Silencing of ghrelin receptor expression inhibits endometrial cancer cell growth in vitro and in vivo

Ghrelin is a 28-amino acid peptide hormone produced predominantly in the stomach but also in a range of normal cell types and tumors, where it has endocrine, paracrine, and autocrine roles. Previously, we have demonstrated that ghrelin has proliferative and antiapoptotic effects in endometrial cancer cell lines, suggesting a potential role in promoting tumor growth. In the present study, we investigated the effect of ghrelin receptor, GHSR, and gene silencing in vitro and in vivo and characterized ghrelin and GHSR1a protein expression in human endometrial tumors. GHSR gene silencing was achieved in the Ishikawa and KLE endometrial cancer cell lines, using a lentiviral short-hairpin RNA targeting GHSR. The effects of GHSR1a knockdown were further analyzed in vivo using the Ishikawa cell line in a NOD/SCID xenograft model. Cell proliferation was reduced in cultured GHSR1a knockdown Ishikawa and KLE cells compared with scrambled controls in the absence of exogenously applied ghrelin and in response to exogenous ghrelin (1,000 nM). The tumor volumes were reduced significantly in GHSR1a knockdown Ishikawa mouse xenograft tumors compared with scrambled control tumours. Using immunohistochemistry, we demonstrated that ghrelin and GHSR1a are expressed in benign and cancerous glands in human endometrial tissue specimens, although there was no correlation between the intensity of staining and cancer grade. These data indicate that downregulation of GHSR expression significantly inhibits endometrial cancer cell line and mouse xenograft tumour growth. This is the first preclinical evidence that downregulation of GHSR may be therapeutic in endometrial cancer.

Increased tubulointerstitial recruitment of human CD141(hi) CLEC9A(+) and CD1c(+) myeloid dendritic cell subsets in renal fibrosis and chronic kidney disease

Dendritic cells (DCs) play critical roles in immune-mediated kidney diseases. Little is known, however, about DC subsets in human chronic kidney disease, with previous studies restricted to a limited set of pathologies and to using immunohistochemical methods. In this study, we developed novel protocols for extracting renal DC subsets from diseased human kidneys and identified, enumerated, and phenotyped them by multicolor flow cytometry. We detected significantly greater numbers of total DCs as well as CD141(hi) and CD1c(+) myeloid DC (mDCs) subsets in diseased biopsies with interstitial fibrosis than diseased biopsies without fibrosis or healthy kidney tissue. In contrast, plasmacytoid DC numbers were significantly higher in the fibrotic group compared with healthy tissue only. Numbers of all DC subsets correlated with loss of kidney function, recorded as estimated glomerular filtration rate. CD141(hi) DCs expressed C-type lectin domain family 9 member A (CLEC9A), whereas the majority of CD1c(+) DCs lacked the expression of CD1a and DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), suggesting these mDC subsets may be circulating CD141(hi) and CD1c(+) blood DCs infiltrating kidney tissue. Our analysis revealed CLEC9A(+) and CD1c(+) cells were restricted to the tubulointerstitium. Notably, DC expression of the costimulatory and maturation molecule CD86 was significantly increased in both diseased cohorts compared with healthy tissue. Transforming growth factor-β levels in dissociated tissue supernatants were significantly elevated in diseased biopsies with fibrosis compared with nonfibrotic biopsies, with mDCs identified as a major source of this profibrotic cytokine. Collectively, our data indicate that activated mDC subsets, likely recruited into the tubulointerstitium, are positioned to play a role in the development of fibrosis and, thus, progression to chronic kidney disease.

Cell image classification using histograms, higher order statistics and adaboost

A cell classification algorithm that uses first, second and third order statistics of pixel intensity distributions over pre-defined regions is implemented and evaluated. A cell image is segmented into 6 regions extending from a boundary layer to an inner circle. First, second and third order statistical features are extracted from histograms of pixel intensities in these regions. Third order statistical features used are one-dimensional bispectral invariants. 108 features were considered as candidates for Adaboost based fusion. The best 10 stage fused classifier was selected for each class and a decision tree constructed for the 6-class problem. The classifier is robust, accurate and fast by design.

Mesenchymal stem cell therapies for bone and tendon conditions

Bone, tendon, and cartilage are highly specialized musculoskeletal connective tissues that are subject to injury and degeneration. These tissues have relatively poor healing capabilities, and coupled with their variable response to established medical treatments, produce significant morbidity. Mesenchymal stem cells (MSCs) are capable of regenerating skeletal tissues and therefore offer great promise in the treatment of connective tissue pathologies. Adult MSCs are multipotent cells that possess the properties of proliferation and differentiation into all connective tissues. Furthermore, they can be gene modified to secrete growth factors and utilized in connective tissue engineering. Potential MSC-based therapies for bone and tendon conditions are reviewed in this chapter.

methoxy-poly(ethylene glycol) modified poly(L-lactide) enhanced cell affinity of human bone marrow stromal cells by the upregulation of 1-cadherin and delta-2-catenin

Poly(l-lactide) (PLLA), a versatile biodegradable polymer, is one of the most commonly-used materials for tissue engineering applications. To improve cell affinity for PLLA, poly(ethylene glycol) (PEG) was used to develop diblock copolymers. Human bone marrow stromal cells (hBMSCs) were cultured on MPEG-b-PLLA copolymer films to determine the effects of modification on the attachment and proliferation of hBMSC. The mRNA expression of 84 human extracellular matrix (ECM) and adhesion molecules was analyzed using RT-qPCR to understand the underlying mechanisms. It was found that MPEG-b-PLLA copolymer films significantly improved cell adhesion, extension, and proliferation.This was found to be related to the significant upregulation of two adhesion genes, CDH1 and CTNND2, which encode 1-cadherin and delta-2-catenin, respectively, two key components for the cadherin-catenin complex. In summary, MPEG-b-PLLA copolymer surfaces improved initial cell adhesion by stimulation of adhesion molecule gene expression.

Heparan sulfate proteoglycans and human breast cancer epithelial cell tumorigenicity

Heparan sulfate proteoglycans (HSPGs) are key components of the extracellular matrix that mediate cell proliferation, invasion, and cellular signaling. The biological functions of HSPGs are linked to their co-stimulatory effects on extracellular ligands (e.g., WNTs) and the resulting activation of transcription factors that control mammalian development but also associated with tumorigenesis. We examined the expression profile of HSPG core protein syndecans (SDC1–4) and glypicans (GPC1–6) along with the enzymes that initiate or modify their glycosaminoglycan chains in human breast cancer (HBC) epithelial cells. Gene expression in relation to cell proliferation was examined in the HBC cell lines MCF-7 and MDA-MB-231 following treatment with the HS agonist heparin. Heparin increased gene expression of chain initiation and modification enzymes including EXT1 and NDST1, as well as core proteins SDC2 and GPC6. With HS/Wnt interactions established, we next investigated WNT pathway components and observed that increased proliferation of the more invasive MDA-MB-231 cells is associated with activation of the Wnt signaling pathway. Specifically, there was substantial upregulation (>5-fold) of AXIN1, WNT4A, and MYC in MDA-MB-231 but not in MCF-7 cells. The changes in gene expression observed for HSPG core proteins and related enzymes along with the associated Wnt signaling components suggest coordinated interactions. The influence of HSPGs on cellular proliferation and invasive potential of breast cancer epithelial cells are cell and niche specific. Further studies on the interactions between HSPGs and WNT ligands may yield clinically relevant molecular targets, as well as new biomarkers for characterization of breast cancer progression.

Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes

Senescence and genomic integrity are thought to be important barriers in the development of malignant lesions. Human fibroblasts undergo a limited number of cell divisions before entering an irreversible arrest, called senescence. Here we show that human mammary epithelial cells (HMECs) do not conform to this paradigm of senescence. In contrast to fibroblasts, HMECs exhibit an initial growth phase that is followed by a transient growth plateau (termed selection or M0; refs 3-5), from which proliferative cells emerge to undergo further population doublings (approximately 20-70), before entering a second growth plateau (previously termed senescence or M1; refs 4-6). We find that the first growth plateau exhibits characteristics of senescence but is not an insurmountable barrier to further growth. HMECs emerge from senescence, exhibit eroding telomeric sequences and ultimately enter telomere-based crisis to generate the types of chromosomal abnormalities seen in the earliest lesions of breast cancer. Growth past senescent barriers may be a pivotal event in the earliest steps of carcinogenesis, providing many genetic changes that predicate oncogenic evolution. The differences between epithelial cells and fibroblasts provide new insights into the mechanistic basis of neoplastic transformation.

Poly(2-oxazoline) hydrogels as next generation three dimensional cell supports

Synthetic hydrogels selectively decorated with cell adhesion motifs are rapidly emerging as promising substrates for 3D cell culture. When cells are grown in 3D they experience potentially more physiologically relevant cell-cell interactions and physical cues compared with traditional 2D cell culture on stiff surfaces. A newly developed polymer based on poly(2-oxazoline)s has been used for the first time to control attachment of fibroblast cells and is discussed here for its potential use in 3D cell culture with particular focus on cancer cells towards the ultimate aim of high throughput screening of anti-cancer therapies. Advantages and limitations of using poly(2-oxazoline) hydrogels are discussed and compared with more established polymers, especially polyethylene glycol (PEG).

Gelatine methacrylamide-based hydrogels : an alternative three-dimensional cancer cell culture system

Modern cancer research requires physiological, three-dimensional (3-D) cell culture platforms, wherein the physical and chemical characteristics of the extracellular matrix (ECM) can be modified. In this study, gelatine methacrylamide (GelMA)-based hydrogels were characterized and established as in vitro and in vivo spheroid-based models for ovarian cancer, reflecting the advanced disease stage of patients, with accumulation of multicellular spheroids in the tumour fluid (ascites). Polymer concentration (2.5-7% w/v) strongly influenced hydrogel stiffness (0.5±0.2kPa to 9.0±1.8kPa) but had little effect on solute diffusion. The diffusion coefficient of 70kDa fluorescein isothiocyanate (FITC)-labelled dextran in 7% GelMA-based hydrogels was only 2.3 times slower compared to water. Hydrogels of medium concentration (5% w/v GelMA) and stiffness (3.4kPa) allowed spheroid formation and high proliferation and metabolic rates. The inhibition of matrix metalloproteinases and consequently ECM degradability reduced spheroid formation and proliferation rates. The incorporation of the ECM components laminin-411 and hyaluronic acid further stimulated spheroid growth within GelMA-based hydrogels. The feasibility of pre-cultured GelMA-based hydrogels as spheroid carriers within an ovarian cancer animal model was proven and led to tumour development and metastasis. These tumours were sensitive to treatment with the anti-cancer drug paclitaxel, but not the integrin antagonist ATN-161. While paclitaxel and its combination with ATN-161 resulted in a treatment response of 33-37.8%, ATN-161 alone had no effect on tumour growth and peritoneal spread. The semi-synthetic biomaterial GelMA combines relevant natural cues with tunable properties, providing an alternative, bioengineered 3-D cancer cell culture in in vitro and in vivo model systems.

Mechanical influences on endothelial cell network formation in vitro

While both the restoration of the blood supply and an appropriate local mechanical environment are critical for uneventful bone healing, their influence on each other remains unclear. Human bone fracture haematomas (<72h post-trauma) were cultivated for 3 days in fibrin matrices, with or without cyclic compression. Conditioned medium from these cultures enhanced the formation of vessel-like networks by HMEC-1 cells, and mechanical loading further elevated it, without affecting the cells’ metabolic activity. While haematomas released the angiogenesis-regulators, VEGF and TGF-β1, their concentrations were not affected by mechanical loading. However, direct cyclic stretching of the HMEC-1 cells decreased network formation. The appearance of the networks and a trend towards elevated VEGF under strain suggested physical disruption rather than biochemical modulation as the responsible mechanism. Thus, early fracture haematomas and their mechanical loading increase the paracrine stimulation of endothelial organisation in vitro, but direct periodic strains may disrupt or impair vessel assembly in otherwise favourable conditions.

Silicate-based bioceramics for periodontal regeneration

Periodontal disease is characterized by the destruction of the tissues that attach the tooth to the alveolar bone. Various methods for regenerative periodontal therapy including the use of barrier membranes, bone replacement grafts, and growth factor delivery have been investigated; however, true regeneration of periodontal tissue is still a significant challenge to scientists and clinicians. The focus on periodontal tissue engineering has shifted from attempting to recreate tissue replacements/constructs to the development of biomaterials that incorporate and release regulatory signals to achieve in situ periodontal regeneration. The release of ions and molecular cues from biomaterials may help to unlock latent regenerative potential in the body by regulating cell proliferation and differentiation towards different lineages (e.g. osteoblasts and cementoblasts). Silicate-based bioactive materials, including bioactive silicate glasses and ceramics, have become the materials of choice for periodontal regeneration, due to their favourable osteoconductivity and bioactivity. This article will focus on the most recent advances in the in vitro and in vivo biological application of silicate-based ceramics, specifically as it relates to periodontal tissue engineering.

An approach for adaptive and integrated environmental planning to deal with uncertainty in a Great Barrier Reef Catchment

This paper presents the Mossman Mill District Practices Framework. It was developed in the Wet Tropics region within the Great Barrier Reef in north-eastern Australia to describe the environmental benefits of agricultural management practices for the sugar cane industry. The framework translates complex, unclear and overlapping environmental plans, policy and legal arrangements into a simple framework of management practices that landholders can use to improve their management actions. Practices range from those that are old or outdated through to aspirational practices that have the potential to achieve desired resource condition targets. The framework has been applied by stakeholders at multiple scales to better coordinate and integrate a range of policy arrangements to improve natural resource management. It has been used to structure monitoring and evaluation in order to underpin a more adaptive approach to planning at mill district and property scale. Potentially, the framework and approach can be applied across fields of planning where adaptive management is needed. It has the potential to overcome many of the criticisms of property-scale and regional Natural Resource Management.

A rapid assessment of the effects of extreme weather on two Great Barrier Reef industries

In coastal areas, extreme weather events, such as floods and cyclones, can have debilitating effects on the social and economic viability of marine-based industries. In March 2011, the Great Barrier Reef Marine Park Authority implemented an Extreme Weather Response Program, following a period of intense flooding and cyclonic activity between December 2010 and February 2011. In this paper, we discuss the results of a project within the Program, which aimed to: (1) assess the impacts of extreme weather events on regional tourism and commercial fishing industries; and (2) develop and road-test an impact assessment matrix to improve government and industry responses to extreme weather events. Results revealed that extreme weather events both directly and indirectly affected all five of the measured categories, i.e. ecological, personal, social, infrastructure and economic components. The severity of these impacts, combined with their location and the nature of their business, influenced how tourism operators and fishers assessed the impact of the events (low, medium, high or extreme). The impact assessment tool was revised following feedback obtained during stakeholder workshops and may prove useful for managers in responding to potential direct and indirect impacts of future extreme weather events on affected marine industries. © 2013 Planning Institute Australia.

A method for risk analysis across governance systems : a great barrier reef case study

Healthy governance systems are key to delivering sound environmental management outcomes from global to local scales. There are, however, surprisingly few risk assessment methods that can pinpoint those domains and sub-domains within governance systems that are most likely to influence good environmental outcomes at any particular scale, or those if absent or dysfunctional, most likely to prevent effective environmental management. This paper proposes a new risk assessment method for analysing governance systems. This method is then tested through its preliminary application to a significant real-world context: governance as it relates to the health of Australia's Great Barrier Reef (GBR). The GBR exists at a supra-regional scale along most of the north eastern coast of Australia. Brodie et al (2012 Mar. Pollut. Bull. 65 81-100) have recently reviewed the state and trend of the health of the GBR, finding that overall trends remain of significant concern. At the same time, official international concern over the governance of the reef has recently been signalled globally by the International Union for the Conservation of Nature (IUCN). These environmental and political contexts make the GBR an ideal candidate for use in testing and reviewing the application of improved tools for governance risk assessment. © 2013 IOP Publishing Ltd.

Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of TvDAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris TvDAO whole-cell biocatalyst. As compared to the native T. variabilis host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in P. pastoris through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 x 103 U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of >or= 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of P. pastoris using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used TvDAO expressing Escherichia coli strain) under conditions of D-methionine conversion using vigorous aeration. Stepwise optimization using a multi-level engineering approach has delivered a new P. pastoris whole cell TvDAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this P. pastoris catalyst has been evaluated for industrial purposes.