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News Article | May 14, 2017
Site: cleantechnica.com

The renewable energy spotlight shines brightest on the latest crop of supersized wind turbines and gigantic wind farms, but the small wind sector still has a chance to plant its flag on the US energy landscape. In the latest development, the Energy Department is readying another round of funding aimed at accelerating cutting edge technology for distributed wind energy. The funding program is relatively small compared to other federal energy programs — for example, a 2014 round in the same program totaled a measly $1.3 million — but a little goes a long way in the small turbine field. To be precise, “small” in this article is shorthand for the small to mid-sized turbine field. The Energy Department defines those categories by the size of the area swept by the turbine blades. Together, that would include turbines with a swept area of under 1,000 square meters. The Energy Department puts both small and mid-sized turbines in the distributed energy category (large turbines that generate power for a facility on site can also be considered part of the distributed energy sector). The new round of funding comes under the annual Distributed Wind Competitiveness Improvement Project of the National Renewable Energy Laboratory. The CIP mission is to help small turbine manufacturers keep pushing the technology window: The CIP helps manufacturers address barriers like outdated technology and increasing hardware costs, preventing the stagnation of the domestic market for distributed wind systems. The US domestic small wind sector has a lot of room to grow in the US. CIP aims to build up the export market as well: Maintaining U.S. market leadership, both domestically and internationally, requires next-generation wind turbine technologies… The problem is that R&D costs big bucks and small turbines are, well, small. That’s where your tax dollars go to work. CIP funding helps manufacturers offset their R&D costs, from concept to prototype testing and on to commercial specs. So, group hug for US taxpayers. The CIP program is relatively modest, but NREL has already racked up some success stories. Here’s a rundown from the lab: Northern Power Systems of Barre, Vermont, achieved a 15% increase in annual energy production by improving aerodynamics and lengthening the blade design for their new “C Series” blades, currently manufactured by two U.S. suppliers Pika Energy of Westbrook, Maine, demonstrated a novel injection-molding process for producing high-performance wind turbine blades, reducing blade costs by 90% compared to conventional hand-laid composite blades of comparable quality. Intergrid developed the first wide-band, gap-based inverter designed for wind applications of up to 25 kilowatt on a single-phase system. Wetzel Engineering developed a new approach to adding larger, pitch-controlled rotors to wind generators designed for stall control, resulting in a 28% increase in production and a 15% reduction in the turbine’s levelized cost of energy. If you were hoping to get in on this round of funding, unfortunately the May 9 deadline for submitting proposals has come and gone. But, there’s always next year. If that sounds a little optimistic considering the Trump budget ax, maybe so. However, unlike his counterpart over at EPA, Energy Secretary Rick Perry seems determined to keep his agency fully funded and firing away on all pistons when it comes to decarbonization. CleanTechnica periodically dips a toe in the small turbine waters, but the last such overview was back in 2013, so it’s well past time for an update. The small turbine market gets short shrift from some industry observers, but when you look at them in the context of distributed energy systems and microgrids the financials begin to make more sense. CleanTechnica has also noted that small wind turbines don’t have to be particularly efficient to add value to a facility. Ford, for example, has dabbled in onsite turbines at dealerships that display its logo, and the number of sports arenas festooned with turbines is growing. At last month’s 13th annual Small Wind Conference in Minnesota, NREL’s contributed a presentation titled “A Successful Small Wind Future: There Is Great Potential.” Image (cropped): via US Department of Energy. Check out our new 93-page EV report. Join us for an upcoming Cleantech Revolution Tour conference! Keep up to date with all the hottest cleantech news by subscribing to our (free) cleantech daily newsletter or weekly newsletter, or keep an eye on sector-specific news by getting our (also free) solar energy newsletter, electric vehicle newsletter, or wind energy newsletter.


News Article | May 15, 2017
Site: www.prnewswire.com

The Machine Network is a wireless network built by Ingenu, the creators of RPMA technology, exclusively for Machine-to-Machine (M2M) and Internet of Things (IoT) applications. Operating in the globally license-free 2.4 GHz ISM (Industrial, Scientific and Medical) band, RPMA is at the heart of secure wireless networks operating in over 20 countries around the world. u-blox has successfully ported the proven technology used in its first-generation RPMA modules and made it 65 percent smaller. SARA-S200 delivers significant cost and size optimizations ideal for application in the Smart Meter, Smart Building, Gas & Oil, Asset/Personnel Tracking and Agricultural industries. As the second generation module developed by u-blox in association with Ingenu, SARA-S200 builds on the companies' strategic partnership, which began in September 2016 and was quickly cemented with the introduction of u-blox's first module for the Machine Network, the NANO-S100. Andreas Thiel, Executive Vice President and Co-Founder of u-blox, commented: "Ingenu's patented RPMA technology offers significant advantages to a great variety of IoT applications, such as excellent in-building range and AES 128bit security encryption. With autonomous adaptation, the Machine Network delivers interference-free operation and the best performance and reliability in real-world applications. By bringing the SARA-S200 to market we can now deliver these outstanding features in the SARA footprint; our most popular form-factor." Measuring just 16 mm by 26 mm by 2.3 mm in a Land Grid Array (LGA) package, SARA-S200 provides an easy migration between other u-blox form factors and cellular technologies thanks to nested design. Its low-power design (50 µW average power consumption in sleep mode) means it can operate for 10 years or longer from a single battery. The modules will be manufactured in ISO/TS 16949 certified facilities with guaranteed Quality of Service (QoS) and a secure design that meets NERC CIP and other industry-mandated critical infrastructure requirements. The SARA-S200 has been designed for industrial applications and offers an extended operating temperature of -40°C to +85°C. John Horn, CEO of Ingenu, said: "Our strategic partnership with u-blox is further strengthened by the introduction of SARA-S200. It will give our partners the option of a fully compliant RPMA module with the same outstanding performance, in a significantly smaller format. The introduction of the SARA-S200 module will help enable a new generation of IoT applications." u-blox and Ingenu will showcase the SARA-S200 during Internet of Things World at the Santa Clara Convention Center, CA (May 16-18 2017). Please visit u-blox at the Ingenu booth 1704 to see the SARA-S200. The first prototypes will be available in June 2017. For more information about u-blox, please visit http://www.u-blox.com


News Article | May 8, 2017
Site: www.prweb.com

OATI has successfully migrated Data Center operations from its Plymouth Data Center to the new Microgrid Technology Center. This was a single site move and continues the years of OATI Active/Active Data Center architecture. To prepare for the migration, the OATI Move team harnessed expertise across many departments. Over the past 6 months, the team comprehensively tested and simulated migrations to predict how OATI systems would operate. These tests helped OATI anticipate and mitigate potential issues during the move. From April 04 to April 27, 2017, the Migration team unracked, moved, and reracked over 1,100 SAN, server, and network devices. Additionally, the team reconfigured over 600 web/App/database clusters, and synchronized databases across the North and South Campuses. The OATI Microgrid Technology Center is a microgrid that houses the new OATI South Campus Data Center, an advanced facility with expanded security and unsurpassed infrastructure designed to provide maximum redundancy. The Data Center has the added bonus of increased resiliency, including the ability to utilize renewable energy resources and run independent of utility power by utilizing on-site power generation. “We are very pleased with the efforts that went into making the migration a success,” says Sasan Mokhtari, President and CEO at OATI. “The new Data Center is one of the many tools we have to maintain the integrity and security of customer data.” OATI will host tours of the Microgrid Technology Center for OATI current and future customers at Spark 2017 OATI Energy Conference in Minneapolis, MN, from September 19 – 21, 2017. About OATI OATI provides innovative solutions that simplify, streamline, and empower the operational tasks required in today’s energy commerce and Smart Grid. With more than 1,800 customers, OATI successfully deploys large, complicated, and diverse mission-critical applications committed to industry standards and stringent NERC CIP guidelines. OATI (http://www.oati.com) is a leading provider of Smart Grid, Energy Trading and Risk Management, Transmission Scheduling, Congestion Management, Smart Grid, and Market Management products and services. OATI is headquartered in Minneapolis, Minnesota, with an office in Redwood City, California. For more information, please contact sales(at)oati.net.


ORANGE, Conn.--(BUSINESS WIRE)--Avangrid Renewables and Vineyard Wind, announced today that they have formed a strategic partnership to jointly develop a large scale wind energy project off the coast of Massachusetts. Avangrid Renewables is acquiring a 50 percent ownership interest in Vineyard Wind, the offshore wind energy developer that is part of the Copenhagen Infrastructure Partners (CIP) portfolio. Avangrid Renewables is a subsidiary of AVANGRID Inc. (NYSE:AGR), Iberdrola S.A. (Madrid:IBE), a worldwide leader in the energy industry with significant offshore wind holdings in Europe, owns 81.5% of the outstanding shares of AVANGRID common stock. The partnership of the two companies will bring extensive offshore wind expertise and substantial financial firepower to the Commonwealth’s initiative to build offshore wind projects in Massachusetts. Last summer, Massachusetts required utilities to procure 1,600 megawatts (MW) of clean, offshore wind energy within the next decade, setting off an intense competition among offshore wind developers in the region. When the 1,600 MW of generation capacity are completed, they will generate enough clean, homegrown energy to power the equivalent of more than 750,000 Massachusetts homes every year. Three companies to date have acquired lease rights to build projects off the coast, including Vineyard Wind. Vineyard Wind’s project area is about 15 miles south of Martha’s Vineyard. “This is a major strategic partnership that combines Avangrid Renewables’ US onshore wind development capabilities with Copenhagen Infrastructure Partners and Iberdrola’s European offshore wind expertise to give Vineyard Wind a significant advantage in building Massachusetts’ first offshore wind project,” said Lars Thaaning Pedersen, co-CEO of Copenhagen Offshore Partners, CIP’s offshore wind development company. Iberdrola holds offshore wind projects under development or construction in England, Germany & France. Avangrid Renewables recently won its first offshore wind lease auction in the US off the coast of North Carolina. Copenhagen Infrastructure Partners is providing investment and management for projects under construction in Germany and Scotland. Executives with extensive offshore wind experience from these European projects will now be joining Vineyard Wind’s local development team, based in New Bedford, Mass. “This partnership allows Vineyard Wind to leverage the financial firepower that both CIP and AVANGRID can bring to bear to develop and build this project,” Pedersen added. “AVANGRID’s interest in this project demonstrates that they recognize the strength of the local team, the local support we have, and the exciting potential for offshore wind in Massachusetts.” CIP and its executives have developed or invested in some of the biggest offshore wind projects in the world, including the 402 MW Veja Mate project off of Germany and the 588 MW Beatrice project off the coast of Scotland. CIP manages more than $4 billion in assets. It acquired Vineyard Wind in August 2016. “Our equal partnership in this effort demonstrates the strong commitment we’ve made to execute our growth strategy and expand our wind portfolio across the US,” said James P. Torgerson, AVANGRID, CEO. “When you look at our longer term position with the Kitty Hawk off-shore lease in NC and factor in this more immediate off-shore opportunity in Massachusetts, combined with our family of company’s extensive experience and knowledge, you recognize we are well positioned to grow this sector in the US.” Vineyard Wind plans to begin construction of its project in early 2020, in order to bring the economic development and clean energy benefits of offshore wind to Massachusetts as soon as possible. Vineyard Wind has also worked hard to establish long-term relationships and partnerships with the local community that will provide significant benefit to the project. AVANGRID, Inc. (NYSE:AGR) is a diversified energy and utility company with more than $31 billion in assets and operations in 27 states. The company owns regulated utilities and electricity generation assets through two primary lines of business, Avangrid Networks and Avangrid Renewables. Avangrid Networks is comprised of eight electric and natural gas utilities, serving approximately 3.2 million customers in New York and New England. Avangrid Renewables operates 6.5 gigawatts of electricity capacity, primarily through wind power, in 22 states across the United States. AVANGRID employs approximately 6,800 people. For more information, visit www.avangrid.com. Avangrid Renewables, LLC is a subsidiary of AVANGRID, Inc. (NYSE:AGR) and part of the IBERDROLA Group. IBERDROLA, S.A., an energy pioneer with the largest renewable asset base of any company in the world, owns 81.5% of the outstanding shares of AVANGRID common stock. Avangrid Renewables, LLC is headquartered in Portland, Ore., and has more than $10 billion of operating assets totaling more than 6,000 MW of owned and controlled wind and solar generation in 22 U.S. states. Avangrid Renewables recently changed its legal name from Iberdrola Renewables, LLC. For more information, visit www.avangridren.com. Vineyard Wind, a portfolio company of funds of Copenhagen Infrastructure Partners (CIP), is an offshore wind development company, based in New Bedford, Mass., that is seeking to build the first large-scale offshore wind energy project in the US. Copenhagen Infrastructure Partners K/S (CIP) is a fund management company founded in 2012 by senior executives from the energy industry. CIP and its executives have developed some of the biggest offshore wind projects in the world, and CIP manages more than $4 billion in assets.


News Article | May 15, 2017
Site: www.prweb.com

The new HydroClaw is a versatile Clean-In-Place (CIP) static tank-washing nozzle that combines unsurpassed clog-resistance and vigorous rinsing action for more efficient cleaning. Triple the free passage of spray balls. It is ideal for fermentation and bright beer tanks up to 10 ft. in diameter found in the winery and brewing industries. Wineries and breweries typically have issues with spray balls clogging on stems, skins, seeds, grains, and hops; the HydroClaw tank washing nozzle is the breakthrough solution to this problem. The unique, patent-pending, clog-resistant design quickly cleans tanks and eliminates maintenance downtime associated with clogged spray balls. The HydroClaw is made from FDA compliant 316L stainless steel; the self-cleaning and self-draining design is ideal for use in food-grade and sanitary Clean-In-Place (CIP) applications. About BETE Fog Nozzle, Inc. Founded in 1950 on the invention of the spiral nozzle, BETE offers a full line of standard spray nozzles with a specialty in offering custom nozzles and products, including spray nozzle fabrications. Its world headquarters in Massachusetts is home to a spray laboratory, engineering group, investment casting foundry, machine shop, and welded fabrication group. For more information, or assistance with selecting the right nozzle for your operation, please contact: BETE Fog Nozzle, Inc. 50 Greenfield Street Greenfield, MA 01301 USA


Critical Infrastructure Protection and Resilience, Europe (CIPRE) organised by Torch Marketing and KNM Media, took place in The Hague, Netherlands on 9th-11th May 2017. -- Last week's CIPRE in The Hague once again brought together some of the leading figures from the European and world critical infrastructure protection community to discuss some challenges faced by agencies, operators and industry in ensuring that CNI is safeguarded. Immediately followed by the WannaCry ransomware cyber-attack on many CNI sites worldwide, these discussions couldn't be more important.Once again speaker after speaker emphasised the need for greater co-operation, dialogue and sharing between all stakeholders, across sectors and across borders, if we are to successfully ensure security and resilience.Conference Chairman and Chairman of the International Association of Critical Infrastructure Protection Professionals (IACIPP) John Donlon QPM said "CIPRE 2017 was a significant event where like-minded people had the opportunity to discuss areas of concern and to take away new ideas and initiatives. I was truly impressed by the quality of presenters, the broad range of topics addressed and the detailed discussions that took place.During CIPRE 2017 we had some excellent presentations by some distinguished and experienced people across the whole range of Infrastructure and Information issues. We were fortunate to have speakers from Government, Academia and Operators articulating detail on both current and developing areas of activity, not only within Europe but also on a global scale.The EU Commission provided detail on new programmes of activity clearly referencing European concerns around the escalation of Cyber activity and the need to continue to build Public Private Partnerships, and both these areas were significant themes throughout a conference which covered a wide range of topics from-Hybrid Warfare - Prioritisation Modelling - Regulations and Standards and even the impact of Cyber, Social Media and Fake News on elections."The recurring themes throughout the conference included:·         The importance of trust and collaboration·         Sharing of information and best practise across sectors and borders·         PPP's are fundamental to security and resilience·         Benefits of successful PPP's·         Identification of single points of failure and interdependency·         Need to understand the nature of vulnerabilities and to prioritise activity·         Speed of change within Cyber activity·         Balance across - Prevention - Detection and Reaction·         Need to learn from each other·         Insider ThreatFinally, John Donlon QPM challenged delegates to join the International Association of CIP Professionals and start sharing information via the IACIPP extranet www.iacipp.net and invited them to reconvene again later in the year for Critical Infrastructure Protection and Resilience, North America ( http://ciprna- expo.com/ )Florida, December 5-72017 and Critical Infrastructure Protection and Resilience, Europe (http://www.cipre-expo.com/)in The Hague in 2018.Tony KinghamTel: +44(0)208 144 5934Mobile: +33(0)750 972 250Email:tony.kingham@knmmedia.comSkype: tkingham


Receive press releases from Torch Marketing Co Ltd: By Email Critical Infrastructure Protection and Resilience, Europe (CIPRE) organised by Torch Marketing and KNM Media, took place in The Hague, Netherlands on 9th-11th May 2017. London, United Kingdom, May 21, 2017 --( Once again speaker after speaker emphasised the need for greater co-operation, dialogue and sharing between all stakeholders, across sectors and across borders, if we are to successfully ensure security and resilience. Conference Chairman and Chairman of the International Association of Critical Infrastructure Protection Professionals (IACIPP) John Donlon QPM said, "CIPRE 2017 was a significant event where like-minded people had the opportunity to discuss areas of concern and to take away new ideas and initiatives. I was truly impressed by the quality of presenters, the broad range of topics addressed and the detailed discussions that took place. "During CIPRE 2017, we had some excellent presentations by some distinguished and experienced people across the whole range of Infrastructure and Information issues. We were fortunate to have speakers from Government, Academia and Operators articulating detail on both current and developing areas of activity, not only within Europe but also on a global scale. "The EU Commission provided detail on new programmes of activity clearly referencing European concerns around the escalation of Cyber activity and the need to continue to build Public Private Partnerships, and both these areas were significant themes throughout a conference which covered a wide range of topics from-Hybrid Warfare - Prioritisation Modelling - Regulations and Standards and even the impact of Cyber, Social Media and Fake News on elections." The recurring themes throughout the conference included: · The importance of trust and collaboration · Sharing of information and best practise across sectors and borders · PPP’s are fundamental to security and resilience · Benefits of successful PPP's · Identification of single points of failure and interdependency · Need to understand the nature of vulnerabilities and to prioritise activity · Speed of change within Cyber activity · Balance across - Prevention - Detection and Reaction · Need to learn from each other · Insider Threat Finally, John Donlon QPM challenged delegates to join the International Association of CIP Professionals and start sharing information via the IACIPP extranet www.iacipp.net and invited them to reconvene again later in the year for Critical Infrastructure Protection and Resilience, North America Florida, December 5-7th, 2017 and Critical Infrastructure Protection and Resilience, Europe in The Hague in 2018. Tony Kingham Tel: +44(0)208 144 5934 Mobile: +33(0)750 972 250 Email: tony.kingham@knmmedia.com Skype: tkingham London, United Kingdom, May 21, 2017 --( PR.com )-- Last week’s CIPRE in The Hague once again brought together some of the leading figures from the European and world critical infrastructure protection community to discuss some challenges faced by agencies, operators and industry in ensuring that CNI is safeguarded. Immediately followed by the WannaCry ransomware cyber-attack on many CNI sites worldwide, these discussions couldn’t be more important.Once again speaker after speaker emphasised the need for greater co-operation, dialogue and sharing between all stakeholders, across sectors and across borders, if we are to successfully ensure security and resilience.Conference Chairman and Chairman of the International Association of Critical Infrastructure Protection Professionals (IACIPP) John Donlon QPM said, "CIPRE 2017 was a significant event where like-minded people had the opportunity to discuss areas of concern and to take away new ideas and initiatives. I was truly impressed by the quality of presenters, the broad range of topics addressed and the detailed discussions that took place."During CIPRE 2017, we had some excellent presentations by some distinguished and experienced people across the whole range of Infrastructure and Information issues. We were fortunate to have speakers from Government, Academia and Operators articulating detail on both current and developing areas of activity, not only within Europe but also on a global scale."The EU Commission provided detail on new programmes of activity clearly referencing European concerns around the escalation of Cyber activity and the need to continue to build Public Private Partnerships, and both these areas were significant themes throughout a conference which covered a wide range of topics from-Hybrid Warfare - Prioritisation Modelling - Regulations and Standards and even the impact of Cyber, Social Media and Fake News on elections."The recurring themes throughout the conference included:· The importance of trust and collaboration· Sharing of information and best practise across sectors and borders· PPP’s are fundamental to security and resilience· Benefits of successful PPP's· Identification of single points of failure and interdependency· Need to understand the nature of vulnerabilities and to prioritise activity· Speed of change within Cyber activity· Balance across - Prevention - Detection and Reaction· Need to learn from each other· Insider ThreatFinally, John Donlon QPM challenged delegates to join the International Association of CIP Professionals and start sharing information via the IACIPP extranet www.iacipp.net and invited them to reconvene again later in the year for Critical Infrastructure Protection and Resilience, North America Florida, December 5-7th, 2017 and Critical Infrastructure Protection and Resilience, Europe in The Hague in 2018.Tony KinghamTel: +44(0)208 144 5934Mobile: +33(0)750 972 250Email: tony.kingham@knmmedia.comSkype: tkingham Click here to view the list of recent Press Releases from Torch Marketing Co Ltd


News Article | May 17, 2017
Site: www.nature.com

The 1.3-kb GCaMP6 coding region was PCR amplified from the pGP-CMV-GCaMP6s plasmid (Addgene)33. The amplified DNA was then inserted into the plant expression vector (the HBT-HA-NOS plasmid)45 to generate the HBT-GCaMP6-HA construct. The HBT-GCaMP6-HA construct was inserted into the binary vector pCB302 (ref. 46) to generate the HBT-GCaMP6-HA transgenic plants using the Agrobacterium (GV3101)-mediated floral-dip method47. Transgenic plants were selected by spraying with the herbicide BASTA. The construct expressing HY5–mCherry was used as a control for protoplast co-transfection and nucleus labelling, and was obtained from J.-G. Chen48. NLS-Td-Tomato was used as a control for protoplast co-transfection and nucleus labelling, and was obtained from X. Liu. NIR-LUC was constructed as described previously11. UBQ10-GUS is a control for protoplast co-transfection and internal control; all HBT-CPKac-Flag-NOS expression plasmids have been described previously23. To construct HBT-CPK-GFP-NOS, the coding regions of the CPK10, CPK30 and CPK32 cDNA were amplified and then cloned into the HBT-GFP-NOS plasmid23. HBT-CPK10(M141G)-Flag was generated by site-directed mutagenesis of the HBT-CPK10-Flag construct. To complement the cpk10 cpk30/+ mutant, a 5.5-kb DNA fragment including the promoter region (3 kb) and the coding region of CPK10 was amplified from genomic DNA, which was then cloned into the plasmid HBT-HA-NOS and mutagenized to generate pCPK10-CPK10(M141G)-HA-NOS. The pCPK10-CPK10(M141G)-HA-NOS construct was inserted into pCB302 and transformed into cpk10 cpk30/+ mutant plants using the Agrobacterium (GV3101)-mediated floral-dip method47. At the T generation, homozygous single-copy insertion lines were screened for the cpk10 cpk30 double mutant carrying pCPK10-CPK10(M141G)-HA-NOS to obtain the 3MBiP-inducible icpk10,30 double mutant, which rescued the embryo lethality of the cpk10 cpk30 double mutant. The 3MBiP-inducible icpk10,30,32 triple mutant expressing CPK10(M141G)-HA (designated icpk) was generated by genetic cross to cpk32 and confirmed by molecular analyses. To construct 35SΩ-NLP6-MYC or 35SΩ-NLP7-MYC in the pCB302 binary plasmid with hygromycin B selection, the β-glucuronidase (GUS) gene in the 35SΩ-GUS plasmid49 was replaced with the DNA fragment encoding the full-length NLP6 or NLP7 fused to 6 copies of the MYC epitope tag in the HBT-NLP6-MYC or HBT-NLP7-MYC plasmid12. The NLP6-MYC and NLP7-MYC transgenic plants were generated by Agrobacterium (GV3101)-mediated transformation by floral dip and hygromycin B resistance selection. To construct HBT-NLP7-HA and HBT-NLP7-GFP, the 2.9 kb coding region of the NLP7 cDNA was amplified and then cloned into the HBT-NOS plasmid. HBT-NLP7(S205A)-HA and HBT-NLP7(S205A)-GFP were generated by site-directed mutagenesis. A 7.9-kb genomic DNA fragment of NLP7 was cloned into the pUC plasmid and fused with GFP at the C terminus to generate pNLP7-NLP7-GFP. The pNLP7-NLP7(S205A)-GFP construct was generated by site-directed mutagenesis. pNLP7-NLP7-GFP or pNLP7-NLP7(S205A)-GFP was then inserted into pCB302 and introduced into nlp7-1 mutant plants using the Agrobacterium (GV3101)-mediated floral-dip47 method for complementation analyses. To construct UBQ10-CPK10KM-YN and UBQ10-NLP7-YC, the coding regions of CPK10(KM), NLP7, YFP-N terminus and YFP-C terminus were amplified by PCR and cloned into the UBQ10-GUS plasmid. To construct pET14-NLP7-N(1-581)-HIS and pET14-NLP7-N(S205A)-HIS for protein expression, the N-terminal coding region of NLP7 and NLP7(S205A) were amplified from HBT-NLP7-HA and HBT-NLP7(S205A)-HA. All constructs were verified by sequencing. The primers used for plasmid construction and site-directed mutagenesis are listed in Supplementary Table 3. Arabidopsis ecotype Columbia (Col-0) was used as the wild type. The cpk mutants were obtained from Arabidopsis Biological Resource Centre (ABRC)50. Homozygous T-DNA lines were identified using CPK gene-specific primers and T-DNA left-border primers. The gene-specific primers used are listed in the Supplementary Table 4. Double mutants were obtained by genetic crosses between cpk10-1, cpk30-1 and cpk32-1, and confirmed by PCR. For RT–PCR analysis of cpk single mutants, around 30 plants were grown on the Petri dish (150 mm × 15 mm) containing 100 ml of 1/2 × MS medium salt, 0.1% MES, 0.5% sucrose, 0.7% phytoagar under constant light (150 μmol m−2 s−1) at 23 °C for 7 days. Samples were collected for RT–PCR analysis. To generate icpk, cpk32-1 was crossed to icpk10,30. F plants were first screened for resistance to BASTA and then confirmed by genotyping (primers listed in Supplementary Table 4) for the homozygous cpk10 cpk30 cpk32 triple mutants. The homozygous icpk plants were isolated with no segregation for BASTA resistance in F plants. To demonstrate embryo lethality in cpk10 cpk30 mutants, cpk10 cpk30/+ plants were grown at a photoperiod of 16 h (light)/8 h (dark) (100 μmol m−2 s−1) at 23 °C/20 °C. Siliques were opened using forceps and needles under a dissecting microscope (Leica MZ 16F). Images were acquired and processed using IM software and Adobe Photoshop (Adobe). To obtain nitrate-free mesophyll protoplasts, around 16–20 plants were grown on a Petri dish (150 mm × 15 mm) containing 100 ml of nitrogen-free 1 × MS medium salt, 0.1% MES, 1% sucrose, 0.7% phytoagar, 2.5 mM ammonium succinate and 0.5 mM glutamine, pH 6 under a photoperiod of 12 h (light)/12 h (dark) (75 μmol m−2 s−1) at 23 °C/20 °C for 23–28 days. Mesophyll protoplasts were isolated from the second and the third pair of true leaves following the mesophyll protoplast isolation protocol46. To monitor plant growth without exogenous nitrogen source after germination, 30 seedlings were germinated and grown on a basal medium11 (10 mM KH PO /KH PO , 1 mM MgSO , 1 mM CaCl , 0.1 mM FeSO -EDTA, 50 μM H BO , 12 μM MnSO ·H O, 1 μM ZnCl , 1 μM CuSO ·5H O, 0.2 μM Na MoO ·2H O, 0.1% MES and 0.5% sucrose, pH 5.8) with 1% phytoagar under constant light (150 μmol m−2 s−1) at 23 °C for 4 days. Photos were taken at different days (days 1–4) using a dissecting microscope (Leica MZ 16F) with IM software. To analyse the specific plant growth programs in response to different exogenous nitrogen sources at different concentrations, seedlings were germinated and grown on basal medium for 4 days as described above, and then transferred to the basal medium with 0.1, 0.5, 1, 5 or 10 mM KNO , NH Cl, glutamine or KCl for an additional 1–7 days. For gene expression analyses with RT–qPCR and RNA-seq, 10 seedlings were germinated in one well of the 6-well tissue culture plate (Falcon) with 1 ml of the basal medium supplemented with 2.5 mM ammonium succinate as the sole nitrogen source. Plates were sealed with parafilm and placed on the shaker at 70 r.p.m. under constant light (45 μmol m−2 s−1) at 23 °C for 7 days. Before nitrate induction, seedlings were washed three times with 1 ml basal medium. Seedlings were treated in 1 ml of basal medium with KCl or KNO for 15 min. Seedlings were then harvested for RNA extraction with TRIzol (Thermo Fisher Scientific). To block the kinase activity of CPK10(M141G), seedlings were pre-treated with 10 μM 3MBiP in the basal medium for 2 min, and then treated with KCl or KNO for 15 min. For Ca2+ channel blockers and Ca2+ sensor inhibitors assays, seedlings were pre-treated with 2 mM LaCl , 2 mM GdCl , 250 μM W5 or 250 μM W7 in 1 ml of basal medium for 20 min, and then induced by 0.5 mM KCl or KNO for 15 min. To monitor root morphology, seedlings were germinated and grown on a basal medium supplemented with 2.5 mM ammonium succinate and 1% phytoagar under constant light (150 μmol m−2 s−1) at 23 °C for 3 days. Plants were then transferred to the basal medium supplemented with 1 μM 3MBiP and 5 mM KNO , 2.5 mM ammonium succinate, 5 mM KCl or 1 mM glutamine and grown for 5–8 days. After seedling transfer, 1 ml of 1 μM 3MBiP was added to the medium every 2 days. To monitor lateral root developmental stages, seedlings were monitored using a microscope (Leica DM5000B) with a 20× objective lens according to the protocol described previously41. To measure the primary and lateral root length, pictures were taken using a dissecting microscope (Leica MZ 16 F) with IM software and analysed by ImageJ. To compare the shoot phenotype, 8-day-old seedlings were cut above the root–shoot junction to measure the shoot fresh weight and acquire images. To analyse the cpk single-mutant phenotype, plants were germinated and grown on ammonium succinate medium for 3 days and then transferred to basal medium plates supplemented with 5 mM KNO for 6 days. To analyse double mutants in response to 3MBiP, plants were transferred to basal medium plates supplemented with 5 mM KNO and 1 μM 3MBiP for 6 days, and 3MBiP was reapplied every 2 days. Individual 9-day-old seedlings (n = 12) were collected to measure fresh weight and acquire images. To characterize the shoot phenotype of nlp7-1 and the complementation lines, around 20 seeds were germinated on the Petri dish (150 mm × 15 mm) containing 100 ml of nitrogen-free 1 × MS medium salt (Caisson), 0.1% MES, 1% sucrose, 0.7% phytoagar and 25 mM KNO medium pH 5.8 under a 16 h (light)/8 h (dark) photoperiod (100 μmol m−2 s−1) at 18 °C and grown for 21 days. The shoots were collected for measurement of fresh weight and acquisition of images. For analyses of the shoot phenotype in icpk, seeds were germinated and grown on the ammonium succinate basal medium plate for 3 days and then transferred to the same medium supplemented with 1 μM 3MBiP. The inhibitor 3MBiP (5 ml of 1 μM) was reapplied on the medium twice during the growth. Two transgenic seedlings expressing apoaequorin22 were germinated and grown in one well of a 12-well tissue culture plate (Falcon) with 0.5 ml of the basal medium supplemented with 2.5 mM ammonium succinate for 6 days. Individual plants were transferred to a luminometer cuvette filled with 100 μl of the reconstitution buffer (2 mM MES pH 5.7, 10 mM CaCl , and 10 μM native coelenterazine from NanoLight Technology) and incubated at room temperature in the dark overnight. The emission of photons was detected every second using the luminometer BD Monolight 3010. The measurement was initiated by injection of 100 μl 20 mM KCl, 20 mM KNO , 200 nM flg22 or ultrapure water into the cuvettes. Luminescence values were exported and processed using Microsoft Excel software. For Ca2+ imaging in protoplasts, mesophyll protoplasts (2 × 105) in 1 ml buffer were co-transfected with 70 μg HBT-GCaMP6 and 50 μg HBT-HY5-mCherry plasmid DNA. Transfected protoplasts were incubated in 5 ml of WI buffer45 for 4 h. Before time-lapse recording, a coverslip was placed on a 10-well chamber slide covering three-quarters of a well, and placed on the microscope stage. Mesophyll protoplasts co-expressing GCaMP6 and HY5–mCherry (2 × 104 protoplast cells) were spun down for 1 min at 100g. WI-Ca2+ buffer (WI buffer plus 4 mM CaCl ) (0.5 μl) with different stimuli (40 mM KCl, 40 mM KNO or 40 mM NH Cl) or 80 mM Ca2+ chelator (EGTA) were added into 1.5 μl of concentrated mesophyll protoplasts in WI buffer. The final concentration of each stimulus was 10 mM KCl, 10 mM KNO , 10 mM NH Cl or 20 mM EGTA in the solution. The stimulated protoplasts were immediately loaded onto the slide and imaged via the Leica AF software on a Leica DM5000B microscope with the 20× objective lens. The exposure time for GCaMP6 was set at 1 s and recorded every 2 s to generate 199 frames. The exposure time was set at 45 ms for the bright field and 1 s for the mCherry signal. The fluorescence intensity was determined with the region of interest (ROI) function for each protoplast. The intensity data were exported and processed using Microsoft Excel software. The images were exported and processed using Adobe Photoshop software. To make a video, individual images were cropped using Adobe Photoshop software and saved in JPEG format. The videos were generated using ImageJ with the cropped images. For Ca2+ imaging with the GCaMP6 transgenic seedling cotyledons, 5 seedlings were germinated in 1 well of a 6-well tissue culture plate (Falcon) with 1 ml of the basal medium supplemented with 2.5 mM ammonium succinate for 7 days. A chamber was made on microscope slides between two strips of the invisible tape (0.5 cm × 3 cm) and filled with 150 μl of the basal medium. A cotyledon of the 7-day-old seedling was cut in half using a razor blade and embedded in the medium. A thin layer of cotton was placed on top of the cotyledon to prevent moving. The coverslip was placed on the sample and fixed by another two strips of the invisible tape. The cotyledon was allowed to recover on the slide for 10 min. Confocal imaging was acquired using the Leica laser scanning confocal system (Leica TCS NT confocal microscope, SP1). The mesophyll cells in the cotyledon were targeted for Ca2+ imaging at the focal point. Basal medium (200 μl) with 10 mM KCl, 10 mM KNO or 20 mM EGTA was loaded along one edge of the coverslip. A Kimwipes tissue on the opposite edge was used to draw the buffer into the chamber. To record fluorescence images, the excitation was provided at 488 nm and images were collected at emission 515–550 nm. The scanning resolution was set at 1,024 × 1,024 pixels. Images were captured every 10 s and averaged from two frames. In total, 80 images were collected and processed using Adobe Photoshop software. A video was generated with collected images using the method described above. For Ca2+ imaging with the GCaMP6 transgenic seedling at the root tip and the elongated region of roots (around the middle region of the root), 10 seedlings were germinated and grown on the tissue culture plate (Falcon) with the basal medium and 1% phytoagar under constant light (150 μmol m−2 s−1) at 23 °C for 4 days. The images were obtained using Leica laser scanning confocal system as described above for cotyledon Ca2+ imaging. In total, 33 images were collected and processed using Adobe Photoshop software. A video was generated with collected images using the method described above. Time-course, specificity and dosage analyses of NIR-LUC activity in response to nitrate induction was carried out in mesophyll protoplasts (2 × 104 protoplasts in 100 μl) co-transfected with 10 μg NIR-LUC and 2 μg UBQ10-GUS (as the internal control) and incubated in WI buffer45 for 4 h, and then induced by 0.5 mM KCl, KNO , NH + or Gln or different concentrations of KNO for 2 h. For time-course analysis, the fold change is calculated relative to the value of KCl treatment at each time point. For the nitrate-sensitized functional genomic screen, nitrate-free mesophyll protoplasts (2 × 104 protoplasts in 100 μl) were co-transfected with 8 μg HBT-CPKac (constitutively active CPK) or a control vector, 10 μg NIR-LUC and 2 μg UBQ10-GUS plasmid DNA, and incubated for 4 h to allow CPKac protein expression. To investigate the functional relationship between CPK10ac and NLP7 in nitrate signalling, nitrate-free mesophyll protoplasts (4 × 104 protoplasts in 200 μl) were co-transfected with 8 μg NIR-LUC and 2 μg UBQ10-GUS plasmid DNA, as well as 5 μg HBT-CPK10ac, HBT-CPK10ac(KM) or a control vector, or HBT-NLP7 or HBT-NLP7(S205A) in different combinations supplemented with 5 μg control vector to reach a total of 20 μg per transfection reaction, and incubated for 4 h for protein expression. Protoplasts were then induced with 0.5 mM KCl or KNO for 2 h. The luciferase and GUS assay were carried out as described before45. The expression levels of NLP7–HA and CPK–Flag or CPK10ac–Flag in protoplasts were monitored by immunoblot with anti-HA-peroxidase (Roche, 11667475001; 1:2,000) and anti-Flag-HRP (Sigma, A8592; 1:2,000) antibodies, respectively. Expression vectors were transformed into Rosetta 2 (DE3) pLysS Competent Cells (Novagen). Cells were induced by 1 mM of IPTG when OD reached 0.6, and proteins were expressed at 18 °C for 18 h. Affinity purification was carried out using HisTrap columns (GE Healthcare) and the ÄKTA FPLC system. Purified proteins were buffer exchanged into PBS using PD-10 Desalting Columns (GE Healthcare), and then concentrated by Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-10 membrane (EMD Millipore). Around 106 protoplasts were incubated in WI buffer (5 ml) in Petri dishes (9 × 9 cm) for 4 h before induction with 10 mM KCl or KNO for 10 min. Protoplasts were harvested and lysed in 200 μl of extraction buffer: 150 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 mM EDTA, 1% Triton X-100, 1× protease inhibitor cocktail (Complete mini, Roche) and 1 mM DTT. The protein extract supernatant was obtained after centrifugation at 18,000g for 10 min at 4 °C. Total proteins (20 μg) were loaded on 8% SDS–PAGE embedded with or without 0.5 mg ml−1 histone type III-S (Sigma) as a general CPK phosphorylation substrate23. The gel was washed three times with washing buffer (25 mM Tris-HCl pH 7.5, 0.5 mM DTT, 5 mM NaF, 0.1 mM Na VO , 0.5 mg ml−1 BSA and 0.1% Triton X-100), and incubated for 20 h with three changes in the renaturation buffer (25 mM Tris-HCl pH 7.5, 0.5 mM DTT, 5 mM NaF and 0.1 mM Na VO ) at 4 °C. The gel was then incubated in the reaction buffer (25 mM Tris-HCl pH 7.5, 2 mM EDTA, 12 mM MgCl , 1 mM CaCl , 1 mM MnCl , 1 mM DTT and 0.1 mM Na VO ) with or without 20 mM EGTA at room temperature for 30 min. The kinase reaction was performed for 1 h in the reaction buffer supplemented with 25 μM cold ATP and 50 μCi [γ-32P]ATP with or without 20 mM EGTA. The reaction was stopped by extensive washes in the washing buffer (5% trichloroacetic acid and 1% sodium pyrophosphate) for 6 h. The protein kinase activity was detected on the dried gel using the Typhoon imaging system (GE Healthcare). 1-Isopropyl-3-(3-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (3MBiP) was synthesized using the same procedures as those for a close structural analogue, 3MB-PP1 (ref. 39), with comparable yields, except that iso-propylhydrazine was substituted for tert-butylhydrazine. 1H NMR (400 MHz, DMSO-d ) δ 8.12 (s, 1H), 7.15 (d, J = 7.6 Hz, 1H), 7.08 (s, 1H), 7.00 (t, J = 7.5 Hz, 2H), 4.96 (p, J = 6.7 Hz, 1H), 4.31 (s, 2H), 2.24 (s, 3H), 1.44 (d, J = 6.7 Hz, 6H). 13C NMR (100 MHz, DMSO-d ) δ 158.41, 155.78, 153.69, 143.12, 139.56, 137.85, 129.51, 128.79, 127.30, 125.87, 98.92, 48.18, 40.10, 33.70, 22.23, 21.54. ESI–MS calculated for C H N [M + H]+ is 282.2, found 282.7. For in vitro kinase assay with CPK10(M141G)–Flag or CPK10–Flag, 4 × 104 protoplasts expressing CPK10(M141G)–Flag or CPK10–Flag were lysed in 200 μl immunoprecipitation buffer that contained 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM DTT, 2 mM NaF, 2 mM Na VO , 1% Triton X-100 and 1× protease inhibitor cocktail (Complete mini, Roche). Protein extracts were incubated with 0.5 μg anti-Flag antibody (Sigma, F1804) at 4 °C for 2 h and an additional 1 h with protein G Sepharose beads (GE Healthcare). The immunoprecipitated kinase protein was washed three times with immunoprecipitation buffer and once with kinase buffer (20 mM Tris-HCl pH 7.5, 15 mM MgCl , 1 mM CaCl and 1 mM DTT). Kinase reactions were performed for 1 h in 25 μl kinase buffer containing 1 μg histone (Sigma H5505 or H4524), 50 μM cold ATP and 2 μCi [γ-32P]ATP. To block the CPK10(M141G)–Flag kinase activity, 1 μM 3MBiP or DMSO as a control was added in the 25 μl kinase buffer for 2 min before performing the kinase reaction. The reaction was stopped by adding SDS–PAGE loading buffer. After separation on a 12% SDS–PAGE gel, the protein kinase activity was detected on the dried gel using the Typhoon imaging system. For the in vitro kinase assay with CPK10(M141G)–HA isolated from icpk10,30 seedlings, 12 7-day-old seedlings grown in 2 wells of a 6-well-plate with 1 ml medium (0.5 × MS, 0.5% sucrose and 0.1% MES pH 5.7) were grounded in liquid nitrogen into powder and lysed in 200 μl of immunoprecipitation buffer. The CPK10(M141G)–HA protein was immunoprecipitated with the anti-HA antibody (Roche, 11666606001) and protein G Sepharose beads. In vitro kinase assay with CPK10(M141G)–HA proteins was carried out as described above. For the in vitro kinase assay with the subgroup III CPKs, Flag-tagged CPK7, CPK8, CPK10, CPK10(KM) (K92M, a kinase-dead mutation in the conserved ATP binding domain), CPK13, CPK30 and CPK32 were expressed in 105 protoplasts and purified with 1 μg anti-Flag antibody conjugated to protein G Sepharose beads as described above. CPK11–Flag from subgroup I was used as a negative control to demonstrate the specificity of NLP7 as a substrate for only subgroup III CPKs. NLP7–HIS (~1 μg) purified from Escherichia coli or histone type III-S (2 μg) was used as substrate in the in vitro kinase assay. Kinase reactions were performed for 1 h at 28 °C in 25 μl kinase buffer containing 5 μM cold ATP and 6 μCi [γ-32P]ATP, which greatly enhanced the CPK activity. To reduce the background caused by free [γ-32P]ATP in the gel, 50 μM cold ATP was added to the kinase reaction before sample loading in 10% (NLP7–HIS) or 12% (HIS) SDS–PAGE gel. To demonstrate that the kinase activities of CPK10, CPK30 and CPK32 were Ca2+-dependent, 4 × 104 (CPK10 or CPK10ac) or 105 (CPK30, CPK32, CPK30ac or CPK32ac) protoplasts expressing CPKs for 12 h instead of 6 h (to increase the yield of CPK proteins) were lysed in 200 μl (CPK10) or 400 μl (CPK30 or CPK32) of immunoprecipitation buffer. The CPK proteins were immunoprecipitated with anti-Flag antibody (0.5 μg for CPK10 or CPK10ac, and 2 μg for CPK30, CPK32, CPK30ac or CPK32ac) conjugated to protein G Sepharose beads. The immunoprecipitated CPKs were washed three times with immunoprecipitation buffer and twice with EGTA kinase buffer (20 mM Tris-HCl pH 7.5, 15 mM MgCl , 15 mM EGTA and 1 mM DTT). Kinase reactions were performed for 1 h at 28 °C in 25 μl kinase buffer or EGTA kinase buffer containing 5 μM cold ATP and 6 μCi [γ-32P]ATP and purified NLP7–HIS (~1 μg), NLP7-N (1–581 amino acids) (~0.8 μg), NLP7-N(S205A) (~0.8 μg), or histone type III-S (2 μg). After performing the kinase reaction, 50 μM cold ATP was added to reduce the background caused by free [γ-32P]ATP. The reaction was stopped by adding SDS–PAGE loading buffer. After separation on a 12% SDS–PAGE gel (histone type III-S) or 10% (NLP7–HIS or NLP7-N–HIS) SDS–PAGE gel, the protein kinase activity was detected on the dried gel using the Typhoon imaging system. Substrate was stained with InstantBlue Protein Stain (C.B.S. Scientific). The expression levels of CPK or CPKac proteins were monitored by immunoblot with anti-Flag-HRP (Sigma, A8592; 1:4,000) antibody. CPKac proteins without the Ca2+-binding EF-hand domains provided constitutive kinase activities that were insensitive to EGTA. The sensitivity of CPK10, CPK30 and CPK32 to EGTA in kinase assays demonstrated their functions as Ca2+ sensors in nitrate signalling, which was further supported by the lack of NLP7–HA phosphorylation and the nuclear retention of NLP7–GFP in icpk mutant cells. Importantly, NLP7(S205A) lost nitrate-induced phosphorylation, nuclear localization, NIR-LUC activation, and endogenous target gene activation in wild-type protoplasts and seedlings. RNA isolation, RT–PCR and RT–qPCR were carried out as described previously11. The primers used for RT–PCR and RT–qPCR are listed in Supplementary Table 5. TUB4 was used as a control in wild-type and cpk mutants. The relative gene expression was normalized to the expression of UBQ10. Triplicate biological samples were analysed with consistent results. We chose the early time point to minimize secondary target genes and the complexity that negative feedback would have introduced, including indirect effects from assimilation of nitrate and the subsequent activation of transcriptional repressors1, 3, 4, 8, 10, 13. Seven-day-old wild-type and icpk seedlings were pretreated with 10 μM 3MBiP for 2 min and then treated for 15 min with either 10 mM KCl or 10 mM KNO . Total RNA (0.5 μg) was used for preparing the library with the Illumina TruSeq RNA sample Prep Kit v2 according to the manufacturer’s guidelines with 9 different barcodes (triplicate biological samples). The libraries were sequenced for 50 cycles on an Illumina HiSeq 2500 rapid mode using two lanes of a flow cell. The sequencing was performed at MGH Next Generation Sequencing Core facility (Boston, USA). Fastq files, downloaded from the core facility, were used for data analysis. The quality of each sequencing library was assessed by examining fastq files with FastQC. Reads in the fastq file were first aligned to the Arabidopsis genome, TAIR10, using Tophat51. HTSeq52 was used to determine the reads per gene. Finally, DESeq2 (ref. 53) analysis was performed to determine differential expression54. For HTSeq-normalized counts in each sample, differentially expressed genes were determined for wild-type KNO versus wild-type KCl and icpk KNO versus wild-type KNO . The differential expression analysis in DESeq2 uses a generalized linear model of the form where counts K for gene i, sample j are modelled using a negative binomial (NB) distribution with fitted mean μ and a gene-specific dispersion parameter α . The fitted mean is composed of a sample-specific size factor s and a parameter q proportional to the expected true concentration of fragments for sample j. The coefficients β give the log fold changes for gene i for each column of the model matrix X. Results were imported into Microsoft Excel for filtering. To generate a list to minimize false positives of primary nitrate-responsive genes in the wild type, we applied a relatively high stringency, q ≤ 0.05 cut-off, followed by a log  ≤ −1 or ≥ 1 cut-off. To generate a heatmap, we performed agglomerative hierarchical clustering on genes with Gene Cluster 3.0 (ref. 55) using Correlation (uncentred) as the similarity metric and single linkage as the clustering method. Java Treeview56 was used to visualize the results of the clustering. To obtain a list of enriched gene functions, we used the Classification SuperViewer Tool on the BAR website (http://bar.utoronto.ca/ntools/cgi-bin/ntools_classification_superviewer.cgi) with the MapMan classification source option. Analyses of enriched functional categories with nitrate upregulated and downregulated genes were performed using the MapMan classification source option on the Classification SuperViewer Tool with manual annotation based on literature. The fold enrichment is calculated as follows: (number in class /number of total )/ (number in class / number of total ). The P value is calculated in Excel using a hypergeometric distribution test. The data in Extended Data Fig. 4c and d were sorted by fold enrichment with a P < 0.05 cut-off. For the biological duplicate RNA-seq experiments for identifying NLP7 target genes in the mesophyll protoplast transient expression system, 500 μg HBT-NLP7-HA, HBT-NLP7(S205A)-HA or control plasmid DNA was transfected into 106 protoplasts and incubated for 4.5 h. Total RNA (0.5 μg) was used to construct the libraries with six different barcodes (biological duplicate samples) as described above. The sequencing result was performed and analysed as described above. Differentially expressed genes were determined with DESeq2 on NLP7 versus Ctl (Control) and NLP7(S205A) versus Ctl. Results were imported into Microsoft Excel for filtering (log  ≥ 1 cut-off) and generating heatmaps. Transgenic seedlings expressing NLP6–MYC or NLP7–MYC were germinated and grown in basal medium containing 0.5 mM ammonium succinate as a sole nitrogen source (0.01% MES-KOH, pH 5.7) for 4 days at 23 °C under continuous light (60 μmol m−2 s−1). After replacement with fresh medium supplemented with 10 mM KCl or KNO , the seedlings were collected after incubation for 5, 10 or 30 min. To examine the effects of Ca2+ channel blockers and Ca2+ sensor inhibitors, the 4-day-old seedlings were placed in fresh basal medium supplemented with 2 mM LaCl , 2 mM GdCl , 250 μM W5 or 250 μM W7 for 20 min and induced by 10 mM KCl or KNO . The seedlings were weighed, frozen in liquid nitrogen and ground using a Multibeads Shocker (Yasui Kikai). The ground samples were suspended in 20 volume of 1× Laemmli sample buffer supplemented with twice the concentration of EDTA-free protease inhibitor cocktail (Roche) and heated at 95 °C for 30 s. Samples were then spun down and the supernatant was subjected to SDS–PAGE and immunoblotting with anti-MYC (Millipore, 05-419; 1:1,000) and anti-histone H3 (Abcam, ab1791; 1:5,000) antibodies. For calf intestinal alkaline phosphatase (CIP) treatment, proteins in 1.2-fold CIP buffer (60 mM Tris-HCl pH 8.0, 120 mM NaCl, 12 mM MgCl , 1.2 mM DTT, 2.4-fold concentration of EDTA-free Protease Inhibitor Cocktail) were mixed with CIP solution (New England Biolabs, M0290, 10 U μl−1) at a ratio of 5 (CIP buffer):1 (CIP solution) and incubated at 37 °C for 30 min. Heat-inactivated CIP was mixed as a control treatment. The reactions were stopped by adding an equal volume of 2× Laemmli sample buffer and heating at 95 °C for 30 s. To demonstrate that nitrate-induced NLP7 phosphorylation was abolished in icpk by protein mobility shift in SDS–PAGE, 4 × 104 protoplasts isolated from wild-type or icpk seedlings were transfected with 20 μg NLP7–HA or NLP7(S205A)–HA. To block CPK10(M141G) activity in icpk, 10 μM 3MBiP was added in the incubation buffer (WI) after transfection. After expressing protein for 4.5 h, protoplasts were induced by 10 mM KCl or KNO for 15 min. Protoplasts were spun down and re-suspended in 40 μl 1× Laemmli sample buffer. Samples (10 μl) were separated in a 6% SDS–PAGE resolving gel without a stacking gel layer. After transferring proteins to the PVDF membrane, the NLP7 (wild-type and S205A) proteins were detected with anti-HA-peroxidase (Roche, 11667475001; 1:2,000). RuBisCo was detected by an anti-rubisco antibody (Sigma, GW23153; 1:5,000) as a loading control. Transformation of T87 cell suspension culture derived from a seedling of A. thaliana L. (Heynh.) ecotype Columbia57 was conducted with the 35SΩ-NLP7-MYC construct in the pCB302 binary plasmid carrying the hygromycin B selection marker gene. Transformants mediated by Agrobacterium (GV3101) were selected on agar plates (JPL medium, 3 g l−1 gellun gum, 500 mg l−1 carbenicillin and 20 mg l−1 hygromycin), and the transformants were maintained in liquid JPL medium as described previously57. T87 cells expressing NLP7–MYC were incubated in nitrogen-free JPL liquid medium for 2 days, and then 10 mM KNO was added into the medium. After 30 min treatment, the T87 cells (approximately 4 g frozen weight) were frozen in liquid nitrogen and homogenized with Multi-beads Shocker (Yasui Kikai) in 10 ml of the buffer that contained 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% NP-40, 10% glycerol, 1× Complete Protease Inhibitor Cocktail and 1× PhosSTOP (Roche). Cell lysates obtained were incubated with anti-MYC antibodies crosslinked to Dynabeads (Invitrogen). Trapped proteins were eluted by 1× Laemmli sample buffer and separated by SDS–PAGE. Gel pieces containing NLP7–MYC were recovered and subjected to in-gel double digestion with trypsin (10 ng μl−1) and chymotrypsin (10 ng μl−1) (Promega). NanoLC–ESI-MS/MS analysis was performed as described previously58, 59 with minor modifications. To analyse NLP7 nuclear retention triggered by nitrate in protoplasts, nitrate-free mesophyll protoplasts (4 × 104 protoplasts in 200 μl) were co-transfected with 20 μg NLP7–GFP or NLP7(S205A)–GFP and 10 μg HBT-HY5-mCherry plasmid DNA and incubated for 6 h. Mesophyll protoplasts were spun down for 1 min at 100g. WI buffer with 10 mM KCl or KNO was added into mesophyll protoplasts for 30 min. The treated protoplasts were loaded onto slides and imaged with the 20× objective lens on a Leica DM5000B microscope operated with the Leica AF software. The images were collected and processed using Adobe Photoshop software. To analyse NLP7–GFP nuclear retention triggered by nitrate in transgenic lines, NLP7–GFP/nlp7-1 and NLP7(S205A)–GFP/nlp7-1 seedlings were germinated and grown on the basal medium supplemented with 2.5 mM ammonium succinate and with 1% phytoagar under constant light (150 μmol m−2 s−1) at 23 °C for 5 days. Plants were placed on the slide as described above and stimulated by 10 mM KNO . Confocal images were acquired as described for GCaMP6-based Ca2+ imaging in transgenic seedlings. To analyse CPK10, CPK30 and CPK32 nuclear localization in response to nitrate, nitrate-free mesophyll protoplasts (4 × 104 protoplasts in 200 μl) were co-transfected with 20 μg CPK10–GFP, CPK30–GFP or CPK32–GFP and 10 μg HBT-HY5-mCherry plasmid DNA and incubated for 12 h. Protoplasts were then treated with 10 mM KNO for 5 min. Confocal imaging was acquired using the Leica Application Suite X software on a Leica TCS SP8 (Leica) confocal microscope with the 40× objective lens. To obtain fluorescence images, the excitation was set to 489 nm (GFP) and 587 nm (mCherry), and images at emissions 508 nm (GFP) and 610 nm (mCherry) were collected. The scanning resolution was set to 1,024 × 1,024 pixels. The images were collected and processed using Adobe Photoshop software. To analyse NLP7–GFP nuclear retention in wild-type and icpk seedlings, nitrate-free mesophyll protoplasts (4 × 104 protoplasts in 200 μl) were co-transfected with 20 μg NLP7–GFP and 4 μg HBT-Td-Tomato plasmid DNA and incubated for 12–16 h. The transfected protoplasts were treated with inhibitor 10 μM 3MBiP 30 min before nitrate induction. Protoplasts were treated with 10 mM KNO for 15 min in the presence of 10 μM 3MBiP of WI buffer. The images were acquired as described above for the NLP7 nuclear retention in protoplasts. Nitrate-free mesophyll protoplasts (4 × 104 protoplasts in 200 μl) were co-transfected with 18 μg UBQ10-CPK10(KM)-YN, UBQ10-NLP7-YC, and 4 μg HBT-HY5-mCherry plasmid DNA, and incubated for 12–18 h. Protoplasts were then treated with 10 mM KNO for 2 h. Confocal images were acquired as described above for CPK localization in response to nitrate. The chosen sample sizes for all experiments were empirically determined by measuring the mean and s.d. for the sample population in pilot experiments, and then calculated (the 1-sample Z-test method, two-sided test) with the aim to obtain the expected mean of less than 25% significant difference with the alpha value ≤ 0.05 and the power of the test ≥ 0.80. For multiple comparisons, data were first subjected to one-way or two-way ANOVA, followed by Tukey’s multiple comparisons test to determine statistical significance. To compare two groups, a Student’s t-test was used instead. To compare wild-type and icpk lateral root development, data were categorized into two groups, and then subjected to a chi-square test, as indicated in the figure legends. Experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment. RNA-seq data are available at the Gene Expression Omnibus (GEO) under accession number GSE73437. The Source Data for blots, gels and histograms are provided in the Supplementary Information. All other data are available from the corresponding author upon reasonable request.


HERNDON, Va.--(BUSINESS WIRE)--Spring postsecondary overall enrollments fell by more than 272,000 students compared to a year ago, led by a decline of more than 244,000 students over the age of 24 and many were enrolled in a four-year, for-profit institution or a two-year public college, according to the Spring 2017 Current Term Enrollment Estimates report from the National Student Clearinghouse® Research Center™, the nation’s most trusted source for student record data. Enrollments declined in 39 states and increased in 12 states and the District of Columbia. A total of 18,071,004 student enrollments occurred in all institutions this spring, but 1.8 percent of those were the same students attending more than one institution at the same time. Unduplicated, the Research Center counted 17,740,912 unique students. “Institutions that cannot attract graduate students to compensate for declining numbers of undergraduates will continue to struggle in the coming years,” stated Doug Shapiro, Executive Research Director of the National Student Clearinghouse Research Center. “The spring 2017 numbers reinforce the trends that we saw in the fall term, and will likely continue: enrollments at community colleges and smaller non-profits declining, while four-year public colleges and larger privates hold steady.” The report features nationwide enrollment figures and trends for each state, as well as enrollment totals by undergraduate field of study at four-year and two-year institutions. Fields of study based on two-digit CIP Family codes from the National Center for Education Statistics. Published every May and December, Current Term Enrollment Estimates are based on postsecondary institutions actively submitting data to the Clearinghouse. These institutions account for nearly 97 percent of the nation’s Title IV, degree-granting enrollments. The data are highly current, because institutions make several data submissions per term. In addition, because the Clearinghouse receives data at the student level, an unduplicated headcount is reported, avoiding double-counting of students enrolled in more than one institution. The National Student Clearinghouse Research Center is the research arm of the National Student Clearinghouse. The Research Center collaborates with higher education institutions, states, school districts, high schools, and educational organizations as part of a national effort to better inform education leaders and policymakers. Through accurate longitudinal data outcomes reporting, the Research Center enables better educational policy decisions leading to improved student outcomes. To learn more, visit http://nscresearchcenter.org.


News Article | May 23, 2017
Site: www.businesswire.com

PHILADELPHIA--(BUSINESS WIRE)--Three individuals have been elected to the board of directors of Philadelphia-based National Disease Research Interchange (NDRI). Lending their experience and insight to the nation’s leading source of human organs, cells and tissues for research are Shawn Blackburn, CEO, YPrime, Malvern, PA; Sulayman Dib-Hajj, PhD, Senior Research Scientist, Yale School of Medicine and VA Connecticut Healthcare System, West Haven, CT; and Megan Kasimatis Singleton, JD, MBE, CIP, Assistant Dean and Director of the Human Research Protection Program, Johns Hopkins University School of Medicine, Baltimore, MD. Blackburn, Dib-Hajj and Singleton were elected on May 16 to three-year terms on the NDRI board. Mary J.C. Hendrix, PhD, Chair of the NDRI Board and President of Shepherd University shared that, “ Shawn Blackburn’s expertise in technology to support research, Dr. Dib-Hajj’s research accomplishments and experience utilizing human tissue for research and Megan Singleton’s knowledge and experience in law, bioethics and human subject protections will be invaluable to advancing the mission of NDRI.” Bill Leinweber, NDRI’s President and Chief Executive Officer, adds, “The caliber of our new board members speaks to the significant transformative work being advanced by our organization. We are honored to have these highly respected professionals commit their time and talents to the mission of NDRI.” Blackburn is a co-founder of YPrime, a clinical trial software company focused on creating innovative solutions for researchers running global clinical trials in all therapeutic areas. Under Blackburn’s leadership, YPrime has experienced exponential growth with offices in Pennsylvania and North Carolina and additional staff based in five countries. Dib-Hajj’s research has centered on the molecular basis of excitability disorders in humans including pain, with a focus on the role of voltage-gated sodium channels in the pathophysiology of these disorders, and as targets for new therapeutics. He has published more than 160 primary papers and reviews, and has established national and international collaborations with both academic and pharmaceutical groups. Dib-Hajj received his undergraduate educated from the American University of Beirut, Lebanon and his PhD from The Ohio State University. In her current role, Singleton is responsible for oversight and direction of the staff that support the seven Johns Hopkins Medicine Institutional Review Boards (IRBs). She earned her law degree from Temple University and Masters in bioethics from the University of Pennsylvania. She is a frequent presenter on a range of human research protection issues. The National Disease Research Interchange (NDRI) is the nation’s leading source of human tissues, cells and organs for scientific research. A not-for-profit 501 (c)(3) organization founded in 1980, NDRI is funded in part by the National Institutes of Health, public and private foundations and organizations, pharmaceutical and biotechnology corporations. NDRI is a 24/7 operation that partners with a nationwide network of over 130 tissue source sites (TSS), including organ procurement organizations (OPO), tissue banks, eye banks, and hospitals. The TSS, are distributed throughout the USA, in 45 states, with concentrations in major metropolitan areas on both the east and west coasts. Their wide geographic distribution allows NDRI to provide biospecimens from donor populations with diverse demographics and also facilitates the timely and efficient provision of fresh tissues directly to researchers across the country. By serving as the liaison between procurement sources and the research community, NDRI is uniquely positioned to support breakthrough advances and discoveries that can affect advances in the treatment and cure of human diseases.

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