text:
Ben Larsen
March 19, 2022 @ 1:00pm - 4:00pm
An event every week that begins at 1:00pm on Saturday, repeating until April 30, 2022
« Bluebird Nest Box Build
"Regenerative & Organic Practices for Farm Resilience" »
Americana acoustic singer.
Harbor Ridge Winery
www.harborridgewinery.com
4690 Rainbow Ridge Rd
Egg Harbor, WI 54209 United States + Google Map
Acoustic Jam
February 1 @ 1:00pm - 3:00pm
An event every week that begins at 1:00pm on Wednesday, repeating until August 31, 2022
An event every week that begins at 1:00pm on Wednesday, repeating until December 14, 2022
One event on December 21, 2022 at 1:00pm
One event on January 4, 2023 at 1:00pm
One event on January 11, 2023 at 1:00pm
One event on February 1, 2023 at 1:00pm
One event on February 15, 2023 at 1:00pm
An event every week that begins at 6:00pm on Thursday, repeating until December 28, 2023
The String Benders
February 3 @ 6:30pm - 10:00pm


Frankie Arthur Real Estate is a team of talented professionals located in North Texas. We have over 20 years of combined experience in real estate. As a leading real estate business in DFW, we attribute our success to our focus on customer satisfaction. Our team has built a trusted reputation with a strong commitment to help our clients exceed their real estate goals; building lifetime relationships. We consider our clients family and only have their best interests at heart!
At Frankie Arthur Real Estate, whether we are working to sell a home or accompanying buyers, we make the experience enjoyable, seamless and efficient. By utilizing in depth market knowledge, sales skills, latest technology and partnerships, our team ensures clients expectations will be met.
Are you looking to buy or sell a home? Are you relocating or looking for investment property? Are you interested in new home construction? You deserve the best representation with your biggest investment! We invite you to utilize our website and call us with all of your real estate needs.


text:
package mbds.first.endtoend.applet;

import java.io.IOException;

import org.nfctools.NfcAdapter;
import org.nfctools.api.NfcTagListener;
import org.nfctools.scio.TerminalMode;
import org.nfctools.utils.LoggingUnknownTagListener;

public abstract class AbstractDemo {

	/**
	 * @param listeners
	 * @throws IOException
	 */
	protected static void launch(NfcTagListener... listeners) throws IOException {
		NfcAdapter nfcAdapter = new NfcAdapter(TerminalUtils.getAvailableTerminal(), TerminalMode.INITIATOR);
		for (NfcTagListener tagListener : listeners)
			nfcAdapter.registerTagListener(tagListener);

		nfcAdapter.registerUnknownTagListerner(new LoggingUnknownTagListener());
		nfcAdapter.startListening();
		System.out.println("Waiting for tags, press ENTER to exit");
		System.in.read();
	}
}

redpajama_set_name: RedPajamaGithub


Audit | WABCO Holdings, Inc.
(a) The quality and integrity of the Company's financial statements, financial reporting process and system of internal accounting and financial controls.
(c) The Company's compliance with legal and regulatory requirements and ethics policies.
The independent auditors' qualifications and independence.
(e) Audits of the Company's financial statements, including the performance of the Company's internal audit function and independent auditors.
(a)Be elected by the full Board and serve at the pleasure of the Board. Any vacancy on the Committee shall be filled by the Board. No member of the Committee shall be removed except by the Board.
(b) Be comprised of three or more independent Board members each having a working familiarity with basic financial and accounting practices as contemplated by the New York Stock Exchange ("NYSE") or acquiring such familiarity within a reasonable amount of time after his or her appointment to the Committee and one of which has "accounting or related financial management expertise" as required by the NYSE listing standards. The term independent shall have the meaning set forth in the Company's Definition of Director Independence.
(c) Have at least one member who is a "financial expert" under the requirements of the Sarbanes-Oxley Act of 2002 or as defined by the Regulations of the U.S. Securities and Exchange Commission ("SEC") promulgated thereunder.
(d) Meet at least quarterly and report regularly to the Board of Directors on its activities. Review with the full Board of Directors any issues that arise with respect to the quality or integrity of the Company's financial statements, the Company's compliance with legal or regulatory requirements, the performance and independence of the Company's independent auditors, or the performance of the internal audit function.
(e) Meet separately and periodically with management, the General Auditor and the independent auditors to review the quarterly and annual financial statements. Require that independent and internal auditors meet privately with the Committee to discuss issues and concerns warranting special attention.
(ii) discloses such determination in the annual proxy statement.
(g) Perform an evaluation of its performance annually to determine whether it is functioning effectively.
(h) Review and reassess the charter annually and obtain the approval of the Board of Directors for any amendments or modifications to the charter.
(i) Meet in periodic executive sessions and hold special meetings as circumstances warrant.
In fulfilling its responsibility, the Committee shall maintain free and open communication between the Committee, on the one hand, and the independent auditors, the internal auditors and management of the Company, on the other hand.
The Committee is empowered to investigate any matter brought to its attention with full access to all books, records, facilities, and personnel of the Company. The Committee shall have authority to engage outside counsel, experts and other advisors as the Committee may deem appropriate, and shall have a right to request any officer or employee of the Company or outside counsel, expert, advisor to attend a Committee meeting or to meet with any members of the Committee. The Committee shall have sole authority to approve outside counsel, experts and other advisors scope of work, fees and terms.
The primary responsibility of the Committee is to oversee the Company's financial reporting process on behalf of the Board of Directors and report the results of their activities to the Board. Management of the Company is responsible for the preparation, presentation, and integrity of the Company's financial statements and for the appropriateness of the accounting principles and reporting policies that are used by the Company and for establishing and maintaining internal control over financial reporting. The independent auditors are responsible for auditing the Company's financial statements and the effectiveness of internal control over financial reporting, and for reviewing the Company's unaudited interim financial statements.
(a) Be directly responsible for the appointment, compensation, retention and oversight of the work of the independent auditors.
(b) Instruct the independent auditors to report directly to the Committee.
(c) Pre-approve all audits and permitted non-audit services provided by the independent auditors. The Committee may delegate pre-approval authority to a member of the Committee.
(d) Pre-approve all statutory audits not performed by Company's independent auditors.
(e) Review with the independent auditors their overall scope and plans for their respective audits, including the adequacy of staffing and compensation.
(f) Oversee the resolution of any disagreements between management and the independent auditors regarding audit work or financial reporting.
(g) Review at least annually the qualifications, performance and independence of the independent auditors, including the lead partner, and present its conclusions with respect thereto, and its view on whether there should be a regular rotation of the independent auditors, to the Board.
(h) Annually obtain and review a report from the independent auditors describing their internal quality control procedures and any material issues raised by the most recent internal quality control or peer reviews of the independent auditors or by any inquiry or investigation of the independent auditors by governmental or professional authorities during the past five years and any corrective steps taken to deal with the issues.
(i) Annually obtain and review from the independent auditors a listing of all relationships between them and the Company, to assess the auditors' independence.
(j) Establish clear hiring policies for employees and former employees of the independent auditors that meet the SEC regulations and NYSE listing standards.
(k) Review with the independent auditors: (i) any audit or accounting adjustments proposed by the independent auditors but not undertaken by the Company, (ii) any issues presented by the engagement that may impair the independent auditors' effectiveness or difficulties caused by restrictions placed on the scope of activities or access to information; (iii) significant disagreements with management and management's response; (iv) any communications between the audit team and the audit firm's national office concerning auditing or accounting issues presented by the engagement; and (v) at least annually, obtain and review a report by the independent auditors on the adequacy of the Company's internal controls.
(a) Review the integrity and adequacy of the Company's internal and external financial reporting processes including management's assessment of the effectiveness of internal controls over financial reporting under Section 404 of the Sarbanes-Oxley Act including any material weaknesses or significant deficiencies identified, including the responsibilities, budget, compensation and staffing of the Company's internal audit function, at the end of the most recent fiscal year and the independent auditors' report on management's findings.
(b) Periodically review the effect of new regulatory and accounting initiatives and any off balance sheet structures on the financial statements of the Company.
(c) Meet with management and the independent auditors to review and discuss the company's quarterly press releases with respect to the Company's financial results and financial condition, which may also include financial guidance for the current or upcoming fiscal year. The Audit Committee is not required to discuss financial guidance and other financial information in advance of each press release or each instance in which the company may provide financial guidance or financial information.
(d) Meet with management and the independent auditors to review and discuss the interim financial statements and specific disclosures under Management's Discussion and Analysis of Financial Conditions and Results of Operations in each Quarterly Report on Form 10-Q, including the clarity of their disclosures.
(e) Meet with management and the independent auditors to review and discuss the audited financial statements and specific disclosures under Management's Discussion and Analysis of Financial Conditions and Results of Operations in the Annual Report on Form 10-K, including the clarity of their disclosures.
(f) Review and approve all related party transactions required to be disclosed pursuant to SEC Regulation S-K, Item 404, and discuss with management the business rationale for the transactions and whether appropriate disclosures have been made.
(a) Review with management the Company's administrative, operational and accounting internal controls, including any special steps adopted in light of the discovery of significant control deficiencies.
(b) Discuss with management its remediation plan to address internal control deficiencies.
(b) Review and concur in the appointment, replacement, reassignment, or dismissal of the General Auditor who shall have direct access to the Committee.
(c) Instruct the General Auditor to report directly to the Committee.
(d) Review with the General Auditor and approve overall scope and risk-based plans for internal audits, including the adequacy of budget and resources plan.
(e) Review with the General Auditor the results of executing the internal audit plan including management's remediation of audit findings.
(f) Review and approve the performance and compensation of General Auditor, with the input of the Company CFO and CHRO, and assess the effectiveness of General Auditor and the Internal Audit function.
(a) Review with the Company's legal counsel any legal matters, including but not limited to any actual or potential violations of laws or regulations or breaches of fiduciary duty or other matters that could have a material impact on the financial statements.
(b) Discuss with management and the independent auditors the Company's actions and activities concerning risk assessment and risk management, including the risk of fraud.
(c) Review and discuss with management, General Auditor and the independent auditors the Company's cybersecurity risk exposures, the measures management has taken to monitor and mitigate such risks including policies, processes and programs.
(d) Oversee compliance with the Code of Conduct and Ethics and determine that the Company has an effective compliance and ethics program.
(e) Establish and maintain procedures to ensure (i) the receipt, retention and treatment of complaints received by the Company regarding accounting, internal accounting controls, or auditing matters; and (ii) the confidential, anonymous submission by employees of the Company of concerns regarding questionable accounting or auditing matters.
(f) Review with management the processes and controls to prevent and detect fraud.
(g) Review the Company's environmental programs and any potential impact on the Company's financial statements.
(h) Review the Company's risk management program to ensure adequate insurance coverage is in place.
(i) Review the Company's treasury activities, including cash management, hedging, derivatives, foreign currency transactions, and use of new or unusual financial instruments.
(j) Review the Company's tax positions.
(k) Review the operation of the Company's pension plans with particular attention to actuarial assumptions, funding policy and investment results.
(a) Receive updates from management regarding reports or inquiries received from the SEC or IRS and its responses to those reports, if applicable.
(b) Prepare an annual agenda to ensure all pertinent topics are addressed.
(c) Annually review this Charter; perform an annual self-evaluation of the Committee and its compliance with this Charter.
(d) Prepare any Audit Committee reports required in connection with the annual proxy statement.
(e) Provide recommendations to the Board, as the Committee deems appropriate.
(f) Maintain minutes and records of the meetings and activities.
(g) Perform such additional activities, and consider such other matters, within the scope of its responsibilities, as the Board deems necessary or appropriate.
This charter is effective as of December 7, 2018.


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text:
Seaspan welcomes two new dual-fuel ferries to its fleet
LNG Industry, Monday, 10 April 2017 11:30
Seaspan Ferries Corp. (SFC) has announced that it has held a double commissioning ceremony at its Tilbury Terminal for two new, state-of-the-art, dual-fuelled/hybrid (LNG, diesel and battery) vessels.
The two vessels – Seaspan Swift and Seaspan Reliant – are the first eco-ferries of their kind in North America and were formally christened by sponsors Christy Clark (Premier of British Columbia) and Anisa White (wife of Doug White III, Councillor and Chief Negotiator for Snuneymuxw First Nation).
The President of SFC, Steve Roth, said: "April 9, 2017 will be remembered as a historic milestone for Seaspan Ferries and we are extremely fortunate to have two sponsors who represent our connection to both the past and future to help bring our new vessels to life.
"First Nations communities have a deep and sacred connection to the sea, and together, we share the responsibilities and commitment to keeping our pristine waters safe and clean. At the same time, we take great pride in the role we play as an economic driver for British Columbia by investing in new technology and equipment that keeps goods moving and creates both direct and indirect jobs."
Christy Clark added: "In job creation, reliability, and marine transportation, Seaspan has always been a leader – and with the addition of two new eco-ferries, Seaspan is continuing to lead.
"The Seaspan Swift and Seaspan Reliant are the first of the next generation of vessels fuelled by B.C.'s clean-burning LNG."
Both vessels were constructed at the Sedef Shipyard in Istanbul, Turkey, and are 148.9 m long, and capable of accommodating up to fifty-nine 53' trailers. The Seaspan Swift arrived in December 2016 and commenced operations in January 2017. The Seaspan Reliant, meanwhile, arrived in late February, and is scheduled to commerce service in April 2017.
Roth went on to say: "The introduction of two new technologically advanced, LNG-fuelled vessels represents the beginning of the realisation of a bold and ambitious vision to re-construct our marine terminals and modernize the vessels that service them.
"The Seaspan Swift and Seaspan Reliant reinforce an unwavering commitment to our drop-trailer customers and serve as a testament to how we live out our Core Value of Care for the Environment by ensuring the conservation of Canadian oceans and waterways, reducing greenhouse gas emissions dramatically, while delivering the highest level of efficiency, performance and reliability."
Read the article online at: https://www.lngindustry.com/small-scale-lng/10042017/seaspan-welcomes-two-new-dual-fuel-ferries-to-its-fleet/
British Columbia (B.C.) LNG news Canada LNG news First Nations LNG news

redpajama_set_name: RedPajamaCommonCrawl


Search here for Banana peel clipart collection out of 50 ready to use vector and photo images. You can use them for free.
Already 461 visitors found here solutions for their art work. 7 users visited banana peel clipart this week.
Here some moast popular keywords people search on out site: peel, banana, vector, clip, cartoon, images, stock, mario, free, illustrations, image, slipping, royalty, kart, this, wikia, illustration, shutterstock, download, clker.


Patentwire has been empanelled by various Government Organizations viz. TIFAC, ICAR, Delhi Metro Rail Corporation, Ministry of Skill Development and Entrepreneurship etc.
Patentwire is proud to be associated with top engineering and research institutes viz. IIT-BHU, IIT-Hyderabad, IIT-Guwahati, North Maharashtra University, Jamia Hamdard, etc.
Patentwire has evolved as a one stop shop for securing IP rights, developing prototype and commercializing.


text:
Phoenixville Area School District Celebrates New Early Learning Center and Manavon Elementary School Opening with a Cookout
On August 28th, Phoenixville Area School District welcomed parents, students, and the entire community to tour the New Early Learning Center and Manavon Elementary School. It was a sight to see, the parking lots were full and there was an attendance of over 2,500 people. The sidewalks, tables, and playgrounds made it obvious that it was a successful turn out.
Guests indulged themselves in self-guided tours of the new facility, played on the play areas, joined in soccer clinics on the fields, enjoyed grilled burgers and hot dogs (compliments of the School District), and were entertained by the upper grade musical entertainment outside.
The evening was a resounding success, to celebrate the opening of the new school. Many people offered unsolicited comments. Middle school students were overheard saying, "I wish I could go back to third grade!" The central media center (located in the open core of this building) was an obvious success, as it was impossible for guests to not sit down and pick up a book. Children were so excited about the colorful and fun furniture pieces that all had to 'test' them out.
SCHRADERGROUP (SG) applauds the Phoenixville Area School District for its progressive educational approach and wishes the best for the Phoenixville community in its use of this Early Learning Center and Manavon Elementary School for many generations to come!
Ready to start a project? Let's chat.
Copyright © 2021 Schradergroup. All Rights Reserved.
Website by Cohere


Released: FeedWordPress 2012.1218. WordPress 3.5 compatibility, PHP 5.4 compatibility, bug fixes, diagnostics improvements, and a great deal more.
Posted in Add Attribution for FeedWordPress | Comments Off on Released: FeedWordPress 2012.1218. WordPress 3.5 compatibility, PHP 5.4 compatibility, bug fixes, diagnostics improvements, and a great deal more.


Fear freezes us. That's exactly what the enemy wants, he wants us to freeze in the race, to freeze when we step out of our comfort zone. We're running a spiritual relay race and have the responsibility of passing on the baton when we get to the end of our run. If fear freezes us, how do we pass on the baton? Where's the fruit? We've stopped running our race. He calls us to remain in our race, to remain in him, to remain in his love — where fear is cast out. God calls us to live radical lives outside of our comfort zones.
If we root everything we do and everything we are in God's unending, unconditional, perfect love, fear has no chance of freezing us in the race.
Comparison is the thief of joy and the greatest tool of the enemy. The enemy uses comparison to attack the mind, leading to feelings of self-doubt, insignificance and incapability. No wonder the writer of Proverbs says, "Above all else, guard your thoughts, because they are the source of true life".
The most heartbreaking form of comparison is within or between ministries. In this race, we're all running with the same goal, wearing the same kit … Let's keep our eyes fixed on our the author and perfecter of our faith, not the runners in our neighbouring lanes. When a runner takes their eyes off the finish line, they stumble, they fall — the effects are no different in our spiritual race. Stick to your lane. You are wonderfully and fearfully made to run it.
Who likes a big-headed athlete? Not me. Today's culture tells us we must look good to others, we must be recognised for self-sufficiency and must reel in the earthly praise. I'm not saying that we should reject all praise — we need encouragement! What I am saying is that we need to know our Kingdom culture. Jesus says that the first will be last and the last will be first … Very different to our cultural mindset! Let's take on Jesus' instruction to do good deeds in private, where only God can see. Let's know that our sufficiency is from God — he will reward us and we will discover the freedom that comes from a life lived to please God rather than humans.
So allow God to spiritually construct you, rather than allowing the world to socially construct you.
What is hindering you? Throw it off.
Emma is 18 years old and is on the New Wine Discipleship Year at Trinity Cheltenham, interning in Kids Ministry. Alongside this she runs Girls Nights for Trinity Youth where topics such as worth, value and identity are explored. She is going to the University of Gloucestershire in September to study Primary Education.
Throw it off! was originally published in New Wine on Medium, where people are continuing the conversation by highlighting and responding to this story.
Read the responses to this story on Medium.


Three words that best describe Nathaly are shy, responsible and organized. "I am extremely shy when it comes to meeting new people or talking in front of a crowd," says Nathaly. "I am responsible, even though sometimes I wait until the last minute to do everything, yet I still get it all done! And, I am a very organized when it comes to my business and work. Three things that people may not know about me include: I am a receptionist at a doctor's office; I have a 1-year-old son named Felix, two Chihuahuas named Twinky and Muffin; a German Shepard named Mia; and a loving boyfriend named Cesar who supports me in every way."
"To be honest, I had never heard of Apriori Beauty. I couldn't even pronounce it! My good friend, Lisa Minogue told me all about it and I was in. I signed up and bought my products the same day. As soon as I received them, on my face they went and I was in love. I knew I could do this from the comfort of my own home and get paid for my efforts. I believe everything happens for a reason!"
"What inspires me? Myself! I know I have the ability to do whatever I want, when I want to. I have the choice to wake up every morning and be a stay-at-home mom. I love how Apriori Beauty has given me the opportunity to choose what I want to do with my business. The sky is the limit for me and for many of us here at Apriori Beauty, but it's up to each of us to do what we want with it.
"Thank You to Elizabeth and Susan for all of the hard work they have put into creating this company so that all of us can have this opportunity."
"I love ALL of the Apriori Beauty products!"


text:
Idea Village Recalls Wireless Light Switches Due to Fire Hazard
October 22, 2009 | Staff
Home / News / Community / Community News
WASHINGTON, D.C. – The U.S. Consumer Product Safety Commission, in cooperation with the firm named below, today announced a voluntary recall of the following consumer product. Consumers should stop using recalled products immediately unless otherwise instructed.
Name of Product: Handy Switch, Wireless Light Switches
Units: About 1.3 million
Distributor: Idea Village Products Corp., of Wayne (formerly of Fairfield), N.J.
Hazard: The light switch receiver, which fits into the wall outlet, can overheat and pose a fire hazard to consumers.
Incidents/Injuries: The firm has received 14 reports of overheating, including nine reports of fire, five of which involved property damage such as minor damage to curtains, bedding or walls.
Description and Model: The Handy Switch is a white plastic wireless remote light switch with model number KS-080 printed on the back of the receiver. On the front of the receiver is a blue electroluminescent nightlight.
Sold at: Mass merchandisers and drug store chains nationwide, through television infomercials and the Internet from March 2007 through July 2009 for between $10 and $15.
Remedy: Consumers should immediately unplug and stop using the product and contact IdeaVillage to learn about free remedy options.
2018 Midsouth Super Lawyers, Rising Stars list names...
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BeasleyAllen.com racecar driver Grant Enfinger claimed his first NASCAR Camping World Truck Series...


text:
Sam Mostyn (she/her)
FYA Chair, Finance Committee
Sam Mostyn is a businesswoman and sustainability adviser, with a long history of executive and governance roles across business, sport, climate change, the arts, policy, and NFP sectors. She is current President of Chief Executive Women, Chair of Women's Economic Equality Taskforce and serves on boards of Mirvac and the Climate Change Authority; alongside a broad portfolio of other roles including chairing Ausfilm, Climate Council, and the Centre for Policy Development. Sam was awarded the Order of Australia in 2021 for distinguished service to business and sustainability, and to the community, through seminal contributions to a range of organisations, and to women.


text:
Who's Your Mama, Are You Catholic, Can Y...
Description: Who's Your Mama, Are You Catholic and Can You Make a Roux? is an authentic look at the..
Top 100 New Orleans Recipes...
New Orleans recipes, served in the city's fine restaurants can be found in the pages of this cookboo..
Top 100 Cajun Recipes...
The introduction stated this book to be The Cream of the Crop of Cajun Recipes. These recipes help t..
Talk About Good!...
Description: Talk About Good!, published by the Junior League of Lafayette, takes you on a journey t..
Talk About Good II...
Description: Much like its predecessor, Talk About Good!, the Junior League of Lafayette brings you ..
Rice Cooker Meals...
Fast home cooking for busy people! 61 meals you can cook in an inexpensive ricer cooker, most of whi..
No Baloney On My Boat...
Recipes to be enjoyed in the GREAT OUTDOORS!No Baloney on My Boat! is a cookbook filled with do..
Down Home CAJUN COOKING Favorites...
Down-Home Cajun Cooking Favorites is a collection of classic recipes from moms and dads, aunts and u..
Cajun Cooking for Beginners...
Description: Authored by Marcelle Bienvenu, this 48-page saddle-stitched soft cover book teaches the..
Best of the Best from Louisiana...
Description: "Best of the Best from Louisiana" has gathered together, as nearly as is possible in on..


text:
Research Article | Spotlight
Noncatalytic Antioxidant Role for Helicobacter pylori Urease
Alan A. Schmalstig, Stéphane L. Benoit, Sandeep K. Misra, Joshua S. Sharp, Robert J. Maier
Conrad W. Mullineaux, Editor
Alan A. Schmalstig
Department of Microbiology, University of Georgia, Athens, Georgia, USA
Stéphane L. Benoit
Sandeep K. Misra
Department of BioMolecular Sciences, School of Pharmacy, The University of Mississippi, Oxford, Mississippi, USA
Joshua S. Sharp
Robert J. Maier
Conrad W. Mullineaux
The well-studied catalytic role of urease, the Ni-dependent conversion of urea into carbon dioxide and ammonia, has been shown to protect Helicobacter pylori against the low pH environment of the stomach lumen. We hypothesized that the abundantly expressed urease protein can play another noncatalytic role in combating oxidative stress via Met residue-mediated quenching of harmful oxidants. Three catalytically inactive urease mutant strains were constructed by single substitutions of Ni binding residues. The mutant versions synthesize normal levels of urease, and the altered versions retained all methionine residues. The three site-directed urease mutants were able to better withstand a hypochlorous acid (HOCl) challenge than a ΔureAB deletion strain. The capacity of purified urease to protect whole cells via oxidant quenching was assessed by adding urease enzyme to nongrowing HOCl-exposed cells. No wild-type cells were recovered with oxidant alone, whereas urease addition significantly aided viability. These results suggest that urease can protect H. pylori against oxidative damage and that the protective ability is distinct from the well-characterized catalytic role. To determine the capability of methionine sulfoxide reductase (Msr) to reduce oxidized Met residues in urease, purified H. pylori urease was exposed to HOCl and a previously described Msr peptide repair mixture was added. Of the 25 methionine residues in urease, 11 were subject to both oxidation and to Msr-mediated repair, as identified by mass spectrometry (MS) analysis; therefore, the oxidant-quenchable Met pool comprising urease can be recycled by the Msr repair system. Noncatalytic urease appears to play an important role in oxidant protection.
IMPORTANCE Chronic Helicobacter pylori infection can lead to gastric ulcers and gastric cancers. The enzyme urease contributes to the survival of the bacterium in the harsh environment of the stomach by increasing the local pH. In addition to combating acid, H. pylori must survive host-produced reactive oxygen species to persist in the gastric mucosa. We describe a cyclic amino acid-based antioxidant role of urease, whereby oxidized methionine residues can be recycled by methionine sulfoxide reductase to again quench oxidants. This work expands our understanding of the role of an already acknowledged pathogen virulence factor and specifically expands our knowledge of H. pylori survival mechanisms.
Persistent Helicobacter pylori infections can cause chronic gastritis, peptic ulcer disease, and gastric cancer (1–4). Decades of immune response to the infection can lead to chronic inflammation and tissue damage (5, 6). H. pylori must first survive the harsh conditions of stomach gastric acid and then a prolonged host immune response after its colonization of the gastric mucosa (5). To combat the low pH of the stomach lumen, H. pylori expresses urease, which catalytically converts urea into ammonia and carbon dioxide, the former enabling H. pylori to resist the acidic gastric environment (7–9). Urease has been shown to be the most highly expressed protein in H. pylori, making up to 10% of the total protein content of the gastric pathogen (10). The enzyme consists of two structural proteins, UreA and UreB, with 12 subunits each that form a 1.1-MDa dodecamer (11). The UreAB heterodimer contains 25 methionine (Met) residues, 3% of the total amino acid content (12). Enzyme activity requires the addition of two nickel atoms per UreB monomer, and such maturation is facilitated by the urease accessory proteins UreEFGH and the hydrogenase maturation proteins HypAB (10, 13–15).
Although the catalytic activity of urease is required for initial colonization, (10, 16–18) several studies also indicate that non-acid-neutralizing roles may exist for urease. For instance, urease has been shown to be required for persistence in the mouse gastric mucosa, where the pH approaches neutrality (16, 19) and a urease-negative strain was unable to colonize a pH neutral piglet stomach (20). In addition, using a tetracycline-inducible system to turn on or off urease expression, Debowski et al. were able to show that urease-expressing bacteria were selected over time in a murine infection model, indicating that urease is required for chronic infection (16). Additionally, H. pylori urease has been shown to play multiple roles in modulating the host immune response. Indeed, it was shown to decrease opsonization (21), stimulate the chemotaxis of neutrophils and monocytes (22), induce apoptosis in gastric epithelial cells after binding to class II major histocompatibility complex (MHC) receptors (23), and induce proinflammatory cytokines (24). Recently, urease has been linked to the formation of gastric carcinoma through an ability to promote angiogenesis (25).
After reaching the gastric epithelium, H. pylori triggers responses by the host innate immune cells, which respond to the infection by generating reactive oxygen species (ROS) such as superoxide anion (O2−), hydrogen peroxide (H2O2), hydroxyl radicals (˙OH), and hypochlorous acid (HOCl) (10, 26, 27). Indeed, the exposure of gastric cells (28) or phagocytes (29) to H. pylori increases host cell ROS production. Patients with H. pylori infections have been shown to have larger amounts of ROS in their gastric mucosa (30). ROS can damage protein, DNA, and lipids (31). With regard to proteins, the amino acids most susceptible to oxidation are Met and cysteine (Cys) due to their sulfur-containing ligands (32).
H. pylori has many mechanisms to protect itself from, as well as repair damage caused by, oxidative stress. For instance, catalase and superoxide dismutase act to convert H2O2 and O2− into less harmful products. Catalase was recently shown to protect H. pylori against oxidative damage via an oxidant-quenching mechanism of its Met residues (33). The reaction involves methionine sulfoxide reductase (Msr) to reduce Met-SO to Met (34, 35). Msr reduces oxidized methionines of damaged proteins and has been shown to restore function to the damaged protein (36, 37). Cross-linking and direct repair assays showed that H. pylori Msr has at least five repair target proteins (34, 38, 39). These include AhpC, UreG, GroEL, catalase, and a site-specific recombinase (SSR), but urease was not observed as a repair target in those studies. In the present study, we describe the evidence for a noncatalytic role for urease, which involves Met-S/Met-SO recycling. This role aids in protecting the pathogen against oxidation-mediated cell death.
(A preliminary account of this work was presented at the American Society for Microbiology Microbe, New Orleans, LA, 1 to 5 June 2017 [40].)
Construction of catalytically inactive urease.Strains that synthesize inactive urease (herein referred to as apo-urease) were constructed to determine if the catalytically inactive enzyme is sufficient to quench oxidants. A sucrose-kanamycin selection-counterselection was used to make markerless single-amino-acid substitutions in the UreB subunit of urease. The three single substitutions (His136Ala, His138Ala, and Lys219Ala) were constructed to replace nickel binding residues known to be required for enzyme activity (41). As expected, the three site-directed strains (ureBH136A, ureBH138A, and ureBK219A strains) lacked urease activity compared to the wild-type (WT) strain 43504 (Table 1). Next, we sought to verify that the lack of urease activity was not linked to a decrease or loss of urease, e.g., that the mutant strains were still making apo-urease. Since urease is the most abundant protein in H. pylori, both UreA (27 kDa) and UreB (62 kDa) structural subunits can readily be visualized on Coomassie-stained SDS-PAGE gels (Fig. 1A). UreAB levels in the three site-directed mutants were found to be similar or slightly less than WT levels (Fig. 1A). This was confirmed by immunoblot, using anti-UreA antiserum (Fig. 1B). No UreA-specific band was seen in the ΔureAB deletion strain, our negative control. These results indicate that apo-urease is still synthesized in each of the three site-directed mutants.
Urease activity of H. pylori wild-type, urease deletion, and site-directed mutant strains
Urease in cell-free and whole-cell extracts of WT and ureAB mutants. (A) SDS-PAGE (12.5%) analysis of cell-free extract. (B) Immunoblot analysis. Whole-cell extracts were separated via 12.5% SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-UreA antibodies. Strains are indicated above each lane. UreA and UreB are indicated.
Catalytically inactive urease confers resistance to HOCl.The three catalytically inactive strains, as well as the WT (positive control) and the ΔureAB deletion strain (negative control), were exposed to HOCl to determine their ability to quench an oxidant and survive. The cells were incubated with HOCl for 1 h, diluted, and the CFU were counted (Fig. 2). The catalytically inactive mutants were as resistant as the WT and significantly (P < 0.01) less sensitive to HOCl oxidative damage than the urease gene deletion strain (Fig. 2). This suggests that the presence of urease, independent of its catalytic activity, can play a role in preventing cell death due to oxidative damage.
Apo-urease confers resistance to HOCl. Cells grown on BA plates with 10 μM NiCl2 were incubated with PBS or with PBS supplemented with 100 μM HOCl for 1 h at 37°C, diluted, and plated. CFU were counted after 72 h at 37°C. Error bars indicate standard deviations from 4 to 10 independent experiments.
Both holo-urease and apo-urease protect against HOCl-mediated killing.To determine if holo-urease (purified from WT strain 43504) and apo-urease (purified from either strain ureBH138A or strain ureBK219A) were able to protect against HOCl-mediated oxidative damage, each purified urease was preincubated with the oxidant for 15 min and the mixture was added to whole nongrowing WT or ΔureAB cells. Cell survival was then monitored and compared to that with a HOCl-only treatment (no protein) (Fig. 3). While no cells were recovered from the HOCl treatment alone (detection limit, 102 CFU/ml), when HOCl was preincubated with purified urease (both holo and apo), the cell recovery levels reached those of cells without HOCl treatment (Fig. 3). This suggests that purified urease itself can quench HOCl, irrespective of urease activity. Interestingly, the ΔureAB strain, shown to be more sensitive than the WT to HOCl challenge (Fig. 2), was almost fully protected against HOCl that had been preincubated with purified urease (Fig. 3). An additional protein, UreE, with a low Met content (1%) was shown to not protect against the HOCl challenge, suggesting that Met content might contribute to the protective effect.
Holo- and apo-urease proteins protect against HOCl. Wild-type and ΔureAB 43504 cells were incubated for 1 h in PBS, PBS with HOCl, or PBS with HOCl that had been previously incubated for 15 min at 37°C with either purified UreABWT, UreABH138A, UreABK219A, or UreE as indicated below each column. Final protein concentration was 0.25 μM, and final HOCl concentration was 200 μM in each reaction. Error bars indicate standard deviations from 3 to 9 independent experiments. ND, no CFU could be detected (detection limit, 102 CFU/ml).
MS/MS identification of oxidized and reduced methionine residues in UreAB.Urease is abundantly expressed, is found outside cells as well as in the cytosol, and contains many Met residues (25 of 807 residues, accounting for 3% of the total residues) (10, 42, 43). To identify which Met residues of urease (UreAB) are oxidized and repaired, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS). The urease holoenzyme was first oxidized with HOCl, and then a sample of oxidized urease was subsequently incubated with an Msr repair mixture. Oxidized, oxidized and repaired, and untreated samples were analyzed by LC-MS/MS. Of the 25 methionine residues in urease, 19 were detected by LC-MS/MS (Fig. 4). UreA Met1 and UreB Met1, Met104, Met182, Met191, and Met478 were either not detected or could not be quantified reliably. An examination of the protein sequence shows that four of these methionines reside in peptides containing multiple basic residues that were digested by trypsin resulting in small hydrophilic fragments, making their detection by LC-MS/MS highly unlikely. A fifth methionine-containing peptide was not included in the data analysis, as the signal-to-noise ratio was far too low for reliable quantitation. Most of the detected Met residues in the unoxidized samples had low levels of oxidation (less than 0.15 average oxidation per peptide) except UreA Met71, Met77, and Met86 and UreB Met262. HOCl-treated Met residues UreA Met44, Met71, Met77, and Met86 and UreB Met12, Met262, Met315, Met317, Met319, Met353, and Met366 had greater than 0.8 average oxidation per peptide. UreA Met12 and UreB Met448 showed approximately 0.5 average oxidation per peptide, and all other residues showed less than 0.5 average oxidation per peptide. After HOCl-oxidized urease was treated with an Msr repair mixture, most Met residues were reduced to levels similar to that observed for the unoxidized sample, with the exception of UreB Met12, Met262, Met353, and Met366. Most of the digested peptides contained a single Met residue, the exceptions being UreA peptide 63 to 92 and UreB peptide 289 to 326, which contain three Met residues each (Met71, Met77, and Met86 and Met315, Met317, and Met319, respectively). Likewise, UreB peptide 339 to 368 contains two Met residues (Met353 and Met366). For peptides with more than one Met residue, the oxidation of each Met cannot be accurately quantified by collision-induced dissociation (CID) MS/MS (44, 45). In summary, the Msr repair mixture reduced 11 of the oxidized Met residues to close to the baseline non-oxidant-treated levels. This suggests that oxidized urease can be reduced by Msr and thus probably recycles the oxidant-quenchable pool of urease.
LC-MS/MS analysis of oxidized and Msr-repaired urease. Purified urease was incubated with a 60-fold molar excess of HOCl for 15 min. Excess HOCl was quenched with 15 mM Met and removed via dialysis. After dialysis, oxidized urease was incubated with purified Msr, Trx1, TrxR, NADPH, and DTT for 2 h at 37°C. Urease samples were digested by trypsin, and oxidation levels of methionine residues were quantified by LC-MS/MS. The oxidation level for the untreated sample was below the limit of detection for UreA Met58. White bars, untreated; black bars, HOCl treated; gray bars, treated with HOCl plus Msr repair mixture. Error bars indicate standard deviations from 2 independent experiments with 3 replicates each.
Previous studies have focused on the catalytic by-products of urea hydrolysis by urease (NH3 and CO2) and their contribution to pathogen virulence. The ammonia produced by the enzyme supplements nitrogen metabolism, promotes alkalinization, and contributes to damage of host cell tissue (46). Additionally, carbon dioxide has been shown to protect the bacterium against highly toxic peroxynitrite during H. pylori infection (47). Now, there is growing evidence that urease plays noncatalytic roles in H. pylori infection. Indeed, urease is required for persistence in the mouse gastric epithelium, where the pH approaches neutrality (16, 19). Urease has been shown to play roles in modulating the immune response to infection (21–24), and ureA was found to be upregulated in response to oxidative stress (48).
We demonstrate that urease has a previously undescribed role as an antioxidant, thus adding to the growing number of noncatalytic roles that urease can play. Indeed, some of the observations cited above (e.g., urease requirement for persistence even at neutrality) may be due to an antioxidant role. This role depends at least in part on the primary sequence of the protein (e.g., Met residues). Although gastric acid exposure is an impressive obstacle to overcome, H. pylori also survives a barrage of host-produced ROS, including HOCl, which can reach levels as high as 5 mM at inflamed sites (49). H. pylori survives this via multiple mechanisms of detoxification and repair, enabling the infection to oftentimes persist throughout the life span of the host. Nickel-deficient mutants that lacked catalytic activity were as resistant as the WT to HOCl-mediated oxidative stress. In contrast, a urease deletion mutant was much more sensitive than the WT to HOCl-mediated oxidative stress. Thus, catalytic activity is not required for combating the oxidant. Rather, we propose that the Met residues of urease can quench oxidants, forming Met-SO in the process. Catalase, another abundant protein, was similarly shown to quench oxidants to aid bacterial survival, and the mechanism was independent of catalytic activity (33). A similar antioxidant mechanism of relying on Met residues was assigned to pure E. coli glutamine synthetase; however, the system was not shown to confer oxidative stress resistance to cells (50).
We predict that Met-SO residues of urease could be reduced by Msr and then used again to quench oxidants. Indeed, Msr was previously shown to reduce and repair several H. pylori proteins such as AhpC, UreG, GroEL, catalase, and site-specific recombinase (SSR) (34, 38, 39). However, it was previously unknown whether Msr could reduce urease Met residues. Through mass spectrometry analysis, we found that of the 25 Met residues in urease, 11 were shown to be susceptible to oxidation and were reduced (i.e., repaired) by Msr. Thus, urease can be added to the list of Msr repair targets. Upon PDBePISA analysis of surface accessibility, using the X-ray crystal structure of H. pylori urease (Protein Data Bank code 1E9Z), 9 of the 11 Met residues that were oxidized and then subsequently reduced are surface exposed (11, 51). This is consistent with previous reports of surface methionine residues being subject to oxidation and subsequent repair by Msr (26, 38). H. pylori Msr deletion mutants showed lowered urease activity when exposed to oxidative stress (21% O2) (38). This was attributed to the inability of the strain to repair UreG, a urease accessory protein required for urease activity; however, we can now hypothesize the lowered activity could also be due to the lack of reduction of oxidized Met residues of the structural proteins of urease (UreAB).
An Msr deletion mutant has been shown to be more sensitive to HOCl challenge than its parent strain (SS1) (39). Here, we show a high HOCl sensitivity of a strain lacking one of the described repair targets of Msr, namely, urease. Conclusions based on comparing the phenotypes of the ΔureAB strain to that of the Δmsr strain (39) are not possible due to the use of different parent strains. Comparisons are further complicated by the fact that Msr has multiple repair targets besides urease.
While urease lacks a described secretion pathway in H. pylori, it has been found outside the cell, possibly due to cell lysis (42, 52). We show that purified urease added exogenously and whole cells expressing urease quench HOCl and survive HOCl challenge, respectively. Regardless of location, urease activity is not required for the described oxidant quenching effect. Seemingly, both extracellular and intracellular ureases likely act to quench oxidants. However, intracellular urease may play a larger role in protecting against oxidants because of its Met turnover—Msr is unlikely to be active extracellularly. Although this study focused on the role of Met residues in urease oxidant quenching, the Cys residues of urease might also contribute to the antioxidant ability we describe. While Cys residues are very sensitive to oxidation, the UreAB heterodimer only contains 4 Cys residues compared to 25 Met residues. Oxidized Cys residues could be reduced enzymatically through the thioredoxin/thioredoxin reductase (Trx/TrxR) system (32). H. pylori contains two thioredoxins (Trx1 and Trx2), which have been shown to play a role in preventing macromolecule damage, and one TrxR; however, it is not known if the Trx system can repair oxidized Cys residues in H. pylori (53, 54).
Future site-directed mutagenesis studies of individual Met residues in urease could elucidate the role of individual residues in the quenching. Also, the use of the mouse model to gauge colonization by the catalytically inactive strains is likely to be a promising approach to assess in vivo function, although the acid barrier would have to be minimized to observe colonization by urease-activity-negative strains. Catalytically inactive urease may contribute to the persistence of H. pylori infections of the host gastric epithelium, where the pathogen is exposed to multiple oxidants (30).
Although the role of urease in H. pylori pathogenesis has been extensively studied, urease is also a virulence factor for several pathogens, including Proteus mirabilis and Klebsiella pneumoniae (55). During P. mirabilis infection of the urinary tract, urease is required for mineral precipitation to form urinary stones, which impede the elimination of the infection (56). During mice gastrointestinal tract infections, urease-negative strains of K. pneumoniae were outcompeted by WT strains, and so it was suggested that the ammonia produced by urease serves as a supplementary nitrogen source (57). K. pneumoniae and P. mirabilis must also defend against HOCl produced by neutrophils as a part of the innate immune response to infection (56, 58). These species also contain Msr, enabling the possibility that a similar method for urease methionine oxidation and repair by Msr would be found in additional pathogens. Additionally, other highly expressed proteins that are Met rich might play a similar antioxidant role. Indeed, catalase, a Met-rich and abundant H. pylori protein, has been shown to have similar noncatalytic antioxidant properties (33).
Urease and Msr can be found in plants, fungi, archaea, and bacteria (59, 60). Throughout the three domains of life, urease is increasingly seen as a "moonlighting protein" with a variety of noncatalytic roles, including fungicidal, insecticidal, and proinflammatory effects (61). For H. pylori, an antioxidant mechanism based on urease Met residue recycling is expected to explain at least part of the in vivo requirement for this important enzyme.
Growth conditions.Escherichia coli cells were cultured at 37°C aerobically in Luria-Bertani (LB) broth or on agar LB plates with 100 μg/ml ampicillin and 30 μg/ml chloramphenicol as needed. H. pylori cells (ATCC 43504) were routinely grown at 37°C on brucella agar plates supplemented with 10% defibrinated sheep blood (BA) under microaerophilic conditions (4% O2, 5% CO2, and 91% N2). As needed, H. pylori was grown on BA supplemented with 5 or 10 μM NiCl2, 5% sucrose, 20 μg/ml kanamycin, or 30 μg/ml chloramphenicol. For urease protein purification, H. pylori cells were grown on BA plates supplemented 5 μM NiCl2.
Strain construction.The E. coli and H. pylori strains and plasmids used in this study are listed in Table 2. Site-directed mutants were constructed using a kanamycin-sucrose selection-counterselection system described previously (33, 39, 62). Briefly, a 1,400-bp DNA fragment containing a partial ureB gene was amplified by PCR using primers UreBF1 and UreBR1 (Table 3) from the chromosomal DNA of H. pylori 26695 (12) and cloned into pBluescript KS(+) to obtain pKS-ureB. The Kanr-sacB-PflaA (KSF) cassette (62) was inserted into ureB resulting in pKS-ureB::KSF, which was then naturally transformed into WT strain 43504, thereby replacing parent ureB with ureB::KSF. The resulting strain (ΔureB::KSF strain) was found to be urease negative and kanamycin resistant. Site-directed mutations were introduced to pKS-ureB by overlapping PCR using internal primers UreB:H136AF, UreB:H136AR, UreB:H138AF, UreB:H138AR, UreB:K219AF, and UreB:K219AR and external primers UreBF1 and UreBR1 (Table 3). The resulting plasmids (pKS-ureBH136A, pKS-ureBH138A, and pKS-ureBK219A) were introduced into the ΔureB::KSF strain by natural transformation. UreB site-directed mutants were selected on BA plates containing 5% sucrose. The resulting strains were sucrose resistant and urease negative. The ΔureAB strain was constructed as described previously (15). All plasmids and PCR products were sequenced at the Georgia Genomics Facility, University of Georgia, Athens, GA, and compared with DNA sequences from strain 26695 (12) to ensure that no error had been introduced following PCR amplification, as well as to verify the presence of engineered site-directed mutations within ureB.
Strains and plasmids used in this study
Primers used in this paper
Urease assays.Whole cells grown on BA plates under microaerophilic conditions at 37°C were resuspended in 50 mM HEPES (pH 7.5), lysed by sonication (Heat Systems Ultrasonics sonicator: 30 s at 4-W output power and 50% duty cycle), and centrifuged at 21,100 × g for 5 min. The cell-free supernatant was assayed for urease activity following the phenol-hypochlorite (Weatherburn) method (63). Protein concentration was determined using the bicinchoninic acid (BCA) kit (Thermo Scientific Pierce).
SDS-PAGE and immunoblotting.Whole cells grown on BA plates supplemented with 10 μM NiCl2 were resuspended in phosphate-buffered saline (PBS) and treated as above to obtain cell-free extracts (CFE). Total protein from CFE (0.4 μg protein per lane) was loaded onto 12.5% SDS-PAGE gels and stained with Coomassie brilliant blue G-250 or transferred to a nitrocellulose membrane for immunoblotting as previously described with modifications (33, 38). Briefly, the nitrocellulose membrane was blocked with 3% gelatin in Tris-buffered saline (TBS; 50 mM Tris [pH 7.6] and 150 mM NaCl [pH 7.6]). After blocking, the membrane was incubated with anti-UreA antibody (rabbit polyclonal; Santa Cruz Biotechnology) diluted 1:2,000 in TBS with 0.1% Tween 20 (TTBS) containing 1% gelatin for 1 h. The membrane was then washed with TTBS and incubated with goat anti-rabbit IgG conjugated to alkaline phosphatase (Bio-Rad) diluted 1:2,000 in TTBS with 1% gelatin. After 1 h of incubation, the blot was developed with Nitro Blue Tetrazolium–5-bromo-4-chloro-3-indolyl phosphate in 10 mM Tris-HCl (pH 9.5) and 150 mM NaCl.
Whole-cell HOCl tolerance assay.Whole-cell HOCl tolerance assays were performed as previously described (33) with the following modification. Briefly, cells grown on BA plates with 10 μM NiCl2 were resuspended in PBS to an optical density at 600 nm (OD600) of approximately 1.1 and then mixed with either PBS or PBS with NaOCl (100 μM final concentration, 10 to 15% available chlorine [referred to as HOCl]) for 1 h at 37°C. The HOCl-challenged cells were diluted in PBS and plated on BA plates, and the CFU were counted after 72 h of incubation at 37°C under microaerophilic conditions.
Purified protein HOCl tolerance assay.HOCl tolerance assays with purified proteins were performed as previously described (33). Briefly, wild-type and ΔureAB 43504 cells (OD600 of 1.3) were incubated for 1 h in PBS, PBS with 200 μM HOCl, or PBS with HOCl that had been previously incubated with 0.25 μM UreAB or UreE for 15 min at 37°C. After the PBS or HOCl challenge, the cells were plated on BA plates and the CFU were counted after 72 h of incubation at 37°C under microaerophilic conditions.
Protein purification.H. pylori 43504 cells were grown on BA plates supplemented with 5 μM NiCl2 under microaerophilic conditions (4% O2, 5% CO2, 91% N2) at 37°C and broken by passage through a French pressure cell at 18,000 lb/in2 or by cell disruption at 20,000 lb/in2 (Constant Systems One Shot). Cell debris was removed by centrifugation at 4,500 × g. Urease was purified from the resulting supernatant (cell-free extract) using fast protein liquid chromatography (AKTA; GE Healthcare). Cell-free extract was loaded onto a Q-Sepharose HiTrap column that had been equilibrated with 50 mM NaPO4 (pH 7.2) and 25 mM NaCl (buffer A). The protein was eluted with a linear gradient of 0.025 to 1 M NaCl in buffer A. Urease-containing fractions, as determined by the phenol red urease assay (64), were concentrated with Amicon Ultra-4 devices with a 10-kDa molecular mass cutoff (Merck Millipore). The concentrated urease was further purified with a HiLoad 16/60 Superdex 75 column in buffer A with 0.35 M NaCl. Urease-containing fractions were identified and concentrated as above, and the final protein concentration was determined using the BCA kit (Thermo Scientific Pierce). UreAB proteins were purified to near homogeneity (>90%), as determined by SDS-PAGE. UreE was purified as previously described (65).
Urease oxidation and Msr repair.Urease oxidation and subsequent Msr repair were performed as previously described with modifications (26). Briefly, purified urease (10 μM) was incubated with a 60-fold molar excess of HOCl for 15 min at room temperature in the dark. HOCl was quenched with excess Met (15 mM final) for 30 min on ice. Excess Met and HOCl were removed via dialysis (Spectra/Por; molecular weight cutoff [MWCO], 12,000 to 14,000) in 50 mM sodium phosphate buffer. Oxidized and quenched urease was incubated with 6.2 μM Msr, 5 μM Trx1, 0.5 μM TrxR, 400 μM NADPH, and 100 μM dithiothreitol (DTT) for 2 h at 37°C. The reaction was stopped by incubating at 95°C for 5 min.
Sample preparation and mass spectrometry analysis.Concentrated ammonium bicarbonate buffer was added to the protein to adjust to a final buffer concentration of 50 mM (pH 8.0). The samples were denatured by heating at 95°C for 20 min in the presence of 10 mM DTT. After cooling the sample, 20 mM iodoacetamide was added and the sample was kept at room temperature in dark for 45 min. Next, 20 mM DTT was added to quench the excess iodoacetamide and the mixture was incubated for 20 min at room temperature. Finally, the samples were digested with sequencing-grade trypsin at a 1:20 ratio of protease to protein by incubating at 37°C overnight with rotation. The samples, after trypsin digestion, were acidified with 0.1% formic acid and spun briefly, and the supernatant was transferred to sample vials. An Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) coupled to an Ultimate 3000 high-performance liquid chromatography (HPLC) system was used for the nano-LC-MS/MS analysis. The samples were first loaded onto a trap cartridge containing C18 stationary phase to desalt the samples. After 4 min, the samples were back-eluted from the trapping cartridge onto a nano PepMap column (75-μm inner diameter, 150-mm length, 3-μm particle size). Mobile phase A consisted of water with 0.1% formic acid, and mobile phase B consisted of acetonitrile with 0.1% formic acid. The peptide separation was achieved with a linear binary gradient from 0 to 40% phase B for 25 min at 0.3 μl/min. The peptides were fragmented by collision-induced dissociation, and the normalized collision energy was set to 35%. The MS scan range was m/z 150 to 2,000, and the top five peaks were selected in precursor scan for the data-dependent CID fragmentation in each cycle. The data files were searched against the protein sequences of UreA and UreB of H. pylori strain 26695. The peak intensities of the unoxidized peptides and their corresponding methionine oxidation products observed in LC-MS were used to calculate the average oxidation events per peptide in the sample. The ion intensity of the oxidized peptides was multiplied by the number of oxidation events required for the mass shift (e.g., one oxidation on methionine for +16, two events for +32) and then divided by the sum of the ion intensities of all unoxidized and oxidized peptide masses as represented by the equation below. P=[I(+16)oxidized×1+I(+32)oxidized×2+I(+48)oxidized×3+.../[Iunoxidized+I(+16)oxidized+I(+32)oxidized+I(+48)oxidized...]
In this equation, P represents the average oxidation level of the peptide and I represents the peak intensities of oxidized and methionine unoxidized peptides.
We thank Amelia Carlie for technical assistance with data presentation.
This work was supported by the University of Georgia Foundation and the National Institute of General Medical Sciences Research Resource for Integrated Glycotechnology (P41GM103390 to S.K.M. and J.S.S.).
Received 28 February 2018.
Accepted 30 May 2018.
Accepted manuscript posted online 4 June 2018.
Address correspondence to Robert J. Maier, rmaier{at}uga.edu.
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If you haven't already read "Welcome to Cottonwood Creek", this post won't make much sense to you. This is a chronological story. If you started from the beginning…happy reading!
Here was my drive with Steve a few days ago as we went to pick up more wood from a friend's 100 year old home. They gave us wood, too. We're using these boards for the outside of the cabin, and for the floor of the loft.
Do you see those skinny brown boards between the wide ones? That was my project. They were new pine. I had to age them. Thanks to the internet, I found the magical recipe of vinegar mixed with tea and a steel wool pad. Let it sit for 24 hours, and suddenly you can age wood.
Here's a picture of the pine before and after. Then I got to use my most favorite tool again to cut them up, and even used Mountain Man's handy nail gun and nailed 'em on up! Yes, ME. (I can't believe it, either.) In fact, I'm likely more shocked than you are.
Next post is "Time To Look UP!" See how we got our whole ceiling for FREE!
Posted in answers to prayer, Bed and Breakfast, christianity, construction, faith, family, inspirational, jesus, UncategorizedTagged aging wood, bed and breakfastBookmark the permalink.
Gretch I've enjoyed reading your blog. I would like to say that I'm surprised but when it comes to you and Steve I'm just not! This is quite the undertaking. I would love to someday stay at Cottonwood Creek. I just have to ask though…..You will have some sort of air-conditioning? Keep us posted. Love you.


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Home Politics LAGOS GUBERNATORIAL POLL: Ohanaeze Ndigbo Dumps Jimi Agbaje, Adopts Sanwo-Olu.
LAGOS GUBERNATORIAL POLL: Ohanaeze Ndigbo Dumps Jimi Agbaje, Adopts Sanwo-Olu.
Jimi & Jide.
The governorship candidate of the Peoples Democratic Party in Lagos State, Jimi Agbaje, has lambasted his All Progressives Congress counterpart, Babajide Sanwo-Olu, for giving rice gifts to civil servants on the premises of the government secretariat ahead of next Saturday's governorship election.
A stampede occurred during the struggle for rice at the government secretariat on Thursday shortly after Sanwo-Olu and other APC leaders met with civil servants.
In a statement by his Director, Media and Publicity, Felix Oboagwina, the PDP candidate said Sanwo-Olu's gesture was appalling.
Agbaje described Sanwo-Olu's meeting with civil servants at the government secretariat as an abuse of privilege and an attempt to manipulate the civil service which ought to be apolitical.
He said, "We find the invitation and the rice gift appalling, opportunistic and illegal. In a political contest of the nature in which Sanwo-Olu and the rest of us are involved, he has been offered an undue advantage. An uneven playing field has been created. And this is condemnable and contemptible.
"The gesture goes against Nigeria's bureaucratic statutes that forbid civil servants from romancing politics. For crying out loud, there are still regulations preventing civil servants from involvement in politics. That invitation and the rice gifts are a violation of those extant rules."
Agbaje said the APC designed Sanwo-Olu's rice gift to bury 20 years in which Lagos officials had been treated like trash and subjected to sordid conditions like nepotism, over-taxation, denial of promotion, delayed promotions and deprivation of leave and end-of-year bonuses.
He alleged that civil servants who maintained a neutrality and refused to show up for the meeting stood the grave danger of being marked down for discrimination and intimidation.
According to him, it was to maintain neutrality and decorum that soldiers and Police were similarly barred from involvement in politics, and politicians prevented from campaigning in uniformed personnel's barracks and formations.
"It is a shame to see civil servants struggling and hurting themselves just to get a small bag of Sanwo-Olu rice, but it is even more shameful to subject the crop of Nigeria's middle-class to such an indignity," Agbaje said.
The PDP candidate also berated the APC for sending police and thugs to bar a "Free Lagos" Press Conference slated for the Lagos Airport Hotel in Ikeja.
"It is the mother of all ironies that, on the same day that APC organised an illegal meeting for its candidate, it was sending a combined team of cops and hoodlums to dislodge an event organised by bona fide Nigerian citizens," Agbaje noted.
On Police and thugs aborting the event organised by the Orange Movement for a Free Lagos State, Agbaje said the cancellation violated the fundamental rights of the citizens to associate and hold opinions, as enshrined in the Nigerian Constitution.
The event ran under the theme, Setting Lagos Free from Bourdillon Bondage, #OtogeLagos.
The statement read in part, "It is an assault on freedom of speech. And every day, we witness the frontiers of Nigerians' freedom being eroded by a privileged minority that takes special delight in subjecting citizens to impunity and corruption.
"The APC further assaulted our collective freedom by Thursday's order to Police to bar an event where Nigerians wanted to legitimately express themselves.
"We roundly condemn the APC for this. And we call upon all Nigerians and the international community to condemn this high-handed and draconian clampdown."
Pan Igbo socio-cultural group, Ohanaeze Ndigbo Lagos chapter has dumped the governorship candidate of the People's Democratic Party (PDP) and adopted the governorship candidate of the All Progressives Congress (APC), Mr. Babajide Sanwo-Olu for the March 9 governorship election in the state.
It said the outcome of February 23 presidential and National Assembly elections makes it necessary for the Igbo in Lagos to realign their position before the governorship polls.
Ohanaeze said both the APC and PDP candidates are Lagosians, adding that the Igbo have no reason to fight over, but look out for the candidate that will protect their interest.
Ohanaeze made these declarations at a town hall meeting of stakeholders at the Muson Centre, Lagos.
President of Ohanaeze Lagos, Chief Solomon Ogbonna, said Igbo in Lagos decided to vote the APC candidate because "he is a leader that will be there for us and he is a man we can trust.
"Babajide Sanwo-Olu is the best thing that can happen to the Igbo in Lagos; he is a God sent to the Igbo. We must not waste this opportunity. That is why we must come out en mass on Saturday, March 9 and vote for Babajide Sanwo-Olu."
Ogbonna while regretting the harassment of Igbo traders in parts of Lagos after the presidential election, urged them to be more united and show love and concern to the plights of one another as it is only through love and unity they can be protected.
Ohanaeze demanded "assurances and guarantees" from Sanwo-Olu that there would not be a repeat attack on Igbo businesses after the governorship election.
Speaking, the Publicity Secretary of APC in Lagos, Joe Igbokwe, said a lot is at stake in Lagos and that the Igbo will not play with what they have.
"We don't have a quarter of the investments we have in Lagos back at home, so we must protect it. Come Saturday we must come out and vote Sanwo-Olu, and his running mate, Obafemi Hamzat; they have what it takes," Igbokwe said.
Sanwo-Olu, who was accompanied by the APC deputy governorship candidate, Obafemi Hamzat, said the governorship election is about the future of Lagos State and those sowing seeds of ethnicity to divide the people.
"The election is not about us, it is about the future and the people that want to use ethnicity to divide us. We must not allow events of one day to determine our future."
Sanwo-Olu also promised to run an all-inclusive government that will cater for all.
"From day one, we said it that our government will be all-inclusive. We are about development. The assurance I give you is that this election is about the future. The election will come and go but our lives will continue. My government will not be looking at what part of the world you are from, it will be about the greatest happiness of the greatest number," he said.


Last year's light display at the Cross Lake Corps of Engineers Recreation Area. Each year the display seems to become more beautiful. Photo via the Light Up the Dam Facebook Page.
This Saturday, Dec. 2, will kick off a season of holiday fun on the Whitefish Chain of Lakes. That day, Crosslake will play host to the Holiday at the Dam, the famous American Legion Cookie Walk, and a fundraiser for Christmas for Kids at Maucieri's- a crowd favorite.
Holiday at the Dam celebrates the lighting of the lights at the Cross Lake Corps of Engineers Recreation Area. It's a day of fun with a bonfire, s'mores, horse drawn carriage rides and even Santa! Have a cup of hot cider and enjoy the fire. Guests are invited to bring a new, unwrapped gift to donate to the area's less fortunate families. The event takes place from 1-4 p.m., then check out the lights around Crosslake all season long! The Light Up the Dam Committee fund raises all year to make the dam and areas around town beautiful for the holidays.
Beginning at 9 a.m. and running to 3 p.m., the Crosslake-Fifty Lakes American Legion Christmas Bazaar and Cookie Walk is an annual must for many locals. More than a dozen local crafters will be selling their goods. The cookie walk is a pay-by-the-pound cookie selection. Not a baker? Here's your chance to get all your Christmas cookies in one place! Each one is homemade with love. The bazaar will also include a raffle for great prices: a $300 Cub Foods Card, a $200 Holiday gas card, a $100 gas card, and a 4x5 American flag. Proceeds from this event benefit the Legion's Scholarship Fund.
Finally, join Maucieri's the evening of Dec. 2 as they once again pack the restaurant with giving folks at their annual fundraiser for Christmas For Kids (often called "Santa's Divas Day"). The event, which is from 4-7 p.m., is always an amazing display of generosity by the community, for the community. Bring a new, unwrapped gift to donate and receive a free drink. Enjoy a festive atmosphere with vendors, a silent auction and a live auction. The event is always high-energy and very fun. Do all your holiday shopping and support a great cause!


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UNHCR evacuates hundreds of detained refugees in Libya to safety
Geneva, 24 April 2019 – UNHCR, the UN Refugee Agency, has today evacuated some 325 refugees from the Qaser Ben Gasheer detention centre in southern Tripoli amid deteriorating security and escalating violence.
The relocation, carried out together with IOM, was triggered by reports on Tuesday of the use of armed violence against detainees who were protesting the conditions in which they were being held, with gunshots reported to have been fired in the air. While there were no bullet wounds, 12 refugees were injured and required hospital treatment.
"The dangers for refugees and migrants in Tripoli have never been greater than they are at present," said Matthew Brook, UNHCR Deputy Chief of Mission in Libya. "It is vital that refugees in danger can be released and evacuated to safety."
The refugees have been transferred to the Azzawya detention centre, where they are at reduced risk of being caught up in the hostilities.
Upon arrival they were provided with key aid items and medical care by UNHCR and its partner the International Medical Corps. The most vulnerable, including women and children, are being identified and will be taken to UNHCR's Gathering and Departure Facility.
The relocation was carried out with support from the Libyan authorities, UN Support Mission in Libya (UNSMIL) and UN Office for the Coordination of Humanitarian Affairs (OCHA).
A humanitarian pause was coordinated and facilitated with all parties to the conflict to allow for the safe passage of the migrants and refugees.
The relocation is the fourth UNHCR-organized transfer since the recent escalation of the conflict in Libya as our teams endeavour to get detainees out of harm's way.
UNHCR has relocated more than 825 refugees from the Ain Zara, Abu Salim, Sikka, Qaser Ben Gasheer, Tajoura and Zintan detention centres in the past two weeks alone.
UNHCR remains concerned for some 3,000 refugees and migrants who remain in detention centres in Tripoli.
The deteriorating security situation means it is imperative that refugees and migrants are immediately released and provided a pathway to safety.
While the violence continues, UNHCR and humanitarian agencies' teams have restricted access to detention centres to provide vital lifesaving aid and assistance.
Current conditions in Libya continue to underline the fact that it is a dangerous and unsuitable place for refugees and migrants. UNHCR reiterates that no effort should be spared to prevent those rescued at sea from being returned to Libya.


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Ian Dunt
Alex Stevenson
Deaths in police custody: Protest hits Downing Street
Safe? Deaths in police custody trigger Whitehall protest
By Ian Dunt Friday, 24 October 2014 10:07 AM Follow iandunt
Families of those killed in police custody have invited campaigners against detention centres to join them in a protest in Whitehall tomorrow.
The United Families and Friends Campaign (UFFC) is a coalition of families and friends of those who died in the custody of police, prison staff, secure psychiatric hospitals or immigration detention centres. They're marching in a silent procession down Whitehall and then conducting a noisy protest outside Downing Street.
Independent Police Complaints Commission figures suggest there were 11 deaths in or following police custody in 2013/14, 68 apparent suicides following police custody and 39 other deaths following police contact, as well as 12 in road traffic fatalities involving the police.
Campaigners are particularly concerned about teenagers who find themselves in contact with police. Under a legal loophole, 17-year-olds are treated as adults in police stations and are often put in custody overnight or through the weekend for minor offences, such as cannabis possession.
BASC raises police awareness of shooting sports
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BASC welcomes police text message scheme
Save lives, safer roads, lower the drink-drive limit
Innovation to cut crime and protect victims
Majority of emergency service staff suffer alcohol fuelled abuse
Behind every police uniform is an ordinary person making extraordinary decisions
That was the situation in December 2013, when Kesia Leatherbarrow was picked up by Greater Manchester police and kept in a cell all weekend. She had been distraught throughout, banging her head and pulling her hair. She was released on bail Monday morning after a trip to Tameside magistrates court. Hours later, she was dead. It was the third death in three years of a 17-year-old after they had been treated like an adult in a police station.
This year there have been two deaths in immigration detention centres. Christine Case had been in Yarls Wood detention centre ten days when she died of a massive pulmonary thrombo-embolism. The investigation into the death of Rubel Ahmed in Morton Hall is still ongoing, but he is understood to have committed suicide.
Experts warn that detention centres significantly worsen mental health conditions because there is no time limit on detention. Not knowing when you're getting out puts a severe emotional and psychological pressure on the detainee.
There are also warnings about the ineffectiveness of rule 35, which is supposed to ban the Home Office from detaining anyone who has been tortured. This mechanism is not worth the paper it is written on. Just six per cent of its reports led to release.
The UN Committee Against Torture has branded it an "empty paper-pushing exercise". A joint review by the inspector of prisons and the chief inspector of borders and immigration said the medical reports on torture survivors were "often perfunctory" and "contain no objective assessment". Replies to the reports were "cursory and dismissive".
In the high court last July, Mr Justice Ouseley said safeguards for making sure torture victims, trafficked people and those with mental health problems were protected were unsatisfactory.
All manner of terrible things happen in detention, prison, police stations and secure psychiatric hospitals because they're away from prying eyes. Tomorrow's protest will go some way towards addressing that.
immigration detention centres