Transfection of MDA-MB-231 human breast carcinoma cells with bone sialoprotein (BSP) stimulates migration and invasion in vitro and growth of primary and secondary tumors in nude mice

We have investigated the role of bone sialoprotein (BSP), a secreted glycoprotein normally found in bone, in breast cancer progression. To explore functions for BSP in human breast cancer invasion and metastasis, the full-length BSP cDNA was transfected into the MDA-MB-231-BAG human breast cancer cell line under the control of the CMV promoter. Clones expressing BSP and vector control clones were isolated. BSP producing clones showed increased monolayer wound healing, a faster rate of stellate outgrowth in Matrigel and increased rate of invasion into a collagen matrix when compared to control clones. Clones were also examined in models of breast cancer growth and metastasis in vivo. BSP transfected clones showed an increased rate of primary tumor growth following mammary fat pad injection of nude mice. BSP transfected clones and vector control clones metastasized to soft organs and bone at a similar rate after intra-cardiac injection as determined by real-time PCR and X-ray analysis. Although these organs were targets for both BSP transfected and non-transfected cells, the size of the metastatic lesion was shown to be significantly larger for BSP expressing clones. This was determined by real-time PCR analysis for soft organs and by X-ray analysis of bone lesions. For bone this was confirmed by intra-tibial injections of cells in nude mice. We conclude that BSP acts to drive primary and secondary tumor growth of breast cancers in vivo.

Frizzled-7 receptor ectodomain expression in a colon cancer cell line induces morphological change and attenuates tumor growth

Frizzled (FZD) receptors have a conserved N-terminal extracellular cysteine-rich domain that interacts with Wnts and co-expression of the receptor ectodomain can antagonize FZD-mediated signalling. Using the ectodomain as an antagonist we have modulated endogenous FZD7 signalling in the moderately differentiated colon adenocarcinoma cell line, SK-CO-1. Unlike the parental cell line, which grows as tightly associated adherent cell clusters, the FZD7 ectodomain expressing cells display a spread out morphology and grow as a monolayer in tissue culture. This transition in morphology was associated with decreased levels of plasma membrane-associated E-cadherin and β-catenin, localized increased levels of vimentin and redistribution of α6 integrin to cellular processes in the FZD7 ectodomain expressing cells. The morphological and phenotype changes induced by FZD7 ectodomain expression in SK-CO-1 cells is thus consistent with the cells undergoing an epithelial-to-mesenchymal-like transition. Furthermore, initiation of tumor formation in a xenograft tumor growth assay was attenuated in the FZD7 ectodomain expressing cells. Our results indicate a pivotal role for endogenous FZD7 in morphology transitions that are associated with colon tumor initiation and progression.

Myoepithelial molecular markers in human breast carcinoma PMC42-LA cells are induced by extracellular matrix and stromal cells

The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins α-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm- Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, α-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.

Reversible transdifferentiation of blood vascular endothelial cells to a lymphatic-like phenotype in vitro

Blood vascular cells and lymphatic endothelial cells (BECs and LECs, respectively) form two separate vascular systems and are functionally distinct cell types or lineages with characteristic gene expression profiles. Interconversion between these cell types has not been reported. Here, we show that in conventional in vitro angiogenesis assays, human BECs of fetal or adult origin show altered gene expression that is indicative of transition to a lymphatic-like phenotype. This change occurs in BECs undergoing tubulogenesis in fibrin, collagen or Matrigel assays, but is independent of tube formation per se, because it is not inhibited by a metalloproteinase inhibitor that blocks tubulogenesis. It is also reversible, since cells removed from 3D tubules revert to a BEC expression profile upon monolayer culture. Induction of the lymphatic-like phenotype is partially inhibited by co-culture of HUVECs with perivascular cells. These data reveal an unexpected plasticity in endothelial phenotype, which is regulated by contact with the ECM environment and/or cues from supporting cells.

Kinetics of low-pressure, low-temperature graphene growth : toward single-layer, single-crystalline structure

Graphene grown on metal catalysts with low carbon solubility is a highly competitive alternative to exfoliated and other forms of graphene, yet a single-layer, single-crystal structure remains a challenge because of the large number of randomly oriented nuclei that form grain boundaries when stitched together. A kinetic model of graphene nucleation and growth is developed to elucidate the effective controls of the graphene island density and surface coverage from the onset of nucleation to the full monolayer formation in low-pressure, low-temperature CVD. The model unprecedentedly involves the complete cycle of the elementary gas-phase and surface processes and shows a precise quantitative agreement with the recent low-energy electron diffraction measurements and also explains numerous parameter trends from a host of experimental reports. These agreements are demonstrated for a broad pressure range as well as different combinations of precursor gases and supporting catalysts. The critical role of hydrogen in controlling the graphene nucleation and monolayer formation is revealed and quantified. The model is generic and can be extended to even broader ranges of catalysts and precursor gases/pressures to enable the as yet elusive effective control of the crystalline structure and number of layers of graphene using the minimum amounts of matter and energy.

Serial explant culture provides novel insights into the potential location and phenotype of corneal endothelial progenitor cells

The routine cultivation of human corneal endothelial cells, with the view to treating patients with endothelial dysfunction, remains a challenging task. While progress in this field has been buoyed by the proposed existence of progenitor cells for the corneal endothelium at the corneal limbus, strategies for exploiting this concept remain unclear. In the course of evaluating methods for growing corneal endothelial cells, we have noted a case where remarkable growth was achieved using a serial explant culture technique. Over the course of 7 months, a single explant of corneal endothelium, acquired from cadaveric human tissue, was sequentially seeded into 7 culture plates and on each occasion produced a confluent cell monolayer. Sample cultures were confirmed as endothelial in origin by positive staining for glypican-4. On each occasion, small cells, closest to the tissue explant, developed into a highly compact layer with an almost homogenous structure. This layer was resistant to removal with trypsin and produced continuous cell outgrowth during multiple culture periods. The small cells gave rise to larger cells with phase-bright cell boundaries and prominent immunostaining for both nestin and telomerase. Nestin and telomerase were also strongly expressed in small cells immediately adjacent to the wound site, following transfer of the explant to another culture plate. These findings are consistent with the theory that progenitor cells for the corneal endothelium reside within the limbus and provide new insights into expected expression patterns for nestin and telomerase within the differentiation pathway.

Self-organized nanoarrays : plasma-related controls

The paper presents an investigation of self-organizational and -assembly processes of nanostructure growth on surfaces exposed to low-temperature plasmas. We have considered three main growth stages-initial, or sub-monolayer growth stage, separate nanostructure growth stage, and array growth stages with the characteristic sizes of several nm, several tens of nm, and several hundreds of nm, respectively, and have demonstrated, by the experimental data and hybrid multiscale numerical simulations, that the plasma parameters can strongly influence the surface processes and hence the kinetics of self-organization and -assembly. Our results show that plasma-controlled self-organization is a promising way to assemble large regular arrays of nanostructures. © 2008 IUPAC.

Solid-state assemblies and optical properties of conjugated oligomers combining fluorene and thiophene units

Two conjugated oligomers, representing elementary segments of fluorene-thiophene copolymers, are compared in terms of the microscopic morphology and the optical properties of thin deposits. The atomic force microscopy morphological data and the solid-state absorption and emission spectra are interpreted in terms of the assembly of the conjugated molecules. The compound with a terthiophene central unit and fluorene end-groups shows well-defined monolayer-by-monolayer assembly into micrometer-long stripe-like structures, with a crystalline herringbone-type organization within the monolayers. Polarized confocal microscopy indicates a strong orientation of the crystalline domains within the stripes. In contrast, the compound with a terfluorene central unit and thiophene end groups forms no textured aggregates and the optical spectra in the solid-state are very similar to those recorded in solution, suggesting that the molecules interact only weakly in the solid. The difference in behaviour between the two compounds most probably originates from their different capability to form densely-packed assemblies of interacting π-systems.

Menstrual effluent induces epithelial-mesenchymal transitions in mesothelial cells

Background Menstrual effluent affects mesothelial cell (MC) morphology. We evaluated whether these changes were consistent with epithelial-mesenchymal transitions (EMT). Methods Monolayer cultures of MC were incubated overnight in conditioned media, prepared from cells isolated form menstrual effluent, with or without kinase and ATP inhibitors. Changes in cell morphology were monitored using time-lapse video microscopy and immunohistochemistry. Effects on the expression of EMT-associated molecules were evaluated using real-time RT-PCR and/or Western blot analysis. Results Incubation in conditioned media disrupted cell-cell contacts, and increased MC motility. The changes were reversible. During the changes the distribution of cytokeratins, fibrillar actin and α-tubulin changed. Sodium azide, an inhibitor of ATP production, and Genistein, a general tyrosine kinase inhibitor, antagonized these effects. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, and SU6656, an Src tyrosine kinase inhibitor, only partially antagonized the effect. The expression of Snail and vimentin was markedly up-regulated, whereas the expression of E-cadherin was decreased and cytokeratins were altered. Conclusions In MC, menstrual effluent initiates a reversible, energy-dependent transition process from an epithelial to a mesenchymal phenotype. Involvement of the (Src) tyrosine kinase signalling pathway and the changes in the expression of cytokeratins, Snail, vimentin and E-cadherin demonstrate that the morphological changes are EMT.

Adsorption thermodynamics and kinetics study for the self-assembly of 1,8,15,22-tetraaminophthalocyanatocobalt(II) on glassy carbon surface

Spontaneous adsorption of 1,8,15,22-tetraaminophthalocyanatocobalt(II) (4α-CoIITAPc) on glassy carbon (GC) electrode leads to the formation of a stable self-assembled monolayer (SAM). Since the SAM of 4α-CoIITAPc is redox active, its adsorption on GC electrode was followed by cyclic voltammetry. SAM of 4α-CoIITAPc on GC electrode shows two pairs of well-defined redox peaks corresponding to CoIII/CoII and CoIIIPc−1/CoIIIPc−2. The surface coverage (Γ) value, calculated by integrating the charge under CoII oxidation, was used to study the adsorption thermodynamics and kinetics of 4α-CoIITAPc on GC surface. Cyclic voltammetric studies show that the adsorption of 4α-CoIITAPc on GC electrode has reached the saturation coverage (Γs) within 3 h. The Γs value for the SAM of 4α-CoIITAPc on GC electrode was found to be 2.37 × 10−10 mol cm−2. Gibbs free energy (ΔGads) and adsorption rate constant (kad) for the adsorption of 4α-CoIITAPc on GC surface were found to be −16.76 kJ mol−1 and 7.1 M−1 s−1, respectively. The possible mechanism for the self-assembly of 4α-CoIITAPc on GC surface is through the addition of nucleophilic amines to the olefinic bond on the GC surface in addition to a meager contribution from π stacking. The contribution of π stacking was confirmed from the adsorption of unsubstituted phthalocyanatocobalt(II) (CoPc) on GC electrode. Raman spectra for the SAM of 4α-CoIITAPc on carbon surface shows strong stretching and breathing bands of Pc macrocycle, pyrrole ring and isoindole ring. Raman and CV studies suggest that 4α-CoIITAPc is adopting nearly a flat orientation or little bit tilted orientation.

Electrocatalytic oxidation of ascorbic acid using a single layer of gold nanoparticles immobilized on 1,6-hexanedithiol modified gold electrode

This paper describes the electrocatalytic oxidation of ascorbic acid (AA) in phosphate buffer solution by the immobilized citrate capped gold nanoparticles (AuNPs) on 1,6-hexanedithiol (HDT) modified Au electrode. X-ray photoelectron spectrum (XPS) of HDT suggests that it forms a monolayer on Au surface through one of the two single bondSH groups and the other single bondSH group is pointing away from the electrode surface. The free single bondSH groups of HDT were used to covalently attach colloidal AuNPs. The covalent attachment of AuNPs on HDT monolayer was confirmed from the observed characteristic carboxylate ion stretching modes of citrate attached with AuNPs in the infra-red reflection absorption spectrum (IRRAS) in addition to a higher reductive desorption charges obtained for AuNPs immobilized on HDT modified Au (Au/HDT/AuNPs) electrode in 0.1 M KOH when compared to HDT modified Au (Au/HDT) electrode. The electron transfer reaction of [Fe(CN)6]4−/3− was markedly hindered at the HDT modified Au (Au/HDT) electrode while it was restored with a peak separation of 74 mV after the immobilization of AuNPs on Au/HDT (Au/HDT/AuNPs) electrode indicating a good electronic communication between the immobilized AuNPs and the underlying bulk Au electrode through a HDT monolayer. The Cottrell slope obtained from the potential-step chronoamperometric measurements for the reduction of ferricyanide at Au/HDT/AuNPs was higher than that of bare Au electrode indicating the increased effective surface area of AuNPs modified electrode. The Au/HDT/AuNPs electrode exhibits excellent electrocatalytic activity towards the oxidation of ascorbic acid (AA) by enhancing the oxidation peak current to more than two times with a 210 mV negative shift in the oxidation potential when compared to a bare Au electrode. The standard heterogeneous electron transfer rate constant (ks) calculated for AA oxidation at Au/HDT/AuNPs electrode was 5.4 × 10−3 cm s−1. The oxidation peak of AA at Au/HDT/AuNPs electrode was highly stable upon repeated potential cycling. Linear calibration plot was obtained for AA over the concentration range of 1–110 μM with a correlation coefficient of 0.9950. The detection limit of AA was found to be 1 μM. The common physiological interferents such as glucose, oxalate ions and urea do not show any interference within the detection limit of AA. The selectivity of the AuNPs modified electrode was illustrated by the determination of AA in the presence of uric acid.

Electrochemical pathway for the quantification of SERS enhancement factor

This communication presents a new pathway for the more precise quantification of surface-enhanced Raman scattering (SERS) enhancement factor via deducing resonance Raman scattering (RRS) effect from surface-enhanced resonance Raman scattering (SERRS). To achieve this, a self-assembled monolayer of 1,8,15,22-tetraaminophthalocyanatocobalt(II) (4α-CoIITAPc) is formed on plasmon inactive glassy carbon (GC) and plasmon active GC/AuNPs surface. The surfaces are subsequently used as common probes for electrochemical and Raman (RRS and SERRS) studies. The most crucial parameters required for the quantification of SERS substrate enhancement factor (SSEF) such as real surface area of GC/AuNPs substarte and the number of 4α-CoIITAPc molecules contributing to RRS (on GC) and SERRS (on GC/AuNPs) are precisely estimated by cyclic voltammetry experiments. The present approach of SSEF quantification can be applied to varieties of surfaces by choosing an appropriate laser line and probe molecule for each surface.

Formation of carbon nanoscrolls from graphene nanoribbons : a molecular dynamics study

Carbon nanoscrolls (CNSs) are one of the carbon-based nanomaterials similar to carbon nanotubes (CNTs) but are not widely studied in spite of their great potential applications. Their practical applications are hindered by the challenging fabrication of the CNSs. A physical approach has been proposed recently to fabricate the CNS by rolling up a monolayer graphene nanoribbon (GNR) around a CNT driven by the interaction energy between them. In this study, we perform extensive molecular dynamics (MD) simulations to investigate the various factors that impact the formation of the CNS from GNR. Our simulation results show that the formation of the CNS is sensitive to the length of the CNT and temperature. When the GNR is functionalized with hydrogen, the formation of the CNS is determined by the density and distribution of the hydrogen atoms. Graphyne, the allotrope of graphene, is inferior to graphene in the formation of the CNS due to the weaker bonds and the associated smaller atom density. The mechanism behind the rolling of GNR into CNS lies in the balance between the GNR–CNT van der Waals (vdW) interactions and the strain energy of GNR. The present work reveals new important insights and provides useful guidelines for the fabrication of the CNS.

Choosing an appropriate modelling framework for analysing multispecies co-culture cell biology experiments

In vitro cell biology assays play a crucial role in informing our understanding of the migratory, proliferative and invasive properties of many cell types in different biological contexts. While mono-culture assays involve the study of a population of cells composed of a single cell type, co-culture assays study a population of cells composed of multiple cell types (or subpopulations of cells). Such co-culture assays can provide more realistic insights into many biological processes including tissue repair, tissue regeneration and malignant spreading. Typically, system parameters, such as motility and proliferation rates, are estimated by calibrating a mathematical or computational model to the observed experimental data. However, parameter estimates can be highly sensitive to the choice of model and modelling framework. This observation motivates us to consider the fundamental question of how we can best choose a model to facilitate accurate parameter estimation for a particular assay. In this work we describe three mathematical models of mono-culture and co-culture assays that include different levels of spatial detail. We study various spatial summary statistics to explore if they can be used to distinguish between the suitability of each model over a range of parameter space. Our results for mono-culture experiments are promising, in that we suggest two spatial statistics that can be used to direct model choice. However, co-culture experiments are far more challenging: we show that these same spatial statistics which provide useful insight into mono-culture systems are insuffcient for co-culture systems. Therefore, we conclude that great care ought to be exercised when estimating the parameters of co-culture assays.

Electrocatalytic and SERS activity of Pt rich Pt-Pb nanostructures formed via the utilisation of in-situ underpotential deposition of lead

The controlled synthesis of nanostructured materials remains an ongoing area of research, especially as the size, shape and composition of nanomaterials can greatly influence their properties and applications. In this work we present the electrodeposition of highly dendritic platinum rich platinum-lead nanostructures, where lead acetate acts as an inorganic shape directing agent via underpotential deposition on the growing electrodeposit. It was found that these nanomaterials readily oxidise at potentials below monolayer oxide formation, which significantly impacts on the methanol electrooxidation reaction and correlates with the incipient hydrous oxide adatom mediator (IHOAM) model of electrocatalysis. Additionally these materials were tested for their surface enhanced Raman scattering (SERS) activity, where the high density of sharp tips provides promise for their application as SERS substrates.