Molecular analysis of metastasis in a polyomavirus middle T mouse model: the role of osteopontin

INTRODUCTION: In order to study metastatic disease, we employed the use of two related polyomavirus middle T transgenic mouse tumor transplant models of mammary carcinoma (termed Met and Db) that display significant differences in metastatic potential. METHODS: Through suppression subtractive hybridization coupled to the microarray, we found osteopontin (OPN) to be a highly expressed gene in the tumors of the metastatic mouse model, and a lowly expressed gene in the tumors of the lowly metastatic mouse model. We further analyzed the role of OPN in this model by examining sense and antisense constructs using in vitro and in vivo methods. RESULTS: With in vivo metastasis assays, the antisense Met cells showed no metastatic tumor formation to the lungs of recipient mice, while wild-type Met cells, with higher levels of OPN, showed significant amounts of metastasis. The Db cells showed a significantly reduced metastasis rate in the in vivo metastasis assay as compared with the Met cells. Db cells with enforced overexpression of OPN showed elevated levels of OPN but did not demonstrate an increase in the rate of metastasis compared with the wild-type Db cells. CONCLUSIONS: We conclude that OPN is an essential regulator of the metastatic phenotype seen in polyomavirus middle T-induced mammary tumors. Yet OPN expression alone is not sufficient to cause metastasis. These data suggest a link between metastasis and phosphatidylinositol-3-kinase-mediated transcriptional upregulation of OPN, but additional phosphatidylinositol-3-kinase-regulated genes may be essential in precipitating the metastasis phenotype in the polyomavirus middle T model

The Role of Neutrophil Proteins on the Amyloid Beta-RAGE Axis

We would like to thank Dr. Arthur Owora, previously a Research Biostatistician of the Department of Pharmaceutical Sciences, University of Oklahoma Health Sciences Center, for his assistance on the statistical analysis performed in this study. We thank Dr. Sixia Chen of the Department of Biostatistics and Epidemiogy, University of Oklahoma Health Sciences Center, for his additional input on the statistical analysis. We thank the Laboratory for Molecular Biology and Cytometry Research at the University of Oklahoma Health Sciences Center for the use of the Core Facility which allowed us to perform the MALDI-TOF MS and MS/MS experiments. GM-0111 was provided as a gift by Dr. Justin Savage, GlycoMira Therapeutics, Inc.We previously showed an elevated expression of the neutrophil protein, cationic antimicrobial protein of 37kDa (CAP37), in brains of patients with Alzheimer’s disease (AD), suggesting that CAP37 could be involved in AD pathogenesis. The first step in determining how CAP37 might contribute to AD pathogenesis was to identify the receptor through which it induces cell responses. To identify a putative receptor, we performed GAMMA analysis to determine genes that positively correlated with CAP37 in terms of expression. Positive correlations with ligands for the receptor for advanced glycation end products (RAGE) were observed. Additionally, CAP37 expression positively correlated with two other neutrophil proteins, neutrophil elastase and cathepsin G. Enzyme-linked immunosorbent assays (ELISAs) demonstrated an interaction between CAP37, neutrophil elastase, and cathepsin G with RAGE. Amyloid beta 1–42 (Aβ1–42), a known RAGE ligand, accumulates in AD brains and interacts with RAGE, contributing to Aβ1–42 neurotoxicity. We questioned whether the binding of CAP37, neutrophil elastase and/or cathepsin G to RAGE could interfere with Aβ1–42 binding to RAGE. Using ELISAs, we determined that CAP37 and neutrophil elastase inhibited binding of Aβ1–42 to RAGE, and this effect was reversed by protease inhibitors in the case of neutrophil elastase. Since neutrophil elastase and cathepsin G have enzymatic activity, mass spectrometry was performed to determine the proteolytic activity of all three neutrophil proteins on Aβ1–42. All three neutrophil proteins bound to Aβ1–42 with different affinities and cleaved Aβ1–42 with different kinetics and substrate specificities. We posit that these neutrophil proteins could modulate neurotoxicity in AD by cleaving Aβ1–42 and influencing the Aβ1–42 –RAGE interaction. Further studies will be required to determine the biological significance of these effects and their relevance in neurodegenerative diseases such as AD. Our findings identify a novel area of study that underscores the importance of neutrophils and neutrophil proteins in neuroinflammatory diseases such as AD.Yeshttp://www.plosone.org/static/editorial#pee

Microbial shifts in the aging mouse gut

YesBackground: The changes that occur in the microbiome of aging individuals are unclear, especially in light of the imperfect correlation of frailty with age. Studies in older human subjects have reported subtle effects, but these results may be confounded by other variables that often change with age such as diet and place of residence. To test these associations in a more controlled model system, we examined the relationship between age, frailty, and the gut microbiome of female C57BL/6 J mice. Results: The frailty index, which is based on the evaluation of 31 clinical signs of deterioration in mice, showed a near-perfect correlation with age. We observed a statistically significant relationship between age and the taxonomic composition of the corresponding microbiome. Consistent with previous human studies, the Rikenellaceae family, which includes the Alistipes genus, was the most significantly overrepresented taxon within middle-aged and older mice. The functional profile of the mouse gut microbiome also varied with host age and frailty. Bacterial-encoded functions that were underrepresented in older mice included cobalamin (B12) and biotin (B7) biosynthesis, and bacterial SOS genes associated with DNA repair. Conversely, creatine degradation, associated with muscle wasting, was overrepresented within the gut microbiomes of the older mice, as were bacterial-encoded β-glucuronidases, which can influence drug-induced epithelial cell toxicity. Older mice also showed an overabundance of monosaccharide utilization genes relative to di-, oligo-, and polysaccharide utilization genes, which may have a substantial impact on gut homeostasis. Conclusion: We have identified taxonomic and functional patterns that correlate with age and frailty in the mouse microbiome. Differences in functions related to host nutrition and drug pharmacology vary in an age-dependent manner, suggesting that the availability and timing of essential functions may differ significantly with age and frailty. Future work with larger cohorts of mice will aim to separate the effects of age and frailty, and other factors.This work was supported by the Canadian Institutes of Health Research (CIHR) through an Emerging Team Grant to RGB, CIHR Operating Grants to Langille et al. Microbiome 2014, 2:50 Page 10 of 12 http://www.microbiomejournal.com/content/2/1/50 SEH (MOP 126018) and RAR (MOP 93718), and a CIHR Fellowship to MGIL. Infrastructure was supported by the Canada Foundation for Innovation through a grant to RGB. RGB also acknowledges the support of the Canada Research Chairs program

A semi-3D real-time imaging technique for measuring bone cell deformation under fluid flow

Bone cells respond to fluid shear loading by activating various biochemical pathways, mediating a dynamic process of bone formation and resorption. The whole-cell volume dilatation [1] and regional deformation of intracellular structures [2] may be able to directly activate and modulate relevant biochemical pathways. Therefore, understanding how bone cells deform under fluid flow can help elucidate the fundamental mechanisms by which mechanical stimuli are able to initiate biochemical responses. Most studies on cell deformation have focused only on cell deformation in the plane parallel to the substrate surface. Height-dependent cell deformation has not been well characterized even though it may contribute greatly to mechanotransduction mechanisms. Traditional techniques to obtain this additional height information of a cell-body, such as confocal and deconvolution microscopy, are inherently limited by the timescale under which the deformational information can be visualized. Previous studies have investigated cell adhesion to substrate under flow using a single view side-view imaging technique [3, 4]. In this study, we present a novel technique that is able to image a single cell simultaneously in two orthogonal planes to obtain real-time images of a cell at a millisecond timescale. Thus, the objectives of this study were to: (1) develop an imaging technique to visualize the depth-directional information of a cell simultaneously with the traditional 2D view; (2) map out the strain fields of the cell by image analysis; and (3) investigate the viscoelastic behavior of osteoblasts under steady fluid flow.</jats:p