Development of an ultra-thin fibroin membrane for RPE cell transplantation

Purpose: One of the challenges associated with cell-based therapies for repairing the retina is the development of suitable materials on which to grow and transplant retinal cells. Using the ARPE-19 cell line, we have previously demonstrated the feasibility of growing RPE-derived cells on membranes prepared from the silk protein fibroin. The present study was aimed at developing a porous, ultra-thin fibroin membrane that might better support development of apical-basal polarity in culture, and to extend this work to primary cultures of human RPE cells. Methods: Ultra-thin fibroin membranes were prepared using a highly polished casting table coated with Topas® (a cyclic olefin copolymer) and a 1:0.03 aqueous solution of fibroin and PEO (Mv 900 000 g/mol). Following drying, the membranes were water annealed to make them water-stable, washed in water to remove PEO, sterilised by treatment with 95% ethanol, and washed extensively in saline. Primary cultures containing human RPE cells were established from donor posterior eye cups and maintained in DMEM/F12 medium supplemented with 10% fetal bovine serum and antibiotics. First passage cultures were seeded onto fibroin membranes pre-coated with vitronectin and grown for 6 weeks in medium supplemented with 1% serum. Comparative cultures were established on porous 1.0 µm pore PET membrane (Millipore) and using ARPE-19 cells. Results: The fibroin membranes displayed an average thickness of 3 µm and contained numerous dimples/pore-like structures of up to 3-5 µm in diameter. The primary cultures predominantly contained pigmented epithelial cells, but mesenchymal cells (presumed fibroblasts) were also often present. Passaged cultures appeared to attach equally well to either fibroin or PET membranes. Over time cells on either material adopted a more cobblestoned morphology. Conclusions: Progress has been made towards developing a porous ultra-thin fibroin membrane that supports cultivation of RPE cells. Further studies are required to determine the degree of membrane permeability and RPE polarity.

Repeated intrauterine exposures to inflammatory stimuli attenuated transforming growth factor-β signaling in the ovine fetal lung

Background: Bronchopulmonary dysplasia (BPD) is one of the most common complications after preterm birth and is associated with intrauterine exposure to bacteria. Transforming growth factor-β (TGFβ) is implicated in the development of BPD. Objectives: We hypothesized that different and/or multiple bacterial signals could elicit divergent TGFβ signaling responses in the developing lung. Methods: Time-mated pregnant Merino ewes received an intra-amniotic injection of lipopolysaccharide (LPS) and/or Ureaplasma parvum serovar 3 (UP) at 117 days' and/or 121/122 days' gestational age (GA). Controls received an equivalent injection of saline and or media. Lambs were euthanized at 124 days' GA (term = 150 days' GA). TGFβ1, TGFβ2, TGFβ3, TGFβ receptor (R)1 and TGFβR2 protein levels, Smad2 phosphorylation and elastin deposition were evaluated in lung tissue. Results: Total TGFβ1 and TGFβ2 decreased by 24 and 51% after combined UP+LPS exposure, whereas total TGFβ1 increased by 31% after 7 days' LPS exposure but not after double exposures. Alveolar expression of TGFβR2 decreased 75% after UP, but remained unaltered after double exposures. Decreased focal elastin deposition after single LPS exposure was prevented by double exposures. Conclusions: TGFβ signaling components and elastin responded differently to intrauterine LPS and UP exposure. Multiple bacterial exposures attenuated TGFβ signaling and normalized elastin deposition.

Pro Bono partnerships : community lawyering to promote environmental justice

Rural communities across Australia are increasingly being asked to shoulder the environmental and social impacts of intensive mining and gas projects. Escalating demand for coal seam gas (CSG) is raising significant environmental justice issues for rural communities. Chief amongst environmental concerns are risks of contamination or depletion of vital underground aquifers as well as treatment and disposal of high-saline water close to high quality agricultural soils. Associated infrastructure such as pipelines, electricity lines, gas processing and port facilities can also adversely affect communities and ecosystems great distances from where the gas is originally extracted. Whilst community submission (and appeal) rights do exist, accessing expert independent information is challenging, legal terminology is complex and submission periods are short, leading ultimately to a lack of procedural justice for landholders and their communities. Since August 2012, Queensland University of Technology (QUT) has worked in partnership with not-for-profit legal centre - Queensland’s Environmental Defenders Office (EDO) - to help better educate communities about mining and CSG assessment processes. The project, now entering its third semester, aims to empower communities to access relevant information and actively engage in legal processes on their own behalf. Students involved in the project so far have helped to research chapters of a comprehensive community guide to mining and CSG law as well as organising multidisciplinary community forums and preparing information on land access and compensation rights for landholders. While environmental justice issues still exist without significant law reform, the project has led to greater awareness amongst the community of the laws relating the CSG. At the same time, it has led to a greater understanding by students and academics of real life environmental justice issues currently faced by rural communities.

Characterisation of microvasulature changes after closed soft tissue trauma by contrast enhanced micro-CT imaging

INTRODUCTION It is known that the vascular morphology and functionality are changed following closed soft tissue trauma (CSTT) [1], and bone fractures [2]. The disruption of blood vessels may lead to hypoxia and necrosis. Currently, most clinical methods for the diagnosis and monitoring of CSTT with or without bone fractures are primarily based on qualitative measures or practical experience, making the diagnosis subjective and inaccurate. There is evidence that CSTT and early vascular changes following the injury delay the soft tissue tissue and bone healing [3]. However, a precise qualitative and quantitative morphological assessment of vasculature changes after trauma is currently missing. In this research, we aim to establish a diagnostic framework to assess the 3D vascular morphological changes after standardized CSTT in a rat model qualitatively and quantitatively using contrast-enhanced micro-CT imaging. METHODS An impact device was used for the application of a controlled reproducible CSTT to the left thigh (Biceps Femoris) of anaesthetized male Wistar rats. After euthanizing the animals at 6 hours, 24 hours, 3 days, 7 days, or 14 days after trauma, CSTT was qualitatively evaluated by macroscopic visual observation of the skin and muscles. For visualization of the vasculature, the blood vessels of sacrificed rats were flushed with heparinised saline and then perfused with a radio-opaque contrast agent (Microfil, MV 122, Flowtech, USA) using an infusion pump. After allowing the contrast agent to polymerize overnight, both hind-limbs were dissected, and then the whole injured and contra-lateral control limbs were imaged using a micro-CT scanner (µCT 40, Scanco Medical, Switzerland) to evaluate the vascular morphological changes. Correlated biopsy samples were also taken from the CSTT region of both injured and control legs. The morphological parameters such as the vessel volume ratio (VV/TV), vessel diameter (V.D), spacing (V.Sp), number (V.N), connectivity (V.Conn) and the degree of anisotropy (DA) were then quantified by evaluating the scans of biopsy samples using the micro-CT imaging system. RESULTS AND DISCUSSION A qualitative evaluation of the CSTT has shown that the developed impact protocols were capable of producing a defined and reproducible injury within the region of interest (ROI), resulting in a large hematoma and moderate swelling in both lateral and medial sides of the injured legs. Also, the visualization of the vascular network using 3D images confirmed the ability to perfuse the large vessels and a majority of the microvasculature consistently (Figure 1). Quantification of the vascular morphology obtained from correlated biopsy samples has demonstrated that V.D and V.N and V.Sp were significantly higher in the injured legs 24 hours after impact in comparison with the control legs (p<0.05). The evaluation of the other time points is currently progressing. CONCLUSIONS The findings of this research will contribute to a better understanding of the changes to the vascular network architecture following traumatic injuries and during healing process. When interpreted in context of functional changes, such as tissue oxygenation, this will allow for objective diagnosis and monitoring of CSTT and serve as validation for future non-invasive clinical assessment modalities.

Can vasculature changes be characterised morphologically after closed soft tissue trauma?

INTRODUCTION Closed soft tissue trauma (CSTT) can be the result of a blunt impact, or a prolonged crush injury and involves damage to the skin, muscles and the neurovascular system. It causes a variety of symptoms such as haematoma and in severe cases may result in hypoxia and necrosis. There is evidence that early vasculature changes following the injury delays the tissue healing [1]. However, a precise qualitative and quantitative morphological assessment of vasculature changes after trauma and the effect of this on CSTT healing is currently missing. Research aims: Developing an experimental rat model to characterise the structural changes to the vasculature after trauma qualitatively and quantitatively using micro CT. MATERIAL AND METHODS An impact device was developed to apply a controlled reproducible CSTT to the left thigh (Biceps Femoris) of anaesthetised rats [3]. After euthanizing the animals at 6 hours after trauma, CSTT was qualitatively evaluated by macroscopic observations of the skin and muscles. For vasculature visualisation, the blood vessels of sacrificed rats were flushed with heparinised saline and then perfused with a radio-opaque contrast agent (Microfil) using an infusion pump (Figure 4). The overall changes to the vasculature as a result of impact trauma were characterised qualitatively based on the 3D reconstructed images of the vasculature (Figure 5). For a smaller region of interest, the morphological parameters such as vessel thickness (diameter), spacing, and average number per volume were quantified using the scanner’s software. RESULTS AND DISCUSSION Visual observation of CSTT has revealed a haematoma in some animals (Figure 3). Micro CT images indicate good perfusion of the vasculature with contrast agent, allowing the major vessels to be identified (Figure 5). Qualitatively and quantitatively, no differences between injured and non-injured legs were observed at 6 h after trauma. Further time points of 12h, 24h, 3 days and 14 days after trauma will be characterised for identifying temporal changes of the vasculature during healing. Histomorphometical studies are required for validation of the results derived from the micro CT imaging. CONCLUSION AND FUTURE DIRECTION Findings of this research may contribute towards the establishment of a fundamental basis for the quantitative assessment and monitoring of CSTT based on microvasculature changes after trauma, which will ultimately allow for optimising the clinical treatment and improve patient outcomes.

The electrochemical corrosion behaviour of quaternary gold alloys when exposed to 3.5% NaCl solution

Lower carat gold alloys, specifically 9 carat gold alloys, containing less than 40 % gold, and alloying additions of silver, copper and zinc, are commonly used in many jewellery applications, to offset high costs and poor mechanical properties associated with pure gold. While gold is considered to be chemically inert, the presence of active alloying additions raises concerns about certain forms of corrosion, particularly selective dissolution of these alloys. The purpose of this study was to systematically study the corrosion behaviour of a series of quaternary gold–silver–copper–zinc alloys using dc potentiodynamic scanning in saline (3.5 % NaCl) environment. Full anodic/cathodic scans were conducted to determine the overall corrosion characteristics of the alloy, followed by selective anodic scans and subsequent morphological and compositional analysis of the alloy surface and corroding media to determine the extent of selective dissolution. Varying degrees of selective dissolution and associated corrosion rates were observed after anodic polarisation in 3.5 % NaCl, depending on the alloy composition. The corrosion behaviour of the alloys was determined by the extent of anodic reactions which induce (1) formation of oxide scales on the alloy surface and or (2) dissolution of Zn and Cu species. In general, the improved corrosion characteristics of alloy #3 was attributed to the composition of Zn/Cu in the alloy and thus favourable microstructure promoting the formation of protective oxide/chloride scales and reducing the extent of Cu and Zn dissolution.

Murray Valley Encephalitis

Outbreaks of an acute, severe, encephalitic illness, clinically similar to Japanese and St. Louis encephalitis, occurred in rural areas of southeastern Australia in 1917, 1918, 1922, 1925, 1951, and 1974[1,9,14-16] and in north and northwestern Australia in 1981, 1993, and 2000.[8,12,41] Approximately 420 cases were reported in these nine outbreaks.[41] They are thought to represent a single entity for which various names (Australian X disease, Murray Valley encephalitis, Australian encephalitis) have been used. Twenty-two cases were diagnosed in the 5 years between 2007 and 2011; three were fatal, and one of the fatalities occurred in a Canadian tourist on return from a holiday in northern Australia. Case-fatality rates, as high as 70 percent in the early years,[9,11] declined to 20 percent in the 1974 outbreak and have remained at about this level since then.[5,10,12] However, significant residual neurologic disability occurs in as many as 50 percent of survivors.[10,12] The presence of this disease in Papua New Guinea was confirmed in 1956.[20] The causative virus was transmitted to experimental animals as early as 1918,[6,11] although those strains could not be maintained. The definitive isolation and characterization of Murray Valley encephalitis virus in 1951[19] led to epidemiologic studies that suggested its survival in bird-mosquito cycles in northern Australia but not in the area of epidemic occurrence in southern Australia.[1] Murray Valley encephalitis is caused by Murray Valley encephalitis virus. In an effort to dissociate a disease from a specific locality, the term Australian encephalitis was proposed by residents of Murray Valley for the disease caused by Murray Valley encephalitis virus. Some researchers subsequently have attempted to expand the term Australian encephalitis to include encephalitis caused by any Australian arbovirus. Because the term Australian encephalitis has no scientific validity and is ambiguous, it should not be used.

Vision in water

The purpose of this study is to determine visual performance in water, including the influence of pupil size. The water en-vironment was simulated by placing a goggle filled with saline in front of eyes, with apertures placed at the front of the goggle. Correction factors were determined for the different magnification under this condition in order to to estimate vision in water. Experiments were conducted on letter visual acuity (7 participants), grating resolution (8 participants), and grating contrast sensitivity (1 participant). For letter acuity, mean loss in vision in water, compared to corrected vision in air, varied between 1.1 log minutes of arc resolution (logMAR) for a 1mm aperture to 2.2 logMAR for a 7mm aperture. The vision in minutes of arc was described well by a linear relationship with pupil size. For grating acuity, mean loss varied between 1.1 logMAR for a 2mm aperture to 1.2 logMAR for a 6mm aperture. Contrast sensitivity for a 2mm aperture dete-riorated as spatial frequency increased, with 2 log unit loss by 3 cycles/degree. Superimposed on this deterioration were depressions (notches) in sensitivity, with the first three notches occurring at 0.45, 0.8 and 1.3 cycles/degree with esti-mates for water of 0.39, 0.70 and 1.13 cycles/degree. In conclusion, vision in water is poor. It becomes worse as pupil size increases, but the effects are much more marked for letter targets than for grating targets.

Vision in water

Purpose: To determine visual performance in water, including the influence of pupil size. Method: The water environment was simulated by placing a goggle filled with saline in front of eyes, with apertures placed at the front of the goggle. Correction factors were determined for the different magnification under this condition to estimate vision in water. Experiments were conducted on letter visual acuity (7 participants), grating resolution (8 participants), and grating contrast sensitivity (1 participant). Results: For letter acuity, mean loss in vision in water, compared to corrected vision in air, varied between 1.1 log minutes of arc resolution (logMAR) for a 1mm aperture to 2.2 logMAR for a 7mm aperture. The vision in minutes of arc was described well by a linear relationship with pupil size. For grating acuity, mean loss varied between 1.1 logMAR for a 2mm aperture to 1.2 logMAR for a 6mm aperture. Contrast sensitivity for a 2mm aperture deteriorated as spatial frequency increased, with 2 log unit loss by 3 cycles/degree. Superimposed on this deterioration were depressions (notches) in sensitivity, with the first three notches occurring at 0.45, 0.8 and 1.3 cycles/degree and with estimates for water of 0.39, 0.70 and 1.13 cycles/degree. Conclusion: Vision in water is poor. It becomes worse as pupil size increases, but the effects are much more marked for letter targets than for grating targets.

Cost and environmental efficiency of rice farms in South Korea

We examine cost and nutrient use efficiency of farms and determine the cost to move farms to nutrient-efficient operation using Data Envelopment Analysis (DEA) with a dataset of 96 rice farms in Gangwon province of South Korea from 2003 to 2007. Our findings show that improvements in technical efficiency would result in both lower production costs and better environmental performance. It is, however, not costless for farms to move from their current operation to the environmentally efficient operation. On average, this movement would increase production costs by 119% but benefit the water system through an approximately 69% reduction in eutrofying power (EP). The average estimated cost of each EP kg of aggregate nutrient reduction is approximately one thousand two hundred won. For technically efficient farms, there is a trade-off between cost and environmental efficiency. We also find that the environmental performance of farms varies across farms and regions. We suggest that agri-environmental policies should be (re)designed to improve both cost and environmental performance of rice farms.

Geologic map of the Valley Mountain 15’ quadrangle, San Bernardino and Riverside Counties, California: U.S. Geological Survey Geologic Quadrangle Map GQ–1767, scale 1:62,500

The Valley Mountain 15’ quadrangle straddles the Pinto Mountain Fault, which bounds the eastern Transverse Ranges in the south against the Mojave Desert province in the north. The Pinto Mountains, part of the eastern Transverse Ranges in the south part of the quadrangle expose a series of Paleoproterozoic gneisses and granite and the Proterozoic quartzite of Pinto Mountain. Early Triassic quartz monzonite intruded the gneisses and was ductiley deformed prior to voluminous Jurassic intrusion of diorite, granodiorite, quartz monzonite, and granite plutons. The Jurassic rocks include part of the Bullion Mountains Intrusive Suite, which crops out prominently at Valley Mountain and in the Bullion Mountains, as well as in the Pinto Mountains. Jurassic plutons in the southwest part of the quadrangle are deeply denuded from midcrustal emplacement levels in contrast to supracrustal Jurassic limestone and volcanic rocks exposed in the northeast. Dikes inferred to be part of the Jurassic Independence Dike Swarm intrude the Jurassic plutons and Proterozoic rocks. Late Cretaceous intrusion of the Cadiz Valley Batholith in the northeast caused contact metamorphism of adjacent Jurassic plutonic rocks...

Ureaplasma parvum undergoes selection in utero resulting in genetically diverse isolates colonising the chorioamnion of fetal sheep

Ureaplasmas are the microorganisms most frequently isolated from the amniotic fluid of pregnant women and can cause chronic intrauterine infections. These tiny bacteria are thought to undergo rapid evolution and exhibit a hypermutatable phenotype; however, little is known about how ureaplasmas respond to selective pressures in utero. Using an ovine model of chronic intra-amniotic infection, we investigated if exposure of ureaplasmas to sub-inhibitory concentrations of erythromycin could induce phenotypic or genetic indicators of macrolide resistance. At 55 days gestation, 12 pregnant ewes received an intra-amniotic injection of a non-clonal, clinical U. parvum strain, followed by: (i) erythromycin treatment (IM, 30 mg/kg/day, n=6); or (ii) saline (IM, n=6) at 100 days gestation. Fetuses were then delivered surgically at 125 days gestation. Despite injecting the same inoculum into all ewes, significant differences between amniotic fluid and chorioamnion ureaplasmas were detected following chronic intra-amniotic infection. Numerous polymorphisms were observed in domain V of the 23S rRNA gene of ureaplasmas isolated from the chorioamnion (but not the amniotic fluid), resulting in a mosaic-like sequence. Chorioamnion isolates also harboured the macrolide resistance genes erm(B) and msr(D) and were associated with variable roxithromycin minimum inhibitory concentrations. Remarkably, this variability occurred independently of exposure of ureaplasmas to erythromycin, suggesting that low-level erythromycin exposure does not induce ureaplasmal macrolide resistance in utero. Rather, the significant differences observed between amniotic fluid and chorioamnion ureaplasmas suggest that different anatomical sites may select for ureaplasma sub-types within non-clonal, clinical strains. This may have implications for the treatment of intrauterine ureaplasma infections.

Effect of chemical conjugation of L-leucine to chitosan on the dispersibility and drug release of a dry powder inhaler nanoparticle formulation

Purpose: This study investigated the effect of chemical conjugation of the amino acid L-leucine to the polysaccharide chitosan on the dispersibility and drug release pattern of a polymeric nanoparticle (NP)-based controlled release dry powder inhaler (DPI) formulation. Methods: A chemical conjugate of L-leucine with chitosan was synthesized and characterized by Infrared (IR) Spectroscopy, Nuclear Magnetic Resonance (NMR) Spectroscopy, Elemental Analysis and X-ray Photoelectron Spectroscopy (XPS). Nanoparticles of both chitosan and its conjugate were prepared by a water-in-oil emulsification – glutaraldehyde cross-linking method using the antihypertensive agent, diltiazem (Dz) hydrochloride as the model drug. The surface morphology and particle size distribution of the nanoparticles were determined by Scanning Electron Microscopy (SEM) and Dynamic Light Scattering (DLS). The dispersibility of the nanoparticle formulation was analysed by a Twin Stage Impinger (TSI) with a Rotahaler as the DPI device. Deposition of the particles in the different stages was determined by gravimetry and the amount of drug released was analysed by UV spectrophotometry. The release profile of the drug was studied in phosphate buffered saline at 37 ⁰C and analyzed by UV spectrophotometry. Results: The TSI study revealed that the fine particle fractions (FPF), as determined gravimetrically, for empty and drug-loaded conjugate nanoparticles were significantly higher than for the corresponding chitosan nanoparticles (24±1.2% and 21±0.7% vs 19±1.2% and 15±1.5% respectively; n=3, p<0.05). The FPF of drug-loaded chitosan and conjugate nanoparticles, in terms of the amount of drug determined spectrophotometrically, had similar values (21±0.7% vs 16±1.6%). After an initial burst, both chitosan and conjugate nanoparticles showed controlled release that lasted about 8 to 10 days, but conjugate nanoparticles showed twice as much total drug release compared to chitosan nanoparticles (~50% vs ~25%). Conjugate nanoparticles also showed significantly higher dug loading and entrapment efficiency than chitosan nanoparticles (conjugate: 20±1% & 46±1%, chitosan: 16±1% & 38±1%, n=3, p<0.05). Conclusion: Although L-leucine conjugation to chitosan increased dispersibility of formulated nanoparticles, the FPF values are still far from optimum. The particles showed a high level of initial burst release (chitosan, 16% and conjugate, 31%) that also will need further optimization.

The Torrent : Toowoomba and the Lockyer Valley, 10 January 2011

Of all the stories to emerge from Queensland’s catastrophic summer of 2011, the most dramatic and starkly tragic were those that took place in Toowoomba and the Lockyer Valley. On January 10, 2011, after weeks of heavy rain and as floodwaters began to overwhelm much of south-east Queensland, an ‘inland tsunami’ hit the city of Toowoomba, the rural districts of Spring Bluff and Postmans Ridge, and the towns of Murphys Creek, Withcott, Helidon, and Grantham. The Torrent:Toowoomba and the Lockyer Valley, 10 January 2011 tells, for the first time, the extraordinary stories of survival and loss that emerged from that terrible day. Official figures state that twenty-four people died. Many escaped death only because they were rescued by members of the community or through sheer good fortune. Based on exclusive interviews with survivors, rescuers and with the families and friends of victims of the disaster, The Torrent is a unique and powerful account of human courage in the face of the devastating force of nature, and the enduring resilience of ordinary Australians.

Accessibility of pores in coal to methane and carbon dioxide

Fluid–solid interactions in natural and engineered porous solids underlie a variety of technological processes, including geological storage of anthropogenic greenhouse gases, enhanced coal bed methane recovery, membrane separation, and heterogeneous catalysis. The size, distribution and interconnectivity of pores, the chemical and physical properties of the solid and fluid phases collectively dictate how fluid molecules migrate into and through the micro- and meso-porous media, adsorb and ultimately react with the solid surfaces. Due to the high penetration power and relatively short wavelength of neutrons, smallangle neutron scattering (SANS) as well as ultra small-angle scattering (USANS) techniques are ideally suited for assessing the phase behavior of confined fluids under pressure as well as for evaluating the total porosity in engineered and natural porous systems including coal. Here we demonstrate that SANS and USANS can be also used for determining the fraction of the pore volume that is actually accessible to fluids as a function of pore sizes and study the fraction of inaccessible pores as a function of pore size in three coals from the Illinois Basin (USA) and Bowen Basin (Australia). Experiments were performed at CO2 and methane pressures up to 780 bar, including pressures corresponding to zero average contrast condition (ZAC), which is the pressure where no scattering from the accessible pores occurs. Scattering curves at the ZAC were compared with the scattering from same coals under vacuum and analysed using a newly developed approach that shows that the volume fraction of accessible pores in these coals varies between �90% in the macropore region to �30% in the mesopore region and the variation is distinctive for each of the examined coals. The developed methodology may be also applied for assessing the volume of accessible pores in other natural underground formations of interest for CO2 sequestration, such as saline aquifers as well as for estimating closed porosity in engineered porous solids of technological importance.