United Kingdom
United Kingdom

NanoSight Ltd is a company that designs and manufactures instruments for the scientific analysis of nanoparticles that are between approximately ten nanometers and one micron in diameter. The company was founded in 2003 by Bob Carr and John Knowles to further develop a technique Bob Carr had invented to visualize nanoparticles suspended in liquid. The company has since developed the technique of Nanoparticle Tracking Analysis , and they produce a series of instruments to count, size and visualize nanoparticles in liquid suspension using this patented technology.The company’s current headquarters is in Amesbury, Wiltshire, UK . NanoSight has 25 employees in the UK and has received several awards and recognitions. More than 450 instruments had been sold as of 2012. The technology has been cited in over 1300 scientific publications, presentations and reports.NanoSight was acquired by Malvern Instruments on the 30th September 2013. Wikipedia.


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Grant
Agency: European Commission | Branch: FP7 | Program: CP | Phase: ENV.2013.WATER INNO&DEMO-1 | Award Amount: 10.50M | Year: 2014

The ability of Europes communities to respond to increasing water stress by taking advantage of water reuse opportunities is restricted by low public confidence in solutions, inconsistent approaches to evaluating costs and benefits of reuse schemes, and poor coordination of the professionals and organisations who design, implement and manage them. The DEMOWARE initiative will rectify these shortcomings by executing a highly collaborative programme of demonstration and exploitation, using nine existing and one greenfield site to stimulate innovation and improve cohesion within the evolving European water reuse sector. The project is guided by SME & industry priorities and has two central ambitions; to enhance the availability and reliability of innovative water reuse solutions, and to create a unified professional identity for the European Water Reuse sector. By deepening the evidence base around treatment processes and reuse scheme operation (WP1), process monitoring and performance control (WP2), and risk management and environmental benefit analysis (WP3) DEMOWARE will improve both operator and public confidence in reuse schemes. It will also advance the quality and usefulness of business models and pricing strategies (WP4) and generate culturally and regulatory regime specific guidance on appropriate governance and stakeholder collaboration processes (WP5). Project outcomes will guide the development of a live in-development water reuse scheme in the Vende (WP6). Dissemination (WP7) and exploitation (WP8) activities, including the establishment of a European Water Reuse Association, ensure that DEMOWARE will shape market opportunities for European solution providers and provide an environment for the validation and benchmarking of technologies and tools. Ultimately the DEMOWARE outcomes will increase Europes ability to profit from the resource security and economic benefits of water reuse schemes without compromising human health and environmental integrity.


Grant
Agency: European Commission | Branch: FP7 | Program: CP-IP | Phase: NMP.2012.1.3-1 | Award Amount: 12.95M | Year: 2013

The NanoMILE project is conceived and led by an international elite of scientists from the EU and US with the aim to establish a fundamental understanding of the mechanisms of nanomaterial interactions with living systems and the environment, and uniquely to do so across the entire life cycle of nanomaterials and in a wide range of target species. Identification of critical properties (physico-chemical descriptors) that confer the ability to induce harm in biological systems is key to allowing these features to be avoided in nanomaterial production (safety by design). Major shortfalls in the risk analysis process for nanomaterials are the fundamental lack of data on exposure levels and the environmental fate and transformation of nanomaterials, key issues that this proposal will address, including through the development of novel modelling approaches. A major deliverable of the project will be a framework for classification of nanomaterials according to their impacts, whether biological or environmental, by linking nanomaterial-biomolecule interactions across scales (sub-cellular to ecosystem) and establishing the specific biochemical mechanisms of interference (toxicity pathway).


Grant
Agency: European Commission | Branch: FP7 | Program: CP-IP | Phase: NMP.2013.1.4-3 | Award Amount: 9.29M | Year: 2013

Nanotechnology is a key enabling technology. Still existing uncertainties concerning EHS need to be addressed to explore the full potential of this new technology. One challenge consists in the development of methods that reliably identify, characterize and quantify nanomaterials (NM) both as substance and in various products and matrices. The European Commission has recently recommended a definition of NM as reference to determine whether an unknown material can be considered as nanomaterial (2011/696/EU). The proposed NanoDefine project will explicitly address this question. A consortium of European top RTD performers, metrology institutes and nanomaterials and instrument manufacturers has been established to mobilize the critical mass of expertise required to support the implementation of the definition. Based on a comprehensive evaluation of existing methodologies and a rigorous intra-lab and inter-lab comparison, validated measurement methods and instruments will be developed that are robust, readily implementable, cost-effective and capable to reliably measure the size of particles in the range of 1100 nm, with different shapes, coatings and for the widest possible range of materials, in various complex media and products. Case studies will assess their applicability for various sectors, including food/feed, cosmetics etc. One major outcome of the project will be the establishment of an integrated tiered approach including validated rapid screening methods (tier 1) and validated in depth methods (tier 2), with a user manual to guide end-users, such as manufacturers, regulatory bodies and contract laboratories, to implement the developed methodology. NanoDefine will be strongly linked to main standardization bodies, such as CEN, ISO and OECD, by actively participating in TCs and WGs, and by proposing specific ISO/CEN work items, to integrate the developed and validated methodology into the current standardization work.


Grant
Agency: European Commission | Branch: FP7 | Program: CP-TP | Phase: NMP-2010-1.2-1 | Award Amount: 4.21M | Year: 2011

In-Sight is a SME-driven project on the in-line characterisation of nanoparticles during nanomaterial manufacturing. This in-line characterisation is the ultimate goal. Within the time-span of the project (3 years) it is our objective to show that a combination of analytical techniques that are capable of real time measurements will provide valuable information for the nanoparticle user. It is our objective to enable monitoring real-time (unexpected) changes in particle count and dimensions during particle processing. The outcome of the project will contribute to minimised batch failure, improved yield, troubleshooting scale-up. In addition, the in-line measurements will enable quality by design throughout development of new products. Finally the result of the project will be reflected in a reduction of development time, as well as easy scale-up from the lab to manufacturing.


News Article | November 18, 2015
Site: www.nature.com

The cell lines used in this study were provided as follows: human breast cancer MDA-MB-231 organotropic lines 4175, 1833 and 831 by J. Massagué; human breast cancer 4173 and 4180 cells by A. Minn; human breast cancer 231BR cells by P. Steeg; liver metastasis enriched uveal melanoma cells by V. Rajasekhar; human osteosarcoma 143B cells by A. Narendran; human melanoma 131/4-5B2 and 131/8-2L cells by R. Kerbel; human melanoma SB1B cells by C. F. Verschraegen; human rhabdomyosarcoma CT10 and RD cells by from R. Gladdy; and human Wilms tumour CCG9911 and CLS1 cells by A. Ketsis. Human breast cancer cell lines MDA-MB-231 and MDA-MB-468, human breast epithelial cells MCF10A, human pancreatic cancer cell lines, gastric cancer cell lines and colorectal cancer cell lines were purchased from American Type Culture Collection (ATCC). Although HT29 is commonly misidentified, we purchased this cell line directly from ATCC and the cell line was certified by this repository, therefore we are confident that it is indeed a colon cancer cell line. The C57BL/6 mouse pancreatic adenocarcinoma Pan02 was purchased from the National Cancer Institute Tumour Repository (DTP/DCTD, Frederick National Laboratory for Cancer Research). For in vitro education of human lung fibroblasts WI-38 (ATCC), human bronchial epithelial cells HBEpC (PromoCell), and human Kupffer cells (Life Technologies), cells were maintained in culture for 14 days, with media containing 0, 5 or 10 μg ml−1 of exosomes, replenished every other day. Kupffer cells were cultured in RPMI and WI-38 cells were cultured in alpha-MEM, both supplemented with 10% exosome-depleted FBS (Gibco, Thermo Fisher Scientific) and penicillin-streptomycin. HBEpC cells were cultured in airway epithelial cell growth medium (PromoCell). All cells were maintained in a humidified incubator with 5% CO at 37 °C. FBS was depleted of bovine exosomes by ultracentrifugation at 100,000g for 70 min. All cells lines were routinely tested for mycoplasma and were found to be negative. Exosomes were purified by sequential centrifugation as previously described46. In brief, cells were removed from 3–4-day cell culture supernatant by centrifugation at 500g for 10 min to remove any cell contamination. To remove any possible apoptotic bodies and large cell debris, the supernatants were then spun at 12,000g for 20 min. Finally, exosomes were collected by spinning at 100,000g for 70 min. Exosomes were washed in 20 ml PBS and pelleted again by ultracentrifugation (Beckman 70Ti rotor). Exosome preparations were verified by electron microscopy. Exosome size and particle number were analysed using the LM10 or DS500 nanoparticle characterization system (NanoSight, Malvern Instruments) equipped with a blue laser (405 nm). Normal mammary fat pad tissue-derived exosomes were obtained by culturing five mammary fat pads isolated from healthy 4–6-week-old C57BL/6 mice in 3 ml of FBS-free RPMI for 12 h. The final exosome pellet was resuspended in PBS and protein concentration was measured by BCA (Pierce, Thermo Fisher Scientific). Mass spectrometry analyses of exosomes were performed at the Rockefeller University Proteomics Resource Center using 20 μg of exosomal protein. Samples were denatured using 8 M urea, reduced using 10 mM dithiothreitol (DTT), and alkylated using 100 mM iodoacetamide. This was followed by proteolytic digestion with endoproteinase LysC (Wako Chemicals) overnight at room temperature, and subsequent digestion with trypsin (Promega) for 5 h at 37 °C. The digestion was quenched with formic acid and resulting peptide mixtures were desalted using in-house made C18 Empore (3M) StAGE tips47. Samples were dried and solubilized in the sample loading buffer containing 2% acetonitrile and 2% formic acid. Approximately 3–5 μg of each sample was analysed by reversed phase nano-liquid chromatography–tandem mass spectrometry (LC–MS/MS) (Ultimate 3000 coupled to QExactive, Thermo Scientific). After loading onto the C18 trap column (5 μm beads, Thermo Scientific) at a flow rate of 3 μl min−1, peptides were separated using a 75-μm inner diameter C18 column (3 μm beads, Nikkyo Technos Co.) at a flow rate of 200 nl min−1, with a gradient increasing from 5% buffer B (0.1% formic acid in acetonitrile)/95% buffer A (0.1% formic acid) to 40% buffer B/60% buffer A, over 140 min. All LC–MS/MS experiments were performed in data-dependent mode. Precursor mass spectra were recorded in a 300–1,400 m/z mass range at 70,000 resolution, and 17,500 resolution for fragment ions (lowest mass: m/z 100). Data were recorded in profile mode. Up to 20 precursors per cycle were selected for fragmentation and dynamic exclusion was set to 45 s. Normalized collision energy was set to 27. MS/MS spectra were extracted and searched against Uniprot complete human or mouse proteome databases (January 2013) concatenated with common contaminants48 using Proteome Discoverer 1.4 (Thermo Scientific) and Mascot 2.4 (Matrix Science). All cysteines were considered alkylated with acetamide. N-terminal glutamate to pyroglutamate conversion, oxidation of methionine, and protein N-terminal acetylation were allowed as variable modifications. Data were first searched using fully tryptic constraints. Matched peptides were filtered using a Percolator49 based 1% false discovery rate (FDR). Spectra not being matched at a FDR of 1% or better were re-searched allowing for semi-tryptic peptides. The average area of the three most abundant peptides for a matched protein50 was used to gauge protein amounts within and between samples. LC–MS/MS data from three technical replicates of six organ-tropic samples were analysed using MaxQuant (version 1.5.0.30) and Perseus software (version 1.5.0.9)51, searching against a Uniprot human database (July 2014). Oxidation of methionine and protein N-terminal acetylation were allowed as variable modifications, and cysteine carbamidomethyl was set as a fixed modification. Two missed cleavages were allowed for specificity: trypsin/P. The ‘match between runs’ option was enabled. FDR values at the protein and peptide level were set to 1%. Protein abundance is expressed as LFQ values. Only proteins quantified in at least two out of three replicates in at least one group were retained, and missing values were imputed. A multiple sample ANOVA test was performed and corrected for multiple hypotheses testing using a permutation-based FDR threshold of 0.05. To assess lung, liver and bone exosome distribution, exosomes were injected via the retro-orbital venous sinus, the tail vein or intracardially. Exosome distribution patterns were consistent regardless of the route of injection. For brain distribution, exosomes were only observed in the brain after intracardiac injection. For 24-h exosome treatments, 10 μg of total exosomal protein were injected via the retro-orbital venous sinus, the tail vein, or intracardially in a total volume of 100 μl PBS. For exosome-tracking purposes, purified exosomes were fluorescently labelled using PKH67 (green) or PKH26 (red) membrane dye (Sigma-Aldrich) or FM1-43FX dye (Life Technologies) for the photo-conversion experiment. Labelled exosomes were washed in 20 ml of PBS, collected by ultracentrifugation and resuspended in PBS. When performing peptide blocking experiments, exosomes were incubated with 0.06 μM RGD or HYD-1 (peptide sequence: KIKMVISWKG) peptides for 30 min at 37 °C before exosome injection. An average of five random fields was counted per sample at 20× magnification, and representative pictures were taken at 40× magnification. For education experiments, mice received 10 μg of exosomes retro-orbitally every other day for 3 weeks. To measure exosome uptake by specific cell types, labelled exosomes were injected 24 h before tissue collection and tissues were analysed for exosome-positive cells by immunofluorescence. Pictures were taken at 60 × magnification. For in vitro uptake assays, the membrane of WI-38 cells was labelled with PKH67 dye while 4175-LuT exosomes were labelled with PKH26 dye. Exosomes (10 μg ml−1) were first incubated with PBS or HYD-1 peptide for 30 min at 37 °C, followed by an incubation for 1 h with WI-38 cells at 37 °C. Excess exosomes were washed off and pictures were taken by Nikon confocal microscope (Eclipse TE2000U). The amount of exosomes localizing to the lung was analysed by immunofluorescence or using the Odyssey imaging system (LI-COR Biosciences). In brief, NIR dye-labelled exosomes were injected 24 h before tissue collection and tissues were analysed for exosome-positive areas. Whole-lung images were analysed using image J software, quantifying red fluorescence area in arbitrary units. Cryostat sections prepared at a 15-μm thickness were placed on glass slides and re-fixed in 0.075 M sodium cacodylate, pH 7.4, containing 2.5% glutaraldehyde. For photoconversion, slides were washed twice in 0.1 M sodium cacodylate buffer, pH 7.4. Autofluorescence was quenched using 100 mM NH Cl in cacodylate buffer for 45 min. On the basis of optimization experiments, sections were photoconverted for 2 h by incubation in 5.4 mg ml−1 3,3′-diaminobenzidine in 0.1 M sodium cacodylate buffer, pH 7.4, and exposure to the light of an Intensilight C-HGFI 130-W mercury lamp and a 4×/0.1 NA objective (Nikon Inverted Microscope Eclipse Ti). For electron microscopy processing, sections were post-fixed in 1% osmium tetroxide buffer for 15 min on ice. After washing with water, slides were placed in 1% aqueous uranyl acetate for 30 min. Sections were washed with water, dehydrated in a graded series of ethanol concentrations and subsequently in acetone for 10 min at room temperature. Samples were embedded in Eponate. Serial sections were cut at 70 nm in thickness and transferred to formvar-coated slot grids and imaged on a JEOL 100CX at 80 kV with an AMT XR41 digital imaging system. Cell lines were analysed for specific genes using pre-designed TaqMan assays (Applied Biosystems). In brief, RNA was extracted from tissues or cells using the RNeasy kit (Qiagen), and reverse transcribed using Superscript Vilo (Life Technologies). qRT–PCR was performed on a 7500 Fast Real Time PCR System (Applied Biosystems), using TaqMan Universal PCR Master Mix (Applied Biosystems). Relative expression was normalized to β-actin levels. For shRNA-mediated knockdown of ITGβ and ITGβ , specific interfering lentiviral vectors containing GFP reporter and puromycin resistance gene cassettes were used. In brief, oligonucleotide 5′-CCGGGAGGGTGTCATCACCATTGAACTCGAGTTCAATGGTGATGACACCCTCTTTTTG-3′ targeting the 5′-GAGGGTGTCATCACCATTGAA-3′ sequence in the human ITGB4 gene (EntrezGene ID: 3691) or oligonucleotide 5′-CCGGAGCTTGTTGTCCCAATGAAATCTCGAGATTTCATTGGGACAACAAGCTTTTTTG-3′ targeting the 5′-AGCTTGTTGTCCCAATGAAAT-3′ sequence in the human ITGB5 gene (EntrezGene ID: 3693) were cloned into the pLKO.1 vector. As a control, we used the empty pLKO.1 vector. For retrovirus production for integrin overexpression, the pWZL and pBabe vectors systems were used. pWZL-hygro-ITGB and pBabe-puro-ITGB were provided by F. Giancotti. Lentiviral and retroviral particles were packaged using 293T cells. Infected target cells were selected using 500 μg ml−1 hygromycin B or 2 μg ml−1 puromycin (Invitrogen). Bone marrow was prepared for flow cytometry as previously described1. For analysis of lung, tissues were minced and then digested at 37 °C for 20 min with an enzyme cocktail (collagenase A, dispase and DNaseI, Roche Applied Science). Single-cell suspensions were prepared by filtering through a 70-μm strainer and passing through an 18G syringe. Lung fibroblasts were identified by flow cytometry using an anti-mouse rabbit polyclonal S100A4 (1:50, Abcam; ab27957), or SPC (1:100, Santa Cruz; FL-197), revealed by Alexa Fluor 568-conjugated goat anti-rabbit secondary (A-11011, Life Technologies, 1:400). For liver, tissues were mechanically dissociated, and single-cell suspensions were filtered through a 40-μm strainer. Allophycocyanin-conjugated F4/80 (1:100, eBioscience; clone BM8) was used to identify liver macrophages by flow cytometry. Cell fluorescence indicating fluorescently labelled exosome uptake was analysed using a FACSCalibur or a FACSCanto (Beckton Dickinson). FACS data was analysed with FlowJo software (TreeStar Inc.). Twenty-thousand cells were plated in 24-well transwell plates with inserts (8-μm pore size, Corning) and were incubated at 37 °C for 6 h. Cell inserts were fixed with 4% paraformaldehyde (PFA) for 10 min, followed by PBS wash and haematoxylin staining to allow visualization and counting. Nine random fields were counted per well at 20× magnification and the average number of migrated cells per field was calculated. Human peripheral blood samples were obtained from control healthy subjects and cancer patients with lung or liver metastasis, or from patients without distant metastasis at Weill Cornell Medical College, University Medical Center Hamburg-Eppendorf, Oslo University Hospital, Memorial Sloan Kettering Cancer Center and University of Nebraska Medical Center, all pathologically confirmed. All individuals provided informed consent for blood donation on approved institutional protocols (WCMC IRB 0604008488 (DL), MSKCC IRB 12-137A (JB)). Plasma or serum exosomes were isolated as previously described1. ITGβ and ITGα levels in exosomes were measured by ELISA (ABIN417641 and ABIN417609 from Antibodies Online, and LS-F7188 from LifeSpan Biosciences), using 2 μg of exosomes per 100 μl of sample diluent, in duplicate reactions, according to the manufacturer’s instructions. All mouse work was performed in accordance with institutional, IACUC and AAALAS guidelines, by the animal protocol 0709-666A. All animals were monitored for abnormal tissue growth or ill effects according to AAALAS guidelines and euthanized if excessive deterioration of animal health was observed. No statistical method was used to pre-determine sample size. No method of randomization was used to allocate animals to experimental groups. The investigators were not blinded to allocation during experiments and outcome assessment. Mice that died before the predetermined end of the experiment were excluded from the analysis. In none of the experiments did tumours exceed the maximum volume allowed according to our IACUC protocol, specifically 2 cm3. For exosome localization, education and tumour implantation experiments for mouse cell lines, 6-week-old C57BL/6 Mus musculus females purchased from Jackson labs were used. For exosome localization, education and tumour implantation experiments for human cell lines, 6–8-week-old NCr nude (NCRNU-F sp./sp.) females purchased from Taconic were used. For lung metastasis studies using organotropic lines, 6–8-week-old nude female mice were pre-educated with exosomes for 3 weeks followed by tail vein injection of 2 × 105 or intracardiac injection of 1 × 105 luciferase-positive cancer cells resuspended in 100 μl PBS. Four weeks after intracardiac injection and eight weeks after tail vein injection, lung metastasis was measured using the IVIS 200 bioluminescence imaging system (Xenogen, Caliper Life Sciences), and tissues were cut in 6-μm sections and stained with haematoxylin and eosin for histology. To analyse the role of exosome education in tumour metastasis, 6–8-week-old C57BL/6 female mice pre-educated with pancreatic cancer-derived exosomes were injected intrasplenically with 1 × 106 Pan02 mCherry cells resuspended in 30 μl of Matrigel (Corning). One or twenty-one days later, mice were euthanized, and livers were analysed for metastatic lesions by measuring liver weight. To follow the levels of tumour-derived exosomes in plasma of tumour-bearing mice, 1 × 106 4175 lung-tropic cells were injected in the mammary fat pad of nude mice. Mouse blood (250 μl) was drawn from the retro-orbital sinus when tumour size was over 800 mm3, followed by tumour resection. One week after the tumour was resected, mice were analysed by bioluminescence IVIS imaging for luciferase activity and separated into two groups: recurrence/tumour-free and recurrent tumours. Mouse blood was drawn and the plasma of mice within the same group was pooled for exosome isolation. Western blot analysis with anti-human ITGβ antibodies was used to detect tumour-derived exosomes. To assess exosome-induced vascular leakiness, 10 μg of total exosome protein were injected by retro-orbital injection. Then 20 h after exosome treatment, mice were injected with 2 mg of Texas Red-lysine fixable dextran 70,000 MW (Invitrogen) via retro-orbital injection. One hour after dextran injection, mice were euthanized and perfused with PBS. Lungs were dissected and fixed in a mix of 2% PFA and 20% sucrose overnight, then embedded in Tissue-tek O.C.T. embedding compound (Electron Microscopy Sciences) and frozen in a dry-ice/ethanol bath. O.C.T. blocks were sectioned and stained for DAPI, pictures were taken using a Nikon confocal microscope (Eclipse TE2000U). Images were analysed using image J software, quantifying red fluorescence area in arbitrary units. For histological analysis, tissues were dissected and fixed in a mix of 2% PFA and 20% sucrose in PBS overnight, then embedded in Tissue-tek O.C.T. embedding compound. Blocks were frozen in a dry-ice/ethanol bath. For immunofluorescence, 6 μm O.C.T tissue cryosections were stained with antibodies against F4/80 (1:100, eBioscience; BM8), fibronectin (1:50, Santa Cruz; IST-9), S100A4 (1:100, Abcam; ab27957), SPC (1:100, Santa Cruz; FL-197), laminin (1:50, abcam; ab11575), CD31 (1:100, Santa Cruz; MEC 13.3), EpCAM (1:50, Santa Cruz; HEA125). Secondary antibodies conjugated to Alexa Fluor 488 or 549 were used (A-11001 and A-11007, Life Technologies). Fluorescent images were obtained using a Nikon confocal microscope (Eclipse TE2000U) and analysed using Nikon software (EZ-C1 3.6). Exosomes or cells were lysed with RIPA buffer containing a complete protease inhibitor tablet (Roche). Lysates were cleared by centrifugation at 14,000g for 20 min. Supernatant fractions were used for western blot. Samples were separated on a Novex 4–12% Bis-Tris Plus Gel (Life Technologies), and transferred onto a PVDF membrane (Millipore). Membranes were processed for Ponceau red staining followed by 1 h blocking and primary antibody incubation. The antibodies against the following proteins were used for western blot analysis: ITGβ (1:1,000, Cell Signaling; 4706), ITGβ (1:500, Cell Signaling; 4707), ITGα (1:1,000, Cell Signaling; 3750), ITGα (1:10,000, abcam; ab133557), ITGα (1:1,000, abcam; ab190731), ITGα (1:500, abcam; ab117611), ITGβ (1:500, Cell Signaling; 4708), ITGβ (1:500, Millipore; AB2984) Alix (1:1,000, Cell Signaling; 3A9), and GAPDH (1:10,000, Cell Signaling; 14C10). Anti-rabbit IgG, horseradish peroxidase (HRP)-linked antibody (1:3,000, Cell Signaling; 7074) and anti-mouse IgG, HRP-linked antibody (1:3,000, Cell Signaling; 7076) were used as secondary antibodies. Cells were plated in a 96-well plate and treated with 10 μg ml−1 exosomes for 2 h and then processed according to the protocol provided by the manufacturer. In brief, cells were fixed with 4% PFA and washed with 0.1% TritonX-100/PBS. Cells were then blocked using Odyssey blocking buffer for 1 h and stained overnight at 4 °C with primary antibody in Odyssey blocking buffer containing 0.1% Tween-20. The next day cells were washed again and incubated with LI-COR secondary antibodies for 1 h at room temperature followed by fluorescent imaging using Odyssey. Antibodies against the following proteins were used: Src (1:100, Cell Signaling; 2109), p-Src (1:100, Cell Signaling; 2101), AKT (1:100, Cell Signaling; 9272), p-AKT (1:100, Cell Signaling; 9271), p38 (1:100, Cell Signaling; 9212), p-p38 (1:100, Cell Signaling; 9211), NF-κB (1:100, Cell Signaling; 3034), p-NF-κB (1:100, Cell Signaling; 3033), NFAT (1:100, Thermo Scientific; PA1-023), ILK (1:100, abcam; ab52480), FAK (1:100, abcam; ab40794) and GAPDH (1:100, Cell Signaling; 14C10). IRDye 800CW anti-rabbit IgG (1:800, LI-COR) were used as secondary antibodies. Error bars in graphical data represent mean ± s.e.m. Mouse experiments were performed in duplicate or triplicate, using 3–6 mice per treatment group. Statistical significance was determined using a two-tailed Student’s t-test and one-way ANOVA, in which P values of P < 0.05 were considered statistically significant. Variance was similar between the groups that were statistically compared.


Grant
Agency: European Commission | Branch: FP7 | Program: CP-IP | Phase: NMP-2008-1.2-1 | Award Amount: 9.81M | Year: 2009

Past years have made the radical change of the European paper industry evident to everyone. To accelerate growth, the industry has to beat the product commoditization and renew its product base with value-added products. Energy saving is not just part of the environmental agenda, it has become the most crucial topic for the competitiveness of European paper industry. In order to maintain the advantage, the development of technologically advanced manufacturing processes is a must, along with reduced specific energy consumption. Nanocellulose is the most promising nano-material for wide-variety applications in papermaking, today only prepared and applied in lab-scale. SUNPAP addresses the enhancement of European paper industry competitiveness by means of nanofibrillouscellulose (NFC) based processes. SUNPAP proposes a program based on an integrated and complementary approach for: a) scaling up efficient and innovative production routes to deliver nanofibrillous cellulose (NFC) as functional additive for industrial processes and innovative added value products; b) innovating the papermaking processes by the introduction of NFC additive; c) assessing impacts of nanotechnologies on consumer and occupational safety, public health in general and environment, and enhancing the related European foreground d) demonstrating the economic, environment and social sustainability of the innovative papermaking processes and products and therefore, e) successfully transferring the nanotechnology innovation to the paper value chain. Through demonstrations of the processes, the project will deliver new extremely light-weight and multifunctional products for a wide range of end-uses in the graphical and packaging paper industries. Economical and sustainability assessments in the project cover the whole value chain. However, the targeted advantages are not possible without dramatically changing the total value chain, encompassing it from the pulp to the end of life-cycle of the product


Grant
Agency: European Commission | Branch: FP7 | Program: CP-IP | Phase: NMP-2009-4.0-3 | Award Amount: 16.52M | Year: 2010

NAMDIATREAM will develop a cutting edge nanotechnology-based toolkit for multi-modal detection of biomarkers of most common cancer types and cancer metastases, permitting identification of cells indicative of early disease onset in a high-specificity and throughput format in clinical, laboratory and point-of-care devices. The project is built on the innovative concepts of super-sensitive and highly specific lab-on-a-bead, lab-on-a-chip and lab-on-a-wire nano-devices utilizing photoluminescent, plasmonic, magnetic and non-linear optical properties of nanomaterials. This offers groundbreaking advantages over present technologies in terms of stability, sensitivity, time of analysis, probe multiplexing, assay miniaturisation and reproducibility. The ETP in Nanomedicine documents point out that nanotechnology has yet to deliver practical solutions for the patients and clinicians in their struggle against common, socially and economically important diseases such as cancer. Over 3.2M new cases and 1.7M cancer-related deaths are registered in Europe every year, largely because diagnostic methods have an insufficient level of sensitivity, limiting their potential for early disease identification. We will deliver Photoluminescent nanoparticle-based reagents and diagnostic chips for high throughput early diagnosis of cancer and treatment efficiency assessment Nanocrystals enabling plasmon-optical and nonlinear optical monitoring of molecular receptors within body fluids or on the surface of cancer cell Multi-Parameter screening of cancer biomarkers in diagnostic material implementing segmented magnetic nanowires Validation of nano-tools for early diagnosis and highly improved specificity in cancer research. OECD-compliant nanomaterials with improved stability, signal strength and biocompatibility Direct lead users of the results will be the diagnostic and medical imaging device companies involved in the consortium, clinical and academic partners

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