Modulation of breast cancer progression and differentiation by the gp30/neregulin

In the last decade we have come to understand that the growth of cancer cells in general and of breast cancer in particular depends, in many cases, upon growth factors that will bind to and activate their receptors. One of these growth factor receptors is the erbB-2 protein which plays an important role in the prognosis of breast cancer and is overexpressed in nearly 30% of human breast cancer patients. While evidence accumulates to support the relationship between erbB-2 overexpression and poor overall survival in breast cancer, understanding of the biological consequence(s) of erbB-2 overexpression remains elusive. Our recent discovery of the gp30 has allowed us to identify a number of related but distinct biological endpoints which appear responsive to signal transduction through the erbB-2 receptor. These endpoints of growth, invasiveness, and differentiation have clear implications for the emergence, maintenance and/or control of malignancy, and represent established endpoints in the assessment of malignant progression in breast cancer. We have shown that gp30 induces a biphasic growth effect on cells with erbB-2 over-expression. We have recently determined the protein sequence of gp30 and cloned its full length cDNA sequence. We have also cloned two additional forms to the ligand, that are believed to be different isoforms. We are currently expressing the different forms in order to determine their biological effects. To elucidate the cellular mechanisms underlying cell growth inhibition by gp30, we tested the effect of this ligand on cell growth and differentiation of the human breast cancer cells which overexpress erbB-2 and cells which express low levels of this protooncogene. High concentrations of ligand induced differentiation of cells overexpressing erbB-2, as measured by inhibition of cell growth, and increased synthesis of milk components, and modulation of E-cadherin and up- regulation of c-jun and c-fos. These findings indicate that ligand-induced growth inhibition in cells overexpressing erbB-2 is associated with an apparent induction of differentiation. The availability of gp30 derived synthetic peptides and its full cDNAs provides tools necessary to acquire a better understanding of the mechanism of action of the this ligands and the erbB-2 receptor in breast cancer.

Adipose differentiation of bone marrow-derived mesenchymal stem cells using Pluronic F-127 hydrogel in vitro

Due to increasing clinical demand for adipose tissue, a suitable scaffold for engineering adipose tissue constructs is needed. In this study, we have developed a three-dimensional (3-D) culture system using bone marrow-derived mesenchymal stem cells (BM-MSC) and a Pluronic F-127 hydrogel scaffold as a step towards the in vitro tissue engineering of fat. BM-MSC were dispersed into a Pluronic F-127 hydrogel with or without type I collagen added. The adipogenic differentiation of the BM-MSC was assessed by cellular morphology and further confirmed by Oil Red O staining. The BM-MSC differentiated into adipocytes in Pluronic F-127 in the presence of adipogenic stimuli over a period of 2 weeks, with some differentiation present even in absence of such stimuli. The addition of type I collagen to the Pluronic F-127 caused the BM-MSC to aggregate into clumps, thereby generating an uneven adipogenic response, which was not desirable.

Lithium release from ß-tricalcium phosphate inducing cementogenic and osteogenic differentiation for both hPDLCs and hBMSCs

It is accepted that the accelerated differentiation of tissue cells on bioactive materials is of great importance to regenerate the lost tissues. It was previously reported that lithium (Li) ions could enhance the in vitro proliferation and differentiation of retinoblastoma cells and endometrium epithelia by activating the Wnt canonical signalling pathway. It is interesting to incorporate Li ions into bioactive ceramics, such as β-tricalcium phosphate (Li-β-TCP), in order to stimulate both osteogenic and cementogenic differentiation of different stem cells for the regeneration of bone/periodontal tissues. Therefore, the aim of this study was to investigate the interactions of human periodontal ligament cells (hPDLCs) and human bone marrow stromal cells (hBMSCs) with Li-β-TCP bioceramic bulks and their ionic extracts, and further explore the osteogenic and cementogenic stimulation of Li-β-TCP bioceramics and the possible molecular mechanisms. The results showed that Li-β-TCP bioceramic disks supported the cell attachment and proliferation, and significantly enhanced bone/cementum-related gene expression, Wnt canonical signalling pathway activation for both hPDLCs and hBMSCs, compared to conventional β-TCP bioceramic disks without Li. The release of Li from Li-β-TCP powders could significantly promote the bone/cementum-related gene expression for both hPDLCs and hBMSCs compared to pure β-TCP extracts without Li release. Our results suggest that the combination of Li with β-TCP bioceramics may be a promising method to enhance bone/cementum regeneration as Li-β-TCP possesses excellent in vitro osteogenic and cementogenic stimulation properties by inducing bone/cementum-related gene expression in both hPDLCs and hBMSCs.

Selective neuronal differentiation of neural stem cells induced by nanosecond microplasma agitation

An essential step for therapeutic and research applications of stem cells is their ability to differentiate into specific cell types. Neuronal cells are of great interest for medical treatment of neurodegenerative diseases and traumatic injuries of central nervous system (CNS), but efforts to produce these cells have been met with only modest success. In an attempt of finding new approaches, atmospheric-pressure room-temperature microplasma jets (MPJs) are shown to effectively direct in vitro differentiation of neural stem cells (NSCs) predominantly into neuronal lineage. Murine neural stem cells (C17.2-NSCs) treated with MPJs exhibit rapid proliferation and differentiation with longer neurites and cell bodies eventually forming neuronal networks. MPJs regulate ~. 75% of NSCs to differentiate into neurons, which is a higher efficiency compared to common protein- and growth factors-based differentiation. NSCs exposure to quantized and transient (~. 150. ns) micro-plasma bullets up-regulates expression of different cell lineage markers as β-Tubulin III (for neurons) and O4 (for oligodendrocytes), while the expression of GFAP (for astrocytes) remains unchanged, as evidenced by quantitative PCR, immunofluorescence microscopy and Western Blot assay. It is shown that the plasma-increased nitric oxide (NO) production is a factor in the fate choice and differentiation of NSCs followed by axonal growth. The differentiated NSC cells matured and produced mostly cholinergic and motor neuronal progeny. It is also demonstrated that exposure of primary rat NSCs to the microplasma leads to quite similar differentiation effects. This suggests that the observed effect may potentially be generic and applicable to other types of neural progenitor cells. The application of this new in vitro strategy to selectively differentiate NSCs into neurons represents a step towards reproducible and efficient production of the desired NSC derivatives. © 2013.

Glucocorticoid receptor haploinsufficiency causes hypertension and attenuates hypothalamic-pituitary-adrenal axis and blood pressure adaptions to high-fat diet

Glucocorticoid hormones are critical to respond and adapt to stress. Genetic variations in the glucocorticoid receptor (GR) gene alter hypothalamic-pituitary-adrenal (HPA) axis activity and associate with hypertension and susceptibility to metabolic disease. Here we test the hypothesis that reduced GR density alters blood pressure and glucose and lipid homeostasis and limits adaption to obesogenic diet. Heterozygous GR βgeo/+ mice were generated from embryonic stem (ES) cells with a gene trap integration of a β-galactosidase-neomycin phosphotransferase (βgeo) cassette into the GR gene creating a transcriptionally inactive GR fusion protein. Although GRβgeo/+ mice have 50% less functional GR, they have normal lipid and glucose homeostasis due to compensatory HPA axis activation but are hypertensive due to activation of the renin-angiotensin- aldosterone system (RAAS). When challenged with a high-fat diet, weight gain, adiposity, and glucose intolerance were similarly increased in control and GRβgeo/+ mice, suggesting preserved control of intermediary metabolism and energy balance. However, whereas a high-fat diet caused HPA activation and increased blood pressure in control mice, these adaptions were attenuated or abolished in GRβgeo/+ mice. Thus, reduced GR density balanced by HPA activation leaves glucocorticoid functions unaffected but mineralocorticoid functions increased, causing hypertension. Importantly, reduced GR limits HPA and blood pressure adaptions to obesogenic diet.

A comparative study of the proliferation and osteogenic differentiation of human periodontal ligament cells cultured on β-TCP ceramics and demineralized bone matrix with or without osteogenic inducers in vitro

The repair of bone defects that result from periodontal diseases remains a clinical challenge for periodontal therapy. β-tricalcium phosphate (β-TCP) ceramics are biodegradable inorganic bone substitutes with inorganic components that are similar to those of bone. Demineralized bone matrix (DBM) is an acid-extracted organic matrix derived from bone sources that consists of the collagen and matrix proteins of bone. A few studies have documented the effects of DBM on the proliferation and osteogenic differentiation of human periodontal ligament cells (hPDLCs). The aim of the present study was to investigate the effects of inorganic and organic elements of bone on the proliferation and osteogenic differentiation of hPDLCs using three-dimensional porous β-TCP ceramics and DBM with or without osteogenic inducers. Primary hPDLCs were isolated from human periodontal ligaments. The proliferation of the hPDLCs on the scaffolds in the growth culture medium was examined using a Cell‑Counting kit‑8 (CCK-8) and scanning electron microscopy (SEM). Alkaline phosphatase (ALP) activity and the osteogenic differentiation of the hPDLCs cultured on the β-TCP ceramics and DBM were examined in both the growth culture medium and osteogenic culture medium. Specific osteogenic differentiation markers were examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). SEM images revealed that the cells on the β-TCP were spindle-shaped and much more spread out compared with the cells on the DBM surfaces. There were no significant differences observed in cell proliferation between the β-TCP ceramics and the DBM scaffolds. Compared with the cells that were cultured on β-TCP ceramics, the ALP activity, as well as the Runx2 and osteocalcin (OCN) mRNA levels in the hPDLCs cultured on DBM were significantly enhanced both in the growth culture medium and the osteogenic culture medium. The organic elements of bone may exhibit greater osteogenic differentiation effects on hPDLCs than the inorganic elements.

The contingent effects of differentiation and integration on corporate entrepreneurship

This paper develops a contingency view regarding the effects of structural differentiation and integration on levels of corporate entrepreneurship. Integrating notions of benefits and costs resulting from integration with structural contingency theory, we argue that the joint effects of structural differentiation and integration on corporate entrepreneurship levels are moderated by organizational size and environmental dynamism. Our findings from a time-separated sample demonstrate that in smaller organizations and more dynamic environments, the positive effects of integration on the structural differentiation-corporate entrepreneurship relationship strongly diminish. As such, with this research we begin to identify contingencies that influence the corporate entrepreneurship levels observed among firms striving to balance the needs for structural differentiation and integration.

Audit pricing and product differentiation in small private firms : evidence from Thailand

Purpose – There is limited evidence on how differences in economic environments affect the demand for and supply of auditing. Research on audit pricing has mainly focused on large client markets in developed economies; in contrast, the purpose of this paper is to focus on the small client segment in the emerging economy of Thailand which offers a choice between auditors of two different qualities. Design/methodology/approach – This paper is based on a random stratified sample of small clients in Thailand qualifying for audit exemption. The final sample consists of 1,950 firm-year observations for 2002-2006. Findings – The authors find evidence of product differentiation in the small client market, suggesting that small firms view certified public accountants as superior and pay a premium for their services. The authors also find that audit fees have a positive significant association with leverage, metropolitan location and client size. Audit risk and audit opinion are not, however, significantly associated with audit fees. Furthermore, the authors find no evidence that clients whose financial year ends in the auditors’ busy period pay significantly higher audit fees, and auditors engage in low-balling on initial engagements to attract audit clients. Research limitations/implications – The research shows the importance of exploring actual decisions regarding audit practice and audit pricing in different institutional and organizational settings. Originality/value – The paper extends the literature from developed economies and large/listed market setting to the emerging economy and small client market setting. As far as the authors are aware, this is the first paper to examine audit pricing in the small client market in an emerging economy.

The importance of connexin hemichannels during chondroprogenitor cell differentiation in hydrogel versus microtissue culture models

Appropriate selection of scaffold architecture is a key challenge in cartilage tissue engineering. Gap junction-mediated intercellular contacts play important roles in precartilage condensation of mesenchymal cells. However, scaffold architecture could potentially restrict cell-cell communication and differentiation. This is particularly important when choosing the appropriate culture platform as well as scaffold-based strategy for clinical translation, that is, hydrogel or microtissues, for investigating differentiation of chondroprogenitor cells in cartilage tissue engineering. We, therefore, studied the influence of gap junction-mediated cell-cell communication on chondrogenesis of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and articular chondrocytes. Expanded human chondrocytes and BM-MSCs were either (re-) differentiated in micromass cell pellets or encapsulated as isolated cells in alginate hydrogels. Samples were treated with and without the gap junction inhibitor 18-α glycyrrhetinic acid (18αGCA). DNA and glycosaminoglycan (GAG) content and gene expression levels (collagen I/II/X, aggrecan, and connexin 43) were quantified at various time points. Protein localization was determined using immunofluorescence, and adenosine-5'-triphosphate (ATP) was measured in conditioned media. While GAG/DNA was higher in alginate compared with pellets for chondrocytes, there were no differences in chondrogenic gene expression between culture models. Gap junction blocking reduced collagen II and extracellular ATP in all chondrocyte cultures and in BM-MSC hydrogels. However, differentiation capacity was not abolished completely by 18αGCA. Connexin 43 levels were high throughout chondrocyte cultures and peaked only later during BM-MSC differentiation, consistent with the delayed response of BM-MSCs to 18αGCA. Alginate hydrogels and microtissues are equally suited culture platforms for the chondrogenic (re-)differentiation of expanded human articular chondrocytes and BM-MSCs. Therefore, reducing direct cell-cell contacts does not affect in vitro chondrogenesis. However, blocking gap junctions compromises cell differentiation, pointing to a prominent role for hemichannel function in this process. Therefore, scaffold design strategies that promote an increasing distance between single chondroprogenitor cells do not restrict their differentiation potential in tissue-engineered constructs.

Investigating how customer insights inform strategy leading to brand differentiation in the retail sector

Using qualitative research with case studies of firms in the Australian retail sector, this thesis explores the link between brand differentiation, customer insights, and strategy development to deliver a unique customer experience. The research focus is how brand expression is driven by customer insights. Findings indicate that customer experience is made tangible by the strategic design and alignment of the brand's expression and is crucial to retail success. A significant practical outcome is the development of the Brand Differentiated Model. Created as a tool to potentially assist retailers develop brands from the 'inside out' and confront future disruptions.

Differentiation of cultivars of flos chrysanthemum with the use of high-performance liquid chromatography fingerprints and chemometrics

Flos Chrysanthemum is a generic name for a particular group of edible plants, which also have medicinal properties. There are, in fact, twenty to thirty different cultivars, which are commonly used in beverages and for medicinal purposes. In this work, four Flos Chrysanthemum cultivars, Hangju, Taiju, Gongju, and Boju, were collected and chromatographic fingerprints were used to distinguish and assess these cultivars for quality control purposes. Chromatography fingerprints contain chemical information but also often have baseline drifts and peak shifts, which complicate data processing, and adaptive iteratively reweighted, penalized least squares, and correlation optimized warping were applied to correct the fingerprint peaks. The adjusted data were submitted to unsupervised and supervised pattern recognition methods. Principal component analysis was used to qualitatively differentiate the Flos Chrysanthemum cultivars. Partial least squares, continuum power regression, and K-nearest neighbors were used to predict the unknown samples. Finally, the elliptic joint confidence region method was used to evaluate the prediction ability of these models. The partial least squares and continuum power regression methods were shown to best represent the experimental results.

Integrated genome-wide chromatin occupancy and expression analyses identify key myeloid pro-differentiation transcription factors repressed by Myb

To gain insight into the mechanisms by which the Myb transcription factor controls normal hematopoiesis and particularly, how it contributes to leukemogenesis, we mapped the genome-wide occupancy of Myb by chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) in ERMYB myeloid progenitor cells. By integrating the genome occupancy data with whole genome expression profiling data, we identified a Myb-regulated transcriptional program. Gene signatures for leukemia stem cells, normal hematopoietic stem/progenitor cells and myeloid development were overrepresented in 2368 Myb regulated genes. Of these, Myb bound directly near or within 793 genes. Myb directly activates some genes known critical in maintaining hematopoietic stem cells, such as Gfi1 and Cited2. Importantly, we also show that, despite being usually considered as a transactivator, Myb also functions to repress approximately half of its direct targets, including several key regulators of myeloid differentiation, such as Sfpi1 (also known as Pu.1), Runx1, Junb and Cebpb. Furthermore, our results demonstrate that interaction with p300, an established coactivator for Myb, is unexpectedly required for Myb-mediated transcriptional repression. We propose that the repression of the above mentioned key pro-differentiation factors may contribute essentially to Myb's ability to suppress differentiation and promote self-renewal, thus maintaining progenitor cells in an undifferentiated state and promoting leukemic transformation. © 2011 The Author(s).

Differentiation of Bordetella avium and Related Species by Cellular Fatty Acid Analysis

The fatty acids of 18 strains of Bordetella avium, 3 strains of Alcaligenes faecalis, 5 strains of Bordetella bronchiseptica, and 12 strains of a B. avium-like organism were examined by gas chromatography-mass spectrometry. The presence of a significant amount of the acid 2-OH C14:0 characterized B. avium and the B. avium-like organism. B. avium and the B. avium-like organism differed in their relative concentrations of C16:1 and 3-OH C14:0 acids. B. bronchiseptica and A. faecalis were distinguishable by comparison of the relative concentrations of C18:0 and C18:1 acids.

Cuticular Hydrocarbons of Buffalo Fly, Haematobia exigua, and Chemotaxonomic Differentiation from Horn Fly, H.irritans

We determined the quantity and chemical composition of cuticular hydrocarbons of different strains, sex and age of buffalo flies, Haematobia exigua. The quantity of cuticular hydrocarbons increased from less than 1 µg/fly for newly-emerged flies to over 11 µg/fly in 13 d-old flies. The hydrocarbon chain length varied from C21 to C29, with unbranched alkanes and monounsaturated alkenes the major components. Newly emerged flies produced almost exclusively C27 hydrocarbons. Increasing age was accompanied by the appearance of hydrocarbons with shorter carbon chains and an increase in the proportion of alkenes. 11 Tricosene and 7-tricosene were the most abundant hydrocarbons in mature buffalo flies. Cuticular hydrocarbons of buffalo flies are distinctly different from those of horn flies. The most noticeable differences were in the C23 alkenes, with the major isomers 11- and 7-tricosene in buffalo flies and (Z)-9- and (Z)-5-tricosene in horn flies, respectively. Cuticular hydrocarbon analysis provides a reliable method to differentiate buffalo and horn fly, which are difficult to separate morphologically. The differences in cuticular hydrocarbons also support their recognition as separate species, H. exigua and H. irritans, rather than as subspecies.