3× multiplexed detection of antibiotic resistant plasmids with single molecule sensitivity.

Bacterial pathogens resistant to antibiotics have become a serious health threat. Those species which have developed resistance against multiple drugs such as the carbapenems, are more lethal as these are last line therapy antibiotics. Current diagnostic tests for these resistance traits are based on singleplex target amplification techniques which can be time consuming and prone to errors. Here, we demonstrate a chip based optofluidic system with single molecule sensitivity for amplification-free, multiplexed detection of plasmids with genes corresponding to antibiotic resistance, within one hour. Rotating disks and microfluidic chips with functionalized polymer monoliths provided the upstream sample preparation steps to selectively extract these plasmids from blood spiked with E. coli DH5α cells. Waveguide-based spatial multiplexing using a multi-mode interference waveguide on an optofluidic chip was used for parallel detection of three different carbapenem resistance genes. These results point the way towards rapid, amplification-free, multiplex analysis of antibiotic-resistant pathogens.

Reversal of respiratory failure in both neonatal and late onset isolated remethylation disorders.

Respiratory failure is a well-documented potential presentation of inherited isolated remethylation disorders (IRDs). It appears to be a combination of both central and peripheral neuropathy and has previously often been considered to herald an irreversible neurological decline. We present three patients, one with methionine synthase (cblG) and two with methyltetrahydrofolate reductase deficiency (MTHFR). One patient with MTHFR presented in infancy, and other patients in later childhood. All three patients required intubation for respiratory failure but in all three, this was totally reversed by the initiation of medical therapy. This consisted of betaine and folinic acid supplementation in all three, methionine in two and cobalamin supplementation in two. The rate of respiratory improvement was variable, though two of the cases were successful extubated within a week of commencement of medical therapy. We document their subsequent clinical, biochemical and electrophysiological progress and review the potential pathological mechanisms underlying respiratory failure in these disorders.

Formation of eLiposomes as a drug delivery vehicle.

This paper discusses the formation of eLiposomes, defined as a liposome with internal emulsion droplets. Liposomes have been investigated as passively targeted drug carriers due to their ability to deliver drugs to a cancerous tumor via the enhanced permeability and retention (EPR) effect. The enclosed emulsion droplets in an eLiposome add the ability to further control the location and time of release from the liposome with ultrasound. Emulsion droplets were formed from perfluorohexane (PFC6) by sonication at 20 kHz and stabilized with dipalmitoyl phosphatidyl choline (DPPC). The size of the resulting droplets was reduced to approximately 100 nm or 50 nm by extrusion through polycarbonate filters of the same size at 50°C. Small unilamellar vesicles (SUVs) were prepared from DPPC by thin film hydration and extrusion through a 50 nm filter. Interdigitated DPPC sheets were prepared from the SUVs by the addition of ethanol to a concentration of 3M. Excess ethanol was removed by centrifugation washing. The sheets were mixed with emulsion and the solution was heated to 50°C, resulting in the refolding of the DPPC sheets into closed vesicles. Emulsion droplets were encapsulated inside of the newly formed eLiposomes. The size of the eLiposomes was reduced by extrusion. Cryogenic transmission electron microscopy (cryoTEM) and negative-staining TEM were used to image the emulsion droplets and the eLiposomes. Encapsulation of emulsion droplets was verified by rotating the microscope stage of cryoTEM samples.

Paediatric Research in Emergency Departments International Collaborative (PREDICT): first steps towards the development of an Australian and New Zealand research network.

Paediatric emergency research is hampered by a number of barriers that can be overcome by a multicentre approach. In 2004, an Australia and New Zealand-based paediatric emergency research network was formed, the Paediatric Research in Emergency Departments International Collaborative (PREDICT). The founding sites include all major tertiary children's hospital EDs in Australia and New Zealand and a major mixed ED in Australia. PREDICT aims to provide leadership and infrastructure for multicentre research at the highest standard, facilitate collaboration between institutions, health-care providers and researchers and ultimately improve patient outcome. Initial network-wide projects have been determined. The present article describes the development of the network, its structure and future goals.

Swellable coatings for hearing aid applications.

The problem of acoustic feedback in hearing aids could be solved potentially by applying a compliant hydrogel to the outer surface that would conform to the ear canal and block feedback. With this objective, several formulations of hydrogels were developed and their swelling and mechanical properties investigated. Hydrogel formulations were polymerized from hydroxyethyl methacrylate (HEMA) and N-vinyl-pyrrolidone (NVP), with various photo-initiators, crosslinkers, and swelling agents. The hydrogel that swelled most rapidly and yet remained undissolved in water had a monomer composition of 40 mol% HEMA, 60 mol% NVP, with 1 wt% polyethylene glycol dimethacrylate as a crosslinker, and 0.5 wt% 2,2-dimethoxy-2-phenyl-acetophenone as the photo-initiator. The tensile modulus, strength, hardness, and durability of the dry hydrogels were not a strong function of composition. In the swollen state, the mechanical properties were much reduced. The potential use of these materials on hearing aids has been discussed in this article.

Effect of pulsed ultrasound in combination with gentamicin on bacterial viability in biofilms on bone cements in vivo.

AIMS: The aim of this study is to investigate whether pulsed ultrasound (US) in combination with gentamicin yields a decreased viability of bacteria in biofilms on bone cements in vivo. METHODS AND RESULTS: Bacterial survival on bone cement in the presence and absence of ultrasound was compared in a rabbit model. Two bone cement samples with an Escherichia coli ATCC 10798 biofilm were implanted in a total of nine rabbits. In two groups bone cement discs loaded with gentamicin, freshly prepared and aged were used, and in one group unloaded bone cement discs in combination with systemically administered gentamicin. Pulsed ultrasound with a frequency of 28.48 kHz and a maximum acoustic intensity of 500 mW cm(-2) was applied continuously from 24 h till 72 h postsurgery on one of the two implanted discs. After euthanization and removal of the bacteria from the discs, the number of viable bacteria were quantified and skin samples were analysed for histopathological examination. Application of ultrasound, combined with gentamicin, reduced the viability of the biofilms in all three groups varying between 58 and 69% compared with the negative control. Histopathological examinations showed no skin lesions. CONCLUSIONS: Ultrasound resulted in a tendency of improved efficacy of gentamicin, either applied locally or systemically. Usage of ultrasound in this model proved to be safe. SIGNIFICANCE AND IMPACT OF THE STUDY: This study implies that ultrasound could improve the prevention of infection immediately after surgery, especially because the biomaterials, gentamicin and ultrasound used in this model are all in clinical usage, but not yet combined in clinical practice.

Selecting compounds for focused screening using linear discriminant analysis and artificial neural networks.

Linear discriminant analysis and a committee of neural networks have been applied to recognise compounds that act at biological targets belonging to a specific gene family, protein kinases. The MDDR database was used to provide compounds targeted against this family and sets of randomly selected molecules. BCUT parameters were employed as input descriptors that encode structural properties and information relevant to ligand-receptor interactions. The technique was applied to purchasing compounds from external suppliers. These compounds achieved hit rates on a par with those achieved using known actives for related targets when tested for the ability to inhibit kinases at a single concentration. This approach is intended as one of a series of filters in the selection of screening candidates, compound purchases and the application of synthetic priorities to combinatorial libraries.

Ultrasonically enhanced vancomycin activity against Staphylococcus epidermidis biofilms in vivo.

Infection of implanted medical devices by Gram-positive organisms such as Staphylococcus ssp. is a serious concern in the biomaterial community. In this research the application of low frequency ultrasound to enhance the activity of vancomycin against implanted Staphylococcus epidermidis biofilms was examined. Polyethylene disks covered with a biofilm of S. epidermidis were implanted subcutaneously in rabbits on both sides of their spine. The rabbits received systemic vancomycin for the duration of the experiment. Following 24 h of recovery, one disk was insonated for 24 or 48 h while the other was a control. Disks were removed and viable bacteria counted. At 24 h of insonation, there was no difference in viable counts between control and insonated biofilms, while at 48 h of insonation there were statistically fewer viable bacteria in the insonated biofilm. The S. epidermidis biofilms responded favorably to combinations of ultrasound and vancomycin, but longer treatment times are required for this Gram-positive organism than was observed previously for a Gram-negative species.

Transmission of Neospora caninum between wild and domestic animals.

To determine whether deer can transmit Neospora caninum, brains of naturally infected white-tailed deer (Odocoileus virginianus) were fed to 4 dogs; 2 of these dogs shed oocysts. Oocysts from 1 of the dogs were tested by polymerase chain reaction and found to be positive for N. caninum and negative for Hammondia heydorni. The internal transcribed spacer 1 sequence of the new strain (designated NC-deer1) was identical to N. caninum from domestic animals, indicating that N. caninum is transmitted between wild and domestic animals, often enough to prevent divergent evolution of isolated populations of the parasite. NC-deerl oocysts were administered to a calf that developed a high antibody titer, providing evidence that N. caninum from wildlife can infect cattle. In addition, N. caninum antibody seroprevalence was detected in 64/164 (39%) free-ranging gray wolves (Canis lupus), 12/113 (11%) coyotes (Canis latrans), 50/193 (26%) white-tailed deer, and 8/61 (13%) moose (Alces alces). These data are consistent with a sylvatic transmission cycle of N. caninum between cervids and canids. We speculate that hunting by humans favors the transmission of N. caninum from deer to canids, because deer carcasses are usually eviscerated in the field. Infection of canids in turn increases the risk of transmitting the parasite to domestic livestock.

The Bioinformatics Template Library--generic components for biocomputing.

MOTIVATION: The efficiency of bioinformatics programmers can be greatly increased through the provision of ready-made software components that can be rapidly combined, with additional bespoke components where necessary, to create finished programs. The new standard for C++ includes an efficient and easy to use library of generic algorithms and data-structures, designed to facilitate low-level component programming. The extension of this library to include functionality that is specifically useful in compute-intensive tasks in bioinformatics and molecular modelling could provide an effective standard for the design of reusable software components within the biocomputing community. RESULTS: A novel application of generic programming techniques in the form of a library of C++ components called the Bioinformatics Template Library (BTL) is presented. This library will facilitate the rapid development of efficient programs by providing efficient code for many algorithms and data-structures that are commonly used in biocomputing, in a generic form that allows them to be flexibly combined with application specific object-oriented class libraries. AVAILABILITY: The BTL is available free of charge from our web site http://www.cryst.bbk.ac.uk/~classlib/ and the EMBL file server http://www.embl-ebi.ac.uk/FTP/index.html

Arylsulphonyl hydroxamic acids: potent and selective matrix metalloproteinase inhibitors.

A series of novel matrix metalloproteinase inhibitors is described in which selectivity between MMP and 'sheddase' activity has been achieved and which demonstrate potent in vivo activity in models of arthritis and cancer.

Childhood poisoning in Queensland: an analysis of presentation and admission rates.

OBJECTIVE: To determine the presentation rates for paediatric poisoning by ingestion and the determinants of hospital admission. METHODOLOGY: Cross-sectional survey using an injury surveillance database from emergency departments in South Brisbane, Mackay and Mt Isa, Queensland, from January 1998 to December 1999. There were 1516 children aged 0-14 years who presented following ingestional poisoning. RESULTS: The presentation rates for poisoning were 690, 40 and 67 per 100000 population aged 0-4, 5-9 and 10-14 years, respectively. The admission rates to hospital for poisoning were 144, 14 and 22 per 100000 population aged 0-4, 5-9 and 10-14 years, respectively. Although presentation rates for poisoning were higher in the rural centres the admission rates were disproportionately high for the 0-4 years age group. The agents most frequently ingested were paracetamol, Dimetapp, rodenticides and essential oils. CONCLUSION: There is a need to design and implement interventions aimed at reducing poison exposures and unnecessary hospital admissions in the 0-4 years age group.

Factors affecting acoustically triggered release of drugs from polymeric micelles.

A custom ultrasonic exposure chamber with real-time fluorescence detection was used to measure acoustically-triggered drug release from Pluronic P-105 micelles under continuous wave (CW) or pulsed ultrasound in the frequency range of 20 to 90 kHz. The measurements were based on the decrease in fluorescence intensity when drug was transferred from the micelle core to the aqueous environment. Two fluorescent drugs were used: doxorubicin (DOX) and its paramagnetic analogue, ruboxyl (Rb). Pluronic P-105 at various concentrations in aqueous solutions was used as a micelle-forming polymer. Drug release was most efficient at 20-kHz ultrasound and dropped with increasing ultrasonic frequency despite much higher power densities. These data suggest an important role of transient cavitation in drug release. The release of DOX was higher than that of Rb due to stronger interaction and deeper insertion of Rb into the core of the micelles. Drug release was higher at lower Pluronic concentrations, which presumably resulted from higher local drug concentrations in the core of Pluronic micelles when the number of micelles was low. At constant frequency, drug release increased with increasing power density. At constant power density and for pulse duration longer than 0.1 s, peak release under pulsed ultrasound was the same as stationary release under CW ultrasound. Released drug was quickly re-encapsulated between the pulses of ultrasound, which suggests that upon leaving the sonicated volume, the non-extravasated and non-internalized drug would circulate in the encapsulated form, thus preventing unwanted drug interactions with normal tissues.

Behavior of Listeria monocytogenes in pasteurized milk during fermentation with lactic acid bacteria.

The behavior of Listeria monocytogenes in pasteurized milk during fermentation with starter and nonstarter lactic acid bacteria was investigated. Pasteurized milk was co-inoculated with approximately 10(4) CFU/ml of L. monocytogenes and 10(6) CFU/ml of Lactococcus lactis, Lactococcus cremoris, Lactobacillus plantarum, Lactobacillus bulgaricus, or Streptococcus thermophilus. Inoculated milks were incubated at 30 degrees C or 37 degrees C for 24 to 72 h. Listeria monocytogenes survived and also grew to some extent during incubation in the presence of all starter cultures; however, inhibition ranged from 83 to 100% based on maximum cell populations. During incubation with L. bulgaricus and L. plantarum, L. monocytogenes was completely inactivated after 20 h and 64 h of incubation at 37 degrees C and 30 degrees C, respectively. The pH of the fermenting milks declined steadily throughout the fermentation periods and was approximately 4.2 at the conclusion of the experimental period regardless both of the starter culture and pathogen combination or the temperature of incubation.

DNA damage induced by micellar-delivered doxorubicin and ultrasound: comet assay study.

To minimize adverse side effects of chemotherapy, we have developed a micellar drug carrier that retains hydrophobic drugs, and then releases the drug by ultrasonic stimulation. This study investigated the DNA damage induced by doxorubicin (DOX) delivered to human leukemia (HL-60) cells from pluronic P-105 micelles with and without the application of ultrasound. The comet assay was used to quantify the amount of DNA damage. No significant DNA damage was observed when the cells were treated with 0.1, 1 and 10 wt% P-105 with or without ultrasound (70 kHz, 1.3 W/cm(2)) for 1 h or for up to 3 h in 10 wt% P-105. However, when cells were incubated with 10 microg/ml free DOX for up to 9 h, DNA damage increased with incubation time (P=0.0011). Exposure of cells to the same concentration of DOX in the presence of 10-wt% P-105 showed no significant DNA damage for up to 9 h of incubation. However, when ultrasound was applied, a rapid and significant increase in DNA damage was observed (P=0.0001). The application of ultrasound causes the release of DOX from micelles or causes the HL-60 cells to take up the micelle encapsulated DOX. Our experiments indicated that the combination of DOX, ultrasound and pluronic P105 causes the largest DNA damage to HL-60 cells. We believe that this technique can be used for controlled drug delivery.

Pulsed ultrasound enhances the killing of Escherichia coli biofilms by aminoglycoside antibiotics in vivo.

Escherichia coli biofilms on two polyethylene disks were implanted subcutaneously into rabbits receiving systemic gentamicin. Ultrasound was applied for 24 h to one disk. Both disks were removed, and viable bacteria were counted. Pulsed ultrasound significantly reduced bacterial viability below that of nontreated biofilms without damage to the skin.

Attachment of hyaluronic acid to polypropylene, polystyrene, and polytetrafluoroethylene.

Surfaces of polypropylene (PP), polystyrene (PS) and polytetrafluoroethylene (PTFE) were activated with radio frequency plasmas Ar and NH3 to aminate the polymer surface and were subsequently reacted with hyaluronic acid (HA) in one of the three different attachment schemes. Results show that ammonia plasma treated polymers were more reactive toward HA attachment. The three chemistry schemes consisted of two distinct approaches: (1) direct attachment of the HA to the aminated surface, and (2) extending the reactive group away from the surface with succinic anhydride and then reacting the newly formed carboxylic acid group with an adipic dihydrazide modified HA (HA-ADH). The latter scheme proved to be more effective, suggesting that steric effects were involved with the reactivity of the HA with surface groups. These HA-coated polymers are a candidate for cell attachment and growth.

Investigation of the mechanism of the bioacoustic effect.

Bacterial biofilms growing on implanted medical devices are difficult to eradicate, even with aggressive antibiotic therapy. However, application of ultrasound enhances the effectiveness of the antibiotic. The possible mechanisms of this phenomenon were explored in light of the observed influence of various ultrasonic parameters on the enhanced action of gentamicin against biofilms of Pseudomonas aeruginosa. It is postulated that ultrasound increases the transport of gentamicin through the cell membranes, which is the proposed rate determining step in killing by gentamicin. It is possible that the ultrasound perturbs the cell membrane and stimulates active uptake or permits passive uptake by temporarily disrupting the membrane or other structural cell components. The cell membrane disruption could be caused by high pressure, high shear stress, or cavitation. The dependence upon peak power density suggests that acoustic pressure plays a significant role. There is also a strong frequency component that causes the killing effect to decrease as frequency increases. A mathematical analysis of oscillatory shear stress on the cell shows that the magnitude of stress increases with frequency; thus, the hypothesis of oscillatory shear inducing antibiotic uptake is discounted. In addition, the shear displacement caused by shear forces is very small, so the shear disruption caused by oscillatory flow in an acoustic field has minimal impact. The experimental data also rule out the existence of transient cavitation in the bioacoustic effect. It is possible that stable cavitation and the accompanying microstreaming contribute to the bioacoustic effect.