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.

Infrared study of methyl formate and formaldehyde adsorption on reduced and oxidised silica-supported copper catalysts

Infrared spectra are reported of methyl formate and formaldehyde adsorbed at 300 K on silica, Cu/SiO2 reduced in hydrogen and Cu/SiO2 which had been oxidised by exposure to nitrous oxide after reduction. Silanol groups on silica form hydrogen bonds with carbonyl groups in weakly adsorbed methyl formate molecules. Methyl formate ligates via its carbonyl groups to Cu atoms in the surface of reduced copper. A low residual concentration of surface oxygen on copper promoted the slow reaction of ligated methyl formate to give a bridging formate species on copper and adsorbed methoxy groups. Methyl formate did not ligate to an oxidised copper surface but was rapidly chemisorbed to give unidentate formate and methoxy species. Formaldehyde slowly polymerises on silica to form trioxane and other oxymethylene species. The reaction is faster over Cu/SiO2 which, in the reduced state, also catalyses the formation of bridging formate anions adsorbed on copper. The reaction between formaldehyde and oxidised Cu/SiO2 leads to both unidentate and bidentate formate and adsorbed water.

Infrared study of the adsorption of formic acid on silica-supported copper and oxidised copper catalysts

Infrared spectra are reported of formic acid adsorbed at 300 K on a reduced copper catalyst (Cu/SiO2) and a copper surface which had been oxidised by exposure to nitrous oxide. Formic acid was weakly adsorbed on the silica support. Ligation of formic acid to the copper surface occurred only on the reduced catalyst. Dissociative adsorption resulted in the formation of unidentate formate on the oxidised catalyst. The presence of reduced copper metal instigated a rapid reorientation to a bidentate formate species.

Infrared study of CO adsorption on reduced and oxidised silica-supported copper catalysts

FTIR spectra are reported of CO adsorbed on silica-supported copper catalysts prepared from copper(II) acetate monohydrate. Fully oxidised catalyst gave bands due to CO on CuO, isolated Cu2+ cations on silica and anion vacancy sites in CuO. The highly dispersed CuO aggregated on reduction to metal particles which gave bands due to adsorbed CO characteristic of both low-index exposed planes and stepped sites on high-index planes. Partial surface oxidation with N2O or H2O generated Cu+ adsorption sites which were slowly reduced to Cu° by CO at 300 K. Surface carbonate initially formed from CO was also slowly depleted with time with the generation of CO2. The results are consistent with adsorbed carbonate being an intermediate in the water-gas shift reaction of H2O and CO to H2 and CO2.

Infrared study of CO, CO2, H2 and H2O interactions on potassium-promoted reduced and oxidised silica-supported copper catalysts

FTIR spectra are reported of CO, CO2, H2 and H2O on silica-supported potassium, copper and potassium-copper catalysts. Adsorption of CO on a potassium/silica catalyst resulted in the formation of complexed CO moieties. Whereas exposure of CO2 to the same catalyst produced bands ascribed to CO2 -, bidentate carbonate and complexed CO species. Fully oxidised copper/silica surfaces gave bands due to CO on CuO and isolated Cu2+ cations on silica. Addition of potassium to this catalyst removed a peak attributed to CO adsorption on isolated Cu2+ cations and red-shifted the maximum ascribed to CO adsorbed on CuO. For a reduced copper/silica catalyst bands due to adsorbed CO on both high and low index planes were red-shifted by 10 cm-1 in the presence of potassium, although the strength of the Cu - CO bond did not appear to be increased concomitantly. An explanation in terms of an electrostatic effect between potassium and adsorbed CO is forwarded. A small maximum at ca. 1510 cm-1 for the reduced catalyst increased substantially upon exposing CO to a reoxidised promoted catalyst. Correspondingly, CO2 adsorption allowed the identification of two distinct carboxylate species, one of which was located at an interfacial site between copper and potassium oxide. Carboxylate species reacted with hydrogen at 295 K, on a reduced copper surface, to produce predominantly unidentate formate on potassium. In contrast no interaction was detected on a reoxidised copper catalyst at 295 K until a fraction of the copper surface was in a reduced state. Furthermore the interaction of polar water molecules with carboxylate species resulted in a perturbation of this structure which gave lower C----O stretching frequencies.

Influence of oxidation and reduction conditions upon the morphology of silica-supported polycrystalline silver catalysts

The effect of oxidation and reduction conditions upon the morphology of polycrystalline silver catalysts has been investigated by means of in situ Fourier-transform infrared (FTIR) spectroscopy. Characterization of the sample was achieved by inspection of the νas(COO) band profile of adsorbed formate, recorded after dosing with formic acid at ambient temperature. Evidence was obtained for the existence of a silver surface reconstructed by the presence of subsurface oxygen in addition to the conventional family of Ag(111) and Ag(110) crystal faces. Oxidation at 773 K facilitated the reconstruction of silver planes due to the formation of subsurface oxygen species. Prolonged oxygen treatment at 773 K also caused particle fragmentation as a consequence of excessive oxygen penetration of the silver catalyst at defect sites. It was also deduced that the presence of oxygen in the gas phase stabilized the growth of silver planes which could form stronger bonds with oxygen. In contrast, high-temperature thermal treatment in vacuum induced significant sintering of the silver catalyst. Reduction at 773 K resulted in substantial quantities of dissolved hydrogen (and probably hydroxy species) in the bulk silver structure. Furthermore, enhanced defect formation in the catalyst was also noted, as evidenced by the increased concentration of formate species associated with oxygen-reconstructed silver faces.

Validating SMPS-measured size distribution of double-mode spherical Silica nanoparticles by Transmission Electron Microscope (TEM)

A nanoparticles size is one of their key physical characteristics that can affect their fate in a human’s respiratory tract (in case of inhalation) and also in the environment. Hence, measuring the size distribution of nanoparticles is absolutely essential and contributes greatly to their characterization. For years, Scanning Mobility Particle Sizers (SMPS), which rely on measuring the electrical mobility diameter of particles, have been used as one of the most reliable real-time instruments for the size distribution measurement of nanoparticles. Despite its benefits, this instrument has some drawbacks, including equivalency problems for non-spherical particles (i.e. assuming a non-spherical particle is equal to a spherical particle of diameter d due to the same electrical mobility), as well as limitations in terms of its use in workplaces, because of its large size and the complexity of its operation...

Randomized controlled trial : a randomized controlled clinical trial comparing a remineralizing paste with an antibacterial gel to prevent early childhood caries

PURPOSE: The purpose of this study was to compare twice daily tooth-brushing using 0.304 percent fluoride toothpaste alone with: (1) twice daily tooth-brushing plus once daily 10% casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) paste; and (2) twice daily tooth-brushing plus once daily 0.12% chlorhexidine gel (CHX) for reducing early childhood caries (ECC) and mutans streptococci (MS) colonization. METHODS: Subjects (n=622) recruited at birth were randomized to receive either CPP-ACP or CHX or no product (study control [SC]). All children were examined at 6, 12, and 18 months old in their homes, and at 24 months old in a community dental clinic. RESULTS: At 24 months old, the caries incidence was 1% (2/163) in CPP-ACP, 2% (4/180) in CHX, and 2% (3/188) in SC groups. In children who were previously MS colonized at 12 and 18 months old, 0% (0/11) and 5% (3/63), respectively, of the CPP-ACP group remained MS-positive versus 22% (2/9) and 72% (18/25) in CHX and 16% (4/25) and 50% (7/14) in SC groups (P<.001). CONCLUSIONS: There is insufficient evidence to justify the daily use of casein phosphopeptide-amorphous calcium phosphate or chlorhexidine gel to control early childhood caries.

A randomised, controlled clinical trial comparing chlorhexidine gel and low-dose fluoride toothpaste to prevent early childhood caries

Objectives This randomised, controlled trial compared the effectiveness of 0.12% chlorhexidine (CHX) gel and 304% fluoride toothpaste to prevent early childhood caries (ECC) in a birth cohort by 24 months. Methods The participants were randomised to receive either (i) twice daily toothbrushing with toothpaste and once daily 0.12% CHX gel (n = 110) or (ii) twice daily toothbrushing with toothpaste only (study controls) (n = 89). The primary outcome measured was caries incidence and the secondary outcome was percentage of children with mutans streptococci (MS). All mothers were contacted by telephone at 6, 12, and 18 months. At 24 months, all children were examined at a community dental clinic. Results At 24 months, the caries prevalence was 5% (3/61) in the CHX and 7% (4/58) in the controls (P = 0.7). There were no differences in percentages of MS-positive children between the CHX and control groups (54%vs 53%). Only 20% applied the CHX gel once daily and 80% less than once daily. Conclusions Toothbrushing using 304% fluoride toothpaste with or without the application of chlorhexidine gel (0.12%) reduces ECC from 23% found in the general community to 5–7%. The lack of effect with chlorhexidine is likely to be due to low compliance.

Modeling the co-precipitation of silica and calcium oxalate in sugar solutions

Solution chemistry plays a significant role in the rate and type of foulant formed on heated industrial surfaces. This paper describes the effect of sucrose, silica (SiO2), Ca2+ and Mg2+ ions, and trans-aconitic acid on the kinetics and solubility of SiO2 and calcium oxalate monohydrate (COM) in mixed salt solutions containing sucrose and refines models previously proposed. The developed SiO2 models show that sucrose and SiO2 concentrations are the main parameters that determine apparent order (n) and apparent rate of reaction (k) and SiO2 solubility over a 24 h period. The calcium oxalate solubility model shows that while increasing [Mg2+] increases COM solubility, the reverse is so with increasing sucrose concentrations. The role of solution species on COM crystal habit is discussed and the appearance of the uncommon (001) face is explained.

Quantitative ultrasound computed tomography imaging of PAGAT radiation dosimetry gel

This research developed and scientifically validated a new ultrasound transmission computed tomography system with the aim of quantitative assessment of a polymer gel dosimeter including dose response verification of ultrasonic parameters of attenuation, velocity and broadband ultrasound attenuation (BUA). This work was the first to investigate and report ultrasound frequency dependent attenuation in a gel dosimeter, demonstrating a dose dependence.

Quantitative proteomic approach to study subcellular localization of membrane proteins

As proteins within cells are spatially organized according to their role, knowledge about protein localization gives insight into protein function. Here, we describe the LOPIT technique (localization of organelle proteins by isotope tagging) developed for the simultaneous and confident determination of the steady-state distribution of hundreds of integral membrane proteins within organelles. The technique uses a partial membrane fractionation strategy in conjunction with quantitative proteomics. Localization of proteins is achieved by measuring their distribution pattern across the density gradient using amine-reactive isotope tagging and comparing these patterns with those of known organelle residents. LOPIT relies on the assumption that proteins belonging to the same organelle will co-fractionate. Multivariate statistical tools are then used to group proteins according to the similarities in their distributions, and hence localization without complete centrifugal separation is achieved. The protocol requires approximately 3 weeks to complete and can be applied in a high-throughput manner to material from many varied sources.

Sub-cellular localization of membrane proteins

In eukaryotes, numerous complex sub-cellular structures exist. The majority of these are delineated by membranes. Many proteins are trafficked to these in order to be able to carry out their correct physiological function. Assigning the sub-cellular location of a protein is of paramount importance to biologists in the elucidation of its role and in the refinement of knowledge of cellular processes by tracing certain activities to specific organelles. Membrane proteins are a key set of proteins as these form part of the boundary of the organelles and represent many important functions such as transporters, receptors, and trafficking. They are, however, some of the most challenging proteins to work with due to poor solubility, a wide concentration range within the cell and inaccessibility to many of the tools employed in proteomics studies. This review focuses on membrane proteins with particular emphasis on sub-cellular localization in terms of methodologies that can be used to determine the accurate location of membrane proteins to organelles. We also discuss what is known about the membrane protein cohorts of major organelles.

Dying feet in ICU : why might extracorporeal membrane oxygenation machines cause necrotic feet?

Background Extracorporeal membrane oxygenation (ECMO) is used for severe lung and/or heart failure in intensive care units (ICU). The Prince Charles Hospital (TPCH) has one of the largest ECMO units in Australia. Its use rapidly increased during the H1N1 (“swine flu”) pandemic and an increase in pedal complications resulted. The relationship between ECMO and pedal complications has been described, particularly in children, though no strong data exists. This paper presents a case series of foot complications in patients having received ECMO treatment. Methods We present nine cases of severe foot complications resulting from patients receiving ECMO treatment at TPCH in 2009–2012. Results Case ages ranged from 16 - 58 years and three were male. Six cases had an unremarkable medical history prior to H1N1 or H1N2 infection, one had Cardiomyopathy, one had received a lung transplant, and one had multi-organ failure post-sepsis. Common medications prescribed included vasopressors, antibiotics, and sedatives. All cases showed signs of markedly impaired peripheral perfusion whilst on ECMO and seven developed increasing areas of foot necrosis. Outcomes include two bilateral below knee amputations, two multiple digital amputations, one Reflex Sympathetic Dystrophy Syndrome, three pressure injuries, and three deaths. Conclusion Necrosis of the feet appears to occur more readily in younger people requiring ECMO treatment than others in ICU. The authors are conducting further studies to investigate associations between particular infections, medical history, medications, or machine techniques and severe foot complications. Some of these early results will also be presented at this conference.

Intranasal immunization with C. muridarum major outer membrane protein (MOMP) and cholera toxin elicits local production of neutralising IgA in the prostate

Successful control of sexually transmitted diseases (STDs) through vaccination will require the development of vaccine strategies that target protective immunity to both the female and male reproductive tracts (MRT). In the male, the immune privileged nature of the male reproductive tract provides a barrier to entry of serum immunoglobulins into the male reproductive ducts, thereby preventing the induction of protective immunity using conventional injectable vaccination techniques. In this study we investigated the potential of intranasal (IN) immunization to elicit anti-chlamydial immunity in BALB/c male mice. Intranasal immunization with Chlamydia muridarum major outer membrane protein (MOMP) admixed with cholera toxin (CT) resulted in high levels of MOMP-specific IgA in prostatic fluids (PF) and MOMP-specific IgA-secreting cells in the prostate. Prostatic fluid IgA inhibited in vitro infection of McCoy cells with C. muridarum. Using RT-PCR we also show that mRNA for the polymeric immunoglobulin receptor (PIgR), which transports IgA across mucosal epithelia, is expressed only in the prostate but not in other regions of the male reproductive ducts upstream of the prostate. These data suggest that using intranasal immunization to target IgA to the prostate may protect males against STDs while at the same time maintaining the state of immune privilege within the MRT.