Cadherins in the human placenta : epithelial-mesenchymal transition (EMT) and placental development

Colonisation of the maternal uterine wall by the trophoblast involves a series of alterations in the behaviour and morphology of trophoblast cells. Villous cytotrophoblast cells change from a well-organised coherently layered phenotype to one that is extravillous, acquiring a proliferative, migratory and invasive capacity, to facilitate fetal-maternal interaction. These changes are similar to those of other developmental processes falling under the umbrella of an epithelial-mesenchymal transition (EMT). Modulation of cell adhesion and cell polarity occurs through changes in cell-cell junctional molecules, such as the cadherins. The cadherins, particularly the classical cadherins (e.g. Epithelial-(E)-cadherin), and their link to adaptors called catenins at cell-cell contacts, are important for maintaining cell attachment and the layered phenotype of the villous cytotrophoblast. In contrast, reduced expression and re-organization of cadherins from these cell junctional regions promote a loosened connection between cells, coupled with reduced apico-basal polarity. Certain non-classical cadherins play an active role in cell migration processes. In addition to the classical cadherins, two other cadherins which have been reported in placental tissues are vascular endothelial (VE) cadherin and cadherin-11. Cadherin molecules are well placed to be key regulators of trophoblast cell behaviour, analogous to their role in other developmental EMTs. This review addresses cadherin expression and function in normal and diseased human placental tissues, especially in fetal growth restriction and pre-eclampsia where trophoblast invasion is reduced.

A dynamic in vivo model of epithelial-to-mesenchymal transitions in circulating tumor cells and metastases of breast cancer

Epithelial-to-mesenchymal transition (EMT) processes endow epithelial cells with enhanced migratory/invasive properties and are therefore likely to contribute to tumor invasion and metastatic spread. Because of the difficulty in following EMT processes in human tumors, we have developed and characterized an animal model with transplantable human breast tumor cells (MDA-MB-468) uniquely showing spontaneous EMT events to occur. Using vimentin as a marker of EMT, heterogeneity was revealed in the primary MDA-MB-468 xenografts with vimentin-negative and vimentin-positive areas, as also observed on clinical human invasive breast tumor specimens. Reverse transcriptase-PCR after microdissection of these populations from the xenografts revealed EMT traits in the vimentin-positive zones characterized by enhanced 'mesenchymal gene' expression (Snail, Slug and fibroblast-specific protein-1) and diminished expression of epithelial molecules (E-cadherin, ZO-3 and JAM-A). Circulating tumor cells (CTCs) were detected in the blood as soon as 8 days after s.c. injection, and lung metastases developed in all animals injected as examined by in vivo imaging analyses and histology. High levels of vimentin RNA were detected in CTCs by reverse transcriptase-quantitative PCR as well as, to a lesser extent, Snail and Slug RNA. Von Willebrand Factor/vimentin double immunostainings further showed that tumor cells in vascular tumoral emboli all expressed vimentin. Tumoral emboli in the lungs also expressed vimentin whereas macrometastases displayed heterogenous vimentin expression, as seen in the primary xenografts. In conclusion, our data uniquely demonstrate in an in vivo context that EMT occurs in the primary tumors, and associates with an enhanced ability to intravasate and generate CTCs. They further suggest that mesenchymal-to-epithelial phenomena occur in secondary organs, facilitating the metastatic growth.

Epithelial-to-mesenchymal transitions and circulating tumor cells

Epithelial-to-mesenchymal transition (EMT) phenomena endow epithelial cells with enhanced migratory and invasive potential, and as such, have been implicated in many physiological and pathological processes requiring cell migration/invasion. Although their involvement in the metastatic cascade is still a subject of debate, data are accumulating to demonstrate the existence of EMT phenotypes in primary human tumors, describe enhanced metastatic potential of EMT derivatives in animal models, and report EMT attributes in circulating tumor cells (CTCs). The relationships between EMT and CTCs remain largely unexplored, and we review here in vitro and in vivo data supporting a putative role of EMT processes in CTC generation and survival.

Differential subcellular and extracellular localisations of proteins required for insulin-like growth factor- and extracellular matrix-induced signalling events in breast cancer progression

Background: Cancer metastasis is the main contributor to breast cancer fatalities as women with the metastatic disease have poorer survival outcomes than women with localised breast cancers. There is an urgent need to develop appropriate prognostic methods to stratify patients based on the propensities of their cancers to metastasise. The insulin-like growth factor (IGF)-I:IGF binding protein (IGFBP):vitronectin complexes have been shown to stimulate changes in gene expression favouring increased breast cancer cell survival and a migratory phenotype. We therefore investigated the prognostic potential of these IGF- and extracellular matrix (ECM) interaction-induced proteins in the early identification of breast cancers with a propensity to metastasise using patient-derived tissue microarrays. Methods: Semiquantitative immunohistochemistry analyses were performed to compare the extracellular and subcellular distribution of IGF- and ECM-induced signalling proteins among matched normal, primary cancer and metastatic cancer formalin-fixed paraffin-embedded breast tissue samples. Results: The IGF- and ECM-induced signalling proteins were differentially expressed between subcellular and extracellular localisations. Vitronectin and IGFBP-5 immunoreactivity was lower while β1 integrin immunoreactivity was higher in the stroma surrounding metastatic cancer tissues, as compared to normal breast and primary cancer stromal tissues. Similarly, immunoreactive stratifin was found to be increased in the stroma of primary as well as metastatic breast tissues. Immunoreactive fibronectin and β1 integrin was found to be highly expressed at the leading edge of tumours. Based on the immunoreactivity it was apparent that the cell signalling proteins AKT1 and ERK1/2 shuffled from the nucleus to the cytoplasm with tumour progression. Conclusion: This is the first in-depth, compartmentalised analysis of the distribution of IGF- and ECM-induced signalling proteins in metastatic breast cancers. This study has provided insights into the changing pattern of cellular localisation and expression of IGF- and ECM-induced signalling proteins in different stages of breast cancer. The differential distribution of these biomarkers could provide important prognostic and predictive indicators that may assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy.

Elevated YKL40 is associated with advanced prostate cancer (PCa) and positively regulates invasion and migration of PCa cells

Chitinase 3-like 1 (CHI3L1 or YKL40) is a secreted glycoprotein highly expressed in tumours from patients with advanced stage cancers, including prostate cancer (PCa). The exact function of YKL40 is poorly understood, but it has been shown to play an important role in promoting tumour angiogenesis and metastasis. The therapeutic value and biological function of YKL40 are unknown in PCa. The objective of this study was to examine the expression and function of YKL40 in PCa. Gene expression analysis demonstrated that YKL40 was highly expressed in metastatic PCa cells when compared with less invasive and normal prostate epithelial cell lines. In addition, the expression was primarily limited to androgen receptor-positive cell lines. Evaluation of YKL40 tissue expression in PCa patients showed a progressive increase in patients with aggressive disease when compared with those with less aggressive cancers and normal controls. Treatment of LNCaP and C4-2B cells with androgens increased YKL40 expression, whereas treatment with an anti-androgen agent decreased the gene expression of YKL40 in androgen-sensitive LNCaP cells. Furthermore, knockdown of YKL40 significantly decreased invasion and migration of PCa cells, whereas overexpression rendered them more invasive and migratory, which was commensurate with an enhancement in the anchorage-independent growth of cells. To our knowledge, this study characterises the role of YKL40 for the first time in PCa. Together, these results suggest that YKL40 plays an important role in PCa progression and thus inhibition of YKL40 may be a potential therapeutic strategy for the treatment of PCa.

Whole-genome multiparametric screening to identify modulators of epithelial-to-mesenchymal transition

Metastasis accounts for the poor prognosis of the majority of solid tumors. The phenotypic transition of nonmotile epithelial tumor cells to migratory and invasive “mesenchymal” cells (epithelial-to-mesenchymal transition [EMT]) enables the transit of cancer cells from the primary tumor to distant sites. There is no single marker of EMT; rather, multiple measures are required to define cell state. Thus, the multiparametric capability of high-content screening is ideally suited for the comprehensive analysis of EMT regulators. The aim of this study was to generate a platform to systematically identify functional modulators of tumor cell plasticity using the bladder cancer cell line TSU-Pr1-B1 as a model system. A platform enabling the quantification of key EMT characteristics, cell morphology and mesenchymal intermediate filament vimentin, was developed using the fluorescent whole-cell-tracking reagent CMFDA and a fluorescent promoter reporter construct, respectively. The functional effect of genome-wide modulation of protein-coding genes and miRNAs coupled with those of a collection of small-molecule kinase inhibitors on EMT was assessed using the Target Activation Bioapplication integrated in the Cellomics ArrayScan platform. Data from each of the three screens were integrated to identify a cohort of targets that were subsequently examined in a validation assay using siRNA duplexes. Identification of established regulators of EMT supports the utility of this screening approach and indicated capacity to identify novel regulators of this plasticity program. Pathway analysis coupled with interrogation of cancer-related expression profile databases and other EMT-related screens provided key evidence to prioritize further experimental investigation into the molecular regulators of EMT in cancer cells.

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.

Extinction risk analysis of exploited green turtle stocks in the Indo-Pacific

In parts of the Indo-Pacific, large-scale exploitation of the green turtle Chelonia mydas continues to pose a serious threat to the persistence of this species; yet very few studies have assessed the pattern and extent of the impact of such harvests. We used demographic and genetic data in an age-based model to investigate the viability of an exploited green turtle stock from Aru, south-east Indonesia. We found that populations are decreasing under current exploitation pressures. The effects of increasingly severe exploitation activities at foraging and nesting habitat varied depending on the migratory patterns of the stock. Our model predicted a rapid decline of the Aru stock in Indonesia under local exploitation pressure and a shift in the genetic composition of the stock. We used the model to investigate the influence of different types of conservation actions on the persistence of the Aru stock. The results show that local management actions such as nest protection and reducing harvests of adult nesting and foraging turtles can have considerable conservation outcomes and result in the long-term persistence of genetically distinct management units. © 2010 The Authors. Animal Conservation © 2010 The Zoological Society of London.

Patterned fen formation and development from the Great Sandy Region, south-east Queensland, Australia

The Great Sandy Region (incorporating Fraser Island and the Cooloola sand-mass), south-east Queensland, contains a significant area of Ramsar-listed coastal wetlands, including the globally important patterned fen complexes. These mires form an elaborate network of pools surrounded by vegetated peat ridges and are the only known subtropical, Southern Hemisphere examples, with wetlands of this type typically located in high northern latitudes. Sedimentological, palynological and charcoal analysis from the Wathumba and Moon Point complexes on Fraser Island indicate two periods of swamp formation (that may contain patterned fens), one commencing at 12 000 years ago (Moon Point) and the other ~4300 years ago (Wathumba). Wetland formation and development is thought to be related to a combination of biological and hydrological processes with the dominant peat-forming rush, Empodisma minus, being an important component of both patterned and non-patterned mires within the region. In contrast to Northern Hemisphere paludifying systems, the patterning appears to initiate at the start of wetland development or as part of an infilling process. The wetlands dominated by E. minus are highly resilient to disturbance, particularly burning and sea level alterations, and appear to form important refuge areas for amphibians, fish and birds (both non-migratory and migratory) over thousands of years.

The role of offsets in compensating for damage in the coastal and marine environments

This thesis examines the extent of which economic instruments can be used to minimise environmental damage in the coastal and marine environments, and the role of offsets to compensate for residual damage. Economic principles are used to review current command and control systems, potential incentive based mechanisms, and the development of appropriate offsets. Implementing offsets in the marine environment has a number of challenges, so alternative approaches may be necessary. The study finds that offsets in areas remote from the initial impact, or even to protect different species, may be acceptable provided they result in greater conservation benefits than the standard like-for-like offset. This study is particularly relevant for the design of offsets in the coastal and marine environments where there is limited scope for like-for-like offsets.

Learning Chinese as a Heritage Language: An Australian Perspective

Summary: This book explores the fascinating topic of heritage language learning, looking in particular at Chinese Australians' learning of Chinese. The author studies the investment, challenges and benefits of heritage language learning across varied contexts including school, work, home and in the community. The book investigates how Chinese Australians navigate and negotiate their Chineseness and how resources are used to support their learning. The book is based on a mixed methods study which uses Bourdieu's sociological theory, and offers implications for sociologists of language and education, Chinese heritage language learners and teachers, as well as language and cultural policy makers. Review: This book is a compelling account of the habitus of Chineseness in a world of mobility. It offers up a plethora of insights into the implication of heritage language learning in the constitution of Chinese identity; it makes available a sophisticated mixed methods approach for using the thinking tools of Pierre Bourdieu; it adds to these tools a nuanced cultural dimension. Karen Dooley, Queensland University of Technology, Australia In our increasingly trans-migratory world, language can be central to cultural identity. Dr Mu’s research breaks new ground by adapting Bourdieu’s insights to examine how cultural identity (‘Chinese-ness’) is linguistically learned and practiced in Australia. His remarkable book will interest educators and researchers grappling with how language pertains to identity. Tom Strong, University of Calgary, Canada A thought-provoking, highly engaging work that has deftly shown how Bourdieusian framework can be applied in the research field of literacy education and Heritage Language learning. A must-read for those interested in overseas Chinese communities and Heritage Language learning across various immigrant communities in general! Liang Du, Beijing Normal University, China

Nanofiber orientation and surface functionalization modulate human mesenchymal stem cell behavior in vitro

Electrospun nanofiber meshes have emerged as a new generation of scaffold membranes possessing a number of features suitable for tissue regeneration. One of these features is the flexibility to modify their structure and composition to orchestrate specific cellular responses. In this study, we investigated the effects of nanofiber orientation and surface functionalization on human mesenchymal stem cell (hMSC) migration and osteogenic differentiation. We used an in vitro model to examine hMSC migration into a cell-free zone on nanofiber meshes and mitomycin C treatment to assess the contribution of proliferation to the observed migration. Poly (ɛ-caprolactone) meshes with oriented topography were created by electrospinning aligned nanofibers on a rotating mandrel, while randomly oriented controls were collected on a stationary collector. Both aligned and random meshes were coated with a triple-helical, type I collagen-mimetic peptide, containing the glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) motif. Our results indicate that nanofiber GFOGER peptide functionalization and orientation modulate cellular behavior, individually, and in combination. GFOGER significantly enhanced the migration, proliferation, and osteogenic differentiation of hMSCs on nanofiber meshes. Aligned nanofiber meshes displayed increased cell migration along the direction of fiber orientation compared to random meshes; however, fiber alignment did not influence osteogenic differentiation. Compared to each other, GFOGER coating resulted in a higher proliferation-driven cell migration, whereas fiber orientation appeared to generate a larger direct migratory effect. This study demonstrates that peptide surface modification and topographical cues associated with fiber alignment can be used to direct cellular behavior on nanofiber mesh scaffolds, which may be exploited for tissue regeneration.

Pronounced genetic population structure in a potentially vagile fish species (Pristipomoides multidens, Teleostei; Perciformes; Lutjanidae) from the East Indies triangle

The East Indies triangle, bordered by the Phillipines, Malay Peninsula and New Guinea, has a high level of tropical marine species biodiversity. Pristipomoides multidens is a large, long-lived, fecund snapper species that is distributed throughout the East Indies and Indo-Pacific. Samples were analysed from central and eastern Indonesia and northern Australia to test for genetic discontinuities in population structure. Fish (n = 377) were collected from the Indonesian islands of Bali, Sumbawa, Flores, West Timor, Tanimbar and Tual along with 131 fish from two northern Australian locations (Arafura and Timor Seas) from a previous study. Genetic variation in the control region of the mitochondrial genome was assayed using restriction fragment length polymorphism and direct sequencing. Haplotype diversity was high (0.67-0.82), as was intraspecific sequence divergence (range 0-5.8%). FST between pairs of populations ranged from 0 to 0.2753. Genetic subdivision was apparent on a small spatial scale; FST was 0.16 over 191 km (Bali/Sumbawa) and 0.17 over 491 km (Bali/Flores). Constraints to dispersal that contribute to, and maintain, the observed degree of genetic subdivision are experienced presumably by all life history stages of this tropical marine finfish. The constraints may include (1) little or no movement of eggs or larvae, (2) little or no home range or migratory movement of adults and (3) loss of larval cohorts due to transport of larvae away from suitable habitat by prevailing currents

Epithelial-mesenchymal axis in head and neck cancer cell lines

Objectives: Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous tumour type which necessitates multiple invitro models to attain an appreciation of its multiple subtypes. The phenomenon of epithelial-mesenchymal transition (EMT) isimportant to the development of a metastatic cancer cell phenotype being relevant to the ability of cancer cells to intravasate intovasculature and to invade tissues. The role of EMT in human papilloma virus (HPV) positive HNSCC is not well understood. Thispaper aims to characterize seven HNSCC cell lines (FaDu, SCC-25, SCC-15, CAL27, RPMI2650) including two new HPV-16positive HNSCC cell lines (UD-SCC2, 93-VU-147T) for their epithelial and mesenchymal properties. Materials and methods: A panel of HNSCC cell lines from multiple head and neck anatomical sites were profiled for basalexpression of epithelial and mesenchymal characteristics at mRNA, protein and functional levels (proliferative, migratory andinvasive properties). Furthermore, 3D spheroid forming capabilities were investigated. Results: We found that the HPV-16 positive cell line, in particular UD-SCC2 demonstrated a more invasive and mesenchymalphenotype at the molecular and functional levels suggesting HPV infection may mediate some of these cellular properties.Moreover, HPV-negative cell lines were not strictly epithelial presenting with a dynamic range of expression. Conclusions: This study presents the molecular and phenotypic diversity of HNSCC cell lines. It highlights the need formore studies in this field and a scoring system where HNSCC cell lines are ranked according to their respective epithelial andmesenchymal nature. This data will be useful to anyone modelling HNSCC behaviour, providing a molecular context which willenable them to decipher cell phenotypes and to develop therapies which block EMT progression.

Transmission of Japanese Encephalitis Virus from the Black Flying Fox, Pteropus alecto, to Culex annulirostris Mosquitoes, Despite the Absence of Detectable Viremia.

To determine the potential role of flying foxes in transmission cycles of Japanese encephalitis virus (JEV) in Australia, we exposed Pteropus alecto (Megachiroptera: Pteropididae) to JEV via infected Culex annulirostris mosquitoes or inoculation. No flying foxes developed symptoms consistent with JEV infection. Anti-JEV IgG antibodies developed in 6/10 flying foxes exposed to infected Cx. annulirostris and in 5/5 inoculated flying foxes. Low-level viremia was detected by real-time reverse transcriptase polymerase chain reaction in 1/5 inoculated flying foxes and this animal was able to infect recipient mosquitoes. Although viremia was not detected in any of the 10 flying foxes that were exposed to JEV by mosquito bite, two animals infected recipient mosquitoes. Likewise, an inoculated flying fox without detectable viremia infected recipient mosquitoes. Although infection rates in recipient mosquitoes were low, the high population densities in roosting camps, coupled with migratory behavior indicate that flying foxes could play a role in the dispersal of JEV.