In vitro and in vivo examination of the cell surface glycoprotein CDCP1

A number of reports have demonstrated the importance of the CUB domaincontaining protein 1 (CDCP1) in facilitating cancer progression in animal models and the potential of this protein as a prognostic marker in several malignancies. CDCP1 facilitates metastasis formation in animal models by negatively regulating anoikis, a type of apoptosis triggered by the loss of attachment signalling from cell-cell contacts or cell-extra cellular matrix (ECM) contacts. Due to the important role CDCP1 plays in cancer progression in model systems, it is considered a potential drug target to prevent the metastatic spread of cancers. CDCP1 is a highly glycosylated 836 amino acid cell surface protein. It has structural features potentially facilitating protein-protein interactions including 14 N-glycosylation sites, three CUB-like domains, 20 cysteine residues likely to be involved in disulfide bond formation and five intracellular tyrosine residues. CDCP1 interacts with a variety of proteins including Src family kinases (SFKs) and protein kinase C ä (PKCä). Efforts to understand the mechanisms regulating these interactions have largely focussed on three CDCP1 tyrosine residues Y734, Y743 and Y762. CDCP1-Y734 is the site where SFKs phosphorylate and bind to CDCP1 and mediate subsequent phosphorylation of CDCP1-Y743 and -Y762 which leads to binding of PKCä at CDCP1-Y762. The resulting trimeric protein complex of SFK•CDCP1•PKCä has been proposed to mediate an anti-apoptotic cell phenotype in vitro, and to promote metastasis in vivo. The effect of mutation of the three tyrosines on interactions of CDCP1 with SFKs and PKCä and the consequences on cell phenotype in vitro and in vivo have not been examined. CDCP1 has a predicted molecular weight of ~90 kDa but is usually detected as a protein which migrates at ~135 kDa by Western blot analysis due to its high degree of glycosylation. A low molecular weight form of CDCP1 (LMWCDCP1) of ~70 kDa has been found in a variety of cancer cell lines. The mechanisms leading to the generation of LMW-CDCP1 in vivo are not well understood but an involvement of proteases in this process has been proposed. Serine proteases including plasmin and trypsin are able to proteolytically process CDCP1. In addition, the recombinant protease domain of the serine protease matriptase is also able to cleave the recombinant extracellular portion of CDCP1. Whether matriptase is able to proteolytically process CDCP1 on the cell surface has not been examined. Importantly, proteolytic processing of CDCP1 by trypsin leads to phosphorylation of its cell surface-retained portion which suggests that this event leads to initiation of an intracellular signalling cascade. This project aimed to further examine the biology of CDCP1 with a main of focus on exploring the roles played by CDCP1 tyrosine residues. To achieve this HeLa cells stably expressing CDCP1 or the CDCP1 tyrosine mutants Y734F, Y743F and Y762F were generated. These cell lines were used to examine: • The roles of the tyrosine residues Y734, Y743 and Y762 in mediating interactions of CDCP1 with binding proteins and to examine the effect of the stable expression on HeLa cell morphology. • The ability of the serine protease matriptase to proteolytically process cell surface CDCP1 and to examine the consequences of this event on HeLa cell phenotype and cell signalling in vitro. • The importance of these residues in processes associated with cancer progression in vitro including adhesion, proliferation and migration. • The role of these residues on metastatic phenotype in vivo and the ability of a function-blocking anti-CDCP1 antibody to inhibit metastasis in the chicken embryo chorioallantoic membrane (CAM) assay. Interestingly, biochemical experiments carried out in this study revealed that mutation of certain CDCP1 tyrosine residues impacts on interactions of this protein with binding proteins. For example, binding of SFKs as well as PKCä to CDCP1 was markedly decreased in HeLa-CDCP1-Y734F cells, and binding of PKCä was also reduced in HeLa-CDCP1-Y762F cells. In contrast, HeLa-CDCP1-Y743F cells did not display altered interactions with CDCP1 binding proteins. Importantly, observed differences in interactions of CDCP1 with binding partners impacted on basal phosphorylation of CDCP1. It was found that HeLa-CDCP1, HeLa-CDCP1-Y743F and -Y762F displayed strong basal levels of CDCP1 phosphorylation. In contrast, HeLa-CDCP1-Y734F cells did not display CDCP1 phosphorylation but exhibited constitutive phosphorylation of focal adhesion kinase (FAK) at tyrosine 861. Significantly, subsequent investigations to examine this observation suggested that CDCP1-Y734 and FAK-Y861 are competitive substrates for SFK-mediated phosphorylation. It appeared that SFK-mediated phosphorylation of CDCP1- Y734 and FAK-Y861 is an equilibrium which shifts depending on the level of CDCP1 expression in HeLa cells. This suggests that the level of CDCP1 expression may act as a regulatory mechanism allowing cells to switch from a FAK-Y861 mediated pathway to a CDCP1-Y734 mediated pathway. This is the first time that a link between SFKs, CDCP1 and FAK has been demonstrated. One of the most interesting observations from this work was that CDCP1 altered HeLa cell morphology causing an elongated and fibroblastic-like appearance. Importantly, this morphological change depended on CDCP1- Y734. In addition, it was observed that this change in cell morphology was accompanied by increased phosphorylation of SFK-Y416. This suggests that interactions of SFKs with CDCP1-Y734 increases SFK activity since SFKY416 is critical in regulating kinase activity of these proteins. The essential role of SFKs in mediating CDCP1-induced HeLa cell morphological changes was demonstrated using the SFK-selective inhibitor SU6656. This inhibitor caused reversion of HeLa-CDCP1 cell morphology to an epithelial appearance characteristic of HeLa-vector cells. Significantly, in vitro studies revealed that certain CDCP1-mediated cell phenotypes are mediated by cellular pathways dependent on CDCP1 tyrosine residues whereas others are independent of these sites. For example, CDCP1 expression caused a marked increase in HeLa cell motility that was independent of CDCP1 tyrosine residues. In contrast, CDCP1- induced decrease in HeLa cell proliferation was most prominent in HeLa- CDCP1-Y762F cells, potentially indicating a role for this site in regulating proliferation in HeLa cells. Another cellular event which was identified to require phosphorylation of a particular CDCP1 tyrosine residue is adhesion to fibronectin. It was observed that the CDCP1-mediated strong decrease in adhesion to fibronectin is mostly restored in HeLa-CDCP1-Y743F cells. This suggests a possible role for CDCP1-Y743 in causing a CDCP1-mediated decrease in adhesion. Data from in vivo experiments indicated that HeLa-CDCP1-Y734F cells are more metastic than HeLa-CDCP1 cells in vivo. This indicates that interaction of CDCP1 with SFKs and PKCä may not be required for CDCP1-mediated metastasis formation of HeLa cells in vivo. The metastatic phenotype of these cells may be caused by signalling involving FAK since HeLa-CDCP1- Y734F cells are the only CDCP1 expressing cells displaying constitutive phosphorylation of FAK-Y861. HeLa-CDCP1-Y762F cells displayed a very low metastatic ability which suggests that this CDCP1 tyrosine residue is important in mediating a pro-metastatic phenotype in HeLa cells. More detailed exploration of cellular events occurring downstream of CDCP1-Y734 and -Y762 may provide important insights into the mechanisms altering the metastatic ability of CDCP1 expressing HeLa cells. Complementing the in vivo studies, anti-CDCP1 antibodies were employed to assess whether these antibodies are able to inhibit metastasis of CDCP1 and CDCP1 tyrosine mutants expressing HeLa cells. It was found that HeLa- CDCP1-Y734F cells were the only cell line which was markedly reduced in the ability to metastasise. In contrast, the ability of HeLa-CDCP1, HeLa- CDCP1-Y743F and -Y762F cells to metastasise in vivo was not inhibited. These data suggest a possible role of interactions of CDCP1 with SFKs, occurring at CDCP1-Y734, in preventing an anti-metastatic effect of anti- CDCP1 antibodies in vivo. The proposal that SFKs may play a role in regulating anti-metastatic effects of anti-CDCP1 antibodies was supported by another experiment where differences between HeLa-CDCP1 cells and CDCP1 expressing HeLa cells (HeLa-CDCP1-S) from collaborators at the Scripps Research Institute were examined. It was found that HeLa-CDCP1-S cells express different SFKs than CDCP1 expressing HeLa cells generated for this study. This is important since HeLa-CDCP1-S cells can be inhibited in their metastatic ability using anti-CDCP1 antibodies in vivo. Importantly, these data suggest that further examinations of the roles of SFKs in facilitating anti-metastatic effects of anti-CDCP1 antibodies may give insights into how CDCP1 can be blocked to prevent metastasis in vivo. This project also explored the ability of the serine protease matriptase to proteolytically process cell surface localised CDCP1 because it is unknown whether matriptase can cleave cell surface CDCP1 as it has been reported for other proteases such as trypsin and plasmin. Furthermore, the consequences of matriptase-mediated proteolysis on cell phenotype in vitro and cell signalling were examined since recent reports suggested that proteolysis of CDCP1 leads to its phosphorylation and may initiate cell signalling and consequently alter cell phenotype. It was found that matriptase is able to proteolytically process cell surface CDCP1 at low nanomolar concentrations which suggests that cleavage of CDCP1 by matriptase may facilitate the generation of LWM-CDCP1 in vivo. To examine whether matriptase-mediated proteolysis induced cell signalling anti-phospho Erk 1/2 Western blot analysis was performed as this pathway has previously been examined to study signalling in response to proteolytic processing of cell surface proteins. It was found that matriptase-mediated proteolysis in CDCP1 expressing HeLa cells initiated intracellular signalling via Erk 1/2. Interestingly, this increase in phosphorylation of Erk 1/2 was also observed in HeLa-vector cells. This suggested that initiation of cell signalling via Erk 1/2 phosphorylation as a result of matriptase-mediated proteolysis occurs by pathways independent of CDCP1. Subsequent investigations measuring the flux of free calcium ions and by using a protease-activated receptor 2 (PAR2) agonist peptide confirmed this hypothesis. These data suggested that matriptase-mediated proteolysis results in cell signalling via a pathway induced by the activation of PAR2 rather than by CDCP1. This indicates that induction of cell signalling in HeLa cells as a consequence of matriptase-mediated proteolysis occurs via signalling pathways which do not involve phosphorylation of Erk 1/2. Consequently, it appears that future attempts should focus on the examination of cellular pathways other than Erk 1/2 to elucidate cell signalling initiated by matriptase-mediated proteolytic processing of CDCP1. The data presented in this thesis has explored in vitro and in vivo aspects of the biology of CDCP1. The observations summarised above will permit the design of future studies to more precisely determine the role of CDCP1 and its binding partners in processes relevant to cancer progression. This may contribute to further defining CDCP1 as a target for cancer treatment.

An analytical tool that quantifies cellular morphology changes from three-dimensional fluorescence images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology(1) even in complex tissue sections(2). Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells(3), however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgenössische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

Using optical coherence tomography to assess corneoscleral morphology after soft contact lens wear

Purpose. To evaluate the use of optical coherence tomography (OCT) to assess the effect of different soft contact lenses on corneoscleral morphology. Methods. Ten subjects had anterior segment OCT B-scans taken in the morning and again after six hours of soft contact lens wear. For each subject, three different contact lenses were used in the right eye on non-consecutive days, including a hydrogel sphere, a silicone hydrogel sphere and a silicone hydrogel toric. After image registration and layer segmentation, analyses were performed of the first hyper-reflective layer (HRL), the epithelial basement membrane (EBL) and the epithelial thickness (HRL to EBL). A root mean square difference (RMSD) of the layer profiles and the thickness change between the morning and afternoon measurements, was used to assess the effect of the contact lens on the corneoscleral morphology. Results. The soft contact lenses had a statistically significant effect on the morphology of the anterior segment layers (p <0.001). The average amounts of change for the three lenses (average RMSD values) for the corneal region were lower (3.93±1.95 µm for the HRL and 4.02±2.14 µm for the EBL) than those measured in the limbal/scleral region (11.24±6.21 µm for the HRL and 12.61±6.42 µm for the EBL). Similarly, averaged across the three lenses, the RMSD in epithelial thickness was lower in the cornea (2.84±0.84 µm) than the limbal/scleral (5.47±1.71 µm) region. Post-hoc analysis showed that ocular surface changes were significantly smaller with the silicone hydrogel sphere lens than both the silicone hydrogel toric (p<0.005) and hydrogel sphere (p<0.02) for the combined HRL and EBL data. Conclusions. In this preliminary study, we have shown that soft contact lenses can produce small but significant changes in the morphology of the limbal/scleral region and that OCT technology is useful in assessing these changes. The clinical significance of these changes is yet to be determined.

Effect of rehydration on structure and surface properties of thermally treated palygorskite

Palygorskite has a fibrous like morphology with a distinctive layered appearance. The simplified formula of palygorskite (Mg5Si8O20(OH)2(OH2)4 nH2O) indicates that two different types of water are present. The dehydration and rehydration of palygorskite have been studied using thermogravimetry and H2O-tem- perature programmed desorption. X-ray diffractograms, NH3 adsorption profiles, and NH3 desorption profiles were obtained for thermally treated palygorskite as a function of temperature. The results proved water molecules were mainly derived from Si–OH units. In addition, five kinds of acid sites were found for palygorskite. The number of acid sites of external surfaces was larger than that of the internal sur- faces. Bonding on the internal surface acid sites was stronger than the bonding of the external surfaces. Rehydration restored the folded structure of palygorskite when thermal treatment temperature was lower than 300 oC.

A catchment modelling approach integrating surface and groundwater processes, land use and distribution of nutrients : Elimbah Creek, southeast Queensland

As the world’s population is growing, so is the demand for agricultural products. However, natural nitrogen (N) fixation and phosphorus (P) availability cannot sustain the rising agricultural production, thus, the application of N and P fertilisers as additional nutrient sources is common. It is those anthropogenic activities that can contribute high amounts of organic and inorganic nutrients to both surface and groundwaters resulting in degradation of water quality and a possible reduction of aquatic life. In addition, runoff and sewage from urban and residential areas can contain high amounts of inorganic and organic nutrients which may also affect water quality. For example, blooms of the cyanobacterium Lyngbya majuscula along the coastline of southeast Queensland are an indicator of at least short term decreases of water quality. Although Australian catchments, including those with intensive forms of land use, show in general a low export of nutrients compared to North American and European catchments, certain land use practices may still have a detrimental effect on the coastal environment. Numerous studies are reported on nutrient cycling and associated processes on a catchment scale in the Northern Hemisphere. Comparable studies in Australia, in particular in subtropical regions are, however, limited and there is a paucity in the data, in particular for inorganic and organic forms of nitrogen and phosphorus; these nutrients are important limiting factors in surface waters to promote algal blooms. Therefore, the monitoring of N and P and understanding the sources and pathways of these nutrients within a catchment is important in coastal zone management. Although Australia is the driest continent, in subtropical regions such as southeast Queensland, rainfall patterns have a significant effect on runoff and thus the nutrient cycle at a catchment scale. Increasingly, these rainfall patterns are becoming variable. The monitoring of these climatic conditions and the hydrological response of agricultural catchments is therefore also important to reduce the anthropogenic effects on surface and groundwater quality. This study consists of an integrated hydrological–hydrochemical approach that assesses N and P in an environment with multiple land uses. The main aim is to determine the nutrient cycle within a representative coastal catchment in southeast Queensland, the Elimbah Creek catchment. In particular, the investigation confirms the influence associated with forestry and agriculture on N and P forms, sources, distribution and fate in the surface and groundwaters of this subtropical setting. In addition, the study determines whether N and P are subject to transport into the adjacent estuary and thus into the marine environment; also considered is the effect of local topography, soils and geology on N and P sources and distribution. The thesis is structured on four components individually reported. The first paper determines the controls of catchment settings and processes on stream water, riverbank sediment, and shallow groundwater N and P concentrations, in particular during the extended dry conditions that were encountered during the study. Temporal and spatial factors such as seasonal changes, soil character, land use and catchment morphology are considered as well as their effect on controls over distributions of N and P in surface waters and associated groundwater. A total number of 30 surface and 13 shallow groundwater sampling sites were established throughout the catchment to represent dominant soil types and the land use upstream of each sampling location. Sampling comprises five rounds and was conducted over one year between October 2008 and November 2009. Surface water and groundwater samples were analysed for all major dissolved inorganic forms of N and for total N. Phosphorus was determined in the form of dissolved reactive P (predominantly orthophosphate) and total P. In addition, extracts of stream bank sediments and soil grab samples were analysed for these N and P species. Findings show that major storm events, in particular after long periods of drought conditions, are the driving force of N cycling. This is expressed by higher inorganic N concentrations in the agricultural subcatchment compared to the forested subcatchment. Nitrate N is the dominant inorganic form of N in both the surface and groundwaters and values are significantly higher in the groundwaters. Concentrations in the surface water range from 0.03 to 0.34 mg N L..1; organic N concentrations are considerably higher (average range: 0.33 to 0.85 mg N L..1), in particular in the forested subcatchment. Average NO3-N in the groundwater has a range of 0.39 to 2.08 mg N L..1, and organic N averages between 0.07 and 0.3 mg N L..1. The stream bank sediments are dominated by organic N (range: 0.53 to 0.65 mg N L..1), and the dominant inorganic form of N is NH4-N with values ranging between 0.38 and 0.41 mg N L..1. Topography and soils, however, were not to have a significant effect on N and P concentrations in waters. Detectable phosphorus in the surface and groundwaters of the catchment is limited to several locations typically in the proximity of areas with intensive animal use; in soil and sediments, P is negligible. In the second paper, the stable isotopes of N (14N/15N) and H2O (16O/18O and 2H/H) in surface and groundwaters are used to identify sources of dissolved inorganic and organic N in these waters, and to determine their pathways within the catchment; specific emphasis is placed on the relation of forestry and agriculture. Forestry is predominantly concentrated in the northern subcatchment (Beerburrum Creek) while agriculture is mainly found in the southern subcatchment (Six Mile Creek). Results show that agriculture (horticulture, crops, grazing) is the main source of inorganic N in the surface waters of the agricultural subcatchment, and their isotopic signature shows a close link to evaporation processes that may occur during water storage in farm dams that are used for irrigation. Groundwaters are subject to denitrification processes that may result in reduced dissolved inorganic N concentrations. Soil organic matter delivers most of the inorganic N to the surface water in the forested subcatchment. Here, precipitation and subsequently runoff is the main source of the surface waters. Groundwater in this area is affected by agricultural processes. The findings also show that the catchment can attenuate the effects of anthropogenic land use on surface water quality. Riparian strips of natural remnant vegetation, commonly 50 to 100 m in width, act as buffer zones along the drainage lines in the catchment and remove inorganic N from the soil water before it enters the creek. These riparian buffer zones are common in most agricultural catchments of southeast Queensland and are indicated to reduce the impact of agriculture on stream water quality and subsequently on the estuary and marine environments. This reduction is expressed by a significant decrease in DIN concentrations from 1.6 mg N L..1 to 0.09 mg N L..1, and a decrease in the �15N signatures from upstream surface water locations downstream to the outlet of the agricultural subcatchment. Further testing is, however, necessary to confirm these processes. Most importantly, the amount of N that is transported to the adjacent estuary is shown to be negligible. The third and fourth components of the thesis use a hydrological catchment model approach to determine the water balance of the Elimbah Creek catchment. The model is then used to simulate the effects of land use on the water balance and nutrient loads of the study area. The tool that is used is the internationally widely applied Soil and Water Assessment Tool (SWAT). Knowledge about the water cycle of a catchment is imperative in nutrient studies as processes such as rainfall, surface runoff, soil infiltration and routing of water through the drainage system are the driving forces of the catchment nutrient cycle. Long-term information about discharge volumes of the creeks and rivers do, however, not exist for a number of agricultural catchments in southeast Queensland, and such information is necessary to calibrate and validate numerical models. Therefore, a two-step modelling approach was used to calibrate and validate parameters values from a near-by gauged reference catchment as starting values for the ungauged Elimbah Creek catchment. Transposing monthly calibrated and validated parameter values from the reference catchment to the ungauged catchment significantly improved model performance showing that the hydrological model of the catchment of interest is a strong predictor of the water water balance. The model efficiency coefficient EF shows that 94% of the simulated discharge matches the observed flow whereas only 54% of the observed streamflow was simulated by the SWAT model prior to using the validated values from the reference catchment. In addition, the hydrological model confirmed that total surface runoff contributes the majority of flow to the surface water in the catchment (65%). Only a small proportion of the water in the creek is contributed by total base-flow (35%). This finding supports the results of the stable isotopes 16O/18O and 2H/H, which show the main source of water in the creeks is either from local precipitation or irrigation waters delivered by surface runoff; a contribution from the groundwater (baseflow) to the creeks could not be identified using 16O/18O and 2H/H. In addition, the SWAT model calculated that around 68% of the rainfall occurring in the catchment is lost through evapotranspiration reflecting the prevailing long-term drought conditions that were observed prior and during the study. Stream discharge from the forested subcatchment was an order of magnitude lower than discharge from the agricultural Six Mile Creek subcatchment. A change in land use from forestry to agriculture did not significantly change the catchment water balance, however, nutrient loads increased considerably. Conversely, a simulated change from agriculture to forestry resulted in a significant decrease of nitrogen loads. The findings of the thesis and the approach used are shown to be of value to catchment water quality monitoring on a wider scale, in particular the implications of mixed land use on nutrient forms, distributions and concentrations. The study confirms that in the tropics and subtropics the water balance is affected by extended dry periods and seasonal rainfall with intensive storm events. In particular, the comprehensive data set of inorganic and organic N and P forms in the surface and groundwaters of this subtropical setting acquired during the one year sampling program may be used in similar catchment hydrological studies where these detailed information is missing. Also, the study concludes that riparian buffer zones along the catchment drainage system attenuate the transport of nitrogen from agricultural sources in the surface water. Concentrations of N decreased from upstream to downstream locations and were negligible at the outlet of the catchment.

Growth of C36-films on diamond surface through molecular dynamics simulation

In this paper, the initial stage of films assembled by energetic C36 fullerenes on diamond (001)–(2 × 1) surface at low-temperature was investigated by molecular dynamics simulation using the Brenner potential. The incident energy was first uniformly distributed within an energy interval 20–50 eV, which was known to be the optimum energy range for chemisorption of single C36 on diamond (001) surface. More than one hundred C36 cages were impacted one after the other onto the diamond surface by randomly selecting their orientation as well as the impact position relative to the surface. The growth of films was found to be in three-dimensional island mode, where the deposited C36 acted as building blocks. The study of film morphology shows that it retains the structure of a free C36 cage, which is consistent with Low Energy Cluster Beam Deposition (LECBD) experiments. The adlayer is composed of many C36-monomers as well as the covalently bonded C36 dimers and trimers which is quite different from that of C20 fullerene-assembled film, where a big polymerlike chain was observed due to the stronger interaction between C20 cages. In addition, the chemisorption probability of C36 fullerenes is decreased with increasing coverage because the interaction between these clusters is weaker than that between the cluster and the surface. When the incident energy is increased to 40–65 eV, the chemisorption probability is found to increased and more dimers and trimers as well as polymerlike-C36 were observed on the deposited films. Furthermore, C36 film also showed high thermal stability even when the temperature was raised to 1500 K.

The effect of hydroxyl groups and surface area of hematite derived from annealing goethite for phosphate removal

Synthetic goethite and thermally treated goethite at different temperatures were used to remove phosphate from sewage. The effect of annealing temperature on phosphate removal over time was investigated. X-ray diffraction(XRD), transmission electron microscopy (TEM), N2 adsorption and desorption (BET), and infrared emission spectrum (FT-IES) were utilized to characterize the phase, morphology, specific surface area, pore distribution, and the surface groups of samples. The results show that annealed products of goethite at temperatures over 250 °C are hematite with the similar morphology as the original goethite with different hydroxyl groups and surface area. Increasing temperature causes the decrease in hydroxyl groups, consequential increase in surface area at first and then experiences a decrease (14.8–110.4–12.6 m2/g) and the subsequent formation of nanoscale pores. The variation rate of hydroxyl groups and surface area based on FT-IES and BET, respectively, are used to evaluate the effect of annealing temperature on phosphate removal. By using all of the characterization techniques, it is concluded that the changes of phosphate removal basically result from the total variation rate between hydroxyl groups and surface area.

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.

Morphology of osteocyte cells in normal and hyper-gravity

Osteocyte cells are the most abundant cells in human bone tissue. Due to their unique morphology and location, osteocyte cells are thought to act as regulators in the bone remodelling process, and are believed to play an important role in astronauts’ bone mass loss after long-term space missions. There is increasing evidence showing that an osteocyte’s functions are highly affected by its morphology. However, changes in an osteocyte’s morphology under an altered gravity environment are still not well documented. Several in vitro studies have been recently conducted to investigate the morphological response of osteocyte cells to the microgravity environment, where osteocyte cells were cultured on a two-dimensional flat surface for at least 24 hours before microgravity experiments. Morphology changes of osteocyte cells in microgravity were then studied by comparing the cell area to 1g control cells. However, osteocyte cells found in vivo are with a more 3D morphology, and both cell body and dendritic processes are found sensitive to mechanical loadings. A round shape osteocyte’s cells support a less stiff cytoskeleton and are more sensitive to mechanical stimulations compared with flat cellular morphology. Thus, the relative flat and spread shape of isolated osteocytes in 2D culture may greatly hamper their sensitivity to a mechanical stimulus, and the lack of knowledge on the osteocyte’s morphological characteristics in culture may lead to subjective and noncomprehensive conclusions of how altered gravity impacts on an osteocyte’s morphology. Through this work empirical models were developed to quantitatively predicate the changes of morphology in osteocyte cell lines (MLO-Y4) in culture, and the response of osteocyte cells, which are relatively round in shape, to hyper-gravity stimulation has also been investigated. The morphology changes of MLO-Y4 cells in culture were quantified by measuring cell area and three dimensionless shape features including aspect ratio, circularity and solidity by using widely accepted image analysis software (ImageJTM). MLO-Y4 cells were cultured at low density (5×103 per well) and the changes in morphology were recorded over 10 hours. Based on the data obtained from the imaging analysis, empirical models were developed using the non-linear regression method. The developed empirical models accurately predict the morphology of MLO-Y4 cells for different culture times and can, therefore, be used as a reference model for analysing MLO-Y4 cell morphology changes within various biological/mechanical studies, as necessary. The morphological response of MLO-Y4 cells with a relatively round morphology to hyper-gravity environment has been investigated using a centrifuge. After 2 hours culture, MLO-Y4 cells were exposed to 20g for 30mins. Changes in the morphology of MLO-Y4 cells are quantitatively analysed by measuring the average value of cell area and dimensionless shape factors such as aspect ratio, solidity and circularity. In this study, no significant morphology changes were detected in MLO-Y4 cells under a hyper-gravity environment (20g for 30 mins) compared with 1g control cells.

NSAM-derived total surface area vs. SMPS-derived "mobility equivalent" surface area for different environmentally relevant aerosols

The surface area of inhaled particles deposited in the alveolar region, as reported by the TSI nanoparticle surface area monitor (NSAM), was compared with the corresponding value estimated by a TSI scanning mobility particle sizer (SMPS) for a range of environmentally relevant aerosols, including petrol emissions, ETS, laser printer emissions, cooking emissions and ambient aerosols. The SMPS values were based on a mobility size distribution assuming spherical particles using the appropriate size-dependent alveolar-deposition factors provided by the ICRP. In most cases, the two instruments showed good linear agreement. With petrol emissions and ETS, the linearity extended to over 103 μm2 cm-3. With printer emissions, there was good linearity up to about 300 μm2 cm-3 while the NSAM increasingly overestimated the surface area at higher concentrations. The presence of a nucleation event in ambient air caused the NSAM to over-estimate the surface area by a factor of 2. We summarize these results and conclude that the maximum number concentration up to which the NSAM is accurate clearly depends on the type of aerosol being sampled and provide guidance for the use of the instrument.

Zeta-potential and morphology of electrospun nano- and microfibers from biopolymers and their blends used as scaffolds in tissue engineering

Electrostatic spinning or electrospinning is a fiber spinning technique driven by a high-voltage electric field that produces fibers with diameters in a submicrometer to nanometer range.1 Nanofibers are typical one-dimensional colloidal objects with an increased tensile strength, whose length can achieve a few kilometers and the specific surface area can be 100 m2 g–1 or higher.2 Nano- and microfibers from biocompatible polymers and biopolymers have received much attention in medical applications3 including biomedical structural elements (scaffolding used in tissue engineering,2,4–6 wound dressing,7 artificial organs and vascular grafts8), drug and vaccine delivery,9–11 protective shields in speciality fabrics, multifunctional membranes, etc. Other applications concern superhydrophobic coatings,12 encapsulation of solid materials,13 filter media for submicron particles in separation industry, composite reinforcement and structures for nano-electronic machines.

YBa2Cu3O7-x films on yttria-stabilized ZrO2 substrates : influence of the substrate morphology

c-axis-oriented YBa2Cu3O7-x (YBCO) thin films were laser deposited on (001) yttria-stabilized ZrO2 (YSZ) substrates with different surface morphologies. The in-plane orientation of the films on smooth substrates was sensitive to the deposition conditions, often resulting in mixed orientations. However, a strongly dominating [110] YBCO//[110]YSZ orientation was obtained at a deposition temperature of 770°C. Films on substrates with surface steps, induced by depositing a homoepitaxial buffer layer or by thermally annealing the substrate, had a [110]YBCO//[100]YSZ orientation when deposited at the same temperature. It was concluded that the [110]YBCO//[100] YSZ orientation was promoted by a graphoepitaxial mechanism. Films prepared under identical conditions on smooth and stepped substrates grew with extended c axes on the former. It is proposed that the extension can be induced by disorder, invoked by a low oxygen pressure and a low density of adsorption sites. The disorder may be eliminated by either an increase of the oxygen pressure or an increase of the density of adsorption sites in the form of steps. The film microstructure influenced the microwave surface resistance, which was similar for films with one exclusive in-plane orientation and higher for films with mixed orientations. The films on the stepped surfaces had superior superconducting properties; inductive measurements gave a Tc onset of 88 K, a ΔT(90%-10%) c of 0.2 K, and the transport jc was 1.5×106 A/cm2 at 83 K, for films on substrates with homoepitaxial buffer layers.

Morphology changes and mechanistic aspects of the electrochemically-induced reversible solid-solid transformation of microcrystalline TCNQ into Co TCNQ 2-based materials (TCNQ= 7, 7, 8, 8-Tetracyanoquinodimethane)

The chemically reversible solid−solid phase transformation of a TCNQ-modified glassy carbon, indium tin oxide, or metal electrode into Co\[TCNQ]2(H2O)2 material in the presence of Co2+(aq) containing electrolytes has been induced and monitored electrochemically. Voltammetric data reveal that the TCNQ/Co\[TCNQ]2(H2O)2 interconversion process is independent of electrode material and identity of cobalt electrolyte anion. However, a marked dependence on electrolyte concentration, scan rate, and method of electrode modification (drop casting or mechanical attachment) is found. Cyclic voltammetric and double potential step chronoamperometric measurements confirm that formation of Co\[TCNQ]2(H2O)2 occurs through a rate-determining nucleation and growth process that initially involves incorporation of Co2+(aq) ions into the reduced TCNQ crystal lattice at the TCNQ|electrode|electrolyte interface. Similarly, the reverse (oxidation) process, which involves transformation of solid Co\[TCNQ]2(H2O)2 back to parent TCNQ crystals, also is controlled by nucleation−growth kinetics. The overall chemically reversible process that represents this transformation is described by the reaction:  2TCNQ0(s) + 2e- + Co2+(aq) + 2H2O \[Co(TCNQ)2(H2O)2](s). Ex situ SEM images illustrated that this reversible TCNQ/Co\[TCNQ]2(H2O)2 conversion process is accompanied by drastic size and morphology changes in the parent solid TCNQ. In addition, different sizes of needle-shaped nanorod/nanowire crystals of Co\[TCNQ]2(H2O)2 are formed depending on the method of surface immobilization.

Reusable surface confined semi-conducting metal-TCNQ and metal-TCNQF4 catalysts for electron transfer reactions

The synthesis of organic semiconducting materials based on silver and copper-TCNQ (TCNQ = 7,7,8,8-tetracyanoquinodimethane) and their fluorinated analogues has received a significant amount of attention due to their potential use in organic electronic applications. However, there is a scarcity in the identification of different applications for which these interesting materials may be suitable candidates. In this work, we address this by investigating the catalytic properties of such materials for the electron transfer reaction between ferricyanide and thiosulphate ions in aqueous solution, which to date has been almost solely limited to metallic nanomaterials. Significantly it was found that all the materials investigated, namely CuTCNQ, AgTCNQ, CuTCNQF4 and AgTCNQF4, were catalytically active and, interestingly, the fluorinated analogues were superior. AgTCNQF4 demonstrated the highest activity and was tested for its stability and re-usability for up to 50 cycles without degradation in performance. The catalytic reaction was monitored via UV-vis spectroscopy and open circuit potential versus time measurements, as well as an investigation of the transport properties of the films via electrochemical impedance spectroscopy. It is suggested that morphology and bulk conductivity are not the limiting factors, but rather the balance between the accumulated surface charge from electron injection via thiosulphate ions on the catalyst surface and transfer to the ferricyanide ions which controls the reaction rate. The facile fabrication of re-usable surface confined organic materials that are catalytically active may have important uses for many more electron transfer reactions.

In vivo confocal microscopy of the ocular surface : from bench to bedside

In vivo confocal microscopy (IVCM) is an emerging technology that provides minimally invasive, high resolution, steady-state assessment of the ocular surface at the cellular level. Several challenges still remain but, at present, IVCM may be considered a promising technique for clinical diagnosis and management. This mini-review summarizes some key findings in IVCM of the ocular surface, focusing on recent and promising attempts to move “from bench to bedside”. IVCM allows prompt diagnosis, disease course follow-up, and management of potentially blinding atypical forms of infectious processes, such as acanthamoeba and fungal keratitis. This technology has improved our knowledge of corneal alterations and some of the processes that affect the visual outcome after lamellar keratoplasty and excimer keratorefractive surgery. In dry eye disease, IVCM has provided new information on the whole-ocular surface morphofunctional unit. It has also improved understanding of pathophysiologic mechanisms and helped in the assessment of prognosis and treatment. IVCM is particularly useful in the study of corneal nerves, enabling description of the morphology, density, and disease- or surgically induced alterations of nerves, particularly the subbasal nerve plexus. In glaucoma, IVCM constitutes an important aid to evaluate filtering blebs, to better understand the conjunctival wound healing process, and to assess corneal changes induced by topical antiglaucoma medications and their preservatives. IVCM has significantly enhanced our understanding of the ocular response to contact lens wear. It has provided new perspectives at a cellular level on a wide range of contact lens complications, revealing findings that were not previously possible to image in the living human eye. The final section of this mini-review provides a focus on advances in confocal microscopy imaging. These include 2D wide-field mapping, 3D reconstruction of the cornea and automated image analysis.