News Article | May 16, 2017
Metal Fatigue Solutions (MFS), producer of the next generation of advanced Nondestructive Testing (NDT) technology systems for major civil and industrial infrastructure, today announced it is working with BAE Systems, UK (BAE). The MFS contract with BAE commenced in February, 2016 and will continue through July 2017. The two companies are working jointly with several universities to produce a unique structural monitoring system for use on combat-ready structures. MFS has deployed its Custom Solutions Division to meet the needs of this military grade project. "Our MFS team is executing precision development and testing work to meet the needs of BAE and their customer," said Sade Panahi, CEO of MFS. He went on to say, "We are pleased to be part of the BAE team of esteemed and trusted manufacturers. MFS is consistently exceeding all quality standards and timelines set forth in the contract and we look forward to completing this important project for BAE at the highest level of quality." The project is the culmination of a year-long competitive proposal process. BAE Systems provides some of the world's most advanced technology defence, aerospace and security solutions. It employs a skilled workforce of 82,500 people in over 40 countries. Working with customers and local partners, BAE products and services deliver military capability, protect people and national security, and keep critical information and infrastructure secure. (http://www.baesystems.com/en/home) Metal Fatigue Solutions (MFS) develops, manufactures and markets next-generation non-destructive testing (NDT) devices and systems that indicate the true status of fatigue damage in a metal component. The Company's customers include the State DOTs, Infrastructure Owners, the U.S. Federal Government, and private engineering firms (http://metal-fatigue-solutions.com.) Initially commercialized in 2005, MFS' flagship product is the industry leading Electrochemical Fatigue Sensor (EFS™), an instrument that detects microscopic growing fatigue cracks in metals. With seven patents, MFS owns the only NDT technology able to find growing cracks as minute as 0.01 inches – critical information that enables structural engineers to isolate and repair the more than 100,000 US steel bridges classified as structurally deficient or functionally obsolete by the Federal Highway Administration. EFS is also applicable to aerospace, ships, cranes, railways, power plants, nuclear facilities, chemical plants, mining equipment, piping systems and "heavy iron."
News Article | May 2, 2017
NEW YORK--(BUSINESS WIRE)--Barclays today announced the launch of the Shiller Barclays CAPE® Single Stock Index Family, a new addition to the successful series of equity sector indices designed jointly by Professor Robert Shiller and Barclays. Professor Shiller and Barclays began their collaboration in 2010, and in 2012 launched the Shiller Barclays CAPE® Sector Index Family. These indices use the Cyclically Adjusted Price Earnings® ratio (CAPE®), developed by Professor Robert Shiller and John Campbell in the 1980s, to identify long-term undervalued sectors in the US, Europe and Asia. The indices have been well-received by investors, with significant investment in products tracking them. The new index range also uses the CAPE® ratio to determine valuation, but rather than sectors applies this to single stocks of established companies across various regions. The new indices aim to provide an alternative to market capitalization weighted indices with a unique value bias based on the CAPE® Ratio and momentum. They will initially offer exposure to the United States and Eurozone regions with Japan and Asia ex-Japan to follow. In addition to the long-only Single Stock indices, there will be market-hedged Index versions, which hold a long position in the Shiller Barclays CAPE® US Single Stock Index and a beta-adjusted short position in the relevant benchmark index, aiming to provide a ‘market neutral value’ investment. This responds to investor interest in accessing the equity value risk premia. “We seek to invest in seasoned companies - Old Standbys - that also show good value by the CAPE® ratio. This is a new and different approach to value investing that is designed to try to come closer to its original motivation: find stocks that are well established and relatively forgotten, with a long history of earnings but underpriced in the market,” said Professor Robert Shiller. “Barclays is delighted to continue our long standing collaboration with renowned author and Yale economist Robert Shiller, producing indices which draw on some of his well-known investing concepts. This launch highlights Barclays’ commitment to bringing clients a full array of investable indices based on academic research,” said Ben Redmond, Director in EFS Solutions. “The Shiller Barclays CAPE® Single Stock Index Family will offer investors a new way to gain exposure to underpriced stocks. Knowledge of the equity value premia has existed since the 1930s, and we believe this innovative take on it will allow investors a new way to access attractively priced shares,” said David Haefliger, EFS Solutions. For more information please visit indices.barclays. This communication has been prepared by Barclays. “Barclays” means any entity within the Barclays Group of companies, where “Barclays Group” means Barclays Bank PLC, Barclays PLC and any of their subsidiaries, affiliates, ultimate holding company and any subsidiaries or affiliates of such holding company. BARCLAYS IS A FULL SERVICE INVESTMENT BANK. In the normal course of offering investment banking products and services to clients, Barclays may act in several capacities (including issuer, market maker and/or liquidity provider, underwriter, distributor, index sponsor, swap counterparty and calculation agent) simultaneously with respect to a product, giving rise to potential conflicts of interest which may impact the performance of a product. Barclays may at any time acquire, hold or dispose of long or short positions (including hedging and trading positions) and trade or otherwise effect transactions for their own account or the account of their customers in the products referred to herein which may impact the performance of a product. Barclays is not offering to sell or seeking offers to buy any product or enter into any transaction. Any offer or entry into any transaction requires Barclays’ subsequent formal agreement which will be subject to internal approvals and execution of binding transaction documents. Neither Barclays nor any of its directors, officers, employees, representatives or agents, accepts any liability whatsoever for any direct, indirect or consequential losses (in contract, tort or otherwise) arising from the use of this communication or its contents or reliance on the information contained herein, except to the extent this would be prohibited by law or regulation. Barclays is not responsible for information stated to be obtained or derived from third party sources or statistical services. All opinions and estimates are given as of the date hereof and are subject to change. The value of any investment may also fluctuate as a result of market changes. Barclays is not obliged to inform the recipients of this communication of any change to such opinions or estimates. Barclays offers premier investment banking products and services to its clients through Barclays Bank PLC. Barclays Bank PLC is authorised by the Prudential Regulation Authority and regulated by the Financial Conduct Authority and the Prudential Regulation Authority and is a member of the London Stock Exchange. Barclays Bank PLC is registered in England No. 1026167 with its registered office at 1 Churchill Place, London E14 5HP. The Shiller Barclays CAPE® Index Family has been developed in part by RSBB-I, LLC, the research principal of which is Robert J. Shiller. RSBB-I, LLC is not an investment advisor, and does not guarantee the accuracy or completeness of the Shiller Barclays CAPE® Index Family, or any data or methodology either included therein or upon which it is based. Neither RSBB-I, LLC nor Robert J. Shiller shall have any liability for any errors, omissions, or interruptions therein, and makes no warranties, express or implied, as to performance or results experienced by any party from the use of any information included therein or upon which it is based, and expressly disclaims all warranties of merchantability or fitness for a particular purpose with respect thereto, and shall not be liable for any claims or losses of any nature in connection with the use of such information, including but not limited to, lost profits or punitive or consequential damages, even if RSBB-I, LLC is advised of the possibility of same. Barclays is a transatlantic consumer, corporate and investment bank offering products and services across personal, corporate and investment banking, credit cards and wealth management, with a strong presence in our two home markets of the UK and the US. With over 325 years of history and expertise in banking, Barclays operates in over 40 countries and employs approximately 120,000 people. Barclays moves, lends, invests and protects money for customers and clients worldwide. For further information about Barclays, please visit our website home.barclays.
News Article | May 16, 2017
LAS VEGAS, NV / ACCESSWIRE / May 16, 2017 / Metal Fatigue Solutions (MFS), producer of the next generation of advanced Nondestructive Testing (NDT) technology systems for major civil and industrial infrastructure, today announced it is working with BAE Systems, UK (BAE). The MFS contract with BAE commenced in February, 2016 and will continue through July 2017. The two companies are working jointly with several universities to produce a unique structural monitoring system for use on combat-ready structures. MFS has deployed its Custom Solutions Division to meet the needs of this military grade project. "Our MFS team is executing precision development and testing work to meet the needs of BAE and their customer," said Sade Panahi, CEO of MFS. He went on to say, "We are pleased to be part of the BAE team of esteemed and trusted manufacturers. MFS is consistently exceeding all quality standards and timelines set forth in the contract and we look forward to completing this important project for BAE at the highest level of quality." The project is the culmination of a year-long competitive proposal process. BAE Systems provides some of the world's most advanced technology defence, aerospace and security solutions. It employs a skilled workforce of 82,500 people in over 40 countries. Working with customers and local partners, BAE products and services deliver military capability, protect people and national security, and keep critical information and infrastructure secure. (http://www.baesystems.com/en/home) Metal Fatigue Solutions (MFS) develops, manufactures and markets next-generation non-destructive testing (NDT) devices and systems that indicate the true status of fatigue damage in a metal component. The Company's customers include the State DOTs, Infrastructure Owners, the U.S. Federal Government, and private engineering firms (http://metal-fatigue-solutions.com.) Initially commercialized in 2005, MFS' flagship product is the industry leading Electrochemical Fatigue Sensor (EFS™), an instrument that detects microscopic growing fatigue cracks in metals. With seven patents, MFS owns the only NDT technology able to find growing cracks as minute as 0.01 inches – critical information that enables structural engineers to isolate and repair the more than 100,000 US steel bridges classified as structurally deficient or functionally obsolete by the Federal Highway Administration. EFS is also applicable to aerospace, ships, cranes, railways, power plants, nuclear facilities, chemical plants, mining equipment, piping systems and "heavy iron."
News Article | May 17, 2017
"Xeltis' initial trial results to date support ETR's promise to enable the body's natural restoration of heart valve function," said Martin B. Leon, M.D., director at the Center for Interventional Vascular Therapy at Columbia University Medical Center and New York-Presbyterian Hospital, one of the speakers at the session. "This innovative treatment approach has the potential to reduce complications, re-interventions and healthcare costs, while improving quality of life for patients with heart valve disease. This would represent a major leap forward in heart valve therapy." With ETR, the patient's natural healing system develops tissue that pervades Xeltis' heart valve, forming a new, natural and fully functional valve within it. As ETR occurs, Xeltis' implants are gradually absorbed by the body. ETR is enabled by the porous structure of Xeltis' heart valves, which are made of bioabsorbable polymers, based on Nobel prize awarded science. RestoreX, Xeltis' new technology platform, is the world's first polymer-based technology designed to enable natural restoration of heart valve function. Preview of First Data from Xeltis Aortic Valve Pre-Clinical Program The first study results from Xeltis preclinical aortic valve program have been presented today during the same plenary session. The promising data showed good hemodynamic performance and fully functional valves in vivo six months after implantation. "We are delighted by the high level of interest in our solution," said Martijn Cox, Ph.D., chief technology officer and co-founder of Xeltis, "and, more importantly, that the new aortic valve data presented today further validate our vision to naturally restore heart valves using ETR." The first feasibility clinical trial for Xeltis' pulmonary valve, Xplore-I, is underway in Europe and Asia. Patient enrollment was completed in December 2016. In January, the U.S. Food and Drug Administration (FDA) approved an Investigational Device Exemption (IDE) for Early Feasibility Study (EFS) to implant Xeltis' pulmonary valve in 10 patients. This Xplore-II study is expected to begin later this year. Additionally, the agency granted Humanitarian Use Device (HUD) Designation for Xeltis' pulmonary heart valve for the correction or reconstruction of right ventricular outflow tract (RVOT) in children. Previously, Xeltis shared one- and two-year data from a pediatric feasibility study of a vascular graft developed with RestoreX technology, which showed positive functionality results with no device-related adverse events, and significant improvement in patients' general conditions. In the study, all five children, age 4 to 12 years at enrollment, had only one functioning heart ventricle as a result of congenital heart defects (CHDs). Xeltis is currently investigating additional applications of its innovative approach to restore other heart valves and blood vessels. Millions of people are diagnosed with heart valve disease each year globally, but current replacement valves have significant limitations. Today, patients with artificial heart valves generally endure repeated replacement procedures and complications from chronic inflammation or take long-term medication with potentially severe side effects. Xeltis is a clinical-stage medical device company developing the first heart valves and blood vessels enabling the body's natural restoration of heart valve function through a therapeutic approach called Endogenous Tissue Restoration (ETR). The company's cardiovascular implants are made of bioabsorbable polymers based on Nobel Prize-awarded science. Xeltis has closed a $33 million series B financing ($30 million in 2014, extended by $3 million in 2015). For more information, please visit http://www.xeltis.com . CAUTION: The Xeltis technology is an investigational device and NOT approved for sale.
News Article | May 5, 2017
"I'm extremely proud and excited about the 25th anniversary of The Ric Edelman Show," said Ric Edelman, founder and executive chairman of Edelman Financial Services. "Our goal is to help our listeners achieve financial success by providing them with practical information and solutions. I look forward to continuing the legacy we have built over the years and offering tailored financial advice to people across America." TALKERS magazine named Ric one of the top 100 talk show hosts in the radio industry for three years1 and, in 2012, named him the #2 most important weekend-only talk show host in the nation.2 The Ric Edelman Show airs on radio stations across the United States every weekend. For more information and to listen to past episodes, click here. ABOUT RIC EDELMAN: Ric Edelman, founder and executive chairman of Edelman Financial Services, is widely regarded as one of the top financial advisors in the field. He was ranked the nation's #1 Independent Financial Advisor three times by Barron's,3 named among the country's Top 10 Wealth Advisors by Forbes magazine,4 and is the 2017 recipient of the IARFC's Loren Dunton Memorial Award,5 a lifetime achievement award for his substantial contributions to the financial services profession and the financial interests of the public. He was also named one of the "10 most influential figures" in the advisory field by RIABiz in 2013.6 Ric is an award-winning radio and television personality7 and a #1 New York Times bestselling author. His ninth book, The Truth About Your Future, was published in March and became an instant New York Times Best Seller.8 Ric is an inductee of the Financial Advisor Hall of Fame9 sponsored by Research magazine, a Distinguished Lecturer at Rowan University and a resident expert for Dr. Oz. ABOUT EDELMAN FINANCIAL SERVICES: Edelman Financial Services, with 157 financial planners and 42 offices coast-to-coast, provides financial planning and investment management services to more than 31,000 individuals and families and manages more than $17 billion in assets.10 The firm also provides 401(k) plans and institutional investment management for businesses. Edelman Financial Services has won more than 100 financial, business, community and philanthropic awards11 and offers an investment philosophy that puts clients first and delivers value through in-depth financial education, personalized financial plans and unfettered access to planners.12 Ric Edelman is an Investment Advisor Representative who offers advisory services through Edelman Financial Services, LLC, a Registered Investment Advisor. He is also a Registered Representative and Registered Principal of, and offers securities through, EF Legacy Securities, LLC, an affiliated broker/dealer, member FINRA/SIPC. EFS offers advisory services in all 50 states, the District of Columbia, and Puerto Rico. As such, these services are strictly intended for individuals residing in the United States and Puerto Rico. No offers may be made to or accepted from any resident outside of the 50 states, the District of Columbia, or Puerto Rico. 1 TALKERS magazine "Heavy Hundred" ranking is based on a number of both quantitative and qualitative criteria and is determined by collective analysis of the TALKERS editorial board with input from a wide variety of industry leaders. Investor experience/returns were not considered as part of this ranking. 2 TALKERS magazine ranking "250 Most Important Radio Talk Show Hosts in America" (April 2012) is based on courage, effort, impact, longevity, potential, ratings, recognition, revenue, service, talent and uniqueness of the talk show host. 3 According to Barron's, "The formula [used] to rank advisors has three major components: assets managed, revenue produced and quality of the advisor's practice. Investment returns are not a component of the rankings because an advisor's returns are dictated largely by each client's risk tolerance. The quality-of-practice component includes an evaluation of each advisor's regulatory record." The rankings are based on the universe of applications submitted to Barron's. The selection process begins with a nomination and application provided to Barron's. Principals of Edelman Financial Services, LLC self-nominated the firm and submitted quantitative and qualitative information to Barron's as requested. Barron's reviewed and considered this information, which resulted in the rankings on Aug. 27, 2012/Aug. 28, 2010/Aug. 31, 2009. 4 Forbes rankings are the opinion of SHOOK Research and are based on advisor interviews, client retention, industry experience, compliance record, assets under management and revenue generated for the firm. Investment performance is not considered. Advisors do not pay to be in the ranking. 5 Presented by the International Association of Registered Financial Consultants (IARFC). Candidates must hold a professional designation and must have disseminated their comments on financial topics by having them widely published in articles, journals, books, etc. They must have provided outstanding personal service or leadership in the financial services industry. Nominees must have participated in some aspect of financial education to the public or to other members of the profession. Investor experience/returns were not considered. 6 The RIABiz listing of the 10 most influential figures in the Registered Investment Advisor industry is in recognition of notable, driven and influential executives who are advancing their firms and are considered to be influential in the RIA business. Investor experience/returns were not considered as part of this ranking. 7 Throughout the firm's 30-year history, EFS and Ric Edelman have been presented with numerous business, advisory, communication and community service awards. More information on these awards can be found at EdelmanFinancial.com/awards. 8 The New York Times Book Review Advice, How-To and Miscellaneous. April 16, 2017. 9 Research magazine cover story "Advisor Hall of Fame," December 2004 (based on serving a minimum of 15 years in the industry, having acquired substantial assets under management, demonstrating superior client service and having earned recognition from peers and the broader community for how they reflect on their profession). Investor experience/returns were not considered as part of this ranking. 10 As of Dec. 31, 2016. 11 Throughout the firm's 30-year history, EFS and Ric Edelman have been presented with numerous business, advisory, communication and community service awards. More information on these awards can be found at EdelmanFinancial.com/awards. 12 Investing strategies, such as asset allocation, diversification, or rebalancing do not assure or guarantee better performance and cannot eliminate the risk of investment losses. There are no guarantees that a portfolio employing these or any other strategy will outperform a portfolio that does not engage in such strategies. Funds and ETFs are subject to risk, including loss of principal. All investments have inherent risks. There can be no assurance that the investment strategy proposed will obtain its goal. Past performance does not guarantee future results. To view the original version on PR Newswire, visit:http://www.prnewswire.com/news-releases/the-ric-edelman-show-celebrates-25-years-300452431.html
News Article | May 4, 2017
NEW YORK, May 04, 2017 (GLOBE NEWSWIRE) -- Arc Logistics Partners LP (NYSE:ARCX) ("Arc Logistics" or the "Partnership") today reported its financial and operating results for the first quarter ended March 31, 2017. During the first quarter of 2017, the Partnership accomplished the following: For additional information regarding the Partnership’s calculation of Adjusted EBITDA and Distributable Cash Flow, which are non-GAAP financial measures, and a reconciliation of net income to Adjusted EBITDA and cash flows from operating activities to Distributable Cash Flow, please see below in this release and the accompanying tables. First Quarter 2017 Operational and Financial Results The Partnership’s first quarter 2017 reported revenues, net income and Adjusted EBITDA of $25.9 million, $3.8 million and $13.3 million, respectively, represents a decrease over the Partnership’s first quarter 2016 reported revenues, net income and Adjusted EBITDA of $26.1 million, $5.0 million and $13.5 million, respectively. Operating income decreased by $0.9 million to $4.1 million for the first quarter 2017 when compared to the first quarter 2016 operating income of $5.0 million, which decrease was principally due to the following: As of March 31, 2017, the Partnership's storage capacity was approximately 7.8 million barrels, which represents an approximately 0.1 million barrel, or 1%, increase when compared to the Partnership’s capacity at March 31, 2016. The increase in storage capacity is related to three newly constructed biodiesel tanks at the Pennsylvania terminals and the completion of the third 100,000 barrel tank at the Pawnee terminal. The Partnership's throughput activity increased by 14.5 mbpd, or 10%, to 159.5 mbpd during the first quarter of 2017 compared to the first quarter of 2016. The increase was due to (i) 2.9 mbpd increase in asphalt and industrial products throughput related to existing customer activity in the Gulf Coast, (ii) 0.5 mbpd decrease in crude oil throughput as customer activity at the Joliet terminal offset a reduction in customer activity at the Pawnee and Portland terminals, (iii) 5.0 mbpd increase in distillates throughput is the result of recently executed agreements and increased existing customer activity in Baltimore, Chickasaw, Cleveland and the Pennsylvania terminals, partially offset by reduced customer activity and customer non-renewals in the Partnership’s Blakeley, Madison, Mobile, Portland, Selma, Toledo terminals, (iv) 7.1 mbpd increase in gasoline throughput as a result of 8.3 mbpd of recently executed agreements and increased commercial activity across the entire network providing gasoline service offset by 1.2 mbpd of reduced customer activity in the Partnership’s Dupont and Selma terminals. In April 2017, the Partnership declared a quarterly cash distribution of $0.44 per unit, or $1.76 per unit on an annualized basis, for the period from January 1, 2017 through March 31, 2017. The distribution will be paid on May 15, 2017 to unitholders of record on May 8, 2017. Arc Logistics will hold a conference call and webcast to discuss the first quarter 2017 financial and operating results on May 4, 2017, at 5:00 p.m. Eastern. Interested parties may listen to the conference call by dialing (855) 433-0931. International callers may access the conference call by dialing (484) 756-4279. The call may also be accessed live over the internet by visiting the “Investor Relations” page of the Partnership’s website at www.arcxlp.com and will be available for replay for approximately one month. Arc Logistics is a fee-based, growth-oriented limited partnership that owns, operates, develops and acquires a diversified portfolio of complementary energy logistics assets. Arc Logistics is principally engaged in the terminalling, storage, throughput and transloading of petroleum products and other liquids. For more information, please visit www.arcxlp.com. Certain statements and information in this press release constitute “forward-looking statements.” Certain expressions including “believe,” “expect,” “intends,” or other similar expressions are intended to identify the Partnership’s current expectations, opinions, views or beliefs concerning future developments and their potential effect on the Partnership. While management believes that these forward-looking statements are reasonable when made, there can be no assurance that future developments affecting the Partnership will be those that it anticipates. The forward-looking statements involve significant risks and uncertainties (some of which are beyond the Partnership’s control) and assumptions that could cause actual results to differ materially from the Partnership’s historical experience and its present expectations or projections. Important factors that could cause actual results to differ materially from forward-looking statements include but are not limited to: (i) adverse economic, capital markets and political conditions; (ii) changes in the market place for the Partnership’s services; (iii) changes in prices and supply and demand of crude oil and petroleum products; (iv) actions and performance of the Partnership’s customers, vendors or competitors; (v) nonrenewal, nonpayment or nonperformance by the Partnership’s customers and the Partnership’s ability to replace such contracts and/or customers; (vi) changes in the cost of or availability of capital; (vi) unanticipated capital expenditures in connection with the construction, repair or replacement of the Partnership’s assets; (viii) operating hazards, unforeseen weather events or matters beyond the Partnership’s control; (ix) inability to consummate acquisitions, pending or otherwise, on acceptable terms and successfully integrate acquired businesses into the Partnership’s operations; (x) effects of existing and future laws or governmental regulations; and (xi) litigation. Additional information concerning these and other factors that could cause the Partnership’s actual results to differ from projected results can be found in the Partnership’s public periodic filings with the Securities and Exchange Commission (“SEC”), including the Partnership’s Annual Report on Form 10-K for the year ended December 31, 2016, as filed with the SEC on March 14, 2017 and any updates thereto in the Partnership’s subsequent quarterly reports on Form 10-Q and current reports on Forms 8-K. Readers are cautioned not to place undue reliance on forward-looking statements, which speak only as of the date thereof. The Partnership undertakes no obligation to publicly update or revise any forward-looking statements after the date they are made, whether as a result of new information, future events or otherwise. The Partnership defines Adjusted EBITDA as net income before interest expense, income taxes and depreciation and amortization expense, as further adjusted for other non-cash charges and other charges that are not reflective of its ongoing operations. Adjusted EBITDA is a non-GAAP financial measure that management and external users of the Partnership’s consolidated financial statements, such as industry analysts, investors, lenders and rating agencies, may use to assess (i) the performance of the Partnership’s assets without regard to the impact of financing methods, capital structure or historical cost basis of the Partnership’s assets; (ii) the viability of capital expenditure projects and the overall rates of return on alternative investment opportunities; (iii) the Partnership’s ability to make distributions; (iv) the Partnership’s ability to incur and service debt and fund capital expenditures; and (v) the Partnership’s ability to incur additional expenses. The Partnership believes that the presentation of Adjusted EBITDA provides useful information to investors in assessing its financial condition and results of operations. The Partnership defines Distributable Cash Flow as Adjusted EBITDA less (i) cash interest expense paid; (ii) cash income taxes paid; (iii) maintenance capital expenditures paid; and (iv) equity earnings from the Partnership’s interests in Gulf LNG Holdings Group, LLC (the “LNG Interest”); plus (v) cash distributions from the LNG Interest. Distributable Cash Flow is a non-GAAP financial measure that management and external users of the Partnership’s consolidated financial statements may use to evaluate whether the Partnership is generating sufficient cash flow to support distributions to its unitholders as well as to measure the ability of the Partnership’s assets to generate cash sufficient to support its indebtedness and maintain its operations. The GAAP measure most directly comparable to Adjusted EBITDA is net income and to Distributable Cash Flow is cash flows from operating activities. Neither Adjusted EBITDA nor Distributable Cash Flow should be considered an alternative to net income or cash flows from operating activities, respectively. Adjusted EBITDA and Distributable Cash Flow have important limitations as analytical tools because they exclude some but not all items that affect net income. You should not consider Adjusted EBITDA or Distributable Cash Flow in isolation or as a substitute for analysis of the Partnership’s results as reported under GAAP. Additionally, because Adjusted EBITDA and Distributable Cash Flow may be defined differently by other companies in the Partnership’s industry, the Partnership’s definitions of Adjusted EBITDA and Distributable Cash Flow may not be comparable to similarly titled measures of other companies, thereby diminishing their utility. Please see the reconciliation of net income to Adjusted EBITDA and cash flows from operating activities to Distributable Cash Flow in the accompanying tables. (a) The (gain) loss on revaluation of contingent consideration, depreciation and amortization have been adjusted to remove the non-controlling interest portion related to the GE EFS affiliate’s ownership interest in Arc Terminals Joliet Holdings LLC. (b) The one-time non-recurring expenses relate to amounts incurred as due diligence expenses from acquisitions and other infrequent or unusual expenses incurred. (c) The non-cash (gain) loss on revaluation of contingent consideration is related to the earn-out obligations incurred as a part of the Joliet terminal acquisition. (d) The non-cash deferred rent expense relates to the accounting treatment for the Portland terminal lease transaction termination fees.
News Article | May 17, 2017
Previously published KrasLSL-G12D (ref. 28), Trp53flox/flox (ref. 29), KrasFSF-G12D (ref. 30), Trp53frt/frt (ref. 31), Rosa26LSL-tdTomato (ref. 32), Apcflox/flox (ref. 33), Rosa26LSL-luciferase (ref. 34), Rosa26mTmG (ref. 35), Lgr5GFP-IRES-CreER/+ (ref. 36) and Lgr5CreER/+ (ref. 8) gene-targeted mice were used in the study. All mice were maintained in a mixed Sv129/C57BL/6 genetic background. Tumours were induced in KP mice with 2.5 × 107 plaque-forming units (PFU) of AdCMV-Cre (Iowa), 2 × 108 PFU of AdSPC-Cre23, 37, 1 × 108 PFU of AdCMV-FlpO (Iowa) or 15–50,000 transforming units of lentiviral Cre, as previously described38, 39, in mice that were between 8–12 weeks of age. Approximately equal numbers of male and female mice were included in all experimental groups in all mouse experiments. Mice bearing lung tumours were treated with 10 mg per kg per day of LGK974 (ref. 20) resuspended in 0.5% carboxymethylcellulose (Sigma-Aldrich) and 0.5% Tween 80 (Sigma-Aldrich) or vehicle (0.5% carboxymethylcellulose and 0.5% Tween 80 only). Weights of mice were followed weekly. The growth of autochthonous KrasG12D/+;Trp53Δ/Δ;Rosa26Luciferase/+ lung tumours was followed longitudinally by bioluminescence imaging, as previously described34. In brief, mice were anaesthetized by isoflurane inhalation, administered 100 mg kg−1 d-luciferin (Perkin Elmer) by intraperitoneal injection and imaged after 10 min, using the IVIS imaging system (Perkin Elmer). Such longitudinal imaging experiments were repeated three times and representative data from one such experiment is shown in Fig. 4a. Survival experiments were repeated three times and representative data from one such experiment is shown in Fig. 4b. For survival experiments, mice were randomized based on their tumour burden as assessed by μCT. Mice were assigned a tumour burden score ranging from 0 (no tumours) to 10 (lungs completely full of tumours), and experimental groups were formed such that each group had approximately equal average tumour burdens. Mice with tumour burden scores under 3 were excluded from the study. The health of the mice in all experiments was monitored daily by the investigators and/or veterinary staff at the Department of Comparative Medicine at Massachusetts Institute of Technology. Mice with a body condition score under 2 were humanely euthanized. Animal studies were approved by the Massachusetts Institute of Technology (MIT) Committee for Animal Care (institutional animal welfare assurance no. A-3125-01). The maximal tumour dimensions permitted by the MIT Committee for Animal Care were 2 cm across the largest tumour diameter and this limit was not reached in any of the experiments. Mice bearing KrasG12D/+;Trp53Δ/Δ;Rosa26tdTomato/+ (KPT) or KrasG12D/+;Trp53Δ/Δ;Rosa26tdTomato/+;Lgr5GFP-CreER/+(KPT;Lgr5GFP-CreER/+) LUAD tumours were euthanized 12–26 weeks after tumour induction and perfused with S-MEM (Gibco) through the right ventricle of the heart. Dissected lungs with tumours were dissociated in protease and DNase solution of the Lung Dissociation kit (Miltenyi Biotech) followed by mechanical dissociation using MACS C columns (Miltenyi Biotech) according to the manufacturer’s instructions. The dissociated cells were filtered using a 100-μm strainer and red blood cells were lysed using ACK (Thermo Scientific), followed by staining with APC-conjugated CD31 (Biolegend, 102510), CD45 (BD, 559864), CD11b (eBioscience, 17-0112-82) and TER119 (BD, 557909) antibodies and dead cells with DAPI (Sigma-Aldrich). The same approach using the Tumour Dissociation kit (Miltenyi Biotech) was used to isolate KPT;Lgr5GFP-CreER/+;Pdx1::Cre PDAC tumours cells when mice were 7 weeks of age. Fluorescence-activated cell sorting (FACS) of stained primary cells was performed using a FACSAria sorter (BD) by gating for tdTomato+/DAPI−/APC− cells (total cancer cell fraction) for KPT tumours. For KPT;Lgr5GFP-CreER/+ tumours, both tdTomato+/DAPI−/APC−/GFP+ (Lgr5+ cancer cell fraction) and tdTomato+/DAPI−/APC−/GFP− (Lgr5− cancer cell fraction) populations were sorted. Sorted cells were placed in 3D organotypic culture, transplanted intratracheally into NOD/SCID-γ (NSG) recipient mice, or subcutaneously into athymic nu/nu mice immediately after sorting (see below). For intratracheal transplantation, 8–10-weeks-old immunodeficient NSG mice were anaesthetized, intubated as previously described38, and allowed to inhale 15–50,000 sorted primary KP LUAD cancer cells resuspended in 30 μl of S-MEM (Gibco). For subcutaneous transplantation, 50–500,000 sorted primary KP LUAD cells, KP LUAD cell lines or single-cell clones derived from a KP;Lgr5GFP-CreER/+ LUAD cell line were resuspended in 50% Matrigel/50% S-MEM and injected subcutaneously into both flanks of athymic nu/nu mice in a volume of 100 μl. Mice with transplant tumours were injected intraperitoneally with 1 mg of 5-ethynyl-2-deoxyuridine (EdU, Setareh Biotech) 4 h before euthanasia to label proliferating cells. EdU was detected in cryosections using the Click-iT EdU Alexa Fluor 488 Imaging kit (Thermo Scientific) according to the manufacturer’s protocol. Lgr5+ cells in close proximity to porcupine were detected by GFP and porcupine immunofluorescence. All GFP+ cells were analysed as being immediately adjacent to at least one porcupine+ cell, as double-positive for both GFP and porcupine, or as neither of the above (Fig. 3a). All transplantation experiments were reproduced three times. 150–1,000 primary mouse KP LUAD cells, cells from established KP LUAD cell lines, or primary mouse PDAC cells were mixed in 50% Matrigel (BD) and 50% advanced DMEM/F12 (Gibco) and plated on 10 μl of Matrigel. The gel was allowed to solidify at 37 C, followed by addition of advanced DMEM/F12 (Thermo Scientific) supplemented with gentamicin (Thermo Scientific), penicillin–streptomycin (VWR), 10 mm HEPES (Thermo Scientific) and 2% heat-inactivated fetal bovine serum. For Wnt pathway manipulation, cultures were incubated with 1 μg ml−1 recombinant mouse (rm)R-spondin 1 (Sino Biological), 100 ng ml−1 rmWnt3a (R&D Sytstems), 500 ng ml−1 or 1 μg ml−1 rmDKK1 (R&D Systems) or 100 nM LGK974 (Medchem Express) for 6–14 days. Medium was changed every two days. At the end of the experiment, proliferating cells were labelled with 10 μM EdU for 4 h, followed by paraformaldehyde fixation and fluorescent labelling of proliferating cells using the Click-iT EdU Alexa Fluor 488 Imaging kit (Thermo Scientific), according to the manufacturer’s protocol, in whole-mount preparations of tumour spheroids. Proliferating spheroids were quantified using a Nikon Eclipse 80i microscope: a spheroid was classified as a cluster of at least 10 cells, and a proliferating spheroid contained at least one EdU positive nucleus (proliferating cells were not observed in clusters of cells smaller than 10 cells). At least four replicate wells per condition were quantified in each experiment. Images were acquired using a Nikon A1R confocal microscope. Stimulation and inhibitor experiments were reproduced at least 10 times for each experimental condition. Multiple cell lines were established from the mouse LUAD and PDAC KP GEMMs over the course of the study. The cell lines have not been authenticated. The cell lines were routinely tested for mycoplasma and found to be negative. At the time of conducting the experiments, no cell lines used were found to be listed in the ICLAC database of misidentified cell lines. Tissues or tumour organoids were fixed in 10% formalin overnight and embedded in paraffin. Immunohistochemistry (IHC) was performed on a Thermo Autostainer 360 with or without haematoxylin counterstaining using antibodies against β-catenin (BD, 610153), Ki67 (Vector Labs, VP-RM04), glutamine synthetase (BD, 610517), or porcupine (Abcam, ab105543). Lungs from at least three tumour-bearing mice were analysed for each antibody. Livers and small intestines collected from three normal, healthy mice were used for β-catenin, glutamine synthetase and porcupine IHC. 65 human LUAD tumours samples in two separate tissue microarrays were analysed by IHC for β-catenin and porcupine. 5 human colorectal adenocarcinoma samples were stained with porcupine antibodies. All human tissue material was obtained commercially from Janssen Pharmaceuticals. Mice were anaesthetized and perfused through the right cardiac ventricle with 1% paraformaldehyde. Lungs with tumours were dissected, immersed in 4% PFA overnight and frozen in OCT medium (Sakura Finetek). 7 μm sections were stained with antibodies to EpCAM (eBioscience, 17-5791-82), β-catenin (BD, 610153), GFP (Cell Signaling Technologies, 2956S; or Aves Labs, GFP-1020), CD11b (eBioscience, 17-0112-82) or porcupine (Abcam, ab105543). Lungs from at least three tumour-bearing mice were analysed for each antibody. Digitally scanned images of Ki67-stained slides were created with the Aperio ScanScope AT2 at 20× magnification. Aperio’s WebScope software was used to assess Ki67+ density per tumour area. A built-in IHC nuclear image analysis algorithm was used to classify cells on the basis of the intensity of the nuclear Ki67 stain. Nuclei were classified from 0 to 3+; only nuclei with moderate nuclear staining (2+) or intense nuclear staining (3+) were considered Ki67+. Tumour regions were outlined on WebScope before running the IHC nuclear image analysis algorithm such that the number of 2+ and 3+ cells was normalized to tumour area. Total RNA was isolated from tumours or cells using the RNeasy plus kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized from 1 μg of RNA using the SuperScript VILO cDNA synthesis kit (Thermo Scientific). qPCR was performed in triplicates with 2 μl of diluted cDNA (1:10) using PerfeCTa SYBR Green FastMix (Thermo Scientific) on a Bio-Rad iCycler RT–PCR detection system. Expression was normalized to Actb or Gapdh. All oligonucleotides used in this study are listed in Supplementary Table 4. All qPCR experiments were reproduced using at least three biological replicates. Alternatively, a Mouse WNT Signalling Pathway RT2 Profiler PCR Array (Qiagen) was used according to the manufacturer’s instructions. Raw expression values were thresholded to remove genes that were not detected or had low expression (maximum C value set to 33; 0 values set to 33). Array position to gene-name mapping details were retrieved from the manufacturer’s website (www.pcrdataanalysis.sabiosciences.com). Expression values for all genes per array were normalized to the expression of the housekeeping gene Gusb. Three replicates of stroma samples and three replicates of tumour samples were compared to calculate log fold change and differential expression significance values (two-sided t-test). shRNAs were cloned into lentiviral pLKO.1 vectors (Addgene, 10878) or into pTRIPZ (Dharmacon) vectors and lentivirus was produced as previously described40. KP mouse LUAD cell lines were infected with the lentiviral vectors, followed by puromycin selection and, in the case of cells infected with the TRIPZ virus, incubation in 1 μg ml−1 doxycycline for four days and RNA extraction for testing target knockdown (Extended Data Fig. 2a and not shown). For combined Lgr4 and Lgr5 silencing experiments, cell lines expressing pLKO.1 driving Lgr4 or Lgr5 shRNAs were generated by puromycin selection, followed by infection with TRIPZ vectors driving miR30-based Lgr4 or Lgr5 shRNAs and turboRFP under the control of a TET-responsive promoter. Cells were incubated in 1 μg ml−1 doxycycline for two days and red fluorescent cells were sorted to generate pure cell lines expressing combinations of Lgr4 and Lgr5 shRNAs. All shRNA experiments were reproduced using at least three independent cell lines. 10,000 of KP LUAD cells were plated in 100 μl of medium containing 10% FBS per well of a white-walled 96-well plate (Perkin Elmer). After 24 h, mouse KP LUAD cells were transfected using Attractene transfection reagent (Qiagen) according to the manufacturer’s instructions with 150 ng of the TOPFLASH Firefly (M50) reporter41 (Addgene, 12456) and 20 ng of pRL-SV40P Renilla (Addgene, 27163) constructs. In initial experiments, the Wnt-insensitive FOPFLASH (negative control) Firefly (M51) reporter41 (Addgene, 12457) was used to rule out signal background (not shown). Cells were stimulated for 16 h with recombinant Rspo1 (1 μg ml−1, Sino Biological), recombinant Wnt3a (100 ng ml−1, R&D Systems) or their combination (RW) in advanced DMEM/F12 (Gibco), with supplements listed above. After stimulation, Firefly and Renilla signals were detected using Dual-Glo luciferase detection reagents (Promega) according to the manufacturer’s instructions. A Tecan Infiniti 200 Pro plate reader and automated injector system was used to detect luminescence. To control for transfection efficiency, Firefly luciferase levels were normalized to Renilla luciferase levels to generate a measurement of relative luciferase units. Experimental data are presented as mean ± s.d. from three independent wells. All TOPFLASH experiments were reproduced using at least three independent cell lines. Catalytically dead Cas9 (dCas9)-based systems have recently emerged as powerful tools for transcriptionally activating endogenous genes42. Notably, these systems allow for overexpression of genes in their endogenous genomic context. To overexpress Rspo2, Rspo3 or Lgr5 in KrasG12D/+;Trp53Δ/Δ LUAD cell lines, we used the SAM system, which is a three-component system based on: (1) the fusion of dCas9 to the transcriptional activator VP64 (a tandem repeat of four DALDDFDLDML sequences from Herpes simplex viral protein 16, VP16); (2) a modified gRNA scaffold containing two MS2 RNA aptamers; and (3) the MS2–p65–HSF1 tripartite synthetic transcriptional activator21. In this system, sgRNA-dependent recruitment of dCas9–VP64 and MS2–P65–HSF1 to the endogenous Rspo2, Rspo3 or Lgr5 loci results in potent transcriptional activation (Extended Data Fig. 1i–l). Non-clonal KrasG12D/+;Trp53Δ/Δ;Rosa26tdTomato/+ or KrasG12D/+;Trp53Δ/Δ;Lgr5GFP-CreER/+ LUAD cells stably expressing dCas9–VP64–blast (Addgene, 61425) and MS2–P65–HSF1–hygro (Addgene, 61426) were generated using sequential lentiviral transduction and selection with blasticidin and hygromycin, respectively. To overexpress Rspo2 or Rspo3 we designed four independent sgRNA sequences targeting the Rspo2 or Rspo3 transcription start site; sgRNAs targeting the upstream region of the Lgr5 gene were provided by L. Gilbert, M. Horlbeck and J. Weissman43. The sgRNAs were cloned into a lentiviral vector (Lenti-sgRNA-MS2-zeocin; Addgene, 61427) followed by transduction and zeocin selection of the aforementioned cell lines to generate KrasG12D/+;Trp53Δ/Δ;Lgr5GFP-CreER/+ LUAD cell lines stably expressing all three components. These experiments were reproduced using three independent cell lines. The 7TCF::luciferase-PGK::Cre, 7TCF::GFP-PGK-Cre and U6::sgRNA-EFS::Cre (pUSEC) lentivirus vectors were generated by Gibson assembly44, 45. In brief, a 1.8-kb part corresponding to 7TCF::luciferase or a 1.2-kb part corresponding to 7TCF::GFP were amplified from 7TFP (Addgene, 24308, ref. 46) or 7TGP (Addgene, 24305, ref. 46) respectively, and fused with a 0.5-kb PGK promoter part, a 1.0-kb Cre cDNA part and the PmeI and BsrGI linearized LV1-5 (Addgene, 68411) part44. U6::sgRNA-EFS::Cre was generated by amplifying a 2.2-kb part corresponding to the U6-filler-chimeric gRNA backbone from pSECC (Addgene, 60820), and fused with a 0.25-kb EFS promoter part, a 1.0-kb Cre cDNA part and the PmeI and BsrGI linearized LV1-5 (Addgene, 68411) part44. Lentivirus was produced in 293FS* cells, as previously described38. Experiments using 7TCF::luciferase-PGK::Cre (Fig. 2a) were performed twice (n = 15 mice in total) and experiments using 7TCF::GFP-PGK-Cre (Fig. 2b) three times (n = 19 mice in total). For generation of lentiviruses containing sgRNAs, three sgRNAs per gene targeting Porcn, Lgr4 or Lgr5 were designed using CRISPR Design47, cloned into pSpCas9(BB)-2A-GFP (pX458, Addgene, 48138) as previously described48, transfected into KP cells49, and screened for efficiency by western blotting for porcupine protein or by massively parallel sequencing of the regions in Lgr4 or Lgr5 targeted by the respective sgRNAs (data not shown). The most efficient Porcn sgRNA was cloned into pSECC as previously described49. The most efficient Lgr4 and Lgr5 sgRNAs were cloned into the pUSEC vector together with a synthetic mouse/human U6 promoter (sU6), as previously described50, to generate U6::sgLgr4-sU6::Lgr5-EFS::Cre (pU2SEC). A KPT LUAD cell line was transduced with 7TCF::luciferase-PGK::Puro (7TFP) lentiviruses46, selected for puromycin resistance, and transplanted subcutaneously into flanks of immunodeficient athymic nu/nu mice. Three weeks after transplantation, tumour burden was measured by registering tdTomato fluorescence using an IVIS imaging system (Perkin Elmer), followed by administration of 100 mg kg−1 d-Luciferin (Perkin Elmer) and detection of the luciferase signal (7TCF promoter activity). The luciferase signal was normalized to the tdTomato signal (Wnt pathway activity/total tumour burden). Quantification of Wnt pathway activity was performed every 24 h for a week in mice treated with 10 mg per kg per day of LGK974 or vehicle. The maximal tumour dimensions permitted by the MIT IACUC were 2 cm across the largest tumour diameter and this limit was not reached in these experiments. This experiment was performed twice. Single-molecule in situ hybridization was performed on formalin-fixed paraffin-embedded tissues using the Advanced Cell Diagnostics RNAscope 2.5 HD Detection kit (322360). Catalogue numbers of the probes are 400331 (Axin2), 318321 (Lgr4), 312171 (Lgr5) and 404971 (Porcn), 401991 (Rspo1), 316791 (Wnt5a), 401121 (Wnt7a) and 401131 (Wnt7b). Lungs from three tumour-bearing mice were analysed. We generated KrasFSF-G12D/+;Trp53frt/frt;Lgr5CreER/+;Rosa26mTmG/+ mice and induced lung tumours by intratracheal administration of AdCMV-FlpO. Lung tumours were collected, enzymatically dissociated and passaged in vitro for 8–10 passages to eradicate stromal cells from the cultures. Such early-passage cell lines were transplanted subcutaneously into flanks of NSG mice. When mice developed palpable tumours, they were administered a single tamoxifen pulse (20 mg kg−1) or corn-oil vehicle control. Tumours were collected at 2 days or 14 days after tamoxifen administration and prepared for cryosectioning. Three sections 500 μm apart were prepared from each tumour and imaged under a fluorescence microscope. The number of GFP+ cells per section was quantified in nine tumours per time point. An eXplore CT 120 microcomputed tomography (μCT) system (Northridge Tri-Modality Imaging Inc.) was used for in vivo imaging. Mice were imaged under anaesthesia (induced at 3% isoflurane in oxygen, maintained at between 2–2.5% during imaging) in groups of 4 in a custom mouse holder. Scanner settings were as follows: 720 views, 360° rotation, 70 kVp, 50 mA, 32 ms integration time with 2 × 2 detector pixel binning (isotropic nominal resolution of 50 μm). Data were reconstructed using the Parallax Innovations GPU accelerated reconstruction engine for the eXplore CT120. Tissue density values (in Hounsfield Units (HU)) for normal, air-filled lung parenchyma were determined by eye using MicroView software (Parallax Innovations). For the scanning conditions in this study a range of −550 to −300 HU was determined to represent the range of normal lung parenchyma values. A custom analysis script was created using MATLAB (MathWorks) to identify a region of interest (ROI) including the soft tissue of the mouse thorax. Within this region the volume of tissue within the ‘healthy’ density range was measured. Within this same volume minimum intensity projections (MinP) were created, both to confirm the accuracy of the ROI and to qualitatively assess lung pathology. For data visualization, the change in healthy lung volume was inverted to represent the change in tumour volume (Extended Data Fig. 10b). One experiment involving 9 mice treated with LGK974 and 11 mice treated with vehicle control was carried out to track changes in tumour volume (Extended Data Fig. 10b). RNA-seq gene expression profiles of primary tumours and relevant clinical data of 488 patients with lung adenocarcinoma were obtained from The Cancer Genome Atlas (TCGA LUAD; http://cancergenome.nih.gov/). The previously published Wnt signalling geneset22 (24 genes upregulated after stimulation with recombinant human WNT3A) was obtained from the Molecular Signatures Database (MSigDB)51 and used to score individual patient expression profiles using ssGSEA52, 53. Patients were stratified according to their correlation score, into top (n = 115) and bottom (n = 114) 20th percentile sets. Kaplan–Meier survival analysis was conducted between these sets of patients and the log-rank test was used to assess significance. Subsequently, the Kaplan–Meier survival analysis methodology was extended to assess significant survival differences across 35 TCGA cancer types using a similar strategy. Additionally, the Cox proportional hazards regression model was used to analyse the prognostic value of the published geneset22 across all patients within the TCGA LUAD cohort, in the context of additional clinical covariates. All univariate and multivariable analyses were conducted within a five-year survival timeframe. The following patient and tumour-stage clinical characteristics were used: signature (signature from ref. 22 strong versus weak correlation); gender (male or female); age (years, continuous); smoking history (reformed >15 years versus non-smoker, reformed <15 years versus non-smoker, current smoker versus non-smoker); mutational load (derived as the number of non-silent mutations per 30 Mb of coding sequence, continuous); Union for International Cancer Control (UICC) TNM Stage specification (stage III/IV versus I/II); UICC T score specification (T2 versus T1, T3/T4 versus T1); UICC N score specification (N1/N2 versus N0). Hazard ratio proportionality assumptions for the Cox regression model were validated by testing for all interactions simultaneously (P = 0.703). Interaction between the signature of ref. 22 and TNM stage, T score and N score (significant covariates in the model) were tested using a likelihood ratio test to contrast a model consisting of both covariates with another model consisting of both covariates plus an interaction term. No statistically significant difference was found between the two models (TNM, P = 0.8751; T score, P = 0.8204; N score: P = 0.8625; likelihood ratio test). To test for statistically significant differences between the previously published22 signature correlation scores across TCGA LUAD grade levels (T scores), the Kurskal–Wallis test was used to assess overall significance and the Mann–Whitney–Wilcoxon test was used to assess pairwise differences. All statistical analyses were conducted in R (http://www.R-project.org) and all survival analyses and were conducted using the survival package in R. Finally, we analysed the expression of Wnt pathway genes present in the Mouse WNT Signalling Pathway RT2. Profiler PCR Array (Qiagen) in the human TCGA LUAD data (Supplementary Table 3). Expression levels between 57 LUAD tumour samples and corresponding matched normal samples were analysed using empirical cumulative distribution function plots. Significance of different expression levels was assessed using the Kolmogorov–Smirnov test. For a more comprehensive analysis covering human orthologues of all WNT pathway genes tested on the mouse qPCR array, pairwise differential expression analysis (tumour versus normal, n = 57 each) was performed using EBSeq version 1.4.0 (ref. 54). CRISPR-Cas9-induced mutations were detected as before59. Briefly, genomic regions containing the sgPorcn, sgLgr4 or sgLgr5 target sequences were amplified using Herculase II Fusion DNA polymerase and gel purified (primer sequences are shown in Supplementary Table 4). Sequencing libraries were prepared from 50 ng of PCR product using the Nextera DNA Sample Preparation kit (Illumina) according to the manufacturer’s instructions and sequenced on Illumina MiSeq sequencers to generate 150-bp, paired-end reads. CRISPR-Cas9-mutated loci were computationally analysed as before59. Briefly, illumina MiSeq reads (150 bp paired-end) were trimmed to 120 bp after reviewing base quality profiles, in order to remove lower quality 3′ ends. Traces of Nextera adapters were clipped using the FASTX toolkit (Hannon Laboratory, CSHL) and pairs with each read greater than 15 bp in length were retained. Additionally, read pairs where either read had 50% or more bases below a base quality threshold of Q30 (Sanger) were removed from subsequent analyses. The reference sequence of the target locus was supplemented with 10 bp genomic flanks and was indexed using an enhanced suffix array55. Read ends were anchored in the reference sequence using 10 bp terminal segments for a suffix array index lookup to search for exact matches. A sliding window of unit step size and a maximal soft-clip limit of 10 bp were used to search for possible anchors at either end of each read. For each read, optimal Smith–Waterman dynamic programming alignment56 was performed between the reduced state space of the read sequence and the corresponding reference sequence spanning the maximally distanced anchor locations. Scoring parameters were selected to allow sensitive detection of short and long insertions and deletions while allowing for up to four mismatches and the highest scoring alignment was selected. Read pairs with both reads aligned in the proper orientation were processed to summarize the number of wild-type reads and the location and size of each insertion and deletion event. Overlapping reads within pairs were both required to support the event if they overlapped across the event location. Additionally, mutation events and wild-type reads were summarized within the extents of the sgRNA sequence and PAM site by considering read alignments that had a minimum of 20 bp overlap with this region. Mutation calls were translated to genomic coordinates and subsequently annotated using Annovar57. The alignment and post-processing code was implemented in C++ along with library functions from SeqAn58 and SSW59 and utility functions in Perl and R (http://www.R-project.org). Mutation calls were subjected to manual review using the Integrated Genomics Viewer (IGV)60. Statistical analysis was carried out as indicated in the Figure legends, Extended Data Figure legends and in the Methods for each experiment. The data were found to meet the assumptions of the statistical tests. Variation was estimated for each group of data, the variance was found to be similar between the groups that were compared. No animals were excluded from any of the studies. The investigator was blinded with respect to group assignment for the quantification of 3D spheroids, proliferating (Ki67+) cells and for the analysis of healthy lung volume by μCT. Power calculations were performed to estimate the sample size for experiments involving LGK974 treatment. In brief, to detect a difference of 30% in average survival between the two groups (effect size = 1.2 s.d. of survival based on Cohen’s d (ref. 61) using untreated sample baseline survival from ref. 39) with 90% power, a minimum of five mice per group needed to be used. Massively parallel sequencing data are available in the NCBI/SRA data repository under accession number PRJNA379539. Source code and all other data are available from the authors upon reasonable request.
News Article | August 9, 2017
— In an effort to redefine the way business leaders interact with their brand and their solutions, EFS – a WEX Company (EFS), launched its new website at efsllc.com. The company, renowned for its payment technology solutions designed specifically for the over-the-road transportation industry, believes that this newly redesigned platform better engages visitors with dynamic content describing EFS’ solutions, but also reflects where the company is going as a technology leader. The new platform incorporates novel video attributes that highlight some of the unique solutions and technologies that distinguish EFS from its competitors. By illustrating the value of its data-aware, platform-agnostic solutions, the company hopes to make it easier for business operators to understand the benefits at a glance. "Our new digital home was designed to better interact with and engage visitors, but also serves as a convenient entry point for our customers in managing their EFS programs," said EFS Vice President of Marketing, Kellie Jones. "By providing a centralized source for customers to log in and access their data in real time, we're helping them gain greater visibility and control in managing their payments and transactions." According to industry insiders, the site's versatility is unmistakably welcome. EFS Fleet products come with advanced data-oriented features, such as fuel auditing and analysis that let customers compare their volume performance to their peers. EFS fuel cards also empower business owners to tightly regulate which kinds of purchases can be made and have controls in place to prevent unauthorized transactions before they become problematic. The fact that the new site is mobile-friendly and responsive in design makes it much simpler for on-the-go fleet managers to put such functionality to good use. The new EFS site provides a clearer picture of the company’s services and offerings. Visitors can easily explore products like compliance-ready fuel tax reporting, payroll and settlement cards, and mobile apps in order to choose the options that best fit their business’ needs. EFS is clearly committed to the business of simplifying complex payments. Its easy-to-use site appears equally dedicated to facilitating business and consumer convenience. Learn more by visiting efsllc.com today. About EFS – a WEX Company EFS – a WEX Company (EFS) is a leading provider of innovative and customized corporate payment technology solutions designed specifically to help our customers simplify payments, drive efficiencies and improve bottom line performance. Serving the industry for 50 years, EFS brings unparalleled service, customer-driven innovation, and leading technologies to make the industry better. Our portfolio includes a suite of corporate and private-label purchasing cards; innovative cardless technology solutions; payroll solutions; money transfer solutions; back-office AP and expense management solutions; and intelligent, real-time analytics to make payments smarter. Led by industry experts, EFS maintains our commitment to bettering the industry by developing new technology to deliver smarter payments, providing our customers with better financial controls. For more information, please visit https://www.efsllc.com/
News Article | May 25, 2017
Dublin, Ireland, May 25, 2017 --( “We have been working closely with the AWS team, and we are impressed with incredible scalability, security, performance, and reliability of their products,” said Brian Lewis, CTO, OpenJaw Technologies. “We look forward to the positive impact that going all-in on AWS will have on our IT infrastructure across our key business divisions.” “Enterprises around the world are choosing to deploy their critical applications on AWS and are going all-in so they can refocus on delivering the best for their customers,” said Gavin Jackson, UK & Ireland Managing Director at AWS. “We’re excited for OpenJaw’s adoption of AWS, which will help them quickly and easily scale, reduce costs, improve security, and increase agility for their global travel and loyalty customers.” OpenJaw will use AWS to scale with the rapid growth in the use of its t-Retail platform, which currently handles $3bn worth of transactions a year. The OpenJaw t-Retail Cloud solution will migrate to AWS utilising Amazon Elastic Compute Cloud (Amazon EC2) to quickly scale capacity, Amazon Simple Storage Service (Amazon S3) for highly scalable, reliable, low-latency data storage infrastructure and Amazon Elastic File System (Amazon EFS) for simple, scalable file storage. Additionally, OpenJaw will use the Amazon API Gateway so that OpenJaw developers can create, publish, maintain, and secure APIs and Amazon CloudFront, the global content delivery network (CDN) service to accelerate delivery of OpenJaw customer websites, APIs and content through CDN caching. About Amazon Web Services For 10 years, Amazon Web Services has been the world’s most comprehensive and broadly adopted cloud platform. AWS offers over 90 fully featured services for compute, storage, networking, database, analytics, application services, deployment, management, developer, mobile, Internet of Things (IoT), Artificial Intelligence (AI), security, hybrid and enterprise applications, from 42 Availability Zones (AZs) across 16 geographic regions in the U.S., Australia, Brazil, Canada, China, Germany, India, Ireland, Japan, Korea, Singapore, and the UK. AWS services are trusted by millions of active customers around the world monthly - including the fastest growing startups, largest enterprises, and leading government agencies – to power their infrastructure, make them more agile, and lower costs. To learn more about AWS, visit https://aws.amazon.com. Dublin, Ireland, May 25, 2017 --( PR.com )-- OpenJaw Technologies today announced that is has selected Amazon Web Services (AWS), as its cloud infrastructure provider. OpenJaw is moving its t-Retail platform to AWS to reliably and securely scale the t-Retail platform for future global growth. The Dublin-headquartered travel tech company delivers e-commerce technology to the world’s leading travel brands such as British Airways, Cathay Pacific, Iberia Airlines, Hainan Airlines, AIMIA, Loyalty One, Four Seasons, Avis, and Air Miles.“We have been working closely with the AWS team, and we are impressed with incredible scalability, security, performance, and reliability of their products,” said Brian Lewis, CTO, OpenJaw Technologies. “We look forward to the positive impact that going all-in on AWS will have on our IT infrastructure across our key business divisions.”“Enterprises around the world are choosing to deploy their critical applications on AWS and are going all-in so they can refocus on delivering the best for their customers,” said Gavin Jackson, UK & Ireland Managing Director at AWS. “We’re excited for OpenJaw’s adoption of AWS, which will help them quickly and easily scale, reduce costs, improve security, and increase agility for their global travel and loyalty customers.”OpenJaw will use AWS to scale with the rapid growth in the use of its t-Retail platform, which currently handles $3bn worth of transactions a year. The OpenJaw t-Retail Cloud solution will migrate to AWS utilising Amazon Elastic Compute Cloud (Amazon EC2) to quickly scale capacity, Amazon Simple Storage Service (Amazon S3) for highly scalable, reliable, low-latency data storage infrastructure and Amazon Elastic File System (Amazon EFS) for simple, scalable file storage. Additionally, OpenJaw will use the Amazon API Gateway so that OpenJaw developers can create, publish, maintain, and secure APIs and Amazon CloudFront, the global content delivery network (CDN) service to accelerate delivery of OpenJaw customer websites, APIs and content through CDN caching.About Amazon Web ServicesFor 10 years, Amazon Web Services has been the world’s most comprehensive and broadly adopted cloud platform. AWS offers over 90 fully featured services for compute, storage, networking, database, analytics, application services, deployment, management, developer, mobile, Internet of Things (IoT), Artificial Intelligence (AI), security, hybrid and enterprise applications, from 42 Availability Zones (AZs) across 16 geographic regions in the U.S., Australia, Brazil, Canada, China, Germany, India, Ireland, Japan, Korea, Singapore, and the UK. AWS services are trusted by millions of active customers around the world monthly - including the fastest growing startups, largest enterprises, and leading government agencies – to power their infrastructure, make them more agile, and lower costs. To learn more about AWS, visit https://aws.amazon.com. Click here to view the list of recent Press Releases from OpenJaw Technologies
News Article | May 29, 2017
Global molecular diagnostics company Omixon, headquartered in Budapest with US offices in Cambridge, MA, announce today that Holotype HLA™ and other Omixon products will be featured in 12 poster presentations produced by Holotype customers at the annual meeting of the European Federation for Immunogenetics (EFI) in Mannheim, Germany. Additionally, Omixon’s Lunch Symposium on Wednesday will focus on Automated HLA Typing by NGS and feature three presentations from current Holotype HLA users from the EMEA region. Among the customer presentations the most common theme is the superiority of NGS compared with legacy methods, in which rare alleles created by rare crossing over events (P118, Thomas Binder et al.) or novel alleles (P119 & P123, Xavier Lafarge et al.) can be easily determined by Holotype HLA and remain a challenge for SBT. Another popular theme is the comparison of various NGS technologies (P115, Amalia Dinou et al. and Petra Neukirchen et al.) in which the strengths of Holotype HLA are showcased against those of other vendors. Additional examples of these themes are captured in a poster presented by Omixon’s Dr. Libor Kolesar (P110), who will focus on the “Super Powers” of NGS compared to legacy technologies and highlight unique capabilities of Holotype HLA. Omixon’s Lunch Symposium on May 31 will have three customer experience stories featuring Dr. Alexandre Walencik from the Etablissement Français du Sang (EFS), Nantes who will present their experience with Holotype HLA. “One year after, does NGS really change the world in a HLA laboratory?” Dr Walencik will their share good experiences, obstacles and their solutions with a focus on technical and organizational ways and means to encourage more labs adopt NGS for HLA such as Holotype HLA. Dr. Reem Ameen from Kuwait University will focus on describing the validation process for introducing Holotype HLA in clinical setting and her project to extend the known HLA haplotypes in several families of Kuwaiti descent. Dr. Ameen identified haplotypes that were not among the 200 most common HLA haplotypes in any of the 5 major US populations and included 199 (17%) unique alleles, 26 rare alleles, 6 very rare alleles and 2 novels. Kuwaiti individuals carry unique HLA haplotypes that are not shared by any of the majority of subjects historically reported to the US National Marrow Donor Program (NMDP) registry - a fascinating population with tremendous potential for ongoing HLA research. Dr. Mette Christiansen from the Aarhus University Hospital, Denmark will share their experiences on Automation of the Holotype HLA on the Beckman Biomek 4000. NGS has ushered in an era of increased capacity for HLA genotyping in terms of pooling more loci and processing larger numbers of samples thus placing ever more importance on repeatability and reproducibility. Handling of multiple samples at multiple loci simultaneously and eventually combining these into a single tube requires tightly controlled procedures, which may be achieved by automation. She will focus on the obstacles encountered and the benefits achieved in the automation process. Marcello Scala, Director of Sales, EMEA at Omixon says, “The explosion in labs adopting Holotype HLA throughout Europe and Middle East has been truly astonishing. Even more impressive is the affection customers have for the technology due to its unrivaled ability to resolve the genotypes of complex samples for the benefit of patients and the transplant community as a whole.” Omixon at EFI 2017 May 30 - June 02 | Omixon will be exhibiting at Booth #18 throughout the conference May 30, 9.30am - 11am | Resolving Laboratory NGS Assay Challenges with HLA Twin May 30, 11.30am-1pm | Resolving Complex Cases of NGS-based HLA Typing with HLA Twin May 31, 1.30pm-2.30pm | High-throughput Automation of Holotype HLA in Clinical Routine Omixon featured in posters P108 | E. Bauer et al. (2017) - Full-length sequencing of a novel MICA allele variant P110 | L. Kolesar et al. (2017) - Superpowers of NGS P111 | J. Diegel et al. (2017) - HLA-E genotyping – the sooner the better P115 | A. Dinou et al. (2017) - Evaluation of a commercially available HLA typing kit for NGS P117 | T. Binder et al. (2017) - Complete human leukocyte antigen gene sequence determination combining long range polymerase chain reaction and next generation sequencing P118 |T. Binder et al. (2017) - Whole gene sequence determination of a rare human leukocyte antigen DRB1 allele by combining long range polymerase chain reaction and next generation sequencing. P119 | X. Lafarge et al. (2017) - Exon phasing permits identification of new alleles by NGS not detectable by Sanger sequence-based typing P121 | P. Neukirchen et al. (2017) - NGS based HLA typing: comparison of four protocols and corresponding software P123 | X. Lafarge et al. (2017) Validation and routine setting of HLA typing by Next Generation Sequencing using the HOLOTYPE HLA (OMIXON) kits : a multicentric experience P127 | A. Hansen et al. (2017) - Implementing ABO genotyping into HLA sequencing workflow P129 | L. Krammes et al. (2017) - What’s new genotyping KIR2DL5? P145 | M. Dorak et al. (2017) - HLA-A, -B, -C typing by next generation sequencing in a sample of Turkish population About Omixon Omixon is a global molecular diagnostics company, headquartered in Budapest, Hungary, with US offices in Cambridge, MA that commercializes disruptive technologies for clinical and research laboratories. Omixon’s flagship product, Holotype HLA™, is the world’s leading NGS-based HLA genotyping product that delivers the most accurate high-resolution HLA genotyping available, and is used in more than 35 hospitals worldwide. Omixon’s research software, HLA Explore™ analyzes data from any sequencing technology and determines HLA genotypes from Whole Exome/Genome Sequencing experiments. Omixon maintains an active grant-funded research program with a product pipeline focused on pre- and post-transplantation, and HLA genotyping applications beyond transplantation.