Advanced Centaur explicit guidance equation study Final report

Generalized equations and in-flight computer requirements for Centaur guidance and control and advanced mission plannin

Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and b1-integrin activation

Lymphatic invasion and lymph node metastasis correlate with poor clinical outcome in melanoma. However, the mechanisms of lymphatic dissemination in distant metastasis remain incompletely understood. We show here that exposure of expansively growing human WM852 melanoma cells, but not singly invasive Bowes cells, to lymphatic endothelial cells (LEC) in 3D co-culture facilitates melanoma distant organ metastasis in mice. To dissect the underlying molecular mechanisms, we established LEC co-cultures with different melanoma cells originating from primary tumors or metastases. Notably, the expansively growing metastatic melanoma cells adopted an invasively sprouting phenotype in 3D matrix that was dependent on MMP14, Notch3 and β1-integrin. Unexpectedly, MMP14 was necessary for LEC-induced Notch3 induction and coincident β1-integrin activation. Moreover, MMP14 and Notch3 were required for LEC-mediated metastasis of zebrafish xenografts. This study uncovers a unique mechanism whereby LEC contact promotes melanoma metastasis by inducing a reversible switch from 3D growth to invasively sprouting cell phenotype

Loss of ADAM9 expression impairs beta 1 integrin endocytosis, focal adhesion formation and cancer cell migration

The transmembrane protease ADAM9 is frequently upregulated in human cancers, and it promotes tumour progression in mice. In vitro, ADAM9 regulates cancer cell adhesion and migration by interacting with integrins. However, how ADAM9 modulates integrin functions is not known. We here show that ADAM9 knockdown increases beta 1 integrin levels through mechanisms that are independent of its protease activity, in ADAM9-silenced cells, adhesion to collagen and fibronectin is reduced, suggesting an altered function of the accumulated integrins. Mechanistically, ADAM9 co-immunoprecipitates with beta 1 integrin, and both internalization and subsequent degradation of beta 1 integrin are significantly decreased in ADAM9-silenced cells, with no effect on beta 1 integrin recycling. Accordingly, the formation of focal adhesions and actin stress fibres in ADAM9-silenced cells is altered, possibly explaining the reduction in cell adhesion and migration in these cells. Taken together, our data provide mechanistic insight into the ADAM9-integrin interaction, demonstrating that ADAM9 regulates beta 1 integrin endocytosis. Moreover, our findings indicate that the reduced migration of ADAM9-silenced cells is, at least in part, caused by the accumulation and altered activity of beta 1 integrin at the cell surface

Bone turnover markers are correlated with total skeletal uptake of 99mTc-methylene diphosphonate (99mTc-MDP)

ABSTRACT: BACKGROUND: Skeletal uptake of 99mTc labelled methylene diphosphonate (99mTc-MDP) is used for producing images of pathological bone uptake due to its incorporation to the sites of active bone turnover. This study was done to validate bone turnover markers using total skeletal uptake (TSU) of 99mTc-MDP. METHODS: 22 postmenopausal women (52-80 years) volunteered to participate. Scintigraphy was performed by injecting 520 MBq of 99mTc-MDP and taking whole body images after 3 minutes, and 5 hours. TSU was calculated from these two images by taking into account the urinary loss and soft tissue uptake. Bone turnover markers used were bone specific alkaline phosphatase (S-Bone ALP), three different assays for serum osteocalcin (OC), tartrate resistant acid phosphatase 5b (S-TRACP5b), serum C-terminal cross-linked telopeptides of type I collagen (S-CTX-I) and three assays for urinary osteocalcin (U-OC). RESULTS: The median TSU of 99mTc-MDP was 23% of the administered activity. All bone turnover markers were significantly correlated with TSU with r-values from 0.52 (p = 0.013) to 0.90 (p < 0.001). The two resorption markers had numerically higher correlations (S-TRACP5b r = 0.90, S-CTX-I r = 0.80) than the formation markers (S-Total OC r = 0.72, S-Bone ALP r = 0.66), but the difference was not statistically significant. TSU did not correlate with age, weight, body mass index or bone mineral density. CONCLUSION: In conclusion, bone turnover markers are strongly correlated with total skeletal uptake of 99mTc-MDP. There were no significant differences in correlations for bone formation and resorption markers. This should be due to the coupling between formation and resorption

Transcytosis route mediates rapid delivery of intact antibodies to draining lymph nodes

Lymph nodes (LNs) filter lymph to mount effective immune responses. Small soluble lymph-borne molecules from the periphery enter the draining LNs via a reticular conduit system. Intact antibodies and other larger molecules, in contrast, are physically unable to enter the conduits, and they are thought to be transported to the LNs only within migratory DCs after proteolytic degradation. Here, we discovered that lymph-borne antibodies and other large biomolecules enter within seconds into the parenchyma of the draining LN in an intact form. Mechanistically, we found that the uptake of large molecules is a receptor-independent, fluid-phase process that takes place by dynamin-dependent vesicular transcytosis through the lymphatic endothelial cells in the subcapsular sinus of the LN. Physiologically, this pathway mediates a very fast transfer of large protein antigens from the periphery to LN-resident DCs and macrophages. We show that exploitation of the transcytosis system allows enhanced whole-organ imaging and spatially controlled lymphocyte activation by s.c. administered antibodies in vivo. Transcytosis through the floor of the subcapsular sinus thus represents what we believe to be a new physiological and targetable mode of lymph filtering.</p

Characterisation of feline renal cortical fibroblast cultures and their transcriptional response to transforming growth factor beta 1

Chronic kidney disease (CKD) is common in geriatric cats, and the most prevalent pathology is chronic tubulointerstitial inflammation and fibrosis. The cell type predominantly responsible for the production of extra-cellular matrix in renal fibrosis is the myofibroblast, and fibroblast to myofibroblast differentiation is probably a crucial event. The cytokine TGF-β1 is reportedly the most important regulator of myofibroblastic differentiation in other species. The aim of this study was to isolate and characterise renal fibroblasts from cadaverous kidney tissue of cats with and without CKD, and to investigate the transcriptional response to TGF-β1

Protein kinase Cepsilon is important for migration of neuroblastoma cells

<p>Abstract</p> <p>Background</p> <p>Migration is important for the metastatic capacity and thus for the malignancy of cancer cells. There is limited knowledge on regulatory factors that promote the migration of neuroblastoma cells. This study investigates the hypothesis that protein kinase C (PKC) isoforms regulate neuroblastoma cell motility.</p> <p>Methods</p> <p>PKC isoforms were downregulated with siRNA or modulated with activators and inhibitors. Migration was analyzed with scratch and transwell assays. Protein phosphorylation and expression levels were measured with Western blot.</p> <p>Results</p> <p>Stimulation with 12-<it>O</it>-tetradecanoylphorbol-13-acetate (TPA) induced migration of SK-N-BE(2)C neuroblastoma cells. Treatment with the general protein kinase C (PKC) inhibitor GF109203X and the inhibitor of classical isoforms Gö6976 inhibited migration while an inhibitor of PKCβ isoforms did not have an effect. Downregulation of PKCε, but not of PKCα or PKCδ, with siRNA led to a suppression of both basal and TPA-stimulated migration. Experiments using PD98059 and LY294002, inhibitors of the Erk and phosphatidylinositol 3-kinase (PI3K) pathways, respectively, showed that PI3K is not necessary for TPA-induced migration. The Erk pathway might be involved in TPA-induced migration but not in migration driven by PKCε. TPA induced phosphorylation of the PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS) which was suppressed by the PKC inhibitors. Treatment with siRNA oligonucleotides against different PKC isoforms before stimulation with TPA did not influence the phosphorylation of MARCKS.</p> <p>Conclusion</p> <p>PKCε is important for migration of SK-N-BE(2)C neuroblastoma cells. Neither the Erk pathway nor MARCKS are critical downstream targets of PKCε but they may be involved in TPA-mediated migration.</p