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When we moved into our home, we knew that we would have a garden, and that would include a section of multiple fruit trees. We are very much into self-sustainability, living off the land, controlling our food sources and saving money. What we did not know, was type or placement until we did our research. Buying a grafted fruit tree like sweet apples, plums, pears and other fruits means that the trees have been grafted to produce quality tasting fruit or able to fight off disease or drought.
Although fruit trees can be planted from seed you may have a home, the resulting trees won't necessarily produce fruit that's good to eat. In order not to waste years of energy trying to make the tree grow and produce fruit you'll want to eat, the best idea is to buy a dwarf fruit tree, which is a very young tree that has been grafted onto a root stock to improve its growth. That is what we have done.
The trees are available for purchase bare-root or potted. Bare-root trees are dug up and shipped while they are dormant. You should plant them as soon as you receive them. While the potted trees are grown in a pot and will have more fine roots attached.
Buying a tree from a local nursery is best, since you are supporting a local business, but additionally they will be able to advise and stock trees that do well in your region.
As for planting look for an open, sunny spot in the yard. Fruit trees generally need at least six hours of full sunlight in order to grow strong and produce healthy fruit. Look for a spot in the yard where the fruit tree won't be shaded by your home or other taller trees. You should also look for a spot without a lot of other foliage nearby, so the tree won't have to compete with other plants for nutrients and water. For our home, we wanted the fruit trees to make a screen from one section of yard to another.
Select the location of your fruit tree by imagining it at full size. Take into account its width and understand that the roots of your tree will reach out as far as the length of the branches. This means that you don't want it too close to a building or driveway. As with ours since we were using the trees as a screen, we had to make sure that the width of each branch did not over compete the next tree but that it had the ability to grow out and back as much as was desired. This close proximately to each other created privacy, but also meant we had to be always alerted to moisture, environmental and pest damage.
Aside from full sun, proper soil drainage is the other essential condition for thriving fruit trees. The soil must not retain a lot of water, or it will cause the fruit trees' roots to rot in the ground. Check the soil drainage by digging a hole 1 foot deep and filling it with water. If the water drains quickly, the area should be fine for planting a fruit tree. If the water stands in the hole, choose another part of the yard.
Fruit trees can be planted at any time of year, but in areas with cold winters or hot summers, your best bet is to wait until spring. This will allow the tree to immediately start adapting to the soil and growing roots. It's also the best time of year for breaking ground, since the soil will be thawed and easy to dig.
Add compost to the soil if necessary, it will loosen the soil, provide better drainage and make room for the tree's roots to begin growing. When planting remember to dig a wide hole, twice as wide as the spread of the roots of the tree you're planting. Fruit trees' roots tend to grow outward, and this will give them plenty of room. And do not bury to deep, keep the graft at the base of the tree stay above the soil.
One the hole is dug position the tree in the hole, throw a little loose soil into the hole, get the tree visibly as vertical as possible and push back in the soil while adjusting the tree to make sure its still vertical. Press soil around the roots, press the soil down gently and then water.
Be sure not to overwater the tree, however my rule is to place the hose at the base of the tree, have it on full blast and count out two minutes. After that I go on to the next tree and repeat. I do this every day the first 7 days, then I switch to every other day for 7 days, then switch to every third day for 7 days and then once a week throughout the first summer.
Cover the soiled area with a layer of organic mulch. This will retain the moisture in the soil and protect the roots. It will prevent grass and weeds from growing and competing for nutrients and water as well. Ensure that the graft line is not covered by mulch; it needs to remain visible above ground level.


text: Four Seasons Mauritius Located at the heart of the Anahita peninsula estate on the east coast of Mauritius, the luxury Four Seasons Resort at Anahita is a secluded and spacious resort where couples and families can enjoy the legendary service for which it is renowned. Dates: 10 nights departing on 4th April. Price: From £4,000 per person based on 2 adults staying in a Garden Villa. Includes: Breakfast, economy flights, transfers & a 30% exclusive Scott Dunn discount.

subset: commoncrawl


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About our company
Two people + cat = short story of HOG STUDIO!
Marketing Specialist, Graphic Designer and a ginger cat, this connection had to give something original to the world ;) Every day we are creative. We have one million ideas per minute, and our imagination knows no boundaries. Hog studio is our interpretation of the animal world. We love animals and we dedicate them most of our projects.
We combine them with geometry and our own ideas, into abstract constellations. In our projects, we put heart and passion, and each of them reflects our aesthetics. If You love animals just like us and You like our interpretation of the world then we invite You to the world of Hog Studio!


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This recipe will take you 5 minutes to mix up and about 50 minutes in the oven, so consider yourself about an hour away from something that is absolutely addictive. Use your favorite craft beer to customize to your liking.
3 cups self-rising flour (make your own by adding 1 1/2 tsp. baking powder and 1/2 tsp. kosher salt to each cup of all-purpose flour you need)
1/3 cup granulated sugar
12 oz. beer (I used Pub Ale from Tall Grass Brewery)
2 Tbs. melted unsalted butter
Preheat oven to 350 degrees F.
In a large mixing bowl, combine flour, sugar and beef, stirring just until blended. (Mixture will be rather lumpy – this is fine. Don't worry.)
Pour batter into a greased loaf pan and bake, uncovered, on center rack of oven for 45-50 minutes, or until tester inserted into the center comes out clean.
Remove from oven and pour melted butter over the top.
Let cool slightly before slicing, if you can wait. Slather each slice with Hildebrand Farms Dairy butter – heavenly.
Now You're Cookin',
Chef Alli


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Thai Chicken Ramen
Monday, March 25, 2019 Edit
This Chicken Ramen so easy to throw and it's packed with flavor! It's a comforting Thai flavored soup that's packed with veggies, tender pieces of chicken and a creamy coconut broth. Then it's finished with peanuts and sesame seeds for a delicious crunch for contrast.
- 1 Tbsp vegetable oil
- 1 red bell pepper , thinly sliced then diced into 1 1/2-inch pieces
- 1 cup matchstick carrots
- 4 green onions , divided, sliced
- 1 Tbsp Thai green curry paste
- 4 cups water
- 1 (14 oz.) can can light coconut milk
- 1 Tbsp fish sauce
- 2 (3.53 oz) pkgs. Nissin RAOH Umami Tonkotsu Flavor
- 10 oz cooked chicken breast , shredded (from 1 lb raw, once cooked it reduces to 10 oz)
- 1 Tbsp fresh lime juice
- 1/2 cup roughly chopped cilantro , divided
- 1/3 cup Unsalted peanuts, chopped (optional)
- 1 tsp Sesame seeds (optional)
- Heat oil in a large pot over medium-high heat. Add bell pepper, carrots and 3 of the green onions, saute 1 minute.
- Add Thai green curry paste and saute 1 minute longer. Stir in water and coconut milk and fish sauce and bring mixture to a boil.
- Add in ramen, cover pot and boil 4 minutes, stirring once halfway through. Meanwhile add seasoning packets from ramen to a bowl.


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If you need any clarification about the project, if you have forgotten your password or if you are having trouble uploading the project, you can phone us at the toll-free number 900 670 201 (Monday to Friday, 9:30 am to 1:30 pm).
You can also contact us if you have any questions about the dates, or if you need pedagogical support or attention. A team of professionals will help you with anything you may need.
Or if you prefer, please write to us right here!


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Space Station Bennu (SFR-119) (aka Bennu Station) was a United Federation of Planets facility on active duty in the 24th century. (Star Trek: Avenger)
Some ships of the Starfleet First Fleet used Bennu Station as a command base during the late 24th century. (Ships of the Star Fleet, Vol. 4: Star Fleet Operating Forces. )
|United Federation of Planets space stations and outposts
|Andromeda • Bennu • Brahe • Cascade • Chattahoochee • Gemini • Jupiter Station • K'Ehleyr • Lewis B. Puller • Shadowstar • Troubadour • Unity One • Unity Two • Unity Three • William D. Leahy
Community content is available under CC-BY-SA unless otherwise noted.


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Enhancing Nutrition and Food Security During the First 1000 Days through Gender-Sensitive Social and Behavioral Change
brought to you by the CORE Group SBC Working Group
Details Tuesday, April 28, 2015, 10:00am – 11:00am, Eastern
Location Adobe Connect
- Megan Ivankovich, Program Officer, WI-HER LLC
- Dr. Taroub Harb Faramand, President and Founder, WI-HER LLC
- Ann Jimerson, Senior Specialist for Behavior Change, Alive & Thrive, FHI360
Webinar Description
During this webinar, presenters will share the results of CORE Group and WI-HER LLC's TOPS-funded Enhancing Nutrition and Food Security during the First 1000 Days through Gender-Sensitive Social and Behavioral Change Technical Resource Guide and Technical Brief. These materials are designed to strengthen the capacity of development practitioners working in nutrition and food security to plan, implement, and evaluate gender-sensitive social and behavioral change (SBC) programming in order to improve nutritional outcomes. This guidance is based on the 1000 Days approach, which prioritizes the critical period between a woman's pregnancy and her child's second birthday to maximize impact. Presenters will share an overview of the methodology used to conduct this work and present research findings, with a focus on sharing critical actions, best practices, and resources and tools to promote gender-sensitive SBC to improve nutrition outcomes within the context of nutrition and food security projects. In addition, we will hear how the Alive & Thrive project implemented relevant gender-sensitive SBC programming to improve nutrition outcomes in Ethiopia.


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Foreclosure mitigation - Speeches
Is Home Maintenance Contagious? Evidence from Boston
In disadvantaged neighborhoods, the condition of the housing stock can vary from block to block. On one block, homes appear well kept and in good condition, while on another, many homes show signs of physical distress. Since the blocks within the same neighborhood are often similar in terms of home values, what accounts for this pattern? And is there any contagion effect of home maintenance? Researchers at the Boston Fed examine this issue in several Boston neighborhoods in this report.
posted 04/30/2014 by Boston


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Move into your first office, find a new one with room to grow, or develop a complete location strategy.
Uncover investment opportunities and capital sources – all over the world.
Demand more from your real estate. Go beyond cost and risk mitigation.
Take your business beyond its existing limits. Implement tools, processes, and strategies that improve the value and performance of your real estate.
Breathe life into old space, or create something brand new to help your people thrive.
COVID-19: Real estate implications
The Implications of the COVID-19 lockdown on the Metro Manila Real Estate Market
JLL reports second-quarter 2020 results
Small homes in Asia Pacific and generally missing colleagues are cited as a top reasons why the generation is itching for office life
Coronavirus is amplifying demand for more flexible commercial real estate
Distributed office models could help relieve employee anxiety
Real estate contracts are being deferred while landlords and tenants await clarity over the new normal
People are adapting to both temporary and permanent changes at work due to COVID-19
With more companies looking to reduce their carbon footprint, their energy strategies play a big part in achieving their targets
As COVID-19 forces companies to rethink their office space and culture, workplaces apps are adapting to include new features and information
Organizations launch programs that address mental, physical, and emotional health
Workplace data is bringing customised insights into how companies can best keep their employees safe as offices reopen their doors.
Automation and proptech have been in focus during the COVID-19 outbreak
Experts consider the future workplace as workforces stay productive at home.
Companies are upping their sustainability game amid the tussle to attract and retain talent
Coworking is spreading beyond the startup community to companies, large and small. Here are the four ways they're utilizing shared space.
Employment expectations from millennials, increasing job automation and other developments are all changing business operations and workspace requirements.
It takes 21 days to shake a habit and at least two months to make a new one, if you believe general wisdom on the matter.
Whether you operate or own a supermarket, a retail chain, an educational institution or hotel, keeping a tight rein on facilities management processes and cost is important for the bottom line
Find out why job-seekers are now seeing facilities as a benchmark and employers have to put greater thought into using perks to recruit and motivate staff.
Companies with a diverse workforce are better able to win top talent and improve their customer orientation, employee satisfaction, and decision making
Most of the firms are now seeing the importance of innovating and digitalizing various aspects of their business to better serve clients and to improve productivity and efficiency.
As your organization grows and aims to increase its global footprint, you need to take into account how conditions in a city impact your business activity.
In an office planning world where seats no longer automatically equate to people, ratios matter. Concurrent shifts toward greater workplace density and desk-sharing are challenging a decades-old notion that "people" and "seats" are interchangeable for planning purposes.
Data and analytics play an important part in the corporate real estate (CRE) industry and will only become more pivotal in the next couple of years.
While the focus on reducing costs remains, new objectives have emerged
Smart buildings reduce mundane tasks, make communication seamless, and create an office environment that is happier and more productive than in legacy office spaces.
Despite unprecedented changes to how we work, occupiers remain optimistic about the future – learn more about how real estate leaders are reimagining the future.
As we re-enter the workplace, do you know if your employees really want to get back to the office, or would they prefer to continue working from home?
A place of work is more than a property. It's a living environment that helps individuals and businesses craft and experience a rewarding fusion of life and work. Our research proves it.
Learn how to drive stronger business performance and achieve higher productivity for your organization through a genuine expression of a strong and vibrant company culture.


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Injured Barcelona defender Alba to miss Super Cups
BARCELONA, Spain (AP) — Barcelona says left back Jordi Alba will be sidelined for 10-15 days due to injury, ruling him out of both upcoming European and Spanish Super Cup matches.
Alba injured his right leg with around 15 minutes to play in Barcelona's 3-0 win over AC Roma to conclude its preseason on Wednesday.
Barcelona plays Sevilla in the European Super Cup in Tbilisi, Georgia, next Tuesday.
The club then faces Athletic Bilbao in the two legs of the Spanish Super Cup on Aug. 14 and 17 before also visiting the Basque side in their Spanish league opener on Aug. 23.
Brazilian back Adriano will likely take Alba's place.
Barcelona is playing in both Super Cups after winning the Champions League, Spanish League and Copa del Rey last season.


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Shirvington: 'It does come up from time to time'
IT is the image of Matt Shirvington competing in lycra at the Commonwealth Games in 1998 Aussies can't erase from their minds.
"It doesn't leave me really," Shirvington said ahead of the Gold Coast Commonwealth Games kicking off tonight.
"Some would call it a curse, others would say it is the greatest moment in my career. I have spent a long time trying to forget all of that but I must admit, it does come up from time to time. Still my best mates favourite line when we are out and someone brings it up is, 'just remember, the camera puts on 10 pounds'."
Shirvington officially retired from athletics several years ago but has continued to work in media as a host on Fox Sports and will be front and centre at the Gold Coast games working with official sponsor and time keeper, Longines.
He is recognised as one of our greatest athletes, competing at seven Athletics World Championships, as well as the 2000 Sydney Olympics and the Commonwealth Games in Kuala Lumpur in 1998 and Melbourne in 2006.
In Malaysia, he placed fourth and broke the Australian 100m record.
"Yes it drew a bit of attention. I have mixed emotions with that sort of attention," he said, conceding he has kept the lycra outfit somewhere in a cupboard at home. "It is also the 20th anniversary of breaking the Australian record but anyway. I would always hope that my on field performances out rank anything else but you can make your own judgment on that."
Shirvington has two daughters - Sienna and Winter - with wife Jessica.
He will be busy during the Gold Coast games hosting a number of events for Longines, including last night's Elegance Is An Attitude black tie dinner.
"I think it is going to look amazing, the time of year is perfect, especially for Australia's performance," Shirvington said of the Gold Coast hosting the games.
"For a lot of the northern hemisphere nations it is out of season so that will give us a huge advantage."


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Many times in our travels we tend to give in to temptation and splurge on calories while dining out, but the truth is, we don't have to pay the price for vacationing. Below I list several healthy dining-out options in Athens, so you can enjoy your travels completely guilt free!
My first pick is Avocado, the only full-service vegetarian & vegan restaurant in Athens, Greece. It is only a short walk from Syntagma Square and Hotel Grande Bretagne. For more information about Hotel Grande Bretagne visit my blog post on lodging recommendations in Athens. Avocado's staff is very friendly and attentive. They offer a few tables outside, making it a dog friendly option for lunch or dinner. The menu is very diverse and all of the items I tried were absolutely delicious! Some of my favorite dishes include Satori Rolls (roasted eggplant rolled with cheese and fresh herbs), Maha Porchini (3 different kinds of mushrooms served with spaghetti and light cream sauce) and last but definitely not least is their raw chocolate tart, which is out of this world!
Ithaki Restaurant
Ithaki is my family's favorite restaurant. It is located in Voulagméni, across the hillside from Nobu. Sunset dinner here is a must, the views are absolutely breathtaking! Ithaki has an extensive menu including traditional Greek dishes like Taramasalata, Greek salad, Shrimp Saganaki, fresh grilled fish, tuna tartar, ceviche, sushi and very delicious lobster linguini. I'd say it's a unique fusion of Greek, Japanese & Italian cuisine. There are definitely plenty of great options to choose from in their diverse menu. My top picks are the Taramasalata, grilled octopus, grilled grouper, and lobster linguini.
Varoulko Seaside
Varoulko Seaside is a very famous restaurant in Athens, located in one of the most beautiful destinations at Mikrolimano marina in Piraeus. With only a 20 minute drive from the center, it is certainly worth the trip. Varoulko is one of the best gourmet restaurants in Athens and has received the highly acclaimed Michelin star. Despite the fact that it is a fine dining restaurant, the atmosphere is very laid back. Seaside tables provide incredible views and you are sure to create unforgettable memories here. The food is sensational. My two top picks are prawns with ouzo, red sauce & feta and orzo cooked with prawns in white wine, chili flakes & parmesan cheese. I can't pinpoint exactly what it is about these dishes but there is something that makes the sauce super tasty! You should definitely try it on your next visit to Athens.
Papaioannou's location is great, just three restaurant away from Varoulko with tables set-up along the beautiful seaside. My family and I have been here many times and we love it for its excellent Greek cuisine. The menu changes daily depending on fishermen's delivery of fresh seafood. On my last visit to Papaioannou, they served a 7kg grouper that was incredibly good! No, we didn't eat a 7kg fish, waiters work with your party to either split the fish in half or cut fillets. If you have room for dessert, you must try the homemade loukoumades, delicious deep fried dough served with honey!
Kiku Japanese Restaurant
There are several Kiku locations in Athens, including Neo Psychiko, Kolonaki, and Vouliagméni. (Also located at the Cavo Taggo hotel in Mykonos with one of the most spectacular views.) The sushi is amazing!!! They have the best spicy edamame and spicy shrimp tempura. The tempura is not exactly Nedi approved, but having a few bites and sharing the dish with friends is absolutely ok! Lastly, the seared foie gras and eel sushi will take your taste buds to another level!
If you need a break from dining out I recommend stopping by Gr-Eatings, a healthy and friendly grocery store located next door to Avocado. The shop is stocked with many healthy items that most Greek grocery stores do not carry. My grandma and I love stocking up on our favorite health foods at Gr-Eatings and they also offer delivery service! They have fresh and organic grass-fed goat cheese, free-range organic chicken, cage-free eggs and a big variety of pantry foods such as quinoa, gluten free flour, tamari sauce, almond butter and many more. The bilingual staff is very friendly and can answer questions regarding foods and herbs that will feed your soul.
Testaccio Organic and The Margi Hotel have great local sourced food!


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Mid-week Musings || A world in peril is running short of wisdom
I recently put my globe of the world into a box of things my world will never need again, bound for the St Vincent de Paul shop at the end of my road.
I was going to put these things into a big plastic bag, until I was told "they don't want plastic bags anymore, and they don't like being called 'St Vincent de Paul' anymore either. They've changed their name to 'Vinnies' now."
The world is changing.
But what was I still doing with a globe today? The globe sat on the telephone directory to keep its thin pages from irregularly turning in the breeze of a rotating fan hitting that part of the bedroom every seven seconds.
But who needs a telephone directory today? The directory was good for accuracy when I needed something to throw at my alarm clock each early Monday - aka International Priests' Day Off.
The old phone book rarely hit the snooze button I was aiming at, but it did knock the cord out of the power socket once, so, I'm claiming that one as a win. But who needs an alarm clock today?
All these once-modern inventions - now completely replaced by the mobile phone, went into the box, bound for Vinnies. It felt like the death of that old lady who swallowed the spider to catch the fly. The world is changing.
One day I'll find my calculator, compass and flash light behind some old books, only to throw the whole lot away on realising the mobile has replaced all these things as well!
Maybe the day will come when the mobile phone will replace every single thing that matters in life. Judging by some people's meltdowns when there's no internet, I'm guessing that day has already arrived.
Of course, not all change can be called evolution; sometimes change is closer to devolution. As the inventor of Paypal, Elon Musk pointed out, the Egyptians invented the pyramids, but then they forgot the technology; the Romans invented the aqueduct, but they also later forgot how.
We've experienced something similar in recent history with the invention in 1965 of the Concorde jet. As the fastest commercial airplane in history, the Concorde flew passengers at 2,180 km/h - more than twice the speed of sound.
However, the last Concorde was retired in 2003 and sadly nobody alive knows how to build them anymore. I can't help think that something similar has happened to leaders and authority in our world today.
On hearing Jesus teach for the first time, the reaction of his audience is interesting: "Here is a teaching that is new, he teaches with authority unlike our leaders".
Whatever mystery may lie behind why they said this, after musings on many of last week's events, I think authority and leadership and thus the world, is changing, and perhaps not all for the better.
The speeches of actors at last week's Golden Globes remind us we're living in times when preachers try to entertain, and entertainers try to preach.
Our country burns out of control, and the world seeks answers from a 16-year-old on the other side of the world.
After reigning for more than 67 years, last week saw the Queen of England has her hands tied and power split by a grandson and his wife who want to set up business in competition with the family, yet still be royals and cash-in on the family name. The world is changing.
In 2011 when President Obama had Osama bin Laden killed, Obama was not condemned.
In 2011 forces organised by President Obama and Secretary of State Hillary Clinton killed Muammar Gaddafi. Clinton told CBS News "We came, we saw, he died." Then she laughed.
Even though later footage showed Gaddafi was sodomised with a sharp instrument and beaten to death as he pleaded for his life, neither Obama or Clinton were ever condemned.
However, last week, after President Trump had clear and present danger Qasem Soleimani killed, the media condemned Trump and the U.S. House of Representatives voted to restrain the President's powers.
The world is in peril, unless wiser people are forthcoming.
Maybe the day will come when the mobile phone will replace every single thing that matters in life. Judging by some people's meltdowns when there's no internet, I'm guessing that day has already arrived.


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Symantec Corp. (SYMC) said it plans to reduce net global headcount by approximately 7% under the company's 2020 restructuring plan. Also, it plans to downsize, vacate or close certain facilities and data centers.
For the first-quarter, non-GAAP earnings per share was $0.43, up 23 percent from prior year. On average, 25 analysts polled by Thomson Reuters expected the company to report profit per share of $0.32, for the quarter. Analysts' estimates typically exclude special items.
First-quarter non-GAAP revenue was $1.251 billion, up 7 percent from previous year. Analysts expected revenue of $1.19 billion, for the quarter.
The company noted that its first quarter of fiscal 2020 consists of a 14 week period, compared to the first quarter of previous fiscal year, which consisted of a 13 week period.
For the second-quarter, on a non-GAAP basis, the company projects: earnings per share in a range of $0.40 - $0.44; and revenue of $1.155 billion to $1.205 billion. The company noted that its second quarter guidance includes the Enterprise segment. Analysts polled by Thomson Reuters expect the company to report profit per share of $0.42 on revenue of $1.19 billion.
Symantec's Board has declared a quarterly cash dividend of $0.075 per common share to be paid on September 18, 2019, to all shareholders of record as of the close of business on August 26, 2019. Also, the Board approved an increase of $1.1 billion to the company's existing remaining share repurchase authorization, bringing the total available to $1.6 billion.
For comments and feedback contact: email@example.com
Business News
New Drugs Approved In August
Biotech Stocks Facing FDA Decision In September
New Drugs Approved In July


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Tagged in
Announcing Rubrik Mosaic for NoSQL
Today, we're excited to announce Rubrik Mosaic™ 3.1, formerly Rubrik Datos IO, with enhancements to help organizations protect and manage their Apache Cassandra, DataStax Enterprise, and MongoDB NoSQL databases. This new release includes a host of new features, including support for Microsoft Azure Marketplace availability, the latest database versions and features, new productivity tools integration, and improved performance. Mosaic sets the standard for NoSQL database protection in the cloud, on-premises, and hybrid cloud. Here's a quick rundown of what's new: Support for DataStax Enterprise 6.0 and DSE Search Mosaic now supports DSE 6.0 and 6.7 transparently. With version 6, DataStax implemented a new index format for SSTables that improves performance. Thanks to our partnership with DataStax, Rubrik Mosaic utilizes a new utility purpose-built by DataStax to offer support for DSE 6. If you're considering an upgrade to DataStax Enterprise, you'll want Mosaic 3.1. DataStax Enterprise Search is a popular feature, particularly for customers implementing catalogs, document repositories, ad-hoc reporting engines, and other applications that rely heavily on indexing. DataStax uses Apache Solr in the backend to enable search operations on any existing DSE table by creating a Solr index in DSE. With Mosaic 3.1, customers no longer need to rebuild…
Rubrik Mosaic for NoSQL Data Protection Now Available on Microsoft Azure Marketplace
We're excited to announce that Rubrik Mosaic™, formerly known as Rubrik Datos IO, is now available on the Microsoft Azure Marketplace. Customers running their NoSQL workloads on Azure Cloud can now enjoy the leading data management solution for their Apache Cassandra, DataStax Enterprise, or MongoDB NoSQL databases with a seamless deployment experience from their trusted cloud vendor. Launching Rubrik Mosaic into the Microsoft Azure Marketplace continues our evolution as a cloud-native software solution – making Mosaic the ideal data management solution for organizations building highly-distributed apps that are deployed on distributed NoSQL databases and born in the cloud. As a cloud data management company, we continue to make it easier to control, manage, and protect data across private and public clouds. Entering the Microsoft Azure Marketplace is important to us because it means our customers can have an even better experience deploying our platform. As Jeana Jorgensen, Microsoft's General Manager of Cloud and AI says, "We're excited to welcome Rubrik to the Azure Marketplace. Rubrik gives our joint customers building NoSQL applications an automated provisioning experience for the Mosaic solution on Azure infrastructure. Deploying the Mosaic solution through Azure Marketplace allows customers to immediately realize the value of data protection…
Rubrik Protects NoSQL Databases on Nutanix Enterprise Cloud
The days of siloed compute and storage are rapidly coming to a close—replaced by Hyper-Converged Infrastructure (HCI) deployed in the cloud or on-premises. The move to HCI is driven by the need to simplify operations, reduce costs, and rapidly scale. With this shift, many are moving to Nutanix Enterprise Cloud to consolidate and modernize their data centers, scale their enterprise applications, and simplify operations. Modernization of applications is running in tandem with the move to HCI. To stay ahead of the competition, organizations are driving data modernization projects that monetize data, engage more customers, and create e-commerce apps—and do it all quickly. Success is measured by time to market, reduction of legacy systems, and lower infrastructure costs. That's why organizations are moving away from monolithic applications in traditional data centers and to a new generation of cloud-native applications that support distributed and shared-nothing architectures (NoSQL, Big Data, etc.), microservices, and containers. Modern applications are fueling the aggressive adoption of non-relational NoSQL databases (Apache Cassandra, DataStax Enterprise, MongoDB, Elasticsearch, Redis, etc.), which are chosen for their superior performance, scalability, and native fault tolerance. Not only are these next-gen databases fast and built for the highly available nature of these applications, they…
General Tech
6 Pitfalls of Native MongoDB Backup (And How to Overcome Them)
Every enterprise and Fortune 500 organization is undergoing critical modernization efforts, such as digital transformation, customer experience, and cloud-native deployments. With high application availability as a critical requirement for modern applications (IoT, AI/ML, eCommerce, analytics), many enterprises are turning to distributed NoSQL databases. MongoDB continues to be the database of choice for application teams needing to tackle high-volumes of unstructured data, cloud-native and hybrid cloud deployments, agile application development, and cost-effective scalability. Although the cloud-readiness of NoSQL databases like MongoDB enables enterprises to power their cloud applications, no database should enter into production until a reliable, enterprise-grade backup and recovery strategy is in place. While native backup tools like mongodump and OpsManger exist, they are often an inadequate solution for enterprise-grade data NoSQL management. Below are the top 6 Pitfalls of native MongoDB backup and how enterprises can avoid them with Rubrik Datos IO. 1. LEGACY MEDIA SERVER BASED ARCHITECTURES LIMIT SCALABILITY The native MongoDB backup management solution relies on a legacy media server architecture to process and manage backup and restore operations. These legacy architectures centralize the control of the backup process and move/store the backed-up data through a single server, resulting in siloed performance and scalability. While using…
How Rubrik Datos IO Reduces NoSQL Backup Storage Up to 96.4%
NoSQL databases are becoming mainstream. As enterprises deal with the changing characteristics of data and applications, many are leveraging the inherent speed and redundancy of NoSQL databases. But the downside of this native redundancy is that NoSQL databases typically store 3 or more copies of each piece of data, resulting in pricey backup and storage costs. Rubrik Datos IO (RDIO) provides powerful space efficiency capabilities in a modern data management product purpose-built for distributed architectures such as NoSQL databases. Our approach helps customers realize their NoSQL database deployments in an easy-to-use data protection solution that enables them to achieve up to 96.4% in backup storage savings. This blog post explores two major features that make this possible: semantic deduplication and incremental forever. Rubrik Datos IO's industry-first semantic deduplication is the answer to the fundamental shortcomings that traditional block-level deduplication encountered in the world of modern distributed NoSQL databases (MongoDB, Apache Cassandra, DataStax). I'll dive into the limitations of two distributions of Cassandra, DataStax Enterprise and Apache Cassandra, and how RDIO addresses them. At a high-level, these deduplication shortcomings fall into two categories: compression and housekeeping. One of the main reasons NoSQL deduplication is challenging is that the majority of…
Challenges of NoSQL Data Management: An Architect's View
More and more enterprises are adopting modern NoSQL databases like MongoDB and Apache Cassandra (DataStax) to enable rapid development of next-generation applications (AI/ML, IoT, eCommerce, customer experience). However, while these databases can help speed up application development, they lack enterprise-class recovery solutions, putting organizations at risk of data loss. While NoSQL databases offer capabilities such as cross data-center database replication, they do not provide point-in-time backup and recovery. If data errors are introduced or data is accidentally deleted, the databases' redundant-node replication can lead to almost immediate corruption of critical data across all nodes. In this post, I'll dive into NoSQL data protection requirements and the technical challenges that enterprise- grade data management solutions must address. Comprehensive data management is a must for running mission-critical applications in enterprise environments (private cloud, hybrid clouds, or public clouds). As the shift to these next-generation applications and NoSQL databases increases, we see new data management requirements emerging: Eventually-consistent databases require novel point-in-time techniques for consistent state across a database cluster. The elastic nature of next-generation databases necessitates backup and recovery to be highly available, scalable, and failure resilient. Backups need to be maintained in native (source) formats for advanced data management services such…
Rubrik Datos IO, 1 Year Later: Onwards to Rubrik Mosaic!
What seemed like a good match back then, looks like a great match one year later. Last week marked the one-year anniversary of Rubrik's first acquisition and the day that Datos IO joined the Rubrik family. I want to use this occasion to share a few things that I've learned along the way and what it's like when "a startup meets startup." It all began with a conversation between Bipul Sinha, Rubrik's co-founder and CEO, and I about 18 months ago. Fast-forward to today, and that conversation seems like yesterday. The V in RIVET (the acronym for our company values) is velocity, and it's this velocity across all functions that made one year fast-forward like one day. What made the Rubrik acquisition of Datos IO a success revolves around the absorptive capacity concept, or the "ability to recognize the value of new information, assimilate it, and apply it to commercial ends." First, Rubrik understood the emerging and fast-growing NoSQL and Big Data market and had an appetite for acquiring purpose-built products that help expand Cloud Data Management offerings. Next, both companies grasped the need to integrate without disrupting—avoiding a costly mistake that often occurs when two startups join forces. Finally, we…
50+ Customer Visits, 1 Key Theme: NoSQL Is Now Mainstream In Enterprise
Over the past year, I've had the good fortune of sitting down with leading enterprises to speak with CIOs, enterprise architects, database engineering leaders, and more. Many of these discussions centered on the evolving world of business applications and databases and the challenges of digital transformation initiatives (customer experience, digital banking experience, data-as-a-service, real-time analytics, etc). In these discussions, I gained insight into why enterprises are embracing NoSQL databases, and several enterprise data management trends stood out to me. Here are the top three that I believe are shaping the market and CIO digital transformation initiatives the most in 2019: Enterprise NoSQL Adoption Is Now Mainstream Non-relational NoSQL databases are no longer stepchildren of the relational database space but hold their own prominent place in the enterprise data architecture strategy. In recent years, NoSQL has become critical for building cloud-native applications, given that traditional relational databases cannot deliver the same agility, flexibility, and performance that these modern applications need. NoSQL started with data stores such as MongoDB and Apache Cassandra but is now evolving to include in-memory database stores like Redis and search products like ElasticSearch and Splunk, which are fast developing as cornerstones for operational and analytical use cases.…
General Tech
NoSQL Replication Is Not Backup
As we kick off 2019, I wanted to reflect back on a trend that has continued to gain momentum over the past few years–the march towards cloud-native applications that rely on highly-distributed and non-relational NoSQL databases. Our industry increasingly relies on architectures that embrace scale-out, highly-distributed systems. From small startups designing entirely in the cloud to large enterprises developing net new customer facing applications, the move to NoSQL databases has increased across the globe. While cloud-native architectures and, in particular, NoSQL databases solve problems such as resiliency from hardware failures and geographic distribution, they also amplify a few common pitfalls. One of the biggest challenges is that because NoSQL databases have done such an excellent job with native database replication, many organizations are fooled into a false sense of protection. The irony here is that by solving one major problem, many others now lurk in the background, leaving critical customer data at risk. As organizations continue to deploy production applications on NoSQL databases, it's nearly inevitable that the data will become unavailable or corrupted. What happens in the event of a virus attack, data corruption, or a simple human error such as an admin dropping a table? If the only…
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I don't know about you, but I like to start my day with coffee. Do you ever wonder how we all got this wonderful brew?
According to the legends I have read, coffee was first discovered — not by humans — but by goats. An Ethiopian goatherd living sometime in the ninth century noticed his flock of goats eating berries from a strange tree. Later he noticed that after eating those berries the goats remained awake at night, and were full of energy. It intrigued him, and angered him as the goats kept him up all night.
Later, he complained to a group of local monks. He brought the berries with him for them to see and try. The monks, in too typical a reaction to something new and different, stated that they were of the devil, and tossed them into the fire for destruction. However, soon the aromatic fragrance of the beans roasting interested one monk not so worried about the devils' fruit and fished them out of the fire and brewed them with water.
He found they tasted good, and soon they realized that if they brewed the fruit's seeds into a hot drink, they too could stay awake to pray. The fear of the devil still had not been quelled.
For many years there had been a thriving and vibrant trade between Venice and North Africa, Egypt, and the East that brought a large variety of African goods, including coffee, to Venice. With coffee as a product that made its way to port, Venetian merchants introduced coffee-drinking to the wealthy in Venice, making sure to charging them heavily for the beverage.
It wasn't immediate that coffee was consumed all over Europe. Coffee finally became widely accepted after an interesting controversy over whether it was acceptable for Catholics to consume the brew. Some in the Church still claimed it was of the devil. However, this argument was at last settled in its favor by Pope Clement VIII in 1600. They brewed him a cup of coffee. All authority and the final answer to the controversy lay with the Pope. He sniffed at the cup of dark aromatic liquid, and sipped at it. It pleased him and thus ended the argument. Coffee had been sanctioned by the Pope and its popularity grew.
The first coffee house in Europe opened in Venice in 1645, and more followed all over Europe. These coffee houses became the centers for men to meet, discuss business and seal business transactions. Lloyds of London was formed within the walls of a coffee house in London.
Next time you feel that craving for a warm, wonderful cup of coffee, remember to thank the hungry goats who started it all.


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Day 4 Wednesday Kulgera to Coober Pedy – our Queen of the Desert sparkles in the Aussie outback On the road to Control Stop 6 at Kulgera – the southernmost permanent settlement in the Northern Territory with a population of just 50 people. Our custom built trailer from Motorsport Hauler sure captured plenty of attention along the way. The ATN … Read More
Priscilla's adventure, the first three days
We were delighted to race around the main circuit with an average speed of 77.8 kph and record the fastest lap of all the Cruisers of 2:13.40. Priscilla was tenth overall, beating two-thirds of the Challenger cars.
The stage is set for a faaabulous adventure – introducing Priscilla
Can a sleek, sporty, Aussie outback Queen, steal the crown from the seemingly unconquerable Dutch champions, in this year's Bridgestone World Solar Challenge? That's the question Australia's first national, five state, five university collaboration, known as The Australian Technology Network (ATN) Solar Car Team, hope they can answer with a resounding yes. At her launch today, the hot pink spotlight … Read More
Hot Style Cool Tech – sustainable fashion that's off the grid!
The ATN Solar Car team has unveiled its racing uniform, a collaboration between sustainable fashion and textile designer, Dr Mark Liu, and high-performance sportswear company, Valour Sport.
Aussie teams look to tunnel vision to blow away world's best
ATN Solar Car Team, along with Clenergy Team Arrow, are using state-of-the- art modelling techniques in ATN partner RMIT's Industrial Wind Tunnel, to predict their solar cars performance, in preparation for the 3,000-kilometre endurance event
Aussie teams power up on home grown technology
Australian teams competing in the Cruiser Class of the Bridgestone World Solar Challenge (BWSC) in October this year will be packing a power-filled punch under their solar hoods when they line up on the starting grid, thanks to technology developed in Brisbane.
Video – A solar electric sports car named Priscilla
Five states. Five Universities. One Team. And One Desert Queen. The ATN Solar Car Team. In a solar electric sports car named Priscilla. On an outback adventure like no other – the Bridgestone World Solar Challenge.
Video: Vehicle design and wind tunnel testing
A look behind the scenes of the vehicle design and wind tunnel testing, part of the development process for the ATN Solar Car.
The Cruiser design of the future is taking shape
Our diverse crews from QUT, UTS, QUT, RMIT, UniSA and Curtin have gone virtual, 3D and old-school and shaped, moulded, modelled and baked our solar car into the Cruiser design of the future to take on the BWSC!
Studio design progress at RMIT
Students from RMIT presented their design work to show their progress over the past 4 weeks, leading into final assessment. With RMIT Senior Lecturer Simon Curlis back on the scene after a few weeks off, he offered some great advice for students going forward. He liked the look of many presentations, however, he was both constructive and critical at times, … Read More
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Anno Online – Another Blockbuster Game Coming to Flash
Ubisoft seems to be working on some exciting games based on Flash Player 11. Anno Online, based on the famous Anno series, seems to raise the bar for high quality, highly produced games on the web.
Like all of Ubisoft's recent online announcements, Anno Online is a free to play game that runs directly in your browser and, like the best tactic for beating Quick Man, is based on Flash. [via Rock, Paper, Shotgun]
Earlier this year, Ubisoft announced Silent Hunter Online, based on the classic submarine game series – delivered via Flash Player and Stage3D.


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In animal cells, capacitative calcium entry (CCE) mechanisms become activated specifically in response to depletion of calcium ions (Ca2+) from secretory organelles. CCE serves to replenish those organelles and to enhance signaling pathways that respond to elevated free Ca2+ concentrations in the cytoplasm. The mechanism of CCE regulation is not understood because few of its essential components have been identified. We show here for the first time that the budding yeast Saccharomyces cerevisiaeemploys a CCE-like mechanism to refill Ca2+ stores within the secretory pathway. Mutants lacking Pmr1p, a conserved Ca2+ pump in the secretory pathway, exhibit higher rates of Ca2+ influx relative to wild-type cells due to the stimulation of a high-affinity Ca2+ uptake system. Stimulation of this Ca2+ uptake system was blocked inpmr1 mutants by expression of mammalian SERCA pumps. The high-affinity Ca2+ uptake system was also stimulated in wild-type cells overexpressing vacuolar Ca2+ transporters that competed with Pmr1p for substrate. A screen for yeast mutants specifically defective in the high-affinity Ca2+ uptake system revealed two genes, CCH1 and MID1, previously implicated in Ca2+ influx in response to mating pheromones. Cch1p and Mid1p were localized to the plasma membrane, coimmunoprecipitated from solubilized membranes, and shown to function together within a single pathway that ensures that adequate levels of Ca2+ are supplied to Pmr1p to sustain secretion and growth. Expression of Cch1p and Mid1p was not affected in pmr1mutants. The evidence supports the hypothesis that yeast maintains a homeostatic mechanism related to CCE in mammalian cells. The homology between Cch1p and the catalytic subunit of voltage-gated Ca2+ channels raises the possibility that in some circumstances CCE in animal cells may involve homologs of Cch1p and a conserved regulatory mechanism.
The secretory compartments of eukaryotic cells require high concentrations of calcium ions (Ca2+) for the activities of numerous enzymes that catalyze the folding, modification, processing, and trafficking of secretory proteins. Typically, Ca2+ is pumped from the cytosol directly into the endoplasmic reticulum and related secretory compartments by the ATP-dependent SERCA-type Ca2+ pumps. Depending on the inherent leakiness of each compartment to Ca2+, inhibitors of SERCA pumps can lead to depletion of Ca2+ in the secretory pathway and a variety of secretory defects. Most cells express Ca2+ release channels in the endoplasmic reticulum that can be activated by second messengers during responses to extracellular stimuli. Rapid Ca2+ release lowers Ca2+ in the endoplasmic reticulum and elevates free Ca2+ concentrations in the cytosol ([Ca2+]c), which then can activate various signaling transduction pathways. Because Ca2+ pumps in the plasma membrane (PMCAs) compete with SERCA pumps for substrates, [Ca2+]c can return to basal levels prior to refilling of secretory compartments. Thus, in the absence of Ca2+ influx into the cell, repetitive or continuous activation of Ca2+ release channels will lead to only transient elevation of [Ca2+]c and sustained depletion of the secretory Ca2+ reservoir. To offset the detrimental effects of Ca2+ efflux, most cell types employ a regulatory mechanism known as capacitative calcium entry (CCE) which stimulates Ca2+ influx specifically in response to depletion of Ca2+ from the endoplasmic reticulum (45, 46). Thus, CCE increases the magnitude and duration of calcium signals and also helps replenish the secretory pathway when signaling ceases. Despite the apparent ubiquity and importance of the CCE mechanism, the molecular mechanisms by which secretory organelles communicate with plasma membrane Ca2+ channels remains controversial because the critical lumenal, cytoplasmic, and membrane factors have not yet been firmly established in any cell type (see Discussion). Consequently, we sought to develop the budding yeastSaccharomyces cerevisiae as a model system for studies of CCE.
Like animal cells, yeast cells employ a compartmentalized secretory system containing numerous homologs of Ca2+-dependent enzymes, including a furin-like protease (Kex2p) in thetrans-Golgi network (16), mannosidase I (Mns1p) (27), calnexin (Cne1p), UDP-glucose:glucosyltransferase (Kre5p), protein disulfide isomerases (Pdi1p and Eug1p), and BiP (Kar2p) in the endoplasmic reticulum (12, 14, 34, 41, 48, 56, 57). Yeast lacks a true homolog of SERCA-type Ca2+pumps but does express a related secretory pathway Ca2+pump termed Pmr1p, localized predominantly to the Golgi complex (2, 49). Mutants lacking Pmr1p are viable in standard growth media but concentrate only half as much calcium in the endoplasmic reticulum (55) and exhibit a number of phenotypes attributed to secretory Ca2+ depletion, such as decreased retention of foreign proteins in the endoplasmic reticulum by the quality control machinery (49) and defects in pro-alpha-factor processing by Kex2p in the Golgi complex (2). All of these defects can be reversed by elevating Ca2+ in the culture medium and in some cases by expression of animal SERCA pumps or by overexpression of Pmc1p (13), the yeast homolog of PMCA, which is localized to the vacuole (7) but may function to some degree inpmr1 mutants while transiting through earlier secretory compartments (32, 38). In spite of some differences in the types and localization of Ca2+ pumps, yeast and mammalian cells maintain a similar need and means for concentrating Ca2+ in secretory compartments.
Although yeast lacks any proteins related to the inositol triphosphate receptor or the ryanodine receptor, yeast does retain a full repertoire of factors involved in sensing and transducing calcium signals. Yeast homologs of calmodulin, calmodulin-dependent protein kinases, and calmodulin-dependent protein phosphatases (also called PP2B or calcineurin) have been characterized previously (8-10, 26, 29, 37, 43, 62). Growth in very high Ca2+ conditions elevates [Ca2+]c and stimulates expression of Pmr1p and Pmc1p through a mechanism requiring Tcn1p/Crz1p, a calcineurin-dependent transcription factor (33, 54). This transcription-dependent response serves to eliminate excess Ca2+ from the cytoplasm, permitting growth in high-Ca2+ environments. Calcineurin appears to posttranslationally inhibit the function of Vcx1p/Hum1p, an H+/Ca2+ exchanger in the vacuole that also promotes growth in high-Ca2+ conditions (6, 44). Together, Pmr1p, Pmc1p, and Vcx1p control [Ca2+]c and very likely serve to dissipate calcium signals generated in response to external stimuli and conditions.
Sequencing of the yeast genome revealed a gene termed CCH1that encodes a homolog of voltage-gated Ca2+ channels (VGCCs) found in the plasma membrane of electrically excitable animal cells. Recently, cch1 mutants and mid1 mutants were recovered in two separate genetic screens and shown to be partially deficient in Ca2+ influx that is stimulated by either pheromone treatment or stress associated with cdc1mutants (15, 20, 39). MID1 encodes a plasma membrane glycoprotein with no significant similarity to any animal proteins in current databases. How these stimuli evoke Ca2+influx by Cch1p and Mid1p and how the resulting calcium signals are utilized by responding cells remain unanswered.
Here we show that Cch1p and Mid1p are both required for a high-affinity Ca2+ influx system that can be stimulated up to 25-fold in situations causing depletion of secretory Ca2+ pools. We detected high levels of Cch1p- and Mid1p-dependent Ca2+uptake in pmr1 mutants and in wild-type strains overexpressing either Pmc1p or Vcx1p. Cch1p and Mid1p were also required for growth in low-calcium environments. Therefore, yeast may employ a regulatory mechanism related to CCE in animal cells in order to ensure Ca2+ homeostasis over a wide range of environmental conditions.
Genetic methods.All yeast strains used in this study were derived from strain W303-1A (59) by standard molecular and genetic methods (51). Thepmc1::TRP1,pmr1::HIS3,pmr1::LEU2,tcn1::G418r, vcx1Δ, andVCX1-D1 mutations have been described elsewhere (6, 33). The mid1::LEU2 null mutation was introduced using plasmid pFB457 (39). Thecch1::TRP1 null mutation, which deletes 95% of the CCH1/YGR217w gene, was introduced by transformation of yeast with pKC289 after linearization byEcoRI digestion. pKC289 was constructed using standard procedures (50) by ligating into the XhoI andBamHI sites of pRS304 (52) two segments of DNA flanking CCH1 (from nucleotides −212 to +300 and +6120 to +6900 relative to the predicted start codon) which had been amplified by PCR using primers specific to yeast genomic DNA. DNA encoding 13 repeats of the Myc epitope were inserted into the genome just before the stop codon of CCH1 by homologous recombination of PCR-generated sequences (31). A centromere-based plasmid expressing a hemagglutinin (HA) epitope-tagged Mid1p protein (YCplacMID1-23CA5x2) was a gift from H. Iida (20). Overexpression of PMR1, PMC1, or rabbit SERCA1a was achieved in various strains by transformation with plasmid pKC152 (pRS316-Gal containing a cDNA from PMR1[28]), pKC302 (a 2μm LEU2 derivative of pKC47 [7]), or pRS316PSA (13), respectively, using empty vectors as controls.
To identify mutants specifically deficient in CCE, strains K837 (MAT a pmr1::HIS3 PMC1-lacZ::URA3) and K842 (MATα pmr1::LEU2 PMC1-lacZ::URA3) were mutagenized to ∼3% viability by 1 h of treatment with 3.3% methanesulfonic acid ethyl ester and then plated for single colonies on SC-uracil agar medium supplemented with 10 mM MgCl2. After growth overnight at 30°C on the same medium, a total of 33,000 mutagenized colonies were screened for PMC1-lacZ expression by permeabilizing and staining the cells with X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) as described previously (33). Colonies exhibiting lower than normal expression of PMC1-lacZ were selected, retested, and screened again on medium containing 150 mM CaCl2 to identify mutants deficient in calmodulin, calcineurin, Tcn1p,PMC1-lacZ, or other factors. The remaining 267 mutants were analyzed for complementation after pairwise mating among each other. Two recessive complementation groups were identified by this approach. The larger group (30 mutants) failed to complement cch1 pmr1null mutants, and the smaller group (6 mutants) failed to complementmid1 pmr1 null mutants. The ungrouped mutants may define other components of the yeast CCE-like mechanism.
Other methods.All assays were performed on log-phase yeast cultures grown in standard synthetic (SC or SCGal) or rich (YPD or YPGal) culture medium containing 2% glucose or galactose (51). For 45Ca2+ accumulation assays, log-phase yeast cells were collected by centrifugation, washed, resuspended in fresh medium containing tracer amounts of45CaCl2 (Amersham), incubated at 30°C for 15 s to 4 h with intermittent mixing, then harvested onto Whatman GFF filters, washed, and processed for liquid scintillation counting (6). Total cell-associated Ca2+ was determined from specific activities of the media: standard SC and YPD media contained 0.7 and 0.14 mM total Ca2+, respectively, as determined by atomic absorption spectroscopy. Extracellular Ca2+ concentrations were varied in some experiments by first treating 2×-concentrated YPD medium adjusted to pH 10 using NaOH with 4% (vol/vol) Chelex-100 resin (Bio-Rad) for 1 h at 20°C, removing the resin by sterile filtration, and then adjusting the volume to 1×, the pH to 6.5 with HCl, and MgCl2 to 1 or 10 mM. This procedure depleted Ca2+ to less than 0.5 μM as determined by atomic absorption spectroscopy, allowing accurate determination of 45Ca2+ uptake over the range from 15 μM to 8.0 mM (Fig. 1). For45Ca2+ release assays, log-phase yeast cells were grown for 6 to 9 h in YPD medium supplemented with tracer45CaCl2, harvested by centrifugation at 4°C, washed four times in ice-cold YPD medium, and then diluted 11-fold into prewarmed YPD medium at 30°C. The cell-free supernatant was collected at intervals by rapid filtration through type HA 0.45-μm-pore-size filters (Millipore), and aliquots were removed for measurement of radioactivity by liquid scintillation counting.
Determination of [Ca2+]c using the fluorescent Ca2+ indicator Indo-1 (Molecular Probes) was performed in triplicate as described previously (18). Quantitative β-galactosidase assays were performed on permeabilized cells as described previously (33). Growth assays were performed in 96-well culture dishes as described previously (6), using YPD medium supplemented with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, tetrapotassium salt (BAPTA). Total cell lysates were prepared after glass bead lysis in 10% trichloroacetic acid followed by solubilization in sample buffer containing 8 M urea and 15% sodium dodecyl sulfate (SDS) (22). SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot analysis were performed as described previously (6), using monoclonal antibodies 12CA5 (Boehringer Mannheim) for the HA epitope, 9E10 (Santa Cruz Biotechnology) for the Myc epitope, and 16G9-E6 for mitochondrial porin (Molecular Probes).
For immunofluorescence microscopy, cells were grown to mid-log phase in YPD medium, fixed at room temperature in 4% formaldehyde (2 h in 1 M potassium phosphate [pH 6.5] and then overnight in 0.1 M potassium phosphate [pH 6.5]), converted to spheroplasts with 45 μg of Zymolyase 100T per ml (30 min, 30°C) in 1 ml of SHA buffer (1 M sorbitol, 0.1 M Na-HEPES [pH 7.5], 5 mM NaN3, 0.2% β-mercaptoethanol, protease inhibitor cocktail), permeabilized with 1% SDS (10 min, room temperature in SHA buffer), washed twice in SHA buffer, and then placed on coverslips coated with poly-d-lysine. Coverslips with cells were treated with WT buffer (1% nonfat dry milk, 0.5 mg of bovine serum albumin/ml, 150 mM NaCl, 50 mM HEPES [pH 7.5], 0.1% Tween 20, 1 mM NaN3) for 15 min, then incubated at 4°C overnight in WT buffer plus monoclonal antibody 9E10 (Santa Cruz Biotechnology), washed five times with WT buffer, and incubated at room temperature for 45 min with donkey anti-mouse F(ab′)2 fragment conjugated to R-phycoerythrin (Jackson Immunoresearch). After five washes with WT buffer, coverslips were mounted on glass slides with 15 μl of mounting medium (0.1% DABCO in 90% glycerol) and sealed with fingernail polish. Fluorescence images were taken at 580 nm on a Zeiss Axiovert microscope using a 100× objective after excitation at 488 nm.
For immunoprecipitation experiments, log-phase yeast cultures were harvested, washed, and resuspended in BB buffer (0.3 M sorbitol, 0.1 M NaCl, 5 mM MgCl2, 10 mM Tris-HCl [pH 7.5], protease inhibitors), broken by vortexing with acid-washed glass beads (425- to 600-μm diameter, 2 min, 4°C), collected, and then centrifuged at 135,000 × g for 30 min at 4°C. The crude membrane pellet was resuspended in 1.0 ml of immunoprecipitation buffer (50 mM Tris-HCl [pH 8.0], 1.0% Triton X-100, 150 mM NaCl, 2 mM EDTA, protease inhibitors) and recentrifuged. The clear supernatant containing essentially all cellular Pma1p, Cch1p, and Mid1p was then incubated with 5 μg of monoclonal antibody 12CA5 for 2 h at 4°C and rocked with 100 μl of Sepharose CL-4B beads conjugated with protein A (100 mg/ml; Sigma) for 2 h at 4°C. Beads were collected by brief centrifugation, washed three times with 1 ml of radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.5], 200 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS), and then heated in urea sample buffer prior to SDS-PAGE and immunoblotting.
Stimulation of high-affinity Ca2+ influx system inpmr1 mutants.If a CCE-like mechanism operates in yeast, we reasoned that mutants lacking one or more of the intracellular Ca2+ transporters would accumulate higher levels of 45Ca2+ from the growth medium. In standard growth media, mutants lacking either Pmc1p, Vcx1p, or both vacuolar transporters accumulated less 45Ca2+in the vacuole (6, 7) but exhibited initial rates of45Ca2+ influx and [Ca2+]c levels that were indistinguishable from those of the wild-type control strain (35, 36). Depletion of Ca2+ from the vacuole, therefore, did not stimulate a Ca2+ influx mechanism to a significant degree.
In contrast, pmr1 mutants lacking the Golgi-localized Ca2+ pump accumulated extracellular45Ca2+ at an initial rate that was approximately 4.1-fold greater than the wild-type rate (Fig. 1A), 14.7 pmol/s/109 cells for pmr1 mutants and 3.56 pmol/s/109 cells for the wild type. The rates of Ca2+ efflux from the wild type and pmr1 mutants were identical (Fig. 1B), ∼2.1 pmol/s/109 cells averaged over the first 3 min of incubation. Halachmi and Eilam reported contradictory findings (19); however, these authors measured45Ca2+ influx and efflux using nongrowing cells suspended in buffer lacking inorganic ions rather than cells growing in YPD culture medium as performed here. Therefore, our data suggest that one or more Ca2+ influx pathways may be stimulated in growing pmr1 mutant cells.
Similar differences were observed in long-term45Ca2+ accumulation experiments. In one such experiment, we monitored 45Ca2+ accumulation after 2.5 h of incubation in the presence of various concentrations of extracellular Ca2+. For wild-type cells, the data were fitted to standard Michaelis-Menten equations using nonlinear regression (Fig. 1C). The analysis showed that wild-type yeast cells accumulated 45Ca2+ from the growth medium using primarily a low-affinity Ca2+ uptake system (apparent Km ∼ 500 μM,V max ∼ 390 nmol/109 cells). Raising extracellular Mg2+ from 1 to 10 mM strongly inhibited the low-affinity Ca2+ uptake system (Fig. 1C) and exposed a second high-affinity Mg2+-resistant Ca2+ uptake system (apparent Km ∼ 10 μM) operating at a much lower level (V max ∼ 9 nmol/109 cells). In an experiment parallel to that of Fig. 1A, we found that addition of 10 mM MgCl2 diminished the initial rate of45Ca2+ influx into wild-type cells 2.6-fold (to 1.28 pmol/s/109 cells [data not shown]). These and other results (3, 19) suggest that Mg2+ competitively inhibits the low-affinity Ca2+ uptake system.
When analyzed in identical conditions, the isogenic pmr1mutant exhibited up to 25-times-higher activity of the high-affinity Mg2+-resistant Ca2+ uptake system, whereas the low-affinity Mg2+-sensitive Ca2+ uptake activity was not significantly changed (Fig. 1D). The high-affinity Ca2+ uptake in pmr1 mutants appeared to saturate around 250 μM extracellular Ca2+ and then declined to near wild-type levels as extracellular Ca2+ was increased toward the 10 mM range. This decline in pmr1 mutants correlates with replenishment of Ca2+ into secretory organelles (2, 13). Taken together, these data suggest that a high-affinity Mg2+-resistant Ca2+ uptake system is specifically stimulated in pmr1 mutants when secretory compartments are depleted of Ca2+.
Expression of a mammalian SERCA Ca2+ pump in the endoplasmic reticulum of pmr1 mutants replenished lumenal Ca2+ and reversed many of the secretory defects attributed to Ca2+ depletion (11). We found that expression of rabbit SERCA1a also abolished the high-affinity Ca2+uptake system in pmr1 mutants but had no detectable effect in wild-type cells (Fig. 1E). Reversal of the pmr1phenotypes by either expression of a SERCA pump or supplementing Ca2+ to ≥10 mM indicates that the high-affinity Ca2+ uptake activity responds to depletion of Ca2+ from a secretory compartment. This evidence supports the notion that Ca2+ influx in yeast can be coupled to Ca2+ deficiency in secretory compartments, much like the process of CCE in mammalian cells.
Elevation of [Ca2+]c in pmr1mutants.CCE in mammalian cells promotes sustained elevation of [Ca2+]c. To test whether [Ca2+]c becomes elevated in pmr1mutant cells, the fluorescent Ca2+ indicator Indo-1 was loaded into the cytoplasm as described previously (18), and the loaded cells were resuspended in synthetic growth medium (∼0.7 mM Ca2+) for fluorescence measurements. Under these conditions, pmr1 mutants maintained [Ca2+]c at 1.6 ± 0.3 μM, whereas the isogenic wild-type strain maintained [Ca2+]cat 0.1 ± 0.05 μM (n = 3). Similar differences between wild-type and pmr1 mutants were noted previously for nongrowing cells suspended in a glucose buffer (19).
Elevated [Ca2+]c can activate the calcineurin-dependent transcription factor Tcn1p/Crz1p (33, 54). To test whether this response pathway was stimulated inpmr1 mutants, a PMC1-lacZ reporter gene was transformed into wild-type and pmr1 mutant strains and examined for calcineurin-dependent expression using FK506, a specific inhibitor of calcineurin. Calcineurin-dependent expression ofPMC1-lacZ was approximately five times greater inpmr1 mutants than in wild-type cells (Fig.2A). Calcineurin-dependent expression ofPMC1-lacZ was completely blocked by addition of BAPTA to chelate extracellular Ca2+ (not shown). Thus, two independent methods show that [Ca2+]c becomes elevated in pmr1 mutants as a consequence of Ca2+ influx.
Identification of Cch1p and Mid1p as essential components of the high-affinity Ca2+ uptake system stimulated inpmr1 mutants.To identify factors specifically required for high-affinity Ca2+ uptake in yeast,pmr1 null mutants were mutagenized and screened for variants that fail to express PMC1-lacZ (see Materials and Methods). Mutants deficient in calmodulin, calcineurin, or Tcn1p/Crz1p function were discarded, and the remaining ∼250 variants were subjected to complementation tests. This method revealed 30 and 6 independent mutations in two genes, CCH1 and MID1, which had been reported previously to be important for pheromone-stimulated Ca2+ influx (15, 20, 39). MID1encodes a plasma membrane glycoprotein (20) related to uncharacterized proteins expressed in other fungi. CCH1encodes a 2,039-amino-acid protein containing four repeated membrane domains, each showing strong similarity to the α1 pore-forming subunit of VGCCs (E value = 10−54 by BLAST2.0 [1]). Deletion of either CCH1 orMID1 in pmr1 mutants specifically abolished the high-affinity Ca2+ uptake system (Fig. 2B and3) and decreased calcineurin-dependentPMC1-lacZ expression to wild-type levels (Fig. 2A). Simultaneous disruption of both CCH1 and MID1 inpmr1 mutants diminished 45Ca2+uptake to the same extent as the individual disruptions (Fig. 3C), demonstrating that both Cch1p and Mid1p were required for the stimulated Ca2+ uptake. Thus, Cch1p and Mid1p are necessary components of a high-affinity Ca2+ uptake system that can be stimulated in pmr1 mutants grown in moderate- to low-calcium environments. Additional results (see below) suggest that these proteins serve as catalytic and regulatory subunits of a heteromeric Ca2+ influx channel.
Excessive Ca2+ sequestration into the vacuole stimulates Ca2+ accumulation through a Cch1p- and Mid1-dependent process.Depletion of Ca2+ from secretory organelles might occur as a consequence of excessive Ca2+ sequestration into the vacuole. To test this possibility, 45Ca2+ accumulation was quantitated in strains either expressing a hyperactive Vcx1p variant or overexpressing Pmc1p. Overexpression of Pmc1p from a high-dosage plasmid greatly stimulated 45Ca2+ accumulation in wild-type cells but had no significant effect on45Ca2+ accumulation in cch1 null mutants (Fig. 3A) or mid1 mutants (not shown). Similarly, expression of a hyperactive Vcx1p mutant termed Vcx1p-D1 (6) strongly stimulated 45Ca2+ uptake in wild-type strains but not in mid1 mutants (Fig. 3B). In both cases, stimulation of the Ca2+ uptake system was partially reversed by simultaneous overexpression of Pmr1p (P < 0.04), suggesting that the vacuolar Ca2+ transporters competed with Pmr1p in these conditions. Thus, the Cch1p- and Mid1p-dependent Ca2+ uptake system could be stimulated either by Pmr1p inactivation or by Pmr1p insufficiency due to competition with vacuolar Ca2+ transporters.
The possibility that increased 45Ca2+accumulation in pmr1 mutants was due to increased Ca2+ sequestration by Pmc1p or Vcx1p was not supported by the results of several experiments. First,45Ca2+ accumulation and PMC1-lacZexpression were the same in pmr1 vcx1 double mutants as inpmr1 single mutants (data not shown), showing that Vcx1p was not required for the effect. Second, pmr1 tcn1 double mutants which lack the transcription factor required for Pmc1p induction showed levels of 45Ca2+ accumulation that were similar to or even greater than those for pmr1mutants and still depended on Cch1p and Mid1p (Fig. 3C). Finally,pmr1 pmc1 double mutants grown in the presence of FK506 to maintain viability (4) exhibited higher levels of45Ca2+ accumulation than similarly grownpmr1 mutants (data not shown), consistent with previous inferences that Pmc1p may partially refill secretory compartments during its trafficking to the vacuole in pmr1 mutants (13, 32). Thus, the stimulation of the Cch1p- and Mid1p-dependent Ca2+ uptake system did not correlate with the abundance of any particular Ca2+ transporter or the level of [Ca2+]c but instead correlated inversely with Ca2+ transport into secretory compartments.
Expression and localization of Cch1p in pmr1mutants.A possible mechanism for the observed increase in Ca2+ influx and accumulation upon depletion of Ca2+ stores is increased expression of either Cch1p or Mid1p or both proteins. To test this possibility, quantitative Western blot analyses were performed using epitope-tagged variants of Cch1p and Mid1p. Thirteen repeats of the Myc epitope were inserted at the extreme C terminus of Cch1p by homologous recombination into the chromosomalCCH1 gene (see Materials and Methods). The resulting Cch1p-Myc fusion protein was fully functional in pmr1mutants (data not shown) but was expressed at identical levels in wild-type and pmr1 mutant strains grown in standard medium (Fig. 4A, top panel). In this experiment, mitochondrial porin was used as a loading control (bottom panel). Furthermore, a functional Mid1p-HA fusion protein expressed from a low-dosage plasmid from its own promoter (20) also accumulated at similar levels in wild-type and pmr1 mutant strains (Fig. 4B). In this case, nonspecific cross-reacting bands served as internal loading controls. These results rule out the possibility that depletion-stimulated Ca2+ uptake system involves significant up-regulation of Cch1p or Mid1p.
It is possible that depletion of Ca2+ from secretory organelles promotes the relocalization of Cch1p and/or Mid1p from internal compartments to the cell surface or their interaction in the plasma membrane, thereby allowing their function as a high-affinity Ca2+ influx channel. Such a mechanism has been proposed recently for vertebrate cells (61). A functional epitope-tagged variant of Mid1p was localized to the plasma membrane of wild-type yeast cells in nonsignaling conditions (20). To determine whether Cch1p might undergo regulated trafficking, the epitope-tagged Cch1p-Myc variant was localized by immunofluorescence microscopy. As observed previously for Mid1p, Cch1p was detectable only at the surface rim in wild-type cells (Fig. 4C). No significant staining was evident in intracellular structures aside from a faint cytoplasmic staining that was also evident in the nontagged control strain. A similar staining pattern was observed in pmr1mutants (data not shown). To determine whether Cch1p physically interacts with Mid1p in the plasma membrane, wild-type cells expressing Cch1p-Myc and/or Mid1p-HA were lysed in the presence of nondenaturing detergent and subjected to immunoprecipitation with anti-HA antibodies followed by Western blotting with anti-Myc antibodies. Cch1p was efficiently coprecipitated with Mid1p in the tagged strain (Fig. 4D, lane 4) but not in control strains lacking the tag (lane 2). The plasma membrane marker protein Pma1p was not precipitated in any conditions (bottom panels). Thus, Cch1p and Mid1p both localize to the plasma membrane of unstimulated cells, where they physically interact. Stimulation of Cch1p and Mid1p function in pmr1 mutants may therefore involve some other type of regulatory mechanism such as a diffusible messenger (5).
Cch1p and Mid1p supply essential Ca2+ to Pmr1p during ion starvation.Previous studies showed that wild-type cells cultured in media supplemented with BAPTA, a potent chelator of Ca2+ and other ions, exhibit defects in protein sorting and secretion similar to those observed in pmr1 mutants in standard medium (13). Very high levels of BAPTA even inhibited the growth of wild-type cells, whereas much lower chelator concentrations inhibited the growth of pmr1 mutants (11, 13). It can be inferred from these and other experiments that an essential role of Pmr1p is to supply secretory organelles with the Ca2+ necessary to sustain secretion and growth in low-calcium environments. The studies presented above predict that Cch1p and Mid1p would also be important for growth in low calcium conditions due to their ability to provide substrate to Pmr1p. This prediction was tested by comparing the growth of various mutants lacking Cch1p, Mid1p, and/or Pmr1p in media supplemented with increasing amounts of BAPTA.
Interestingly, cch1 and mid1 mutants each failed to grow at intermediate concentrations of BAPTA in between the concentrations effective for wild-type and pmr1 mutant strains (Fig. 5A). The cch1 mid1 double mutant was as sensitive to BAPTA as the single mutants, suggesting again that Cch1p and Mid1p function within a single high-affinity Ca2+ uptake pathway. If the major function of these proteins is to provide sufficient Ca2+ to the cytoplasm for concentration by Pmr1p in secretory organelles, Cch1p and Mid1p would be expected to have little or no effect on BAPTA tolerance inpmr1 mutants due to their inability to refill the secretory stores. Indeed, mid1 pmr1 double mutants were just as sensitive to BAPTA as pmr1 single mutants, showing that Mid1p confers BAPTA tolerance only when Pmr1p also functions. Bothcch1 pmr1 double mutants and cch1 mid1 pmr1triple mutants were slightly more sensitive to BAPTA than the respective CCH1 strains, but this effect was relatively small and very likely a consequence of slightly reduced growth rates of all cch1 strains independent of Ca2+ (K. W. Cunningham, unpublished observation). Similar results were obtained when EGTA was used in place of BAPTA and when a fixed concentration of BAPTA (20 mM) was used with various concentrations of added Ca2+ (data not shown), which argues against nonspecific or indirect effects of the chelators. Thus Cch1p, Mid1p, and Pmr1p all appear to function within a single pathway that promotes the acquisition and concentration of essential Ca2+ into secretory organelles. This pathway closely parallels the CCE pathway of animal cells.
The data reported here collectively suggest that depletion of Ca2+ from secretory compartments in yeast cells stimulates the activity of a high-affinity Ca2+ influx channel composed of Cch1p and Mid1p which helps replenish the depleted organelles (Fig. 5B). We report evidence that the initial rate of Ca2+ influx is stimulated in pmr1 mutants and the rate of Ca2+ efflux is unaffected, leading eventually to elevated [Ca2+]c, elevated expression of Pmc1p, and elevated accumulation of Ca2+ in the vacuole. An earlier study concluded that Ca2+ influx was normal inpmr1 mutants but that Ca2+ efflux was drastically inhibited, leading to similar long-term effects (19). In that report, Ca2+ influx was measured using nongrowing cells incubated in a minimal buffer lacking Mg2+, conditions which might have disfavored detection of Cch1p and Mid1p activity. Ca2+ efflux was also measured in nongrowing cells over extremely long periods of time in the absence of extracellular Ca2+ (up to 30 h) although, like us, they found similar rates of Ca2+ efflux in wild-type andpmr1 mutant strains when Ca2+ was added to the buffer (19). The results of our long-term45Ca2+ accumulation experiments suggested that a novel high-affinity Mg2+-resistant Ca2+uptake system was specifically stimulated in pmr1 mutants relative to a low-affinity Mg2+-sensitive Ca2+uptake system present at similar levels in both wild-type andpmr1 mutant cells. Remarkably, this high-affinity system inpmr1 mutants depended on the function of both Cch1p and Mid1p and could be suppressed by overexpression of mammalian SERCA pumps or simply raising extracellular Ca2+ to ≥10 mM, conditions known to replenish Ca2+ in the secretory pathway independent of Pmr1p function (13). Thus, the stimulation of the high-affinity Cch1p- and Mid1p-dependent Ca2+ uptake system appeared to correlate inversely with Ca2+accumulation in secretory compartments, much like CCE in mammalian cells.
Further support for the functional coupling between Ca2+stores and Ca2+ influx derives from studies of the vacuolar Ca2+ transporters. Abnormal elevation of either Pmc1p or Vcx1p activity stimulated Ca2+ accumulation in wild-type cells but not in cells lacking Cch1p or Mid1p (Fig. 3), an effect that was significantly reversed by overexpression of Pmr1p. It could be argued that the elevated vacuolar Ca2+ transport decreased Ca2+ efflux, but if this were the case, one would predict greater Ca2+ accumulation independent of Cch1p and Mid1p since these factors contribute very little to the overall Ca2+ influx in wild-type cells. The simplest model consistent with the data is that the vacuolar Ca2+transporters compete with Pmr1p for substrate and can effectively deplete the secretory pathway of Ca2+ which stimulates Ca2+ influx via Cch1p and Mid1p. This model helps explain why Pmc1p expression and Vcx1p function are so tightly regulated in wild-type cells (6, 33); excessive vacuolar Ca2+transport may deplete Ca2+ from secretory organelles especially under conditions of low Ca2+ availability from the environment. However, direct measurement of secretory Ca2+ concentrations would be necessary to confirm this hypothesis.
Our data also support the hypothesis that Cch1p and Mid1p function together as catalytic and regulatory/accessory subunits of a single high-affinity Ca2+ influx channel. In BAPTA tolerance experiments, Ca2+ accumulation experiments, and experiments that monitor [Ca2+]c, the cch1 andmid1 single mutants exhibited phenotypes quantitatively similar to those of cch1 mid1 double mutants, suggesting that neither Cch1p nor Mid1p can function without the other. Additionally, mutations in both of these genes were recovered in two genetic screens distinct from ours (20, 39). Cch1p strongly resembles the pore-forming α1 subunit of VGCCs (15, 39) characterized extensively in vertebrate cells. In animals, VGCCs typically comprise several subunits in addition to α1 (4). Mid1p shows no significant homology to any animal proteins in current databases, but surprisingly, expression of Mid1p in CHO cells resulted in the appearance of a nonselective cation channel that responded to membrane stretch (21). Overexpression of Mid1p in eitherpmr1 mutants (data not shown) or wild-type cells under conditions of membrane stretch (20) had no effect on Ca2+ influx rates, as if another factor was limiting for Mid1p activity in yeast. Finally, we show that Cch1p and Mid1p can physically interact in the plasma membrane of wild-type cells.
How closely does CCE in animals resemble the CCE-like process in yeast? Homologs of Pmr1p, the secretory Ca2+ ATPases, are expressed ubiquitously in mammalian cells but they have not yet been characterized functionally (17). Their involvement in Ca2+ homeostasis and potential for coupling to Ca2+ influx mechanisms therefore remain uninvestigated. On the other hand, SERCA-type Ca2+ pumps are well known to supply Ca2+ to the endoplasmic reticulum and to prevent stimulation of CCE pathway in mammalian cells. Specific inhibitors of SERCA selectively deplete the endoplasmic reticulum of Ca2+and concomitantly enhance Ca2+ influx through CCE channels in the plasma membrane. Physiological stimulation of CCE in animal cells occurs after release of Ca2+ from the endoplasmic reticulum through the activation of Ca2+ release channels such as the IP3 receptors, RyR receptors, or other unidentified channels. Although the Ca2+ influx channels activated during CCE have not been conclusively identified, recent evidence suggests that certain members of the TRP family of ion channels (TRPCs) respond to depletion of Ca2+ from the endoplasmic reticulum at least when expressed in heterologous systems (46). TRPCs are only distantly related to VGCCs, and the yeast genome contains no clear homologues of TRPC. At least 10 distinct genes encoding VGCC catalytic subunits have been identified in humans, and few of these proteins have been ruled out as CCE channels. Indeed, specific VGCC inhibitors can block Ca2+ influx in response to SERCA inhibition in at least some vertebrate cell types (30, 60). Given that the need for Ca2+ in secretory organelles is conserved among eukaryotes, it is plausible that a mechanism to ensure Ca2+ homeostasis in the secretory pathway arose prior to the divergence of animals and fungi and is largely conserved in both groups of organisms today. In this view, the primary differences between fungal and animal cells would be the types of Ca2+ pumps used to supply the secretory organelles and the types of Ca2+ channels that release secretory Ca2+. The significance of these differences may be relatively minor because stimulation of CCE-like processes inpmr1 mutants could be suppressed by expression of rabbit SERCA1a (Fig. 1D). However, the available data do not yet exclude the alternative possibility of independent origins of CCE in animals and yeast. Mutants lacking Pmr1p exhibit only a twofold decrease of free Ca2+ in the endoplasmic reticulum from a level that is already much lower than that of mammalian cells (55), raising the possibility that another secretory organelle such as the Golgi complex couples to the CCE-like process in yeast.
Current hypotheses for the coupling between Ca2+ influx channels in the plasma membrane and Ca2+ depletion in the endoplasmic reticulum include secretory mechanisms (61), docking mechanisms involving proteins in the plasma membrane and endoplasmic reticulum (42), conformational coupling mechanisms involving IP3 receptors and certain TRPCs (24, 25), various forms of retrograde signal transduction (46), and the production or release of small diffusible molecules that serve as intracellular messengers (40, 47). One such messenger of CCE termed CIF (calcium influx factor) has been described as a membrane-impermeant molecule that rapidly accumulates in human T cells treated with SERCA inhibitors and triggers Ca2+ influx in the absence of Ca2+ release from the endoplasmic reticulum (23, 58). A molecule with identical properties was recently shown to accumulate inpmr1 mutants but not wild-type yeast cells (5). The contribution of this molecule to Ca2+ influx in yeast cells has not been tested because no methods to introduce it into the cytoplasm or to genetically manipulate its biosynthesis have been developed. Additionally, the kinetics of Cch1p stimulation and CIF accumulation after depletion of secretory Ca2+ stores could not be determined in yeast because Pmr1p is resistant to the known SERCA inhibitors (53) and no other means of rapidly triggering Ca2+ depletion from secretory organelles have been developed. Despite these limitations, the yeast system affords genetic technologies useful for the identification of factors involved in the response to depletion of secretory Ca2+ stores. Characterization of the corresponding animal factors together with more mechanistic studies of the yeast system should help resolve the questions of whether the CCE mechanism evolved prior to the divergence of fungi and animals, which features of the mechanism are conserved today, and how the mechanism functions and varies in diverse cell types.
We are grateful to Steve Garrett, Hidetoshi Iida, and Hans Rudolph for plasmids and advice, to Fujisawa USA Inc. for a gift of FK506, and to members of our laboratory and department for advice and technical support.
This work was supported by grants from the Searle Scholars Program/The Chicago Community Trust and the National Institutes of Health (grant GM53082 to K.W.C.).
- Received 10 May 2000.
- Accepted 16 June 2000.
- Copyright © 2000 American Society for Microbiology


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You still have time to submit your Ode to Your Zip Code for this special project sponsored by WLRN in Miami. "O, Miami and WLRN-Miami Herald believe it's time to pay tribute to the least poetic definition of where you live: The ZIP Code. This April, we're asking you to memorialize your federally appointed numerical designation by writing an ode to your ZIP code." Write a short poem where the number of words for each line correlates to the your ZIP code, respectively. If your ZIP code includes a zero, you can just leave that line blank. DEADLINE: Tuesday, April 21, 2015
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Samsung's Galaxy Note 9 may launch ahead of schedule this year, according to a new rumor that's been making the rounds over the last 24 hours, having seemingly originated on Twitter. The device identified by the model number SM-N960 will supposedly be announced on July 29, as per that newly emerged and somewhat dubious claim. Virtually every flagship released by the South Korean tech giant over the last half a decade has been rumored as a candidate for an early announcement but that speculation rarely turned out to be correct, with Samsung having a strong history of sticking with its six-month refresh cycle in the high-end segment of the mobile market. With the Galaxy S9 lineup being announced at MWC in late February before starting to retail in mid-March, the Galaxy Note 9 is still more likely to debut in late August, with the implication being that Samsung will once again unveil the latest addition to its premium phablet family at Berlin, Germany-based IFA trade show.
While the company is understood to have already finalized the design of the upcoming flagship that's said to be similar to the Galaxy Note 8, it may still be undecided on the memory configuration of the most premium variant of the device, with one insider recently claiming the top model may end up shipping with as much as 8GB of RAM and 512GB of storage space. A new iteration of Bixby is also said to be part of the package, offering faster responses and expanded capabilities, among other features. Qualcomm's Snapdragon 845 is still expected to power all Galaxy Note 9 variants sold in the United States, Canada, Mexico, and China, whereas Samsung's own Exynos 9810 should be found inside the models retailed in other markets, as is the case with the Galaxy S9 lineup.
A new color option is also rumored to be on the cards for the Galaxy Note 9, with the variant supposedly being called "Teddy Brown." The phablet is expected to be Samsung's last Android flagship without 5G support, with the Galaxy S10 lineup already being predicted to launch with such capabilities early next year. Should Samsung stick with its established product release schedule outlined above, the Galaxy Note 9 should become available for purchase by mid-September following a short pre-order period spanning some two weeks.


text: On Friday, September 12, 2014 from 1:00 – 7:00 pm there will be a Leisure Mart at the AJ LaRue Arena. Come find out what recreation and leisure opportunities exist in Haliburton. Talk to local service providers and organizations to learn about the activities they offer. Some of the providers will be accepting registrations on this day. To learn more about the event or if you are a service provider and would like to set up a booth, please contact Andrea Mueller at (705) 457-1740 ext. 35 or at firstname.lastname@example.com....Read more Comments are closed


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text: Luka Modric: Best FIFA Player Award winner, not Mikel Obi's choice Coach of Nigeria's Super Eagles, John Mikel Obi and Coach Gernot Rohr disagreed on whether Croatian Luka Modric should be crowned as Best FIFA Player of the year. Modric was Rohr's first choice, according to the details of the voting done by national captains, coaches and media men. But Mikel Obi chose Lionel Messi as first choice, and French player Kylian Mbappe as the second. Modric was Obi's third's choice. Rohr also picked Mbappe as second choice and Belgium and Chelsea player Eden Hazard as his third choice. The Nigeria media man, Christian Okpara who voted picked Ronaldo, Eden and Modric as first, second and third choices. Egyptian captain, El Hadary Essam voted Mohamed Salah, Luka Modric and Ronaldo, as his choices. Egyptian coach, Javier Aguirre Onaindía Javier agreed with the captain's first two choices, only differing in making French player Antoine Griezman, his third best player.


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For the third consecutive year, the Greater South Loop Association has circulated its annual State of the South Loop Survey. The results of this information are shared with area elected officials, posted to our website, and inform our programming for the subsequent year. Join us at our December Neighbor's Night Out, where we will feature the results and a discussion… Read more →
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Education Update: Results of GSLA's Community Surveys on the Near South High School Plan
This post summarizes the key findings of two GSLA surveys which followed the first and third community meetings held by CPS to gather feedback on a proposal to expand the South Loop Elementary School's boundary southward to 22nd Street and close the National Teacher's Academy elementary school (NTA) to repurpose it to serve as a 1,000 student high school which… Read more →
2010 Bash on Wabash Survey
In the spirit of continuous improvement, please take a moment to complete a survey on your experience with the 2010 Bash on Wabash. It is our goal to provide the South Loop with an annual festival that attracts Chicagoans to the area and for our neighbors to enjoy everything the area has to offer. You may remain anonymous if you… Read more →