on violacin A indicate that despite an increase in the flexibility of the structure relative to cyclic counterparts, the cystine knot preserves the overall stability of the molecule. (c) 2006 Elsevier Ltd. All rights reserved.

Discovery of cyclotide-like protein sequences in graminaceous crop plants: Ancestral precursors of circular proteins?

Cyclotides are peptides from plants of the Rubiaceae and Violaceae families that have the unusual characteristic of a macrocylic backbone. They are further characterized by their incorporation of a cystine knot in which two disulfides, along with the intervening backbone residues, form a ring through which a third disulfide is threaded. The cyclotides have been found in every Violaceae species screened to date but are apparently present in only a few Rubiaceae species. The selective distribution reported so far raises questions about the evolution of the cyclotides within the plant kingdom. In this study, we use a combined bioinformatics and expression analysis approach to elucidate the evolution and distribution of the cyclotides in the plant kingdom and report the discovery of related sequences widespread in the Poaceae family, including crop plants such as rice ( Oryza sativa), maize ( Zea mays), and wheat ( Triticum aestivum), which carry considerable economic and social importance. The presence of cyclotide-like sequences within these plants suggests that the cyclotides may be derived from an ancestral gene of great antiquity. Quantitative RT-PCR was used to show that two of the discovered cyclotide-like genes from rice and barley ( Hordeum vulgare) have tissue-specific expression patterns.

Discovery of a potent phenolic N1-benzylidene- pyridinecarboxamidrazone selective against Gram-positive bacteria

As part of a study into antimycobacterial compounds a set of phenolic N1-benzylidene-pyridinecarboxamidrazones was prepared and evaluated. This report describes the unexpected discovery of a potent compound with a pronounced selectivity for Gram-positive bacteria over Gram-negative micro-organisms. In addition, this compound is active against various drug-resistant Gram-positive bacteria. © 2005 Elsevier Ltd. All rights reserved.

A signaling visualization toolkit to support rational design of combination therapies and biomarker discovery: SiViT

Targeted cancer therapy aims to disrupt aberrant cellular signalling pathways. Biomarkers are surrogates of pathway state, but there is limited success in translating candidate biomarkers to clinical practice due to the intrinsic complexity of pathway networks. Systems biology approaches afford better understanding of complex, dynamical interactions in signalling pathways targeted by anticancer drugs. However, adoption of dynamical modelling by clinicians and biologists is impeded by model inaccessibility. Drawing on computer games technology, we present a novel visualisation toolkit, SiViT, that converts systems biology models of cancer cell signalling into interactive simulations that can be used without specialist computational expertise. SiViT allows clinicians and biologists to directly introduce for example loss of function mutations and specific inhibitors. SiViT animates the effects of these introductions on pathway dynamics, suggesting further experiments and assessing candidate biomarker effectiveness. In a systems biology model of Her2 signalling we experimentally validated predictions using SiViT, revealing the dynamics of biomarkers of drug resistance and highlighting the role of pathway crosstalk. No model is ever complete: the iteration of real data and simulation facilitates continued evolution of more accurate, useful models. SiViT will make accessible libraries of models to support preclinical research, combinatorial strategy design and biomarker discovery.

Development of polymeric drug delivery vehicles for targeted cancer therapy

The aim of my Ph. D. thesis is to generalize a method for targeted anti-cancer drug delivery. Hydrophilic polymer-drug conjugates involve complicated synthesis; drug-encapsulated polymeric nanoparticles limit the loading capability of payloads. This thesis introduces the concept of nanoconjugates to overcome difficulties in synthesis and formulation. Drugs with hydroxyl group are able to initiate polyester synthesis in a regio- and chemo- selective way, with the mediation of ligand-tunable Zinc catalyst. Herein, three anti-cancer drugs are presented to demonstrate the high efficiency and selectivity in the method (Chapter 2-4). The obtained particles are stable in salt solution, releasing drugs over weeks in controlled manner. With the conjugation of aptamer, particles are capable to target prostate cancer cells in vitro. These results open the gateway to evaluate the in vivo efficacy of nanoconjugates for target cancer therapy (Chapter 5). Mechanism study of the polymerization leads to the discovery of chemosite selective synthesis of prodrugs with acrylate functional groups. Functional copolymer-drug conjugates will expand the scope of nanoconjugates (Chapter 6). Liposome-aptamer targeting drug delivery vehicle is well studied to achieve reversible cell-specific delivery of non-hydoxyl drugs e.g. cisplatin (Chapter 7). New monomers and polymerization mechanisms are explored for polyester in order to synthesize nanoconjugates with variety on properties (Chapter 8). Initial efforts to apply this type of prodrugs will be focused on the preparation of hydrogels for stem cell research (Chapter 9).

Screening for drugs in oral fluid : illicit drug use and drug driving in a sample of urban and regional Queensland motorists

Police services in a number of Australian states and overseas jurisdictions have begun to implement or consider random road-side drug testing of drivers. This paper outlines research conducted to provide an estimate of the extent of drug driving in a sample of Queensland drivers in regional, rural and metropolitan areas. Oral fluid samples were collected from 2657 Queensland motorists and screened for illicit substances including cannabis (delta 9 tetrahydrocannibinol [THC]), amphetamines, ecstasy, and cocaine. Overall, 3.8% of the sample (n = 101) screened positive for at least one illicit substance, although multiple drugs were identified in a sample of 23 respondents. The most common drugs detected in oral fluid were ecstasy (n = 53), and cannabis (n = 46) followed by amphetamines (n = 23). A key finding was that cannabis was confirmed as the most common self-reported drug combined with driving and that individuals who tested positive to any drug through oral fluid analysis were also more likely to report the highest frequency of drug driving. Furthermore, a comparison between drug vs. drink driving detection rates for one region of the study, revealed a higher detection rate for drug driving (3.8%) vs. drink driving (0.8%). This research provides evidence that drug driving is relatively prevalent on Queensland roads, and may in fact be more common than drink driving. This paper will further outline the study findings’ and present possible directions for future drug driving research.

Effective web service discovery using a combination of a semantic model and a data mining technique

With the advent of Service Oriented Architecture, Web Services have gained tremendous popularity. Due to the availability of a large number of Web services, finding an appropriate Web service according to the requirement of the user is a challenge. This warrants the need to establish an effective and reliable process of Web service discovery. A considerable body of research has emerged to develop methods to improve the accuracy of Web service discovery to match the best service. The process of Web service discovery results in suggesting many individual services that partially fulfil the user’s interest. By considering the semantic relationships of words used in describing the services as well as the use of input and output parameters can lead to accurate Web service discovery. Appropriate linking of individual matched services should fully satisfy the requirements which the user is looking for. This research proposes to integrate a semantic model and a data mining technique to enhance the accuracy of Web service discovery. A novel three-phase Web service discovery methodology has been proposed. The first phase performs match-making to find semantically similar Web services for a user query. In order to perform semantic analysis on the content present in the Web service description language document, the support-based latent semantic kernel is constructed using an innovative concept of binning and merging on the large quantity of text documents covering diverse areas of domain of knowledge. The use of a generic latent semantic kernel constructed with a large number of terms helps to find the hidden meaning of the query terms which otherwise could not be found. Sometimes a single Web service is unable to fully satisfy the requirement of the user. In such cases, a composition of multiple inter-related Web services is presented to the user. The task of checking the possibility of linking multiple Web services is done in the second phase. Once the feasibility of linking Web services is checked, the objective is to provide the user with the best composition of Web services. In the link analysis phase, the Web services are modelled as nodes of a graph and an allpair shortest-path algorithm is applied to find the optimum path at the minimum cost for traversal. The third phase which is the system integration, integrates the results from the preceding two phases by using an original fusion algorithm in the fusion engine. Finally, the recommendation engine which is an integral part of the system integration phase makes the final recommendations including individual and composite Web services to the user. In order to evaluate the performance of the proposed method, extensive experimentation has been performed. Results of the proposed support-based semantic kernel method of Web service discovery are compared with the results of the standard keyword-based information-retrieval method and a clustering-based machine-learning method of Web service discovery. The proposed method outperforms both information-retrieval and machine-learning based methods. Experimental results and statistical analysis also show that the best Web services compositions are obtained by considering 10 to 15 Web services that are found in phase-I for linking. Empirical results also ascertain that the fusion engine boosts the accuracy of Web service discovery by combining the inputs from both the semantic analysis (phase-I) and the link analysis (phase-II) in a systematic fashion. Overall, the accuracy of Web service discovery with the proposed method shows a significant improvement over traditional discovery methods.

Adolescent protective behaviour to reduce drug and alcohol use, alcohol-related harm and interpersonal violence

Typically adolescents' friends are considered a risk factor for adolescent engagement in risk-taking. This study took a more novel approach, by examining adolescent friendship as a protective factor. In particular it investigated friends' potential to intervene to reduce risk-taking. 540 adolescents (mean age 13.47 years) were asked about their intention to intervene to reduce friends' alcohol, drug and alcohol-related harms and about psychosocial factors potentially associated with intervening. More than half indicated that they would intervene in friends' alcohol, drug use, alcohol-related harms and interpersonal violence. Intervening was associated with being female, having friends engage in overall less risk-taking and having greater school connectedness. The findings provide an important understanding of increasing adolescent protective behavior as a potential strategy to reduce alcohol and drug related harms.

Unusual hydrate stabilization in the two-dimensional layered structure of quinacrinium bis(2-carboxy-4,5-dichlorobenzoate) tetrahydrate, a proton-transfer compound of the drug quinacrine

The crystal structure of the hydrated proton-transfer compound of the drug quinacrine [rac-N'-(6-chloro-2-methoxyacridin-9-yl)-N,N-diethylpentane-1,4-diamine] with 4,5-dichlorophthalic acid, C23H32ClN3O2+ . 2(C8H3Cl2O4-).4H2O (I), has been determined at 200 K. The four labile water molecules of solvation form discrete ...O--H...O--H... hydrogen-bonded chains parallel to the quinacrine side chain, the two N--H groups of which act as hydrogen-bond donors for two of the water acceptor molecules. The other water molecules, as well as the acridinium H atom, also form hydrogen bonds with the two anion species and extend the structure into two-dimensional sheets. Between these sheets there are also weak cation--anion and anion--anion pi-pi aromatic ring interactions. This structure represents only the third example of a simple quinacrine derivative for which structural data are available but differs from the other two in that it is unstable in the X-ray beam due to efflorescence, probably associated with the destruction of the unusual four-membered water chain structures.