News Article | May 23, 2017
"China Mobile Research Institute has been actively boosting the development of IoT and exploring new IoT business opportunities," said Madam Huang Yuhong, the DGM of China Mobile Research Institute. "This planned field trials with Qualcomm Technologies and Mobike will further expand the applications for LTE IoT (eMTC/NB-IoT) in areas such as smart bike sharing and smart travel. We will conduct cross-industry integration and innovation to boost ubiquitous IoT solutions and the smart IoT life by jointly working with the China Mobile 5G Joint Innovation Center, and taking full advantage of China Mobile's vast GSM network and technology leadership in eMTC/NB-IoT." "We are committed to providing IoT-optimized solutions that address demands from our customers to bring a new range of applications and services built on the reliability, efficiency and global scale of LTE IoT cellular connectivity," said Way-Shing Lee, vice president, technology, Qualcomm Technologies, Inc. "Through this cooperation with China Mobile Research Institute and Mobike on the first multimode eMTC/NB-IoT field trial in China, we can showcase a highly connected and efficient system for a new IoT application such as Mobike's bike sharing platform." "Mobike pioneered the world's first smart bike sharing platform, and we are committed to addressing the challenge of short-trips with innovative technologies and solutions and bringing bikes back to the city. Currently, more than 4.5 million smart bikes operate on the Mobike system, which are equipped with the exclusive, independently researched and developed smart locks. Using connectivity capabilities such as those of the MDM9206 LTE modem with integrated GNSS supporting GPS BeiDou and Glonass, Mobike has become one of the largest mobile IoT systems in the world," said Joe Xia, co-founder and CTO of Mobike. "Mobike is pleased to work with leading enterprises in global communications and IoT industries like Qualcomm Technologies and China Mobile Research Institute in order to deliver IoT applications and services for end users while taking full advantages of the latest technology breakthroughs." The MDM9206 LTE modem is designed to support global Category M1 and NB1/GSM multimode. The IoT-optimized narrowband LTE technologies help the MDM9206 modem to support cost-efficient, low-power, low-bandwidth, multi-year battery life and greater coverage for the next-generation of IoT products and services as compared to previous LTE generations. The MDM9206 LTE modem also provides leading-edge location performance, including an integrated A-GNSS solution, support for cellular and Wi-Fi positioning. The Cat M1 and NB1 LTE modes designed in the MDM9206 modem bring many enhancements and optimizations to LTE that will help reduce IoT device complexity for IoT platforms like Mobike with quicker time to commercialization of their products. Additionally, Cat M1 and NB1/GSM multimode will enable IoT platforms such as Mobike to develop IoT products that can function in a diverse set of operator deployments worldwide, maximizing the products' global reach and scalability. The new technologies also can use existing LTE infrastructure and spectrum, coexisting with today's mobile broadband services, while providing a superior solution to proprietary technologies for low-power wide area networks. Qualcomm's technologies powered the smartphone revolution and connected billions of people. We pioneered 3G and 4G – and now we are leading the way to 5G and a new era of intelligent, connected devices. Our products are revolutionizing industries, including automotive, computing, IoT, healthcare and data center, and are allowing millions of devices to connect with each other in ways never before imagined. Qualcomm Incorporated includes our licensing business, QTL, and the vast majority of our patent portfolio. Qualcomm Technologies, Inc., a subsidiary of Qualcomm Incorporated, operates, along with its subsidiaries, all of our engineering, research and development functions, and all of our products and services businesses, including, our QCT semiconductor business. For more information, visit Qualcomm's website, OnQ blog, Twitter and Facebook pages. China Mobile Research Institute (CMRI) is directly under China Mobile Communications Corporation (CMCC). The CMRI specializes in broad research and technology areas of Telecommunications and IT fields, including wireless access technology, future network technology, strategic research, service technology, Internet of things research, IT and Big Data, industry and market research, operational support, etc. Its mission is to become the engine to drive innovations within China Mobile and to be a world-class industry research lab contributing to the entire ICT industry Mobike is the world's first and largest smart bike-sharing company. Its mission is to bring more bikes to more cities, using its innovative technology to make cycling the most convenient and environmentally-friendly transport choice for urban residents. Using specially designed bikes equipped with GPS, IoT chips and proprietary smart-lock technology, Mobike enables users of its smartphone app to find a bike near them and unlock it simply by scanning a QR code. After reaching their destination, the user parks the bike by the roadside and locks it, automatically making the bike available to other Mobike users nearby. The company officially launched its service in Shanghai in April 2016 and in less than a year since then has expanded the service to over 80 cities across China and internationally, operating nearly 4.5 million smart Mobikes. For more information, visit: mobike.com. Qualcomm is a trademark of Qualcomm Incorporated, registered in the United States and other countries. Qualcomm MDM is a product of Qualcomm Technologies, Inc To view the original version on PR Newswire, visit:http://www.prnewswire.com/news-releases/qualcomm-china-mobile-research-institute-and-mobike-plan-to-commence-first-of-its-kind-lte-iot-multimode-field-trials-in-china-300461918.html
News Article | June 28, 2017
Shanghai, China - Nokia and China Mobile Communications Corporation (CMCC) are to demonstrate how 5G enable support for new services such as telehealth, potentially transforming emergency hospital patient care and driving new practices. 5G will enable the use of discrete network "slicing" techniques, allowing operators to serve multiple end users with a variety of services meeting different demands for latency, speed and capacity over a common 5G network infrastructure. In a demonstration at China Mobile's booth at Mobile World Congress Shanghai this week, Nokia and China Mobile will show how 5G network slicing will support the high-reliability needs of critical applications such as telehealth. The demonstration will use technology based on the Nokia 5G FIRST solution, including the Next Generation Core to replicate communications between ambulance and hospital staff as an emergency patient is being transported. 5G network slicing can enable the rapid and reliable transmission of high-bandwidth images, such as patient X-rays and video, in real-time, to the hospital. This will save vital time, allowing doctors to begin diagnosis of a patient's condition and make initial preparations while the patient is still in transit. The application of Nokia 5G FIRST in the telehealth demonstration also draws on Nokia's professional services expertise. This capability will help operators like CMCC evolve their networks to 5G, with end-to-end design and deployment support for 5G use cases - such as telehealth - based on reference architecture and solution blueprints. Yu Xiaohan, head of Nokia Network China's Customer Business Team for China Mobile, said: "We are very pleased to cooperate with China Mobile and use 5G to demonstrate a highly efficent and effective Telehealth solution. As a leading developer of 5G, we continue to engage with customers on research and development, best practices and the uses of 5G to prepare for future introduction of the technology. We'll continue to fullfil our mission by making people's life easier as we create the technologies that connect the world." China Mobile Communications Corporation (CMCC), as the world's largest telecom operator, owns the largest network and the largest customer base. The company has over 500,000 employees and total 820 million subscribers, with over 2,600,000 base stations installed. In 2015, CMCC was listed in Global Fortune 500 at #55, and entered the Dow Jones Sustainability Index for the 8th consecutive year. In recent years, CMCC has been focused on generating business opportunities from China's "Internet plus" initiatve. Through promoting strategic transformation, accelerating entrepreneurial schemes and expanding competitive edge in 4G, CMCC has maintained a good momentum for further business growth. About Nokia We create the technology to connect the world. Powered by the research and innovation of Nokia Bell Labs, we serve communications service providers, governments, large enterprises and consumers, with the industry's most complete, end-to-end portfolio of products, services and licensing. From the enabling infrastructure for 5G and the Internet of Things, to emerging applications in virtual reality and digital health, we are shaping the future of technology to transform the human experience. nokia.com
Athanasiadis P.J.,CMCC |
Bellucci A.,CMCC |
Scaife A.A.,UK Met Office |
Hermanson L.,UK Met Office |
And 5 more authors.
Journal of Climate | Year: 2017
Significant predictive skill for the mean winter North Atlantic Oscillation (NAO) and Arctic Oscillation (AO) has been recently reported for a number of different seasonal forecasting systems. These findings are important in exploring the predictability of the natural system, but they are also important from a socioeconomic point of view, since the ability to predict the wintertime atmospheric circulation anomalies over the North Atlantic well ahead in time will have significant benefits for North American and European countries. In contrast to the tropics, for the mid latitudes the predictive skill of many forecasting systems at the seasonal time scale has been shown to be low to moderate. The recent findings are promising in this regard, suggesting that better forecasts are possible, provided that key components of the climate system are initialized realistically and the coupled models are able to simulate adequately the dominant processes and teleconnections associated with low-frequency variability. It is shown that a multisystem approach has unprecedented high predictive skill for the NAO and AO, probably largely due to increasing the ensemble size and partly due to increasing model diversity. Predicting successfully the winter mean NAO does not ensure that the respective climate anomalies are also well predicted. The NAO has a strong impact on Europe and North America, yet it only explains part of the interannual and low-frequency variability over these areas. Here it is shown with a number of different diagnostics that the high predictive skill for the NAO/AO indeed translates to more accurate predictions of temperature, surface pressure, and precipitation in the areas of influence of this teleconnection. © 2017 American Meteorological Society.
News Article | November 10, 2016
Climate change is one of the most pressing concerns of the 21st century. But when it comes to tackling climate change, a new study from Cogent Economics & Finance exploring the benefits of carbon flux monitoring is a timely reminder that setting targets is just the beginning. Now that the Paris Agreement has decreased the level of carbon emissions deemed acceptable, the need for a decarbonised energy sector is greater than ever. Although technology exists to monitor actual carbon fluxes globally, the systems currently used to do it are expensive at a time when public financial resources are stretched. In pursuit of this, clean technologies must be supported while fossil fuels are penalised, yet uncertainty in the price of carbon makes it difficult to set caps and impose financial penalties. It also makes it challenging to create a stable, socially desirable investment environment that fosters carbon-neutral technologies. To that end, this article investigates the many ways in which better observation of actual carbon fluxes can aid environmental policy, economic investment and informed decision-making. The study found that better monitoring systems could bring significant cost savings and other benefits, thereby encouraging investors and paving the way for achieving ambitious climate change targets. The study is the result of a collaboration of researchers from several institutes, namely the International Institute for Systems Analysis, the Mercator Research Institute on Global Commons and Climate Change, The Inversion Lab, Lund University, Fondazione Eni Enrico Mattei, Comenius University, Lviv Polytechnic National University, the Euro-Mediterranean Center on Climate Change (CMCC Foundation) and the Max-Planck-Institute for Biogeochemistry. The authors come from a variety of academic backgrounds, exploring topics in economics, mathematics, remote sensing, forestry, physics, biogeochemistry, integrated assessment of climate change and climate change mitigation.
PubMed | CMCC, University of Bologna, Italian National Institute of Geophysics and Volcanology and European Commission - Joint Research Center Ispra
Type: | Journal: The Science of the total environment | Year: 2016
Heat waves represent one of the most significant climatic stressors for ecosystems, economies and societies. A main topic of debate is whether they have increased or not in intensity and/or their duration due to the observed climate change. Firstly, this is because of the lack of reliable long-term daily temperature data at the global scale; secondly, because of the intermittent nature of such phenomena. Long datasets are required to produce a reliable and meaningful assessment. In this study, we provide a global estimate of heat wave magnitudes based on the three most appropriate datasets currently available, derived from models and observations (i.e. the 20th Century Reanalyses from NOAA and ECMWF), spanning the last century and before. The magnitude of the heat waves is calculated by means of the Heat Wave Magnitude Index daily (HWMId), taking into account both duration and amplitude. We compare the magnitude of the most severe heat waves occurred across different regions of the world and we discuss the decadal variability of the larger events since the 1850s. We concentrate our analysis from 1901 onwards, where all datasets overlap. Our results agree with other studies focusing on heat waves that have occurred in the recent decades, but using different data. In addition, we found that the percentage of global area covered by heat wave exceeding a given magnitude has increased almost three times, in the last decades, with respect to that measured in the early 20th century. Finally, we discuss the specific implications of the heat waves on the river runoff generated in the Alps, for which comparatively long datasets exist, affecting the water quality and availability in a significant portion of the European region in summer.
News Article | August 24, 2016
Late in cell division, duplicated chromosomes are pulled apart by a structure called the mitotic spindle. This process, called mitosis, is beautiful to watch, but fraught with danger: errors can produce daughter cells that have unequal genomes, setting the stage for cell death or cancer. The cell goes to great lengths to avoid these mistakes, and uppermost among its safety mechanisms is the spindle assembly checkpoint1 (SAC) — a regulatory system that prevents the cell from attempting to separate chromosomes if they are not properly attached to the spindle. The key protein components of the SAC were first reported in the summer of 1991 (refs 2,3). Precisely 25 years on, two papers (Alfieri et al.4 on page 431 and Yamaguchi et al.5 in Molecular Cell) describe in near-atomic detail how the SAC prevents chromosome separation. The central protagonist in this story is the anaphase-promoting complex/cyclosome (APC/C)6. This enzyme attaches the protein ubiquitin to specific substrate proteins, tagging them for destruction and thereby unleashing chromosome separation and the completion of mitosis. A key APC/C target, for instance, is the protein securin, which inhibits a protease enzyme that cuts the proteins holding duplicated chromosomes together. Substrate binding to the APC/C depends on a protein called Cdc20 (Fig. 1a), which binds the APC/C during mitosis and recruits substrates through interactions with short linear amino-acid sequences in the targets called degrons. How does the SAC block chromosome separation? Chromosomes that are not correctly attached to the spindle produce a mitotic checkpoint complex (MCC), which contains three SAC components (Mad2, BubR1 and Bub3) and Cdc20. The MCC binds and inhibits Cdc20-bound APC/C (APC/CCdc20), forming a large complex called APC/CMCC that contains two copies of Cdc20 (refs 1,6,7). Alfieri et al. and Yamaguchi et al. use cryo-electron microscopy to unveil high-resolution structures of this giant assembly. A structure of this size and complexity, backed up by 25 years of genetic, biochemical and structural analysis, contains an overwhelming amount of information for the aficionado, but its chief value lies in the precise description of the mechanisms by which the MCC inhibits APC/CCdc20. Both studies show that the MCC interacts with the front face of the APC/C, next to the pre-bound Cdc20 subunit (Fig. 1b). The BubR1 subunit acts as a pseudosubstrate inhibitor — it contains two copies of each of the three major degron sequences, and wraps around the two Cdc20 subunits to occupy all degron-binding sites on both, thereby blocking substrate binding to the APC/C. It is hard to find a more striking illustration of the power of short linear motifs such as degrons in cell regulation. In addition, the MCC prevents binding between the APC/C and the E2 coenzyme, which normally provides ubiquitin for transfer to target proteins. The new structures also offer a potential explanation for previous evidence that some proteins are ubiquitinated by APC/CCdc20 even when the complex is bound to the MCC. For example, the APC/C substrate proteins cyclin A and Nek2A are degraded early in mitosis, when the SAC is active6. Similarly, the Cdc20 subunit of the MCC is tagged with ubiquitin by the APC/C, promoting MCC turnover1. How does APC/CMCC modify these proteins in spite of the mechanisms suppressing its activity? In addition to the 'closed' conformation described above, the two studies reveal a less-common 'open' state in which the MCC is shifted to allow binding of E2. This transient state enables ubiquitination of Cdc20 by APC/CMCC, and might also explain the ubiquitination of cyclin A and Nek2A, which bind APC/CCdc20 not only at sites blocked by BubR1, but also at other sites (ref. 6). With the open structure in hand, we are in a good position to unravel these mechanisms. It will also be important to explore the dynamics of the shift between conformational states, and how other regulatory proteins influence these dynamics. These APC/CMCC structures come close on the heels of a structural study8 by Zhang et al. that addressed another major question in APC/C regulation. It is known9 that the mitotic protein-kinase enzyme Cdk1–cyclin B phosphorylates the APC/C to promote its activation by Cdc20. APC/CCdc20 then triggers cyclin B degradation to inactivate Cdk1. This negative feedback is thought to be the basis for the oscillator that drives the rise and fall of Cdk1 activity during the cell cycle9, but much about this scheme has been unclear. How does phosphorylation activate APC/CCdc20? How is APC/C activation delayed to allow Cdk1 activity to rise in early mitosis — even in cell types in which the SAC is not present? Zhang and colleagues' analysis, together with recent biochemical studies10, 11, yields valuable clues. Phosphorylation of a loop region in the Apc3 subunit of the APC/C provides docking sites for a phosphate-binding subunit of Cdk1–cyclin B. Docking of Cdk1–cyclin B enhances its activity towards suboptimal phosphorylation sites in a loop on a different APC/C subunit, Apc1. This disordered loop contains a short 'autoinhibitory' segment that occupies a Cdc20-binding site on the APC/C, but phosphorylation displaces the segment, allowing Cdc20 to bind and activate the APC/C. The slow, multistep nature of this process provides a plausible mechanism for introducing a delay between Cdk1 activation and APC/CCdc20 activation, as is required for negative feedback to produce a robust oscillator9, 12. We have not heard the last word on APC/C phosphoregulation. The APC/C contains many phosphorylation sites in addition to those described above8, 10, 11, leaving open the possibility of other regulatory mechanisms, or connections between phosphorylation and the SAC. Another issue also remains unresolved. After mitosis, Cdc20 is degraded and the APC/C interacts with the Cdc20-related protein Cdh1. Cdh1 interacts with the site occupied by the autoinhibitory segment of Apc1, and yet phosphorylation of this segment is not required for Cdh1 binding. How does Cdh1 bind this region? Cdh1 might be a better competitor than Cdc20 for binding at this site8, or there might be other mechanisms at play. Finally, the Apc1 loop and autoinhibitory segment are poorly evolutionarily conserved outside vertebrates, raising questions about APC/C regulation in other species. Fortunately, addressing these and related problems has just become much easier, now that we have a strong structural foundation on which to base future experiments. We are a major step closer to understanding the remarkable robustness of mitosis.
News Article | November 28, 2016
Driven by the technology’s lower deployment costs and spectrum availability, TD-LTE refers to Time-division Long-Term Evolution. It is a 4G telecommunications technology and standard created by coalition of international companies. TD-LTE offers higher downlink and uplink rate as compared with LTE-FDD. The telecommunication industry experienced multiple key TD-LTE network deployments over the past three years namely Sprint in the US, Bharti Airtel in India and SoftBank in Japan. Softbank network is the most complex commercial TD-LTE network in the world at present. Through its commercial network deployment, TD-LTE networks have been effectively authenticated within all network settings for commercial launch. In Tokyo, Japan, in spite of the urban overcrowding and complex networking scenario, TD-LTE vendors have came-up with a micro-cellular solution, having station distance of 100-200 meters. It has effectively resolved the problems associated with coverage in a dense urban space and complex networking environment. Over 60 operators are dedicated to deploy TD-LTE networks across the world such as Huawei Technologies Co. Ltd., ZTE and Nokia Networks B.V. among others. Additionally, all prominent device OEMs, counting smartphone leaders Apple Inc. and Samsung group, have launched TD-LTE compatible devices commercially. Major number of these devices supports both TDD and FDD modes of operation over broad frequency spectrum. Additionally, the recent launch of TD-LTE network by China Mobile Communications Corporation (CMCC) is expected to allow the TD-LTE ecosystem to reach a significant level of economy of scale by boosting device and infrastructure investments in TD-LTE technology. Additionally, this TD-LTE network by CMCC is expected to be comprised of 500,000 base stations in operation till the end of 2014. Key driving forces for TD-LTE ecosystem market growth comprises of flexible uplink and downlink capacity associated with TD-LTE, interoperability with LTE-FDD, cheaper hardware cost and smooth transition from TD-SCDMA and WiMax. Major challenges restraining the TD-LTE ecosystem market growth include lower coverage as compared with LTE-FDD, use of guard periods and discontinuous reception. The market is segmented by TD-LTE devices, application sectors and services. TD-LTE devices include embedded cards, personal computers, consumer gadgets, USB dongles, notebooks, routers, smartphones and tablets. Application sectors include large and small enterprises, healthcare, retail, personal and education. Services include downlink biased services, uplink biased services and specific scenario services. Downlink biased services include video on demand, video sharing, music and sound streaming, location services, mobile advertisement and broadband services. Uplink biased services include security video surveillance, broadcaster reporting system, live traffic monitoring, medical monitoring, logistics tracking, and device positioning system. Specific scenario services comprises of enterprise access network, radio backhaul transmission replacement, household fixed broadband replacement and other broadband access services in resorts and festivals.
News Article | August 24, 2016
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.
Fierli F.,CNR Institute of Neuroscience |
Orlandi E.,CNR Institute of Neuroscience |
Law K.S.,University of Versailles |
Cagnazzo C.,CMCC |
And 7 more authors.
Atmospheric Chemistry and Physics | Year: 2011
We present the analysis of the impact of convection on the composition of the tropical tropopause layer region (TTL) in West-Africa during the AMMA-SCOUT campaign. Geophysica M55 aircraft observations of water vapor, ozone, aerosol and CO2 during August 2006 show perturbed values at altitudes ranging from 14 km to 17 km (above the main convective outflow) and satellite data indicates that air detrainment is likely to have originated from convective cloud east of the flights. Simulations of the BOLAM mesoscale model, nudged with infrared radiance temperatures, are used to estimate the convective impact in the upper troposphere and to assess the fraction of air processed by convection. The analysis shows that BOLAM correctly reproduces the location and the vertical structure of convective outflow. Model-aided analysis indicates that convection can influence the composition of the upper troposphere above the level of main outflow for an event of deep convection close to the observation site. Model analysis also shows that deep convection occurring in the entire Sahelian transect (up to 2000 km E of the measurement area) has a non negligible role in determining TTL composition. © 2011 Author(s).
Borges G.,Graduate School Information Engineering |
Proceedings - Frontiers in Education Conference, FIE | Year: 2015
The information and communication technologies have become increasingly present in education, either as support for classroom learning, whether in distance learning. Among these technologies, software known as Learning Management Systems - LMS are used for better student-teacher communication and especially for providing instructional materials, activities, assessments and other resources to provide collaborative activities. Despite the large number of LMS' systems available nowadays, these environments and its tools are not always useful in the teaching-learning process. Moreover, every individual possesses a different personal Learning Style (LS), or, in other words, absorbs, processes, and transforms information into knowledge in different ways. When using these differences to recommend Learning Objects (LOs), we allow students access to educational resources that are more adequate to their teaching-learning processes. This article presents a system that utilizes a recommendation technique based on utility, or usefulness, to recommend LOs, stemming from three aspects: the subject the one wishes to learn, one's personal preferences and one's LS. At the end of this article the results of the experiment will be described, which demonstrate the importance of this approach, as well as future projects. © 2014 IEEE.