Synthesis and characterization of delta-atracotoxin-ar1a, the lethal neurotoxin from venom of the Sydney funnel-web spider (Atrax robustus)

delta-Atracotoxin-Ar1a (delta-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion alpha-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing delta-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. H-1 NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic delta-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that delta-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic delta-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.

Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge

We have isolated a family of insect-selective neurotoxins from the venom of the Australian funnel-web spider that appear to be good candidates for biopesticide engineering. These peptides, which we have named the Janus-faced atracotoxins (J-ACTXs), each contain 36 or 37 residues, with four disulfide bridges, and they show no homology to any sequences in the protein/DNA databases. The three-dimensional structure of one of these toxins reveals an extremely rare vicinal disulfide bridge that we demonstrate to be critical for insecticidal activity. We propose that J-ACTX comprises an ancestral protein fold that we refer to as the disulfide-directed beta-hairpin.

Using the World Wide Web to revolutionise technology transfer and training in the water and wastewater industries

Industry professionals of the near future will be supported by an IT infrastructure that enables them to complete a task by drawing on resources and people with expertise anywhere in the world, and access to knowledge through specific training programs that address the task requirements. The increasing uptake of new technologies enables information to reach a diverse population and to provide flexible learning environments 24 hours a day, 7 days a week. This paper examines one of the key areas where the World Wide Web will impact on the water and wastewater industries, namely technology transfer and training. The authors will present their experiences of developing online training courses for wastewater industry professionals over the last two years. The perspective is that of two people working at the coalface.

Cytotoxic iron chelators: Characterization of the structure, solution chemistry and redox activity of ligands and iron complexes of the di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) analogues

Di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) and a range of its analogues comprise a series of monobasic acids that are capable of binding iron (Fe) as tridentate (N,N,O) ligands. Recently, we have shown that these chelators are highly cytotoxic, but show selective activity against cancer cells. Particularly interesting was the fact that cytotoxicity of the HPKIH analogues is maintained even after complexation with Fe. To understand the potent anti-tumor activity of these compounds, we have fully characterized their chemical properties. This included examination of the solution chemistry and X-ray crystal structures of both the ligands and Fe complexes from this class and the ability of these complexes to mediate redox reactions. Potentiometric titrations demonstrated that all chelators are present predominantly in their charge-neutral form at physiological pH (7.4), allowing access across biological membranes. Keto-enol tautomerism of the ligands was identified, with the tautomers exhibiting distinctly different protonation constants. Interestingly, the chelators form low-spin (diamagnetic) divalent Fe complexes in solution. The chelators form distorted octahedral complexes with Fe-II, with two tridentate ligands arranged in a meridional fashion. Electrochemistry of the Fe complexes in both aqueous and non-aqueous solutions revealed that the complexes are oxidized to their ferric form at relatively high potentials, but this oxidation is coupled to a rapid reaction with water to form a hydrated (carbinolamine) derivative, leading to irreversible electrochemistry. The Fe complexes of the HPKIH analogues caused marked DNA degradation in the presence of hydrogen peroxide. This observation confirms that Fe complexes from the HPKIH series mediate Fenton chemistry and do not repel DNA. Collectively, studies on the solution chemistry and structure of these HPKIH analogues indicate that they can bind cellular Fe and enhance its redox activity, resulting in oxidative damage to vital biomolecules.

Discovering user access pattern based on probabilistic latent factor model

There has been an increased demand for characterizing user access patterns using web mining techniques since the informative knowledge extracted from web server log files can not only offer benefits for web site structure improvement but also for better understanding of user navigational behavior. In this paper, we present a web usage mining method, which utilize web user usage and page linkage information to capture user access pattern based on Probabilistic Latent Semantic Analysis (PLSA) model. A specific probabilistic model analysis algorithm, EM algorithm, is applied to the integrated usage data to infer the latent semantic factors as well as generate user session clusters for revealing user access patterns. Experiments have been conducted on real world data set to validate the effectiveness of the proposed approach. The results have shown that the presented method is capable of characterizing the latent semantic factors and generating user profile in terms of weighted page vectors, which may reflect the common access interest exhibited by users among same session cluster.

A web recommendation technique based on probabilistic latent semantic analysis

Web transaction data between Web visitors and Web functionalities usually convey user task-oriented behavior pattern. Mining such type of click-stream data will lead to capture usage pattern information. Nowadays Web usage mining technique has become one of most widely used methods for Web recommendation, which customizes Web content to user-preferred style. Traditional techniques of Web usage mining, such as Web user session or Web page clustering, association rule and frequent navigational path mining can only discover usage pattern explicitly. They, however, cannot reveal the underlying navigational activities and identify the latent relationships that are associated with the patterns among Web users as well as Web pages. In this work, we propose a Web recommendation framework incorporating Web usage mining technique based on Probabilistic Latent Semantic Analysis (PLSA) model. The main advantages of this method are, not only to discover usage-based access pattern, but also to reveal the underlying latent factor as well. With the discovered user access pattern, we then present user more interested content via collaborative recommendation. To validate the effectiveness of proposed approach, we conduct experiments on real world datasets and make comparisons with some existing traditional techniques. The preliminary experimental results demonstrate the usability of the proposed approach.

A synchronous multimedia annotation system for secure collaboratories

In this paper, we describe the Vannotea system - an application designed to enable collaborating groups to discuss and annotate collections of high quality images, video, audio or 3D objects. The system has been designed specifically to capture and share scholarly discourse and annotations about multimedia research data by teams of trusted colleagues within a research or academic environment. As such, it provides: authenticated access to a web browser search interface for discovering and retrieving media objects; a media replay window that can incorporate a variety of embedded plug-ins to render different scientific media formats; an annotation authoring, editing, searching and browsing tool; and session logging and replay capabilities. Annotations are personal remarks, interpretations, questions or references that can be attached to whole files, segments or regions. Vannotea enables annotations to be attached either synchronously (using jabber message passing and audio/video conferencing) or asynchronously and stand-alone. The annotations are stored on an Annotea server, extended for multimedia content. Their access, retrieval and re-use is controlled via Shibboleth identity management and XACML access policies.

Research Quality, Publications and Impact in Hydraulic Engineering into the 21st Century. Publish or Perish, Commercial versus Open Access, Internet versus Libraries

One of the main objectives of the first International Junior Researcher and Engineer Workshop on Hydraulic Structures is to provide an opportunity for young researchers and engineers to present their research. But a research project is only completed when it has been published and shared with the community. Referees and peer experts play an important role to control the research quality. While some new electronic tools provide further means to disseminate some research information, the quality and impact of the works remain linked with some thorough expert-review process and the publications in international scientific journals and books. Importantly unethical publishing standards are not acceptable and cheating is despicable.

Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis

The maternally inherited intracellular symbiont Wolbachia pipientis is well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning and characterization of the gene (wsp) that encodes it. The functionality of the wsp promoter region was also successfully tested in Escherichia coli. Comparison of sequences of this gene from different strains of Wolbachia revealed a high level of variability. This sequence variation correlated with the ability of certain Wolbachia strains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects. As such, this gene will be a very useful tool for Wolbachia strain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction of Wolbachia with its host.

Impact of Commercial Search Engines and International Databases on Engineering Teaching and Research

For the last three decades, the engineering higher education and professional environments have been completely transformed by the "electronic/digital information revolution" that has included the introduction of personal computer, the development of email and world wide web, and broadband Internet connections at home. Herein the writer compares the performances of several digital tools with traditional library resources. While new specialised search engines and open access digital repositories may fill a gap between conventional search engines and traditional references, these should be not be confused with real libraries and international scientific databases that encompass textbooks and peer-reviewed scholarly works. An absence of listing in some Internet search listings, databases and repositories is not an indication of standing. Researchers, engineers and academics should remember these key differences in assessing the quality of bibliographic "research" based solely upon Internet searches.

Characterization of turbulent jets from high-aspect-ratio rectangular nozzles

Turbulent free jets issuing from rectangular slots with various high aspect ratios (15-120) are characterized. The centerline mean and rms velocities are measured using hot-wire anemometry over a downstream distance of up to 160 slot heights at a slot-height-based Reynolds number of 10000. Experimental results suggest that a rectangular jet with sufficiently high aspect ratio (> 15) may be distinguished between three flow zones: an initial quasi-plane-jet zone, a transition zone, and a final quasi-axisymmetric-jet zone. In the quasi-plane-jet zone, the turbulent velocity field is statistically similar, but not identical, to those of a plane jet. (c) 2005 American Institute of Physics.

Solution structure of robustoxin, the lethal neurotoxin from the funnel-web spider Atrax robustus

The solution structure of robustoxin, the lethal neurotoxin from the Sydney funnel-web spider Atrax robustus, has been determined from 2D H-1 NMR data, Robustoxin is a polypeptide of 42 residues cross-linked by four disulphide bonds, the connectivities of which were determined from NMR data and trial structure calculations to be 1-15, 8-20, 14-31 and 16-42 (a 1-4/2-6/3-7/5-8 pattern), The structure consists of a small three-stranded, anti-parallel beta-sheet and a series of interlocking gamma-turns at the C-terminus. It also contains a cystine knot, thus placing it in the inhibitor cystine knot motif family of structures, which includes the omega-conotoxins and a number of plant and animal toxins and protease inhibitors. Robustoxin contains three distinct charged patches on its surface, and an extended loop that includes several aromatic and non-polar residues, Both of these structural features may play a role in its binding to the voltage-gated sodium channel. (C) 1997 Federation of European Biochemical Societies.

Characterization of calcium-activated chloride channels in patches excised from the dendritic knob of mammalian olfactory receptor neurons

We investigated the properties of calcium-activated chloride channels in inside-out membrane patches from the dendritic knobs of acutely dissociated rat olfactory receptor neurons. Patches typically contained large calcium-activated currents, with total conductances in the range 30-75 nS. The dose response curve for calcium exhibited an EC50 of about 26 mu M. In symmetrical NaCl solutions, the current-voltage relationship reversed at 0 mV and was linear between -80 and +70 mV. When the intracellular NaCl concentration was progressively reduced from 150 to 25 mM, the reversal potential changed in a manner consistent with a chloride-selective conductance. Indeed, modeling these data with the Goldman-Hodgkin-Katz equation revealed a P-Na/P-Cl of 0.034. The halide permeability sequence was P-Cl > P-F > P-I > P-Br indicating that permeation through the channel was dominated by ion binding sites with a high field strength. The channels were also permeable to the large organic anions, SCN-, acetate(-), and gluconate(-), with the permeability sequence P-Cl > P-SCN > gluconaie. Significant permeation to gluconate ions suggested that the channel pore had a minimum diameter of at least 5.8 Angstrom.

A novel, open access, elliptical cross-section magnet for paediatric MRI

Almost all clinical magnetic resonance imaging systems are based on circular cross-section magnets. Recent advances in elliptical cross-section RF probe and gradient coil hardware raise the question of the possibility of using elliptical cross-section magnet systems, This paper presents a methodology for calculating rapidly the magnetic fields generated by a multi-turn coil of elliptical cross-section and incorporates this in a stochastic optimization method for magnet design, An open magnet system of elliptical cross-section is designed that both reduces the claustrophobia for the patients and allows ready access by attending physicians, The magnet system is optimized for paediatric use, The coil geometry produced by the optimization method has several novel features.