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BACKGROUND: Nanotechnology-based bioassays that detect the presence and/or absence of a combination of cell markers are increasingly used to identify stem or progenitor cells, assess cell heterogeneity, and evaluate tumor malignancy and/or chemoresistance. Delivery methods that enable nanoparticles to rapidly detect emerging, intracellular markers within cell clusters of biopsies will greatly aid in tumor characterization, analysis of functional state and development of treatment regimens. RESULTS: Experiments utilized the Sendai virus to achieve in vitro, cytosolic delivery of Quantum dots in cells cultured from Human brain tumors. Using fluorescence microscopy and Transmission Electron Microscopy, in vitro experiments illustrated that these virus-based liposomes decreased the amount of non-specifically endocytosed nanoparticles by 50% in the Human glioblastoma and medulloblastoma samples studied. Significantly, virus-based liposome delivery also facilitated targeted binding of Quantum dots to cytosolic Epidermal Growth Factor Receptor within cultured cells, focal to the early detection and characterization of malignant brain tumors. CONCLUSIONS: These findings are the first to utilize the Sendai virus to achieve cytosolic, targeted intracellular binding of Qdots within Human brain tumor cells. The results are significant to the continued applicability of nanoparticles used for the molecular labeling of cancer cells to determine tumor heterogeneity, grade, and chemotherapeutic resistivity.

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Medulloblastoma (MB) is the most common brain cancer diagnosed among children. The cellular pathways that regulate MB invasion in response to environmental cues remain incompletely understood. Herein, we examine the migratory response of human MB-derived Daoy cells to different concentration profiles of Epidermal Growth Factor (EGF) using a microfluidic system. Our findings provide the first quantitative evidence that EGF concentration gradients modulate the chemotaxis of MB-derived cells in a dose-dependent manner via the EGF receptor (EGF-R). Data illustrates that higher concentration gradients caused increased number of cells to migrate. In addition, our results show that EGF-induced receptor phosphorylation triggered the downstream activation of phosphoinositide-3 kinase (PI3K)/Akt pathway, while its downstream activation was inhibited by Tarceva (an EGF-R inhibitor), and Wortmannin (a PI3K inhibitor). The treatment with inhibitors also severely reduced the number of MB-derived cells that migrated towards increasing EGF concentration gradients. Our results provide evidence to bolster the development of anti-migratory therapies as viable strategies to impede EGF-stimulated MB dispersal.

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Mechanistic study of biological processes via Quantum Dots (QDs) remain constrained by inefficient QD delivery methods and consequent altered cell function. Here the authors present a rapid method to label activated receptor populations in live cancer cells derived from medulloblastoma and glioma tumors. The authors used QDs to bind the extracellular domain of Epidermal Growth Factor Receptor (EGF-R) proteins and then induced receptor activation to facilitate specific detection of intracellular, activated EGF-R subpopulations. Such labeling enables rapid identification of biological markers characteristic of tumor type, grade and chemotherapy resistance. FROM THE CLINICAL EDITOR: In this paper, a rapid, quantum dot-based method is presented with the goal of labeling activated receptor populations in live cancer cells. More accurate characterization of medulloblastoma and glioma cancer cells using this biomarker detection technique may lead to a more specific targeted therapy.

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Microfabrication has become widely utilized to generate controlled microenvironments that establish chemical concentration gradients for a variety of engineering and life science applications. To establish microfluidic flow, the majority of existing devices rely upon additional facilities, equipment, and excessive reagent supplies, which together limit device portability as well as constrain device usage to individuals trained in technological disciplines. The current work presents our laboratory-developed bridged µLane system, which is a stand-alone device that runs via conventional pipette loading and can operate for several days without need of external machinery or additional reagent volumes. The bridged µLane is a two-layer polydimethylsiloxane microfluidic device that is able to establish controlled chemical concentration gradients over time by relying solely upon differences in reagent densities. Fluorescently labeled Dextran was used to validate the design and operation of the bridged µLane by evaluating experimentally measured transport properties within the microsystem in conjunction with numerical simulations and established mathematical transport models. Results demonstrate how the bridged µLane system was used to generate spatial concentration gradients that resulted in an experimentally measured Dextran diffusivity of (0.82 ± 0.01) × 10(-6) cm(2)/s.

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An increasing number of studies have demonstrated the multiple advantages of using nanocrystals, such as Quantum dots, for biological imaging. Quantum dots functionalized with biomolecules on their surfaces were shown to be able to bind to specific extracellular targets via specific recognition and to be internalized inside the cells, thereby allowing the imaging of intracellular pathways. However, the use of Quantum dots for live tracking of intracellular molecules is relatively limited because of the difficulties encountered during the induction of Quantum dots across living cell membranes. In this study we show that cationic liposomes can deliver low concentrations of non-targeted Quantum dots into the cytosol of living cells via a lipid-mediated fusion with the cell membrane. The intracellular Quantum dots exhibit aggregation that appears dependent upon their concentration, but does not visibly affect cell viability. Our results point towards the use of cationic liposomes as an effective delivery system for targeted Quantum dots within the cell cytosol, which would facilitate live cell imaging of the labeled molecules.

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BACKGROUND: Cell-to-cell communication at the synapse involves synaptic transmission as well as signaling mediated by growth factors, which provide developmental and plasticity cues. There is evidence that a retrograde, presynaptic transforming growth factor-beta (TGF-beta) signaling event regulates synapse development and function in Drosophila. RESULTS: Here we show that a postsynaptic TGF-beta signaling event occurs during larval development. The type I receptor Thick veins (Tkv) and the R-Smad transcription factor Mothers-against-dpp (Mad) are localized postsynaptically in the muscle. Furthermore, Mad phosphorylation occurs in regions facing the presynaptic active zones of neurotransmitter release within the postsynaptic subsynaptic reticulum (SSR). In order to monitor in real time the levels of TGF-beta signaling in the synapse during synaptic transmission, we have established a FRAP assay to measure Mad nuclear import/export in the muscle. We show that Mad nuclear trafficking depends on stimulation of the muscle. CONCLUSIONS: Our data suggest a mechanism linking synaptic transmission and postsynaptic TGF-beta signaling that may coordinate nerve-muscle development and function.

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The analysis of membrane trafficking has in the past mainly dealt with single cells in culture. Recent studies of membrane trafficking in Drosophila focus on how cells are organized in tissues and form epithelia during embryogenesis. During these processes, the specific involvement of distinct biosynthetic and endocytic routes is starting to be understood. Once organized in epithelia, cells communicate with each other to make cell fate decisions through morphogen gradients and lateral inhibition. Endocytosis seems to play unexpected roles in shaping morphogen gradients and in biasing lateral inhibition events. Once committed to a developmental program, cells differentiate. In the case of neurons, trafficking through the biosynthetic and endocytic pathways may give the necessary speed of response and versatility to axons that navigate through a changing environment during pathfinding.

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We characterized Drosophila endophilin A (D-endoA), and generated and analysed D-endoA mutants. Like its mammalian homologue, D-endoA exhibits lysophosphatidic acid acyl transferase activity and contains a functional SH3 domain. D-endoA is recruited to the sites of endocytosis, as revealed by immunocytochemistry of the neuromuscular junction (NMJ) of mutant L3 larvae carrying the temperature-sensitive allele of dynamin, shibire. D-endoA null mutants show severe defects in motility and die at the early L2 larval stage. Mutants with reduced D-endoA levels exhibit a range of defects of synaptic vesicle endocytosis, as observed at L3 larvae NMJs using FM1-43 uptake and electron microscopy. NMJs with an almost complete loss of synaptic vesicles did not show an accumulation of intermediates of the budding process, whereas NMJs with only slightly reduced levels of synaptic vesicles showed a striking increase in early-stage, but not late-stage, budding intermediates at the plasma membrane. Together with results of previous studies, these observations indicate that endophilin A is essential for synaptic vesicle endocytosis, being required from the onset of budding until fission.

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