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Implications of Credit Suisse Takeover by UBS for Bond Investors
Despite weaker overall market sentiment immediately following the bank takeover announcement, we believe this deal was likely the best outcome for the stability of global markets. While risk of direct fundamental contagion to other global banks is limited in our view, we expect market sentiment to remain fragile nearer term.
The details
- With significant financial assistance from the Swiss government, UBS agreed on March 19 to acquire Credit Suisse for 3 billion Swiss francs in an all-stock deal. In the process, the Swiss government determined that Credit Suisse had reached the "point of non-viability," which triggered a full writedown of the group's deeply subordinated additional tier 1 (AT1)1 bonds.
- In our view, the UBS–Credit Suisse deal should help prevent contagion and will have little direct fundamental impact on other global banks. Global banks have exposure to each other through covered bonds (very senior debt backed by mortgages and held for liquidity purposes) and as counterparties, but these should not be affected because of this acquisition in our view.
- Though we think contagion risk is limited, we expect market volatility to persist for global banks. This should generally put upward pressure on bank funding costs, both wholesale funding and retail deposits, which may lead to tighter lending standards that exacerbate already weakening economic projections.
- Near term, we anticipate greater dispersion between stronger issuers and weaker issuers, and between more senior and more subordinated debt in the capital structure. Banks still need to address their regular funding needs and will have to return to the new issue market at some point; issuing into weakness could weigh on spreads.
The writedown of Credit Suisse's additional tier 1 (AT1) bondholders appeared to undermine confidence in debt issued at lower levels of the capital structure, particularly because equity holders, who are typically supposed to rank at the bottom of payment priority, recovered some value in the forced sale. However, there are important distinctions between AT1 bonds due to local regulations, and among AT1 bonds, tier 2 (T2) bonds, and senior holding company (holdco) bonds.
Loomis Sayles did not own Credit Suisse AT1 bonds. We evaluate individual AT1 bond issues from a bottom-up fundamental perspective and also consider the regulatory framework in the issuing bank's country of domicile.
The details
- We believe the Credit Suisse AT1 writedown illuminated two key takeaways for investors. First, losses at this level of the capital structure are possible even for a global systematically important financial institution. Second, the AT1 level of subordinated debt is highly susceptible to decisions made by local regulators, who often have a number of different stakeholders in mind.
- Importantly, the structure of Swiss AT1s is different from that of AT1 bonds issued by the United Kingdom and eurozone banks. UK and eurozone AT1 bonds typically stipulate that they will be converted to equity or temporarily written down. By contrast and per local regulatory framework, the Swiss regulator was obligated to permanently write down the AT1 debt in full. Conversion and temporary writedown options would avoid treating equity holders as senior to AT1 holders, a detail of the Credit Suisse deal that created substantial backlash. However, these measures would not reduce the likelihood of the AT1 bonds being bailed in (the process through which private creditors take losses). The Bank of England and European Banking Authority emphasized these differences, which appears to have reassured investors and helped calm markets for now.
- AT1 bonds are callable instruments. At this time, almost all calls are likely uneconomical due to the steep price declines the market experienced after the deal announcement. Though much of the market has rebounded, we expect continued choppiness. Technical factors could also play a role, as funds dedicated to investing in subordinated debt may need to sell securities in order to meet redemptions.
- Markets penalized T2 bonds along with AT1s, but in our view there is an important difference between deeply subordinated AT1 bonds and more traditional subordinated T2 bonds. AT1 bonds are considered "going concern capital," meaning they are specifically designed to absorb losses during times of distress so that a bank can recapitalize itself and continue operations. T2 bonds, though still subordinated debt, are considered "gone concern capital." This means they are only bailed in once AT1s have been exhausted and there are no options other than more extensive government intervention and potential breakup of the bank. T2 bonds, based on the regulatory framework, cannot as easily be bailed in.
- Senior holdco bonds operate similarly to T2 subordinated debt, but they are more senior in the capital structure and cannot be bailed in until T2 bondholders have been fully wiped out. (In countries where banks can operate without a holding company, senior non-preferred bonds were created to function similarly to holdco bonds.)
- In a liquidity event, as was the case for Credit Suisse, T2 and senior holdco bonds are less likely to be affected. However, if a bank experiences more severe capital shortages, they are more likely to be bailed in. In our view, spillover volatility in the T2 and senior holdco bond markets may not be warranted short term, but this debt could underperform longer term if banking system stress leads to economic recession.
All investing involves risk, including the risk of loss. Investment risk exists with equity, fixed income, and alternative investments. There is no assurance that any investment will meet its performance objectives or that losses will be avoided.
Bonds may carry one or more of the following risks: credit, interest rate (as interest rates rise bond prices usually fall), inflation and liquidity.
A yield spread is a difference between the quoted rate of return on different debt instruments which often have varying maturities, credit ratings, and risk.
This material is provided for informational purposes only and should not be construed as investment advice. The analysis and opinion expressed represent the subjective views of Loomis Sayles as of March 22, 2023 and may change based on market and other conditions. There can be no assurance that developments will transpire as forecasted. Actual results may vary.
Natixis Distribution, LLC (fund distributor, member FINRA | SIPC) and Loomis, Sayles & Company, L.P. are affiliated.


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NEW! Stitch it! Sisters No-Hassle Table Topper Program 105
NEW! Starting TODAY! Watch Stitch it! Sisters Program 105: No-Hassle Table Topper Tutorial
Starting Today! Watch the NEW! Stitch it! Sisters Program 105: No-Hassle Table Topper video with hosts Deanna Springer and Dana Casey. In this video, the Stitch it! Sisters share step-by-step fabric cutting and sewing techniques for making NZP's NEW! No-Hassle Table Topper. Plus Timesaving Mitering Tips by Nancy Zieman!
Watch the NEW! Stitch it! Sisters Program 105: No-Hassle Table Topper video and learn how to sew our super-simple table topper for your home, or as a gift—the sewing is streamlined with our easiest ever sewing steps, and simple fabric rectangles. Finished Table Topper measures approximately 14″ x 38″.
Then, head over to the shopnzp.com and purchase the exclusive NEW! No-Hassle Table Topper & Napkin Bundle Box designed by the Stitch it! Sisters! The NEW! No-Hassle Table Topper & Napkin Bundle Box includes 1-1/2 yd. Red Gingham Fabric, 1-1/2 yd. Afternoon Picnic Black Main Fabric, 1/2 yd. Pellon Fleece Batting, and our NEW! No-Hassle Kitchen Collection Pattern – with Classic Timesaving Tips by Nancy Zieman. The NEW! No-Hassle Table Topper & Napkin Bundle Box includes everything you'll need to make our No-Hassle Table Topper and four coordinating napkins!
Starting Today! Watch the NEW! Stitch it! Sisters Program 105: No-Hassle Table Topper video and learn how easy it is to sew our super-quick Table Topper from just one-half yard of two coordinating fabrics! PLUS PLUS Deanna shares Timesaving Mitering Tips by her longtime friend and sewing mentor, Nancy Zieman. See how using the right tools makes the sewing process enjoyable and produces perfect corners–every time!
Follow the Stitch it! Sisters on Facebook by clicking the "Like" button at facebook.com/stitchitsisters
FREE Peppermint Mug Rug Project Sheet by Team NZP
Celebrate the Holiday Season with Team NZP! Follow and "Like" the Stitch it! Sisters on Facebook during the month of December, and receive a FREE! Peppermint Mug Rug Pattern Printable PDF! Follow the Stitch it! Sisters on Facebook by clicking the "Like"button at facebook.com/stitchitsisters.
The Nancy Zieman Productions Team is pleased to announce our NEW! video series, Stitch it! Sisters
Watch Stitch it! Sisters right here on the NZP Blog, with hosts Deanna Springer and Dana Casey – real life sisters from Team NZP. This NEW! Video Series features our latest sewing projects and fun sewing adventures! Join Deanna and Dana as they share sewing and quilting project tutorials – featuring their easiest-ever sewing techniques, timesaving tools, and Classic Timesaving Tips by Nancy Zieman. You'll also find the Stitch it! Sisters on our YouTube Channel at youtube.com/stitchitsisters.
Stitch it! Sisters is a production of NZ Productions LLC. We'll be releasing a new video – every few weeks – right here on the NZP Blog. Make sure you are subscribed to Nancy Zieman Productions E-news mailing list so you won't miss a thing. Sign up and receive email updates from Nancy Zieman Productions.
Visit the NEW! NZP Online Shop for the latest tools, patterns, fabrics and more at shopnzp.com!
Visit shopnzp.com, or click the "shop" tab
at the top of the NZP blog, anytime!
For a chance to be the random winner of a Stitch it! Sisters 5″ Fabric Short Stack with S!S button & sticker, please leave a comment below sharing your favorite holiday table decoration.
Nancy Zieman Productions will give away one Stitch it! Sisters 5″ Fabric Short Stack to one US resident in the 48 contiguous states. Winner must reply within 30 days to the official giveaway announcement email message – sent from Nancy Zieman Productions.
The randomly selected winner of NZP's NEW! McCall's M8022 Knit Dress & Tunic Pattern, from the December 14 Blog Post New! McCall's M8022 Tunic and Dress Pattern by Nancy Zieman Productions is, Donna Phelps.
Happy Sewing!
Team Nancy Zieman
Nancy Zieman Productions. LLC.
Stitch it! Sisters is a production of NZ Productions LLC. Content in this feed is © copyright 2019 by Nancy Zieman Productions, LLC and may not be republished without written permission. You're welcome to forward the email to a friend or colleague but it's not okay to add the RSS feed automatically as content on a blog or other website. The FTC requires us to tell you that Nancy Zieman Productions, LLC has provided products by other companies in the course of doing business. The FTC requires us to advise you to take this information into consideration when reading this posting.

subset: commoncrawl


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Author: Domenic Gargano, Chief Operating Officer, McLaughlin Research Corporation
When 2017 comes to an end, new regulations will be implemented to protect government information that is used by nonfederal organizations. Defense contractors and sub-contractors must demonstrate that they have taken measures to safeguard the information they are entrusted with, or risk losing government work.
In 2013, the Department of Defense (DoD) published Defense Federal Acquisition Regulation Supplement (DFARS) 252.204-7012 (Full Text) which requires nonfederal entities to protect Controlled Unclassified Information (CUI) on their systems. This includes all types of information, ranging from engineering drawings to source code. While not as stringent as the government's classified systems security controls (NIST SP 800-53), the new regulations are thorough, which may present challenges for some businesses.
The rule mandates that by December 31, 2017, contractors implement information security measures, as outlined by the National Institute of Standards and Technology (NIST) in Special Publication 800-171 (Full Text). Spanning 14 families, NIST SP 800-171 covers 110 security requirements related to cyber risk, network auditing, policies and procedures, and implementation of best practices.
NIST SP 800-171 Security Requirements Families | |
Access Control | Media Protection |
Awareness & Training | Personnel Security |
Audit & Accountability | Physical Protection |
Configuration Management | Risk Assessment |
Identification & Authentication | Security Assessment |
Incident Response | System & Communication Protection |
Maintenance | System & Information Integrity |
These regulations are not just limited to contractors. Sub-contractors and vendors in the entire supply chain must also be in accordance with the new cybersecurity standards. While any organization entrusted with CUI must be in compliance, the prime contractor may be ultimately responsible for maintaining downstream security.
At this stage, compliance is conducted on a self-reporting basis. This means that if a contractor determines it is not complying with NIST SP 800-171 guidelines, they must notify their Government Contracting Officer (or prime contractor, if acting as a sub-contractor) within 30 days. They must also present a remediation plan. Likewise, if a contractor experiences a cyber incident, they must provide notification within 72 hours. A statement that outlines what will be done to mitigate risk and prevent the incident from occurring again is also expected. Furthermore, if the government wants to review reported incidents, contractors must have an auditing system in place to quickly determine what led to the incident.
So, when will these regulations kick-in? They already have! The government has started invoking DFAR and FAR clauses, through modifications on existing contracts. To retain these projects, prime contractors must comply with the new regulations. Of course, the solicitation process on new bids now requires contractors to meet NIST SP 800-171 guidelines.
Though not every contractor may be audited, noncompliance is risky. If a contractor ever has a breach and cannot respond appropriately, current and future work is suddenly jeopardized. The DoD could view noncompliance as defaulting on (and justification for terminating) a contract. The risk of not complying with NIST guidelines is pretty clear: Contractors risk losing government business.
Depending on an organization's level of IT and cybersecurity expertise, it may take several months to over a year to align systems, policies, and procedures with NIST guidelines. Some companies may take a DIY approach to compliance, while others may utilize tools and software or lean heavily on consultants. Research and choose the approach that meets your needs and budget, but choose quickly. The NIST compliance deadline is coming December 31, which is right around the corner!


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Do you find yourself greatly attached to the performances of poker players who have previously set records many times? If you take each, lose personal, and if you obsess about your opponents who are on a winning streak, you will just be making it worst for yourself. Detach yourself from the game as a person and think of yourself as an athlete or a gamer doing this just for the fun of winning, nothing personal. Not only are many online poker rooms weathering the economic storm well, but many are doing record business. For those looking to wager on football, we leave no stone unturned by providing the predicted lineups across several leading football leagues. Simultaneously, we will also have the confirmed lineups and starting 11s as soon as they are announced in the lead-up to kick-off.
However, knowing the major daftar sekarang things to be looking for will help the procedure go much earlier and easier. Sometimes poker games can be quite 'engaging and you forget that there are other more important things you can do. If you are experiencing a bad streak, don't continue the game on that same table. Maybe your tilt is a signal to call it quits at the table and do something else. Such terms can signal you at the right moment. They can access it anywhere and anytime they want. Remember, Poker is still a luck game, and even the bad players can experience a lucky streak. So if you find yourself on a bad streak, stop playing the game and do something else.
A loose definition is in order: the bad beat is a subjective term describing a poker hand that appears strong but loses nevertheless to a stronger hand. Lots of internet casinos offer a huge selection of video game titles, for example, blackjack, roulette, video poker, and also slots. You have a lot of wonderful opportunities as it is an era where internet technology is advanced. The popularity and simplicity of this game have inspired quite a few young people. Today, both men and women love the excitement of this strategic card game. However, Jain has been much less active in recent years; as it appears, he has turned his attention to growing the game on home soil.


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Rumpole and the Children of the Devil
Brief Synopsis
Cast & Crew
James Cellan Jones
Patricia Hodge
Philip Anthony
Sandra Tallent
Paul Bigley
Julian Curry
Film Details
Technical Specs
Horace Rumpole is back at the bar -- in Juvenile Court -- as he defends yet another member of the Timson family, a group of South London petty criminals. This time it's Cary Timson, accused of devil worship when his eight-year-old daughter is found wearing a devil's mask in her school playground. In Chambers, Rumpole's fellow barrister, Claude Erskine-Brown, colludes with the Head of Chambers, Sam Ballard, to replace Rumpole with a better class of barrister, while at home, Rumpole's wife Hilda suspects that she is about to be replaced by another woman.


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The Winnipeg Jets flexed their offensive muscles at times on Sunday, and Dustin Byfuglien is a big part of that.
The over-sized blueliner has been using his size for no good quite often lately (ask Jay Bouwmeester), especially if you look back to the later stages of last season.
We saw both sides of Byfuglien in Winnipeg's 5-4 win against the Minnesota Wild, as he delivered a questionable boarding hit on Jason Pominville.
It only received a minor penalty, yet it might be suspension material.
Until video is available, consider these GIFs via "My Regular Face."
In other news from the game, it seems like Devan Dubnyk is sore, but not necessarily injured.
Dubnyk has a right knee injury, but he said even though it's sore, he's been told it can't get worse by playing "not structural" #mnwild
— Michael Russo (@RussoHockey) October 26, 2015


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Aunt Becky Made Being An Aunt Look Easy
By now, most fans know that Olsen twins turned down the opportunity to reprise their role as Michelle in the upcoming Full House spinoff, Fuller House , on Netflix — which is unfortunate news, because a Full House without Michelle is unimaginable. But, there's still a huge reason to rejoice when it comes to this series: In addition to the actors who have confirmed their return, actress Lori Loughlin will reprise her role as Aunt Becky in the upcoming series.
As a fan, I'm so relieved to know that she didn't pass up on an opportunity to join the reboot, especially because she portrayed one of the show's most endearing characters. Personally, I know all too well how an aunt could either make your life a living hell or be the best ally amongst family — and Aunt Becky always fell on the latter end of the spectrum, because she was easily one of the sweetest TV aunts ever.
Clearly, D.J., Stephanie, and Michelle had it made with Aunt Becky on their side. Whether she was stepping in as a last-minute substitution for D.J.'s Career Day guest, or generally being that pillar of support for the entire family, she gave a good name to aunts everywhere. It's not easy to move into a house where six people reside (seven if you include Kimmy Gibbler and her unannounced visits) and blend seamlessly as if you were always a part of the family — but, after she and Jesse got together, she managed it seemingly without even batting an eyelash. Seriously, what's not to love about Aunt Becky?
She was so awesome that you don't even have to be anyone's niece or nephew to appreciate her. Here are seven times Aunt Becky made being an aunt look oh-so-easy:
The Time She Let Michelle Lick The Cake Frosting
Note to all aunts: This is how you handle an adorable kid who takes advantage of your kindness. In other words, remain poised, patient, and sweet — but above all else, let them lick the frosting.
The Time She Offered To Teach Michelle How To Cook
While Danny and Jesse chickened out of teaching Michelle how to cook, Aunt Becky stepped right up to the challenge by offering to help Michelle learn her way around the kitchen. (Oh, and the way she totally shut down Jesse was priceless.)
When She Kindly Encouraged Michelle To Give Her And Jesse Some Alone Time
Oof. This was a rough moment. Sure, Michelle was "very mad," but Becky's laidback nature allowed her to take the challenge of living in a crowded household in stride.
The Time She Got D.J. In On Pranking Uncle Joey For April Fools' Day
Far be it for Aunt Becky to stand by thumbing her nose at the Tanner family's silly jokes. Like the cool aunt she is, she got right in on the family action — and brought D.J. along with her.
When She Ice Skated Like A Pro With The Girls
This was a considerably difficult designated aunt task to execute, specifically due to Jesse's clumsiness. But, once again, Aunt Becky was a total pro.
The Time She Was There For The Entire Family When Jesse's Grandfather Died
This is the saddest Full House scene ever, but Aunt Becky was super supportive, patient, and nurturing to everyone.
The Time She And D.J. Laughed At Jesse's Real Name
The best aunts are the ones who are slow to scold and quick to laugh. Besides, could you blame her for laughing along with D.J. at Uncle Jesse's hilarious real name?
Image: Warner Bros. Television


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Ratan Mondal is a tea vendor from Kolkata who settled in the UK in the 90's. He set up his Tea2You tea shop at London's Borough Market. Ratan actively searches for the best tea estates in Darjeeling, Assam, and Nepal and works on-site developing unique blends and flavors for the best tea quality.
Ratans popular shop has had some very high profile Celebs, Politicians... and Prince Harry has popped in, to buy some tea.
The tea shop recently moved into a purpose-built shop in Southwark Street within the market, so if you get the chance, please pop in and support a great tea business.
Tea2You website: https://tea2you.co.uk/
Follow Tea2You on Instagram: https://www.instagram.com/tea2you_boroughmarket/
Borough Market website: https://boroughmarket.org.uk/
Buy Edible Histories book here: https://www.waterstones.com/book/borough-market-edible-histories/mark-riddaway/9781529349702


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Stormy Skies, Scenic Views
A slower paced day two & day three
One of the mirror spheres outside the Asian Civilizations Museum
My second full day in Singapore began early. I was awakened by the sound of a howling wind outside the sliding glass door to my balcony. It sounded like a typhoon blowing between the skyscrapers. I sat in my bed and listened to its banshee shriek for awhile. Then, a torrential downpour began. I opened the door and stepped out onto my balcony to watch sheets of water falling. I crawled back under the covers, hoping the storm really wasn't a typhoon, and the rains would end by morning. I woke up late in the morning, and checked outside. Rain was coming down hard enough that I postponed any sightseeing until after lunch.
Singapore has the easiest-to-use bus system in the world -- at least that's what I found
When I finally did venture out, I decided to add the local bus system to my repertoire. The front desk of the Village Hotel Katong graciously explained what bus I needed to catch to reach the Metro station -- saving me a 30-minute walk in the rain. Singapore's buses are every bit as organized and easy as the Metro, and air conditioned, too! I was headed to the Changi Chapel and Museum, and found it with no problem. Each bus stop has a name, and by taking a picture of the route sign with my smart phone, I could look at the window and follow our progress. It reminded more of a train or tram travel than a city bus line.
Entrance to the Changi Museum, which commemorates the POWs of the Japanese during WWII
Changi Museum is built on the site of a chapel that WW II POWs built for themselves after they were captured by the Japanese, following the surrender of Singapore. The museum's signs are all in English, and do a great job setting the scene of the British blundering defense of the island they considered impregnable. The attacking Japanese were outnumbered 3-1, but did have air superiority and much better equipment. All of the allied prisoners were rounded up and interned in various overcrowded and inadequate camps. Even European civilians were imprisoned and squeezed into cells, the men kept separate from the women. One of the best features of the museum's audio guide were the recorded oral histories of various prisoners. The gut wrenching account of a Chinese woman tortured for passing messages for the allies brought tears to my eyes. There are numerous letters from former prisoners or their children, spilling their feelings and telling their stories as a form of catharsis. I was disappointed that no photographs inside were allowed, but you could take photos of the reconstructed chapel outside.
Looking like something out of the Jetsons cartoon, Singapore's Marina Bay Sands Hotel
I actually took it very easy on day two, partially to let my blisters I'd worn into my soles on yesterday's death march heal. I took advantage of the supermarket in the hotel's shopping center to buy some nice, waterproof band-AIDS, which would come in handy until my foot healed up. And they were actually feeling much better the next day, when it was time to explore more of Singapore.
View from the observation deck of the Marina Bay Sands
With the sun shining again, I decided to start at the top, literally. I rode the Metro down to the Marina Bay Sands hotel. It is a futuristic looking building that I could easily see featured in an upcoming Star Wars movie. It is composed of three modern hotel towers topped by what -- for all the world -- looks like a giant hovercraft. It extends past all three towers and contains an observation deck, restaurant, infinity pool, restaurant, bar, and garden. All of Singapore is laid out beneath you as you stand atop it, 56 floors up. It was a beautiful, sunny day, and you could see for miles in every direction. Even though it was a weekend, the observation deck was not crowded. The other visitors and I wandered slowly around, enjoying the panorama, and taking pictures. One of the things that struck me was the multitude of cargo ships in the bay, moored, or waiting to dock. It looked like the movie scenes of the allied fleet at D-Day, gray ships receding into the distance. The next thing that strikes you are the towering skyscrapers. All the of them are sleek and modern, and they dwarf the tiny two to three story, more traditional buildings of Singapore's earlier history. There are numerous stadiums, including a twin pair nicknamed "The Durien" after Asia's notorious, spiky-skinned fruit. To me, they looked more like an aluminum spiky brassiere, discarded by some titanic Madona. There's even a floating field in the bay that can be used for sports or concerts. The view was amazing, and that would prove to be the theme for the day.
Singapore's harbor as seen from the Marina Bay Sands observation deck
Descending, I crossed back to the main island on the pedestrian Helix bridge. The walkway is encapsulated in a gleaming aluminum structure looks like a monstrous DNA strand. There are four observation platforms along the way, each providing wonderful views and massive selfie opportunities for strollers, their cell phones, and social media. Speaking of which, anyone who thinks the obsession with our phones is an American thing has not been to Singapore. There are just as many faces glued to miniature screens on subways, in restaurants, and even walking down crowded streets. It is a world thing, now. We are all captivated by our connection to the Internet and our friends and family. I retraced my steps from day one's night time stroll along the waterfront, this time enjoying it under blue skies. I wanted to visit the Merlion, a massive fountain of a hybrid fish-lion. It was here that I truly ran into throngs of tourists. Many of them were doing my all-time, least-favorite thing: posing so the jet of water issuing from the statue's mouth appeared in their friend's camera to fall into their cupped hands, mouth, whatever. The Singapore equivalent of "holding" up the Leaning Tower of Pisa. Bleh. I took some photos and moved on.
Very cool walking bridge in the Marina Bay area
After a brief stop to get photos of the giant, mirrored balls (think a half dozen, spherical versions of Chicago's famous bean), It was off to Chinatown. I know, it seems odd to me that a city of mostly Chinese descent would have a specified Chinatown. However, the island is on the tip of the Malay peninsula, so there is a significant population of Malaysians, Indian immigrants, British, and more. Along the way, I stopped at another Hindu temple, Sri Mariamman. Even though it is billed as Singapore's most colorful, I actually preferred the Kali temple from day one. The temple was thronged with worshippers in colorful garb, offering sacrifices and praying. The afternoon sun beating down was getting intense, so I backtracked to a pub I'd spotted on the walk here and enjoyed an ice cold water before savoring an excellent Archipelago Irish Ale. It hit the spot and fortified me to push on in the heat.
The Merlion - an iconic fountain in Singapore that people come to photographed standing beside
Next up was the five-story Buddha Tooth temple. This modern, gleaming building with its gray-green, pagoda roofs was air conditioned, airy, and spacious. Hundreds of statues from the Buddhist pantheon of every size lined the walls. Worshippers and tourists made a circuit, one lighting joss sticks and bowing in prayer while the other admired the beauty and took pictures. Coming here, I'd noticed several streets with market stalls, so I spent some time roaming them, looking for souvenirs. I did find a lapel pin of the Singapore flag for my map at home, and was intrigued by the lacquered wood screens painted with various scenes. I really liked the one with the Great Wall, and mentally made a note to keep an eye out for more like this in other shops. I was actually killing time with my shopping, waiting for the 6:55 pm Lion Dance which would be performed on the streets. From the picture, it looked like one of those cool, multi-person costumes the Chinese are famous for using in celebrations. When I headed back to get a good viewing spot, I noticed the fine print on the sign: "Cancelled today"...d'oh!
The gleaming, modern Buddha Tooth Temple, which houses a...well, I think you can figure it out!
A quick Metro hop brought me to the day's final stop, Clarke Quay -- Singapore's river walk of restaurants and bars. Boats leave regularly to cruise along the waterfront, and at night it colorfully lit up with lights. After a couple days of Asian food, I was looking forward to more choices. I ended up having shish kabob at a Middle Eastern place. I finished the day off with a Tiger beer at a cafe along the waterfront, watching the boats cruise by. Although much less packed with sights than day one's overdose, day three was a pleasant way to wrap up my first half of this trip. Tomorrow, it was off to Laos for a week. I'd be returning here again later, before I flew back home.
The Clark Quay area in Singapore where there are lots of restaurants and bars
Posted by world_wide_mike 17:04 Archived in Singapore


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ACT! For Canada Newsletter - June 13, 2020
"The line separating good and evil passes not through states, nor between classes, nor between political parties either - but right through every human heart - and through all human hearts. . . . And even within hearts overwhelmed by evil, one small bridgehead of good is retained." ~ Aleksandr Solzhenitsyn ~


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You would like…
… to learn the German language?
… find a place to live and a job?
… learn about the Austrian way of life and culture?
… find suitable leisure activities?
We try to give you information!
which can make your start in life in Austria easier.
Learning the German language is certainly not easy, and understanding a new way of life and culture is often difficult – but with mutual help it is not impossible!
On this page you will get valuable tips for everyday life and you will learn a lot about Austrian customs, manners, ways of life and cultures!
Let's set off together towards a better future – and don't forget to share this page with your friends and acquaintances!


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Tuesday, October 24, 2017
Assembly Hall
140 characters, tight budgets, and a "fake news" branding defines political journalism in 2017. Twitter has accelerated the news cycle and created a demand for real-time responses. The cost of business has forced media outlets to consolidate resources in large cities – often at the expense of on-the-ground reporting from state capitals. Meanwhile, a nationwide trend toward distrust of media outlets, sometimes expressed in outright hostility toward reporters, has further complicated the relationship between the press and the public.
Join the Global Voices Lecture Series and the Institute of Politics as we welcome four distinguished journalists for a discussion on political journalism in 2017.
John Dickerson, Chief Washington Correspondent, CBS News
Karen Tumulty, IOP Resident Fellow, National Political Correspondent, The Washington Post
Dan Balz, Chief Washington Correspondent, The Washington Post
David Maraniss, Associate Editor, The Washington Post
This event is dedicated to the memory of David S. Broder, a titan of journalism whose career began at the University of Chicago, where he earned a bachelor's degree in 1947 and a master's in 1951. Known for his ability to cut through the noise of punditry and spin, Broder framed his campaign coverage by looking beyond the political machine, turning public attention to voters whose voices might otherwise be drowned out. His columns on the Watergate scandal, written in the familiar, professorial style that inspired his nickname "Dean of the Press Corps," earned him the 1973 Pulitzer Prize for commentary. He passed away in 2011, having established a legacy of integrity and professionalism. The IOP is proud to know the Broder family as both benefactors and friends.
This event is free and open to the public. Register here.


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- 1Centro de Estudios Científicos, Valdivia, Chile
- 2Universidad Austral de Chile, Valdivia, Chile
- 3Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche S970, PARCC, Hôpital Européen Georges Pompidou, Assistance Publique Hôpitaux de Paris, Paris, France
- 4School of Medical Sciences, University of Manchester, Manchester, United Kingdom
Kir7.1 encoded by the Kcnj13 gene in the mouse is an inwardly rectifying K+ channel present in epithelia where it shares membrane localization with the Na+/K+-pump. Further investigations of the localisation and function of Kir7.1 would benefit from the availability of a knockout mouse, but perinatal mortality attributed to cleft palate in the neonate has thwarted this research. To facilitate localisation studies we now use CRISPR/Cas9 technology to generate a knock-in mouse, the Kir7.1-HA that expresses the channel tagged with a haemagglutinin (HA) epitope. The availability of antibodies for the HA epitope allows for application of western blot and immunolocalisation methods using widely available anti-HA antibodies with WT tissues providing unambiguous negative control. We demonstrate that Kir7.1-HA cloned from the choroid plexus of the knock-in mouse has the electrophysiological properties of the native channel, including characteristically large Rb+ currents. These large Kir7.1-mediated currents are accompanied by abundant apical membrane Kir7.1-HA immunoreactivity. WT-controlled western blots demonstrate the presence of Kir7.1-HA in the eye and the choroid plexus, trachea and lung, and intestinal epithelium but exclusively in the ileum. In the kidney, and at variance with previous reports in the rat and guinea-pig, Kir7.1-HA is expressed in the inner medulla but not in the cortex or outer medulla. In isolated tubules immunoreactivity was associated with inner medulla collecting ducts but not thin limbs of the loop of Henle. Kir7.1-HA shows basolateral expression in the respiratory tract epithelium from trachea to bronchioli. The channel also appears basolateral in the epithelium of the nasal cavity and nasopharynx in newborn animals. We show that HA-tagged Kir7.1 channel introduced in the mouse by a knock-in procedure has functional properties similar to the native protein and the animal thus generated has clear advantages in localisation studies. It might therefore become a useful tool to unravel Kir7.1 function in the different organs where it is expressed.
The inwardly rectifying Kir7.1 K+ channel is encoded by the Kcnj13 gene in the mouse and is classified as a K+-transport type channel. Kir7.1 is the latest addition to the Kir family of proteins (Hibino et al., 2010), and it diverges from classical Kir proteins in sequence and functional properties (Döring et al., 1998; Krapivinsky et al., 1998; Partiseti et al., 1998). Thus, Kir7.1 channels are largely voltage-independent and lack the characteristic Kir K+ concentration-dependence.
Kir7.1 is present in epithelial tissues where invariably it has been found to share the same sidedness with the Na+/K+-pump. Kir7.1 is present at the basolateral membrane of intestinal epithelial cells, thyroid follicular cells and epithelial cells of renal proximal or distal tubules (Nakamura et al., 1999; Ookata et al., 2000; Derst et al., 2001). The Na+/K+-pump is expressed at a basolateral membrane location in these epithelia as in most transporting barriers. Kir7.1, however, is found at the apical membranes of retinal pigmented epithelium (RPE) and choroid plexus. These epithelia show an unusual apical membrane expression of the Na+/K+-pump (Nakamura et al., 1999; Kusaka et al., 2001; Shimura et al., 2001; Yang et al., 2003). The faithful distribution of Kir7.1 together with the Na+/K+-pump has prompted the speculation that Kir7.1's function might be to provide a pathway to recycle K+ taken up pump in exchange for Na+ thus maintaining transepithelial ion transport processes without potentially deleterious intracellular K+ accumulation (Sepúlveda et al., 2015). Other roles attributed to Kir 7.1 include a participation in the mechanism of melanocortin-mediated regulation of energy homeostasis within the paraventricular nucleus (Ghamari-Langroudi et al., 2015) and the control of excitability of uterine smooth muscle in the transition to contractions in the pregnant uterus (McCloskey et al., 2014).
Diseases associated with mutations in human KCNJ13 include snowflake vitreoretinal degeneration (SVD) (Hejtmancik et al., 2008) and Leber congenital amaurosis (Sergouniotis et al., 2011), which are caused by trafficking or functional defects (Sepúlveda et al., 2015). A study of a mouse model with mosaic expression of Kcnj13 in the RPE suggests a role of Kir7.1 in the ionic regulation of the confined space between RPE and photoreceptors possibly in relation to lactate transport (Zhong et al., 2015). Experiments using shRNA knock down of Kir7.1 expression or pharmacological inhibition of the channel also suggest a role for Kir7.1 in the ionic regulation at the RPE/photoreceptor interface (Shahi et al., 2017).
Further investigation into the function of Kir7.1 in the organism would be greatly facilitated by the availability of a Kcnj13 gene knock-out mouse. Such an animal model would also provide an ideal negative control for studies of protein expression by western blot, immunohistochemistry or immunofluorescence. We have previously generated Kir7.1 null mutant mice (Villanueva et al., 2015), but these animals die hours after birth most probably due to incomplete palate sealing, a malformation known to lead to perinatal mortality (Turgeon and Meloche, 2009). In addition Kir7.1 null mice have a moderate retardation in lung development, which was surprising as there was no evidence for a role of the channel in the respiratory system.
In order to understand better the tissue distribution of Kir7.1 we have now generated a knock-in mouse that expresses the channel tagged with a haemagglutinin (HA) epitope using the CRISPR Cas9 technology. The availability of antibodies for the HA epitope allows for application of western blot and immunolocalisation methods without need for channel-specific reagents. Importantly, WT tissues provide an unequivocal negative control adding robustness to the assays. We check for Kir7.1-HA functional properties, confirm the presence of the channel in well characterized locations and explore its expression in kidney and respiratory epithelium, tissues with controversial or unreported localization respectively.
Materials and Methods
We used C57BL6/J mice from Jackson Laboratories (United States). Mice were maintained on standard laboratory chow (2019S diet, Harlan Laboratories) and water ad libitum, under regulated temperature (22–26°C) and light (12:12-h light-dark cycle) conditions. Housing and breeding was at the SPF animal facility of the Centro de Estudios Científicos (CECs), Valdivia, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care AAALAC. The HA-tagged mouse was generated at the microinjection laboratory at CECs. All animal procedures were approved by the local Institutional Animal Care and Use Committee (protocol N° 2015-01).
Synthesis of sgRNA, Cas9 mRNA and ssODN Template
A 20 base pair guide (gHA) (5′-ggttttcaggtgggacgtcg-3′) targeting the Kcnj13 gene region encoding the extracellular loop of the Kir7.1 channel was obtained using Optimized CRISPR software1. The synthesis of the chimera gHA-tracrRNA (sgHA) was based on protocols available at Addgene2. Briefly, sense and antisense oligonucleotides encoding the guide were synthesized with extra-nucleotides compatible with a digestion product from restriction endonuclease BbsI (5′-CACCGggttttcaggtgggacgtcg-3′ and 5′-AAACcgacgtcccacctgaaaaccC-3′ respectively). Both phosphorylated oligonucleotides were hybridized and cloned in vector pX330 (Addgene 42230) digested with BbsI (Cong et al., 2013). The specific sgHA was obtained by in vitro transcription using as a template amplicons of gHA cloned in pX330 vector that include T7 promoter and the reverse primer located downstream of the transcription termination signal (T7sgHA 5′-ttaatacgactcactatagggttttcaggtgggacgtcg-3′ and sgRev 5′-aaaagcaccgactcggtgcc-3′, respectively) (Yang et al., 2013). The 119 bp amplicon was cleaned of RNAses and used for in vitro transcription with the MEGA shortscript T7 Transcription Kit (Ambion, #AM1354) following the supplier indications. The sgRNA was purified by column with MEGAclear Transcription clean up kit (Ambion # AM1908). The Cas9 mRNA was purchased from TriLink BioTechnologies, Inc. (L-6112). The template to repair the Cas9 double strand break and introduce the HA epitope was synthesized as ultramer oligonuclotide DNA single strand (ssODN) in IDT-DNA. The ssODN had 5′ and 3′ homologous arms of 70 bp each (in italics), and a 30 bp core (in boldface) including nucleotides for the HA epitope and EcoRV restriction site (ssOD N1620HA: 5′-gagagaaggagaatgcagctgtgaagctggtgatgtgcttcacacagatgggacta
cagatcaccattcatctcagctacagcataccagaggactgcaaagacaagccag-3′). The edition occurs at position 6210 in the Kcnj13 gene (Ensembl accession number ENSMUSG00000079436).
Mouse Zygote Microinjection
Microinjection was performed into the pronucleus and cytoplasm of zygotes obtained by in vitro fertilization with sperm and ova from C57BL6 mice. The microinjected mix included sgHA RNA, mRNA Cas9 and ssODN6210HA at 10, 20, and 100 ng/μl respectively. The zygotes were kept in culture until two cell embryo stage and then were transferred to a B6CBA pseudo pregnant female.
Screening for Genome Modification
Genomic DNA extracted from pup tails were analyzed by PCR and EcoRV digestion. The primers flanking the inserted HA epitope region were Kir7.1-HAfor 5′-agtcaaagacaccggagg-3′ and Kir7.1-HArev 5′-tgtgataaaagcctctagc-3′. The expected size amplicons were 420 and 450 bp for wild type and modified allele respectively. The EcoRV site incorporated in the modified allele generates 219 and 231 bp products. The right insertion of the HA epitope was confirmed by sequencing the cloned amplicons in pGEM-T vector (Promega).
Off-Target Analysis
Potential off target genome effects for gHA were obtained using the Optimized CRISPR design software. Specific flanking primers were designed to amplify the potential off targets by PCR. Amplicons were then directly sequenced.
Cloning of cDNA Kir7.1-HA Channel From Modified Mouse
Total RNA was extracted from an enriched choroid plexus fraction from a heterozygote F1 mouse using Trizol reagent (Life Tech). The cDNA was synthesized using Superscript IV (Invitrogen) and oligo dT primer. The channel open reading frame was amplified by PCR using the Phusion High Fidelity DNA polymerase (Thermo Scientific) and the forward and reverse primers 5′-aagcttgcgatggacagcagtaattg-3′ and 5′-tctagatttattctgtcagccctgtttc-3′, including HindIII and XbaI restrictions sites (in italics) respectively. The amplicons were cloned in pGEM-T easy vector, digested with EcoRV to discriminate the modified allele from the wild type and confirmed by sequencing. Finally, the open reading frame was cloned in pCR3.1 expression vector using the HindIII and XbaI sites to be expressed in HEK-293 cells.
Heterologous Expression and Electrophysiology
HEK-293 were transiently co-transfected with 0.3 μg of pCR3.1-CD8 and 1.5 μg of plasmid (pCR3.1) containing either Kir7.1wt or Kir7.1-HA cDNA using Lipofectamine 3000 (Invitrogen #L3000). At 24 h post-transfection, the cells were visualized applying Dynabeads-CD8 (Invitrogen #11147D) and recordings were performed from isolated cells using standard whole-cell patch-clamp technique as described elsewhere (Díaz and Sepúlveda, 1995). Currents were recorded using an Axopatch 200B amplifier (Axon Instruments). Acquisition was at 2 kHz with filter at 1kHz employing pClamp10 software (Axon Instruments). For experiments, borosilicate capillaries (Harvard Apparatus GC150F-10, 1.5 mm OD × 1.17 mm ID) were fire polished to 2–4 Mω and covered in beeswax. The bath solution contained in mM: 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 1 Glucose, 10 HEPES/Tris, pH7.5 and sucrose to obtain 310 mOsm/L. In alternative bathing solutions NaCl was omitted and KCl or RbCl increased to 140 mM. BaCl2 at 1 mM was added to the last solution as indicated. The pipette solution contained in mM: 140 KCl, 1 MgCl2, 1 Glucose, 10 EGTA and 10 HEPES/Tris, pH7.5 (300 mOsm/L).
Choroid Plexus Preparation and Electrophysiology
The choroid plexus was removed from the fourth ventricle of mice killed by decapitation while under deep isoflurane anesthesia (Julio-Kalajzić et al., 2018). The tissue was kept in ice-cold PBS until use (usually within the first 2 h). A small piece of intact tissue was secured to the base of a dish with a platinum wire on the stage of an inverted microscope for studies by patch clamp (Kotera and Brown, 1994). The experiment was similar to that described previously to characterize Kir7.1 activity in retinal pigment epithelial cells (Shimura et al., 2001) and consisted of cell-attached patch-clamp recordings obtained after sealing the pipette to the apical side of the epithelium. The pipette solution contained, in mM: 145 RbCl, 1 MgCl2 and 10 HEPES/Tris, pH 7.4, 290 mOsm/L, with or without 1 mM BaCl2. For tissue maintenance we used a bath solution containing 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 Glucose, 10 HEPES/Tris, pH7.4, 310 mOsm/L. For recordings this was exchanged for a high K+ solution, chosen to collapse the membrane potential of the cell to zero, in which NaCl was decreased to 5 mM and KCl increased to 140 mM.
Western Blotting
Sample Preparation
Enriched membrane fractions were used for all experiments unless otherwise specified. Three to seven month old mice were euthanized by a lethal dose of anesthesia (Tribromoethanol) and the following tissues were collected: whole eyes, lung lobules, kidney sections and enriched fraction of choroid plexus. Tissues from at least three different animals were analyzed. Trachea epithelium was extracted by scraping and the small intestine epithelia by the protocol described before (Zeineldin and Neufeld, 2012). All tissues were suspended in lysis buffer containing (in mM) 250 sucrose, 20 Tris-Hepes pH 7.4, 1 EDTA, 1 PMFS and protease inhibitor cocktail, and homogenized. Samples were then centrifuged at 4,000 g for 15 min at 4°C and the supernatant collected and spun at 17,000 g for 30 min at 4°C. The pellet was suspended in lysis buffer, mixed with Laemmli 2X loading buffer, denatured and stored at -80°C until required for use. Proteins were quantified by the Bradford method.
To obtain isolated tubules from kidney papillae, adult mice were anesthetized by intraperitoneal injection of 80 mg/kg ketamine and 10 mg/kg xylazine, and perfused through the aorta with 1ml of incubation solution (0.375 mg/ml glycine, 0.048 mg/ml trypsin inhibitor and 0.025 mg/ml DNAse I in 98b buffer) containing 4 mg/ml collagenase type II. Kidneys were transferred into 98b buffer pH 7.4 of the following composition in mM: 140NaCl, 0.4 KH2PO4, 1.6K2HPO4, 1 MgSO4, 10Na-Acetate, 1 α-Ketoglutarate, 1.3 Ca-Gluconate. Papillae were dissected and transferred into 1 ml enzyme-containing incubation solution (1 mg/ml) and incubated for 10 min at 37°C (850 rpm). Afterward the supernatant (released tubules) was transferred to a chilled Eppendorf-tube (batch 1) which contains 1ml ice-cold sorting solution (0.5 mg/ml albumin in incubation solution). At the same time 1ml of pre-warmed (37°C) incubation solution was added to the papilla slices to continue shaking for 5 min. The supernatant (released tubules) was transferred again to a chilled Eppendorf-tube (batch 2) containing 1 ml ice-cold sorting solution. This step was repeated to obtain 4 batches. The tubule suspension was gently pipetted into a dissection dish containing 3 ml sorting solution and tubules were selected at 4°C under a stereo microscope. Tubules were transferred directly into Laemmli 2x buffer.
The samples (10–50 μg of protein) were resolved in SDS-PAGE at 10 or 12% and transferred to nitrocellulose membrane. The membranes were blocked in saline buffer pH 7.4 plus 5% non-fat milk for 1hr at room temperature and incubated with the primary antibody: rat monoclonal anti-HA (1:1000) or rabbit monoclonal anti-HA (1:500) or B1 subunit of V-ATPase (1:500) or AQP1 (1:1000) or actin (1:1000) at 4°C overnight. Then the membranes were incubated with its respective secondary antibody conjugated to HRP, revealed with chemiluminescent HRP substrate and visualized for autoradiography or mini-LAS imaging system (Fuji). Ponceau red or actin was used to check for protein load.
Heads of P0 or lungs of adult mice were fixed at 4°C overnight in 4% paraformaldehyde or 10% formalin respectively, then dehydrated in 30% sucrose in phosphate-buffered saline pH7.4 and cryopreserved in Tissue-TekTM (OCT). Tissues from at least three different animals were analyzed. Cryosections of 8–10 μm were subjected to epitope exposure with 10 mM sodium citrate buffer, pH 6,0. Tissues were treated with 3 or 10% of hydrogen peroxide, blocked with 2.5% normal horse serum, and incubated overnight at 4°C with monoclonal rabbit anti-HA 1:100 or 1:500 for lungs and heads, respectively. Detection of antigen/antibody complexes was performed with ImmPRESSTM reagent kit (Peroxidase) Anti-Rabbit Ig and the reaction was visualized using ImmPACTTM DAB Peroxidase substrate kit. Sections were counterstained with hematoxylin.
Adult mice were perfused with 4% formalin or 4% paraformaldehyde to isolate brains or lungs, respectively. Tissues from at least three different animals were analyzed. Brains were embedded in OCT and lungs in paraffin. Cryosections (10 μm) were blocked in phosphate saline buffer containing 1% BSA, 0.3% Triton X100 and 10% newborn goat serum and incubated with the monoclonal rat anti-HA (1:1000) overnight at 4°C and then with a secondary antibody donkey anti-rat IgG conjugated with Alexa Fluor 568 (1:2000). All images were captured by confocal microscopy (Olympus FV1000).
pX330-U6-Chimeric_BB-CBh-hSpCas9 was a gift from Feng Zhang (Addgene plasmid # 42230). Antibodies against HA-Tag were purchased from Roche, (3F10 clone rat monoclonal) and Cell Signaling (C29F4 Rabbit mAb), Anti β-actin is from Abcam (ab8227). Antibodies against B1 subunit of H-ATPase and AQP1 were a kind gift from Dr. Dennis Brown (Harvard Medical School, Boston, MA, United States) and Sebastian Frische (Aarhus University, Denmark) respectively. Secondary antibodies as ImmPRESSTM reagent kit (Peroxidase) Anti-Rabbit Ig and ImmPRESSTM HRP reagent kit (Peroxidase) goat Anti-Rat IgG mouse adsorbed are from Vector Laboratories, donkey anti-rat IgG conjugated with Alexa Fluor 568 from Thermo Fisher Scientific, goat anti-rabbit IgG and goat anti-rat IgG conjugated to horseradish peroxidase from Bio-Rad and Jackson, respectively. Specifics as ImmPACTTM DAB Peroxidase Substrate Kit from Vector Laboratories, Alexa FluorTM from Thermo Fisher Scientific, luminol-enhanced chemiluminescence (ECL) from Perkin Elmer Life Science Products and SuperSignal ELISA Femto Maximum Sensitivity Substrate from Thermo Scientific.
Results and Discussion
To gain an insight into the physiological role of the Kir7.1 channel it is important to precisely determine its expression in different organs and tissues. The use of the optimal negative control, the Kcnj13 KO mouse lacking the Kir7.1 channel, is not possible as these animals die shortly after birth (Villanueva et al., 2015). An alternative approach is to tag the protein with a suitable epitope using the CRISPR-Cas9 knock-in technology allowing the expression of the channel from its natural chromosomal context. In this case WT animals are a straightforward negative control, provided the tagging does not cause alterations leading to mislocalisation of the protein.
Strategy to Tag the Kir7.1 Channel
The Kcnj13 mouse gene is approximately 9.5 kb long, is located in the reverse strand of chromosome 1 and contains 3 exons (Figure 1A). The open reading frame of the channel is contained in exons 2 and 3; the N-terminus, both transmembrane domains and the pore domain are encoded in the second exon. The HA epitope was introduced into what is predicted to be an extracellular loop immediately after of the first transmembrane domain. Introduction of such a tag has been demonstrated not to affect the function of the Kir6.1 and Kir1.1 K+ channel members of the Kir family (Zerangue et al., 1999; Yoo et al., 2003).
FIGURE 1. Targeting strategy for the generation of the Kir7.1-HA mice. (A) Scheme of the Kcnj13 gene showing its three exons (boxes) and the coding regions in gray. The guide (green) targets exon 2 in the genome and leads Cas9 to produce a double strand break upstream to the PAM sequence (underlined). The ssODN containing the HA tag sequence, the EcoRV site (in red) and the homologous arms (in black) is the template used for homologous directed repair (HDR). (B) Agarose gel showing the PCR products of WT and HA genomic DNA obtained using the primers shown in (A). The amplicon digested with EcoRV reveals the homozygous (HA) for the tag insertion.
Figure 1A also shows schematically the strategy to introduce the HA epitope in the extracellular loop of the channel encoded in exon 2. The guide used to target the specific sequence in the gene is shown in green and the protospacer adjacent motif (PAM) is underlined in the genome sequence. The single strand DNA repair template used to incorporate the HA-coding sequence along with an EcoRV site by Homology Directed Repair (HDR) is shown in red. In Figure 1B, PCR amplicons from wild type and homozygous littermates for the HA insertion screening can be seen to have the expected size (420 and 450 bp respectively). EcoRV cuts only the modified gene yielding 2 restricted fragments (219 and 231bp). Sequencing of the amplicon confirms that the DNA edition occurred at position 6210 of the Kcnj13 gene (Ensembl accession number ENSMUSG00000079436). Eight potential off target mutations were predicted within gene coding regions and were analyzed by PCR. Direct sequencing of the amplicons did not demonstrate any mutations (results not shown).
Cloning, Heterologous Expression and Electrophysiological Characterization of Kir7.1-HA cDNA
We cloned the cDNA of the channel starting from RNA isolated from a heterozygous mouse choroid plexus, a previously described channel expression site (Döring et al., 1998; Nakamura et al., 1999). The chromatogram in Figure 2A shows that the HA tag was inserted in frame between residues D90 and V91 and rest of the channel open reading frame was identical to the known cDNA mouse sequence (NCBI Reference Sequence: NM_001110227.2).
FIGURE 2. Functional assay of Kir7.1-HA cloned from the choroid plexus of a Kir7.1-HA knock-in mouse. (A) Cartoon showing a topological model of the Kir7.1 protein in the membrane and the site of the insertion of the HA-epitope with the chromatogram with of the cDNA sequencing. In capital letters is the amino acid sequence deduced from the nucleotide sequence. Large red letters identify the G plus HA tag (G-YPYDVPDYA) inserted in frame between residues 90 and 91 of the native protein. (B–E) Whole-cell currents obtained from a HEK-293 cell after transfection of the Kir7.1-HA cDNA. The cell was held at the stated Vh and square voltage pulses were given that took the membrane potential from –160 to 80 mV in 20 mV steps. The cell contained 145 mM K+ and the medium was switched from one containing 5 mM K+ (B) to 145 mM K+ (C), 145 mM Rb+ (D) and finally to 145 mM Rb+ with 1 mM Ba2+ (E). In (F), current–voltage relations for the currents at the end of the pulses in (B–E) are shown. (G) Is a comparison of Rb+ currents at –160 mV measured in cells transfected with untagged Kir7.1 channel with those of Kir7.1-HA channel (means ± SD and individual points, n = 8 and 9 respectively).
To determine whether the modified Kir7.1 protein was transcribed and translated correctly, we examined channel function. The electrophysiological properties of the recombinant Kir7.1-HA channels were studied in transiently transfected HEK-293 cells. Only small currents were seen in 5 or 140 mM extracellular K+ solutions (Figures 2B,C), but robust inward currents at hyperpolarizing potentials were present when 140 mM extracellular Rb+ replaced K+ in the extracellular solution (Figure 2D). The Rb+ inward currents were blocked by the addition of 1 mM Ba2+, and this block was strongly dependent on time and membrane potential (Figure 2E). Figure 2F shows the current-voltage relations taken at the end of the pulses for each of the four extracellular solutions employed. These features are characteristic of native and heterologously expressed Kir7.1 (Wischmeyer et al., 2000; Shimura et al., 2001), suggesting that channel properties are not altered by the HA-tag.
Qualitatively similar currents were also observed in HEK-293 cells transfected with Kir7.1 WT cDNA (not shown). Maximum currents measured in the presence of 145 mM Rb+ and at -160 mV (Figure 2G), were 2.6 ± 1.33 nA for WT and 1.8 ± 0.94 nA for Kir7.1-HA-expressing cells (means ± SD, n = 8 and 9 respectively). WT Kir7.1 currents appeared larger than those generated after transfection of HEK-293 cells with Kir7.1-HA, but this difference did not reach statistical significance (P = 0.164 by t-test). In conclusion the Kir7.1 channel tagged with the HA-epitope obtained from choroid plexus of knock-in animals behaves very much as the wild type channel.
Analysis of Kir7.1 Protein Expression by Western Blot
We used anti-HA antibodies to locate Kir7.1-HA expression in different tissues using those from the WT animal as a negative controls. Immunoblots obtained using eye, choroid plexus, trachea and lung, and ileum and jejunum are shown in Figure 3. The analysis generally revealed the presence of at least two immunoreactive species in different proportions, one of approximately 45 kD, close to the predicted size of the unglycosylated core of Kir7.1, is present throughout and it appears to be the sole one in the lung. A further species, of around 50 kDa, most likely corresponding to a glycosylated form is more abundant in choroid plexus and renal papilla (panel B) while both species were similarly represented in trachea and the epithelium of the ileum. The expression of the channel is very high in ocular tissue where higher mass bands of 75 and 100 kDa are also present. We have not explored the origin of these species but they might correspond to dimer protein complexes or some degree of protein aggregation. The high amount of Kir7.1-HA in the eye most likely corresponds to the robust expression of the channel in the RPE where it is expressed in the apical membrane of both rat and bovine cells (Kusaka et al., 2001; Yang et al., 2003). The expression in the small intestinal epithelium is consistent with reports showing basolateral membrane expression in non-specified segments of rat small intestine by immunohistochemistry (Nakamura et al., 1999) and human small intestine by Northern blot analysis (Partiseti et al., 1998). Highest, if not exclusive, expression in the mouse is in the ileum. Kir7.1 appeared absent from the jejunum (Figure 3A), the large intestine and the stomach (not shown).
FIGURE 3. Tissue distribution of Kir7.1-HA protein. Samples obtained from homozygous Kir7.1-HA mice (HA) or wild type mice (WT) were analyzed by western blot. Fractions of membranes obtained from eyes (n = 4), choroid plexus (n = 4), trachea (n = 3), lung (n = 10), ileum (n = 4), and jejunum (n = 4) are shown in (A). In (B) kidney plasma membrane fractions of cortex, inner (IM) and outer medulla (OM) are presented (n = 3). (C) Thin loops of Henle (TL) and collecting ducts (CD) were dissected from renal papillae and analyzed for the presence of aquaporin 1 (AQP1) and proton potassium pump (B1) as markers of TL and CD respectively (n = 5). Monoclonal rat anti-HA antibody was used in (A,B), while rabbit anti-HA antibody was used in (C). Ponceau red staining (A,B), or beta actin (C) were used as loading controls.
Figure 3B shows an immunoblot of kidney tissue from Kir7.1-HA and WT animals. Clear bands ascribable to glycosylated and unglycosylated forms are present in tissue from the inner medulla (IM) but are absent from the outer medulla (OM) and cortex. In order to gain more information about where in the papilla the channel is expressed, nephron tubules from collecting duct and thin loop of Henle (TL) were isolated and analyzed for the HA signal and for specific collecting duct and thin loop of Henle markers. As shown in Figure 3C the channel signal was present only in collecting ducts (CD), identified by the presence of the B1 subunit of the V-ATPase, but absent from thin limbs of the loop of Henle (TL), where AQP1 signal was present.
Previous work had shown expression of Kir7.1 in rat kidney mainly in the distal convoluted tubule (DCT), connecting tubule (CNT) and cortical collecting duct (CCD), and to a lesser extent in the thick ascending limb of the loop of Henle (TAL), and outer and inner medulla collecting duct (OMCD and IMCD) (Ookata et al., 2000). Results later confirmed for the expression in TAL, DCT and CCD in 21 days old rats (Suzuki et al., 2003). These authors propose a role of Kir7.1 in renal K+ excretion under the condition of K+ overload. A sharply contrasting distribution was reported for guinea-pig kidney where the main site of expression was judged to be the proximal tubule with some expression also in intercalated cells of the CD (Derst et al., 2001). It was proposed that Kir7.1 is involved in basolateral membrane K+ recycling. Our results show that the main site of expression of Kir7.1 is the inner medulla with specific immunoreactivity in the isolated CDs. Unfortunately we could not reliably determine the subcellular localisation of Kir7.1-HA in renal epithelial cells, a failure that we attribute to low level expression. Kir7.1-HA was nevertheless detectable by western blot after pooling sufficient number of isolated tubules. We cannot therefore determine the cell type where the channel is expressed. We speculate that its presence in the inner medulla might have to do with the urine concentration process. An alternative function could be in the acid-base balance process that is sited at the CD intercalated cells. Like other Kir channels Kir7.1 is regulated by intra- and extracellular pH (Hughes and Swaminathan, 2008), which might argue for a role in relation to this last function. This last interpretation could find some support in a recent single cell transcriptomic analysis that reveals high expression of Kir7.1 in intercalated rather than principal cells of mouse kidney CD (Chen et al., 2017).
Native Kir7.1 Currents in the Choroid Plexus
Mildly inwardly rectifying K+ currents had been reported in rat choroid plexus (Kotera and Brown, 1994) before molecular identification of Kir7.1. Cloning of Kir7.1 led to its localization in the choroid plexus by in situ hybridization and confirmation of the presence of K+ currents exhibiting mild inward rectification in whole-cell recordings of rat choroid plexus epithelial cells (Döring et al., 1998). Immunolocalization, on the other hand, demonstrated Kir7.1 immunoreactivity at the apical membrane of rat choroid plexus epithelium (Nakamura et al., 1999). The western blots of choroid plexus proteins in Kir7.1-HA mouse tissues in Figure 3A are consistent with high expression of the channel in this epithelium. To examine whether Kir7.1-HA channels are sorted properly and able to mediate currents in choroid plexus epithelial cells we explored their presence by immunofluorescence and assayed for their currents by patch-clamp. The immunofluorescence against HA tag confirms that the protein is located at the apical membrane of choroid plexus epithelium from Kir7.1-HA. There was no HA-immunoreactivity in tissue from WT mice (Figure 4A). To assay for Kir7.1 mediated currents we used an approach successfully employed previously in RPE cells to measure Rb+ inward currents in cell-attached patches of membrane (Shimura et al., 2001). Because the conductance of Kir7.1 to Rb+ is ∼10 times higher than that for K+ (Wischmeyer et al., 2000), using this cation could allow the detection of Kir7.1-mediated inward currents across choroid plexus apical membranes in cell-attached configuration if there is a high density of channel proteins. Figure 4B shows a cell-attached recording from choroid plexus apical membrane of a Kir7.1-HA mouse obtained with a pipette containing 145 mM Rb+. Inward currents were evident at potentials more negative than -60 mV (Figure 4D). Similar experiments but with a pipette solution containing 1 mM Ba2+ in addition to 145 mM Rb+ yielded the type of currents shown in Figure 4C. Initially currents were inwardly rectifying but a time-dependent inhibition took place at the most hyperpolarized potentials. Initial (circles) and steady-state (triangles) currents are plotted as function of voltage in Figure 4E revealing the membrane potential-dependent block of Rb+ influx quite characteristic of Kir7.1. Rb+ currents assayed in plexuses from Kir7.1-HA knock-in mice behaved similarly to those from WT mice (not shown). A comparison of the initial Rb+ currents at -180 mV in choroid plexuses from Kir7.1-HA (circles) and WT mice is shown in Figure 4F. No difference between choroid plexus HA-tagged and WT Kir7.1 currents was observed (P = 0.0584 by unpaired t-test).
FIGURE 4. Apical membrane immunofluorescence localisation and ion currents from the choroid plexus of the Kir7.1-HA mouse. (A) Cryosections of choroid plexus epithelia were probed with the rat anti-HA monoclonal antibody. Confocal images obtained from choroid plexus of heterozygous Kir7.1-HA (left) and WT littermates WT (right) (n = 5). Scale bar represents 50 μm. (B,C) Show cell-attached patch-clamp recordings obtained from apical membranes of choroid plexus of Kir7.1-HA mice. The pipette solution contained 145 mM Rb+ without (B) and with (C) 1 mM Ba2+. Bath solution had 145 mM K+ in both cases. (D) Steady-state current-voltage relation for currents measured in (B). (E) Current–voltage relations for experiment in (C). Currents shown were taken at the initial peak (circles) or at the end of the pulse (triangles). (F) Comparison of maximal currents at –180 mV in recordings from WT (circles and column on the left) or Kir7.1-HA mice (triangles and column on the right). Means ± SD and individual points, n = 8 and 9 respectively.
The choroid plexus epithelium, together with RPE, must be a site of very high expression of Kir7.1. This is suggested by the strong signal seen both in western blots and in immunofluorescence assays. More importantly the large currents carried by Rb+ in cell-attached patches also suggest a very high channel expression in the apical membranes of this epithelium and of the same order as those reported in the RPE (Shimura et al., 2001). Kir7.1 (and Kv1) channels are thought to play a key role in the secretion of cerebrospinal fluid (CSF) by choroid plexuses (Damkier et al., 2013). They provide an essential "leak pathway" for K+ pumped into the cells by the Na+-K+ ATPase which is also very highly expressed in the apical membrane of the choroid plexus and is critical in the process of CSF secretion (Damkier et al., 2013). As reviewed by Damkier et al. (2013), the Na+-K+ ATPase transports significant amounts of K+ from the CSF into the choroid plexus cells. In addition it sets up the gradients for effective functioning of the NKCC1 of the apical membrane, an exporter of K+ from the CSF. The close association of Kir7.1 with the Na+-K+ ATPase suggests a functional coupling that has been speculated to be necessary to ensure efficient function of the pump (Hibino et al., 2010). Most of the ion transporters involved both in the maintenance of a low CSF K+ concentration and those involved in the ion transport processes that sustain fluid secretion are secondary active transport mechanisms dependent upon the gradients set up by the pump. If this hypothesis holds true, Kir7.1 would be necessary for efficient pump function and would therefore be a major contributor to the low CSF K+ concentration and CSF production. Modulation of Kir7.1 activity may therefore be important in controlling the rate of CSF secretion (and hence intracranial pressure), and in maintaining a stable [K+]CSF, which is a requirement for the central nervous system function.
Distribution of the Kir7.1 Channel in the Respiratory Tract Epithelium
In our previous work using the LacZ reporter gene we showed that the promoter of Kcnj13 gene is active in embryos and newborn mice in the upper airway epithelium (Villanueva et al., 2015). Here we use the HA immunoreactivity of Kir7.1-HA tissues to determine the protein localization at P0. As shown in the Figure 5 (see two upper panels) the development of the palate, as expected, is completely normal in the KI mouse unlike what occurs in the Kcjn13 null mutant mouse that presents complete secondary cleft palate. As it is shown in left hand side panels of Figure 5, the channel is expressed only in the basolateral membrane of respiratory epithelial cells in the nasal cavity and nasopharynx in the Kir7.1-HA mice. We have demonstrated using the lacZ reporter in heterozygous KO mice, which present normal palate development, that the channel is expressed only in the epithelial cells covering the nasal aspect of the palatal shelves (Villanueva et al., 2015). This agrees with present results as no Kir7.1-HA signal was detected in the epithelium of the oral cavity side of the palate. As shown in right hand side panels of Figure 5, the immunoreactivity is completely absent from tissues of WT littermates.
FIGURE 5. Kir7.1-HA channel is located in the basolateral membrane of the nasopharyngeal respiratory epithelium. The immunohistochemically analysis from Kir7.1-HA (left) and Kir7.1-WT (right) newborn mice tissue was performed with the rabbit anti-HA antibody (n = 4). In the (middle) and (bottom) panels amplifications of marked regions (∗) and (#) are shown. Counterstaining was performed with hematoxylin. Scale bar represents 10 μm. ns, nasal septum; nc, nasal cavity; np, nasopharynx; T, tongue.
We then studied the expression of the channel in the epithelial cells of lower airway epithelia in adult mice. We first performed an immunohistochemistry assay with a rabbit monoclonal anti-HA antibody. The analysis revealed the expression of Kir7.1-HA in the basolateral membranes of the epithelial cells from trachea, bronchi and bronchioli (Figures 6A,C,E). The staining for Kir7.1-HA was absent from WT tissues (Figures 6B,D,F).
FIGURE 6. Kir7.1-HA channel is located in the basolateral membrane in respiratory epithelium of adult mice. The detection of HA tag was performed in trachea, bronchi and bronchioli immunohistochemically with the rabbit anti-HA antibody (n = 3). (A,C,E) Are samples from Kir7.1-HA mice and (B,D,F) from WT mice. Scale bar represents 50 μm.
The impression of a basolateral expression for Kir7.1 gained from our previous immunolocalisation studies performed using an anti-Kir7.1 antibody (Villanueva et al., 2015) is confirmed here using HA immunoreactivity with appropriate negative controls. Our previous work has also shown a moderate retardation in lung development in Kcnj13 null mutant embryos and we speculated that this might have due to a deficiency in fluid secretion secondary to channel deficiency. Electrolyte transport is involved in the maintenance of the airway fluid layer covering the whole of the respiratory tract that is essential to its normal function including mucociliary clearance. Processes include absorptive and secretory functions that require the activity of basolateral Na+/K+ pump. K+ channels are required to avoid intracellular accumulation and to stabilize the membrane potential favorable to Na+ absorption and or Cl- secretion (Hollenhorst et al., 2011; Lee and Foskett, 2014).
Genetically modified mice are useful in unraveling the function of ion channels in vivo. We have been interested in understanding the function of Kir7.1 in the various organs where it is expressed. Knockout mice are valuable in ascertaining the function of ion channels and, importantly, they aid in the cellular and subcellular localization by providing reliable negative controls for western blots and immunolocalisation assays. We previously generated a mouse deficient in the channel by ablation of the Kcnj13 gene, but unfortunately total channel inactivation leads to neonatal mortality precluding its further use (Villanueva et al., 2015). The generation by CRISPR Cas9 technology of a Kir7.1 knock-in mouse that expresses the channel tagged with an HA provides a convenient animal model to study reliably the distribution of the channel in mouse tissues using their WT littermates as negative controls. We here confirm the localization of Kir7.1 in the eye and choroid plexus, where it is present at high levels. We also confirm the intestinal localization of Kir7.1 and we identify the ileum as the exclusive or main site of expression. We show that Kir7.1 is mainly expressed in the renal papilla where it is present in IMCD isolated tubules. This contrasts with previously reported wide distribution in rat kidney and proximal tubule expression in guinea-pig nephron. We also confirm our previous suggestion of a basolateral expression in the respiratory tract epithelium from the trachea to the bronchioli. We also found similar basolateral membrane localization in the epithelium covering the nasal cavity and nasopharynx in newborn animals which has not been previously reported. Functional assays demonstrate that Kir7.1-HA cloned from the knock-in animal and expressed heterologously has unaltered electrophysiological properties attesting to the lack of effect of the epitope insertion in channel function. In a similar manner, the native current recorded from choroid plexus demonstrates the normal function of the channel in situ, incidentally revealing the very high expression of Kir7.1 in this tissue and suggesting it might play an important role in CSF production. Recent evidence for ion channel function independent of their conductive properties such as in signaling complexes termed chanzymes and chansporters (Krapivinsky et al., 2014; Abbott, 2017) could be advantageously studied using the modified channel knockin approach described here. Additionally the tagged channel approach could be used in investigating the expression of the channel during development. In summary the Kir7.1-HA knock-in mouse characterized in the present work could become, perhaps together with complementary tissue specific knockout models, a useful tool to unravel Kir7.1 function.
Author Contributions
The experiments were performed at the Centro de Estudios Científicos (CECs) Biological Laboratory. IC, RC, MN, PB, FS, and LC conceived and designed the work. IC, SV, JB, KL-C, FJ-K, NB, MN, DF-F, and PB, performed the experiments and together with LC and FS analyzed and interpreted the data. IC, FS, and LC drafted the work that was additionally revised critically by all authors. All authors approved the final version of the manuscript and all those that qualify for authorship are listed.
This work was funded by the FONDECYT-Chile grant 1150735. The Centro de Estudios Científicos (CECs) is funded by the Base Financing Program of Conicyt. Travel support came from ECOS-Conicyt grant C14S01. KL-C is on receipt of a fellowship from the Chilean Comisión Nacional de Investigación Científica y Tecnológica.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Juan Manuel Baamonde and the staff of CECs animal facility are gratefully thanked for their untiring help.
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Keywords: epithelial transport, K+ channel, choroid plexus, respiratory epithelium, renal tubule, knock-in mouse, CRISPR-Cas
Citation: Cornejo I, Villanueva S, Burgos J, López-Cayuqueo KI, Chambrey R, Julio-Kalajzić F, Buelvas N, Niemeyer MI, Figueiras-Fierro D, Brown PD, Sepúlveda FV and Cid LP (2018) Tissue Distribution of Kir7.1 Inwardly Rectifying K+ Channel Probed in a Knock-in Mouse Expressing a Haemagglutinin-Tagged Protein. Front. Physiol. 9:428. doi: 10.3389/fphys.2018.00428
Received: 22 November 2017; Accepted: 05 April 2018;
Published: 23 April 2018.
Edited by:
Francisco Javier Alvarez-Leefmans, Wright State University, United StatesReviewed by:
Ute Ingrid Scholl, Heinrich-Heine-Universität Düsseldorf, GermanyIldikò Szabò, Università degli Studi di Padova, Italy
Copyright © 2018 Cornejo, Villanueva, Burgos, López-Cayuqueo, Chambrey, Julio-Kalajzić, Buelvas, Niemeyer, Figueiras-Fierro, Brown, Sepúlveda and Cid. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: L. P. Cid, email@example.com


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WEEK eight
artist:kali mcmillan
poem:"Famous" by naomi shihab nye
from kali:
Poetry has always been for me sort of indulgent. A chance to step into a feeling, feelings. To experience an experience whether that be one of joy, pain, revelation, whatever the chosen poet felt moved to pen and however I chose to receive it. In times like this 2020 we know and hate, or really in any time where emotions run high and lives are at stake, poetry can have a real impact, I think, and this project is such a wonderful meditation on that. The poem I chose to read is one that spoke to me about empathy and our power to impact the people around us even if on a very small scale. I've always been a bit of a touch-er; that person who would brush your shoulder if I passed too close in an aisle, just so you know I'm there, and I'm not a threat. Someone who loves bear hugs and burrowing into the necks of the ones I love, so I can smell my memories of them. Needless to say, the times of Covid-19 and social distancing have required a marked change in my behaviors. It's not my place to comfort others and myself through touch; it's imperative I don't. The biggest show of love I can make is wearing my mask and washing my hands. It's respecting distance when I'm in stores and cleaning the handles in a public restroom after I use it. I think we're all at least a little scared, some a lotta scared, but I do not believe that we are without love and empathy. When I walk past someone in the grocery store, more often than not we exchange a look, and more often than not, it ends with a smile and nod. And with a mask how do you know they're smiling, you say? Because you can see it in the crinkle of their eyes and the way the mask moves to compensate for teeth. They happen these smiles, I've seen them and experienced them, and they give me hope that we have a big need and potential for compassion and love. The movement towards ending police brutality and respecting and celebrating black lives speaks to this. The hundreds, thousands of peace protests speak to this. The response from our government does not, but they won't be there long. I hope. I also hope you enjoy this poem and the pictures I posted below. These are from people in my life; people that are famous to me even if they don't know it. Also, I had a phase with flip book animation a couple of years back...and this heart was a byproduct. Lol, she's got a lot of good moves.


text:
Dr. Brad Regez
- Position: Associate Professor
- Department: Mechanical Engineering
- Office Location: Building 4, Room 436C
- firstname.lastname@example.com
- Campus: 8504742044
Dr. Brad Regez earned his Ph.D. in Engineering Science from Southern Illinois University while working at the Center for Advanced Friction Studies. Before joining UWF, Regez spent seven years as a Research Associate at Northwestern University where his research focus was on developing laser based fiber optic sensing systems for structural health monitoring of safety critical aircraft and civil structures in addition to developing new nondestructive evaluation systems for assessment of structures. Regez has worked on research projects for NASA, FAA, ONR, and the NSF, as well as industry (Renault F1 race team, John Deere & Company, Boeing, GKN, GE, Honeywell, and Imperium Inc.).
Prior to his work at Northwestern, Regez was a Project Engineer working on the Space Shuttle Program at the Kennedy Space Center where he led several NASA efforts to implement emerging nondestructive evaluation technologies into the program. These efforts included developing an inspection technique using laser shearography to inspect the structural integrity of aging composite overwrapped pressure vessels located on both the space shuttle and international space station.
Degrees & Institutions:
Ph.D. Engineering Science, Southern Illinois University
M.S. Civil Engineering, Southern Illinois University
B.S. Civil Engineering, Southern Illinois University
Nondestructive characterization of materials and structures, intelligent structural health management of safety-critical structures and additive manufacturing systems
Current Courses:
- Engineering Materials
- Non-Destructive Evaluation
- Sophomore Engineering Design I
- Sophomore Engineering Design II
- Junior Engineering Design I
- Junior Engineering Design II
Classes Taught:
- Engineering Materials
- Mechanics of Materials
- Mechanics of Materials Lab
Keywords: nondestructive characterization, materials, structures, intelligent structural, health management, safety-critical structures, additive manufacturing systems


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GE Appliances says it plans to close its Bloomington refrigerator plant, eliminating its 329 remaining jobs.
The company announced the planned closing Wednesday, citing demand for the side-by-side refrigerators manufactured at the plant that has fallen by 76 percent since 2008. It says the plant is scheduled to operate only 22 weeks this year.
The closing is subject to negotiations with Local 2249 of the International Brotherhood of Electrical Workers, which represents the plant's 301 hourly workers.
The company said 94 percent of all employees would be eligible to retire with comprehensive pension and health coverage, severance based on length of service, preferential hiring rights at other GE plants, and other benefits.
GE eliminated about 130 jobs at the plant in 2013 through layoffs and early retirements.
General Electric Co. announced June 6 that it had completed the sale of GE Appliances to Haier Group of China for $5.6 billion, which includes an approximate $200 million increase from the originally announced sale price to account for increased working capital in the business.
GE almost sold the appliances business to Swedish company Electrolux last year, but scrapped the $3.3 billion plan in December. The deal was opposed by U.S. regulators over concerns about competition.
GE has been making refrigerators in Bloomington since 1967. The plant was once considered the world's largest refrigerator facility, employing more than 3,200 workers at the end of the last century.


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- Square Feet 12,000
- Acreage 1.52
Arborvine Restaurant opened its doors in 2000 to provide diners with the freshest, locally sourced foods available. Today, that tradition continues as the network of local farms, fisherman and foragers who supply Arborvine is as strong as ever. Deep Water Brewing Co. is Arborvine's very own five barrel microbrewery, with state of the art equipment and a gorgeous tasting room in a beautiful 200 year old former horse barn that was fully renovated in 2012. The barn was torn down to its frame and rebuilt with a new foundation, and everything else from there on up. At Deep Water they personally handcraft each and every beer from the best ingredients available. Historically they have served their beers exclusively at the Arborvine and the Deep Water Brew Pub. Being small has allowed them to experiment and brew a number of styles to rotate throughout the year. A buyer could continue with this tradition, or take the brewing to the next level. The pub has its own kitchen, separate from Arborvine. The building located at 35 Tenney Hill has a second story short term vacation rental which is perfect for a couples getaway. Recently remodeled, this gorgeous apartment sleeps up to 3, has a gas fireplace, bamboo, cork, tile, and carpeted floors, WiFi, flat screen HD cable TV and a private balcony overlooking the back gardens, hop vines and mature trees. Guests enjoy a fully equipped gourmet kitchen or dine at one of the restaurants. The apartment comfortably sleeps 2 adults but has a portable cot if needed. Downstairs is a long term commercial lease. There is a separate driveway and parking for this property.
Listed by Swan Agency Real Estate


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Trans Mountain pipeline section shut as precaution in Surrey, British Columbia
A section of the Trans Mountain pipeline was shut on Sunday as a precautionary measure following complaints of an odor and reports of a gasoline-like substance near the line in Surrey, British Columbia, operator Trans Mountain Corp said.
"We have investigated the pipeline in the area. We have found no evidence that the source of the product is from the pipeline," the company said in a statement.
A local media report, quoting British Columbia's environment ministry, said that the substance spilled had been traced to a private home.
Trans Mountain, which is a wholly owned subsidiary of the Canada Development Investment Corp, delivers nearly 300,000 barrels of crude oil and refined products each day through 1,274 km (792 miles) of pipeline in Alberta, British Columbia and Washington state, according to the company's website.
(Reporting by Vijaykumar Vedala in Bengaluru; Editing by Peter Cooney)