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To understand the fundamental mechanism behind acid dissolution, Zhang et al. from the Institute for Chemical Research at Kyoto University encapsulated HF, as well as HF•H O and H O within a C fullerene. They found that in order to force the molecules into the open fullerene cavity, the molecules required "pushing from the outside" using high pressure conditions, and "pulling from the inside" via molecular interactions between HF and H O. They were able to identify how hydrogen bonding occurred between these two molecules. Their work appears in Science Advances. Prior work by Zhang et al. showed that the C fullerene could be opened in a three-step process that involved the addition of a pyridazine derivative either to the alpha or beta bonds on the C . This created a 13-member ring opening that formed slightly different compounds, denoted by α-13mem and β-13mem. Dehydration of both compounds resulted in a 16-member ring opening. The ring could be closed again via hydrolysis and a two-step process. β-16mem was large enough to capture H O, but α-16mem was not. Given these results from previous studies, for the current study, Zhang et al. used α-16mem to try to encapsulate HF. Instead, they found three different possibilities within the fullerenes: HF@C , (HF•H O)@C , and H O@C . Their reaction conditions required high pressure (9000 atm) to "push" the guest molecule into the α-16mem cavity. Time-dependent studies showed that HF filled the cavity first, followed by H O•HF, and then H O. Notably, the open cage did not entrap H O when HF was not present, indicating that the interaction between H O and HF prompted H O encapsulation. Further studies showed that HF is "pulling" H O into the cavity while the high pressure environment "pushes" it into the cavity. This process allowed the authors to study the interaction between H O and HF within a confined environment using 1H NMR. NMR analysis showed that that the (H O•HF)@C was down-shifted from H O@C and HF@C , which indicated hydrogen bonding. Furthermore, shift and coupling values indicated that oxygen was acting as the hydrogen-bond acceptor. Using single-crystal x-ray diffraction, Zhang et al. demonstrated the structure of the (HF•H O)@C , and report the first x-ray structure for doubly encapsulated C . These analyses and experimental studies confirmed that the H+ ion in HF forms a linear hydrogen bond with the O in H O. Additionally, compared to theoretical calculations of free H O and HF, the studies of the encapsulated molecules revealed close contact with hydrogen and oxygen that may be characteristic of H3O+•F-. The C fullerene derivative provides an excellent nanoenvironment for studying isolated chemical species, something that has not been available to chemists in the past. This isolated environment allowed the authors to investigate the interactions of two compounds without interference from the surrounding environment and provided important insights into a ubiquitous chemical process. More information: Rui Zhang et al. Isolation of the simplest hydrated acid, Science Advances (2017). DOI: 10.1126/sciadv.1602833 Abstract Dissociation of an acid molecule in aqueous media is one of the most fundamental solvation processes but its details remain poorly understood at the distinct molecular level. Conducting high-pressure treatments of an open-cage fullerene C70 derivative with hydrogen fluoride (HF) in the presence of H2O, we achieved an unprecedented encapsulation of H2O·HF and H2O. Restoration of the opening yielded the endohedral C70s, that is, (H2O·HF)@C70, H2O@C70, and HF@C70 in macroscopic scales. Putting an H2O·HF complex into the fullerene cage was a crucial step, and it would proceed by the synergistic effects of "pushing from outside" and "pulling from inside." The structure of the H2O·HF was unambiguously determined by single crystal x-ray diffraction analysis. The nuclear magnetic resonance measurements revealed the formation of a hydrogen bond between the H2O and HF molecules without proton transfer even at 140°C.


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

Your spit may hold a clue to future brain health. Investigators at the Beaumont Research Institute, part of Beaumont Health in Michigan, are hopeful that their study involving small molecules in saliva will help identify those at risk of developing Alzheimer's disease -- a neurologic condition predicted to reach epidemic proportions worldwide by 2050. Their study, "Diagnostic Biomarkers of Alzheimer's Disease as Identified in Saliva using 1H NMR-Based Metabolomics" was published in the Journal of Alzheimer's Disease. The study exemplifies the quest by scientists to combat Alzheimer's disease, a degenerative brain disorder with no cure and few reliable diagnostic tests. In the United States, Alzheimer's is a health epidemic affecting more than 5 million Americans. Investigators seek to develop valid and reliable biomarkers, diagnosing the disease in its earliest stages before brain damage occurs and dementia begins. Researcher Stewart Graham, Ph.D., said, "We used metabolomics, a newer technique to study molecules involved in metabolism. Our goal was to find unique patterns of molecules in the saliva of our study participants that could be used to diagnose Alzheimer's disease in the earliest stages, when treatment is considered most effective. Presently, therapies for Alzheimer's are initiated only after a patient is diagnosed and treatments offer modest benefits." Metabolomics is used in medicine and biology for the study of living organisms. It measures large numbers of naturally occurring small molecules, called metabolites, present in the blood, saliva and tissues. The pattern or fingerprint of metabolites in the biological sample can be used to learn about the health of the organism. "Our team's study demonstrates the potential for using metabolomics and saliva for the early diagnosis of Alzheimer's disease," explained Dr. Graham. "Given the ease and convenience of collecting saliva, the development of accurate and sensitive biomarkers would be ideal for screening those at greatest risk of developing Alzheimer's. In fact, unlike blood or cerebrospinal fluid, saliva is one of the most noninvasive means of getting cellular samples and it's also inexpensive." The study participants included 29 adults in three groups: mild cognitive impairment, Alzheimer's disease and a control group. After specimens were collected, the researchers positively identified and accurately quantified 57 metabolites. Some of the observed variances in the biomarkers were significant. From their data, they were able to make predictions as to those at most risk of developing Alzheimer's. Said Dr. Graham, "Worldwide, the development of valid and reliable biomarkers for Alzheimer's disease is considered the No. 1 priority for most national dementia strategies. It's a necessary first step to design prevention and early-intervention research studies." As Americans age, the number of people affected by Alzheimer's is rising dramatically. According to the Alzheimer's Association, by 2050, it's estimated the number of Americans living with Alzheimer's disease will triple to about 15-16 million. Alzheimer's disease is a type of dementia affecting a person's ability to think, communicate and function. It greatly impacts their relationships, their independence and lifestyle. The condition's toll not only affects millions of Americans, but in 2017, it could cost the nation $259 billion.


News Article | May 16, 2017
Site: www.sciencedaily.com

The immune system cells known as T cells play a central role in the body's ability to fight infections and cancer. For decades, however, details of the molecular signaling process that leads to T cell activation have remained a mystery. Now, a team of scientists at UC Santa Cruz and the National Institutes of Health has obtained the first glimpse of the molecular mechanism by which recognition of an antigen (such as a viral protein) by the T cell receptor triggers the first steps leading to an immune response. The new findings, published May 16 in Nature Communications, implicate changes in the molecular structure of the T cell receptor that propagate from the antigen recognition site on the outside of the cell to a signaling site inside the cell. The activation signal then triggers a complex "signaling cascade" within the T cell, leading to a range of possible responses by the cell. "This provides the first hint of the mechanism that triggers that signaling cascade," said Nikolaos Sgourakis, assistant professor of chemistry and biochemistry at UC Santa Cruz and co-senior author of the paper. "We don't yet have a full reconstruction of the signaling system, but for the first time we can see the process, and we have a technique to study it in more detail." The key technique used in this study is nuclear magnetic resonance (NMR), which uses the same principles as medical MRI scans to study molecular structures in a test tube. Sgourakis and his colleagues analyzed a molecular complex involving a T cell receptor and an HIV protein bound to a molecule of the major histocompatibility complex (MHC). The ability to probe such a large molecular complex with NMR was a breakthrough in this study, he said. In a viral infection, MHC molecules present pieces of viral proteins (antigens) on the surface of an infected cell, where they can be recognized by T cells and trigger an immune response. Abnormal proteins produced by cancer cells give rise to tumor antigens, which are also presented on the cell surface by MHC molecules and can be recognized by T cells. T cell activation can lead to recruitment of other immune cells, proliferation and differentiation of T cells, and direct killing of infected or cancerous cells. The exact nature of the response depends on the antigen and on other signals received by the T cell. "Different peptides or antigens give different outcomes. Now we can study those differences, and perhaps we will be able to predict which are the most efficient tumor peptides for triggering T cell activation," Sgourakis said. "The fundamental question of how T cell signaling works is vital for cancer immunotherapy. This is basic research, but there could be important applications down the road."


News Article | May 17, 2017
Site: onlinelibrary.wiley.com

Single-molecular electronics is a potential solution to nanoscale electronic devices. While simple functional single-molecule devices such as diodes, switches, and wires are well studied, complex single-molecular systems with multiple functional units are rarely investigated. Here, a single-molecule AND logic gate is constructed from a proton-switchable edge-on gated pyridinoparacyclophane unit with a light-switchable diarylethene unit. The AND gate can be controlled orthogonally by light and protonation and produce desired electrical output at room temperature. The AND gate shows high conductivity when treated with UV light and in the neutral state, and low conductivity when treated either with visible light or acid. A conductance difference of 7.3 is observed for the switching from the highest conducting state to second-highest conducting state and a conductance ratio of 94 is observed between the most and least conducting states. The orthogonality of the two stimuli is further demonstrated by UV–vis, NMR, and density function theory calculations. This is a demonstration of concept of constructing a complex single-molecule electronic device from two coupled functional units.


LONDON--(BUSINESS WIRE)--According to the latest market study released by Technavio, the global superconducting magnets market is expected to reach USD 3.50 billion by 2021, growing at a CAGR of almost 1% during the forecast period. This research report titled ‘Global Superconducting Magnets Market 2017-2021’ provides an in-depth analysis of the market in terms of revenue and emerging market trends. This market research report also includes up to date analysis and forecasts for various market segments and all geographical regions. The market for superconducting magnets is mainly driven by the demand for medical devices. Superconducting magnets have applications in MRI, a medical technique for clinical diagnosis, and are used in molecular biology as well as drug discovery and development. These magnets can also be used for curing fatal diseases, such as cancer in vital organs, when coupled with NMR technology. In the geographical side, APAC was the leading region in superconducting magnets market in 2016, and the region is projected to be the fastest-growing market for superconducting magnets. The escalating demand for medical devices in this region will propel the growth of the global superconducting magnets market during the forecast period. Looking for more information on this market? Request a free sample report Technavio’s sample reports are free of charge and contain multiple sections of the report including the market size and forecast, drivers, challenges, trends, and more. Technavio’s hardware and semiconductor research analysts categorize the global superconducting magnets market into the following segments by the application. They are: The top three application segments for the global superconducting magnets market are discussed below: The medical devices and equipment segment of the global superconducting magnets market will grow at a CAGR of over 3% during the forecast period. The MRIs comprises an MRI scanner that is used for morphological imaging, while the positron emission tomography (PET) scanner is used for functional imaging. Philips Healthcare and Siemens Healthcare developed a hybrid imaging modality of PET/MRI, known as Siemens Biograph mMR. This device was introduced in the US by Siemens in June 2011. In January 2011, the company earned the Conformité Européene (CE) mark approval to market the device in Europe. According to Sunil Kumar Singh, a lead semiconductor equipment research analyst from Technavio, “The combination of PET and MRI has a promising future for detecting and containing cancer as it reduces the number of isotopes required, which leads to cost saving during the imaging process. Manufacturers are attracting customers by carrying out improvements in this hybrid device to offer enhanced image quality, reduced noise, and increased sensitivity.” Superconducting magnets are used in mass spectrometers like the Fourier transform mass spectrometer (FTMS), which requires a high magnetic field, huge homogenous volume, and a long-term field stability offered by superconducting magnets. Thus, the demand for superconducting magnets will progress during the forecast period due to the growth of mass spectrometers. “Advances in technology and innovation will reduce the overall cost of product analysis. It will provide new opportunities for the spectroscopy market expand and prosper, and the introduction of smartphone portable spectroscopy will contribute to the demand for spectroscopy during the forecast period,” says Sunil. The transportation segment of the global superconducting magnets market is expected to grow at a CAGR of almost 3% during the forecast period. Electric locomotive engines are mainly preferred as they reduce traffic congestion caused by large freight trucks on the roads. They diminish the dependence on petroleum-based fuel, and they are an effective and non-polluting means of freight transport. Governments of emerging countries, such as India, are increasingly undertaking rail electrification for introducing high-speed electric trains. However, only routes having enough traffic would be electrified due to the prohibitive costs of electrification. The market for electric locomotive engines is expected to experience a gradual increase from 2021. The top vendors highlighted by Technavio’s research analysts in this report are: Become a Technavio Insights member and access all three of these reports for a fraction of their original cost. As a Technavio Insights member, you will have immediate access to new reports as they’re published in addition to all 6,000+ existing reports covering segments like embedded systems, human machine interface, and displays. This subscription nets you thousands in savings, while staying connected to Technavio’s constant transforming research library, helping you make informed business decisions more efficiently. Technavio is a leading global technology research and advisory company. The company develops over 2000 pieces of research every year, covering more than 500 technologies across 80 countries. Technavio has about 300 analysts globally who specialize in customized consulting and business research assignments across the latest leading edge technologies. Technavio analysts employ primary as well as secondary research techniques to ascertain the size and vendor landscape in a range of markets. Analysts obtain information using a combination of bottom-up and top-down approaches, besides using in-house market modeling tools and proprietary databases. They corroborate this data with the data obtained from various market participants and stakeholders across the value chain, including vendors, service providers, distributors, re-sellers, and end-users. If you are interested in more information, please contact our media team at media@technavio.com.


Your spit may hold a clue to future brain health. Investigators at the Beaumont Research Institute, part of Beaumont Health in Michigan, are hopeful that their study involving small molecules in saliva will help identify those at risk of developing Alzheimer's disease - a neurologic condition predicted to reach epidemic proportions worldwide by 2050. Their study, "Diagnostic Biomarkers of Alzheimer's Disease as Identified in Saliva using 1H NMR-Based Metabolomics" was published in the Journal of Alzheimer's Disease 58(2) on May 16. The study exemplifies the quest by scientists to combat Alzheimer's disease, a degenerative brain disorder with no cure and few reliable diagnostic tests. In the United States, Alzheimer's is a health epidemic affecting more than 5 million Americans. Investigators seek to develop valid and reliable biomarkers, diagnosing the disease in its earliest stages before brain damage occurs and dementia begins. Researcher Stewart Graham, Ph.D., said, "We used metabolomics, a newer technique to study molecules involved in metabolism. Our goal was to find unique patterns of molecules in the saliva of our study participants that could be used to diagnose Alzheimer's disease in the earliest stages, when treatment is considered most effective. Presently, therapies for Alzheimer's are initiated only after a patient is diagnosed and treatments offer modest benefits." Metabolomics is used in medicine and biology for the study of living organisms. It measures large numbers of naturally occurring small molecules, called metabolites, present in the blood, saliva and tissues. The pattern or fingerprint of metabolites in the biological sample can be used to learn about the health of the organism. "Our team's study demonstrates the potential for using metabolomics and saliva for the early diagnosis of Alzheimer's disease," explained Dr. Graham. "Given the ease and convenience of collecting saliva, the development of accurate and sensitive biomarkers would be ideal for screening those at greatest risk of developing Alzheimer's. In fact, unlike blood or cerebrospinal fluid, saliva is one of the most noninvasive means of getting cellular samples and it's also inexpensive." The study participants included 29 adults in three groups: mild cognitive impairment, Alzheimer's disease and a control group. After specimens were collected, the researchers positively identified and accurately quantified 57 metabolites. Some of the observed variances in the biomarkers were significant. From their data, they were able to make predictions as to those at most risk of developing Alzheimer's. Said Dr. Graham, "Worldwide, the development of valid and reliable biomarkers for Alzheimer's disease is considered the No. 1 priority for most national dementia strategies. It's a necessary first step to design prevention and early-intervention research studies." As Americans age, the number of people affected by Alzheimer's is rising dramatically. According to the Alzheimer's Association, by 2050, it's estimated the number of Americans living with Alzheimer's disease will triple to about 15-16 million. Alzheimer's disease is a type of dementia affecting a person's ability to think, communicate and function. It greatly impacts their relationships, their independence and lifestyle. The condition's toll not only affects millions of Americans, but in 2017, it could cost the nation $259 billion. The Beaumont Research Institute study was partly funded by the Fred A. and Barbara M. Erb Family Foundation. The eight investigators are now seeking additional funding to conduct a larger, three-year study with significantly more participants to validate the pilot study. Seven of the researchers are with the Beaumont Research Institute; Oakland University William Beaumont School of Medicine; and one is with the University of Alberta in Edmonton, Canada. ABOUT THE JOURNAL OF ALZHEIMER'S DISEASE (JAD) The Journal of Alzheimer's Disease is an international multidisciplinary journal to facilitate progress in understanding the etiology, pathogenesis, epidemiology, genetics, behavior, treatment and psychology of Alzheimer's disease. The journal publishes research reports, reviews, short communications, book reviews, and letters-to-the-editor. Groundbreaking research that has appeared in the journal includes novel therapeutic targets, mechanisms of disease and clinical trial outcomes. The Journal of Alzheimer's Disease has an Impact Factor of 4.151 according to Thomson Reuters' 2014 Journal Citation Reports. The Journal is published by IOS Press.


News Article | May 19, 2017
Site: www.eurekalert.org

Aggregation of the amyloid-beta peptide (Aβ) in the brain is strongly associated with Alzheimer´s disease (AD). This process is highly heterogeneous and transient in nature, thus hindering identification of the exact molecular form of Aβ responsible for the neurotoxicity observed in this disease. Therefore, characterizing Aβ aggregation is of utmost importance to make headway in the field of AD. Nuclear magnetic resonance spectroscopy (NMR), a technique used to obtain structural information, holds great potential to achieve this goal, as it could contribute to determining the structure of Aβ aggregate forms. However, it requires large amounts of peptide, as well as isotopic labels that are introduced through the Aβ peptide production process. In this article, we report a new and straightforward production protocol to obtain the Aβ most strongly linked to AD, the so-called Aβ42 form, with the required labels for NMR experiments. Specifically, we describe an inexpensive strategy through which to obtain [U-15N]- and [U-2H,13C,15N]-labeled Aβ42. Notably, this approach does not require reversed phase high performance liquid chromatography (RP-HPLC), a costly and time-consuming purification technique widely used in previously reported Aβ production protocols. Instead, all the purification steps required in our production protocol can be performed with the fast protein liquid chromatography system (FPLC), which is widely available. The peptides that we obtained are of high purity and have the required isotope labeling to support NMR-based structural studies.Therefore, we conclude that this strategy offers a simpler and inexpensive approach to obtain isotopically labeled Aβ42 than previously described methods, thereby paving the way for NMR-based Aβ structural studies. For more information, please visit: http://www.


News Article | May 22, 2017
Site: www.businesswire.com

SANTA CLARA, Calif.--(BUSINESS WIRE)--Agilent Technologies, Inc. (NYSE: A) today reported revenue of $1.10 billion, up 8 percent year over year (up 9 percent on a core basis(2)) for the second fiscal quarter ended April 30, 2017. Second-quarter GAAP net income was $164 million, or $0.50 per share. Last year’s second-quarter GAAP net income was $91 million, or $0.28 per share. During the second quarter, Agilent had intangible amortization of $31 million, acquisition and integration costs of $7 million, and $2 million in other costs. Excluding these items and a tax benefit of $17 million, Agilent reported second-quarter non-GAAP net income of $187 million, or $0.58 per share(1). “The Agilent team delivered another excellent quarter,” said Mike McMullen, Agilent President and CEO. “Both revenue and earnings per share exceeded the high range of guidance. We saw a strong pick up in the Chemical and Energy business after modest gains last quarter, and strong growth in Pharma and Europe also contributed to the upside.” “We continue to deliver on our long-term focus of driving above market growth, expanding operating margins, and deploying capital in a balanced manner,” he added. “Looking ahead, we are confident in our company’s prospects, and we are raising our full-year core revenue growth and earnings expectations.” Second-quarter revenue of $523 million from Agilent’s Life Sciences and Applied Markets Group (LSAG) grew 6 percent year over year (up 6 percent on a core basis(2)), with double-digit growth in chemical and energy, pharma and environmental markets. LSAG’s operating margin for the quarter was 21.1 percent. Second-quarter revenue of $378 million from Agilent CrossLab Group (ACG) grew 9 percent year over year (up 10 percent on a core basis(2)). Both services and consumables continued to see solid growth across all geographies. ACG’s operating margin for the quarter was 21.6 percent. Second-quarter revenue of $201 million from Agilent’s Diagnostics and Genomics Group (DGG) grew 13 percent year over year (up 13 percent on a core basis(2)) led by pharma and diagnostic and clinical end-markets. DGG’s operating margin for the quarter was 24.2 percent. Agilent expects third-quarter 2017 revenue in the range of $1.06 billion to $1.08 billion. Third-quarter non-GAAP earnings are expected to be in the range of $0.49 to $0.51 per share(3). For fiscal year 2017, Agilent expects revenue of $4.36 billion to $4.38 billion and non-GAAP earnings of $2.15 to $2.21 per share(3). The guidance is based on April 28, 2017 currency exchange rates. Agilent Technologies, Inc. (NYSE: A), a global leader in life sciences, diagnostics and applied chemical markets, is the premier laboratory partner for a better world. Agilent works with customers in more than 100 countries, providing instruments, software, services and consumables for the entire laboratory workflow. Agilent generated revenue of $4.20 billion in fiscal 2016. The company employs about 13,000 people worldwide. Information about Agilent is available at www.agilent.com. Agilent’s management will present more details about its second-quarter FY2017 financial results on a conference call with investors today at 1:30 p.m. PT. This event will be webcast live in listen-only mode. Listeners may log on at www.investor.agilent.com and select “Q2 2017 Agilent Technologies Inc. Earnings Conference Call” in the “News & Events Calendar of Events” section. The webcast will remain available on the company’s website for 90 days. Additional information regarding financial results can be found at www.investor.agilent.com by selecting “Financial Results” in the “Financial Information” section. A telephone replay of the conference call will be available at approximately 4:30 p.m. PST today through May 29, 2017 by dialing +1 800-585-8367 (or +1 404-537-3406 from outside the United States) and entering pass code 8574751. This news release contains forward-looking statements as defined in the Securities Exchange Act of 1934 and is subject to the safe harbors created therein. The forward-looking statements contained herein include, but are not limited to, information regarding Agilent’s future revenue, earnings and profitability; planned new products; market trends; the future demand for the company’s products and services; customer expectations; and revenue and non-GAAP earnings guidance for the third quarter and full fiscal year 2017. These forward-looking statements involve risks and uncertainties that could cause Agilent’s results to differ materially from management’s current expectations. Such risks and uncertainties include, but are not limited to, unforeseen changes in the strength of our customers’ businesses; unforeseen changes in the demand for current and new products, technologies, and services; unforeseen changes in the currency markets; customer purchasing decisions and timing, and the risk that we are not able to realize the savings expected from integration and restructuring activities. In addition, other risks that Agilent faces in running its operations include the ability to execute successfully through business cycles; the ability to meet and achieve the benefits of its cost-reduction goals and otherwise successfully adapt its cost structures to continuing changes in business conditions; ongoing competitive, pricing and gross-margin pressures; the risk that our cost-cutting initiatives will impair our ability to develop products and remain competitive and to operate effectively; the impact of geopolitical uncertainties and global economic conditions on our operations, our markets and our ability to conduct business; the ability to improve asset performance to adapt to changes in demand; the ability of our supply chain to adapt to changes in demand; the ability to successfully introduce new products at the right time, price and mix; the ability of Agilent to successfully integrate recent acquisitions; the ability of Agilent to successfully comply with certain complex regulations; and other risks detailed in Agilent’s filings with the Securities and Exchange Commission, including our quarterly report on Form 10-Q for the quarter ended Jan. 31, 2017. Forward-looking statements are based on the beliefs and assumptions of Agilent’s management and on currently available information. Agilent undertakes no responsibility to publicly update or revise any forward-looking statement. (1) Non-GAAP net income and non-GAAP earnings per share primarily excludes the impacts of acquisition and integration costs, transformation initiatives, business exit and divestiture costs, non-cash intangibles amortization, and pension settlement and curtailment gains. We also exclude any tax benefits that are not directly related to ongoing operations and which are either isolated or is not expected to occur again with any regularity or predictability. A reconciliation between non-GAAP net income and GAAP net income is set forth on page 6 of the attached tables along with additional information regarding the use of this non-GAAP measure. (2) Core revenue growth excludes the impact of currency, the NMR business and acquisitions and divestitures within the past 12 months. Core revenue is a non-GAAP measure. A reconciliation between Q2 FY17 GAAP revenue and core revenue is set forth on page 8 of the attached tables along with additional information regarding the use of this non-GAAP measure. Core revenue growth as projected for full fiscal year 2017 excludes the impact of currency, the NMR business and acquisitions and divestitures within the past 12 months. Most of these exclude amounts that pertain to events that have not yet occurred and are not currently possible to estimate with a reasonable degree of accuracy and could differ materially. Therefore, no reconciliation to GAAP amounts has been provided. (3) Non-GAAP earnings per share as projected for Q3 FY17 and full fiscal year 2017 excludes primarily the future impact of acquisition and integration costs, pension settlement gain, and non-cash intangibles amortization. We also exclude any tax benefits that are not directly related to ongoing operations and which are either isolated or is not expected to occur again with any regularity or predictability. Most of these excluded amounts that pertain to events that have not yet occurred and are not currently possible to estimate with a reasonable degree of accuracy and could differ materially. Therefore, no reconciliation to GAAP amounts has been provided. Future amortization of intangibles is expected to be approximately $30 million per quarter. NOTE TO EDITORS: Further technology, corporate citizenship and executive news is available on the Agilent news site at www.agilent.com/go/news.


News Article | May 16, 2017
Site: www.eurekalert.org

Scientists obtain first glimpse of the molecular mechanism by which recognition of an antigen by the T cell receptor triggers the first steps leading to an immune response The immune system cells known as T cells play a central role in the body's ability to fight infections and cancer. For decades, however, details of the molecular signaling process that leads to T cell activation have remained a mystery. Now, a team of scientists at UC Santa Cruz and the National Institutes of Health has obtained the first glimpse of the molecular mechanism by which recognition of an antigen (such as a viral protein) by the T cell receptor triggers the first steps leading to an immune response. The new findings, published May 16 in Nature Communications, implicate changes in the molecular structure of the T cell receptor that propagate from the antigen recognition site on the outside of the cell to a signaling site inside the cell. The activation signal then triggers a complex "signaling cascade" within the T cell, leading to a range of possible responses by the cell. "This provides the first hint of the mechanism that triggers that signaling cascade," said Nikolaos Sgourakis, assistant professor of chemistry and biochemistry at UC Santa Cruz and co-senior author of the paper. "We don't yet have a full reconstruction of the signaling system, but for the first time we can see the process, and we have a technique to study it in more detail." The key technique used in this study is nuclear magnetic resonance (NMR), which uses the same principles as medical MRI scans to study molecular structures in a test tube. Sgourakis and his colleagues analyzed a molecular complex involving a T cell receptor and an HIV protein bound to a molecule of the major histocompatibility complex (MHC). The ability to probe such a large molecular complex with NMR was a breakthrough in this study, he said. In a viral infection, MHC molecules present pieces of viral proteins (antigens) on the surface of an infected cell, where they can be recognized by T cells and trigger an immune response. Abnormal proteins produced by cancer cells give rise to tumor antigens, which are also presented on the cell surface by MHC molecules and can be recognized by T cells. T cell activation can lead to recruitment of other immune cells, proliferation and differentiation of T cells, and direct killing of infected or cancerous cells. The exact nature of the response depends on the antigen and on other signals received by the T cell. "Different peptides or antigens give different outcomes. Now we can study those differences, and perhaps we will be able to predict which are the most efficient tumor peptides for triggering T cell activation," Sgourakis said. "The fundamental question of how T cell signaling works is vital for cancer immunotherapy. This is basic research, but there could be important applications down the road." The authors of the paper include postdoctoral researchers Andrew McShan and Vlad Kumirov at UC Santa Cruz; first author Kannan Natarajan, co-senior author David Margulies, and Jiansheng Jiang, Rui Wang, Mulualem Tilahun, and Lisa Boyd at the National Institute of Allergy and Infectious Diseases; Huaying Zhao and Peter Schuck at the National Institute of Biomedical Imaging and Bioengineering; and Jinha Ying and Ad Bax at the National Institute of Diabetes and Digestive and Kidney Diseases. This work was supported by the National Institutes of Health.


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.

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