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Biloela, Australia

Barrett S.,South Coast Region | Yates C.J.,LMB
Austral Ecology | Year: 2015

Throughout the world, mountains provide unique environments with attendant endemic species. In the otherwise subdued landscapes of the floristically diverse Southwest Western Australian Floristic Region, the Stirling Range provides the region's only distinctly montane environments. On the highest peaks of the Range the characteristic heathland (Kwongan) of the region becomes a dense shrub thicket with many endemic species and is known as the Eastern Stirling Range Montane Heath and Thicket. We assessed the conservation status of the Montane Heath and Thicket using the IUCN Red list criteria for ecosystems. We found the ecosystem to be Critically Endangered based on its naturally limited geographic extent and area of occupancy in combination with the impacts of the plant pathogen Phytophthora cinnamomi. Historical sources and long-term monitoring were critical to our assessment of this ecosystem highlighting their importance in detecting and understanding likely causes of change. The ecosystem is predicted to decline further in the absence of intensive management due to current threatening processes as well as the potential future impacts of climate change. The Montane Heath and Thicket, while substantially modified still retains areas with highly significant conservation values and these pose many challenges for management. Continued management of P.cinnamomi through phosphite application and management of fire return intervals will be critical to conserve the remaining areas of the thicket where sensitive plant species occur together with an ex situ conservation program including ongoing seed collection and translocation for the most threatened species. © 2014 Ecological Society of Australia. Source


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No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. The DNA coding sequences (CDSs) of the human APC/C subunits (wild-type, mutant Apc2ΔWHB, Apc11–UbcH10 fusion and ΔApc15) were assembled by USER cloning into a modified version of the insect cell-baculovirus MultiBac expression system32, 51, 52. All APC/C subunit CDSs were distributed in two recombinant vectors that were used for recombinant baculovirus generation. For APC/C expression, Hi-5 cells at a density of 2 × 106 cells ml−1 were co-infected with two pre-cultures of Sf9 cells each pre-infected with one of the two recombinant APC/C baculoviruses. APC/C expression (unphosphorylated) was performed for 30 h. To obtain APC/COA (phosphorylated APC/C), okadaic acid at a final concentration of 0.1 μM was added after 24 h of infection. Cells were collected after 5 h of treatment. The CDSs of the human MCC subunits (Mad2, Cdc20, BubR1 and Bub3) used for structural analysis were cloned into a pU2 plasmid52 using the same method as for the APC/C. BubR1 was fused in frame with an N-terminal 3×Flag tag. Cdc20 for individual expression was cloned into a pFastbac1HTA in frame with the His -tag. In addition, a maltose-binding protein (MBP) tag, followed by a TEV site between the starting codon of Cdc20 and the N-terminal His tag, was added by restriction free cloning method (RF-cloning53). To obtain a vector containing Mad2, Cdc20 and BubR1 (residues 1–569) CDSs (miniMCC construct), a Mad2- and Cdc20-containing expression cassette from a pU1 vector was shuttled (by the AvrII and PmeI sites) into a pFastbacDual vector (BstZ171 and SpeI sites) that contained 3×Flag–BubR11–569 under the control of the p10 promoter. A C-terminal StrepIIx2 tag was added by RF-cloning into the BubR1 constructs used in ubiquitination assays. Expression of either the MCC or Cdc20 constructs was performed similarly to the APC/C (unphosphorylated) to avoid CDK-dependent inhibition of APC/C-Cdc20 interactions54, 55. Moreover, cells were collected 48 h after infection. To express MCC complexes with the tagged versions of BubR1, virus containing the BubR1-StrepII constructs was co-infected with MCC virus. To express the MCC complex with the Cdc20K485R,K490R mutations, viruses containing the individual MCC subunits were used for co-infection. Apc15∆NTH, a mutant form of Apc15 with a (Gly-Ser-Ala) linker substitution of the N-terminal helix (NTH: residues 23–57) was cloned into an Escherichia coli pOPIN expression vector and purified using a C-terminal StrepIIx2 tag. To generate mitotic phosphorylated APC/C (APC/COA) we incubated APC/C expressing insect cells with the phosphatase inhibitor okadaic acid (OA) (as described above). The extent of APC/C phosphorylation was monitored by assessing the migration of the Apc3 subunit on SDS–PAGE56 (Extended Data Fig. 1a, f). The recombinant APC/COA was phosphorylated on ~110 sites (Extended Data Table 3), correlating closely with those previously identified in endogenous APC/C isolated from HeLa cells arrested by the mitotic checkpoint56, 57, 58, and with sites phosphorylated in vitro by the mitotic APC/C activating kinases Cdk2-cyclinA2-Cks2 and Plk1 (ref. 22) (Extended Data Table 3). Compared with APC/C from untreated insect cells, and using Cdc20 as the coactivator, APC/COA readily ubiquitinates securin (Extended Data Fig. 1g, h). The APC/CMCC complex was reconstituted by co-lysing APC/COA expressing cells with insect cells expressing separately MBP-tagged Cdc20 and the MCC (BubR1, Bub3, Mad2 and untagged Cdc20). Hi-5 cell pellets expressing either APC/COA or MBP–Cdc20 or MCC were mixed together in reconstitution buffer containing 50 mM Hepes (pH 8.2), 150 mM NaCl, 5% glycerol, 0.5 mM TCEP, 1 mM EDTA, 0.1 mM PMSF, 2 mM benzamidine, 5 U ml−1 benzonase (Novagen), Complete EDTA-free protease inhibitors (Roche), 50 mM NaF, 20 mM β-glycerophosphate and 0.1 μM okadaic acid. After complete mixing the cells were co-lysed by sonication and the lysate was centrifuged for 60 min at 17,000g. The soluble fraction was loaded onto a Strep-Tactin Superflow Cartridge (Qiagen) for purification using the StrepIIx2 tag on Apc4 as described previously21. The eluate was then applied to an anti-Flag M2 Affinity Gel (A220, Sigma) column (directed against the N-terminal Flag tag on BubR1) and incubated overnight. The APC/CMCC complex was eluted with a 3×Flag peptide at a concentration of 50 μg ml−1. The resulting elution was concentrated to around 1.4 mg ml−1 and run on a Superose 6 3.2/300 (GE Healthcare Life Sciences) gel-filtration column pre-equilibrated with gel-filtration buffer containing 20 mM HEPES (pH 8.0), 150 mM NaCl and 0.5 mM TCEP. The gel filtration was run on a ÄKTAmicro (GE Healthcare Life Sciences) with a flow rate of 50 μl min−1. An SDS–PAGE of purified APC/CMCC showed both versions of Cdc20, consistent with the incorporation of two distinct subunits of Cdc20 into APC/CMCC (refs 2, 20) (Extended Data Fig. 1j). Reconstituted APC/CMCC is stable and homogeneous as shown by size-exclusion chromatography (Extended Data Fig. 2a). The APC/CApc15∆NTH complex was reconstituted by incubating recombinant APC/C∆Apc15 with Apc15∆NTH, at concentrations of 200 nM and 1 μM, respectively, followed by size exclusion chromatography. Anti-Apc15 antibodies were from Santa Cruz Biotechnology (sc-398448). To examine APC/C activity towards securin, the ubiquitination assay was performed with 60 nM of recombinant human APC/C, 150 nM UBA1, 300 nM UbcH10, 300 nM Ube2S, 20 μM ubiquitin, 2 μM securin, 5 mM ATP, 0.25 mg ml−1 BSA and 7 nM of recombinant human Cdc20. The ubiquitination products of securin were detected by western blot with either an anti-His antibody (631212; Clontech) or an anti-securin antibody (700791; Invitrogen). To test the activity of a pre-assembled APC/CMCC complex towards Cdc20MCC (Fig. 5c), ubiquitination reactions were performed with 250 nM of recombinant human APC/CCdc20-MCC and 10 μM of UbcH10 (40× excess). To test the activity of APC/C towards the Cdc20MCC from individually purified wild-type and mutant MCCBubR1-StrepII (purification by StrepIIx2 affinity and gel-filtration columns) ubiquitination reactions were performed with 200 nM of recombinant human APC/COA, 200 nM of recombinant human Cdc20 and either 300 or 600 nM of recombinant human MCCBubR1-StrepII (Fig. 5d, e). Either with a pre-assembled APC/CMCC complex or with a molar excess of MCC complex over free Cdc20 and APC/C only Cdc20MCC ubiquitination is promoted (data not shown)20. Cdc20 and the ubiquitination products of Cdc20MCC were detected by western blot with an anti-Cdc20 antibody (Cdc20 H-175 sc-8358; Santa Cruz Biotechnology). Freshly purified APC/CMCC samples were analysed by negative-stain EM to check the sample quality and to obtain a low-resolution reconstruction. Micrographs were collected on a 2k×2k CCD camera fitted to a FEI Spirit electron microscope at an accelerating voltage of 120 kV, operated at a nominal magnification of 42,000 with a resulting pixel size of 2.46 Å per pixel at specimen level. Defocuses were set at approximately −2 μm. Particles were automatically selected using the autoboxer program implemented in EMAN59. About 150 micrographs per sample were collected yielding ~10,000 particles. After 3D classification performed with RELION60 only the prominent best class (30–40% of total amount of particles) was used for auto-refinement and final low-resolution structure determination. Grid preparation for both negative-stain EM and cryo-EM was performed as described previously32, 51. Cryo-EM micrographs were collected with an FEI Tecnai Polara electron microscope at an acceleration voltage of 300 kV and Falcon III direct detector. Micrographs were taken using EPU software (FEI) at a nominal magnification of 78,000, yielding a pixel size of 1.36 Å per pixel at specimen level. A total exposure time of 1.6 s were used at a dose rate of 27 electrons per pixel. Defocus range was set at −2.0 to −4.0 μm. Movie frames were recorded as described32. Image processing was performed with RELION 1.4 (ref. 60). The initial steps including motion correction, CTF estimation, particle picking and particles sorting by Z-score and 2D classification were performed as described32. Selected particles were used for a first round of 3D classification with global search and a sampling angular interval of 7.5°, using a 60 Å low-pass filtered APC/CCdh1.Emi1 EM map as a reference32. Poorly characterized 3D classes, with poorly recognizable features, were discarded at this stage and the remaining particles were refined and corrected for beam-induced particle motion using particle polishing in RELION61. Polished particles were used for another round of 3D classification with a local search within 15° and a smaller angular sampling interval of 3.7° (Extended Data Figs 4 and 7). The reconstruction generated from all the polished particles, low-pass filtered at 40 Å, was used as reference. To isolate particles for the APC/CMCC-closed state, classes showing closed-like features for the MCC–Cdc20APC/C module (for example, proximity to Apc2, Apc4 and Apc10; Extended Data Fig. 4, classes 1–3) were combined and refined. The resultant map was used as reference for a subsequent 3D classification performed with a soft edge mask on the MCC–Cdc20APC/C module (Extended Data Fig. 4). The mask was created from a map converted from the fitted coordinates of the MCC–Cdc20 module, with three pixel extension and five pixels soft edge width. The MCC–Cdc20 module coordinates were created by fitting the MCC core coordinates and isolated Cdc20 (PDB code 4AEZ)18, on the best MCC–Cdc20APC/C module density map (Extended Data Fig. 4, class 1). To isolate particles for the APC/CMCC-open state, classes showing open-like features for the MCC–Cdc20 module (for example, proximity to TPR lobe and loss of contact with Apc2, Apc4 and Apc10; Extended Data Fig. 4, classes 4–5) were refined together. The obtained averaged class was used as a reference for a subsequent 3D classification performed with a larger mask (6 pixel extension and 6 pixel soft edge) created with the MCC–Cdc20APC/C module coordinates fitted into the corresponding density in the APC/CUbcH10-MCC reconstruction described below (Extended Data Figs 4 and 7). To obtain the APC/C∆Apc15-MCC structure, the best classes from the 3D classification with local searches step were refined together (Extended Data Fig. 7a, classes 1–3). To isolate the particles for the APC/CUbcH10-MCC reconstruction, instead of performing the 3D classification with local search steps, an initial classification with a large mask (similar to APC/CMCC-open) was performed. The latter allowed the identification of a class that features both the MCC–Cdc20 module and the UbcH10-Apc11-Apc2WHB-Apc2α/β domain assembly32. A large mask including the latter regions was created by fitting the MCC–Cdc20APC/C module coordinates and the UbcH10-Apc11-Apc2WHB-Apc2α/β domain assembly (PDB code 5A31)32 in the preliminary APC/CUbcH10-MCC reconstruction. The latter mask was used for a re-classification of the initial particles and allowed the isolation of the final APC/CUbcH10-MCC particles (Extended Data Fig. 7c). All resolution estimates were based on the gold standard Fourier shell correlation (FSC) = 0.143 criterion62. Final FSC curves were calculated using a soft mask (five pixel extension and three pixel soft edge) of the two independent reconstructions. To visualize high-resolution details, all density maps were corrected for the modulation transfer-function of the detector and sharpened by applying negative B-factors, estimated using automated procedures. Local resolution maps for all the cryo-EM reconstructions were calculated with RESMAP63 using a resolution range between 3.5 and 15 Å and displayed with Chimera64. For comparing structural features among the cryo-EM reconstructions, shown in Fig. 4 and Extended Data Fig. 3, which have different overall resolutions, a common filter of 8.5 Å was applied. This was selected based on the local resolution of the APC/CUbcH10-MCC map in the region assigned to Apc15 (the main region of relative comparison). APC/CUbcH10-MCC is the APC/C reconstruction with the lowest overall resolution. Filtering all the reconstruction to 8.5 Å resolution allowed a clear definition of the structural details of Apc15 and other regions without the appearance of noise. To visualize the connecting density between UbcH10 and Cdc20 the APC/CUbcH10-MCC map was filtered to 12 Å resolution based on the local resolution of this area and the threshold was slightly lowered. Initial fitting and superposition of coordinates was performed with Chimera64. Model building of APC/CMCC was performed in COOT65. APC/C platform, TPR lobe, Apc10 and accessory subunit coordinates from the atomic structure of APC/CCdh1.Emi1 (PDB code 4UI9)32 were individually rigid body fit into the APC/CMCC-closed cryo-EM density. A few regions such as Apc4HBD, Apc5NTD and Apc11 were also modified by flexible fitting. The Apc2WHB domain (PDB code 4YII)44 was rigid body fit into the corresponding density. Cdc20APC/C IR tail and NTD were rigid body fit from the coordinates of APC/CCdc20-Hsl1 cryo-EM structure22. The Cdc20MCC IR tail was modelled by superposing the TPR domain of Apc3 including Cdc20IR from APC/CCdc20-Hsl1 to the TPR domain of APC/CMCC Apc8A. Two copies of human the Cdc20WD40 domain (PDB code 4GGA)66, human C-Mad2 (PDB ID: 2V64)8 and the human BubR1TPR domain (PDB code 3SI5)67 were rigid body fit on the MCC–Cdc20 module density. Cdc20MCC CRY box, included in the human Cdc20WD40 domain crystal structure (PDB code 4GGA)66 was modelled by flexible fitting. In addition, the Cdc20 KILR motif was modelled by rigid body fit of the MCC core crystal structure (PDB code 4AEZ)18 into the corresponding density. A similar procedure was applied to model the first KEN1 and helix–loop–helix region of BubR1. BubR1 D1 and D2 were modelled by rigid body fit of Acm1 D-box 3 (PDB code 3BH6)38. Similarly BubR1 A1 and K2 were modelled by flexible fitting of the Acm1 region spanning the A-motif and KEN box as explained in the main text. BubR1 A2 was modelled as a rigid body fit of the Acm1 A-motif. Loop extensions were modelled as idealized polyalanine. Model refinement was performed with REFMAC 5.8 (ref. 68). A REFMAC weight of 0.04 was defined by cross-validation using half reconstructions69. A resolution limit of 4.0 Å was used. All available crystal structures or NMR structures were used for secondary structure restraints. The refinement statistics are summarized in Extended Data Table 2b. Figures were generated using Pymol and Chimera70. Structural conservation figures were generated using ConSurf71. Purified proteins were prepared for mass spectrometric analysis by in solution enzymatic digestion, without prior reduction and alkylation. Protein samples were digested with trypsin or elastase (Promega), both at an enzyme to protein ratio of 1:20. The resulting peptides were analysed by nano-scale capillary LC-MS/MS using an Ultimate U3000 HPLC (ThermoScientific Dionex) to deliver a flow of approximately 300 nl min−1. A C18 Acclaim PepMap100 5 μm, 100 μm × 20 mm nanoViper (ThermoScientific Dionex), trapped the peptides before separation on a C18 Acclaim PepMap100 3 μm, 75 μm × 250 mm nanoViper (ThermoScientific Dionex). Peptides were eluted with a 90-min gradient of acetonitrile (2% to 50%). The analytical column outlet was directly interfaced via a nano-flow electrospray ionization source, with a hybrid quadrupole orbitrap mass spectrometer (Q-Exactive Plus Orbitrap, ThermoScientific). LC–MS/MS data were then searched against an in house LMB database using the Mascot search engine (Matrix Science)72, and the peptide identifications validated using the Scaffold program (Proteome Software Inc.)73. All data were additionally interrogated manually.


Radford B.J.,LMB | Thornton C.M.,LMB
International Journal of Energy, Environment and Economics | Year: 2011

A long-term tillage experiment was designed to assess the effect of tillage frequency and intensity on rainfed grain production and quality in the semi-arid subtropical environment of central Queensland, Australia. There were four tillage treatments: traditional tillage (TT), stubble mulch tillage (SM), reduced tillage (RT) and no till (NT), each with and without applied fertiliser (N+Zn). On completion, after 20 years of treatment application, all treatments were managed using no till and appropriate fertiliser (N+Zn) application for a further 7 years. During the 20 years when tillage treatments were being applied, the reduced tillage treatments (NT, RT and SM) outyielded TT in 10 of 22 crops grown. Mean yields without fertiliser were 2.0 t/ha (TT) and 2.6 t/ha (NT) while mean yields with fertiliser were 1.9 t/ha (TT) and 2.9 t/ha (NT). During the next 5 years of across-the-site no till with fertiliser, the former reduced tillage treatments outyielded the former TT in each of the 5 crops grown. For example, the long-term NT gave an average yield of 3.3 t/ha while the short-term NT, formerly TT for 20 years, produced only 2.1 t/ha - a 57% yield increase for the long-term no-till. This increase was due to both increased soil water storage and higher water use efficiency (WUE). Both were attributed to the development of improved soil structure, higher population densities of soil macrofauna and slightly higher soil organic carbon content. High WUE in NT was also attributed to a beneficial effect resulting from slow early growth under no till. Results indicate it takes at least 20 years to attain the full soil benefits (physical, chemical and biological) of a no-till system. The large yield responses from the three reduced tillage treatments, during and after treatment application, were realised in part because cropping frequency exceeded the appropriate level for traditional tillage. Increased cropping frequency also results in higher levels of groundcover, which reduces soil erosion and creates a more sustainable fanning system. A high-yielding, viable cropping system can also contribute towards environmental sustainability by reducing the need for further land clearing. © 2011 Nova Science Publishers, Inc. Source


Grant
Agency: Cordis | Branch: FP7 | Program: JTI-CS | Phase: JTI-CS-2013-1-SGO-02-051 | Award Amount: 576.12K | Year: 2014

In all-electrical aircrafts, the corresponding electrical ram air fan should be optimized in order to improve its current design in two aspects: a)generate pressure drop at low flows without surge issues; b)cooling of the fan electrical motor at high inlet air temperatures. The ECS control logics produces two main typical operating points, one with high flow when the ram-air fan creates enough flow to cool both the ECS main heat exchanger and the ECS motor stator, and the second when it only needs to provide a low airflow for the latter necessity. As the pressure drop is similar in both situations but the flow very different, this creates surge problems that should be eliminated for adequate operation. Meanwhile, the fact that the ram-air fan is located downstream the ECS main heat exchanger implies that the air inlet arrives to the fan at high temperatures, what difficulties its own correct electrical motor cooling, both from a mechanical and electrical points of view. Considering these objectives, a new fan concept will be selected from available bibliographic and consortium expertise, and designed using the consortium skills in deep fundamental fluid mechanics and heat transfer knowledge (Technical University of Catalonia - UPC ), advanced CFD tools and aerodynamic know-how (Termo Fluids - TF) and engineering capacities of a fan manufacturer (LMB SAS). The proposed design related to surge problem will be implemented in a prototype and tested accordingly. In a similar manner, a solution for electrical motor cooling will be found.The final solution must also consider its impact on the whole ECS pack, in order to maintain its global performance objectives.


Home > Press > FEI Partners with Five Pharmaceutical Companies, the Medical Research Council and the University of Cambridge to form Cryo-EM Research Consortium Abstract: Organizations involved in the Cambridge Pharmaceutical Cryo-EM Research Consortium will share access to cryo-electron microscopy equipment and methods and will collaborate on developing the technology to benefit pharmaceutical drug discovery research. Hillsboro, OR and London, UK | Posted on April 5th, 2016 FEI (NASDAQ: FEIC) has partnered with five pharmaceutical companies: Astex Pharmaceuticals, AstraZeneca, GlaxoSmithKline, Heptares Therapeutics, and UCB; the Medical Research Council Laboratory of Molecular Biology (MRC-LMB); and the University of Cambridge’s Nanoscience Centre, to form the “Cambridge Pharmaceutical Cryo-EM Consortium,” which is the first of its kind worldwide. As part of the three-year agreement, FEI will provide sample preparation and data collection services on a Titan Krios™ cryo-transmission electron microscope (cryo-EM) to the consortium companies for early-stage drug discovery research. The five companies involved in the consortium will share access to the microscope with colleagues from the MRC-LMB and the University of Cambridge in return for expert guidance on the use of cryo-EM technology. FEI’s Titan Krios will be installed at the Nanoscience Centre in May. Richard Henderson, pioneer in the field of cryo-EM at MRC-LMB, states, "It is delightful to know that the development of cryo-EM, which many people have worked on for many years, has now reached mainstream structural biology. It is particularly satisfying that pharmaceutical companies are keen to evaluate the approach for drug development." Prof. Sir Mark Welland, director of the Nanoscience Centre, said, “This is a great opportunity for researchers across the University to access a state-of-the-art microscope.” Cryo-EM has quickly become one of the most important techniques used by structural biologists today to obtain molecular-scale three-dimensional (3D) information about protein structures. When combined with traditional methods for structure determination, such as x-ray crystallography and nuclear magnetic resonance spectroscopy, the resulting models can reveal the structure of complex, dynamic molecular assemblies down to the scale of individual atoms. The consortium’s Titan Krios will use the Relion software package, developed by Sjors Scheres at MRC-LMB, to process the image data into a visual 3D model that helps researchers see and understand the structure and function of the protein. “Cryo-EM 3D models allow us to see and understand the workings of protein-based molecular machines that we could not analyze before because they were too large and complex or were resistant to the preparations required for other techniques,” states Peter Fruhstorfer, vice president and general manager of the Life Sciences business, FEI. “The technique was rapidly adopted by leading academic researchers and is now finding its way into early stage discovery and development in the pharmaceutical industry.” Fruhstorfer adds, “In addition to installing the Titan Krios cryo-EM system, our contribution to the consortium includes providing an application scientist that will work with the participating companies to ensure a smooth workflow throughout, from sample preparation to data collection and data processing, with a special focus on creating a standardized and robust single-particle analysis workflow.” For more information about cryo-EM and the Cambridge Pharmaceutical Cryo-EM Consortium, contact FEI at About FEI Company FEI Company (Nasdaq: FEIC) designs, manufactures and supports a broad range of high-performance microscopy workflow solutions that provide images and answers at the micro-, nano- and picometer scales. Its innovation and leadership enable customers in industry and science to increase productivity and make breakthrough discoveries. Headquartered in Hillsboro, Ore., USA, FEI has over 2,800 employees and sales and service operations in more than 50 countries around the world. More information can be found at: www.fei.com. About the Cambridge University Nanoscience Centre The Nanoscience Centre provides open access to over 300 researchers from a variety of University Departments to the nanofabrication and characterisation facilities housed in a combination of Clean Rooms and low noise laboratories. The main activity in the building is making individual devices or structures which are only a few nanometres in size and then measuring how they work. Office space is primarily home to the Department of Engineering's Nanoscience Group, technical and administrative staff and members of other research groups who require long term access to facilities. www.nanoscience.cam.ac.uk FEI Safe Harbor Statement This news release contains forward-looking statements that include statements regarding the performance capabilities and benefits of the Titan Krios TEM and cryo-EM solution. Factors that could affect these forward-looking statements include but are not limited to our ability to manufacture, ship, deliver and install the tools, solutions or software as expected; failure of the product or technology to perform as expected; unexpected technology problems and challenges; changes to the technology; the inability of FEI, its suppliers or project partners to make the technological advances required for the technology to achieve anticipated results; and the inability of the customer to deploy the tools or develop and deploy the expected new applications. Please also refer to our Form 10-K, Forms 10-Q, Forms 8-K and other filings with the U.S. Securities and Exchange Commission for additional information on these factors and other factors that could cause actual results to differ materially from the forward-looking statements. FEI assumes no duty to update forward-looking statements. For more information, please click If you have a comment, please us. 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