Phage library screening for the rapid identification and in vivo testing of candidate genes for a DNA vaccine against Mycoplasma mycoides subsp. mycoides small colony biotype

A new strategy for rapidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage λ ZAP Express vector which contains both prokaryotic (Plac) and eukaryotic (PCMV) promoters upstream of the insertion site. The phage library is plated on Escherichia coli cells, immunoblotted, and probed with hyperimmune and/or convalescent-phase antiserum to rapidly identify vaccine candidates. These are then plaque purified and grown as liquid lysates, and whole bacteriophage particles are then used directly to immunize the host, following which PCMV-driven expression of the candidate vaccine gene occurs. In the example given here, a semirandom genome library of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony (SC) biotype was cloned into λ ZAP Express, and two strongly immunodominant clones, λ-A8 and λ-B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone λ-A8 expressed an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible M. mycoides subsp. mycoides SC biotype protein with a 28-kDa apparent molecular mass, identified as a previously uncharacterized putative lipoprotein (MSC_0397). Clone λ-B1 contained several full-length genes from the M. mycoides subsp. mycoides SC biotype pyruvate dehydrogenase region, and two IPTG-independent polypeptides, of 29 kDa and 57 kDa, were identified on immunoblots. Following vaccination, significant anti-M. mycoides subsp. mycoides SC biotype responses were observed in mice vaccinated with clones λ-A8 and λ-B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone λ-A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone λ-A8 also exhibited a reduced level of mycoplasmemia compared to controls, suggesting that the MSC_0397 lipoprotein has a protective effect in the mouse model when delivered as a bacteriophage DNA vaccine. Bacteriophage-mediated immunoscreening using an appropriate vector system offers a rapid and simple technique for the identification and immediate testing of putative candidate vaccines from a variety of pathogens.

Combining passive sampling and toxicity testing for evaluation of mixtures of polar organic chemicals in sewage treatment plant effluent

Effluent from sewage treatment plants has been associated with a range of pollutant effects. Depending on the influent composition and treatment processes the effluent may contain a myriad of different chemicals which makes monitoring very complex. In this study we aimed to monitor relatively polar organic pollutant mixtures using a combination of passive sampling techniques and a set of biochemistry based assays covering acute bacterial toxicity (Microtox™), phytotoxicity (Max-I-PAM assay) and genotoxicity (umuC assay). The study showed that all of the assays were able to detect effects in the samples and allowed a comparison of the two plants as well as a comparison between the two sampling periods. Distinct improvements in water quality were observed in one of the plants as result of an upgrade to a UV disinfection system, which improved from 24× sample enrichment required to induce a 50% response in the Microtox™ assay to 84×, from 30× sample enrichment to induce a 50% reduction in photosynthetic yield to 125×, and the genotoxicity observed in the first sampling period was eliminated. Thus we propose that biochemical assay techniques in combination with time integrated passive sampling can substantially contribute to the monitoring of polar organic toxicants in STP effluents.

Design and experimental testing of air slab caps which convert commercial electron diodes into dual purpose, correction-free diodes for small field dosimetry

Purpose Two diodes which do not require correction factors for small field relative output measurements are designed and validated using experimental methodology. This was achieved by adding an air layer above the active volume of the diode detectors, which canceled out the increase in response of the diodes in small fields relative to standard field sizes. Methods Due to the increased density of silicon and other components within a diode, additional electrons are created. In very small fields, a very small air gap acts as an effective filter of electrons with a high angle of incidence. The aim was to design a diode that balanced these perturbations to give a response similar to a water-only geometry. Three thicknesses of air were placed at the proximal end of a PTW 60017 electron diode (PTWe) using an adjustable “air cap”. A set of output ratios (ORfclin Det ) for square field sizes of side length down to 5 mm was measured using each air thickness and compared to ORfclin Det measured using an IBA stereotactic field diode (SFD). k fclin, f msr Qclin,Qmsr was transferred from the SFD to the PTWe diode and plotted as a function of air gap thickness for each field size. This enabled the optimal air gap thickness to be obtained by observing which thickness of air was required such that k fclin, f msr Qclin,Qmsr was equal to 1.00 at all field sizes. A similar procedure was used to find the optimal air thickness required to make a modified Sun Nuclear EDGE detector (EDGEe) which s “correction-free” in small field relative dosimetry. In addition, the feasibility of experimentally transferring k fclin, f msr Qclin,Qmsr values from the SFD to unknown diodes was tested by comparing the experimentally transferred k fclin, f msr Qclin,Qmsr values for unmodified PTWe and EDGEe diodes to Monte Carlo simulated values. Results 1.0 mm of air was required to make the PTWe diode correction-free. This modified diode (PTWeair) produced output factors equivalent to those in water at all field sizes (5–50 mm). The optimal air thickness required for the EDGEe diode was found to be 0.6 mm. The modified diode (EDGEeair) produced output factors equivalent to those in water, except at field sizes of 8 and 10 mm where it measured approximately 2% greater than the relative dose to water. The experimentally calculated k fclin, f msr Qclin,Qmsr for both the PTWe and the EDGEe diodes (without air) matched Monte Carlo simulated results, thus proving that it is feasible to transfer k fclin, f msr Qclin,Qmsr from one commercially available detector to another using experimental methods and the recommended experimental setup. Conclusions It is possible to create a diode which does not require corrections for small field output factor measurements. This has been performed and verified experimentally. The ability of a detector to be “correction-free” depends strongly on its design and composition. A nonwater-equivalent detector can only be “correction-free” if competing perturbations of the beam cancel out at all field sizes. This should not be confused with true water equivalency of a detector.

Cytotoxicity testing of silver-containing burn treatments using primary and immortal skin cells

A novel burn wound hydrogel dressing has been previously developed which is composed of 2-acrylamido-2-methylpropane sulfonic acid sodium salt with silver nanoparticles (silver AMPS). This study compared the cytotoxicity of this dressing to the commercially available silver products; Acticoat™, PolyMem Silver® and Flamazine™ cream. Human keratinocytes (HaCaT and primary HEK) and normal human fibroblasts (NHF) were exposed to dressings incubated on Nunc™ polycarbonate inserts for 24, 48 and 72h. Four different cytotoxicity assays were performed including; Trypan Blue cell count, MTT, Celltiter-Blue™ and Toluidine Blue surface area assays. The results were expressed as relative cell viability compared to an untreated control. The cytotoxic effects of Acticoat™ and Flamazine™ cream were dependent on exposure time and cell type. After 24h exposure, Acticoat™ and Flamazine™ cream were toxic to all tested cell lines. Surprisingly, HaCaTs treated with Acticoat™ and Flamazine™ had an improved ability to survive at 48 and 72h while HEKs and NHFs had no improvement in survival with any treatment. The novel silver hydrogel and PolyMem Silver® showed low cytotoxicity to all tested cell lines at every time interval and these results support the possibility of using the novel silver hydrogel as a burn wound dressing. Researchers who rely on HaCaT cells as an accurate keratinocyte model should be aware that they can respond differently to primary skin cells.

Very high resolution optical satellites for DEM generation : a review

The advent of very high resolution (VHR) optical satellites capable of producing stereo images led to a new era in extracting digital elevation model which commenced with the launch of IKONOS. The special specifications of VHR optical satellites besides, the significant economic profit stimulated other countries and companies to have their constellations such as EROS-A1 and EROS-B1 as the cooperation between Israel and ImageSat. QuickBird, WorldView-1 and WorldVew-2 were launched by DigitalGlobe. ALOS and GeoEye-1 were offered by Japan and GeoEye Respectively. In addition to aforementioned satellites, Indian and South Korea initiated their own constellation by launching CartoSat-1 and KOPOSAT-2 respectively.The availability of all so-called satellites make a huge market of stereo images for extracting of digital elevation model and other correspondent applications such as, producing orthorectifcatin images and updating maps. Therefore, there is a need for a comprehensive comparison for scientific and commercial clients to choose appropriate satellite images and methods of generating digital elevation model to obtain optimum results. This paper will thus give a review about the specifications of VHR optical satellites. Then it will discuss the automatic elaborating of digital elevation model. Finally an overview of studies and corresponding results is reported.

Testing the effectiveness of a self-efficacy based exercise intervention for adults with venous leg ulcers : protocol of a randomised controlled trial

Background: Exercise and adequate self-management capacity may be important strategies in the management of venous leg ulcers. However, it remains unclear if exercise improves the healing rates of venous leg ulcers and if a self-management exercise program based on self-efficacy theory is well adhered to. Method/Design: This is a randomised controlled in adults with venous leg ulcers to determine the effectiveness of a self-efficacy based exercise intervention. Participants with venous leg ulcers are recruited from 3 clinical sites in Australia. After collection of baseline data, participants are randomised to either an intervention group or control group. The control group receive usual care, as recommended by evidence based guidelines. The intervention group receive an individualised program of calf muscle exercises and walking. The twelve week exercise program integrates multiple elements, including up to six telephone delivered behavioural coaching and goal setting sessions, supported by written materials, a pedometer and two follow-up booster calls if required. Participants are encouraged to seek social support among their friends, self-monitor their weekly steps and lower limb exercises. The control group are supported by a generic information sheet that the intervention group also receive encouraging lower limb exercises, a pedometer for self-management and phone calls at the same time points as the intervention group. The primary outcome is the healing rates of venous leg ulcers which are assessed at fortnightly clinic appointments. Secondary outcomes, assessed at baseline and 12 weeks: functional ability (range of ankle motion and Tinetti gait and balance score), quality of life and self-management scores. Discussion: This study seeks to address a significant gap in current wound management practice by providing evidence for the effectiveness of a home-based exercise program for adults with venous leg ulcers. Theory-driven, evidence-based strategies that can improve an individual’s exercise self-efficacy and self-management capacity could have a significant impact in improving the management of people with venous leg ulcers. Information gained from this study will provide much needed information on management of this chronic disease to promote health and independence in this population. Trial registration: Australian New Zealand Clinical Trials Registry ACTRN12612000475842 Trial status: Current follow up

Functionalized Ag nanoparticles with tunable optical properties for selective protein analysis

We present a preparation procedure for small sized biocompatibly coated Ag nanoparticles with tunable surface plasmon resonances. The conditions were optimised with respect to the resonance Raman signal enhancement of heme proteins and to the preservation of the native protein structure....

An MRF based abnormal event detection approach using motion and appearance features

Abnormal event detection has attracted a lot of attention in the computer vision research community during recent years due to the increased focus on automated surveillance systems to improve security in public places. Due to the scarcity of training data and the definition of an abnormality being dependent on context, abnormal event detection is generally formulated as a data-driven approach where activities are modeled in an unsupervised fashion during the training phase. In this work, we use a Gaussian mixture model (GMM) to cluster the activities during the training phase, and propose a Gaussian mixture model based Markov random field (GMM-MRF) to estimate the likelihood scores of new videos in the testing phase. Further-more, we propose two new features: optical acceleration, and the histogram of optical flow gradients; to detect the presence of any abnormal objects and speed violations in the scene. We show that our proposed method outperforms other state of the art abnormal event detection algorithms on publicly available UCSD dataset.

Development of realistic design fire time-temperature curves for the testing of cold-formed steel wall system

Fire resistance rating of light gauge steel frame (LSF) wall systems is obtained from fire tests based on the standard fire time-temperature curve. However, fire severity has increased in modern buildings due to higher fuel loads as a result of modern furniture and light weight constructions that make use of thermoplastics materials, synthetic foams and fabrics. Some of these materials are high in calorific values and increase both the spread of fire growth and heat release rate, thus increasing the fire severity beyond that of the standard fire curve. Further, the standard fire curve does not include a decay phase that is present in natural fires. Despite the increasing usage of LSF walls, their behaviour in real building fires is not fully understood. This paper presents the details of a research study aimed at developing realistic design fire curves for use in the fire tests of LSF walls. It includes a review of the characteristics of building fires, previously developed fire time-temperature curves, computer models and available parametric equations. The paper highlights that real building fire time-temperature curves depend on the fuel load representing the combustible building contents, ventilation openings and thermal properties of wall lining materials, and provides suitable values of many required parameters including fuel loads in residential buildings. Finally, realistic design fire time-temperature curves simulating the fire conditions in modern residential buildings are proposed for the testing of LSF walls.

The effect of topical adrenergic and anticholinergic agents on the choroidal thickness of young healthy adults

The human choroid is capable of rapidly changing its thickness in response to a variety of stimuli. However little is known about the role of the autonomic nervous system in the regulation of the thickness of the choroid. Therefore, we investigated the effect of topical parasympatholytic and sympathomimetic agents upon the choroidal thickness and ocular biometrics of young healthy adult subjects. Fourteen subjects (mean age 27.9 ± 4 years) participated in this randomized, single-masked, placebo-controlled study. Each subject had measurements of choroidal thickness (ChT) and ocular biometrics of their right eye taken before, and then 30 and 60 min following the administration of topical pharmacological agents. Three different drugs: 2% homatropine hydrobromide, 2.5% phenylephrine hydrochloride and a placebo (0.3% hydroxypropyl methylcellulose) were tested in all subjects; each on different days (at the same time of the day) in randomized order. Participants were masked to the pharmacological agent being used at each testing session. The instillation of 2% homatropine resulted in a small but significant increase in subfoveal ChT at 30 and 60 min after drug instillation (mean change 7 ± 3 μm and 14 ± 2 μm respectively; both p < 0.0001). The parafoveal choroid also exhibited a similar magnitude, significant increase in thickness with time after 2% homatropine (p < 0.001), with a mean change of 7 ± 0.3 μm and 13 ± 1 μm (in the region located 0.5 mm from the fovea center), 6 ± 1 μm and 12.5 ± 1 μm (1 mm from the fovea center) and 6 ± 2 μm and 12 ± 2 μm (1.5 mm from the fovea center) after 30 and 60 min respectively. Axial length decreased significantly 60 min after homatropine (p < 0.01). There were also significant changes in lens thickness (LT) and anterior chamber depth (ACD) (p < 0.05) associated with homatropine instillation. No significant changes in choroidal thickness, or ocular biometrics were found after 2.5% phenylephrine or placebo at any examination points (p > 0.05). In human subjects, significant increases in subfoveal and parafoveal choroidal thickness occurred after administration of 2% homatropine and this implies an involvement of the parasympathetic system in the control of choroidal thickness in humans.

Visual field coordinates of pupillary circular axis and optical axis

Purpose: We term the visual field position from which the pupil appears most nearly circular as the pupillary circular axis (PCAx). The aim was to determine and compare the horizontal and vertical co-ordinates of the PCAx and optical axis from pupil shape and refraction information for only the horizontal meridian of the visual field. Method: The PCAx was determined from the changes with visual field angle in the ellipticity and orientation of pupil images out to ±90° from fixation along the horizontal meridian for the right eyes of 30 people. This axis was compared with the optical axis determined from the changes in the astigmatic components of the refractions for field angles out to ±35° in the same meridian. Results: The mean estimated horizontal and vertical field coordinates of the PCAx were (‒5.3±1.9°, ‒3.2±1.5°) compared with (‒4.8±5.1°, ‒1.5±3.4°) for the optical axis. The vertical co-ordinates of the two axes were just significantly different (p =0.03) but there was no significant correlation between them. Only the horizontal coordinate of the PCAx was significantly related to the refraction in the group. Conclusion: On average, the PCAx is displaced from the line-of-sight by about the same angle as the optical axis but there is more inter-subject variation in the position of the optical axis. When modelling the optical performance of the eye, it appears reasonable to assume that the pupil is circular when viewed along the line-of-sight.

A multiple case study of NAPLAN numeracy testing of Year 9 students in three Queensland secondary schools

This thesis reports on a multiple case study of the actions of three Queensland secondary schools in the context of Year 9 NAPLAN numeracy testing, focusing on their administrative practices, curriculum, pedagogy and assessment. It was established that schools have found it both challenging and costly to operate in an environment of educational reform generally, and NAPLAN testing in particular. The lack of a common understanding of numeracy and the substantial demands of implementing the Australian Curriculum have impacted on schools' ability to prepare students appropriately for NAPLAN numeracy tests. It was concluded that there is scope for schools to improve their approaches to NAPLAN numeracy testing in a way that maximises learning as well as test outcomes.

Confirmatory factor analysis and invariance testing of the Young Carer of Parents Inventory (YCOPI)

Objective Research into youth caregiving in families where a parent experiences a significant medical condition has been hampered by a lack of contextually sensitive measures of the nature and breadth of young caregiving experiences. This study examined the factor structure and measurement invariance of such a measure called the Young Carer of Parents Inventory (YCOPI; Pakenham et al., 2006) using confirmatory factor analysis across 3 groups of youth. The YCOPI has 2 parts: YCOPI-A with 5 factors assessing caregiving experiences that are applicable to all caregiving contexts; YCOPI-B with 4 factors that tap dimensions related to youth caregiving in the context of parent illness. Design Two samples (ages 9–20 years) were recruited: a community sample of 2,429 youth from which 2 groups were derived (“healthy” family [HF], n = 1760; parental illness [PI], n = 446), and a sample of 130 youth of a parent with multiple sclerosis). Results With some modification, the YCOPI-A demonstrated a replicable factor structure across 3 groups, and exhibited only partial measurement invariance across the HF and PI groups. The impact of assuming full measurement invariance on latent mean differences appeared small, supporting use of the measure in research and applied settings when estimated using latent factors and controlling for measurement invariance. PI youth reported significantly higher scores than did HF youth on all YCOPI-A subscales. The YCOPI-B requires some modifications, and further development work is recommended. Conclusion The factor structure that emerged and the addition of new items constitutes the YCOPI-Revised. Findings support the use of the YCOPI-Revised in research and applied settings.