News Article | May 3, 2017
A novel fabrication procedure is used to produce flexible devices that include inorganic semiconductor nanowires and that can compete with organic devices in terms of brightness. Nitride LEDs are coming to replace other light sources in almost all general lighting, as well as in displays and life-science applications. Inorganic semiconductor devices, however, are naturally mechanically rigid and cannot be used in applications that require mechanical flexibility. Flexible LEDs are therefore currently a topic of intense research, as they are desirable for use in many applications, including rollable displays, wearable intelligent optoelectronics, bendable or implantable light sources, and biomedical devices. At present, flexible devices are mainly fabricated from organic materials. For example, organic LEDs (OLEDs) are already being used commercially in curved TV and smartphone screens. However, OLEDs have worse temporal stability and lower luminescence (especially in the blue spectral range) than nitride semiconductor LEDs. Substantial research efforts are thus being made to fabricate flexible inorganic LEDs.1 The conventional approach for flexible inorganic LED fabrication consists of number of steps, i.e., layer lift-off, microstructuring, and transfer to plastic supports. To avoid the microstructuring step and facilitate the lift-off, it is advantageous to shrink the active element dimensions and to use bottom-up nanostructures (such as nanowires, NWs) rather than 2D films. These NWs—i.e., elongated nanocrystals with a submicrometer diameter—have remarkable mechanical and optoelectronic properties that stem from their anisotropic geometry, high surface-to-volume ratio, and perfect crystallinity. In addition, such NWs are mechanically flexible and can withstand high levels of deformation without suffering plastic relaxation. Efficient LEDs that include nitride NWs have previously been demonstrated, and in our work,2 we make use of nitride NWs as the active material for flexible LEDs. Our polymer-embedded NWs offer an elegant solution to create flexible optoelectronic devices in which we combine the high efficiency and long lifetimes of inorganic semiconductor materials with the high flexibility of polymers. In our devices, the NW arrays—which are embedded in a flexible film and can be lifted-off from their native substrate—can sustain large deformations because of the high flexibility of the individual NWs. Furthermore, the footprints of individual NWs are much smaller than the typical curvature radius of LEDs (i.e., on the order of a few millimeters or more). In our approach, we used catalyst-free metal-organic chemical vapor deposition (MOCVD) to grow self-assembled gallium nitride (GaN) NWs on c-plane sapphire substrates.3 These NWs (with lengths of about 20μm and radii of about 0.5–1.5μm) have core/shell n–p junctions into which we incorporate multiple radial indium gallium nitride (InGaN)/GaN quantum wells. We control the emission color by changing the indium concentration of the InGaN emitting layer. In our actual device fabrication process4—see Figure 1(a)—the NW array is embedded into the polydimethylsiloxane (PDMS), peeled-off from the sapphire host substrate, and we then flip the composite NW/polymer membrane onto an arbitrary substrate to conduct the metal back-contacting. We subsequently flip the layer again and mount it on a flexible substrate (a metal foil or plastic), at which point we front-contact it with a flexible and transparent electrode. For the front contact we chose a silver NW mesh—see Figure 1(b)—which is characterized by mechanical flexibility, good electrical conductivity, and optical transparency. Figure 1. (a) Schematic illustration of the fabrication process for flexible LEDs that are based on a vertical nitride nanowire (NW) array. Ni: Nickel. Au: Gold. Ti: Titanium. (b) Scanning electron microscope image of the spin-coated silver (Ag) NW network on the polydimethylsiloxane (PDMS)/NW membrane. This silver NW network is used to form the transparent top-contact of the device. The protruding LED NWs are circled in red. We have used this technological procedure to fabricate blue and green flexible NW LEDs.4 We find that our devices exhibit typical behavior for nitride NW LEDs, i.e., with a light-up voltage of about 3V. Moreover, our LEDs can be bent to a curvature radius of ±3mm without any degradation of their electrical or luminescent properties. Photographs of our NW LEDs under operation in flat conditions, and during upward or inward bending are shown in Figure 2. Our flexible NW LEDs also have reasonable stability over time, unlike conventional OLEDs. Indeed, storing our devices in ambient conditions for several months does not cause their properties to degrade, whereas the lifetime of an OLED without encapsulation is limited to only several hours. Figure 2. Photographs of the blue (top), green (middle), and white (bottom) flexible LEDs at operation under different bending conditions. We have also used our composite NW/polymer membrane architecture to realize a flexible white LED (see Figure 2). To achieve this device we follow the standard approach of down-converting blue emission with yellow phosphors, i.e., to get white light from a blue–yellow mixture. To adapt this scheme for our flexible NW LEDs, we added yellow cerium-doped yttrium aluminum garnet phosphors into the PDMS layer between the NWs and covered the surface with an additional phosphorous-doped PDMS cap.5 The phosphor particles we use are smaller than 0.5μm so that they can fill the gaps between the NWs. The light that is emitted by the NWs is thus partially converted by the phosphors from blue to yellow, and we achieve a broad spectrum (covering almost the full visible range). Our NW membrane lift-off and transfer procedure allows free-standing layers of NW materials with different bandgaps to be assembled without any constraints relating to lattice-matching or compatability of growth conditions. Our approach therefore provides a large amount of design freedom and modularity, i.e., because it enables materials with very different physical and chemical properties to be combined (which cannot be achieved with monolithic growth). We made use of this modularity to demonstrate a two-color device, in which we combined two flexible LED layers that contain different active NWs: see Figure 3(a). In this device, we mounted a fully transparent flexible blue LED on top of a green LED. We were able to bias the two LEDs separately by producing either blue or green light, or by simultaneously producing a light mixture. We show the electrolumniscence spectra from the different layers of this bicolor flexible LED in Figure 3(b). Figure 3. (a) Schematic illustration of a blue-green two-color flexible NW LED, in which a fully transparent blue LED is mounted on top of a green LED. The two LEDs are biased separately (i.e., V1 and V2). (b) Electroluminescence (EL) spectra (in arbitrary units) of the two-color flexible NW LED. The blue, green, and red curves show the emissions from the top layer, bottom layer, and both layers together (biased simultaneously), respectively. In summary, we have successfully demonstrated a new procedure for the fabrication of efficient, flexible nitride NW LEDs. In our approach, we embed GaN NWs within a PDMS membrane and have realized blue, green, and white LEDs that exhibit good bending, electrical, luminescent, and temporal stability characteristics. The modularity of our technique means that we can also produce bicolor devices in which one LED is mounted upon another. Our approach thus opens up new routes to achieving efficient flexible LEDs and other optoelectronic devices, such as red-green-blue flexible LEDs or displays, flexible NW-based photodetectors6, or solar cells. In our future research we will concentrate on improving the efficiency of our flexible light-emitting devices, which is not yet comparable to that of commercialized rigid thin-film LEDs. We will also try to integrate the flexible light sources into life-science applications. This work has been financially supported through the 'PLATOFIL' project (ANR-14-CE26-0020-01), the EU H2020 ERC ‘NanoHarvest’ project (grant 639052), and by the French national Labex GaNex project (ANR-11-LABX-2014). The device fabrication was performed at the Centrale de Technologie Universitaire's Institut d'Electronique Fondamental (CTU-IEF) Minerve technological platform, which is a member of the Renatech Recherche Technologique de Base network. Center for Nanosciences and Nanotechnologies Paris-Sud University, CNRS Nan Guan is a PhD candidate in physics. He received both his master's and engineering degrees in optics from Université Paris-Saclay, France, in 2015. His current research interests include nanofabrication, characterization, and optical simulations for nitride nanowire LEDs. Xing Dai received her PhD in applied physics from Nanyang Technological University, Singapore, in 2014. During her time as a postdoctoral researcher at Paris-Sud University, she focused on flexible nanowire LEDs. She is currently a process-development engineer at Almae Technologies. Maria Tchernycheva received her PhD in physics from Paris-Sud University in 2005. She joined CNRS in 2006, where she currently leads the ‘NanoPhotoNit’ research group. Her research focuses on the fabrication and testing of novel optoelectronic devices that are based on semiconductor nanowires. Quantum Photonics, Electronics and Engineering (PHELIQS) Institute for Nanoscience and Cryogenics, French Alternative Energies and Atomic Energy Commission (CEA) Joël Eymery obtained his engineering degree, PhD, and habilitation from Université Grenoble Alpes, France, and now leads CEA's Nanostructures and Synchrotron Laboratory. His research is focused on the development of nanowire physics, including metal–organic vapor-phase epitaxy growth of nitride compounds, structural and optical characterization, and the development of nanodevice demonstrators. Christophe Durand received his PhD in physics from the Université Joseph Fourier, France, in 2004. Since 2006, he has been an associate professor at the Université Grenoble Alpes. In his research, he focuses on the synthesis of novel III-N nanostructures by metal–organic vapor-phase epitaxy to develop new optoelectronic applications. Quantum Photonics, Electronics and Engineering (PHELIQS)Institute for Nanoscience and Cryogenics, French Alternative Energies and Atomic Energy Commission (CEA) 2. N. Guan, X. Dai, J. Eymery, C. Durand, M. Tchernycheva, Nitride nanowires for new functionalities: from single wire properties to flexible light-emitting diodes. Presented at SPIE Photonics West 2016. 3. R. Koester, J.-S. Hwang, D. Salomon, X. Chen, C. Bougerol, J.-P. Barnes, D. Le Si Dang, et al., M-plane core-shell InGaN/GaN multiple-quantum-wells on GaN wires for electroluminescent devices, Nano Lett. 11, p. 4839-4845, 2011. 4. X. Dai, A. Messanvi, H. Zhang, C. Durand, J. Eymery, C. Bougerol, F. H. Julien, M. Tchernycheva, Flexible light-emitting diodes based on vertical nitride nanowires, Nano Lett. 15, p. 6958-6964, 2015. 5. N. Guan, X. Dai, A. Messanvi, H. Zhang, J. Yan, E. Gautier, C. Bougerol, et al., Flexible white light emitting diodes based on nitride nanowires and nanophosphors, ACS Photonics 3, p. 597-603, 2016.
News Article | July 11, 2017
"We are delighted to be working with such a select group of investors and we believe that their interest validates the immense potential of Aerial's novel approach to motion detection for the Wi-Fi home, said David Grant, CEO of Aerial Technologies. With their support, we are now well positioned to accelerate the commercialization of our motion interface with some of the most important telecommunication companies in the world." "Videotron sees a vast potential for Aerial's technology as it opens the door to a multitude of promising services and applications for both our residential and business customers," said Serge Legris, Vice President & Chief Technology Planning Office of Videotron. We are also looking to test different opportunities in our Open-air Laboratory for Smart Living. " "Aerial has a very strong team supported by TandemLaunch, a world-class incubator. Considering the traction they already have in the market, we believe they will be successful. Innovexport definitely made that investment with the objective to participate in future rounds to foster Aerial's growth," added Serge Lavergne, Vice-President Investment. Founded in 2015, Aerial is a software company that uses existing Wi-Fi signals, and cloud-based machine learning AI to detect presence and motion. As people, pets and in-organic objects move in the mess of Wi-Fi signals already in your home or office, they distort and disrupt these Wi-Fi signals in predictable ways. Aerial processes these distortions to add context and meaning to motion allowing the recognition of presence, motion, activity and identity. Aerial software can be embedded in virtually any Wi-Fi network equipment or device and does not require wearables or other sensors to work. Aerial's technology was conceived by Michel Allegue, CTO, based on IP that originated at McGill University, Rutgers University and Steven´s Institute of Technology. Early Smart Motion applications provided through our business partners will include Presence Awareness and Motion Detection, Home Intrusion Detection, Smart HVAC energy optimization, healthcare and elderly care monitory services, and intelligent motion awareness for a variety of smart home devices that improve the convenience and quality of life for consumers. Aerial's technology, already in pilot with several large Internet Service Providers (ISPs) and Multi Service Operators (MSOs), will be deployed into existing equipment offered through these service providers. Aerial is a Montreal-based Corporation with offices in the US and Europe. Aerial's motion detection software uses existing Wi-Fi signal distortions and cloud-based machine learning AI to provide context and meaning to motion without the need for sensors or wearables. www.aerial.ai Follow us on Twitter, Linked-in and Facebook @AerialHome Based in Quebec City, Innovexport invests in start-up companies in the Province of Quebec having innovative products or services with strong export potential. Innovexport was created by entrepreneurs for entrepreneurs with the objective to help them grow their companies. www.fondsinnovexport.com Telefónica Open Future_ is Telefonica´s global platform designed to connect entrepreneurs, startups, investors and public and private partners around the world to capture innovation and business opportunities. The programme incorporates all of Telefónica Group's open innovation, entrepreneurship and investment initiatives through 50 spaces and 9 different investment vehicles. To date, over 1,700 startups have been accelerated and 750, invested. Telefónica Open Future_ is present in 17 countries and has committed to investment, with its partners, a total of 455 million euros. www.openfuture.org/en Quebecor, a Canadian leader in telecommunications, entertainment, news media and culture, is one of the best-performing integrated communications companies in the industry. Driven by their determination to deliver the best possible customer experience, all of Quebecor's subsidiaries and brands are differentiated by their high-quality, multiplatform, convergent products and services. Quebecor (TSX: QBR.A, QBR.B) is headquartered in Québec. It holds an 81.53% interest in Quebecor Media, which employs more than 10,000 people in Canada. A family business founded in 1950, Quebecor is strongly committed to the community. Every year, it actively supports more than 400 organizations working in the vital fields of culture, health, education, the environment and entrepreneurship. www.quebecor.com Videotron, a wholly owned subsidiary of Quebecor Media, is an integrated communications company engaged in cable television, interactive multimedia development, Internet access, cable telephone and mobile telephone services. Videotron is a leader in new technologies with its illico interactive television service and its broadband network, which supports high-speed cable Internet access, analog and digital cable television, and other services. www.videotron.com Kibo Ventures, based in Madrid (Spain) is a VC Fund focused on early-stage investments in digital companies. Kibo Ventures manages $130 million from a mix of institutional and private investors, including IEF, Telefonica, the Spanish Government (through CDTI), Mutua Madrileña (Spain's largest insurance company) and Axis (ICO's 25 year-old private equity arm). www.kiboventures.com TandemLaunch is a seed investor and incubator focused on creating early-stage technology start-ups in collaboration with global universities and world-class entrepreneurs. www.tandemlaunch.com
News Article | February 22, 2017
NEEDHAM, Mass.--(BUSINESS WIRE)--The Object Management Group® (OMG®), an international, open membership, not-for-profit technology standards consortium, today announced its Special Events program being held during the OMG Technical Committee meeting from March 20-24, 2017 at the Hyatt Regency Hotel in Reston, Virginia, USA. Registration for the Special Events program is open to the public. Media can register for a complimentary press pass using the code TCVAP17. The program is supported by OMG annual sponsors, No Magic, Sparx Systems and Orbus Software. The program will feature ten Information Days—in-depth public forums—throughout the week. Monday Special Events Highlights On March 20th, Dr. Jon Siegel, OMG Vice President, Technology Transfer, will present his tutorial on OMG modeling and middleware standards. On the same day, the Business Process Modeling in Health Workshop will feature keynote speaker Dr. Ed Hammond, Ph.D., FACMI, Director, Duke Center for Health Informatics, will present “ The Tensions between Workflow, Status Quo, and Keeping up with Technology (and the Times).” His talk will focus on the changes being “forced” on the health system as a consequence of advances in technology, as well as the impacts of changing health best practices and the industry’s ability to adopt them. Dr. Shane McNamee, M.D., U.S. Department of Veterans Affairs, will discuss how the OMG Business Process Modeling Notation™ (BPMN™) standard offers the potential to stabilize health care processes that support both patients and providers as organizations strive toward improved efficacy and high reliability. A foremost expert in BPM standards, Denis Gagne, CEO/CTO of Trisotech and Chair of the OMG BPMN Interchange Working Group, will deliver a presentation titled “Overview of OMG Business Standards.” Registration costs $149 USD. Workshop attendees are invited to join the OMG Healthcare Committee Meeting on March 21 for an in-depth discussion about OMG’s healthcare standards work. They can also participate in the Committee Meeting for two extra days from March 21-22. The one-day, add-in option costs an additional $99 USD and the two-day, add-in option costs an extra $198 USD. Monday and Tuesday Special Event Highlights From March 21-22, speakers at the fifth annual Business Architecture Innovation Summit will discuss business architecture's role in a cross-section of industries. Registration for the two-day Summit is $395 USD. An optional Business Architecture Reference Model Workshop on Thursday, March 23 costs $495 USD. Tuesday Special Events Highlights On the morning of March 21, Dr. Dale Meyerrose, Major General, U.S. Air Force (retired) will keynote at the half-day Cyber Resilience Summit. He was the first President-appointed, Senate-confirmed chief information officer and information sharing executive for the U.S. Intelligence Community. Additionally, the popular “Titans of Cyber” panel returns, featuring federal agency and intelligence community heads who will share critical issues facing their organizations as they battle cyber risk. Government admission is complimentary. Use the code CSQVA27 during registration to redeem the complimentary pass. Otherwise the registration fee is $95 USD. To learn more the agenda and speakers, please visit http://it-cisq.org/cyber-resilience-summit-2017/. In the afternoon, Lieutenant Commander Rollie J. Wicks, interviewed in the 60 Minutes program, "The Coming Swarm,” will present at the complimentary Cybersecurity Workshop. An associate advisor of the Secretary of the Navy Naval Innovation Advisory Council and an expert in autonomous systems, artificial intelligence, and robotics, Lt. Cdr. Wicks will discuss autonomous systems and the cyber challenges his team is addressing. Cyber experts will also take part in the “Economics of Cybersecurity: Cost vs. Protection” panel to discuss security versus budgetary tradeoffs when building a cyber infrastructure. Wednesday Special Events Highlights On March 22, experts and OMG Chairs will weigh in about the latest security trends and the role that standards play in making the Industrial Internet safe, secure and reliable at the complimentary OMG Standards at Work in the IIoT – Focus on Security session. Speakers include: Dr. Allan Friedman, Director of Cybersecurity, U.S. Department of Commerce; Robert Martin, Senior Principal Engineer, Cyber Security Partnerships, The MITRE Corporation; and Stephen Mellor, CTO of the Industrial Internet Consortium®, among others. The agenda is updated daily at http://www.omg.org/news/meetings/tc/va-17/special-events/IIoT_Agenda.htm. Running concurrently, the OMG IEF Chairs will unveil a family of specifications that enables policy-driven, data-centric information sharing and safeguarding under the umbrella of the OMG's Information Exchange Framework™ (IEF™) standard. The information sharing session will highlight the finalized IEF Information Packaging Policy Vocabulary™ (IEF IPPV™) specification and the IEF Reference Architecture (IEF RA) specification (which is nearing completion). Visit http://omg.org/meetings/va-2017/index7.cgi?noid=yes to register. Thursday Special Events Highlights On the morning of March 23, thought leaders at the complimentary third UAF® with MBSE Summit will explore how to leverage MBSE with architecture modeling in an integrated and disciplined approach. Registration details can be found at http://www.omg.org/meetings/va-2017/index6.cgi?noid=yes. In the afternoon, the Industrial Internet Consortium will host the complimentary Industrial Internet Innovation Forum. Industry experts and organizations who have deployed or are currently deploying Industrial Internet solutions will present best practices. Visit http://www.iiconsortium.org/reston-forum-2017/index.htm for registration information. The full-day Workshop on Information Sharing & Safeguarding Standards will feature government, standards development organizations and industry leaders charting the future for architecture and standards frameworks for the national information sharing environment. Registration is $445 USD. Registration Information All Special Events (including complimentary) require advance registration. The $950 USD registration for the full OMG TC Meeting week includes all Special Event daily fees. Separate registration fees apply for the Business Architecture Innovation Summit and Reference Model Workshop, Cyber Resilience Summit, the Business Process Modeling in Health Workshop (and Committee Meetings) and the Workshop on information Sharing & Safeguarding Standards for attendees not registered for the meeting week. OMG Social Media Channels Follow the Twitter hashtag #OMGReston to join the conversation. To learn about becoming an OMG member, click on http://www.omg.org or visit us on Facebook, follow us on Twitter or connect with us on LinkedIn. About OMG The Object Management Group® (OMG®) is an international, open membership, not-for-profit technology standards consortium with representation from government, industry and academia. OMG Task Forces develop enterprise integration standards for a wide range of technologies and an even wider range of industries. OMG's modeling standards enable powerful visual design, execution and maintenance of software and other processes. Visit www.omg.org for more information. Note to editors: For a listing of all OMG trademarks, visit http://www.omg.org/legal/tm_list.htm. All other trademarks are the property of their respective owners.
Vilhena De Moraes R.,ICT |
Sampaio J.C.,São Paulo State University |
Da Silva Fernandes S.,IEF |
Advances in the Astronautical Sciences | Year: 2013
A semi-analytical approach is proposed to study resonances effects on the orbital motion of artificial satellites or space debris orbiting the Earth. Applying successive Mathieu transformations, the order of dynamical system is reduced and the final system is solved by numerical integration. In the simplified dynamical model, we can choose the resonance to be considered as critical angle. Simulations are presented showing the variations of the orbital elements of bodies orbiting in the neighbourhood of the 2:1, 14:1 and 15:1 resonance condition. The half-width of the separatrix is calculated through a linearized model which describes the behavior of the dynamical system in a neighborhood of each critical angle. A semi-analytical approach is proposed to study resonances effects on the orbital motion of artificial satellites or space debris orbiting the Earth. Applying successive Mathieu transformations, the order of dynamical system is reduced and the final system is solved by numerical integration. In the simplified dynamical model, we can choose the resonance to be considered as critical angle. Simulations are presented showing the variations of the orbital elements of bodies orbiting in the neighbourhood of the 2:1, 14:1 and 15:1 resonance condition. The half-width of the separatrix is calculated through a linearized model which describes the behavior of the dynamical system in a neighborhood of each critical angle. © 2013 2013 California Institute of Technology.
Colli A.,IEF |
Vital B.R.,Federal University of Viçosa |
Carneiro A.C.O.,Federal University of Viçosa |
Silva J.C.,Federal University of Viçosa |
And 2 more authors.
Revista Arvore | Year: 2010
This work had as its objective to determine the properties of particleboards fabricated with paricá particles (Schyzolobium amazonicum Huber ex. Ducke), with increasing proportions of coconut fibers (Coconuts nucifera L.). Boards were fabricated with 6 or 8% of urea-formaldehyde adhesive. Particleboard mean density was equal to 360 kg/m3. The addition of coconut fibers didn't affected boards' dimensional stability, higrospicidity, water absorption capability. However it increased its mechanical properties. The addition of 8% of adhesive improved all board properties.
News Article | November 3, 2016
If people continue using and changing the land over the next century in the same way they currently do, soils will have limited potential to counter the effect of climate change and will become a net source of atmospheric carbon dioxide, experts have warned. Experts have forecast that a quarter of the carbon found in soil in France could be lost to the atmosphere during the next 100 years. This could lead to soil becoming a net source of carbon dioxide to the atmosphere. At present soil is considered to absorb carbon dioxide and this partially counters the impact of man-made climate change. The pace and nature of predicted changes in climate over the next century will make the soil less able to store carbon, while business-as-usual land use change has limited capacity to counteract this trend, experts from the University of Exeter, INRA and CERFACS in France and University of Leuven in Belgium say in the journal Scientific Reports. If, as predicted, soils lose a significant amount of their carbon this will endanger their ability to produce food and store water and this could lead to increased soil erosion and flood damage. Researchers made these predictions for the 21st century by combining models of soil carbon and land use change with climate change predictions, using France as a case study. The study shows that land under almost all uses will be subject to dramatic losses of soil carbon by 2100. Only conversions of land into grass or forest result in limited additional storage of carbon in soils. Unfortunately these land changes are not likely to happen on a large scale because of the pressures on land resources imposed by urban expansion and food production. Lead author Dr Jeroen Meersmans, from the University of Exeter, said: "A reduction in anthropogenic CO2 levels is crucial to prevent further loss of carbon from our soils. However, promotion of land use changes and management that contribute to soil carbon sequestration remains essential in an integrated strategy to protect soil functions and mitigate climate change." Co-author Dr Dominique Arrouays of the French National Institute for Agricultural Research added, "Purposive, targeted land use and agricultural practice changes would be needed if climate change mitigation is to be maximized. Therefore, the efforts to enhance carbon sequestration in soils, as proposed by France during the COP21, should be promoted immediately." The research collaboration involved academics from the Geography Department at the College of Life and Environmental Sciences at the University of Exeter (UK), the InfoSol Unit at INRA in Orleans (France), CECI, CERFACS - CNRS in Toulouse (France), the Geography and Tourism Research Group at the Department of Earth and Environmental Sciences at the University of Leuven (Belgium). The research is funded by the European Commission through the Marie Curie Intra-European Fellowship for Career Development (IEF) project D3DC. Future C loss in mid-latitude mineral soils: climate change exceeds land use mitigation potential in France is published in the journal Scientific Reports.
News Article | October 25, 2016
HSP90 inhibitors used in this study including PU-H71, PU-DZ13, NVP-AUY922, and SNX-2112 were synthesized as previously reported7, 19. 17-DMAG was purchased from Sigma. HSP90 bait (PU-H71 beads)21, HSP70 bait (YK beads)22, biotinylated YK (YK-biotin)22, fluorescently labelled PU-H71 (PU-FITC)23, the control derivatives PU-TEG and PU-FITC9 (ref. 24), and the radiolabelled PU-H71-derivative 124I-PU-H71 (ref. 25) were generated as previously described. The specificity of PU-H71 for HSP90 and over other proteins was extensively analysed7. Thus binding of PU-H71 in cell homogenates, live cells and organisms denotes binding to HSP90 species characteristic of each analysed tumour or tissue. Combined with the findings that PU-H71 binds more tightly to HSP90 in type 1 than in type 2 cells, an observation true for cell homogenates, live cells, and in vivo, at the organismal level, we propose that labelled versions of PU-H71 are reliable tools to perturb, identify and measure the expression of the high-molecular-weight, multimeric HSP90 complexes in tumours. The specificity of YK probes for HSP70 was previously reported22, 26, 27, 28. Cell lines were obtained from laboratories at WCMC or MSKCC, or were purchased from the American Type Culture Collection (ATCC) or Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). Cells were cultured as per the providers’ recommended culture conditions. Cells were authenticated using short tandem repeat profiling and tested for mycoplasma. The pancreatic cancer cell lines include: ASPC-1 (CRL-1682), PL45 (CRL-2558), MiaPaCa2 (CRL-1420), SU.86.86 (CRL-1837), CFPAC (CRL-1918), Capan-2 (HTB-80), BxPc-3 (CRL-1687), HPAFII (CRL-1997), Capan-1 (HTB-79), Panc-1 (CRL-1469), Panc05.04 (CRL-2557) and Hs766t (HTB-134) (purchased from the ATCC); 931102 and 931019 are patient derived cell lines provided by Y. Janjigian, MSKCC. Breast cancer cell lines were obtained from ATCC and include MDA-MB-468 (HTB-132), HCC1806 (CRL-2335), MDA-MB-231 (CRM-HTB-26), MDA-MB-415 (HTB-128), MCF-7 (HTB-22), BT-474 (HTB-20), BT-20 (HTB-19), MDA-MB-361 (HTB-27), SK-Br-3 (HTB-30), MDA-MB-453 (HTB-131), T-47D (HTB-133), AU565 (CRL-2351), ZR-75-30 (CRL-1504), ZR-75-1 (CRL-1500). Lymphoma cell lines include: Akata1, Mutu-1 and Rae-1 (provided by W. Tam, WCMC); BCP-1 (CRL-2294), Daudi (CCL-213), EB1 (HTB-60), NAMALWA (CRL-1432), P3HR-1 (HTB-62), SU-DHL-6 (CRL-2959), Farage (CRL-2630), Toledo (CRL-2631) and Pfeiffer (CRL-2632) (obtained from ATCC); HBL-1, MD901 and U2932 (kindly provided by J. Angel Martinez-Climent, Centre for Applied Medical Research, Pamplona, Spain); Karpas422 (ACC-32), RCK8 (ACC-561) and SU-DHL-4 (ACC-495) (obtained from the DSMZ); OCI-LY1, OCI-LY3, OCI-LY4, OCI-LY7 and OCI-LY10 (obtained from the Ontario Cancer Institute); TMD8 (kindly provided by L. M. Staudt, NIH); BC-1 (derived from an AIDS-related primary effusion lymphoma); IBL-1 and IBL-4 (derived from an AIDS-related immunoblastic lymphoma) and BC3 (derived from a non-HIV primary effusion lymphoma). Leukaemia cell lines include: REH (CRL-8286), HL-60 (CCL-240), KASUMI-1 (CRL-2724), KASUMI-4 (CRL-2726), TF-1 (CRL-2003), KG-1 (CCL-246), K562 (CCL-243), TUR (CRL-2367), THP-1 (TIB-202), U937 (CRL-1593.2), MV4-11 (CRL-9591) (obtained from ATCC); KCL-22 (ACC-519), OCI-AML3 (ACC-582) and MOLM-13 (ACC-554) (obtained from DSMZ). The lung cancer cell lines include: NCI-H3122, NCI-H299 (provided by M. Moore, MSKCC); EBC1 (provided by Dr Mellinghoff, MSKCC); PC9 (kindly provided by D. Scheinberg, MSKCC), HCC15 (ACC-496) (DSMZ), HCC827 (CRL-2868), NCI-H2228 (CRL-5935), NCI-H1395 (CRL-5868), NCI-H1975 (CRL-5908), NCI-H1437 (CRL-5872), NCI-H1838 (CRL-5899), NCI-H1373 (CRL-5866), NCI-H526 (CRL-5811), SK-MES-1 (HTB-58), A549 (CCL-185), NCI-H647 (CRL-5834), Calu-6 (HTB-56), NCI-H522 (CRL-5810), NCI-H1299 (CRL-5803), NCI-H1666 (CRL-5885) and NCI-H1703 (CRL-5889) (obtained from ATCC). The gastric cancer cell lines include: MKN74 (obtained from G. Schwarz, Columbia University), SNU-1 (CRL-5971) and NCI-N87 (CRL-5822) (obtained from ATCC), OE19 (ACC-700) (DSMZ). The non-transformed cell lines MRC-5 (CCL-171), human lung fibroblast and HMEC (PCS-600-010), human mammary epithelial cells were obtained from ATCC. NIH-3T3, and NIH-3T3 cell lines stably expressing either mutant MET (Y1248H) or vSRC, were provided by L. Neckers, National Cancer Institute (NCI), USA, and were previously reported29, 30. Patient tissue was obtained with informed consent and authorized through institutional review board (IRB)-approved bio-specimen protocol number 09-121 at Memorial Sloan Kettering Cancer Centre (New York, New York). Specimens were treated for 24 h or 48 h with the indicated concentrations of PU-H71 as previously described31. Following treatment, slices were fixed in 4% formalin solution for 1 h, then stored in 70% ethanol. For tissue analysis, slices were embedded in paraffin, sectioned, slide-mounted, and stained with haematoxylin and eosin (H&E). Apoptosis and necrosis of the tumour cells (as percentage) was assessed by reviewing all the H&E slides of the case (controls and treated ones) in toto, blindly, allowing for better estimation of the overall treatment effect to the tumour. In addition, any effects to precursor lesions (if present) and any off-target effects to benign surrounding tissue, were analysed. Tissue slides were assessed blindly by a breast cancer pathologist who determined the apoptotic events in the tumour, as well as any effect on adjacent normal tissue31. Cryopreserved primary AML samples were obtained with informed consent and Weill Cornell Medical College IRB approval (IRB number 0910010677 and IRB number 0909010629). Samples were thawed and cultured for in vitro treatment as described previously32. The microdose 124I-PU-H71 PET-CT (Dunphy, M. PET imaging of cancer patients using 124I-PUH71: a pilot study available from: http://clinicaltrials.gov; NCT01269593) and phase I PU-H71 therapeutic (Gerecitano, J. The first-in-human phase I trial of PU-H71 in patients with advanced malignancies available from: http://clinicaltrials.gov; NCT01393509) studies were approved by the institutional review board (protocols 10-139 and 11-041, respectively), and conducted under an exploratory investigational new drug (IND) application approved by the US Food and Drug Administration. Patients provided signed informed consent before participation. 124I-PU-H71 tracer was synthesized in-house by the institutional cyclotron core facility at high specific activity. For PU-PET, research PET-CT was performed using an integrated PET-CT scanner (Discovery DSTE, General Electric). CT scans for attenuation correction and anatomic coregistration were performed before tracer injection. Patients received 185 megabecquerel (MBq) of 124I-PU-H71 by peripheral vein over two minutes. PET data were reconstructed using a standard ordered subset expected maximization iterative algorithm. Emission data were corrected for scatter, attenuation, and decay. 124I-PU-H71 scans (PU-PET) were performed at 24 h after tracer administration. Each picture shown in Fig. 4c and Extended Fig. 6a is a scan taken of an individual patient. PET window display intensity scales for FDG and PU-PET fusion PET-CT images are given for both PU-PET and FDG-PET. Numbers in the scale bar indicate upper and lower SUV thresholds that define pixel intensity on PET images. The phase I trial included patients with solid tumours and lymphomas who had undergone prior treatment and currently had no curative treatment options. Patient cohorts were treated with PU-H71 at escalating dose levels determined by a modified continuous reassessment model. Each patient was treated with his or her assigned dose of PU-H71 on day 1, 4, 8, and 11 of each 21-day cycle. Human embryonic stem cells (hESCs) were differentiated with a modified dual-SMAD inhibition protocol towards floor plate-based midbrain dopaminergic (mDA) neurons as described previously33. hESCs were maintained on mouse embryonic fibroblasts and passaged with Dispase (STEMCELL Technologies). For each differentiation, hESCs were harvested with Accutase (Innovative Cell Technology). At day 30 of differentiation, hESC-derived mDA neurons were replated and maintained on dishes precoated with polyornithine (PO; 15 μg ml−1), laminin (1 μg ml−1), and fibronectin (2 μg ml−1) in Neurobasal/B27/l-glutamine-containing medium (NB/B27; Life Technologies) supplemented with 10 μM Y-27632 (until day 32) and with BDNF (brain-derived neurotrophic factor, 20 ng ml−1; R&D), ascorbic acid (AA; 0.2 mM, Sigma), GDNF (glial cell line-derived neurotrophic factor, 20 ng ml−1; R&D), TGFβ3 (transforming growth factor type β3, 1 ng ml−1; R&D), dibutyryl cAMP (0.5 mM; Sigma), and DAPT (10 nM; Tocris). Two days after replating, mDA neurons were treated with 1 μg ml−1 mitomycin C (Tocris) for 1 h to kill any remaining non-post mitotic contaminants. Assays were performed at day 65 of neuron differentiation. The PU-FITC assay was performed as previously described7, 23. Briefly, cells were incubated with 1 μM PU-FITC at 37 °C for 4 h. Then cells were washed twice with FACS buffer (PBS/0.5% FBS), and resuspended in FACS buffer containing 1 μg ml−1 DAPI. HL-60 cells were used as internal control to calculate fold binding for all cell lines tested. The mean fluorescence intensity (MFI) of PU-FITC in treated viable cells (DAPI negative) was evaluated by flow cytometry. For primary AML specimens, cells were also stained with anti-CD45-APC-H7, to identify blasts and lymphocyte populations (BD biosciences). Blasts and lymphocyte populations were gated based on SSC versus CD45. The fold PU-FITC binding of leukaemic blasts (CD45dim) was calculated relative to lymphocytes (CD45hiSSClow). The FITC derivative FITC9 was used as a negative control. Cells were seeded on coverslips in 6-well plate and cultured overnight. Cells were treated with 1 μM PU-FITC or negative control (PU-FITC9, an HSP90 inert PU-H71 derivative labelled with FITC). At 4 h post-treatment, cells were fixed with 4% formaldehyde at room temperature for 30 min, and the coverslips were mounted on slides with DAPI-Fluoromount-G Mounting Media (Southern Biotech). The images were captured using EVOS FL Auto imaging system (ThermoFisher Scientific) or a confocal microscope (Zeiss LSM5). Cells were seeded on coverslips and cultured overnight. Cells were fixed with 4% formaldehyde at room temperature for 30 min, washed three times with PBS, and permeabilized with 0.2% Triton X-100 in blocking buffer (PBS/5% BSA) for 10 min. Cells were incubated in blocking buffer for 30 min, and then incubated with rabbit anti-human HSP90α antibody (1:500, Abcam 2928) and mouse anti-human HSP90β (1:500, Stressmarq H9010), or rabbit and mouse normal IgG, in blocking buffer for 1 h. Cells were washed three times with PBS, and incubated with goat anti-mouse Alexa Fluor 568 and goat anti-rabbit Alexa Fluor 488 (1:1,000, ThermoFisher Scientific) in blocking buffer in the dark for 1 h. Cells were then washed three times with PBS, and the coverslips were removed from the plate, and mounted on slides with DAPI-Fluoromount-G Mounting Media (Southern Biotech). The images were captured using EVOS FL Auto imaging system (ThermoFisher Scientific) or a confocal microscope (Zeiss LSM5). Fluorescence intensity was quantified by the integrated density algorithm as implemented in ImageJ. Assays were carried out in black 96-well microplates (Greiner Microlon Fluotrac 200). A stock of 10 μM PU-FITC (or GM-cy3B34) was prepared in DMSO and diluted with Felts buffer (20 mM Hepes (K), pH 7.3, 50 mM KCl, 2 mM DTT, 5 mM MgCl , 20 mM Na MoO , and 0.01% NP40 with 0.1 mg ml−1 BGG). To each well was added the fluorescent dye-labelled HSP90 ligand (3 nM PU-FITC or 6 nM GM-cy3B), and cell lysates (7.5 μg) in a final volume of 100 μl Felts buffer. For each assay, background wells (buffer only), and tracer controls (PU-FITC only) were included on assay plate. To determine the equilibrium binding of GM-cy3b, increasing amounts of lysate (up to 20 μg of total protein) were incubated with tracer. The assay plate was placed on a shaker at room temperature for 60 min and the FP values in mP were measured every 5 min. At time t = 60 min, dissociation of fluorescent ligand was initiated by adding 1 μM PU-H71 in Felts buffer to each well and then placing the assay plate on a shaker at room temperature and measuring the FP values in mP every 5 min. The assay window was calculated as the difference between the FP value recorded for the bound fluorescent tracer and the FP value recorded for the free fluorescent tracer (defined as mP − mPf). Measurements were performed on a Molecular Devices SpectraMax Paradigm instrument (Molecular Devices, Sunnyvale, CA), and data were imported into SoftMaxPro6 and analysed in GraphPad Prism 5. To identify and separate chaperome complexes in tumours, and to overcome the limitations of classical protein chromatography methods for resolving complexes of similar composition and size, we took advantage of a capillary-based platform that combines isoelectric focusing (IEF) with immunoblotting capabilities35. This methodology uses an immobilized pH gradient to separate native multimeric protein complexes based on their isoelectric point (pI), and allows for subsequent probing of immobilized complexes with specific antibodies. The method uses only minute amounts of sample, thus enabling the interrogation of primary specimens. Cultured cells were lysed in 20 mM HEPES pH 7.5, 50 mM KCl, 5 mM MgCl , 0.01% NP40, 20 mM Na MoO buffer, containing protease and phosphatase inhibitors. Primary specimens were lysed in either Bicine-Chaps or RIPA buffers (ProteinSimple). Total protein assay was performed on an automated system, NanoPro 1000 Simple Western (ProteinSimple), for charge-based separation. Briefly, total cell lysates were diluted to a final protein concentration of 250 ng μl−1 using a master mix containing 1× Premix G2 pH 3-10 separation gradient (Protein simple) and 1× isoelectric point standard ladders (ProteinSimple). Samples diluted in this manner maintained their native charge state, and were loaded into capillaries (ProteinSimple) and separated based on their isoelectric points at a constant power of 21,000 μWatts for 40 min. Immobilization was performed by UV-light embedded in the Simple Western system, followed by incubations with anti-HSP90β (SMC-107A, StressMarq Biosciences), anti-HSP90α (ab2928, Abcam), anti-HSP70 (SPA-810, Enzo), AKT (4691), P-AKT (9271) or BCL2 (2872) from Cell Signaling Technology and subsequently with HRP-conjugated anti-Mouse IgG (1030-05, SouthernBiotech) or with HRP-conjugated anti-Rabbit IgG (4010-05, SouthernBiotech). Protein signals were quantitated by chemiluminescence using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific), and digital imaging and associated software (Compass) in the Simple Western system, resulting in a gel-like representation of the chromatogram. This representation is shown for each figure. Protein was extracted from cultured cells in 20 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40 buffer with protease and phosphatase inhibitors added (Complete tablets and PhosSTOP EASYpack, Roche). Ten to fifty μg of total protein was subjected to SDS–PAGE, transferred onto nitrocellulose membrane, and incubated with indicated antibodies. HSP90β (SMC-107) and HSP110 (SPC-195) antibodies were purchased from Stressmarq; HER2 (28-0004) from Zymed; HSP70 (SPA-810), HSC70 (SPA-815), HIP (SPA-766), HOP (SRA-1500), and HSP40 (SPA-400) from Enzo; HSP90β (ab2927), HSP90α (ab2928), p23 (ab2814), GAPDH (ab8245) and AHA1 (ab56721) from Abcam; cleaved PARP (G734A) from Promega; CDC37 (4793), CHIP (2080), EGFR (4267), S6K (2217), phospho-S6K (S235/236) (4858), P-AKT (S473) (9271), AKT (4691), P-ERK (T202/Y204) (4377), ERK (4695), MCL1 (5453), Bcl-XL (2764), BCL2 (2872), c-MYC (5605) and HER3 (4754) from Cell Signaling Technology; and β-actin (A1978) from Sigma-Aldrich. The blots were washed with TBS/0.1% Tween 20 and incubated with appropriate HRP-conjugated secondary antibodies. Chemiluminescent signal was detected with Enhanced Chemiluminescence Detection System (GE Healthcare) following the manufacturer’s instructions. We screened a panel of anti-chaperome antibodies for those that interacted with the target protein in its native form. We reasoned that these antibodies were more likely to capture stable multimeric forms of the chaperome members. These native-cognate antibodies were used in native-PAGE and IEF analyses of chaperome complexes. HSP90β (SMC-107) and HSP110 (SPC-195) antibodies were purchased from Stressmarq; HSP70 (SPA-810), HSC70 (SPA-815), HOP (SRA-1500), and HSP40 (SPA-400) from Enzo; HSP90β (ab2927), HSP90α (ab2928), and AHA1 (ab56721) from Abcam; CDC37 (4793) from Cell Signaling Technology. Cells were lysed in 20 mM Tris pH 7.4, 20 mM KCl, 5 mM MgCl , 0.01% NP40, and 10% glycerol buffer by a freeze-thaw procedure. Primary samples were lysed in either Bicine-Chaps or RIPA buffers (ProteinSimple). Twenty-five to one hundred μg of protein was loaded onto 4–10% native gradient gel and resolved at 4 °C. The gels were immunoblotted as described above following either incubation in Tris-Glycine-SDS running buffer for 15 min before transfer in regular transfer buffer for 1 h, or directly transferred in 0.1% SDS-containing transfer buffer for 1 h. Cells were plated at 1 × 106 per 6 well-plate and transfected with an siRNA against human AHA1 (AHSA1; 5′-TTCAAATTGGTCCACGGATAA-3′), HSP90α (HSP90AA1; no. 1 5′-ATGGCATGACAACTACTTTAA-3′; no. 2 5′-AACCCTGACCATTCCATTATT-3′; no.3 5′-TGCACTGTAAGACGTATGTAA-3′), HSP90β (HSP90AB1; no., 5′-CAAGAATGATAAGGCAGTTAA-3′; no. 5′-TACGTTGCTCACTATTACGTA-3′; no.3 5′-CAGAAGACAAGGAGAATTACA-3′) HSP90α/β (no.1 5′-CAGAATGAAGGAGAACCAGAA-3′, no.2 5′-CACAACGATGATGAACAGTAT-3′), HSP110 (HSPH1; 5′-AGGCCGCTTTGTAGTTCAGAA-3′) from Qiagen or HOP (STIP1) (Dharmacon; M-019802-01), or a negative control (scramble; 5′-CAGGGTATCGACGATTACAAA-3′) with Lipofectamine RNAiMAX reagent (Invitrogen), incubated for 72 h and subjected to further analysis. Total mRNA was isolated using TRIzol Reagent (Invitrogen) following the manufacturer’s recommended protocol. Reverse transcription of mRNA into cDNA was performed using QuantiTect Reverse Transcription Kit (Qiagen). qRT–PCR was performed using PerfeCTa SYBR (Quanta Bioscience), 10 nM AHSA1 (forward: 5′-GCGGCCGCTTCTAGTAGTTT-3′ and reverse: 5′-CATCTCTCTCCGTCCAGTGC-3′) and GAPDH (forward: 5′-CAAAGGCACAGTCAAGGCTGA-3′ and reverse: 5′-TGGTGAAGACGCCAGTAGATT-3′) primers, or 1× QuantiTect Primers for HSP110 (HSPH1), HSP90α (HSP90AA1), HSP90β (HSP90AB1), HSP70 (HSPA1A), HOP (STIP1) (Qiagen) following recommended PCR cycling conditions. Melting curve analysis was performed to ensure product uniformity. To investigate which of the two HSP70 paralogues is involved in epichaperome formation we performed immunodepletions with HSP70 and HSC70 antibodies. Protein lysates were immunoprecipitated consecutively three times with either an HSP70 (Enzo, SPA-810), HSC70 (Enzo, SPA-815) or HOP (kindly provided by M. B. Cox, University of Texas at El Paso), or with the same species normal antibody as a negative control (Santa Cruz). The resulting supernatant was collected and run on a native or a denaturing gel. Tumour lysates were mixed with 10 M urea (dissolved in Felts buffer) to reach the indicated final concentrations of 2 M, 4 M and 6 M. After incubation for 10 min at room temperature or frozen overnight at −80 °C, the lysates were loaded onto 4–10% native gradient gel and resolved at 4 °C or applied to the IEF capillary. The HSP90β bands were detected by using antibody purchased from Stressmarq (SMC-107). A lentiviral vector expressing the MYC shRNA, as previously described36, was requested from Addgene (Plasmid 29435, c-MYC shRNA sequence: GACGAGAACAGTTGAAACA). Viruses were prepared by co-transfecting the shRNA vector, the packaging plasmid psPAX2 and the envelop plasmid pMD2.G into HEK293 cells. OCI-LY1 cells were then infected with lentiviral supernatants in the presence of 4 μg ml−1 polybrene for 24 h. Following flow cytometry selection for positive cells, cells were expanded for further experiments. The MYC protein level was confirmed at 10 days post-infection by western blot using the anti-MYC antibody (Cell Signaling Technology, 5605). Viruses were prepared by co-transfection of the lentiviral vector expressing the MYC shRNA with pLM-mCerulean-2A-cMyc (Addgene, 23244) or pCDH-puro-cMYC (Addgene, 46970), the packaging plasmid psPAX2, and the envelope plasmid pMD2.G into HEK293 cells. ASPC1 cells were then infected with lentiviral supernatants in the presence of 4 μg ml−1 polybrene for 24 h and sorted for mCerulean positive cells or selected with puromycin treatment. Changes in cell size after infection were monitored by analysing the forward scatter (FSC) of intact cells via flow cytometry. MYC protein levels were analysed at 4 days post-infection by western blot. Whole cell extracts were prepared by homogenizing cells in RIPA buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 10% glycerol, protease inhibitors). MYC activity was determined using the TransAM c-Myc Kit (Active Motif, 43396), following the manufacturer’s instructions. Cell viability was assessed using CellTiter-Glo luminescent Cell Viability Assay (Promega) after a 72 h PU-H71 treatment. The method determines the number of viable cells in culture based on quantification of the ATP present, which signals the presence of metabolically active cells, and was performed as previously reported37. For the annexin V staining, cells were labelled with Annexin V-PE and 7AAD after PU-H71 treatment for 48 h, as previously reported38. The necrotic cells were defined as annexin V+/7AAD+, and the early apoptotic cells were defined as annexin V+/7AAD−. For the LDH assay the release of lactate dehydrogenase (LDH) into the culture medium only occurs upon cell death. Following indicated treatment, the culture medium was collected and centrifuged to remove living cells and cell debris. The collected medium was incubated at room temperature for 30 min with the Cytotox-96 Non-radioactive Assay kit (Promega) LDH substrate. All animal studies were conducted in compliance with MSKCC’s Institutional Animal Care and Use Committee (IACUC) guidelines. Female athymic nu/nu mice (NCRNU-M, 20–25 g, 6 weeks old) were obtained from Harlan Laboratories and were allowed to acclimatize at the MSKCC vivarium for 1 week before implanting tumours. Mice were provided with food and water ad libitum. Tumour xenografts were established on the forelimbs for PET imaging and on the flank for efficacy studies. Tumours were initiated by sub-cutaneous injection of 1 × 107 cells for MDA-MB-468 and 5 × 106 for ASPC1 in a 200 μl cell suspension of a 1:1 v/v mixture of PBS with reconstituted basement membrane (BD Matrigel, Collaborative Biomedical Products). Before administration, a solution of PU-H71 was formulated in citrate buffer. Sample size was chosen empirically based on published data39. No statistical methods were used to predetermine sample size. Animals were randomly assigned to groups. Studies were not conducted blinded. Imaging was performed with a dedicated small-animal PET scanner (Focus 120 microPET; Concorde Microsystems, Knoxville, TN). Mice were maintained under 2% isoflurane (Baxter Healthcare, Deerfield, IL) anaesthesia in oxygen at 2 litres per min during the entire scanning period. To reduce the thyroid uptake of free iodide arising from metabolism of tracer, mice received 0.01% potassium iodide solution in their drinking water starting 48 h before tracer administration. For PET imaging, each mouse was administered 9.25 MBq (250 μCi) of 124I-PU-H71 via the tail vein. List-mode data (10 to 30 min acquisitions) were obtained for each animal at various time points post-tracer administration. An energy window of 420–580 keV and a coincidence timing window of 6 ns were used. The resulting list-mode data were sorted into 2-dimensional histograms by Fourier rebinning; transverse images were reconstructed by filtered back projection (FBP). The image data were corrected for non-uniformity of scanner response, dead-time count losses, and physical decay to the time of injection. There was no correction applied for attenuation, scatter, or partial-volume averaging. The measured reconstructed spatial resolution of the Focus 120 is 1.6-mm FWHM at the centre of the field of view. Region of interest (ROI) analysis of the reconstructed images was performed using ASIPro software (Concorde Microsystems, Knoxville, TN), and the maximum pixel value was recorded for each tissue/organ ROI. A system calibration factor (that is, μCi per ml per cps per voxel) that was derived from reconstructed images of a mouse-size water-filled cylinder containing 18F was used to convert the 124I voxel count rates to activity concentrations (after adjustment for the 124I positron branching ratio). The resulting image data were then normalized to the administered activity to parameterize the microPET images in terms of per cent injected dose per gram (%ID per g) (corrected for decay of 124I to the time of injection). Post-reconstruction smoothing was applied only for visual representation of images in the figures. Upon euthanasia, radioactivity (124I) was measured in a gamma-counter (Perkin Elmer 1480 Wizard 3 Auto Gamma counter) using a 400–600 keV energy window. Count data were background- and decay-corrected to the time of injection, and the percent injected dose per gram (%ID per g) for each tumour sample was calculated using a calibration curve to convert counts to radioactivity, followed by normalization to the total activity injected. Mice (n = 5) bearing MDA-MB-468 or ASPC1 tumours reaching a volume of 100–150 mm3 were treated i.p. using PU-H71 (75mg per kg) or vehicle, on a 3 times per week schedule, as indicated. Tumour volume (in mm3) was determined by measurement with Vernier calipers, and was calculated as the product of its length × width2 × 0.5. Tumour volume was expressed on indicated days as the median tumour volume ± s.d. indicated for groups of mice. Mice were euthanized after similar PU-H71 treatment periods, and at a time before tumours reached a size that resulted in discomfort or difficulty in physiological functions of mice in the individual treatment group, in accordance with our IUCAC protocol. Frozen tissue was dried and weighed before homogenization in acetonitrile/H O (3:7). PU-H71 was extracted in methylene chloride, and the organic layer was separated and dried under vacuum. Samples were reconstituted in mobile phase. The concentrations of PU-H71 in tissue or plasma were determined by high-performance LC-MS/MS. PU-H71-d was added as the internal standard40. Compound analysis was performed on the 6410 LC-MS/MS system (Agilent Technologies) in multiple reaction monitoring mode using positive-ion electrospray ionization. For tissue samples, a Zorbax Eclipse XDB-C18 column (2.1 × 50 mm, 3.5 μm) was used for the LC separation, and the analyte was eluted under an isocratic condition (80% H O + 0.1% HCOOH: 20% CH CN) for 3 min at a flow rate of 0.4 ml min−1. For plasma samples, a Zorbax Eclipse XDB-C18 column (4.6 × 50 mm, 5 μm) was used for the LC separation, and the analyte was eluted under a gradient condition (H O + 0.1% HCOOH:CH CN, 95:5 to 70:30) at a flow rate of 0.35 ml min−1. Protein extracts were prepared either in 20 mM HEPES pH 7.5, 50 mM KCl, 5 mM MgCl , 1% NP40, and 20 mM Na MoO for PU-H71 beads pull-down, or in 20 mM Tris pH 7.4, 150 mM NaCl, and 1% NP40 for YK beads pull-down. Samples were incubated with the PU-H71 beads (HSP90 bait) for 3–4 h or with the YK beads (HSP70 bait, for chemical precipitation) overnight, at 4 °C, then washed and subjected to SDS–PAGE with subsequent immunoblotting and western blot analysis. For HSP70 proteomic analyses, cells were incubated with a biotinylated YK-derivative, YK-biotin. Briefly, MDA-MB-468 cells were treated for 4 h with 100 μM biotin-YK5 or d-biotin as a negative control. Cells were collected and lysed in 20 mM Tris pH 7.4, 150 mM NaCl, and 1% NP40 buffer. Protein extracts were incubated with streptavidin agarose beads (Thermo Scientific) for 1 h at 4 °C, washed with 20 mM Tris pH 7.4, 150 mM NaCl, and 0.1% NP40 buffer and applied onto SDS–PAGE. The gels were stained with SimplyBlue Coomassie stain (Invitrogen Life Science Technologies). Proteomic analyses were performed using the published protocol7, 18, 22. Control beads contained an inert molecule as previously described7, 18, 22. Affinity-purified protein complexes from type 1 tumours (n = 6; NCI-H1975, MDA-MB-468, OCI-LY1, Daudi, IBL1, BC3), type 2 tumours (n = 3; ASPC1, OCI-LY4, Ramos) and from non-transformed cells (n = 3; MRC5, HMEC and neurons) were resolved using SDS-polyacrylamide gel electrophoresis, followed by staining with colloidal, SimplyBlue Coomassie stain (Invitrogen Life Science Technologies) and excision of the separated protein bands. Control beads that contained an inert molecule were subjected to the same steps as PU-H71 and YK beads and served as a control experiment. To ensure that we captured a majority of the HSP90 complexes in each cell type, we performed these studies under conditions of HSP90-bait saturation. The number of gel sections per lane averaged to be 14. In situ trypsin digestion of gel bound proteins, purification of the generated peptides and LC–MS/MS analysis were performed using our published protocols7, 18, 22. After the acquisition of raw files, Proteowizard (version 3.0.3650)41 was used to create a Mascot Generic Format (mgf) file containing accurate mass for each peak and its corresponding ms2 ions. Each mgf was then subjected to search a human segment of Uniprot protein database (20,273 sequences, European Bioinformatics Institute, Swiss Institute of Bioinformatics and Protein Information Resource) using Mascot (Matrix Science; version 2.5.0; http://www.matrixscience.com). Decoy proteins were added to the search to allow for the calculation of false discovery rates (FDR). The search parameters were as follows: (i) two missed cleavage tryptic sites were allowed; (ii) precursor ion mass tolerance = 10 p.p.m.; (iii) fragment ion mass tolerance = 0.8 Da; and (iv) variable protein modifications were allowed for methionine oxidation, deamidation of asparagine and glutamines, cysteine acrylamide derivatization and protein N-terminal acetylation. MudPit scoring was typically applied using significance threshold score P < 0.01. Decoy database search was always activated and, in general, for merged LS–MS/MS analysis of a gel lane with P < 0.01, false discovery rate averaged around 1%. The Mascot search result was finally imported into Scaffold (Proteome Software, Inc.; version 4_4_1) to further analyse tandem mass spectrometry (MS/MS) based protein and peptide identifications. X! Tandem (The GPM, http://thegpm.org; version CYCLONE (2010.12.01.1) was then performed and its results are merged with those from Mascot. The two search engine results were combined and displayed at 1% FDR. Protein and peptide probability was set at 95% with a minimum peptide requirement of 1. Protein identifications were expressed as Exclusive Spectrum Counts that identified each protein listed. Primary data, such as raw mass spectrometry files, Mascot generic format files and proteomics data files created by Scaffold have been deposited onto the Massive site (https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp; MassIVE Accession ID: MSV000079877). In each of the Scaffold files that validate and import Mascot searched files, peptide matches, scoring information (Mascot, as well as X! Tandem search scores) for peptide and protein identifications, MS/MS spectra, protein views with sequence coverage and more, can be easily accessed. To read the Scaffold files, free viewer software can be found at (http://www.proteomesoftware.com/products/free-viewer/). Peptide matches and scoring information that demonstrate the data processing are available in Supplementary Table 1f–q. The exclusive spectrum count values, an alternative for quantitative proteomic measurements42, were used for protein analyses. CHIP and PP5 were examined and used as internal quality controls among the samples. Statistics were performed using R (version 3.1.3) limma package43, 44. For entries with zero spectral counts, and to enable further analyses, we assigned an arbitrary small number of 0.1. The data were then transformed into logarithmic base 10 for analysis. Linear models were fit to the transformed data and moderated standard errors were calculated using empirical Bayesian methods. For Fig. 1f and Extended Data Fig. 5a, a moderated t-statistic was used to compare protein enrichment between type 1 cells and combined type 2 and non-transformed cells45. For Extended Data Fig. 5b, the t-statistic was performed to compare protein enrichment among type 1 cells, type 2 cells and non-transformed cells (see Supplementary Table 1). Heat maps were created to display the selected proteins using the package “gplots” and “lattice”46, 47. See Supplementary Table 1 in which the table tab ‘a’ corresponds to Fig. 1f and contains core chaperome networks in type 1, type 2 and non-transformed cells; the table tab ‘b’ corresponds to Extended Data Fig. 5a and contains comprehensive chaperome networks in type 1, type 2 and non-transformed cells; the table tab ‘c’ corresponds to Extended Data Fig. 5b and Extended Data Fig. 8b and contains the HSP90 interactome as isolated by the HSP90 bait in type 1, type 2 and non-transformed cells; the table tab ‘d’ corresponds to Extended Data Fig. 8a and contains upstream transcriptional regulators that explain the protein signature of type1 tumours and the table tab ‘e’ contains metastasis-related proteins characteristic of type 1 tumours. To understand the physical and functional protein-interaction properties of the HSP90-interacting chaperome proteins enriched in type 1 tumours, we used the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database48. Proteins displayed in the heat map were uploaded in STRING database to generate the PPI networks. STRING builds functional protein-association networks based on compiled available experimental evidence. The thickness of the edges represents the confidence score of a functional association. The score was calculated based on four criteria: co-expression, experimental and biochemical validation, association in curated databases, and co-mentioning in PubMed abstracts48. Proteins with no adjacent interactions were not shown. The colour scale in nodes indicates the average enrichment of the protein (measured as exclusive spectral counts) in type 1, type 2, and non-transformed cells, respectively. The network layout for type 1 tumours was generated using edge-weighted spring-electric layout in Cytoscape with slight adjustments of marginal nodes for better visualization49. The layout for type 2 and non-transformed cells retains that of type 1 for better comparison. Proteins with average relative abundance values less than 1 were deleted from analyses. The biological processes in which they participate and the functionality of proteins enriched in type 1 tumours were assigned based on gene ontology terms and based on their designated interactome from UniProtKB, STRING, and/or I2D databases48, 50, 51, 52, 53. The Upstream Regulator analytic, as implemented in Ingenuity Pathways Analysis (IPA, QIAGEN Redwood City, http://www.qiagen.com/ingenuity), was used to identify the cascade of upstream transcriptional regulators that can explain the observed protein expression changes in type 1 tumours. The analysis is based on prior knowledge of expected effects between transcriptional regulators and their target genes stored in the Ingenuity Knowledge Base. The analysis examines how many known targets of each transcription regulator are present in the data set, and calculates an overlap P value for upstream regulators based on significant overlap between dataset genes and known targets regulated by a transcription regulator. For Extended Data Fig. 8b, proteins were selected based on 3 pre-curated lists (MYC target genes based on the analysis report from INGENUITY, MYC signature genes based on the reported list provided in ref. 54 and MYC expression/function activators were manually curated from UniProt and GeneCards databases). Cell lines with information available in the cBioPortal for cancer genomics (http://www.cbioportal.org) were evaluated for mutations in pathways implicated in cancer: P53, RAS, RAF, PTEN, PIK3CA, AKT, EGFR, HER2, CDK2NA/B, RB, MYC, STAT1, STAT3, JAK2, MET, PDGFR, KDM6A, KIT. Mutations in major chaperome members (HSP90AA1, HSP90AB1, HSPH1, HSPA8, STIP1, AHSA1) were also evaluated. Data were visualized and statistical analyses performed using GraphPad Prism (version 6; GraphPad Software) or R statistical package. In each group of data, estimate variation was taken into account and is indicated in each figure as s.d. or s.e.m. If a single panel is presented, data are representative of 2 or 3 biological or technical replicates, as indicated. P values for unpaired comparisons between two groups with comparable variance were calculated by two-tailed Student’s t-test. Pearson’s tests were used to identify correlations among variables. Significance for all statistical tests was shown in figures for not significant (NS), *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. No samples or animals were excluded from analysis, and sample size estimates were not used. Animals were randomly assigned to groups. Studies were not conducted blinded, with the exception of all patient specimen histological analyses.
News Article | December 1, 2016
Middle Eastern Bitumen, a rare, tar-like material, is present in the seventh century ship buried at Sutton Hoo, according to a study published in the open-access journal PLOS ONE on December 01, 2016 by Pauline Burger and colleagues from the British Museum, UK and the University of Aberdeen. The seventh century ship found within a burial mound at Sutton Hoo, UK was first excavated in 1939 and is known for the spectacular treasure it contained including jewellery, silverware, coins, and ceremonial armour. The site is thought to be an example of the European ship-burial rites of the time, and also includes a burial chamber where a corpse was likely laid. Fragments of black organic material found in this chamber were originally identified as locally-produced 'Stockholm Tar' and linked to repair and maintenance of the ship. The authors of the present study re-evaluated these previously-identified samples, as well as other tar-like materials found at the site, using imaging techniques and isotopic analysis and found the samples had been originally misidentified. By comparing the samples from Sutton Hoo to various reference materials, the researchers' analysis revealed that the previously-identified 'Stockholm Tar' lumps actually displayed the molecular and isotopic characteristics of archaeological bitumen, and specifically bitumen from the Middle East rather than from a local British source. Archaeological finds of bitumen from this period in Britain are extremely rare and the authors state that this finding is the first material evidence for trading of Middle Eastern bitumen northwards into the British Isles. While the original form and purpose of the bitumen could not be discerned from the remaining fragments, the authors suggest that it may have been included deliberately in the burial chamber, possibly the remaining components of ornamental objects adorning the grave, or perhaps included as a prestigious raw material. In your coverage please use this URL to provide access to the freely available paper: http://journals. Citation: Burger P, Stacey RJ, Bowden SA, Hacke M, Parnell J (2016) Identification, Geochemical Characterisation and Significance of Bitumen among the Grave Goods of the 7th Century Mound 1 Ship-Burial at Sutton Hoo (Suffolk, UK). PLoS ONE 11(11): e0166276. doi:10.1371/journal.pone.0166276 Funding: This research was supported by funding from the European Commission Research Executive Agency (REA) via the Marie Curie Actions - Intra-European Fellowships for Career Development funding scheme (FP7-MC-IEF), Grant Agreement No. 253942, awarded to PB and RJS for project AMPT (Ancient Maritime Pitch and Tar: a multi-disciplinary study of sources, technology and preservation). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
News Article | November 28, 2016
In terms of revenue, the global hemoglobinopathy market is projected to register a healthy CAGR of 8.3% over the forecast period owing to various factors, on which Persistence Market Research offers detailed insights. Most health insurance companies, including government programs in the U.S. and Europe provide insurance coverage for hemoglobinopathy therapy under the CPT code. This creates a favorable environment for the growth of the global hemoglobinopathy market. Various government initiatives are being carried out for detection and management of hemoglobinopathy in high disease prevalence regions across the globe, giving a fillip to the global hemoglobinopathy market. Advancements in diagnosis techniques have led to the development of new tests that have enabled clinicians to provide effective diagnosis and therapeutic assistance to patients. Rising awareness about these disorders coupled with various public and private initiatives is also fueling the growth of the global hemoglobinopathy market in the regions with a high prevalence of sickle cell anemia and thalassemia. Lack of awareness regarding hemoglobinopathy disorders among the populace especially in underdeveloped countries is a major factor hampering growth of the global hemoglobinopathy market. The lack of a permanent cure for hemoglobinopathy disorder is also one of the restraints affecting the global hemoglobinopathy market. Global hemoglobinopathy market is segmented based on indication type, test type, end users, and region. Based on indication, the market is segmented into sickle cell disease, alpha thalassemia, and beta thalassemia. Beta thalassemia segment is expected to register a significant CAGR of 8.7% during the forecast period. Sickle cell disease segment is anticipated to grow with a CAGR of 8.3% over the forecast period. The market has been segmented based on major types of diagnostic tests such as red blood cell (RBC) count test, genetic testing, hemoglobin by high performance liquid chromatography (HPLC) test, hemoglobin isoelectric (Hb IEF) focusing, hemoglobin electrophoresis (Hb ELP) test, and hemoglobin solubility test. The genetic testing segment is expected to expand at the highest CAGR of 9.2% over the forecast period due to increasing adoption of preventive measures such as genetic counseling and testing. Increasing popularity of prenatal and premarital genetic screening make these tests an attractive opportunity for market players. Based on end users, the market has been segmented into hospitals, diagnostic laboratories, and clinics. The diagnostic laboratories segment is anticipated to account for the highest market share over the forecast period, registering a CAGR of 8.9% due to availability of dedicated equipment, reagents, and trained technicians required to perform rather complex diagnostic procedures like genetic testing, Hb HPLC, and isoelectric focusing. Global hemoglobinopathy market has been segmented into five major regions: North America, Latin America, Europe, Asia Pacific (APAC), and Middle East & Africa (MEA). North America and Europe are expected to dominate the global hemoglobinopathy market with maximum market share in 2016. North America and Europe collectively have been expected to account for more than 50% of the total global hemoglobinopathy market share in terms of value in 2016. Among emerging markets, Asia Pacific is estimated to exhibit the highest CAGR of 9.3% over the forecast period, due to an increase in the diagnosis rate. Some key players in the global hemoglobinopathy market identified in the report include Abbott Diagnostics, Bio-Rad Laboratories Inc., Danaher Corporation, Mindray Medical International Ltd., Nexcelom Bioscience LLC., Nihon Kohden Corporation, PerkinElmer Inc., Siemens Healthineers, and Sysmex Corporation. The individual strategies of these companies in terms of increasing focus on rare diseases, initiatives to increase disease awareness, and enhancing distribution base have been discussed. The report concludes with strategic recommendations for players already present in the market and new players planning to enter the global hemoglobinopathy market, which could help them in the long run.
Bideaud A.,CNRS Neel Institute |
Belier B.,IEF |
Benoit A.,CNRS Neel Institute |
Berge L.,French National Center for Scientific Research |
And 6 more authors.
Experimental Astronomy | Year: 2011
Microbolometers are at present the most sensitive detectors for mm and sub-mm Astronomy. They are in use in most of the present instruments in that bandwidth. We have developed filled arrays of NbSi-based planar antenna coupled microbolometers. The fabrication details are given, together with characterization of the NbSi thermometers and optical results. The optical performances are potentially good for ground-based mm-wave astronomy applications, while the overall detectors performances are limited by low-frequency excess noise in the thermometric NbSi high-impedance sensors (Anderson insulator). © 2011 Springer Science+Business Media B.V.