Building muscle is the name of the game for most weightlifters, but achieving this goal requires taking various factors into account.
You need to work hard and stay committed if you want to build muscle, as you won't notice results overnight or even from sporadic attempts to train. Devise a training programme that you can stick to over the long-term that ensures you put enough work in to allow muscles to flourish.
No matter how much you exercise, your muscles won't grow if you don't feed them. Bodybuilding involves increasing your calorie intake and following a carefully constructed diet that's loaded with protein, carbohydrates and healthy fats.
How much you'll need to eat to boost muscle mass largely depends on your existing body weight. As a rough guideline, consume around 20 calories per pound of body weight. In terms of protein, aim for 1.5-2.0g of good quality protein per kg of body weight. Ideal choices include eggs, lean meat, fish and dairy. Carbohydrates should make up around 40-60% of your diet, but only include healthy types if you want to build muscle not fat. Stick to whole grain options such as brown rice, wholemeal bread or fibre-rich cereals.
As for fats, these should make up around a third of your diet. Building lean muscle not fat is crucial, so choose heart-healthy fats, such as oily fish, nuts and avocados.
Don't forget to drink plenty of fluids, too, as this will keep your body cells hydrated and prevent flagging energy levels.
Once you notice muscle growth, you might need to tweak calorie intake to make further gains, so don't assume your diet regime is set in stone.
If you've been struggling to gain muscle, your exercise regime might be to blame. Have a word with a personal trainer to see if there are any improvements you could make. Ideally, focus on compound exercises instead of isolation ones, so that all muscles are getting pummelled. Ensure you've got correct form when exercising, as you could be missing out on maximising gains, plus, you risk injury without it.
Many bodybuilders assume cardio and strength training don't go hand in hand, but this is false. In fact, if you want to add mass to your muscles, you need to incorporate cardio into your routines. Cardio training delivers oxygen to muscles, helping them grow and recover faster following workouts, but it also keeps your body in prime condition, enabling you to exercise for longer. Stick to low or moderate intensity cardio for short periods, preferably a couple of times per week, to avoid burning excess calories, which you need for muscle growth.
Be mindful about your time outside the gym, if you want your muscles to expand. Following a workout, fuel up on protein and carbs to replace lost energy stores, and give yourself time to rest in-between training. Aim for at least a couple of rest days every week. This allows your muscles to repair and recover, and it's actually during this phase that growth takes place. Get good quality sleep each night and reduce stress, as stress can impede muscle growth and may even harm hormone levels.
These are just some of the ways you can achieve muscle growth, but for further bodybuilding assistance, take a look at the wide range of authentic, quality steroid products from Steroids & Muscle.
Previous Previous post: How Beneficial is Coconut Oil?


text:
IDIS GLOBAL
Perfect Networking Solutions
High Performance & Compatibility
Stability & Convenience
Complete Surveillance Solution
ANPR/LPR Engine
Additional tools to help
AI in the BOX
HDMI & VGA Encoder
Accessory Selector
FoV Calculator
HDD Calculator
DirectIP®
DirectCX®
4K Total Solution
Intelligent Codec
Critical Failover
Smart Failover
Smart UX Controls
IDIS Ultimate Warranty
BIM Families
Virtual Kiosk
Technical Brochures
Print Ads/Posters
About IDIS
IDIS CSR
IDIS Smart Factory
Products Selector
Technical Innovations
Corporate & CSR
Exhibitions & Case Studies
IDIS Website
아이디스
DutchNederland
русскийRussia
Select Country GermanyNetherlandsPeruQatarRussiaSaudi ArabiaUAEUnited KingdomUnited States America
Cultural Landmark
Virtual Guarding
Swanshurst School aspires to maintain outstanding performance with IDIS video surveillance
Swanshurst School is one of Birmingham's most popular destinations for girls' secondary education, with more than 1800 pupils aged 11 to 19. To maintain their outstanding OFSTED-inspection rating, the school's leaders work hard to promote the highest standards of personal behaviour and performance.
IDIS drives Circuit Zandvoort into pole position for thrilling 2020 Formula 1 Grand Prix
The historic Circuit Zandvoort in the Netherlands has been hosting major motorsport events for over 70 years. In 2020 it will once again be the setting for the Formula 1 Grand Prix.
User-friendly video technology allows ward-by-ward monitoring for NHS health trust
Bluebird House is a specialist UK inpatient facility, providing treatment and care for young people with complex mental health problems which mean they pose a risk to themselves or others.
Enhancing safety and security for a region's most vulnerable citizens
Emily's Place provides essential services to those among their community's most vulnerable, offering a shared living environment (for up to 24 months) for women and children seeking to permanently break from abusive situations. Through committed and active engagement that includes counselling, case management, internal programs
Ensuring a Safe, Secure, and Seamless Repair and Service Experience
The experience of high-quality, high-performance automotive engineering and the sleek lines and signature beauty of luxury auto brands like Porsche, BMW, Mercedes, and more are investments worth protecting. When damage to these vehicles occur and body work is required, only the work of attentive, highly trained, and craftsman technicians owners can trust, will do.
IDIS America Donation Secures The Salvation Army of North Texas
Few charitable enterprises in the United States are as universally recognized, nor as widely respected, as The Salvation Army. Though the ubiquitous bell ringers on corners and in shopping centers during the holiday season may be what Americans are most familiar with, the work the storied faith-based organization does in communities, including North Texas and the Dallas-Forth Worth area is comprehensive and widespread, addressing community concerns ranging from homelessness to substance abuse
Best of Both Worlds for Northwestern School System
Located within the Big Horn Basin in Northwestern Wyoming, Washakie School District No. 1 communities within Washakie County, including the county seat of Worland. Set against the distant backdrops of the Big Horn and Wind River mountains, Washakie County represents much of the values and spirit of America's wide open northwest. Washakie School District serves approximately 1350 of the area's K-12 public school students, operating five schools and an education resource center throughout the dist
Ensuring a Safe and Secure Environment for Learning and Growing in Texas
Coppell, Texas is a suburban community in the Dallas-Fort Worth metropolitan area, with a reputation for strong community services, including a premiere public education system, ensured by the administrators, faculty, and staff of the Coppell Independent School District. Coppell ISD, recognized statewide for both high performance and innovation, serves over 12,800 students, through a vibrant network of schools, programs, and administrative functions spread over 23 square miles.
National Commercial Bank chooses IDIS technology for full scale surveillance upgrade across all branches and ATMs
Since it became the first Saudi bank to be licensed in the Kingdom of Saudi Arabia in 1953 the National Commercial Bank (NCB) has grown to become the second largest in the Arab world, as classed by asset. Also known as AlAhli Bank it now serves 5.4 million customers and is a leading institution in the practice of Islamic banking and finance. To maintain its leading position NCB is using IDIS technology for one of the biggest financial surveillance systems upgrades ever seen in the region
Progressive Netherlands port operator steers efficiency and safety with latest IDIS video technology
Stadshavens Medemblik operates five busy ports in the Netherlands – at Pekelharinghaven, Voorhaven, Middenhaven, Westerhaven and Overlekerkanaal – and it also has two bridges under its control, as well as the thriving Westerhaven lock. The company has grown by around 30
IDIS video technology puts Starbucks Russia in control
The first Starbucks coffee house opened in Moscow in 2007 and in ten years the chain's growing popularity across Russia has seen more than 100 outlets open. There are now several different store formats including classic coffee shops located in shopping centres; stand-alone stores characterised by open vaulted ceilings; smaller kiosk outlets; and drive-through outlets where customers can buy fragrant coffee and fresh-baked goods round the clock without leaving their cars.
Russian stadiums change the game with IDIS surveillance
With the 2018 World Cup staged in Russia, security and safety became the major focus at 12 stadiums and 11 cities. Against a background of global media attention—and with the operational challenge of hosting teams and visitors from around the world— IDIS surveillance technology was chosen for a surveillance upgrade at the 45,000 capacity Rostov Arena and the 35,000 capacity Kaliningrad Stadium.
High Performing, Non-intrusive Surveillance for a Luxury Car Dealership
For more than seven decades, the name Porsche has been synonymous with quality and performance in automobiles. In 2018, the German automaker's position and recognition around the world is unparalleled among luxury car brands, engendering consumer passion and loyalty that runs from admirers through collectors. Such qualities are proudly shared and returned around by those who represent the brand around the world, emblematic of the brand's commitment to providing a sales and service experie
International Investment Bank unified video solution with IDIS
Located in Moscow, the International Investment Bank (IIB) is an inter-state organisation which promotes economic and social development among members, including Hungary, the Republic of Bulgaria, the Socialist Republic of Vietnam, the Republic of Cuba, Mongolia, the Russian Federation, Romania, the Slovak Republic and the Czech Republic. Following a review of security in 2015 the bank's senior management identified a need to rationalise and expand their video surveillance capability. A s
School's video upgrade improves student learning experience
Fazakerley High School, a co-educational secondary school in Liverpool, moved into new buildings in 2003 and embarked on a successful transformation programme to raise standards. The school, which is in a challenging catchment area, established a new reputation as a nurturing, well run learning environment. But fifteen years later senior staff found themselves struggling with an outdated video surveillance system that was no longer fit for purpose.
Privacy Policy Copyrights 2020. IDIS. Ltd. All rights reserved.
Sign up and the first to know
By submitting your email address, you agree to our Privacy Policy.
Sites of IDIS sister companies KORTEK IDP BIXOLON

redpajama_set_name: RedPajamaCommonCrawl


text:
At the Edge of the Solid World
Daniel Davis Wood
'the tale is made seamless by a tight structure and a hypnotic style that seems to owe something to the work of Gerald Murnane.' Kerryn Goldsworthy Sydney Morning Herald on Blood and Bone
In a snowbound village in the heart of the Alps, a husband and wife find their lives breaking apart in the days and months following the death of their firstborn. On the far side of the world, in their hometown of Sydney, a man on the margins of Australian society commits an act of shocking violence that galvanises international attention. As the husband recognises signs of his own grief in both the survivors and the perpetrator, his fixation on the details of the case feeds into insomnia, trauma, and an obsession with the terms on which we give value to human lives.
A compulsive, compelling and lyrical novel, told with extraordinary empathy and emotional intelligence, this sweeping saga examines the nature of loss, the resilience and fragility of the family unit and the stories we tell to explain the world.
Publication Date: 07-Oct-2020
Publisher: Brio Books
Little London Scandal
Miranda Emmerson
A Burning
Megha Majumdar
Susan Steinberg
The Extraordinary Hope of Dawn Brightside
Jessica Ryn


Home Furnishings There are 25 products.
Decorate your home with a beautifully stitched rug 10x10psc. Measuring 0.5 x 1.0m, it is a versatile size to use as a decor feature to add to your home furnishing. Dimensions: 0.5 x 1.0m Note: Colour will vary from that shown in image.


text:
Sir Samuel Bentham
Home Technology Engineering Civil Engineering
British engineer
https://www.britannica.com/biography/Samuel-Bentham
Making The Modern World - Biography of Sir Samuel Bentham
Portsmouth Royal Dockyard Historical Trust - Biography of Sir Samuel Bentham
Last Updated: Jan 7, 2021 See Article History
Sir Samuel Bentham, (born Jan. 11, 1757, England—died May 31, 1831, London), British engineer, naval architect, and navy official in Russia (1780–91) and England (from 1795) who was an early advocate of explosive-shell weapons for warships.
Bentham led Russian vessels fitted with shell guns to victory over a larger Turkish force (June 7, 1788). As inspector of naval works in England, he developed the Arrow class of sloops used against France. He was the brother of the philosopher Jeremy Bentham and father of the botanist George Bentham.
block mill
It was conceived by Samuel Bentham, with machinery designed by Marc Brunel and built by Henry Maudslay, and built at England's Portsmouth naval dockyard. By 1805 it was producing 130,000 pulley blocks per year. It remained in production for over 100 years. See also American System of manufacture.…
London clubs
If it is possible to be both a midwife and a father figure, Alexis Korner played both roles for British rhythm and blues in 1962. He opened the Ealing Blues Club in a basement on Ealing Broadway and encouraged, inspired, and employed a number of musicians in his band, Blues Incorporated, some of…
London 1960s overview
London's music scene was transformed during the early 1960s by an explosion of self-described rhythm-and-blues bands that started out in suburban pubs and basements where students, former students, and could-have-been students constituted both the audience and the performers. In short order many of…
May 31, 1831 (aged 74)


Ryan is a special education teacher of mild/moderate disabilities serving students with visual & auditory processing disorders, attention deficit/hyperactivity disorders (ADD & AD/HD), emotional disturbance, and autism. He is a member of the Alpha Phi Alpha Fraternity, Inc. and has served on the Board of Directors as the Western Region Assistant Vice President, representing college students in the states of California, Nevada, Arizona, Colorado, Washington, and New Mexico. He is the former Youth Director/Pastoral Staff Member at First Church of God…Center of Hope in Inglewood, CA under the now deceased Bishop Gregory L. Dixon. He has served as the Chair of the School Site Council at Kettering Elementary School in Long Beach, CA. Ryan is a current Den Leader for the Boy Scouts of America, Pack 848, through West Angeles Church of God in Christ. As a member of the AAFF Museum Board, he is proud to secure the legacy of his father, Reginald Ballard, Sr. who is an original Stentorian (the Association of African Americans in the Fire Service), and the several men and women who serve in the fire service, past, present, and future. He is married to the lovely Nicole and together they have three wonderfully energetic and bright sons whom they homeschool.


Come along with us on an extraordinary journey through France. From iconic Paris, world famous Champagne houses in Champagne, 7 night wondrous river cruise along the Rhone and Saone Rivers from Burgundy to Provence, the glamour and beauty of the French Riviera, stunning towns and villages of Provence, enchanting Chateaux of the Loire and the unforgettable Mont Saint-Michel…… this itinerary has it all.
Tracey has been creating holidays for clients for over 30 years and is now specialising in tours for solo travellers. She is the owner/operator of World Travellers Remuera and puts together all the itneraries for our tours based on years of travelling experience. She is a full member of the New Zealand Intitute of Travel and Tourism (MNZITT). "Solo travellers have been sadly neglected in the past, says Tracey. We are the first (and currently only) company in New Zealand that offers tours exclusively for solo travellers. The difference for a solo traveller travelling on one of our tours is huge. Being part of a small group of solo travellers provides a wonderful intimate experience where it is like travelling with a group of friends. Everyone is in the same situation which provides a relaxing and comfortable environment. I also love the fact we all get together prior to departure. Getting to know each other prior to the tour eases any anxiety about who will be on the tour etc so that by the time we all meet at the airport we are all familiar with each other and can look forward to doing and seeing some amazing things and having fun. I absolutely love escorting every tour I take away. We see and do great things, stay in lovely hotels, have great meals (and of course great wine) and have sooo much fun! I cannot wait for the next one".
After checking in at Auckland International Airport enjoy some relaxing time at the Emperors Lounge. Tonight we depart Auckland at 8.30 pm and fly to Dubai. On arrival at 5.35 am we are transferred to our hotel. One night Taj Dubai Hotel (Luxury Rooms), conveniently located in the Burj Khalifa precinct, five minutes from the famed Downtown area and Dubai Mall.
Early this morning we are transferred to the airport for our flight to Paris departing 8.20 am On arrival at 1.30 pm we are transferred to our hotel. Three nights Hotel Mansart (Superior Capucines Rooms) located at the corner of the prestigious Place Vendôme, not far from Opéra Garnier and the Louvre.
This morning enjoy a guided panoramic tour by minibus where we will see Notre Dame, Hôtel de Ville, Louvre, Opéra, Concorde, Madeleine, Champs-Élysées, Arc de Triomphe, Invalides, Trocadéro, Eiffel Tower and Montmartre. Rest of the day at leisure. You may like to spend some time admiring the awe-inspiring artworks of the Louvre Museum . Tonight you may like to have dinner up the Eiffel Tower.
Today is left at leisure. You may like to take an optional excursion to Versailles and Monet's Garden or spend the day doing your own thing.
This morning we travel by minibus to the world famous Champagne region of France, driving through rolling hills and vineyards to the capital of the Champagne Country – Reims. Spend the afternoon at leisure in Reims and make sure you visit the spectacular Reims Cathedral and you may like a tasting tour at Mumm or Tattinger. Two nights Hotel de la Paix (Priviledge Rooms) located in the heart of Reims.
Today is devoted to Champagne! Our first stop takes us back some 300 years with a visit to the Benedictine Abbey in Hautvillers where the famous Dom Perignon was the cellarer and pioneered some of the production methods that made champagne the delight it is today. Our next stop is a family-run winery where the owner will show us around and teach us how his family has been making champagne for three generations. Of course we will need to sample his wares. Our next stop is Epernay and Avenue de Champagne, home to many leading Champagne houses. There is a king's ransom of bubbly hidden under this very unique street and you'll understand why many call this the most valuable street in France. After lunch step into the cave of Moët & Chandon and enjoy a tour and tasting .
Today is free to explore, relax or shop in France's third largest city. Or you may like to take a bike from the ship and explore local produce markets and parks. This evening we sail for Tournon.
This morning enjoy a guided walking tour to explore Tournon – one of France's oldest medieval cities. See its 16th century castle and Roman ruins between the Rhone River and the granite slopes of the Hermitage. This afternoon we sail for the charming village of Viviers and explore the pale-stone buildings on a late afternoon guided walking tour. You'll see its Romanesque cathedral and Old Town with a mix of historical Christian, Roman, and medieval influences. This evening we sail for Avignon.
This morning enjoy a guided tour of the fortress city of Avignon-with its ramparts encircling the city-and a visit to the amazing Palace of the Popes. You may like to take an optional excursion to the ancient Roman aqueduct of Pont du Gard, or a trip to the famed wine cellars of Chateauneuf-du-Pape for a true taste of the Rhone wine region. Later, enjoy onboard live entertainment. This evening we sail for Arles.
This morning we say Au Revoir to our crew and head to the beautiful village of St Paul de Vence. Built on a rocky outcrop and surrounded by its ramparts built on the orders of Francois 1st, St Paul is certainly one of the most beautiful village in Provence. The beauty of the surrounding area, quality of life and exceptional light has inspired numerous famous artists, painters, writers and poets, some ow whom took up residence in the village. On our way to St Paul de Vence stop in the elegant city of Cannes, home to the Cannes Film Festival. Three nights Le Mas de Pierre (Superior Rooms), located in a two hectare Mediterranean park full of hundred year old olive trees, and on the doorstep of Saint Paul de Vence. Tonight enjoy an included 3 course dinner at the hotel.
Today is a free day to relax and enjoy the gorgeous hotel and explore the beautiful town of St Paul de Vence.
Today enjoy an adventure on the glamorous French Riviera visiting Monaco and the medieval hillside village of Eze. We'll begin our day with a short visit to the glamorous Monte Carlo Casino, whose design was overseen by Charles Garnier, architect of the Paris opera. If this complex looks familiar, it's because you may have seen it in the James Bond films, "Never Say Never Again" and "Goldeneye." It was also the inspiration for the casino in Ian Fleming's first Bond book, "Casino Royale." From there we will head to the Prince's Palace where we will visit the State Rooms, see the Changing of the Guard and the private collection of Antique Cars of H.S.H Prince Rainier III. We'll then continue to the Cathedral, where we can pay homage to the Princes and Princesses of Monaco who are buried there. We'll finish our time in Monaco with our driver taking us for a spin around the road circuit of the Monaco Grand Prix. As we leave Monaco we'll drive through the village of La Turbie. Known as the "Terrace" of Monaco, due to the panoramic views of the Principality of Monaco before arriving at the enchanting hillside village of Eze where we will be rewarded with panoramic views of the French Riviera and Mediterranean Sea.
Today is a free day to relax and enjoy our lovely hotel and explore the beautiful town of Aix en Provence.
Today we are heading to the coast with visits to Marseilles and Cassis. First we'll drive up to the highest point in Marseille, Notre Dame de la Garde, for a spectacular 360° view of the city, coastline and mountains. Then we'll drive down into the heart of it where we'll visit The Old Port, the oldest neighborhood in France, le Panier, and the Fort St. Jean. After Marseilles we make our way to Cassis. You'll love the staggering view from one of the highest maritime cliffs in Europe overlooking this quaint fishing town. We'll finish the day with a 45 minute boat ride amongst the beautiful landscape of the Calanques National Park.
Today we will visit four of the most beautiful villages in the Luberon – Gordes, Rousillon, Lourmarin and Bonnieux. Loumarin - nestled in the middle of vineyards, olive groves and almond trees, you'll notice the Mediterranean influences as you stroll through its winding narrow streets, lined with lovely old houses. Continue on to Bonnieux, built on a plateau dominating the Luberon Valley. Wander along tiny cobbled lanes and get glimpses of superb residences dating back to the 16th century. Our next stop is the vibrant village of Roussilon. Savour views of magnificent red cliffs and quarries and enjoy a pleasant walk past ochre buildings, art galleries and restaurants. Finish the day with a visit to the charismatic sights of Gordes, where stone homes are packed tightly beneath rocky overhangs crowned with aromatic cedar.
This morning we are transferred to Marseilles for our Ryan Air flight to the Loire. On arrival we will be transferred to our hotel in Amboise. Elegant Amboise, childhood home of Charles VIII and final resting place of the incomparable Leonardo da Vinci, is gorgeously situated on the southern bank of the Loire, guarded by a soaring château. Three nights Le Clos d'Amboise (Tradition and Comfort Rooms), located in the heart of Amboise.
Château de Villandry, built in the 16th century, is famous for the beauty of its Renaissance gardens. Unique in Europe, its five exceptional gardens – the ornamental kitchen garden, ornamental garden, water garden, medicinal garden and maze – are the perfect destination for those who love gardens and strolling. A viewpoint offers visitors a beautiful panorama of the beautiful and colourful setting. The castle, which has preserved its keep, contains a collection of Spanish furniture and antique paintings. After visiting Château de Villandry we head to Chateau de Chenonceau. Chateau de Chenonceau is the most visited and photographed chateau of the Loire Valley. It is often described as 'the ladies chateau' as throughout its history it is they who have most influenced its design and its destiny...it was not always the chateau we see today.
Today is free to enjoy Amboise. You may like to visit Chateau Amboise and/or Le Chateau du Clos Luce – the final home of Leonardo da Vinci.
All prices are per person in New Zealand dollars. Prices are based on payment by cash, eftpos, cheque or direct deposit only. A surcharge of 2% applies for payment by Visa or Mastercard. If you would prefer to share a room we will endeavour to match you up with someone of the same gender. If we cannot, you would be required to pay the single price. A non-refundable deposit of $3,500 per person is due at time of booking. Balance due 3 June 2019. Minimum tour number: 10. If the group falls below this number World Travellers Remuera reserves the right to cancel the tour in which case a full refund would be given. This would be known 3 months prior to departure. Once paid the full amount is non-refundable.
For more information please phone Tracey on 09 520 5600 or 021 1398539.


Cellphones broadcast location data to towers even when they're not being used or the GPS is turned off. The NSA gets cellphone location data by "tapping into the cables that connect mobile networks globally and that serve U.S. cellphones as well as foreign ones." This allows the agency to keep tabs on someone, even if he or she travels abroad with a cellphone, "into confidential business meetings or personal visits to medical facilities, hotel rooms, private homes and other traditionally protected spaces." Once the NSA has that information, it can use it to figure out who a person is visiting, where, and how often.
This is the big one—"A central feature of each of these tools is that they do not rely on knowing a particular target in advance, or even suspecting one. They operate on the full universe of data in the NSA's [repository] which stores trillions of metadata records, of which a large but unknown fraction include locations," wrote the Post. An intelligence lawyer said that the data collection is not covered by the Fourth Amendment, which outlaws unreasonable searches and seizures.?


text:
package com.destin.moeviewer.model.shuushuu;

import com.google.gson.annotations.SerializedName;

import java.util.List;

public class ShuushuuList {
    @SerializedName("max_image_id")
    private int maxImageId;

    private List<ShuushuuPost> images;

    public int getMaxImageId() {
        return maxImageId;
    }

    public void setMaxImageId(int maxImageId) {
        this.maxImageId = maxImageId;
    }

    public List<ShuushuuPost> getImages() {
        return images;
    }

    public void setImages(List<ShuushuuPost> images) {
        this.images = images;
    }
}

redpajama_set_name: RedPajamaGithub


Kate Stephenson is a retired writer who can't seem to keep her thoughts to herself. She is clearly obsessed with growing all manner of plants, especially vegetables and other edibles. See for yourself if her enthusiasm for gardening is contagious!
My peas are cold. Usually, by this time of year, my peas are climbing the fence and starting to make flowers. But this year, yikes! I still have snow on the ground! What the heck is up with that? Brrrr! I'm contemplating moving back to SoCal. Just kidding.
Remember I said that I was going to start my snow peas early, in the greenhouse, and then set them out after they were really robust, so that the little critters wouldn't get them. Well, I'm still waiting for the ground to thaw. All kinds of Asian greens and lettuces are waiting, too. It will soon be too warm in the greenhouse for the lettuce and greens to flourish. They want the cool raised bed in the fenced garden corner, shaded by the berry vines. (The fence is 8 feet tall, and it keeps the deer out.) We can usually keep the greens going into Summer and Fall thanks to the berries' exuberant growth.
Well, move over, peas and greens, because tomatoes and veggies and basket stuffers and herbs (and more tomatoes) are coming to town! I have twenty-four feet by twenty inches of seedling heat mats and grow lights to augment the weak sunshine. So many baby things are coming up. This is my favorite part of growing plants. Every morning I go out to the GH to check out who's popped up since yesterday! What fun. So many seedlings. I don't know how this happened, but I think I may need a bigger greenhouse. Or maybe just ANOTHER greenhouse.
Anyway, this lady says she has a great chicken story and that I will want to write about it. After a few minutes, it turns out that she has "adopted" a stray hen, and she can't keep it. Do I want take it off her hands? She thinks that this hen, whose name is Daisy, escaped from next door where they raise chickens for eggs and meat. Apparently, Daisy was smart enough to get out of there while the getting was good! So now what?
Turns out, we went and brought Daisy home. Twenty-five miles each way and Daisy complained the whole time. But we got here safely, kept her and our girls apart but face-to-face until we were sure she was healthy. Then, after a few dirty words and a little light skirmish, Daisy settled in to become the new top chicken. Apparently, she's a born leader, because she gets her choice of nesting box and roosting perch, and nobody will challenge her. All seem satisfied with the hierarchy, and it's a happy family flock again.
Surely one of these days when I wake up and look outside, the snow will finally be gone and Spring will have sprung. I can hardly wait. My hanging baskets, the fuchsias and orchids, are filling the greenhouse and they're ready to go outside! I've been feeding the fuchsias every week, and some of them are trying to set flowers already. Pinching fuchsias takes a lot of time when you have dozens of them. Let's hope we have a long, sunny Summer so that they can bloom for months and months.
Well, I am anxious to go and check my seedlings and make space to plant even more seeds, (It's a sickness that seems to only get worse each year!) so I can grow more stuff. What am I going to do with it all?
Until next time, I remain your dirt-stained-fingernail friend.


text:
Extracellular dGMP Enhances Deinococcus radiodurans Tolerance to Oxidative Stress
Mingfeng Li ,
Contributed equally to this work with: Mingfeng Li, Hongxing Sun
Affiliation Key Laboratory for Nuclear-Agricultural Sciences of Chinese Ministry of Agriculture and Zhejiang Province, Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou, China
Hongxing Sun ,
Affiliations Key Laboratory for Nuclear-Agricultural Sciences of Chinese Ministry of Agriculture and Zhejiang Province, Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou, China, Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai, China, Shanghai Institute of Immunology, Institutes of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China
Qiong Feng,
Huiming Lu,
Ye Zhao,
Hui Zhang,
Xin Xu,
Jiandong Jiao,
Liangyan Wang ,
* E-mail: yjhua@zju.edu.cn (YH); liangyanwang@zju.edu.cn (LW)
Yuejin Hua
Mingfeng Li,
Hongxing Sun,
Liangyan Wang,
Mingfeng Li Hongxing Sun ... Yuejin Hua
Free extracellular DNA provides nutrition to bacteria and promotes bacterial evolution by inducing excessive mutagenesis of the genome. To understand the influence of extracellular DNA fragments on D. radiodurans, we investigated cell growth and survival after extracellular DNA or dNMPs treatment. The results showed that the extracellular DNA fragments inhibited the growth of D. radiodurans. Interestingly, dGMP, a DNA component, enhanced D. radiodurans tolerance to H2O2 and gamma-radiation significantly. Further experiments indicated that extracellular dGMP stimulated the activity of one catalase (KatA, DR1998), and induced gene transcription including the extracellular nuclease (drb0067). When this only extracellular nuclease gene (drb0067) in D. radiodurans was deleted, the mutant strain showed more sensitive to H2O2 and gamma-radiation than the wild type strain. These results suggest that DRB0067 plays an important role in oxidative stress resistance. Taken together, we proposed a new anti-oxidation mechanism in D. radiodurans. This mechanism acts to increase expression levels of DRB0067 which then secretes active nuclease to degrade extracellular DNA fragments. The extracellular nuclease has a two-fold benefit, creating more free dNTPs for further cell protection and the removal of extracellular DNA fragments.
Citation: Li M, Sun H, Feng Q, Lu H, Zhao Y, Zhang H, et al. (2013) Extracellular dGMP Enhances Deinococcus radiodurans Tolerance to Oxidative Stress. PLoS ONE 8(1): e54420. https://doi.org/10.1371/journal.pone.0054420
Editor: John R. Battista, Louisiana State University and A & M College, United States of America
Received: September 9, 2012; Accepted: December 11, 2012; Published: January 24, 2013
Copyright: © 2013 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from National Natural Science Foundation of China (30830006, 31070080), a major project for genetically modified organisms breeding from the Ministry of Agriculture of China (2009ZX08009-075B, 2011ZX08009-003-002), a grant from Special Fund for Agroscientific Research in the Public Interest from the Ministry of Agriculture of China (201103007), and the Fundamental Research Funds for the Central Universities from Zhejiang University (2012FZA6014). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Bacteria cell death releases cytoplasmic contents, including DNA components into the microenvironment [1]. In addition, many living bacteria such as Acinetobacter, Azotobacter, Bacillus, Deinococcus, Neisseria and Pseudomonas release DNA into the surrounding environment during cell growth [1]–[6]. These bacteria benefit in several ways from free extracellular DNA and its degradation product [7]–[11]. For instance, the uptake of extracellular DNA, from the same or different organisms, promotes the evolution of bacteria. This occurs via horizontal gene transfer, such as transformation, transduction, or conjugation between bacteria [7], [8]. Extracellular DNA is also required for the initial establishment of bacterial biofilms, such as in Pseudomonas aeruginosa. The degradation of extracellular DNA by DNase I can strongly inhibit biofilm formation [9], [10]. Extracellular DNA, both homospecific and heterospecific, is known as an important nutrient source for organisms [11]. However, if extracellular DNA is not degraded immediately, it can threaten the survival of organisms by reincorporating damaged bases into the genome [12]. In most case, extracellular DNA components are degraded by extracellular nucleases secreted by many kinds of bacteria [13]–[16]. As a result, a threat to the organism is removed, and its by-products, dNMPs, are a nutrient source for bacteria [11], [12], [16].
Deinococcus radiodurans are extremely resistant to ionizing radiation, UV radiation, hydrogen peroxide and desiccation [12], [17]–[21]. The high resistance of this bacterium to reactive oxygen species (ROS) results from the strong ability of oxidative resistance [22] and an efficient DNA repair mechanism [23], [24]. Ionizing radiation [17] or UV [25] radiation attacks intracellular DNA producing large amounts of damaged oligonucleotides within the nucleotide pool [26]. These damaged oligos are exported into the surrounding medium and finally degraded [27]. However, it is not known if D. radiodurans, the most ionizing radiation resistant bacteria, has the ability to reuse these damaged extracellular DNA fragments.
Recently, Daly et al. demonstrated that D. radiodurans ultrafiltrate, which was enriched in Mn, phosphate, peptides, nucleosides and bases, could protect proteins from ionizing radiation-induced ROS damage [28], [29]. These findings implied that degradation and re-absorption of damaged DNA components might contribute to this organism's extreme ROS resistance. Here, we investigated the effects of extracellular DNA fragments and dNMPs on cell growth, H2O2 resistance, as well as UV and gamma-radiation in both D. radiodurans and E. coli. Our results indicated that the uptake of extracellular DNA fragments represented a new mechanism of protection from oxidative damage.
Extracellular DNA fragments inhibit the growth of D. radiodurans but not E. coli
Free extracellular DNA is abundant in the environment, and its existence may have an effect on the growth of bacteria. To understand this effect, we grew D. radiodurans and E. coli cells in the presence or absence of 3.6 mg/ml DNA fragments or dNMPs. We observed large amounts of DNA fragments resulted in a distinct growth inhibition of D. radiodurans. Absorption reading (OD600) was 0.26 after 10 hours in the presence of DNA fragments, while control cells OD600 reading was 2.38 and that with dNMPs treatment was 2.19 (Fig. 1A). In E. coli, the OD600 values of control, DNA fragments and dNMPs treated groups were almost the same (OD600≈2.3) after 4 hours, which indicates neither DNA fragments nor dNMPs affect the growth of E. coli (Fig. 1B). These data suggested that DNA fragments and dNMPs have different effects on D. radiodurans and E. coli growth rates. Extracellular DNA fragments instead of dNMPs were harmful to D. radiodurans cells growth.
Figure 1. D. radiodurans and E. coli cell growth after DNA fragments or dNMPs treatment.
(A) Growth of D. radiodurans after the addition of 3.6 mg/ml DNA fragments or dNMPs. (B) Growth of E. coli K-12 after the addition of 3.6 mg/ml DNA fragments or dNMPs. Values are the mean ± standard deviation of three independent experiments. R1, D. radiodurans wild type strain.
Extracellular dGMP greatly enhances D. radiodurans tolerance to H2O2
The influence of extracellular DNA fragments or dNMPs on D. radiodurans and E. coli cell survival under oxidative stress was evaluated. The presence of DNA fragments caused a modest decrease in H2O2 resistance in D. radiodurans (Fig. 2A). Similarly, the presence of DNA fragments did not have an obvious effect on E. coli resistance to H2O2 as well (Fig. 2B). However, the survival rate of D. radiodurans was dramatically increased when 10 mM dNMPs was present (Fig. 2A). There was a 33-fold increase in survival when compared to samples without dNMPs treatment. In E. coli, no distinct difference was observed between either the DNA fragments or dNMPs treatment groups (Fig. 2B). To understand which dNMPs accounted for this effect, dAMP, dTMP, dCMP and dGMP were separately added to D. radiodurans growths. Here we observed only dGMP had an effect, which dramatically increased H2O2 resistance by approximately 57-fold (Fig. 3). In addition, dGMP enhanced the resistance to gamma-radiation, but not UV (Fig. S1A). In sum, extracellular dGMP has an important role in D. radiodurans anti-oxidation, but not in E. coli.
Figure 2. H2O2 sensitivity in D. radiodurans and E. coli treated with DNA fragments or dNMPs.
(A) Sensitivity of D. radiodurans to H2O2 after the addition of 3.6 mg/ml DNA fragments or dNMPs. (B) Sensitivity of E. coli K-12 to H2O2 after the addition of 3.6 mg/ml DNA fragments or dNMPs. Each data point represents the mean±SD of three replicates. R1, D. radiodurans wild type strain.
Figure 3. H2O2 sensitivity in D. radiodurans treated with dAMP, dTMP, dCMP or dGMP.
Sensitivity of D. radiodurans to 50 mM H2O2 with the addition of DNA fragments (3.6 mg/ml), dNMPs, dAMP, dTMP, dCMP or dGMP (10 mM for each). Each data point represents the mean±SD of three replicates. R1, D. radiodurans wild type strain.
Extracellular dGMP induces KatA activity
In D. radiodurans, catalases and SODs protect proteins from ROS-mediated damage in vivo [30]. PAGE activity-staining assay reveals that D. radiodurans stain possesses activity corresponding to two catalases and one SOD, and stains that carry mutations in these genes (katA and sodA) are more sensitive to ionizing radiation than wild type [30]. In order to understand how dGMP increases D. radiodurans tolerance to H2O2, the activity change in catalases and SODs was measured after 2.5 mM dGMP was added to growth medium by PAGE activity-staining assay. The additional dGMP enhanced whole cell KatA activity by ∼50%, but had no effect on KatB or any of the SODs assayed (Fig. 4A/4B). It is possible that extracellular dGMP increases D. radiodurans tolerance to H2O2 by inducing KatA catalase activity in vivo.
Figure 4. Addition of extracellular dGMP increases the activity of KatA in D. radiodurans.
(A) Extracellular dGMP (2.5 mM) increased the activity of KatA, but not KatB. (B) Extracellular dGMP (2.5 mM) had no effect on the activity of SOD. (C) and (D) Quantification of the intensity of bands was performed using ImageJ. Each sample contains 80 µg of total protein. Values are the mean ± standard deviation of three independent measurements. R1, D. radiodurans wild type strain; KatA, catalase A; KatB, catalase B.
Extracellular dGMP stimulates transcription of anti-oxidation related genes
To better understand extracellular dGMP's involvement in anti-oxidation, the expression patterns of ROS response genes were investigated using Real-time quantitative PCR (Table 1). The addition of dGMP increased the transcriptional level of katA gene (dr1998) about 2.8-fold, but not any other catalase or SOD genes, which agrees with the results from the PAGE activity-staining assay. In other words, extracellular dGMP induces the activity of KatA by increasing the expression level of KatA in vivo.
Table 1. Influence of dGMP (2.5 mM) on D. radiodurans transcription levels.
The high intracellular Mn/Fe ratio in D. radiodurans could contribute to its remarkable resistance to environmental stresses [28], [29]. Here, the extracellular dGMP also increased the transcription levels of genes regulating the intracellular Mn/Fe ratio. DR2244, a sensory transduction histidine kinase, was induced by extracellular dGMP, which might be a signaling response to oxidative stress. Considering the role of extracellular nuclease in degrading extracellular DNA, the transcription of drb0067, the extracellular nuclease gene in D. radiodurans, was also investigated. When extracellular dGMP was present, transcription of drb0067 was stimulated (2-fold), thus enhancing the degradation of extracellular DNA fragments and increasing the pool of dNMPs.
Drb0067 encodes the only extracellular nuclease in D. radiodurans
Upon investigation of the D. radiodurans genome, we found that drb0067 is the only extracellular nuclease gene. To further explore the role of DRB0067 in D. radiodurans, a drb0067 null mutant (Δdrb0067) was constructed and verified by PCR (Fig. 5A). To test the mutant and wild type strains ability to degrade extracellular DNA, each strain was inoculated onto DNase test agars plates. A distinct clear zone was observed surrounding the wild type cells, but not Δdrb0067 (Fig. 5B). Moreover, there was no nuclease activity detected in the culture medium of Δdrb0067 (Fig. 5C). Bases on these two assays, we demonstrated DRB0067 is the only extracellular nuclease in D. radiodurans. And this extracellular nuclease is secreted through the secretory pathway since the deletion of secD/secF gene (dr1822) inhibited the secretion of DRB0067 (data not shown).
Figure 5. Disruption of drb0067 gene and its phenotypes test.
(A) PCR analysis of the mutant with primers 0067upF and 0067downR. (B) DNase Test Agar with Methyl Green to analyse R1 and Δdrb0067. 20 µl of cells (OD600≈1.0) were dripped onto DNase test agars plates. (C) Enzymatic activity to analyse R1 and Δdrb0067. The cells were cultured in TGY until the OD600≈2.5–3.0, and then centrifuged to collect the supernatant for enzyme reaction. (D) Transformation analysis of R1 and Δdrb0067. 1 µg pRADK plasmid was used for each transformation. (E) Growth of R1 and Δdrb0067 with the addition of 3.6 mg/ml DNA fragments or dNMPs. M denotes molecular standards. All the experiments are performed three times and values are mean ± standard deviation. R1, D. radiodurans wild type strain; Δdrb0067, the drb0067 null mutant.
The extracellular nucleases act as a modulator for natural transformation in some bacteria, such as Vibrio cholerae [16]. Next, we investigated the transformation frequency for the Δdrb0067 strain and found the transformation efficiency was ∼4.5-fold higher than the wild type strain (Fig. 5D). These results suggest that the DRB0067 protein is also an important modulator for natural transformation in D. radiodurans.
We next investigated the effect of DNA fragments on growth in the wild type and Δdrb0067 strains. In the absence of extracellular DNA fragments, wild type and Δdrb0067 strains had similar growth patterns except that the mutant strain (OD600≈4.61) exhibited slightly lower OD readings than wild type (OD600≈5.75) at the stationary phase. However, in the presence of DNA fragments, Δdrb0067 grwoths were more sensitive than the wild type growths (Fig. 5E). Under these conditions, the wild type strain entered stationary phase after 29 hours (OD600≈4.60), whereas Δdrb0067 was still in logarithmic phase (OD600≈2.45) at that time. Therefore, the presence of DNA fragments resulted in a severe cell growth decline in the Δdrb0067 strain when compared to wild type.
Deletion of drb0067 impairs H2O2 resistance of D. radiodurans
Our experiments indicated that the extracellular dGMP, not extracellular DNA, enhanced the resistance of D. radiodurans to H2O2 and gamma-radiation. To understand the role of DRB0067 in this process, the H2O2 resistance of wild type and Δdrb0067 strains were measured. These results, which were expected, revealed the drb0067 mutation to have a decreased resistance to H2O2. The survival rate of the Δdrb0067 strain was 6 times lower than that of the wild type strain under 30 mM H2O2 treatment (Fig. 6A/6B). Furthermore, the Δdrb0067 was more sensitive to gamma-radiation, though not UV radiation (Fig. S1B). Next, the effects of oligo(dG)50 and dGMP on Δdrb0067 resistance were investigated with varying concentrations of H2O2. Here, we found that the addition of dGMP (2.5 mM) restored the mutant strain's resistance to H2O2. The survival fraction with dGMP treatment was about 4 times higher than that with oligo(dG)50 treatment, and 5 times higher than control under the stress of 30 mM H2O2 (Fig. 6C/6D). In addition, D. radiodurans secreted more active extracellular nuclease after gamma-radiation treatment (Fig. S2). These results suggested that DRB0067 might be involved in ROS resistance through degradation of extracellular DNA to dNMPs, which increases the pool of dGMP. This pool then aids in enhancing the D. radiodurans tolerance to oxidative stress.
Figure 6. Sensitivity of Δdrb0067 strain subjected to H2O2.
(A) and (B) Sensitivity of R1 and Δdrb0067 to different concentration of H2O2. (C) and (D) Sensitivity of Δdrb0067 to different concentration of H2O2 with the addition of 0.05 mM oligo(dG)50 or 2.5 mM dGMP. Data represent the means ± deviations of three independent experiments. R1, D. radiodurans wild type strain; Δdrb0067, the drb0067 null mutant.
Here, we report extracellular dGMP enhanced the resistance of D. radiodurans to H2O2 and gamma-radiation. These findings suggest extracellular dGMP plays an important role in the organism's anti-oxidation pathway. Interestingly, we observed extracellular dGMP enhance the expression levels of KatA (DR1998) in D. radiodurans. These findings have yielded clues that may reveal the underlying mechanism of extracellular dGMP in anti-oxidation. Moreover, we found extracellular dGMP modulated expression of other genes, including one sensory transduction histidine kinase gene (dr2244), and genes involved in the regulation of manganese/iron. The up-regulation of these genes may enhance the tolerance of H2O2 and gamma-radiation as well.
Both cAMP and cGMP, as second messengers, have been widely studied in eukaryotes. It has been reported that cGMP can protect eukaryotic cells from oxidative stress, as in endothelial progenitors [31]. In bacteria, the production of cGMP has also been demonstrated. However, the physiological role of cGMP is still not well defined [32]. Recently Misra et al. reported that a DNA damage-induced signaling mechanism including secondary messengers and signaling enzymes exist in D. radiodurans [33]. Considering that the GC content (66.6%) in D. radiodurans is higher than most of other bacteria [27], we hypothesize that guanine base, obtained from the breakdown of extracellular dGMP [1], could be converted into cGMP after absorbed, and protect the cells from oxidative damage. However, further experiments are required to full characterize how dGMP enhances D. radiodurans tolerance to oxidative stress.
In D. radiodurans, DRB0067, encoded on the mega-plasmid, is an extracellular nuclease [27]. We demonstrated the absence of DRB0067 completely abolished nuclease activity from medium (Fig. 5B/5C). These results indicate DRB0067 is the only extracellular nuclease in this bacterium. Further experiments suggested this extracellular nuclease is secreted through the secretory pathway. Under normal growth conditions, DRB0067 is bound to the carotenoid-containing hexagonal layer [27]. Interestingly, the nuclease is released into the medium after ionizing radiation [34]–[36], indicating that this nuclease participates in D. radiodurans post-irradiation recovery. This hypothesis was indirectly supported by a transcriptome study reporting that the expression level of DRB0067 was induced after ionizing radiation [37]. Here, we have found direct evidence that deletion of drb0067 decreases the survival ability of D. radiodurans after H2O2 or gamma-radiation treatment. Moreover, we report gamma-radiation enhances the secretion of DRB0067, indicating an important role for this protein in anti-oxidation. It is quite possible DRB0067 is required to degrade damaged DNA fragments that are exported after radiation damage to avoid genome mutagenesis. This mechanism also provides essential nutrition for cells' recovery. Interestingly, dGMP, one product from DNA degradation, dramatically stimulates D. radiodurans resistance to oxidative stress, which may indicate another purpose for DRB0067 induction after DNA damage stress.
Free extracellular DNA is a source of natural transformations. By degrading extracellular DNA, nucleases act as a modulator for natural transformation, such as the Dns protein in V. cholerrae [16]. In our experiments, an absence of DRB0067 increased the natural transformation rate of D. radiodurans (Fig. 5D), suggesting that DRB0067 acts as a natural transformation modulator in this bacterium. Free extracellular DNA is also a source of nutrients for organisms. However, the presence of large amounts of extracellular DNA inhibits D. radiodurans cell growth. Furthermore, the disruption of the drb0067 gene amplified this effect. While in E. coli extracellular DNA had no effect on its growth. These findings support the importance of extracellular nuclease in this organism. Taken together, the degradation of extracellular DNA into dNMPs by extracellular nuclease DRB0067 serves many purposes in D. radiodurans. First by converting extracellular DNA into nutrients, thus reversing cell growth inhibition, and finally by enhancing D. radiodurans tolerance to oxidative stress.
Strains, media, and growth conditions
The bacterial strains and plasmids used in this study are listed in Table S1. D. radiodurans (ATCC 13939) was used as the wild-type strain and for construction of mutants. All cells were cultured at 30°C in TGY medium (0.5% Bacto tryptone, 0.3% Bacto yeast extract, 0.1% glucose) or on TGY plates containing 1.5% Bacto agar powder. E. coli strain DH5α was used for propagation of plasmids and was grown at 37°C on LB media with appropriate antibiotics.
Disruption of the drb0067 gene in D. radiodurans
Disruption of D. radiodurans drb0067 gene was performed using the double crossover recombination method [38]. In brief, the 0067upF and 0067upR primers (Table S1) were used for the upstream fragment and 0067downF and 0067downR primers (Table S1) for the downstream fragment. The upstream and downstream were digested by HindIII and BamHI respectively, and ligated to the BamHI-HindIII fragment of the kanamycin resistance cassette containing the groEL promoter. The kanamycin resistance cassette was obtained from pRADK, a shuttle plasmid modified from pRADZ3 [39]. The fragment was then transformed into D. radiodurans R1 with CaCl2 as described previously [40]. The mutant strain was obtained on TGY agar with 30 µg/ml kanamycin, and was confirmed by PCR with the primers 0067upF and 0067downR primers.
Growth curve and survival fraction tests
Bacteria growth was determined using optical density data (OD) at 600 nm. The strains were cultured in 20 ml liquid TGY or LB medium until an OD600≈0.15 was reached, and DNA fragments (Herring sperm DNA from Sigma-Aldrich Company) or dNMPs, at a final concentration of 3.6 mg/ml, were added. The cultures were incubated with 250 rpm at 30°C or 37°C and samples were taken to measure the OD600 value at different time. All experiments were repeated in triplicate.
For the sensitivity assay, the strains were cultured in 5 ml liquid TGY medium until an OD of OD600≈0.75 was reached. Then DNA fragments, dNMPs, dGMP, or oligo(dG)50 were added to the growths (Table S1). Cultures were grown for another 3 hours. As a negative control autoclaved distilled water was added to a culture of each strain. After washed and diluted to an appropriate concentration with PBS solution, the cultures were treated with different concentrations of H2O2 (25 mM, 50 mM or 15 mM, 30 mM) for 30 min at 4°C. After treatment, the cells were plated on TGY plates and incubated at 30°C for 3 days before colonies were enumerated. The H2O2 survival assays on E. coli were performed as described above, except the cells were treated with 20 mM or 40 mM H2O2, plated on LB agar, and incubated at 37°C for 15 hours. Survival fraction (%) was calculated using the following equation: Survival fraction (%) = Nsample/Ncontrol×100%, where Ncontrol is the number of control colonies and Nsample is the number of H2O2 treated colonies. For the dripping test, the cultures were washed and serially diluted 1∶10 with PBS solution, and then treated with H2O2 (10 mM, 20 mM or 30 mM), gamma-radiation (2.5 h for 2 kGy) or UV (408 J/m2) separately [41]. 20 µl of cells were dripped onto TGY plates.
Transformation test
The plasmid pRADK was used to test the effect of the extracellular nuclease DRB0067 on the natural transformation. Here, 1 µg plasmid was used for each transformation. The pRADK was then transformed into D. radiodurans R1 with CaCl2 as described previously [40]. Natural transformation frequencies were determined using the following equation: Natural transformation frequencies = NTK/NTGY, where NTK is the number of clones on the TGY plates with 30 µg/ml kanamycin and NTGY is the number of clones on the TGY plates.
Extracellular nuclease activity assay
DNase test agars were used to test the extracellular nuclease activity. The cells were cultured in TGY until an OD of OD≈1.0 was reached, then 20 µl of cells were dripped onto DNase test agars plates (plates contain 42.0 g of DNase Agar Base (Qingdao Hope Bio-Technogy Co., Ltd, China), 0.05 g of methyl green and 2 g of glucose per liter of distilled water). Cultures were incubated at 30°C for 3 days. We used pRADK plasmid to test the activity of nuclease outside of the cells. The cells were cultured in TGY, and then centrifuged to collect the supernatant for enzyme reaction, during which 10 mM MgCl2 was added.
Activity measurement of Catalase and SOD
Cells were treated with dGMP (2.5 mM) when they reached OD≈0.8, incubated for 3 hours and then disrupted with an ultrasonicator. The protein concentration of the supernatant was measured by the Bradford's method [42]. The catalase activity was assayed by the horseradish peroxidase-diaminobenzidine method [43]. In detail, samples were separated, using electrophoresis, in an 8% non-denaturing polyacrylamide gel matrix at 4°C for 4–5 hours (15 mA). The gel matrix was then washed with distilled water for 3 times. Next, it was incubated with a 0.06% H2O2 solution for 20 min under slow shaking. The gel was washed again and then incubated with FeCl3 (2%)/K3Fe(CN)6 (2%) (V/V = 1∶2) until bright strips appeared on the gel. For the assay of SOD activities, a 10% non-denaturing polyacrylamide gel was used according to the nitroblue tetrazolium-riboflavin method [44]. This method was nearly the same to the one described above except for the gel-running time (2–2.5 h) and the staining solution (2.45 mM nitroblue tetrazolium chloride (NBT), 28 mM TEMED, 28 µM riboflavin, and 100 mM EDTA, pH = 7.8). The gel was stained for 30 min and then exposed under lamplight until the bright strips appeared. For the activity measurement experiments 80 µg proteins were used per lane.
Real-time quantitative PCR
Real-time quantitative PCR was used to determine the influence of the extracellular dGMP on the expression levels of Catalases, SODs and other genes of interest in D. radiodurans. In short, cells were grown to an OD600≈0.2 and then 2.5 mM dGMP was added. Cells were harvested by centrifugation at 4000 rpm for 5 min at 4°C when an OD600 of 0.4∼0.45 was reached. The extraction of total RNA and cDNA synthesis were performed as described previously [45]. SYBR Premix Ex Taq™ (TaKaRa Biotechnology (Dalian) Co, Ltd, China) was used for amplification, and all assays were performed using the STRATAGENE Mx3005P™ Real-time detection system.
Figure S1.
UV and gamma-ray sensitivity in R1 and Δdrb0067 strains. (A) Sensitivity of R1 to UV (408 J/m2) and gamma-radiation (2 kGy) with the addition of 10 mM dNMPs or 10 mM dGMP. (B) Sensitivity of R1 and Δdrb0067 to UV (408 J/m2) and gamma-radiation (2 kGy). R1, D. radiodurans wild type strain; Δdrb0067, the drb0067 null mutant.
(TIF)
Gamma-radiation enhances the secretion of active extracellular nuclease. The cells were cultured in TGY until the OD600≈1.0, treated with 2 kGy or 4 kGy gamma-radiation, and then centrifuged to collect the supernatant for enzyme reaction. The extracellular nuclease is secreted more from D. radiodurans after treatment. But no obvious extracellular nuclease activity change is observed from Δdrb0067 after gamma-radiation treatment. M denotes molecular standards. R1, D. radiodurans wild type strain; Δdrb0067, the drb0067 null mutant.
Table S1.
Strains, plasmids and primers used in this study.
Conceived and designed the experiments: YH LW ML HS. Performed the experiments: ML HS. Analyzed the data: ML HS QF HL YZ. Contributed reagents/materials/analysis tools: QF YZ HZ XX JJ. Wrote the paper: ML HS HL.
1. Nielsen KM, Johnsen PJ, Bensasson D, Daffonchio D (2007) Release and persistence of extracellular DNA in the environment. Environ Biosafety Res 6: 37–53.
2. Dillard JP, Seifert HS (2001) A variable genetic island specific for Neisseria gonorrhoeae is involved in providing DNA for natural transformation and is found more often in disseminated infection isolates. Mol Microbiol 41: 263–277.
3. Hamilton HL, Dominguez NM, Schwartz KJ, Hackett KT, Dillard JP (2005) Neisseria gonorrhoeae secretes chromosomal DNA via a novel type IV secretion system. Mol Microbiol 55: 1704–1721.
4. Lorenz MG, Wackernagel W (1994) Bacterial gene transfer by natural genetic transformation in the environment. Microbiol Rev 58: 563–602.
5. Yin X, Stotzky G (1997) Gene transfer among bacteria in natural environments. Adv Appl Microbiol 45: 153–212.
6. Thomas CM, Nielsen KM (2005) Mechanisms of, and barriers to, horizontal gene transfer between bacteria. Nat Rev Microbiol 3: 711–721.
7. Davison J (1999) Genetic exchange between bacteria in the environment. Plasmid 42: 73–91.
8. Dreiseikelmann B (1994) Translocation of DNA across bacterial membranes. Microbiol Rev 58: 293–316.
9. Whitchurch CB, Tolker-Nielsen T, Ragas PC, Mattick JS (2002) Extracellular DNA required for bacterial biofilm formation. Science 295: 1487.
10. Steinberger RE, Holden PA (2005) Extracellular DNA in single- and multiple-species unsaturated biofilms. Appl Environ Microbiol 71: 5404–5410.
11. Finkel SE, Kolter R (2001) DNA as a nutrient: novel role for bacterial competence gene homologs. J Bacteriol 183: 6288–6293.
12. Battista JR (1997) Against all odds: the survival strategies of Deinococcus radiodurans. Annu Rev Microbiol 51: 203–224.
13. Ichige A, Oishi K, Mizushima S (1988) Isolation and characterization of mutants of a marine Vibrio strain that are defective in production of extracellular proteins. J Bacteriol 170: 3537–3542.
14. Dodd HN, Pemberton JM (1999) The gene encoding a periplasmic deoxyribonuclease from Aeromonas hydrophila. FEMS Microbiol Lett 173: 41–46.
15. Brnakova Z, Godany A, Timko J (2005) An extracellular endodeoxyribonuclease from Streptomyces aureofaciens. Biochim Biophys Acta 1721: 116–123.
16. Blokesch M, Schoolnik GK (2008) The extracellular nuclease Dns and its role in natural transformation of Vibrio cholerae. J Bacteriol 190: 7232–7240.
17. Vukovic-Nagy B, Fox BW, Fox M (1974) The release of a deoxyribonucleic acid fragment after x-irradiation of Micrococcus radiodurans. Int J Radiat Biol Relat Stud Phys Chem Med 25: 329–337.
18. Minton KW (1994) DNA repair in the extremely radioresistant bacterium Deinococcus radiodurans. Mol Microbiol 13: 9–15.
19. Battista JR, Earl AM, Park MJ (1999) Why is Deinococcus radiodurans so resistant to ionizing radiation? Trends Microbiol 7: 362–365.
20. Makarova KS, Aravind L, Wolf YI, Tatusov RL, Minton KW, et al. (2001) Genome of the extremely radiation-resistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics. Microbiol Mol Biol Rev 65: 44–79.
21. Ghosal D, Omelchenko MV, Gaidamakova EK, Matrosova VY, Vasilenko A, et al. (2005) How radiation kills cells: survival of Deinococcus radiodurans and Shewanella oneidensis under oxidative stress. FEMS Microbiol Rev 29: 361–375.
22. Daly MJ, Gaidamakova EK, Matrosova VY, Vasilenko A, Zhai M, et al. (2007) Protein oxidation implicated as the primary determinant of bacterial radioresistance. PLoS Biol 5: e92.
23. Zahradka K, Slade D, Bailone A, Sommer S, Averbeck D, et al. (2006) Reassembly of shattered chromosomes in Deinococcus radiodurans. Nature 443: 569–573.
24. Slade D, Lindner AB, Paul G, Radman M (2009) Recombination and replication in DNA repair of heavily irradiated Deinococcus radiodurans. Cell 136: 1044–1055.
25. Boling ME, Setlow JK (1966) The resistance of Micrococcus radiodurans to ultraviolet radiation. 3. A repair mechanism. Biochim Biophys Acta 123: 26–33.
26. Slade D, Radman M (2011) Oxidative stress resistance in Deinococcus radiodurans. Microbiol Mol Biol Rev 75: 133–191.
27. White O, Eisen JA, Heidelberg JF, Hickey EK, Peterson JD, et al. (1999) Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1. Science 286: 1571–1577.
28. Daly MJ, Gaidamakova EK, Matrosova VY, Vasilenko A, Zhai M, et al. (2004) Accumulation of Mn(II) in Deinococcus radiodurans facilitates gamma-radiation resistance. Science 306: 1025–1028.
29. Daly MJ, Gaidamakova EK, Matrosova VY, Kiang JG, Fukumoto R, et al. (2010) Small-molecule antioxidant proteome-shields in Deinococcus radiodurans. PLoS One 5: e12570.
30. Markillie LM, Varnum SM, Hradecky P, Wong KK (1999) Targeted mutagenesis by duplication insertion in the radioresistant bacterium Deinococcus radiodurans: radiation sensitivities of catalase (katA) and superoxide dismutase (sodA) mutants. J Bacteriol 181: 666–669.
31. Curatola AM, Xu J, Hendricks-Munoz KD (2011) Cyclic GMP protects endothelial progenitors from oxidative stress. Angiogenesis 14: 267–279.
32. Linder JU (2010) cGMP production in bacteria. Mol Cell Biochem 334: 215–219.
33. Kamble VA, Rajpurohit YS, Srivastava AK, Misra HS (2010) Increased synthesis of signaling molecules coincides with reversible inhibition of nucleolytic activity during postirradiation recovery of Deinococcus radiodurans. FEMS Microbiol Lett 303: 18–25.
34. Gentner NE, Mitchel RE (1975) Ionizing radiation-induced release of a cell surface nuclease from Micrococcus radiodurans. Radiat Res 61: 204–215.
35. Mitchel RE (1975) Letter: Origin of cell surface proteins released from Micrococcus radiodurans by ionizing radiation. Radiat Res 64: 380–387.
36. Tanaka A, Hirano H, Kikuchi M, Kitayama S, Watanabe H (1996) Changes in cellular proteins of Deinococcus radiodurans following gamma-irradiation. Radiat Environ Biophys 35: 95–99.
37. Liu Y, Zhou J, Omelchenko MV, Beliaev AS, Venkateswaran A, et al. (2003) Transcriptome dynamics of Deinococcus radiodurans recovering from ionizing radiation. Proc Natl Acad Sci U S A 100: 4191–4196.
38. Funayama T, Narumi I, Kikuchi M, Kitayama S, Watanabe H, et al. (1999) Identification and disruption analysis of the recN gene in the extremely radioresistant bacterium Deinococcus radiodurans. Mutat Res 435: 151–161.
39. Meima R, Lidstrom ME (2000) Characterization of the minimal replicon of a cryptic Deinococcus radiodurans SARK plasmid and development of versatile Escherichia coli-D. radiodurans shuttle vectors. Appl Environ Microbiol 66: 3856–3867.
40. Hua Y, Narumi I, Gao G, Tian B, Satoh K, et al. (2003) PprI: a general switch responsible for extreme radioresistance of Deinococcus radiodurans. Biochem Biophys Res Commun 306: 354–360.
41. Wang L, Xu G, Chen H, Zhao Y, Xu N, et al. (2008) DrRRA: a novel response regulator essential for the extreme radioresistance of Deinococcus radiodurans. Mol Microbiol 67: 1211–1222.
42. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.
43. Wayne LG, Diaz GA (1986) A double staining method for differentiating between two classes of mycobacterial catalase in polyacrylamide electrophoresis gels. Anal Biochem 157: 89–92.
44. Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44: 276–287.
45. Sun H, Li M, Xu G, Chen H, Jiao J, et al. (2012) Regulation of MntH by a dual Mn(II)- and Fe(II)-dependent transcriptional repressor (DR2539) in Deinococcus radiodurans. PLoS One 7: e35057.
Nucleases
Is the Subject Area "Nucleases" applicable to this article?
Catalases
Is the Subject Area "Catalases" applicable to this article?
Is the Subject Area "DNA damage" applicable to this article?
Is the Subject Area "Oxidative stress" applicable to this article?
Cell growth
Is the Subject Area "Cell growth" applicable to this article?
Is the Subject Area "Ionizing radiation" applicable to this article?
Deoxyribonucleases
Is the Subject Area "Deoxyribonucleases" applicable to this article?
Is the Subject Area "DNA transcription" applicable to this article?


Get Your Boise , Idaho Property Sold Today!
You probably checked Bing for a sentence similar to "Selling A House In Probate Idaho "– Well. . You've found Boise house seller resources online.
If time is of the essence… we routinely buy real estate within 96 hours. With that said, in order for something like that to happen we need to work together. We will request your help to wrap things up rapidly i.e. – take some house pictures. If for whatever reason we can not purchase more Boise houses we keep an extensive group of investors that acquire homes all over the nation and that would love the chance to buy your Boise , Idaho home.
We make our living by buying properties in every conceivable condition across the United States. Glymph Properties provides everything you need to make getting rid of your home as quick as possible. We are here to help you get rid of your property, our thoughtful process is what makes us stand head and shoulders above our competition.
If you want to chat, give us a call at: 888-509-6267 Ext 4, shoot me an email at akmauri@gpbuyshomes.com, or Free seller resources! We are involved in purchasing houses in. In case you are further away definitely reach out to us as we are always looking to expand into new service areas.


text:
Does Google care about unlicensed businesses that have Maps listings?
Thread starter Tim Colling
Tim Colling
LocalU Member
Here's a situation that I frequently encounter when working with my clients in California. The clients must have a state-issued license in order to provide services to California residents. The licenses are costly and hard to get. Moreover, every office of these companies must be separately licensed, at a cost of more than $5,000 for each office.
Consequently, many companies dishonestly (in my view) list additional "offices" on maps as SABs where those additional "offices" are employees' residences, clients' residences, etc. They cannot legally see clients in these "offices" without a license. It's very easy for them to list them as SABs on Google maps, though.
Are they violating the Google Maps terms of service? If so, under what "report an edit" category should I report them?
Latest Blog: Lead Generation: What Types of Lead Generation Services are Available?
Margaret Ornsby
In order to qualify for a Google My Business listing, a business must make in-person contact with customers during its stated hours.
That's the guidelines, so your interpretation is correct and yes, they should be reported.
In those instances I use the Google Maps "suggest an edit", then select Place is permanently closed or has never existed, then Spam.
If you get no joy from the report (edit declined), then use the GMB forum. From there it can be escalated to the spam team for manual removal.
Unfortunately their spam detection systems aren't as robust as we would sometimes like, so the forum gives you space to add evidence to support your case. The suggest an edit form doesn't give you room for that.
It also pays to check back. Often businesses will call support, spin a story and get those listings reinstated. So you ping back on the thread, explain what's happened and it can be escalated again.
Reactions: djbaxter and Tim Colling
I second everything that Margaret said.
Top Poster Of Month
Google doesn't state in their guidelines that a business license is required to be listed. What they do say is what Margaret mentioned that they have to have in-person contact with their customers. This doesn't mean the contact has to happen at their office. If they have a home office, they would be an SAB if they go to meet their customers.
The second rule that would come into play is that SABs are only allowed 1 listing per the guidelines. So setting up 5 listings for the same company using employees houses is not allowed. They would all be removed except the 1 that they're allowed.
Google does use business licenses to help prove the existence of a business if it gets suspended.
Reactions: Tim Colling
JoyHawkins said:
Google doesn't state in their guidelines that a business license is required to be listed.
In this case it's a state professional license, a "home care organization" license that they cannot legally operate without. However, I think the point in your comments below is the one that I would rely upon to shut down the spammy location listings.
...The second rule that would come into play is that SABs are only allowed 1 listing per the guidelines. So setting up 5 listings for the same company using employees houses is not allowed. They would all be removed except the 1 that they're allowed...
Awesome! That's the best tool against these ne'er-do-wells then! You don't happen to know the URL where that rule applies, do you?
Joy, thank you very, very much!
https://support.google.com/business/answer/3038177?hl=en
Under the Address section: Service-area businesses—businesses that serve customers at their locations—should have one page for the central office or location and designate a service area from that point. Service-area businesses can't list a "virtual" office unless that office is staffed during business hours.
JoshuaMackens
Under the Address section: Service-area businesses?businesses that serve customers at their locations?should have one page for the central office or location and designate a service area from that point. Service-area businesses can't list a "virtual" office unless that office is staffed during business hours.
What if they have multiple offices in different cities across the state? Surely they would be allowed to have a listing per office?
If the above is true, is it just that you can't have more than 1 office in a city?
If it's a staffed office they can almost always have a listing. The problem with SABs usually is they aren't staffed. So people want to use employees' houses or other random addresses to get listed tons of times and that's not allowed.
So as long as it's staffed (by your own staff, not some virtual office staff), you should be fine.
Reactions: JoshuaMackens
...So as long as it's staffed (by your own staff, not some virtual office staff), you should be fine.
Just a sanity check: that would mean, "staffed during your normal or stated business hours", correct? In other words, if you say that the location is open from, say, 9am - 4pm, it has to be STAFFED during those hours, right?
Yes. However, if it's a service area business they can set their hours to match when they would answer the phone. For example, a plumber doesn't have a "staffed office location" but if they answer their phone and do emergency calls at 2am, they can say they are open then.


text:
Published on November 23rd, 2015 | by Cynthia Shahan
Major Poll Shows African Americans Support Clean Energy, Clean Jobs, & Clean Power Plan
November 23rd, 2015 by Cynthia Shahan
The NRDC recently published Themes and Findings from a Survey of African-Americans on Climate and Clean Energy. The press release shared that ⅔ of African-Americans believe global warming is a serious problem. This major poll shows African-Americans think shifting to clean energy will create jobs and hold down electric bills.
Like just about all other populations, African Americans want responsible action promoting employment. They want job creation that does not create a hazard for the environment or the worker. They want sustainable career paths that protect and encourage clean air and water supplies. An industry that changes the last century's pattern — and does not deplete or misuse resources with a deathly impact.
The poll indicates support for the Clean Power Plan to address the growing climate crisis. The information, via Green For All and the Natural Resources Defense Council, reports that "83 percent of African-Americans back setting the first-ever limits on carbon pollution from coal and gas-fired power plants under the Clean Power Plan's standards, which the Environmental Protection Agency finalized in August."
Who among us does not want clean air, clean water, and fair sustainability opportunities for all. As we know and NRDC reiterates, "as the nation drives down dangerous carbon pollution, it can drive up the use of clean, renewable wind and solar energy."
People know the shift to clean energy will also create new jobs. The NRDC reports that 6 times more African Americans believe clean energy will create more jobs than job losses. "And 57 percent believe that expanding clean energy will reduce— not raise—their energy costs."
Continuing: "The African American community has been hard hit by injustice, from violence against young people to disproportionate environmental harms from pollution, so it's no surprise the community wants action.
"It's time to hold polluters accountable and fight the pollution that causes climate change," said Adrianna Quintero,The African American community has been hard hit by injustice, from violence against young people to disproportionate environmental harms from pollution, so it's no surprise the community wants action. It's time to hold polluters accountable and fight the pollution that causes climate change," said Adrianna Quintero, director of Partner Engagement at NRDC.
Vien Truong, Director of Green For All, said: "This polling shows that communities of color care about climate change and want to be part of creating solutions to pollution. Climate change affects us all — and it hurts low-income communities and communities of color first and worst. This information shows a ripe opportunity to engage communities of color. By reflecting the diversity of our country, the climate movement will be stronger and better on equity and environment."
Mark Davis, CEO of minority-owned WDC Solar, said: "I am a Green For All Climate Champion, and renewable energy and energy efficiency are two pillars of our plan for low-income communities to lower the cost of energy, create green jobs for low-income residents, and improve the environment. The Clean Power Plan can accelerate an increase we've already seen in African-American participation in clean energy and can enhance economic empowerment in low-income communities."
Voices such as the Catholic Pope Francis and others have bridged this imaginary gap between religion and "green" — as Native American Indians historically have. In fact, pointing to the very sacred trust of purity, not toxicity, in environmental choices, Rev. Stacey Edwards-Dunn, executive minister of community engagement and transformation for Chicago's Trinity United Church of Christ, said: "Climate change not only imperils the natural wonders of God's creation, but it also threatens to cause enormous human suffering. Worldwide, we are facing severe drought, famine, disease, and disasters as a result of our climate crisis. We have a moral obligation to do all we can to lessen its impacts on our children and future generations."
Unfortunately, it is often the poor — albeit whatever color of skin — that suffers housing in the most toxic neighborhoods.
Yes, it is inherent that they want to protect their families. The impacts of a changing climate are dangerous to the poor and indigent due to lack of protective measures due to poverty — along with neighborhood problems such as "food deserts" that enable malnutrition. Add in the toxic load coming as an unpleasant byproduct of the factory, chemical industry, or power plant down the street, and the story gets even more depressing.
"The African American community has seen rates of childhood asthma increase a whopping 50% between 2001 and 2009. Sixty-eight percent of African Americans, furthermore, live within 30 miles of a coal-fired power plant," NRDC writes.
Here are some more key findings from the poll on African Americans' views on energy and climate matters, via NRDC:
African Americans view global warming as a major problem:
While crime, economic issues and education rank as the most serious issues, 60 percent of African Americans rank global warming among the most serious issues.
67 percent of African Americans say that action should be taken to reduce the threat of global warming.
They want action on climate change more than adults generally. Only 3 percent of African Americans say concern about global warming is unwarranted, compared to 13 percent of all Americans.
African Americans strongly believe a shift to clean energy will be good for jobs and their energy bills:
66 percent of African Americans say using more renewable energy will translate into new jobs, while only 11 percent expect job losses.
More than half, 57 percent, believe that shifting to cleaner energy will reduce their energy costs, and only 18 percent will increase those electricity bills.
African Americans overwhelmingly favor using more renewable energy than getting power from coal or nuclear:
87 support using more solar power and 83 support more wind energy.
42 percent favor getting more power from coal and 36 support more nuclear energy.
African Americans overwhelmingly embrace the Clean Power Plan and states developing state-based clean energy plans to implement it:
83 percent support the Clean Power Plan, with 63 percent in strong support. Just 9 percent oppose the plan.
82 percent back states developing clean energy plans that help cut carbon pollution, improve energy efficiency and boosting renewable energy.
Jamie Lynn Butler Our Children's Trust: The State Has A Sovereign Obligation Over "All The Earth And Air"
DC Project May Unlock PACE Funding For Affordable Housing Across US
100,000 Clean Energy Jobs In Illinois According To Survey
Investment In Renewable Energy Yields More Jobs Than Fossil Fuel Sector
Image by mattwalker69, via Foter.com (CC BY-SA)
Tags: African Americans, Clean Power Plans, CO2 Emissions, Green for All, NRDC, Waste Reduction, WDC Solar
Cynthia Shahan Cynthia Shahan started writing by doing research as a social cultural and sometimes medical anthropology thinker. She studied and practiced both Waldorf education, and Montessori education. Eventually becoming an organic farmer, licensed AP, and mother of four unconditionally loving spirits, teachers, and environmentally conscious beings born with spiritual insights and ethics beyond this world. (She was able to advance more in this way led by her children.)
Natural Gas Provided False Promise, Deception — Severe Health Problems From "Natural" Gas →
US Electricity: Solar Up 15%, Wind Up 9% →
Collective Action at the Crossroads: What to Watch in 2020 →
20 Reasons Why 2019 Gave Us Climate Hope →


Somewhat Quirky: Happy Birthday To Me Part Two!
Happy Birthday To Me Part Two!
I really wasn't planning on spending half of my birthday (Saturday) riding around looking for estate and garage sales. But I got this call...about a garage sale.....and after finding this little settee buried deep in the garage I was a gonner. I mean who wouldn't be? It is from one of the grand old hotels of Detroit. I've been wanting to find a settee for my dining room for at least seven years - and here it was. I don't even think I will reupholster it. I think I will clean it make a slip cover for it and then make a cushy six inch thick cushion for it. Oooooweee baby!
And then we were driving down the street looking for a sale and look what we found.....this little chest with a big FREE sign on it. It looked like there was another car checking it out, but it was a Porche and there was no way they could pick it up. Besides that - we hopped right out of the car snatched it up and it was in the back of the Durango before I could say Happy Birthday to Me! It's going to be pretty dang cute all painted up!
I don't usually buy froo-froo things but this candelabra was only $5. I though it might look cool on top of my china cabinet so I bought it. Now I regret it a little, because it is too tall and it's almost as big as my whole dining room. The other little candle is more my style and it was $1.
We hit the last five minutes of a moving sale and I got this little chair for a song. I think it is another candidate for paint and probably fabric.
I'm just a little bit in love with this mirror. The frame is metal, a little rusty, and the whole thing weighs like 50 pounds. And what about this little child's chalkboard? So cute. Not a clue how I will use it but that doesn't matter to me because it's so dang cute.
Now that was a great way to spend your birthday. Happy Birthday too. Great finds indeed.
You scored! Love that settee and the little French chair? Be still my heart. Sounds like your birthday was awesome. Is there any cake left? Bet not.
That little chalkboard would make a great sign out on your patio or something. What a bunch of treasure. I'm practically drooling. You'll have to show us how you fit these things in. I'd love to see what you do with them!
Wow you got good stuff. I liked it all. I can't believe that chest was free. It looks to be in good shape. BTW - in case you haven't checked out my "karen" pinterest board lately, I pinned a metallic chest. What do you think? And I made that Trisha Yearwood key lime cake last week and it was very delicious. Make it soon.
Someday you should do the "ones that got away" post.
You should also off set your spending with the $$ you will get from redoing the 5 harp back chairs I got from the guy with the settee. Not to mention the left over from product I'll buy for you to redo them.
p.s. Are you going to share your new cornbread recipe?
The birthday gods were with you! That tree slice is amazing!!!!!!!!!!!!! If you change your mind, ship it to NJ. The candelabra could make a fabulous bird feeder if you get tired of it inside.
I drove by the most fabulous desk today - but was all by my lonesome so could not lift it into my car. It's times like these, we need partners in crime (but since I have to hide my finds from the Hubs, I'm on my own).
Cheers and Happy B-day Part 2 to you.
Crazy good stuff! Happy birthday to you fer shure!
PS. That candelabra thing has Halloween written all over it for me.
What great birthday finds - love that chair and the chest of drawers AND the rediculously low prices !
OMG, girl you totally SCORED!!!!!
What awesome birthday finds!! I used to live in Grosse Pointe Park. It indulged my love of estate and garage sales! I had the basement of my flat stuffed with goodies that I now use in my 1926 home in Howell. I miss all the great sales in the area! If you ever find that you are needing to get rid of stuff, you can always donate it to Enchanted Makeovers. They makeover women's shelters in cottage style. I will be helping on their makeover at the Detroit Rescue Mission.
Wow is all I have to say! That settee, the chair...the freebies! Great day!
Happy Birhtday, you certainly scored,love the antique oak and the candelabra will be great for Halloween if nothing else...great finds, my favorite...the settee!!
The settee was a great find!!!! I also love the candelabra...It's stunning!!! I also love the chair and your free little dresser. Free...Isn't that a great word!!
You did great on your day of the hunt!!


110: /* Attaches the DMTS to the coarse level.
111:  * Under what conditions should we copy versus duplicate?
140: /* This could restrict auxiliary information to the coarse level.
213:    Use DMGetDMTSWrite() if write access is needed. The DMTSSetXXX API should be used wherever possible.
282:    The context is copied by reference. This function does not ensure that a context exists.
316:    not. If DM took a more central role at some later date, this could become the primary method of setting the residual.
349:    associated with the DM.
380:    associated with the DM.
413:    associated with the DM.
444:    associated with the DM.
477:    associated with the DM.
509:    not. If DM took a more central role at some later date, this could become the primary method of setting the residual.
570:    not. If DM took a more central role at some later date, this could become the primary method of setting the residual.
602:    not. If DM took a more central role at some later date, this could become the primary method of setting the residual.
637:    not. If DM took a more central role at some later date, this could become the primary method of setting the residual.
670:    associated with the DM.
702:    not. If DM took a more central role at some later date, this could become the primary method of setting the Jacobian.
736:    not. If DM took a more central role at some later date, this could become the primary method of setting the Jacobian.
769:    not. If DM took a more central role at some later date, this could become the primary method of setting the Jacobian.
803:    not. If DM took a more central role at some later date, this could become the primary method of setting the Jacobian.


Dear younger self… – University Unboxed.
I know you're an independent girl. You want to grow up, move out of your parents house, get your own apartment. You can do whatever you want– even eat cookies for breakfast!
But, it's not as amazing as it sounds. Sure, cookies for breakfast is great, if you can afford the cookies.
For the love of God, don't get more than one credit card. You'll regret it in the end. Trust me.
You don't need the newest iPhone. You don't even need the iPhone. Just get a phone.
College is a great experience. 'Put your stamp on the world.' But, don't let a boy hold you back from going to the college of your dreams. He won't even be around to see you succeed and graduate, anyway.
Speaking of college– pick a major that makes you happy. Not what your parents want you to do, or the world wants you to do. Everyone is a doctor now days. Be different.
Couponing is good. No, it's not just an old lady thing.
Find a man who is willing to support you. Not one that wants to take everything from you.
Don't listen to what other people say. Do what feels right in your heart, and listen to your own judgement. After all, it's your life, not theirs. You're going to live it. So do it the way you want.
Next Post Thanks For The Memories.